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Tobacco smoke is the leading preventable cause of death in the world and treatments aimed to increase success rate in smoking cessation by reducing nicotine dependence are sought. Activation of peroxisome proliferator-activated receptor-alpha (PPARα) by synthetic or endogenous agonists was shown to suppress nicotine-induced activation of mesolimbic dopamine system, one of the major neurobiological substrates of nicotine dependence, and nicotine-seeking behavior in rats and monkeys. An alternative indirect way to activate PPARα is inhibition of N-acylethanolamine acid amidase (NAAA), one of the major hydrolyzing enzyme for its endogenous agonists palmitoylethanolamide (PEA) and oleoylethanolamide (OEA). We synthetized a novel specific brain permeable NAAA inhibitor, AM11095. We administered AM11095 to rats and carried out brain lipid analysis, a functional observational battery (FOB) to assess toxicity, in vivo electrophysiological recording from dopamine cells in the ventral tegmental area, brain microdialysis in the nucleus accumbens shell and behavioral experiments to assess its effect on nicotine -induced conditioned place preference (CPP). AM11095 (5 and 25 mg/kg, i.p.) was devoid of neurotoxic and behavioral effects and did not affect motor behavior and coordination. This NAAA inhibitor (5 mg/kg i.p.) increased OEA and PEA levels in the hippocampus and cortex, prevented nicotine-induced activation of mesolimbic dopamine neurons in the ventral tegmental area, nicotine-induced elevation of dopamine levels in the nucleus accumbens shell and decreased the expression of nicotine CPP. Our results indicate that NAAA inhibitors represent a new class of pharmacological tools to modulate brain PEA/PPARα signalling and show potential in the treatment of nicotine dependence. |
Ischemia, a severe metabolic stress, increases adenosine levels and causes the suppression of synaptic transmission through adenosine A<sub>1</sub> receptors. Although temperature also regulates extracellular adenosine levels, the effect of temperature on ischemia-induced activation of adenosine receptors is not yet fully understood. Here we examined the role of adenosine A<sub>1</sub> receptors in mild hypothermia-mediated neuroprotection during the acute phase of ischemia. Severe ischemia-induced neurosynaptic impairment was reproduced by oxygen-glucose deprivation at normothermia (36 °C) and assessed with extracellular recordings or whole-cell patch clamp recordings in acute hippocampal slices in mice. Mild hypothermia (32 °C) induced the protection of synaptic transmission by activating adenosine A<sub>1</sub> receptors. Stricter hypothermia (28 °C) caused additional neuroprotective effects by extending the onset time to anoxic depolarization; however, this effect was not associated with adenosine A<sub>1</sub> receptors. The response of exogenous adenosine-induced inhibition of hippocampal synaptic transmission was increased by lowering the temperature to 32 °C or 28 °C. Hypothermia also reduced the function of dipryidamole-sensitive nucleoside transporters. These findings suggest that an increased response of adenosine A<sub>1</sub> receptors, caused by a reduction in the function of nucleoside transporters, is one mechanism by which therapeutic hypothermia (usually used within the mild range) mediates neurosynaptic protection in the acute phase of stroke. |
In the last decade it became clear that a previously consolidated memory can be modified during the plastic state induced by retrieval. This updating process opens the possibility to adapt undesired memory. Here we investigated whether fear memory could be updated to less-aversive/positive level by inserting hedonic information during retrieval. Considering that methylphenidate has strong rewarding propriety, we injected 3 or 10 mg/kg pre or post-reactivation in rats previously trained in contextual fear conditioning. We found that memory reactivation under effect of methylphenidate attenuates fear memory within-session and in subsequent tests in a drug-free condition, without presenting spontaneous recovery. Interestingly, methylphenidate impaired memory extinction when injected before, but not after a long reactivation session. We also showed that methylphenidate induces place preference and increases motor activity. Thus, this study provides new insights in the memory updating process and suggests that a previously consolidated fear memory can be attenuated by inserting appetitive information during retrieval. |
Previous studies have shown that injection of the mGlu5 receptor positive allosteric modulator (PAM) VU0360172 into either the thalamus or somatosensory cortex markedly reduces the frequency of spike-and-wave discharges (SWDs) in the WAG/Rij model of absence epilepsy. Here we have investigated the effects of VU0360172 on GABA transport in the thalamus and somatosensory cortex, as possible modes of action underlying the suppression of SWDs. Systemic VU0360172 injections increase GABA uptake in thalamic synaptosomes from epileptic WAG/Rij rats. Consistent with this observation, VU0360172 could also enhance thalamic GAT-1 protein expression, depending on the dosing regimen. This increase in GAT-1 expression was also observed in the thalamus from non-epileptic rats (presymptomatic WAG/Rij and Wistar) and appeared to occur selectively in neurons. The tonic GABA<sub>A</sub> receptor current present in ventrobasal thalamocortical neurons was significantly reduced by VU0360172 consistent with changes in GAT-1 and GABA uptake. The in vivo effects of VU0360172 (reduction in tonic GABA current and increase in GAT-1 expression) could be reproduced in vitro by treating thalamic slices with VU0360172 for at least 1 h and appeared to be dependent on the activation of PLC. Thus, the effects of VU0360172 do not require an intact thalamocortical circuit. In the somatosensory cortex, VU0360172 reduced GABA uptake but did not cause significant changes in GAT-1 protein levels. These findings reveal a novel mechanism of regulation mediated by mGlu5 receptors, which could underlie the powerful anti-absence effect of mGlu5 receptor enhancers in animal models. |
Depression is a common mental illness and leading cause of disability. Most current antidepressants are associated with significant limitations, and in particular, a delayed onset and low rate of efficacy. Consequently, there remains an ongoing need for antidepressants that are either more effective or better tolerated than existing standards. We previously identified ZY-1408 as a drug with a novel chemical structure and potential anti-depressant-like activity. Specifically, ZY-1408 is a novel serotonin 2C (5-HT<sub>2C</sub>) receptor antagonist and serotonin/norepinephrine (5-HT/NE) reuptake inhibitor. In this study, we further investigated the antidepressant-like efficacy of ZY-1408 using in vitro and in vivo behavioral tests. ZY-1408 showed 5-HT<sub>2C</sub> receptor antagonist and 5-HT/NE reuptake inhibitor properties in vitro. Meanwhile, ZY-1408 decreased immobility in vivo in a dose-dependent manner in rats (via the forced-swim test) and mice (via the tail-suspension test). The behavioral test results do not appear to result from stimulation of locomotor activity. In chronically stressed rats, repeated ZY-1408 treatment significantly reversed depressive-like behavior, including reduced sucrose preference, decreased locomotor activity, and prolonged time to begin eating. Furthermore, in vivo microdialysis showed that administration of ZY-1408 significantly increased extracellular concentrations of 5-HT and NE in the hippocampus of freely moving rats. Thus, ZY-1408 is a potent and orally active 5-HT<sub>2C</sub> receptor antagonist and 5-HT/NE reuptake inhibitor with antidepressant-like activity in rodents. |
We used fMRI to examine the functional correlates of syntactical processing in the first (L1) and second (L2) languages of non-proficient, late bilinguals. Subjects either covertly read words or produced sentences from them. Syntactical production during sentence production activated regions including left inferior frontal (LIFG) gyrus and the supplementary motor area in both languages. Analyses performed on the LIFG activation identified on a subject-by-subject basis revealed greater activation in L2 compared to L1 during sentence production and during word reading, consistent with previous work suggesting that greater cognitive effort may be subserved by less well-tuned neural representations that require greater neuronal activity. Remarkably, there was a greater separation in the LIFG activations in L1 versus L2 in less compared to more proficient bilinguals during syntax production, suggesting a functional reorganisation of regions involved in syntactical production as a function of syntactical proficiency. |
The processing of biological motion is a critical, everyday task performed with remarkable efficiency by human sensory systems. Interest in this ability has focused to a large extent on biological motion processing in the visual modality (see, for example, Cutting, J. E., Moore, C., & Morrison, R. (1988). Masking the motions of human gait. Perception and Psychophysics, 44(4), 339-347). In naturalistic settings, however, it is often the case that biological motion is defined by input to more than one sensory modality. For this reason, here in a series of experiments we investigate behavioural correlates of multisensory, in particular audiovisual, integration in the processing of biological motion cues. More specifically, using a new psychophysical paradigm we investigate the effect of suprathreshold auditory motion on perceptions of visually defined biological motion. Unlike data from previous studies investigating audiovisual integration in linear motion processing [Meyer, G. F. & Wuerger, S. M. (2001). Cross-modal integration of auditory and visual motion signals. Neuroreport, 12(11), 2557-2560; Wuerger, S. M., Hofbauer, M., & Meyer, G. F. (2003). The integration of auditory and motion signals at threshold. Perception and Psychophysics, 65(8), 1188-1196; Alais, D. & Burr, D. (2004). No direction-specific bimodal facilitation for audiovisual motion detection. Cognitive Brain Research, 19, 185-194], we report the existence of direction-selective effects: relative to control (stationary) auditory conditions, auditory motion in the same direction as the visually defined biological motion target increased its detectability, whereas auditory motion in the opposite direction had the inverse effect. Our data suggest these effects do not arise through general shifts in visuo-spatial attention, but instead are a consequence of motion-sensitive, direction-tuned integration mechanisms that are, if not unique to biological visual motion, at least not common to all types of visual motion. Based on these data and evidence from neurophysiological and neuroimaging studies we discuss the neural mechanisms likely to underlie this effect. |
Unilateral spatial neglect due to right brain damage (RBD) can occur in several different sensory modalities in the same patient. Previous studies of the association between auditory and visual neglect have yielded conflicting outcomes. Most such studies have compared performance on relatively simple clinical measures of visual neglect, such as target cancellation, with that on more sophisticated measures of auditory perception. This is problematic because such tasks are typically not matched for the cognitive processes they exercise. We overcame this limitation by using equivalent visual and auditory versions of extinction and temporal-order judgment (TOJ) tasks. RBD patients demonstrated lateralized deficits on both visual and auditory tasks when compared with same-aged, healthy controls. Critically, a significant association between the severity of visual and auditory deficits was apparent on the TOJ task but not the extinction task, suggesting that even when task demands are matched across modalities, dissociations between visual and auditory neglect can be apparent. Across the auditory tasks, patients showed more pronounced deficits for verbal stimuli than for non-verbal stimuli. These findings have implications for recent models proposed to explain the role of spatial attention in multimodal perception. |
A haptic curvature aftereffect is a phenomenon in which the perception of a curved shape is systematically altered by previous contact to curvature. In the present study, the existence and intermanual transfer of curvature aftereffects for dynamic touch were investigated. Dynamic touch is characterized by motion contact between a finger and a stimulus. A distinction was made between active and passive contact of the finger on the stimulus surface. We demonstrated the occurrence of a dynamic curvature aftereffect and found a complete intermanual transfer of this aftereffect, which suggests that dynamically obtained curvature information is represented at a high level. In contrast, statically perceived curvature information is mainly processed at a level that is connected to a single hand, as previous studies indicated. Similar transfer effects were found for active and passive dynamic touch, but a stronger aftereffect was obtained when the test surface was actively touched. We conclude that the representation of object information depends on the exploration mode that is used to acquire information. |
In a series of four experiments we assessed whether functional properties of the human face, such as signaling an object through eye gaze, influence face processing in 3- and 4-month-old infants. Infants viewed canonical and scrambled faces. We found that 4- but not 3-month-old infants' ERP showed an enhanced face-sensitive N170 component for the scrambled stimulus. Furthermore, when canonical and scrambled faces were gazing toward an object, 4-month-olds displayed an enhanced Negative central (Nc) component, related to attentional processes, for the scrambled face. Three-month-olds did not display any of these effects. These results point to important transition in the first months of infancy and show that triadic cues influence the processing of the human face. |
We used two established methods for analysing the EEG response of the neurotypical adult human brain to examine the execution and observation of simple motor actions. In one, execution or observation of a button-press in response to a tone caused a decrease in the power at 8-13 Hz ("mu") frequencies. In the other, the response preparation (or the inferred response preparation when these actions are observed in another person) was measured by the averaged response time-locked potentials measured over motor cortex--the "readiness potential". Results indicated that the mirrored readiness potentials were bilaterally generated. We found sex differences for both measures. However, whereas females showed a greater degree of response for the mu power measure during the observation of movement only, males showed larger readiness potentials during both movement performance and observation. Both measures have been claimed to be neural correlates of mirror systems in the brain where processes responsible for actions are linked to the perception of such actions. Such mirror systems have also been implicated in higher order social cognition such as empathy. However, we found no correlations between either of our EEG measures and self-report scales of social cognition. The results imply sex differences in the measured systems and for mirroring that are not directly related to social cognition. We suggest that the results may indicate two dissociable motor mirroring systems that can be measured by induced and evoked EEG. |
Outcome evaluation is a cognitive process that plays an important role in our daily lives. In most paradigms utilized in the field of experimental psychology, outcome valence and outcome magnitude are the two major features investigated. The classical "independent coding model" suggests that outcome valence and outcome magnitude are evaluated by separate neural mechanisms that may be mapped onto discrete event-related potential (ERP) components: feedback-related negativity (FRN) and the P3, respectively. To examine this model, we presented outcome valence and magnitude sequentially rather than simultaneously. The results reveal that when only outcome valence or magnitude is known, both the FRN and the P3 encode that outcome feature; when both aspects of outcome are known, the cognitive functions of the two components dissociate: the FRN responds to the information available in the current context, while the P3 pattern depends on outcome presentation sequence. The current study indicates that the human evaluative system, indexed in part by the FRN and the P3, is more flexible than previous theories suggested. |
Neurophysiological studies of creativity thus far have not allowed for clear conclusions to be made regarding the specific neural underpinnings of such complex cognition due to overgeneralizations concerning the creativity construct, heterogeneity in the type of creativity tasks used, and the questionable efficacy of the employed comparison tasks. A novel experimental design was developed in the present fMRI study which rendered it possible to investigate a critical facet of creative cognition - that of conceptual expansion - as distinct from general divergent thinking, working memory, or cognitive load. Brain regions involved in the retention, retrieval and integration of conceptual knowledge such as the anterior inferior frontal gyrus, the temporal poles and the lateral frontopolar cortex were found to be selectively involved during conceptual expansion. The findings go against generic ideas that argue for the dominance of the right hemisphere during creative thinking and indicate the necessity to reconsider the functions of regions such as the anterior cingulate cortex to include more abstract facets of cognitive control. This study represents a new direction in the investigation of creativity in that it highlights the necessity to adopt a process based perspective in which the multifaceted nature of creativity can be truly grasped. |
Research has shown poor performance on verbal memory tasks in patients with major depressive disorder relative to healthy controls, as well as structural abnormalities in the subcortical structures that form the limbic-cortical-striatal-pallidal-thalamic circuitry. Few studies, however, have attempted to link the impairments in learning and memory in depression with these structural abnormalities, and of those which have done so, most have included patients medicated with psychotropic agents likely to influence cognitive performance. This study thus examines the relationship between subcortical structural abnormalities and verbal memory using the California Verbal Learning Test (CVLT) in unmedicated depressed patients. A T1 weighted magnetic resonance imaging scan and the CVLT were obtained on 45 subjects with major depressive disorder and 44 healthy controls. Using the FMRIB's Integrated Registration and Segmentation Tool (FIRST) volumes of selected subcortical structures were segmented and correlated with CVLT performance. Depressed participants showed significantly smaller right thalamus and right hippocampus volumes than healthy controls. Depressed participants also showed impaired performance on global verbal learning ability, and appeared to depend upon an inferior memory strategy (serial clustering). Measures of serial clustering were correlated significantly with right hippocampal volumes in depressed participants. Our findings indicate that depressed participants and healthy controls differ in the memory strategies they employ, and that while depressed participants had a smaller hippocampal volume, there was a positive correlation between volume and use of an inferior memory strategy. This suggests that larger hippocampal volume is related to better memory recall in depression, but specifically with regard to utilizing an inferior memory strategy. |
The most consistent negative cortical component of somatosensory evoked potentials (SEPs), namely the frontal N30, can be considered more multidimensional than a strict item of standard somatosensory investigation, dedicated to tracking the afferent volley from the peripheral sensory nerve potentials to the primary somatosensory cortex. In this review, we revisited its classical sensorimotor implication within the framework of the recent oscillatory model of ongoing electroencephalogram (EEG) rhythms. Recently, the N30 component was demonstrated to be related to an increase in the power of beta-gamma EEG oscillation and a phase reorganization of the ongoing EEG oscillations (phase locking) in this frequency band. Thanks to high density EEG recordings and the inverse modeling method (swLORETA), it was shown that different overlapping areas of the motor and premotor cortex are specifically involved in generating the N30 in the form of a beta gamma oscillatory phase locking and power increase. This oscillatory approach has allowed a re-investigation of the movement gating behavior of the N30. It was demonstrated that the concomitant execution of finger movements by a stimulated hand impinges the temporal concentration of the ongoing beta/gamma EEG oscillations and abolished the N30 component. It was hypothesized that the involvement of neuronal populations in both the sensorimotor cortex and other related areas were unable to respond to the phasic sensory activation so could not phase-lock their oscillatory signals to the external sensory input during the movement. In this case, the actual movement has primacy over the artificial somatosensory input. The contribution of the ongoing oscillatory activity in the N30 emergence calls for a reappraisal of fundamental and clinical interpretations of the frontal N30 component. An absent or reduced amplitude of the N30 can now be viewed not only as a deficit in the activation of the somatosensory synaptic network in response to sensory input, but also as a global alteration of the beta-gamma ongoing oscillation and/or of the phase-locking mechanism itself. In addition, it has lately been shown that the N30 amplitude increases during the observation of another person's hand movement. A new paradigm in which the experimenter's hand movement, observed by the participant, triggered the electric stimulation of the subject's hand has been introduced. This has allowed the identification of different cortical areas which are closely related to those involved in the mirror neuron system. This contribution of N30 behavior has paved the way for future investigation of the integration of sensory input into cognitive context. |
Sentences, musical phrases and goal-directed actions are composed of elements that are linked by specific rules to form meaningful outcomes. In goal-directed actions including a non-canonical element or scrambling the order of the elements alters the action's content and structure, respectively. In the present study we investigated event-related potentials of the electroencephalographic (EEG) activity recorded during observation of both alterations of the action content (obtained by violating the semantic components of an action, e.g. making coffee with cola) and alterations of the action structure (obtained by inverting the order of two temporally adjacent pictures of sequences depicting daily life actions) interfering with the normal flow of the motor acts that compose an action. Action content alterations elicited a bilateral posterior distributed EEG negativity, peaking at around 400 ms after stimulus onset similar to the ERPs evoked by semantic violations in language studies. Alteration of the action structure elicited an early left anterior negativity followed by a late left anterior positivity, which closely resembles the ERP pattern found in language syntax violation studies. Our results suggest a functional dissociation between the processing of action content and structure, reminiscent of a similar dissociation found in the language or music domains. Importantly, this study provides further support to the hypothesis that some basic mechanisms, such as the rule-based structuring of sequential events, are shared between different cognitive domains. |
Beliefs profoundly affect people's lives, but their cognitive and neural pathways are poorly understood. Although previous research has identified the ventromedial prefrontal cortex (vmPFC) as critical to representing religious beliefs, the means by which vmPFC enables religious belief is uncertain. We hypothesized that the vmPFC represents diverse religious beliefs and that a vmPFC lesion would be associated with religious fundamentalism, or the narrowing of religious beliefs. To test this prediction, we assessed religious adherence with a widely-used religious fundamentalism scale in a large sample of 119 patients with penetrating traumatic brain injury (pTBI). If the vmPFC is crucial to modulating diverse personal religious beliefs, we predicted that pTBI patients with lesions to the vmPFC would exhibit greater fundamentalism, and that this would be modulated by cognitive flexibility and trait openness. Instead, we found that participants with dorsolateral prefrontal cortex (dlPFC) lesions have fundamentalist beliefs similar to patients with vmPFC lesions and that the effect of a dlPFC lesion on fundamentalism was significantly mediated by decreased cognitive flexibility and openness. These findings indicate that cognitive flexibility and openness are necessary for flexible and adaptive religious commitment, and that such diversity of religious thought is dependent on dlPFC functionality. |
To answer the question of how brain pathology affects reasoning about negative emotional content, we administered a disjunctive logical reasoning task involving arguments with neutral content (e.g. Either there are tigers or women in NYC, but not both; There are no tigers in NYC; There are women in NYC) and emotionally laden content (e.g. Either there are pedophiles or politicians in Texas, but not both; There are politicians in Texas; There are no pedophiles in Texas) to 92 neurological patients with focal lesions to various parts of the brain. A Voxel Lesion Symptom Mapping (VLSM) analysis identified 16 patients, all with lesions to the orbital polar prefrontal cortex (BA 10 & 11), as being selectively impaired in the emotional reasoning condition. Another 17 patients, all with lesions to the parietal cortex, were identified as being impaired in the neutral content condition. The reasoning scores of these two patient groups, along with 23 matched normal controls, underwent additional analysis to explore the effect of belief bias. This analysis revealed that the differences identified above were largely driven by trials where there was an incongruency between the believability of the conclusion and the validity of the argument (i.e. valid argument/false conclusion or invalid argument/true conclusion). Patients with lesions to polar orbital prefrontal cortex underperformed in incongruent emotional content trials and over performed in incongruent neutral content trials (compared to both normal controls and patients with parietal lobe lesions). Patients with lesions to parietal lobes underperformed normal controls (at a trend level) in neutral trials where there was a congruency between the believability of the conclusion and the validity of the argument (i.e. valid argument/true conclusion or invalid argument/false conclusion). We conclude that lesions to the polar orbital prefrontal cortex (i) prevent these patients from enjoying any emotionally induced cognitive boost, and (ii) block the belief bias processing route in the neutral condition. Lesions to parietal lobes result in a generalized impairment in logical reasoning with neutral content. |
Pseudoneglect refers to a tendency of neurologically healthy individuals to produce leftward perceptual biases during spatial tasks, which is traditionally measured using line bisection tasks. This behavioral asymmetry is often explained as a consequence of right hemispheric dominance for visuospatial attention. The present study directly tested this notion by comparing line bisection performance between left-handers with either right hemispheric dominance (RVSD, N = 40) or "atypical" left hemispheric dominance (LVSD, N = 23) for visuospatial attention as determined by fMRI. Although we expected a reversal of pseudoneglect in participants with LVSD, our results show that they equally often err to the left of the true center compared to RVSD controls (74% of LVSD participants and 80% of RVSD participants). However, the magnitude of misbisections was found to be slightly, but significantly, smaller in the LVSD subgroup.We conclude that hemispheric asymmetry for visuospatial attention is not the main determinant of pseudoneglect as is commonly thought, but rather only constitutes one of the multiple factors which (subtly) contributes to its direction and magnitude. |
Array-centred and subarray-centred neglect were disambiguated in a group of 116 patients with left neglect by means of a modified version of the Albert test in which the central column of segments was deleted so as to create two separate sets of targets grouped by proximity. The results indicated that neglect was more frequent in array- than subarray-centred coordinates and that, in a minority of cases, neglect co-occurred in both coordinate-systems. The two types of neglect were functionally but not anatomically dissociated. Presence of visual field defects was not prevalent in one type of neglect with respect to the other. These data contribute further evidence to previous single-case and small-group studies by showing that neglect can occur in single or multiple reference frames simultaneously, in agreement with current neuropsychological, neurophysiological and computational concepts of space representation. |
The neuropsychological features of the primary progressive aphasia (PPA) syndromes continue to be defined. Here we describe a detailed neuropsychological case study of a patient with a mutation in the progranulin ( GRN ) gene who presented with progressive word-finding difficulty. Key neuropsychological features in this case included gravely impoverished propositional speech with anomia and prolonged word-finding pauses, impaired speech repetition most marked for sentences, and severely impaired verbal (with preserved spatial) short-term memory. There was a dissociated profile of performance on semantic processing tasks: visual semantic processing was intact, while within the verbal domain, verb comprehension was impaired and processing of nouns was intact on tasks requiring direct semantic processing but impaired on tasks requiring associative or inferential processing. Brain MRI showed asymmetric left cerebral atrophy particularly affecting the temporo-parietal junction, supero-lateral temporal and inferior frontal lobes. This case most closely resembles the PPA syndrome known as the logopenic/phonological aphasia variant (LPA) however there were also deficits of grammar and speech repetition suggesting an overlap with the progressive non-fluent aphasia (agrammatic) variant (PNFA). Certain prominent features of this case (in particular, the profile of semantic impairment) have not been emphasised in previous descriptions of LPA or PNFA, suggesting that GRN may cause an overlapping PPA syndrome but with a distinctive cognitive profile. This neuropsychological evidence suggests that GRN -PPA may result from damage involving the temporo-parietal junction and its functional connections in both the dorsal and ventral language networks, with implications for our understanding of language network pathophysiology.
## Introduction
Progressive language impairment as a primary feature of neurodegenerative disease was initially described by in the late 19th century and such cases continued to be described intermittently in the early 20th century. However, recent decades have seen a resurgence of research in this field. In a series of studies, described a group of patients with “primary progressive aphasia” (PPA) who had a variety of different impairments of language. Independently, in the mid 1970s described patients with progressive impairment of semantic memory, which was later to be called semantic dementia (SD) ( ). Although language impairment dominated the presentation in these groups it was observed that many of these patients developed behavioural features similar to frontotemporal dementia and hence in the “Neary criteria” of 1998 ( ) the term ‘frontotemporal lobar degeneration’ (FTLD) was introduced to cover three disorders—the behavioural syndrome of frontotemporal dementia (FTD or behavioural variant FTD, bvFTD) and two syndromes presenting with language impairment: progressive non-fluent aphasia (PNFA), a disorder of speech production with agrammatism, and SD, a disorder of semantic knowledge which commonly presents with fluent aphasia and loss of vocabulary. However, it has long been recognised that a number of patients exhibit language syndromes that do not fit clearly into either the PNFA or SD category as originally defined, particularly in the non-fluent aphasia category which is more heterogeneous. More recently, have described a third syndrome designated the “logopenic/phonological variant” of PPA (LPA) and characterized by slow speech rate with long word-finding pauses and impaired verbal short-term memory. These authors proposed modified criteria for “PNFA” emphasizing the motor speech impairment (apraxia of speech) and agrammatism. This tripartite division of the PPA spectrum underlines the inadequacy of the fluent/non-fluent dichotomy as a descriptor of progressive aphasias. However, definition of the LPA syndrome remains challenging, and ‘logopenia’ is itself a clinical descriptor which requires further neuropsychological analysis.
Until recently, descriptions of the PPA syndromes had been purely clinical but recent genetic and pathological studies have shed light on the molecular basis of PPA. In the majority of studies, SD is chiefly associated with TDP-43 pathology ( ). PNFA is more frequently associated with tau-positive pathology at post-mortem ( ) however non-tau pathologies are well documented (e.g., ): it has been proposed that patients with motor speech impairment are more likely to have tau pathology while those without motor speech impairment may be more likely to have TDP-43 pathology ( ). There are currently few studies of LPA with histopathological correlation, however early work has emphasised an association with Alzheimer's disease (AD) pathology ( ). Consistent with this and in a parallel theme in the literature, an atypical language variant of AD overlapping closely with the LPA syndrome has been described, largely based on retrospective correlation with post-mortem data ( ). In the face of this strong association with AD, other studies have shown that patients with LPA may have TDP-43 pathology ( ) suggesting that the clinico-pathological correlation of LPA with AD should not be considered universal. A key recent finding has been the discovery that mutations in the progranulin ( GRN ) gene can cause FTLD and in particular PPA ( ). Early descriptions suggested that these patients had a “non-fluent aphasia”: detailed case studies have described progressive anomia without motor speech impairment and subsequent development of repetition and reading deficits ( ). Based on the documented association of LPA with TDP-43 pathology ( ) the phenotypic range of GRN mutations might also include LPA-like syndromes, however the true nosological place of LPA within the PPA spectrum and the core features of the LPA syndrome and the aphasic syndrome(s) that accompany GRN mutations have not been clarified.
Here we present a detailed clinical, neuropsychological and linguistic analysis of the language syndrome exhibited by a patient with a GRN mutation who presented with PPA. Our motivation for undertaking this study was twofold. Firstly, we wished to characterise the GRN -associated PPA syndrome in detail, and to assess the extent to which it is similar to or diverges from other PPA clinical syndromes: this speaks to the important nosological issue of commonality and diversity within the PPA spectrum. Secondly, we wished to put on record a new case with the neurolinguistic signature of a defined molecular lesion: this speaks to the broader issue of ‘molecular network-opathies’ in neurodegenerative disease ( ).
## Clinical details
A 62-year-old right-handed male retired shopkeeper, GAA, presented with a 3-year history of progressive word-finding difficulty. He would break off in mid-sentence, unable to find the words to finish, and would often say the opposite of what he meant (e.g., ‘yes’ for ‘no’, ‘left’ for ‘right’, ‘small’ for ‘big’). His speech became very sparse and he would overuse stereotyped phrases such as ‘at some stage’ and ‘it's aggravation’. He had difficulty repeating things told to him, understanding complex instructions and remembering messages. Early on in the illness he developed problems with arithmetic and subsequently also with reading, writing and spelling. He had no other cognitive symptoms. However, his family had noted he had become more socially withdrawn in recent years and less motivated. There was no family history of dementia in his parents (his mother died at the age of 80 of cancer and his father died at 70 of cardiac disease) however two of his mother's sisters developed dementia in their 80s and his mother's father had died after some time in a psychiatric hospital.
On examination he scored 19/30 on the MMSE ( ) and 13/18 on the Frontal Assessment Battery ( ). There was mild bilateral ideomotor and ideational limb apraxia. The general neurological examination was unremarkable. He had a Clinical Dementia Rating (CDR)—total of 0.5 and CDR—sum of boxes of 4.0 ( ). On a behavioural assessment, his total Neuropsychiatric Inventory score ( ) was 13, scoring 6 on depression/dysphoria, 2 on anxiety, 3 on apathy/indifference and 2 on irritability/lability subscales.
Brain MRI was performed 3 years after symptom onset ( ). This showed asymmetric atrophy predominantly involving the left cerebral hemisphere and accentuated in the temporal lobe (particularly the superior and lateral temporal cortex) and parietal lobe (supramarginal and angular gyri) with additional left prefrontal lobe atrophy. Changes of cerebrovascular disease were minimal. Following this study, he required a permanent pacemaker for cardiac conduction disease, precluding serial MR imaging.
A blood sample was obtained as part of a study into the genetics of young-onset dementia. All 13 exons of the GRN gene were sequenced in at least 1 direction. Analysis of electropherogram traces revealed the Arg493X mutation, the most common GRN mutation reported to date ( ).
Neuropsychological and neurolinguistic functions were investigated in detail between 36 and 42 months following symptom onset.
## General neuropsychology
There was a large discrepancy between GAA's very impaired verbal IQ score and average performance IQ score (on WAIS-III, ) (see ). He was tested on four separate tests from The Camden Memory Tests battery ( ): his performance was below the 5th percentile on a test of verbal memory whereas visual memory was intact (10th to 25th percentile on a recognition memory test for faces, 95th percentile on a topographical recognition memory test and an errorless performance on a pictorial recognition memory test). Executive functions were relatively intact on two separate tests and performance was normal on tests of visuoperceptual and visuospatial skills ( ). However he was unable to score on the graded difficulty calculation test ( ).
## Speech assessment
### Propositional speech
GAA's propositional speech was gravely impaired. He volunteered little spontaneous speech. At his first clinical assessment he was asked to describe his last holiday:
“I went to… the USA… for… (long pause) Boston… round there… we did round there… (long pause) we you-sted the… (long pause) all.” (48 seconds)
When asked to describe the Cookie Theft Scene from the Boston Diagnostic Aphasia Examination ( ) he volunteered:
“This is falling out… they wanted that… they falling that… this was water… (long pause) that's about it I think… this was… this was along there… that's about it.” (30 seconds)
Analysis of these two short samples of spontaneous speech (total time 1.3 min) revealed a speech rate of 33 words/minute (in nine cognitively normal male controls with mean age 68, who spoke for an average of 2.6 min, the range was 102–148 words/minute). The mean log frequency of the words (based on the CELEX database, ) used was 3.41 (control range 2.24–2.73), mean log frequency of nouns (also based on CELEX database) used was 2.58 (control range 1.63–1.97) and noun imageability (based on the MRC database) was 596 (control range 509–574). There were no features of speech apraxia and the speech diadochokinetic rate was normal (Apraxia Battery for Adults-2 subtest 1: ). There were relatively few speech production errors although there were rare phonemic and semantic errors. Although GAA's spontaneous speech was sparse and assessment for the presence of agrammatism was therefore difficult, there were nevertheless occasional clearly agrammatic errors, e.g., “we did round there” and “they falling that”. GAA was unable to perform sentence completion tasks of either high or low probability where he was given a sentence frame (e.g., he loosened the tie around his…) and asked to complete it with a single word (i.e., neck). On a second assessment 6 months after the initial assessment, GAA's spontaneous speech was even more severely impoverished—attempting to describe his last holiday he said:
“It's aggravation… (long pause) it's… can’t do the… (long pause) along there… can’t do… it's aggravation” (45 seconds)
Describing the Cookie Theft picture he said:
“That along there… along there, that's… that's… (long pause) see I don’t these… (long pause) I know what it is but I can’t do it, you know, it's aggravation” (35 seconds)
## Detailed linguistic assessment
### Naming
GAA was severely anomic scoring below the 1st percentile on the Graded Naming Test ( ) (see ). On a category naming test comprising high frequency nouns ( ) he had more difficulty with body parts than with animals, objects or colours. On a test comparing the naming of nouns (objects) and verbs (action pictures) matched for frequency using the CELEX database, performance was more impaired for verbs than nouns ( χ = 4.33, p = 0.04). On analysis of errors made, he would commonly provide no answer, but when attempting an answer made mainly phonemic errors (e.g., ‘cheet’ for sheep; ‘flad’ for flag, ‘theeze’ for tweezers) and only occasional semantic (descriptive) errors (e.g., ‘red bits’ for bird (robin)).
### Speech repetition
GAA's repetition of both single words and sentences was impaired ( ). He was able to repeat 78/120 words from a list comprising high and low frequency words and words of one, two or three syllables. Single word repetition showed a small but non-significant frequency effect (43/60 high frequency; 35/60 low frequency, χ = 2.34, p = 0.13) and a significant effect of syllable length (31/40 one-syllable words; 28/40 two-syllable words; 19/40 three-syllable words, χ = 8.57, p = 0.01) (see ). Analysis of the 42 repetition errors revealed 11 items with no response (26%) and 31 phonological errors (11 substitutions (26%), 11 omissions (26%), 3 additions (7%), 1 transposition (2%) and 5 with multiple errors). GAA was able to repeat only 13/20 nonsense words. Sentence repetition was severely impaired: he was unable to repeat any of 10 short sentences or 10 clichés. In general he provided no response, however examples of errors made included:
### Single word comprehension
GAA's performance was assessed on a series of single word comprehension tests, some of which involved direct matching between a word and target, and other tests which involved a degree of associative or inferential knowledge; performance was also compared on the visual versions of the associative tasks. GAA showed evidence of dissociated performance on these comprehension tasks (see ). Thus his performance on the verbal (spoken and written input) version of the Pyramids and Palm Trees test ( ) was impaired, and furthermore significantly inferior to his performance on the visual version of the task which was within the normal range (sign test: N = 11, x = 2, p = 0.03). Similarly he had difficulty on the verbal version of the Camels and Cactus test ( ) compared to his normal score on the visual version (sign test: N = 17, x = 4, p = 0.03). GAA also attempted the short version of the British Picture Vocabulary Scale ( ), and he scored below the 5th percentile with both written word and spoken word presentation. By contrast, on a test of semantic knowledge that probed attributes of size and weight in animals and objects respectively ( ) he scored at a normal level on both the verbal and visual versions of the test. He was also assessed on the Category Specific Names Test assessing single word comprehension ( ): this test comprises arrays of five pictures selected from four categories, graded in difficulty so that the range of items encompasses very low frequency objects: on each section of this test (both spoken and written name to picture matching), he scored above the average level. He also attempted four graded two-choice (spoken and written) synonym comprehension tests, involving concrete and abstract nouns and verbs ( ). He was clearly impaired on both the verb versions of the test (scores near chance) but within the normal range for both concrete and abstract nouns. He performed well on the Graded Naming Test presented as a forced three-choice recognition task in which he was presented simultaneously with a spoken and written definition for each item (e.g., “What is the large canvas covered frame upon which children can bounce and jump? – TARPAULIN, TAMBOURINE or TRAMPOLINE”).
### Sentence comprehension and grammar
GAA's performance was below the 5th percentile on the Test of Reception of Grammar (TROG, ). On a further set of 24 sentences taken from PALPA55 ( ) his performance was significantly worse on reversible than nonreversible sentences and on passive than active sentences. Furthermore, performance did not benefit from a semantic variable (directionality). We explored GAA's comprehension of verb tense using an adapted version of the Lesser/Pizzamiglio and Parisi syntax test ( ) comprising 20 pairs of pictures which differ in whether the agent is doing something/has done something (present/past comparison, 10 items) or whether the agent is doing something/is about to do something (present/future comparison, 10 items). He scored 16/20 on this task scoring equally on the present/past and present/future items (healthy controls score at or near ceiling on this test). GAA was also tested on a grammaticality judgment test which was an adapted version of the test for syntactic abilities ( ): this test entails a two-alternative forced choice on two sentences (presented simultaneously both visually and aurally), one of which is grammatical and the other agrammatical. The agrammatical sentences contained a variety of errors including incorrect verb tense, addition/substitution/deletion of function words and incorrect word order. GAA scored 79% on this test making 23 errors of which 15 were errors made on incorrect verb tense (see ).
### Reading
GAA was able to read single letters fairly competently with only 1 error from 25 letters (see ). However he had great difficulty reading both real words and nonwords ( ). Investigating his real word reading further, he had similar difficulty in reading regular and irregular words. He had greater difficulty with abstract words than concrete words and with increasing word length. A battery of 275 three-letter words was also administered to examine reading errors: he read 77% of the words correctly, with 62 errors in total. Included in this test were 55 three-letter function words: there were errors on 29% of these words (compared to 21% errors on the other 220 content words). There was a mixture of error types across the reading subtests, comprising mainly phonological (e.g., ‘opperosite’ for opposite) and visual (e.g., ‘December’ for decent) errors but also occasional regularisation (e.g., ‘gem’ with hard ‘g’ for gem), and semantic errors (e.g., ‘salt’ for sour).
### Writing and spelling
GAA's spelling was severely impaired. He was unable to score on the written graded difficulty spelling test ( ) (see ). His attempts for the first four items were ‘ONE’ for TWO, ‘BULL’ for WORLD, ‘SEA’ for SAID and ‘NICE’ for SHOE. On a further set of three-letter words he scored equally poorly on both regular and irregular words and oral and written spelling were comparably affected. He made seven errors on oral spelling, comprising five no responses and the errors ‘SIK’ for SEA and ‘SAT’ for CAP; six errors on written spelling, comprising single letters: ‘S’ for SON and SAW, ‘M’ for CUP, ‘W’ for LOG and ‘M’ for BAR. On attempting to write single letters to dictation he was able to produce only 5 of 25 letters.
GAA was asked to construct grammatical sentences containing each of 10 written target words. He made no attempt for three words (‘new’, ‘radio’, ‘tree’), and for the remaining seven words produced the following:
#### Short-term memory
GAA's digit span, assessed as part of the WAIS-III, was severely impaired (see ). We subsequently compared his auditory–verbal digit span, auditory–verbal letter span, auditory–verbal word (three-letter, one-syllable) span, visual–verbal digit span and spatial span. In each condition, eight trials were presented with one, two or three items. GAA was unable consistently to repeat more than one item for spoken digits, letters or words. Performance was better for visually presented digits, for which he was occasionally able to repeat three items. Furthermore, in stark contrast to his performance on the auditory tasks, his spatial span (assessed with the Corsi block-tapping test, ) was within the normal range—he was able to point without error to three blocks, scored 4/8 completely correct trials (24/32 positions) with four blocks and 1/8 completely correct trial (22/40 positions) with five blocks.
## Discussion
Here we have described in detail the pattern of neuropsychological and linguistic deficits in a patient with GRN -associated PPA. The salient clinical features were sparse, slow and impoverished spontaneous speech with word-finding pauses. The profile of neuropsychological deficits comprised severe anomia, poor verbal short-term memory and impaired sentence comprehension, associated with dyslexia, dysgraphia and dyscalculia. By contrast certain (non-associative) aspects of single word comprehension, non-verbal memory and visual perceptual skills were well preserved. The constellation of neuropsychological findings in GAA constitutes a distinctive pattern of cognitive impairment and preservation. The clear verbal modality specificity of GAA's language deficits indicates preferential involvement of the dominant hemisphere, while the association of dyslexia, dysgraphia and dyscalculia constitutes a classical left parietal syndrome; the lobar localisation for other features, such as anomia and impaired phonological memory, is less clear. This neuropsychological syndrome overlaps in a number of respects with previous descriptions of the LPA syndrome ( ) while the presence of grammatical errors in spontaneous speech and markedly impaired speech repetition suggests an additional overlap with the PNFA syndrome. However, the cognitive profile exhibited by GAA should not be regarded simply as a variant or a composite of other PPA syndromes: key features of this profile in relation to LPA and PNFA are summarised in . Anatomically, although detailed correlation was not possible, cerebral atrophy in this case involved the left posterior temporal/anterior parietal region and also left inferior frontal areas ( ). According to the current dual stream model of cortical language processing, a ventral pathway involved in processing word meaning links the superior temporal gyrus to middle and inferior temporal gyri, temporal pole and inferior frontal cortex; while a dorsal pathway involved in articulation-to-sound mapping links the superior temporal gyrus with inferior parietal and inferior frontal cortices ( ). Following this formulation, and taking the neuropsychological and neuroimaging evidence into account, we propose that GRN -associated PPA in this case is likely to reflect involvement of both the dorsal and ventral language pathways, with a key site of overlap in the region of the temporo-parietal junction. We now consider the evidence for this claim in more detail.
GAA had progressive anomia. While this is likely to be attributable at least in part to impaired word retrieval, a verbal semantic deficit may also have contributed. The pattern of GAA's performance on single word comprehension tests is relevant both to neuropsychological theories of semantic knowledge as well as how such a syndrome would fit into current PPA classifications. He had no difficulty with the Size/Weight Attribute Test of conceptual knowledge and more impressively he scored at a high level on both the spoken and written word versions of the Category Specific Names Test probing knowledge of low frequency items. Furthermore, on a synonyms test of concrete noun comprehension his performance was at an average level. By contrast, on word–picture matching tests such as the British Picture Vocabulary Scale where the mapping between word and target picture is less direct, his performance was impaired. He was also impaired on verbal (spoken and written word to word) matching tasks such as the verbal versions of the Pyramids and Palm Trees and Camels and Cactus tests whilst exhibiting normal performance on the visual versions. How can we explain the profile of dissociated verbal semantic impairments observed in GAA?
Considering the noun comprehension tests, we suggest that GAA's weaker performance is observed on those tasks involving some degree of associative (or inferential) rather than direct semantic processing. Associative tasks are likely to involve executive control processes, as suggested by . However, a primary deficit in executive control would not easily explain the difference between GAA's performance on verbally and visually mediated versions of these associative tasks. This visual advantage is in contrast to the pattern of performance described in stroke patients ( ), and belies the equal semantic control demands of the visual and verbal versions of this task. Another possibility is that GAA has mildly impaired lexical semantics, such that response selection among closely related alternatives is required to expose degraded semantic representations; or alternatively, an intact semantic store but a deficit in linking phonological representations of words with their meanings, which is exposed when the semantic targets are closely related. Picture–picture matching might provide additional information or cues unavailable from the written or spoken word, with correspondingly better performance on visual than verbal matching tasks. An explanation of this kind would be in line with evidence from studies of focal lesions such as stroke affecting associative cortical areas in the region of the temporo-parietal junction ( ). Moreover, degraded access to verbal semantic stores resulting from posterior temporal-inferior parietal lobe atrophy would be consistent with functional imaging evidence in healthy subjects suggesting that the extraction of meaning from both spoken and written language may require connectivity between posterior and anterior temporal lobe areas in the ventral language stream ( ).
A test such as Pyramids and Palm Trees seems to call for manipulation of concepts and contexts (e.g., in order to decide whether “cat” or “dog” is the correct answer when presented with “mouse”, one must not only comprehend individual concepts but also activate the salient relationships between target and response, i.e., “hunter/hunted” rather than “both animals” or “do not bark”, etc.). We therefore raise the further possibility that the dissociation between verbal and non-verbal comprehension performance observed in GAA may arise from a selective deficit of verbal reasoning. ‘Verbal reasoning’ is itself an under-specified term: we use it here to embrace several potentially relevant processes, in particular inference or abstraction of a semantic relationship that is not directly implied by the stimuli. That such processes can be specific to the verbal modality is supported by the existence of a selective deficit of verbal message formulation in patients with so-called “dynamic aphasia” ( ). The present study does not disambiguate any deficit in verbal reasoning from a mild deficit of lexical semantics (indeed, that distinction is difficult even in principle). However, processes such as verbal inference are likely to involve fronto-parietal circuitry ( ), raising the possibility that the associative verbal semantic deficit we have identified in GAA might implicate either the dorsal or the ventral language pathway (or indeed, a conjoint deficit attributable to temporo-parietal junction damage).
In detailed descriptions of LPA, patients have performed well on the visual version of the Pyramids and Palm Trees test, leading to the suggestion that semantic memory is intact in patients with LPA ( ). The present evidence suggests a qualification of this position, in that at least some patients may have intact performance on this visual test, yet still perform poorly on certain other tests of single word comprehension, in particular those requiring associative or inferential verbal semantic processing. It is unclear whether this is a distinct feature of a GRN -associated PPA syndrome or an effect of disease progression or worsening severity in the LPA syndrome where progressive left hemispheric atrophy encroaches on posterior semantic areas. There is some evidence that patients with LPA show impaired single word comprehension with disease progression and the overlapping pattern of anatomical involvement of the temporo-parietal junction in both LPA associated with AD and GRN mutations suggests that this may be a feature of the neuroanatomy rather than the underlying molecular substrate ( ). More detailed longitudinal studies of LPA and GRN disease progression will be necessary to investigate this further.
GAA showed evidence of an impaired phonological store (poor verbal short-term memory). His auditory–verbal span was not entirely intact even for single items (digits, letters or words), while visual–verbal span was only marginally better. This contrasted with his normal visuospatial span. In addition, GAA's performance was impaired on tests not only of receptive grammar (e.g., TROG, PALPA55) but also grammaticality judgement tests (e.g., test of syntactic abilities). Previous evidence suggests that although they may cause deficits in sentence comprehension tasks, auditory–verbal span deficits are neither necessary nor sufficient to produce such deficits in receptive grammar and grammaticality judgements (e.g., ). We propose that GAA has a double deficit affecting both his auditory–verbal short-term memory and the systems mediating receptive grammar. This would also be consistent with the distributed pattern of left cerebral atrophy with left temporo-parietal emphasis in this case: the phonological store is likely to be mediated by anterior inferior parietal and posterior superior temporal areas whilst sentence and grammatical processing are associated with inferior frontal and posterior superior temporal areas ( ). Sentence comprehension has been studied in LPA with suggestions that deficits are secondary purely to phonological store deficits ( ). However, there have been no previous studies attempting to dissociate a true receptive grammatical deficit from a phonological store deficit in LPA (e.g., on a grammaticality judgment test). Similarly, it has been difficult to characterise any expressive agrammatism in LPA, as speech tends to be sparse with prolonged pauses. In this study there was some evidence for agrammatism in GAA's spontaneous speech and further evidence in his production of very simple or agrammatic sentences in writing. This may represent a further distinction from the LPA syndrome (suggesting an overlap with the classical PNFA syndrome), but again, will require further study, particularly with detailed quantitative analysis of spontaneous speech and writing in this group.
With further regard to his deficit of receptive grammar processing, GAA had particular difficulty with comprehension of verb tense which, in conjunction with poor performance on verb naming and verb comprehension tasks, suggests a relatively selective deficit of verb (versus noun) processing. Anatomically, verb processing is thought to rely on left dorsal language pathway areas including left prefrontal cortex ( ) and posterolateral temporal cortex ( ), consistent with the pattern of atrophy seen here. Of note, a selective deficit in verb processing has been previously described in a familial ubiquitin-positive inclusion dementia ( ): although the genetic diagnosis in this previous case was not defined, considered together these observations raise the possibility that defective verb processing may be a signature of GRN mutations in PPA.
GAA exhibited additional deficits of literacy skills that provide further evidence of deficient phonological processing. His reading deficit shows the typical pattern of deep/phonological dyslexia affecting regular and irregular real words as well as nonwords, the errors produced being a mixture of phonological, visual and more rarely regularisation and semantic errors, with better performance reading concrete compared to abstract words ( ). Similarly his pattern of spelling deficits indicates phonological dysgraphia in both oral and written modes. The presence of phonemic errors would be consistent with a deficit of phonological transcoding, which may result from damage to the left temporo-parietal junction. Patients with LPA have previously been described as having phonological dyslexia ( ) and a more general deficit of phonological processing ( ).
It is worth considering how this neurolinguistic and anatomical formulation may relate to other clinical features in this case and in previous descriptions of GRN -associated disease. GAA did not exhibit neurological signs of parkinsonism (described in around a third of GRN mutation cases) or motor neurone disease (a rare feature) ( ). However, GAA did display evidence of apathy and depression as well as increased anxiety and irritability: such behavioural changes have been previously reported with GRN mutations ( ) and indeed, the most common clinical phenotype of GRN mutations is progressive personality change (behavioural variant frontotemporal dementia). Similar behavioural symptoms have been described in association with both PNFA and LPA ( ). In anatomical terms, such complex behaviours are likely to depend on distributed circuitry and might therefore be vulnerable to disease processes that strike long intra-hemispheric pathways linking frontal and anterior temporal cortices with more posterior areas, as we propose may underpin the GRN -associated aphasic syndrome here.
Beyond demonstrating a molecular and anatomical association, aphasia associated with GRN mutations suggests a pathophysiological mechanism that may underpin certain key features of the LPA syndrome. Broadly, a number of features can be understood as the consequence of breakdown of phonological processing due to dysfunction of the left temporo-parietal junction and its connections. However, this case has highlighted certain neuropsychological differences with respect to previous descriptions of the LPA syndrome ( , see ), in particular, the early occurrence of single word comprehension deficits (also a feature in our previously described case of GRN -associated PPA, ) and receptive (and also mild expressive) agrammatism. Detailed longitudinal single case analysis of GRN -associated FTLD has shown a strikingly asymmetric pattern of involvement of functionally connected but distributed cortical areas within a cerebral hemisphere ( ), and the finding of markedly asymmetric left temporal and parietal atrophy in the present case would be consistent with this. Involvement of the key left temporo-parietal junction zone is predicted to correlate with involvement of functionally connected regions in the left inferior frontal and anterior temporal lobes via the dorsal and ventral speech processing pathways demonstrated in functional imaging studies in healthy subjects ( ) and implicated in clinical aphasia syndromes of vascular disease ( ). Any conclusions based on the detailed analysis of a single case must be extrapolated with care, in order to assess their relevance to the wider population of patients with the syndrome. The key unresolved issue raised by this case is whether the features here typify a discrete syndrome of GRN -associated aphasia, or rather, one instance of a broader continuum of non-fluent aphasia cases with different molecular substrates. It will be important to conduct further group and detailed single case studies in patients with GRN -associated PPA to define the full clinico-pathological and clinico-genetic spectrum of the disorder, to establish the extent to which GRN -associated PPA, AD-LPA and other non-fluent cases can be distinguished on neuropsychological grounds, and to address in detail the anatomical and pathophysiological basis of the GRN -associated language ‘network-opathy’.
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Previous studies have shown that appetitive motivation enhances episodic memory formation via a network including the substantia nigra/ventral tegmental area (SN/VTA), striatum and hippocampus. This functional magnetic resonance imaging (fMRI) study now contrasted the impact of aversive and appetitive motivation on episodic long-term memory. Cue pictures predicted monetary reward or punishment in alternating experimental blocks. One day later, episodic memory for the cue pictures was tested. We also investigated how the neural processing of appetitive and aversive motivation and episodic memory were modulated by dopaminergic mechanisms. To that end, participants were selected on the basis of their genotype for a variable number of tandem repeat polymorphism of the dopamine transporter (DAT) gene. The resulting groups were carefully matched for the 5-HTTLPR polymorphism of the serotonin transporter gene. Recognition memory for cues from both motivational categories was enhanced in participants homozygous for the 10-repeat allele of the DAT, the functional effects of which are not known yet, but not in heterozygous subjects. In comparison with heterozygous participants, 10-repeat homozygous participants also showed increased striatal activity for anticipation of motivational outcomes compared to neutral outcomes. In a subsequent memory analysis, encoding activity in striatum and hippocampus was found to be higher for later recognized items in 10-repeat homozygotes compared to 9/10-repeat heterozygotes. These findings suggest that processing of appetitive and aversive motivation in the human striatum involve the dopaminergic system and that dopamine plays a role in memory for both types of motivational information. In accordance with animal studies, these data support the idea that encoding of motivational events depends on dopaminergic processes in the hippocampus.
Highlights
Effect of reward and punishment on episodic memory is modulated by DAT genotype.
Enhanced recognition memory for motivational cues in DAT 10-repeat homozygotes.
Higher activation at encoding in striatum and hippocampus in DAT 10-repeat homozygotes.
## Introduction
Reward improves episodic memory formation in humans ( ). Functional imaging studies have shown that memory encoding of reward-associated stimuli involves a network of dopaminergic midbrain areas, ventral striatum and hippocampus ( ). Evidence from animal studies suggests that this reward-related modulation of long-term memory could be mediated by dopamine release in the hippocampus ( ; for a review of dopamine effects on hippocampal long-term potentiation, see ). This is supported by studies in humans indicating that dopamine binding potential in the hippocampus is correlated with memory performance ( ).
In contrast to the memory effects of monetary reward, little is known about the effects of monetary punishments on episodic memory formation. For emotional stimuli, it has been shown that negative emotional events are remembered better than emotionally neutral events, and that this effect involves the amygdala (for a review see ). For aversive motivation, there have been inconsistent reports across a range of human memory tasks. Whereas aversive electrical stimulation impaired memory in a human version of the Morris water maze ( ), threat of shocks enhanced memory for scene images when participants were tested 24 h later, an effect that was based on amygdala-hippocampal interaction at encoding ( ). When monetary rewards and punishments were dependent on memory performance, threat of monetary loss enhanced source memory retrieval in a similar manner to reward when tested immediately after learning ( ). This was associated with a correlation of activity in striatum and hippocampus during successful source retrieval. In contrast, punishment cues during an incidental memory task had no effect on item recollection or recognition when tested immediately after learning ( ). These contrasting results suggest that the effect of punishment on memory may be dependent on contextual influences. The current study investigated whether monetary punishment affects memory consolidation through a dopaminergic network.
Appetitive and aversive motivation have been suggested to be processed in opponent brain systems, with rewards eliciting dopaminergic activity and punishments eliciting serotonergic activity ( ). More recent data indicate that punishments can also induce firing of dopaminergic neurons in rats ( ) and monkeys ( ), although other data suggest that punishment-responsive SN/VTA neurons are GABAergic ( ). By combining fMRI with genetics, the current study investigated transmitter specificity of midbrain signals in humans. In humans, striatal activity has been shown to correlate with aversive predictions ( ). Current models propose that the interaction of appetitive and aversive motivation in the dopamine and serotonin systems could depend on the overall motivational value of the context and on action requirements ( ). A recent study supports these models by demonstrating the relevance of action requirements for activation of SN/VTA and the striatum in humans ( ). Thus, when investigating the effects of dopamine-related polymorphisms on episodic memory for appetitive and aversive events, it is important to stratify and match populations for polymorphisms that influence serotonergic neurotransmission. After non-synaptic sources, transporter concentration is the most important factor in neurotransmitter homeostasis ( ). The genes for the serotonin transporter, SLC6A4/SERT, and the dopamine transporter, SLC6A3/DAT1, both contain length variations in their promoter regions that regulate expression of their respective transporters. As transporters both influence speed of reuptake from the synapse and increase presynaptic neurotransmitter availability, they may be expected to shape phasic neuromodulation seen in reward and punishment.
The current study investigated (i) whether anticipation of monetary punishments modulates episodic memory, (ii) whether reward and punishment related anticipation and memory are modulated by dopamine transporter genotype under conditions when groups are matched for serotonin transporter genotype, and (iii) the common and dissociable fMRI correlates of these processes. Subjects were genotyped for common polymorphisms in the dopamine transporter (DAT1 VNTR) and serotonin transporter (5-HTTLPR) and scanned during a motivational anticipation task, followed one day later by a memory test outside the scanner. In line with previous studies ( ), we expected reward-predicting stimuli to activate the SN/VTA system and enhance episodic memory. Based on reports of activations in the dopaminergic system for aversive stimuli, we hypothesized that punishment prediction would also activate the mesolimbic system. Increased dopaminergic transmission was expected to lead to improved episodic memory performance.
## Experimental procedures
### Participants
A total of 24 healthy adults (all right-handed, mean age [±SD] 25.3±3.9 years; 8 men) participated in the study. They were screened for neurological conditions and past psychiatric disorders using the Mini International Neuropsychiatric Inventory ( ) and provided blood samples for genotyping. The study was designed to compare DAT 9-repeat carriers and 10-repeat homozygotes based on previous reports of a role of the DAT VNTR in dopaminergic modulation of memory ( ). We here report comparisons of the DAT 10-repeat homozygotes with DAT 9/10-repeat heterozygotes. There were no DAT 9-repeat homozygotes in the participant sample. The two DAT groups were matched for age, gender and 5-HTTLPR genotype. In relation to 5-HTTLPR, only short allele homozygotes (SS) and long allele homozygotes (LL) were included in this study. The final sample included 12 participants from each DAT group. Half of the participants in each group were SS homozygotes and half were LL homozygotes. The majority of participants were invited based on their genotype. Additionally, some participants were genotyped after scanning and excluded if they were heterozygous for 5-HTTLPR (seven participants). Because the overall sample was non-random, we did not calculate Hardy–Weinberg equilibrium statistics. Twenty-one participants were Caucasian, three participants were Asian (two 10-repeat homozygotes, one 9/10-repeat heterozygous). To address possible effects of ethnicity, second-level analyses were performed excluding the three Asian participants. Since there was no significant change in the result, we report analyses of the combined group. All participants gave written informed consent, and the study was approved by the local ethics committee.
### Behavioural task
We obtained fMRI data while participants were completing alternating blocks of a reward and punishment task (modified from ). Before entering the scanner, participants received written instructions and completed a practice version of each block type. The anticipation task was presented in alternating blocks of reward and punishment. In each block, motivational stimuli were randomly mixed with neutral stimuli in an event-related design. This design allowed a contrast of each motivational category with corresponding neutral items from the same block as well as a direct contrast of appetitive and aversive motivational processes. At the beginning of each block, participants were informed of the motivational block type (reward/punishment). At the beginning of each trial, the motivational status of the trial (motivational/neutral) was indicated by the category of a picture on the screen (indoor/outdoor scene). In motivational trials, participants were rewarded or punished for their performance on a rapid number detection task. One day after scanning, participants performed a recognition memory task on the cue pictures.
On day 1, participants engaged in three fMRI sessions of 8–9 min length, each consisting of one reward block followed by one punishment block ( ). Each block contained 38 (reward blocks) or 32 (punishment blocks) trials of 4.3–11.1 s duration, half of which were potentially rewarded/punished. Picture category (indoor or outdoor) indicated the motivational status of each trial. One category predicted neutral trials (neither reward nor punishment). The other category predicted reward in the reward blocks and punishment in the punishment blocks. Data from the first three motivational and first three neutral trials of each block were discarded to allow for switching effects. Additionally, the last six trials in reward blocks were discarded to eliminate a potential confound of different block lengths, which were necessary to ensure overall monetary gain for participants. During each trial, participants saw a greyscale landscape photograph for 1500 ms, responded to it with a button press (right index or middle finger) indicating the motivational status of the trial (reward/neutral in reward blocks, punishment/neutral in punishment blocks), waited a variable interval (delay, 200–3000 ms duration), and then responded to a number (target, 100 ms) by button press. Visual feedback (1000 ms duration) was given 1000 ms after presentation of the target. A variable fixation phase (500–4500 ms) followed. The speeded number comparison task ( ) required participants to decide whether the target number (1, 4, 6 or 9) was lower or higher than 5. They responded as quickly as possible by button press with their right index or middle finger. A response time limit was used to determine trial outcome.
In reward trials, participants received no-win feedback (£0, yellow downward arrow) if their response to the target number was incorrect or exceeded the response time limit. After correct decisions within the time limit, they received win feedback (£1, green upward arrow). In punishment trials, participants received loss feedback (−£1, red downward arrow) if their response to the target number was incorrect or exceeded the response time limit. After correct decisions within the time limit, they received no-loss feedback (£0, yellow upward arrow). The time limit was adjusted individually in a staircase procedure to ensure reward and punishment rates of ~66%. In neutral trials, uninformative feedback was given. Participants were informed of the speed-accuracy requirements and cue categories. Frequency of target buttons and numbers was counterbalanced for each session. Participants were asked to pay attention to the cues to ensure awareness of the reward/punishment status of each trial, but not told that a memory test would follow.
In the memory test given one day after the study session, participants were shown all images from the study phase randomly mixed with newly presented distractor images. Participants received written instructions and additional examples detailing the difference between ‘remember’ and ‘know’ responses. First, participants indicated whether they recognized the image (‘Old/New’). For images classified as old, they then distinguished between recollection and familiarity according to the remember/know procedure (‘Remember/Know/Guess’) ( ). For images classified as new, participants indicated whether their decision was confident (‘Sure/Guess’). Response time limits were set at 3 s for each decision. A fixation phase of 1.5 s followed. Every 96 trials, the task was paused until participants were ready to continue.
### Behavioural analysis
Participants’ reaction times and hit rates during the study task were analysed in repeated-measures ANOVAs. Adding the remember and know rates obtained in the memory test for old stimuli (percentage of studied items classified as remembered or known) and subtracting the corresponding false alarm rate for distractors (percentage of unstudied items classified as remembered or known) yielded corrected hit rates. We also calculated a corrected remember rate and a corrected know rate separately by subtracting the corresponding false alarm rates. Note that these response rates excluded trials in which participants guessed.
### fMRI acquisition
Magnetic resonance images were acquired on a 3 T Allegra head scanner (Siemens Medical Systems, Erlangen, Germany) with a head coil for RF transmission and signal reception. A field map was acquired with a double echo gradient echo field map sequence (TE, 10.0 and 12.46 ms; TR, 1020 ms; matrix size, 64×64), using 64 slices covering the whole head (voxel size, 3×3×3 mm), to improve distortion correction of the functional images. For functional images, we used BOLD signal sensitive T2⁎-weighted transverse single-shot gradient-echo echo-planar imaging (EPI). Each volume contained 35 slices of 1.5 mm thickness and 1.5 mm in-plane resolution (TR 3.5 s, TE 30 ms, matrix size, 128×128). Coverage was obtained from the base of the orbitofrontal cortex and the medial temporal lobe (MTL) to the superior border of the anterior cingulate cortex. Possible BOLD sensitivity losses in the hippocampus due to susceptibility artifacts were minimized by applying a z -shim gradient moment of 0.6 mT m ms and a slice orientation of −30° to the AC-PC line ( ). For normalization, a whole-brain image (100 slices) with the same EPI parameters was used. In each scanning session, ~150 functional whole brain volumes were acquired. Scanner noise was reduced with ear plugs, and participants’ head movements were minimized with foam pads. Additionally, anatomical scans were collected using multi-echo 3D FLASH for mapping proton density, T1 and magnetization transfer (MT) at 1 mm resolution ( ).
### fMRI analysis
Preprocessing and data analysis were performed using Statistical Parametric Mapping software implemented in Matlab (SPM5; Wellcome Trust Center for Neuroimaging, London, UK). Using the FieldMap toolbox ( ), field maps were estimated from the phase difference between the images acquired at the short and long TE. The EPI images were corrected for distortions based on the field map ( ) and the interaction of motion and distortion using the Unwarp toolbox ( ). EPI images were then spatially normalized to the Montreal Neurological Institute (MNI) template by warping the acquired whole-head EPI to the SPM template and applying these parameters to the functional images (voxel size 1×1×1 mm), and smoothed using a 4 mm Gaussian kernel. A high-pass filter with a cutoff of 128 s was applied to the data.
For statistical analysis, trial-related activity for each participant was assessed by convolving a vector of trial onsets with a canonical hemodynamic response function ( ). A general linear model (GLM) was specified for each participant to model the effects of interest and six covariates capturing residual motion-related artefacts. After creating statistical parametric maps for each participant by applying linear contrasts to the parameter estimates, a random effects analysis was performed to assess group effects. The relevant contrasts were: Reward-predicting vs. neutral cue, punishment-predicting vs. neutral cue, reward-predicting vs. punishment-predicting cues and the reverse contrast. We also analyzed activity in the encoding phase with respect to subsequent memory performance on a trial-by-trial basis (difference due to memory, dm) for recognised vs. forgotten rewarded, punished and neutral items.
The statistical threshold for the imaging results was set to p <0.05, family-wise error (FWE) rate corrected for spherical search volumes in pre-defined areas. The areas of interest were chosen based on experimental results from the reward-based memory paradigm: The putamen and substantia nigra were chosen based on , the anterior hippocampus was chosen based on and the amygdala was chosen based on and because of its relevance for aversive memory formation ( ). Spherical SVC was centered on peak voxels identified in these regions. The radius of each SVC corresponded to the anatomical volumes of the a priori regions as reported in anatomical studies. These were: 9 mm for activations in the ventral striatum (see ), 6 mm for activations in the anterior hippocampus (see ), 7.5 mm for activations in the amygdala (see ) and 4.5 mm for activations in the substantia nigra (see ). Activations are displayed at a threshold of p <0.005 (uncorrected) with 15 contiguous voxels unless stated otherwise. All stereotaxic coordinates are given in MNI space. All brain images are shown in neurological orientation. All behavioural averages are given as mean values±SE.
To better localize SN/VTA activity, relevant activation maps were superimposed on the mean image of the spatially normalized MT maps. MT imaging is based on the transfer of energy between protons in free water and highly bound protons within macromolecules ( ). Thus MT saturation is thought to be a more direct measure to image myelin and improves contrast between SN and surrounding white matter tracts ( ) without the geometric distortion present in iron-based imaging such as susceptibility and R2 mapping. It has been shown to allow distinguishing the SN from surrounding structures as a bright area, which has been confirmed to be coextensive with the SN as delineated histologically by tyrosine hydroxylase immunohistochemistry ( ). It has also been shown to provide a measure of nigral degeneration in clinical populations such as Parkinson’s disease ( ). However, we will refer to BOLD activity from the entire SN/VTA complex throughout this paper because dopamine neurons are dispersed throughout the SN/VTA complex and form a functional continuum in primates ( ). This is underlined by recordings showing that dopamine neurons in the SN and VTA respond to reward ( ).
### Genotyping
DNA was extracted from blood samples and genotyped by gene sizing. Primer sequences were chosen based on ( ) and ( ) and were checked on on Primer3 software ( ) found and blasted by electronic polymerase chain reaction (PCR) on the UCSC genome browser NCBI build 36.1 ( ). Genotyping was performed through PCR followed by restriction digest and subsequent capillary electrophoresis. PCR with Taq polymerase (Molzym) involved initial denaturation at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 61 °C for 30 s and elongation at 72 °C for 60 s, followed by 72 °C for 7 min. This was heat denatured to single-stranded fragments in formamide and run with a ROX500 ladder on a 3730xl DNA Analyser (Applied Biosystems). Individual genotypes were called according to peak size on GeneMapper software version 4.0.
## Results
### Behavioural effects
Participants successfully categorized the motivational and neutral cues (mean hit rate neutral: 96±1%, hit rate reward-predicting: 95±1%, hit rate punishment-predicting: 95±1%). As expected, a one-way repeated-measures ANOVA (three motivational levels and two between-subjects factors for 5-HTT and DAT genotype) on reaction times (RT) in the picture category task revealed a main effect of motivation ( F =5.9, p <0.01). Post-hoc one-tailed t -tests confirmed shorter RTs for reward-predicting ( t =2.1, p <0.05) and punishment-predicting ( t =4.1, p <0.001) compared to neutral cues (mean RT reward 673±38 ms, punishment 655±27 ms, neutral 722±26 ms).
There was a significant main effect of motivation on RTs to the number targets ( F =23.5, p <0.001) and no effect of or interaction with DAT genotype. Post-hoc one-tailed t -tests confirmed shorter RTs for reward ( t =2.1, p <0.05) and punishment trials ( t =4.0, p <0.001) compared to neutral trials (mean RT reward 431±18 ms, punishment 432±21 ms, neutral 506±21 ms). In the number task, rates of accuracy/reinforcement differed slightly from the targeted 0.67 rate because of participants’ effort in the two motivational categories (mean rate of win feedback in reward trials 0.7±0.05; neutral correct [in the absence of feedback for participants] 0.67±0.004; rate of loss feedback in punishment trials 0.65±0.003).
A previous study reported an effect of motivational outcome on memory for the cue ( ). Since there was no memory difference based on motivational outcomes in the current study, trials were grouped for analysis based on cue type only. For the delayed memory test ( A), a three-way ANOVA (reward/punishment block, motivational/neutral trials, remember/know responses) with two between-subjects factors (5-HTT and DAT genotype) revealed that memory performance was better for pictures from reward blocks than for pictures from punishment blocks (main effect of block; F =14.5, p <0.01) and better for motivational (rewarded or punished) items than neutral items (main effect of motivation; F =5.7, p <0.05). Based on our hypotheses, we performed post-hoc one-tailed paired t-tests on memory performance for rewarded and punished items compared to neutral items from the same block. Memory performance was significantly better for rewarded items compared to neutral items ( t =2.0, p <0.05) and for punished items compared to neutral items ( t =2.2, p <0.05). However, tested across all participants, punished items were not recognized better than neutral items from the reward block ( t =−0.4, p >0.5) and worse than rewarded items ( t =−2.2, p <0.05).
ANOVA results also showed interactions between memory performance and genotype. There was a significant interaction of DAT genotype with block, motivational status and remember/know judgment ( F =4.8, p <0.05). We then performed separate three-way ANOVAs on DAT 10-repeat homozygotes ( B) and on 9/10-repeat heterozygotes ( C) to explore this interaction effect. There was a main effect of motivation in 10-repeat homozygotes ( F =21.2, p <0.01) but not in 9/10-repeat heterozygotes ( F =0.1, p =0.76). Post-hoc paired t -tests on homozygous participants confirmed that memory for rewarded items was higher than for neutral items from reward blocks ( t =3.8, p <0.01) and memory for punished items was higher than for neutral items from punishment blocks ( t =3.0, p <0.05). In contrast to memory effects of the whole group, memory for punishment cues in 10-repeat homozygotes was also significantly better than memory for neutral items from the reward block ( t =2.4, p <0.05). In addition, the ANOVA showed a main effect of block in 9/10-repeat heterozygotes ( F =17.8, p <0.01) that only achieved trend-level significance in 10-repeat homozygotes ( F =3.7, p =0.08). Post-hoc paired t-tests revealed that items from the reward block were remembered better than items from the punishment block in 9/10-repeat heterozygotes ( t =3.7, p <0.01) but not in 10-repeat homozygotes ( t =1.9, p =0.09). There was no effect of remember/know judgment in either of the DAT groups. There was no effect of 5-HTTLPR on overall memory performance for punishment cues.
### fMRI results
Reward and punishment anticipation elicited overlapping activations in the SN/VTA-striatal system, as shown by inclusive masking of the reward anticipation contrast with the punishment anticipation contrast ( A, ). A direct contrast of reward-predicting vs. punishment-predicting pictures revealed a small cluster in the left ventral striatum ( B), whereas the reverse contrast did not reveal any significant activations. At outcome time, there was no difference in activations in our a priori regions between reward and punishment.
Brain activity in response to both reward and punishment was influenced by genotype. DAT genotype affected responses to motivational items and motivational memory encoding. In comparison with 9/10-repeat heterozygotes, participants homozygous for the 10-repeat allele showed increased striatal activations for motivational compared to neutral cues ( A and B). For punishment-predicting cues, activity was also higher in right hippocampus ( B). Activity related to subsequent memory for reward-predicting items was higher in striatum and anterior hippocampus for homozygous participants compared to heterozygous participants ( C). Later memory for punishment-predicting items was associated with activation of SN/VTA and bilateral hippocampus ( D). The reverse contrasts revealed no significantly higher activations in heterozygotes compared homozygotes.
## Discussion
These results suggest that the dopaminergic system is involved in the neural processing of appetitive and aversive motivation in humans and in memory formation for motivational stimuli. The main findings of the current study are (i) that anticipation of monetary punishments enhances long-term memory for punishment-predictive items, (ii) that dopamine genotype modulates recognition memory for motivational stimuli, and (iii) that dopamine genotype modulates striatal activity to reward and punishment anticipation and striatal and hippocampal activity related to subsequent memory for motivational items. These data are consistent with our hypothesis that dopaminergic action in the hippocampus is associated with higher memory for stimuli eliciting appetitive and aversive motivational processes.
Memory for punishment-predictive stimuli was enhanced in comparison to neutral stimuli. Such an effect of incentive motivation has previously been shown for reward-associated stimuli ( ). In the context of reward, memory enhancement has been suggested to be mediated by dopamine release in the hippocampus. This is supported by data showing that hippocampal dopamine is necessary for the late phase of long-term potentiation, which it prolongs and enhances ( ; for a review see ). In humans, integrity and activity of the dopamine system is correlated with individual memory performance ( ). Consistent with these data, punishment-related and reward-related memory enhancement in the current study were stronger in DAT 10-repeat homozygotes compared to DAT heterozygous participants. Successful encoding of punishment-predictive items was associated with higher midbrain, striatal and hippocampal activity, supporting the idea that punishment anticipation elicited dopaminergic activity and thereby increased hippocampal encoding of cue stimuli. Successful encoding of reward-predictive items was associated with higher activity in striatum and hippocampus, but not in SN/VTA. Why there was an absence of a SN/VTA subsequent memory effect in the reward condition is unclear, since previous studies reported DM effects in SN/VTA both in motivational and non-motivational paradigms ( ). However, another study that presented appetitive and aversive pictures also did not find this reward effect ( ), suggesting that it could be influenced by the inclusion of an aversive category. In line with previous findings ( ), activations in this study were located in ventral striatum and anterior hippocampus.
Previous studies investigating the effect of aversive motivation on memory reported inconsistent effects. In a human version of the Morris water maze ( ), aversive electrical stimulation at incorrect platforms impaired learning. Memory was improved, however, when cues indicated aversive electrical stimulation if the current stimulus on display was not remembered at test 24 h later ( ). This effect was mediated by amygdala-hippocampal interactions during the study phase of the task. The different network in comparison to the current study could result from the use of an intentional memory task, where punishment avoidance was dependent on successful memory formation. It is also possible that primary punishment such as shocks depends more strongly on an amygdala-based emotional system than secondary motivation elicited by monetary losses. This interpretation is supported by the finding that in an intentional memory task, threat of monetary loss enhanced source memory performance in an immediate memory test via a retrieval network of striatum and hippocampus ( ), although fMRI results of the encoding phase were not reported. A behavioural study, in contrast, found no effect of punishment cues on immediate incidental memory for items presented as subsequent targets ( ). Instead, the motivational effect on memory depended on the motivational outcome of each trial. This difference could be due to the 2–4 s delay between cue and encoded item, which would prevent dopaminergic modulation of hippocampal processing because of the phasic nature of dopaminergic activity to motivational cues. Another crucial difference between the two studies is the probability of punishment indicated by the cue, which was approx. 65% in the current study in contrast to a rate of approx. 33% in . In the context of the current study, therefore, results are consistent with the hypothesis that the high punishment probability indicated by the cues elicited phasic dopaminergic activity associated with the items at encoding and increased long-term memory via a striatal-hippocampal network.
The DAT1 VNTR polymorphism has been shown to affect expression of the dopamine transporter. The 9-repeat allele was found to be associated with higher levels of DAT in vivo ( ), while a post-mortem study reported the reverse association ( ). Results from in vitro and in vivo studies have so far remained inconsistent ( ), and the impact of the DAT1 polymorphism on dopamine transmission cannot reliably be inferred. If we assume a higher DAT expression in 9-repeat carriers ( ), dopamine levels would be expected to be higher in 10-repeat homozygotes, which is supported by the higher activation in the reward system in homozygous participants in the current study. In contrast, several previous functional imaging studies found that 9-repeat carriers showed higher striatal activity in reward tasks ( ), while one study reported that genotype effects depended on an interaction with the personality trait reward sensitivity ( ). Differences between these studies could be due to variations in task protocols. Additional insight could be gained from studies that investigate the cumulative impact of multiple dopaminergic polymorphisms (e.g. ) and interactions between several polymorphisms (e.g. ), although the inconsistent literature on each individual genotype currently still presents some difficulties for this approach. In the context of the current study, however, DAT genotype had consistent effects on anticipation of motivational outcomes and on memory for motivational outcomes. The current findings therefore support the possibility that dopamine contributes to striatal processing during anticipation of punishment and to hippocampal processing related to memory formation for motivational items.
A number of recent studies found activity in the dopaminergic system related to punishment. In anesthetized rats, dopamine neurons in the ventral VTA are excited by the onset of footshocks, whereas neurons in the dorsal VTA are inhibited by noxious stimulation ( ). In monkeys, midbrain dopamine neurons respond to aversive air puffs by an increase in firing rate ( ), and there are indications that the neuronal populations coding for appetitive and aversive events are spatially separated (for a review see ). Analysis of distinct subpopulations within the SN/VTA was not possible in the current study given the spatial resolution of our fMRI protocol. The block design in combination with event-related within-block contrasts of motivational and neutral items, however, enabled us to eliminate factors that potentially confound findings of aversive dopaminergic responses: The contrast with neutral items from the same block eliminates general effects of a rewarding or punishing context on responses to all stimuli presented in that context. A generalized carry-over of neuronal responding from rewarded to punished items is additionally prevented by the temporal separation into rewarding and punishing blocks and by excluding the first three motivational and first three neutral stimuli in each block from the analysis, corresponding to about one minute at the beginning of each block. Although we cannot completely rule out the possibility of carryover effects, our data are consistent with other reports that have eliminated this confound behaviourally ( ) and with fMRI studies reporting an involvement of the striatum in active avoidance ( ). We observed activation in the dopaminergic system to punishment cues but not outcomes. In line with previous results, this supports the conclusion that punishment-related activity is not caused by relief after termination of an aversive stimulus ( ), and suggests that neural activity was specific to the anticipation of motivational trials. This is also supported by the absence of striatal activation to reward compared to neutral or punishment outcomes, which is in line with the coding of prediction errors by the striatum ( ). Since both reward and punishment were largely predicted by the cues in our task, we did not expect significant outcome-related activity in the dopaminergic system.
The DAT results support the hypothesis that striatal activation in fMRI studies on punishment is modulated by dopaminergic processes. Recent models of incentive learning and experimental findings suggest that the involvement of the dopaminergic system in punishment signals is related to action requirements ( ). In the current study, participants were instructed to try to avoid punishments by fast button presses. Although the staircase procedure ensured that the punishment rate remained above 65%, thus making it impossible to effectively avoid punishments, the lower reaction times in punishment compared to neutral trials indicate that participants were highly motivated and applied considerable effort towards punishment avoidance. The involvement of dopamine in punishment avoidance is hypothesized to depend on a shift of the motivational baseline in aversive contexts ( ). In the current study, reward and punishment were presented in separate, alternating blocks. It is therefore possible that an adjustment of the baseline anticipation contributed to the striatal punishment signal. However, even in a punishment context, neutral trials would be expected to elicit more positive anticipation signals than punishment trials. The higher striatal activation to punishment cues in comparison to neutral cues can therefore not be fully explained by a shift in the motivational baseline. Our data suggest that striatal dopamine signals are equally involved in the anticipation of appetitive and aversive events when action is required to either obtain a reward or avoid a punishment ( ).
In conclusion, the present study showed that anticipation of monetary punishments and anticipation of monetary rewards exhibit overlapping patterns of neural activity and of long-term memory modulation. These effects were influenced by dopamine transporter genotype, suggesting that dopamine is involved in aversive motivation in humans. Our results provide support to the idea that dopamine enhances human long-term memory via a midbrain-striatal-hippocampal network and extend this network to include processing of punishment incentives.
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Excess neural activity in the CA3 region of the hippocampus has been linked to memory impairment in aged rats. We tested whether interventions aimed at reducing this excess activity would improve memory performance. Aged (24 to 28 months old) male Long-Evans rats were characterized in a spatial memory task known to depend on the functional integrity of the hippocampus, such that aged rats with identified memory impairment were used in a series of experiments. Overexpression of the inhibitory neuropeptide Y 13-36 in the CA3 via adeno-associated viral transduction was found to improve hippocampal-dependent long-term memory in aged rats, which had been characterized with impairment. Subsequent experiments with two commonly used antiepileptic agents, sodium valproate and levetiracetam, similarly produced dose-dependent memory improvement in such aged rats. Improved spatial memory with low doses of these agents was observed in both appetitve and aversive spatial tasks. The benefits of these different modalities of treatment are consistent with the concept that excess activity in the CA3 region of the hippocampus is a dysfunctional condition that may have a key role underlying age-related impairment in hippocampal-dependent memory processes. Because increased hippocampal activation occurs in age-related memory impairment in humans as observed in functional neuroimaging, the current findings also suggest that low doses of certain antiepileptic drugs in cognitively impaired elderly humans may have therapeutic potential and point to novel targets for this indication. |
Some individuals with bipolar disorder transition directly from major depressive episodes to manic, hypomanic, or mixed states during treatment, even in the absence of antidepressant treatment. Prevalence and risk factors associated with such transitions in clinical populations are not well established, and were examined in the Systematic Treatment Enhancement Program for Bipolar Disorder study, a longitudinal cohort study. Survival analysis was used to examine time to transition to mania, hypomania, or mixed state among 2166 bipolar I and II individuals in a major depressive episode. Cox regression was used to examine baseline clinical and sociodemographic features associated with hazard for such a direct transition. These features were also examined for interactive effects with antidepressant treatment. In total, 461/2166 subjects in a major depressive episode (21.3%) transitioned to a manic/hypomanic or mixed state before remission, including 289/1475 (19.6%) of those treated with antidepressants during the episode. Among the clinical features associated with greatest transition hazard were greater number of past depressive episodes, recent or lifetime rapid cycling, alcohol use disorder, previous suicide attempt, and history of switch while treated with antidepressants. Greater manic symptom severity was also associated with risk for manic transition among both antidepressant-treated and antidepressant-untreated individuals. Three features, history of suicide attempt, younger onset age, and bipolar subtype, exhibited differential effects between individuals treated with antidepressants and those who were not. These results indicate that certain clinical features may be associated with greater risk of transition from depression to manic or mixed states, but the majority of them are not specific to antidepressant-treated patients. |
Disrupted-in-schizophrenia-1 (DISC1) is associated with mental disorders, including major depression. We previously showed that DISC1-Q31L mutant mice have depression-like behaviors and can therefore be used to study neurobiological mechanisms of depression and antidepressant (AD) medication action. First, we found reduced levels of dopamine, serotonin and norepinephrine in the nucleus accumbens (NAC) of DISC1-Q31L mutants. Next, we assessed social-conditioned place preference as a reward-dependent task and the capacity of distinct ADs to correct impaired social behavior in DISC1-Q31L mice. Bupropion, but not fluoxetine or desipramine, was able to correct deficient social facilitation, social reward, and social novelty in DISC1-Q31L mutants, whereas all three ADs were able to improve social motivation and behavioral despair in DISC1-Q31L mutants. Furthermore, we sought to correlate social anhedonia with molecular and cellular features including dendritic spine density, β-arrestin-1,2, and cAMP-response-element-binding protein (CREB) in the NAC as biomarkers related to depression and the DISC1 pathway. DISC1-Q31L mutants showed reduced levels of β-arrestin-1,2, CREB, and spine density in the NAC, further supporting the construct validity of the genetic model. Bupropion induced the greatest effect on CREB in DISC1-Q31L mutants, whereas all studied ADs corrected the reduced levels of β-arrestin-1,2 and modestly ameliorated deficient spine density in this brain region. Overall, we find neurobiological changes accompanying social anhedonia in the NAC of DISC1-Q31L mutant mice, consistent with a role for DISC1 in regulating social reward as an endophenotype of depression. |
Aging is associated with declines in memory and cognitive function. Here, we evaluate the effects of HT-0712 on memory formation and on cAMP response element-binding protein (CREB)-regulated genes in aged mice. HT-0712 enhanced long-term memory formation in normal young mice at brain concentrations similar to those found to increase CRE-mediated gene expression in hippocampal neurons. Aged mice showed significantly poorer contextual and trace conditioning compared with young-adult mice. In aged mice, a single injection of HT-0712 significantly boosted contextual and trace long-term memory. Additional effects of HT-0712 were seen in a spatial memory task. Our parallel biochemical experiments revealed that inductions of the CREB-regulated genes, cFos, Zif268, and Bdnf, after fear conditioning were diminished in aged mice. HT-0712 facilitated expression of these CREB-regulated genes in aged hippocampus, indicating that the drug engages a CREB-regulated mechanism in vivo. These findings corroborate and extend our previous results on the mechanism of action of HT-0712 and its efficacy to enhance memory formation. Our data also indicate that HT-0712 may be effective to treat age-associated memory impairment in humans. |
Mesolimbic α6* nicotinic acetylcholine receptors (nAChRs) are thought to have an important role in nicotine behavioral effects. However, little is known about the role of the various α6*-nAChRs subtypes in the rewarding effects of nicotine. In this report, we investigated and compared the role of α6*-nAChRs subtypes and their neuro-anatomical locus in nicotine and cocaine reward-like effects in the conditioned place preference (CPP) paradigm, using pharmacological antagonism of α6β2* nAChRs and genetic deletion of the α6 or α4 subunits in mice. We found that α6 KO mice exhibited a rightward shift in the nicotine dose-response curve compared with WT littermates but that α4 KO failed to show nicotine preference, suggesting that α6α4β2*-nAChRs are involved. Furthermore, α6β2* nAChRs in nucleus accumbens were found to have an important role in nicotine-conditioned reward as the intra-accumbal injection of the selective α6β2* α-conotoxin MII [H9A; L15A], blocked nicotine CPP. In contrast to nicotine, α6 KO failed to condition to cocaine, but cocaine CPP in the α4 KO was preserved. Intriguingly, α-conotoxin MII [H9A; L15A], blocked cocaine conditioning in α4 KO mice, implicating α6β2* nAChRs in cocaine reward. Importantly, these effects did not generalize as α6 KO showed both a conditioned place aversion to lithium chloride as well as CPP to palatable food. Finally, dopamine uptake was not different between the α6 KO or WT mice. These data illustrate that the subjective rewarding effects of both nicotine and cocaine may be mediated by mesolimbic α6β2* nAChRs and that antagonists of these receptor subtypes may exhibit therapeutic potential. |
The neurobiological underpinnings of intermittent explosive disorder (IED) are traditionally linked to deficiencies in the serotonergic system. In this study, we investigated the effects of escitalopram, a selective serotonin reuptake inhibitor (SSRI), on brain activation during face processing. We expected that escitalopram would reduce amygdala activity in IED and in addition, we explored the effect in other social-emotional-related brain regions. A total of 17 subjects with current IED and 14 healthy controls participated in a randomized, double-blind, placebo-controlled, counterbalanced fMRI face processing study. The analysis focused on the faces compared to a fixation baseline contrast, and a factorial model with Group as between-subject and Drug as within-subject factor was tested. Group × Drug interaction effects were found in the amygdala (small volume corrected) and the left temporal parietal junction (TPJ; whole-brain corrected). Escitalopram increased amygdala activation in controls, but surprisingly not in IED. However, the TPJ showed increased activity in IED on escitalopram compared with placebo. The TPJ is associated with social-cognitive processes, such as perspective taking and empathy. The TPJ findings suggest that SSRI administration may reduce aggressive tendencies towards other people by enhancing these social-cognitive processes. Future research should further elucidate the long-term effects of SSRIs on various social-emotional tasks in IED. |
Activin, a member of the transforming growth factor-β family, exerts multiple functions in the nervous system. Originally identified as a neurotrophic and -protective agent, increasing evidence implicates activin also in the regulation of glutamatergic and GABAergic neurotransmission in brain regions associated with cognitive and affective functions. To explore how activin impacts on ethanol potentiation of GABA synapses and related behavioral paradigms, we used an established transgenic model of disrupted activin receptor signaling, in which mice express a dominant-negative activin receptor IB mutant (dnActRIB) under the control of the CaMKIIα promoter. Comparison of GABAA receptor currents in hippocampal neurons from dnActRIB mice and wild-type mice showed that all concentrations of ethanol tested (30-150 mM) produced much stronger potentiation of phasic inhibition in the mutant preparation. In dentate granule cells of dnActRIB mice, tonic GABA inhibition was more pronounced than in wild-type neurons, but remained insensitive to low ethanol (30 mM) in both preparations. The heightened ethanol sensitivity of phasic inhibition in mutant hippocampi resulted from both pre- and postsynaptic mechanisms, the latter probably involving PKCɛ. At the behavioral level, ethanol produced significantly stronger sedation in dnActRIB mice than in wild-type mice, but did not affect consumption of ethanol or escalation after withdrawal. We link the abnormal narcotic response of dnActRIB mice to ethanol to the excessive potentiation of inhibitory neurotransmission. Our study suggests that activin counteracts oversedation from ethanol by curtailing its augmenting effect at GABA synapses. |
Chronic early-life stress increases vulnerability to alcoholism and anxiety disorders during adulthood. Similarly, rats reared in social isolation (SI) during adolescence exhibit augmented ethanol intake and anxiety-like behaviors compared with group housed (GH) rats. Prior studies suggest that disruption of dopamine (DA) signaling contributes to SI-associated behaviors, although the mechanisms underlying these alterations are not fully understood. Kappa opioid receptors (KORs) have an important role in regulating mesolimbic DA signaling, and other kinds of stressors have been shown to augment KOR function. Therefore, we tested the hypothesis that SI-induced increases in KOR function contribute to the dysregulation of NAc DA and the escalation in ethanol intake associated with SI. Our ex vivo voltammetry experiments showed that the inhibitory effects of the kappa agonist U50,488 on DA release were significantly enhanced in the NAc core and shell of SI rats. Dynorphin levels in NAc tissue were observed to be lower in SI rats. Microdialysis in freely moving rats revealed that SI was also associated with reduced baseline DA levels, and pretreatment with the KOR antagonist nor-binaltorphimine (nor-BNI) increased DA levels selectively in SI subjects. Acute ethanol elevated DA in SI and GH rats and nor-BNI pretreatment augmented this effect in SI subjects, while having no effect on ethanol-stimulated DA release in GH rats. Together, these data suggest that KORs may have increased responsiveness following SI, which could lead to hypodopaminergia and contribute to an increased drive to consume ethanol. Indeed, SI rats exhibited greater ethanol intake and preference and KOR blockade selectively attenuated ethanol intake in SI rats. Collectively, the findings that nor-BNI reversed SI-mediated hypodopaminergic state and escalated ethanol intake suggest that KOR antagonists may represent a promising therapeutic strategy for the treatment of alcohol use disorders, particularly in cases linked to chronic early-life stress. |
Acute administration of gamma-aminobutyric acid B (GABAB) receptor agonists decreased nicotine, cocaine, ethanol, and heroin self-administration. GABAB receptor agonists also decreased cue-induced cocaine craving or seeking in humans and animals, respectively. The present study investigated the effects of repeated subcutaneous administration of the GABAB receptor agonist CGP44532 on nicotine- and food-maintained responding under a fixed ratio 5 schedule of reinforcement. The second part of the study determined whether contingent presentation of previously nicotine-associated cues reinstated extinguished nicotine-seeking behavior, and whether acute subcutaneous CGP44532 administration affected cue-induced reinstatement of extinguished nicotine-seeking behavior. The results indicated that repeated administration of 0.25 mg/kg CGP44532 selectively decreased nicotine self-administration compared to food-maintained responding during the first 7 days of treatment. Repeated administration of 0.5 mg/kg/day CGP44532 nonselectively decreased both nicotine- and food-maintained responding. Contingent presentation of previously nicotine-associated cues reinstated extinguished nicotine-seeking behavior. Further, acute CGP44532 administration (0.125 and 0.25 mg/kg) decreased cue-induced reinstatement of nicotine-seeking behavior. In summary, the present results indicated that 0.25 mg/kg/day CGP44532 selectively decreased nicotine self-administration compared to food-maintained responding, and acute administration of CGP44532 (0.125 and 0.25 mg/kg) dose-dependently decreased cue-induced reinstatement of nicotine-seeking behavior. |
In this paper, we present new estimates for the risk of becoming cocaine dependent within 24 months after first use of the drug, and study subgroup variation in this risk. The study estimates are based on the National Household Survey on Drug Abuse conducted during 2000-2001, with a representative sample of US residents aged 12 years and older (n=114 241). A total of 1081 respondents were found to have used cocaine for the first time within 24 months prior to assessment. Between 5 and 6% of these recent-onset users had become cocaine dependent since onset of use. Less [corrected] risk of recent cocaine dependence soon after onset of cocaine use was found for female subjects, young adults aged 21-25 years, and non-Hispanic Black/African-Americans. Use of crack-cocaine and taking cocaine by injection were associated with having become cocaine dependent soon after onset of use. These epidemiologic findings help to quantify the continuing public health burden associated with new onsets of cocaine use in the 21st century. |
Dehydroepiandrosterone (DHEA) or their sulfate conjugate (DHEAS) (together abbreviated DHEA(S)) exert multiple effects in the central nervous system, and may be involved in the pathophysiological processes in schizophrenia. This prospective study aimed to investigate whether serum cortisol/DHEA(S) molar ratios are associated with response to antipsychotic treatment during the exacerbation of schizophrenia. Serum DHEA(S) and cortisol were determined at baseline, and 2 and 4 weeks later for 43 medicated schizophrenia inpatients with acute exacerbation. The patients were treated with stable doses of antipsychotic agents up to 2 weeks prior to entering the study and for the 4-week duration of the study after which they were classified as either responders or nonresponders to treatment. Findings suggest that responders had significantly higher serum cortisol levels and cortisol/DHEA(S) ratios compared with nonresponders. These differences remained significant at three time points controlling for gender, age, severity of symptoms and emotional distress, benzodiazepines, type or dosage of antipsychotic agents, and background variables. The logistic regression model shows advantages of both cortisol/DHEA(S) molar ratios vs serum cortisol and DHEA(S) concentrations for prediction of responsivity to antipsychotic treatment. No significant canonical correlations were observed between changes from baseline through end-of-study in hormonal values and severity of symptoms and emotional distress among responders and nonresponders. Thus, these data provide evidence that elevated serum cortisol and cortisol/DHEA(S) ratios may serve as markers of biological mechanisms that are involved in responsivity of schizophrenia patients to antipsychotic treatment. |
Withdrawal symptoms are observed upon cessation of cannabis use in humans. Although animal studies have examined withdrawal symptoms following exposure to delta-9-tetrahydrocannabinol (THC), difficulties in obtaining objective measures of spontaneous withdrawal using paradigms that mimic cessation of use in humans have slowed research. The neuromodulator dopamine (DA) is affected by chronic THC treatment and plays a role in many behaviors related to human THC withdrawal symptoms. These symptoms include sleep disturbances that often drive relapse, and emotional behaviors like irritability and anhedonia. We examined THC withdrawal-induced changes in striatal DA release and the extent to which sleep disruption and behavioral maladaptation manifest during abstinence in a mouse model of chronic THC exposure. Using a THC treatment regimen known to produce tolerance, we measured electrically elicited DA release in acute brain slices from different striatal subregions during early and late THC abstinence. Long-term polysomnographic recordings from mice were used to assess vigilance state and sleep architecture before, during, and after THC treatment. We additionally assessed how behaviors that model human withdrawal symptoms are altered by chronic THC treatment in early and late abstinence. We detected altered striatal DA release, sleep disturbances that mimic clinical observations, and behavioral maladaptation in mice following tolerance to THC. Altered striatal DA release, sleep, and affect-related behaviors associated with spontaneous THC abstinence were more consistently observed in male mice. These findings provide a foundation for preclinical study of directly translatable non-precipitated THC withdrawal symptoms and the neural mechanisms that affect them.
## Introduction
### Cannabis use and abuse
Cannabis derivatives are currently the most widely used illicit drugs in the world [ ], with reported use increasing, at least in part, due to efforts towards its legalization [ ]. While adverse effects of cannabis use are often mild, a subpopulation of chronic cannabis users experience hallmarks of a substance use disorder (SUD), including use despite adverse consequences, craving, tolerance to the drug’s effects, and withdrawal symptoms [ – ]. Experiencing these symptoms can lead to diagnosis of cannabis use disorder (CUD) as defined by the DSM-5 [ ].
The main psychoactive component of cannabis is Δ-9-tetrahydrocannabinol (THC). This compound is primarily responsible for the drug-induced “high” via actions at the cannabinoid-type 1 receptor (CB1) [ , ]. Indeed, behavioral effects of chronic THC treatment are absent in mice genetically engineered to lack the CB1 receptor [ , ], and CB1 antagonists prevent self-administration of synthetic cannabinoids [ , ].
### Cannabis withdrawal symptoms
Cessation of chronic cannabis or THC use causes withdrawal symptoms in a significant population of users [ – ]. In humans, cannabis withdrawal symptoms (CWS) may include: irritability/aggression, nervousness/anxiety, disrupted sleep, hypophagia and weight loss, restlessness, depressed mood, and uncomfortable somatic symptoms e.g., abdominal pain, shakes, sweating, fever/chills, or headache [ ]. One of the most prominent CWS is disrupted sleep, and poor sleep quality is a major risk factor towards cannabis relapse [ – ]. Human studies show that acute THC produces sleep facilitation including shorter sleep latency, less difficulty falling asleep and more time spent asleep [ – ]. Our group has shown that endocannabinoid (eCB) activity contributes to non-rapid eye movement, (NREM) stability [ ]. The sleep-inducing effects of acute THC may dissipate during chronic exposure in humans [ ], and abstaining from chronic THC or cannabis use in humans is associated with decreased sleep time and efficiency [ – ].
At the preclinical level, while some studies have modeled CWS using spontaneous withdrawal (abrupt cessation of drug treatment), the far more prevalent model is withdrawal precipitated by CB1 antagonist/inverse agonist administration, which immediately blocks the effects of the chronically administered cannabinoid drug [ ]. An obvious caveat of precipitated withdrawal is that humans undergo spontaneous withdrawal, so the translational relevance of precipitated CWS is unclear. While spontaneous cannabis or THC withdrawal does reliably induce somatic withdrawal symptoms (e.g., wet dog and head shaking, front paw tremor, hunched posture, body tremor, etc.) [ – ], indicating its potential to drive CWS, previous studies have struggled to provide strong evidence for spontaneous THC withdrawal symptoms that more closely model human CWS.
### Dopamine, CUD and CWS
The neuromodulator dopamine (DA) has well established roles in SUDs [ , ], behavioral alterations during drug withdrawal [ , ], and sleep [ – ]. There is considerable evidence that exogenous cannabinoids indirectly modulate DA activity [ – ] with ramifications for development of CUD and CWS. The striatum, comprised of the nucleus accumbens (NAc), dorsal medial (DMS) and dorsal lateral striatal (DLS) subregions, is a major site of DA action in the brain and is involved in many behaviors associated with drug abuse and withdrawal symptoms [ ], as well as sleep [ ], making it an interesting nexus THC action and withdrawal phenomena.
### Aim of study
In the present study, we modeled chronic cannabis use using a well established procedure to induce THC tolerance in mice [ ] to examine whether spontaneous THC withdrawal drives: (1) alterations in striatal DA release, (2) sleep disruption, and (3) translationally relevant behavioral maladaptation. To examine effects of acute and chronic THC treatment on behavior and sleep, we compared vehicle injections to the first and last injections of the chronic THC or vehicle control (VEH) treatment. To examine maladaptive behavior and sleep during withdrawal, we compared changes in baseline metrics (2 days pretreatment) to early (1–2 days) and late (5–6 days) abstinence in mice treated with THC or VEH (Supplementary Fig. ). Both male and female mice were examined independently, as accumulating evidence suggests sex differences in CUD and severity of CWS [ ].
## Methods and materials
### Subjects
All experiments were conducted in the same facility at the National Institute on Alcohol Abuse and Alcoholism using 8 to 10-week-old wildtype male and female mice (Mus musculus, C57BL/6 J strain) at the time of electroencephalogram/electromyogram (EEG/EMG) implantation or behavioral studies. For full description of subjects see supplementary information. All methods used in this work were approved by the Animal Care and Use Committee of the National Institute on Alcohol Abuse and Alcoholism (protocol #: LIN-DL-1) and were within the guidelines described in the NIH Guide to the Care and Use of Laboratory Animals. Note that all times of day are expressed in terms of hours from lights on, i.e., zeitgeber time (ZT). Separate cohorts of mice were used in all experiments.
### Drugs
THC stock solution (100 mg/mL in ethanol) was obtained from the U.S. National Institute on Drug Abuse. Ethanol was evaporated using nitrogen gas and the remaining THC was re-dissolved in dimethysulfoxide (DMSO) and aliquoted for storage at −20 . THC at the various doses used in these studies was made fresh before each administration in a vehicle consisting of 1:1:18 (DMSO : Cremaphor : 0.9% Saline) (Cat# D2650 and C5135 for DMSO and Cremaphor EL, respectively, Sigma Aldrich, St. Louis, MO).
### Sleep recordings
Sleep experiments were performed as previously described [ , ]. See supplementary information for full description of surgical procedures, recording environment, polysomnographic acquisition, and vigilance state scoring (e.g., NREM vs REM sleep).
### THC treatment and behavioral paradigms
#### Sleep effects during and after THC treatment
Recordings and treatment began after the 7-day acclimatization period. First, we collected 2 separate days of baseline polysomnographic measurements (pretreatment). Following stoppage of recording on the 2nd day of baseline, all mice received injection of VEH. After stoppage of recording for this VEH treatment, mice were treated with either VEH or 10 mg/kg of THC in vehicle and EEG/EMG were recorded for another ~22 h. After stoppage of recording for the first injection, the animals received injections according to their assigned group twice daily for four more days – once before the dark phase, between ZT11:00 and ZT12:00, and the second injection between ZT1:00 and ZT2:00. Treatments were assigned randomly such that each recording chamber with five individually housed mice had roughly half the subjects receiving either THC or VEH. Injections for Vehicle-only session and first injection occurred within the last hour before the start of the dark phase, between ZT11:00 and ZT12:00. Finally, the mice received one last injection, again between ZT11:00 and ZT12:00, and EEG/EMG were recorded for ~22 h. EEG/EMG recording continued for the next 6 full days of abstinence (ABST 1–6). Each ABST recording began between ZT11:00 and ZT12:00, and lasted ~22 h. To measure effects from chronic low dose THC, the same procedure as above was used, but a 1 mg/kg dose of THC was given. To measure sleep effects during abstinence from an acute dose of THC, the same procedure as chronic 10 mg/kg THC experiments was used except a single THC treatment was immediately followed by ABST 1–6.
#### Behavioral effects of chronic 10 mg/kg dose of THC
For all awake behaviors, mice were treated with the chronic 10 mg/kg dose of THC using the treatment procedure described above in sleep experiments. All mice were initially group housed, four animals per cage. Before all experiments mice were transported to respective behavioral rooms and handled daily for 3 days.
See supplementary information for full description of sucrose preference test, operant conditioned sucrose seeking, intake and locomotor activity metrics, and bottle brush test procedures.
### Fast scan cyclic voltammetry
Following isoflurane anesthesia, brains were removed and 300 µm-thick coronal sections through the striatum were prepared (Leica VT1200S, Leica Biosystems, IL) in ice-cold carbogen-saturated (95% O /5% CO ) cutting solution (in mM: Sucrose 194, NaCl 30, KCl 4.5, MgCl 1, NaHCO 26, NaH PO 1.2, Glucose 10). Slices were then transferred to a chamber filled with oxygenated artificial cerebrospinal fluid (aCSF) (pH 7.4) containing (in mM): NaCl (126), KCl (2.5), NaHCO (25), NaH PO (1.2), dextrose (10), HEPES (20), CaCl (2.4), MgCl (1.2), and L-ascorbic acid (0.4) kept at 32 °C and allowed to recover for 1 h until used for recordings. After the equilibration period, brain slices were transferred to the recording chamber and perfused at a rate of ~1.5 mL/min with aCSF. Once the brain slice was in place, a bipolar stainless-steel stimulating electrode (Plastics One, Roanoke, VA) was placed in the region of interest and a carbon fiber electrode was placed ~300 µm from the stimulating electrode. Cylindrical carbon fibers (T650 carbon fiber, 7 μm diameter, 100–150 μm exposed length; Goodfellow, PA) were inserted into a glass pipette [ ]. The carbon fiber electrode was held at −0.4 V, and the potential was increased to 1.2 V and back at 400 V/s every 100 ms using a triangle waveform. DA release was evoked by rectangular, electrical pulse stimulation (250–800 µA; 2 ms, monophasic) applied every 5 min with a NL 800 A Current Stimulus Isolator (Digitimer, Hertfordshire, UK). Data collection and analysis were performed using the Demon Voltammetry and Analysis software suite [ ]. The maximum amplitudes of extracellular DA transients were obtained from input/output (I/O) curves constructed by plotting stimulus current versus peak DA concentration calculated from the peak amplitude of the stimulus-induced transient over a range of stimulus intensities measured in three areas of the striatum: DLS, DMS, and NAc. Carbon fiber electrodes were calibrated using 1.0 uM DA after recordings. All tissue was harvested between ZT2:00 – ZT4:00 on the abstinence day of interest. When possible, all three regions of interest were measured within each slice across animals.
### Plasma corticosterone quantification
Blood was collected via lateral tail vein bleeding between ZT10-12. See supplementary information for full description of plasma corticosterone collection procedures. Plasma corticosterone concentration was determined using enzyme-linked immunosorbent assay as per manufacturer’s instructions for small sample volume (ELISA part # ADI-901-097, Enzo Life Sciences, Inc., NY). The assay was performed in duplicate and read using a SpectraMax190 Microplate Reader. Standard curves and corticosterone concentration extrapolation were performed using an open-access online analysis service (MyAssays Ltd., USA).
### Statistical analysis
Data were analyzed using GraphPad Prism (version 9; GraphPad Software, La Jolla, CA, USA) statistics software. All behavioral data and CORT measurements were transformed to change, “Δ”, from vehicle injection (when investigating effects of first and last treatments) or pretreatment (when investigating effects during early and late abstinence). For sleep, intake/locomotion, sucrose preference test, and operant tasks, the pretreatment, early, and late withdrawal metrics were the average of the 2 pretreatment sessions, first 2, and last 2 days of abstinence sessions, respectively. For all other experiments we only recorded metrics on one pretreatment day and the 2nd and 6th days of abstinence, so analysis reflects those single sessions. Sex differences are known to exist in most of the metrics we examine, so we decided, a priori, to analyze male and female mice independently. See supplementary information for full description of statistical analysis.
## Results
### Striatal dopamine release in early and late abstinence after acute and chronic THC treatment
#### Changes in DA release during abstinence following acute THC treatment
We first used fast scan cyclic voltammetry to measure electrically stimulated DA release in the NAc, DMS, and DLS during early and late abstinence following acute THC treatment. Male mice showed a significant increase in peak DA release in DMS following a single, i.e., acute, injection of THC (Supplementary Fig. ). This increase was observed during early (Supplementary Fig. ) and late abstinence (Supplementary Fig. ). However, there were no differences in peak DA release in the DLS or NAc after an acute THC injection. In contrast, female mice showed decreased peak DA levels in DMS and NAc following a single THC dose in early abstinence. However, this same THC administration protocol elicited an increase in peak DA levels in DLS (Supplementary Fig. ). In late abstinence, after acute THC treatment, no differences in peak DA release were observed (Supplementary Fig. ). THC treatments did not elicit changes in uptake rate (data not shown).
#### Changes in DA release during abstinence following chronic THC treatment
In male mice exposed to chronic THC treatment (Fig. ), a significant increase in DMS DA release was present during late abstinence but not early abstinence (Fig. ). In females, during early abstinence we found a robust increase in DA levels in all three striatal regions (Fig. ), but no such increase during late abstinence (Fig. ). Effects of chronic THC treatment on striatal DA release in acute brain slices using FSCV.
A Schematic illustrating chronic treatment and timepoints of FSCV recordings in striatal subregions where a carbon fiber recording electrode and stimulating electrode were placed to record electrical stimulation-elicited DA release. Purple rectangles indicate abstinence timepoints used in recordings. B DA release in males (♂) during early abstinence following chronic treatment. No differences detected in DLS (F (1,72) = 0.02, p = 0.8712), DMS (F (1,84) = 1.01, p = 0.3168), or NAc (F (1,84) = 3.07, p = 0.0834). C DA release in males during late abstinence following chronic treatment. No differences detected in DLS (F (1,186) = 0.45, p = 0.5019) or NAc (F (1,87) = 0.64, p = 0.4270). In DMS THC treated mice had elevated DA release (F (1,71) = 13.56; p = 0.0004). D DA release in females (♀) during early abstinence following chronic treatment. DA release was elevated in DLS (F (1,72) = 51.61, p = <0.0001), DMS (F (1,72) = 33.82, p = <0.0001) and NAc (F (1,60) = 11.69, p = 0.0011). E DA release in females during late abstinence following acute treatment. No differences detected in DLS (F (1,84) = 2.54, p = 0.1151), DMS (F (1,84) = 1.68, p = 0.1981), or NAc (F (1,72) = <0.01, p = 0.9976).
### Sleep and vigilance state architecture during acute and chronic THC treatment and abstinence
We next examined if THC and VEH treatments changed percent time spent in NREM and REM sleep and sleep architecture (i.e., bout number and duration) referenced to baseline metrics (i.e., vehicle treatment or pretreatment epochs). Note that the sleep and behavioral data that follow are reported as a change-score (i.e., delta “∆”) from either vehicle treatment or pretreatment epochs. Additionally, we refer to light-photoperiod as lights on (LON) and dark-photoperiod as lights off (LOFF) throughout the remainder of this report.
#### Acute and chronic THC administration alters sleep in both sexes
We first examined how VEH or THC treatments altered sleep after the first and last injections of the chronic treatment regimen compared to the vehicle treatment session (Supplementary Fig. ). In male mice, the first THC injection increased percent time spent in NREM compared to VEH during LOFF. After the last injection, percent time in NREM was lower in THC treated compared to VEH treated male mice during both LOFF and LON. During LOFF, THC treated male mice showed a lesser change in percent time in NREM as compared to the first injection (Supplementary Fig. ). No significant effects were detected for NREM bout duration (Supplementary Fig. ). However, an increased number of NREM bouts was observed during LOFF in THC treated male mice compared to VEH treated mice. After the last injection, the number of NREM bouts during both LOFF and LON was decreased regardless of treatment when compared to the first injection (Supplementary Fig. ).
Small changes in percent time in REM sleep were observed following the first THC injection, but post-hoc testing found no significant differences between treatments or within treatment groups across epochs (Supplementary Fig. ). In contrast, after the last injection, THC treated male mice showed larger increases in REM bout duration compared to VEH treated mice during LOFF. THC treated male mice also had longer REM bout durations after the last injection compared to first injection during both photoperiods (Supplementary Fig. ). THC treated male mice also exhibited increased number of REM bouts during LOFF after the first injection compared to VEH treated mice, while the number of REM bouts in THC treated mice decreased after the last injection when compared to the first injection for both LOFF and LON (Supplementary Fig. ).
Female mice also exhibited increased percent NREM sleep after acute THC (Supplementary Fig. ), that was abolished by the end of chronic treatment. In contrast to male mice, NREM sleep time did not differ between THC and VEH treated females at the end of chronic treatment. Neither acute nor chronic THC administration altered NREM bout duration in female mice (Supplementary Fig. ). While there was an overall increase in the number of NREM bouts, there were no pair-wise differences between groups (Supplementary Fig. ). Also, in contrast to male mice, we observed no effect of THC injections on REM sleep time or architecture (Supplementary Fig. ).
#### Chronic THC transiently disrupts sleep in male mice at early abstinence timepoints
Because sleep disturbances are commonly reported by individuals with CUD during abstinence [ , , ], we obtained electrographic measures of sleep after the 6-day chronic treatment regimen and normalized these to the pretreatment baseline for each subject (Fig. ). In male mice, we observed reduced NREM sleep following chronic THC treatment, evidence of acute, spontaneous withdrawal symptoms in early abstinence (Fig. ). These effects were specific to LOFF in early abstinence, and they recovered by late abstinence, when THC treated males slept no differently than the VEH group. Reduced sleep during early abstinence was largely due to a reduction in the duration of NREM bouts with THC treated subjects exhibiting decreased bout duration during LOFF in early but not late abstinence (Fig. ). While interaction effects for NREM bout number were observed in late-LOFF compared to early-LOFF in male THC treated mice, post-hoc tests found no significant differences related to treatment (Fig. ). Effects of chronic THC administration on sleep in male and female mice during early and late abstinence.
A Cartoon illustrating chronically tethered mouse during epochs where no injection is given, i.e., pretreatment and abstinence. B Timeline illustrating treatment regimen and epochs contributing to analysis. Dotted purple line indicates average of two pretreatment epochs that average of days 1 and 2 of early and days 5 and 6 of late abstinence are compared. C Comparison between effects of THC or VEH on changes from pretreatment in percent time spent in NREM sleep in male (♂) mice. There is a significant treatment effect (F (1,35) = 20.64; p = <0.0001), with post-hoc tests finding differences between-groups during Early-LOFF ( p **** = <0.0001). Both groups had significant within group difference between Early and Late abstinence LOFF (THC p = <0.0001; VEH p = 0.0030). D Comparison between effects of THC or VEH on changes from pretreatment in NREM bout duration in male mice. There is a significant treatment effect (F (1,35) = 11.14; p = 0.0020), with post-hoc tests finding differences between-groups during Early-LOFF ( p * = 0.0113). E Comparison between effects of THC or VEH on changes from pretreatment in number of NREM bouts in male mice. There is a significant interaction (F (1,37) = 12.64; p = 0.0011). F Comparison between effects of THC or VEH on changes from pretreatment in percent time spent in REM sleep in male mice. There is a significant treatment effect (F (1,33) = 8.048; p = 0.0077), with post-hoc tests finding differences between-groups during Late-LOFF ( p ** = 0.0017) and Late-LON ( p ** = 0.0057). G Comparison between effects of THC or VEH on changes from vehicle injection in REM bout duration in male mice. No significant effects were detected (F (1,33) = 3.447; p = 0.0732). H Comparison between effects of THC or VEH on changes from pretreatment in number of NREM bouts in male mice. There is a significant interaction (F (1,33) = 5.744; p = 0.0224), with post-hoc tests finding differences between-groups during Late-LOFF ( p * = 0.0228). ( I – N ) Same metrics as ( C–H ) but in female (♀) mice. No significant effects were detected. I F (1,42) = 1.191; p = 0.2814. J F (1,33) = 3.881; p = 0.0581. K F (1,42) = 6.279; p = 0.4326. L F (1,30) = 3.260; p = 0.0810. M F (1,42) = 3.649; p = 0.0630. N F (1,42) = 1.816; p = 0.1879.
Chronic THC treatment in male mice led to increased time spent in REM sleep compared to VEH treatment in LOFF and LON periods late in abstinence (Fig. ). We did not detect any treatment related effects on REM bout duration (Fig. ). THC treated male mice had greater increases in REM bout number compared to VEH treated mice during LOFF in late abstinence (Fig. ).
In contrast to male mice, no significant treatment related main or interaction effects were detected for NREM or REM metrics during abstinence in females (Fig. ).
#### Low dose chronic treatment does not strongly alter sleep during administration or abstinence
We next performed separate experiments where mice were chronically treated with a lower dose (1.0 mg/kg) of THC. (Supplementary Fig. ). During the treatment period we only detected a decrease in NREM bout duration and number in THC treated relative to VEH treated mice during LON after the last injection (Supplementary Fig. ). A significant interaction in number of REM bouts was detected, but no significant differences between treatments or across injection epochs was found in post-hoc tests (Supplementary Fig. ). We found no significant main or interaction effects of low dose chronic THC treatment in female mice for NREM sleep metrics, nor percent time in REM or number of REM bouts. We did detect an interaction effect on REM bout duration in these groups, but post-hoc tests found no significant treatment related effects (Supplementary Fig. ). Low dose chronic THC treatment did not alter any sleep metrics during early or late abstinence when compared to the pretreatment epoch in either sex (Supplementary Fig. ).
#### Acute THC treatment does not alter sleep during abstinence
Profound sleep disturbances were only observed in male mice after chronic 10 mg/kg THC treatment. These changes could be due to effects of the final THC treatment in the chronic regimen rather than changes accrued during chronic treatment. To examine this possibility, we gave male mice a single 10 mg/kg THC treatment and examined sleep in abstinence. A significant interaction was detected for changes in NREM and REM bout duration, but no significant differences between treatments or across abstinence epochs were observed (Supplementary Fig. ). Thus, the effects of chronic 10 mg/kg THC in male mice are likely due to accrued effects of chronic treatment.
### Sustenance intake and locomotor activity during chronic THC treatment and abstinence
Accumulating evidence indicates humans experience altered hunger and thirst during cannabis withdrawal. These symptoms are often accompanied by restlessness which might manifest as increased locomotor behavior in general contexts. Thus, we examined how food and water intake and locomotor behavior are altered during early and late abstinence following chronic THC treatment in mice (Supplementary Fig. ). For both males and females, significant treatment related effects were detected for food and water intake, and locomotion (Supplementary Fig. ). Additionally, THC treated animals showed decreased food and water intake during LOFF after the first injection of the chronic treatment regimen, which differed from VEH treated mice. Changes from vehicle treatment did not differ between groups after the last chronic treatment, but after the last injection both sexes showed normalization of intake back towards vehicle treatment levels during LOFF. Similarly, THC treated mice of both sexes had reduced changes in locomotion compared to VEH treated mice during LOFF after the first treatment. While VEH treated mice showed reduced locomotion after the last treatment compared to first treatment, an increase was found in THC treated mice. THC treatment did not alter food or water intake and locomotion during early or late abstinence when compared to the pretreatment epoch in either sex (Fig. ). Measurements of changes in food and water intake, and locomotion, during early and late abstinence.
A Cartoon illustrating home cage intake and locomotion monitoring platform. B Timeline illustrating treatment regimen and epochs contributing to analysis. Dotted purple line indicates average of two pretreatment epochs that average of days 1 and 2 of early and days 5 and 6 of late abstinence are compared. C – E Comparison between effects of THC or VEH on changes from pretreatment during early and late abstinence epochs in food and water intake, and locomotion in male (♂) mice. No significant effects were detected. C F (1,18) = 0.7414; p = 0.4005. D F (1,18) = 3.369; p = 0.0830. F (1,18) = 1.514; p = 0.7018. F – H Same as ( C – E ) for female mice (♀). No significant effects were detected. F F (1,18) = 1.201; p = 0.2876. G F (1,18) = 0.8053; p = 0.3814. H F (1,18) = 1.295; p = 0.1041. I Percent of Vehicle treatment weights during chronic THC treatment and abstinence in male mice. There is a significant treatment effect (F (1,18) = 11.08; p = 0.0037), with post-hoc tests finding differences between-groups ( p * = <0.05, p ** = < 0.01, p *** = < 0.001, p **** = < 0.0001). J Percent of Vehicle treatment weights during chronic THC treatment and abstinence in female mice. There is a significant treatment effect (F (1,18) = 5.577; p = 0.0297), with post-hoc tests finding differences between-groups ( p * = < 0.05, p ** = < 0.01).
THC treated mice of both sexes showed significantly lower body weight compared to VEH treated mice during the first 2 days of treatment. This effect normalized by the final THC treatment but reemerged during abstinence (Fig. ).
### Altered reward seeking and conditioned cue discrimination during chronic THC abstinence
We hypothesized that various aspects of reward seeking and consumption would be altered during THC abstinence (Fig. ), consistent with observed alterations in striatal DA release and sleep disturbances. We examined these behaviors by training mice to respond on levers that led to the presentation of auditory stimuli signaling whether-or-not sucrose-solution reward would be delivered (Fig. ). Over five pretraining sessions, all mice displayed increased latency to enter the sucrose port upon lever press-contingent presentation of an auditory stimulus signaling no reward (CS−), while maintaining relatively short latency when presented with the stimulus signaling the reward (CS+) (Fig. ) indicating that the mice learned to discriminate CS+ from CS−. Changes in reward seeking behaviors during early and late abstinence from chronic THC treatment in male and female mice.
A Timeline illustrating treatment regimen and epochs contributing to analysis. Dotted purple line indicates average of two pretreatment epochs that average of days 1 and 2 of early and days 5 and 6 of late abstinence are compared. B Cartoon illustrating operant chamber for reward seeking and conditioned auditory cue presentation. C Diagram showing operant sucrose seeking procedure with cue discrimination. D Cue discrimination during last five pretraining sessions as measured by latency to enter reward port after cue onset. There is a significant effect of cue (2 × 5 RM-ANOVA; F (1,45) = 182.9; p = <0.0001). CS + = cue predicting reward; CS− = cue predicting absence of reward; ♂ = male; ♀ = female. E – I Changes in behavioral metrics related to reward seeking and cue discrimination in male mice. There is a significant treatment effect for change in latency to approach sucrose port after CS + ( G ; F (1,10) = 8.616; p = 0.0149) with post-hoc tests finding group differences during Early ( p * = 0.0261) and Late ( p * = 0.0131) abstinence, and significant treatment effects in cue discrimination (I; F (1,10) = 5.830; p = 0.0364) with post-hoc tests finding group differences during Late abstinence ( p ** = 0.0045). Non-significant effects – (E) F (1,10) = 2.516; p = 0.1438. F F (1,10) = 3.883; p = 0.0771. H F (1,10) = 4.201; p = 0.0675. J – N Same as E - I but for female mice. There is a significant treatment effect for change in latency to approach sucrose port after CS + ( L ; F (1,12) = 5.787; p = 0.0332) with post-hoc tests finding group differences during Early abstinence ( p ** = 0.0014). Non-significant effects – ( J ) F (1,12) = 4.161; p = 0.0640. K F (1,12) = 1.837; p = 0.1879. M F (1,12) = 3.110; p = 0.1032. N F (1,12) = 1.237; p = 0.2879. O Cartoon depicting sucrose preference test. P , Q Change in sucrose preference score during Early and Late abstinence in male ( P ) and female ( Q ) mice. No significant effects were detected. P F (1,38) = 3.451; p = 0.0710. Q F (1,26) = 1.085; p = 0.3071.
Comparing behavior in male mice during pretreatment and following chronic THC or VEH treatment indicated no significant differences in rewards earned (Fig. ). THC treatment did not alter sucrose consumption behavior (i.e., ‘licks’) upon reward delivery (Fig. ). The most striking effect was the behavioral response to reward predictive cues, where THC treated male mice were slower to approach the sucrose reward port after a lever press indicating reward availability, i.e., CS+ latency (Fig. ), in both early and late abstinence. This effect was not present for non-rewarded (i.e., CS−) lever press latency (Fig. ). Interestingly, THC treated male mice also differed from VEH treated male mice in their change in cue discrimination during late abstinence (Fig. ). Here, the VEH mice increased the ratio of CS− latency to CS+ latency, while the THC mice showed ratios similar to pretreatment and early abstinence levels, indicating THC treatment attenuated the enhanced discrimination between the cues that VEH treated mice experience during late abstinence. Like male mice, THC treated female mice showed no significant differences in rewards earned (Fig. ), or reward consummatory behaviors (Fig. ), and had slower CS+ latency during early abstinence (Fig. ) with no difference in CS− latency (Fig. ). Unlike males, no effect on cue discrimination was observed in female mice for either abstinence epoch (Fig. ).
To examine whether differences in reward seeking behaviors were due to altered hedonic characteristics of sucrose reward, we tested mice in a sucrose preference test (Fig. ). No differences in sucrose preference during early or late abstinence were observed for THC treated mice of either sex compared to VEH treated mice (Fig. ).
### Irritability measurements and circulating stress hormones
#### Altered irritability behaviors in male mice during THC abstinence
Clinical evidence suggests some cannabis users display enhanced aggression and irritability during abstinence [ , , ]. We used the bottle brush test [ , ] (Fig. ) to assess changes in irritability-related behaviors during abstinence in chronic THC vs VEH treated mice (Fig. ). Effects of chronic THC administration on irritability-related behaviors during Early and Late abstinence in male and female mice.
A Cartoon illustrating bottle brush test procedure for measuring irritability-related responses. B Timeline illustrating treatment regimen and epochs contributing to analysis. Dotted purple line indicates pretreatment session that Early and Late abstinence sessions are compared. C – I Change in irritability-related behavioral metrics during Early and Late abstinence in male (♂) mice. Significant effect of treatment was detected for boxing behavior ( D F (1,14) = 12.47; p = 0.0015) with post-hoc tests finding significant group differences in Early and Late abstinence ( p * = 0.0187). Significant effect of epoch was detected for escaping behavior ( F F (1,14) = 8.211; p = 0.0125) with post-hoc tests finding within group difference in THC treated mice ( p = 0.0096). Non-significant effects – ( C ) F (1,14) = 2.541; p = 0.1333. E F (1,14) = 0.7368; p = 0.4051. G F (1,14) = 0.3984; p = 0.5381. H F (1,28) = 3.478; p = 0.0727. I F (1,14) = 0.0596; p = 0.8107. J – P Same metrics as above but for female (♀) mice. No significant effects were detected. J F (1,14) = 1.246; p = 0.2830. K F (1,28) = 0.9020; p = 0.3504. L F (1,14) = 3.500; p = 0.0824. M F (1,14) = 0.0392; p = 0.8459. N F (1,14) = 0.3751; p = 0.5500. O F (1,14) = 0.2166; p = 0.1632. P F (1,14) = 0.0777; p = 0.7845.
We scored metrics related to aggressive (smelling, biting, boxing, following, exploring, tail rattling) and defensive/stress-induced behaviors (escaping, digging, jumping, climbing, defecating, vocalizing, grooming) (Fig. ). As these behaviors may be correlated within these categories, i.e., aggressive or defensive/stress-induced, we first calculated correlation matrices for each pair of behaviors for each condition (epoch, sex, and treatment). Since few significant correlations were detected (Supplementary Table ) we report all behaviors independently. Male THC treated mice showed enhanced boxing behavior during both early and late abstinence (Fig. ). In addition, male THC but not VEH treated mice had a greater change in escape behaviors in late compared to early abstinence (Fig. ). No significant changes in irritability behaviors were detected in female mice. Biting, exploring, tail rattling, jumping, climbing, and vocalizing were scored, but were scarce or absent in most mice, so were not further analyzed.
#### THC withdrawal symptoms are not likely driven by changes in stress as measured by circulating corticosterone
Stress is known to have profound effects on sleep and behavior [ ]. We assessed whether the THC treatment itself acted as a stressor in a manner that might alter sleep and wake behaviors during abstinence. We measured circulating plasma corticosterone (CORT) as an index of perceived stress and assessed changes relative to pretreatment CORT during early and late abstinence (Supplementary Fig. ). While THC treated male mice displayed increased CORT during early abstinence compared to VEH treated mice (Supplementary Fig. ), this increase (27.26 ng ± 13.17 ng; mean ± SEM) was an order of magnitude smaller than CORT increases from chronic mild stress [ ]. Changes in CORT did not differ between THC treated and VEH treated female mice (Supplementary Fig. ).
## Discussion
### Summary
We examined how spontaneous withdrawal from chronic THC administered to naïve mice drives changes in striatal DA release, sleep disruptions, and behavioral maladaptation. Overall, our experiments show that mice can be used to study translatable THC withdrawal symptoms and neural mechanisms that might drive these symptoms. Of particular interest are apparent sex differences in nearly all metrics during spontaneous THC withdrawal. These studies open the door for further investigation into the neurophysiological changes mediating CWS and potential therapeutic interventions to treat CUD/CWS. Key findings and implications are discussed below.
### Striatal DA release and THC abstinence
Striatal DA has well established roles in drug dependence [ ], including CUD [ , ] and DA dysregulation following chronic cannabis use, is a known factor in the manifestation of CWS [ ]. The implications of differing DA release in males and females during abstinence after acute THC are unclear. Perhaps the increased DA release in males makes them susceptible to enhanced sensitivity to salient or rewarding stimuli after their first drug experience, while females are resistant to such changes. Future studies are required to test whether the increased DA release in males contributes to behavioral and sleep alterations following chronic THC administration.
More pertinent to CUD/CWS are the different observations in males and females regarding DA release during abstinence after chronic THC treatment. In early abstinence, female mice had far greater DA release across the striatum, while males showed no such effects. However, while in females these measurements returned to control level in late abstinence, the relative lack of change in DA release in males may result in susceptibility to changes in reward-associated stimuli. These findings may be related to effects of altered dopaminergic transmission in disrupted cognitive function due to dysregulation in signal to noise processing [ ]. Again, further investigation into the role of altered DA release in wake behaviors and sleep is needed.
Previous studies have mainly examined effects of chronic cannabis or THC on firing of ventral tegmental area DA neurons that project to striatum, with less information about changes during abstinence. Furthermore, it is unclear how these firing changes relate to alterations in striatal DA release, as that has not been measured in past studies. Diana and coworkers found that rat midbrain DA single-unit activity was reduced 24 h after the last THC injection using a treatment regimen similar to the one we used [ ]. Based on this finding one might not expect the increased striatal DA release that we observed during abstinence. A possible explanation for this contradiction is the recent evidence that striatal DA activity does not necessarily reflect midbrain DA neuron activity [ ], and this dissociation can be explained by intrastriatal microcircuitry. Indeed, the endocannabinoid (eCB) system can modulate striatal DA release independent of postsynaptic action potentials [ ], suggesting prolonged alterations in striatal eCB action due to THC administration as a potential contributor to increased DA release. Imaging studies in human cannabis users abstaining for 1–7 days found a reduction in stimulant-induced DA response [ ], which also contrasts with our findings of increased DA release at similar timepoints during abstinence. Technical differences can potentially reconcile these divergent findings, as stimulant-mediated increases involve altered DA transporter function while electrical stimulation induces DA release directly via DA afferent stimulation and indirectly via cholinergic interneuron activation [ ]. Examining the role of cholinergic drive on DA release may thus also indicate mechanisms underlying THC actions. Likewise, our data were collected postmortem in brain slice preparations taken at precise timepoints during abstinence. Future in-vivo studies using genetically coded DA sensors [ ] can also shed light on the relationship between striatal DA activity and precise timepoints during abstinence from THC.
### A mouse model for sleep disruption during THC withdrawal
Treating sleep pathologies is often given as a reason for THC use both recreationally and medically [ , ]. Indeed, many individuals list poor sleep as a major factor leading to their relapse to cannabis use [ ], and those subjects showing sleep disruption, poor sleep quality in particular, relapse more readily [ , , , ]. However, sleep disruption is one of the most consistent and problematic aspects of cannabis withdrawal, with altered sleep observed in the majority of regular users who attempt to quit [ , ].
The observed effects on sleep, especially in male mice, are generally consistent with our hypothesis that chronic THC administration would cause sleep disruption that mirrors the transient changes observed in the clinical setting, and are consistent with a study using the synthetic CB1 agonist AM2389 [ ] that was published while the present study was under consideration for publication. The observations that acute THC treatment enhanced NREM while REM sleep was fragmented in male mice during abstinence are consistent with previous reports of single THC or cannabis exposures in animal models [ – ] and humans [ , – ]. The finding that THC tolerance following chronic treatment diminishes the drug’s effect on sleep is also consistent with past findings in animals [ , , – ] and regular human cannabis users [ , , , – ]. We also found that male mice experience disruptions during THC abstinence similar to human cannabis users [ , , , , ] while female mice appear more resilient to the overall effects of spontaneous THC withdrawal on sleep. The absence of significant differences in female mice in our study was surprising given that female cannabis users report sleep difficulty during cannabis abstinence [ ]. However, in clinical polysomnographic studies measuring sleep during cannabis abstinence the participants are primarily male, making comparison to our findings in female mice difficult. Future clinical studies should be properly powered to examine sleep physiology in female cannabis abstainers. Additionally, if female cannabis users are indeed more resilient to sleep changes as measured by polysomnography, there are other psychological manifestations occurring during cannabis or THC abstinence that may drive self-report of poor sleep quality despite no profound changes in sleep physiology. We believe overall our study provides a valuable back-translational model to investigate the neural mechanisms mediating sleep disruption during cannabis withdrawal.
### Mouse as a back-translational model for CWS during wake-behaviors
Past research using mice to study spontaneous CWS primarily report negative findings for behaviors beyond typical somatic symptoms and cannabis tetrad metrics [ ]. This is potentially confounded by typical behavioral paradigms used in preclinical settings that may not adequately probe the most pronounced human CWS. We focused our behavioral tasks to measure changes in behaviors that directly map to some of the strongest CWS; (1) reduced food and water intake, (2) restlessness (i.e., heightened locomotion), (3) amotivation, (4) attention deficits, and (5) irritability.
We observed profound hypophagia, hypodipsia, and reduced locomotion after the first THC injection, and our chronic THC treatment clearly induces behavioral tolerance to the effects of the drug as evidenced by the normalization of these measures to control levels by the final THC injection. However, the lack of change in these metrics in abstinence following chronic THC exposure suggests home cage intake and locomotion may not serve as an appropriate back-translational model for human CWS. Perhaps this paradigm does not recapitulate other environmental and cognitive factors that manifest in the human population, e.g., additional life stressors, or situations that when compounded with cannabis abstinence affect food and water intake. Nonetheless, we observed significant differences between treatment groups in body weight as early as day 2 of abstinence. This finding is particularly intriguing as it suggests chronic THC treatment alters metabolic function that may contribute to alterations in body weight despite no differences in intake or locomotion – a notion ripe for future investigation.
Our observation that both male and female mice did not differ in rewards earned indicates that CWS in mice does not involve strong changes in motivation or drive. However, the slower retrieval of reward in THC treated mice of both sexes may indicate decreased motivation. Responses to the CS+ and CS− cues that followed operant responding provide insight into attention during this goal-directed task. In particular, poorer cue discrimination in male mice treated with THC in late abstinence indicates a deficit in attention or focus that emerges later in abstinence. While this operant responding paradigm provided evidence of impairment in several cognitive processes related to goal-directed action during chronic THC abstinence, future studies should directly target specific aspects of reward seeking behaviors and attention processes in an effort to decipher the neural mechanisms mediating these specific disruptions.
Increased irritability is prevalent during cannabis abstinence in humans [ , ]. We were surprised to find that relatively few irritability-like behaviors were altered in our bottle brush test assay. Nonetheless, male mice showed enhancement of some irritability-like metrics, i.e., boxing and escape behaviors. These behaviors are interesting in that they demark disparate types of irritability behaviors – aggressive and defensive, respectively, indicating that male mice experience alterations in irritability behavior in general, though these alterations manifest in somewhat specific behavioral outputs in the bottle brush test.
### Conclusions and caveats
Technical limitations to our studies, primarily that metrics were gathered from separate cohorts of animals, make it difficult to draw conclusions about correlations between various behaviors and, in particular, DA voltammetry measurements. Nonetheless, a general observation is that female mice appear more resilient to effects of chronic THC treatment on sleep and maladaptive behaviors during abstinence. A speculative explanation is the elevation in striatal DA release we observe in female mice during early abstinence may play a protective role in attenuating withdrawal symptoms, and the clearest indicator of this effect in our studies is in the relationship between lack of sleep disruption and striatal DA release in early abstinence in female mice. Striatal DA is known to play a role in sleep-state architecture [ ], so further studies are needed to investigate the causal role of enhanced striatal DA release observed in female mice in alleviating sleep disruption during early chronic THC abstinence.
Beyond the changes in striatal DA release we observed in female mice, we can speculate about other physiological mechanisms that may contribute to the lack of abstinence effects in female sleep or wake behavior in our spontaneous THC withdrawal assays. In several of our assays, female data appear more variable than males. This could be due to influences of sex-hormone cycling, which are known to influence sleep [ ] and motivated behaviors [ ]. Likewise, THC is lipophilic and can be stored in adipose tissue [ ]. In general, male rodents have larger fat stores than females which could influence behaviors during abstinence, due to transient THC release from this tissue reserve [ ]. However, for several of our assays, we use an average of 2 days of measurements which may mitigate any such effects. Another factor might be innate differences in the eCB system between males and females [ , ]. We hypothesize, and have shown here for sleep measurements, that neurophysiological changes that drive tolerance are critical for the manifestations of CWS, and as such females may express these changes differently than males, despite both sexes showing evidence of tolerance in our treatment procedure.
Additionally, it is clear that the light cycle plays a role in CWS manifestation in mice, particularly with respect to sleep. Several of our assays were conducted at one timepoint (either during dark phase or light phase) and further studies should be done to address the role of circadian rhythms in the strength of CWS in mice.
Our findings provide models of significant and translatable CWS in rodents using a protocol that does not require antagonist-precipitated withdrawal. Note that withdrawal from misused drugs is a complex psychophysiological phenomenon, and our aim here is to present preclinical assays that have both successfully and unsuccessfully modeled distinct CWS. Future studies based on the treatment protocol and timepoints used in our study can unravel the likely complex neural mechanisms that drive sleep disruption and behavioral maladaptation prevalent in CWS – crucial first steps towards therapeutics to combat CUD.
## Supplementary information
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Sensitivity of neurons to estrogen in down-regulation of estrogen receptor alpha (ERalpha) can be thought to make a sex difference in regulatory system of reproductive activities. In this study, to investigate the sex difference of expression of ERalpha in the hypothalamus and midbrain, the number of ERalpha immunoreactive (-ir) cells was counted in orchidectomized (OCX) and ovariectomized (OVX) rats with or without treatment with estrogen. A week after the gonadectomy, 5 rats in each female and male were injected with 1mg estradiol benzoate (EB). The remaining 5 rats in both sexes did not receive EB. The brain was fixed 24h after EB-injection and 50 microm-serial frozen sections were made. After immunohistochemical staining for ERalpha, the number of ERalpha-ir cells was counted in a 0.2-mm2 frame in the anteroventral periventricular nucleus (AVPvN), the ventrolateral part of the ventromedial hypothalamic nucleus (vlVMN), the arcuate nucleus (ARCN), and the lateral mesencephalic central gray (lMCG) in 2 or 3 sections. The total number of ERalpha-ir cells was changed to a density value (number per 1mm3). As the results, in EB-treated rats, the density of ERalpha-ir cells in all regions, except the male AVPvN and male lMCG, were lower than those in untreated rats of both sexes. In the vlVMN, the density of ERalpha-ir cells in OVX rats was higher than in OCX rats. These results suggest that there are sex and regional differences in the mechanisms of down-regulation of ERalpha by estrogen in the rat brain. |
The ascending nociceptive control (ANC), a novel spinostriatal pain modulation pathway, mediates a form of pain-induced analgesia referred to as noxious stimulus-induced antinociception (NSIA). ANC includes specific spinal cord mechanisms as well as circuitry in nucleus accumbens, a major component of the ventral striatum. Here, using the trigeminal jaw-opening reflex (JOR) in the rat as a nociceptive assay, we show that microinjection of the nicotinic acetylcholine receptor (nAChR) antagonist mecamylamine into the rostral ventral medulla (RVM) blocks NSIA, implicating RVM as a potentially important link between ANC and the PAG-RVM-spinal descending pain modulation system. A circuit connecting nucleus accumbens to the RVM is proposed as a novel striato-RVM pathway. |
This study was designed to assess the association between stress, positive affect and catecholamine levels in meditation and control groups. The meditation group consisted of 67 subjects who regularly engaged in mind-body training of "Brain-Wave Vibration" and the control group consisted of 57 healthy subjects. Plasma catecholamine (norepinephrine (NE), epinephrine (E), and dopamine (DA)) levels were measured, and a modified form of the Stress Response Inventory (SRI-MF) and the Positive Affect and Negative Affect Scale (PANAS) were administered. The meditation group showed higher scores on positive affect (p=.019) and lower scores on stress (p<.001) compared with the control group. Plasma DA levels were also higher in the meditation (p=.031) than in the control group. The control group demonstrated a negative correlation between stress and positive affects (r=-.408, p=.002), whereas this correlation was not observed in the meditation group. The control group showed positive correlations between somatization and NE/E (r=.267, p=.045) and DA/E (r=.271, p=.042) ratios, whereas these correlations did not emerge in the meditation group. In conclusion, these results suggest that meditation as mind-body training is associated with lower stress, higher positive affect and higher plasma DA levels when comparing the meditation group with the control group. Thus, mind-body training may influence stress, positive affect and the sympathetic nervous system including DA activity. |
We want to know how the growth of neural stem/progenitor cells and their differentiation are affected by reactive oxygen species evolved in photosensitizing reaction, because of the similarity between the stem cells and the tumor cells in central nervous system. We investigated the effects of two photosensitizers (rhodamine 123 and hematoporphyrin) on the mouse neural stem/progenitor cells cultured in vitro under the illumination. ABC transporters were expressed in the cells, and could pump rhodamine 123 and hematoporphyrin out of the cells. Under the illumination of strong actinic light with those photosensitizers, reactive oxygen species was evolved to injure the cells. Number of viable cells decreased under illumination through apoptosis and necrosis. Those cell-killing activities were not clearly dependent on the presence of inhibitors for ABC transporters. Immunocytochemical staining with showed that immature cells with markers of neural stem/progenitor cells (Sox 2, CD133, nestin) were more sensitive to the reactive oxygen species than the differentiated cells. |
To investigate whether anorectal malformations (ARMs) were associated with a global neuromuscular maldevelopment of the lower gastrointestinal (GI) tract and anorectum, the distribution of neuronal markers protein gene product (PGP9.5), nitric oxide synthases (NOs), neuromuscular junction markers (synaptophysin, SYP), interstitial cells of Cajal (ICC) marker (c-kit) within the terminal rectum were analyzed by immunohistochemistry and Western blot in rat embryos with ethylenethiourea (ETU) induced ARMs. From Gestational day16 (Gd16) to Gd21, neural crest-derived cells (NCC) migrated from the proximal gut into the terminal colon, colonising it along its entire length, gradually proliferated and differentiated to innervate the distal gut. From Gd19 to Gd21, significant gross-morphological differences of the anorectum of normal (n=90) and ARMs (n=90) embryos were found. Different myenteric plexus (MPs) development of the anorectum suggested that ARMs were associated with a global abnormal innervation patterns in the anorectum in gestational course and might have some postoperative effect. |
The protein kinase AKT1 belongs to the Akt family and is a potent mediator of cell growth and survival and fully activated when phosphorylated. The AKT family has been found to be phosphorylated to a lesser extent in the dopaminergic cells of Parkinson's disease patients compared to control individuals, which might influence cell survival. Several publications support the implication of AKT1 in disorders of the dopaminergic system including bipolar disease and schizophrenia. In 2008 an association study performed in a Greek Parkinson's disease case-control material reported the identification of a protective AKT1 haplotype. Based on their work we have performed a replication study in a Swedish Parkinson's disease cohort. We genotyped the four single nucleotide polymorphims (SNPs): rs2494743, rs2498788, rs2494746 and rs1130214 in a case-control material consisting of 243 Parkinson patients and 315 controls. We did not find any associations with Parkinson's disease for either the individual SNPs or any of the haplotypes. In contrast to previously published results, our data do not support the hypothesis of genetic variants in AKT1 confering protection against Parkinson's disease. |
Badminton players of varying skill levels viewed normal and point-light video clips of opponents striking the shuttle towards the viewer; their task was to predict in which quadrant of the court the shuttle would land. In a whole-brain fMRI analysis we identified bilateral cortical networks sensitive to the anticipation task relative to control stimuli. This network is more extensive and localised than previously reported. Voxel clusters responding more strongly in experts than novices were associated with all task-sensitive areas, whereas voxels responding more strongly in novices were found outside these areas. Task-sensitive areas for normal and point-light video were very similar, whereas early visual areas responded differentially, indicating the primacy of kinematic information for sport-related anticipation. |
Normally long-term depression (LTD) is difficult to be induced in naïve adult rats in vivo, but it can be induced in the juvenile females and acute-stressed adult males. Using these rats as LTD models, we find in our previous study that LTD induction by the classical low-frequency stimuli (LFS) may be associated with sleep. During sleep, endogenous field potential oscillations presented in the neocortical and hippocampal circuits play important roles in synaptic downscaling as well as memory consolidations. Generally, LTD can be considered as a special synaptic downscaling and the classical LFS is very similar to such endogenous oscillations. Thus, we speculate whether we can design a new LFS which is more similar to such oscillations and whether LTD can be induced by it in naïve adult rats? In this study, we found that in the naïve adult rats anesthetized in sleep stage, the classical LFS could not induce LTD, however, a low-intensity LFS, an endogenous oscillation-like one, could induce LTD. Furthermore, in the rats anesthetized in wakefulness stage, neither the classical nor the low-intensity LFS could induce LTD. Our study showed that in the naïve adult rats, LTD could be induced by the oscillation-like LFS in the sleep stage anesthesia, suggesting that LTD may physiologically occur during sleep and be inhibited in wakefulness stage. Our study suggested that in the hippocampus LTD may be a potential long-term synaptic plasticity underlying sleep-dependent memory consolidations. |
Retinal progenitor cells (RPCs) are an excellent resource for retinal replacement therapy, because they show enormous potential to differentiate into retinal-specific cell types. While the differentiating influence of serum has long been appreciated, the effects of serum concentration on RPC differentiation into specified retinal neural cells have not been investigated. Using cultured murine RPCs, this study compared the effects of different levels of fetal bovine serum (FBS) (1%, 5%, 10% and 20%) on RPC differentiation in vitro. RPC multipotentiality was assessed by using quantitative polymerase chain reaction (qPCR) to determine the relative expression levels of 10 genes involved in retinal development. In addition, analyses of cell morphology and retinal development-related protein expression were performed using microscopy and immunocytochemistry. The data revealed that 1% FBS-induced cultures preferentially generated rhodopsin- and PKC-α-positive cells. Calbindin and AP2α expression levels were greater in 5% FBS-induced cultures. Brn3a was expressed at similar levels in 1%, 5% and 10% FBS treatment conditions but diminished in 20% FBS conditions. Twenty percent FBS induced more glial fibrillary acid protein (GFAP)-immunoreactive cells corresponding to glia populations. These findings suggest that the concentration of FBS plays an important role in RPC differentiation in vitro. Treatment with low levels of FBS favors differentiation of rhodopsin-positive photoreceptors, interneurons and retinal ganglion cells (RGCs), while high FBS concentrations preferentially induce differentiation of glia cells. These results are expected to facilitate research in the treatment of neurodegenerative retinal diseases. |
The cannabinoid CB2 receptor has been under investigation as a potential target for neuroprotection with the suppression of neuroinflammation as the proposed mechanism of action. Several studies have now reported that CB2 agonists are neuroprotective in models of cerebral ischemia. However, these studies have tended to measure brain infarctions in rodents 1-3 days after drug administration and have not assessed behavioral outcomes. As it has been shown that apparent protection soon after injury is not necessarily correlated with improved outcome after several weeks, we tested the CB2 selective agonist GW405833 in a model of cerebral hypoxia-ischemia, and assessed histological and behavioral outcomes 15 days after injury. Many putatively neuroprotective drugs have failed to translate from promising preclinical results to clinical success. We designed our experiments to not only stringently test CB2 mediated neuroprotection, but also to test several drug administration regimens to maximize the chance of detecting any therapeutic effect. However, GW405833 failed to provide neuroprotection in any of our experiments. These results challenge how far the results of earlier studies into CB2 mediated neuroprotection as measured at early time points may be extrapolated to later time points or to other models. |
Cisplatin is a widely used chemotherapeutic agent for the treatment of various malignancies. However, its maximum dose is often limited by severe ototoxicity. Cisplatin ototoxicity may require the production of reactive oxygen species (ROS) in the inner ear by activating enzymes specific to the cochlea. Molecular hydrogen was recently established as an antioxidant that selectively reduces ROS, and has been reported to protect the central nervous system, liver, kidney and cochlea from oxidative stress. The purpose of this study was to evaluate the potential of molecular hydrogen to protect cochleae against cisplatin. We cultured mouse cochlear explants in medium containing various concentrations of cisplatin and examined the effects of hydrogen gas dissolved directly into the media. Following 48-h incubation, the presence of intact auditory hair cells was assayed by phalloidin staining. Cisplatin caused hair cell loss in a dose-dependent manner, whereas the addition of hydrogen gas significantly increased the numbers of remaining auditory hair cells. Additionally, hydroxyphenyl fluorescein (HPF) staining of the spiral ganglion showed that formation of hydroxyl radicals was successfully reduced in hydrogen-treated cochleae. These data suggest that molecular hydrogen can protect auditory tissues against cisplatin toxicity, thus providing an additional strategy to protect against drug-induced inner ear damage. |
Increasing body of evidence suggests that inflammatory and neurotrophic factors might be important for epileptogenesis. Most animal studies demonstrated altered levels of these mediators in drug-induced models of seizures and epilepsy. In the present study, we investigated the production of cytokines and a neurotrophin in the brain of Wistar Audiogenic Rats (WAR), a genetic model of epilepsy, stimulated with high-intensity sound. Four hours after stimulation, animals were decapitated and the hippocampus, inferior colliculus, striatum and cortex were removed for evaluation of the levels of interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α and brain derived neurotrophic factor (BDNF). All the cytokines and BDNF levels were increased in the cortex. Increased levels of TNF-α and IL-6 were also observed in the striatum. Finally, TNF-α also increased in the inferior colliculus after the seizures induced by high-intensity sound. Although different studies have demonstrated that the levels of cytokines and BDNF increase in animal models of epilepsy induced by chemical stimuli, we provided here evidence that these mediators are also increased in WAR, a genetic model of epilepsy. Thus, the observed increase in these mediators might be involved in the pathophysiology of epilepsy. |
Parkinson's disease (PD) is the second most common neurodegenerative disease in the elderly caused by dopaminergic neuronal cell death. Human neuroblastoma SH-SY5Y cells differentiated by retinoic acid have been used to study the in vitro PD model induced by 1-methyl-4-phenyl pyridinium (MPP<sup>+</sup>). In this study, pretreatment of insulin inhibited MPP<sup>+</sup>-induced cell membrane damages, which also inhibited the Cox-2 and α-synuclein levels. In addition, MPP<sup>+</sup> and/or insulin enhanced the autophagy LC3. Furthermore, MPP<sup>+</sup>-induced neurotoxicity diminished the integrins β3, αV and induced the syndecan-1 and -3. Insulin pretreatment enhanced the phosphorylation of integrin-linked kinase and further induced the integrin and syndecan molecules. These findings suggest that insulin prevents MPP<sup>+</sup>-induced α-synuclein apoptosis through the activation of integrin and syndecan pathways in SH-SY5Y+RA cells. |
The eyeblink conditioning paradigm captures an elementary form of associative learning in a neural circuitry that is understood to an extraordinary degree. Cerebellar cortical Purkinje cell simple spike suppression is widely regarded as the main process underlying conditioned responses (CRs), leading to disinhibition of neurons in the cerebellar nuclei that innervate eyelid muscles downstream. However, recent work highlights the addition of a conditioned Purkinje cell complex spike response, which at the level of the interposed nucleus seems to translate to a transient spike suppression that can be followed by a rapid spike facilitation. Here, we review the characteristics of these responses at the cerebellar cortical and nuclear level, and discuss possible origins and functions. |
Preclinical and clinical studies suggest that brain-derived neurotrophic factor and nerve growth factor are involved in the pathogenesis of schizophrenia (SCZ). However, the roles of other neurotrophic factors in SCZ remain unclear. The aim of this study was to investigate the blood levels of FGF2 and ADNP in first-episode, drug-free SCZ patients compared with healthy control subjects. 20 SCZ patients, and 20 age and sex matched controls were recruited in this study. Serum FGF2 and ADNP protein levels were measured by ELISA assay, and the results showed that FGF2 levels were significantly increased in patients with SCZ when compared with controls, whereas ADNP protein levels did not significantly associated with SCZ. However, we found that blood ADNP mRNA levels were significantly increased in the patients with SCZ when compared with controls. In addition, subgroup analyses suggested that FGF2 levels were significantly increased in female patients of SCZ, but not in male patients of SCZ. Correlation analyses suggested that age and disease severity (PANSS score) did not have moderating effects on the serum FGF2 levels. Taken together, our results for the first time demonstrated that blood FGF2 was up-regulated in first-episode, drug free-SCZ patients, therefore enhancing the knowledge of neurotrophic factor profile in patients with SCZ. |
Alzheimer's disease (AD) is one of the most devastating conditions affecting elderly in Western World. Unfortunately, there are no effective treatments, and patients diagnosed with AD face an uncertain future, caused by the current inability to predict the course of the disease. This is mainly due to the poor comprehension of AD pathophysiology and of patients' clinical heterogeneity. In recent years, several evidences supported the concept that loss of synaptic density could be an early event and precede neuronal degeneration, suggesting that the impairment of synaptic transmission should play a key role in the pathogenesis of different forms of dementia, including AD, frontotemporal dementia and Lewy body dementia. Despite this emerging background it has not been possible to quantify synaptic functioning (or dysfunction) directly in vivo in AD patients. Transcranial magnetic stimulation (TMS) has been recently introduced as a novel approach able to identify the early signatures of synaptic dysfunction characterizing the different forms of AD. We review the novel emerging neurophysiological signatures of AD and how this information may be used as biomarkers for differential diagnosis, disease progression and response to therapy. Finally, we also consider novel therapeutic approaches based on the clinical use of repetitive TMS. |
Focal cortical dysplasia (FCD) is one of the main causes of medically intractable epilepsy. Some studies have reported that transient receptor potential canonical channel 3 (TRPC3) may play an important role in the occurrence of seizures. In this study, we investigated the expression patterns of TRPC3 in different types of FCD. Forty-five FCD specimens and 12 control samples from autopsies were used in our study. Western blotting, immunohistochemistry, and immunofluorescence staining were employed to detect protein expression and distribution. The amount of TRPC3 protein was markedly elevated in the FCD group. The immunohistochemistry results revealed that TRPC3 staining was strong in the malformed cells and microcolumns. Most of the TRPC3-positive cells were colabeled with glutamatergic and GABAergic markers. The overexpression and altered cellular distribution of TRPC3 in the FCD samples suggest that TRPC3 may be related to epileptogenesis in FCD. |
The Glucagon Like Peptide 1 Receptor (GLP1R) plays a critical role in selective death of dopaminergic neurons and development of Parkinson's disease (PD). However, little is known about genetic associations of GLP1R gene polymorphisms with PD susceptibility. Therefore, this study aimed to verify whether GLP1R polymorphisms contribute to PD risk in a Chinese Han population. We recruited 518 individuals comprising 259 sporadic PD patients and 259 healthy controls. All of the participants were genotyped for two possibly functional polymorphisms located in GLP1R (rs3765467 and rs6923761) using the Sequenom MassARRAY platform. The frequency of the rs3765467 GG genotype was significantly higher in the PD group compared with that in the control group (OR = 1.444, 95 % CI: 1.015-2.055, p =  0.041). Subgroup analysis revealed that male patients and late-onset patients with the rs3765467 GG genotype suffered an increased risk of PD compared with healthy controls (p =  0.021 and p =  0.012, respectively). However, the genotype and allele frequencies for rs6923761 were not significantly different between PD and healthy subjects. Our results indicate that the GLP1R rs3765467 GG genotype is a potential risk factor for PD, especially for male and late-onset PD patients in the Chinese Han population. |
Lycium ruthenicum Murray is widely used in traditional Chinese medicine and is believed to have antimicrobial, antioxidant, and anti-fatigue effects. Anthocyanins are considered to be one of the main active components. The previous work by our research team found that the anthocyanins in Lycium ruthenicum extract (ALRM) produce a stable anti-anxiety effect. The mechanisms of action include reducing the level of corticotropin-releasing factor (CRF) as well as regulating extracellular signal-regulated kinase/mitogen activation, protein kinase (ERK/MAPK) pathways, and others, all of which are related to the mechanisms of nicotine addiction. To investigate the effects of ALRM on anxiety and craving behavior after nicotine withdrawal, the components of ALRM were analyzed using the UPLC-Orbitrap MS method. The effects of ALRM on anxiety behavior induced by nicotine withdrawal were investigated in mice using the elevated plus maze (EPM) and light-dark box (LDB) tests. The effects of ALRM on craving behavior after nicotine withdrawal were further investigated using the conditional place preference (CPP) test. The EPM and LDB tests demonstrated that ALRM could alleviate the anxiety behavior induced by nicotine withdrawal and reduce nicotine craving in mice. Based on the identified ALRM components, the network pharmacology method was used to predict the mechanism of ALRM alleviating anxiety after nicotine withdrawal in mice. It was speculated that ALRM was involved in the production and transmission of dopamine, choline, and other nervous system functions and exhibited a potential role in treating nicotine addiction. |
Alzheimer's disease (AD) is the most common neurodegenerative disease. SUMOylation, a post-translational modification, has been found to be dysregulated in the AD brain and to exacerbate learning and memory disabilities and increase amyloid beta (Aβ) expression further. To investigate whether exercise-induced alleviation of AD was associated with SUMOylation, which still remains unknown, 3-month-old C57BL/6 mice and APP/PS1 transgenic mice were randomly divided into the wild-type control (WC), wild-type exercise (WE), APP/PS1 control (AC), and APP/PS1 exercise (AE) groups. Mice in the exercise groups underwent a 3-month treadmill exercise regimen. We observed impaired learning and memory abilities in APP/PS1 mice, but the 3-month treadmill exercise regimen improved spatial learning and memory abilities in wild-type and APP/PS1 mice. In addition, senile plaques, SUMO1 mRNA, and SENP1 mRNA levels increased in the hippocampi of APP/PS1 mice. However, 3-month treadmill exercise decreased the levels of senile plaques, SUMO1 mRNA and SENP1 mRNA as well as may reduce SUMO1 modification in 6-month-old APP/PS1 mice, but SUMO2 mRNA expression, SUMO2/3 modification, and overall SUMOylation levels did not significantly change. Our results suggest that the impaired learning and memory abilities and aggregations of Aβ may relate to increased hippocampal SUMO1 transcription levels; the beneficial effects of treadmill exercise on learning and memory performances and AD pathogenesis may associated with the abatement of SUMO1 modification, but may not with SUMO2/3 modification. |
Psychological studies have shown that music has an impact on human cognitive function. We aimed to compare the performance and neural activity of pianists and non-musicians during a non-musical motor-planning task. In addition, we investigated the effect of task complexity on the characteristics of the behavioral and neural responses. The participants had to grasp a hexagonal knob with their right hand and rotate it 60° or 180° clockwise (CW) or counterclockwise (CCW). We examined the groups in terms of the amplitude of the P2 component in the event-related potential (at the neural level) and the planning time, grasping time, releasing time, and planning pattern for initial grip selection (at the behavioral level). At the behavioral level, we observed no significant difference between groups, while at the neural level; we found an interaction between direction and group indicating that pianists showed lower P2 amplitude in the CW directions. However, there was no significant difference between groups in the CCW direction. A significant main effect of rotation was revealed at both the neural and behavioral levels; increasing the rotation angle led to an increase in the planning time and the P2 amplitude, indicating a complexity effect. In conclusion, we observed that pianists had lower P2 amplitude in lateral movements than non-musicians; however, due to the lack of behavioral group differences, further research is warranted to support the far-transfer theory in this field. |
The cranial window technique has proven to be an effective method for in vivo imaging of cortical activity. However, given the invasive nature of this procedure, possible side effects could be expected in the nervous system. In this study, we evaluated the effects of unilateral cranial window surgery on auditory function in C57BL6 mice using electrophysiological and behavioral approaches. We found that one week after implantation, mice exhibited both increased thresholds and decreased amplitudes of their auditory brainstem responses. These changes were accompanied by a decrease in distortion product otoacoustic emissions, indicating a deterioration in cochlear function. In addition, behavioral testing of these mice revealed reduced suppression of their acoustic startle response by gap prepulse, suggesting a deficit in auditory processing or possibly the presence of tinnitus. The changes in auditory function appeared to be only partially reversible within four weeks after surgery. Thus, our findings suggest that cranial window implantation causes long-term functional changes in the auditory system that should be considered when interpreting data from optical imaging techniques. |
Aging is associated with a substantial decline in the expression of social behavior as well as increased neuroinflammation. Since immune activation and subsequent increased expression of cytokines can suppress social behavior in young rodents, we examined age and sex differences in microglia within brain regions critical to social behavior regulation (PVN, BNST, and MEA) as well as in the hippocampus. Adult (3-month) and aged (18-month) male and female F344 (N = 26, n = 5-8/group) rats were perfused and Iba-1 immunopositive microglia were assessed using unbiased stereology and optical density. For stereology, microglia were classified based on the following criteria: (1) thin ramified processes, (2) thick long processes, (3) stout processes, or (4) round/ameboid shape. Among the structures examined, the highest density of microglia was evident in the BNST and MEA. Aged rats of both sexes displayed increased total number of microglia number exclusively in the MEA. Sex differences also emerged, whereby aged females (but not males) displayed greater total number of microglia in the BNST relative to their young adult counterparts. When morphological features of microglia were assessed, aged rats exhibited increased soma size in the BNST, MEA, and CA3. Together, these findings provide a comprehensive characterization of microglia number and morphology under ambient conditions in CNS sites critical for the normal expression of social processes. To the extent that microglia morphology is predictive of reactivity and subsequent cytokine release, these data suggest that the expression of social behavior in late aging may be adversely influenced by heightened inflammation. |
Autism Spectrum Disorder (ASD) and epilepsy are two neurodevelopmental disorders that have a high comorbidity rate, suggesting that a common neurodevelopmental mechanism exists. However, to date there is no conclusive way to predict whether a child will develop either syndrome or both and to what degree associated phenotypes will be affected. Failure to consistently identify predictive patterns of ASD and/or epilepsy diagnosis stems from the fact that they are etiologically heterogeneous conditions and research into their neuropathological mechanisms becomes challenging. Whole genome/exome sequencing has advanced our understanding of the genetic causes of ASD and epilepsy to an extent that currently about half of all ASD as well as epilepsy cases are known to have a genetic basis. In fact, a picture is emerging of both conditions as a collection of distinct genetically defined disorders, although the role of environmental factors has also been established. A plethora of animal models, most of them based on identified human genetic mutations and a few on known environmental causes, have been developed. Animal models provide a major experimental avenue for studying the underlying cellular and molecular mechanisms of human disorders. They also provide invaluable preclinical tools that can be used to test therapeutic approaches. In this review, we first summarize the methods for validating mouse models of ASD and epilepsy. Second, we present the current models validated for the comorbidity and finally, we recapitulate the common pathomechanisms identified in these models with special emphasis on synaptic plasticity. |
Mechanisms initiated by traumatic brain injury (TBI), leading to the development of progressive secondary injury are poorly understood. MicroRNAs (miRNAs) have a proposed role in orchestrating the post-injury aftermath as a single miRNA can control the expression of several genes. We hypothesized that the post-injury level of circulating brain-enriched miR-124-3p explains the extent of post-TBI cortical lesion. Three separate cohorts of adult male Sprague-Dawley rats (total n = 57) were injured with lateral fluid-percussion-induced TBI. The miR-124-3p levels were measured in whole blood and/or plasma in cohorts 1 and 2 before TBI as well as at 2 d, 7 d, 2 months or 3 months post-TBI. The third cohort (22/57) was imaged with T2-weighted magnetic resonance imaging (MRI) at 2 months post-TBI to quantify cortical lesion area and perilesional T2-enhancement volume. Our data shows that miR-124-3p levels were elevated at 2 d post-TBI in both blood (FC 4.63, p < 0.01) and plasma (FC 1.39, p < 0.05) as compared to controls. Receiver operating curve (ROC) analysis indicated that plasma miR-124-3p level of 34 copies/µl or higher differentiated TBI animals from controls [area under curve (AUC) 0.815, p < 0.05]. The data was validated in the third cohort (FC 1.68, p < 0.05). T2-weighted MRI revealed inter-animal differences in cortical lesion area. Linear regression analysis revealed that higher the plasma miR-124-3p level at 2 d post-TBI, larger the lesion area at chronic time point (R<sup>2</sup> = 0.327, p < 0.01). Our findings indicate that the extent of lateral fluid-percussion injury-induced chronic cortical pathology associated with the acutely elevated plasma miR-124-3p level. |
The brain opioid system plays an important role in cocaine reward. Altered signaling in the opioid system by chronic cocaine exposure contributes to cocaine-seeking and taking behavior. The current study investigated concurrent changes in the gene expression of multiple components in rat brain opioid system following cocaine self-administration. Animals were limited to 40 infusions (1.5 mg/kg/infusion) within 6 h per day for five consecutive days. We then examined the mRNA levels of opioid receptors including mu (Oprm), delta (Oprd), and kappa (Oprk), and their endogenous opioid peptide precursors including proopiomelanocortin (Pomc), proenkephalin (Penk), prodynorphin (Pdyn) in the dorsal striatum (CPu) and the prefrontal cortex (PFC) 18 h after the last cocaine infusion. We found that cocaine self-administration significantly increased the mRNA levels of Oprm and Oprd in both the CPu and PFC, but had no effect on Oprk mRNA levels in either brain region. Moreover, cocaine had a greater influence on the mRNA levels of opioid peptide precursors in rat CPu than in the PFC. In the CPu, cocaine self-administration significantly increased the mRNA levels of Penk and Pdyn and abolished the mRNA levels of Pomc. In the PFC, cocaine self-administration only increased Pdyn mRNA levels without changing the mRNA levels of Pomc and Penk. These data suggest that cocaine self-administration influences the expression of multiple genes in the brain opioid system, and the concurrent changes in these targets may underlie cocaine-induced reward and habitual drug-seeking behavior. |
Feedback on success or failure is critical to increase rewards through behavioral adaptation or learning of dependencies from trial and error. Learning from reward feedback is thereby treated as embedded in a reinforcement learning framework. Due to temporal discounting of reward, learning in this framework is suspected to be vulnerable to feedback delay. Together, investigations of reinforcement learning in learned decision making tasks show that performance and learning impairments due to feedback delay vary as a function of task type. Performance in tasks that require implicit processing is affected by the delayed availability of feedback compared to tasks that can be accomplished with explicit processing. At the same time, the feedback related negativity, an event related potential component in the electroencephalogram that is associated with feedback processing, is affected by feedback delay similarly independent of task type. With the idea of fully implicit or explicit processing as opposite endpoints of a continuum of reciprocal shares of the implicit and explicit processing systems with feedback delay as the determinant of where on this continuum processing can be located, a common explanatory approach of both, behavioral and electrophysiological findings, is suggested. |
Perineuronal nets (PNNs) are extracellular matrix (ECM) chondroitin sulfate proteoglycan (CSPG)-containing structures that surround the soma and dendrites of various mammalian neuronal cell types. PNNs appear during development around the time that the critical periods for developmental plasticity end and are important for both their onset and closure. A similar structure - the perinodal ECM - surrounds the axonal nodes of Ranvier and appears as myelination is completed, acting as an ion-diffusion barrier that affects axonal conduction speed. Recent work has revealed the importance of PNNs in controlling plasticity in the CNS. Digestion, blocking or removal of PNNs influences functional recovery after a variety of CNS lesions. PNNs have further been shown to be involved in the regulation of memory and have been implicated in a number of psychiatric disorders. |
In the version of this article initially published, the footnote number 17 was missing from the author list for the two authors who contributed equally. Also, the authors have added a middle initial for author Justin R. Fallon and an acknowledgement to the Babraham Institute Imaging Facility and Sequencing Core Facility. The errors have been corrected in the HTML and PDF versions of the article. |
Zika virus (ZIKV) is a flavivirus linked to multiple birth defects including microcephaly, known as congenital ZIKV syndrome. The identification of host factors involved in ZIKV replication may guide efficacious therapeutic interventions. In genome-wide transcriptional studies, we found that ZIKV infection triggers aryl hydrocarbon receptor (AHR) activation. Specifically, ZIKV infection induces kynurenine (Kyn) production, which activates AHR, limiting the production of type I interferons (IFN-I) involved in antiviral immunity. Moreover, ZIKV-triggered AHR activation suppresses intrinsic immunity driven by the promyelocytic leukemia (PML) protein, which limits ZIKV replication. AHR inhibition suppressed the replication of multiple ZIKV strains in vitro and also suppressed replication of the related flavivirus dengue. Finally, AHR inhibition with a nanoparticle-delivered AHR antagonist or an inhibitor developed for human use limited ZIKV replication and ameliorated newborn microcephaly in a murine model. In summary, we identified AHR as a host factor for ZIKV replication and PML protein as a driver of anti-ZIKV intrinsic immunity. |
To characterize the dysregulation of chromatin accessibility in Alzheimer's disease (AD), we generated 636 ATAC-seq libraries from neuronal and nonneuronal nuclei isolated from the superior temporal gyrus and entorhinal cortex of 153 AD cases and 56 controls. By analyzing a total of ~20 billion read pairs, we expanded the repertoire of known open chromatin regions (OCRs) in the human brain and identified cell-type-specific enhancer-promoter interactions. We show that interindividual variability in OCRs can be leveraged to identify cis-regulatory domains (CRDs) that capture the three-dimensional structure of the genome (3D genome). We identified AD-associated effects on chromatin accessibility, the 3D genome and transcription factor (TF) regulatory networks. For one of the most AD-perturbed TFs, USF2, we validated its regulatory effect on lysosomal genes. Overall, we applied a systematic approach to understanding the role of the 3D genome in AD. We provide all data as an online resource for widespread community-based analysis. |
Huntington's disease (HD) is an inherited neurodegenerative disorder with adult-onset clinical symptoms, but the mechanism by which aging drives the onset of neurodegeneration in patients with HD remains unclear. In this study we examined striatal medium spiny neurons (MSNs) directly reprogrammed from fibroblasts of patients with HD to model the age-dependent onset of pathology. We found that pronounced neuronal death occurred selectively in reprogrammed MSNs from symptomatic patients with HD (HD-MSNs) compared to MSNs derived from younger, pre-symptomatic patients (pre-HD-MSNs) and control MSNs from age-matched healthy individuals. We observed age-associated alterations in chromatin accessibility between HD-MSNs and pre-HD-MSNs and identified miR-29b-3p, whose age-associated upregulation promotes HD-MSN degeneration by impairing autophagic function through human-specific targeting of the STAT3 3' untranslated region. Reducing miR-29b-3p or chemically promoting autophagy increased the resilience of HD-MSNs against neurodegeneration. Our results demonstrate miRNA upregulation with aging in HD as a detrimental process driving MSN degeneration and potential approaches for enhancing autophagy and resilience of HD-MSNs. |
<b>Background:</b> Autophagy-related gene 7 (ATG7) plays a key role in autophagy and is strongly implicated in Parkinson's disease (PD). This study investigated the associations of rs1375206 polymorphism in <i>ATG7</i> gene promoter and plasma ATG7 levels with late-onset sporadic PD in a cohort of Han Chinese from southern China.<b>Methods:</b> Variant genotypes were identified using polymerase chain reaction-restriction fragment length polymorphism and gene sequencing in 124 patients with late-onset sporadic PD, as well as in 105 age- and sex-matched healthy controls. Plasma ATG7 levels were determined using an enzyme-linked immunosorbent assay.<b>Results:</b> No significant differences in genotype distributions were found between the two groups. Stratification analyses by sex and clinical motor subtypes revealed that the differences remained non-significant in each subgroup (all <i>p</i> > 0.05). Plasma ATG7 protein levels were significantly higher in the PD group than in the control group (<i>p</i> = 0.000). Haplotype analysis demonstrated that the A-T haplotype was significantly associated with late-onset sporadic PD (<i>p</i> = 0.045).<b>Conclusion:</b> Our study suggests that the rs1375206 polymorphism in <i>ATG7</i> may not be associated with late-onset sporadic PD; however, high plasma ATG7 levels and the A-T haplotype may be associated with susceptibility to late-onset sporadic PD in the Han population from Zhejiang and Guangdong provinces. |
Neuronal activity in the prefrontal cortex (PFC) controls dominance hierarchies in groups of animals. Dopamine (DA) strongly modulates PFC activity mainly through D1 receptors (D1Rs) and D2 receptors (D2Rs). Still, it is unclear how these two subpopulations of DA receptor-expressing neurons in the PFC regulate social dominance hierarchy. Here, we demonstrate distinct roles for prefrontal D1R- and D2R-expressing neurons in establishing social hierarchy, with D1R<sup>+</sup> neurons determining dominance and D2R<sup>+</sup> neurons for subordinate. <i>Ex vivo</i> whole-cell recordings revealed that the dominant status of male mice correlates with rectifying AMPAR transmission and stronger excitatory synaptic strength onto D1R<sup>+</sup> neurons in PFC pyramidal neurons. In contrast, the submissive status is associated with higher neuronal excitability in D2R<sup>+</sup> neurons. Moreover, simultaneous manipulations of synaptic efficacy of D1R<sup>+</sup> neurons in dominant male mice and neuronal excitability of D2R<sup>+</sup> neurons of their male subordinates switch their dominant-subordinate relationship. These results reveal that prefrontal D1R<sup>+</sup> and D2R<sup>+</sup> neurons have distinct but synergistic functions in the dominance hierarchy, and DA-mediated regulation of synaptic strengths acts as a powerful behavioral determinant of intermale social rank.<b>SIGNIFICANCE STATEMENT</b> Dominance hierarchy exists widely among animals who confront social conflict. Studies have indicated that social status largely relies on the neuronal activity in the PFC, but how dopamine influences social hierarchy via subpopulation of prefrontal neurons is still elusive. Here, we explore the cell type-specific role of dopamine receptor-expressing prefrontal neurons in the dominance-subordinate relationship. We found that the synaptic strength of D1 receptor-expressing neurons determines the dominant status, whereas hyperactive D2-expressing neurons are associated with the subordinate status. These findings highlight how social conflicts recruit distinct cortical microcircuits to drive different behaviors and reveal how D1- and D2-receptor enriched neurocircuits in the PFC establish a social hierarchy. |
The structural plasticity of the axon initial segment (AIS) contributes to the homeostatic control of activity and optimizes the function of neural circuits; however, the underlying mechanisms are not fully understood. In this study, we prepared a slice culture containing nucleus magnocellularis from chickens of both sexes that reproduces most features of AIS plasticity <i>in vivo</i>, regarding its effects on characteristics of AIS and cell-type specificity, and revealed that microtubule reorganization via activation of CDK5 underlies plasticity. Treating the culture with a high-K<sup>+</sup> medium shortened the AIS and reduced sodium current and membrane excitability, specifically in neurons tuned to high-frequency sound, creating a tonotopic difference in AIS length in the nucleus. Pharmacological analyses revealed that this AIS shortening was driven by multiple Ca<sup>2+</sup> pathways and subsequent signaling molecules that converge on CDK5 via the activation of ERK1/2. AIS shortening was suppressed by overexpression of dominant-negative CDK5, whereas it was facilitated by the overexpression of p35, an activator of CDK5. Notably, p35(T138A), a phosphorylation-inactive mutant of p35, did not shorten the AIS. Moreover, microtubule stabilizers occluded AIS shortening during the p35 overexpression, indicating that CDK5/p35 mediated AIS shortening by promoting disassembly of microtubules at distal AIS. This study highlights the importance of microtubule reorganization and regulation of CDK5 activity in structural AIS plasticity and the tuning of AIS characteristics in neurons.<b>SIGNIFICANCE STATEMENT</b> The structural plasticity of AIS has a strong impact on the output of neurons and plays a fundamental role in the physiology and pathology of the brain. However, the mechanisms linking neuronal activity to structural changes in AIS are not well understood. In this study, we prepared an organotypic culture of avian auditory brainstem, reproducing most AIS plasticity features <i>in vivo</i>, and we revealed that activity-dependent AIS shortening occurs through the disassembly of microtubules at distal AIS via activation of CDK5/p35 signals. This study emphasizes the importance of microtubule reorganization and regulation of CDK5 activity in structural AIS plasticity and tonotopic differentiation of AIS structures in the brainstem auditory circuit. |
People are particularly sensitive to injustice. Accordingly, deeper knowledge regarding the processes that underlie the perception of injustice, and the subsequent decisions to either punish transgressors or compensate victims, is of important social value. By combining a novel decision-making paradigm with functional neuroimaging, we identified specific brain networks that are involved with both the perception of, and response to, social injustice, with reward-related regions preferentially involved in punishment compared with compensation. Developing a computational model of punishment allowed for disentangling the neural mechanisms and psychological motives underlying decisions of whether to punish and, subsequently, of how severely to punish. Results show that the neural mechanisms underlying punishment differ depending on whether one is directly affected by the injustice, or whether one is a third-party observer of a violation occurring to another. Specifically, the anterior insula was involved in decisions to punish following harm, whereas, in third-party scenarios, we found amygdala activity associated with punishment severity. Additionally, we used a pharmacological intervention using oxytocin, and found that oxytocin influenced participants' fairness expectations, and in particular enhanced the frequency of low punishments. Together, these results not only provide more insight into the fundamental brain mechanisms underlying punishment and compensation, but also illustrate the importance of taking an explorative, multimethod approach when unraveling the complex components of everyday decision-making.<b>SIGNIFICANCE STATEMENT</b> The perception of injustice is a fundamental precursor to many disagreements, from small struggles at the dinner table to wasteful conflict between cultures and countries. Despite its clear importance, relatively little is known about how the brain processes these violations. Taking an interdisciplinary approach, we combine methods from neuroscience, psychology, and economics to explore the neurobiological mechanisms involved in both the perception of injustice as well as the punishment and compensation decisions that follow. Using a novel behavioral paradigm, we identified specific brain networks, developed a computational model of punishment, and found that administrating the neuropeptide oxytocin increases the administration of low punishments of norm violations in particular. Results provide valuable insights into the fundamental neurobiological mechanisms underlying social injustice. |
We previously found that low-frequency stimulation of direct temperoammonic (TA) inputs to hippocampal area CA1 depotentiates previously established long-term potentiation in the Schaffer collateral (SC) pathway through complex signaling involving dopamine, endocannabinoids, neuregulin-1, GABA, and adenosine, with adenosine being the most distal modulator identified to date. In the present studies, we examined mechanisms contributing to the effects of adenosine in hippocampal slices from male albino rats. We found that extracellular conversion of ATP to adenosine via an ectonucleotidase contributes significantly to TA-mediated SC depotentiation and the depotentiation resulting from block of adenosine transport. Adenosine-mediated SC depotentiation does not involve activation of c-Jun N-terminal protein kinase, serine phosphatases, or nitric oxide synthase, unlike homosynaptic SC depotentiation. Rather, adenosine-induced depotentiation is inhibited by specific antagonists of p38 MAPK, but not by a structural analog that does not inhibit p38. Additionally, using antagonists with relative selectivity for p38 subtypes, it appears that TA-induced SC depotentiation most likely involves p38 MAPK β. These findings have implications for understanding the role of adenosine and other extrahippocampal and intrahippocampal modulators in regulating SC synaptic function and the contributions of these modulators to the cognitive dysfunction associated with neuropsychiatric illnesses.<b>SIGNIFICANCE STATEMENT</b> Low-frequency stimulation of temperoammonic (TA) inputs to stratum lacunosum moleculare of hippocampal area CA1 heterosynaptically depotentiates long-term potentiation of Schaffer collateral (SC) synapses. TA-induced SC depotentiation involves complex signaling including dopamine, endocannabinoids, GABA, and adenosine, with adenosine serving as the most downstream messenger in the cascade identified to date. The present results indicate that TA-induced depotentiation requires intact inputs from entorhinal cortex and that adenosine ultimately drives depotentiation via activation of p38 MAPK. These studies have implications for understanding the cognitive dysfunction of psychiatric illnesses and certain abused drugs. |
Knowledge about objects encompasses not only their prototypical features but also complex, atypical, semantic knowledge (e.g., "Pizza was invented in Naples"). This fMRI study of male and female human participants combines univariate and multivariate analyses to consider the cortical representation of this more complex semantic knowledge. Using the categories of food, people, and places, this study investigates whether access to spatially related geographic semantic knowledge (1) involves the same domain-selective neural representations involved in access to prototypical taste knowledge about food; and (2) elicits activation of neural representations classically linked to places when this geographic knowledge is accessed about food and people. In three experiments using word stimuli, domain-relevant and atypical conceptual access for the categories food, people, and places were assessed. Results uncover two principles of semantic representation: food-selective representations in the left insula continue to be recruited when prototypical taste knowledge is task-irrelevant and under conditions of high cognitive demand; access to geographic knowledge for food and people categories involves the additional recruitment of classically place-selective parahippocampal gyrus, retrosplenial complex, and transverse occipital sulcus. These findings underscore the importance of object category in the representation of a broad range of knowledge, while showing how the cross recruitment of specialized representations may endow the considerable flexibility of our complex semantic knowledge.<b>SIGNIFICANCE STATEMENT</b> We know not only stereotypical things about objects (an apple is round, graspable, edible) but can also flexibly combine typical and atypical features to form complex concepts (the metaphorical role an apple plays in Judeo-Christian belief). In this fMRI study, we observe that, when atypical geographic knowledge is accessed about food dishes, domain-selective sensorimotor-related cortical representations continue to be recruited, but that regions classically associated with place perception are additionally engaged. This interplay between categorically driven representations, linked to the object being accessed, and the flexible recruitment of semantic stores linked to the content being accessed, provides a potential mechanism for the broad representational repertoire of our semantic system. |
Neuron activity accompanies synapse formation and maintenance, but how early circuit activity contributes to behavior development is not well understood. Here, we use the <i>Caenorhabditis elegans</i> egg-laying motor circuit as a model to understand how coordinated cell and circuit activity develops and drives a robust two-state behavior in adults. Using calcium imaging in behaving animals, we find the serotonergic hermaphrodite-specific neurons (HSNs) and vulval muscles show rhythmic calcium transients in L4 larvae before eggs are produced. HSN activity in L4 is tonic and lacks the alternating burst-firing/quiescent pattern seen in egg-laying adults. Vulval muscle activity in L4 is initially uncoordinated but becomes synchronous as the anterior and posterior muscle arms meet at HSN synaptic release sites. However, coordinated muscle activity does not require presynaptic HSN input. Using reversible silencing experiments, we show that neuronal and vulval muscle activity in L4 is not required for the onset of adult behavior. Instead, the accumulation of eggs in the adult uterus renders the muscles sensitive to HSN input. Sterilization or acute electrical silencing of the vulval muscles inhibits presynaptic HSN activity and reversal of muscle silencing triggers a homeostatic increase in HSN activity and egg release that maintains ∼12-15 eggs in the uterus. Feedback of egg accumulation depends upon the vulval muscle postsynaptic terminus, suggesting that a retrograde signal sustains HSN synaptic activity and egg release. Our results show that egg-laying behavior in <i>C. elegans</i> is driven by a homeostat that scales serotonin motor neuron activity in response to postsynaptic muscle feedback.<b>SIGNIFICANCE STATEMENT</b> The functional importance of early, spontaneous neuron activity in synapse and circuit development is not well understood. Here, we show in the nematode <i>Caenorhabditis elegans</i> that the serotonergic hermaphrodite-specific neurons (HSNs) and postsynaptic vulval muscles show activity during circuit development, well before the onset of adult behavior. Surprisingly, early activity is not required for circuit development or the onset of adult behavior and the circuit remains unable to drive egg laying until fertilized embryos are deposited into the uterus. Egg accumulation potentiates vulval muscle excitability, but ultimately acts to promote burst firing in the presynaptic HSNs which results in egg laying. Our results suggest that mechanosensory feedback acts at three distinct steps to initiate, sustain, and terminate <i>C. elegans</i> egg-laying circuit activity and behavior. |
A current view proposes that the right inferior frontal cortex (IFC) is particularly responsible for attentive decoding and cognitive evaluation of emotional cues in human vocalizations. Although some studies seem to support this view, an exhaustive review of all recent imaging studies points to an important functional role of both the right and the left IFC in processing vocal emotions. Second, besides a supposed predominant role of the IFC for an attentive processing and evaluation of emotional voices in IFC, these recent studies also point to a possible role of the IFC in preattentive and implicit processing of vocal emotions. The studies specifically provide evidence that both the right and the left IFC show a similar anterior-to-posterior gradient of functional activity in response to emotional vocalizations. This bilateral IFC gradient depends both on the nature or medium of emotional vocalizations (emotional prosody versus nonverbal expressions) and on the level of attentive processing (explicit versus implicit processing), closely resembling the distribution of terminal regions of distinct auditory pathways, which provide either global or dynamic acoustic information. Here we suggest a functional distribution in which several IFC subregions process different acoustic information conveyed by emotional vocalizations. Although the rostro-ventral IFC might categorize emotional vocalizations, the caudo-dorsal IFC might be specifically sensitive to their temporal features. |
Depression and impulse control disorders (ICD) are two common neuropsychiatric features in Parkinson's disease (PD). Studies have revealed that both phenomena are associated with aberrations in ventral striatal dopamine signaling and concomitant dysfunction of the reward-related (limbic) cortico-striatal-thalamocortical (CSTC) circuit. Depression in PD seems associated with decreased activity in the limbic CSTC circuit, whereas ICD seem associated with increased limbic CSTC circuit activity, usually after commencing dopamine replacement therapy (DRT). Not all DRT using PD patients, however, develop symptoms of ICD, suggesting an additional underlying neurobiological susceptibility. Furthermore, the symptoms of depression and ICD frequently coincide even though they are related to seemingly contrasting limbic CSTC circuit activation states. The aim of this review is to provide an overview of the currently available literature on the neurobiology of PD-related depression and ICD and discusses possible susceptibility factors. Finally, we propose a neurobiological model that identifies ventral striatal dopaminergic denervation as a common underlying neurobiological substrate of depression and ICD and subsequent dysfunction of reward and motivation-related brain areas. |
The G4C2 repeat expansion within C9orf72 has been recently identified as the most common genetic cause of frontotemporal dementia and amyotrophic lateral sclerosis. This mutation has also been detected in a variety of other neurological diseases with distinct clinical manifestations. The exact mechanisms of how this mutation leads to the wide spectrum of clinical syndromes remain unknown. A series of molecular changes together with some potential modifiers may play a key role. Nucleolar stress, nucleocytoplasmic transport defect, oxidative damage, inhibited stress granules assembly, activated endoplasmic reticulum stress, and inhibited proteasome activity are mechanisms that contribute to the pathogenesis of these diseases. Additional mutations, epigenetic modifiers, and repeat size are potential modifiers that modulate specific phenotypes on the basis of the molecular changes. Here, we summarize distinct C9orf72-related neurological disorders and their corresponding neuropathological changes. Then, we elucidate the existing molecular knowledge and the potential modifiers. Finally, we detail the main target of treatment aiming at controlling expanded RNA transcripts. |
DNA methylation (DNAm) - an epigenetic process that regulates gene expression - may represent a mechanism for the biological embedding of early traumatic experiences, including childhood maltreatment. Here, we conducted the first systematic review of human studies linking childhood maltreatment to DNAm. In total, 72 studies were included in the review (2008-2018). The majority of extant studies (i) were based on retrospective data in adults, (ii) employed a candidate gene approach (iii) focused on global maltreatment, (iv) were based on easily accessible peripheral tissues, typically blood; and (v) were cross-sectional. Two-thirds of studies (n = 48) also examined maltreatment-related outcomes, such as stress reactivity and psychiatric symptoms. While findings generally support an association between childhood maltreatment and altered patterns of DNAm, factors such as the lack of longitudinal data, low comparability across studies as well as potential genetic and 'pre-exposure' environmental confounding currently limit the conclusions that can be drawn. Key challenges are discussed and concrete recommendations for future research are provided to move the field forward. |
## Background
Images of perfusion estimates obtained with the continuous arterial spin labelling technique are characterized by variation between single acquisitions. Little is known about the spatial determinants of this variation during the acquisition process and their impact on voxel-by-voxel estimates of effects.
## Results
We show here that the spatial patterns of covariance between voxels arising during the acquisition of these images uncover distinct mechanisms through which this variance arises: through variation in global perfusion levels; through the action of large vessels and other, less well characterized, large anatomical structures; and through the effect of noisy areas such as the edges of the brain.
## Conclusions
Knowledge of these covariance patterns is important to experimenters for a correct interpretation of findings, especially for studies where relatively few acquisitions are made.
## Background
Arterial spin labelling (ASL, [ ]) is a non-invasive technique for the measurement of cerebral blood flow (CBF), enabling investigators to study brain perfusion with magnetic resonance techniques (see [ , ] for general introductions). Recent advances in arterial spin labelling techniques allow the practical acquisition of CBF estimates with wide spatial coverage of the brain using multi-slice or 3D acquisitions [ ]. This has made of ASL a very promising technique for the systematic investigation of the physiology and functional determinants of brain perfusion, and of individual differences in baseline CBF at rest [ - ].
Several studies have described the spectral components of time series in ASL data [ - ]. This study is concerned with the spatial patterns of covariation in the residual images of linear models fitted to quantitative perfusion images obtained with the continuous arterial spin labelling (CASL) technique, as implemented by [ ]. A previous study investigated the principal components associated to subject-to-subject spatial variance, and described acquisition-to-acquisition variance [ ]. Study of spatial covariation is of interest for two reasons. Firstly, it gives insight on the interplay of brain physiology with respect of vascularisation, and the signal acquired with this imaging technique. Secondly, knowledge of spatial covariation is important to experimenters because it constitutes a violation of the stationarity assumption on the random field that models spatial covariation of residuals after smoothing [ , ]. Under this assumption, residuals are spatially distributed like smoothed white noise. Concretely, the violation of this assumption means that under the null hypothesis the estimated effects are likely to reflect the pattern determined by the most important patterns of covariation, rather than a random set of blobs scattered across the brain. Therefore, when inspecting images of the estimated effects, knowledge of the form these images are likely to take even if the null hypothesis is true can assist in evaluating the spatial patterns produced by an experiment or an observational study, and distinguishing between sources of variation due to vascularisation and those due to the variable of interest.
Here, we refer to acquisitions as to images of CBF values computed from applying a compartment model to two scans, one with and one without labelled spins [ ]. In a study of baseline perfusion at rest, acquisition-to-acquisition variance arises from variation from one estimated perfusion image to the other within subjects. In a study in which participants carry out a task in the scanner, acquisition-to-acquisition variance is constituted by the residual variance arising from one estimated perfusion image to the other within experimental conditions/factor levels. Figure displays maps of acquisition-to-acquisition variance, collected from participants resting quietly in the scanner with closed eyes for 8 min, and therefore originated from a study of the first type. One can see that this variance shows differences of one order of magnitude across the brain, and that these differences follow anatomical features such as the course of large vessels [ ]. High variance also occurs at the edges of the brain and in ventricles, where the model used to estimate perfusion may not hold (because quantified CBF is estimated from a ratio, misspecification of the model may amplify the variation in the denominator). Because the reasons for high variance in these areas and near large vessels may differ, it is conceivable that spatial covariance may show different intensities depending on the mechanism at the basis of variance. Specifically, we would expect high variance at the edges of the brain to arise because of high variation from one voxel to the next leading to low spatial covariance, while near large vessels groups of voxels may tend to assume largely different values from one acquisition to the other, giving rise to larger spatial correlations.
Acquisition-to-acquisition variance maps . Acquisition-to-acquisition variance, axial slices, overlaid on a template brain. Very large variance (in red) is present at the edges of the brain and in correspondence of the course of large vessels (MCA: middle cerebral artery; PCA: posterior cerebral artery; ACA: anterior cerebral artery and frontal arteries; IA: insular arteries; CA: middle occipital and choroidal arteries; RS: sinus rectus). Most of the brain parenchyma, in contrast, displays lower variance (in yellow, green, and blue), especially in white matter. On the right, the histogram of the variance values shows a long right tail; isolated values reach σ = 30 000.
In this study, patterns of spatial covariation will be identified by carrying out a principal component analysis of the estimated acquisition-to-acquisition variance shown in Figure . The sample consists of a total of 13 680 images collected from 228 participants, and because of its size should yield a quite reliable eigendecomposition. To characterize the origin of these components further, the relation of the main components of variation with global CBF levels will be investigated.
## Results
### Principal component analysis
Figure shows the first four principal spatial pattern of acquisition-to-acquisition variation obtained through principal component analysis. The relative weights of the first 20 components, expressed by the proportion of variance captured in the data, are displayed in Figure , left. Even if individual acquisition images may be characterized by variance at high spatial frequency [ ], the common modes of variation displayed in these images tend to be spatially smooth (very similar components, not shown here for brevity, were obtained by analysing non-smoothed data).
Principal components maps . From top to bottom, component images of the first four components of acquisition-to-acquisition variance, overlaid on template brains. On the right, histograms of the component scores (unitless). The scale and sign of these images is arbitrary. The coefficients of the first component are positive across the brain; for better contrast, a different colour scale was used than for the remaining components.
Screeplot . On the left, screeplot of the first 20 principal components. On the right, scatterplot of the first component score on global CBF values (differences from the mean subject global CBF in ml/100 gr/min). Component scores refer to individual volumes. To increase the clarity of the plot, global CBF was ranked, and sampled at intervals of 200 rank scores.
The first component explained about 10% of the variance, and contained variation shared by all voxels, shown by the coefficients of the component being of the same sign over the whole volume. This mode of variation was determined by the perfusion level of the acquisition in the whole volume, as is shown by the high correlation of the score of this component with global CBF values ( R = 94%, p < 0.001, Figure ). Areas of high acquisition-to-acquisition variance, visible in Figure , were represented in the spatial pattern of this component, which loaded with different intensity depending on overall acquisition-to-acquisition variance. Taken together, the evidence presented in these Figures shows that when global CBF of the acquisition is high, the computed regional CBF at the large vessels is even higher than the average level; furthermore, also perfusion in the cortex increases more than in the white matter. However, the variation of the first component appears to affect the calcarine and medial occipital cortex more than other cortical areas and as anticipated by their vicinity to large vessels (slices at z = 0 and 24, top row, Figure ). Furthermore, the high variance at the edges of the brain and in ventricles of Figure is underrepresented. Outside the brain parenchyma, high spatial covariation is observed in the space between the hemispheres and in the Sylvian scissure.
The second and third components, each accounting for about 5% of the variance, captured variation located along the course of main vessels. The dissociation between anterior and posterior areas visible in the comparison between these two components reflects the main subdivision of branches stemming from the internal carotids and the vertebral artery. The fourth component, accounting for about 2.5% of the overall variance, reflected dissociation between the brain parenchyma as a whole and the high-variance edges. In these components, association with global CBF was negligible ( R was 1.5, 2.4 and 0.3% for the second, third, and fourth components), in contrast to the first component. No component up to the 10 was noted to reflect right-left asymmetries in perfusion.
## Discussion
In this study, the pattern of spatial covariation in the acquisition-to-acquisition variance in CASL images was investigated using principal component analysis in a large sample of images. The results obtained here reflect the effectiveness and accuracy with which this technique could be implemented in our laboratory, and are certainly of limited generalizability to future techniques that may seek a more accurate estimation of CBF. However, the CASL imaging protocol implemented here represents a pragmatic approach to CBF estimation (that is frequently adopted in fMRI ASL studies), and its adoption may be informed by the results presented here.
The first component of variation captured changes in the overall intensity of the quantitative CBF estimate in each acquisition. These changes affected gray more than white matter and large vessel areas more than the rest of the brain parenchyma. An important aspect of this component of variation is its high correlation with global perfusion levels (which does not arise by necessity). Even if the spatial pattern of covariance is somewhat different, this component is in this respect analogous to the first component obtained by the principal component analysis of subject-to-subject variance, which also captures changes of global perfusion levels [ ]. The finding that the first component is associated with global perfusion levels in both subject-to-subject and acquisition-to-acquisition sources of variance suggests that many factors, now affecting variation between subjects, now affecting variation between acquisitions, may have a common effect on global perfusion levels. What one then sees is that differences in global perfusion levels affect brain regions with distinct but characteristic spatial patterns, depending on whether these factors have acted between acquisitions or between subjects. This observation is relevant for the application of procedures that, with the aim of reducing this source of variance and increase testing power, correct for global CBF values [ - ].
The second and third components reflected a vascular factor, consistent with the predominant dissociation between the anterior and posterior regions due to the two main sources of arterial supply, internal carotids and cerebral artery. This source of variation may ensue from labeled spins that have not yet reached the capillary bed, or from interactions between the timing of the labeling pulse and the rhythm of blood flow. These components reflect the interplay of brain physiology, specifically of large vessels, and the signal acquired with this imaging technique. In contrast, variation at the edges of the brain is underrepresented in covariance patterns, loading strongly only on the fourth component. This is consistent with the observation of the noisy character of the data at the brain edges.
The relative importance of the first three components, accounting together for over 20% of the total variance, shows that residual variation across voxels in this type of images is affected by important violations of the stationarity assumption on the Gaussian random field modeling residual variation. Furthermore, the spatial pattern of these components follows anatomical boundaries, such as the anterior and medial temporal lobe, the medial occipital and calcarine cortex, the insula (especially posteriorly), and the subgenual portion of the anteromedial prefrontal cortex. When averaging these images, some combination of these patterns, apparently following anatomical structures, may arise just by chance, as was demonstrated in the case studies. The anteromedial part of the temporal lobe and the lower half of the medial aspect of the brain hemispheres appear to be particularly affected. Note also that the apparently homogenous high variance of the medial aspect of the brain hemispheres visible in the sagittal slice of Figure is in fact produced by the superposition of spatially characterized patterns.
## Conclusions
Using principal component analysis, we investigated the patterns of spatial covariation arising from changes in the global estimated perfusion levels and their interplay with vascular anatomy.
## Methods
### Data acquisition
Perfusion images at rest (8 min., 120 scans giving 60 acquisitions of perfusion estimates) were acquired using continuous arterial spin labeling [ ] from 228 right-handed participants (101 males) aged between 17 and 52 years at the time of the scan (mean age 24.7, std. dev. 5.4) who gave informed consent. The study protocol was approved by the local ethical committee and was in compliance with national legislation and the Code of Ethical Principles for Medical Research Involving Human Subjects of the World Medical Association. Exclusion criteria were neurological or medical conditions, use of medication, or a history of mental illness, and subclinical structural abnormalities.
All magnetic resonance imaging (MRI) data were obtained with a 3-Tesla Magnetom Allegra (Siemens, Erlangen, Germany) MRI system equipped with a head volume coil. All participants were scanned at the Department of Psychiatry of the University of Ulm. A standard T2-weighted structural brain scan from the clinical screening routine in use in our hospital (TR 4120, TE 82) was taken on all participants to exclude subclinical structural abnormalities. A continuous arterial spin-labelling technique was used as described in ref. [ ]. Interleaved images with and without labelling were acquired for 8 min by using a gradient-echo echo-planar imaging (EPI) sequence with a field of view of 22 cm. Image size was 64 × 64 × 15 voxels, slice thickness 6 mm with a gap of 1.5 mm, giving a voxel size of 3.44 × 3.44 × 7.50 mm. The images were acquired with TR 4000, TE 17, anterior-to-posterior phase encoding, a flip angle of 90°, and a bandwidth of 3005 Hz/Pixel. A delay of 1 sec was inserted between the end of a 2 sec labelling pulse and image acquisition to reduce transit artefacts. The SPM2 package was used (Wellcome Department of Cognitive Neurology, London; online at ) for realignment and stereotactic normalization to an EPI template (Montreal Neurological Institute, resampling size: 2 × 2 × 2 mm). Reconstruction of CBF values at each voxel was obtained using the Perf_resconstruct_V02 SPM add-on software by H. Y. Rao and J. J. Wang, from the Department of Radiology and Center for Functional Neuroimaging at University of Pennsylvania (online at ). The software implements eq. (1) of ref. [ ], or, equivalently, eq. (1) of ref. [ ]. The 'simple subtraction' method was used. No scaling procedures such as 'grand mean scaling' were applied to the data. All volumes were smoothed using an isotropic Gaussian kernel of full width half-maximum (FWHM) of 6 mm prior to the principal component analysis. An explicit mask was obtained by combining an a priori thresholded tissue probability maps provided by the SPM package at 0.25 for gray or white matter with another mask thresholding the standard deviation of the mean images to less than 25 (as described in ref. [ ]). Furthermore, slices lower than z = -24 mm. were excluded, since very low slices have very large variance in our data (these slices are close to where the labelling pulse was given). We also excluded slices above z = 48 mm. to prevent lack of coverage of the top of the brain in some individuals to influence the outcome of the principal component analysis. After masking, each volume contained 158 856 voxels.
### Statistical analysis
The data of the present sample contain two sources of variance: subject-to-subject, and acquisition-to-acquisition. The former is the variance arising from differences in brain perfusion among individuals, while the latter is constituted by variation due to unaccounted effects and experimental error in the data collected within each individual. Formally, in an experiment in which j = 1,... m baseline perfusion images are acquired in a single session from i = 1,... n individuals, the data at each voxel may be modelled by a simple random effects ANOVA:
where y is the voxel signal, A is the average regional CBF in subject i , and ε is an experimental error term at each acquisition containing the unaccounted effects. A and ε are assumed to be independent random variables with means μ and zero, and variance (the subject-to-subject variance) and σ (the acquisition-to-acquisition variance), respectively. Voxel-by-voxel estimates of acquisition-to-acquisition variance were obtained by standard ANOVA estimators; in the present case, the estimator of acquisition-to-acquisition variance is given simply by the variance of images centred relative to the subject mean image.
Principal component analysis was carried out as described in the Appendix of ref. [ ] on the estimated images . The analysis was carried out by directly calling the relevant BLAS and LAPACK routines in the version of these packages provided with MATLAB R2006b (The Mathworks, Natick, MA) installed on a machine equipped with a 64-bit Athlon processor (Advanced Micro Devices, Sunnyvale, CA) running Windows XP (Microsoft, Redmond, WA). Data were single-centered voxel by voxel, thus considering voxels to be 'variables' and the average individual volumes to be 'observations' in the usual principal component terminology [ ]. A nm × nm covariance matrix was then computed from the outer product of the data acquired in each voxel. In the present case, n = 228 and m = 60, giving a covariance matrix of size 13 680 × 13 680. A singular value decomposition of this matrix gave the principal directions of variation in time, i.e. a set of orthonormal vectors of size 13 680. The spatial components ('eigenimages') were obtained as the coefficients of the voxel-by-voxel regression of all volumes on this set of vectors. The principal component analysis also delivers a principal component score, one for each volume. This score is the inner product between the eigenimages and the CBF values in each volume, and represents the extent to which each volume displays the pattern identified by the components. The correlation of this score with global CBF values provides a summary measure of how much the pattern of variation is associated with changes in global perfusion. All images were generated with the software package MRIcroN, obtained from .
## Authors' contributions
RV conceived of the study and its design, carried out scans, wrote software for the statistical analysis, analyzed data, and drafted the manuscript. PB, HL, NO, JT recruited participants, carried out scans, and contributed to writing the manuscript. EJS coordinated the study, carried out scans, and contributed to writing the manuscript. All authors read and approved the final manuscript.
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The Carnot cycle is the most energy efficient process known in nature. Energy efficiency is also a hallmark of biological systems – from sub-cellular processes to the organizational level of the composite organism. Lastly, extremely fundamental connections and equivalences are known to exist between computation and thermodynamics [ ]. This paper extends previous work [ - ] to develop a simple and detailed model of the 4 phases of operation of the Carnot cycle of the cortical neuron. In the entropy-temperature ( S - T ) plane, these phases correspond to (1) entropy reduction via information acquired through dendritic inputs, (2) storage of this information within the soma, (3) use of this information in making firing decisions thereby expending this information and increasing the system entropy, and (4) the resetting of the somatic memory thereby allowing it to acquire new information. The last phase corresponds to the restoration of Na and K ion concentrations across the cell membrane during its refractory period. Memory resetting is often confused with memory erasure which is a misinterpretation of Landauer’s original findings [ ] and is the fundamental reason why non-reversible computation and causal systems are dissipative.
The neural system entropy is shown to be exactly n +1 bits where n is the number of synaptic inputs and the increment 1 arises from the ability of the neuron to fire – or not. The neuron cyclically operates by reducing its entropy (uncertainty) by approximately 1 bit to n upon acquiring input information and then restoring it to n +1 after a firing decision. The storage of information within the soma and its resetting are shown to correspond to phase state transitions due to variations in its computational temperature during its Carnot cycle thereby altering its partition function and computational degrees of freedom, e.g., during its refractory period it cannot fire.
In pursuit of information processing efficiency [ ], it is also shown that this equates to energy processing efficiency. Since the area of a Carnot cycle operating in refrigeration mode is its area in the S - T plane, the neuron tries to minimize the range of computational temperatures over which it operates. This is accomplished by maximizing, subject to physical and metabolic constraints, the number n of its synaptic input-derived signals influencing its outputs. Simultaneous morphological (spatial) adaptation of dendritic and axonal trees in forming new connections with pre- and post-synaptic neurons is shown to increase the morphological entropy of the neuron in a conjugate and synergistic fashion by both guiding and being guided by computational adaption and operation. This requires defining the neural system entropy in terms of two independent functional (computational) and structural (morphological) components. Since independent, and noting the extensive nature of entropy, this means that the system entropy is the sum of these two entropies as mathematically captured by the factorability of its conjoint system partition function.
It should be recalled that [ - ] describe how to reverse-engineer ostensibly all known architectural and computational aspects of cortical neurons including space-time codes, dendrites, soma, axon, Hebb’s rule, logistic function-based decision-making and many other properties.
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Ever since the proposal that brain activity is organized into networks of coherent activity in the framework of functional connectivity (FC, [ ]), a great amount of research has been conducted to characterize these networks, their inter-relations, and their differential participation in sensory-motor and cognitive processes, as well as their alteration in brain states such as pathological diseases, sleep, etc. The main techniques to study FC are functional magnetic resonance imaging (fMRI) and positron emition tomography (PET), but it has also been described using other techniques, such as MEG and EEG [ ]. The classic approach to obtain FC networks is to calculate the correlation between the low-pass filtered time series of brain nodes (i.e., voxels, anatomical areas, etc.) along the scanning period. In this way the studies on FC commonly assume a rather static correlation pattern. Although such studies unraveled important features of brain large-scale functioning, FC is in fact highly variable in time and across subjects [ ]. Recent work digs deeper into this variability and brings up important caveats to many previous assumptions. For instance, new findings suggest that networks may reorganize in time, weakening their intra-network connectivity strengh and enforcing the inter-network connectivities, and that there may exist discrete states of multistability over time [ ].
In the present work the dynamic functional connectivity was analyzed in a group of patients with disorders of consciousness (DOC) as well as in healthy subjects, during a resting state (RS) fMRI paradigm. The correlation matrices ( R ) between predefined brain areas were obtained in sequential time windows. The temporal variability of different metrics of the R matrices was examined. Furthermore, main components (i.e., hidden states of frequent brain configurations) were identified from DOC or Control groups' R temporal series by employing singular value decomposition (SVD). SVD is a dimensionality reduction technique that allows for obtaining a latent correlation structure model, and has been previously applied by Leonardi et al. [ ] to RS fMRI data. In the present work we investigated whether the main SVD components differ between DOC and Control groups and in which temporal manner. Preliminary results show that the main components present dissimilarities between the groups in specific brain areas. Furthermore, the temporal variation of the largest eigenvalue (that is interpreted as the collective motion of all areas) of the R matrices is different in DOC compared to Control group.
The results may contribute to the understanding of the process of consciousness in so far as brain dynamic functionality and have potential impact in the understanding of consciousness disorder.
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Adaptive technology holds great promise for sensorimotor rehabilitation in people suffering from spinal cord injury, neuromuscular disease and stroke. With a long-term goal of developing adaptive technology for diagnosis and rehabilitation of neuromuscular dysfunction, we begin the development of a self-organizing neural network (SNN) that compensates for reduced neural drive. We suggest that the self-organizing architecture that adds or deletes nodes online to generate suitable compensatory muscle excitation (Figure ) is an apt mechanism to emulate the motor pool behavior of recruitment and de-recruitment of motor units during muscle force generation. Using a virtual muscle [ ] resembling the human biceps brachii, we demonstrate the augmentation of neural excitation by the SNN to compensate for abnormal muscle force due to change in the number of motor units.
A. Schematic showing the virtual muscle-SNN system; Φ , Φ , .. Φ are radial basis functions and w , w , ..w are weights for summation . B . Simulation of normal (Slow-Fast motor unit ratio - 2:4), abnormal (Slow-Fast motor unit ratio - 3:3) muscle force and, compensation by SNN.
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## Background
It is generally thought that granule-mitral cell synapses in the olfactory bulb function to inhibit mitral cell firing, and that this inhibition can underlie such functionally important phenomena as lateral inhibition and synchronization [ ]. Recent electrophysiology [ ] and imaging [ ] studies indicate that the location of the dendrodendritic synapse must be close to the soma to impact the mitral cell's firing.
## Materials and methods
Our objective was to survey the effect of dendrodendritic synapses on firing of pairs of mitral cells sharing a granule cell using a standard, computational mitral cell model [ ].
## Results
We show that depending on the location of the dendrodendritic synapses along the mitral cell lateral dendrite, three types of inhibitory effects can be described between mitral cell pairs: 1) A "bidirectional gate" arises when the granule cell induces a discernible inhibitory response in both mitral cell somas. 2) A "unidirectional gate" occurs when the granule cell induces a discernible inhibitory response in only one mitral cell soma. 3) An "inconsequential gate" occurs when the granule cell does not induce a discernible inhibitory response in either mitral cell soma.
## Conclusion
Preliminary results indicate that most of the lateral dendrite contains unidirectional or inconsequential gates. This is important as most olfactory bulb models effectively treat the mitral-granule dendrodendritic synapse as a bidirectional gate and may need to account for other gating behaviors created by considering the spatial extent of dendrodendritic synapses.
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## Research aim
To study the RBCs functional and metabolic parameters and the microcirculatory brain structure at traumatic brain injury (TBI) under the action of 2-ethyl-6-methyl-3-hydroxypyridine succinate.
## Methods
A closed TBI was modeled by the free fall of a load on the parietooccipital regions of head. We made studies of the influence of 2-ethil-6-methil-3-hydroxipiridin succinate on aggregation and electrophoretic mobility of RBCs, catalase activity, malonic dialdehyde concentration, adenosine triphosphate and 2.3-biphosphoglycerate (2.3 – BPG) concentrations in RBCs. The state of parenchyma and microcirculatory brain mainstream in post-traumatic period of TBI have been studied on micro-preparations.
## Results
The use of 2-ethyl-6-methyl-3-hydroxypyridine succinate under conditions of head injury leads to a decrease in MDA concentration and in aggregation of RBCs, to an increase in the 2.3—BPG concentration and RBC electrophoretic mobility compared to the control (group value). The most pronounced changes under the action of 2-ethyl-6-methyl-3-hydroxypyridine succinate were observed 3–7 days after the TBI. Significant indicators of the restoration of the microvasculature and brain tissue provoked by the use of 2-ethyl-6-methyl-3-hydroxypyridine succinate of were evident from the 7th day unlike the control group, where the restoration of structural morphological parameters was observed only on the 12th day of the post-traumatic period. Fast recovery of blood flow under the action of 2-ethyl-6-methyl-3-hydroxypyridine succinate ensured effective restoration of neurons and glia in comparison with the control group.
## Conclusions
Early and long-term cytoprotective correction intensifies the oxygen transport function of the blood, prevents and / or reduces disorders of microvessels, neurons and glia in the post-traumatic period, thereby provides correction of hypoxic state and drives to the restoration of brain tissues homeostasis.
## Background
Traumatic brain injury (TBI) is the most difficult multidisciplinary, medical, biological, and social problem due to a persistent high prevalence of death and disablement of patients of the most active working age [ – ]. In multiple researches, devoted to TBI problem, the role of secondary mechanisms of brain trauma is shown. Their trigging provokes the broadening of primary lesion foci and formation of new pathological processes abruptly making worse the clinical course and trauma outcome [ , ].
In post-traumatic period, the quickest interrelated changes with oxygen status, hemorheology, acid—basis and water-electrolytic states take place [ ]. Great influence on perfusion and brain oxygenation in early post-traumatic period is caused by micro-rheological blood defection [ ], which define its fluidity on capillary level and depend on form, sizes and also erythrocyte membrane condition [ ]. It is also known that in the basis of pathological changes, appearing at TBI, there is an activation of free radical oxidation processes, parallel with the decrease in antioxidant protection system activity [ ].
The decrease or interruption of continuous generation of antioxidant ability provokes abnormal accumulation of reactive oxygen intermediate implementing the devastating effects at all levels of organisms vital activity [ ]. The development of vascular and morphological disturbances are supposed to be one of the most important mechanisms of secondary trauma at that [ , ]. That is why to stop negative changes, appearing in case of TBI, it is necessary to provide the early correction of hypoxic state and the restoration of cell and subcell membrane homeostasis.
Now a derivative of 3-oxipiridin 2-ethil-6-methil-3-hydroxiperidin succinate is used as a preparation which combines both antihypoxic and antioxidant properties [ ]. It is a coordination complex of emoxipin (the derivative of oxipiridin) with amber acid (succinate). This complex is used for correction of neurological and cardiovascular pathologies [ – ]. The research results received in previous studies showed that 2-ethyl-6 -methil-3-hydroxiperidin succinate unlike other succinate antihypoxic drugs enter the cells considerably quicker and then it dissociates in cytosol in two components. Each of them makes an independent positive impact on brain and myocard in ischemia reperfusion thanks to high penetration properties of emoxypine. Emoxypine provokes the inhibition of free-radical processes. Amber acid allows to sustain the processes of high-energy compounds formation [ , ]. Taking into account this fact it’s possible to suppose that 2-ethil-6-methil-3-hydroxiperidin succinate may play any role in the correction of RBC membrane molecular mechanisms and of cell metabolic status which is the response to the homeostasis system destabilization provoked by TBI. So, it’s impact may improve the perfusion of microvessels. However, the therapeutic potential of this compound to influence the structural functional state of erythron and microcirculation in TBI is not yet studied. As macro- and microrheological disorders of blood–vascular system play a considerable role in extracranial disturbances in TBI the search of drugs which would make a correction of RBCs and of microcirculation is feasible and of grand importance.
The intention of this research was to study if the 2-ethil-6-methil-3-hydroxiperidin succinate injection capable to reduce the risk of claudication in brain cortex as a response to the restoration of functional and metabolic indices of RBCs. The rat model of TBI was used. As it’s difficult to study a mono exposure in medical condition the experiments are made with animals. Such models provide a practical platform for initial evaluations and studies that can inform clinical practice and provide a better understanding of the effects of drugs in the post-traumatic period. In numerous studies it’s proved that the distinction of TBI consequences depend on the sex of animals. It’s shown that women incur a higher risk of adverse outcome in brain commotion than men. So, though men incur a higher risk to receive a brain commotion because they make more often activities associated with risk of brain commotion, women as a rule have more grave consequences of the brain commotion. Nowadays the most studies of people received TBI concern adult men [ , ]. The preclinical models of trauma concern adult males of animals. It makes a gap concerning the study of female brain and the elaboration of personalized and efficacious methods of TBI treatment.
In this regard, our goal was to study the RBCs functional and metabolic parameters and the brain microcirculatory bloodstream structure under the action of 2-ethil-6-methil-3-hydroxiperidin succinate in order to limit secondary faults modeling female TBI.
## Results
### The analysis of RBCs functional indexes under the action of 2-ethil-6-methil-3-hydroxipiridin succinate.
The animals suffered the experimental TBI had disorders of functional RBC indexes in the post-traumatic acuity (Table ). From the first day on the background of TBI the rats from the control group demonstrated a considerable decrease in RBC electrophoretic mobility by 14.3% and the increase in RBC aggregation degree by 131.3% relative to the indicators of the intact group of animals. During all the post-traumatic period the RBC electrophoretic mobility increased step by step and the aggregation decreased. But the restoration of the indexes up to the intact group value did not take place. In the groups where 2-ethil-6-methil-3-hydroxipiridin succinate was injected the decrease in RBC electrophoretic mobility was by 24% lower than in that of the control animals from the very first day of the post-traumatic period. On the 3rd day of experiment caused the increase in RBC electrophoretic mobility of EPME by 25.8% and decrease in aggregation degree by 36% in comparison with the control. On the 12th day of the study the RBC electrophoretic mobility and aggregation indices restored up to the normal value.
The change in functional indexes of RBCs (erythrocytes) in the TBI post-traumatic period of rats in the investigated groups
Data are means ± SD. “*” statistically important differences regarding the intact group data, p ≤ 0.05, “ ” statistically important differences of experiment from control, p ≤ 0.05
These changes were accompanied by the increase in lipid peroxidation (LPO) products quantity and the decrease in catalase activity (by 187.5% and 27.5%, respectively) of the rats from the control group in comparison with the animals from the intact group. Within the following 7 days of post-traumatic period reliably high activity of LPO processes in comparison with the animals from the intact group. This was accompanied by a further decrease in catalase activity by 41.1% on the 7th day after the injury relative to the the intact group animal value. The injection of 2-ethil-6-methil-3-hydroxipiridin succinate provoked the decrease in MDA concentration up to the normal value on the 3rd day of the experiment. Catalase activity was increasing from the 1st day of the post-traumatic period and exceeded the level of the intact group values. The maximal increase in catalase activity (by 144.4%) relative to the control group value was registered on the 3rd day of the post-traumatic period.
The study of ATP and 2.3-BPG concentration in RBCs showed the increase in the indices in all the comparison groups. But it was strongly pronounced where 2-ethil-6-methil-3-hydroxipiridin succinate was injected. On the first day after the trauma, the ATP concentration in the RBCs in the control group was by 28.7% lower and on the 3rd day, it was by 21.9% higher than that of the intact group. The maximal increase of ATP concentration was registered on the 7th day after the trauma. The 2.3-BPG concentration in the RBCs of the control group on the 1st day of the experiment was by 74.5% higher than that of the intact group. On the 1st and 3rd day of the post-traumatic period, after the 2-ethil-6-methil-3-hydroxipiridin succinate injection the ATP concentration value in RBCs was lower than that of the intact animal group. On the 7th and 12th day the ATP concentration was higher by 34.1% and 26.8% respectively than that of the intact group. From the 3rd day of the experiment till its end the 2.3-BPG concentration was higher. So, On the 3rd, 7th, and 12th day of the experiment it was higher by 123.4%, 90% and 49.2% respectively than that of the intact animal group.
### Microcirculatory mainstream morphofunctional changes at TBI and under the action 2-ethil-6-methil-3-hydroxipiridin succinate
The of histological investigations of animals in the post-traumatic period showed that one day after the TBI there were small-focal hemorrhages, pronounced perivascular edema, vasodilation and fullness of blood vessels in the area of the primary trauma (Fig. a–c). In particular, vascular fullness with manifestations of stagnation was observed in the vessels of the microcirculatory bed and veins (Fig. b, c). 70% of venules and 30% of the arterioles were occluded by microthrombs. Outside the focus of primary damage, there was an uneven blood filling of blood vessels, a complete decline in the lumen of some and overflow of blood of others, edema of the vascular wall and endothelial cells. The above changes were visualized at the level of arterioles, veins, and capillaries.
The structure of brain microcirculatory mainstream of the rats from control group after traumatic brain injury. ( a Arteriole. One day after the TBI vascular fullness, single perivascular hemorrhages, aggregates of erythrocytes were observed and perivascular edema was highly expressed, b —Venule. One day after the TBI, diapedesis, stored erythrocytes were observed and as well as highly expressed perivascular edema, c —Capillary. One day after the TBI in brain spawn capillaries the erythrocytes were absent. In some capillaries they were like sludges. Also everywhere there was highly expressed perivascular edema, d —Arteriole. 3 days after the TBI in arterioles clearance there were pink hyaline thrombuses, highly expressed perivascular edema was formed, e —Venule. 3 days after the TBI sludge erythrocytes and hyaline like masses, highly expressed endothelium edema and prevascular edema were noted in venules clearances, f —Capillary. 3 days after the TBI sludge erythrocytes, endothelium edema, highly expressed perivascular edema were noticed in the clearance, g —Arteriole. 7 days after the TBI in arterioles there were the parietal erythrocytes aggregations. Moderate endothelium edema was observed and perivascular edema was highly expressed, h —Venule. 7 days after the TBI in venules there was moderate endothelium edema, in the clearance between them there were protein masses, slagged of erythrocytes and blood clots of erythrocytes, moderately expressed perivascular edema was defined everywhere, i —Capillary. 7 days after the TBI in capillaries plethora, highly expressed endothelium and perivascular edema were noted, j —Arteriole. 12 days after the TBI in arteriole lumen there were erythrocytes and moderate perivascular edema, k —Venule. 12 days after the TBI free laying erythrocytes, endothelium edema was not expressed, moderately expressed perivascular edema was defined were defined, l —Capillary. 12 days after the TBI in the capillary lumen there were free laying erythrocytes, moderate endothelium edema and moderate perivascular edema). Scale bar is 100 μm, original magnification × 200 for arteriole and venule. Scale bar is 50 μm, original magnification × 400 for capillary. Abbreviations: Ag—aggregates from red blood cells, Ed edema, En endothelium, H—hyaline blood clots, RBC—red blood cells, T thrombuses
In some cases, there was a rupture of the integrity of the vascular wall and the exit of erythrocytes outside the vascular bed diapedesis (Fig. a, b). In the lumen of the arterioles, there were blood clots and aggregates from erythrocytes, and strongly expressed perivascular edema (Fig. a). In the lumen of the venules, diapedesis, slags and parietal aggregates from red blood cells were observed, and strongly expressed perivascular edema (Fig. b). The area of perivascular edema of venules and arterioles relative to the norm was higher by 5.6 and 3.8 times, respectively, with the predominance of changes in the zone of primary damage (Table ).
Characteristics of morphometric indicators of the microcirculatory bed of the brain in the investigated groups of rats (M + m)
Data are means ± SD. “*” statistically important differences regarding the intact group data, p < 0,05, “ ” statistically important differences of experiment from control, p < 0,05
In the dormant capillaries there were no RBCs, in some fields of vision in their lumen, red blood cells were located in the form of aggregates and slags (Fig. c). In all capillaries, an increase in the area of pericapillary edema by 2.7 times relative to the norm was observed. In comparison with intact rats, there was a decrease in capillary density by 15–20%, a decrease in their diameter by 40%, and as a result, a decrease in the area of the capillary bed in halve.
The maximum changes in the morphological state of the vascular bed of the brain were observed on the 3rd day after the injury. The arterioles showed a visible edema of the endothelium, and red and hyaline blood clots were found in their lumen (Fig. d). The venules detected pronounced endothelial edema and stored RBCs and hyaline-like masses were detected in the lumen (Fig. e). At the same time, the area of perivascular edema of arterioles and venules was 14% and 13% larger, respectively, compared to the 1st day after the injury. The microcirculatory bed retained narrowed capillaries with strongly pronounced pericapillary edema (Fig. f), but in comparison with the 1st day after injury, there was an increase in the area of the capillary bed.
7 days after the injury, there was a significant tendency in the control group of animals to decrease the area of the capillary bed against the background of an increase in the average diameter of capillaries relative to the 3rd day value. At the same time, the capillaries remained full of blood, strongly expressed endothelial and perivascular edema (Fig. i) took place. Parietal aggregates of RBCs were found in arterioles, moderate edema of the endothelium and strongly expressed perivascular edema were determined (Fig. g). In the lumen of some venules, free-lying red blood cells were detected, but in most venules, protein masses, slagged red blood cells and blood clots, moderate endothelial edema and perivascular edema were detected (Fig. h).
On the 12th day of the study, the control group of animals showed a restoration of blood circulation in the cerebral cortex. Free-lying red blood cells were detected in the lumen of arterioles (Fig. j) and venules (Fig. k), endothelial edema was not expressed, but single blood clots and moderate perivascular edema persisted everywhere. The area of perivascular edema of arterioles decreased in halve, and venules by 2.24 times relative to the values for 1 day. When examining the capillary bed, it was found that free-lying red blood cells were observed in the lumen of the capillaries, moderate endothelial edema and moderate perivascular edema were preserved (Fig. l). The average diameter of capillaries was higher by 8% relative to the indicators of the intact group, the density of
the capillary bed was lower by 28% relative to the indicators on the 7th day, but was 10% lower than normal (Fig. m).
1 day after the TBI in animals of the experimental group were observed vacuolation of the intercellular space, vessels of the microcirculatory bed with less pronounced aggregation of red blood cells, but strongly expressed edema of the endothelium and perivascular edema, as well as in the control group (Fig. a–c). Free-lying red blood cells were detected in the lumen of most arterioles (Fig. a), wall aggregates of red blood cells and microthrombs were observed in 10% of arterioles. In the lumen of the venules, aggregates of red blood cells, parietal located hyaline-like masses were recognized (Fig. b). The area of perivascular edema of arterioles and venules was 3.7 and 5.4 times higher than those of the intact group, but by 2% and 5% lower than that of the control group (Table ). In comparison with the animals of the intact group, there was a decrease in the average capillary diameter (Table , Fig. a). It should be noted that despite of the narrowing of the capillary lumen after the injury, most capillaries are in a functionally active state, which is confirmed by morphometric analysis, indicating that the area of the capillary bed was statistically significantly higher after the treatment with the action of 2-ethyl-6-methyl-3-hydroxypyridine succinate compared to similar values in the control group of rats (Table , Fig. b).
The structure of brain microcirculatory mainstream of the rats after the traumatic brain injury under the action of 2-ethil-6-methil-3-hydroxiperidin succinate. (a Arteriole. Within 1 days after TBI in arteriole clearance there were singular erythrocytes, moderate endothelium edema, highly expressed perivascular edema, b —Venule. 1 day after the TBI in venule lumen there were erythrocyte aggregations, moderate endothelium edema, highly expressed perivascular edema, c —Capillary. 1 day after TBI in capillary lumen there were sludge from erythrocytes, moderate endothelium edema, highly expressed perivascular edema, d —Arteriole. 3 days after the TBI there were the parietal erythrocytes aggregations in arterioles. Moderate endothelium edema and perivascular edema were defined, e —Venule. 3 days after the TBI there were moderate endothelium edema, in the lumen between them they were defined, f —Capillary. 3 days after the TBI in capillaries erythrocytes sludges, moderate perivascular edema were defined, g —Arteriole. 7 days after the TBI the arteriole lumen partially collapsed, with singular erythrocytes in its lumen. Poorly expressed perivascular edema was observed, h —Venule. 7 days after the TBI here were distinguished free laying count blood elements in the lumen of the most of venules. Everywhere poorly expressed perivascular edema was observed, i —Capillary. 7 days after the TBI there were free laying erythrocytes in the capillary lumen, perivascular edema was not expressed, j —Arteriole. 12 days after the TBI there were singular count blood elements in the most of arteriole lumens; endothelium was without the features of edema. Perivascular edema was poorly expressed, k —Venule. 12 days after the TBI there were no count blood elements in the lumens of the most of venules or it was possible to meet singular free laying erythrocytes. Endothelium was without or with poorly expressed edema. Perivascular edema was poorly expressed, l —Capillary. 12 days after the there were free laying erythrocytes in capillary lumens. Around the most of them perivascular edema was absent). Scale bar is 100 μm, original magnification × 200 for arteriole and venule. Scale bar is 50 μm, original magnification × 400 for capillary. Abbreviations: Ag—aggregates from red blood cells, Ed edema, En endothelium, H—hyaline blood clots, RBC—red blood cells, T thrombuses
Effect of 2-ethil-6-methil-3-hydroxiperidin succinate treatment on the average capillary diameter (a) and area of the capillary bed (b). Data are means ± SD. “*” statistically important differences regarding the intact group data, p < 0,05, “▲ “ statistically important differences of experiment from control, p < 0,05
On the 7th day of the post-traumatic period, the experimental group showed a visible reparative changes with a tendency to normalize the structure of the microcirculatory bed. Free-lying red blood cells were detected in the lumen of arterioles and venules, edematous endothelium was observed of vessels, and weakly expressed perivascular edema was detected everywhere (Fig. g, h). The diameter of the capillaries and the area of the capillary bed were restored to normal values in spite of the general tendency on against the background of a decrease in pericapillary edema (Fig. i; Table ). Reduction of pericapillary edema is associated with restoration of the structural integrity of the endothelial lining. By the 12th day of the study, the lumen of the arterioles had no formed blood elements, vascular endothelium without signs of edema, and perivascular edema was poorly expressed (Fig. j). In the lumen of most venules, there were no shaped blood elements or there were single free-lying red blood cells, the endothelium was slightly edematous, and perivascular edema was weakly expressed (Fig. k). In the capillaries, the diameter did not differ significantly from the norm, free-lying red blood cells were detected in the lumen, and there was no pericapillary edema (Fig. l).
### Morphofunctional changes of brain tissue with traumatic brain injury and action of 2-ethil-6-methil-3-hydroxipiridin succinate.
Morphofunctional changes after the TBI were characterized by degenerative-dystrophic changes in the control group of rats (Fig. ). The maximum changes were observed on the 1st and 3rd days. One day after the TBI, the total number of neurons was lower than that in the intact group of animals. Expressed dystrophic changes were observed in neurons, part of them were swollen, very few shrunken, with poorly distinguished nucleus and nucleolus, homogeneous cytoplasm and with twisted deformed scions. There were also detected single neurons with indistinct contours, with progressive karyoplasmocytolysis and almost lost nucleus (Fig. A). The area of neuron edema was 80% larger compared to the norm (Table , Fig. B). There was also visible and pronounced pericellular edema around the glia elements.
Effects of 2-ethil-6-methil-3-hydroxypiridin succinate on Neurons and glial cells of rat brain tissue after the traumatic brain injury. (A) Neurons and glial cells of control rats and rat under the action of 2-ethil-6-methil-3-hydroxypiridin succinate. Scale bar is 50 μm, magnification × 400. (B) Quantification of pericellular edema around the neurons and glia cells. ( 1 on the first day of post-traumatic period, there was a strongly pronounced pericellular edema around neurons and glia cells. The intercellular substance had an almost frothy appearance; 2 on the 3rd day of post-traumatic period there was a strongly expressed pericellular edema around neurons and glia cells. Moderate amount of vacuoles was in the intercellular substance; 3 on the 7th day of the post-traumatic period edema around the neurons and glia cells was moderate. Small number of vacuoles was in the intercellular substance; 4 on the 12th day of the post-traumatic period there was was a moderate pericellular edema around neurons and glia cells. Single vacuoles were in the intercellular substance; 5 on the 1st day of the post-traumatic period there was a severe pericellular edema around neurons and glia cells. Moderate amount of vacuoles was in the intercellular substance; 6 on the 3st day of the post-traumatic period there was a moderate pericellular edema around neurons and glia cells. Moderate amount of vacuoles was in the intercellular substance; 7 on the 7th day of the post-traumatic period there was a weakly expressed pericellular edema around neurons and glia cells. Single vacuoles were in the intercellular substance; 8 —on the 12 day of the post-traumatic period the pericellular edema around the neurons and glia cells was absent. The intercellular substance was not vacuolated). Examples of pericellular edema around the neurons and glia cells are indicated by arrows
On the 3rd day of the post-traumatic period pronounced dystrophic changes were recognized in neurocytes: neurons with karyolysis, in the state of neurocytolysis, with vacuolated cytoplasm, deformed cell membrane and strongly expressed pericellular edema. General neuron density was low, but the number of shrivelled hyperchromic neurons and cells-shadows increased. Glial cells are light of rounded form with intensively colored nucleolus, the pericellular edema persisted around them (Fig. A). It should be noted that the bulk of the altered neurons was located in areas which were far from the capillaries. In the brain tissue in the area of primary damage vacuolation and expansion of perivascular and perineuronal spaces was observed, which reflected the phenomena of diffuse brain edema.
On the 7th day in the control group of animals a part of neurons were preserved in a swallen state, with karioreksis in the form of “melting” neurons and cells-shadows. The area of pericellular edema decreased by 12% compared to the 1st day value. Glial cells preserved the correct body form, sometimes have the jagged edges, their nucleus was colored intensively, severily expressed pericellular edema was noted around them (Fig. A). On the 12th day of the investigation the restoration of brain tissue was observed, the decrease in pericellular spaces, edema spaces around neurons by 30%, the number of hyperchromic shriveled neurons and shadow cells relative to 3rd day of the study took place (Fig. A).
On the 1st day after TBI the animals from the experimental group demonstrated hydropic changes in brain cells. In the clarified cytoplasm there was spongy foam, separate vacuoles, the nuclei were under the influence of erosion, of a changed form; clarified or pictonic, some of them lost the nucleolus. The general neuron density was lower relative to the norm, but compared to the control, the loss of neurons was less pronounced. Highly expressed pericellular edema was formed around glial cells and neurons (Fig. A).
On the 3rd day of the post-traumatic period the structural changes of brain tissues were less expressed in the experimental group in comparison with the control group: poorly expressed perinuclear edema and moderate pericellular edema took place. Neurons were with well expressed nucleus and nucleolus, there were long protrusions, light glial cells of rounded form with intensively colored nucleolus (Fig. A). On the 7th day after the trauma, the processes of regenerative reparation in different intensity were observed.
This is evidenced by the preservation of most neurons with a well-defined nucleus, nucleolus and long processes. The area of pericellular edema decreased by 20% relative to the 1st day value and by 23% relative to the control group value (Fig. B). In addition, there were signs of compensatory-restorative reactions of preserved neurons and glia cells, which were structurally manifested at the neuron level by restoring the tinctorial properties of the cytoplasm, hypertrophy of the soma and nucleus with a decrease in the nuclear-cytoplasmic ratio and quality characteristics of supporting cells (astrocytes). In the absence of widespread foci of destruction and hemorrhagic infection, hyperchromic shrunken neurons and shadow cells were found only in places. Around the neurons and glial cells moderate and poorly expressed pericellular edema was noted (Fig. A).
On the 12th day of the post-traumatic injury neurons and glial cells the reconstruction of neurons and glial cells in animals was observed. Here and there poorly expressed perinuclear edema was preserved (Fig. A).
### Animal motion activity analysis
Tonic and clonic seizures had been observing just after the TBI for 2–4 s. They lost sensitivity and they had been in lateral position for 10-20 s. The study of motor response by method of moving on bar showed the worsening of motor function up to the 1st of the experiment (Table ).
Motion activity analysis in the TBI post-traumatic period of rats in the investigated groups
Data are means ± SD. “*” statistically important differences regarding the intact group data, p < 0.05, “ ” statistically important differences of experiment from control, p < 0.05
2-ethyl-6-methyl-3-hydroxypyridine succinate injection provoked the normalization of standing balance and walking ability. It manifested itself in the decrease in paw sliding frequency as well as in time spent for moving on bar. The paw sliding frequency and time of moving on bar decreased by 55.5% and 61.7% correspondingly by the end of the first day in the group treated with 2-ethyl-6-methyl-3-hydroxypyridine succinate. The positive dynamics in the motion activity indices was evident to the end of the first day after the 2-ethyl-6-methyl-3-hydroxypyridine succinate injection. A considerable improvement of motion reactions were registered by the 3 day after the injection. The level of these indices achieved the intact animal value to the 7th day after the 2-ethyl-6-methyl-3-hydroxypyridine succinate injection. In the control group recovery was observed only in 12 days.
## Discussion
The research results demonstrates that the application of 2-ethil-6-methil-3-hydroxipiridin succinate after TBI already on the 3rd day increased RBC electrophoretic mobility, decreased aggregation level and the intensity of LPO processes in RBCs. The decrease in RBC aggregation is caused by the increase in their electronegativity, which is restored under the influence of 2-ethil-6-methil-3-hydroxipiridin succinate. May be it’s possible that one of the mechanism of the charge conservation which took place in the experiment is the increase in the stability of membrane structure caused by the use of drug, it has been shown for several times that the stability of the membrane is largely recognized by LPO processes [ , ]. It is discovered that emoxipin which is the constituent of a given preparation, inhibit free radical oxygenation, cooperates actively with peroxide lipid radicals, hydroxyl peptide radicals, stabilizes cells membranes. Besides, the effects of 2-ethil-6-methil-3-hydroxipiridin succinate may be mediated, as we believe, by the presence of succinate in the preparation. In particular, it has been shown that the use of succinate reduces the formation of malondialdehyde [ ]. In addition, it has been shown that when the intracellular oxygen concentration decreases and/or the rise of succinate concentration HIF-1a factor activity increases [ ]. HIF-1a is an oxygen-sensitive protein complex that triggers the expression of a number of peptides, including erythropoietin (EPO), glucose Transporter proteins (GLUT 1, 3), and glycolysis enzymes [ ]. We suppose that in this case, there may be an increase in erythropoiesis and the formation of erythroblasts with high energetic potential on the early stages of erythropoiesis with the following way out of mature RBCs resistant to hypoxia to the vascular bed. It is also demonstrated that an increase in extracellular succinate concentrations through the interaction of GPR91-receptors associated with G-proteins leads to the activation of intracellular metabolism [ ].
The results given in the Table prove the given statement. The content of 2.3-BPG and ATP under the action of 2-ethil-6-methyl-3-hydrospiridin succinate from the 3rd and the 7th day of the experiment was higher than that of the control group respectively (p ≤ 0,05). The content of ATP and 2.3-BPG defines the deformation of RBCs which influenced the RBCs aggregation [ ], which blocks the blood flow and provokes the disorder of transcapillary exchange [ , ] 2-ethil-6-methil-3-hydrospiridin succinate caused the increase of metabolic and the restoration of oxidizing processes, which defined the decrease in RBC aggregation.
Besides, RBCs transporting oxygen to brain tissues, depending on their functional activity, influence greatly the degree of tissue hypoxia expression [ ]. We note that 2.3-BPG acts as an important allosteric regulator of binding oxygen with hemoglobin [ ]. The increase in 2.3-BPG production of RBCs, obtained in our experiments, facilitate the release of oxygen in tissues which promotes the content of pO in blood and tissues on the adequate level.
It should be noted that with the action of 2-ethyl-6-methyl-3-hydroxypyridine succinate, there is an increase in the area of the capillary bed, probably due to the inclusion of plasma capillaries, which were involved in response to the increased oxygen demand of the brain in the post-traumatic period. In addition, an increase in succinate concentration activates HIF-1a, which causes an increase in endothelial growth factor (VEGF) [ ]. VEGF acts selectively on vascular endothelium, ensuring its stability, promoting proliferation, migration and formation of endothelial cell tubules, that affects angiogenesis. Reduced VEGF levels cause endothelial apoptosis, leading to vascular lumen obstruction [ ]. Maintaining the endothelium in a stable state also contributes to NO. VEGF interacts with endothelial NO-synthase (eNOS) in the caveoles of endothelial cells, regulating its activity and thereby contributing to the production of NO. NO is a powerful vasodilator [ ]. It is likely that the marked pathways of succinate influence through VEGF may also occur with the action of 2-ethyl-6-methyl-3-hydroxypyridine succinate in the post-traumatic period of TBI.
The increase in oxygen-transport blood function and of the capillary bed in post-traumatic period of traumatic brain industry revealed in the group of animals in which 2-ethyl-6-methyl-3-hydroxypyridine succinate was injected is an important result of the investigation as it is demonstrated that a distinctive feature of secondary disorders of TBI is microvascular disorders which happens immediately after the primary mechanical trauma [ , , ].
At the same time, rapid and timely restoration of blood flow in the penumbra zone ensures cell survival and restoration of neurological deficits. In its turn, in spite of the fact that the structural and functional integrity of the endothelial lining restored, there was a decrease in edema and restoration of neurons and glia in the post-traumatic period. Probably, the positive effect of succinate on neurons after TBI can be realized through GPR91 receptors, which are found in the kidneys, liver, blood cells, adipose tissue, retina, and nervous tissue [ , ] The expression of GPR91 mRNA and the protein itself is recognized in neurons and astrocytes of the cortex [ ]. Activation of GPR91 leads to an increase in intracellular Ca [ ], which is a secondary messenger and largely determines the metabolic activity of cells.
The investigations of microcirculatory mainstream and brain tissues proves the validity of a given statement. The comparative analysis of the results of the functional state of RBCs, morphological investigation of microcirculatory mainstream and brain tissues of rats suffering from TBI, allows to come to a conclusion that intraperitoneal continuous injection of 2-ethil-6-methil-3-hydroxipiridin succinate on the early stages prevents or / and decreases micro vessels, neurons and glia disorder in a post-traumatic period. It is expressed in an earlier hemorrhage disappearance, the decrease in pericellular, intracellular and pericircular edema development, the preservation of morphology of microcirculatory mainstream and nervous tissue in comparison with control animals.
Taking into account, the widespread prevalence of TBI and the fact that today there is no adequate treatment [ ], the results of our experimental work show the effectiveness of the use of 2-ethyl-6-methyl-3-hydroxypyridine succinate in the correction of traumatic brain injuries. Confirmation of the restoration of brain homeostasis was obtained in the study of the functional state of animals: the study of motor reactions of rats in the test "method of movement on a bar" revealed a significant reduction in the time of manifestation of motor dysfunction when using 2-ethyl-6-methyl-3-hydroxypyridine succinate compared to the control compared to the control. From the point of view of further directions of research, it would be advisable to investigate whether the revealed effects of 2-ethyl-6-methyl-3-hydroxypyridine succinate are specific for females with TBI or are they stereotyped. Because on the one hand, the composition suggests the non-specificity of the action of 2-ethyl-6-methyl-3-hydroxypyridine succinate on erythrocytes and the morphology of the microvasculature of animals, on the other hand, a marked more aggressive neuroinflammatory profile in males compared to females during acute and subacute phases after injury suggests a different intensity of the effects [ ].
### Findings
The use of 2-ethil-6-methil-3-hydroxipiridin succinate determined the decrease in oxidative stress in animals and RBC aggregation, the increase in electro negativity and ATP and 2.3-BPG concentration from the 3rd till the 7th day of TBI.
The use of 2-ethil-6-methil-3hydroxipiridin succinate demonstrated the restoration of the state of microcirculatory mainstream beginning on the 7th day of post-traumatic injury whereas in control the restoration processes were registered on the 12th day.
The animals protected by 2-ethil-6-methil-3-hydroxipiridin succinate demonstrated the restoration of the structures of brain nervous tissue after TBI quicker than those of the control group.
## Conclusions
The improvement of RBC functional metabolic indices and of histologic indices of microcirculatory bloodstream and of brain tissues in the post traumatic period of TBI under the influence of 2-ethyl-6-methyl-3-hydroxypyridine succinate injection is an important research result.
Restoring blood flow reduces damage to brain tissue and has a positive effect on angio- and cytoarchitectonics of the cerebral cortex in the post-traumatic period. Revealing the mechanisms of action of 2-ethyl-6-methyl-3-hydroxypyridine succinate using visualization methods allows to substantiate the direction of action of 2-ethyl-6-methyl-3-hydroxypyridine succinate. Thus, the results of the study showed that 2-ethyl-6-methyl-3-hydroxypyridine succinate, having energy-synthesizing, antiradical and oxygen-supplying effects, in the post-traumatic period caused by TBI, has a corrective effect not only on microcirculation, but also on the vascular system, which largely prevents secondary brain damage.
## Materials and methods
### Animals
White females rats weighting 180 ± 20 g the age 14 weeks were obtained from the SPF the vivarium Institute of Biology and Biomedicine of Lobachevsky State University. Rats were kept in a vivarium equipped in accordance with the requirements of the «Sanitary rules for the design, equipment and maintenance of experimental and biological clinics (vivariums)" (SR 2.2.1.3218–14) (Resolution of the Chief State Sanitary Doctor of the Russian Federation of 29.08.2014 № 51). The animals were kept in the same plastic cages with drinkers, and were provided with complete extruded mixed fodder and a sufficient amount of water. Rat were housed in groups of 4–5, with standard rodent chews and water freely accessible during the entire experimental period. Animals were maintained in an artificial 12-h day/night lighting cycle (lights on at 07:00) at a constant temperature of 21 ± 2 °C (50 ± 5% humidity).
The research was conducted in accordance with the rules of work and the use of experimental animals (Annex to the Order of the Ministry of Health of the USSR of 12.08.77 № 775), the European Convention for the Protection of Vertebrate Animals Used for Experimental or Other Scientific Purposes of 18 March 1986 and the requirements of the Order of the Ministry of Health of the Russian Federation of April 1, 2016 № 199n "On Approval of the Rules of Good Laboratory Practice". The research protocol was approved by the Local Ethics Committee for conducting scientific research involving animals as research objects of the Lobachevsky State University on July 4, 2014.
### Animal model and drug administration
One of the most widely used models to replicate focal cerebral contusion as well as diffuse brain injury characterized by axonal damage is weight drop models in rodents. This model induces a TBI by a standardized weight-drop device inducing a focal blunt injury over an intact skull without pre-injury manipulations [ ]. Such a mechanical impact on the closed skull, rather than on the exposed dura, certainly mimics, e.g., a fall, a motor vehicle ejection or a motorcycle crash, much more closely than initial trephination of the skull with prolonged exposure of the meninges before impact, cryogenic brain injury, direct suction injury, or a fluid wave striking the exposed brain [ ]. In order to simulate the TBI, rats, no anesthetized were situated under a device consisting of a metal tube (inner diameter 20 mm) placed vertically over the animal’s head. Rats were fixed on a tablet, but the head was not fixed. The fall of the load (100 gr) was guided by a cylindrical tube 100 cm long, which was rigidly fixed on a tripod by two holders and centered over the head of the rat [ ]. Immediately after the injury, rats were transferred to a special plastic cage, and they were monitored until normal behavioral patterns were restored. The application of such mechanical energy provided simulation of concussion, focal injuries, including brain injury, which is accompanied by the formation of epidural and subdural hematomas. The mortality rate of falling weight was 0–10% and a righting reflex time was 2—4 min. This indicates the development of minor trauma [ ]. After the injury, the animals experienced asphyxia, convulsions, bleeding, etc. 30 to 40 min after the injury, the animals returned to normal life and nutrition. The animals that died (n = 2 in total) during the experiments were discarded from this study.
The animals (n = 72) were randomly divided into: the rats that had been being introduced to 2-ethil-6-methil- 3hydroxipiridin succinate (commercial name—mexicor, which is the solution for intra- vein and intra-muscular application, OOO “EcoFarmInvest”, Moscow) at 8 mg/kg intro-abdominally for 10 days every day (experimental group, n = 32) and rat introduced the physiological solution in the same volume (control group, n = 32) after the TBI. The first application of the preparation was 1 h after causing the TBI. The values of the studied parameters in the intact animal group were accepted as values of the physiological norm (n = 8 rats).
### Blood collection and RBCs preparation
Blood samples were collected at each time point from 8 randomly selected rats 1, 3, 7, and 12 days after the TBI. Approximately 2.0 ml whole blood was drawn from the sublingual vein. The first blood sampling was carried out 1 h after the injury. Blood samples were collected in conical tubes containing 1.0 ml of 3.2% buffered sodium citrate solution. For example, in the tube labeled “C” there was blood from one rat suffered the TBI and received the physiological solution, “M”—blood from one rat suffered the TBI and received 2-ethil-6-methil- 3hydroxipiridin succinate; and “I”—another one rate which did not receive any exposure. Cells and plasma had been being separated by centrifugation at 1000 × rpm for 10 min with 0.9% sodium chloride solution.
### RBCs functional indexes analysis
RBC electrophoretic mobility (EPME) was defined with the use of micro-electrophoresis method (n = 8/group/time point) [ ]. The time of the distance 100 mkm in Tris–HCL-buffer with pH 7.4 with currant force 8 mA covering was registered. The value of RBC electrophoretic mobility was calculated with the formulae U = S/TH, where S—the distance of cells movement, T—the time of cells movement on the distance S, H—gradient potential. The value of the gradient potential was defined by the formulae H = I/gχ, where I—amperage, g—camera cross section, χ—electrical conductivity of the medium.
RBC aggregation was studied by optic microscopy method by calculating solar erythrocytes and their aggregation (n = 8/group/time point) [ ]. As the aggregation stimulator the solution of blue dextran-T-2000 (GE Healthcare firm, 20 mg/ml) was used in Tris HCL- buffer (pH-7.4) was used. Drench RBC diluted with the dextran solution (in proportion 1:10 volume units) and counted the number of non-aggregated RBC in hemocytometer. The total number of RBC in a probe counted in the isotonic solution of NaCl. A level aggregation counted by formulae A = 100%—(a number of free (non-aggregated) RBC, x—the total number of RBC − 1 × 100%).
The concentration of malonic dialdehyde (MDA) was defined by the formation of tined trimethine complex with the absorption maximum at 530 nm under the reaction with thiobarbituric acid (n = 8/group/time point). To calculate the concentration of MDA the coefficient of molar extinction E = 1,56 × 10-5 M-1sm-1 [ ] was used. Catalase activity was analyzed by the decrease in peroxide in the probe. The measurements were made by spectrophotometry on Shimadzu XRD-700 immediately after the introduction of H O in the pan with the probe and 20 s after the introduction at the wave length 240 nm [ ].
ATP and 2.3-BPG concentration measurement was produced by non-enzymatic method (n = 8/group/time point) [ ]. ATP and 2.3-BPG concentration was defined in trichloroacetic acid (TCA) filtrate of hemolyzed RBC. When defining ATP equal volumes of TCA filtrate were mixed with 2H HCl and 2H NaOH. Inorganic phosphorus (Pn) in the composition of which there were Pn separated from ATP after hydrolysis and Pn which had been there before the hydrolysis. To define 2.3-BPG the nucleotides (ATP, ADP, AMP) were removed from trichloroacetic acid filtrate of hemolyzed RBC. It was perfomed by the adsorption on activated carbon with the further centrifugation. In supernatant, inorganic phosphate (Pn) was defined (test tube 1). Another part of TCA filtrate was subjected to the process of ashing, with the addition of 5% solution of Mg nitrate and 0.36 N H SO . Pn was measured in supernatant (test tube 2). Pn was defined in each test tube, registering coloring density on KFK-3 photoelectric photometer (λ 660 nm). Pn concentration was defined by gauge curve, using standard solution KH PO . 2.3-BPG concentration calculation was according to the formulae (Pn (test tube 1) × 100 − Pn (test tube 2) × 10) / 2 [ ].
### Histopathology
Histological examination were carried out on the 1st, 3rd, 7th and 12th days with rats suffered TBI after blood sampling. Decapitation animals took place under Na thiopental anesthesia, in a dose 100 mg/kg animal mass. After decapitation (n = 8/group/time point), the brain was quickly removed from each rat, dissected, and was put into 10% buffered neutral formalin solution. Material fixation had been lasting for 72–96 h, then bits of parietal occipital regions of the brain was cut from the fixed material for the following histological investigation. For this purpose they were embedded into paraffin (the media Histomix-extra, “Biovitrum”, Russia were used). The slices 5–7 mkm thick on the rotation microtome Leica 450 RM (Leica Microsystems, Germany) from the received blocks were produced. The slices were colored with hematoxylin and eosin.
A senior pathologist performed an initial histopathological analysis, while blind to the RBCs functional indexes results. He analysis the area (S, mm ) of arterioles, venules and capillaries (on cross sections), the area of perivascular edema around the examined vessels (S, mm ) and the diameter of capillaries (d, mm ) were visualized under a lightmicroscope. In total about 30 brain sections per rat were analyzed. Using 400 × magnification, 20 randomized field were selected randomly and 10 vessels and / or 30 cells were counted in each field. Histological preparations were studied with the light microscope Leica DM1000 (Leica Microsystems, Germany), micro photos were got with the help of digital camera Leica DFC290 (Leica Microsystems, Germany).
### Analysis of animal motion activity
The presence and degree of intensity of motor disorders were defined by using the method of moving on bar [ ]. The ability to balance and to stay at the bar (in numbers), time spent for moving on the bar from the bright light source to the darkroom (in seconds), and rats paw sliding frequency (in numbers) were defined in the posttraumatic period.
### Statistical analysis
The received data were processed with the use of the application package BIOSTAT (Analystsoft < USA) and Microsoft Excel (Microsoft, USA) applying the methods of one-dimensional statistics. The results are given as M + SD, where M—arithmetic average, m—standard error of the mean. The authenticity of average differences was defined by Student's t -test. The differences were supposed to be reliable when the level of importance was p < 0.05.
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## Background
Dyslexia is one of the most common learning disabilities, especially among children. Type 2 diabetes is a metabolic disorder that affects a large population globally, with metabolic disorders. There have been several genes that are identified as causes of Dyslexia, and in recent studies, it has been found out that some of those genes are also involved in several metabolic pathways. For several years, it has been known that type 2 diabetes causes several neurodegenerative disorders, such as Alzheimer’s disease and Parkinson’s disease. Furthermore, in several studies, it was suggested that type 2 diabetes also has some associations with learning disabilities. This raises the question of whether “ Is there a connection between type 2 diabetes and dyslexia ?”. In this study, this question is elaborated by linking their developmental processes via bioinformatics analysis about these two diseases individually and collectively.
## Result
The literature review for dyslexia and type two diabetes was completed. As the result of this literature review, the genes that are associated to type 2 diabetes and dyslexia were identified. The biological pathways of dyslexia, and dyslexia associated genes, type 2 diabetes, and type 2 diabetes associated genes were identified. The association of these genes, regarding to their association with pathways were analysed, and using STRING database the gene associations were analysed and identified.
## Conclusion
The findings of this research included the interaction analysis via gene association, co-expression and protein–protein interaction. These findings clarified the interconnection between dyslexia and type 2 diabetes in molecular level and it will be the beginning of an answer regarding to the relationship between T2D and dyslexia. Finally, by improving the understanding this paper aims to open the way for the possible future approach to examine this hypothesis.
## Background—Introduction
### Hallmarks of dyslexia, psychology, and behaviour
Dyslexia is a neurobiological disorder that causes a learning disability. It is the most common type of learning disability; however, there are still many unknowns about its neurobiological pathways and how it is related to other risk factors and conditions. Unlike other learning disabilities, Dyslexia does not reflect on the IQ level of the patient [ ]. It causes difficulty in reading and writing. Also, if not diagnosed, Dyslexia may lead to low self-esteem in children [ ]. It was firstly identified in 1878 by Adolf Kussmaul, who introduced the world with the concept of ‘Word blindness’ through his observations with his stroke patients, who seem to have selectively lost their ability to read but remain their verbal and non-verbal reasoning skills [ ]. This condition was later named Dyslexia by Rudolph Berlin, and the information on this condition was later extended by Pringle Morgan, who explained ‘congenital word blindness’ through his studies [ ]. Until the mid-twentieth century, it was thought that this condition was due to a hereditary defect, however, later, it was found out though it might also be due to hereditary defects, it can also be due to non-hereditary genetic problems. There are several symptoms of Dyslexia, which are observed as linguistic, sensory, and motor signs [ , ]. The most significant symptom of Dyslexia is difficulty in reading and writing. In developmental Dyslexia cases, the symptoms can be delayed speech or other speech problems such as stuttering, learning and writing difficulty, and visual disturbances while reading, such as blurred vision, double vision, eye strain, headaches and eye fatigue [ ].
In 1986, Hansen et al. reported a case study with a group of diabetic patients to find out if there is any association between Dyslexia and Diabetes. They identified the history of Dyslexia in Type 1 Diabetic patients. While some of those patients have been identified as dyslexic, some were not. But they had family members who were dyslexic [ ]. In 1987, Hansen, Nerup, and Holbek published a follow up on their research, in which they performed their experiment again to compare the accuracy of their findings. The results were very similar to each other that they concluded their study output, stating that 21% of dyslexic relatives were among 114 diabetics, and in those 114 patients, four were dyslexic. On their findings, there must be a common dominantly inherited autosomal genetic error, which causes both disorders [ ]. Upon their research, several other case studies supported their findings by stating there might be some minor or major deficits that may lead to both dyslexia and diabetes [ , ]. In contrast, those findings were not sufficient enough for them to make a decisive conclusion to state that there is a relationship between Diabetes and Dyslexia.
In the normal human brain, while reading, the left hemisphere of the brain is actively working. The functional difference in the brain of a dyslexia patient, due to the hypoactivity in left temporal, parietal, and fusiform regions sensitivity in reading decreases. It is associated with both anatomical and functional abnormalities in the left hemisphere due to failures in neural migration [ ]. In addition to this, there is also a structural difference in the brain of a dyslexia patient, which is the colocalization of the gray matter in lobule VI of the cerebellum, which is involved in the memory and voluntary movements, and the region that has functional differences with normal brain [ ]. It affects the daily life of the patient. Nevertheless, there is no functional medicine as a treatment of Dyslexia. However, there are several exercises for the patients with Dyslexia as a treatment, such as reading and writing therapies and spelling therapies.
### Hallmarks of type 2 diabetes, pathophysiology and biology
Diabetes mellitus (DM) is the most common disease among the people in the world [ ]. Type 2 diabetes (T2D) Mellitus is one of the most common diabetes types, and nowadays, it has started to spread among young people due to unhealthy lifestyles and inheritance. T2D can be caused by either genetic factors or/and environmental factors. The leading genetic factors behind T2D are impaired insulin secretion or insulin resistance, or it can even be both due to impaired pancreatic β-cells [ , ]. These lead to a metabolic disorder that can also be caused by dysregulation of carbohydrate, lipid, and protein metabolism in the body [ , ]. The main symptom of T2D is hyperglycaemia, which is a condition with excessive levels of glucose in the blood. This is followed by frequent urination and thirst, weight change, skin irritation, headache, and vision problems such as blurred vision, dry eye and retinopathy [ , ]. The leading environmental factors behind T2D can be irresponsible high-calorie diets, excessive glucose consumption, or it can also be due to the lack of physical activity. Because of these conditions, the glucose homeostasis system in the body can be affected, which shows itself as insulin resistance at first; if it is not treated with a healthy diet and physical activity, the condition can develop T2D [ ]. However, these factors might also cause a functional problem in pancreatic β-cell secretion or directly on β-cell functioning, leading to T2D [ ].
Insulin is secreted by pancreatic β-cells, and due to an impairment in these cells, the insulin secretion is dysregulated. As a result of this condition, the insulin secretion is blocked, and it leads to a hyperglycaemic condition and T1D via pathogenic mechanisms. When the body cannot produce enough insulin to absorb excessive amounts of blood glucose, the body goes into a hyperglycaemic state. When this happens in the patients with T2D, the kidneys and liver play an essential role in compensating or regulating the glucose homeostasis, in which the kidneys and liver try to balance the insulin and glucagon in the blood to maintain blood glucose levels [ ]. Another malfunction of T2D is insulin-mediated whole-body glucose utilization, which is due to the defective glucose disposal in the muscle; the amount of glucose intake significantly decreases [ , ]. These are the two most important problems among T2D patients, controlled by therapeutic diabetes drugs combined with dietary restrictions and increased physical activity. T2D is correlated with aging because as the person ages, the frequency and the intensity of the islet amyloids increase [ ]. This increase is mostly associated with the malfunctioning of pancreatic β-cells.
## The hypothesis
According to the initial literature research that was discussed in the introduction, the first similarity between Dyslexia and type 2 diabetes is that they have an important common symptom, vision impairment. Although the reason behind the vision impairment in dyslexia is not clear yet it is hypothesised that the efficiency of ocular motor tracking or other parameters of visual functioning may be the possible cause of it [ ]. It has been identified that the visual impairment in T2D is related to microvascular complications, which may cause low blood and fluid flow to the eyes that may lead blurred vision, dry eyes or retinopathy according to the severity of the condition [ ]. Due blurred vision that is caused by microvascular complications, reading difficulties can be developed which may trigger Dyslexia, however, it requires further research within the dyslexic patients and their microvascular system. Headache can be a linked cause of blurred vision; however, it could also be the reason of hyperglycaemic condition within the body of T2D patients.
## Molecular mechanisms and related genes
### Physiology and molecular mechanism of dyslexia
The efforts on Dyslexia are currently increasing and evolving. However, we have not yet had sufficient information on Dyslexia. According to the current researches, we are aware that there are some functional and structural differences in the brain of patients with Dyslexia compared to the average population. Norton et al. stated that there are hypoactivation in the left temporal, parietal, and fusiform regions of the brain. Most cases are due to lack of differential sensitivity. Even though it differs among the people according to their age, gender, ethnic diversity, it’s mostly seen in the left hemisphere, especially the left inferior frontal and angular and temporal gyrus [ , , ]. According to Ramus et al., the post-mortem survey, showed that there are structural differences between a person with dyslexic and a control brain, which are seen in the left perisylvian cortex, underlying white matter, thalamus, corpus callosum, and cerebellum. As a result of the researches, the neural signature of Dyslexia is identified as the failure of the left-hemisphere posterior of the brain to function correctly [ ].
The findings from several studies indicate that these functional abnormalities are the results of neuronal migration. While in some patients, this abnormal neuronal migration is seen in microgyria as a disturbed organization of all layers of the cortex, in some patients, cytoarchitectonic anomalies, disorganized magnocellular layers with small neuronal cell bodies, in the thalamus is observed [ ]. It was observed that the neuronal cell bodies in the magnocellular layers of the lateral geniculate nucleus are smaller in the dyslexic patient with respect to a healthy individual, and the neuronal asymmetry of the primary visual cortex wasn’t seen in dyslexic patients [ ]. Another structural difference in the brain of the patients with Dyslexia is the structural white and gray matter difference; in which it has been observed that the gray matter colocalizes with the regions that show functional differences (left hemisphere) as well as the cerebellum, especially in lobule VI [ ]. According to the researches that have been done on the white matter of dyslexic patients, the local white matter changes in the left temporoparietal regions and the left interior frontal gyrus [ ]. Peterson et al. stated that the gray matter density decreases in the left medial temporal gyrus of dyslexic patients. White matter also decreases in the left frontal and parietal portions of the arcuate fasciculus and other left hemisphere sites in the dyslexic patients. While these decreases result in reading disability, according to researches that have been done until now, there has been no indication of it causing any mental disability.
Over the last few years, several genes that are linked with developmental Dyslexia have been identified. There are six key genes for developing Dyslexia, including DYX1C1 , ROBO1 , DCDC2 , K1AA0319 , SLIT1 , and SRGAP1 . In 1983, Smith et al. represented a new locus, DYX1 locus, linked with the reading disability, which is later discovered that the gene on that locus ( DYX1C1 gene) is a genetic risk factor for developmental dyslexia [ , ]. ROBO1 is a crucial gene for Dyslexia because it encodes an integral membrane protein, which functions in axon guidance as well as neuronal cell migration. It is known as axon guidance receptor gene. During brain development, two functional copies of ROBO1 are required to have an average reading ability; however, in the event when there is a partial haploinsufficiency for ROBO1 , the patient develops dyslexia [ ]. K1AA0319 gene encodes a transmembrane protein. It regulates the neuronal migration and cell adhesion, which plays a part in the development of the cerebral cortex. DCDC2 gene belongs to a superfamily of doublecortin domain-containing proteins that bind to microtubules, which is involved in neuronal migration, and it is localized to the primary cilium in the primary neurons [ ]. Massinen et al. states that while the overexpression of DCDC2 affects the ciliary length and Shh signalling, which is a signalling pathway that functions in the cell differentiation during embryonic development; downregulation of DCDC2 expression enhances WNT signalling, which is a signalling pathway during the embryonic development and the development of the nervous system (neurogenesis). As a result of a disruption in the DCDC2 gene, migration of developing neurons via these pathways from the ventricular zone to their cortical plate is also disrupted and impaired [ ]. K1AA0319 is also responsible for the development of Dyslexia, and it functions with DCDC2 because the evidence states that there is a gene–gene interaction between K1AA0319 and DCDC2 [ ]. Also, SLIT1 is thought to be associated with the development of dyslexia because it is responsible for preventing unwanted midline crossing of axons by acting as the molecular guidance cue in cellular migration. SRGAP1 is a protein-coding gene which is involved in GTPase activator activity, and it is associated with ROBO1 and SLIT protein family. However, it is not yet clear how SLIT1 and SRGAP1 are directly involved in the development of dyslexia. DYX1C1 , ROBO1 , DCDC2 , and KIAA0319 , are effectively functional in the neuronal migration and axon guidance, and these four genes collectively work while they also co-regulate each other [ ]. DYX1C1 and DCDC2 genes are related to ciliary Dyslexia. They are regulated by Regulatory Factor (RF) X transcription factors, whereas DYX1C1 , DCDC2 , and KIAA0319 are linked to the cytoskeletal organisation, which causes them to work together in the neuronal migration in the brain [ ]. As to complement their work, ROBO1 acts as a neuronal axon guidance gene through the neuronal migration [ ]. These four genes were the basis of the susceptibility of developing dyslexia genes. In several studies, such as Anthoni et al., they are used as a comparison to analyse other dyslexia-related genes [ ].
### Physiology and molecular mechanism of type 2 diabetes
Type 2 diabetes mellitus is the most common type of diabetes disorder. The reasons behind T2D are either defective insulin secretion or loss of glucose uptake in the glucose consuming tissues. As previously mentioned in this article, pancreatic β-cell death is the leading cause of type 2 diabetes. As the functionality of the β-cell decreases, glucose intolerance starts to develop; however, if it progresses drastically, type 2 diabetes is developed [ ]. Several studies [ , ] show that it is also linked with lipotoxicity or glucotoxicity, resulting from over-usage of pancreatic β-cells due to the excessive amount of glucose in the blood. In both cases, hyperglycaemia is the key response element for type 2 diabetes. For glucotoxicity , due to the over-usage, the phenotype of β-cells, or glucose stimulus-secretion coupling and even gene expression, can change, which leads to apoptosis of the pancreatic β-cells [ , ]. In the case of lipotoxicity , it has been observed that abdominal obesity is correlated with loss of β-cell function, which leads to glucose intolerance, which also results in inflammatory response [ , ]. This apoptosis can also be a result of inflammatory reactions when there are high concentrations of proinflammatory cytokinesis because of the excessive levels of glucose in the blood [ ].
Hyperglycaemia is the leading condition of type 2 diabetes, and as it was previously mentioned that hyperglycaemia is affected by the volume of fat tissue in the body. Glucose homeostasis is the balance between the insulin and the glucagon in the blood to preserve the blood glucose levels, and it is associated with peripheral insulin resistance. For glucose homeostasis, the adipose tissue is vital because by producing cytokines and several other inflammatory pathway reagents, the white adipose tissue is the primary source of inflammatory markers in T2D [ ]. The free fatty acids lead to chronic metabolic inflammation, which leads to endoplasmic reticulum (ER) stress, TLR4 signalling, protein kinase Cε or protein kinase R(PKR), and insulin resistance [ ]. In order to preserve the homeostasis, inflammation responses are triggered, which results in apoptosis [ ].
Aggregating researches provided us with the genetic information of type 2 diabetes. There are several risk-factor genes involved in the development and the progress of type 2 diabetes. T2D can be caused by a mutation in genes involved in metabolic pathways, such as a single polymorphism (SNP) in TCF7L2 gene [ ]. T2D also be caused by chronic inflammation due to proteins in inflammatory pathways, such as interleukin-6 (IL-6) and tumour necrosis factor (TNF) [ ]. TCF7L2 is the most known of those genes, which are associated with type 2 diabetes. It encodes a high mobility group (HMG) box-containing transcription factor, which is involved in regulating blood glucose homeostasis, and it is associated with the increased risk of developing T2D. It is also essential for the WNT signalling pathway, which is a key pathway that functions in organogenesis and the development and the process of tumours during embryonic development, and it is also involved in regulating gene expression during the adulthood [ ]. Transcription factor β-catenin (β-catenin)/TCF acts as an effector for the canonical signalling pathway; in the absence of WNT signalling, HMG box TCF proteins functions as the transcriptional corepressors of the WNT target genes in the nucleus [ , ]. β-catenin governs the development of pancreatic islet and with the adipocyte-derived WNT molecules induces the pancreatic β-cell proliferation, and insulin secretion [ ]. It has been observed and reported in several studies that due to polymorphism in TCF7L2 , the risk of developing type 2 diabetes significantly increases. According to the researches, SNPs on TCF2L7 cause insulin secretion level reduction [ ]. However, according to the study of Shu et al., when TCF7L2 is overexpressed, the islets are prevented from glucose and cytokine-mediated apoptosis of pancreatic β-cells.
Another key gene for type 2 diabetes is CTNNB1 . It is responsible for encoding β-catenin, and it is involved in cadherin-mediated intracellular adhesion and cell growth. It is actively involved in the WNT signalling pathway. Upon the activation of WNT/CTNNB1 signalling, Pyruvate kinase muscle isoform 2 (PKM2), a potential modulator of insulin secretion in pancreatic β-cells, inhibits cell apoptosis and simultaneously promotes cell proliferation, as well as insulin secretion [ ]. In addition to this, KIF3A, which is a kinesin motor protein and an essential subunit for the transportation along microtubules in cilium and cytoplasm in the neurons, is also a critical gene in the development of type 2 diabetes [ ]. The critical component of KIF3 is necessary for GLUT4 translocation, and by forming a complex with Axin and TNKS, KIF3A is directly involved in glucose transportation by promoting GLUT4 translocation [ ].
## Evaluation of the hypothesis
### Interconnection between dyslexia and T2D molecular mechanisms
According to Massinen et al., overexpression of DCDC2 may enhance KIF3A mediated translocation of the transmembrane protein Smoothened (Smo) to the cilium and leads to overactivation of the Shh signalling pathway. As it was mentioned before, KIF3A is involved in the regulation of phosphorylation and stabilization of β-catenin, which interacts with TCF family proteins, which are associated with the increased risk of developing type 2 diabetes [ ]. With this finding, the knowledge about the association between type 2 diabetes and Dyslexia deepened. However, the literature mentioned above, didn’t provide detailed information because there is insufficient data about this association [ ].
As previously mentioned, with the literature research on both the common and rare genetic backgrounds of Dyslexia and type 2 diabetes, it was identified that both disorders are genetically linked with the canonical WNT signalling pathway. WNT signalling pathway is an evolutionarily conserved pathway, which regulates cell migration, cell fate determination, cell polarity, neural patterning, and organogenesis during embryonic development, and it is divided into two types as canonical and non-canonical pathways [ , ]. The canonical pathway is essential for cell fate determination during the early embryogenesis, and the accumulation and translocation of the adherents junction-associated β-catenin into the nucleus is the hallmark of this pathway [ ]. Regulation of β-catenin is essential for both dyslexia and type 2 diabetes. While the overexpression of DCDC2 inhibits β-catenin-dependent WNT signalling, the upregulation of β-catenin levels may lead to an increase in the INS-1 cell. As it was mentioned before, DCDC2 is an essential gene for developmental Dyslexia, and it is localized to the ciliary axoneme and the mitotic spindle fibres in a cell-cycle-dependent manner [ ]. When DCDC2 is knocked down, the number of cilia in the cell culture reduces [ ], leading to developmental Dyslexia. It was also discovered previously that β-catenin/TCF7L2-dependent WNT signalling is involved in the islet function, the development of the pancreas, and the production and the secretion of insulin [ , ]. When TCF7L2 increases in the islets, insulin secretion can be inhibited, which leads to an increased risk of diabetes [ ]. In addition to this modulation of β-cell growth is affected by E-cadherin, which forms a link with actin by β-catenin. Due to the problem in β-catenin, the risk of developing type 2 diabetes can increase as well [ , ].
### Interaction analysis—results
Upon the findings from the literature, the genes involved in the development of Dyslexia and diabetes, separately, were gathered and divided into two gene sets as Dyslexia genes and T2D genes. In order to understand their protein–protein interactions, the genes that are shown in Fig. were the inputs and the predicted associations. According to Fig. , ROBO1 , Dyslexia related gene, and CTNNB1 , type 2 diabetes-related gene, are associated via co-expression; with the known interactions from the curated database. In addition to that, it is also seen in Fig. there is another association between SLIT1 , which acts as a molecular guidance cue in cellular migration, and CTNNB1 via interactions from both curated database and experimentally determined; as well as SLIT1 and ROBO1 via co-expression with known interactions from both a curated database and experimentally determined. All of the mentioned interactions were identified in textmining too. Figure is a proof that there is protein–protein interaction between these two gene sets via several genes.
Protein interaction network obtained from STRING database. In the left side of the figure the genes that are related to dyslexia are given, and on the right side of the figure the genes that are associated with type 2 diabetes are shown. The light-blue lines between the genes indicates interactions from curated database, while the pink lines between the genes indicates interactions that are experimentally determined. Green lines between the genes indicates predicted interactions of gene neighbourhood, while red lines indicate gene fusions and dark-blue lines indicates gene co-occurrence. Finally, the yellow lines indicate textmining co-occurrence, black lines indicate co-expression and blue-greyish lines indicates protein homology. These indications are also explained at the legend of the figure. As it is shown in a light blue box, the important connection between dyslexia and type 2 diabetes is between ROBO1 and CTNNB1, from curated database, textmining and co-expression
In addition to the protein–protein interaction network, co-expression of these genes is also queried via the STRING database, which results is represented in Fig. . Since these co-expression levels are defined in the STRING database as a heat map, the red squares indicate the co-expression. As it is seen here, there is no strong co-expression evidence between these genes. However, on the left side of the figure, in Homo sapiens , a light-pink box is seen between KIAA0319 and KIF3A . This indicates a weak co-expression level. In addition to this, on the right side of the figure, the observed co-expressions in other organisms are shown. As in S. mansoni , between ROBO1 and CNTNB1 , a weak co-expression is shown, while in M. musculus , between KIF3A and DYX1C1 and KIF3A and CTNNB1, there are light-pink boxes, which indicates a weak co-expression between those genes. Finally, in M. domestica , between DCDC2 and DYX1C1 , and between DCDC2 and KIAA0319 , weak co-expression is shown as light-pink boxes. This figure can also be supportive evidence for the idea of this study and it provides information for possible further research with animal models.
Dyslexia and T2D gene co-expression analysis data from STRING Database. The co-expression of genes that are related to dyslexia and the genes that are related to type 2 diabetes results from STRING Database are shown in this figure. The co-expression of the genes is represented as a heat map. In this figures, light-pink boxes, indicate weak co-expressions between the genes KIAA0319 and KIF3A in Homo sapiens, in the left side of the figure. On the right side of the figure, the co-expression between in other organisms are shown. S. mansoni shows a co-expression of ROBO1 and CNTNB1; M. musculus shows co-expression of KIF3A, DYX1C1 and CTNNB1; M. domestica shows co-expression of DCDC2, DYX1C1 and KIAA0319. The heat map illustrates Co-expression scores based on RNA expression patterns and protein co-regulation, provided by ProteomeHD database and visualized by string database
### Flowchart of the research
First, a careful literature search for both diseases was done. More than 30 research papers were screened for each condition individually. During the literature research, the focus was on type 2 diabetes, so the other types of diabetes are not included in this examination. Out of the genes that were mentioned, analysed, and searched in those papers, which are the references, the genes that were related to the developmental stages of each disorder were separated. They are explored in detail, the pathways they are involved in, their functions and their molecular background were searched. With these data, the genes that are associated with Dyslexia and genes that are associated with type 2 diabetes are sorted and listed. These genes are entered into the STRING database for protein–protein interactions [ ]. After the initial input, predicted functional partners were shown. The network settings are chosen as network edges to represent the type of evidence. The active interaction sources are chosen as text mining, experiments, database, co-expression, neighbourhood, gene fusion, and co-occurrence. The minimum required interaction score was chosen as 0.400 for medium confidence. Finally, the max number of interactions to show are selected as no more than ten interactions.
## Consequences of the hypothesis, discussion and further aspects
In the past, it was thought that Dyslexia is caused by an underlying deficit in phonological representations. Still, now it is known that a single phonological deficit is not enough to diagnose a patient as dyslexic [ ]. There are several clinical features of Dyslexia, such as attention and hyperactivity, motor integration disorder, arithmetic, mild oral language, and the severity of these deficits’ changes in patients [ ]. On the other hand, Dyslexia is often confused with attention deficit hyperactivity disorder (ADHD) [ ]. There are significant differences between these two diseases. For instance, ADHD patients have short focus periods, whereas dyslexia patients can maintain their focus; however, they have difficulty in reading, writing a word, or a number correctly.
In this research, the aim was to analyse if there is an association between type 2 diabetes and Dyslexia at the molecular level since type 2 diabetes is associated with several neurodegenerative disorders and learning disabilities. According to the literature study, some genes that are associated with developmental dyslexia, such as ROBO1 , DCDC2 , DYX1C1, and KIA0319 , and some genes that are associated with type 2 diabetes CTNNB1 , TCF7L2, and KIF3A had shown some connections with each other. When these connections are searched further with STRING analyses, the outcome was promising, even though it was small evidence. In Fig. , the interactions between KIAA0319 and ROBO1 are text mining and co-expression, but they are not experimentally validated, which means that this interaction is less likely to be considered. However, the interaction between SLIT1 and CTNNB1 is not only from the curated database, but it is experimentally validated. So, this might be useful evidence for continuing of this research. As it was mentioned above, in the previous studies by Hansen and other researchers, a connection between type 1 diabetes and Dyslexia, was also mentioned. For the future aspect of this research, the association might be improved into the association between diabetes mellitus and Dyslexia upon gathering enough evidence. This research will continue with in vivo analysis.
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Active forgetting occurs in many species, but how behavioral control mechanisms influence which memories are forgotten remains unknown. We previously found that when rats need to retrieve a memory to guide exploration, it reduces later retention of other competing memories encoded in that environment. As with humans, this retrieval-induced forgetting relies on prefrontal control processes. Dopaminergic input to the prefrontal cortex is important for executive functions and cognitive flexibility. We found that, in a similar way, retrieval-induced forgetting of competing memories in male rats requires prefrontal dopamine signaling through D receptors. Blockade of medial prefrontal cortex D receptors as animals encountered a familiar object impaired active forgetting of competing object memories as measured on a later long-term memory test. Inactivation of the ventral tegmental area produced the same pattern of behavior, a pattern that could be reversed by concomitant activation of prefrontal D receptors. We observed a bidirectional modulation of retrieval-induced forgetting by agonists and antagonists of D receptors in the medial prefrontal cortex. These findings establish the essential role of prefrontal dopamine in the active forgetting of competing memories, contributing to the shaping of retention in response to the behavioral goals of an organism.
SIGNIFICANCE STATEMENT Forgetting is a ubiquitous phenomenon that is actively promoted in many species. The very act of remembering some experiences can cause forgetting of others, in both humans and rats. This retrieval-induced forgetting process is thought to be driven by inhibitory control signals from the prefrontal cortex that target areas where the memories are stored. Here we started disentangling the neurochemical signals in the prefrontal cortex that are essential to retrieval-induced forgetting. We found that, in rats, the release of dopamine in this area, acting through D receptors, was essential to causing active forgetting of competing memories. Inhibition of D receptors impaired forgetting, while activation increased forgetting. These findings are important, because the mechanisms of active forgetting and their linkage to goal-directed behavior are only beginning to be understood.
## Introduction
Neuroscientific accounts of forgetting often have focused on the passive decay of memory traces ( ). However, recent neurobiological studies indicate that active forgetting mechanisms also can dictate the fate of a memory ( ; ; ; ; ; ). A common feature of both active forgetting processes and passive decay is that they are indifferent to memory content, but there is the question of whether the forgetting of particular traces may be adaptively prioritized to benefit the goals of the organism. Selective forgetting mechanisms have been described that adaptively tune the accessibility of memories to the behavioral demands of the organisms ( ; ). When people and rats retrieve a past event, other memories that compete with and hinder retrieval are more likely to be forgotten ( ; ). This “retrieval-induced forgetting” (RIF) occurs for a broad range of stimuli and contexts ( ; ). In humans, retrieval-induced forgetting arises because trying to retrieve a specific memory triggers an inhibitory control mechanism mediated by the lateral prefrontal cortex that focuses retrieval on goal-relevant traces by suppressing distracting memories ( ; ). Paralleling these findings, rats can also engage this active forgetting mechanism to inhibit competing memories. As in humans, RIF in rats requires prefrontal engagement during the selective retrieval practice (RP) phase ( ; ), and yields long-lasting forgetting that generalizes across multiple retrieval cues ( ). In mammals, the prefrontal cortex facilitates flexible behavior ( ; ; ; ) via control mechanisms that suppress habitual responses that might otherwise dominate goal-directed action and have also been associated with attentional processes ( ; ; ). In rodents, the medial prefrontal cortex (mPFC) has been associated with attentional and inhibitory control processes ( ; ; ; ). We have proposed that the mPFC also suppresses competing memories, initiating a key signal that triggers active forgetting ( ).
Dopamine is essential for cognitive control mechanisms mediated by the prefrontal cortex of humans, monkeys, and rodents ( ). In the mPFC, dopamine modulates processes such as working memory ( ; ; ; ), attention, and behavioral flexibility ( ; ). The rodent mPFC receives a dopaminergic input from neurons in the ventral tegmental area (VTA) that innervates both pyramidal cells and interneurons. In particular, D dopamine receptors (D Rs) in the mPFC are critical for mediating dopamine effects on cognitive functioning ( ). Interestingly, an imaging genetics study in humans has linked genetic variation in prefrontal dopamine levels to differences in the engagement of lateral prefrontal cortex during selective retrieval and, correspondingly, to adaptive forgetting ( ). Here, we investigated whether dopamine-mediated control processes in the mPFC contribute to adaptive forgetting of competing memories in our rodent model of retrieval-induced forgetting. We found that the blockade of D Rs in mPFC of rats abolished retrieval-induced forgetting of object memories. To strengthen our hypothesis that control processes are involved in retrieval-induced forgetting, we also showed that the same manipulation of the D Rs prevented animals from switching to a new rule in a set-shifting task that involves control processes to change a course of action ( ; ). Inactivating VTA activity also impaired forgetting, and we could reverse this impairment by concurrently activating D Rs in mPFC. Importantly, we show that dopaminergic modulation of adaptive forgetting is bidirectional, as activation of D R in mPFC significantly enhances retrieval-induced forgetting. Our results suggest that dopamine-dependent mechanisms of cognitive control over memory are conserved across species and are essential for adaptive forgetting in the mammalian brain.
## Materials and Methods
### Ethics statement
All experimental procedures were conducted in accordance with institutional regulations (Institutional Animal Care and Use Committee of the School of Medicine, University of Buenos Aires, ASP #49527/15) and government regulations (SENASAARS617.2002). All efforts were made to minimize the number of animals used and their suffering.
### Subjects
Two hundred forty-five male adult Wistar rats (weight range, 180–250 g) were housed up to five per cage and were kept with water and food available ad libitum under a 12 h light/dark cycle (lights on at 7:00 A.M.) at a constant temperature range of 21–23°C. Separate groups of animals were used for the different experiments. Experiments took place during the light phase of the cycle. The experimental protocol for this study followed guidelines of the National Institutes of Health in the Guide for the Care and Use of Laboratory Animals . The number of animals used is stated for each experiment (see below).
### Apparatus
Different arena contexts were used during the experiments.
The design for most of the experiments was mixed factorial designs with a within-subjects manipulation of drug versus vehicle (Veh) and a between-subjects behavioral manipulation, except for the experiment depicted in a figure (see C–E ), which was a full within-subjects design. All animals were exposed to at least four contexts during the experiment in which they participated. Animals in the within-subjects behavioral designs were exposed to a total of six contexts. All contexts were assigned pseudorandomly to each experimental phase, except for contexts 5, 7, and 8, which were used exclusively to habituate animals to the objects presented as contextually novel during the practice phase. All animals that underwent the retrieval practice paradigm went through a shaping phase (see explanation below) and then started the experiment.
Arena 1 was 50 cm wide × 50 cm long × 39 cm high with black plywood walls and floor, divided into nine squares by white lines. Arena 2 was an acrylic box 60 cm wide × 40 cm long × 50 cm high. The floor was white as well as two of its walls, which had different visual cues, geometric forms, or strips made with self-adhesive paper tape of different colors. The front wall was transparent, and the back wall was hatched. Arena 3 was 50 cm in diameter × 50 cm high, round with brown acrylic walls and black plywood floor, divided into nine squares by white lines. Arena 4 was a box 50 cm wide × 50 cm long × 40 cm high that was constructed with white Plexiglas. The floor was made of white Plexiglas as well. Each wall had different visual cues, geometric forms, or strips made with self-adhesive paper tape of different colors. Arena 5 was round, 40 cm in diameter × 50 cm high, with brown acrylic walls and sky-blue floor. Arena 6 was a bow-tie-shaped maze made of opaque white Plexiglas. The maze was 94 cm long, 50 cm wide, and 50 cm high. Each end of the apparatus was triangular, the apexes of which were joined by a narrow corridor (14 cm wide). Arena 7 was a Y-shaped apparatus constructed from Plexiglas. All walls were 40 cm high, and each arm was 27 cm in length and 10 cm wide. Arena 8 was an equilateral triangular 40 cm side × 40 cm high made of white semirigid PVC with a white floor made of the same material.
### Objects
All experiments used numerous junk objects, each differing in shape, texture, size, and color. The height of the objects ranged from 8 to 24 cm, and they varied with respect to their visual and tactile qualities. All objects had duplicates so that identical objects could be used at the same time. All objects were affixed to the floor of the apparatus with an odorless reusable adhesive to prevent them for being displaced during each session. Specific objects were never repeated across different conditions for a given animal. All objects were cleaned with 50% alcohol wipes after each session.
### Memory test for retrieval-induced forgetting
#### Overview
Rats as well as many other species innately prefer novel objects to familiar ones and, in displaying this preference, reveal memory for the familiar object ( ; ; ; ; ; ). As in our previous study, we capitalized on this tethering of innate behavior and cognition to show that remembering a prior encounter with one object caused rats to forget other objects seen in the same setting ( ). We modified the spontaneous object recognition procedure to include three phases equivalent to the ones present in human studies of retrieval-induced forgetting ( ; ; ; ): encoding, retrieval practice, and test. In addition to this theoretically critical RP condition, there were two control conditions in which the intervening retrieval practice phase was replaced either by returning the rat to its home cage [time control (TC)] or by giving the rat the same number of exploration trials on entirely new objects [the interference control (IC)]. For each experiment, different cohorts of animals were used. For all the experiments (with the exception seen in C–E ), animals were randomly assigned to one of the three possible conditions after the shaping phase (see below). The order in which they were exposed to each treatment (drug/vehicle or practice length) was pseudorandomly assigned, and experiments were conducted over a span of 2 weeks. Once we finished evaluating the animal for one of the treatments, we waited at least 4 d to start testing the other treatment. For the experiment described in , D and E , animals were exposed to each condition (RP, IC, and TC). The order in which they were exposed to each condition was pseudorandomly assigned, and experiments were conducted over a span of 3 weeks. Once we finished evaluating the animal in one condition (e.g., retrieval practice) we waited 4 d to start testing the following condition (e.g., interference control).
##### The general retrieval practice paradigm.
Our new retrieval practice paradigm generally involved the following three conditions: RP, IC, and TC ( ). All the conditions followed the same basic sequence across 3 d, as follows: day 1, habituation to the contexts; day 2, habituation to “distractor” objects to be used during the retrieval practice phase of the experiment; day 3, the main memory task (during the main memory task, encoding and practice phases took place in a single session); and day 4, test phases.
##### Habituation.
We incorporated a shaping procedure that included four sessions of object exposure. During shaping, rats were first habituated to two different contexts (10 min each, not described in the Apparatus section), and 3 h later rats were exposed to two pairs of novel objects in two contexts. The animals were exposed twice to each context (four sessions) with a delay of 20 min. In each session that lasted 5 min, the rats encountered the same two pairs of different objects in distinct locations. The objects were novel during the first exposure, but familiar during the next three. Each rat saw the four objects twice in both contexts. For each context, the location of the objects was different between the first and the second exposure. The shaping phase was conducted only once during the first week of the experiment independently of the condition assigned for that particular week. We added this procedure to familiarize rats with the possibility that the very same objects could be presented in different locations within a context or across contexts ( ). All experiments started 72 h after shaping.
On the first day of the experiment, animals were habituated to two arena contexts (e.g., contexts 1 and 2) and were allowed to explore each context for 10 min. On the second day, each animal was exposed to three pairs of identical novel objects (X, Y, and Z) in context 2 in three consecutive (30 min apart) sessions, for 5 min each. The following day, the task was conducted in context 1.
##### RP condition.
The sample phase consisted of two consecutive sessions separated by 25 min. In these sample sessions, the animal was allowed to freely explore for 5 min two identical copies of two novel objects [e.g., Object A (session 1) and Object B (session 2)]. The practice phase took place 60 min after the last sample session. This phase consisted of three 3 min sessions with an intersession interval of 15 min. In each session, the animal was exposed to a copy of one of the two encoded objects (e.g., Object A) presented during the sample phase, accompanied by one copy of objects X, Y, or Z, respectively, across the three trials (e.g., A and X; then A and Y; then A and Z across the three sessions). We pseudorandomly assigned which object was presented during the retrieval practice phase from the two objects that were sampled in the sampling phase (either A or B), so the practiced object could either be the first or the second one that was encoded in the sampling phase. Moreover, the location (right or left) in which the studied object appeared during retrieval practice was randomly assigned for each trial. The test phase was conducted 24 h after the last practice session. The animal was exposed for 3 min to a copy of a nonpracticed competitor object presented only during the sample phase (e.g., Object B) and one completely novel object (Object C). Thirty minutes later the animals were reintroduced to the context and exposed for 3 min to a copy of a practiced object (Object A) and one completely novel one (Object D). These two test sessions are defined in the Results section as a “competing object” and a “practiced object,” respectively. For both test sessions, the locations of the novel and familiar objects (right or left) were randomly assigned. The letters used in these descriptions and in our diagrams meant to indicate the nature of the item ( i.e., the practiced object, competitor object, novel object, or distractor). Repetitions of the same letter across conditions do not indicate that the same object was used across conditions: in fact, different objects were used for the different conditions—RP, IC, or TC—of the task. Thus, Object A used in the RP condition is different from Object A used in the IC or TC conditions.
##### IC condition.
On the first day, the animals were habituated to two contexts (e.g., contexts 3 and 4) and allowed to explore them for 10 min each. On the second day, each animal was exposed to three novel objects (X, Y, and Z) in three consecutive sessions (30 min apart), and in context 4 for 5 min each. On the third day, the main memory task was conducted in context 3. On this final day, during the sample phase each rat was allowed to freely explore for 5 min two identical copies of two novel objects (Objects A and B) in two consecutive sessions separated by 25 min. The practice phase took place 60 min after the sample phase. During this phase, the animal was allowed to explore two copies of Objects X, Y, and Z in context 3 during three consecutive 3 min sessions with a delay of 15 min between each session. The test phase (24 h after the last practice session) consisted of a 3 min exposure to a copy of Object B and one completely novel object (Object C). The time the animals spent exploring the objects in each trial was manually recorded using hand chronometers. The order in which the sample objects were tested was pseudorandomly assigned, and the position in which the sample objects appeared on the final test was randomly determined.
##### TC condition.
On the first day, the animals were habituated to one context (e.g., arena context 5), and allowed to explore it for 10 min. On the second day, the animals were transferred to the behavioral testing room but were allowed to stay in their home cage for the duration of time that the animals assigned to the other two conditions were habituated to the novel objects. On the third day, the main memory task was conducted in context 5. The sample phase consisted of two consecutive sessions separated by 25 min. In these sessions, the animal was allowed to freely explore for 5 min two identical copies of two novel objects: Object A (session 1) and Object B (session 2). Unlike in the RP and IC conditions, however, there were no practice trials; instead, the rats spent the same interval of time in their home cages in between the sample phase and the test. The test phase took place 24 h later. During this phase, the animal was exposed to a copy of Object B and a completely novel object (Object C) for 3 min. The order in which the sample objects were tested was pseudorandomly assigned, and the position in which the sample objects appeared on the final test was randomly determined.
##### Quantification of behavior.
The behavioral responses of the animals for all experiments were analyzed given the following criteria. We defined exploration of an object as the rat directing its nose to the object at a distance of <2 cm and/or touching it with its nose. Turning around or sitting on the object was not considered exploratory behavior. Encoding, practice, and test phases were recorded using cameras (model HMX-F80, Samsung). The cameras were located on top of each arena, allowing the visualization of the complete space. Offline analysis was performed using Stopwatch software (Center for Behavioral Neuroscience, Emory University, Atlanta, GA) by a trained person. The test phase was analyzed by an experimenter who was blind to the conditions of the experiment.
Based on these criteria, we calculated a discrimination index (DI) for each trial of each session on each condition, as follows.
###### Practice trials
A discrimination index was calculated as the difference in time spent exploring the contextually novel and familiar objects divided by the total time spent exploring the objects (i.e., [(contextually novel – familiar)/total exploration time]).
###### Test trials
A discrimination index was calculated as the difference in time spent exploring the novel and familiar objects divided by the total time spent exploring the objects (i.e., [(novel – studied)/total exploration time]). In our experiments, we treat discrimination indices that exceed 0 in a given condition as evidence for memory of the previously presented object, as is common with the spontaneous object recognition procedure (for a detailed consideration of alternative factors that may contribute to this measure, see the study by ). The process of retrieval-induced forgetting is evidenced by lower discrimination index scores (i.e., worse memory) of the competitor object in the RP condition compared with the IC and TC conditions in which there is no retrieval practice.
##### Criteria of exclusion.
Animals that explored the objects for <10 s during any of the phases were excluded from the experiments. However, no rats had to be excluded from the study based on this criterion.
### Specific design features of individual experiments
#### Surgery and drug infusions
Rats were deeply anesthetized with ketamine (60 mg/kg) and xylazine (8 mg/kg) and put in a stereotaxic frame (Stoelting). The skull was exposed and adjusted to place bregma and λ on the same horizontal plane. After small holes were drilled, a set of 22 g guide cannulae were implanted bilaterally into the mPFC [anteroposterior (AP), +3.20 mm; left lateral (LL), ±0.75 mm; dorsoventral (DV), −3.50 mm] and/or the VTA (AP, −7.20 mm; LL, ±0.75 mm; DV, −5.30 mm; ). Cannulae were fixed to the skull with dental acrylic. A dummy cannula was inserted to each cannula to prevent clogging. At the end of surgery, animals were injected with a single dose of meloxicam (0.2 mg/kg) as an analgesic and gentamicin (0.6 mg/kg) as antibiotic.
Behavioral procedures commenced 5–7 d after surgery. On the experimental day, the dummy cannulae were removed before the injection and an injection cannula extending 1 mm below the guide cannula was inserted. The injection cannula was connected to a 10 µl Hamilton syringe. Cannulated rats received bilateral 0.5 µl infusions of the corresponding drug/vehicle. Muscimol (Mus; 0.1 mg/ml in saline; catalog #2763–96-4, Sigma-Aldrich) infusions into the VTA occurred 15 min before the retrieval practice phase. Injections were also made before exposure to the interpolated objects (equivalent to the “practice phase”) in the IC condition or before returning rats to their home cages for the TC condition.
SCH 23389 (SCH; 3 mg/ml in saline, 0.5 µl/side; catalog #0925/10, Tocris Bioscience) and SKF 38393 (SKF; 8.41 mg/ml in saline, 0.5 µl/side; catalog #0922/100, Tocris Bioscience) occurred 10 min before the retrieval practice phase (or at the corresponding points in TC conditions). We conducted the final test 24 h later. Doses were chosen based on previous studies ( ) and solubility data.
#### Cannulae placement
To check cannulae placement, 24 h after the end of the behavioral experiments, animals were infused with 1 µl of methylene blue through the dummy cannulae, and 15 min later were deeply anesthetized and killed. Histologic localization of the infusion sites was established using magnifying glasses. Five animals were excluded because of cannulae misplacement.
To control for VTA coordinates of infusions, three stereotaxically cannulated rats were infused with Green Beads (1:1000 dilution of concentrated 1-μm-diameter fluorescent beads; Bangs Laboratories). Seven days after the infusion, animals were deeply anesthetized with ketamine/xylazine and transcardially perfused with 10 ml of 0.04% heparin cold saline followed by 20 ml of 4% paraformaldehyde in 0.1 PBS. Brains were removed and immersed overnight in the same fixative. Then brains were stored in a 0.1 PBS 30% sucrose solution at 4°C until processed.
#### Immunohistochemistry assay
Thirty-five-micrometer-thick coronal brain sections were cut in a cryostat (Leica). Sections containing the VTA region were preserved in 0.1 PBS. Dopamine neurons were confirmed by immunohistochemical detection of tyrosine hydroxylase (TH). Briefly, sections were blocked for 2 h at room temperature and then incubated with mouse anti-TH antibody (1:1000; catalog #MAB318, Sigma-Aldrich) overnight at 4°C, washed three times, and incubated with a conjugated Cy3 goat anti-mouse secondary antibody (1:500; catalog #115–165-146, Jackson ImmunoResearch) for 2 h at room temperature. Finally, the slides were incubated with DAPI and mounted.
### Experimental design and statistical analysis
Statistical analyses were performed using GraphPad version 6.01. In the experiments in which we used drug infusions, the “drug” variable was analyzed within a subject and the “condition” variable (i.e., RP, IC, and TC), between subjects. Each subject was tested in one condition, with vehicle and with drug, in a pseudorandomized way. This type of analysis corresponds to the experiments from C , C , and B , F , and G . For the experiment in B , each subject was assigned a single condition (one practice or two practice sessions were treated as distinct conditions). For the experiments that did not involve drug infusions, the “condition” variable was analyzed within subject; each subject experienced all three conditions in a pseudorandomized way, which this corresponds to experiments in , D and E . For all retrieval-induced forgetting experiments, individual object exploration times during test phase were analyzed using a paired t test (see tables). Discrimination indexes calculated from the test phase object exploration times were analyzed using a two-way repeated-measures ANOVA followed by Bonferroni's post hoc comparisons in the experiments with drugs or vehicle and using a repeated-measures one-way ANOVA followed by Bonferroni's post hoc comparisons in the experiments without drug or vehicle infusion (in Results). Asterisks shown in graphs represent p -values for the post hoc analysis (** p < 0.01, *** p < 0.001, and **** p < 0.0001). In all cases, p -values were considered to be statistically significant at p < 0.05. Discrimination indexes calculated from the retrieval practice phase sessions object exploration times were analyzed using one-tailed unpaired t test (see tables; total exploration times are compared in Results). Absolute exploration times between vehicle-infused and drug-infused animals for each retrieval practice session (e.g., RP group: drug A + X mean vs vehicle A + X mean; IC group: drug X1 + X2 mean vs vehicle X1 + X2 mean) were compared using an unpaired Student's t test (see tables).
Exploration times and discrimination indexes during the practice phase in the retrieval practice condition for experiment depicted in A
Exploration times during the final test phase for experiment depicted in A
For the set-shifting experiment, the Acquisition criterion and Trials criterion were analyzed using a one-way ANOVA; Tukey's post hoc comparisons are indicated by asterisks. Comparisons between response and visual conditions were made considering the response group as two distinct groups (two groups of n = 5 each), segregating the animals that later performed the visual cue training with vehicle or with SCH (in Results) and comparing, respectively. Total Perseverative Errors, Perseverative Errors, Regressive Errors, and Never Reinforced Errors were compared for the visual cue training between the vehicle and SCH treatments using an unpaired t test (see tables). For data details, see the tables.
### Set-shifting task
#### Apparatus
The cross-maze was a four-arm maze made of 1-cm-thick black Plexiglas ( F ). The maze was placed on the floor. Each arm was 52 cm long and 9 cm wide; the height of the arm wall was 40 cm. Each arm contained a food well (diameter, 3 cm; height, 2.5 cm) that was 3.2 cm from the end wall.
#### Habituation procedure
The habituation procedure was similar to that described in the study by . Rats were allowed 7–10 d to recover from surgery before the habituation procedure commenced. Rats were food restricted to 85% of their original ad libitum weight. During food restriction, rats were handled for 10 min/d. On the first day of habituation, three pieces of Fruit Loops cereal (Kelloggs) were placed in each arm, with two pieces in the food well. A rat was placed in the maze and allowed to freely navigate and consume cereal pieces for 15 min. If a rat consumed all 12 cereal pieces before 15 min, then the rat was placed in a holding cage, the maze was rebaited, and the rat was placed back in the maze; this process was repeated a total of three times (if a rat did not consume all 12 cereal pieces before 15 min, then the habituation day 1 was repeated the next day until the rat reached criterion). On the second habituation day, the procedure was similar except that after a rat consumed two cereal pieces per arm, the rat was picked up and placed in a different arm. This acclimated the rat to being handled in the maze after consuming cereal. On subsequent habituation sessions, the procedure was the same as that on day 2, except that there were only two half-pieces of cereal put in each food well. Each time a rat consumed all the cereal pieces after being placed in the maze was considered one trial. This procedure continued until a rat consumed cereal from all food wells for four trials or more in a 15 min session. On the last day of habituation, the turn bias for a rat was determined. The maze was arranged such that a white Plexiglas block (9 × 40 × 1 cm) was placed at the center entrance of one of the arms so that it prevented entry into that arm, giving the maze a T shape. A rat was started from the stem arm and allowed to turn left or right to obtain a half-piece of cereal. In one of the choice arms, a white-blue piece of posterboard (8 × 48 × 0.3 cm) was placed on the floor ( F ). After a rat made a turn and consumed a cereal piece, the rat was picked up, placed in the stem arm, and allowed to make a choice. If the rat chose the same arm as in the initial choice, it was returned to the stem arm until it chose the other arm and consumed the cereal piece. After choosing both arms, the rat was returned to the holding cage, the block and visual cue were moved to different arms, and a new trial was begun. Thus, a trial for the turn-bias procedure consisted of entering both choice arms and consuming both cereal pieces. This procedure continued for seven trials. The turn that a rat made first during the initial choice of a trial was recorded and counted toward its turn bias. Whatever direction (right or left) a rat turned, four or more times during these seven trials was considered its turn bias. During response discrimination testing, a rat was required to turn in the opposite direction of its turn bias. Behavioral testing was started the next day.
#### Response–visual cue testing procedure
The testing procedure was similar to that described in the study by except that all testing was carried across two consecutive sessions. For each discrimination, three start arms were used. In this experiment, each rat started on the response version. A rat started from the arms designated west (W), south (S), and east (E), leaving the north (N) arm unused as a starting arm. The visual cue was placed pseudorandomly in one of the choice arms such that for every consecutive set of 12 trials it occurred an equal number of times in each choice arm. During the acquisition session, a rat had to turn in the opposite direction of its turn bias to receive a half-piece of Froot Loops cereal. F (top) illustrates an example of the correct navigation patterns for a rat that was required to always make a turn to the right. Between trials, a rat was placed back in the holding cage, which sat on a shelf next to the maze. The intertrial interval was <20 s. To minimize the use of intramaze cues from the apparatus, every six trials the maze was turned 90° clockwise relative to the experimenter. A rat reached criterion when it made 10 correct choices consecutively. There was no limit for the number of trials prearranged for a rat to reach this criterion. Once a rat made 10 correct choices consecutively, a probe trial was given. The probe trial consisted of starting the rat from the fourth arm (N) that was not used during testing. If a rat correctly turned the same direction as on testing, then the response procedure was completed. If a rat made an incorrect turn, then response testing was continued until a rat made an additional five correct choices consecutively, at which time another probe trial was administered. This procedure was continued until a rat made a correct choice on the probe trial. The following measures were taken for each rat: (1) acquisition criterion, defined as the total number of test trials to complete 10 consecutive correct choices in a session; (2) trials to criterion, defined as the total number of test trials completed before a correct choice on the probe trial was made; and (3) probe trials, defined as the total number of probe trials to get one correct. The day after reaching criterion on the response version, rats were switched to the visual cue version. Each rat was injected with SCH or Veh into the mPFC 15 min before the beginning of the visual cue learning session. In the visual cue version, a similar procedure was used as in the response version. However, in this test the rat always had to enter the arm with the visual cue. The visual cue was pseudorandomly varied in the left and right arms such that it occurred in each arm an equal number of times for every consecutive set of 12 trials. F (bottom) shows an example of a rat that learned to always enter the visual cue arm. A rat reached criterion when it made 12 correct choices consecutively. There was no limit on the number of trials a rat was allotted to reach this criterion. Once a rat made 12 correct choices consecutively, a probe trial was given. If a rat correctly turned following the visual cue, then the response procedure was completed. If a rat made an incorrect turn (error), then visual testing was continued until a rat made an additional six correct choices consecutively, at which time another probe trial was administered..
Additional parameters were analyzed on the switch to determine whether treatments altered perseveration. Perseveration involved continuing to make the same egocentric response as required on the response version, when the trial required turning the opposite direction to enter the visual cue arm. For every consecutive 12 trials in a session, half the trials consisted of these trials. These trials were separated into consecutive blocks of four trials each ( ). Perseveration was defined as entering the incorrect arm in three or more trials per block. This is a similar criterion as used in previous experiments measuring perseveration ( ; ). Once a rat made less than three errors in a block the first time, all subsequent errors were no longer counted as perseverative errors.
## Results
To test whether control processes regulated by dopamine in the mPFC participate in adaptive forgetting, we studied how exploratory behavior in a rodent object recognition task was affected by manipulation of the dopaminergic system.
The D R is one of the main dopamine receptors in the mPFC ( ; ). Thus, in experiment 1 we studied the role of mPFC D Rs in retrieval-induced forgetting. Each rat, assigned to the RP, IC, or TC condition, was tested twice: once with saline and once with the D R antagonist SCH. We injected SCH into the mPFC bilaterally ( B ) 10 min before the first retrieval practice session, and at the same time point in the IC and TC conditions. Thus, in drug studies, treatment (drug or saline) was done within subject, but the condition (RP, IC, or TC) was compared between subjects.
D receptors in medial prefrontal cortex mediate retrieval-induced forgetting. A , Schematic representation of the behavioral protocol. After the acquisition, animals were divided into the three different conditions, RP, IC, and TC. The syringe indicates the infusion of the drug or its vehicle 10 min before the practice phase. Animals were assigned to one condition and subjected to a different pharmacological treatment each week. B , Histology. Left, Diagram of the coronal section of the rat brain, showing the placement of the markings produced by methylene blue infusion for all the rats that received infusions of dopaminergic (or vehicle) drugs in the mPFC. The sections of the brain correspond to the atlas by . Right, Safranin staining showing an example of the cannula track left. C , Discrimination indexes for the three sessions of the practice phase for the RP and IC groups in drug conditions and their vehicle ( ). D , Discrimination indexes ± SEM for the testing phase. Animals performed the task twice, once with the drug and once with the vehicle in a pseudorandomized way and for the same condition ( ), two-way ANOVA, n = 8–11, Bonferroni's post hoc comparisons are shown indicated by asterisks. E , Exploration times ± SEM for each individual object in the test phase ( ) compared by a paired t test, shown with asterisks. F , Training schemes for the set-shifting task, with the Response Cue (left, egocentric) and the Visual Cue (right, visual). The arrows indicate the correct turn expected for each example trial. G , Trials to criterion ± SEM are the number of trials conducted to complete a Criterion test correctly. Ordinary one-way ANOVA, n = 5; Tukey's post hoc comparisons are shown indicated by asterisks. H , Perseverative errors ± SEM, each trial in which the animal responded according to the self-centered key. Perseverative errors were defined as entering the wrong arm in three or more trials per block. Unpaired t test comparisons are shown by asterisks. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.
Infusing animals with saline or SCH did not alter their total exploration times during the retrieval practice phase (total exploration times: RP Veh, 51.12 s ±5.74; RP SCH, 59.21 s ±6.02; n = 9; paired t test: p = 0.24, t = 1.27, df = 8). For the RP group, both the saline and SCH treatments rats preferred the novel objects during practice trials, indicating that retrieval of the practiced object was not affected by SCH infusion ( C , ). Although SCH injection could have affected retrieval practice performance, we observed no evidence of this in any of the conditions.
On the final test, we scored the time rats spent exploring the old object versus the novel object ( E ). Our dependent variable was a discrimination index that reflects the bias in the time they spent exploring the novel item instead of the old one ( D ). If the discrimination index at test was significantly lower for the RP condition compared with the IC and TC conditions, we considered there was significant retrieval-induced forgetting. We found that saline-infused rats explored the competitor Object B as if it was new, as shown by the lower discrimination index in the RP condition compared with the IC and TC groups ( D , ). Critically, however, rats infused with SCH showed high discrimination indexes (two-way ANOVA; Interaction: p = 0.0013, F = 6.65; Drug: p = 0.0008, F = 13.77; Condition: p < 0.0001, F = 10.05; Subjects: p = 0.356, F = 1.14). Bonferroni's corrected comparisons confirmed that the discrimination indexes of rats for competitors were lower when infused with saline than with SCH, which is consistent with the possibility that SCH had prevented competitors from being forgotten. Indeed, infusing SCH abolished evidence for retrieval-induced forgetting completely ( D ). The discrimination index in the RP group was indistinguishable from that of the IC or TC groups. It is worth mentioning that the pharmacological manipulations were made right before the retrieval practice phase, after encoding had concluded. Thus, all groups encoded the objects in the absence of any drug. Since memory was evaluated 24 h after the retrieval practice phase, it is very unlikely that the drug itself affected retrieval during the test phase. So, the animals from the three groups (RP, TC, and IC) should have attempted to retrieve Object B in the same motivational, attentional, and perceptual state. Any changes in memory at test had to be the product of what happened during the practice phase. This phase is very similar in the RP and IC conditions. In both cases, the animals are exposed to contextually novel objects. However, retrieval-induced forgetting is only observed in the RP condition. In addition, we did not observe any differences between saline-infused and drug-infused animals in exploration during the retrieval practice phase. This indicates that perception, motivation, attention, or reactivity to novelty were not altered by the drugs.
To verify that the dose of SCH we used for our experiments was sufficient to impair cognitive control in a nonmemory task, a different group of rats infused with SCH or Veh was evaluated in a set-shifting task that requires the organism to exert inhibitory control over the tendency to engage in a previously relevant behavioral strategy ( ; ; ). Blockade of D R in mPFC has been shown to impair performance in the set-shifting task ( ; ).
SCH-injected rats produced significantly more errors than Veh-injected animals in the probe trials and required significantly more trials to reach criterion ( G , , Acquisition Criterion; one-way ANOVA; treatment: p < 0.0001, F = 57.09; Response vs Visual Veh, p < 0.0001; Response vs Visual SCH, p < 0.0001; Visual Veh vs SCH, p < 0.0001). In addition, animals infused with SCH increased the number of trials to achieve the criterion relative to vehicle-infused animals ( G , , trials to criterion; one-way ANOVA; treatment: p < 0.0001, F = 95.25; Response vs Visual Veh, p < 0.0001; Response vs Visual SCH, p < 0.0001; Visual Veh vs SCH, p < 0.0001) and made a greater number of perseverative errors ( H , ; unpaired t test; perseverative errors: p = 0.0173, t = 2.990, df = 8; total perseverative errors: p < 0.0001, t = 9.856, df = 8). Thus, the blockade of D R receptors in the mPFC impaired shifting from an egocentric strategy to a visual strategy. This treatment equally affected cognitive control and retrieval-induced forgetting.
Set-shift parameters for the experiment depicted in F
The main prefrontal dopamine source is the VTA, which projects directly to the mPFC ( ). We designed experiment 2 to establish whether dopamine release from VTA terminals into mPFC was required for retrieval-induced forgetting. We injected bilaterally Mus or Veh directly into VTA 15 min before the first retrieval practice session ( A ). Unlike permanent lesions, this treatment causes a transient silencing of the structure ( ), allowing the final object recognition test to occur in the absence of the drug.
VTA projections to mPFC are necessary for retrieval-induced forgetting. A , Diagram of the coronal section of rat brain, showing the site of infusion of fluorescent green beads for all rats injected with muscimol (or vehicle) in the VTA. The sections of the brain correspond to the atlas by . The orange immunofluorescence corresponds to TH detection; the green color corresponds to green beeds infused through the implanted cannulae. Scale bar, 200 μm. B , Discrimination indexes ± SEM for the three sessions of the practice phase for the RP and IC groups under both conditions. C , Discrimination indexes ± SEM for the test phase after Mus or Veh injection into the VTA. Two-way ANOVA with Bonferroni's corrected post hoc analysis. There was a significant drug × condition interaction. Muscimol impaired the forgetting of the competitor object. D , Discrimination indexes ± SEM for the test phase of the “restoration of forgetting” experiment by infusion of SKF 38393 in mPFC. The animals performed the task twice, once with the drug and once with the vehicle in a pseudorandomized way for the condition to which they were pseudorandomly assigned after the training phase. All animals were infused with muscimol in the VTA. Two-way ANOVA followed by a Bonferroni's post hoc analysis indicated a significant drug × condition interaction. ** p < 0.01, *** p < 0.001, and **** p < 0.0001.
Mus infusion in VTA did not affect total object exploration during the retrieval practice phase (total exploration times: RP Veh: 92.99 ± 9.35 s, n = 10; RP SCH: 85.97 ± 8.63 s, n = 12; unpaired t test: p = 0.58, t = 0.55, df = 20; ). Critically, during the test phase, in the Veh-injected animals the discrimination index was significantly lower for the RP condition compared with the IC and TC groups, whereas we did not observe any difference between the RP and the control groups in Mus-infused animals ( C ; two-way ANOVA: Interaction: p < 0.0001, F = 16.29; Drug: p = 0.0002, F = 16.49; Condition: p < 0.0001, F = 11.95; Bonferroni's post hoc multiple comparisons) and exploration times ( ). Given that Mus had no effect in the IC or TC conditions ( C , ), this suggests that silencing the VTA did not modify recognition memory, but rather that VTA activity during the retrieval practice phase specifically affected object recognition at testing in the RP condition. These findings are consistent with our hypothesis that dopamine release into mPFC during selective retrieval practice was important for successful control processes that inhibited competing memories and produced retrieval-induced forgetting.
Exploration times and discrimination indexes during the practice phase in the retrieval practice condition for experiment depicted in , muscimol into the VTA
Exploration times during the final test phase for experiment depicted in , muscimol into the VTA
In experiment 3, we sought to elucidate whether VTA projections to the mPFC were important to modulate activity in this structure and cause retrieval-induced forgetting; we thus combined Mus injections into the VTA with injection of the D R agonist SKF into the mPFC in a new set of animals. Mus was injected bilaterally into the VTA in all animals 15 min before retrieval practice (or the equivalent phase in the IC and TC conditions). Injection of SKF or Veh into the mPFC was performed 10 min before retrieval practice (or the equivalent phase in the IC and TC conditions). SKF infusion did not produce any changes in exploration or recognition of the familiar object during the practice phase (total exploration times: RP Veh, 52.17 ± 8.50 s; RP SKF, 48.89 ± 4.14 s; unpaired t test: p = 0.73, t = 0.35, df = 12; ). Critically, SKF administration into mPFC caused significant reductions in the discrimination index in the RP group on the final test, compared with Veh-infused animals. Thus, SKF completely reversed the effect of silencing VTA with Mus ( D ; two-way ANOVA; Interaction: p = 0.01, F = 4.59; Drug: p = 0.002, F = 10.84; Condition: p = 0.01, F = 4.72; Bonferroni's post hoc multiple-comparisons test) and exploration times at test ( ). No differences in discrimination indexes were found between Veh-infused and SKF-infused animals in the IC and TC groups ( D , ). To control that any fluid injection into the VTA could modify the mPFC response to SKF injection, we compared an RP group and an IC group when injecting Veh into the VTA and Veh or SKF into the mPFC. Again, the within-subject variable was the drug (Veh or SKF) and the between-subject variable was the condition (RP or IC). Both RP groups had lower discrimination indexes than the IC groups during the test phase, independent of the injected drug into the mPFC (RP Veh: 0.04 ± 0.01, N = 5; RP SKF: 0.07 ± 0.02, N = 5; IC Veh: 0.35 ± 0.05, N = 4; IC SKF: 0.52 ± 0.08, N = 4). There was a significant effect of the condition ( p < 0.0001, F = 136.9, repeated-measures two-way ANOVA), but no interaction ( F = 0.003, p = 0.95). Thus, in the absence of activity within the VTA, the activation of mPFC D Rs was sufficient to lower the discrimination index selectively in the RP condition, suggesting that the activation of mPFC D Rs via dopamine release from VTA is one of the main mechanisms required for retrieval-induced forgetting in rats.
Exploration times and discrimination indexes during the practice phase in the retrieval practice condition for experiment depicted in , muscimol into the VTA and SKF 38393 into the mPFC
Exploration times during the final test phase for experiment depicted in , muscimol into the VTA and SKF 38393 into the mPFC
In humans, higher prefrontal dopamine availability has been associated with greater retrieval-induced forgetting ( ). To evaluate whether the activation of D Rs improved retrieval-induced forgetting, we injected the D R agonist SKF into mPFC in a new group of animals before a modified retrieval practice phase consisting of only one practice trial ( A ). We reasoned that whereas only one practice trial would likely be insufficient to produce retrieval-induced forgetting on its own, it might do so given the activation of D Rs in mPFC, which could magnify the impact of inhibitory processes.
Bidirectional modulation of retrieval-induced forgetting. A , Schematic representation of the behavioral protocol. After the acquisition, the animals were divided into three conditions: RP, IC, and TC. Both RP and IC were subdivided into two groups: (1) a group that performed a practice phase with only one retrieval practice session and (2) another group that did two retrieval practice sessions. Only the RP group is schematized; the IC group performed the equivalent to the practice phase with two copies of identical objects (XX or XX and then YY). The syringe indicates the infusion of SKF or its vehicle (saline) 10 min before the practice phase. B , Discrimination rates for the test phase. The animals performed the task twice, once with the SKF and once with saline in a pseudorandomized way and for the same condition. Two-way ANOVA followed by a Bonferroni's post hoc test. C , Schematic representation of the behavioral protocol. The protocol consisted of an acquisition phase with double training for each object (strong acquisition). D–G , After the acquisition, the animals were divided in three conditions, RP, IC, and TC; the top panels ( D , E ) correspond to two groups of animals that performed the protocol without infusion of any drug, and the bottom panels ( F , G ) correspond to the other two groups of animals that were cannulated and infused with the D R agonist and antagonist. The syringe indicates the infusion of the drug or its vehicle 10 min before the practice phase (extended practice, one-way ANOVA for D and E , and two-way ANOVA for F and G ). * p < 0.05; ** p < 0.01; *** p < 0.001; and **** p < 0.0001.
A single retrieval practice did not yield significant memory impairment during the later test phase either in the Veh-infused or SKF-infused animals ( A , , , ; two-way ANOVA; Interaction: p < 0.90, F = 0.25; Drug: p = 0.71, F = 0.13; Condition: p < 0.0001, F = 10.06; Bonferroni's post hoc multiple-comparisons test). The impact of inhibition arising from one practice session may have not been strong enough to produce retrieval-induced forgetting. In prior work, we had already observed that exposure to two retrieval practice sessions during the practice phase produced retrieval-induced forgetting that was measurable in a test session 30 min after the practice phase comparing the RP and IC conditions ( ). However, in the present study, the final test took place 24 h after the practice phase. Thus, we tested our hypothesis again, but with a protocol in which the animals were exposed to two practice sessions, as in our prior work ( ), and injected with Veh or SKF ( A ). In this case, we found no differences between Veh-infused or SKF-infused animals in discrimination indexes on the final test, consistent with both groups showing similar and significant levels of retrieval-induced forgetting ( B , ). Decreasing the number of trials proved not to be a sensitive strategy to evaluate positive modulation of retrieval-induced forgetting.
Exploration times and discrimination indexes during the practice phase in the retrieval practice condition for experiment depicted in A , single practice session
Exploration times and discrimination indexes during the final test phase for experiment depicted in A , single practice session
Exploration times and discrimination indexes during the practice phase in the retrieval practice condition for experiment depicted in A , double practice sessions
Exploration times during the final test phase for experiment depicted in A , double practice sessions
We found an alternative approach to potentially observe a positive modulation of retrieval-induced forgetting. We introduced a longer delay in between the encoding phase, the practice phase, and the final test, a manipulation that significantly reduced the size of retrieval-induced forgetting. We extended the delay between the encoding and final test phase to 48 h, and the delay between the encoding and the retrieval practice phases to 24 h ( C , scheme) with the aim of weakening the overall effect so that positive modulation could be observed. To ensure that memory performance was adequate to measure retrieval-induced forgetting after 48 h, we modified our encoding protocol to create stronger memories. Preliminary work indicated that control animals required two separate exposures to each pair of objects during encoding to remember these objects 48 h later. Thus, we slightly modified the protocol for the particular mnemonic demands of longer-lasting object memories.
Using this protocol, the discrimination index in the RP group injected with Veh was not significantly different from that of the IC or TC groups after three practice sessions ( D , ; one-way ANOVA; Condition: p = 0.212, F = 4.055; Animals: p = 0.36, F = 1.12; multiple comparisons). Critically, however, injection of the D R agonist SKF into mPFC 10 min before the beginning of the retrieval practice session produced a robust reduction in the discrimination index in the RP condition compared with the control groups ( F , , ; two-way ANOVA; Interaction: p < 0.0001, F = 25.50; Drug: p = 0.0016, F = 12.85; Condition: p = 0.013, F = 3.41; Bonferroni's post hoc multiple-comparisons test). These findings are consistent with the possibility that SKF amplified the capacity of the mPFC to hinder competing memories, enabling retrieval-induced forgetting even after 48 h.
Exploration times and discrimination indexes during final test for experiment depicted in C , normal practice phase
Retrieval practice exploration times and discrimination indexes for experiment depicted in F , normal practice phase with SKF 38393 infusion into the mPFC
Exploration times during the final test phase for experiment depicted in C , normal practice phase with SKF 38393 infusion into the mPFC
To confirm that retrieval-induced forgetting could also occur in this longer protocol, we added two extra retrieval practice sessions to the practice phase ( C , scheme, extended practice). In Veh-infused rats, five retrieval practice trials induced significant reductions in the discrimination index during the final test for the RP condition, even at the 48 h postencoding delay compared with matched IC and TC control groups ( E , ; one-way ANOVA; Condition: p = 0.0002, F = 17.47; Animals: p = 0.092, F = 2.25; multiple comparisons). Injection of the D R antagonist SCH into mPFC 10 min before the first of the five practice trials completely prevented this reduction in the discrimination index on the final test of RP items, as performance was indistinguishable from that of the IC and TC groups ( G , , ; two-way ANOVA; Interaction: p < 0.038, F = 3.18; Drug: p = 0.0009, F = 13.48; Condition: p < 0.0001, F = 11.51; Bonferroni's post hoc multiple-comparisons test). Together, these findings are consistent with a bidirectional modulation of retrieval-induced forgetting by manipulation of dopaminergic signaling through D Rs in the mPFC.
Exploration times and discrimination indexes during final test for experiment depicted in C , extended practice phase
Retrieval practice exploration times for experiment depicted in G , extended practice phase with SCH 23389 infusion into the mPFC
Exploration times during the final test phase for experiment depicted in C , extended practice phase with SCH 23389 infusion into the mPFC
## Discussion
Memory enables organisms to draw on past experiences to improve their choices and actions. Because of their relational nature and richness, episodic memories are flexible in the way that past events can be retrieved as needed to guide future behavior ( ). Experience modifies behavior by restructuring access to memories or directly modifying the memory traces themselves ( ; ; ). Dopamine plays important functions in the ability to change a learned rule and to select appropriate behaviors ( ) by biasing action selection and even by modifying neural plasticity in regions of memory storage ( ; ). In this work, we expand the functions of dopamine to include a mechanism of adaptive forgetting of competing memories. Although the role of dopamine has been studied mainly in the motivation of goal-directed behaviors, here we argue that dopamine-dependent mechanisms are related to adaptive forgetting even in the absence of explicit reward or instructions. We propose that retrieval-induced forgetting of competing object memories is enabled by mechanisms similar to those engaged during rule switching and selection in the mPFC of rodents. This dopaminergic modulation of control processes enables access to memory content in the face of retrieval competition, supporting the behavioral demands of organisms.
Remarkably, retrieval-induced forgetting in rats resembles the corresponding process in humans ( ). The mPFC in rats is essential to forget competing object memories, paralleling results observed for the lateral prefrontal cortex in humans. These results point to the key role of inhibitory control in retrieval-induced forgetting. We provide strong causal evidence favoring a dopamine-dependent mechanism of inhibitory control for retrieval-induced forgetting. Blockade of D Rs in the mPFC of rats during the practice phase abolished retrieval-induced forgetting of a competing object memory. This manipulation did not have any effect when it preceded the encoding of different interfering materials (interference control) or when it preceded rest in the home cage of rats, indicating that it affected processes specifically associated with retrieval practice and not nonspecific factors such as novelty salience or mood, which would have affected performance in all three conditions. The function of D Rs in the prefrontal cortex has been extensively investigated. D R blockade in nonhuman primates disrupts task performance and spatial working memory activity in the dorsolateral prefrontal cortex ( , ; ). Importantly, D R blockade also disrupts prefrontal cognitive rule-related selectivity ( ). In this work, we found that the same dose and place of infusion of the D R antagonist that prevented retrieval-induced forgetting also impaired performance in a set-shifting task in which rats are required to inhibit a prepotent response associated with a learned rule. The parallel impact of a D R antagonist on the need to inhibit prepotent actions and memories is consistent with human studies indicating that retrieval-induced forgetting is triggered by inhibitory control processes shared with action stopping ( ; ; ). It also provides new evidence in favor of a general function of dopamine in cognitive processes related to flexible and adaptive behavior.
We provided causal evidence that the critical source of dopamine for retrieval-induced forgetting in the mPFC is the VTA, because silencing this structure impaired retrieval-induced forgetting. This effect was reversed by concomitant activation of D Rs in mPFC during the practice phase, indicating that, in the absence of dopamine release from VTA, the activation of D Rs in the mPFC is sufficient for retrieval-induced forgetting. Critically, dopaminergic modulation of retrieval-induced forgetting is bidirectional. Activation of D Rs in the mPFC just before the retrieval practice phase caused retrieval-induced forgetting in a protocol that does not reliably induce it without D R activation. No anxiety, movement, or perception changes were observed after any of the infusions, as rats did not significantly modify their exploratory behavior after the infusion of any of the drugs.
There is a strong link between dopamine availability in the brain and cognitive abilities. Many studies point at a function of dopamine in adaptive behavior in humans. For example, the administration of -DOPA to Parkinson's disease patients improved the ability to alter behavior according to changes in the dimensional relevance of stimuli in a task that resembles the set-shifting paradigm used in our study ( ). Impairments in this form of higher-level attentional control have also been associated with lesions of the monkey lateral PFC ( ) and significant activation of the dorsolateral prefrontal cortex in humans ( ; ). In addition, the enzyme COMT, which degrades catecholamines, appears to play a pivotal role in the modulation of frontostriatal networks. The COMT gene presents an evolutionarily recent functional single nucleotide polymorphism (Val158Met). The Met allele produces an enzyme that has only a quarter the activity of the Val-containing polypeptide ( ). Several studies found that the low-activity Met allele allows for better performance on cognitive tasks that have a working memory component and the high-activity Val allele was associated with poorer performance on the Wisconsin Card Sorting Test, a putative measure of “executive” function (for review, see ). Interestingly, in humans, retrieval-induced forgetting increased linearly with Met allele load, suggesting a positive relationship between cortical dopamine availability and inhibitory control over memory ( ). Mirroring the linear effect of genotype on behavior, functional imaging data revealed that the beneficial effects of memory suppression, as assessed by a decrease in prefrontal activity across retrieval practice blocks, a sign of efficient suppression of competing memories ( ; ; ), also increased with Met allele load. In agreement with these results, the present study supports a general contribution of dopamine in the mPFC in the control of memory and, in particular, establishes causality between dopamine availability and retrieval-induced forgetting. Greater dopamine availability may lead to greater activation of D Rs, improving the suppression of competing memories.
What are the mechanisms by which dopamine participates in retrieval-induced forgetting? Activation of D Rs in mPFC could initiate active circuit-level inhibition over competing memory traces in the medial temporal lobe. Given that top-down connections from the mPFC to the medial temporal lobe are mainly excitatory ( ; ) projections from the mPFC would not directly enact inhibition over the competing memory trace. A possible mechanism could involve excitatory projections from the prefrontal cortex that directly excite local inhibitory neurons in the medial temporal lobe, which then inhibit a distracting stimulus, or unwanted representation or process ( ), but this remains highly speculative. Since there are no direct projections from the mPFC to the hippocampus, the activation of the mPFC could induce inhibition of the competing traces in the hippocampus via nucleus reuniens (RE; ). also developed this mPFC-RE-Hippocampus model in the context of memory inhibition during extinction.
Regardless of the circuit involved in retrieval-induced forgetting, we made the surprising discovery that dopaminergic modulation of retrieval-induced forgetting seems to be independent of any mechanisms of retrieval itself (i.e., D R blockade in mPFC does not affect retrieval during the practice phase but impairs retrieval-induced forgetting). This suggests that dopamine modulates retrieval-induced forgetting by specifically acting on the future availability of the competing memory trace (i.e., at the test phase), without affecting the retrieval processes during the practice phase. Thus, we argue that retrieval control and retrieval-induced forgetting mechanisms are intrinsically distinct. During retrieval practice, activity in the mPFC would be required for inhibition of the competing memory, but not for the mechanism of retrieval itself. Lesions to the mPFC in rats do not normally impair object recognition when the task relies on the identity of the object ( ). However, what we found is that even if the mPFC is not implicated in object memory retrieval, it does not mean that the structure does not participate in memory retrieval at all. In particular, D Rs would be essential for high-level function of the mPFC. As would be expected given the plethora of diffuse ascending inputs from the major monoaminergic and cholinergic neurotransmitter systems, the PFC needs to be highly sensitive to neurochemical state. In particular, in set-shifting tasks, the modulation of noradrenaline usually produces similar effects to the modulation of the dopaminergic system ( ; ). Thus, it is possible that noradrenaline is also involved in retrieval-induced forgetting. However, we found that the activation of D Rs after silencing the VTA restored retrieval-induced forgetting, indicating that dopaminergic release may be a key step for this process. It is clear that acetylcholine and serotonin can also modulate mPFC activity, but their manipulation does not seem to produce the same behavioral effects as that of dopamine or noradrenaline in tasks that involve attention and control processes, although there is some complex interaction between dopamine and serotonin to modulate PFC function ( ; ). So, the neurochemical processes involved in retrieval-induced forgetting require a thorough evaluation.
Two limitations of our study could be addressed in the future. First, for simplicity, we only analyzed the effect of a single dose of each ligand. Because dopamine exerts a complex modulation of cortical function ( ; ; ) in future studies, it will be helpful to analyze the effects of other doses to examine whether the modulation of retrieval-induced forgetting follows the same pattern as has been observed in other cognitive functions. Second, we only studied male subjects. Our current work uses both sexes, which will generalize the conclusions that we might obtain. The present study is just one of the first steps toward understanding the biological mechanisms underlying retrieval-induced forgetting.
In agreement with an adaptive and evolutionarily conserved role in memory and behavior, dopamine has been recently implicated in forgetting mechanisms in both invertebrates ( ) and vertebrates ( ; ). Modulation of a small subset of dopaminergic neurons in Drosophila regulates the rate of forgetting of aversive and rewarding experiences. In particular, forgetting appears to depend on signaling through a specific type of receptor in the mushroom bodies of the fly brain ( ). On the other hand, inhibition of D Rs in the VTA during training of a conditioned place preference task in rats, increases memory duration, while activation of these receptors produces forgetting of already consolidated memories ( ). In the absence of any type of retrieval practice, blockade of mPFC D Rs did not produce forgetting of the conditioned place preference memory. Although they did not evaluate the function of D Rs in retrieval-induced forgetting, it does contribute to an increasing accumulation of evidence for the involvement of the dopaminergic system in the different mechanisms of forgetting linked to adaptive behavior.
According to our results, dopamine acting on D Rs in the mPFC modulates control processes required for adaptive forgetting in the mammalian brain. Thus, across species, dopaminergic transmission may be essential to suppress competing memories by sculpting the mnemonic and behavioral repertoire of an organism according to their goals and the demands of the environment.
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Subjective inner experiences, such as mind-wandering, represent the fundaments of human cognition. Although the precise function of mind-wandering is still debated, it is increasingly acknowledged to have influence across cognition on processes such as future planning, creative thinking, and problem-solving and even on depressive rumination and other mental health disorders. Recently, there has been important progress in characterizing mind-wandering and identifying the associated neural networks. Two prominent features of mind-wandering are mental time travel and visuospatial imagery, which are often linked with the hippocampus. People with selective bilateral hippocampal damage cannot vividly recall events from their past, envision their future, or imagine fictitious scenes. This raises the question of whether the hippocampus plays a causal role in mind-wandering and, if so, in what way. Leveraging a unique opportunity to shadow people (all males) with bilateral hippocampal damage for several days, we examined, for the first time, what they thought about spontaneously, without direct task demands. We found that they engaged in as much mind-wandering as control participants. However, whereas controls thought about the past, present, and future, imagining vivid visual scenes, hippocampal damage resulted in thoughts primarily about the present comprising verbally mediated semantic knowledge. These findings expose the hippocampus as a key pillar in the neural architecture of mind-wandering and also reveal its impact beyond episodic memory, placing it at the heart of our mental life.
SIGNIFICANCE STATEMENT Humans tend to mind-wander ∼30–50% of their waking time. Two prominent features of this pervasive form of thought are mental time travel and visuospatial imagery, which are often associated with the hippocampus. To examine whether the hippocampus plays a causal role in mind-wandering, we examined the frequency and phenomenology of mind-wandering in patients with selective bilateral hippocampal damage. We found that they engaged in as much mind-wandering as controls. However, hippocampal damage changed the form and content of mind-wandering from flexible, episodic, and scene based to abstract, semanticized, and verbal. These findings expose the hippocampus as a key pillar in the neural architecture of mind-wandering and reveal its impact beyond episodic memory, placing it at the heart of our mental life.
## Introduction
Even when in the same place and involved in the same activity, at any given moment people can experience the world in different ways. Recently, there have been advances in delineating the various forms of spontaneous inner experiences and their neural correlates ( ; ). Self-generated thinking typically refers to the ability to mentally decouple from current perceptual surroundings and generate independent internal thoughts ( ). These thoughts can either be task related, such as actively thinking about how this manuscript should be structured, or task unrelated, where there is a spontaneous inner focus, such as suddenly remembering what a nice time I had yesterday with my friends ( ). These latter thoughts are the focus of the current study and have been variously described as task-unrelated, self-generated thoughts; daydreaming; or mind-wandering ( ).
It has been shown that humans tend to mind-wander ∼30–50% of waking time, regardless of the current activity ( ; ). Nevertheless, mind-wandering frequency is particularly pronounced during restful periods and low-demanding tasks ( ). The latter is often exploited by experimentalists examining mind-wandering. Although the precise function of mind-wandering is still debated, it is increasingly acknowledged to have influence across cognition on processes such as future planning, creative thinking, and problem-solving ( , ) and even on depressive rumination and other mental health disorders ( ; ). Furthermore, the content of mind-wandering seems wide-ranging, including episodic memory recall (which involves a sense of re-experiencing and is specific in time and place), future planning, mentalizing, and simulation of hypothetical scenarios, and involves a variety of emotions and different sensory modalities ( ; ). Interestingly, two of the most prominent features of mind-wandering are mentally traveling forward and backward in time and visual imagery, which are functions usually associated with the hippocampus ( , ; ).
The default mode network (DMN), within which the hippocampus is a node, has been associated with self-generated thoughts such as mind-wandering ( ; ). Of particular relevance here, stronger hippocampal connectivity with other regions of the DMN was observed in individuals who experienced more episodic details and greater flexibility in mental time travel during mind-wandering episodes ( ; ). Unfortunately, causal evidence for hippocampal involvement in mind-wandering is lacking ( ). Behavioral studies of patients with lesions are crucial because they permit examination of the causal effects of regional brain damage on the networks established by neuroimaging work. People with hippocampal damage cannot vividly recall events from their past ( ), envision their future ( ), or imagine fictitious scenes ( ). Therefore, whether they experience mind-wandering and if they do what form does it take are important and timely questions that we addressed by examining mind-wandering in patients with selective bilateral hippocampal damage.
Previous studies have examined the effects of hippocampal damage during demanding tasks, such as autobiographical memory retrieval ( ), designed to challenge the patients' cognitive abilities. In contrast, to establish what patients with hippocampal damage think about spontaneously when there is no concurrent task, our focus was on what they do in their mentally “free” time. We initially asked whether or not patients with hippocampal damage were able to mentally decouple from the current perceptual input. If yes, we then had a series of additional questions. First, would they engage in mental time travel? Second, what form would their mind-wandering take: spontaneous episodic, detailed thoughts or semantic, abstract thoughts? Finally, we asked whether they experienced spontaneous visual imagery similar to that typically reported by control participants during mind-wandering ( ).
## Materials and Methods
###
#### Participants
Six patients [all right-handed males; mean age, 57.0 years (SD, 16.9); age range, 27–70] with selective bilateral hippocampal lesions and selective episodic memory impairment took part in the study (see and for demographic information and neuropsychological profiles). Of note, these patients were the same high-functioning individuals that took part in our previous studies ( , ). Hippocampal damage (see example in a ) resulted in all cases from voltage-gated potassium channel complex antibody-mediated limbic encephalitis. Two of the patients had bilateral signal hyperintensities in the hippocampi on presentation, but hippocampal atrophy was observed in all patients. Testing took place a median of 7 years after onset of hippocampal damage. In line with previous reports of this patient population ( ; ), manual (blinded) segmentation of the hippocampi from high-resolution structural MRI scans confirmed that our patients showed volume loss confined to the left [hippocampal-damaged patients (HPC), 2506 mm (mean) ± 394 (SD); control participants (CTL), 3173 mm ± 339; W = 4.0, p = 0.002] and right (HPC, 2678 mm ± 528; CTL, 3286 mm ± 301; W = 8.0, p = 0.01) hippocampus. To rule out pathological differences between patients and controls elsewhere in the brain, an automated voxel-based morphometry (VBM; ) analysis was performed on whole-brain T1-weighted MRI images and, in line with previous reports on patients of this sort ( ; ; ), did not result in any significant group differences outside of the hippocampus even at a liberal uncorrected p value of <0.001.
Summary of demographic information
Summary of neuropsychological information
Neuropsychologically, the patients displayed an impairment in immediate and delayed recall on the Logical Memory (short stories) test ( ), and they recollected significantly fewer episodic (“internal”) but not semantic (“external”) details on the Autobiographical Interview ( ), as detailed in . All other cognitive and emotional aspects of cognition were intact in these patients. In summary, these patients seemed to have a selective difficulty in reconstructing internal events. Importantly, their working memory capacity did not differ from that of controls, suggesting that the differences in mind-wandering episodes we report here are unlikely to be attributable to an inability to remember the thoughts.
Twelve healthy control participants also took part (all male, one left-handed, mean age, 57.2 (16.6) years, age range from 25 to 77). In addition to comparing the two groups, we ensured that each patient was matched closely to two of the control subjects on sex, age, and general cognitive ability (measured by the Matrix Reasoning and Similarities subtests of the Wechsler Abbreviated Scale of Intelligence–WASI; ). There were no significant differences between patients and controls on age, general cognitive ability and on neuropsychological tests assessing semantic memory, language, perception, executive functions and mood [see which includes: the Autobiographical Interview ( ); Wechsler Memory Scale III ( ); Rey-Osterrieth complex figure ( ); Warrington Graded Naming Test ( ; ); Delis-Kaplan Executive Function System ( ); Hayling Sentence Completion Test ( ); Visual Object and Space Perception Battery ( ; ), Hospital Anxiety and Depression Scale ( )].
#### Characterization of hippocampal damage
##### High-resolution T2-weighted structural MRI scans of the medial temporal lobes.
Five of the patients and 10 of the control participants underwent structural MR imaging limited to a partial volume focused on the temporal lobes using a 3.0 T whole-body MR scanner (Magnetom TIM Trio; Siemens Healthcare) operated with a radiofrequency (RF) transmit body coil and 32-channel head RF receive coil. These structural images were collected using a single-slab, 3D T2-weighted turbo spin echo sequence with variable flip angles ( ) in combination with parallel imaging to simultaneously achieve a high-image resolution of ∼500 μm, high sampling efficiency, and short scan time while maintaining a sufficient signal-to-noise ratio (SNR). After excitation of a single axial slab, the image was read out with the following parameters: resolution, 0.52 × 0.52 × 0.5 mm; matrix, 384 × 328; partitions, 104; partition thickness, 0.5 mm; partition oversampling, 15.4%; field of view, 200 × 171 mm ; TE, 353 ms; repetition time (TR), 3200 ms; GRAPPA × 2 in phase-encoding (PE) direction; bandwidth, 434 Hz/pixel; echo spacing, 4.98 ms; turbo factor in PE direction, 177; echo train duration, 881; averages, 1.9. For reduction in signal bias caused by, for example, spatial variation in coil sensitivity profiles, the images were normalized using a prescan, and a weak intensity filter was applied as implemented by the scanner's manufacturer. It took 12 min to obtain a scan.
##### High-resolution T1-weighted structural MRI scans of the whole brain at 3.0 tesla.
In addition, five of the patients and 11 of the control participants underwent a whole-brain structural T1-weighted sequence at an isotropic resolution of 800 μm ( ), which was used for the automated VBM analysis (one control participant could not be scanned). These images had a field of view of 256 mm head–foot, 224 mm anteroposterior (AP), and 166 mm right–left (RL). This sequence was a spoiled multi-echo 3D fast low angle shot acquisition with a flip angle of 21° and a TR of 25 ms. To accelerate the data acquisition, partially parallel imaging using the GRAPPA algorithm was used in each phase-encoded direction (AP and RL) with 40 reference lines and a speedup factor of 2. Gradient echoes were acquired with alternating readout polarity at eight equidistant echo times ranging from 2.34 to 18.44 ms in steps of 2.30 ms using a readout bandwidth of 488 Hz/pixel ( ). The first six echoes were averaged to increase SNR ( ), producing a T1-weighted image with an effective echo time of 8.3 ms.
##### High-resolution T1-weighted MRI scans of the whole brain at 7.0 tesla.
One patient could not be scanned at our center because of recent dental implants. We, therefore, used a whole-brain T1-weighted image acquired previously on a 7.0 tesla MRI scanner, a three-dimensional whole-brain T1-weighted phase-sensitive inversion recovery sequence ( ) at an isotropic resolution of 600 μm, with a tailored inversion pulse for magnetization inversion at ultrahigh field ( ), providing inherent bias field correction.
##### Hippocampal segmentation.
To improve the SNR of the anatomical images, two or three T2-weighted high-resolution scans were acquired for a participant. Images from each participant were coregistered and denoised following the Rician noise estimation ( ). The denoised images were averaged and smoothed with a full-width at half-maximum (FWHM) kernel of 2 × 2 × 2 mm. In each case, left and right hippocampi were manually (blindly) segmented and volumes were extracted using the ITK Snap software version 3.4.0 ( ).
##### VBM analysis.
An automated VBM analysis was performed using SPM12 (Statistical Parametric Mapping; Wellcome Centre for Human Neuroimaging, London, UK). The averaged T1-weighted images were segmented into gray and white matter probability maps using the unified segmentation approach ( ). Intersubject registration of the tissue classes was performed using Dartel, a nonlinear diffeomorphic algorithm ( ). The resulting Dartel template and deformations were used to normalize the tissue probability maps to the stereotactic space defined by the MNI (Montreal Neurological Institute) template. For VBM analysis, the normalization procedure included modulating the gray matter tissue probability maps by the Jacobian determinants of the deformation field and smoothing with an isotropic Gaussian smoothing kernel of 8 mm FWHM. The normalized gray matter from controls and the patients with hippocampal damage were contrasted using a two-sample t test and thresholded at p < 0.001 uncorrected and a cluster extend of 50 voxels.
#### Experimental design and procedure
We had the opportunity to shadow the patients with selective bilateral hippocampal damage over 2 days during daytime hours, and so we adapted for use a well established method, descriptive experience sampling (DES), in which participants are asked frequently over an extended period of time to describe what was on their minds just before they were aware of being asked ( ; ; ). DES has the advantage that thought probes can extend over a long period of time and the sampling interval can be more extensive than alternative approaches in which a few thought samples are taken while participants perform low-demanding distractor tasks ( ; ). Furthermore, using DES, participants are encouraged to describe freely what is on their minds, rather than categorizing thoughts into prespecified classes.
To mitigate any potential difficulties the hippocampal-damaged patients may have had with remembering task instructions over longer timescales, we made a number of adaptations to the original DES protocol. For example, we changed the type of reminder. The reminder is an important tool as it identifies the precise moment of sampling and happens externally to the participant, meaning that the participant does not have to remember to track his own thoughts ( ). Usually, DES participants carry a beeper and receive frequent sampling reminders while going about their everyday life ( ). However, we adapted this sampling method to suit an extended experimental setting over 2 days in which patients and controls experienced the same structured days (three MRI scans, various cognitive tasks, breaks, lunches, etc.). In our case, the experimenter provided the external cue for the participant. Equally important as the reminder is the exact time point of the sample. Whereas previous studies have used a random sampling schedule ( ; ), our main goal was to examine the general ability to perceptually decouple and the content of spontaneous thoughts of these rare patients. We, therefore, tried to maximize our chances of catching perceptually decoupled thoughts. Hence, we probed 20 times over the course of 2 structured research days (8 h each) at prespecified times in restful moments. To keep the experimental context of the sampling time points as closely matched across participants as possible, thoughts for all participants were probed in the same rooms of our center, around the same times of day, and in approximately the same experimental situations. This procedure resulted in schedules whereby some samples were separated by several hours (e.g., during which the participant underwent MRI scanning) and other samples that were relatively close in time (e.g., a few minutes). In addition, to ascertain that all participants, especially the patients, could remember time spans long enough to report their thoughts, we asked them to describe two experiments unrelated to the current study shortly after completion. All participants were able to provide accurate accounts of those experiments.
During sampling moments, such as after obtaining consent, and at the beginning of the tea break, the experimenter would allow for a moment of quiet to emerge. That is, the experimenter would fill out some forms or naturally disengage from any conversation. When there was an appropriate time of silence, the experimenter would ask the participant “What were you thinking about just before I asked you?” The participant was encouraged to briefly describe the current thought in one or two sentences. On a prepared note sheet, the participant's response was written down verbatim. In a follow-up question, the experimenter established whether the thought had been a visual image (if yes, scene or object) or a verbal thought. Then, the experimenter clarified whether that thought had concerned the past, present, or future (and if it had been past or future, how far into the past or future). The sampling procedure lasted no longer than ∼1 min to prevent lengthy post hoc elaboration. Finally, divergent from other DES reports, we opted not to train our participants before the start of the study. Although the training may have provided useful guidance in monitoring one's own thoughts for the control participants, we felt that patients might not find this as beneficial. Therefore, because the experimenter was present for all samples, none of the participants was required to remember the follow-up questions themselves but were instead cued by the experimenter. Of note, control participants reported equal numbers of decoupled, scene-based thoughts in the first and second half of the samples (first half, 5.8 ± 0.9; second half, 6.3 ± 1.2; W = 11.0, p = 0.42), suggesting that there was no significant training effect. A lack of monitoring was further confirmed by the control participants, because they anticipated the sampling probe for only 3 of a total of 240 sampled thoughts.
Whereas previous research has examined the frequency and content of mind-wandering episodes in healthy participants for features such as goal orientation and emotional valence ( , ; ), we focused here on examining the effect of hippocampal damage on the frequency, time range, representational content, and form of mind-wandering, which are key to understanding hippocampal function.
In summary, our adapted sampling protocol permitted us to leverage the naturalistic approach of the typical DES reports that sample over an extended period of time and allowed participants to report their thoughts freely, while equating the daily activities and the sampling moments of patients and control participants to maximize our chances of catching perceptually decoupled thoughts in an experimentally rigorous manner.
#### Scoring
##### Perceptually coupled or decoupled thoughts and mind-blanking.
An episode was considered mind-wandering when the response indicated that the mind was disengaged from the external world (perceptually decoupled; ). For example, the thought “I see your watch” was considered perceptually coupled, whereas the thought “Time is sometimes slow and sometimes fast” was considered perceptually decoupled. In a few instances, patients and control participants reported thinking about nothing (i.e., mind-blanking; ). The frequency of this mind-blanking did not differ between the groups (CTL mean, 0.4 ± 0.88; HPC mean, 1.8 ± 2.8; Mann-Whitney U test (MWU) = 23; p = 0.19), and we therefore excluded these samples from further analysis.
##### Temporal range.
After each sample, we clarified directly with participants whether that thought had concerned the past, present, or future and, if past or future, how distant into the past or future. We also sorted participants' responses from the “present” category based on the observation that patients and control participants reported very different types of thoughts. Consequently, we classified each mind-wandering episode that was labeled by participants as concerning the present moment as either an atemporal scenario or not, in line with the protocol of . A mind-wandering episode was considered present related if the thought was perceptually decoupled but concerned the now, e.g., “I'm thinking that you are right-handed” or “I wonder whether I should eat another grape.” On the other hand, a thought was classified as an atemporal scenario if the participant reported a mental event that had no clear temporal direction. For example, a control participant's thought was, “I noticed this apparatus (EEG box) and I just imagined a picture in my mind in which that box was being used in a horror setting.” By contrast, a patient reported while noticing the same EEG box, “I wonder what this box with all these cables does. But I have no idea.” We display a detailed characterization of the temporal range of mind-wandering episodes in . For statistical analysis, thoughts were binned into four main time categories, namely past (any thought related to earlier than the present moment), present (now), future (any thought related to later than the present moment), and atemporal thoughts.
##### Representation type.
Thoughts were classified as either semantic or episodic (in line with established methods; ; ), and, in addition, whether they contained self-referential thinking or not ( , ). A thought was classified as semantic if it contained mentalization, or general knowledge about the world or the participant. For example, a semantic, self-referential thought of a patient was “I am self-pondering. Am I a creative person?” A thought was classified as episodic if it contained specificity of time and place and a feeling of re-experiencing or pre-experiencing ( , ). For example, an episodic, self-referential thought of a control participant was “I am remembering a discussion I had with my friend at King's Cross concourse a few weeks ago. I can see the scene clearly in front of me.” Of note, we also classified thoughts as episodic that had reference to a specific place and time, even if one or both were fictitious (time was more often fictitious). For example, an atemporal, episodic, non-self-referential thought of a control was “I'm thinking about my friend. He's traveling around giving lectures. I imagine an auditorium and see my friend speaking.”
##### Form of thoughts.
We asked participants after each sample whether the thought had been verbal or visual and, if visual, whether it had been a scene or an object. Each thought was sorted into only one of these categories. Some participants reported that some of the visual scenes also contained verbal aspects; however, they regarded the visual scene as being more dominant. Therefore, these thoughts were classified as scenes. This classification was accomplished in agreement with each participant.
#### Interrater reliability
To avoid potential rater biases, a second rater, who was blind to group membership, scored all thoughts from the patients and the control participants (except for one control dataset that was used as a training set). Interrater reliability was calculated as the direct correspondence between the two raters. That is, thoughts that were scored identically in a category were given a “1”; otherwise, they were given a “0.” The reliability was then established as the sum divided by the total amount of rated thoughts. Therefore, a value of 0.99 indicates that in 99% of samples, the raters categorized them identically. The overall agreement between raters ranged between 84 and 99% across the thought categories (i.e., atemporal, 88%; coupled/decoupled, 99%; semantic, 84%; episodic, 85%; self-referential, 87%).
#### Statistical analyses
Since most of the dependent variables did not meet the assumptions for parametric statistics, nonparametric tests were used for all within- and between-group analyses. Within-group analyses with more than two dependent variables were first conducted using Friedman tests (the nonparametric equivalent of repeated-measures ANOVAs) and followed up with two-tailed Wilcoxon signed rank tests (the nonparametric equivalent of paired t tests). Between-group analyses with more than two dependent variables were first conducted using Kruskal–Wallis tests (the nonparametric equivalent of one-way ANOVAs) and followed up with two-tailed Mann–Whitney U tests (the nonparametric equivalent of two-sample t tests). Analyses with two dependent variables were directly compared using two-tailed Wilcoxon signed rank tests (within-group effects) or Mann–Whitney U tests (between-group effects). In all cases, we considered p values <0.05 as statistically significant. For significant results, we also report, where appropriate, the effect size (using nonparametric Cohen's d ), and we show the data of every participant.
## Results
### Frequency of mind-wandering
We first examined whether or not patients with hippocampal damage were able to mentally decouple from the current perceptual input ( b , c ). We found that the percentage of perceptually decoupled thoughts was greater than perceptually coupled thoughts in the controls ( W = 78.0, p = 0.0005) and patients ( W = 21.0, p = 0.03; ). Notably, we found no difference between the two groups in the frequency of coupled (MWU = 19.5, p = 0.12) or decoupled (MWU = 19.5, p = 0.12) thoughts.
Hippocampal damage and the frequency of mind-wandering. a , A T2-weighted structural MR image of an example patient with selective bilateral hippocampal damage and an age-, gender-, and IQ-matched healthy control participant. Images are displayed in native space corresponding approximately to the position of y = −10 in the MNI coordinate system. b , Examples of mind-wandering experiences from CTL and HPC. c , The average percentage of perceptually coupled and decoupled spontaneous thoughts (minus “blank” thoughts) during quiet restful moments for individual patients with hippocampal damage (red symbols) and healthy control participants (blue circles). Both groups reported a high level of mind-wandering experiences, with no differences between patients and control participants.
Summary of mind-wandering data
### Temporal range of mind-wandering
Since mental time travel seems to occur frequently during mind-wandering ( ), we next examined whether the patient and control groups spontaneously thought about the past, present, or future. After each thought sample, we asked participants whether the thought concerned the present moment, past, or future and, if the latter two, how distant was it from the present moment (see for a detailed visualization of multiple time bins). As described above, we also included an atemporal category in our analyses, comprising thoughts where a participant reported a mental event that had no clear temporal direction.
The temporal range of mind-wandering. Mean percentages of mind-wandering thoughts of HPC (red circles with a dot) and CTL (blue circles) for the past, present, and future are shown. For display purposes, thoughts are classified into time bins according to the past (including earlier today), the present (now), and the future (including later today). m, Months; y, years. Control participants reported more thoughts related to the past than patients. In contrast, patients reported more thoughts related to the present than controls. Inset, The percentage of thoughts during which patients (red symbols) and controls (blue circles) engaged in the imagining of atemporal scenarios.
Examining the results for control participants in the first instance, we found that there was a significant effect of time category (Friedman statistic = 19.99, df = 3, p = 0.0002). Post hoc analyses showed that controls spent more of their mind-wandering time thinking about the past than the present ( W = −62.0, p = 0.002) or future ( W = −72, p = 0.002). They also spent more time simulating atemporal scenarios than thinking about the present ( W = 62, p = 0.01). In contrast, there was no overall effect of time category for patients (Friedman statistic = 6.86, df = 3, p = 0.07).
Direct comparison between the two groups revealed overall differences (Kruskal–Wallis statistic = 31.93,df = 7, p < 0.0001). Post hoc analyses showed that the patients thought less often than controls about past events (MWU = 14.0, p = 0.04, Cohen's d = 1.1). By contrast, the patients thought more often about the present moment than control participants (MWU = 7.0, p = 0.0034, Cohen's d = 1.7). There was no difference between the groups in the percentage of future-thinking, which was generally low for both groups (MWU = 18.0, p = 0.09). Finally, we found that controls more often than the patients imagined atemporal events and hypothetical scenarios that concerned a fictitious reality, which was not attached to any temporal dimension ( , inset; MWU = 3.0, p = 0.0007, Cohen's d = 2.1).
### Representation type
We next investigated what the patients mind-wandered about ( ; ). Focusing first on the control participants, we found that they reported significantly more episodic than semantic thoughts ( W = 78.0, p = 0.0005) and more self-related than non-self-related thoughts ( W = 78, p = 0.0005). The patients with hippocampal damage, on the other hand, experienced more semantic than episodic thoughts ( W = −20.0, p = 0.04) and more self-related than non-self-related thoughts ( W = 21.0, p = 0.03).
Semantic and episodic thinking during mind-wandering. Percentages of mind-wandering samples classified as semantic, episodic, self-referential, or non-self-referential for HPC (red symbols) and CTL (blue circles) are shown. The patients had predominantly semantic thoughts, whereas the thoughts of the control participants were mainly episodic.
Directly comparing the participant groups revealed that the controls reported more episodic thoughts than the patients (MWU = 0.0, p = 0.0001, Cohen's d = 2.6) and the patients reported more semantic thoughts than the controls (MWU = 0.0, p = 0.0001, Cohen's d = 2.6). As expected, there was no significant difference in the percentage of self-referential (MWU = 35.0, p = 0.95) or non-self-referential (MWU = 35.0, p = 0.95) thinking between the groups. Together, these results show striking differences in the representational nature of spontaneous inner experiences between control participants and hippocampal-damaged patients.
### Form of thoughts
Finally, after each sample, we asked participants whether the thought had been verbal or visual and, if visual, whether it had been a scene or an object ( ; ). For controls, we found overall differences in the frequency of the different forms of thought (Friedman statistic = 21.83, df = 2, p < 0.0001). Post hoc analyses showed that control participants reported that the majority of their thoughts involved visual scenes, more so than visual objects ( W = −78.0, p = 0.0005) or verbal thoughts ( W = −78.0, p = 0.0005), but more verbal thoughts than visual objects ( W = 58.0, p = 0.007). For the patients, too, there were overall differences in the frequency of the different forms of thought (Friedman statistic = 9.48, df = 2, p = 0.005). In striking contrast to controls, patients thought almost entirely verbally. They reported more verbal thoughts than visual scenes ( W = 21, p = 0.03) and visual objects ( W = 21, p = 0.03), with no difference between visual scenes and objects ( W = −3, p = 0.81).
Cumulative percentages of visual and verbal mind-wandering thoughts. The average percentage of verbal thoughts is depicted per group (HPC and CTL) as an orange bar; the individual data points are illustrated with orange symbols. The average cumulative percentage of thoughts containing visual objects is depicted as a gray bar above the average percentage of the verbal thoughts. The individual data points of thoughts containing visual objects (gray symbols) are illustrated as cumulative percentages above the orange data points (i.e., the patient represented as a square symbol reported ∼70% verbal and ∼25% visual object thoughts). Finally, the average cumulative percentage of thoughts containing visual scenes is depicted as a green bar on top of the gray bar (green symbols all adding up to 100%). Whereas patients with hippocampal damage reported thinking in words for the majority of samples, healthy control participants' thoughts were predominantly in the form of visual scenes.
These differences in the experiential form of mind-wandering were confirmed by directly comparing the participant groups (Kruskal–Wallis statistic = 56.33, df = 7, p < 0.0001). Whereas controls reported more visual scenes than patients (MWU = 0.0, p = 0.0001, Cohen's d = 2.6), patients reported more verbal thoughts than controls (MWU = 0.0, p = 0.0001, Cohen's d = 2.6), with no difference between participant groups for visual objects (MWU = 30.0, p = 0.59).
## Discussion
Mind-wandering is pervasive in humans and likely has an important role to play across cognition, influencing processes such as future planning, creative thinking, and problem-solving ( , ; ). Here we showed that patients with hippocampal damage were able to perceptually decouple from the external world and experience spontaneous thoughts. Nevertheless, the small, selective lesions of their hippocampi dramatically affected the nature of their mind-wandering. Whereas healthy participants thought about the past, present, and future, primarily in terms of episodic, detail-rich visual scenes, the patients mainly experienced verbally mediated semantic thoughts anchored in the present. Previous studies have examined episodic thought processes in patients with hippocampal damage using explicit tasks, such as the Autobiographical Interview ( ) or the scene construction task ( ), that were designed to challenge the patients' ability. In contrast, our findings show that even when there is no direct cognitive demand, the thought structure of people with hippocampal damage is strikingly different from healthy controls.
We first consider whether our results can be explained by a memory deficit that caused the patients to rapidly forget their mind-wandering thoughts before they could be accurately reported. We do not think is the case for a number of reasons. First, the patients had intact working memory and could retain task instructions during neuropsychological tests ( ) over longer timescales than those in the current study. Second, we asked participants to describe two experiments unrelated to the current study shortly after completion, thus mirroring the timescale of reporting their mind-wandering experiences. All participants, including the patients, were able to provide accurate accounts of those experiments. Third, in previously published studies involving the same patients and control participants using different paradigms, the patients were able to maintain information over time periods that were longer than those required for generating the current mind-wandering samples ( , ). Fourth, our sampling method did not involve any delay or distraction that might have affected the patients, nor did our protocol allow for increased post hoc elaboration on the part of the control participants. Finally, if patients did not remember what they had been thinking about, the frequency of their mind-wandering would have been lower and they would have reported more mind-blanking, which was not the case. Thus, we are confident the patients were able to accurately report what was on their mind within seconds of the sampling cue.
Previous reports have estimated that humans tend to mind-wander ∼30–50% of waking time ( ; ). Here we report percentages nearer 80–90%. However, we specifically aimed to catch restful periods, and so our higher percentage of mind-wandering thoughts suggests that we were successful at probing time points when mind-wandering levels were high.
Numerous studies have focused on delineating different aspects of inner experiences. For example, self-generated thinking (either intentional or unintentional; ) typically refers to the ability to mentally decouple from the current perceptual surroundings and generate independent internal thoughts ( ), which is a dichotomous definition that we used in the current study. In reality, these self-generated thoughts align on a continuum ranging from closely task related to totally task unrelated ( ). What was most important for our research question was whether patients could decouple perceptually from their immediate surroundings in a completely task-free context. We found that they were able to do so and that the frequency of their mind-wandering did not differ from that of the control group. This result is especially noteworthy, given a recent study that found reduced frequency of mind-wandering in patients with ventromedial prefrontal cortex (vmPFC) lesions ( ), a brain region with dense functional and anatomical connections with the hippocampus ( ; , ; ). Although there were differences in the experimental setup between our study and that involving the vmPFC patients, the difference in mind-wandering frequency observed in these two studies might indicate that the vmPFC is critical for the initiation of endogenous spontaneous thought and the hippocampus for its form and content.
At first glance, our finding of group differences in the temporal extent of mind-wandering is not surprising given the difficulty patients with hippocampal damage are known to have with recalling recent and remote episodic memories and imagining the future ( ; ). However, these previous results were based on active and cognitively demanding tasks. To the best of our knowledge, this is the first indication that hippocampal-damaged patients experience reduced mental time travel even in their spontaneous thoughts. Of note, we did not replicate previous reports suggesting a near future-thinking bias in the mind-wandering of healthy participants ( ; ; ). The current experimental procedure and the older age of our participants may have influenced these results ( ). For example, instead of sampling during low-demanding computer tasks or in natural environments that may encourage thoughts about the near future (e.g., “Where am I going after I'm finished here?”), we sampled thoughts across a structured day of stimulating research activities. This may have provided more opportunities to think about the recently completed cognitive tasks or MRI scans. In addition, many previous studies have not included an atemporal category of thoughts, and it has been argued that thoughts labeled as future oriented might, in some instances, be more accurately characterized as atemporal ( ). Indeed, in line with our results, it has been reported that healthy older adults experience more atemporal than future-oriented mind-wandering episodes ( ).
Recently, there have been increased efforts to map the complex cognitive processes that support mind-wandering to specific brain regions. Although it is has been established that the DMN is associated with mind-wandering ( ; ; ), the contributions of specific brain areas within the DMN to mind-wandering remain unclear. Our results provide novel evidence that the hippocampus plays a causal role in episodic mind-wandering. These findings align with recent neuroimaging work that focused on a subsystem of the DMN, of which the hippocampus (and vmPFC) are nodes ( ), and illustrated that functional and structural connectivity is stronger in individuals who report many detail-rich mental time travel experiences during mind-wandering ( ; ). Our results further accord with network analyses in patients with hippocampal damage that showed altered hippocampal–neocortical connectivity patterns ( ; ; ), which were associated with worse episodic memory capacity ( ). Of note, to the best of our knowledge, ours is the first report of a concomitant increase in spontaneous semantic thoughts associated with hippocampal damage. This may help to explain previous findings of increased connectivity between brain areas involved in semantic processing in resting-state fMRI studies involving similar patients ( ; ).
In line with previous studies, our results demonstrate that mind-wandering episodes of control participants typically comprise visual imagery ( ). We expand on existing studies by showing that visual imagery in task-unrelated mind-wandering of healthy controls primarily consists of spatially coherent visual scenes. In striking contrast, the patients with bilateral hippocampal damage no longer reported visualizing mental scenes, relying instead on a verbal thought structure. A scene construction deficit has been implicated in the impaired autobiographical memory and future thinking of patients with hippocampal damage ( ; ; ). Our findings support this link between episodic thought and scene imagery. Importantly, this deficit also extends to scene perception tasks ( ; ; ), suggesting that the lack of mental scenes is not because of faster visual degradation of imagery ( ), but rather is attributable to an online scene construction problem. Thus, our results strongly suggest that hippocampal-supported scene construction is also central to the content and form of mind-wandering and that without it spontaneous thought seems to be reliant on verbal semantics.
Although the precise definition of mind-wandering is still debated, our results show that selective bilateral lesions to the hippocampus impair perceptually decoupled inner thoughts in specific ways, thus informing the nature of mind-wandering and how it is realized at the neural level. That individuals with hippocampal damage experience mind-wandering but very little detail-rich mental imagery is an important new insight that indicates the hippocampus is not necessary for the instigation of spontaneous thought per se. Instead, it seems to be crucial for processing the form and content of mind-wandering. Our results also speak to the functions of the hippocampus. By showing it plays a causal role in a phenomenon as ubiquitous as mind-wandering, this exposes the impact of the hippocampus beyond its traditionally perceived role in episodic memory, placing it at the center of our everyday mental experiences.
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Overly stable visual perception seen in individuals with autism spectrum disorder (ASD) is related to higher-order core symptoms of the condition. However, the neural basis by which these seemingly different symptoms are simultaneously observed in individuals with ASD remains unclear. Here, we aimed to identify such a neuroanatomical substrate linking perceptual stability to autistic cognitive rigidity, a part of core restricted, repetitive behaviors (RRBs). First, using a bistable visual perception test, we measured the perceptual stability of 22 high-functioning adults with ASD and 22 age-, IQ-, and sex-matched typically developing human individuals and confirmed overstable visual perception in autism. Next, using a spontaneous task-switching (TS) test, we showed that the individuals with ASD were more likely to repeat the same task voluntarily and spontaneously, and such rigid TS behavior was associated with the severity of their RRB symptoms. We then compared these perceptual and cognitive behaviors and found a significant correlation between them for individuals with ASD. Finally, we found that this behavioral link was supported by a smaller gray matter volume (GMV) of the posterior superior parietal lobule (pSPL) in individuals with ASD. Moreover, this smaller GMV in the pSPL was also associated with the RRB symptoms and replicated in two independent datasets. Our findings suggest that the pSPL could be one of the neuroanatomical mediators of cognitive and perceptual inflexibility in autism, which could help a unified biological understanding of the mechanisms underpinning diverse symptoms of this developmental disorder.
SIGNIFICANCE STATEMENT Behavioral studies show perceptual overstability in autism spectrum disorder (ASD). However, the neural mechanisms by which such sensory symptoms can coexist and often correlate with seemingly separate core symptoms remain unknown. Here, we have identified such a key neuroanatomical substrate. We have revealed that overstable sensory perception of individuals with ASD is linked with their cognitive rigidity, a part of core restricted, repetitive behavior symptoms, and such a behavioral link is underpinned by a smaller gray matter volume in the posterior superior parietal lobule in autism. These findings uncover a key neuroanatomical mediator of autistic perceptual and cognitive inflexibility and would ignite future studies on how the core symptoms of ASD interact with its unique sensory perception.
## Introduction
Studies of autism spectrum disorder (ASD) have mainly investigated two core aspects of the condition: sociocommunicational difficulties and restricted, repetitive behaviors (RRBs) ( ). In addition to this trend, behavioral and clinical studies in the last decade have accumulated evidence for connections between these core symptoms of autism and its unique sensory perception ( ): the core symptoms of ASD are correlated with hypersensitivity ( , ), overstability ( ; ; ), and prediction error ( ) in visual perception. Moreover, the core symptoms are associated with overevaluation of auditory information ( ) and unique interpretation of smell ( ).
Little is known, however, about the neural substrates underpinning the coexistence of such altered sensory perception with more complex, higher-order core symptoms in individuals with ASD ( ). Neuroimaging studies identified larger signal variability in the visual, auditory, and somatosensory brain areas of individuals with ASD ( ; ), and the disruption of GABAergic inhibitory neuronal systems in their early visual cortex ( ). However, these studies did not report associations between such altered neural responses in the sensory areas and autistic behaviors. Another study found that ASD core symptoms are correlated with the size of population visual receptive fields in the primary visual cortex ( ), but this work did not identify relationships between the neural architecture in autism and its sensory symptoms. Although a recent population-based genetics study found genes that were commonly related to core autistic traits and unique perceptual sensitivity in typically developing (TD) individuals ( ), this research neither directly examined these genetic observations in cohorts with ASD nor investigated neural mechanisms involving the overlapping genetic patterns. Currently, therefore, the neurobiological bases that link sensory symptoms to core symptoms in autism remain unclear ( ).
Here, we aimed to identify such a biological link by focusing on overly stable visual perception ( ; ; ) and cognitive rigidity, an aspect of the RRB symptoms ( ; ; ). We chose these perceptual and cognitive tendencies in ASD because our previous studies imply that both of them could be supported by overly stable brain dynamics in autism: although it has not been confirmed in ASD, perceptual stability during the bistable perception was predicted by less frequent transitions of large-scale brain activity patterns in TD individuals ( ); likewise, the unique cognitive skills of high-functioning adults with ASD were explained by overly stable transitory brain dynamics ( ). Based on these observations, we hypothesized that the stable visual perception in ASD and its rigid cognitive styles should share neuroanatomical substrates.
We examined this hypothesis by searching for neuroanatomical features of cortical gray matter that were correlated with both the perceptual stability and the cognitive rigidity in high-functioning adults with ASD.
## Materials and Methods
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##### Overall study design.
First, we behaviorally compared the stability of visual perception and cognitive rigidity. The stability of visual perception was measured in a test of bistable visual perception with a structure-from-motion stimulus ( , ; ; ) ( A ). We used this stimulus because it contains no explicit social/biological information, which enabled us to evaluate perception with the least effects of the social symptoms of ASD.
Cognitive rigidity was evaluated by observing spontaneous task switching (TS) between two cognitive tasks (shape and brightness tasks; B ). As in previous studies ( ; ), for each trial, participants were asked to freely choose one of the two tasks and to conduct the task accurately and quickly ( C ). We counted how many times participants repeated the same task and used the task repetition length as an index of cognitive rigidity.
As a control, the participants also performed instructed TS tests, in which they were given explicit instructions indicating which of the two tasks they had to conduct on each trial ( D ). The order of instructions (i.e., the task order) was determined by the record of that participant's behavior in the previous spontaneous TS tests, so that the participants had seemingly the same task-switching (TS) experience in both the instructed and spontaneous TS tests ( E ). This experimental paradigm allowed us to determine whether the cognitive rigidity seen in the spontaneous TS test could be simply explained by basic cognitive/sensorimotor skills seen in the instructed TS test.
Using these behavioral data, we first quantified perceptual stability in ASD ( ) and the basic skills for these TS behaviors ( ). Then, we examined the randomness and spontaneity of participants' responses during the spontaneous TS tests ( ) and investigated associations between perceptual and cognitive inflexibility ( ).
By combining these behavioral observations with structural MRI data, we conducted a voxel-based morphometry (VBM) and searched for brain areas with cortical gray matter volumes (GMVs) that were commonly correlated with perceptual stability and with cognitive rigidity in individuals with autism ( , , ) and controls ( ).
Finally, to confirm the associations between the GMVs of the focal brain regions and cognitive rigidity in ASD, we also compared the GMVs and the severity of the RRB symptoms in our dataset and two independent neuroimaging datasets ( ) ( ).
##### Participants.
We recruited 27 high-functioning right-handed adults with ASD and 27 age-, IQ-, and sex-matched TD individuals. This sample size was determined based on previous studies that examined perceptual stability in autism ( , ). Participants were diagnosed by independent clinicians according to DSM-IV or ICD-10, and the severity of their core symptoms was assessed by a qualified administrator using ADOS ( ). To reduce the heterogeneity of the participants, we focused on high-functioning adults with ASD (Full IQ/Verbal IQ/Practice IQ ≥ 85). The IQ was assessed by the Wechsler Adult Intelligence Scale (third edition, UK). We also excluded individuals with any other neuropsychiatric disorders, those whose were taking psychiatric medicines at the time of the experiments, and those who could not undergo all the experiments. In total, five ASD and five TD individuals were excluded for incompletion of the experiments due to technical problems or their visibly noisy MRI data probably due to head motion. We analyzed behavioral and MRI data of the remaining 22 ASD (three females) and 22 TD (four females) individuals ( ).
Demographic data
This study was approved by the University College London (UCL) ethics committee. All participants provided written informed consent and were financially compensated for their participation and travel expenses.
##### Test of bistable visual perception.
In the test of bistable visual perception, the participants were presented with a structure-from-motion stimulus ( A ), a sphere consisting of 200 moving white dots in a black background ( ). The dots moved sinusoidally upward and downward (angular velocity, 120°/s) with a fixation cross (0.1°×0.1°) at the center of the 21.5-inch LCD monitor (Samsung Sync Master 2233, resolution: 1680 × 1050). In each run, the participants were asked to look at this stimulus for 90 s with their chins put on a chin rest. They were instructed to push one of the three buttons according to their visual perception: one for upward rotation, another for downward rotation, and the other for unsure/mixture perception. After sufficient training sessions, the participants repeated this run five times. We conducted the stimulus presentation and response recording with PsychToolbox 3 in MATLAB (The MathWorks).
Consistent with our previous studies ( ; ), mixture perception was rare in both participant groups (1.7 ± 0.3% of all stimulus presentation times, mean ± SD). Therefore, this study focused on the time during which participants had clear awareness about the direction of the rotation. For each participant, we measured the duration of such clear perception and calculated the median of the duration to evaluate their perceptual stability. We used the median duration because perceptual durations showed long-tailed distributions in both participant groups ( A ).
##### Spontaneous TS test.
In the spontaneous TS test ( ; ), participants were presented with visual stimuli consisting of four figures with different shapes and brightness ( B ), and were asked to perform a shape task or a brightness task. In the shape task, they had to identify a specific shape (here, a circle), whereas in the brightness task, they were asked to identify the brightest figure. They were asked to press one of four buttons to indicate which figure in the display they chose for each task as accurately and quickly as possible. If they did not press any button in 3 s, the trial automatically ended and the next trial started.
Participants practiced the two types of task separately by repeating a 30 s trial until they could respond correctly (≥95% of accuracy) and quickly [reaction time (RT) ≤2 s]. Then, the participants underwent the spontaneous TS test consisting of 5 3 min runs.
For each trial, participants could freely choose which task they would conduct. As in previous studies ( ; ), we gave them the following instructions: “You have to choose which task to perform on each trial. Ideally, you should perform each task randomly but on about half of the trials. Sometimes you will repeat the same task and sometimes you will switch from one to another. We don't want you to count the number of times you've done each task or alternate strictly between tasks to make it sure you do each one half the time. Just try to do them randomly.”
Based on the chosen figures, we retrospectively inferred the selected tasks and classified each trial into a shape or brightness trial. Participants rarely chose a figure that could not be classified into either a shape or brightness task (the proportion of such unclassifiable trials ≤1.5%; E ). Therefore, we excluded such unclassifiable trials for the following analysis.
##### Instructed TS test.
In the instructed TS test, participants were given clear instructions about which task to choose. In each trial, a letter appeared at the center of the screen, and indicated the instruction. For example, “C” meant a shape task in which the participants should choose a circle, whereas “B” meant a brightness task ( D ). Participants were asked to perform a task according to this instruction accurately and quickly.
The sequence of the instruction letters was determined based on the behaviors in the spontaneous TS test that the participants underwent right before ( E ). The unclassifiable trials in the spontaneous TS task were simply omitted in the instructed TS task. Thus, the participants were supposed to have seemingly the same task repetition/switching experience as in the spontaneous TS test.
##### Behavioral analyses for TS tests.
For both the TS tests, we calculated the mean RT for task repetition, mean RT for task switching, and switch cost (mean RT for tasks switching − mean RT for task repetition) for each participant. The response accuracy was also estimated in the instructed TS test, and the median of the task repetition length was calculated in the spontaneous TS test. The mean values were used to represent the RT because RT showed normal distributions ( p ≥ 0.24 in Shapiro–Wilk test), whereas the median values were chosen as a representative metric for the task repetition length because they showed long-tailed distributions ( E ). All of these behavioral indices were first estimated for each experiment and then averaged across the five runs.
We used behavioral data during the instructed TS test to evaluate sensorimotor skills to perform TS. We compared the RT for task repetition, RT for task switch, switch cost, and response accuracy between the ASD and TD groups. Then, to examine the spontaneity in the spontaneous TS test, we compared the RT for the task repetition, RT for the task switch, and switch cost between the instructed and spontaneous TS tests.
##### Validation of the randomness in the spontaneous TS test.
To examine the randomness in the spontaneous TS test, we compared the TS/repetition behaviors in the TS test with the state-switching/duration patterns in a simple random walk model.
First, we conducted a one-dimensional Markov-chain random walk simulation ( G ). The randomness of this simulation was iterated by changing the transition probability, P , between 0.5 and 0.9 by 0.1 ( P = 0.5: the most random movements, P = 0.9: the most systematic and regular movements). The number of the hidden states between the extreme states (state h in G ) was also iterated between one and five. For each parameter set, we simulated this random walk for 10 steps and counted the state duration by measuring the number of the steps taken to move from one extreme to the other (e.g., movements from the state A to the state B in G ). Then, we calculated the distribution of the state duration and compared it with that of the task repetition length in the spontaneous TS test. Kullback–Leibler (KL) divergence was used to quantify the difference between the two distributions. To directly compare the shape of the distributions, we normalized the distributions before assessing KL divergence.
This normalization procedure also lessened a potentially confounding effect that would be induced by the following constraint in the random walk simulation. For simplicity, we did not consider the possibility of self-recurrent movements in the random walk. This constraint would allow continuous staying in a hidden state over time, and, theoretically, the state duration in this random walk would be shorter compared with the otherwise case. However, such reduction in the state duration should be nearly cancelled out by the normalization of the resultant distribution. Thus, this random walk constraint is considered to have no significant effects on the calculation of KL divergence.
Finally, for each participant group, we examined associations between the KL divergence and the randomness of the random walk (i.e., P ) and identified which P could produce the most similar state duration distribution compared with that of the task repetition length.
##### Comparison of cognitive rigidity.
After confirming the behavioral randomness and spontaneity in the spontaneous TS test, we compared the median task repetition length in the test between the ASD and TD groups.
The task repetition length was sufficiently short compared with the length of one run. In every 3 min run, both the ASD and TD participants completed ≥200 trials, whereas the median task repetition length was approximately three trials in both the groups and the ASD–TD gap was less than one trial ( A ). Therefore, even if the ASD individuals showed longer task repetition length, they experienced task switching as often (∼65 times per run, ∼330 times per participant) as TD individuals did (∼68 times per run, ∼340 times per participant). Given this, we could assume that the longer TS length in the ASD group did not reduce the size of the behavioral data sampling and thus, did not affect the accuracy of the statistical analyses using the behavioral records of the ASD individuals.
##### Behavioral associations.
In each participant group, we calculated the Pearson correlation between the percept duration in the bistable perception test and the task repetition length in the spontaneous TS test.
We also estimated associations between these behavioral metrics and the clinical scores. For the social symptoms, we calculated correlation coefficients between the experimental indices and ADOS social and communication scores. For RRB symptoms, we compared the behavioral indices between individuals with different ADOS RRB scores using an ANOVA.
##### Neuroimaging experiment and analysis.
We obtained T1-weighted brain images using a 3 T Trio MRI scanner (Siemens Medical Systems) with the 32-channel head coil at the Wellcome Trust Centre for Neuroimaging at UCL (TR 7.92 ms, TE 2.48 ms, Flip angle 16°, spatial resolution 1 mm cubic). These MRI images were preprocessed in SPM12 in the following steps. First, the images were segmented into gray matter, white matter, and CSF in native space by the New Segment Toolbox ( ). Then, the segmented gray matter images were aligned, warped to a template space (ICBM space template for European), resampled down to 1.5 mm isotropic voxels, and registered to a participant-specific template by the DARTEL Toolbox ( ). Using deformation parameters calculated by the DARTEL toolbox, the gray matter images were normalized to MNI space and smoothed with a Gaussian kernel (FWHM = 8 mm). We set FWHM at 8 mm because the value was widely used (for review, see ) and previous studies demonstrated that this size of spatial smoothing would improve the accuracy of VBM ( ) without increasing false-positive rates ( ). This preprocessing using DARTEL Toolbox included a modulation process to preserve the volume of a particular tissue within a voxel ( ). Thus, signal values in the preprocessed images should indicate GMVs.
We analyzed these preprocessed MRI images of the ASD group with a multiple regression model in SPM12. In VBM, we separately searched for brain regions with GMVs that were correlated with both the median percept duration and the median task repetition length. Both the regression analyses used individual ages and Full IQ scores as covariates. We then identified brain regions whose GMVs were positively/negatively correlated with each behavioral index in the ASD data ( P = 0.05). The anatomical labels for the brain regions were determined based on the Automated Anatomical Labeling atlas ( ) and the Harvard–Oxford Atlas.
Next, we conducted a conjunction analysis ( ) to specify brain regions with GMVs that were correlated with both of the behavioral indices. Technically, we searched for the overlaps between the two type of statistical brain map calculated above. The statistical threshold of this conjunction analysis was equivalent or more stringent compared with those in previous studies adopting this analysis approach ( ; ).
We then investigated whether the GMV of the overlapping region (here, posterior superior parietal lobule, pSPL) was significantly different between the TD and ASD groups.
##### Brain–behavior associations.
We conducted a partial correlation analysis to examine the associations between the GMV of the pSPL, perceptual stability, cognitive rigidity, and RRB symptoms.
We then performed a nonparametric mediation analysis to test whether the pSPL was a mediator linking the perceptual overstability to cognitive rigidity in autism. The GMV of the pSPL was set as a mediator variable, whereas the median percept duration and median task repetition length were used as an independent and dependent variable, respectively. We also conducted a whole-brain voxelwise nonparametric mediation analysis using the same independent/dependent variables ( P < 0.05).
Finally, we performed a structural equation modeling analysis to determine whether the GMV of the pSPL was related to domain general behavioral/mental flexibility. The model had a latent variable representing a domain general flexibility and was fitted to the behavioral and GMV data with a maximum likelihood method. As in a previous study ( ), goodness of fit was evaluated based on adjusted goodness of fit index (AGFI), comparative fit index (CFI), root mean square error of approximation (RMSEA), and standardized root mean square residual (SRMR).
To validate results of the above neuroanatomical analysis, we repeated the same VBM and brain–behavior analyses using data collected from only male individuals with ASD. Also, we repeated the neuroanatomical analyses using data of the TD individuals.
##### Reproducibility of GMV alteration in autism.
We examined the reproducibility of the smaller GMV of the right pSPL using two independent MRI datasets (the University of Utah and ETH Zürich) shared in ABIDE and ABIDE II ( ) ( ). The participants were selected with the same criteria as in the original analysis and their T1-weighted brain images were preprocessed in the same manner as stated above.
Demographic data for reproducibility tests
We extracted the GMV of the right pSPL from each participant using the gray matter mask identified in the original analysis (yellow area in B ). We then compared the GMV of the pSPL between the ASD and TD groups and investigated whether the individuals with ASD showing less GMV in the pSPL had severer RRB symptoms.
To examine the specificity of the pSPL, we conducted whole-brain voxelwise search for brain regions with GMVs that were significantly smaller in the ASD group compared with the controls ( P < 0.05).
For every brain region found this VBM, we then examined associations between its GMV and the severity of RRB symptoms. First, we calculated the average GMV for each significant cluster for each individual with ASD. Next, we conducted two-sample t tests of the mean GMV between the individuals with ASD with larger RRB scores (ADOS RRB score ≥ 1) and the other individuals with ASD (i.e., ADOS RRB score = 0).
##### Statistics.
All post hoc t tests were adjusted using Bonferroni correction. We set α = 0.05/3 when comparing three behavioral indices between the two TS tests ( A–C ) and α = 0.05/2 when examining correlations between the task repetition length and two types of ADOS score ( C ) and when evaluating associations between the GMV of pSPL and two behavioral indices ( C ). When comparing GMV in the four different brain regions between the ASD and TD groups, we set α = 0.05/4 ( G ). In the whole-brain neuroimaging analyses, FDR correction was adopted. Correlations were evaluated as Spearman's coefficients when either of them was nonparametric (e.g., ADOS score). Otherwise, they were calculated as Pearson correlation coefficients.
## Results
### Perceptual stability
First, using the bistable perception test ( A ), we found a significantly longer percept duration in the ASD group compared with the controls ( t = 3.0, p = 0.0049 in a two-sample t test; A , B ). This perceptual stability in autism was consistently observed throughout the test [ F = 7.6, p = 0.0068 in a repeated-measures two-way ANOVA with an (ASD/TD) × (five runs) structure; C ].
Experimental design. A , In a bistable visual perception test, participants were presented with a structure-from-motion stimulus to both their eyes and asked to report when their perception switched between downward and upward rotating with a button press. B , TS tests consisted of shape and brightness tasks. On each trial, participants had to choose the circle (shape task) or the brightest figure (brightness task). C , In the spontaneous TS test, participants could freely choose which task to perform for each trial. We quantified cognitive rigidity as how long participants repeated the same task continuously. D , In the instructed TS test, participants were given instructions about which task to perform for each trial. E , Order of the instruction in the instructed TS tests was determined based on their own responses during the spontaneous TS tests. Therefore, the participants performed the same TS pattern in the two TS tests.
Behavior in the bistable visual perception test. Both the ASD and TD groups showed a skewed distribution of percept duration in a bistable perception test ( A ). The median duration was longer in the ASD group ( B ), which was observed throughout the experiment ( C ). Error bars indicate SEM.
### Sensorimotor skill for task switching
Next, we quantified cognitive rigidity of autism by comparing behaviors between the instructed and spontaneous TS tests ( B–E ).
Behaviors during the instruction-based TS test were not different between the ASD and TD groups. Both the groups accurately performed the two tasks (response accuracy > 93.5%; no group difference, t = 0.02, p = 0.98 in a two-sample t test; A ) and showed equivalent RT for task repetition and for task switching ( t < 0.83, p ≥ 0.41 in two-sample t tests; B ). Thus, the switch cost (RT for task switching − RT for task repetition) was almost the same between the two groups ( t = 0.56, p = 0.58 in a two-sample t test; C ). This seemingly intact TS ability of the individuals with ASD did not change throughout the test ( F = 0.041, p = 0.84 in a repeated-measures two-way ANOVA; D ).
Behavior in instructed TS test. In the instructed TS test, both groups showed similar behavior. The ASD and TD groups responded accurately ( A ) with almost the same RT ( B ). The switch cost was significantly larger than zero in both the groups ( p < 10 in one-sample t tests; *** in C ) with no significant difference between the cohorts ( p = 0.58 in a two-sample t test; C ) throughout the test ( D ). The switch cost of the individuals with ASD was not related to the severity of their RRB symptoms ( p = 0.27 for the main effect of ADOS RRB scores in a one-way ANOVA; E ). Error bars indicate SEM.
In addition, the switch cost, a widely used index for cognitive flexibility in TD-participant studies ( ; ; ), did not show significant differences between individuals with different severity of RRB symptoms ( F = 1.4, p = 0.27, one-way ANOVA; E ).
These results suggest that high-functioning adults with ASD had sufficient sensorimotor skills to accurately perform the two tasks and smoothly switch between them according to the instructions.
### Spontaneous and random TS
Participants experienced the same TS sequence in the spontaneous and instructed TS tests, because the timing of the switches in the instructed TS test was yoked to previous per-participant spontaneous switch timings ( E ). However, behavioral responses during the spontaneous TS test were distinct from those during the instructed TS test mainly in the following two points ( ), each of which indicates the spontaneity and randomness of the TS behavior, respectively.
Validation of spontaneous TS test. A – C , In the spontaneous TS test, ASD and TD participants repeated the tasks more quickly ( A ) and switched them more smoothly ( B ) than in the instructed TS test. The switch cost was not significantly different from zero and significantly less than in the instructed TS test ( C ). These results suggest that the participants repeated and switched the tasks spontaneously. * P < 0.05. D , The ASD and TD participants performed the two types of task at almost equal frequencies. Error bars indicate SEM. E , The mean proportion of the unclassified trials in the spontaneous TS test was <1.5% throughout the test and not different between the ASD and TD groups. Error bars indicate SD. F – I , In both the participant groups, the task repetition length showed a skewed, long-tailed distribution ( F ). To evaluate the randomness of the task switch, we compared the distribution with that seen in one-dimensional Markov-chain random walk simulations ( G ). When the simulation adopted more random transition probability (e.g., P = 0.5), the difference between the distributions (KL divergence) became smaller ( H ). The lines in the H show the mean KL divergence across different numbers of the hidden states varying between 1 and 5 shown in the I . Error bars indicate SEM.
First, the spontaneity was supported by RT analyses. Compared with the instructed TS test, the ASD and TD participants repeated tasks in the spontaneous TS test more quickly [ F = 8.6, p = 0.004 in a repeated-measures two-way ANOVA with a (ASD/TD) × (spontaneous/instructed) structure; A ] and switched them more smoothly ( F = 15.0, p = 0.0002 in a repeated-measures two-way ANOVA; B ) with significantly less switch cost ( F = 21.0, p < 0.0001; C ). These behaviors are reasonable if the participants switched and repeated the tasks spontaneously.
Second, the randomness was supported by the TS frequencies and intervals. The participants performed the two tasks for almost equal frequencies ( D ) with a small proportion of unclassified trials (mean ≤ 1.5%; E ), which is reasonable if the participants switched tasks randomly. Moreover, the task repetition length showed a skewed distribution with a long tail ( F ), which was similar to the distribution of state-switching intervals in a random walk ( G ). That is, when a one-dimensional Markov-chain random walk was determined by more random transitions (e.g., P = 0.5 in G ), the resultant distribution of the state-switching intervals was more similar to the actual task repetition length distribution seen in this spontaneous TS test ( H , I ).
These observations suggest that the participants performed the spontaneous TS test as spontaneously and randomly as possible.
### Cognitive rigidity
Such spontaneous TS behaviors enabled us to detect cognitive rigidity in ASD. The ASD and TD groups showed equivalent RTs for task repetition, RTs for task switching, and the switch cost ( p > 0.55 in two-sample t tests; A–C ), but the task repetition length was significantly longer in the ASD group ( t = 4.5, p < 0.0001, P < 0.05 in a two-sample t test; A ). This was observed consistently throughout the test ( F = 8.6, p = 0.0038 in a repeated-measures two-way ANOVA; B ).
Associations between perceptual stability and cognitive rigidity. A – C , The task repetition length in the spontaneous TS test was significantly larger in the ASD group ( A ), which was consistently seen throughout the test ( B ). In the individuals with ASD, the task repetition length was specifically related to the severity of the RRB symptoms ( C ). In A and C , the y -axis shows the average of the median task repetition length across the five experimental runs for each participant. In B , the y -axis represents the mean of the median task repetition length across participants. D , E , We compared the perceptual stability with the cognitive rigidity in the ASD group. The deviations from the neurotypical responses were almost the same in the two types of behavioral indices ( D ), and these two indices were correlated with each other in the ASD group ( E ). d , Cohen's effect size. Error bars indicate SEM.
The prolonged task repetition length in the ASD group could not be explained by basic cognitive/sensorimotor skills: the task repetition length was not significantly correlated with the RT for task repetition, the RT for task switching, or the switch cost in either type of TS test (| r | < 0.19, p > 0.40); neither the IQ scores (Full IQ, Performance IQ, Verbal IQ) nor the age of the participants were associated with this cognitive rigidity index (| r | < 0.21, p > 0.34).
Instead, this longer task repetition in the ASD group was associated with the severity of the RRB symptoms ( F = 6.2, p = 0.009 for the main effect of ADOS RRB scores in a one-way ANOVA; C ), but was not correlated with the severity of the sociocommunicational symptoms (Spearman's ρ = 0.13, p = 0.56; C ).
Together, the behavioral results from the two TS tests indicate that prolonged task repetition length in individuals with ASD is not a consequence of their sensorimotor skills or basic cognitive abilities, but is related to the RRB symptoms. This observation supports our assumption that, at least in high-functioning individuals with ASD, task repetition lengths in the spontaneous TS test are associated with their cognitive rigidity.
### Associations between perceptual and cognitive inflexibility
We then assessed links between perceptual stability and cognitive rigidity by comparing the percept duration in the bistable perception test and the task repetition length in the spontaneous TS test. We found that, in the individuals with ASD, both behavioral indices were deviated from those of TD individuals to almost the same degree ( F = 0.82, p > 0.37 in a repeated-measures two-way ANOVA; D ) and were significantly correlated with each other ( r = 0.45, p = 0.035; E ).
### Neuroanatomical substrates for perceptual and cognitive inflexibility
We then conducted a VBM and searched for a neuroanatomical substrate underlying this behavioral link between the perceptual stability and cognitive rigidity in autism ( ).
Results of VBM using ASD data
First, we found that perceptual stability in the individuals with ASD was positively correlated with the GMV of the inferior occipital cortex (IOC) ( t = 4.6, P < 0.05; red in A ) and negatively correlated with the GMV of the pSPL ( t = 5.1, P < 0.05; red in B ).
Neuroanatomical results. A , B , Red areas indicate voxels with GMVs that were significantly correlated with the median percept duration in the bistable perception test; blue areas indicate those with GMVs that were significantly correlated with the median task repetition length in the spontaneous TS test. The yellow area was an overlap between the red and blue regions (center, [36, −53, 44] in MNI coordinates, pSPL). For presentation purposes, the statistic brain maps adopted t = 3.0 as their thresholds. See also for details. C , The GMV of the overlapping pSPL region was negatively correlated with both the percept duration and task repetition length. D , The GMV of the overlapping pSPL was significantly smaller in ASD individuals with severer RRB symptoms. E , The whole-brain GMV was marginally larger in the ASD group. Error bars indicate SEM. F , The relative GMV of the pSPL, a ratio of the GMV of the pSPL to the whole-brain GMV, still shows significant negative correlations with the median percept duration in the bistable perception test (left) and the median task repetition length in the spontaneous TS test (right). Each circle represents each individual with ASD, G , Among the four regions, the pSPL was the only region with a GMV that was significantly different between the ASD and TD groups. Error bars indicate SEM. * P < 0.05. H , The relative GMV of the pSPL was significantly smaller in the individuals with ASD compared with the controls. Error bars indicate SEM.
In contrast, the cognitive rigidity in autism showed positive correlations with the GMVs of the anterior cingulate cortex (ACC) and middle frontal gyrus (MFG) ( t ≥ 4.4, P < 0.05; red in A ), and a negative correlation with the GMV of the pSPL ( t = 4.8, P < 0.05; red in B ).
That is, the pSPL was the only region whose GMV was significantly associated with both perceptual stability and cognitive rigidity in autism. In fact, the GMV in the overlapping pSPL area (yellow area in B ) was correlated with both perceptual and cognitive rigidity in the ASD group ( r ≤ –0.62, p ≤ 0.003, P < 0.05; C ). In addition, the GMV of the pSPL was smaller when the individuals with ASD had severer RRB symptoms ( t = 3.1, p = 0.0062 in a two-sample t test; D ).
These brain–behavior associations seen in the pSPL were not explained by differences in the whole-brain GMV. The whole-brain GMV in the ASD group was marginally larger compared with the TD group ( t = 2.1, p = 0.048, two-sample t test; E ), but was not correlated with either perceptual stability or cognitive rigidity (| r | ≤ 0.1, p > 0.64). Moreover, even the relative GMV of the pSPL, a ratio of the pSPL GMV to the whole-brain GMV, was still associated with both the behavioral indices ( r ≤ −0.48, p ≤ 0.01; F ).
We then compared GMVs of brain regions found in the above VBM (i.e., IOC, pSPL, ACC, and MFG) between the ASD and TD groups. Despite logical independence between this GMV comparison and the above VBM ( A , B ), we found that the pSPL was, again, the only region with a significant difference in the GMV ( t = 4.2, P = 0.0012, P < 0.05 in two-sample t tests; G ). This difference survived even when we considered effects of the whole-brain GMV ( t = 9.6, p < 10 in a two-sample t test; H ).
Together, these findings suggest that the perceptual overstability and cognitive rigidity in autism share some neural bases and the diminished GMV of the pSPL constitutes such brain mechanisms.
### Associations among the pSPL, perceptual stability, and cognitive rigidity
Based on the above findings, we then investigated whether the pSPL was a key neural substrate linking perceptual overstability to cognitive rigidity in autism.
First, we found that this notion was consistent with results of a partial correlation analysis ( A ). The correlation between the percept duration and the task repetition length ( E ) was explainable by the associations between the GMV of the pSPL and these two autistic behaviors.
Brain–behavior associations. A , Partial correlation analysis implies that the smaller GMV of the pSPL in autism linked the perceptual overstability to the cognitive rigidity and, consequently, RRB symptoms. B , A nonparametric mediation analysis suggests that the smaller GMV of the pSPL would be a mediator linking perceptual stability to cognitive rigidity. This analysis used the median percept duration, GMV of the pSPL, and median task repetition length as an independent variable, mediator variable, and dependent variable, respectively. α indicates a regression coefficient of the percept duration on the GMV of the pSPL, and β denotes that of the GMV of the pSPL on the task repetition length. γ represents the direct effect of the percept duration on the task repetition length. α × β indicates the indirect effect of the percept duration on the task repetition length via the pSPL. C , Colored clusters are brain regions with GMVs that had significant indirect effects (α × β) in a whole-brain nonparametric mediation analysis with the same independent and dependent variables as in B . For presentation purposes, the statistic brain map adopted p = 0.01 as its threshold. A significant cluster was found in the pSPL ( p = 0.0003 in [34, −52, 44] in MNI coordinates). D , A structural equation modeling analysis, in which domain general flexibility was introduced as a latent variable, indicates that the pSPL is related to domain general behavioral/mental flexibility and that the smaller GMV of the pSPL in autism would attenuate such flexibility and induce perceptual overstability and cognitive rigidity. Note that the arrows did not indicate causal relationships between the variables. λ, path coefficients; e and ζ, residual terms.
A nonparametric mediation analysis provided more direct evidence. This analysis showed that the GMV of the pSPL had a significantly large indirect effect (α × β = 0.54, p = 0.009; B ), which suggests that the pSPL would be a mediator between the perceptual and cognitive rigidity. In addition, a whole-brain nonparametric mediation analysis demonstrated that a region in the pSPL was the only brain area that had a significant indirect effect (α × β = 0.62, P = 0.0003, P < 0.05; C ).
Furthermore, a structural equation modeling analysis indicates that the pSPL area could be related to a neural mechanism supporting domain general behavioral/mental flexibility ( D ).
These findings suggest that the pSPL is one of the key brain regions linking the perceptual stability to the cognitive inflexibility in autism and the smaller GMV of the pSPL would result in the two seemingly different symptoms of autism. These observations were preserved even when we conducted the analyses using only the data of the males with ASD ( ).
Neuroanatomical results based on male data. We repeated the neuroanatomical analyses using only the data recorded from the males with ASD ( n = 19) and the male controls ( n = 18). A , B , In the VBM, we found almost the same pSPL region (yellow area in A ) with a GMV that was inversely correlated with both the percept duration and task repetition length ( B ). C , The GMV of this pSPL was larger in the ASD individuals with smaller RRB scores compared with those with larger RRB score. D , The GMV of the pSPL area was smaller in the ASD group than in the TD group. E , A nonparametric mediation analysis showed that the pSPL is a mediator linking the percept duration to the task repetition length. For the details of the analysis and abbreviations, see the legend for B . F , A structural equation modeling analysis indicates that the pSPL area could be related to domain general behavioral/mental flexibility. For the details of the analysis and abbreviations, see the legend for D .
### Neuroanatomical results in TD individuals
We then investigated whether these brain–behavior associations observed in the pSPL were specific to individuals with ASD or they were also seen in the TD individuals.
First, we directly tested this question: we calculated the GMV–behavior correlations by applying the pSPL area determined in the above analyses of the ASD data (yellow area in B ) to the TD dataset as an anatomical mask. We found that, even in the TD dataset, the GMV of this pSPL area was inversely correlated with both the percept duration ( r = −0.42, p = 0.04) and the task repetition length ( r = −0.49, p = 0.015).
Second, we conducted a more thorough examination by repeating the entire VBM with the TD dataset only. Compared with the observations based on the ASD data ( , A , B ), similar brain regions were associated to the perceptual stability and/or cognitive rigidity ( A , B , ; P < 0.05), and, again, the pSPL was the only region whose GMV was commonly correlated with the two behavioral indices.
Neuroanatomical results based on TD data. A , B , The GMVs in the red areas were correlated with the median percept duration in the bistable perception test, whereas those in the blue areas were correlated with the median task repetition length in the spontaneous TS test. The yellow area was an overlap between the red and blue clusters. The overlapping area ( B ) was located around [34, −46, 40] in MNI coordinates. For presentation purpose, the statistic brain maps adopted t = 3.0 as their thresholds. aSPL/pSPL, anterior/posterior superior parietal lobule; SFG, superior frontal gyrus. See also for details. C , A nonparametric mediation analysis suggests that, even in TD individuals, the pSPL is a mediator linking perceptual stability to cognitive rigidity. For the details of the analysis and abbreviations, see the legend for B . D , Colored clusters are brain regions with GMVs that had significant indirect effects (α×β) in a whole-brain nonparametric mediation analysis. For presentation purposes, the statistic brain map adopted p = 0.01 as its threshold. A significant cluster was found in the pSPL ( p = 0.0008 in [36, −52, 38] in MNI coordinates). E , A structural equation modeling analysis indicates that the pSPL could be related to domain general behavioral/mental flexibility even in TD individuals. For the details of the analysis and abbreviations, see the legend for D .
Results of VBM analysis using TD data
In fact, the GMV of the overlapping pSPL area (yellow region in B ) showed significant negative correlations with both the percept duration ( r = −0.50, p = 0.01) and the task repetition length ( r = −0.54, p = 0.006). Moreover, as seen in the analysis of the ASD data, a nonparametric mediation analysis indicates that the pSPL area would be a mediator linking the perceptual stability to the cognitive rigidity (α×β = 0.50, p = 0.009; C ). This pSPL area was also found in a whole-brain nonparametric mediation analysis ( P < 0.05; D ). Furthermore, a structural equation modeling analysis implies that the pSPL could be related to domain general behavioral/mental flexibility ( E ).
These results suggest that the associations between the pSPL and the perceptual/cognitive rigidity are not specific to autism and the pSPL could be one of the essential brain regions supporting human behavioral/mental flexibility.
### Replication of smaller GMV of pSPL in autism
Finally, we examined reproducibility for the smaller GMV of the pSPL in autism and its association with the RRB symptoms. This test was conducted using two independent neuroimaging datasets in ABIDE ( ) (datasets recorded in University of Utah and ETH Zürich; ).
In both datasets, the GMV of the pSPL (yellow area in B ) was significantly smaller in high-functioning adults with ASD compared with demographically matched TD individuals ( p ≤ 0.003 in two-sample t tests; A ). Within the ASD groups, the GMV of the pSPL was decreased when the individuals with ASD had larger ADOS RRB scores ( p ≤ 0.04 in two-sample t tests; B ), which is consistent with the observation shown in D .
Reproducibility of smaller GMV of pSPL in autism. We examined the reproducibility of the diminished GMV seen in the pSPL in autism and its link with the RRB symptoms ( D , G ) using two independent neuroimaging datasets of high-functioning adults with ASD and age-, IQ-, and sex-matched controls (University of Utah and ETH Zürich; ). In both datasets, the GMV of the pSPL (yellow area in B ) was decreased in the ASD individuals ( A ) and showed smaller values when the individuals with ASD had more severe RRB symptoms ( B ).
In addition, this smaller GMV and association with the RRB symptoms were specific to the pSPL. In the both datasets, voxelwise neuroanatomical comparisons between the TD and ASD groups identified significant decreases in the GMV in other brain regions, such as right hippocampus and amygdala, in autism ( P < 0.05; ); however, none of them showed significant associations with the severity of the RRB symptoms ( t ≤ 0.84, p ≥ 0.41 in two-sample t tests; right two columns in ). Only in the pSPL was the GMV smaller in the ASD individuals with larger RRB scores (RRB ≥ 1) compared with those with smaller ones (RRB = 0) ( t ≥ 2.7, p ≤ 0.022 in two-sample t tests).
Brain regions with GMVs smaller in ASD and associated with RRB symptoms
These results add evidence for associations between the diminished GMV in the pSPL and the cognitive inflexibility in autism.
## Discussion
This case–control study has shown that overly stable visual perception of high-functioning adults with ASD is linked to their cognitive rigidity, and this behavioral link is supported by the smaller GMV of the pSPL. We first found that overstable sensory perception seen in the bistable perception test in individuals with ASD is correlated with cognitive rigidity measured by the spontaneous TS test. Then, we identified the smaller GMV in the pSPL as a neuroanatomical substrate filling the gap between these seemingly separate behaviors in individuals with ASD.
The current behavioral findings can be seen as evidence for a link between overstable visual perception of high-functioning adults with ASD and (a part of) their RRB symptoms. Multiple previous studies reported behavioral associations between autistic sensory perception and ASD core symptoms ( ; ; ; ); however, the core symptoms examined in these earlier researches were sociocommunicational symptoms or total ASD severity, not RRB symptoms. Some behavioral studies reported an association between hyper-/hyposensitivity in visual perception and RRB in children with autism using fine clinical scales for the core symptoms such as Repetitive Behavior Scales–Revised ( , ), but the relationship has not been demonstrated in an adult population. As a consequence, unlike hyper-/hyposensitivity, perceptual inflexibility has not been yet included in a subcategory of the RRB symptoms even in the latest diagnosis criteria for ASD ( ).
This may be due to difficulty in finely quantifying the nonsocial features of autism in adults with ASD. In clinical settings, the RRB symptoms of adults with autism are measured on a relatively coarse scale. For example, in ADOS ( ), the severity of RRB is ranked using a limited range of integers (e.g., 0, 1, or 2 in this study and 0, 1, 2, or 3 in the ABIDE datasets used here; and ). ADOS has been repeatedly validated as a tool to aid diagnosis ( ) and index severity ( ; ) of this autism, but such a relatively sparse scoring system of the RRB symptoms raises the possibility that this scale could not capture nuanced individual differences in cognitive rigidity. Even in behavioral experiments, autistic cognitive rigidity is known to be difficult to detect ( ; ; ; ). In particular, high-functioning adults with ASD often easily adapt themselves to instruction-based TS tests, and occasionally outperform neurotypical individuals ( ; ). In fact, the current instructed TS test found no significant behavioral differences between the ASD and TD groups ( A–D ).
In contrast, the spontaneous TS test detected cognitive rigidity, a part of the RRB symptoms ( ; ; ), in the high-functioning adults with ASD with a relatively large effect size (Cohen's d = 1.4; A , D ). The task repetition length recorded in this TS test provided more detailed information about individual differences in cognitive rigidity and was related to the severity of the RRB symptoms ( C ). In addition, given that previous studies also reported unique behavioral patterns in autistic people using a similar test ( ; ), this psychological paradigm appears to have a robust sensitivity to detect cognitive rigidity in autism. This improved detectability may support our ability to identify neuroanatomical bases related to cognitive rigidity in autism.
In the neuroanatomical investigation, we found that the smaller GMV in the pSPL was associated with both autistic stable perception and cognitive rigidity. In terms of the relationship between the brain area and bistable visual perception, this finding is consistent with previous reports about dissociable functions of the anterior and posterior SPLs. A series of behavioral, neuroimaging, and brain stimulation studies show that the posterior SPL destabilizes visual perception when viewing the structure-from-motion stimulus, whereas the anterior SPL stabilizes it ( , ; ; ). Therefore, the smaller GMV in pSPL should stabilize visual perception, which is consistent with the current observation. The findings in this work strengthen our knowledge about the functional anatomy of the SPL and its role in bistable perception.
The association between the GMV of the pSPL and cognitive rigidity is also consistent with previous reports about neural mechanisms underlying human cognitive flexibility ( ; ; ). The frontoparietal network, including this region, is considered essential for flexible coordination of different types of cognitive skill ( ; ), and several studies reported associations between autistic cognitive inflexibility and atypical neural activity of this brain network ( ; ; ). In addition, a meta-analysis demonstrated a critical role of the SPL in a wide range of TS behaviors ( ), and another neuroimaging study using similar voluntary TS test also suggested the importance of SPL in a voluntary action ( ). These previous findings are consistent with the results of the current structural equation modeling analysis ( G ), in which the pSPL appears to be involved in domain general mental flexibility.
This study found diminished GMV in the pSPL in high-functioning adults with ASD in three independent datasets ( G , A , ), which are consistent with previous findings on the neuroanatomy of autism. Although we have to carefully consider effects of the heterogeneity of ASD ( ; ; ; ), multiple meta-analyses reported significant decreases in the GMVs of the SPL ( ; ) and neighboring parietal regions ( ; ; ) in individuals with ASD. The current findings can be seen as further biological evidence implying the critical role of the pSPL in autism but now linking this finding to specific behaviors.
One of the major limitations of this study is that we did not examine functional brain architecture underlying the autistic perceptual and cognitive inflexibility. Methodologically, this work focused on establishing of a psychological paradigm to detect a link between perceptual and cognitive inflexibility in autism. Based on this behavioral observation, we then searched for preliminary neuroanatomical evidence for the behavioral link. Future research should examine how the pSPL interacts (or fails to interact) with other brain areas during spontaneous task switching in individuals with ASD.
Another limitation is in the relatively small sample size used for the main experiment. The sample size was determined by previous behavioral studies on perceptual stability in autism where the number of participants was sufficient to reject the null hypothesis ( , ). No previous study provided data about how strongly the spontaneous TS test can detect the cognitive rigidity in high-functioning individuals with ASD, but the effect sizes reported here provide a principle basis for determining future sample sizes necessary to identify neural bases for such cognitive rigidity.
It remains unknown how the atypical neuroanatomical development of the SPL in autism ( ) is related to the development of cognitive flexibility. In addition, our findings are not necessarily directly applicable to other sensory symptoms in autism. Seemingly lower-level sensory symptoms, such as hyper-/hyposensitivity, might not involve the SPL area. For example, typical auditory perception might be underpinned by atypical neural architectures in the superior temporal sulcus. At the same time, the SPL may also play a key role in these sensory symptoms because the area is known to integrate a wide range of sensory information ( ) and be involved in the top-down allocation of attention over primary sensory regions (for review, see ). The smaller GMV in adults with ASD reported here may affect how low-level sensory inputs are constrained by top-down expectations ( , ; ). Future studies will be necessary to clarify whether feedback processing from the SPL to sensory regions is associated with perceptual sensitivity.
This case–control study has found a behavioral association between overly stable visual perception and cognitive rigidity in high-functioning adults with ASD. Moreover, we have identified the pSPL as one of the major neuroanatomical bases supporting this behavioral association. These findings should lead to future studies on how the ASD core symptoms interact with perceptual difficulties experienced by individuals with autism, which will help us to comprehensively understand the cognitive and behavioral styles of this disorder.
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Autism is a condition manifested as abnormalities of relatedness, communication, range of interests, and repetitive behaviors. Despite alarming prevalence estimates and exhortations to research, little is known regarding its pathophysiology. Recent reports of a putative minicolumnopathy explain changes in brain size, gray/white matter ratios, and interareal connectivity. This article summarizes possible links between minicolumns and other topics-cortical modularity, age of onset, gliosis, and genetics-relevant to the pathophysiology of autism. |
Recently developed fMRI can map small functional structures noninvasively and repeatedly without any depth limitation. However, there has been a persistent concern as to whether the high-resolution fMRI signals actually mark the sites of increased neural activity. To examine this outstanding issue, the authors used iso-orientation columns of isoflurane-anesthetized cats as a biological model and confirmed the neural correlation of fMRI iso-orientation maps by comparing them with intrinsic optical imaging maps. The results suggest that highest fMRI signals indeed indicate the sites of increased neuronal activity. Now fMRI can be used to determine plastic and/or developmental change of functional columnar structure possibly on a layer-to-layer basis. In this review, the authors focus mainly on what technical aspects should be considered when mapping functional cortical columns, including imaging techniques and experimental design. |
[(18)F]Fluoro-3,4-dihydroxyphenyl-L-alanine (FDOPA) was one of the first successful tracers for molecular imaging by positron emission tomography (PET), and has proven immensely valuable for studies of Parkinson's disease. Following intravenous FDOPA injection, the decarboxylated metabolite [(18)F] fluorodopamine is formed and trapped within terminals of the nigrostriatal dopamine neurons; reduction in the simple ratio between striatum and cerebellum is indicative of nigrostriatal degeneration. However, the kinetic analysis of dynamic FDOPA-PET recordings is formidably complex due to the entry into brain of the plasma metabolite O-methyl-FDOPA and due to the eventual washout of decarboxylated metabolites. Linear graphical analysis relative to a reference tissue input function is popular and convenient for routine clinical studies in which serial arterial blood samples are unavailable. This simplified approach has facilitated longitudinal studies in large patient cohorts. Linear graphical analysis relative to the metabolite-corrected arterial FDOPA input yields a more physiological index of FDOPA utilization, the net blood-brain clearance. Using a constrained compartmental model, FDOPA-PET recordings can be used to calculate the relative activity of the enzyme DOPA decarboxylase in living brain. We have extended this approach so as to obtain an index of steady-state trapping of [( 18)F]fluorodopamine in synaptic vesicles. Although simple methods of image analysis are sufficient for the purposes of routine clinical studies, the more complex approaches have revealed hidden aspects of brain dopamine in personality, healthy aging, and in the pathophysiologies of Parkinson's disease and schizophrenia. |
The prevalence of vascular cognitive impairment (VCI) is likely to increase as the population ages and cardiovascular disease survival improves. We provide an overview of the definition and disease mechanisms of VCI and present a systematic literature review of the current evidence for the pharmacologic and nonpharmacologic therapies used to treat the VCI symptoms of cognitive dysfunction or to modify VCI through primary and secondary prevention. The Cochrane Database of Systematic Reviews was searched from 2005 to October 2010 using the keywords "vascular dementia" or "vascular cognitive impairment and therapy." MEDLINE was searched for English-language articles published within the last 10 years using the combined Medical Subject Headings (MeSH) "therapeutics and dementia," "vascular" or "vascular cognitive impairment." Although cholinesterase inhibitors and memantine produce small cognitive improvements in patients with VCI, these drugs do not improve global clinical outcomes and have adverse effects and costs. Selective serotonin reuptake inhibitors and dihydropyridine calcium channel blockers may improve short-term cognitive function in patients with VCI. Anti-hypertensive therapy with an ACE inhibitor-based regimen and statins may prevent the major subtype of VCI known as poststroke cognitive decline. Clinical and effectiveness studies with long-term follow-up are needed to determine the benefits and risks of pharmacologic and nonpharmacologic therapies to prevent and treat VCI. Given its growing health, social, and economic burden, the prevention and treatment of VCI are critical priorities for clinical care and research. |
The human genome encodes tens of thousands of long non-coding RNAs (lncRNAs), a novel and important class of genes. Our knowledge of lncRNAs has grown exponentially since their discovery within the last decade. lncRNAs are expressed in a highly cell- and tissue-specific manner, and are particularly abundant within the nervous system. lncRNAs are subject to post-transcriptional processing and inter- and intra-cellular transport. lncRNAs act via a spectrum of molecular mechanisms leveraging their ability to engage in both sequence-specific and conformational interactions with diverse partners (DNA, RNA, and proteins). Because of their size, lncRNAs act in a modular fashion, bringing different macromolecules together within the three-dimensional context of the cell. lncRNAs thus coordinate the execution of transcriptional, post-transcriptional, and epigenetic processes and critical biological programs (growth and development, establishment of cell identity, and deployment of stress responses). Emerging data reveal that lncRNAs play vital roles in mediating the developmental complexity, cellular diversity, and activity-dependent plasticity that are hallmarks of brain. Corresponding studies implicate these factors in brain aging and the pathophysiology of brain disorders, through evolving paradigms including the following: (i) genetic variation in lncRNA genes causes disease and influences susceptibility; (ii) epigenetic deregulation of lncRNAs genes is associated with disease; (iii) genomic context links lncRNA genes to disease genes and pathways; and (iv) lncRNAs are otherwise interconnected with known pathogenic mechanisms. Hence, lncRNAs represent prime targets that can be exploited for diagnosing and treating nervous system diseases. Such clinical applications are in the early stages of development but are rapidly advancing because of existing expertise and technology platforms that are readily adaptable for these purposes. |
Patient-funded research has started to emerge in multiple sclerosis studies, such as low-dose naltrexone and stem-cell therapy. While these represent greater opportunities for the physician, scientist, and patient, ethical concerns concerning protocol review, conflict of interests, and protection of subjects are reviewed. |
Repeated sessions of cerebellar anodal transcranial direct current stimulation (tDCS) have been suggested to modulate cerebellar-motor cortex (M1) connectivity and decrease ataxia severity. However, therapeutic trials involving etiologically homogeneous groups of ataxia patients are lacking. The objective of this study was to investigate if a two-week regimen of daily cerebellar tDCS sessions diminishes ataxia and non-motor symptom severity and alters cerebellar-M1 connectivity in individuals with spinocerebellar ataxia type 3 (SCA3). We conducted a randomized, double-blind, sham-controlled trial in which twenty mildly to moderately affected SCA3 patients received ten sessions of real or sham cerebellar tDCS (i.e., five days per week for two consecutive weeks). Effects were evaluated after two weeks, three months, six months, and twelve months. Change in Scale for the Assessment and Rating of Ataxia (SARA) score after two weeks was defined as the primary endpoint. Static posturography, SCA Functional Index tests, various patient-reported outcome measures, the cerebellar cognitive affective syndrome scale, and paired-pulse transcranial magnetic stimulation to examine cerebellar brain inhibition (CBI) served as secondary endpoints. Absolute change in SARA score did not differ between both trial arms at any of the time points. We observed significant short-term improvements in several motor, cognitive, and patient-reported outcomes after the last stimulation session in both groups but no treatment effects in favor of real tDCS. Nonetheless, some of the patients in the intervention arm showed a sustained reduction in SARA score lasting six or even twelve months, indicating interindividual variability in treatment response. CBI, which reflects the functional integrity of the cerebellothalamocortical tract, remained unchanged after ten tDCS sessions. Albeit exploratory, there was some indication for between-group differences in SARA speech score after six and twelve months and in the number of extracerebellar signs after three and six months. Taken together, our study does not provide evidence that a two-week treatment with daily cerebellar tDCS sessions reduces ataxia severity or restores cerebellar-M1 connectivity in early-to-middle-stage SCA3 patients at the group level. In order to potentially increase therapeutic efficacy, further research is warranted to identify individual predictors of symptomatic improvement. |
Episodic ataxia type 1 is a paroxysmal neurological disorder characterized by short-lived attacks of recurrent midline cerebellar dysfunction and continuous motor activity. Mutations in KCN1A, the gene encoding Kv1.1, a voltage-gated neuronal potassium channel, are associated with the disorder. Although rare, the syndrome highlights the fundamental features of genetic ion-channel diseases and serves as a useful model for understanding more common paroxysmal disorders, such as epilepsy and migraine. This review examines our current understanding of episodic ataxia type 1, focusing on its clinical and genetic features, pathophysiology, and treatment. |
## Introduction
The field of movement disorder management and therapeutics continues to evolve and change at a remarkable pace. Advances in our understanding of the underlying biology of diseases, new capabilities in genetics, and improved approaches to diagnosis, including biomarkers and imaging, provide clinicians today with a growing array of options to help improve the lives and outcomes of our patients (see Table ). For this special edition of Neurotheraputics , we are fortunate to include twenty-seven contributions from experts leading the development and application of these new therapeutic options in movement disorders. Each can stand alone to serve as a comprehensive resource for a particular topic, but together they provide an outstanding and detailed synopsis of current therapeutics in the field. Many of the chapters focus on Parkinson’s disease, but more than a third of the reviews highlight the remarkable transformation in therapeutic options for other conditions - perhaps most notably Huntington’s disease and the tauopathies. Despite the progress highlighted, there still remains a great unmet need for improved treatments. The diseases we manage still lack neuroprotective or disease-modifying approaches, let alone cures. With an aging world population and prevalence increasing for the most common of these conditions [ ], the urgency has never been greater to move beyond current therapeutics to relieve suffering and the burden of disease.
US FDA approvals for drugs and devices for movement disorders, 1/1/2016–11/30/2020
New drugs, new class III devices, new drug formulations and expanded use indications for movement disorders approved for use in the USA between January 2016 and November 2020 are presented in the table
5HT = 5 hydroxytryptamine; VMAT = vesicular monoamine transporter; MAOB = monoamine oxidase B; DBS = deep brain stimulation; COMT = catechol-O-methyltransferase
FDA CDER review designations: First in class—drugs with a new and unique mechanism for treating a medical condition; Orphan—drugs approved for small populations of patients with rare diseases. Regulatory methods to expedite development and approval of novel drugs: Fast track—drugs that can treat unmet medical needs; Breakthrough—a drug with preliminary clinical evidence demonstrating that it may result in substantial improvement on at least one clinically significant endpoint over other available therapies; Priority Review—is given if the drug could potentially provide a significant advance in medical care; Accelerated approval—early approval based on markers that predict a reasonable benefit, with more testing to confirm clinical benefit after approval. Other: First indication; Expanded use
Source:
## Parkinson’s Disease Therapeutics
This issue of the Journal first focuses on nonsurgical approaches to treatment of the motor features of Parkinson’s disease. With the ever growing therapeutic options available to treat PD, determining the optimal therapeutic regimen for an individual patient has become increasingly complicated. Sy and Fernandez [ ] review pharmacological strategies to consider when motor symptoms are sufficient to warrant treatment. There are many approved monotheraputic agents to choose from in early PD, requiring an individualized approach to optimize risk reduction while providing effective relief from symptoms. Next Aradi and Hauser [ ] discuss the clinical features, epidemiology, and risk factors for the development of motor fluctuations and dyskinesia, which are commonly seen with short-acting medications as the disease advances. They also provide a helpful review of strategies to consider to maximize motor function and limit dyskinesia—a common clinical dilemma. Marras and colleagues [ ] review current approaches to understanding PD subtypes including motor phenotype, age, nonmotor dominant symptoms, and genetic forms. There is much enthusiasm for these emerging data-driven approaches to better define PD subtypes based on clinical features and biomarkers with the hope of translating this into patient-specific therapeutic approaches. Schneider et al. [ ] follow with a more focused description of emerging targeted therapeutics for specific genetic subtypes of parkinsonism. The first precision medicine approaches for treatment in PD may apply to GBA-associated and LRRK2-associated forms of the disease. An update on other emerging PD therapeutic approaches being explored that may influence disease progression is provided by Drs. Schwarzschild and Hung [ ]. Novel immunotherapies and drugs targeting misfolded α-synuclein, c-Abelson tyrosine kinase (cABl), Glucagon-like peptide-1 (GLP-I) agonists, calcium channel blockers, nicotine pathways, and reactive oxygen species are all under development and offer hope to the field despite the long history of multiple failed disease-modifying therapeutic compounds to date. Next, our current understanding about the understudied, yet likely important, influence of the gut microbiome in PD is highlighted by Brown and Goldman including strategies now under study to modulate the microbiome to improve the effects of PD medications absorbed through the gut [ ]. Schootemeijer et al. [ ] provide a wonderful summary of the role of aerobic exercise in PD. They present evidence including a meta-analysis, which supports the notion that aerobic exercise has general health benefits, improves physical fitness, and attenuates motor symptoms in PD. Dosing of exercise is important and compliance issues remain a challenging barrier. Complementary and alternative medicine approaches have never been more popular in the PD community, including acupuncture, tai qi, yoga, meditation, and cannabis. Deuel and Seeberger [ ] review for us the latest in clinical trials evaluating the effectiveness of these complementary treatments, which have a newly appreciated role in management of PD. The tremendous benefit of palliative care methods in Parkinson’s spectrum disorders is thoughtfully reviewed by Katz [ ]. Rather than reserving palliative approaches for late-stage disease, she emphasizes the value of this multidisciplinary approach for patients and caregivers at any stage of disease.
The PD field has increasingly recognized the enormous impact nonmotor PD symptoms have on quality of life and the historically limited therapeutic options available to manage them. We have included four articles that each address current and emerging management of aspects of nonmotor symptoms that greatly increase the burden of disease in PD. The broad clinical spectrum of autonomic dysfunction is discussed by Pfeiffer et al. [ ]. Importantly, if these symptoms are identified, they can often be managed with the many available non-PD specific approaches. Sleep disorders are common in PD, but under-diagnosed, as Zuzuarregui and During report in their comprehensive review [ ]. They cover the current understanding and approaches to treatment of REM sleep behavior disorders (RBD), restless legs syndrome (RLS), obstructive sleep apnea (OSA) and circadian rhythm dysfunction, noting that treating these disorders can significantly improve quality of life. Perhaps one of the greatest unmet needs in the field of PD is the prevention and management of cognitive decline and dementia. Uc et al. describe strategies to diagnose and treat this very difficult nonmotor aspect of PD [ ]. Weintraub discusses the increasingly complicated topic of management of psychiatric disorders in PD, another area with significant impact on quality of life [ ]. Psychiatric disorders are still under-recognized and undertreated in PD, and even though psychotropic medications are commonly used, we lack randomized controlled trials demonstrating efficacy and tolerability specific to PD.
Rounding out the chapters on Parkinson’s disease therapeutics are two that focus on surgical approaches. Surgical treatment approaches have transformed the management of moderate to advanced PD care and, besides the discovery of levodopa, represent one of the greatest therapeutic breakthroughs for the field. Sharma et al. review the latest in DBS and lesioning approaches in PD including indications, patient selection, and management strategies and help us to appreciate how this rapidly changing area is becoming more complex with application of multiple DBS systems, improved technology, and leveraging a more sophisticated understanding of PD pathophysiology [ ]. Buttery and Barker end the PD section on a hopeful note, with a wonderful review of where we are now with gene- and cell-based therapies [ ], many of which are in ongoing high-profile clinical trials. They highlight the great promise for these approaches, some of which may allow for slowing progression of the disease or medication reduction, without the need for implanted hardware.
## Therapeutics in Other Movement Disorders
The next section of this issue of the Journal includes manuscripts that highlight current therapeutics for movement disorders other than Parkinson’s disease. Treatment of other forms of neurodegenerative parkinsonism has long been fraught with challenges. Many of these disorders are relatively rare, and clinical syndromes may be varied despite similar pathology, making both diagnosis and treatment challenging. An example is the 4 repeat tauopathies, where clinical syndromes may be predominantly motor or behavioral, or any combination. Following an excellent pragmatic overview of currently available options for symptomatic treatment in the four-repeat tauopathies, VandeVrede and colleagues provide hope for disease-modifying therapies in their comprehensive review of clinical trials [ ]. Treatment of multiple system atrophy (MSA), an α-synucleinopathy, provides similar challenges in clinical heterogeneity. Burns and McFarland provide a useful guide to diagnosis and treatment of the core motor symptoms of the several clinical syndromes of MSA, importantly emphasizing the value of a multidisciplinary care team in providing effective therapy that integrates nonpharmacologic management and drug treatment. Their review of current trials to identify disease-modifying therapies provides hope for more effective interventions in the future [ ]. Hopfner and Deuschl review the two-axis Movement Disorder Society (MDS) classification for essential tremor syndrome, providing a foundational approach to determining therapeutics in this common but heterogeneous movement disorder [ ]. They provide a comprehensive overview of nonpharmacologic, pharmacologic, and surgical treatments, and a useful framework for determining a patient-specific therapeutic plan. Dystonia comprises a heterogeneous group of syndromes with diverse etiologies. Therapeutic approaches to dystonia are equally broad. Bledsoe et al. provide a masterful overview of the many existing and emerging therapies for dystonia, ranging from pathogenesis-targeted therapies to symptomatic treatments with drugs or botulinum toxin, surgical therapies, noninvasive stimulation, and rehabilitation [ ]. They highlight the need for an individualized approach to each patient and provide a helpful starting point for clinicians. Next, Dash and Mestre discuss the rapidly developing therapeutic options for Huntington’s disease including symptomatic treatments and treatments that target specific pathways in the HD biology [ ]. Novel huntingtin protein–lowering therapies using antisense oligonucleotides, RNA interference, small-molecule splicing modulators, and zinc-finger protein transcription factor approaches are likely to bring the first ever disease-modifying therapy to patients with HD. They also address the need for specific biomarkers, improved rating scales, and development of other tools to identify disease progression necessary to move therapeutic advances for the disease forward. Then we turn to the latest medication strategies and emerging therapies for ataxia, which are highlighted by Perlman [ ]. While there still remain no FDA-approved therapies for ataxia, a hopeful pipeline of disease-modifying therapies is discussed. In the meantime, multiple drugs are available for symptomatic management of coexisting symptoms, and there are many medications that are used to treat ataxia off-label. Pena and Caviness then review a physiology-based treatment of myoclonus, a symptom that is often resistant to treatment. Myoclonus is currently best managed by identifying the underlying etiology and using medications that best address the neurophysiological subtype [ ]. Tourette syndrome is a heterogeneous neurobehavioral disorder, which develops in childhood, presenting with motor and phonic tics but frequently accompanied by behavioral comorbidities that require a tailored management approach. Billnitzer and Jankovic highlight current behavioral, pharmacologic, and surgical treatment strategies and also review therapeutics in development for Tourette syndrome [ ]. In recent years, new strategies have emerged for management of tardive syndromes (TS), which are highlighted in an excellent review by Factor [ ]. Covered are the pathogenesis, clinical features, and epidemiology, followed by a comprehensive discussion of use of the new vesicular monoamine transporter type 2 inhibitors recently approved by the FDA. Other medications, botulinum toxin and surgical therapies can also contribute in the management of this common syndrome, but hopefully, future efforts will focus on the need for prevention and/or reversal of this iatrogenic movement disorder. Deputy and Tilton review common management strategies for pediatric movement disorders not covered in previous chapters, largely focusing on disorders of tone and cerebral palsy but also highlighting disorders representative of these heterogeneous disorders, including glucose transporter type 1 (Glut-1) deficiency, Sydenham’s chorea and neurodegeneration with brain iron accumulation (NBIA) [ ]. They provide a useful discussion of the unique aspects of management of children with movement disorders.
## Other Novel Factors and Approach to Therapeutics
The two final reviews in this special edition address and highlight other novel factors that influence today’s clinical trials. Vaswani et al. describe the critical issue of overcoming barriers to Parkinson’s disease trial participation by using novel trial designs for recruitment and ways to increase participation and diversity [ ]. While they focus on Parkinson’s disease, their insights are generally applicable to all movement disorders. Lastly, Artusi et al. provide a fascinating look at current mobile health technologies, their use in clinical trials today, and the great potential for these approaches to revolutionize the field of movement disorders clinical care and clinical trials in the near future [ ].
## Conclusion/Final Words
Overall, the reviews in this issue provide a hopeful outlook for movement disorder therapeutics in the next decade. Improved symptomatic therapies, disease-modifying interventions, and approaches to disease prevention are in reach for many disorders. We wish to thank all the authors for their outstanding contributions to this issue, especially during the challenging times of the COVID-19 pandemic and its impact. We also thank the editorial team at Neurotherapeutics and, in particular, Maral Mouradian and Linda Powell, for their advice and support. We are humbled and honored to serve as coeditors.
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## Supplementary Information
|
Chemotherapy-induced neuropathy (CIN) is a major dose-limiting side effect of anticancer therapy that can compel therapy discontinuation. Inadequate analgesic efficacy of current pharmacological approaches requires the identification of innovative therapeutics and, hence, the purpose of this study is to conduct a preclinical evaluation of the efficacy of DDD-028, a versatile pentacyclic pyridoindole derivative, against paclitaxel-induced neuropathic pain. In two separate experiments, DDD-028 was administered per os acutely (1–25 mg kg ) or repeatedly (10 mg kg ) in paclitaxel-treated rats. The response to mechanical noxious stimulus (paw pressure) as well as to non-noxious mechanical (von Frey) and thermal (cold plate) stimuli was investigated. Acute administration of DDD-028 induced a dose-dependent anti-neuropathic pain effect in all tests performed. Further, repeated daily treatment for 18 consecutive days (starting the first day of paclitaxel administration) significantly reduced the development of pain over time without the development of tolerance to the anti-hyperalgesic effect. Ex vivo analysis showed that DDD-028 was able to reduce oxidative damage of dorsal root ganglia as evidenced by the increase in the level of carbonylated proteins and the decrease in catalase activity. In the lumbar spinal cord, periaqueductal gray matter, thalamus, and somatosensory cortex 1, DDD-28 significantly prevented the activation of microglia and astrocytes. The pharmacodynamic study revealed that the pain-relieving effects of DDD-028 were fully blocked by both the non-selective nicotinic receptor (nAChR) antagonist mecamylamine and by the selective α7 nAChR antagonist methyllycaconitine. In conclusion, DDD-028 was active in reducing paclitaxel-induced neuropathic pain after single or repeated administrations without tolerance development and displaying a double symptomatic and neuroprotective profile. DDD-028 could represent a valuable candidate for the treatment of CIN.
## Supplementary Information
The online version contains supplementary material available at 10.1007/s13311-021-01069-8.
## Introduction
The success of cancer treatments is frequently limited by neuropathies which represent a major health concern [ ]. Neuropathy resulting from chemotherapy can be disabling, causing a significant functional loss and decreasing the quality of life. Moreover, neuropathy is the predominant reason for dose modification and discontinuation of treatment, and may thereby affect overall survival [ ]. Paclitaxel is an antineoplastic agent originally derived from the bark of the western yew tree, Taxus brevifolia , and one of the most effective and widely used drug in several solid tumors, including neck, lung, breast, head, and ovarian cancers and AIDS-related Kaposi’s sarcoma [ – ]. Unfortunately, paclitaxel induces sensory peripheral neuropathy characterized by burning pain symptoms, allodynia, hyperalgesia, tingling, and numbness. Neuropathy is positively correlated with increasing number of paclitaxel doses per cycle, total cumulative dose, and duration of infusion and can persist for months or years following the cessation of treatments [ ]. Grade 3 or 4 sensory neuropathy occurs in 20–35% of patients receiving 250 mg/m paclitaxel every 3 weeks [ ]. Several attempts have been made to treat or prevent CIN with various neuroprotective drugs, but the results are contradictory and most of them are either ineffective or caused adverse effects such as nausea, reflex dysfunctions, treatment-emergent nervousness, insomnia, tremor, anorexia, or stomach burning [ – ]. Commonly used anti-neuropathic treatments, such gabapentin, lamotrigine, or pyridoxine plus pyridostigmine, have not shown efficacy in random clinical trials [ ]. Amitriptyline or oxycodone only diminishes the pain symptom associated with taxane-induced neuropathy. Therefore, it is crucial to identify novel, safe therapeutic strategies that may efficiently prevent or suppress both the painful condition and damage to the nervous system.
Accordingly, our extensive work on pentacyclic pyridoindole heterocycles such as scaffold A (Fig. ) [ – ] resulted in the identification of a potent, non-opioid analgesic, DDD-028, for the potential treatment of CIN. The chemical properties of DDD-028 are summarized in Table [ – ]. The purpose of the study was to evaluate the effect of acute and sub-chronic administrations of DDD-028 on paclitaxel-induced neuropathic pain in rats. The relief of pain hypersensitivity as well as the protective effects on the peripheral and central nervous system along with the pharmacodynamic profile will be presented herein.
Structure of DDD-028
Chemical and preliminary safety properties of DDD-028
## Materials and Methods
### Animals
For all the experiments described below, male Sprague–Dawley rats (Envigo, Varese, Italy) weighing approximately 200–250 g at the beginning of the experimental procedure were used. Animals were housed in CeSAL (Centro Stabulazione Animali da Laboratorio, University of Florence) and used at least 1 week later after their arrival. Four rats were housed per cage (size 26 × 41 cm ), kept at 23 ± 1 °C with a 12-h light/dark cycle, light at 7 a.m, and were fed with standard laboratory diet and tap water ad libitum. All animal studies were carried out according to the Directive 2010/63/EU of the European parliament and of the European Union council (22 September 2010) on the protection of animals used for scientific purposes. The ethical policy of the University of Florence complies with the Guide for the Care and Use of Laboratory Animals of the US National Institutes of Health (NIH Publication No. 85–23, revised 1996; University of Florence assurance number: A5278-01). Formal approval to conduct the experiments described was obtained from the Italian Ministry of Health (No. 498/2017-PR) and from the Animal Subjects Review Board of the University of Florence. Experiments involving animals have been reported according to ARRIVE guidelines [ ]. All efforts were made to minimize animal suffering and to reduce the number of animals used.
### Paclitaxel Rat Model of Neuropathy
Paclitaxel was dissolved in a mixture of 10% saline solution and Chremophor EL, a derivative of castor oil and ethylene oxide that is clinically used as paclitaxel vehicle. Rats were injected intraperitoneally (i.p.) with paclitaxel (2.0 mg kg ) on four alternate days (days 1, 3, 5, and 8) [ , ]. Control animals received an equivalent volume of the vehicle.
### DDD-028 Administration and Study of the Pharmacodynamic Mechanisms
To evaluate its symptomatic efficacy, DDD-028 [ – ] was suspended in 1% carboxymethylcellulose (CMC) and acutely administered per os with the dose ranging from 1 to 25 mg kg on day 10, when paclitaxel neuropathy was well established. Thereafter, to demonstrate a protective effect, repeated per os administrations of DDD-028 10 mg kg were carried out daily from the beginning of the paclitaxel administration (day 1) to the end of the experiment (day 18). Control animals were treated with vehicles.
To determine possible pharmacodynamic mechanisms, the following compounds were administered along with DDD-028: pan nicotinic receptor antagonist mecamylamine (MECA; 2 mg kg , i.p.) [ , ], selective α7 nAchR antagonist methyllycaconitine (MLA; 6 mg kg , i.p.)[ ], Kv7 channel blocker XE991 (1 mg kg , i.p.) [ , ], and σ σ agonist (method described below). For MECA, 2 protocols were adopted: in the first, a single dose of MECA was administered 15 min before DDD-028 injection, an in the second, two doses of MECA were given, 15 min before DDD-028 and 45 min after DDD-028 treatment. MLA and XE991 were administered once, 15 min before DDD-028 treatment. Control animals were treated with vehicles. To assess the effect of σ /σ receptors on the analgesic activity of DDD-028, the σ modulation of movements and posture were used. It is known that σ receptors are concentrated in brain structures that control movement, such as the red nucleus and substantia nigra. The unilateral microinjection of σ receptor agonists, such as 1,3-di-(2-tolyl)guanidine (DTG), ( +)-N-allylnormetazocyne [( +)-SKF-10,047], and ( +)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine [( +)-3-PPP], into the red nucleus induces neck dystonia in rats. The antidystonic effect observed in rats was shown to be related to σ receptor antagonism [ ]. The neck dystonia was quantified by measuring the torsion of the neck. Briefly, the torticollis was quantified by measuring the torsional deviation of the head from the horizontal plane, using the eyes of the animals as a reference. Each rat was tested only once to minimize the damage to brain tissue. Rats were anesthetized with a combination of ketamine and xylazine and placed in a stereotaxic apparatus. An incision was made at the midline of the head from slightly posterior to the eyes toward the base of the skull to sufficiently expose the cranium for identification of the landmarks, bregma and lambda . The “flat skull” position was achieved by adjusting the incisor piece until bregma and lambda were of similar height (± 0.2 mm), after which bregma was used to establish the stereotaxic position of the red nucleus. A small hole in the cranium was made with a dental drill, and the dura mater was carefully removed. A guide cannula was place 2.0 mm above the left red nucleus, using the following coordinates: 5.8 mm posterior to bregma, 0.7 mm lateral, 7.2 mm below the cortical surface [ ]. A stainless steel wire was used to plug the guide cannula that was secured with dental cement and skull screws.
After at least a 24-h recovery time, an injection cannula was insert into the red nucleus through the guide cannula. The location of injection cannula probes was confirmed histologically by an examination of brain slice sections. Fifteen min after the administration of 25 mg kg DDD-028 or vehicle, rats received a single microinjection of DTG (Sigma-Aldrich) 5 nmol/0.5 µl and were photographed every 5 min for 30 min. The volume of 0.5 µl was injected over a 1-min period.
### Paw Pressure Test
The nociceptive threshold in the rat was determined with an analgesimeter (Ugo Basile, Varese, Italy) according to the method described by Leighton et al. [ ]. Briefly, a constantly increasing pressure was applied to a small area of the dorsal surface of the hind paw using a blunt conical mechanical probe. Mechanical pressure was increased until vocalization or a withdrawal reflex occurred while rats were lightly restrained. Vocalization or withdrawal reflex thresholds were expressed in grams. These limits assured a more precise determination of mechanical withdrawal threshold in experiments aimed to determine the effect of treatments. An arbitrary cutoff value of 100 g was adopted.
### von Frey Test
The animals were placed in 20 × 20 cm Plexiglas boxes equipped with a metallic mesh floor, 20 cm above the bench. A habituation of 15 min was allowed before the test. An electronic Von Frey hair unit (Ugo Basile, Varese, Italy) was used: the withdrawal threshold was evaluated by applying force ranging from 0 to 50 g with an accuracy of 0.2 g. Punctuate stimulus was delivered to the mid-plantar area of each anterior paw from below the mesh floor through a plastic tip, and the withdrawal threshold was automatically displayed on the screen. Paw sensitivity threshold was defined as the minimum pressure required to elicit a robust and immediate withdrawal reflex of the paw. Voluntary movements associated with locomotion were not taken as a withdrawal response. Stimuli were applied on each anterior paw with an interval of 5 s. The measure was repeated 5 times, and the final value was obtained by averaging the 5 measures [ ].
### Cold Plate Test
Thermal allodynia was assessed using the cold plate test. With minimal animal-handler interaction, rats were taken from home-cages, and placed onto the surface of the cold-plate (Ugo Basile, Varese, Italy) maintained at a constant temperature of 4 ± 1 °C. Ambulation was restricted by a cylindrical Plexiglas chamber (diameter 10 cm, height 15 cm), with open top. A timer controlled by foot pedal began timing response latency from the moment the mouse was placed onto the cold plate. Pain-related behavior (licking of the hind paw) was determined by recording the time (seconds) of the first sign of licking of the hind paw. The cutoff time of the latency of paw lifting or licking was set at 30 s [ , ].
### Open Field Test
Rats were placed into the center of the arena, brightly lit (1000 lx). The total distance travelled, the mobility time, the time spent by animals in the center, and in the periphery of the arena within 10 min of observation were recorded [ ].
### Tissue Collection
On day 18 (i.e., the end of the experiments on the assessment a protective effect of DDD-028), animals were sacrificed by decapitation. L4-L5 dorsal root ganglia (DRG), sciatic nerve, lumbar spinal cord, and brain were collected, frozen using liquid nitrogen or fixed by immersion in 4% neutral buffered formalin.
### Immunohistochemistry of Brain and Spinal Cord
Formalin fixed cryostat Sects. (10 μm for brain and 5 μm for spinal cord) were incubated for 1 h in blocking solution (Bio-Optica; Italy) at room temperature, and thereafter, sections were incubated for 24 h at 4 °C in PBST containing primary antisera and 5% normal donkey serum. The primary antibody was directed against Iba1 (rabbit antiserum, 1:500; Wako Chemicals, USA [ ]) for microglial staining and against glial fibrillary acidic protein (GFAP; rabbit antiserum, 1:500; Dako, USA [ ]) for astrocyte staining. After rinsing in PBST, sections were incubated in donkey anti-rabbit IgG secondary antibody labelled with Alexa Fluor 488 or 568 (1:1000, Invitrogen, USA) at room temperature for 1 h.
For all immunohistochemical studies, negative control sections (no exposure to the primary antisera) were processed concurrently with the other sections.
A single optical density value for the dorsal horns in each rat was obtained by averaging the two sides, and this value was compared to the homologous average values from the vehicle-treated animals.
### Quantitative Analyses of Iba1 and GFAP Immunohistochemistry
Images were acquired using a motorized Leica DM6000B microscope equipped with a DFC350FX camera. Morphological examination of microglia and astrocytes was assessed by inspection of at least three fields (× 40 0.75NA objective) in the dorsal horn of the spinal cord and brain areas per section. The full specimen thicknesses were acquired as z-stack series, deconvolved using Huygens Professional software (SVI, The Netherlands) and displayed using ImageJ software.
Quantitative analysis of GFAP- and Iba1-positive cells was performed by collecting at least three independent fields through a × 20 0.5NA objective. GFAP-positive cells were counted using the “cell counter” plugin of ImageJ, whereas Iba1-positive cells were quantified by means of the automatic thresholding and segmentation features of ImageJ. Quantification of GFAP signal in immunostained sections was also performed using FIJI software by automatic thresholding images with the aid of the “Moments” algorithm, which delivered the most consistent pattern recognition across all acquired images.
### Carbonylated Protein Evaluation
Sciatic nerve and DRGs protein extracts were quantified by bicinchoninic acid. Five micrograms of each sample were denatured by 6% SDS and derivatized by 15-min incubation with 2, 4 dinitrophenyl hydrazine (DNPH; Sigma-Aldrich, Italy) at room temperature. Samples were separated on a 4–12% sodium dodecyl sulfate (SDS)-polyacrylamide gel by electrophoresis and transferred onto nitrocellulose membranes (Biorad, Italy). Membranes were blocked with 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) containing 1% Tween 20 (PBST) and then probed overnight with primary antibody specific versus DNPH (Sigma-Aldrich, Italy) 1:5000 in PBST/1% BSA. After washing with PBST, the membranes were incubated for 1 h in PBST containing the appropriate horseradish peroxidase-conjugated secondary antibody (1:5000; Cell Signalling, USA) and again washed. ECL (Pierce, USA) was used to visualize the peroxidase-coated bands. Densitometric analysis was performed using the “Image J” analysis software, and the density of all bands displayed in the lane is reported as a mean. Ponceau-stained membranes were used as loading control [ ].
### Catalase Activity
Enzymatic activity in both DRGs and sciatic nerve was measured in PBS using the homogenated tissues: the suspension was sonicated in ice using three 10 s bursts at high intensity with a 10-s cooling period between each burst and then centrifuged (13.000 × g for 15 min at 4 °C). Catalase activity was measured in the supernatant by Amplex Red Catalase Assay Kit (Invitrogen, Monza, Italy) following the manufacturer’s instructions. Protein concentration was quantified by bicinchoninic acid assay (Sigma-Aldrich, Milan, Italy). Catalase activity for each sample was normalized to protein concentration. Control conditions in the absence of treatment were set as 100% [ ].
### Statistical Analysis
The results were expressed as mean ± SEM, and data were analyzed using the “Origin 9.1” software. Statistical analysis was performed using one-way ANOVA followed by post hoc Bonferroni’s significant difference procedure. P values of less than 0.05, 0.01, or 0.001 were considered significant. All data were collected by an observer who was blinded to the treatments.
### Results
Acute analgesic effects (mechanical hyperalgesia, thermal and mechanical allodynia) of DDD-028 are shown in Fig. a–c. For these studies, DDD-028 was acutely per os administered when neuropathy was well established (day 10), 48 h after the last chemotherapeutic drug injection. On day 10, paclitaxel-treated rats showed a significant reduction of the weight tolerated on posterior paws with respect to the control animals (43.2 ± 0.5 g vs 66.5 ± 0.7 g, respectively) (Paw pressure test; Fig. a). Increasing doses of DDD-028 (1–25 mg kg ) reduced mechanical hypersensitivity in a dose-dependent manner starting 30 min after treatment. The highest dose of DDD-028 (25 mg kg ) completely abrogated paclitaxel-induced mechanical hyperalgesia with maximum analgesic effect occurring at 30 min. The effect persisted for at least 90 min and then vanished 120 min after treatment. Strong, albeit slightly reduced, analgesic effect was also observed at the doses of 10, 5, and 1 mg kg , but the duration of the analgesic effect was reduced from about 30–90 min at 25 mg kg to about 30–60 min at 1 mg kg (Fig. a). In the same way, acute administration of DDD-028 counteracted paclitaxel-induced mechanical allodynia in a dose-dependent manner in the von Frey test (Fig. b). The highest dose again showed a complete reversal of paclitaxel-induced neuropathy with a long-lasting effect starting from 30 up to 90 min after treatment. All the lower doses of DDD-028 displayed a shorter anti-hyperalgesic efficacy (Fig. b). Finally, DDD-028 also counteracted paclitaxel-induced thermal allodynia in a dose-dependent manner in the Cold Plate test (Fig. c). As shown in Fig. c, paclitaxel alone significantly enhanced the sensitivity to cold after 10 days of treatment. Thermal allodynia was fully alleviated by DDD-028 (25 mg kg ) administration. The result obtained with the higher dose was more effective and long-lasting with respect to the lower doses that were capable anyway to reach the statistical significance peaking 45 min after administration (Fig. c).
Effect of single DDD-028 administrations on pain behavior induced by paclitaxel. Sensitivity to a noxious mechanical stimulus as measured by the paw pressure test ( a ). Pain threshold to a non-noxious mechanical stimulus as measured by the von Frey test ( b ). Pain threshold to a non-noxious thermal stimulus as measured by the cold plate test ( c ). Paclitaxel (2.0 mg kg , i.p.) was administered on four days (1, 3, 5, and 8). Starting from day 10, DDD-028 was acutely per os administered (1–25 mg kg ) and measurements assessed before treatment and 15, 30, 45, 60, 90, and 120 min after injection. Results were expressed as mean ± SEM of 8 rats analyzed in 2 different experimental sets. ** P < 0.01 vs vehicle + vehicle; ^^ P < 0.01 vs paclitaxel + vehicle
To study the pharmacodynamics of DDD-028, three possible targets were hypothesized based on previous evidence obtained by binding studies [ ]: particularly the nicotinic receptor (nAChR) of the cholinergic system, the voltage-gated potassium channel subtype Kv7, and sigma 1 (σ ) and 2 (σ ) receptors. In the cold plate test, as shown in Fig. a, DDD-028 (25 mg kg , per os) increased the licking latency of paclitaxel-treated animals starting 15 min after administration and lasting up to 105 min. Pre-treatment of the animals with the nAChR antagonist MECA (2 mg kg , i.p.) 15 min before DDD-028 administration completely abolished the pain-relieving effect of the compound up to 60 min. However, when MECA was administered the second time at 45 min after DDD-028 treatment, the effect of DDD-028 was entirely blocked for all the times observed.
Study of DDD-028 pharmacodynamic profile. The nAChRs ( a ) and α7 nAChR ( b ) involvement in DDD-028 effects. Pain was induced by repeated treatment with paclitaxel. The hypersensitivity to a cold stimulus was measured by the Cold plate test. DDD-028 was administered per os at 25 mg/kg. The nAChR antagonist MECA (2 mg kg ) was administered intraperitoneally 15 min before DDD-028 administration. In a separate experiment, mecamylamine was administered for a second time 45 min after DDD-028. The α7 nAChR antagonist MLA (6 mg kg ) was administered intraperitoneally 15 min before DDD-028 injection. Results were expressed as mean ± SEM of 8 rats analyzed in 2 different experimental sets. ** P < 0.01 vs vehicle + vehicle; ^^ P < 0.01 vs paclitaxel + vehicle; °° P < 0.01 vs paclitaxel + DDD-028
The anti-hyperalgesic effect induced by DDD-028 was also substantially reduced by the pre-treatment with the α7 nAchR antagonist MLA (6 mg kg , i.p.) (Fig. b), which suggests that this receptor subtype is very likely involved in the mechanism of action of DDD-028.
The relevance of the Kv7 channels in the DDD-028 mechanism was studied by using the selective Kv7 antagonist XE991 (1 mg/kg, i.p.) (Supplementary Fig. a). XE991 administered 15 min before DDD-028 was not able to alter the efficacy of DDD-028 over the time of observation (Supplementary Fig. a).
Finally, based on previous studies, that σ receptor is involved in the pain pathway, DDD-028 was subjected to the modulation of the σ receptor-mediated neck dystonia [ ]. Accordingly, the σ agonist DTG was injected in the red nucleus. DTG induced postural changes characterized by a marked deviation in the head angle (neck dystonia), peaking at 25–30 min after microinjection (Supplementary Fig. b). DDD-028 (25 mg kg ) was administered per os 15 min before DTG infusion without preventing DTG-induced neck dystonia (Supplementary Fig. b).
Thereafter, to assess the protective profile of DDD-028, the compound was subjected to repeated administrations over an 18-day period. Paclitaxel-treated animals were administered daily with DDD-028 (10 mg kg , p.o.) starting from the same day of paclitaxel injection. The response to mechanical noxious stimulus was measure on days 10, 12, and 18, 24 h after the last treatment. As shown in Fig. a, DDD-028 significantly increased the pain threshold of paclitaxel-injected rats at all-time points considered without development of tolerance to the anti-hypersensitivity effect (Fig. a). Repeated administration of DDD-028 induced similar results in reducing paclitaxel-induced mechanical and thermal allodynia as shown by Fig. b, c. In both measurements, DDD-028 increased the withdrawal latency and the licking latency of the animals at all-time points as evidenced by the Von Frey and the cold plate tests, respectively. The efficacy of DDD-028 was not different among 30 min and 24 h after treatment suggesting a stable improvement of the pain threshold (Supplementary Fig. ). Moreover, non-reflexive measures, as evaluation of spontaneous pain, were also performed by the Open field test. On day 18, the total distance travelled, the time spent in the center and in the periphery of the arena and the mobility time of each animal was recorded. No statistically significant differences between groups were found analyzing these parameters (Supplementary Table ).
Effects of repeated administration of DDD-028 on pain behavior induced by paclitaxel. Sensitivity to a noxious mechanical stimulus as measured by the paw pressure test ( a ). Pain threshold to a non-noxious mechanical stimulus as measured by the von Frey test ( b ). Pain threshold to a non-noxious thermal stimulus as measured by the cold plate test. Behavioral tests were performed on days 10, 12, and 18 after the beginning of paclitaxel and DDD-028 administrations, 24 h after the last treatment. Paclitaxel (2.0 mg kg , i.p.) was administered on four days (1, 3, 5, and 8) while DDD-028 (10 mg kg , p.o.) was daily administered, starting from day 1 of paclitaxel injection. Results were expressed as mean ± SEM of 8 rats analyzed in 2 different experimental sets. ** P < 0.01 vs vehicle + vehicle; ^^ P < 0.01 vs paclitaxel + vehicle
To evaluate the capability of DDD-028 to intervene against direct damages and against the maladaptive plasticity of the nervous system induced by paclitaxel, nervous tissues (brain, spinal cord, DRGs, and sciatic nerve) were collected and analyzed at the end of the repeated treatment with the compound (day 18). Oxidative stress, a typical signature of chemotherapy-induced neurotoxicity [ , ], was measured in the peripheral nervous system. As shown in Fig. , paclitaxel induced an oxidative damage of DRG as indicated by three-fold increase in carbonylation of proteins. Treatment with DDD-028 resulted in a significant prevention of the damage as evidenced by protein carbonylation values similar to the control group (Fig. , densitometric analysis and representative plot). The sciatic nerve was not affected by this kind of oxidative damage suggesting a stronger toxicity on DRG (Supplementary Fig. , densitometric analysis and representative plot).
Carbonylated protein. Dorsal root ganglia. Densitometric analysis, data were normalized on the expression of beta-actin as housekeeping and expressed as mean ± SEM of 6 samples from 6 different animals analyzed twice. Representative western blot was also showed (3 samples of each treatment are shown). ** P < 0.01 vs vehicle + vehicle; ^^ P < 0.01 vs paclitaxel + vehicle
Catalase activity, another marker of the state of functionality of peroxisome, is shown in Fig. a; paclitaxel-treated animals showed a 40% decrease of catalase activity at the DRG, suggesting an impairment of the organelle. Treatment with DDD-028 significantly rescued the enzymatic activity. On the other hand, the sciatic nerve, which is damaged to a lesser extent than DRG induced by paclitaxel as indicated in Fig. b, showed an increase of catalase activity that can be explained as a detoxifying effort of cells of the sciatic nerve to counteract paclitaxel toxicity. DDD-028 did not interfere in this survival mechanism (Fig. b).
Catalase activity. Dorsal root ganglia ( a ) and sciatic nerve ( b ) were analyzed. Enzymatic activity was expressed as percentage of control (vehicle + vehicle was considered as 100%). Data were expressed as mean ± SEM of 6 samples from 6 different animals analyzed in triplicate. * P < 0.05 vs vehicle + vehicle; ^ P < 0.05 vs paclitaxel + vehicle
To determine whether neurochemical reorganization in the spinal cord occurs following DDD-028 repeated treatment, we examined the lumbar spinal cord sections by immunohistochemistry using antibodies against GFAP and Iba1 to label astrocytes and microglia, respectively, which are non-neuronal cells strongly involved in chemotherapy-induced neuropathic pain [ , ]. Astrocyte activation was measured as an increase in the number of GFAP-expressing cells in the dorsal horn of the spinal cord of treated rats. GFAP-positive cell number in superficial laminae of paclitaxel-treated rats was significantly greater than the vehicle-treated cell number at day 18 (Fig. a). Moreover, spinal astrocytes presented altered morphology showing hypertrophy of the cell body and processes (Fig. a). Animals treated with paclitaxel + DDD-028 showed a lower number of astrocytes characterized by a reactive phenotype. Cell density increase was also significantly prevented (Fig. a).
Glial cells analysis. Spinal cord. Astrocytes ( a ) were studied by immunohistochemistry performed with a GFAP antibody. Representative image of the dorsal horn (lumbar level) at × 20 magnification; × 40 images were shown to highlight morphological alterations. Quantitative analysis was reported as number of GFAP-positive cells, and data were expressed as mean ± SEM of 3 different fields of 3 specimens for each of 6 samples from 6 different animals. * P < 0.05 vs vehicle + vehicle; ^ P < 0.05 and ^^ P < 0.01 vs paclitaxel + vehicle. Microglia ( b ) was studied by immunohistochemistry performed with a Iba1 antibody. Representative image of the dorsal horn (lumbar level) at × 20 magnification. Quantitative analysis was reported as number of Iba1-positive cells, and data were expressed as mean ± SEM of 3 different fields of 3 specimens for each of 6 samples from 6 different animals. * P < 0.05 vs vehicle + vehicle; ^ P < 0.05 vs paclitaxel + vehicle. Brain. Astrocytes ( c ) and microglia ( d ) were studied by immunohistochemistry performed with GFAP and Iba1 antibodies, respectively. Analysis was performed on periaqueductal grey (PAG), thalamus, and somatosensory area 1 (S1). Quantitative analysis was reported as number of GFAP- and Iba1-positive cells, and data were expressed as mean ± SEM of 3 different fields of 3 specimens for each of 6 samples from 6 different animals. * P < 0.05 vs vehicle + vehicle; ^ P < 0.05 vs paclitaxel + vehicle
Microglia activation was measured by the quantification of Iba1-positive cells in the spinal cord of treated rats. On day 18, paclitaxel treatment produced increased density of Iba1-positive cells in the dorsal horns of the lumbar spinal cord (Fig. b). However, no hypertrophy of this type of glia cells was observed, and microglia possessed a highly ramified morphology similar to microglia in saline rats. On the other hand, day 18 can be considered a late phase for microglia activation, that is, according to the literature, strongly involved in the first days of treatment. Animals treated with paclitaxel + DDD-028 showed a significant prevention of microglia activation (Fig. b).
To probe the effect of DDD-028 in the brain regions involved in pain sensation, a topographic analysis of microglia and astrocyte cells in three areas, the periacqueductal gray (PAG; involved in endogenous pain modulatory system), the thalamus and the somatosensory cortex (S1) were examined. As shown in Fig. c, d, paclitaxel induced a significant numerical increase of both glial cell populations in brain areas, but a higher astrocyte activation was observed. DDD-028 reduced both microglia and astrocyte cell number increase (numerical activation) in PAG, thalamus and S1 (Fig. c, d). The morphological analysis of Iba1 and GFAP immunopositive brain cells did not reveal any sign of reactivity (usually the activated microglia cells are characterized by a ramified aspect with a cell body that was always the most intensely labeled and with slender and radially projecting processes; activated astrocytes showed multiple branched processes extending in different directions from an elongated cell body).
## Discussion
The results of this study highlighted that DDD-028, a pentacyclic pyridoindole heterocycle, was able to exert anti-neuropathic and protective effects in the paclitaxel-induced neuropathic pain animal paradigm. Pain relieving efficacy was mediated by the α7 nAChR subtype.
In the last decade, early detection of tumor with concomitant success of anticancer therapies has led to an increase in cancer survival rate. Unfortunately, cancer chemotherapy is also beset with iatrogenic adverse effects, and neuropathies [ ] are unavoidable toxicity of chemotherapy treatments that are endured by patients in exchange for a life extension offered by these drugs. Moreover, no preventive or therapeutic options are currently available for the management of neuropathic pain [ ], and new disease-modifying approaches to treat neuropathies remains a critical unmet need.
DDD-028 emerged from binding and functional studies as promising candidate. The Lipinski-Veber rules for drug-like properties [ ], and the central nervous system multiparameter optimization (CNS MPO) [ ] value of 4.5 places it in the high desirability range for drugs that are purported to target the CNS. In vivo experiments, DDD-028 displayed potent acute anti-hyperalgesic activity in both of the widely used rodent models of neuropathic pain such as chronic constriction injury (CCI) and spinal nerve ligation (SNL) at oral doses between 1 and 5 mg kg [ ]. Moreover, its efficacy was also confirmed in an inflammatory pain model such as that induced by the intra-articular injection of the complete Freund’s adjuvant (CFA) [ ]. Preliminary pharmacokinetic study in rats indicated that DDD-028 elicited pain relief at low peak plasma concentration (C , 1.91 ng/mL), which suggests a wide safety window (c.a. ~ 500 based on hERG binding) for the expected dose regimen. In our work, we explored the pain relieving properties of the molecule in neuropathic pain condition induced by the antitumor drug paclitaxel. DDD-028 was able to recover, dose dependently, paclitaxel-induced hypersensitivity as well as to prevent the development of neuropathic symptoms when was co-administered with the chemotherapeutic drug. Moreover, the sub-chronic administration of DDD-028 did not develop tolerance to the antinociceptive effect exerted in paclitaxel-treated mice, in contrast to other known antinociceptive or analgesic drugs, such as morphine, which induces tolerance after repeated administration both in naïve animals [ , ] and in mice and rats treated with paclitaxel representing one of the most limiting side effects of opioids [ , ]. It has been previously reported that DDD-028 dose not bind to any of the opioid, cannabinoid, histamine, or dopamine receptors [ ]. As it binds to σ σ receptors with moderate affinity, we tested DDD-028 in vivo because of the relevant role of σ antagonists in pain control [ , ]. As mentioned before, to evaluate the σ-mediated effect, we measured the neck dystonia induced by the selective σ agonist DTG after injection in the red nucleus [ ]: DTG efficacy was unmodified by DDD-028. Two other pharmacodynamic mechanisms were also analyzed. Opening of Kv7 may regulate neuron excitability showing efficacy against neuropathic pain [ , ], but the anti-hypersensitive effect of DDD-028 was not blocked by the Kv7 blocker XE991. Finally, nAChRs, a family of channel receptor strongly implied in pain regulation and candidate to be interesting drug targets [ ]. The acute effect of DDD-028 was fully blocked by the both nAChR non-specific antagonist MECA, and by the selective α7 nAChR antagonist MLA. In several models of neuropathic pain (induced by toxic, traumatic, or metabolic events), the α7 subtype stimulation emerged as relevant target for both pain control and neuroprotection [ – ]. In this context, it appears that α7 nAChR may be involved in the mechanism of action of DDD-028, and the identification of precise downstream pathway is under investigation. Regarding neuroprotective properties, it is well established that in PNS, in general, and in DRGs, in particular, are the first targets for the dysregulation of antioxidant enzymes following paclitaxel treatment [ , ]. Among the multiplicity of the pathophysiologic mechanisms, this oxidative stress plays a crucial role in the generation of paclitaxel-induced neuropathy.
ROS act in normal cellular processes and the concentration of these compounds is controlled by the antioxidant system that involves numerous non-enzymatic molecules and enzymes such as superoxide dismutase (SOD) and catalase. The unbalance of redox mechanisms provokes alterations to proteins, lipids, and DNA as highlighted also in chemotherapy-induced neuropathies [ , ]. As a consequence, antioxidants were investigated as a possible treatment. TEMPOL (4-hydroxy-2,2,6,6-tetramethyl piperidinoxyl), a superoxide dismutase mimetic, inhibited the development and maintenance of paclitaxel-induced mechanical hypersensitivity [ ], phenyl-N-t-butyl nitrone (PBN), a non-specific ROS scavenger, inhibited the development of paclitaxel-induced mechanical hypersensitivity [ ] while another SOD mimetic, MnL4, reduced mechanical hyperalgesia and thermal allodynia induced by oxaliplatin administration [ ].
DDD-028 was able to prevent the development of hypersensitivity, and to reduce paclitaxel-related damages to PNS and CNS when repeatedly administered with the anticancer drug. DDD-028 demonstrated detoxifying properties as indicated by the enhancing the activity of the peroxisomal enzyme, catalase, and reducing the protein oxidation in DRGs.
The protective properties of DDD-028 are extended also to CNS, where a complex maladaptive response of neuronal and non-neuronal cells orchestrates the chronicization of pain. Glial cells, in particular, have been shown to contribute to the development of chronic pain in various conditions including surgery, inflammation, and nerve injury [ , ]. Pharmacological treatments including minocycline [ ] and fluorocitrate [ , ] were able to prevent glial activation and reduce neuropathic pain. According to [ ] and to [ ], we observed an increased number of Iba1- (microglia) and GFAP- (astrocytes) positive cells in the dorsal horn of the lumbar spinal cord after a cumulative dose of 8 mg kg paclitaxel indicating an astrocytes activation on day 18 after the beginning of treatment. This increase was also detected in supraspinal pain stations like PAG, thalamus, and somatosensory cortex 1. Microglia and astrocyte activation in all these areas was prevented by DDD-028 repeated treatment. Although it is generally believed that paclitaxel does not penetrate blood–brain barrier [ , ], low concentrations of paclitaxel can be detected in spinal cord after systemic treatment and this could be the explanation regarding the central effects recorded on astrocytes [ ]. Thus, further study is needed to evaluate whether the activation of spinal astrocytes is induced by the direct effect of paclitaxel (probably due to its transport along the centrifugal and centripetal branches of the DRG neuron axons, as suggested by Cavaletti et al. [ ]) or through its effects on peripheral targets.
In summary, DDD-028 seems to be a promising candidate for the management of paclitaxel-induced neuropathy. Its anti-hyperalgesic effect is mediated by the nicotinic system, particularly by the α7 nAChR subtype. Further, DDD-028’s profile appears to be a disease-modifying agent that is able to counteract oxidative damages of PNS and to reduce the maladaptive plasticity of spinal and supraspinal glial cells.
## Supplementary Information
Below is the link to the electronic supplementary material.
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RNA virus polymerases must initiate replicative RNA synthesis with extremely high accuracy to maintain their genome termini and to avoid generating defective genomes. For the single-stranded negative-sense RNA viruses, it is not known how this accuracy is achieved. To investigate this question, mutations were introduced into the 3' terminal base of a respiratory syncytial virus (RSV) template, and the RNA products were examined to determine the impact of the mutation. To perform the assay, RNA replication was reconstituted using a modified minireplicon system in which replication was limited to a single step. Importantly, this system allowed analysis of RSV RNA generated intracellularly, but from a defined template that was not subject to selection by replication. Sequence analysis of RNA products generated from templates containing 1U-C and 1U-A substitutions showed that, in both cases, replication products were initiated with a nontemplated, WT A residue, rather than a templated G or U residue, indicating that the polymerase selects the terminal NTP independently of the template. Examination of a template in which the position 1 nucleotide was deleted supported these findings. This mutant directed efficient replication at approximately 60% of WT levels, and its product was found to be initiated at the WT position (-1 relative to the template) with a WT A residue. These findings show that the RSV replicase selects ATP and initiates at the correct position, independently of the first nucleotide of the template, suggesting a mechanism by which highly accurate replication initiation is achieved. |
How G protein-coupled receptors (GPCRs) evoke specific biological outcomes while utilizing a limited array of G proteins and effectors is poorly understood, particularly in native cell systems. Here, we examined signaling evoked by muscarinic (M<sub>2</sub>R) and adenosine (A<sub>1</sub>R) receptor activation in the mouse sinoatrial node (SAN), the cardiac pacemaker. M<sub>2</sub>R and A<sub>1</sub>R activate a shared pool of cardiac G protein-gated inwardly rectifying K<sup>+</sup> (GIRK) channels in SAN cells from adult mice, but A<sub>1</sub>R-GIRK responses are smaller and slower than M<sub>2</sub>R-GIRK responses. Recordings from mice lacking Regulator of G protein Signaling 6 (RGS6) revealed that RGS6 exerts a GPCR-dependent influence on GIRK-dependent signaling in SAN cells, suppressing M<sub>2</sub>R-GIRK coupling efficiency and kinetics and A<sub>1</sub>R-GIRK signaling amplitude. Fast kinetic bioluminescence resonance energy transfer assays in transfected HEK cells showed that RGS6 prefers Gα<sub>o</sub> over Gα<sub>i</sub> as a substrate for its catalytic activity and that M<sub>2</sub>R signals preferentially via Gα<sub>o</sub>, while A<sub>1</sub>R does not discriminate between inhibitory G protein isoforms. The impact of atrial/SAN-selective ablation of Gα<sub>o</sub> or Gα<sub>i2</sub> was consistent with these findings. Gα<sub>i2</sub> ablation had minimal impact on M<sub>2</sub>R-GIRK and A<sub>1</sub>R-GIRK signaling in SAN cells. In contrast, Gα<sub>o</sub> ablation decreased the amplitude and slowed the kinetics of M<sub>2</sub>R-GIRK responses, while enhancing the sensitivity and prolonging the deactivation rate of A<sub>1</sub>R-GIRK signaling. Collectively, our data show that differences in GPCR-G protein coupling preferences, and the Gα<sub>o</sub> substrate preference of RGS6, shape A<sub>1</sub>R- and M<sub>2</sub>R-GIRK signaling dynamics in mouse SAN cells. |
Current models of mental effort in psychology, behavioral economics, and cognitive neuroscience typically suggest that exerting cognitive effort is aversive, and people avoid it whenever possible. The aim of this research was to challenge this view and show that people can learn to value and seek effort intrinsically. Our experiments tested the hypothesis that effort-contingent reward in a working-memory task will induce a preference for more demanding math tasks in a transfer phase, even though participants were aware that they would no longer receive any reward for task performance. In laboratory Experiment 1 (<i>n</i> = 121), we made reward directly contingent on mobilized cognitive effort as assessed via cardiovascular measures (β-adrenergic sympathetic activity) during the training task. Experiments 2a to 2e (<i>n</i> = 1,457) were conducted online to examine whether the effects of effort-contingent reward on subsequent demand seeking replicate and generalize to community samples. Taken together, the studies yielded reliable evidence that effort-contingent reward increased participants' demand seeking and preference for the exertion of cognitive effort on the transfer task. Our findings provide evidence that people can learn to assign positive value to mental effort. The results challenge currently dominant theories of mental effort and provide evidence and an explanation for the positive effects of environments appreciating effort and individual growth on people's evaluation of effort and their willingness to mobilize effort and approach challenging tasks. |
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