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Functional MRI measured with blood oxygen dependent (BOLD) contrast in the absence of intermittent tasks reflects spontaneous activity of so-called resting state networks (RSN) of the brain. Group level independent component analysis (ICA) of BOLD data can separate the human brain cortex into 42 independent RSNs. In this study we evaluated age-related effects from primary motor and sensory, and, higher level control RSNs. One hundred sixty-eight healthy subjects were scanned and divided into three groups: 55 adolescents (ADO, 13.2 ± 2.4 years), 59 young adults (YA, 22.2 ± 0.6 years), and 54 older adults (OA, 42.7 ± 0.5 years), all with normal IQ. High model order group probabilistic ICA components (70) were calculated and dual-regression analysis was used to compare 21 RSN's spatial differences between groups. The power spectra were derived from individual ICA mixing matrix time series of the group analyses for frequency domain analysis. We show that primary sensory and motor networks tend to alter more in younger age groups, whereas associative and higher level cognitive networks consolidate and re-arrange until older adulthood. The change has a common trend: both spatial extent and the low frequency power of the RSN's reduce with increasing age. We interpret these result as a sign of normal pruning via focusing of activity to less distributed local hubs. |
Visual information is paramount to space perception. Vision influences auditory space estimation. Many studies show that simultaneous visual and auditory cues improve precision of the final multisensory estimate. However, the amount or the temporal extent of visual information, that is sufficient to influence auditory perception, is still unknown. It is therefore interesting to know if vision can improve auditory precision through a short-term environmental observation preceding the audio task and whether this influence is task-specific or environment-specific or both. To test these issues we investigate possible improvements of acoustic precision with sighted blindfolded participants in two audio tasks [minimum audible angle (MAA) and space bisection] and two acoustically different environments (normal room and anechoic room). With respect to a baseline of auditory precision, we found an improvement of precision in the space bisection task but not in the MAA after the observation of a normal room. No improvement was found when performing the same task in an anechoic chamber. In addition, no difference was found between a condition of short environment observation and a condition of full vision during the whole experimental session. Our results suggest that even short-term environmental observation can calibrate auditory spatial performance. They also suggest that echoes can be the cue that underpins visual calibration. Echoes may mediate the transfer of information from the visual to the auditory system. |
Traumatic brain injury (TBI) is a heterogeneous disorder with many factors contributing to a spectrum of severity, leading to cognitive dysfunction that may last for many years after injury. Injury to axons in the white matter, which are preferentially vulnerable to biomechanical forces, is prevalent in many TBIs. Unlike focal injury to a discrete brain region, axonal injury is fundamentally an injury to the substrate by which networks of the brain communicate with one another. The brain is envisioned as a series of dynamic, interconnected networks that communicate via long axonal conduits termed the "connectome". Ensembles of neurons communicate via these pathways and encode information within and between brain regions in ways that are timing dependent. Our central hypothesis is that traumatic injury to axons may disrupt the exquisite timing of neuronal communication within and between brain networks, and that this may underlie aspects of post-TBI cognitive dysfunction. With a better understanding of how highly interconnected networks of neurons communicate with one another in important cognitive regions such as the limbic system, and how disruption of this communication occurs during injury, we can identify new therapeutic targets to restore lost function. This requires the tools of systems neuroscience, including electrophysiological analysis of ensemble neuronal activity and circuitry changes in awake animals after TBI, as well as computational modeling of the effects of TBI on these networks. As more is revealed about how inter-regional neuronal interactions are disrupted, treatments directly targeting these dysfunctional pathways using neuromodulation can be developed. |
Dyslexia is a multifactorial reading deficit that involves multiple brain systems. Among other theories, it has been suggested that cerebellar dysfunction may be involved in dyslexia. This theory has been supported by findings from anatomical and functional imaging. A possible rationale for cerebellar involvement in dyslexia could lie in the cerebellum’s role as an oscillator, producing synchronized activity within neuronal networks including sensorimotor networks critical for reading. If these findings are causally related to dyslexia, a training regimen that enhances cerebellar oscillatory activity should improve reading performance. We examined the cognitive and neural effects of Quadrato Motor Training (QMT), a structured sensorimotor training program that involves sequencing of motor responses based on verbal commands. Twenty-two adult Hebrew readers (12 dyslexics and 10 controls) were recruited for the study. Using Magnetoencephalography (MEG), we measured changes in alpha power and coherence following QMT in a within-subject design. Reading performance was assessed pre- and post-training using a comprehensive battery of behavioral tests. Our results demonstrate improved performance on a speeded reading task following one month of intensive QMT in both the dyslexic and control groups. Dyslexic participants, but not controls, showed significant increase in cerebellar oscillatory alpha power following training. In addition, across both time points, inter-hemispheric alpha coherence was higher in the dyslexic group compared to the control group. In conclusion, the current findings suggest that the combination of motor and language training embedded in QMT increases cerebellar oscillatory activity in dyslexics and improves reading performance. These results support the hypothesis that the cerebellum plays a role in skilled reading, and begin to unravel the underlying mechanisms that mediate cerebellar contribution in cognitive and neuronal augmentation.
## Introduction
Reading is a basic ability necessary in every-day life. Failure to acquire literacy early in the schooling years may have serious consequences for an individual’s academic achievements, well-being and employment prospects. Dyslexia, which is characterized by difficulties with accurate and fluent word recognition, poor spelling and decoding abilities, is the most common learning disability, with a prevalence rate of about 10% in school-age children (Deffenbacher et al., ). Longitudinal studies further indicate that dyslexia is a chronic condition that persists into adulthood (Shaywitz et al., ).
Difficulties in learning to read are commonly thought to derive from impaired phonemic representations and phonological processing (Bradley and Bryant, ; Ramus, ). This phonological deficit has been associated with aberrant cortical responses and altered asymmetry of activity in frontal and temporal language- and reading-related areas (Ramus, ; Dufor et al., ; Gabrieli, ), as well as with structural and functional abnormalities of the cerebellum (Pernet et al., ).
The involvement of the cerebellum in higher cognitive functions such as language was once a controversial issue (Leiner et al., ; Rao et al., ). However, much evidence has been gathered in recent years to support this view. Initially, studies in patients with cerebellar disease reported significant deficits in verbal fluency (Akshoomoff et al., ; Appollonio et al., ). Later, cerebellar involvement was found in other aspects of language, such as phonological and semantic processing (for reviews see Stoodley and Schmahmann, ; Stoodley and Stein, ). In addition, structural imaging demonstrated that lower cerebellar declive volumes are associated with impaired reading abilities, suggesting that the cerebellum may be a biomarker of dyslexia (Pernet et al., ). In fact, the cerebellar and frontal differences between dyslexics and controls are the most consistent (for reviews see Pernet et al., , ). For example, Eckert et al. ( ) found that the volume of the right anterior lobe of the cerebellum significantly distinguished dyslexic from control participants, and was correlated with reading measured by a single-word reading task.
According to broader theories, dyslexia is not limited to phonological difficulties but encompasses a wide range of neurodevelopmental deficits that can be traced back to the sensorimotor systems (Stein, ; Galaburda et al., ). It follows that difficulties in phonological processing related to dyslexia are secondary to impairments in basic sensory and motor processing. Some posit an impairment at an early stage in which fast incoming sensory information is processed in the magnocellular system (Stein and Walsh, ), while others have suggested a fundamental deficit in the integration of rapidly successive transient signals (Tallal et al., ) or in the detection of regularities in sound sequences (Oganian and Ahissar, ). These approaches all put forward the premise that sensorimotor alterations might be the source of the core reading impairments observed in dyslexia.
Although the role of the motor system in dyslexia is still controversial, it is by no means a novel proposal that dyslexia involves a motor component. Already in the 1930’s, Orton observed abnormal clumsiness in dyslexic children. He suggested that clumsy children could have difficulties in learning complex body movements as well as movements which are necessary for speech and writing (Orton, ). Studies of dyslexic participants have found impaired motor performance in a variety of tasks such as speed of tapping, heel-toe placement, rapid successive finger opposition, accuracy in copying, learning and execution of motor sequence (Nicolson et al., ; De Kleine and Verwey, ). This body of evidence supports the claims regarding the functional interactions between motor control systems, language and reading (for reviews see Hickok et al., ; Buckner, ).
The importance of cerebellar oscillatory function in neuroplasticity (Swinnen, ; De Zeeuw et al., ) and its role in motor acquisition, such as bimanual skills (e.g., Andres et al., ), have long been acknowledged in studies related to motor learning. Since impaired motor skills were often observed in dyslexics, some researchers attributed dyslexics’ cognitive and motor deficiencies to abnormal development and functioning of the cerebellum (Nicolson et al., , ). These findings lead to the claim that the role of the cerebellum is not limited to regulating the rate, force, rhythm, and accuracy of movements, but also the speed, capacity, consistency and appropriateness of cognitive processes (Schmahmann, ; Hölzel et al., ; Buckner, ).
Consequently, several training studies aimed to improve reading through integrated sensory stimulation, incorporating visuomotor and vestibular home-based exercise program lasting 6 months (Reynolds et al., ; Reynolds and Nicolson, ). In their study, the intervention group improved in a range of motor skills, such as cerebellar/vestibular and eye movement tests, as well as in the Dyslexia Screening Test, more than the control group. Although the authors could only speculate about the neural mechanisms underlying these motor and cognitive improvements, they pointed to the involvement of cerebellar function in mediating these behavioral changes. In order to improve reading and spelling in dyslexia, other studies investigated the effect of normalizing oscillatory activity on reading and spelling using neurofeedback (Breteler et al., ). Following 10 weeks of neurofeedback training, the intervention group showed improved spelling in contrast to the control group; however, no improvement was found in reading performance in either group. In addition, a significant increase in alpha coherence was found, which was interpreted as an indication that attentional processes account for the observed improvement in spelling, while no correlation was found between the two measures. So far, the link between training-induced changes in cerebellar alpha oscillatory activity and reading skills remained unexplored.
In the current study, we explore the possible potential interactions between sensorimotor and reading systems, and the role of the cerebellum as a mediator between them. In a preliminary attempt to understand the causal relationship between these constructs and their role in dyslexia, we examined how reading skills change as a result of a highly-structured form of sensorimotor training. We applied Quadrato Motor Training (QMT), a new sensorimotor whole-body training that involves following a structured set of simple oral instructions, by stepping to the instructed corner in a square. Recently, we demonstrated that one session of QMT can improve cognitive function, including creativity and spatial cognition, in comparison to two alternative training regimens that did not combine motor and cognitive aspects (Ben-Soussan et al., , ). In the current study, the QMT is applied for a period of one month, in order to test its efficacy in inducing plasticity.
We have chosen magneto-encephalography (MEG) as the main tool for assessing changes in brain activity, due to its excellent resolution in the temporal domain, as well as its superiority to EEG in terms of effective spatial resolution (Kanda et al., ; Genow et al., ). In fact, it has been explicitly argued that MEG could be an excellent tool for evaluating the neural correlates of training-induced changes in dyslexia because of its ability to localize the sources of the alpha activation in parallel to the examination of long-distance alpha coherence (Salmelin, ). Further theoretical motivation for this choice is provided by the temporal sampling framework (TSF), which has been recently proposed to connect the observed sensorimotor deficits in dyslexia to temporal alterations in neuronal oscillations (Goswami, ). We therefore set out to examine the effects of QMT in a group of adult dyslexics and matched controls using MEG alpha power and coherence as electrophysiological dependent measures, as well as reading performance and verbal fluency as cognitive measures.
We hypothesized that dyslexics would show reduced alpha power, altered alpha coherence and lower reading skills at baseline in comparison to controls. We further hypothesized that QMT would increase cerebellar alpha power due to the important role of cerebellar alpha power in voluntary action (Tesche and Karhu, ; Ivry et al., ). Increased cerebellar alpha power would then serve to normalize alpha coherence (Basar et al., ; Andres et al., ; Silberstein et al., ; Silberstein, ; Güntekin and Basar, ) and improve reading (Goswami, ). We therefore tested whether a 4-week period of daily QMT would: (a) Enhance alpha power and normalize alpha coherence in dyslexic adults; (b) Enhance reading performance. Finally, we tested whether changes in alpha power and inter-hemispheric alpha coherence would correlate with behavioral changes in reading.
## Methods
### Participants
Twenty-two native Hebrew speakers participated in the study: 12 dyslexic participants (5 females and 7 males; mean age = 30 (±6); years of education = 15 (±1)) and 10 controls (7 females and 3 males; mean age = 27 (±5); years of education = 14(±2)). We recruited volunteers who had been previously diagnosed as dyslexic by a clinical or educational psychologist and had a documented history of reading and spelling difficulties. We excluded participants who were further diagnosed with comorbid disorders, including Attention Deficit Hyperactivity Disorder (ADHD), Attention Deficit Disorder (ADD) and developmental coordination disorder (Ramus et al., ). All participants provided written informed consent to take part in the study.
### Procedure
The study included three phases: pre-training assessment, QMT training, and post-training assessment (See Figure ). The pre-training session (Day 1) included the following components in this fixed order: (a) Cognitive testing (about 30 min, see Section Cognitive Tasks below); (b) MEG measurements (about 15 min, see Section MEG Data Acquisition below); and (c) QMT training (about 7 min, see Section Quadrato Motor Training below). Post-training assessment took place at the lab on Day 29, and included the cognitive and MEG components as on Day 1 in the same order using matched versions of the cognitive tasks (see Section Cognitive Tasks). Due to technical problems, one dyslexic participant did not complete the behavioral tasks; additionally, the behavioral data for the post-training verbal fluency tasks was incomplete for two dyslexic and one control participants. In those cases where participants failed to complete certain behavioral tests, their MEG measurements were still included in the analysis of the MEG data in order to increase statistical power given the small sample size.
Study design and Quadrato Motor Training. (A) Experimental protocol. MEG and behavioral measurements were conducted before and after 4 weeks of QMT training (see text for details). Two different versions of the cognitive tests were presented pre- and post-training in a counterbalanced order. (B) The spatial layout of the training space. (C) Practice setup. The trainee listens to recorded instructions and takes a step towards the target point. Figure adapted from Ben-Soussan et al. ( ). Quadrato Motor Training = QMT; Magnetoencephalography = MEG.
### Quadrato motor training
The participant stood in a quiet room at one corner of a 0.5 m × 0.5 m square and made movements in response to verbal instructions given by an audio tape recording. Participants were instructed to keep the eyes focused straight ahead and their hands loose at the side of the body. They were also told to immediately continue with the next instruction and not to stop due to mistakes. At each corner, there are three possible directions to move. The training thus consists of 12 possible movements (Figure ). The daily training consisted of a sequence of 69 commands, lasting 7 min. Two variables that were addressed in other studies of motor learning are limb velocity and the decision regarding the responding limb (Criscimagna-Hemminger et al., ; Donchin et al., ). In order to control these parameters, we used a movement-sequence paced at a rate of an average of 0.5 Hz (similar to a slow walking rate), and we instructed the participants to begin all movements with the leg closest to the center of the square. Starting on day 2, daily QMT sessions were conducted by the participants at home. Home training lasted 27 consecutive days (from Day 2 to Day 28), and lasted 7 min each day.
### Cognitive tasks
The cognitive tasks were performed before the MEG measurement, and lasted for about 30 min. The order of tasks was fixed, starting with the reading test, category-based fluency and then letter-based fluency task. Each task had two different versions, and each of these versions was assigned to the pre- or post-training session in a counterbalanced manner across subjects.
#### Reading test
This test examines single-word reading speed and accuracy. A list of forty five written Hebrew words of increasing difficulty was presented and participants were asked to accurately read as many words as possible from the list in 1 min. The level of difficulty of the words was controlled in terms of word length and number of syllables. In order to minimize learning effects from the pre-test to the post-test, two non-overlapping word lists were created. Each list was presented either before or after training, in a counterbalanced manner. The two lists of 45 words were sampled from a database of rated Hebrew words (Levy-Drori and Henik, ), and were matched item-by-item for concreteness, availability of context, familiarity, number of letters and number of syllables. Since several participants from the control group finished the list of words in less than 1 min, the final score represents the number of words which were read correctly in the first 30 s.
#### Category-based fluency task
Participants were asked to say in 1 min as many words as possible belonging to a given semantic category. Two semantic categories were used alternately: (a) Animals; (b) Fruits and vegetables. One category was presented in the pre-training session (Day 1) and the other in the post-training session (Day 29), and the order of the categories was counterbalanced across subjects. Fruits and vegetables were treated as one category in order to avoid the ambiguity between botanical definitions and common usage (as in “avocado”). These categories were chosen because they have comparable norms, and in order to avoid test-retest influence by repeating the same category (Kavé, ).
#### Letter-based fluency task
Participants were asked to say in one minute as many words as possible that start with a given letter. We used two Hebrew letters: Bet (/b/) and Gimel (/g/). One letter served for pre-training and the other for post-training, and the order of the categories was counterbalanced across subjects. These letters were chosen because they have comparable norms, and in order to avoid test-retest influence by repeating the same letter (Kavé, ).
### MEG data acquisition
Power and coherence measures were collected using the MEG at the beginning and at the end of the month, after performing the cognitive tasks. MEG recordings were conducted with a whole-head 248-channel magnetometer array (4-D Neuroimaging, Magnes 3600 WH) in a magnetically shielded room. During the Rest condition, the participants were asked to refrain from moving and from falling asleep. In addition, the participants were asked to keep their eyes closed, in order to reduce ocular artifacts in the measured signals and to facilitate the localization of potential generator regions of the alpha resting–state oscillations (Goldman et al., ). Data acquisition took 15 min. We also collected MEG data using two active tasks which will be reported elsewhere. Before acquiring the data, the head-shape of each subject was digitized. Reference coils located approximately 30 cm above the head oriented by the x , y and z axes were used to record environmental noise. Three accelerometers, one for each axis, attached to the MEG gantry were used to record building vibrations in order to remove artifacts caused by them. The data were digitized at a sampling rate of 1017.25 Hz, and a 0.1 to 400 Hz band-pass filter was used online. The 50 Hz line power fluctuations were recorded directly from the power-line in order to remove the artifact on the MEG sensors.
### Preprocessing MEG data
Power line, heartbeat, and vibration artifacts were removed automatically (Tal and Abeles, ). The data were then divided into 1 s epochs. Muscle artifact was estimated by examining the absolute value of all the MEG channels for every epoch, after applying a 20 Hz high-pass filter. For each epoch, the mean absolute value was computed. These values were then converted to z-scores, and epochs with z -scores greater than 3 standard deviations were rejected. Eye blinks were not considered as possible artifact for the alpha power processing, because the participants had their eyes closed and did not blink. Some eye movement artifact was still present in the data, but this was in a lower frequency range and was negligible in the alpha frequency range. Two of the 248 channels were noisy (one of the channels registered a constant zero value and the other exceeded 1 nanotesla); these channels were therefore excluded from all sensor and source level analysis. For left-right coherence computation, their homolog channels were omitted as well.
### Source localization
Source localization was applied for the alpha (7–13 Hz) frequency band. Synthetic Aperture Magnetometry (SAM) beamforming (Robinson and Vrba, ) was used with multiple spheres forward solution based on the digitized headshape. The neural activity was estimated for a grid of points covering the volume of the brain with 5 mm intervals. The power of activity was calculated for every grid point and for every epoch. Since raw beamforming results are biased toward deep sources it was necessary to normalize the images in order to keep the noise level equal throughout the whole volume of the brain. For this purpose, a pseudo- z score was calculated by averaging the power of every location, across epochs, divided by its noise estimate. The noise estimate was determined by the weights (the spatial filter). Deep sources generally have weights with higher values and are therefore noisier. Dividing the power of activity by the square of the weight norm can compensate for this bias (see Equation 3 in Sekihara et al., ). The absolute value of the weights of a particular location serves as a noise estimate. The resulting images represent the increase of alpha compared to noise, without being biased to deep sources. The pseudo- z value for each location was visualized as the color of voxels in the resulting functional images. The images were transformed to Talairach space by fitting a template MRI to the individual headshapes using SPM8 (Friston et al., ) and FieldTrip (Open Source Software for Advanced Analysis of MEG, Oostenveld et al., ) packages used with Matlab® R2010b. In order to control for multiple comparisons, a simulation was applied using an Analysis of Functional NeuroImages (AFNI) function (AlphaSim) which determines the probability to get significant clusters of different sizes at random. According to the simulation, at current parameters (given the template brain and spatial resolution used), clusters of voxels with a p -value smaller than 0.05 and exceeding one cubic cm (8 voxels) do not count as random noise. We decided to be even more conservative and to take only clusters containing more than 20 voxels at a threshold of p < 0.005 (Bunge et al., ).
### Coherence
The coherence between left and right sensors was computed using FieldTrip. The data were first baseline-corrected by subtracting the mean of every epoch from the MEG traces. Eighteen channels located along the midline of the helmet were omitted from the coherence analysis, because these channels are likely to present with high coherence based on proximity, since spatially close sensors are likely to pick up very similar activities (Lehnertz et al., ).
Fourier transform was then computed using a spectral smoothing box of 1 Hz (meaning that the 10 Hz bin includes 9 to 11 Hz oscillations). The frequencies per time window were computed using a DPSS bell-shaped window. The resulting complex spectrum was used to assess coherence between each channel and its homolog. The coherence value of the 18 channels located along the mid-line of the helmet was set to one, representing perfect coherence between each channel and itself (the vertical red line in Figure ). The coherence was projected onto a two-dimensional map of the sensor array, using the same left-right coherence value for the left as well as the right sensor, thus creating symmetrical maps of coherence. After the creation of the maps, channels close to the midline were excluded from the statistical analysis (in addition to the midline channels). This step was necessary because channels near the midline had high coherence values which did not represent cortical coherence, but simply the fact that the activity of one brain region was measured by two nearby channels. The channel pairs chosen were at least 11 cm apart in order to avoid “false coherence” resulting from close channels that pick up the same source and covered the lateral area of increased coherence (greater than 0.3; Figure ). This procedure resulted in 59 channel pairs for which left-right coherence was statistically evaluated. Usually when studying inter-hemispheric differences, coherence is computed for one artifact-free channel for each region (e.g., from frontal, central, parietal, and temporal regions) and is computed for channels located in the corresponding regions of the two hemispheres (Osipova et al., ; Kikuchi et al., ). In fact, the number of chosen pairs is conventionally determined by apriori assumptions and therefore restricted to particular regions of interest. However, due to the exploratory nature of the current study, it was important to expand the search across multiple sensors.
Reading performance pre- and post-training . Number of words read correctly (mean ± SEM) in 30 s, pre- and post-training in the dyslexic (red) and control (blue) groups.
Changes in alpha power. (A) and (B) demonstrate the significant clusters resulting from the Group (dyslexics, controls) by Training (pre-training, post-training) interaction. Voxels are colored by the F statistics, overlaid on coronal (A) and sagittal (B) views. The statistical map is thresholded at p < 0.0025 in addition to a cluster size threshold of 20 voxels. The focus point (green cross) is positioned in the right culmen (Talairach coordinate: 12, −37, −22). (C) The bar graph shows alpha power as a function of Group and Training (mean + SEM). * p = 0.01; ** p = 0.001.
Alpha coherence in the dyslexic and control groups .≢wline (A) Group differences in inter-hemispheric alpha coherence. The coherence was higher over temporal channels in the dyslexic group compared with the control group (* p < 0.01, uncorrected). (B) Temporal alpha coherence as a function of Group and Training (mean + SEM), demonstrating significant group differences between the dyslexic and control groups (* p < 0.01, uncorrected), as well as a null effect of the training in the dyslexic group and a trend toward an increase in alpha coherence in the control group.
### Statistical analysis
Mixed design ANOVA was used to test the effects of QMT on performance in the reading and verbal fluency tasks, with Training (pre-training, post-training) as a within-subject factor and Group (dyslexic, control) as a between-subjects factor. Statistical parametric maps were produced from MEG data using the AFNI package (Cox, ). Mixed design ANOVA was used to test the effects of QMT on alpha activity, i.e., alpha power and alpha coherence. Pearson correlation was used to test the association between behavioral and neuronal changes. The correlation threshold was p < 0.05. Post hoc comparisons were conducted using t -tests.
## Results
### Cognitive results
#### Reading task
Performance on the speeded reading task was entered into a 2 (Group) by 2 (Training) ANOVA. First, a significant main effect for Group [ F = 7.80, p < 0.05] was observed, indicating that, across both time points, the number of words correctly read by the controls ( M = 38.5, SD = 6.3) was higher than the number of words correctly read by the dyslexic participants ( M = 27.6, SD = 10.7). Secondly, the analysis yielded a main effect for Training [ F = 6.89, p < 0.05], showing that QMT improved single-word reading performance across both groups (see Figure ). Finally, the interaction between Training and Group was not significant ( p > 0.9).
#### Verbal fluency
We conducted two separate analyses for the category-based and letter-based fluency tasks using a 2 (Group) by 2 (Training) ANOVA. No significant main effects or interactions were found, for either the category-based or the letter-based fluency task. The mean scores (i.e., number of words generated in 1 min) of the control participants for the category-based or letter-based fluency were similar to the norms (Kavé, ).
Based on previous studies demonstrating significant differences in phonological fluency between dyslexic and controls (Rack et al., ; Reid et al., ), we conducted a planned comparison between the groups for letter-based fluency, separately for pre- and post-QMT. While a marginally significant difference in phonological fluency was found pre-training between the dyslexic and control groups [ t = 2.08, p = 0.051], no such difference was found following the training. In addition, no differences were found between the groups in semantic fluency, neither before nor after training. See Table .
Mean scores of letter-based and category-based fluency tasks as a function group and training .
### MEG results
#### Between-group differences in cerebellar alpha power
We first examined the effect of QMT on alpha power using a mixed design ANOVA, with Training as a within-subject factor and Group (Dyslexia, Control) as a between-subjects factor, for each voxel. The ANOVA revealed a significant Group × Training interaction in a cluster in the right cerebellum. The center of mass of this cluster was located in the right culmen (Talairach coordinates (in mm): 12, −37, −22; F = 13.3, p < 0.0025; See Figure ). Before training, cerebellar alpha power was significantly lower in the dyslexic group compared to the control group ( t = 3.88, p = 0.001). Following 4 weeks of daily QMT, cerebellar alpha power significantly increased in the dyslexic group ( t = 3.08, p = 0.01) in contrast to the control group which showed no significant change following training (see Figure ).
The ANOVA also revealed a significant Group × Training interaction for three frontal clusters, located in the right superior frontal gyrus (SFG) (Talairach coordinates in (mm): 23, 53, 17) [ F = 16.08, p < 0.001], supplementary motor area (SMA) (Talairach coordinates in (mm): 13, 18, 42) [ F = 22.41, p < 0.001] and the left middle frontal gyrus (Talairach coordinates in (mm): −27, 8, 57) [ F = 16.01, p < 0.001]. While there were no significant differences between the groups in these areas prior to training, the control group showed a significant decrease in alpha power in the left medial frontal gyrus (MFG) ( t = 4.54, p < 0.005), right SFG ( t = 3.73, p < 0.005) and SMA ( t = 3.69, p < 0.005) following 4 weeks of daily QMT. On the other hand, the opposite pattern was observed in the dyslexic group in which alpha power increased in the right SFG ( t = 2.66, p < 0.05).
#### Coherence
Inter-hemispheric alpha coherence was tested using a mixed design 2-way ANOVA for each of the 59 channels, with Training as within-subject factor and Group as between-subjects factor. Across both time points, inter-hemispheric alpha coherence was significantly higher in the dyslexic group compared to the control group for five channel pairs ( F > 8.35, p < 0.01, uncorrected; See Figure ). No main effect for Training or interaction was found.
#### Neuro-cognitive correlations
In order to study the possible associations between change in alpha activity and change in reading performance, we calculated Pearson correlations between behavioral and neuronal change within each group. This analysis was motivated by previous studies relating reading, cerebellar activity and alpha coherence (Nicolson et al., ; Weiss and Mueller, ; Arns et al., ). Change in speeded reading was calculated as the difference between the number of words read correctly in 30 s before and after training. Change in cerebellar alpha power was calculated as the difference between pre- and post- training cerebellar alpha power of the cluster which was found to have the significant Group × Training interaction. Change in inter-hemispheric alpha coherence was calculated as the difference between pre- and post-training values of the bilateral temporal alpha coherence. No significant correlation was found between change in cerebellar alpha power and change in reading in the two groups. Yet, as can be seen in Figure , change in temporal alpha coherence was positively correlated with the change in reading score ( r = 0.58, p < 0.05, n = 11; uncorrected) in the dyslexic group but not in the control group. Using the Fisher r -to- z transformation, we calculated the z value to assess the significance of the difference between two correlation coefficients. The results indicated a significant difference between the two correlation values ( z = 1.87, p < 0.05).
Correlation between change in temporal alpha coherence and change in reading performance in the dyslexic group . Change in alpha coherence, calculated by the subtraction of pre- from post-training, was positively correlated with the change in number of words read correctly in 30 s ( r = 0.58, p < 0.05).
## Discussion
Our results contribute two novel findings with regards to the cerebellar involvement in dyslexia: First, we show that cerebellar alpha activity prior to training is lower in dyslexics compared to controls. Second, a 4-week training program enhanced cerebellar alpha activity in dyslexics, but not in controls. Two other important findings are reported here for the first time: First, QMT over a period of 4 weeks improves reading speed in adults, and second, the improvement in reading performance is associated with increase in temporal alpha coherence in the dyslexic group. Below, we discuss these results in the context of different approaches to dyslexia and examine the possible role of the cerebellum in this neurodevelopmental disorder.
### QMT enhances performance in a speeded reading task
This study was inspired by the controversial body of research that examines the connection between reading and the sensorimotor systems (e.g., Flöel et al., ; Pulvermüller, ), in the context of novel discoveries about the benefit of daily sensorimotor practice for cognition (Ben-Soussan et al., ). We found improved reading skills in both groups as a result of 4-week QMT. Commonly, experimental studies attempt to enhance reading abilities and phonological functions by providing a training program for the target skill. Here we show that sensorimotor training might be beneficial for improving reading skills even though the practice relies on faculties that are not directly related to reading. Indeed, the QMT is based on a series of motor responses to verbal commands, which involve functions such as spatial cognition and response inhibition (Ben-Soussan et al., ). QMT-related improvement in the speeded reading task was found using different stimulus lists at each time point (pre- and post-training) and is thus considered to result from QMT and not from test-retest effects. This finding is also in agreement with previous results showing cognitive improvement in non-dyslexics adults even following short term QMT (Ben-Soussan et al., , ).
In addition to group differences in reading score measured at baseline, a trend of a lower score in the phonological fluency task was observed pre-training in dyslexics compared to controls. This trend was not observed following 4 weeks of daily QMT, suggesting that QMT helped in normalizing the performance on this task. Again, the use of different categories at each time point (pre- and post-training) provided support to the interpretation that it is probably the QMT that normalized performance and not the repetition of the task. In addition, no differences were found between the groups in semantic fluency, neither before nor after training. This supports the view that dyslexia is more related to phonological than to semantic impairment (Leggio et al., ).
### QMT enhances cerebellar alpha power in dyslexic adults
In line with our hypothesis, cerebellar alpha power was significantly lower in the dyslexic group prior to training in comparison to the control group (see Figure ). Importantly, following 4 weeks of daily QMT, we found that cerebellar alpha significantly increased in the dyslexic group in the right culmen, a region which has been previously reported to be related to language processing (Luke et al., ; Pernet et al., , ; Rudner et al., ). This finding may reflect the role of the cerebellum as a general timing mechanism for both sensorimotor and cognitive processes (Ivry, ; Tesche and Karhu, ; Ivry et al., ; Tesche et al., ), such as the acquisition of sensorimotor skills and response readiness (Martin et al., ). It might also be linked to the critical involvement of the cerebellum in the coordination of smooth movements, maintenance of balance and posture, visually guided movements and motor learning (for review see Manto et al., ), which are inherent components of the QMT.
In addition to cerebellar changes, we found differences between dyslexics and controls in frontal alpha activity following 4 weeks of daily QMT. While alpha power in the left MFG and SMA significantly decreased in the control group, the dyslexic group showed increased alpha power in right SFG. These regions have been previously reported to be related to movement and language processing (Binder et al., ; Eckert et al., ; Neumann et al., ). Contrary to the dyslexics, the control group showed a trend towards a reduction in cerebellar alpha power; however, there was a notable increase of the control group’s dispersion around the mean, which might account for the lack of statistical significance of this effect. In line with previous findings on motor practice, it is possible that the reduction of frontal and cerebellar activity indicates that practice became simpler as control of movement and coordination improve (Lacourse et al., ).
Previous work based on one session of training reported decreased frontal alpha activity in healthy young subjects (Ben-Soussan et al., ). This decreased frontal activity was mostly observed following simple motor training, indicating that changes in these regions might be related to motor learning as well as action observation and intention understanding (Exner et al., ; Dapretto et al., ). These results are compatible with Goldberg et al. ( ) who reported a complete segregation between self-related and sensorimotor activity in relevant cortical regions using functional neuroimaging. Their results showed that frontal regions were functionally inactive during sensorimotor tasks and active during self-engaged tasks. It is therefore possible that reduced activity of frontal regions at rest in controls signifies automaticity of sensorimotor components as a result of the repetition of the same sequence of QMT for 4 weeks.
### Alpha coherence in dyslexic adults compared to controls
Alpha coherence is important for cognitive and sensory processing (Weiss and Mueller, ; Ben-Soussan et al., ). Previously, EEG studies revealed increased coherence in dyslexic children, especially between temporal areas during rest (Shiota et al., ; Arns et al., ). Contrary to prior results showing increased inter-hemispheric alpha coherence following a single session of QMT (Ben-Soussan et al., ), in the current study neither group showed a significant increase in alpha coherence following one month of daily QMT. It should be noted that the previous results were obtained using EEG and not MEG. In fact, calculating connectivity from sensor level recordings is not straightforward, as these recordings are highly dependent on the effects of field spread. In other words, coherence measured by MEG reflects fewer sources because the spatial scale of the MEG sensors is smaller resulting in inflated estimates. Moreover, EEG and MEG are different in their sensitivity to radial and tangential dipoles (Srinivasan et al., ). This points to the necessity to integrate different methods in the study of training-induced plasticity.
Importantly, in the current study coherence analysis confirmed increased inter-hemispheric alpha coherence in the dyslexic group compared to the control group across time points. The increased inter-hemispheric coherence, especially between the temporal areas, may reflect the connection between left and right superior temporal sulci, which are considered to be necessary for phonological processing (Hickok and Poeppel, ). These findings converge with independent data from diffusion imaging showing that children with lower phonological and reading skills have higher anisotropy in temporal-callosal fiber tracts (Ben-Shachar et al., ; Dougherty et al., ).
Consequently, we propose that the increased coherence found in the dyslexic group may reflect a compensation mechanism (Roberts and Kraft, ; Arns et al., ). This suggestion further accords with the view that both left and right posterior superior temporal cortices are required for phonological processing (Hickok and Poeppel, ). Indeed, earlier models of dyslexia promoted the premise that complex cognitive functions, such as the translation of graphic symbols into a phonemic code, depend on component processes from both cerebral hemispheres, and that at least some subtypes of dyslexia may be due to abnormal inter-hemispheric communication (Gazzaniga, ; Gladstone and Best, ; Wolff et al., ). The association between change in coherence and reading performance revealed a significant positive correlation only in the dyslexic group, suggesting that the underlying mechanisms of improved reading observed in this study are connected with increased inter-hemispheric communication in the alpha range. Due to the low power of the correlation analysis ( N = 11) and the non-significant effect of training on alpha coherence, this finding should be treated as suggestive, and should be tested in future larger MEG studies of developmental dyslexia. Nonetheless, the positive correlation reveals that participants who showed higher improvement of speeded reading also demonstrated increased bilateral temporal alpha coherence, in addition to the general increase in coherence observed in dyslexia. Some researchers aimed at normalizing brain activity (and consequently ameliorating behavioral and cognitive deficits) in various developmental disorders by suppressing hyper-connectivity (Pineda et al., ). Similarly, in stroke rehabilitation, applying brain stimulation to inhibit inappropriate activity of non-specialized areas has been argued to offer an effective avenue of treatment (Naeser et al., ). However, ameliorating cognitive deficits in developmental disorders may not necessarily be achieved through suppressing abnormal connectivity, because the observed hyper-connectivity does not necessarily reflect a dysfunction (Arns et al., ). Indeed, findings from neurofeedback training show that, contrary to the expected effects, 6 months of training induced an increase in alpha coherence, which might be related to improved attention (Breteler et al., ).
### Towards a new approach to understanding and treating dyslexia
Existing methods of treating dyslexia usually rely on phonetic and reading materials which aim at dealing directly with the linguistic impairments. Nevertheless, dyslexia, as well as other developmental disorders, should not be interpreted as being impairments in a single cognitive process (Castles and Coltheart, ; Pernet et al., ). These cognitive impairments should rather be regarded as the endpoint of an abnormal developmental process, reflecting the interactions of multiple potentially deficient processes as well as compensatory processes (Thomas and Karmiloff-Smith, ). The current study attempted to investigate dyslexia-related differences in specific regions, in inter-hemispheric coherence, and in response to intervention. In this way, the differences between groups in the training-induced electrophysiological effects may provide further insight into the deficient and compensatory processes that characterize dyslexia.
In line with our results, we suggest that both the deficient cerebellar alpha power and possibly compensatory alpha coherence may be connected to the cerebellum’s role as a generator of alpha activity, and that sensorimotor training may lead to cerebellar plasticity which could eventually rebalance the system. In this respect, altered cerebellar oscillatory activity may be the source of the deficit in dyslexia since it could be viewed as a neural system that mediates cortical communication (Andres et al., ; Silberstein et al., ). Our preliminary findings also disclose that sensorimotor training can be a practical intervention in dyslexia because of its potential to facilitate cerebellar oscillatory activity. Exploring how neuronal oscillation and cerebellar function change as a result of training may have valuable implications for educational neuroscience.
### Limitations
The current study is a preliminary attempt to examine empirically the question of system modulation, which is required for improving reading in dyslexia. The main limitations of the current study are the small sample size and the use of only one training paradigm. The choice of QMT was made based on previous studies in which it was demonstrated that cognitive changes, namely increased creativity and improved spatial cognition, are QMT specific, and are not observed in two control groups (Ben-Soussan et al., , ). In the future, a study on a larger sample that includes several training regimes may extend the current results. In future research, it would be important to include a passive control group; in particular, a dyslexic passive control group would ensure that any test-retest effects that were controlled for with normal-readers and with the two different versions of the tasks are not different in participants with dyslexia.
So far, EEG studies have generally avoided studying cerebellar function because of the complex folding of the cerebellar cortex. As for MEG, signals can be obtained from the cerebellum especially within the alpha range (Ivry, ; Park et al., ). However, source localization makes it difficult to distinguish between signals arising in the cerebellar cortex and deep nuclei. Thus, our results should be interpreted keeping these limitations in mind. Regarding the coherence analysis, the statistical significance was not corrected for 59 comparisons and should be therefore evaluated with caution. However, since no previous study has shown similar left-right coherence effects it was impossible for us to focus on channels of interest and reduce the number of multiple comparisons. The results we report here can therefore be considered as exploratory, and should be confirmed in future studies. We expect that future studies will utilize independent imaging methods in order to examine the role of the cerebellum in reading and dyslexia, and the impact of QMT on cerebellar activity and connectivity.
## Conclusions
The current MEG study is in line with previous studies suggesting that dyslexia may be related to cerebellar dysfunction. Four weeks of daily QMT enhances reading performance and cerebellar alpha oscillations in dyslexic participants. In addition, improved reading performance in dyslexics correlates with inter-hemispheric temporal alpha coherence. Our results suggest that cerebellar impairment in dyslexia can be modulated by sensorimotor training. Most importantly, the investigation of training-induced effects on reading performance provides a unique opportunity to gain insight into the relation between behavioral and neuronal changes. A better understanding of the functional coordination between cortical regions and the cerebellum would be necessary to pinpoint some of the underlying sources of dyslexia and to create training paradigms for clinical purposes. The current study provides an important step in bringing together different approaches to study the sources and treatment of dyslexia and the scientific value of sensorimotor training.
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There is a rising medical need for novel therapeutic targets of physical activity. Physical activity spans from spontaneous, low intensity movements to voluntary, high-intensity exercise. Regulation of spontaneous and voluntary movement is distributed over many brain areas and neural substrates, but the specific cellular and molecular mechanisms responsible for mediating overall activity levels are not well understood. The hypothalamus plays a central role in the control of physical activity, which is executed through coordination of multiple signaling systems, including the orexin neuropeptides. Orexin producing neurons integrate physiological and metabolic information to coordinate multiple behavioral states and modulate physical activity in response to the environment. This review is organized around three questions: (1) How do orexin peptides modulate physical activity? (2) What are the effects of aging and lifestyle choices on physical activity? (3) What are the effects of aging on hypothalamic function and the orexin peptides? Discussion of these questions will provide a summary of the current state of knowledge regarding hypothalamic orexin regulation of physical activity during aging and provide a platform on which to develop improved clinical outcomes in age-associated obesity and metabolic syndromes.
## Introduction: physical activity and the orexin neuropeptide system
Physical activity can improve overall health. For example, it can prevent obesity and reduce age-associated cognitive decline. There is wide variation between individuals in their drive to be physically active. The drive for physical activity is operationally defined as spontaneous physical activity (SPA). In humans, SPA includes time spent standing and ambulating, but not voluntary exercise. The energy expended by SPA is termed “nonexercise activity thermogenesis” or NEAT. Exercise is a necessary part of a healthy lifestyle but many people cannot or do not exercise. New treatments to target exercise-independent aspects of achieving and maintaining a healthy weight are greatly needed. Spontaneous physical activity is an excellent candidate, but our understanding of the brain mechanisms driving SPA is incomplete. Therapies that enhance SPA will contribute to better clinical outcomes for obesity and metabolic syndrome, diseases of high prevalence in the developed world. This review describes recent advances in our understanding of neuronal processes that regulate SPA, with a specific focus on changes that occur in the orexin neuropeptide system during normal and pathological aging.
The orexin (hypocretin) neurons are a group of hypothalamic neurons defined by expression of the orexin peptides. The orexin signaling system regulates a variety of complex behaviors, including sleep/arousal, reward, food intake and SPA, with an overall effect of increasing energy expenditure. Orexin neuron activity is affected by multiple environmental and physiological variables like fasting and circadian rhythms. Function of the orexin system varies with lifestyle and age (see Figure ), as does its ability to influence factors that contribute to pathological weight gain in humans and animals. Clarifying how these two variables impact orexin-induced SPA will facilitate development of improved obesity prevention and treatment programs.
Prepro-orexin, SPA, and body weight during aging . Relative levels of prepro-orexin (green), SPA (blue), and body weight (red) throughout the mammalian life span.
### Orexin neuropeptides and receptors
The orexin signaling system consists of two orexin peptides (orexin A and orexin B) and two G-protein coupled receptors (orexin receptor 1, OXR1 and orexin receptor 2, OXR2) (de Lecea et al., ; Sakurai et al., ). Orexin A and orexin B are 33- and 28-amino acid peptides cleaved from a single gene product, prepro-orexin (Sakurai et al., ). Orexin A has equal affinity for both orexin receptors, while orexin B preferentially binds to OXR2 (Sakurai et al., ; Ammoun et al., ). Both OXR1 and OXR2 couple to the G -alpha subunit to activate phospholipase C and induce cation influx, thereby depolarizing neurons and increasing excitability (de Lecea et al., ; Zhu et al., ). When overexpressed in cultured cells, OXR2 also signals through the pertussis-toxin sensitive G -alpha subunit to reduce cAMP production (van den Pol et al., ; Zhu et al., ). Electrophysiological studies of cell types that endogenously express a single OXR subtype in vivo confirm that orexin receptors are generally excitatory in nature and can affect neuronal activity via both presynaptic and post-synaptic mechanisms (Zhu et al., ; Aracri et al., ; Schöne et al., ). Like the other neuropeptide systems lacking known reuptake transporters, it is believed that orexin signaling is terminated through diffusion, receptor sequestration, and enzymatic degradation.
The expression pattern of the orexin receptors differs widely among brain sites but is often complimentary in nature. Most brain sites investigated thus far predominately express a single receptor subtype and those that express both subtypes typically do so in separate cell types (Trivedi et al., ; Marcus et al., ). Functional differences between the two orexin receptor subtypes are not clearly delineated. Many studies are limited by the use of methods that affect both receptor populations, as is the case with exogenous administration of orexin A and genetic manipulations of the prepro-orexin gene. Direct comparison of OXR1 and OXR2 knockout mice report contributions of both subtypes to body weight and sleep patterns, albeit with one receptor subtype typically displaying a greater effect (Funato et al., ; Mieda et al., ).
Orexin signaling takes on a modulatory nature in many experimental paradigms. Behavioral or physiological effects differ depending on the brain site of action. In other words, the function of the brain area in which orexin signaling is being manipulated is the primary determinant of the particular orexin-dependent effects that are observed at both the behavioral and cellular levels. For example, orexin A signaling via OXR1 in the periaquaductal gray area induces analgesia through cannabinoid-mediated retrograde inhibition whereas OXR1 signaling in the dorsal hippocampus facilitates excitatory LTP and formation of new associative memories (Ho et al., ; Riahi et al., ; Yang et al., ). Thus, while it is tempting to assign distinct functions to each receptor subtype, the currently available body of data does not fully support a simple, dichotomous characterization. A more refined understanding is needed of functional dissociations in brain-site specific receptor subtypes and the molecular mechanisms underlying them.
### Orexin neurons
In the mammalian brain, orexin neurons are concentrated in the lateral hypothalamus (LH), perifornical area, and dorsomedial hypothalamus (Peyron et al., ). Orexin fibers are found throughout the central nervous system (CNS), including nuclei in cortical and limbic areas, basal ganglia, midbrain, brainstem, and spinal cord (de Lecea et al., ; Peyron et al., ; Taheri et al., ; Colas et al., ). In addition to orexin, these neurons synthesize glutamate, as well as other neuropeptides, notably dynorphin (Chou et al., ; Rosin et al., ; Torrealba et al., ). Orexin neuron activity is affected by a variety of metabolic signaling molecules (i.e., glucose, leptin, amino acids) and environmental factors which will be discussed in more detail below (Yamanaka et al., ; Karnani and Burdakov, ; Karnani et al., ; Leinninger et al., ). For example, activity levels of orexin neurons, as measured by the immediate early gene Fos, increased during the waking phase of the circadian cycle and during fasting or caloric restriction (Sakurai et al., ; Estabrooke et al., ). Orexin neurons, in turn, regulate physiological and behavioral processes that have major impacts on energy balance and metabolic state, physical activity, blood glucose levels, and food intake (Sakurai et al., ; Akiyama et al., ; Alam et al., ; Kotz et al., ; Inutsuka et al., ).
As the orexin neurons are known to modulate multiple behaviors, it has been suggested there are functionally specialized subpopulations of orexin neurons, yet this critical issue remains unresolved. The most well-known hypothesis proposes that orexin neurons located in the lateral portion of the LH mediate reward behaviors and those located more medially in the perifornical/dorsomedial areas are involved in arousal and stress (Harris and Aston-Jones, ; Harris et al., ). This theory is in part supported by the observation that circadian fluctuations in Fos expression in orexin neurons are most pronounced in the medial LH and less so in the more lateral portions, as well as, by differential activation of orexin neurons in reward behavioral paradigms (Estabrooke et al., ; Harris and Aston-Jones, ; Harris et al., ). However, orexin neurons send collateral projections throughout the CNS, indicating that anatomical location of orexin cell bodies is unlikely to be the most informative criterion when attempting to identify or predict functional specialization of orexin neurons. Accordingly, subpopulations of orexin neurons have been described based on electrophysiological and morphological variables (España et al., ; Oldfield et al., ; Schöne et al., ). Analysis of orexin neuron projections to the ventral tegmental area and locus coeruleus revealed that differences in electrophysiological properties and neuronal architecture are better parameters compared to location of soma when attempting to categorize distinct subpopulations of orexin neurons (Schöne et al., ; González et al., ). While there is some degree of specialization of orexin neurons, the characteristics that define specific subpopulations and whether they have overlapping or unique functions remain poorly defined.
### Orexin and energy expenditure
Orexin peptides modulate energy metabolism, arousal, and physical activity (Chemelli et al., ; Hara et al., ; Kiyashchenko et al., ; Mileykovskiy et al., ; Adamantidis et al., ; Takahashi et al., ; Sasaki et al., ; Inutsuka et al., ). Orexin system activity is positively associated with activity levels in animals and humans (Kiyashchenko et al., ; Wu et al., ; Kok et al., ). Orexin signaling promotes obesity resistance via enhanced SPA and energy expenditure (Perez-Leighton et al., ). Animal models lacking a functional orexin system develop obesity despite consuming fewer calories than their wildtype counterparts (Hara et al., , ). Pathological weight gain in these animals is most likely due to energy imbalance resulting from reduced physical activity. Animals in which there is progressive loss of orexin neurons display more severe obesity phenotypes than mice who are only deficient in prepro-orexin, indicating that multiple factors and signaling systems coalesce in orexin neurons to regulate body weight (Hara et al., ). To complement genetic ablation approaches, pharmacological studies of repeated orexin A injection into the brain result in body weight loss and protection against obesity (Novak and Levine, ; Perez-Leighton et al., ; Teske et al., ). Indeed, selective activation of orexin neurons in the LH via Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) stimulates SPA, food intake, and energy expenditure (Inutsuka et al., ).
Orexin-dependent modulation of SPA involves several brain sites with site-specific participation of OXR subtypes (Kiwaki et al., ; Thorpe and Kotz, ; Kotz et al., ). Data from our laboratory and others show that a major effect of orexin A signaling is to promote SPA and NEAT (Kotz et al., ; Inutsuka et al., ). Increased SPA and NEAT are observed following injection of the orexin peptides directly into the rostral LH, hypothalamic paraventricular nucleus, nucleus accumbens, locus coeruleus, dorsal raphe nucleus, tuberomammillary nucleus, and substantia nigra (Kotz et al., , ; Kiwaki et al., ; Thorpe and Kotz, ; Novak and Levine, ; Perez-Leighton et al., ; Teske et al., ). Of these sites, our work suggests that orexin A in the rostral LH has the greatest effect on SPA. As this brain area has been the focus of previous reviews the reader is referred to those reviews for additional information (Kotz et al., , ; Teske et al., ). It is worth emphazing that the effect of orexin A on SPA is a primary outcome that occurs within minutes whereas effects on body weight are considerably more delayed (Teske et al., ; Perez-Leighton et al., ).
#### Orexin, energy expenditure, and obesity
The strong correlation between orexin signaling, SPA, and NEAT, makes orexin an attractive anti-obesity target. Indeed, selective activation of orexin neurons is sufficient to drive increased SPA and energy expenditure in mice (Inutsuka et al., ). Many reports exist implicating reduced physical activity and NEAT in the etiology of obesity in humans (Levine et al., , ). Our work using two different animal models of obesity reveals a strong link between endogenous orexin function, SPA, and body weight. In rats selectively bred for their weight gain in response to high-fat diet (HFD), obesity resistant rats have higher sensitivity to the behavioral effects of orexin A (Levin, ; Teske et al., , ). Over time, HFD decreases SPA in obesity prone animals, whereas obesity resistant rats maintain pre-HFD levels of SPA and sensitivity to orexin-induced SPA (Perez-Leighton et al., , ). Additionally, higher SPA in obesity resistant rats predicts lower fat mass gain throughout their lifetime (Teske et al., ). Consistent with these findings, non-selectively bred rats that display greater levels of SPA are significantly more resistant to pathological weight gain induced by a HFD compared to animals with naturally lower SPA (Perez-Leighton et al., , ). Animals who are resistant to diet induced obesity also exhibit higher expression of prepro-orexin in the LH and enhanced sensitivity to effects of orexin A in rostral LH on SPA (Perez-Leighton et al., , ). Importantly, 10 daily treatments of orexin A administration into the rostral LH prevented HFD induced obesity without altering caloric intake (Perez-Leighton et al., ). Together, these data implicate orexin signaling in determining sensitivity to diet induced obesity and provide clear evidence that orexins regulate energy expenditure through SPA and NEAT.
Animal models of diet-induced-obesity consistently display attenuated levels of orexin signaling molecules in both the CNS and peripheral tissues (Kotz et al., ; Zhang et al., , ; Sellayah and Sikder, ). Similarly, obese humans have lower circulating levels of orexin and impaired orexin receptor activity in adipose tissue (Adam et al., ; Digby et al., ). No comparable studies have been performed investigating differences in the orexin system in the CNS of obese and healthy humans. Unlike in animal studies, we are unable to distinguish between the contributions of individual differences in orexin signaling that predispose humans to develop obesity, and the consequences of environmental effects of calorie-rich diets and sedentary lifestyles (Kotz et al., ; Perez-Leighton et al., , ). Nonetheless, physical activity is a promising candidate for improving clinical outcomes in aged humans at both the metabolic and neurological levels (Castaneda et al., ; Larson et al., ).
#### Orexin, energy expenditure, and narcolepsy
There is a near complete loss of central orexin production in human narcolepsy with cataplexy, as measured by orexin immunoreactivity in post-mortem brain slices (Nishino et al., ; Peyron et al., ). Human narcoleptic patients suffer from extreme episodes of daytime sleepiness. In both humans and animals, narcolepsy is accompanied by higher BMI, increased prevalence of obesity, and reduced physical activity levels (Daniels, ; Hara et al., ; Kok et al., ; Heier et al., ). It should be noted that some research groups have attempted to correlate BMI with orexin levels in blood or CSF, samples which can be relatively easily obtained in a clinical setting. However, studies of circulating orexin, either in serum or CSF, should be interpreted with caution, as one study reported no correlation between orexin A concentrations in serum and CSF samples in either control or narcoleptic patients (Dalal et al., ). Here, narcoleptic individuals had normal serum levels of orexin A yet CSF levels were below detectable levels, in agreement with post-mortem tissue analysis showing a widespread loss of orexin production in the hypothalamus (Nishino et al., ; Dalal et al., ). Perhaps of greater consequence is the issue that measures of freely available orexin neuropeptides do not effectively capture orexin neuropeptide concentrations at important sites of action in the CNS or peripheral tissues nor will this approach fully appreciate the dynamic changes that may be occurring in the signaling system as a whole, including changes in receptor efficacy and cellular excitability (Estabrooke et al., ; Kiyashchenko et al., ; Wu et al., ). Despite these methodological limitations, selective optogenetic or DREADD stimulation of orexin neurons unmistakably rescues deficits in sleep and wake patterns in mouse models of narcolepsy (Adamantidis et al., ; Hasegawa et al., ).
#### Central orexin and peripheral physiology
As described above, a critically important function of the orexin system is its ability to maintain a healthy energy balance by driving physical activity. Orexins act at sites both in the brain and peripheral tissues to regulate physiological processes that contribute to body weight, notably, glucose mobilization, utilization, and adipocyte differentiation (Cai et al., ; Sellayah et al., ; Tsuneki et al., ). The overwhelming majority of orexin production occurs in the hypothalamus, yet orexin signaling is not limited to the CNS (Sakurai et al., ). Small amounts of orexins produced by the enteric nervous system and secretory organs result in circulating plasma levels that are a fraction of those observed in the brain (Sakurai et al., ; Kirchgessner and Liu, ). Importantly, orexin A given intravenously or intranasally to non-human primates is able to rescue cognitive impairments due to sleep-deprivation, indicating central action of systemically administered neuropeptides and viability of clinical applications (Deadwyler et al., ).
Orexin receptors are found in a number of tissues outside of the brain, including adipose tissue, gonads, and the gut (Jöhren et al., ; Digby et al., ; Ducroc et al., ). While most tissues display relatively low levels of orexin receptor expression there is approximately four-fold higher expression of OXR2 in the adrenal glands of rats than of that in the brain (Jöhren et al., ). This is consistent with our understanding of the orexin system being involved in HPA-activation and the responses to physiological and environmental stressors. Although the functional significance is unclear, it is worth noting that orexin receptor levels in the adrenal cortex are dysregulated in an animal model of diabetes (Jöhren et al., ).
Numerous studies indicate a clear relationship between central orexin signaling and pathological changes in peripheral physiology. Selective loss of orexin neurons in the hypothalamus of mice increases susceptibility to diet-induced obesity and age-related weight gain, despite having an intact orexin system in peripheral tissues (Hara et al., , ). As expected, transgenic mice engineered to over-express prepro-orexin, thereby increasing orexin signaling tone, exhibit improved insulin-sensitivity and protection against the negative effects of a HFD on adiposity (Funato et al., ). Furthermore, DREADD-dependent activation of orexin neurons in food-deprived mice promoted glucose mobilization into the blood stream, suggesting enhanced ability to access energy stores during a state of energy imbalance (Inutsuka et al., ). As a whole, the studies described above demonstrate the importance of orexin signaling in promoting healthy energy balance through coordinated mechanisms in both the CNS and in the periphery.
## Effects of life-style choices on physical activity and the orexin system
Evidence that moderate, aerobic physical activity has positive effects on health and body weight is well established. One of the most well characterized phenomena is the ability of physical activity to improve cognitive performance (Colcombe et al., , ; Lindwall et al., ; Erickson et al., ; Miller et al., ). This is a two-way interaction, as choices made throughout life and aging, either directly or indirectly, impact physical activity levels. This section focuses on how excessive calorie consumption (i.e., over-nutrition), which commonly results in obesity and metabolic syndrome, affects physical activity, in particular, SPA, and the orexin system.
In the current climate of rising obesity trends, a great deal of focus has been given to the deleterious effects of sedentary lifestyles on body weight and overall health. Studies have reported that obese individuals spend significantly less time engaged in physical activity. Lean people spend an extra 150 min per day moving compared to obese people, while obese patients sat for 2 h longer per day than lean individuals (Levine et al., ). This difference in SPA equates to an additional energy expenditure of 5 kcal/kg in non-obese participants, indicating excellent therapeutic potential for treating pathological body weight (Levine et al., ). Severity of obesity (measured as accumulation of fat mass) is negatively correlated with NEAT, although this effect only appears in humans after long-term overfeeding (Levine et al., ; Schmidt et al., ). These data reinforce the view that obesity decreases physical activity, but there is large inter-individual variability in this effect.
Animal studies support the idea that higher SPA prior to overfeeding, as well as increased SPA during overfeeding, protects against obesity (Teske et al., ; Perez-Leighton et al., , ). Similarly, development and maintenance of obesity is associated with decreased levels of physical activity in rodents (Bjursell et al., ). The question then becomes, what brain mechanisms contribute to obesity via regulation of physical activity levels? Different lines of evidence support the orexin peptides as key modulators of physical activity, especially in response to nutrition levels and energy availability.
The orexin system is well-placed to both modulate and be influenced by metabolic state. Overall, orexin signaling is suppressed in an obese state (Kok et al., ; Perez-Leighton et al., ). Caloric restriction, as occurs during food deprivation in animals or dieting in humans, increases orexin mRNA and orexin receptor expression (Mondal et al., ; Komaki et al., ; Alam et al., ). Furthermore, orexin neurons act as adaptive glucosensors and are inhibited directly at higher glucose concentrations, suggesting that hyperglycemia results in decreased orexin signaling (Burdakov et al., ; Williams et al., ; Gonzàlez et al., ). This would promote lower SPA and energy expenditure, contributing to the development of obesity, but there are currently no reported electrophysiological studies comparing orexin neuron activity in lean and obese states.
The short- and long-term consequences of diet and lifestyle on orexin neuron activity merit further investigation. It must be emphasized that orexin neurons are part of a local (intra-hypothalamic) and global (across the brain) network involved in the control of behavior and energy balance (Peyron et al., ; Burt et al., ; Kotz et al., ). Thus, when considering specific mechanisms that contribute to obesity, orexin signaling is but one part of an interconnected system influenced by multiple genetic and environmental factors.
## Aging and the orexin system
A number of physiological functions controlled by the hypothalamus vary with age, including SPA, circadian rhythms, and cognitive function. Weight is typically gained throughout early and middle age, followed by gradual, age-associated anorexia (Figure , Chumlea et al., ; Schoenborn et al., ; Sullivan et al., ). The evidence reviewed above indicates orexin signaling is an important driver of energy expenditure and modulates energy metabolism via blood glucose levels and food intake. Simply put, increases in physical activity are generally accompanied by greater energy needs. Anecdotally, one might consider the diet of a professional athlete when training compared to off-season calorie consumption. In line with this reasoning, reduced physical activity levels observed in studies of aged humans and animals may underlie decreased appetite and changes in body weight observed in these populations (Meijer et al., ; Schoenborn et al., ; Kotz et al., ; Bordner et al., ). Many patients near the end of life undergo precipitous weight loss, suggesting severe dysregulation of mechanisms that normally maintain a healthy body weight (Aziz et al., ). Moreover, elderly populations experience a greater prevalence of sleep disturbances and cognitive decline/dementia (Foley et al., ; Corrada et al., ). The diminished physical activity, blunted circadian rhythms, and cognitive deficits associated with aging could be readily explained by compromised orexin signaling in the aged brain.
### Aging in humans
Reductions in the orexin system are observed in humans under a variety of conditions in which symptom onset and severity are strongly tied to aging (Drouot et al., ; Fronczek et al., , ; Karakus et al., ). Dramatic drops in body weight often precede the rapid cognitive and physical decline seen in age-related neurodegenerative diseases, clearly indicating disruption of neurological and physiological processes that promote healthy energy balance (Fronczek et al., , ; Aziz et al., ). While it is clear that patients with Parkinson’s and Alzheimer’s disease display significant loss of orexin neurons in post-mortem exams, analysis of CSF levels in living patients do not always bear this pattern, suggesting there may be a progressive and possibly sudden loss of central orexin synthesis or compensatory peripheral production (Ripley et al., ; Drouot et al., ; Baumann et al., ; Fronczek et al., , ). Some animal studies suggest a tentative link between neurodegenerative disease symptoms and deficits in orexin signaling in monoaminergic and cholinergic neurons in the brainstem and forebrain (Drouot et al., ; Wu et al., ; Sakurai et al., ; Zhang et al., , ; Downs et al., ; Stanley and Fadel, ; Fadel et al., ; Yang et al., ).
Orexin plasma levels are correlated with body weight in postmenopausal females, such that individuals with more circulating orexin A in their blood have lower BMI (Karakus et al., ). However, other studies have failed to identify a clear relationship between changes in orexin CSF and plasma levels. For instance, in narcolepsy, where there is a well-known loss of orexin-producing neurons in the brain, there are reports of patients with low orexin CSF, yet normal orexin plasma levels (Peyron et al., ; Dalal et al., ). It should be noted that assessments of circulating orexin neuropeptides provide very limited insight into the orexin system as a whole, as they do not accurately reflect the complex minutia of events occurring at vital sites of action in the CNS (see Section Orexin, Energy Expenditure, and Narcolepsy for further discussion). Measuring absolute levels of orexin peptide also fails to capture dynamic changes in orexin receptor signaling or changes in somatodendritic excitability of orexin neurons, which are important factors when considering the overall function of the orexin signaling system. Evidence from non-human primates is in line with this reasoning. There was no detectable difference in orexin B labeling in the LH or serum levels of aged rhesus macaques (25–32 years old) compared to mature adults (9–13 years old), yet there was significantly reduced innervation of orexin B fibers in the locus coeruleus (Downs et al., ). Increased levels of orexin in the periphery may be a compensatory response to reduced production in the brain. Therefore, even if peripheral levels of orexin do not decline in aged humans, there may be undetected alterations in prepro-orexin production and/or efficacy of orexin receptor activation in the brain. Unfortunately, given the present lack of investigations using post-mortem human brain tissue or functional imaging, it is still unknown whether age-dependent alterations in physical activity and body composition observed in humans can be attributed to decreased orexin signaling in the CNS.
### Aging in animal models
Animal models exhibit clear age-related reductions in the orexin system in the hypothalamus and other brain regions (Brownell and Conti, ; Sawai et al., ; Kessler et al., ). Aging appears to have a uniform effect on orexin production throughout the hypothalamus as orexin A labeling is reduced to a similar degree in both medial and lateral portions of the hypothalamus (Kessler et al., ). Although there is no overt neuronal loss or degeneration in the hypothalamus of aged rats, there is a substantial age-related decrease of both orexin A and orexin B peptides (Sawai et al., ; Kessler et al., ). Aging also results in reduction of one or both of the orexin receptors in the brain, with some species-specific differences in orexin receptor expression throughout life (Terao et al., ; Zhang et al., ; Porkka-Heiskanen et al., ; Takano et al., ). As expected, transgenic mice with enhanced orexinergic tone exhibit resistance to both age-related weight gain and diet-induced obesity (Funato et al., ; Willie et al., ).
Research groups consistently report reduced behavioral efficacy of orexin-neuropeptides in aged rodents. Intraventricular and intrahypothalamic administration of orexin A increased food consumption in adult rats less than 1 year old, but not in aged, 2-year old rats (Kotz et al., ; Akimoto-Takano et al., ). The ability of both orexin A and orexin B to alter circadian rhythms and increase time-spent awake was also diminished in aged animals (Morairty et al., ). Furthermore, age-related loss of prepro-orexin mRNA production in the LH of rodents is accompanied by reduced orexinergic innervation in the hippocampus, basal forebrain, and locus coeruleus, brain regions associated with cognitive decline in neurodegenerative diseases (Zhang et al., , ; Downs et al., ; Stanley and Fadel, ).
Central orexin signaling modulates aspects of peripheral physiology (e.g., blood sugar regulation and adipocity), which are critically linked to obesity and often become dysregulated with age (Cai et al., ; Tsuneki et al., , ; Sellayah et al., ; Inutsuka et al., ). Animals that do not produce prepro-orexin in the brain develop insulin sensitivity, hyperglycemia, and increased susceptibility to diet-induced obesity, all of which escalate in severity with age (Cai et al., ; Hara et al., ; Tsuneki et al., , ; Sellayah et al., ). Age-associated impairments in brown adipose tissue thermogenesis, which contribute to energy imbalance and weight gain, can be rescued by systemic orexin administration (Sellayah and Sikder, ). Aging-dependent reductions in brown adipose tissue thermogenesis are further exacerbated in mice lacking orexin neurons (Sellayah and Sikder, ). Importantly, dysregulation of insulin signaling is detected in the hypothalamus of prepro-orexin knockout mice before abnormal metabolic symptoms occur in the periphery (Tsuneki et al., ). Together, these studies indicate that central orexin neuron dysfunction precedes development of overt changes in peripheral tissues that result in metabolic disorders and pathological weight gain.
The studies described above indicate that orexin release and receptor activation in the brain declines with age, but additional studies are needed to determine if this occurs in a consistent, uniform fashion or if some projections are spared or possibly increased in a compensatory manner (Zhang et al., , , ; Stanley and Fadel, ). This will be an important factor to consider when developing therapies that target orexin signaling, as some treatments may be more or less effective with age.
## Summary
The hypothalamus is an important regulator of energy balance. Orexin neuropeptide-producing neurons in the hypothalamus integrate metabolic cues (energy availability) and physical activity (energy expenditure). Orexin neurons alter their activity in response to metabolic signals from the periphery, including leptin, glucose, and insulin (Håkansson et al., ; Moriguchi et al., ; Tsuneki et al., , ; Yamanaka et al., ; Burdakov et al., ; Karnani and Burdakov, ; Leinninger et al., ). Orexin signaling is positively correlated with physical activity and negatively correlated with adiposity in both humans and animals (Hara et al., ; Adam et al., ; Perez-Leighton et al., ). Aging has an overall inhibitory effect on orexin signaling, which is likely exacerbated by unhealthy lifestyle choices (Kok et al., ; Hara et al., ; Brownell and Conti, ; Sawai et al., ; Kessler et al., ).
While much has been done in animal models and in humans to show that SPA significantly impacts body weight, metabolic and cognitive health, more work is needed to fully understand the neurocircuitry and molecular mechanisms which regulate SPA, in particular, what happens to this network during aging. Given our current knowledge, therapies should be developed that aim at producing behavioral and lifestyle changes that prevent or ameliorate age-associated declines in physical activity. There is a clear need for multifaceted approaches to altering SPA that include targeted manipulations of the neural systems that drive SPA. Knowing that aging is associated with an altered metabolic and hormonal milieu, an important future research direction is to understand how these molecular changes directly impact orexin signaling and SPA.
In summary, hypothalamic orexin activity fluctuates over the lifespan to impact physical activity and body weight throughout the aging process (Figure ). Aged animals have reduced levels of orexin peptides and receptors, although the magnitude is species dependent. Consistent with a loss of signaling molecules are diminished behavioral, cognitive, and metabolic responses to administration of OXR agonists; a significant issue to consider when developing therapeutics to enhance orexinergic tone. Elevating orexin system activity during aging has the potential to improve both physiologic and cognitive status. A significant strategy in moving forward will be to focus on developing treatments that selectively enhance orexin neuron activity and/or receptor function.
## Conflict of interest statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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Adaptation and homeostasis, the ability to reach stable attractor states within changing environments, are the most typical characteristics of biological systems. A recent study by Benucci et al. ( ) reported that the primary visual cortex (V1) counteracts biases in a rapidly changing stimulus ensemble by introducing the appropriate opposing biases in the responsiveness and selectivity of neurons. Using sequences of differently oriented gratings (32 ms frame duration) and biasing the input statistics toward one orientation (the “adaptor”), such that the selected orientation occurred three to four times more often than all other orientations, a remarkable adaptive behavior was found: decreased activities in response to the adapter were exactly counterbalanced such that the average population signal was kept constant. This was attributed to homeostatic mechanisms “that work toward two simple goals: to maintain equality in the time-averaged responses across the population and to enforce independence in selectivity across the population.” When calculating the necessary time span to show these effects, the authors concluded that V1 needs ~1.7 s in order to catch up with the actual statistical input properties and, thus, adapt adequately to the bias within the sequence of input.
While it is an intriguing idea, assigning V1 a role as a probability detector that integrates incoming information over a considerable time of multiple seconds, our own data (Nortmann et al., ) revealed a much smaller time window for a similar effect; that is, responses to the adapting orientation and to all other orientations balanced each other within 100 ms (Figure , hatched areas in bottom graph). In this study, we used voltage-sensitive dye imaging to capture V1 population dynamics and applied unbiased 10-Hz sequences of oriented stimuli. Moreover, our data suggest that this mechanism is effective for gratings as well as for natural stimuli, and even for single isolated switches. In the example shown, vertically and horizontally filtered natural images were presented, depicted here as gratings for simplicity. Because superimposed orientations were also embedded (cf. plaid in Figure marked purple) in our sequences, a switch from a single orientation to superposition could be analyzed with respect to its underlying adapting component (marked red) and the newly added orientation (i.e., the changing component, blue). Comparing the calculated superimposed component responses with the measured superposition responses revealed that population tuning amplitude for adapted orientation decreased (see purple line and downward arrow) while activity of the changing orientation was facilitated in the opposite direction (upward arrow). Strikingly, the effect was also valid for switches in opposite direction; that is, when a single orientation was removed from superposition, most likely due to increased contributions from OFF responses. In addition, these results suggest that V1 encoded orientation differences rather than current orientations (see Eriksson et al., for similar findings) and hence, reduced input redundancies in accordance with predictive encoding principles (Rao and Ballard, ). These immediate dynamics might be mediated by tuned “push–pull”-like mechanisms (Hirsch et al., ) involving synaptic depression (Nelson, ) and post-inhibitory rebound (Creutzfeld and Struck, ; Sanchez-Vives et al., ).
(A) In Nortmann et al. ( ), pseudorandom stimulus sequences of 17 stimuli (vertically and horizontally filtered natural images, their superpositions, and isoluminant gray image) were presented at 10 Hz (>64 shuffled repetitions). For switch-triggered averaging, sequences were aligned to a specific switch between a pair of stimuli, here to a switch from a single orientation to superimposed horizontal and vertical orientations (see sketch in gray box at top). Plot depicts fitted V1 population tuning curves for adaptive component (red), changing component (blue), and composite switch (purple curve, median across 12 different experiments). Hatched areas indicate deviations (~20%) from the component average (gray). (B) Introducing a bias in one orientation (“adapter”) across random sequences of differently oriented gratings. A simple permutation test was done: adapter bias was set to 30% probability, 12 different orientations were simulated (100 repetitions), single frame = 32 ms, overall stimulation time was 20 s (#625 frames), as used in Benucci et al. ( ). Number of adapter occurrences as single, doublet, triplet, quadruple, and quintuple (black, gray, red, blue, and green, respectively). Inset: number of counts (black horizontal line represents N = 1) for 96 and 128 ms periods of adapter stimulation after 3 s of sequence presentation. After 0.6 s, probability is enhanced to include at least one triplet (red; cf. start of adaptation effect in Supplementary Figure 5 of Benucci et al., ). After 1.7 s, a presence of quadruple is likely (blue); variance was smaller than line width.
Although the stimulation protocols in the studies outlined here have differed in several aspects, we think a link between them exists. The biased stimulation protocol in Benucci et al. ( ) inevitably promotes occurrence of doublets, triplets, etc. and thus repeated frames, leading to longer adapter durations of effectively 64, 96 ms etc., rather than independent presentations of single adapter frames (Figure ). Particularly, the onset of adaptation may vary with the introduced amount of statistical bias. Hence, the reported 1.7 s window might comprise a stimulation-specific time span needed for collecting enough responses to long-duration adapters to reach the signal detection threshold (Figure , inset) within the particular regime of boundary conditions. To be conclusive however, several open questions remain to be addressed in future experiments. For example, in Benucci et al. ( ), adaptation occurs exponentially, whereas probability for long duration adapters increases linearly. Thus, it is not clear if the counterbalancing effects, as observed in Nortmann et al. ( ), are of sufficient magnitude to explain homeostasis as found by Benucci et al. ( ). Moreover, in Benucci et al. ( ) the phase of the gratings was varied while Nortmann et al. ( ) used stationary images. The latter could promote contributions of simple cells, whereas variation in phase averages these out and may enhance nonlinear complex cell contributions.
Regardless of the underlying neural mechanisms: which benefits are provided by cortical adaptation in the hundreds of milliseconds up to second ranges? One may ask the question the other way around: which disadvantages are brought about by longer adaptation times? As often, the answer depends on the task. We suggest that this is when eye movements come into play. For inspection of fine details within natural scenes, high-frequency sampling is useful (Rucci et al., ), requiring ongoing cortical encoding (Benucci et al., ; Nortmann et al., ), and rapid transmission of information along with further adaptive cortical mechanisms acting at tens of milliseconds (Felsen et al., ). In contrast, for saccades occurring at much lower frequencies and on larger spatial scales, it may be advantageous to emphasize stimulus differences to past input (Movshon and Lennie, ; Müller et al., ; Dragoi et al., ), as natural scene statistics predict distant image structure sampled by saccades, to be weakly correlated (Dragoi et al., ).
In the study by Benucci et al. ( ), the dense sampling over different orientations (up to 12) allowed a comprehensive modeling account for cortical homeostasis and decorrelation effects across fine-scaled orientation space. In our work (Nortmann et al., ), we showed counterbalancing effects already after 100 ms adapter times, without probabilistic accumulation of external stimulus statistics over seconds. In fact, the underlying internal processing dynamics may have been implemented in neuronal functioning via adaptation to embodied sensorimotor regularities, such as provided by eye movements. As emphasized in Benucci et al. ( ), adaptation operates on multiple timescales (Wark et al., ; Haak et al., ). In this way, indeed, input statistics experienced during a lifetime may guide manifold cortical network properties while “homeostasis” acts as a dynamic attractor that maintains the ability of the cortex to perform lively deviations from baseline across populations of neurons.
## Conflict of interest statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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## Introduction
Human intelligence (i.e., the ability to consistently solve problems successfully) has evolved through the need to adapt to changing environments. This is not only true of our past but also of our present. Our brain faculties are becoming more sophisticated by cooperating and interacting with technology, specifically digital communication technology (Asaro, ).
When we consider the matter of brain function augmentation, we take it for granted that the issue refers to the human brain as a distinct organ. However, as we live in a complex technological society, it is now becoming clear that the issue is much more complicated. Individual brains cannot simply be considered in isolation, and their function is no longer localized or contained within the cranium, as we now know that information may be transmitted directly from one brain to another (Deadwyler et al., ; Pais-Vieira et al., ). This issue has been discussed in detail and attempts have been made to study the matter within a wider and more global context (Nicolelis and Laporta, ). Recent research in the field of brain to brain interfaces has provided the basis for further research and formation of new hypotheses in this respect (Grau et al., ; Rao et al., ). This concept of rudimentary “brain nets” may be expanded in a more global fashion, and within this framework, it is possible to envisage a much bigger and abstract “meta-entity” of inclusive and distributed capabilities, called the Global Brain (Mayer-Kress and Barczys, ; Heylighen and Bollen, ; Johnson et al., ; Helbing, ; Vidal, ).
This entity reciprocally feeds information back to its components—the individual human brains. As a result, novel and hitherto unknown consequences may materialize such as, for instance, the emergence of rudimentary global “emotion” (Garcia and Tanase, ; Garcia et al., ; Kramera et al., ), and the appearance of decision-making faculties (Rodriguez et al., ). These characteristics may have direct impact upon our biology (Kyriazis, ). This has been long discussed in futuristic and sociology literature (Engelbart, ), but now it also becomes more relevant to systems neuroscience partly because of the very promising research in brain-to-brain interfaces. The concept is grounded on scientific principles (Last, ) and mathematical modeling (Heylighen et al., ).
## Augmenting brain function on a global scale
It can be argued that the continual enhancement of brain function in humans, i.e., the tendency to an increasing intellectual sophistication, broadly aligns well with the main direction of evolution (Steward, ). This tendency to an increasing intellectual sophistication also obeys Ashby's Law of Requisite Variety (Ashby, ) which essentially states that, for any system to be stable, the number of states of its control mechanisms must be greater than the number of states in the system being controlled. This means that, within an ever-increasing technological environment, we must continue to increase our brain function (mostly through using, or merging with, technology such as in the example of brain to brain communication mentioned above), in order to improve integration and maintain stability of the wider system. Several other authors (Maynard Smith and Szathmáry, ; Woolley et al., ; Last, ) have expanded on this point, which seems to underpin our continual search for brain enrichment.
The tendency to enrich our brain is an innate characteristic of humans. We have been trying to augment our mental abilities, either intentionally or unintentionally, for millennia through the use of botanicals and custom-made medicaments, herbs and remedies, and, more recently, synthetic nootropics and improved ways to assimilate information. Many of these methods are not only useful in healthy people but are invaluable in age-related neurodegenerative disorders such as dementia and Parkinson's disease (Kumar and Khanum, ). Other neuroscience-based methods such as transcranial laser treatments and physical implants (such as neural dust nanoparticles) are useful in enhancing cognition and modulate other brain functions (Gonzalez-Lima and Barrett, ).
However, these approaches are limited to the biological human brain as a distinct agent. As shown by the increased research interest in brain to brain communication (Trimper et al., ), I argue that the issue of brain augmentation is now embracing a more global aspect. The reason is the continual developments in technology which are changing our society and culture (Long, ). Certain brain faculties that were originally evolved for solving practical physical problems have been co-opted and exapted for solving more abstract metaphors, making humans adopt a better position within a technological niche.
The line between human brain function and digital information technologies is progressively becoming indistinct and less well-defined. This blurring is possible through the development of new technologies which enable more efficient brain-computer interfaces (Pfurtscheller and Neuper, ), and recently, brain-to-brain interfaces (Grau et al., ).
We are now in a position expand on this emergent worldview and examine what trends of systems neuroscience are likely in the near-term future. Technology has been the main drive which brought us to the position we are in today (Henry, ). This position is the merging of the physical human brain abilities with virtual domains and automated web services (Kurzweil, ). Modern humans cannot purely be defined by their biological brain function. Instead, we are now becoming an amalgam of biological and virtual/digital characteristics, a discrete unit, or autonomous agent, forming part of a wider and more global entity (Figure ).
Computer-generated image of internet connections world-wide (Global Brain) . The conceptual similarities with the human brain are remarkable. Both networks exhibit a scale-free, fractal distribution, with some weakly-connected units, and some strongly-connected ones which are arranged in hubs of increasing functional complexity. This helps protect the constituents of the network against stresses. Both networks are “small worlds” which means that information can reach any given unit within the network by passing through only a small number of other units. This assists in the global propagation of information within the network, and gives each and every unit the functional potential to be directly connected to all others. Source: The Opte Project/Barrett Lyon. Used under the Creative Commons Attribution-Non-Commercial 4.0 International License .
## Large scale networks and the global brain
The Global Brain (Heylighen, ; Iandoli et al., ; Bernstein et al., ) is a self-organizing system which encompasses all those humans who are connected with communication technologies, as well as the emergent properties of these connections. Its intelligence and information-processing characteristics are distributed, in contrast to that of individuals whose intelligence is localized. Its characteristics emerge from the dynamic networks and global interactions between its individual agents. These individual agents are not merely the biological humans but are something more complex. In order to describe this relationship further, I have introduced the notion of the noeme, an emergent agent, which helps formalize the relationships involved (Kyriazis, ). The noeme is a combination of a distinct physical brain function and that of an “outsourced” virtual one. It is the intellectual “networked presence” of an individual within the GB, a meaningful synergy between each individual human, their social interactions and artificial agents, globally connected to other noemes through digital communications technology (and, perhaps soon, through direct brain to brain interfaces). A comparison can be made with neurons which, as individual discrete agents, form part of the human brain. In this comparison, the noemes act as the individual, information-sharing discrete agents which form the GB (Gershenson, ). The modeling of noemes helps us define ourselves in a way that strengthens our rational presence in the digital world. By trying to enhance our information-sharing capabilities we become better integrated within the GB and so become a valuable component of it, encouraging mechanisms active in all complex adaptive systems to operate in a way that prolongs our retention within this system (Gershenson and Fernández, ), i.e., prolongs our biological lifespan (Kyriazis, ; Last, ).
## Discussion
This concept is a helpful way of interpreting the developing cognitive relationship between humans and artificial agents as we evolve and adapt to our changing technological environment. The concept of the noeme provides insights with regards to future problems and opportunities. For instance, the study of the function of the noeme may provide answers useful to biomedicine, by coopting laws applicable to any artificial intelligence medium and using these to enhance human health (Kyriazis, ). Just as certain physical or pharmacological therapies for brain augmentation are useful in neurodegeneration in individuals, so global ways of brain enhancement are useful in a global sense, improving the function and adaptive capabilities of humanity as a whole. One way to augment global brain function is to increase the information content of our environment by constructing smart cities (Caragliu et al., ), expanding the notion of the Web of Things (Kamilaris et al., ), and by developing new concepts in educational domains (Veletsianos, ). This improves the information exchange between us and our surroundings and helps augment brain function, not just physically in individuals, but also virtually in society.
Practical ways for enhancing our noeme (i.e., our digital presence) include:
Cultivate a robust social media base, in different forums.
Aim for respect, esteem and value within your virtual environment.
Increase the number of your connections both in virtual and in real terms.
Stay consistently visible online.
Share meaningful information that requires action.
Avoid the use of meaningless, trivial or outdated platforms.
Increase the unity of your connections by using only one (user)name for all online and physical platforms.
These methods can help increase information sharing and facilitate our integration within the GB (Kyriazis, ). In a practical sense, these actions are easy to perform and can encompass a wide section of modern communities. Although the benefits of these actions are not well studied, nevertheless some initial findings appear promising (Griffiths, ; Granic et al., ).
## Concluding remarks
With regards to improving brain function, we are gradually moving away from the realms of science fiction and into the realms of reality (Kurzweil, ). It is now possible to suggest ways to enhance our brain function, based on novel concepts dependent not only on neuroscience but also on digital and other technology. The result of such augmentation does not only benefit the individual brain but can also improve all humanity in a more abstract sense. It improves human evolution and adaptation to new technological environments, and this, in turn, may have positive impact upon our health and thus longevity (Solman, ; Kyriazis, ).
In a more philosophical sense, our progressive and distributed brain function amplification has begun to lead us toward attaining “god-like” characteristics (Heylighen, ) particularly “omniscience” (through Google, Wikipedia, the semantic web, Massively Online Open Courses MOOCs—which dramatically enhance our knowledge base), and “omnipresence” (cloud and fog computing, Twitter, YouTube, Internet of Things, Internet of Everything). These are the result of the outsourcing of our brain capabilities to the cloud in a distributed and universal manner, which is an ideal global neural augmentation. The first steps have already been taken through brain to brain communication research. The concept of systems neuroscience is thus expanded to encompass not only the human nervous network but also a global network with societal and cultural elements.
### Conflict of interest statement
The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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The groundbreaking work of Hubel and Wiesel in the 1960’s on ocular dominance plasticity instigated many studies of the visual system of mammals, enriching our understanding of how the development of its structure and function depends on high quality visual input through both eyes. These studies have mainly employed lid suturing, dark rearing and eye patching applied to different species to reduce or impair visual input, and have created extensive knowledge on binocular vision. However, not all aspects and types of plasticity in the visual cortex have been covered in full detail. In that regard, a more drastic deprivation method like enucleation, leading to complete vision loss appears useful as it has more widespread effects on the afferent visual pathway and even on non-visual brain regions. One-eyed vision due to monocular enucleation (ME) profoundly affects the contralateral retinorecipient subcortical and cortical structures thereby creating a powerful means to investigate cortical plasticity phenomena in which binocular competition has no vote.In this review, we will present current knowledge about the specific application of ME as an experimental tool to study visual and cross-modal brain plasticity and compare early postnatal stages up into adulthood. The structural and physiological consequences of this type of extensive sensory loss as documented and studied in several animal species and human patients will be discussed. We will summarize how ME studies have been instrumental to our current understanding of the differentiation of sensory systems and how the structure and function of cortical circuits in mammals are shaped in response to such an extensive alteration in experience. In conclusion, we will highlight future perspectives and the clinical relevance of adding ME to the list of more longstanding deprivation models in visual system research.
## Introduction
The capacity of the mammalian brain to rewire and physiologically modify neural connections in response to environmental changes is an intriguing and evolutionary conserved feature. Plastic modifications can have distinct causes and purposes but in general the brain operates in a state of ongoing plasticity (Pascual-Leone et al., ). Given the diversity of its functions, various types of plasticity including synaptic, homeostatic and structural plasticity, are present across distributed neural networks in both juveniles and adults. They typically operate in parallel to allow specific changes at the molecular, cellular, systems and behavioral level as well as to allow compensational or homeostatic changes at the network level (Turrigiano and Nelson, ; Citri and Malenka, ; Holtmaat and Svoboda, ).
When a sensory system fails, as in blindness or deafness, the remaining senses can recruit the “non-stimulated” brain areas by making new or by potentiating existing connections. At the same time, they can strengthen their own functional processing of sensory input to compensate for the loss of the other sense. This phenomenon is defined as cross-modal plasticity. Indeed, extensive reports in rodents and higher-order mammals like cat, monkey, and humans describe cross-modal plasticity in response to complete loss of a sensory modality early in life (for review see Bavelier and Neville, ; Merabet and Pascual-Leone, ). Nevertheless, accumulating evidence also supports the presence of such plasticity in adulthood and even after partial sensory deprivation (Newton et al., ; Allman et al., ; Meredith et al., ; Maslin et al., ) substantiating the notion of the capacity for brain plasticity throughout life.
The visual system of many mammals has been extensively studied to unravel the basic working principles of neuronal physiology, development and plasticity. Reports on the visual cortex are numerous and it is one of the best-described brain areas in relation to the principle of structure-function coupling. Also, the clear dissimilarity of sensory response properties between cortical and subcortical brain regions allows the identification of exclusive cortical attributes (Espinosa and Stryker, ). Since experience acts as a potent force to shape neural circuits and ultimately behavior, the relatively straightforward manipulations of vision in different animal models continue to help decipher the driving forces behind experience-dependent neuronal plasticity. Popular experimental paradigms for the study of visual impairment range from invasive methods such as eyelid suture (mouse: Gordon and Stryker, ; Levelt and Hübener, ), splitting the optic chiasm (Berlucchi and Rizzolatti, ; Yinon and Hammer, ) and intraocular injection of tetrodotoxin (TTX; Frenkel and Bear, ) to non-invasive methods including dark rearing from birth (Morales et al., ; Gianfranceschi et al., ; Kreczko et al., ; Yang et al., ), dark exposure (shorter period with varying starting point: He et al., , ; Huang et al., ; Montey and Quinlan, ; Guo et al., ; Duffy and Mitchell, ; Petrus et al., ), eye patching (Zapasnik and Burnat, ; Laskowska-Macios et al., ), stimulus-specific exposure like stripe (Blakemore and Cooper, ; Blasdel et al., ) or strobe rearing (Humphrey and Saul, ) and the application of prism goggles (Yoshitake et al., ). These studies have led to the characterization and timing of critical periods for ocular dominance and also for direction and orientation selectivity.
Monocular enucleation (ME) or the surgical removal of one eye can be considered a model for unilateral sensory deafferentation where half of the normal visual input is lost. In this type of drastic vision loss even low contrast vision, which still occurs through sutured eye lids, as well as any form of retinal spontaneous activity from the manipulated eye is lost. It has been used for the first time in neonatal rabbits as early as 1870 to trace the course and destination of eye-specific neuronal projections across the visual system (Gudden, ). These primordial observations together with subsequent studies in rats, cats and dogs consistently revealed histological alterations and marks of degeneration along the ascending retino-geniculate and—collicular pathway following ME early in life. Numerous studies in the following century collectively contributed to our current understanding of enucleation-induced subcortical structural alterations, including the extension of retinal afferents originating from the remaining eye in the lateral geniculate nucleus (LGN), and superior colliculus (SC) (for review see Toldi et al., ). Overall, they revealed the topographic maturation of distinct retinal projections to the contra- and ipsilateral target regions inside the LGN and SC. In comparison, the contralateral visual cortex appeared less prone to the anterograde degenerative mechanisms of ME (Tsang, ). Nevertheless, visual areas, especially ipsilateral to the remaining eye, displayed an enlarged distribution of callosal connections in adulthood such that they are no longer limited to the strip across the border between primary and extrastriate cortices (Wree et al., ; Olavarria et al., ). These observations are in relation to less extensive pruning of axons of callosal neurons due to the early lack of input from one eye.
Experience-dependent neuroplasticity occurs throughout life and as such has gained ample interest from the 1970’s onwards (Wall and Egger, ). Indeed, a vast body of literature has demonstrated that the mature neocortex is not a fixed entity but retains substantial malleability, which is exemplified in primates (Kaas et al., ; Kaas, ; Donoghue, ; Gilbert, ; Qi et al., ), cat (Chino et al., ; Darian-Smith and Gilbert, ; Hu et al., , , ), ferret (Erisir and Harris, ; Allman et al., ), raccoon (He et al., ), rat (Siucinska and Kossut, ; Kossut, ; Zhou et al., ; Tandon et al., ) and mouse (Keck et al., ; Lehmann and Löwel, ; Maya-Vetencourt et al., ; Van Brussel et al., ). In this context ME applied later in life has been a valuable research model since it could reveal additional plasticity and physiological modifications in the mature sensory cortex as compared to the other invasive and non-invasive vision impairment models (Newton et al., ; Paulussen et al., ; Van Brussel et al., ; Nys et al., ).
This review will mainly emphasize on the effects of ME within the visual system of mammals. Since age at the time of surgical intervention is a decisive factor for the subsequent alterations, including a different degree of plasticity in subcortical structures and sensory cortex, we will highlight how age next to the type of visual manipulation is paramount towards understanding the multifaceted aspects of developmental, visual and cross-modal plasticity. Observations in ME animal models and humans will be compared and contrasted with observations in the blind.
## Monocular Enucleation as a Tool to Map Eye-Input Specific Subdivisions Within Visual Cortex
The areal map for the mouse visual cortex, including the number and identity of different visual areas is still constantly being refined (Garrett et al., ). A decade ago, no clear consensus about the number of areas and their functional organization, including the specific location of monocular and binocular driven zones, was available. Historical efforts to adequately delineate the different visual areas entail neuroanatomical methods like (immuno)histology (Caviness, ; Van der Gucht et al., ; Paulussen et al., ) and tracer-based mapping (Olavarria and Montero, ; Wang and Burkhalter, ) next to in vivo electrophysiological (Wagor et al., ; Wang and Burkhalter, ; Van den Bergh et al., ; Vreysen et al., ) and intrinsic optical imaging approaches (Schuett et al., ). In this context ME was specifically applied to characterize the eye-input specific subdivisions within the mouse visual cortex. Indeed, the analysis of visually driven molecular activity patterns in the brain of mice with one or two eyes enucleated based on the expression of activity reporter genes like zif268 and c - fos , identified the full spatial extent of the visual cortex along the anterior-posterior and medio-lateral axes of the brain as well as the different monocular and binocular driven subregions therein (Van Brussel et al., ; Aerts et al., ). In fact, the monocular and binocular zones derived from that study correspond well with the representations of the monocular and binocular visual field(s) in the topographic map of mouse visual cortex by Wang and Burkhalter ( ). Meanwhile, advanced analysis by intrinsic optical imaging (Kalatsky and Stryker, ; Garrett et al., ), two-photon calcium imaging methods (Andermann et al., ; Marshel et al., ; Roth et al., ; Glickfeld et al., ) and modern anatomical studies combined with network analysis (Wang et al., , ; Wang and Burkhalter, ) made it possible to study the functional and connectional properties of V1 and each of the extrastriate areas in the mouse in more detail.
A major difference between the visual cortex of rodents and higher-order mammals is the salt-and-pepper configuration or “intermingled” organization of functionally linked neurons instead of the cortical columns of neurons with a similar ocular dominance, orientation and direction selectivity found in carnivores and primates (Dräger, ; Niell and Stryker, ; Gao et al., ). Actually, by virtue of the high percentage of crossing-over of the anatomical connections at the optic chiasm and the lack of ocular dominance columns in mouse and rat visual cortex, ME results in the irreversible loss of visual input to distinct contralateral monocular cortical target regions as elegantly shown in the mentioned ME investigations of rodent visual cortex structure (Toldi et al., ; Van der Gucht et al., ; Van Brussel et al., ).
In higher order mammals like primates and cats, this explains why ME can only result in complete loss of vision in those cortical regions that represent the monocular crescent of the peripheral visual field that is normally provided by the nasal retina of the removed eye (Eysel, ; Horton and Hocking, ). Monocular enucleation nevertheless revealed evolutionary differences in the functional ocular dominance columns in primary visual areas of distinct primate species. A comparative study based on the examination of cytochrome oxidase activity patterns after ME in adulthood revealed the absence of ocular dominance columns across layer III, IV and VI of V1 in the New World squirrel monkey in contrast to a clear eye-input specific organization in the Old World macaque monkey (Hendrickson and Tigges, ; Takahata et al., ). Other studies using intrinsic optical imaging of V1 confirmed the existence of ocular dominance columns in the New World owl monkey (Kaskan et al., ) and the Prosimian Bush baby (Xu et al., ). Using a similar approach of ME and cytochrome oxidase histochemistry in macaque monkeys, Horton and Hocking ( ) could demonstrate the presence of intrinsic variability in the periodicity of ocular dominance columns in layer IVc from animal to animal of the same species. In a subsequent study, they compared the effects of ME with eyelid suture and retinal laser lesions on cytochrome oxidase activity in the striate cortex of macaque monkeys. They revealed an additional functional parcellation of monocular core zones alternating with binocular border strips outside layer IVc in both monkey and human visual cortex (Horton and Hocking, ). Others have assessed neuronal activity in the Vervet monkey by means of expression analysis of activity reporter genes. Similar to cytochrome oxidase activity patterns, Zif268 and c-Fos immunoreactivity after monocular deprivation (lid suture, enucleation and TTX injections) revealed ocular dominance columns as well as their respective size and density (Chaudhuri et al., , ; Van der Gucht et al., ).
## Monocular Enucleation as a Brain Plasticity Model
### Monocular Enucleation vs. Monocular Deprivation—Impact on Binocular V1
An important difference between ME and the frequently used MD paradigms, like lid suturing or eye patching, is that in case of ME, all retinal activity from one eye, including spontaneous waves and light-driven patterns, is instantly and irreversibly eliminated. Upon MD, the well-structured spontaneous retinal waves from one eye are replaced by uncorrelated noise and transferred via the LGN towards the visual cortex where it induces synaptic long-term depression (LTD; for review see Cooper and Bear, ). Upon ME no retinal input is left yet spontaneous synchronous bursting can still occur within the LGN, preventing cortical LTD and likely originating from cortico-thalamic feedback (Weliky and Katz, ). There is a complete removal of inhibitory binocular interactions following ME, which is responsible for the absence of binocular competition as a factor contributing to subsequent experience-dependent cortical plasticity (Hübener, ; Van Brussel et al., ). Consequently, the MD paradigms have been particularly instrumental in understanding the contribution of the correlation of binocular inputs as well as of high quality patterned vision in sculpting cortical circuits during development (Morales et al., ; Konur and Yuste, ; Burnat et al., ; Espinosa and Stryker, ; Zapasnik and Burnat, ; Chen et al., ). They will therefore remain the dominant methods of deprivation to understand ocular dominance and its plasticity, in relation to diseases like amblyopia (lazy eye) (Hofer et al., ; Morishita and Hensch, ; Levelt and Hübener, ; Sengpiel, ).
ME on the other hand allows to model other aspects of long-term vision loss, which are more difficult to accomplish and to maintain using for example pharmacological injections (i.e., TTX) in the eye. It is noteworthy that, in response to ME, histological alterations have been documented in subcortical vision centers (see Sections Effect of monocular enucleation at birth and Subcortical effects of monocular enucleation in adulthood), yet in the mouse visual cortex, only a negligible influence of injury artifacts of enucleation has been found. For instance, Smith and Trachtenberg ( ) demonstrated that pharmacological silencing of one eye without deafferentation of the optic nerve, results in a similar reduction of contralateral cortical activity as ME (Smith and Trachtenberg, ).
Ever since the pioneering studies of Cynader and colleagues (Shaw et al., ; Prasad and Cynader, ; Prasad et al., ), there has been an intense focus on identifying potential mechanisms that regulate plasticity and could control critical periods. In this context, gene expression profiling studies in both mouse (Majdan and Shatz, ) and old-World monkey (Lachance and Chaudhuri, ) did recognize ME as a proper and robust deprivation paradigm to elucidate candidate “plasticity genes” that are particularly sensitive to alterations in visual input during the traditional critical period of ocular dominance plasticity. In these studies, ME was exactly chosen because it induces robust changes in the eye-specific circuitry and it has been proven to globally change visual cortex gene expression (Chaudhuri et al., ). Although MD was shown to be useful in similar molecular studies (Rietman et al., ), gene expression changes turned out more reproducible upon ME than upon MD or monocular inactivation with TTX exactly because of variable levels of residual retinal activity in these visual deprivation paradigms (Majdan and Shatz, ).
The overall effect of MD and ME on binocular neurons in V1 is comparable, namely an ocular dominance shift towards the open eye (Faguet et al., ). Since ME induces the most robust intraocular activity imbalance possible, the signal to noise ratio in, for example, molecular activity mapping studies is maximal, in line with the above mentioned gene expression studies (Kanold et al., ; Van Brussel et al., ). Indeed, arc (activity-regulated cytoskeletal-associated protein) is one of the frequently used IEGs that can be specifically induced in V1 neurons by visual stimulation (Syken et al., ; Tagawa et al., ). When ME is performed in mice during the critical period (age P28), the arc expression in contralateral V1 in response to stimulation of the non-deprived open eye expands into closed-eye territory after a few days reflecting a spatial representation of the robust ME-induced ocular dominance plasticity (Tagawa et al., ; Syken et al., ; Datwani et al., ; Kanold et al., ).
Nevertheless in MD and ME different activity-dependent (synaptic and homeostatic) changes will likely occur in the contralateral visual cortex. For example, the amount of homosynaptic LTD of deprived connections, which is stronger when asynchronous (de-correlated) afferent activity is present, is probably less abundant upon ME. Hence, similar to monocular inactivation with TTX, ME will induce less LTD in the binocular visual cortex (Rittenhouse et al., ; Frenkel and Bear, ; Coleman et al., ). Furthermore, it is expected that activity-dependent modifications in both local and long-range intracortical connectivity patterns of GABAergic and pyramidal neurons, respectively (Trachtenberg et al., ; Calford et al., ; Erisir and Harris, ; Allman et al., ; Keck et al., ; Vasconcelos et al., ), are differentially modulated upon ME and MD. Especially after long time periods, these two deprivation methods will likely cause a different recalibration of the excitation-inhibition balance, inside binocular V1, and certainly also in adjacent monocular cortical territories. Pronounced effects in the monocular cortex would not depend upon the mechanisms that underlie ocular dominance plasticity but rather implicate a broad contingent of distinct plasticity mechanisms, such as homeostatic synaptic scaling operating across the visual cortex after an altered regime of neural activity (Turrigiano et al., ; Goel et al., ; Mrsic-Flogel et al., ).
Summarized, complementary to MD studies in the critical period, different results in the ME model can reveal additional information regarding deprivation-specific mechanisms at play across visual areas whereas similar results between ME and MD could illustrate general mechanisms that take place after the loss of visual input, regardless of the severity of input removal.
### Effect of Monocular Enucleation at Birth
Enucleation of one eye at birth obviously interferes with the development of vision. Drastic structural rearrangements and changes in synaptic efficiency are induced along the subcortical, thalamocortical and cortico-cortical pathways, especially contralateral to the removed eye (for review see Toldi et al., ). In subcortical (Lund et al., ; Yagi et al., ; Chan et al., ; Furman and Crair, ) and cortical (Toldi et al., , ; Hada et al., ; Yagi et al., ) structures of the rodent visual system, the ME-induced rerouting of retinogeniculate, retinotectal and geniculocortical afferents and callosal inputs corresponds with the recruitment of deafferented neurons and the functional modifications in favor of the remaining eye. This enucleation-dependent reorganization of the uncrossed, ipsilateral visual pathway during development mirrors the perceptual learning ability of enucleated rats exposed to a black-white and horizontal-vertical discrimination task. Once the task has been learned, a lesion in the contralateral cortex, the ipsilateral cortex or the contralateral optic tract relative to the remaining eye was performed. An ipsilateral lesion resulted in retained learning skills in both the neonatal ME and late ME group whereas in the case of a contralateral lesion, only the neonatal ME rats were able to preserve memory (Yagi and Sakai, ; Sakai et al., ). In addition, visual acuity of the remaining eye in neonatally enucleated rats is significantly enhanced at 3 months of age (Sakai et al., ).
Remarkably, if a neonatal induced lesion in the visual cortex of kittens is combined with ME, the retrograde severe loss of X-type retinal ganglion cells, with high spatial resolution and low contrast thresholds and linked to the form-sensitive visual pathway, in the remaining eye is prevented. This retinal rescue suggests an ME-induced neuroprotection of retrograde cells normally degenerated by cortical damage (Illig et al., ). This is in line with the reduced apoptosis of retinal ganglion cells and preservation or even expansion of their connections in the remaining eye observed following early monocular vision (Guillery, ; Steeves et al., ).
Next to systems level changes, early ME in animals has uncovered certain molecular players involved in the development of afferent visual pathways. It is proposed that BDNF (and its TrkB receptor) levels are altered across retinotopic targets upon early ME. In this scenario, BDNF or other neurotrophic factors could initially decrease due to the loss of retinal input but, after long-term survival, are produced or secreted by different sets of local cells or delivered by anterograde or retrograde trafficking through neuronal pathways (Frost et al., ). Moreover, the transcription factor CREB (Vierci et al., ) and the matrix metalloproteinase 9 (Oliveira-Silva et al., ) have been implicated in the establishment and plasticity of retinotectal projections in rat and mouse upon ME.
The metabolic and biochemical mechanisms that accompany the ME-induced plasticity at early ages consist of changes in glucose utilization (Vargas et al., ; Wang et al., ) and neurotransmitter levels (Nakamura et al., ; Riback and Robertson, ). Recently, they have been evaluated in vivo using proton magnetic resonance spectroscopy of the visual cortex 3 weeks post-enucleation. The metabolic outcome likely reflects cortical reorganization associated with a general neural activity loss, the elimination of neurons and retraction of axon terminals (Chow et al., ).
The cytoarchitectonic structure of the visual cortex of neonatal ME mice, assessed by Golgi and histological methods, undergoes a reduction in the neuropil volume, an increase in neuronal densities, a higher variation in the dendrite orientation of stellate cells with ascending projections and a decrease in the number of dendritic spines of layer V pyramidal neurons (Valverde, ; Heumann and Rabinowicz, ). Furthermore, it appears that supragranular layers II and III of both contra- and ipsilateral visual cortex are most affected by neonatal enucleation (Heumann and Rabinowicz, ). In newborn ferrets that underwent ME the formation of orientation, spatial frequency and retinotopic maps is unaffected, but their structure and spatial relationships are altered compared with normal development in binocular intact animals (Farley et al., ).
### Subcortical Effects of Monocular Enucleation in Adulthood
At the cellular level, a glial response prevails across different subcortical direct retinal target structures in the adult mouse upon ME, as an early marker of neuronal injury (Cuyvers et al., ). Instant denervation-induced microglial activation precedes astrogliosis mainly in contra- but also in ipsilateral subcortical structures, including the LGN and SC (Wilms and Bähr, ; Gonzalez et al., ). In general, activated glial cells are known to clean up axonal debris, in this case of lost retinal ganglion cells, to restore tissue homeostasis and to release growth factors and cytokines to stimulate neuronal sprouting (Bechmann and Nitsch, ). Yet in adulthood, enucleation triggers a reduction of trophic influences in direct retinal targets in the brain. For example, BDNF levels in the LGN and SC of adult enucleated rats are significantly decreased (Avwenagha et al., ). Reactive oxygen species, which at non-toxic levels act as messenger molecules to mediate structural remodeling, are also apparent in subcortical structures of the adult rat visual system upon ME (Hernandes et al., ). Other manipulations at eye level, which involve retinal ganglion cell loss, and mimic visual disorders characterized by RGC death, also induce a glial response in the brain. For example, laser-induced monocular hypertension (mOHT), a mouse model for glaucoma, induces astrogliosis in the left and right SC and LGN of the mouse (Dekeyster et al., ), just as observed in primate OHT models (Lam et al., ; Shimazawa et al., ) and in optic nerve heads of human glaucomatous eyes (Prasanna et al., ) and this could correlate with the neurodegeneration and atrophy observed in the LGN of glaucoma patients (Gupta et al., ).
## Cross-Modal Plasticity: An Intriguing Response to Sensory Input Loss at Subcortical and Cortical Level
Cortical reorganization upon complete sensory deprivation does not only occur within the affected sensory system but is also present in other modalities. It is a popular belief that profound deprivation or denervation of one sense early in life can modify the structural and functional development of the remaining modalities and recruit these to drive the deprived cortical areas. In the human visual system, perceptual tasks, electrophysiological and neuroimaging experiments have principally concentrated on congenital or neonatal blind subjects to study this cross-modal type of plasticity (Sadato et al., , ; Cohen et al., ; Hamilton and Pascual-Leone, ; Lessard et al., ; Pascual-Leone et al., ; Ptito et al., ; Collignon et al., ; Lazzouni and Lepore, ).
### Lessons from Blind Mammals
In early blind (binocular enucleated) mammals, territories associated with somatosensory and auditory functions appear expanded and recruit the former visual areas, including V1, to perform increased multimodal processing (Rauschecker et al., ; Toldi et al., ; Izraeli et al., ; Laemle et al., ; Laramée et al., ; Charbonneau et al., ).
At the systems level, one possible mechanism is the rewiring of long-range subcortical connectivity patterns (Karlen et al., ). Indeed, the inferior colliculus (IC), a midbrain auditory nucleus that normally projects to the primary auditory thalamic nucleus (medial geniculate nucleus, MGN) in sighted animals, can additionally connect with the dorsal LGN and thereby convey non-visual information to V1 in experimentally blind animals (Piché et al., , ; Chabot et al., , ) as observed in the naturally blind mole rat Spalax ehrenbergi (Bronchti et al., ). Similarly, somatosensory afferents can form an alternative route to innervate the LGN in order to transfer tactile inputs to regions normally devoted to visual processing (Asanuma and Stanfield, ). In addition, the higher-order lateral posterior thalamic nucleus can also constitute to an anatomical pathway for the transmission of somatosensory-driven responses to the rat visual cortex upon neonatal binocular enucleation (Négyessy et al., ).
In the mature brain, severe deprivation will also cause a time-dependent cascade of reorganization across allocated neural networks. Preexisting connections carrying information of the other senses are rapidly unmasked and strengthened, leading to long-term structural adjustments including new synapses. Although the examples of subcortical involvement are mostly present following early deprivation, it is proposed that hearing impairment in adult ferrets results in cross-modal cortical reorganization originating from alterations in the brainstem, which in normal animals already receives multimodal inputs (Shore et al., ; Allman et al., ). In support of the latter, hearing loss also enhances somatosensory innervation of the dorsal cochlear nucleus (auditory brainstem) (Shore et al., ).
Other system-level hypotheses have been described that could prevail in both juvenile and adult blind subjects. The first implies changes in cortico-cortical feedback, in which existing projections from multimodal higher-order cortices (i.e., frontal, parietal and temporal association cortex) increase their influence onto primary sensory cortices (Newton et al., ; Lippert et al., ; Lingnau et al., ). Additionally, changes in direct and indirect cortico-cortical connections, between different primary sensory cortices can account for a cross-modal aspect of plasticity, at least at the functional level (Wang et al., ; Sieben et al., ). Multimodal neurons in primary cortices that work as information hubs that regulate multisensory cortical recruitment under different conditions of sensory stimulation or deprivation, could add to these existing connections modulating cross-modal changes (Vasconcelos et al., ). Apart from tracer studies in V1 of adult opossums enucleated at birth (Karlen et al., ), evidence for large-scale structural changes of cortical afferents to V1 is lacking in mouse models of congenital or neonatal blindness (Laramée et al., , ; Charbonneau et al., ; Wang et al., ; Sieben et al., ). This is in agreement with intermodal connections between primary sensory cortices whose presence was already shown in intact rodents (Larsen et al., ; Campi et al., ; Iurilli et al., ; Stehberg et al., ; Henschke et al., ) and suggests that their development is mainly unaffected by early blindness. Recently, cross-modal potentiation of thalamocortical axons in non-deprived primary sensory cortices of the mouse is put forward as a general mechanism of adult synaptic plasticity in response to short sensory deprivation (Petrus et al., ). Dark exposure and cochlear inactivation were used as sensory deprivations in adult mice. Functional changes in the non-deprived primary cortex were evaluated using in vivo single unit recordings to characterize tuning properties and in vitro optogenetic activation of thalamocortical axons combined with mEPSCs in layer IV neurons to dissect alterations in synaptic transmission. Dark exposure altered the tuning properties of auditory neurons and increased the synaptic responsiveness in layer IV neurons of A1 upon optogenetic activation of MGB neurons. However, it did not affect the strength of V1 layer IV inputs originating from geniculate neurons since the critical period for geniculocortical axon plasticity within the visual system had already passed. Deafening induced specific potentiation of geniculocortical inputs of the visual cortex without affecting granular neurons in A1. Together, these findings suggest that deprivation of one sensory input results in the subsequent strengthening of thalamocortical projections in the non-deprived primary cortices in adulthood.
What kind of molecular mechanisms mediate this experience-dependent plasticity and which neural networks are susceptible to cross-modal reorganization at any given age remains largely unknown. These queries deserve attention because they are essential for understanding the specific development of each sensory system and their multimodal interactions (Bavelier and Neville, ). At the molecular level, cross-modal homeostatic plasticity in the primary sensory cortices of juvenile mice (P28) has been associated with changes in AMPA receptor subunits. Excitatory postsynaptic transmission was scaled up in V1 upon dark exposure while opposite changes in mEPSC amplitudes and AMPA receptor 1 expression, phosphorylation and rectifying properties were discerned in S1 (Goel et al., ). The latter likely reflect a homeostatic response to increased activity of the spared senses upon transient blindness. In addition, synapse-specific strengthening or LTP of layer IV to layer II/III inputs in the barrel cortex of juvenile rats occurs after visual deprivation (dark exposure for 2 days) and is mediated by serotonin-signaling-dependent delivery of the AMPA receptor 1 subunit to the synapse. These cross-modal alterations ultimately sharpen the tuning of barrel neurons in response to principal whisker stimulation (Jitsuki et al., ). Epigenetic changes, namely H4 deacetylation, are additional mechanisms that orchestrate the expansion of the barrel cortex following binocular enucleation in rats (Fetter-Pruneda et al., ). Together, these advances have led to subsequent whole cell recording experiments in juvenile mice with a different degree of visual deprivation (dark exposure, binocular enucleation and bilateral lid suturing) that confirmed distinct, independent functions and sensory requirements of unimodal vs. cross-modal synaptic plasticity. Complete loss of vision is necessary to induce unimodal scaling whereas loss of patterned vision is sufficient to induce cross-modal alterations in synaptic scaling (He et al., ).
### Monocular Enucleation can also Induce Cross-Modal Reorganization
Given that the timing or age of vision loss, in addition to the degree (complete or partial) and type (natural or experimental) of deprivation, has a strong influence on the nature of cross-modal plasticity, the effects of ME during development, adolescence and adulthood will be discussed separately.
#### Early Monocular Enucleation (At Birth, P0)
Apart from unimodal changes in visually evoked response maps, in neonatal rats ME triggers cross-modal changes including the invasion of somatosensory cortex in the contralateral visual cortex (V1 and V2) since bimodal neurons are more frequently found within these visual areas (Toldi et al., , , ). Multisensory interaction experiments confirmed that somatosensory-evoked potentials are generated within the visual cortex and are not passively conducted from the somatosensory cortex (Toldi et al., ). In contrast, auditory activation maintains its territory as in normally sighted rats and does not invade the visual cortex upon ME at birth (Toldi et al., ). Massive cross-modal plasticity was further explored by combining the electrophysiological and autoradiographic detection of tactile responses in the visual cortex evoked by both electrical and mechanical whisker stimulation. A widespread expansion of the somatosensory responsive area is observed along the antero-posterior axis (Toldi et al., , ), indicating that neonatal ME also exerts a strong influence on the somatosensory system itself. These cross-modal interactions will likely provide the neural basis for behavioral compensation(s).
#### Effects of Monocular Enucleation in Adulthood (Mouse, P120)
So far, only few studies could prove that cross-modal changes are also manifested at the cortical level in partially deprived adults. Indeed, functional reorganization by unmasking of mature but silent intermodal connections in adult monocularly enucleated rabbits (P140) has been demonstrated (Newton et al., ). Moreover, in adult ferrets (P189–240) with moderate hearing loss new multisensory neurons, yet a few that show multisensory integration, are detected in the deprived core auditory cortex (Meredith et al., ). However, their functional meaning and consequences to behavior remain largely unclear and could even be responsible for maladaptive perceptual effects such as tinnitus (Allman et al., ; Meredith et al., ).
In line with these studies, Van Brussel et al. ( ) discerned a partial non-visual contribution to the restoration of cortical activity in the visual cortex of adult mice (P120) following long-term ME (7 weeks) (Figure ). Establishing expression maps for the activity reporter gene zif268 allowed comparison of neuronal activity between the monocular and binocular contralateral visual cortex of control and ME mice. ME first induced the potentiation of ipsilateral open-eye input leading to the reactivation and expansion of the binocular territory. Next, a slower unmasking of preexisting long-range cross-modal projections occurred, facilitating the transfer of tactile information to the extrastriate cortex. This interpretation was further substantiated at the functional level by ipsilateral whisker deprivation as well as whisker stimulation experiments that respectively reduced and increased visual cortex activity, especially in the medial monocular cortex of long-term ME mice (Figure ; Van Brussel et al., ). Indeed, in control mice such whisker manipulations only influence molecular activity in the barrel cortex and not in the visual cortex. The lack of changes in molecular activity in the visual cortex of control mice combined with the clear effect in adult ME mice put the conversion of silent or subthreshold multimodal input into suprathreshold input forward as a substrate of the ME induced cross-modal plasticity (Van Brussel et al., ).
Spatiotemporal reactivation of the contralateral visual cortex by visual and cross-modal inputs after monocular enucleation (ME) in adult mice. (A) Layer- and time-specific recovery of neuronal activity in the left visual cortex subsequent to removal of the right eye at an adult age of P120 is illustrated. Molecular activity profiles of the visual cortex have been assessed by the zif268 mRNA expression analysis around Bregma level −3.40 mm. For each section, the original autoradiogram displaying the deprived (left) visual cortex is shown in gray and its matching pseudo-colored mirror image. The medial and lateral extent of the left visual cortex is marked by the two large arrowheads whereas small arrowheads delineate the interareal boundaries. The activity in the central binocular cortex starts to expand supragranularly (asterisk first and second panel) between 1 and 3 weeks post-ME. Between 3 and 5 weeks, infragranular layers also start to show increased reactivation. (B) Anterograde and retrograde transport of fluororuby upon injection in V2M of a 7wME mouse. b: Detail of Fluororuby signal at the location of somatosensory cortex: tracer is transported in an anterograde way to axon terminals in layers V and VI, while supragranular layers II/III contain retrogradely labeled cell bodies and dendrites. c: Detail of anterogradely labeled fibers in contralateral V2M. d: Detail of retrogradely labeled pyramidal cells in layers II and III of ipsilateral/adjacent somatosensory cortex. (C) Subsequent whisker manipulations in 7wME mice were employed in order to verify the functional relevance of the intermodal connections in the ME-induced reactivation profile. Somatosensory deprivation (SD) by trimming the right-side vibrissae results in decreased visual cortex activity whereas somatosensory stimulation (SS) through exposure to toys and novel objects in the dark increased activity, especially in V2M. Adapted from Van Brussel et al. ( ).
A high incidence of multisensory neurons has been detected in so-called “transition zones” between the primary areas of different modalities (Toldi et al., ; Wallace et al., ). In light of these multisensory zones, it was not surprising that the multimodal reactivation of molecular activity in adult mice appeared starting from the anterior and lateral borders of visual cortex with somatosensory and auditory cortex.
#### Monocular Enucleation in Adolescence (Mouse P28–P60)
In mouse and rat, eye opening occurs around the age of 12–14 days (P12–14). It is assumed that puberty starts around P28 and adolescence lasts until approximately the 56th postnatal day. This transition period between P28 and adulthood (P120) corresponds to the physiological age window of human adolescence (Han et al., ; Brenhouse and Andersen, ). Despite massive synaptic rearrangements, functional changes and gene expression modifications upon ME (Majdan and Shatz, ), this age interval is often overlooked in typical visual plasticity research, including ocular dominance plasticity (but see Daw et al., ; Majdan and Shatz, ; Lehmann and Löwel, ; Huang et al., ). Important structural alterations occur in the adolescent brain, including the visual cortex, and this without a large effect on overall volume. For example, all layers in V1 of the macaque monkey undergo intensive synaptogenesis during early postnatal life, followed by a slow decrease in synaptic density in the next years (Bourgeois and Rakic, ). Thereafter, a rapid reduction of excitatory synapses situated on dendritic spines is observed around the age of puberty. This period of synaptic pruning seems to manifest itself more quickly in layer IV compared with supra- and infragranular layers (Bourgeois and Rakic, ). Likewise, certain perceptual abilities (i.e., contour integration) related to the ventral stream of the human visual system do not develop until well into adolescence (Kovács, ), and it is likely that a gradual maturation of particular molecular and cellular characteristics largely support these late aspects of visual cortex functioning (Bourne and Rosa, ).
When ME is applied to adolescent mice (P45) an incomplete reactivation of the deprived visual cortex was detected, even after 7 weeks, due to the lack of a clear take-over of the visual cortex by somatosensation (Nys et al., ). This was a quite surprising observation since different types of plasticity, including ocular dominance plasticity, are often more elaborate in young animals compared to older ones. The current identification of a pre-adult period for cross-modal plasticity resulting from ME is quite remarkable but may not be generalized to cross-modal plasticity following other deprivation paradigms. There are undoubtedly multiple critical periods for different types of plasticity across different subdivisions of the visual cortex, and in fact this is what we want to bring forward with this review.
## Consequences of ME on the Human Visual System
Most of the studies of plasticity in humans have focused on the effect of ME during early development whereas studies on late ME subjects are rather limited. From a medical perspective, negative consequences of ME have been described in adult patients where unilateral enucleation can cause complex visual hallucinations even if they have excellent vision in the remaining eye (Ross and Rahman, ). Phantom pain or sensation is also frequently encountered after eye amputation (i.e., ME) in adult patients, whereas it is rather unlikely in children (Flor et al., ; Rasmussen et al., ).
### Effects of Early ME on the Human Visual System
Just like in ME animals, the loss of stereoscopic vision has been studied in unilateral enucleation patients (for review see Steeves et al., ). Upon early ME in humans, the location of the visual egocenter is altered due to a shift towards the open ipsilateral eye, inducing an asymmetric bias. However, after a prolonged period with one-eyed vision, the spatial processing system recalibrates to adapt to their new monocular world. Hence, the egocenter is restored to an anatomically symmetrical location (Hoover et al., ). In relation to intramodal changes of visual processing upon early enucleation, some features of ventral stream functions, such as visual spatial (contrast-defined visual) abilities, are enhanced in enucleated individuals compared with monocular viewing controls or are at least equal to binocular viewing controls (Nicholas et al., ; González et al., ). In contrast, visuo-spatial memory (Cattaneo et al., ) and dorsal stream functions such as motion perception, oculomotor behavior and speed perception are negatively affected by early ME since these functions strongly rely on normal binocular experience early in life (Steeves et al., ; Burnat et al., ; Kelly et al., ; Zapasnik and Burnat, ; González et al., ). A strong age-at-enucleation effect is present since it determines the amount of behavioral compensation achieved during monocular vision (Marotta et al., ; Nicholas et al., ). In addition, morphological changes in subcortical structures such as the optic nerve, optic chiasm, optic tract and LGN, reported in early enucleated subjects differ from the ones detected after late enucleation (Horton and Hocking, ; Kelly et al., ). Although, improvements in low- to mid-level spatial abilities are observed, early ME seems to impair development of higher-spatial functions such as face perception in one-eyed humans (Kelly et al., ). Other visual processing parameters such as horizontal saccade dynamics are unchanged in monocular viewing people compared with those with normal binocularity suggesting that the afferent (sensory) and efferent (motor) pathways from the saccadic system are not functionally impaired (González et al., ).
Aside from the unimodal, within-visual system effects, the complete loss of one eye additionally recruits cross-modal adjustments in the auditory system that support improved sound localization in monocular blind subjects (Hoover et al., ). Many perceptual skills do not merely rely on one sense but are established via the integration of different congruent sensory stimuli (visual, tactile, auditory or olfactory) to maximally extract information from the environment (Meredith, ). In additional behavioral experiments, people with one eye show no Colavita effect (visual dominance and auditory ignorance in a bimodal stimulation task) but instead reveal equal preference for visual and auditory stimuli (Moro and Steeves, ). When Moro and Steeves ( ) adapted the stimulation protocol of the Colavita task in favor of audition by increasing the temporal processing (repetitive stimuli), the expected reverse Colavita effect was absent in one-eyed people (Moro and Steeves, ). Accordingly, the enhanced auditory localization capacity following early ME is not sufficient to allow an auditory dominance in the temporal version of the Colavita task suggesting impartial multisensory processing.
## Neural Mechanism of Cross-Modal Plasticity: Importance of Multimodal Regions and Relation to ME-Induced Plasticity
### Observations in the Blind: Cortical Function Specificity and Age-Effects
Multisensory integration is shown to occur widely along the neuroaxis, including primary sensory areas which are often regarded as unisensory (Shimojo and Shams, ; Wallace et al., ; Cappe and Barone, ; Kayser and Logothetis, ; Vasconcelos et al., ; Henschke et al., ) and has an essential role in the following “supramodal” skills: spatial localization, shape recognition and motion detection. Multimodal associative areas (Lingnau et al., ) and to a lesser extent homologous neuronal populations in early cortical areas that subserve these “supramodal” abilities are exactly the ones that mediate cross-modal reorganization and enhanced performances of intact modalities after the loss of a sense. It thus seems that cross-modal plasticity is not a global phenomenon but rather induces specific changes in functional abilities while leaving others unaltered. In other words, specific circuits of the deprived visual cortex in the early blind will use their repertoire of computational properties (laid down by early development and genetics) to perform similar functions for audition, only now they have a different input source (Oliveira-Silva et al., ; Bavelier and Hirshorn, ; Renier et al., ). Specifically deactivating the functionally homologous regions of the deprived cortex by transmagnetic stimulation (Cohen et al., ; Vargas et al., ; Wang et al., ; Merabet and Pascual-Leone, ) or cryogenic cooling in cats (Nakamura et al., ; Riback and Robertson, ; Lomber et al., ) abolishes the better performance in multimodal skills using the remaining senses. These manipulations corroborate the hypothesis that the behavioral function of cross-modal plasticity in a specific area is related to its role in normally hearing/sighted individuals as recently shown by single unit recordings in cats (Chow et al., ; Meredith et al., ) and by functional magnetic resonance imaging in humans (Valverde, ; Heumann and Rabinowicz, ; Renier et al., ; Lingnau et al., ).
Considering the theory of “preserved function”, it should be noted that the preservation of visual perceptual properties could guide cross-modal plasticity presumably if vision is lost early in life (Illig et al., ; Collignon et al., ). In agreement with this finding, some studies indicate a critical period for cross-modal plasticity in the blind based on the performance in non-visual perception and cognitive tasks (Buchel et al., ; Cohen et al., ; Sadato et al., ; Steeves et al., ). For example, mathematical modeling estimated that auditory activation of V1 in congenital blind subjects is mediated by direct functional connections between A1 and V1 whereas auditory-driven activity in V1 from late blind subjects is largely derived from feedback projections of the parietal cortex (Collignon et al., ).
Only a few extrastriate areas (bilateral cuneus) involved in depth perception were significantly more activated in congenital compared with the late blind suggesting also region-specific cross-modal plasticity (Avwenagha et al., ; Collignon et al., ). Complementary to the extensive functional and behavioral studies in blind subjects, a recent study addressed the age-dependent structural and topological modifications in cortical networks to determine at which age the brain network properties are affected by visual deprivation (Li et al., ). Following the comparison of four age groups, namely congenital, early, adolescent and adult blind human subjects, it was shown that early blindness decreases global network efficiency while late-onset blindness was characterized by a diminished local efficiency. The largest differences compared with sighted controls were found after congenital blindness and the smallest between adolescent and late blind subjects. The authors conclude that the overall differences in structural alterations mirror the complexity of neurodevelopment, plasticity and disuse in blind people.
The described enhancement of certain perceptual abilities in the congenital or early blind should not lead to the misconception that blind subjects can compensate everything through increased sensitivity of the remaining senses. Still many aspects of tactile and auditory processing are impaired because early sight loss disrupts cross-sensory calibration during development (Gori et al., , ).
### Putative Mechanisms of ME-Induced Plasticity: Outlooks
Table shows an overview of the candidate mechanisms underlying ME-induced reorganization early (around birth) or later in life (around adolescence and adulthood). It is plausible that a cortico-cortical framework is responsible for the observed plasticity later in life, in line with what has been described in blind subjects (Klinge et al., ) and well characterized for cortical map reorganization (Darian-Smith and Gilbert, , ) after less extensive partial deprivations. The amount of anatomical input does not always correctly reflect the strength and significance of cortical pathways. Together with studies examining the synaptic properties and functional activation of cortical networks, a more accurate characterization of the cortico-cortical connections has been established in mouse. This has been done within a modality (i.e., V1 and V2 (De Pasquale and Sherman, ; Ko et al., ); A1 and A2v (Covic and Sherman, )) as well as between numerous intra- and interhemispheric cortical areas using the stimulation of arbitrary neuronal populations by optogenetics combined with voltage-sensitive dye imaging as a high resolution readout (Lim et al., ). In the context of putative alterations in hierarchical cortical processing within and between senses, information from lower cortical areas (i.e., primary cortex) is not only transferred directly to higher-order cortical areas via cortico-cortical connections (Felleman and Van Essen, ) but also indirectly through cortico-thalamo-cortical projections (for review see Guillery and Sherman, ; Sherman and Guillery, ). Indeed, a tracer study of Négyessy et al. ( ) uncovered cross-modal plasticity across a cortico-thalamo-cortical pathway, which transmits somatosensory information from the barrel cortex via the LP nucleus to V1 in enucleated rats (Négyessy et al., ).
Candidate mechanisms at different levels underlying visual (U) and cross-modal (CM) plasticity following early and late-onset ME and in comparison with binocular enucleation (BE) or dark exposure (DE) effects .
On top of cortico-cortical connections and transthalamic loops, a recent study by Petrus et al. ( ) puts cross-modal potentiation of thalamocortical synapses in the non-deprived primary cortices forward as a general mechanism of functional adaptation in the adult cortex upon a short loss of one sensory input. The combined and balanced alterations in thalamocortical and intracortical circuits may support both enhanced feed-forward processing along the non-deprived senses and efficient unmasking of multimodal connections in the sensory deprived areas (Toldi et al., ; Wallace et al., ; Yu et al., ).
Despite lack of direct evidence, it is very likely that structural plasticity is part of the response to ME. In the somatosensory cortex of adult mice, long-term (8 weeks) vibrissectomy (whisker trimming but in this case with one row intact) increases the spine density on basal dendrites of layer V pyramidal neurons and induces the elongation and higher level of branching of axons resident in the spared barrel column compared with the deprived column (Kossut, ). Given the 7 weeks time course of the adult ME-induced reactivation (see Section Effects of monocular enucleation in adulthood (mouse, P120); Figure ), it is also likely that after unmasking and strengthening of open-eye and multimodal inputs, sprouting of cortico-cortical afferents is a structural mediator of the visual and cross-modal reorganization (Table ). The tracer study in Van Brussel et al. ( ) revealed no large-scale differences in connectivity patterns of V2M in long-term enucleated adult mice or control animals, which is in agreement with the findings of Charbonneau et al. ( ) in intact adult mice. However, it is conceivable that ME-specific structural changes are present but can only be uncovered in a more detailed analysis of axon boutons after anterograde tracer injections or high-resolution fluorescent microscopy in transgenic mice and analysis of spine density and dendritic morphology for instance by means of Golgi-cox impregnation (Aerts et al., ).
These reports and future work with respect to connections will not only expand our knowledge regarding age-dependent and laminar-specific mechanisms of plasticity but will also contribute to a better understanding of mouse (Paperna and Malach, ; Zingg et al., ) and rat (Paperna and Malach, ) cortical networks, including the inhibitory microcircuitry (Pfeffer et al., ), and the whole-brain connectome (Oh et al., ; Sporns and Bullmore, ). In light of these efforts about the structural and functional characterization of intramodal, intermodal and callosal connections, studies that will link the stability and plasticity of these connections with specific behavior and experiences will definitely accelerate future discoveries in the healthy and the diseased brain.
The variation in adult (see Section Effects of monocular enucleation in adulthood (mouse, P120)) and pre-adult plasticity (see Section Monocular enucleation in adolescence (mouse P28-P60)) in reaction to ME in the mouse (Van Brussel et al., ; Nys et al., ) can in part be explained by the fact that the networks of neurons, neurotransmitter systems (Herlenius and Lagercrantz, ), gene regulation patterns as well as their extracellular environment may still change over time (Berardi et al., ; Karmarkar and Dan, ; Putignano et al., ). The GABAergic network, on one side of the excitation-inhibition balance, is a well-know factor in controlling the age-dependent expression of diverse types of plasticity (Hensch, ; Keck et al., ). It tightly regulates the activity of cortico-(thalamo-)cortical inputs (Callaway, ) and it has been suggested to be involved in multisensory integration (Meredith, ; Friedel and van Hemmen, ; Olcese et al., ). However, the limited knowledge regarding the organization of GABA microcircuits across sensory cortices hampers the constructive prediction of cross-modal changes between inhibitory neurons or between inhibitory and excitatory neurons in the mouse. Two hypotheses were described so far. First, studies in the ferret (Heumann and Rabinowicz, ; Pallas, ) and hamster (Desgent et al., ; Farley et al., ) indicated a non-stereotypical but modality-specific organization in the primary sensory areas, A1 and V1, suggesting that early cross-modal plasticity may require experience-dependent adjustments in the number and distribution of specific interneuron subtypes to shape the receptive fields of newly acquired inputs (Desgent et al., ). Alternatively, they may adopt a new GABAergic configuration to control or amplify oscillatory activity carrying multisensory information (Lakatos et al., ). A second study by Clemo et al. ( ) in cat anterior ectosylvian cortices on the other hand revealed a similar distribution of GABAergic markers across higher-order cortices representing different modalities, suggesting a canonical circuit for sensory processing (Clemo et al., ). The different results found in the two studies can be traced back to differences in species, cortical areas and hierarchical level and subset of GABA-related proteins investigated. The presence of multiple types of molecularly and functionally divergent inhibitory interneurons (for a review see Markram et al., ) also imposes another degree of variation and complexity in this matter.
Slow working pharmacological manipulations could test the hypothesis that shifting the excitation-inhibition balance supports the ME-induced reorganization in mouse visual cortex. Diazepam (Hensch et al., ) or muscimol (Caleo et al., ) have often been used as GABA receptor agonists to increase inhibitory function, whereas picrotoxin (PTX, a GABA receptor antagonist) and mercaptopropionic acid (MPA, an inhibitor of GABA synthesis) (Harauzov et al., ) decrease inhibition. More refined techniques such as muscimol-releasing Elvax implants can furthermore reveal the cortical (sub)regions involved and the physiological and behavioral underpinnings of age-specific reactivation profiles (Smith et al., ). These agents are interesting since some features of ME-induced cortical plasticity indeed occur on a longer time-scale. However, the presence of multiple types of inhibitory interneurons as well as the fast millisecond time scales which neurons communicate with, limit the investigative power of these pharmacological receptor agonists. A more sensitive neuromanipulation technique such as optogenetics, a technique incorporating light-inducible channel proteins into specific neuronal cell types, is showing great promise to causally investigate cell type specific functions in awake, behaving animals, offering the possibility to combine this approach with electrophysiological or behavioral readouts to acquire functional information (Yizhar et al., ).
## Contribution of the ME Model to the Clinical Relevance of Cortical Plasticity
From a clinical perspective, the relevance of fundamental research using vision impairment models is reflected in the high prevalence of vision-impaired and blind patients worldwide. 285 million people globally suffer from some form of vision deficiency, of which 8% are blind (Pascolini and Mariotti, ). Notwithstanding the fact that many eye-diseases can be treated at eye level, it is becoming increasingly evident that frequently occurring vision impairments such as glaucoma can be caused by or are associated with cortical changes (Baroncelli et al., ). In this regard, ME studies increase our understanding of how the brain copes with altered vision and age-dependent cortical deficits or functional alterations underlying distinct visual disorders. ME also replicates unilateral vision loss of human patients following ophthalmic trauma, inflammation, injury or enucleation as a common treatment for end-stage glaucoma, retinoblastoma or Phthisis bulbi (a shrunken, non-functional eye) (Moshfeghi et al., ; Setlur et al., ).
Being able to modulate plasticity in a certain direction could attain the best functional and behavioral outcome in a given patient or situation. It will offer great promises in the quest for new therapeutic strategies for neurological disorders or brain injuries although in general, caution is warranted for interventions that tap into brain structure and function to enhance or lower plasticity. At any given time point, an optimal balance between plasticity and stability must be retained.
In this context, the mouse has emerged as the model of choice as it offers unique advantages including molecular and genetic tools to monitor, label and manipulate specific neuronal subtypes or circuits (Huberman and Niell, ). Furthermore, great strides have been made in supporting the idea that plasticity mechanisms in mammals can be studied in mice and that its sensory systems are more complex than originally believed.
In relation to ocular dominance shifts, adult visual cortex plasticity following MD can be elevated by impinging on the cortical excitation-inhibition balance and molecular or structural brakes that were established and maintained in a use-dependent manner during postnatal development (Di Cristo, ; Hensch and Bilimoria, ). Indeed, pharmacological (Pizzorusso et al., ; Maya-Vetencourt et al., ; Harauzov et al., ), genetic (Hensch et al., ; Fagiolini and Hensch, ; Syken et al., ; Carulli et al., ) and housing (He et al., ; Sale et al., ; Huang et al., ; Tognini et al., ) interventions revealed the possibility to rapidly restore ocular dominance plasticity in adult rodents by circumventing the inhibitory and extracellular matrix limitations on binocular visual cortex plasticity. This explosion of invasive and noninvasive interventions that induce adult ocular dominance plasticity or restore visual acuity in adulthood have moved the field of visual plasticity research and clinical interventions for amblyopia forward apace. As a result, the combination of targeted pharmacological (invasive) manipulation, action video game training to enhance neuromodulation (non-invasive) (Green et al., ) and even brain stimulation (Fregni and Pascual-Leone, ) to directly tap into the excitation-inhibition balance could reinstate a sensitive period and improve low-level as well as high-level vision in the weak eye. This in turn could guide the treatment of cortical deficits that accompany amblyopia developed early in life, and glaucoma, cataract or macular degeneration often manifested later in life (Dekeyster et al., ). Several of the molecular principles governing plasticity outcome in amblyopia are conserved and have been found to occur in the injured brain (Imbrosci and Mittmann, ). Insights in age-dependent plasticity gathered by both animal and human research also opens the door to develop new strategies for enhanced learning and memory, for the treatment of mental illness, and for functional rehabilitation following cortical injuries ( stroke, ischemia or trauma). When damage to the brain occurs due to cancer, stroke or lesions, post-operative or post-lesion training (non-invasive) of sensorimotor and cognitive functions can enable recovery-based plasticity to improve the quality of life for the patient.
With regard to cross-modally driven plasticity, the larger proportion of intrinsic multimodal connections found in the lissencephalic rat and mouse neocortex, even in primary areas, (Paperna and Malach, ; Budinger et al., ; Wang et al., ; Olcese et al., ; Hishida et al., ) compared to the cat, ferret (Meredith et al., ) or monkey, has two important consequences. First, it is plausible to observe ample cross-modal reorganization along the antero-posterior and medio-lateral extent of the visual cortex after visual input loss, especially in higher extrastriate visual areas. Second, widespread cross-modal changes in rodents are likely mediated by the unmasking or signal amplification of latent or subthreshold multisensory circuits that were already tuned by multimodal experiences before deprivation (human: Lee et al., ; mouse: Olcese et al., ). Accordingly, also upon ME the age of deprivation and the location within the visual cortex will determine the relative expression of certain plasticity mechanisms at the systems, synaptic and molecular level that may partially overlap with those active following binocular enucleation or blindness (Karlen et al., ; Qin and Yu, ; see Table ). Many studies have focused on the effects of early loss of sensory input while in relation to the human population; partial sensory deprivation in terms of progressive hearing and vision loss and corresponding cross-modal changes are frequently encountered later in life. Therefore, studying adult cross-modal plasticity in the visual (and other sensory) systems is of equal importance.
Although a large focus has been put on the positive aspects of plasticity, it can also be the origin of pathological conditions (Johnston, ; Fernandez et al., ) or the cause of maladaptation in light of rehabilitation efforts (Sandmann et al., ). For instance, cross-modal plasticity is one factor that is responsible for the absence of or reduced success after cochlear implantation, especially when a long time-interval of deafness was present before the implantation (Harrison et al., ). This negative outcome is mediated by the cross-modal functional improvement of non-auditory cortices at the cost of the auditory cortex ability to process electrical stimulation originating from the cochlear device (Lee et al., ). Therefore, effective therapy should include the suppression of cross-modal alterations to permit the recruitment of auditory cortex by the new auditory inputs from the implant and to obtain desirable recovery of auditory functions. Likewise, suppression of non-visual processing in the visual cortex of the blind could be required after introducing a retinal implant. Although electrical implants, such as the recently developed Argus IITM epiretinal prosthesis system, may partially restore vision and allow the identification of letters and words (da Cruz et al., ), further research is still needed to improve the interpretation of input signals to the visual cortex. It is exactly in that context that plasticity research in laboratory animals will be of great support.
## Conflict of Interest Statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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## Introduction
Cortical/cerebral visual impairment (CVI) is the leading cause of pediatric visual impairment in children in developed countries and has become a significant public health concern (Kong et al., ). CVI is clinically defined as significant visual dysfunction resulting primarily from perinatal injury to visual pathways and structures rather than ocular pathology alone (Dutton, ). Perinatal hypoxia is the most common cause resulting in impaired maturation of key visual pathways such as the optic radiations; a general condition referred to as white matter damage of immaturity (WMDI). In preterm infants, this maldevelopment is often associated with periventricular leukomalacia (PVL), which is characterized by an enlargement of the lateral ventricles and focal gliosis of surrounding white matter pathways coursing on to the visual cortex (Good et al., ; Hoyt, ). Depending on the location and extent of the damage, children with CVI often present with a broad range and combination of visual dysfunctions such as decreased visual acuity, visual field deficits, and also impairments in oculomotor, visuomotor, and cognitive visual processing (Good et al., ; Dutton, ; Hoyt, ). The variability in the location and extent of brain injury across individuals makes the prediction of visual functional outcomes and recovery in CVI patients particularly challenging (McKillop and Dutton, ).
Despite the increasing prevalence of this condition, the relationship between observed visual deficits in CVI and the underlying structural and functional changes resulting from damage to key visual pathways, remains poorly understood. Specifically, it remains unknown how the maldevelopment of key visual pathways relates to the organization of the visual cortex and further, how these structural and functional changes relate to visual impairments observed within the clinical setting. Standard clinical neuroimaging techniques such as computerized tomography (CT) and magnetic resonance imaging (MRI) can help characterize gross changes in cerebral structure. However, the underlying micro-architecture of key white matter pathways (such as the optic radiations) cannot be fully ascertained, nor can the function of visual cortical areas be assessed. Advances in diffusion based imaging (i.e., diffusion MRI) modalities such as high angular resolution diffusion based imaging (HARDI) combined with tractography analysis techniques can be used to reveal the organization of specific white matter projections (Jones, ) see also (Ffytche et al., ). At the same time, retinotopic mapping using functional magnetic resonance imaging (fMRI) can be employed to assess the organizational and functional integrity of early visual cortical areas (Wandell, ).
In this study, we used a combined structural and functional multi-modal neuroimaging approach to characterize the underlying maldevelopment of the geniculo-striate pathway in an adolescent with CVI. The patient presented here had a documented inferior visual field deficit determined on clinical ophthalmic examination. Despite her diagnosis of CVI and associated visual impairments, she was able to participate in formal testing and provide reliable data (including maintaining fixation during perimetry and retinotopic stimulation) and also remain immobile in the scanner environment without the need of anesthesia. Thus, (and contrary to prior imaging studies with CVI individuals), we had the opportunity to obtain high quality structural and functional imaging data on the same subject in order to investigate the relationship between the structural integrity of the optic radiations and the functional organization of early visual cortical areas with respect to her clinical visual field impairment. We demonstrate the feasibility of combining this structural and functional imaging approach in a patient with CVI along with an age/gender matched normal developed control for comparison. By combining these imaging modalities, it is possible to provide further insight regarding the functional manifestations of early onset developmental damage to key visual pathways and their relation to specific impairments of visual function.
## Case history
The CVI patient (and age/gender matched control subject) and parents provided written informed consent prior to participating in the study. The protocol was approved by the investigative review board of the Massachusetts Eye and Ear Infirmary (Boston, MA, USA) and the study was carried out according to the tenants of the Declaration of Helsinki and conformed to the requirements of the United States Health Insurance Portability and Privacy Act (HIPPA). Ophthalmological examination of the patient was conducted by an experienced pediatric neuro-ophthalmologist. At the time of study, the CVI patient was a 17-year-old girl, born prematurely at 32 weeks gestational age. In the perinatal period, she developed a grade III intra-ventricular hemorrhage with subsequent post-hemorrhagic hydrocephalus, periventricular leukomalacia (PVL), and spastic diplegia. She underwent bilateral strabismus surgery at 11 months to correct an esotropia. Best corrected visual acuity (Snellen) at the time of testing was 20/50 (right eye) and 20/40 (left eye). Her sensorimotor exam was notable for latent nystagmus and a residual microtropia. Funduscopic examination revealed evidence of optic atrophy in each eye, but was otherwise unremarkable. Visual field assessment was performed with automated visual field testing on a Humphrey Field Analyzer using a SITA-Fast 24-2 protocol (Humphrey Field Analyzer 750i, Zeiss Humphrey Systems; Dublin, CA).
As a control, an age and gender matched subject (female, 17 years old) with normal visual acuities and no history of prematurity or neurological/ophthalmic disorders was also recruited for comparison.
## Visual field assessment
Automated perimetry using the Humphrey Field Analyzer revealed a stable, bilateral visual field defect involving the inferior visual fields (Figure ). Inspection of the pattern deviation plot confirmed the presence of an inferior visual field defect with an apparent greater impairment on the left side. Record review confirmed that the bilateral, inferior visual field loss was non-progressive and identified as early as age 5; when the patient was first able to participate in formal visual field testing.
(A) Visual field assessment of CVI patient obtained by Humphrey automated visual perimetry (see text for test details). A bilateral field defect involving the inferior visual fields (with a more dense defect involving the left side) in each eye evident on the gray scale plot (top panel) and confirmed on the pattern deviation plot (bottom panel). (B) Axial T -weighted MRI images (MP-RAGE pulse sequence) in a normally sighted control (top panel) and CVI patient (lower panel). Enlarged lateral ventricles with irregular posterior borders are apparent in the CVI patient (black arrow). (C) Corresponding white matter tractography of the optic radiations (sagittal view) revealed with HARDI in the same individuals. Note in the control subject, the complete arborization of the superior and inferior banks of the optic radiations extending from the thalamus to the occipital cortex in both hemispheres (open arrows). In contrast, the CVI patient shows markedly fewer projections and in particular, along the superior bank of the optic radiations (white arrows). The paucity of connections along the superior bank along with a greater deficit of connections in the right (R) compared to the left (L) hemisphere correspond to the location of the visual field deficit of the CVI patient characterized by automated perimetry.
## Structural and diffusion weighted imaging and white matter tractography
All imaging was carried out using a 3 Tesla Philips Achieva system and an eight-channel head coil. Conventional T1 weighted structural images were acquired using an MP-RAGE pulse sequence (TE 3.1 ms, TR 6.8 ms, flip angle 9 degree, 1 × 1× 1.2 mm voxel size). For white matter tract reconstruction, high angular resolution diffusion imaging (HARDI) was chosen given its superior ability in revealing intravoxel white matter fiber heterogeneity and delineation of multiple fiber orientations within an individual voxel (Tuch et al., ). HARDI images were acquired using a single shot EPI sequence (TE 73 ms, TR 17844 ms, 64 directions, Bmax 3000, Bmin 0, 2 mm isotropic voxel size). White matter fiber tracking and reconstruction were performed using DSI Studio software ( ) with diffusion decomposition and sparse solution of the fiber orientation distribution function (ODF). HARDI images were aligned to the anatomical data using boundary based registration (Greve and Fischl, ). The optic radiations were defined using a two-seed approach. For each hemisphere, the thalamus (composed of the thalamus proper and the ventral diencephalon containing the lateral geniculate nucleus; Desikan et al., ) was used as the start seed, while the white matter adjacent to the pericalcarine cortex was used as the second seed. Termination criteria were based on a subject-specific threshold of quantitative anisotropy and an angle change of 75 degree, enabling the capture of the full extent of the optic radiations. Both T1-weighted and HARDI images were acquired in the same scanning session. HARDI images were acquired within a 22 min scan period.
Standard T1-weighted MRI imaging of the CVI patient revealed markedly enlarged ventricles characteristic of PVL as compared to the age/gender matched control (Figure ). However, no details regarding the structural integrity of the optic radiations could be ascertained by standard MRI alone. By comparison, white matter reconstruction of the optic radiations obtained by HARDI revealed a generalized reduction in geniculo-striate projections in the CVI patient compared to the normal developed control (sagittal view; Figure ). Furthermore, the optic radiations along the superior bank qualitatively appeared to be markedly reduced than in the ventral bank, consistent with the inferior visual field deficit characterized on clinical examination. Finally, the marked reduction of optic radiations in the right hemisphere (compared to left) was also consistent with the laterality of the visual deficit suggested by perimetry findings.
## Functional neuroimaging and visual retinotopy
The functional organization of occipital visual cortex was characterized using static retinotopic mapping techniques allowing for rapid and robust identification of early visual area borders (Rajimehr and Tootell, ; Nasr et al., ). Boundaries between primary (area V1), secondary (area V2), and tertiary (area V3) visual areas were functionally identified in both hemispheres. Flashing high-contrast colored checkboard stimuli (8 Hz) were presented in sub regions of the visual field and two spatially complementary stimuli were contrasted: (1) a horizontal meridian wedge (8.6° radius and 30° angle) vs. a vertical meridian wedge (8.6° radius and 60° angle) to identify early visual area borders and (2) an upper-field wedge (8.6° radius, 15° angle) vs. a lower-field wedge (8.6° radius, 15° angle) to differentiate activation between the upper and lower visual field. Each pair of stimuli (i.e., meridians as well as upper and lower wedges) were presented in two runs of 16, 16-s blocks comprised of six blocks of each stimulus and four blocks of fixation per run. Retinotopy was acquired with a single-shot EPI sequence (TE 28 ms, TR 2000 ms, flip angle 90 degree, 3 mm isotropic resolution with no slice gap). Each run of the scan was 256 s in duration. For all retinotopic runs, the subjects were instructed to maintain fixation on a central 0.9° fixation stimulus throughout the run and respond using a button box when the central target changed its luminance (TR-wise probability of a luminance change = 30%). Fixation performance (detection accuracy and reaction time) was scored after the scanning session. Stimuli were viewed under binocular viewing conditions.
All structural and functional data were analyzed with FreeSurfer and FS-FAST packages ( ). Using standard FreeSurfer methods (Fischl et al., ; Greve and Fischl, ), the surface of each cerebral cortical hemisphere was extracted via image processing based on the anatomical gray-white matter boundary and computationally reconstructed as a 3D mesh of vertices. In order to permit visualization of multiple visual cortical areas, each cortical hemisphere was computationally inflated to reveal buried sulcal regions and an occipital lobe “flat patch” was created for each subject hemisphere by making cuts in the vertex mesh (along the calcarine sulcus and the anterior extent of the occipital lobe) and unfurling this mesh into a 2D representation (see Figure for further details). Each functional run was rigidly registered to the anatomical data using gray-white matter boundary based registration (Greve and Fischl, ). Functional data was motion corrected and spatially smoothed using a 3D Gaussian kernel (fwhm = 3.0), separately by run and by hemisphere. Voxel-wise statistical tests were based on a univariate general linear model (GLM), and the significance levels (inverse log p -value) were visualized on the inflated and flattened cortical surfaces.
Activation of early retinotopic areas using fMRI. (A) Cortical flattening of an occipital cortical patch from the right hemisphere of a normally sighted control. For reference, colored dashed lines are in the same position in both the inflated and flattened views (orange, calcarine sulcus; red, lateral occipital; green, medial, dorsal occipital; and blue, medial ventral occipital). The boundaries of early visual areas (V1–V3) are shown and separated in dorsal (d) and ventral (v) parts. (B) Occipital patch projections showing cortical activation in the control (upper panel) and CVI patient (lower panel) in response to visual stimulation using meridians as well as upper and lower wedges (blue and yellow colors correspond to location of activation in response to visual stimulation). Overall, the organization of early visual areas appears largely intact in the CVI patient. However, selectivity for stimulation within the lower visual field was reduced compared to the sighted control (note the left hemisphere is flipped for easier visual comparison). (C) Comparison of relative activation in V1 between the CVI patient and the control subject for the cortical representation of each visual field quadrant (expressed as a percentage) confirms that the largest impairment in activation was in dorsal V1 (V1d) and in the right hemisphere (RH) corresponding the left inferior visual field deficit obtained on perimetry.
Dorsal (d) and ventral (v) regions of interest (ROI) were identified in each hemisphere and in each participant using block-design retinotopic fMRI analysis (e.g., Nasr et al., ). Briefly, the V1/V2 border lies along the center of the vertical meridian response, as it runs both dorsally and ventrally, and the center of the horizontal meridian representation in the calcarine sulcus was used to define dorsal and ventral subdivisions in each cerebral hemisphere. Each ROI represents the diametrically opposed region of the visual field (e.g., dorsal V1 of the right hemisphere represents the lower-left quadrant of the visual field). It is important to note that identifying the boundaries between early visual areas permits us to define ROIs for V1 and V2, but leaves the anterior border of V3 undefined. Since the visual cortical areas just anterior to V3d and V3v (V3A and hV4) contain hemifield representations rather than quadrant representations, the quadrant analysis could be seriously contaminated if the anterior borders are not well localized. For this reason, we quantitatively examined the quadrant results only in areas V1 and V2.
We then determined the BOLD percent signal change to the upper visual field wedge (relative to passive fixation) and to the lower visual field wedge, within each ROI for each subject for each run. Since the dorsal parts of V1 and V2 are responsive to lower visual field stimulation and ventral parts are responsive to the upper visual field stimulation, this allowed us to create an in-field vs. out-of-field metric for each ROI and for each subject, normalized by the standard deviations of the blood oxygen level dependent (BOLD) signals. This in vs. out metric was computed as follows:
Where “preferred” is the BOLD percent signal change evoked by the preferred stimulus for each ROI (i.e., the upper visual field wedge in ventral areas and the lower field wedge in dorsal areas) and “nonpreferred” is the BOLD percent signal change evoked in the ROI by the other, nonpreferred wedge. Standard deviations in BOLD percent signal change were computed within subjects and across runs. The in vs. out metric was contrasted between the CVI and control subject for each visual quadrant ROI as a ratio. A ratio of 100 indicates that the CVI patient and the control demonstrate similar responsivity profiles, while a ratio less than 100 indicates that the representation of the upper/lower visual field is less strongly segregated in the CVI patient than in the control subject.
Both the CVI patient and the control subject were able to maintain adequate central fixation during the fMRI scans allowing for the identification of retinotopically specific cortical activation patterns (CVI patient fixation accuracy: 80%, mean reaction time 640 ms; control subject fixation accuracy: 58%, mean reaction time 729 ms). Functional MRI assessments resulting from the stimulation of horizontal and vertical visual field meridians were used to reveal the boundaries between visual cortical areas and the boundaries of early visual areas (i.e., V1–V3, see Figure ). These areas were robustly identified in each hemisphere in both the CVI and control participants suggesting that the overall organization of early visual areas is largely intact in the CVI patient despite the maldevelopment of geniculo-striate projections (compare upper and lower panels of Figure ). However, the upper and lower visual field responses showed the most marked difference in the CVI patient mirroring the visual field deficit identified by automated perimetry (described above). Specifically, activation within primary visual cortical representations of the lower visual field (V1d) in the right and left hemisphere of the CVI patient exhibited respectively only 21.5 and 22.2% of the activation observed in the same lower visual field representations in the control participant (Figure ). In contrast to those strongly attenuated for the lower visual field, cortical representations of the upper visual field (V1v) exhibited minimal differences in activation between participants (right hemisphere: 91.9% activation of control, left hemisphere: 89.1% activation of control). Again, this pattern is consistent with the inferior visual field deficit characterized by perimetry and the observed reduction in optic radiation tracts along the dorsal branch corresponding to the inferior visual field representation. Finally, in terms of laterality, a greater reduction in activation in the right lower visual field (V1d) compared to the left lover field (V1d) was also observed. This is further consistent with the lateralized visual field deficit in the left side as well as marked reduction in optic radiations observed in the right hemisphere. For comparison, we also examined regional activations in V2 and found a similar pattern to that of V1. Specifically, dorsal areas (lower field representations) exhibited much lower activations in the CVI subject as compared to control (right hemisphere: 33.1% activation of control, left hemisphere: 35.3% activation of control), while the ventral (upper field representations) were similar between CVI and control subjects (right hemisphere: 147.4% activation of control, left hemisphere: 93.7% activation of control). We further noted that the activation ratios between the CVI and control subjects were somewhat smaller in V1 than in V2 (approximately 22% compared to 34%, respectively). This result is consistent with the fact that V2 neurons have larger receptive fields than V1 neurons and thus V2d receives more upper field visual stimulation than does V1d (Zeki, ; Smith et al., ).
## Discussion
The combination of advanced structural and functional neuroimaging methodologies allows for the characterization of the maldevelopment of visual pathways in relation to assessments of visual function obtained in the clinical setting. In the case presented here, there was a structural-functional correspondence between the clinically observed inferior (and greater on the left) visual field deficit, damage to superior (and greater in the right hemisphere) branches of the optic radiations, and the reduced activation of early visual cortical areas within the inferior visual field (greater in left). This correspondence is in accordance to the known anatomical and functional organization of visual pathways and geniculo-cortical representation of visual field space (Wandell, ). To our knowledge, this is the first report combining a multi-modal imaging approach revealing an anatomical and functional correspondence to a visual field deficit in a patient with CVI.
Previous studies have employed various neuroimaging methodologies to investigate the impact of pathology and/or the maldevelopment of the visual system (see Haak et al., ; Raz and Levin, for reviews). For example, Slotnick et al. ( ) reported a case study of a patient with congenital cortical dysgenesis using fMRI retinotopic mapping to characterize a large scale displacement in her cortical visual retinotopic map representation (Slotnick et al., ). In another study, Bridge et al. ( ) used a combined structural and diffusion-weighted MRI approach to demonstrate preservation of visual cortex architecture in case of congenital anophthalmia (Bridge et al., ). In a unique study by Levin et al. ( ), a combined structural and functional imaging approach was used to characterize white matter tracts and visual cortical maps in an adult individual blinded at the age of three, but who had sight restoration surgery following a corneal and limbal stem-cell transplant procedure in one eye. Even after many years following the surgery, the visual abilities of this individual remained severely limited, and corroborative evidence obtained from combined imaging measurements revealed a number of abnormal visual cortical responses as well as structural (i.e., diffusivity) abnormalities of key whiter matter pathways (Levin et al., ).
Regarding CVI, previous studies using diffusion based MRI have identified marked alterations in white matter structure and have further proposed associations between the maldevelopment of key visual pathways and the visual dysfunctions observed in this condition (Ortibus et al., ; Bauer et al., ; Lennartsson et al., ). With regards to the case reported here, a recent review revealed that many individuals with early periventricular damage to the optic radiations (i.e., during third trimester of gestation) often showed normal development of visual field function, perhaps as a result of compensatory neuroplastic reorganization (Guzzetta et al., ). These studies suggest that CVI may be associated with a generalized vulnerability in numerous key pathways supporting the developing visual system. However, neuroplastic changes within the developing brain (such as the “re-wiring” of key geniculo-cortical or cortico-cortical connections) may support the sparing of visual function in certain individuals with CVI. Continued studies using advanced multi-modal imaging approaches will likely help in further characterizing these structural-functional associations including the identification of key developmental factors such as the timing, location, and degree of insult.
Apart from the limited observations that can be drawn from a single case study, it is also important to note that the descriptions provided here regarding white matter projections and the degree of functional activation of early visual cortical areas are largely qualitative in nature. Specifically, a reduction in the number of tracts revealed by diffusion-based reconstruction techniques do not necessarily equate to an absolute absence of these connections. Thus, it is important to consider the possibility of uncharacterized reorganization of white matter connections and/or possible false negatives related to the reconstruction process (Johansen-Berg and Behrens, ). At the same time, activation measured by fMRI is an indirect measure of brain activity and thus, may not fully characterize underlying physiological and morphological changes. As a result, future studies will require large sample populations in order to fully establish a clear relationship between structural and functional changes in the brain with respect to various outcomes of visual dysfunction.
The combination of structural and functional imaging modalities such as presented here may serve as a key approach in helping to broaden our understanding of brain anatomical-functional relationships as they relate to developmental disorders such as CVI. Observed differences in activation (both in terms of visual areas and between the CVI and control subjects) may also reflect the relative contributions of top-down (i.e., feed-back projections) to both striate and extra-striate cortices from higher order visual areas. At this juncture, it is reasonable to speculate that the optic radiation damage characterized in this CVI subject is responsible for the focal visual field deficit observed. There is evidence however that children with CVI can successfully undergo intensive rehabilitative training and recover a certain degree of visual function (Farrenkopf et al., ; McKillop and Dutton, ); see also (Poggel et al., ). Thus, with functional improvements in overall visual field function, we would suspect changes in the relative activation ratios within these same early visual areas; possibly due to greater top down/feedback influence from higher order areas. To further investigate and confirm this hypothesis, longitudinal scanning, and phase-encoded retinotopic mapping (i.e., to map the retinotopic organization beyond early visual areas) would be needed.
Further, research is needed to fully understand how the developing brain reorganizes itself in relation to sensory and functional recovery and provide a neurological rationale for individually tailored rehabilitative strategies for these patients. Thus, fully characterizing the associations between underlying structural and functional changes with clinical assessments of visual dysfunction may ultimately help us understand how individuals develop, and adapt, in response to early damage to the visual system.
## Author contributions
Designed the study: LM and DS. Collected data: CB and LM. Interpretation and Analysis of data: all authors. Contributed to preparation of the manuscript: all authors.
### Conflict of interest statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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Homonymous hemianopia (HH) is the most common cortical visual impairment leading to blindness in the contralateral hemifield. It is associated with many inconveniences and daily restrictions such as exploration and visual orientation difficulties. However, patients with HH can preserve the remarkable ability to unconsciously perceive visual stimuli presented in their blindfield, a phenomenon known as blindsight. Unfortunately, the nature of this captivating residual ability is still misunderstood and the rehabilitation strategies in terms of visual training have been insufficiently exploited. This article discusses type I and type II blindsight in a neuronal framework of altered global workspace, resulting from inefficient perception, attention and conscious networks. To enhance synchronization and create global availability for residual abilities to reach visual consciousness, rehabilitation tools need to stimulate subcortical extrastriate pathways through V5/MT. Multisensory bottom-up compensation combined with top-down restitution training could target pre-existing and new neuronal mechanisms to recreate a framework for potential functionality.
## From Cortical Blindness to Awareness: Understanding Blindsight Beyond The First Sight
### Cortical Blindness
Normal vision in humans is primarily mediated by the geniculo-striate pathway where the visual information is processed in a hierarchical order via the retina, the lateral geniculate nucleus (LGN) and the striate cortex. Once the primary characteristics of visual information are processed, visual connections are sent to the parietal cortex involved in spatial attention and action (dorsal pathway) and to the temporal cortex involved in recognition and identification (ventral pathway). Following post-chiasmatic lesions inducing an alteration in the geniculo-striate pathway, a contralateral cortical blindness (CB) occurs, either as a result of a neurophysiologic disorder requiring surgical interventions of V1, or mainly subsequent to a stroke affecting the posterior visual cortex (Sand et al., , for review, see Goodwin, ). Depending on the extent of the lesioned cortex, the visual field deficit may correspond to a CB of a few degrees (scotoma), a quarter of a hemifield (quadranopsia) or an entire hemifield (hemianopsia; for review see Swienton and Thomas, ). The homonymous hemianopsia (HH) is the most common visual cortical deficit representing 10% of stroke cases, with little more than 70,000 new cases per year in the US (Zhang et al., ; Mozaffarian et al., ). Moreover, more than 500,000 Americans live with a HH and this deficit reduces significantly their quality of life, for example preventing them from driving and, decreasing their reading, orientation and exploration visuo-spatial abilities (Perez and Chokron, ). In addition, due to comorbidity, HH significantly reduces the prognosis and the possibility of recovery from other damaged functions after stroke, including motor skills (Patel et al., ). As only a small minority experiences spontaneous recovery, possible within the first 6 months (Duquette and Baril, ), it is crucial to rehabilitate these individuals during this time. Unfortunately, with the exception of the vision restitution therapy (VRT) approved by the FDA due to its therapeutic potential (Sabel et al., ; Kasten et al., ), yet controversial on the benefits in terms of pure visual restitution (Horton, , ; Bouwmeester et al., ; Melnick et al., ), there are very few available resources for clinical interventions. This may be due to a lack of consensus in the literature caused by inconsistent results across the target population, differences between protocols used in research and an inefficient vision recovery (Pollock et al., ; for reviews see Riggs et al., ; Pouget et al., ). Furthermore, there is so much inter-individual variability in the origin and extent of lesions that most probably plasticity in the visual system is heterogeneous throughout the population. This leads us to the question: is visual recovery in cortically blind individuals possible with strategic rehabilitation? In theory, it would be conceivable, in the light of a well-documented preserved visual ability in CB referred to as blindsight, where visual information is processed in the blindfield without the knowledge of visual awareness (Weiskrantz et al., ).
In a behavioral perspective, blindsight is the dissociation between what is reported subjectively, for example, the subject states not seeing anything in the usual scale of binary report (seen, not seen), and what is measured objectively, i.e., the rate of correct answers in the two-alternative forced-choice paradigm is above the chance level. The first extended precepts of the phenomenon are described from the results obtained on GY who had a trauma affecting the striate cortex at a young age (Barbur et al., ; de Gelder et al., ; Kentridge et al., ) and DB who required a removal of V1 at an adult age (Weiskrantz, ; Tamietto et al., ). Blindsight includes the unconscious ability to be able to locate random targets by reaching or pointing at them, to determine the presence or absence of visual targets, to have a considerable visual acuity mainly for low spatial frequencies, to discriminate directions (Weiskrantz, ), to recognize colors (Brent et al., ), to detect global movement, to distinguish between coherence (Alexander and Cowey, ; Pavan et al., ) and to recognize facial expressions (de Gelder et al., ). However, it has been found that high contrast and fast movement stimuli could induce sensations described as something elusive that happened in the blind hemifield. Accordingly, a distinction has been made between type I blindsight, i.e., absolute blindness without conscious awareness, and type II blindsight, i.e., blindness with awareness but no visual qualia (Lau and Passingham, ). In fact, attempting to understand the intrinsic process governing this unconscious vision has been the foundation of many theories (Smythies, ; Kanemoto, ; Zeman, ).
### Fine Line between Type I and Type II Blindsight
It was in 1917 that the first evidence of a residual visual ability in the blindfield emerged when a patient reported visual sensation specifically to motion. This non absolute blindness sustained a form of visual qualia called the Riddoch Syndrome (Riddoch, ). Keeping this in mind, the notion of visual qualia is quite important to consider in blindsight studies, especially when considering blindsight type II because even if some kind of awareness remains it has to be dissociated from visual awareness (Ko and Lau, ) which we will discuss further on. Subsequently, the characterization and understanding of the dissociation between type I blindsight and type II is controversial, due to the fact that only a few studies have been able to demonstrate a correlation between the loss of the striate cortex, the takeover of secondary visual pathways and the state of visual consciousness (for review see Leopold, ). The problem that arises with respect to blindsight is to figure out which hypothesis could best explain the phenomenon: (1) a degraded normal vision; (2) an unconscious vision; and (3) a degraded abnormal vision.
Degraded normal vision occurs when visual stimuli are processed through the primary visual pathway, but do not reach the threshold of full visual awareness. In fact, in some cases, spared islands of the striate cortex explain the residual visual capabilities found in HH (Fendrich et al., , ). However, several patients may present blindsight in the absence of a functional striate cortex (Morland et al., ; Ajina et al., ; Mazzi et al., ), regardless of the state of awareness (Ffytche and Zeki, ). Though we should not overlook the importance of targeting vestiges of V1 in rehabilitation strategies, we must be able to stimulate the secondary visual pathways bypassing V1 potentially responsible for type I and II blindsight. Therefore, we need to understand the mechanisms governing the two forms of this phenomenon.
Unconscious vision has been showcased by proving that residual abilities in HH do not follow the same rules as it is qualitatively different from that of the conscious normal vision (Weiskrantz, ). In fact, for certain visual stimulations, the performance in the blind side is better than the one in the normal side (Trevethan et al., ). For example, unlike normal vision, performance in a task of exclusion is inversely correlated to the stimuli’s contrasts (Persaud and Cowey, ), there is a clear abnormal distinction between choice-forced and detection performances (Azzopardi and Cowey, ) and some physical attributes are processed in the blind hemifield, while others are not (Morland et al., ; Kentridge et al., ). Taken together, these studies provide robust evidences to the hypothesis that blindsight is different from normal vision and is not simply a form of degraded normal vision. However, they have assumed that this abnormal vision is unconscious, whereas an abnormal degraded vision could also explain the behavioral results. In fact, unconscious and degraded abnormal vision can both make reference to a vision qualitatively different from normal vision mediated by secondary neurophysiological correlates, but that differ in terms of conscious subjectivity and the nature of the sensation.
Some authors refute the theory of unconscious vision by stating that the residual visual capabilities are due to a degraded abnormal vision that does not reach the threshold of detection (Overgaard and Grünbaum, ; Mazzi et al., ). In GR and SL case studies, with complete lesion to the striate cortex, the perceptual awareness scale (PAS) was used to allow a subjective finer report based on four indices, instead of the usual scale of binary report (seen, not seen). This showed that patients tend to have a higher threshold to acknowledge that something is conscious if the criterions are not based on a scale of consciousness. In fact, after using the PAS, awareness was better than what the theory of unconsciousness would have predicted (Overgaard et al., ; Mazzi et al., ). They concluded that type I blindsight can be wrongly considered as unconscious; instead it seems that above chance level performance comes with conscious perception. Therefore GR and SL do not have blindsight, rather they have conscious vision. Their results agree with the continuum perception theory, where there is a correlation between increased visual sensitivity and higher brain activity. In the normal population, results are contradictory depending on the paradigm used. For example, when using masked stimuli, results tend to suggest that performance can’t exist without awareness and that if the reverse is often inferred, it is due to visual bias induced by inappropriate methodological tools measuring awareness. Moreover, even among the ideal model, performance is greater than awareness in a non-linear relationship where the threshold for perception is inferior to the one for awareness which could explain why in altered perception, the state of consciousness decreases more rapidly than the performance. Thus, even if performance is accompanied by awareness, the latter can be wrongly underestimated without the appropriate tools, since thresholds for explicit visual consciousness is not reached (Peters and Lau, ). Therefore, is it possible that blindsight consists of an abnormal conscious degraded vision mediated by secondary visual pathways? If this extrapolation is accurate, we nevertheless disagree with the conclusion of Mazzi et al. ( ) that SL’s residual abilities are due to conscious vison inducing visual qualia, thus inferring that in such case there is no such thing as blindsight. First, blindsight was employed to explain an ability which was phenomenologically different from blindness and sight, and can be referred to as the loss of visual function that is accompanied by altered “sight”. Second, SL had the feeling that something happened in her blindfield; however she couldn’t visually describe what she saw. Can we say that she showed a form of consciousness? Yes. Can we conclude that the nature of the feeling is visual? Not so much. The scale evaluated the following perceptual judgments as: “(1) no experience of the stimulus; (2) brief glimpse; (3) almost clear experience; and (4) clear experience” (Mazzi et al., ). There was never a reference to the nature of the visual stimuli, or even to what was seen, contrarily to the Riddoch phenomenon where visual qualia of motion was described (Riddoch, ). While we agree that the use of the PAS allows patients to “pay more attention”, subsequently giving more insight on residual abilities and potential tools to rehabilitation, it’s nevertheless insufficient to conclude on the nature of the awareness and the continuum scale of perception. In fact, others have also used a continuous scale to assess visual awareness and showed that either the visual stimuli presented in the attentional blink was completely perceived or not detected at all independently of stimuli visibility (Sergent and Dehaene, ). The non-perceived stimuli in the attentional blink were correlated with suppression of the P300 wave, and a dynamic change in brain oscillations indicating that perception without consciousness has distributed neuronal correlates (Kranczioch et al., ). Unfortunately, the neuronal correlates underlying theses controversial residual abilities has yet to be explained.
### An Unsynchronized Framework for Blindsight
This article supports the global neuronal workspace framework as a model for conscious and unconscious vision (Sergent and Dehaene, ). Thus, in alignment with promising views on “local” and “global” visual functions in blindsight (Silvanto, ), blindsight can be understood as a lack of synchronization in neuronal activity (Melloni et al., ) and rapid globalization for specific visual properties between altered perception processors (neuronal networks implicated in bottom-up activity and visual performances), attention processors (systems of complex neuronal association that allow perceptual information to access consciousness) and conscious processors (workspace neurons for awareness via top-down activity). Moreover, because the attention network can interact with the perception network without creating any kind of visual awareness, it is most probable that the perception workspace can send projections to the attention workspace without creating any attentional awareness. This phenomenon recently called attentional unawareness was hypothesized in blindsight patient for emotional stimuli, subsequent to studies in normal vision (for review see Diano et al., ). In this context, it would be conceivable to induce learning effect resulting in awareness, and moreover visual consciousness, if attentional and perceptual feedforward and feedback connections were simultaneously stimulated. This would lead to synchronization enhancement and would allow cascading amplification resulting in long-distance reciprocal connections and global availability (Dehaene et al., ). Indeed, attention is necessary to visual consciousness even if not sufficient (Kentridge et al., ; Schurger et al., ; Yoshida et al., ). Visual consciousness would be mediated by top-down activity through connections between higher and lower perception processors, as well as between perception, attention and conscious processors creating a global workspace. As a result, the inability of cortically blind people to describe what is presented in their blindfield could be linked to a lack of global availability of the global workspace due to inefficient looping among the altered perception and attention processors and interaction with the conscious network. Hence, according to the global workspace, blindsight could be mediated by secondary visual pathways that activate the neuronal perception and attention networks insufficiently and only locally, without sending long ranging connections to other networks in the brain therefore suppressing visual qualia which could explain the above-chance visual performances in choice-forced paradigms. Consequently, in type I blindsight, the neuronal network generates sufficient activity to process the stimulus, however the neuronal pattern required for phenomenal consciousness is insufficient. In type II blindsight, activity is sufficient to create a sense of awareness, perhaps due to the activation of local conscious processors, but it doesn’t reach the threshold for global availability (see Figure ).
An illustrative schematic of the proposed model for unsynchronized framework for blindsight (inspired from Dehaene et al., ). The gray circles represent neuronal processors that are activated in normal vision and the gray lines their respective connections. The black circles illustrates the neuronal processors that underlies blindsight and the black lines their respective connections. Blindsight can be understood as an alteration in the perception and attentional systems, therefore inactivating the long-range workspace connectivity, global availability and conscious visual perception. The lack of visual awareness is due to a non-efficient global workspace. Awareness found in blindsight type II, could be linked to some long-range connectivity between the perception, attention and consciousness workspaces without activating the global workspace.
Nonetheless, all residual visual abilities found in CB are not necessarily due to blindsight, in the contrary it could be linked to degraded normal or abnormal vision, as we discussed previously. In reality multiple networks can co-exist, vary in function of the lesions and express themselves depending on the stimulation or given paradigm. In literature, the term blindsight lacks clarity because it refers to several types of visions, mechanisms and correlates all at once. Residual visual abilities are found only in a few individuals with CB. However, it is more than possible that co-existent residual secondary pathways arise together (Tamietto and Morrone, ), but when cortical alterations are too diffuse residual pathways aren’t activated strongly enough to induce residual vision. Therefore, a same individual could have multiple types of residual visual abilities or the potential to develop them with training. Thus, we propose the following terminology:
Degraded visual abilities consist of a degraded normal vision caused by vestiges of the striate cortex. It is linked to reduced performances and/or visual awareness that are qualitatively similar to normal vision but quantitatively poorer.
Blindsight consists of an unconscious vision mediated by secondary visual pathways bypassing V1 independent of visual awareness. It could be explained by inexistent (blindsight type I) or not optimal synchronization (blindsight type II) between the perception, attention and conscious networks.
Alternative visual abilities consist of an abnormal degraded vision which is qualitatively different from normal vision but is associated with visual awareness mediated by secondary visual pathways that can’t be explained by the activity of the striate cortex.
The idea is to understand how we can pass from blindsight type I, to blindsight type II to an alternative visual ability. In other terms how can we pass from a state of no awareness to a state of awareness and finally to visual awareness by stimulating secondary visual pathways?
## Neuronal Substrates Underlying The Framework of Blindsight
### Geniculo-Extrastriate Pathway: A Door to Perception
The geniculo-extrastriate pathway is a perfect candidate to our altered perception workspace. Its existence implies that V1 lesions do not lead to a complete degeneration of the LGN, and that koniocellular projections are sent to the secondary extrastriate regions, such as MT (Warner et al., ). In macaques with no striate cortex (eliminating the possibility of V1 islands) there is a causal link between the LGN and blindsight (Schmid et al., ). In fact, by presenting high contrasts stimuli in the blindfield, the authors have observed visual processing corresponding to blindsight, correlated with fMRI activations in several areas including extrastriate region MT. By inactivating the LGN, the neuronal activations and the residual detection skills were abolished (Schmid et al., ). Also in macaques, direct koniocellular projections were found between the LGN and MT corroborated by a retrograde technique of tracing and histological sections. In addition, a new neuronal population, not belonging to the koniocellular system, has been discovered in the intercalated layers of the LGN (Sincich et al., ). Interestingly, in humans MT (hMT+) acts similarly to V1 when presented with global motion (Ajina et al., ). This highlights the role of existing subcortical visual pathways in blindsight, which was specifically and exclusively correlated with the presence of the geniculo-extrastriate pathway (Ajina et al., ). However, in this study, blindsight was assessed with a motion task; it is possible that the correlation existed just for the geniculo hMT+ pathway because the psychophysical measure was specific to this pathway. Blindsight negative individuals were categorized as such using the same task, nonetheless they could have exhibited blindsight using saccadic localization of a brief visual flash, or using indirect methods where reaction time to stimuli presented in the normal field are enhanced by stimuli presented in the blindfield. We extrapolate a possible correlation between blindsight positive individuals derived from such paradigms and the colliculo-extrastriate pathway or interhemispheric connections between hMT+, respectively.
### Colliculo-Pulvinar-Extrastriate Pathway: A Door to Integration and Attention
The implication of the superior colliculus (SC) in blindsight is strongly corroborated with behavioral data. More specifically, the physical parameters of stimuli inducing blindsight correspond specifically to the inherent characteristics of the SC neurons (Leh et al., ; Tamietto et al., ). For example, the lack of projections from the short-wavelength sensitive cones of the retina towards SC neurons is associated with blindness to blue (Leh et al., ; Tamietto and de Gelder, ). Consequently, when the color blue is used instead of red or an achromatic visual stimulation, then blindsight and activations of the SC disappears. From a functional point of view, an association between the collicular pathway and type I blindsight was found in a hemispherectomized patient, as well as interhemispheric connections extending from the SC to the visual, parietal and prefrontal, areas (Leh et al., ). Even if the SC could relay to the extrastriate cortex via colliculo-geniculate projections (Harting et al., ), Lyon et al. ( ), have demonstrated projections from the SC to V3 and V5/MT throughout the pulvinar in macaques assessing the possibility that blindsight could be mediated by relays ranging from the SC to the pulvinar and the dorsal pathway similar to the magnocellular pathway involved in movement and ocular orientations. We postulate that the subcortical extrastriate pathway passing by the SC and the pulvinar serves attentional workspaces and can be enhanced with multisensory stimulations. In fact, bimodal neurons of the SC respond to audio-visual stimuli by fortifying extrastriate pathways (Stein and Rowland, ; Paraskevopoulos et al., ). Moreover, the SC is responsible for ocular movements in the centers of attention becoming faster and more accurate with repetitive multisensory stimulations (Corneil et al., ; Bell et al., ; Gingras et al., ). Training of the oculomotor track could allow a potential increase in allocation of attention in the blind hemifield, which is necessary to perception and visual consciousness. On another note, it seems that the pulvinar can perform higher order visual processing, as motion-selectivity and emotional processing for the former (Villeneuve et al., ; Maior et al., ) so can the SC, as Gestalt like analysis associated with faster responses for stimuli with specific configuration and numerosity (Celeghin et al., ; Georgy et al., ). Other studies demonstrated activations, projections and connections from the SC and the pulvinar to the amygdala when unconscious visual emotional stimuli occurred in hemispherectomized patients (de Gelder et al., ; Morris et al., ; Tamietto et al., ; Celeghin et al., ). Therefore specific perceptual training could directly target these structures and reinforce subcortical pathways bypassing V1, hence the idea of combining different types of visual training.
### Multiple Workspaces of Consciousness
We hypothesize that consciousness and moreover visual consciousness is mediated by multiple workspaces interacting together. Conscious processors can be mediated by interactions of the fronto-parietal and prefrontal network (Zeman, ; Persaud et al., ) with higher visual areas (Dehaene and Changeux, ), and visual conscious processors by the thalamic reticular network (Min, ). Consciousness, and more specifically visual consciousness can be achieved with feedforward and feedback connections from higher to lower visual areas (for review see Urbanski et al., ). An alteration in feedback loops and synchronization between high cognitive areas and visual areas could lead to a lack of awareness (blindsight type I). Between higher and lower visual areas inefficient long ranging connections could lead to the lack of visual awareness found in type II blindsight. This blindsight model is subsequently the result of altered local workspaces that take over when a normal global network degenerates (Silvanto, ). Therefore, connectivity between new workspaces of perception and abnormal workspace of consciousness are weak and non-specific, due to a lack of visual learning reflected by a lack of appropriate synchronization. This unsynchronized framework between posterior and more anterior areas diminishes the visual sensitivity for motion stimuli in healthy subjects demonstrating precisely the effects of synchronization on V5 (Romei et al., ). Hence, the idea is to employ neurorehabilitation to target residual pathways passing by V5/MT, induce new connectivity between interhemispheric V5/MT areas (Bridge et al., ; Silvanto et al., ) and functional interactions within the lesioned hemisphere (Huxlin, ).
## A Model of Combined Interventions Reinforcing The Global Framework of Blindsight
### Importance of the Subacute Period
This section reports the estimated tools to promote plasticity following a CB and increase potential recovery of functional vision. First, future researches should emphasize the importance of stimulating visual pathways in the subacute period following the lesion (Alber et al., ) to notably reduce the degenerations of subcortical tracks (Nijboer et al., ) and increase the chances of visual improvement (Keller and Lefin-Rank, ). In fact, spontaneous restoration in the subacute phase is associated with a reactivation of V1, a restoration of the ipsilateral optical radiations and a progressive recovery of visual functions (for review see Matteo et al., ). An increase in spontaneous restoration could be induced with reinforcement of the residual tracks and recruitment of new ones by activating interhemispheric connections.
### The Value of Interhemispheric Connections
It has been demonstrated that subsequent to a striate lesion, reorganization of the cerebral cortex in favor of the intact hemisphere induces V5/MT of the ipsilateral hemifield to project to V5/MT of the contralateral hemifield after stimulation of the blind field (Bridge et al., ). Moreover, in the lesioned hemisphere, there are areas that respond to stimulations presented in the normal hemifield, but not to stimulations in the blind hemifield (Kavcic et al., ). Simultaneous stimulation of the two hemifields could produce an effect of learning, allow for ipsilesional reorganization (Celeghin et al., ) and would be essential to regain visual perception (Silvanto et al., ). Contralesional V5/MT activation induced by repetitive stimulation of the normal hemifield would allow reorganization and potentiation of the ipsilesional V5/MT via interhemispheric connections, and therefore gain new normal functionality instead of the “V1 like functions”. In fact, we postulate that connections from the contralesional to the ipsilesional V5/MT could lower the threshold to induce global availability within the lesioned hemisphere by activating new processors in the lesioned hemisphere and/or by interhemispheric synchronization between workspaces. These results added to the proposed neuronal substrates of the global workspace seem to lead to the notion that V5/MT could be considered as the crossroad of residual abilities and should therefore be the central key to rehabilitation. Multisensory bottom-up activations mediated by the colliculo-extrastriate pathway could lead to V5/MT enhancement without the need of attentional processes.
### Enhancing Attention with Audio-Visual Training
Reorganization following audio-visual stimulations allows a decrease of the ipsilesional attentional bias showed by a reduction in P300 amplitude (Dundon et al., ), potentially moving the attentional capacities towards the blind hemifield. The role of the SC in this attentional process is particularly important, since such an effect is obtained by the intermediary of saccadic movements. This enhancement of attentional capacities, without prior visual attention needed, is exactly why rehabilitation tools should include audio-visual stimulation training (for review see Grasso et al., ) which has been associated with an improvement in visual detection and exploration (Bolognini et al., ; Leo et al., ; Passamonti et al., ), and in life quality (Roth et al., ). Although compensation therapies proved their reliability over more than two decades (Kerkhoff et al., ), they are still underestimated, due to very little clinical evidence of their impact (Pollock et al., ). For this reason, the use of multisensory bottom-up training in association with top-down training could lead to a higher chance of improving visual detection, localization and recognition.
### Re-Establishing Perception with Restitution Training
Restitution techniques are effective if they aim typical visual attributes training specific to blindsight to expand over a large spectrum of visual characteristics and functions. For example, a transfer of information can be achieved by presenting simultaneous and diversified stimulations in the blindfield accompanied by temporal and spatial cues (Kentridge et al., ). As well, it would be possible to improve conscious visual detection performances with training of residual visual abilities (Chokron et al., ), to improve visual functions that are initially outside of the spatiotemporal band of blindsight by using double stimulations including complex motion and static stimuli presented in different positions in the blind field (Das et al., ). This improvement in perception is obtained when a transfer of information processing happens between different stimuli and experimental conditions (Huxlin et al., ), implying that the perception workspace is capable of great plasticity when it is targeted via different mechanisms. Restitution tools must therefore target multiple functions used in perception, that is to say: detection, localization, identification and discrimination, as well as functions used in consciousness, that is to say: a judgment on the nature and the level of visual consciousness (Sahraie et al., ). Moreover, it is possible with repeated stimulation to increase visual sensitivity (Sahraie et al., , ; Trevethan et al., ). This change in subjective awareness linked to the performance, highlights the possibility of a transfer from an unconscious vision (type I blindsight), to a state of awareness (type II blindsight), hence to a potential visual qualia (vision), which is encouraging in regards of rehabilitation tools (Sahraie et al., ). Taken together, these results imply that to gain vision, we have to trigger long-term plasticity by targeting multiple pathways and mechanisms together creating a synchronous activity through multiple processors of the blindsight framework. Thus, we endorse a combined strategy using multisensory compensation and restitution.
### Potential Effects of a Combined-Training within a Global Subcortical Framework of Blindsight
Audio-Visual Scanning Training could allow feedforward interactions between the SC and V5/MT (Dundon et al., ), causing V5/MT to increase its functional activity and potential to make stronger connections in the attentional workspace. When applied with restitution training, the increase in functionality could optimally reinforce the tracks between the LGN and V5/MT in the perception workspace (Ajina et al., ), leading to more efficient interactions with lower and higher visual areas resulting in long-distance reciprocal connections and cascading amplification in the conscious workspace (see Figure ). Therefore, a stimulation of the altered blindsight framework would allow attention and perception to enhance each other leading to a better access to consciousness by a decrease in the threshold of visual attention and discrimination. Lowering these thresholds implicates that the visual properties of a stimulus are prompt to be accessible to different areas of the brain making them more easily perceived and processed permitting awareness. This will be reflected by higher synchronization of neural activity in visual and higher cognitive areas which will induce global availability and possibly lead to conscious visual perception (Melloni et al., ). Finally, although the main focus of this review covered visual training, we can’t omit the potential benefit of pharmacological interventions (Gratton et al., ) and novel tools for neuromodulation used alone (Gall et al., ) or combined with vision restauration strategies, e.g., VRT with dtCS (Plow et al., ; Alber et al., ), that could target in different ways the global workspace. However, let’s keep in mind that prior to using any kind of neurostimulation it would be essential to use an efficient visual training that could facilitate rehabilitation at home.
An illustrative schematic of the proposed hypothesis of the pathways involved in blindsight within the model of global workspace. In peach and green are represented the normal and lesioned hemispheres and subcortical areas projecting towards their respective hemispheres. The brown lines represent feedforward and feedback projections between workspaces. Enhancing the projections from the superior colliculus (SC)/pulvinar and the Lateral Geniculate Nucleus (LGN) to V5/MT and interhemispheric connections between V5/MT could allow synchronization between different areas, including the extrastriate regions, the dorsal pathways and the frontal areas, thus leading to more efficient interactions between lower and higher visual areas resulting in long-distance reciprocal connections and cascading amplification in the conscious workspace.
## Conclusion
The problem is to know how visual therapies can target residual visual abilities when neurophysiological correlates are so divergent between patients. Can we really use what we know of blindsight to develop rehabilitation tools? Our review explains how combined rehabilitation tools using visual training can enhance blindsight by targeting an inefficient global framework. Blindsight, defined as an unconscious residual visual ability, can come with or without awareness, but except in rare cases, doesn’t elicit visual awareness (Balsdon and Azzopardi, ). The reason why some patients may not present residual vision or awareness could include an inability to allocate sufficient attention to the information presented in the blind hemifield and to access their own state of consciousness. By understanding blindsight within the global workspace theory (Sergent and Dehaene, ), we can define the lack of visual awareness as a lack of neuronal synchrony and global availability between inefficient workspaces of attention, perception and consciousness that we can target and optimize with rehabilitation tools. Therefore, it would be possible to pass from a state of no awareness (type I blindsight) to a state of awareness (type II blindsight) to a state of visual awareness (alternative visual abilities) by moving the thresholds of attention, perception and consciousness via stimulation of the colliculo and geniculo-extrastriate pathways and creating connections between different processors. By doing so, we could target higher visual areas as V5/MT, induce loops with higher cognitive areas, synchronization of neuronal activity and global availability, and potentially it would lead to visual consciousness. These mechanisms can be targeted optimally in the subacute phase, using interhemispheric stimulations, Audio-Visual Scanning Training and combined restitution strategies, where several processes are enhanced at the same time inducing learning transfer and promoting the brain reorganization. The establishment of new guidelines in rehabilitation tools targeting the global framework of blindsight can lead to clinical intervention tools applicable to the majority of CB patients.
## Author Contributions
FL and VH: equal contribution for the literature search and article preparation.
## Conflict of Interest Statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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Neurotransmitters released at synapses mediate Ca2+ signaling in astrocytes in CNS grey matter. Here, we show that ATP and glutamate evoke these Ca2+ signals in white matter astrocytes of the mouse optic nerve, a tract that contains neither neuronal cell bodies nor synapses. We further demonstrate that action potentials along white matter axons trigger the release of ATP and the intercellular propagation of astroglial Ca2+ signals. These mechanisms were studied in astrocytes in intact optic nerves isolated from transgenic mice expressing enhanced green fluorescent protein (EGFP) under control of the human glial fibrillary acidic protein promoter (GFAP) by Fura-2 ratiometric Ca2+ imaging. ATP evoked astroglial Ca2+ signals predominantly via metabotropic P2Y1 and ionotropic P2X7 purinoceptors. Glutamate acted on both AMPA- and NMDA-type receptors, as well as on group I mGlu receptors to induce an increase in astroglial [Ca2+]i. The direct Ca2+ signal evoked by glutamate was small, and the main action of glutamate was to trigger the release of the "gliotransmitter" ATP by a mechanism involving P2X7 receptors; propagation of the glutamate-mediated Ca2+ signal was significantly reduced in P2X7 knock-out mice. Furthermore, axonal action potentials and mechanical stimulation of astrocytes both induced the release of ATP, to propagate Ca2+ signals in astrocytes and neighboring EGFP-negative glia. Our data provide a model of multiphase axon-glial signaling in the optic nerve as follows: action potentials trigger axonal release of ATP, which evokes further release of ATP from astrocytes, and this acts by amplifying the initiating signal and by transmitting an intercellular Ca2+ wave to neighboring glia. |
Poly(ADP-ribose) polymerase-1 (PARP-1) is a ubiquitous nuclear enzyme involved in genomic stability. Excessive oxidative DNA strand breaks lead to PARP-1-induced depletion of cellular NAD(+), glycolytic rate, ATP levels, and eventual cell death. Glutamate neurotransmission is tightly controlled by ATP-dependent astrocytic glutamate transporters, and thus we hypothesized that astrocytic PARP-1 activation by DNA damage leads to bioenergetic depletion and compromised glutamate uptake. PARP-1 activation by the DNA alkylating agent, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), caused a significant reduction of cultured cortical astrocyte survival (EC(50) = 78.2 +/- 2.7 microM). HPLC revealed MNNG-induced time-dependent reductions in NAD(+) (98%, 4 h), ATP (71%, 4 h), ADP (63%, 4 h), and AMP (66%, 4 h). The maximal [(3)H]glutamate uptake rate (V(max)) also declined in a manner that corresponded temporally with ATP depletion, falling from 19.3 +/- 2.8 in control cells to 2.1 +/- 0.8 nmol/min/mg protein 4 h post-MNNG. Both bioenergetic depletion and loss of glutamate uptake capacity were attenuated by genetic deletion of PARP-1, directly indicating PARP-1 involvement, and by adding exogenous NAD(+) (10 mM). In mixed neurons/astrocyte cultures, MNNG neurotoxicity was partially mediated by extracellular glutamate and was reduced by co-culture with PARP-1(-/-) astrocytes, suggesting that impairment of astrocytic glutamate uptake by PARP-1 can raise glutamate levels sufficiently to have receptor-mediated effects at neighboring neurons. Taken together, these experiments showed that PARP-1 activation leads to depletion of the total adenine nucleotide pool in astrocytes and severe reduction in neuroprotective glutamate uptake capacity. |
CD47 is a membrane receptor that plays pivotal roles in many pathophysiological processes, including infection, inflammation, cell spreading, proliferation, and apoptosis. We show that activation of CD47 increases proliferation of human U87 and U373 astrocytoma cells but not normal astrocytes. CD47 function-blocking antibodies inhibit proliferation of untreated U87 and U373 cells but not normal astrocytes, suggesting that CD47 may be constitutively activated in astrocytoma. CD47 expression levels were similar in our three cell types. CD47 couples to G-proteins in astrocytes and astrocytoma and especially to the Gβγ dimer. Downstream signaling following CD47 activation involves Gβγ dimer-dependent activation of the PI3K/Akt pathway in astrocytoma cells but not in normal astrocytes. This pathway is known to be deregulated in astrocytoma, leading to cell proliferation and enhanced survival signals. Putative PLIC-1 interaction with CD47 in astrocytoma cells but not astrocytes may contribute to the proliferative effect observed upon activation of CD47. Our data indicate that CD47 receptors have a stimulatory role in cell proliferation and demonstrate for the first time that CD47 signals via the PI3K/Akt pathway in cancerous cells but not normal cells. |
In the adult mammalian brain, neurogenesis originates from astrocyte-like stem cells. We generated a transgenic mouse line in which the tetracycline dependent transactivator (tTA) is expressed under the control of the murine GFAP promoter. In this mouse line, inducible gene expression targets virtually all GFAP-expressing stem-like cells in the dentate gyrus and a subset of GFAP-expressing progenitors located primarily in the dorsal wall/dorsolateral corner of the subventricular zone. Outside the neurogenic zones, astrocytes are infrequently targeted. We introduce a panel of transgenic mice which allow both inducible expression of candidate genes under control of the murine GFAP promoter and, at the same time, lineage tracing of all cells descendant from the original GFAP-positive cell. This new mouse line represents a versatile tool for functional analysis of neurogenesis and lineage tracing. |
The technology to generate autologous pluripotent stem cells (iPS cells) from almost any somatic cell type has brought various cell replacement therapies within clinical research. Besides the challenge to optimize iPS protocols to appropriate safety and GMP levels, procedures need to be developed to differentiate iPS cells into specific fully differentiated and functional cell types for implantation purposes. In this article, we describe a protocol to differentiate mouse iPS cells into oligodendrocytes with the aim to investigate the feasibility of IPS stem cell-based therapy for demyelinating disorders, such as multiple sclerosis. Our protocol results in the generation of oligodendrocyte precursor cells (OPCs) that can develop into mature, myelinating oligodendrocytes in-vitro (co-culture with DRG neurons) as well as in-vivo (after implantation in the demyelinated corpus callosum of cuprizone-treated mice). We report the importance of complete purification of the iPS-derived OPC suspension to prevent the contamination with teratoma-forming iPS cells. |
The histaminergic neurons located in the posterior hypothalamus modulate whole brain activity in a manner dependent on behavioral state. We have investigated their influence on high-frequency oscillation (200-Hz ripples) in the hippocampal CA1 region of freely moving rats. The occurrence of these ripples, assumed to be involved in memory trace formation, was markedly enhanced after injection of the H1-antagonists pyrilamine and ketotifen in a lateral ventricle, indicating a tonic activity of the histaminergic system. The H2- and H3-antagonists cimetidine and thioperamide were ineffective. We suggest a mediation of these effects through blocking the known histaminergic excitation of septal neurons. Histamine administered by the intracerebroventricular route had an inhibitory action on ripples. H1-receptor activation, which has been shown to inhibit learning and memory, thus shifts hippocampal activity away from high-frequency oscillation toward theta activity. |
In examining the role of Class 3 secreted semaphorins in the prenatal and postnatal development of the septohippocampal pathway, we found that embryonic (E14-E16) septal axons were repelled by the cingulate cortex and the striatum. We also found that the hippocampus exerts chemorepulsion on dorsolateral septal fibers, but not on fibers arising in the medial septum/diagonal band complex, which is the source of septohippocampal axons. These data indicate that endogenous chemorepellents prevent the growth of septal axons in nonappropriate brain areas and direct septohippocampal fibers to the target hippocampus. The embryonic septum expressed np-1 and np-2 mRNAs, and the striatum and cerebral cortex expressed sema 3A and sema 3F. Experiments with recombinant semaphorins showed that Sema 3A and 3F, but not Sema 3C or 3E, induce chemorepulsion of septal axons. Sema 3A and 3F also induce growth cone collapse of septal axons. This indicates that these factors are endogenous cues for the early guidance of septohippocampal fibers, including cholinergic and gamma-aminobutyric acid (GABA)ergic axons, during the embryonic stages. During postnatal stages, when target cell selection and synaptogenesis take place, np-1 and np-2 were expressed by septohippocampal neurons at all ages tested. In the target hippocampus, pyramidal and granule cells expressed sema 3E and sema 3A, whereas most interneurons expressed sema 3C, but few expressed sema 3E or 3A. Combined tracing and expression studies showed that GABAergic septohippocampal fibers terminated preferentially onto sema 3C-positive interneurons. In contrast, cholinergic septohippocampal fibers terminated onto sema 3E and sema 3A-expressing pyramidal and granule cells. The data suggest that Class 3 secreted semaphorins are involved in postnatal development. Moreover, because GABAergic and cholinergic axons terminate onto neurons expressing distinct, but overlapping, patterns of semaphorin expression, semaphorin functions may be regulated by different signaling mechanisms at postnatal stages. |
The firing of place cells in the rodent hippocampus is reliable enough to infer the rodent's position to a high accuracy; however, hippocampal firing also reflects the stages of complex tasks. Theories have suggested that these task-stage responses may reflect changes in reference frame related to task-related subgoals. If the hippocampus represents an environment in multiple ways depending on a task's demands, then switching between these cell assemblies should be detectable as a switch in spatial maps or reference frames. Place cells exhibit extreme temporal variability or "overdispersion," which Fenton et al. suggest reflects changes in active cell-assemblies. If reference-frame switching exists, investigating the relationship of the single cell variability described by Fenton and colleagues to network level processes provides an entry point to understanding the relationship between cell-assembly-like mechanisms and an animal's behavior. We tested the cell-assembly explanation for overdispersion by recording hippocampal neural ensembles from rats running three tasks of varying spatial complexity: linear track (LT), cylinder-foraging (CF), and cylinder-goal (CG). Consistent with the reports by Fenton and colleagues, hippocampal place cells showed high variance in their firing rates across place field passes on the CF and CG tasks. The directional firing of hippocampal place cells on LT provided a test of the reference-frame hypothesis: ignoring direction produced overdispersion similar to the CF and CG tasks; taking direction into account produced a significant decrease in overdispersion. To directly examine the possibility of a network modulation of cell-assemblies, we clustered the firing patterns within each pixel and chained them together to construct whole-environment spatial firing maps. Maps were internally self-consistent, switching with mean rates of several hundred milliseconds. There were significant increases in map-switching rates following reward-related events on the LT and CG tasks, but not on the CF task. Our results link single cell variability with network-level processes and imply that hippocampal spatial representations are made up of multiple, continuous sub-maps, the selection of which depends on the animal's goals when reward is tied to the animal's spatial behavior. |
Posttraumatic stress disorder (PTSD) is characterized by the presence of anatomo-functional hippocampal alterations. To date, the ability to orient within the environment, which relies on hippocampal integrity, has never been investigated in PTSD. We hypothesized that the ability to form a cognitive map of the environment would be impaired in PTSD. Moreover, spatial memory consolidation benefits from postlearning sleep. Because PTSD individuals often complain about sleep disturbances, we hypothesized that any sleep effect on memory performance would be hampered in these subjects. Twenty-two subjects, all survivors of the L'Aquila 2009 earthquake, were divided into a PTSD and a control group, based on clinical evaluation. After an acquisition phase, they were tested twice ("test" and "retest") on a virtual navigation task. In addition, participants were administered the Digit Span and Task Switching. Subjective sleep quality and sleep disturbances were also assessed. The two testing sessions were on consecutive mornings, interspersed with a night of sleep. During the acquisition phase, the PTSD group took more than twice as long to form a cognitive map of the environment compared to the control group. However, once this phase was successfully completed, the two groups did not differ at test, but they tendentially differed at postsleep retest. Additional analyses comparing performances between groups on test-retest difference scores confirm that sleep-dependent consolidation may be differentially affected in the two groups. Our findings are strictly confined to the navigation performance, excluding a generalized cognitive deficit. PTSD also reported more subjective sleep disturbances and shorter sleep time than controls, which were correlated to worse performance at retest. The specific deficit in the formation of a cognitive map reported in PTSD may be related to hippocampal dysfunctions as well as to the sleep disturbances experienced by these patients. The possible deficiency of sleep-dependent spatial performance improvement should however be confirmed by further studies comprising a wake control group. |
The precise timing of pre-postsynaptic activity is vital for the induction of long-term potentiation (LTP) or depression (LTD) at many central synapses. We show in synapses of rat CA1 pyramidal neurons in vitro that spike timing dependent plasticity (STDP) protocols that induce LTP at glutamatergic synapses can evoke LTD of inhibitory postsynaptic currents or STDP-iLTD. The STDP-iLTD requires a postsynaptic Ca(2+) increase, a release of endocannabinoids (eCBs), the activation of type-1 endocananabinoid receptors and presynaptic muscarinic receptors that mediate a decreased probability of GABA release. In contrast, the STDP-iLTD is independent of the activation of nicotinic receptors, GABAB Rs and G protein-coupled postsynaptic receptors at pyramidal neurons. We determine that the downregulation of presynaptic Cyclic adenosine monophosphate/protein Kinase A pathways is essential for the induction of STDP-iLTD. These results suggest a novel mechanism by which the activation of cholinergic neurons and retrograde signaling by eCBs can modulate the efficacy of GABAergic synaptic transmission in ways that may contribute to information processing and storage in the hippocampus. |
Adult neurogenesis is necessary for proper cognition and behavior, however, the mechanisms that underlie the integration and maturation of newborn neurons into the pre-existing hippocampal circuit are not entirely known. In this study, we sought to determine the role of action potential (AP)-dependent synaptic transmission by adult-generated dentate granule cells (DGCs) in their survival and function within the existing circuitry. We used a triple transgenic mouse (NestinCreER<sup>T2</sup> :Snap25<sup>fl/fl</sup> : tdTomato) to inducibly inactivate AP-dependent synaptic transmission within adult hippocampal progenitors and their progeny. Behavioral testing in a hippocampal-dependent A/B contextual fear-discrimination task revealed impaired discrimination learning in mice harboring SNAP-25-deficient adult-generated dentate granule cells (DGCs). Despite poor performance on this neurogenesis-dependent task, the production and survival of newborn DGCs was quantitatively unaltered in tamoxifen-treated NestinCreER<sup>T2</sup> :Snap25<sup>fl/fl</sup> : tdTomato SNAP compared to tamoxifen-treated NestinCreER<sup>T2</sup> :Snap25<sup>wt/wt</sup> : tdTomato control mice. Although SNAP-25-deficient adult DGCs displayed a small but statistically significant enhancement in proximal dendritic branching, their overall dendritic length and distal branching complexity was unchanged. SNAP-25-deficient newborn DGCs also displayed robust efferent mossy fiber output to CA3, with normal linear density of large mossy fiber terminals (LMTs). These studies suggest that AP-dependent neurotransmitter release by newborn DGCs is not essential for their survival or rudimentary structural maturation within the adult hippocampus. |
The anatomy and physiology of monosynaptic connections in rodent hippocampal CA1 have been extensively studied in recent decades. Yet, the resulting knowledge remains disparate and difficult to reconcile. Here, we present a data‐driven approach to integrate the current state‐of‐the‐art knowledge on the synaptic anatomy and physiology of rodent hippocampal CA1, including axo‐dendritic innervation patterns, number of synapses per connection, quantal conductances, neurotransmitter release probability, and short‐term plasticity into a single coherent resource. First, we undertook an extensive literature review of paired recordings of hippocampal neurons and compiled experimental data on their synaptic anatomy and physiology. The data collected in this manner is sparse and inhomogeneous due to the diversity of experimental techniques used by different groups, which necessitates the need for an integrative framework to unify these data. To this end, we extended a previously developed workflow for the neocortex to constrain a unifying in silico reconstruction of the synaptic physiology of CA1 connections. Our work identifies gaps in the existing knowledge and provides a complementary resource toward a more complete quantification of synaptic anatomy and physiology in the rodent hippocampal CA1 region.
## INTRODUCTION
The hippocampal formation, notably the CA1 region, is one of the most exhaustively studied regions in the mammalian brain and is thought to play a role, for example, in the acquisition of memory, recognition of place and language (Bliss & Collingridge, ; Buzsáki, ). Neuronal microcircuits in the hippocampal CA1 region process and store information through a myriad of cell‐type‐specific monosynaptic connections.
Previous studies have shown that hippocampal cell types are connected through multiple synaptic contacts, which are positioned across distinct axo‐dendritic domains with a wide diversity in their physiology. Despite the wealth of data, we lack an integrative framework to reconcile the diversity of synaptic physiology, and therefore, identify knowledge gaps. There have been several noteworthy attempts to integrate knowledge on the cellular and synaptic components of hippocampal CA1 microcircuitry, which have provided a solid foundation to bring together anatomical properties and kinetic parameters of cell‐type‐specific connections—including the number of synapses per connection, connection probabilities, neurotransmitter release probabilities, and amplitudes of synaptic responses (Bezaire & Soltesz, ; Moradi & Ascoli, ; Wheeler et al., ). As a complementary endeavor, we extended a previously developed framework to reconstruct neocortical microcircuitry at the cellular and synaptic levels of detail (Markram et al., ), by integrating disparate data on the physiology of short‐term dynamics of depression and facilitation of cell‐type‐specific synaptic transmission in hippocampal CA1. Using this framework, we identified and extrapolated organizing principles to predict missing knowledge for a repertoire of connection types, for example, the short‐term dynamics and peak conductance of synaptic connections between inhibitory interneurons (Klausberger & Somogyi, ; Pelkey et al., ), which remain largely uncharacterized, and could, therefore, require high‐throughput strategies that employ multiple whole‐cell patch‐clamp recordings to surmount the relatively low yield obtained through conventional paired recordings (Espinoza, Guzman, Zhang, & Jonas, ; Jiang et al., ; Perin, Berger, & Markram, ).
We accounted for the dynamic and probabilistic nature of synaptic transmission by fitting experimental traces using a stochastic generalization of the Tsodyks–Markram (TM) short‐term plasticity (STP) model (Fuhrmann, Segev, Markram, & Tsodyks, ; Markram, Wang, & Tsodyks, ; Tsodyks & Markram, ), and also considered temperature and extracellular calcium concentration ( ) differences, which were adjusted using Q10 and Hill scaling factors, respectively.
Measuring peak quantal conductances directly at individual synaptic contacts remains very difficult, if not impossible with current experimental techniques. While theoretically, the peak synaptic conductance can be calculated from voltage‐clamp recordings by simply dividing the peak postsynaptic current (PSC) by the liquid junction potential (LJP)‐corrected driving force, this approach does not take into account the space‐clamp artifact (Gulyás, Freund, & Káli, ; Spruston, Jaffe, Williams, & Johnston, ; Williams & Mitchell, ). We have recently demonstrated that space‐clamp corrected peak synaptic conductances in neocortical connections are at least twofold to threefold higher than estimated previously (Markram et al., ). As a connection is formed by several synaptic contacts, each subject to a different space‐clamp effect, a purely theoretical correction is challenging. We, therefore, used an alternative approach, where we calibrated peak synaptic conductances in the in silico model of connected pairs such that the resulting postsynaptic potential (PSP) amplitudes match in vitro recordings. This yielded an estimate of peak synaptic conductance since other factors affecting the PSP amplitude—such as number and location of synapses, release probability and reversal potential, depression, facilitation, and synaptic conductance rise and decay time constant—were independently validated beforehand.
The resulting models for a subset of hippocampal connection types were applied predictively to the remaining uncharacterized connection types by clustering them into nine groups based on synapse types and neuronal biomarkers and applying the estimated parameters within each group. Curated and predicted parameters presented here should serve as a resource to researchers aiming to model hippocampal synapses at any level, while the detailed methodology intends to give a guideline to utilize such a framework to integrate data from other brain regions or species.
## METHODS
### Circuit building and synapse anatomy
A detailed model of the rat hippocampal CA1 area was built by adapting a previously described pipeline for reconstructing neocortical microcircuitry (Markram et al., ). In brief, detailed axo‐dendritic morphological reconstructions and electrophysiological traces obtained from the dorsal part of hippocampal CA1 were used to build single cell‐type‐specific computational models (Migliore et al., ) (see Supplementary Methods). The resulting single‐cell models were assembled in an atlas‐based volume corresponding to the dimensions of the hippocampal CA1 region (Ropireddy, Bachus, & Ascoli, ), cell‐densities and proportions, which yielded a tissue model consisting about 400,000 cells, 90% pyramidal cells (PCs), and 10% interneurons comprising 11 distinct morphology types (m‐types; see Supplementary Methods and Supplementary Figure ) (Bezaire & Soltesz, ). Structural appositions between axons and dendrites were detected based on touch distance criteria and subsequently pruned to yield a functional connectome through an algorithmic process, which was constrained with experimentally reported bouton density, number of synapses per connections, and connection probability (Reimann, King, Muller, Ramaswamy, & Markram, ). A previous study suggests targeted innervation of interneurons from PCs (Takács, Klausberger, Somogyi, Freund, & Gulyás, ). Therefore, to recreate this tendency, touch distances from PCs to interneurons were set to 6 μm as against 1 μm for connections between PCs. Furthermore, touch distances of 6 μm for connections between all interneurons and 1 μm for connections between interneurons and PCs were assumed. In this manner, the number and location of synapses for each cell‐types specific connection were derived in a data‐driven manner. When reproducing paired recordings in silico (see below), monosynaptically connected pairs of neurons were sampled from this reconstructed circuit based on their intersomatic distance as sampling criterion.
### Dendritic features of single cell models
Detailed, multicompartmental morphoelectrical models with 3D reconstructed dendrites from Migliore et al. ( ) were used in the present study (see Supplementary Methods and Supplementary Figure ). The attenuation of synaptic responses along the dendrites with varying diameters was validated against experimental data from Magee and Cook ( ) using the HippoUnit framework (see Supplementary Methods). To this end, excitatory PSC (EPSC) like currents were injected into the apical trunk of PCs with varying distance from the soma and PSPs were simultaneously measured at the local site of the injection and in the soma.
### Model of postsynaptic conductance and current
Synaptic conductances were modeled with biexponential kinetics: where (nS) is the peak synaptic conductance and and (ms) are PSC rise and decay time constants, respectively. The normalization constant ensures that synapses reach their peak conductance at (ms). (Equation ) is modified below to take stochastic release of multiple vesicles into account.) AMPAR and GABAR synaptic currents are then computed as: where (mV) is the membrane potential and (mV) is the reversal potential of the given synapse. NMDAR currents depend also on block: where is the LJP‐corrected (see below) Jahr–Stevens nonlinearity (Jahr & Stevens, ): where (mM) is the extracellular magnesium concentration and = 0.062 (1/mV) and (mM) are constants (the difference from the original Jahr and Stevens ( ) constant is because the authors did not correct for the LJP offset of mV). PC‐to‐PC NMDAR rise and decay time constants are Q10 corrected (see below) ( ms for rise and 1.7 ms for decay time constants (Hestrin, Sah, & Nicoll, ; Korinek, Sedlacek, Cais, Dittert, & Vyklicky, )) values from Andrasfalvy and Magee ( ): = 3.9 ms and = 148.5 ms. All, but the CCK+ interneuron excitatory afferents have the same NMDAR time constants as the PC‐to‐PC ones, while the PC to CCK+ interneuron NMDAR conductance decays with a slower time constant: = 298.75 ms (Cornford et al., ; Le Roux, Cabezas, Böhm, & Poncer, ; Matta et al., ). Peak NMDAR conductance (nS) is calculated from the AMPAR one by multiplying it with NMDAR/AMPAR peak conductance ratio. PC‐to‐PC NMDAR/AMPAR peak conductance ratio = 1.22 was taken from Groc, Gustafsson, and Hanse ( ) and Myme, Sugino, Turrigiano, and Nelson ( ).
PC to CCK+ interneuron NMDAR/AMPAR ratio was set to 0.86, as against 0.28 for PC to other interneurons (Le Roux et al., ; Matta et al., ). Synaptic currents are individually delayed based on axonal path length and conduction velocity of 300 μm/ms (Stuart, Schiller, & Sakmann, ) and an additional 0.1 ms delay of neurotransmitter release (Ramaswamy et al., ).
### STP parameter fitting
STP of synapse dynamics was fit by the TM model (Markram et al., ; Tsodyks & Markram, ). The model assumes that each synapse has a pool of available neurotransmitter resources ( R ) that is utilized by a presynaptic action potential (AP) with a release probability ( U ). The utilization of resources leads to postsynaptic conductance that is proportional to the amount utilized. R decreases and U increases after an AP and both R and U recover between spikes to a steady‐state (SS) value. The speed of recovery is parameterized by time constants D and F (ms) that together determine the short‐term dynamics of the synapse. This is described by the following differential equations: where is the utilization of synaptic efficacy or absolute release probability (also known as the release probability in the absence of facilitation), is the Dirac delta function and indicates the timing of a presynaptic spike. Each AP in a train elicits an amplitude PSC, where is the absolute synaptic efficacy. and are assumed before the first spike.
The , D , F , and free parameters of the model were fit to amplitudes of experimentally recorded trains of PSCs. In the case of Losonczy, Zhang, Shigemoto, Somogyi, and Nusser ( ), amplitudes were already extracted by the authors, while in the case of Kohus et al. ( ) custom‐written Python routines were used to extract them from the averaged postsynaptic traces. Fitting the 10 + 1 recovery spikes was done by using a multiobjective genetic algorithm from BluePyOpt (Van Geit et al., ). For Kohus et al. ( ), different frequency stimulations (10, 20, and 40 Hz) were fit together for better generalization. Thus, the optimized error function contained 3 (frequencies) × 11 (peak amplitudes) points. For the event‐based version of the equations above, see Maass and Markram ( ) and Supplementary Methods. The Python source code fitting amplitudes from multiple frequencies is available on GitHub under /BluePyOpt/examples.
Data inclusion and exclusion criteria
### Stochastic TM model with multivesicular release
For the simulation of synapses, the canonical TM model (introduced above and used for fitting experimental traces) was modified to include stochastic release of multiple vesicles, and connected to the model of postsynaptic conductance described above. To take multivesicular release (MVR) into account in the postsynaptic conductance model, the classical “quantal model” of Del Castillo and Katz ( ) was used. In this model, synapses are assumed to be composed of (size of the readily releasable pool) release sites, each of which has a probability of release U (see deterministic TM model above) and contributes a quanta to the postsynaptic response (Barros‐Zulaica et al., ; Loebel et al., ; Markram et al., ; Ramaswamy et al., , ). Unlike in the deterministic TM model above, individual quanta were assumed to be released in an all‐or‐none fashion with probability U ( t ) (Fuhrmann et al., ). Vesicle availability is also an all‐or‐none process where only available vesicles can be released. To this end, synaptic vesicles were implemented as two‐state (available: 1 and unavailable: 0) Markov processes. After release, the state is set to unavailable and the probability of staying in the unavailable state at time t was described as a survival process, with the time constant D . The state transitions are described by the following set of equations:
The above‐described model converges to the canonical TM model in the limit (number of trials ). In this formalism, a presynaptic AP releases only a fraction fraction of vesicles, which follows a Bernoulli distribution. Equation ) is thus updated as follows: where r and d are the rising and decaying components of the postsynaptic conductance, respectively. The implementation of the above described stochastic synapse model is available at the open‐access NMC portal (Ramaswamy et al., ).
These changes to the canonical TM model introduce variability of the postsynaptic traces, where the magnitude of the variability depends on the additional parameter (Barros‐Zulaica et al., ; Loebel et al., ). In vitro this variability is typically assessed by the coefficient of variation (CV, SD /mean) of the peak PSC (or PSP) amplitudes. Therefore, the was calibrated to match the CVs of the first PSCs extracted from the raw traces of Kohus et al. ( ). For a better comparison, artificial membrane noise was added to the simulated traces (see Barros‐Zulaica et al. ( ) and Supplementary Methods).
### Calibrating peak synaptic conductances through in silico paired recordings
Paired recordings were replicated in silico as follows: First, pairs were selected from the circuit based on pathway specific distance criteria used by experimentalist (100 μm for cells in the same layer and 200 μm for cell pairs from different layers). Second, postsynaptic cells were current clamped to match the LJP‐corrected (see below) SS potential specified in the experiments. It is important to note, that in the case of PCs sodium channels were blocked ( in silico TTX application) when clamping above −58 mV to avoid spontaneous firing of the cell models (see Migliore et al. ( ), figure 5), whereas sodium channels were not blocked in in vitro experiments. Next, the presynaptic cell was stimulated by somatic current injection, which resulted in a PSP recorded in the soma of the postsynaptic neuron. This protocol was repeated for 50 monosynaptic connections of the same pre‐post combination with 35 repetitions for each neuron pair. Finally, the mean PSP amplitude was compared against experimentally data and the peak conductance value was calibrated using the formula: where (mV) and (mV) are the experimental and modeled PSPs amplitudes respectively and (mV) is the driving force. For all the experiments we aimed to reproduce, mV was calculated for excitatory connections, while mV for inhibitory connections (Moradi & Ascoli, ). All simulations were run using the NEURON simulator as a core engine (Hines & Carnevale, ) with the Blue Brain Project's collection of hoc and NMODL (Hines & Carnevale, ) templates for parallel execution on supercomputers (Hines, Eichner, & Schürmann, ; Hines, Markram, & Schürmann, ). The default temperature in all simulations was set to 34°C and the integration time step to 0.025 ms.
### Correcting for calcium ion concentration, temperature, and LJP
Before integrating published parameters from different sources into the in silico synapse model, they were corrected for differences in experimental protocols. This included scalings for levels different from 2 mM, temperatures different from 34°C and the correction of holding and SS potentials by the theoretical LJP.
Levels of impact the neurotransmitter release probability. The corresponding in silico correction was applied by scaling the absolute release probability parameter (see above) of the synapses, using the Hill isotherm with n = 4 (Hill, ). The Hill equation below describes the nonlinear increase in release probability as a function of increasing : where is the maximum value of the release probability ( ) at high and is the at which is one‐half of . and parameters can be fit to data points (e.g., an indicator of release probability—the ratio between PSP amplitudes) measured at different s. values were taken from Rozov, Burnashev, Sakmann, and Neher ( ), 2.79 (mM) for steep and 1.09 (mM) for shallow calcium dependence and were shown to generalize well for other characterized pathways of the neocortex (see Markram et al. ( ), supplementary figure S11). In the absence of hippocampus specific data, we followed the approach of Markram et al. ( ) and assumed a steep dependence in PC to PC and PC to distal dendrite targeting inhibitory (O‐LM) cells, and a shallow dependence between PC to proximal targeting cells (PVBC (PV+ basket cell), CCKBC (CCK+ basket cell), and axo‐axonic cell). For experimentally uncharacterized pathways, an intermediate calcium dependence was used, as the average of the steep and shallow ones. This intermediate curve was in agreement with the few relevant data points for specific hippocampal synaptic connections (Price, Scott, Rusakov, & Capogna, ; Tyan et al., ). The temperature dependence of kinetic parameters such as rise and decay time constants was corrected by dividing them with Q10 scaling factors: where is the time constant, Q10 is an empirically determined, receptor‐specific parameter, is the temperature used in the simulations, while is the temperature of the experiment. The Q10 correction was only needed for the NMDA current between connected PCs (see above) because all other kinetic values that we used were recorded at near physiological temperature ( ).
Holding and SS potentials were corrected by the theoretical LJP (Neher, ). These potentials arise from the differences in solutions in the pipette and bath and are in 2–12 mV range for the standard solutions. Theoretical LJPs, calculated from the reported pipette and bath solutions were obtained from Moradi and Ascoli ( ).
### Statistical analysis
R values for validating matching experimental and model values are Pearson correlations. Data are presented as to yield comparable values to the experimental ones. , D , F distributions from two different sources (e.g., found in the literature vs. fitted here) are said to be comparable if the mean of the second distributions is not further away than one‐half of the SD of the first distribution.
## RESULTS
### Literature curation
First, we undertook an extensive literature review of paired recording experiments, and compiled data on the various parameters (Figure , Step 1; Tables and for the data inclusion and exclusion criteria, and a list of data and modeling assumptions, respectively; see also Supplementary Table for voltage‐clamp data from rat hippocampal CA1, and for current‐clamp recordings). The data collected in this manner is sparse and inhomogeneous, due to the disparate experimental conditions used by different groups and were, therefore, corrected for various aspects (Figure , Step 2). For example, is known to affect release probability and, therefore, an additional Hill scaling had to be considered while parameterizing STP models (see Section ). Rise and decay time constants of synaptic currents are influenced by temperature differences but can be corrected with Q10 factors (see Section ). For electrophysiological recordings, patch pipettes have become the method of choice over sharp electrodes, which necessitates applying an LJP correction for voltage traces (see Section ).
In silico data integration pipeline. (1) 51 peer‐reviewed papers, spanning 21 years were used to compile data on various parameters of connected neurons in rat CA1 including connection probability, number of synapses per connections, axo‐dendritic innervation profile, kinetics, STP profiles, calcium and temperature sensitivity. (2) Parameters were integrated into a common framework and experimental paradigm, including temperature, and liquid junction potential (LJP) corrections. TM models of STP were fit to publicly available raw traces. (3) In silico paired recordings were run to correctly adjust the unitary peak conductance of connections with experimentally characterized postsynaptic potential (PSP) amplitudes. (4) The resulting parameters were averaged within each of the nine classes of synapses and used predictively to describe experimentally uncharacterized pathways [Color figure can be viewed at ]
List of assumptions. All the assumptions that were made to arrive at model parameters from a sparse set of raw data and published values
### Synaptic model parameters
We integrated the collected and corrected data into a model of synaptic transmission that includes STP and stochastic neurotransmitter release. We found that for some connection types the parameters of this model could be fully determined by employing in silico paired recordings (Figure , Step 3). Yet, for the majority of connection types parameters had to be extrapolated (Figure , Step 4). We use “synapse” to refer to a single anatomical synaptic contact and “connection” to indicate the collection of all synaptic contacts between a given presynaptic and postsynaptic neuron, comprising one or more synapses.
The underlying synapse model consisted of several parts, each with their associated parameters, which we determined in a six‐step procedure: We modeled synaptic connections with biexponential conductances requiring 8 parameters. Three parameters ( , , ) were directly obtained from the literature (see Supplementary Table for AMPAR and GABAR rise and decay time constants, methods for NMDAR time constants, and Supplementary Table for reversal potentials (Moradi & Ascoli, )). In particular, for the (Supplementary Table ) with the exception of Maccaferri, Roberts, Szucs, Cottingham, and Somogyi ( ) who used either single or weighted biexponential fits, none of the other studies we considered explicitly reported how was extracted. Therefore, we extrapolated single exponential fits of all pathways, which were measured through somatic voltage‐clamp recordings. We used these measurements directly as dendritic PSC time constants without any correction for attenuation (Table ). STP was modeled with the TM model, which added three parameters , D , F ) to a synaptic connection type. They were fit in conjunction to the experimentally observed STP behavior (Figure , Step 4; see Section ). Stochastic synaptic transmission was modeled by extending the TM model to include quantal release from multiple sites. This added another parameter ( ) that was fit to the observed variability of PSC amplitudes of experimental traces in terms of their CV ( SD /mean; Figure , Step 5; see Section ). Finally, the mean amplitude of PSPs depended on three of the parameters and thus could be fit to the peak synaptic conductance ( ) only after the other two parameters had been determined (Figure , Step 6).
In silico synapse model and parameter fitting: Properties of the network (left) and the parameters synapse model (right) determine certain features of the emergent postsynaptic potentials (PSPs) (middle). (These PSP features are schematized at the bottom of the figure. Individual trials are shown in gray and their average postsynaptic voltage trace in black.) These dependencies between properties/parameters and PSP features (indicated by arrows, and dots where they join and continue as a single arrow) were used to fit the synapse model parameters to data in six steps. Left: Parts of the network model that affect these features such as biophysical and anatomical neuron models via dendritic attenuation (1) as well as dendritic innervation and the number of synapses per connection (2) are independently validated. Top right: Parameters of the model of postsynaptic conductance are taken from averaged experimental PSC traces (3). Middle right: The TM model of STP adds three parameters that are fit to observed STP behavior (4). Bottom right: The model of stochastic quantal release adds another parameter fit to the observed CV of PSP amplitudes (5). In the last step, peak synaptic conductances are calibrated to match PSP amplitudes from data (6). Numbers on arrows indicate that the given parameter was validated against—or fitted to data, while numbers on boxes indicate that the parameters were taken from literature and directly plugged in into the model [Color figure can be viewed at ]
In addition to the parameters of synaptic models, the physiology of PSPs is also dependent on several anatomical parameters, which result from the single‐cell and tissue modeling workflow (see Section ; Supplementary Figure ). To ensure the accuracy of the fitted synaptic parameters we independently validated aspects of the modeled anatomy (Figure , Steps 1 and 2). In the following sections, we present the results of the anatomical validations, followed by the results of the various fits of synaptic parameters.
### Validation of synaptic anatomy and dendritic attenuation
The anatomical properties of synaptic connections such as number of synapses per connection and axo‐dendritic innervation patterns, along with the dendritic properties of single cell models were validated against experimental data (Figure ). Pairs of synaptically connected neurons were sampled from a dense tissue‐level reconstruction of the rat hippocampal CA1 region (see Section , Supplementary Figure , Figures and ). The number of synapses per connection for the handful of experimentally characterized pathways (Ali, ; Biró, Holderith, & Nusser, ; Buhl, Halasy, & Somogyi, ; Buhl, Han, et al., ; Deuchars & Thomson, ; Földy, Lee, Morgan, & Soltesz, ; Maccaferri et al., ; Sik, Penttonen, Ylinen, & Buzsáki, ; Vida, Halasy, Szinyei, Somogyi, & Buhl, ) was consistent with biological data ( ; Figure and Supplementary Table ). The mean number of synapses per connection for the in silico pathways that have been experimentally characterized are as follows: Excitatory to excitatory (E‐E): 1.26 0.6; inhibitory to excitatory (I‐E): 8.2 2.1; excitatory to inhibitory (E‐I); only connections between PC to O‐LM cells): 2.8 1.2; inhibitory‐inhibitory (I‐I): 2.8 0.2 (Supplementary Table ). A systematic, quantitative characterization of axo‐dendritic innervation profiles for hippocampal CA1 synaptic connections is largely lacking. Therefore, although we derived many predictions of axo‐dendritic innervation profiles from in silico synaptic pathways, these could, however, only be validated based on anecdotal evidence (Figures and ). In addition, we sampled neuron pairs at intersomatic distances of 0–200 to predict their connection probability and number of synapses per connection (Figure ). The upper bound of 200 ensured that we obtained a sufficient number ( ) of pairs for all connections, even where the pre–post neurons were in different layers, for example, Schaffer collateral‐associated and OLM cells to PC connections. Although the perforant path‐associated cell to PC connections occur in our model, they were excluded in these analyses since their somata are farther apart than the general 200 distance criteria chosen for these predictions.
Finally, we also validated the dendritic attenuation profile of PSPs in single neuron models of PCs, which were also found to be consistent with experimental data (Magee & Cook, ) ( , , Supplementary Figure ).
In silico synapse anatomy. (a) A representative in silico O‐LM (purple) to PC (blue) pair, with synapses visualized in red. 3D morphologies were reconstructed with the Neurolucida software by the members of the Thomson/Mercer lab (Migliore et al., ). (a1) Branch order distribution ( connections) of the presynaptic (O‐LM) axons. (a2) Branch order distribution of the postsynaptic (PC) tuft dendrites. (a3) Distribution of the number of synapses per connection of the in silico O‐LM to PC pathway. In vitro experimental data is indicated in red. (a4) Distance‐dependent connection probability of the in silico O‐LM to PC pathway. (b) Validation of the number of synapses per connection against experimental data. (E: excitatory, I: inhibitory, e.g.,: I‐E: inhibitory to excitatory pathways.) Dashed gray line represents perfect correlation between experimental and model values. (c) Predicted mean number of synapses per connections for all pathways in the full‐scale CA1 network model. Only connections with intersomatic distance were used to calculate the average. Averages were calculated from pairs. White boxes represent connections that are not present in the circuit model due to the lack of axo‐dendritic overlap (given the intersomatic distance sampling criteria). Experimentally measured values (same as on its left) are highlighted with black rectangles. Layer abbreviations: SR, stratum radiatum; SP, stratum pyramidale; SO, stratum oriens. M‐type abbreviations: AA, axo‐axonic cell; BP, back‐projecting cell; BS, bistratified cell; CCKBC, CCK+ basket cell; Ivy, ivy cell; OLM, oriens‐lacunosum moleculare cell; PC, pyramidal cell; PVBC, PV+ basket cell; PPA, performant path‐associated cell; SCA, Schaffer collateral‐associated cell; Tri, trilaminar cell (see Supplementary Methods). (d) Predicted mean connection probability (within 200 intersomatic distance) for all pathways in the CA1 network model. M‐type abbreviations, white boxes, black rectangles, and number of pairs are as in (c) [Color figure can be viewed at ]
In silico synapse physiology. (a) In silico paired recording experiment with the STP protocol used in Kohus et al. ( ). Presynaptic (PVBC) voltage trace is shown on top. In silico PVBC (green) to PC (blue) pair, with synapses visualized in red in the middle. 3D morphologies were reconstructed with the Neurolucida software by the members of the Thomson/Mercer lab (Migliore et al., ). Postsynaptic (PC) experimental traces recorded in vitro (in gray) and their mean in red, as well as model traces recorded in silico (in gray) and their mean in blue, are presented at the bottom panel. Insets show the variance of the first IPSCs. (b) Validation of the CV of the first PSC amplitudes (excluding failures) against experimental data. (E: excitatory, I: inhibitory, e.g.,: I‐E: inhibitory to excitatory pathways.) Dashed gray line represents perfect correlation between experimental and model values. (c) Validation of the postsynaptic potential (PSP) amplitudes against experimental data. (d) Predicted CVs of first PSC amplitudes (excluding failures) for all pathways in the CA1 network model after synapse parameter generalization. As in Figure , only connections with intersomatic distance were used to calculate the average postsynaptic response from pairs with 35 repetitions for each pair. Postsynaptic cells were held at −65 mV in in silico voltage‐clamp mode. M‐type abbreviations, white boxes, and black rectangles are as in Figure . (e) Predicted PSP amplitudes for all pathways in the CA1 network model after synapse parameter generalization. Then, 20 pairs with 35 repetitions for every possible connection. Postsynaptic cells were held at −65 mV steady‐state potential in in silico current‐clamp mode. Consistent with Gulyás et al. ( ), PC to interneurons are the strongest. M‐type abbreviations, white boxes, black rectangles and number of pairs are as in (d). (f) Properties of postsynaptic (PC) IPSPs from 100 PVBC to PC pairs with 35 repetitions each. (f1) Distribution of in silico PSP amplitudes. In vitro experimental data from Pawelzik et al. ( ) is indicated in red. (f2) Distribution of in silico PSP 10–90% rise times. (10–90% rise time constants of PSCs are fixed to 0.2 ms in the model, but the PSP rise times wary.) (f3) Distribution of in silico PSP decay time constants (single exponential fit). (f4) Distribution of in silico PSP latencies. (f5) Distribution of the CVs of the first in silico PSP amplitudes (excluding failures). (f6) Distribution in silico failures (0 measurable PSP amplitude from 35 repetitions) [Color figure can be viewed at ]
### STP of synapses
The synaptic physiology of hippocampal CA1 connections expresses a rich diversity of STP profiles in response to presynaptic AP trains at different stimulus frequencies (Ali, Bannister, & Thomson, ; Ali, Deuchars, Pawelzik, & Thomson, ; Ali & Thomson, ; Éltes, Kirizs, Nusser, & Holderith, ; Kohus et al., ; Losonczy et al., ; Pouille & Scanziani, ). However, to the best of our knowledge, only Losonczy et al. ( ) reported TM model parameters for CA1 pathways and used an additional recovery spike elicited about 500–100 ms after the last spike in the train, which is crucial to characterize frequency‐dependent STP profiles of depression and facilitation (Gupta, Wang, & Markram, ). Published STP parameters from Losonczy et al. ( ) were used for PC to BC pathways, after refitting a subset of their data, and ensuring their consistency with our resulting , D , F values (see Section ). The dataset from Kohus et al. ( ) were obtained in the mouse CA3 region at 1.6 mM , which differs from the rest of the datasets we considered, we nevertheless made use of this resource due to the availability of their raw data, which was subsequently used in our procedure of fitting TM model parameters (see Section ; Table for data inclusion and exclusion criteria; Table for a list of data and modeling assumptions). The resulting TM model parameters following the fitting procedure were consistent with those in the source dataset (Kohus et al., ). In addition, we were able to match the CVs of the first PSC amplitudes ( ; Figure , Supplementary Table ), by calibrating (see Loebel et al. ( ); Barros‐Zulaica et al. ( ) and Section ) with the resulting values of in a biologically plausible range. An elegant study demonstrated that under experimental conditions to induce high neurotransmitter release probability (high Mg/Ca) CCKBC to PC connections in CA3 are characterized by MVR (with vesicles) (Biró, Holderith, & Nusser, ). However, univesicular release (UVR, ) is more prevalent under physiological conditions (Biró et al., ). The in silico CV of CCKBC to PC PSCs with compared well against experimental data obtained under physiological conditions. In the cases of synaptic connections from PVBC to PC and PVBC, a value larger than 1 ( ) vesicles were required (see Section ; Figure ). For pathways not present in the Kohus et al.'s ( ) dataset, the could not be calibrated and was thus assumed. The assumption of MVR with vesicles at each excitatory to excitatory connections was used in this study (Barros‐Zulaica et al., ; Christie & Jahr, ; Conti & Lisman, ; Tong & Jahr, ), while UVR was assumed at all other noncalibrated pathways (see Gulyás et al. ( ); Biró et al. ( ) suggesting UVR for certain PC to interneuron connections).
Based on the literature and our model fitting, we identified several rules to group STP profiles. The mapping of STP profiles for all pathways is as follows: PC to O‐LM cells (Ali & Thomson, ; Biró et al., ; Losonczy et al., ; Pouille & Scanziani, ) and other interneurons in stratum oriens (Éltes et al., ) E1 (excitatory facilitating). PC to PC (Deuchars & Thomson, ), PC to all SOM interneurons (Ali et al., ; Losonczy et al., ; Pouille & Scanziani, ) E2 (excitatory depressing). CCK+ interneurons to CCK+ interneurons (Ali, , ; Kohus et al., ) I1 (inhibitory facilitating), PV+ and SOM+ interneurons to PC (Ali et al., , ; Bartos et al., ; Buhl, Cobb, Halasy, & Somogyi, ; Daw, Tricoire, Erdelyi, Szabo, & McBain, ; Kohus et al., ; Maccaferri et al., ; Pawelzik et al., ) as well as interneurons to interneurons (except the CCK+ ones) (Bartos et al., ; Daw et al., ; Elfant, Pal, Emptage, & Capogna, ; Karayannis et al., ; Kohus et al., ; Price et al., ) I2 (inhibitory depressing). CCK+ and NOS+ (only Ivy cells, since we lack NGF morphologies) to PC (Fuentealba et al., ; Kohus et al., ; Price et al., ) I3 (inhibitory pseudo linear). The parameters of the groups and the resulting dynamics are summarized in Table and Figure .
Parameters and generalization to nine classes
Summary of synapse diversity in the CA1 network model. Panels represent exemplar in silico pairs from the nine generalized pathways (two for PC to SOM− interneurons). Presynaptic voltage traces are shown in the upper traces of each panel (a–j), while the postsynaptic potentials elicited in 35 trials (in gray) and the average of these trials are superimposed in the lower traces of each panel. Postsynaptic cells were held at −65 mV steady‐state potential in in silico current‐clamp mode. Physical dimensions are as follows: decay time constant and D , F depression and facilitation time constants: ms, peak synaptic conductance : nS, while the absolute release probability and NMDA/AMPA conductance ratios are dimensionless. (a) PC to PC (E2). (b) PC to O‐LM cell (E1). (c) PC to (SP) bistratified cell (E2). (d) PC to CCKBC (E2). (e) O‐LM cell to PC (I2). (f) CCKBC to CCKBC (I1). (g) Ivy cell to PC (I3). (h) CCKBC to PC (I3). (i) PVBC to PC (I2). (j) PVBC to PVBC (I2). Vertical scale bars on each panel represent 0.25 mV. Connectivity in the schematic CA1 microcircuit in the middle is simplified for clarity (e.g., most of the interneuron to interneuron connections are missing). Simplified synapses of the pathways shown in the panels around are indicated with gray circles. M‐type abbreviations are as in Figure [Color figure can be viewed at ]
Neurotransmitter release probability and the STP profile are not only sensitive to the recording temperature and the developmental age but also (Guzman, Schlögl, Frotscher, & Jonas, ; Rozov et al., ; Williams & Atkinson, ). Therefore, we modeled sensitivity with a highly nonlinear scaling of (absolute release probability) values (see Section ). As an exemplar result of this additional modeling detail, the PC‐to‐PC pathway exhibits an E3 (excitatory pseudolinear) STP profile characterized by low PSP amplitudes with high trial‐by‐trial variability and failures at in vivo like levels (1.1–1.3 mM) compared to the in vitro levels (2–2.5 mM) E2 (excitatory depressing) profile (Supplementary Figure b). values are scaled by a Hill isotherm (see Section ) parameterized with data from PSP amplitudes in neocortex (Markram et al. ( ), supplementary figure S11), which is an indirect measure of the release probability. Here, we have shown that applying this Hill isotherm directly to the values indeed results in the same scaling profile of PSP amplitudes in the case of PC‐to‐PC connection (Supplementary Figure a).
### Calibration of peak synaptic conductances to match PSP amplitudes
There is a dearth of studies characterizing both the PSC and PSP amplitudes for the same connections in rat hippocampal CA1 (compare Supplementary Tables and ). Therefore, we only used PSP amplitudes that were measured experimentally to calibrate the in silico peak synaptic conductances in order to match the in vitro PSPs (Ali et al., ; Ali & Thomson, ; Cobb et al., ; Deuchars & Thomson, ; Fuentealba et al., ; Pawelzik et al., ; Pawelzik, Bannister, Deuchars, Ilia, & Thomson, ) (see Figure and Table ). Having parameterized all relevant anatomical and physiological synaptic properties including the number of synapses per connections, axo‐dendritic innervation patterns, PSC rise and decay time constants, STP parameters, , NMDA/AMPA peak conductance ratio, and reversal potential, we undertook in silico paired recordings by following a sequence of steps. A connected pair of neurons within a pathway specific intersomatic distance (usually ~100 μm) for a given pathway was sampled from the hippocampal CA1 model, the postsynaptic neuron was current clamped to a pathway‐specific SS potential (see Supplementary Table ), an AP was elicited in the presynaptic neuron, which caused a postsynaptic response, measured in the soma. After repeating this sequence for multiple pairs of the same pathway ( ) with many trials ( ), we derived the peak synaptic conductance value that yielded the reference mean experimental PSP amplitude (see Section ). Next, we repeated the same protocol on a set of 50 randomly selected pairs with the calibrated peak conductance values as a validation of our approach ( ; Figure and Supplementary Table ).
As an independent external validation of the peak conductances, we compared them against sparse published data estimating single‐receptor conductance and receptor numbers in excitatory synapses on PCs. Hippocampal CA1 PCs receive most of their inputs from CA3 PCs through the Schaffer collaterals (Megías, Emri, Freund, & Gulyás, ; Takács et al., ), whereas in this study we only considered intrinsic connections—for example, excitatory connections between local CA1 PCs—and not long‐range extrinsic projections. Thus, single‐receptor conductances and receptor number estimates from the Schaffer collateral synapses were assumed to generalize for the intrinsic PC‐to‐PC connections. Using nonstationary fluctuation analysis on EPSCs recorded in outside‐out dendritic membrane patches, (Spruston, Jonas, & Sakmann, ) estimated peak single‐receptor conductances of 10.2 pS and 43.5 pS for AMPARs and NMDARs, respectively. Based on these numbers, our calibrated values resulting in a peak AMPAR conductance of 0.6 0.1 nS is the net result of AMPARs per synaptic contact. Based on an experimentally measured NMDAR/AMPAR peak conductance ratio of 1.22 (Myme et al., ), we predict that there are about NMDARs constituting a single synaptic contact between CA1 PCs. Our in silico predictions are consistent with experimental studies that estimate 58–70 AMPA and 5–30 NMDA receptors (Jonas, Major, & Sakmann, ; Matsuzaki et al., ; Nusser et al., ; Spruston et al., ). Taken together, these experimental datasets enable an independent validation of the calibrated peak conductance of PC‐to‐PC connections in CA1. In addition, we also predict an average GABA peak conductance of 2 1 nS at a single inhibitory synaptic contact comprising 100 GABAergic receptors, which is also in good agreement with previous estimates (Mody & Pearce, ).
### Parameter extrapolation
By integrating all the synaptic parameters and performing paired recordings in silico , we procured a dataset of 16 pathways (Table ). The number of theoretically possible pathways (based on 12 m‐types) in our CA1 circuit model is 144; however, only 102 of these are biologically viable based on the extent of axo‐dendritic overlap (Figure ). Therefore, the parameters of the remaining 90% of the pathways had to be extrapolated. We generalized the anatomical properties of synapses (number of synapses per connection, connection probability, bouton density, innervation profile) obtained from the fraction of characterized to the remainder of uncharacterized pathways as shown previously (Markram et al., ; Reimann et al., ). However, for STP profiles of hippocampal connections obtained from studies that reported measurements of paired‐pulse ratios, but did not provide the raw experimental traces with presynaptic spikes (Ali & Thomson, ; Deuchars & Thomson, ; Fuentealba et al., ), we applied analogous parameters from the somatosensory cortex (Markram et al., ). We performed a prior consistency check of the parameter ranges for similar connection types—perisomatic inhibitory (BCs) to PC, and inhibitory to inhibitory—that have been experimentally characterized in both somatosensory cortex and hippocampus and found them to be comparable. Therefore, our rationale to generalize four sets of , D , F values from the somatosensory cortex to the hippocampus (Table ) could be justified. Thereafter, we approximated the missing parameters with averaged values across specific connection types that were grouped according to neurochemical markers that appear to have similar STP parameters and peak conductances (Table ). For example, it is known that excitatory synapses on distal dendrite targeting interneurons, which predominantly express SOM—such as PC to O‐LM connections—are mostly facilitating, and on the contrary inhibitory synapses from SOM+ neurons to PCs are strongly depressing (Ali & Thomson, ). This exercise resulted in nine synaptic classes, covering all connection types in the CA1 region (Table and Figure ). Most of these classes contain few experimentally characterized examples, especially between inhibitory interneurons (Table ). We have previously shown that averaging STP parameters and peak conductances within synaptic classes is a valid method to extrapolate missing values (Markram et al., ; Ramaswamy et al., ).
With the integrated and calibrated, but mostly generalized set of parameters ( , , D , F parameters of STP and ; Figure ) for all pathways in the CA1 model we predicted the CVs of the first PSCs (Figure ) and the first PSP amplitudes (Figure ), based on previously published cell models (Migliore et al., ) and statistically derived connectivity. In addition, we performed in silico paired recordings in all possible pre‐post combination of m‐type‐specific pathways ( biologically viable pathways) to generate detailed predictions of the physiological properties of synaptic transmission including PSP amplitudes, 10–90% rise times, decay time constants, latencies, CV of first PSP amplitude, and percentage of failures (Figure ). Although these predictions could provide preliminary insights into the organizing principles of synaptic transmission in hippocampal CA1—in particular, inhibitory pathways, which remain mostly uncharacterized—they require further validation through targeted experiments, for example, employing state‐of‐the‐art multiple whole‐cell patch‐clamp recordings (Espinoza et al., ; Guzman et al., ; Perin et al., ).
## DISCUSSION
Recent advances in high‐performance computing have enabled biologically detailed, data‐driven reconstructions and large‐scale simulations of brain regions (Bezaire, Raikov, Burk, Vyas, & Soltesz, ; Bezaire & Soltesz, ; Markram et al., ; Wheeler et al., ). Here, we demonstrate that a data‐driven workflow grounded in biological first‐principles, which was used to reconstruct a biologically detailed model of rat neocortical tissue digitally, can be extended to model other brain regions such as the hippocampal CA1, to reconcile disparate cellular and synaptic data, and to extrapolate from the sparse set of experimentally obtained parameters to predict those of synaptic connections not yet characterized experimentally. In this study, we chose a previous implementation of the phenomenological TM model of STP, which is based on the quantal model of neurotransmitter release. The approach was able not only to extract relevant parameters from raw experimental traces, but scaled well to simulate dynamic transmission (Markram et al., ; Ramaswamy et al., , ). In addition, this version of the TM model also enabled us to simulate trial‐to‐trial fluctuations to recreate, validate, and predict a broad spectrum of synaptic properties for cell‐type‐specific hippocampal connections including amplitudes, rise and decay times, latency, variability, and response failures (Figure ). It is known that regulates the neurotransmitter release probability, and therefore, the amplitudes of PSPs. In this study, we adapted the existing data‐driven digital reconstruction workflow to reconcile differences in synaptic dynamics that were characterized at different levels. Therefore, we scaled the neurotransmitter release probabilities for all pathways that were characterized at 1.6–2 mM (Kohus et al., ; Losonczy et al., ; Markram et al., ) before calibrating peak conductances to match PSP amplitudes that were measured at 2.5 mM , which is more representative of baseline values for hippocampal slice experiments (Ali et al., ; Ali & Thomson, ; Deuchars & Thomson, ; Fuentealba et al., ; Pawelzik et al., , ).
In the continuing spirit of bringing together, hippocampal synaptic electrophysiology from published literature a recent complementary study leveraged text‐mining techniques to extract the properties of synaptic connections in hippocampal CA1, including PSP amplitudes and peak conductances (Moradi & Ascoli, ). The authors have also open‐sourced their collection of papers and parameters alongside useful cloud‐based tools to calculate reversal potentials and LJPs, of which we took advantage for this paper. However, our approach to data integration from literature demonstrates that synaptic properties reported in the literature such as peak conductances should not be interpreted at face value but require further corrections to account for inadequate space‐clamp errors, which could severely underestimate their value by twofold to threefold (Markram et al., ). Furthermore, when integrating data from whole‐cell patch‐clamp recordings, the interaction between the extracellular bath and intracellular pipette solutions, and their influence on the kinetics of ion channel mechanisms used in the in silico single‐cell models becomes paramount.
The results we report, to the best of our knowledge, constitute a comprehensive resource, not only for the anatomy but also for the physiology of synaptic connections in the rat hippocampal CA1 region. Consolidation of the state of the literature not only facilitates building detailed models but also highlights knowledge gaps and could help in prioritizing the identification of missing data on CA1 connections, such as PC to interneurons, and between interneurons, which could form diverse presynaptic–postsynaptic combinations of potential CA1 connection types that are crucial in regulating hippocampal oscillations (Klausberger & Somogyi, ; Pelkey et al., ). Our modeling approach predicts relatively high connection probabilities for interneuron to interneuron connections, and low IPSP amplitudes (see Figures and ). However, these predictions need further experimental validation, probably through multiple patch‐clamp recordings, which have enabled high‐throughput mapping of inhibitory circuits not only in the neocortex (Jiang et al., ), but also in the dentate gyrus of the hippocampal formation (Espinoza et al., ). Indeed, the parameter set presented here should be considered a first draft, with many assumptions and limitations. For example, we assume somatically measured PSC decay time constants for dendritic synapses without any correction for attenuation, use , D , F values obtained in CA3, generalize NMDA/AMPA peak ratios characterized between PCs to all other excitatory pathways, and do not model receptors. We plan to refine these assumptions systematically in future versions of our model and overcome limitations by integrating new experimental data when available (see Table for all data inclusion criteria and Table for all explicit limitations). The presence of blockers such as TTX, QX314, cesium, and gluconate among many others, alter the kinetics of dendritic ion channels, which are active in the subthreshold regime, and thus, are key factors in governing the attenuation of PSPs in active dendrites. However, in our study, the core experimental dataset that was used to calibrate the peak synaptic conductances (Supplementary Tables and ) were derived exclusively from sharp‐electrode recordings where the intracellular medium is devoid of any of the above blockers, and therefore, the subthreshold regime of the single‐cell models are not unduly influenced. Indeed, the effects of blockers on the subthreshold regime will not only become important for future refinements of single‐cell models but also when more experimental data from whole‐cell patch clamp recordings are available.
By detailing all the integration steps in this study, we had two main objectives. First, we aimed to demonstrate that published parameters should not be taken at face value without rigorously checking their consistency within any modeling framework and the necessity of being abreast of the state‐of‐the‐art experimental techniques. Second, we attempted to emphasize the fact that a growing diversity of experimental standards combined with published literature that provides access to only processed data sets but not raw experimental traces could lead to an inconsistent picture of a fundamental mechanism such as synaptic transmission. The bottom‐up modeling framework presented as a resource in this article could facilitate the integration of disparate datasets and provide a platform within which a community‐driven consensus of the synaptic organization of the hippocampal formation could develop.
## CONFLICT OF INTEREST
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
## AUTHOR CONTRIBUTIONS
Srikanth Ramaswamy, András Ecker, and Eilif Muller conceptualized the study. Srikanth Ramaswamy supervised the study. Joanne Falck and Sigrun Lange reconstructed single cells in Neurolucida. Audrey Mercer and Alex M. Thomson provided experimental datasets. Sára Sáray, Szabolcs Káli, and Michele Migliore optimized and validated single cell models. Armando Romani built the CA1 circuit with inputs from all authors. András Ecker performed literature curation, simulations, analyses, and generated the figures with inputs from Szabolcs Káli, Michael W. Reimann, and Srikanth Ramaswamy. András Ecker, Michael W. Reimann, and Srikanth Ramaswamy wrote the manuscript with inputs from all authors.
## Supporting information
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Implicit, abstract knowledge acquired through language experience can alter cortical processing of complex auditory signals. To isolate prelexical processing of linguistic tones (i.e., pitch variations that convey part of word meaning), a novel design was used in which hybrid stimuli were created by superimposing Thai tones onto Chinese syllables (tonal chimeras) and Chinese tones onto the same syllables (Chinese words). Native speakers of tone languages (Chinese, Thai) underwent fMRI scans as they judged tones from both stimulus sets. In a comparison of native vs. non-native tones, overlapping activity was identified in the left planum temporale (PT). In this area a double dissociation between language experience and neural representation of pitch occurred such that stronger activity was elicited in response to native as compared to non-native tones. This finding suggests that cortical processing of pitch information can be shaped by language experience and, moreover, that lateralized PT activation can be driven by top-down cognitive processing. |
Estimating the thickness of the cerebral cortex is a key step in many brain imaging studies, revealing valuable information on development or disease progression. In this work, we present a framework for measuring the cortical thickness, based on minimizing line integrals over the probability map of the gray matter in the MRI volume. We first prepare a probability map that contains the probability of each voxel belonging to the gray matter. Then, the thickness is basically defined for each voxel as the minimum line integral of the probability map on line segments centered at the point of interest. In contrast to our approach, previous methods often perform a binary-valued hard segmentation of the gray matter before measuring the cortical thickness. Because of image noise and partial volume effects, such a hard classification ignores the underlying tissue class probabilities assigned to each voxel, discarding potentially useful information. We describe our proposed method and demonstrate its performance on both artificial volumes and real 3D brain MRI data from subjects with Alzheimer's disease and healthy individuals. |
The linguistic relativity hypothesis proposes that speakers of different languages perceive and conceptualize the world differently, but do their brains reflect these differences? In English, most nouns do not provide linguistic clues to their categories, whereas most Mandarin Chinese nouns provide explicit category information, either morphologically (e.g., the morpheme "vehicle" che1 in the noun "train" huo3che1) or orthographically (e.g., the radical "bug" chong2 in the character for the noun "butterfly" hu2die2). When asked to judge the membership of atypical (e.g., train) vs. typical (e.g., car) pictorial exemplars of a category (e.g., vehicle), English speakers (N = 26) showed larger N300 and N400 event-related potential (ERP) component differences, whereas Mandarin speakers (N = 27) showed no such differences. Further investigation with Mandarin speakers only (N = 22) found that it was the morphologically transparent items that did not show a typicality effect, whereas orthographically transparent items elicited moderate N300 and N400 effects. In a follow-up study with English speakers only (N = 25), morphologically transparent items also showed different patterns of N300 and N400 activation than nontransparent items even for English speakers. Together, these results demonstrate that even for pictorial stimuli, how and whether category information is embedded in object names affects the extent to which typicality is used in category judgments, as shown in N300 and N400 responses. |
Voxel-based morphometry (VBM) has been used repeatedly in single-center studies to investigate regional gray matter (GM) atrophy in multiple sclerosis (MS). In multi-center trials, across-scanner variations might interfere with the detection of disease-specific structural abnormalities, thereby potentially limiting the use of VBM. Here we evaluated longitudinally inter-site differences and inter-site comparability of regional GM in MS using VBM. Baseline and follow up 3D T1-weighted magnetic resonance imaging (MRI) data of 248 relapsing-remitting (RR) MS patients, recruited in two clinical centers, (center1/2: n = 129/119; mean age 42.6 ± 10.7/43.3 ± 9.3; male:female 33:96/44:75; median disease duration 150 [72-222]/116 [60-156]) were acquired on two different 1.5T MR scanners. GM volume changes between baseline and year 2 while controlling for age, gender, disease duration, and global GM volume were analyzed. The main effect of time on regional GM volume was larger in data of center two as compared to center one in most of the brain regions. Differential effects of GM volume reductions occurred in a number of GM regions of both hemispheres, in particular in the fronto-temporal and limbic cortex (cluster P corrected <0.05). Overall disease-related effects were found bilaterally in the cerebellum, uncus, inferior orbital gyrus, paracentral lobule, precuneus, inferior parietal lobule, and medial frontal gyrus (cluster P corrected <0.05). The differential effects were smaller as compared to the overall effects in these regions. These results suggest that the effects of different scanners on longitudinal GM volume differences were rather small and thus allow pooling of MR data and subsequent combined image analysis. |
Somatoform disorder patients suffer from impaired emotion recognition and other emotional deficits. Emotional empathy refers to the understanding and sharing of emotions of others in social contexts. It is likely that the emotional deficits of somatoform disorder patients are linked to disturbed empathic abilities; however, little is known so far about empathic deficits of somatoform patients and the underlying neural mechanisms. We used fMRI and an empathy paradigm to investigate 20 somatoform disorder patients and 20 healthy controls. The empathy paradigm contained facial pictures expressing anger, joy, disgust, and a neutral emotional state; a control condition contained unrecognizable stimuli. In addition, questionnaires testing for somatization, alexithymia, depression, empathy, and emotion recognition were applied. Behavioral results confirmed impaired emotion recognition in somatoform disorder and indicated a rather distinct pattern of empathic deficits of somatoform patients with specific difficulties in "empathic distress." In addition, somatoform patients revealed brain areas with diminished activity in the contrasts "all emotions"-"control," "anger"-"control," and "joy"-"control," whereas we did not find brain areas with altered activity in the contrasts "disgust"-"control" and "neutral"-"control." Significant clusters with less activity in somatoform patients included the bilateral parahippocampal gyrus, the left amygdala, the left postcentral gyrus, the left superior temporal gyrus, the left posterior insula, and the bilateral cerebellum. These findings indicate that disturbed emotional empathy of somatoform disorder patients is linked to impaired emotion recognition and abnormal activity of brain regions responsible for emotional evaluation, emotional memory, and emotion generation. |
Diffusion tensor imaging (DTI) studies have revealed group differences in white matter between patients with obsessive-compulsive disorder (OCD) and healthy controls. However, the results of these studies were based on average differences between the two groups, and therefore had limited clinical applicability. The objective of this study was to investigate whether fractional anisotropy (FA) of white matter can be used to discriminate between patients with OCD and healthy controls at the level of the individual. DTI data were acquired from 28 OCD patients and 28 demographically matched healthy controls, scanned using a 3T MRI system. Differences in FA values of white matter between OCD and healthy controls were examined using a multivariate pattern classification technique known as support vector machine (SVM). SVM applied to FA images correctly identified OCD patients with a sensitivity of 86% and a specificity of 82% resulting in a statistically significant accuracy of 84% (P ≤ 0.001). This discrimination was based on a distributed network including bilateral prefrontal and temporal regions, inferior fronto-occipital fasciculus, superior fronto-parietal fasciculus, splenium of corpus callosum and left middle cingulum bundle. The present study demonstrates subtle and spatially distributed white matter abnormalities in individuals with OCD, and provides preliminary support for the suggestion that that these could be used to aid the identification of individuals with OCD in clinical practice. |
Multi-atlas based methods have been recently used for classification of Alzheimer's disease (AD) and its prodromal stage, that is, mild cognitive impairment (MCI). Compared with traditional single-atlas based methods, multiatlas based methods adopt multiple predefined atlases and thus are less biased by a certain atlas. However, most existing multiatlas based methods simply average or concatenate the features from multiple atlases, which may ignore the potentially important diagnosis information related to the anatomical differences among different atlases. In this paper, we propose a novel view (i.e., atlas) centralized multi-atlas classification method, which can better exploit useful information in multiple feature representations from different atlases. Specifically, all brain images are registered onto multiple atlases individually, to extract feature representations in each atlas space. Then, the proposed view-centralized multi-atlas feature selection method is used to select the most discriminative features from each atlas with extra guidance from other atlases. Next, we design a support vector machine (SVM) classifier using the selected features in each atlas space. Finally, we combine multiple SVM classifiers for multiple atlases through a classifier ensemble strategy for making a final decision. We have evaluated our method on 459 subjects [including 97 AD, 117 progressive MCI (p-MCI), 117 stable MCI (s-MCI), and 128 normal controls (NC)] from the Alzheimer's Disease Neuroimaging Initiative database, and achieved an accuracy of 92.51% for AD versus NC classification and an accuracy of 78.88% for p-MCI versus s-MCI classification. These results demonstrate that the proposed method can significantly outperform the previous multi-atlas based classification methods. |
Clinical research suggests that imitating meaningless hand postures and pantomiming tool-related hand shapes rely on different neuroanatomical substrates. We investigated the BOLD responses to different tasks of hand posture generation in 14 right handed volunteers. Conjunction and contrast analyses were applied to select regions that were either common or sensitive to imitation and/or pantomime tasks. The selection included bilateral areas of medial and lateral extrastriate cortex, superior and inferior regions of the lateral and medial parietal lobe, primary motor and somatosensory cortex, and left dorsolateral prefrontal, and ventral and dorsal premotor cortices. Functional connectivity analysis revealed that during hand shape generation the BOLD-response of every region correlated significantly with every other area regardless of the hand posture task performed, although some regions were more involved in some hand postures tasks than others. Based on between-task differences in functional connectivity we predict that imitation of novel hand postures would suffer most from left superior parietal disruption and that pantomiming hand postures for tools would be impaired following left frontal damage, whereas both tasks would be sensitive to inferior parietal dysfunction. We also unveiled that posterior temporal cortex is committed to pantomiming tool grips, but that the involvement of this region to the execution of hand postures in general appears limited. We conclude that the generation of hand postures is subserved by a highly interconnected task-general neural network. Depending on task requirements some nodes/connections will be more engaged than others and these task-sensitive findings are in general agreement with recent lesion studies. |
Psychopathy is a personality disorder characterized by callous lack of empathy, impulsive antisocial behavior, and criminal recidivism. Here, we performed the largest diffusion tensor imaging (DTI) study of incarcerated criminal offenders to date (N = 147) to determine whether psychopathy severity is linked to the microstructural integrity of major white matter tracts in the brain. Consistent with the results of previous studies in smaller samples, we found that psychopathy was associated with reduced fractional anisotropy in the right uncinate fasciculus (UF; the major white matter tract connecting ventral frontal and anterior temporal cortices). We found no such association in the left UF or in adjacent frontal or temporal white matter tracts. Moreover, the right UF finding was specifically related to the interpersonal features of psychopathy (glib superficial charm, grandiose sense of self-worth, pathological lying, manipulativeness), rather than the affective, antisocial, or lifestyle features. These results indicate a neural marker for this key dimension of psychopathic symptomatology. |
For successful motor control, the central nervous system is required to combine information from the environment and the current body state, which is provided by vision and proprioception respectively. We investigated the relative contribution of visual and proprioceptive information to upper limb motor control and the extent to which structural brain measures predict this performance in youth (n = 40; age range 9-18 years). Participants performed a manual tracking task, adopting in-phase and anti-phase coordination modes. Results showed that, in contrast to older participants, younger participants performed the task with lower accuracy in general and poorer performance in anti-phase than in-phase modes. However, a proprioceptive advantage was found at all ages, that is, tracking accuracy was higher when proprioceptive information was available during both in- and anti-phase modes at all ages. The microstructural organization of interhemispheric connections between homologous dorsolateral prefrontal cortices, and the cortical thickness of the primary motor cortex were associated with sensory-specific accuracy of tracking performance. Overall, the findings suggest that manual tracking performance in youth does not only rely on brain regions involved in sensorimotor processing, but also on prefrontal regions involved in attention and working memory. Hum Brain Mapp 38:5628-5647, 2017. © 2017 Wiley Periodicals, Inc. |
Concussion pathophysiology in humans remains incompletely understood. Diffusion tensor imaging (DTI) has identified microstructural abnormalities in otherwise normal appearing brain tissue, using measures of fractional anisotropy (FA), axial diffusivity (AD), and radial diffusivity (RD). The results of prior DTI studies suggest that acute alterations in microstructure persist beyond medical clearance to return to play (RTP), but these measures lack specificity. To better understand the observed effects, this study combined DTI with neurite orientation dispersion and density imaging (NODDI), which employs a more sophisticated description of water diffusion in the brain. A total of 66 athletes were recruited, including 33 concussed athletes, scanned within 7 days after concussion and at RTP, along with 33 matched controls. Both univariate and multivariate methods identified DTI and NODDI parameters showing effects of concussion on white matter. Spatially extensive decreases in FA and increases in AD and RD were associated with reduced intra-neurite water volume, at both the symptomatic phase of injury and RTP, indicating that effects persist beyond medical clearance. Subsequent analyses also demonstrated that concussed athletes with higher symptom burden and a longer recovery time had greater reductions in FA and increased AD, RD, along with increased neurite dispersion. This study provides the first longitudinal evaluation of concussion from acute injury to RTP using combined DTI and NODDI, significantly enhancing our understanding of the effects of concussion on white matter microstructure. |
This work investigates the transfer of motor learning from the eye to the hand and its neural correlates by using functional magnetic resonance imaging (fMRI) and a sensorimotor task consisting of the continuous tracking of a virtual target. In pretraining evaluation, all the participants (experimental and control group) performed the tracking task inside an MRI scanner using their right hand and a joystick. After which, the experimental group practiced an eye-controlled version of the task for 5 days using an eye tracking system outside the MRI environment. Post-training evaluation was done 1 week after the first scanning session, where all the participants were scanned again while repeating the manual pretraining task. Behavioral results show that the training in the eye-controlled task produced a better performance not only in the eye-controlled modality (motor learning) but also in the hand-controlled modality (motor transfer). Neural results indicate that eye to hand motor transfer is supported by the motor cortex, the basal ganglia and the cerebellum, which is consistent with previous research focused on other effectors. These results may be of interest in neurorehabilitation to activate the motor systems and help in the recovery of motor functions in stroke or movement disorder patients. |
Extinction of appetitive conditioning is regarded as an important model for the treatment of psychiatric disorders like addiction. However, very few studies have investigated its neural correlates. Therefore, we investigated neural correlates of appetitive extinction in a large human sample including all genders ( N = 76, 40 females) to replicate and extend results from a previous study. During differential appetitive conditioning, one stimulus (CS+) was paired with the chance to win a monetary reward, whereas another stimulus (CS−) was not. During appetitive extinction on the next day, neither the CS+ nor the CS− were reinforced. After successful acquisition of appetitive conditioning, the extinction phase elicited significant reductions of valence and arousal ratings toward the CS+ and a significant reduction in skin conductance responses to the CS+ from early to late extinction. On a neural level, early extinction showed significant differential (CS+ − CS−) activation in dACC and hippocampus, whereas involvement of the vACC and caudate nucleus did not replicate. The differential activation of amygdala and nucleus accumbens during late extinction was replicated, with the amygdala displaying significantly higher differential activation during the late phase of extinction as compared to the early phase of extinction. We show discernible signals for reward learning and extinction in subregions of amygdala and nucleus accumbens after extinction learning. This successful replication underlines the role of nucleus accumbens and amygdala in neural models of appetitive extinction in humans that was previously only based on animal findings.
## INTRODUCTION
Appetitive extinction is regarded as a key mechanism for the treatment of psychiatric disorders like addiction (Millan, Marchant, & McNally, ). During extinction training, a stimulus (CS+; e.g., blue rectangle) associated with a reward (unconditioned stimulus or UCS; e.g., money) during an acquisition training is no longer paired with that reward. Subjects are then assumed to form an extinction memory trace that inhibits the CS‐UCS association (Quirk & Mueller, ). However, studies of its neural correlates in humans are scarce (Konova & Goldstein, ). In a previous study, we identified the nucleus accumbens (NAcc) and the amygdala as key structures involved in extinction learning when it has taken place (Kruse, Tapia León, Stark, & Klucken, ). This study was the first to investigate main effects of appetitive extinction with functional magnetic resonance imaging (fMRI); therefore, its findings need to be replicated in an independent sample to build a neural model of appetitive extinction and to further study neural mechanisms and moderators.
Acquisition of appetitive conditioning is investigated by repeatedly pairing one neutral stimulus (CS+; e.g., blue rectangle) with the chance to win a reward (UCS; e.g., money). Another neutral stimulus (CS−; e.g., yellow rectangle) is never paired with reward. After few pairings, presenting the CS+ elicits conditioned responses as compared to the CS−. These include higher subjective ratings of valence and arousal, increased skin conductance responses (SCRs), and increased BOLD responses in brain areas associated with conditioning and reward processing (Andreatta & Pauli, ; Tapia León, Kruse, Stalder, Stark, & Klucken, ).
Brain regions associated with appetitive conditioning mainly include the amygdala, nucleus accumbens (NAcc), dorsal and ventral anterior cingulate cortex (dACC/vACC), orbitofrontal cortex (OFC), and caudate nucleus (Chase, Kumar, Eickhoff, & Dombrovski, ; Klucken, Tabbert et al., ; Klucken, Wehrum et al., ; Martin‐Soelch, Linthicum, & Ernst, ). In the context of acquisition of appetitive conditioning, the amygdala is thought to encode the association of the CS with the unconditioned reaction, whereas the NAcc has been associated with subjective CS/UCS‐association and the reward prediction error (O'Doherty, Dayan, Friston, Critchley, & Dolan, ; Tapia León et al., ). Within the anterior cingulate cortex, the vACC is thought to mediate early discriminatory learning in contrast to the dACC, which is thought to encode outcome expectancy (Alexander & Brown, ). The OFC has been linked to the subjective value of the CS and was found to be a key region in reversal learning (Finger, Mitchell, Jones, & Blair, ). The caudate as part of the dorsal striatum has been associated with instrumental reward learning (O'Doherty et al., ).
In the context of extinction learning, central roles have been ascribed to the amygdala, NAcc, and vACC (or ventromedial prefrontal cortex, depending on the exact location), whereas the dACC is regarded a key region in the recall of conditioning (Konova & Goldstein, ). In our first study, we found an involvement of the vACC, caudate nucleus, and hippocampus during the early phase of extinction, whereas during the late phase of extinction, amygdala and NAcc were involved (Kruse et al., ). As mentioned before, the brain areas associated with extinction learning are also involved in the acquisition of appetitive conditioning. Animal studies using multi‐cell‐recordings have shown that although some neurons in amygdala and NAcc fire during anticipation of conditioned reward and cease to fire after extinction, other populations of neurons in these regions begin to fire with successful extinction learning (Janak, Chen, & Caulder, ; Tye, Cone, Schairer, & Janak, ).
The aim of the present study is to replicate and extend the previous results in a large, independent sample of all genders, as opposed to the smaller sample of male subjects in the previous study. During the early phase of extinction, we assumed increased activation of vACC and dACC to the CS+ as compared to the CS−. In the late phase, we assumed increased activation in NAcc and amygdala to the CS+ as compared to the CS−. In an extension of the previous results, we also tested for increased differential activation in the late phase as compared to differential activation during the early phase, utilizing the increased power of the greater sample.
## MATERIALS AND METHODS
### Subjects
The final sample included in the analyses consisted of 76 subjects (40 female, 36 male; age: M = 23.76, S.D. = 3.73). A total of 90 subjects took part, 14 subjects were excluded because (a) they could not correctly name the CS+ immediately before extinction in a free recall question which colored rectangle had been associated with a chance to win a reward on the previous day ( n = 6), (b) they expected rewards after the CS− as well as indicated in equally high UCS‐expectancy ratings immediately following the acquisition phase ( n = 1), (c) technical difficulties during data collection ( n = 2), (d) anomalies in the fMRI‐data indicated by more than 10% outlying volumes ( n = 2), and (e) reported familiarity with the paradigm after finishing the study ( n = 3). All subjects were right‐handed, German native speakers and had normal or corrected‐to‐normal vision. Past or current mental or neurological problems, consumption of psychotropic drugs, chronic illnesses or treatments and conditions preventing them from entering the MRI scanner were exclusion criteria. There was no overlap between this sample and the data reported in Kruse et al. ( ). All subjects gave written informed consent. The study was conducted in accordance with the Declaration of Helsinki (2008) and approved by the local ethics committee.
### Procedure
The appetitive conditioning paradigm took place on two consecutive days with acquisition on the first day and extinction on the second, roughly about 24 hr later. Data collection took place throughout the day between 8 a.m. and 7 p.m.
#### Acquisition training
We used an adapted version of the monetary incentive delay task (MID; Knutson, Westdorp, Kaiser, & Hommer, ) as a conditioning procedure in the MRI. The same paradigm has been used before and is described in detail in Kruse et al. ( ). In short, it consisted of 21 CS+ with partial reinforcement (62% reinforcement) and 21 CS− trials. Subjects were not instructed about the explicit CS‐UCS contingencies. The first trials of the CS+ and the CS− were later excluded from the analyses because learning could not have taken place yet (Kruse, Tapia León, Stalder, Stark, & Klucken, ). In each trial, a CS+ or CS− (blue or yellow rectangle) was presented for 6 s, followed by a fixation cross for a variable (1–3 s) interstimulus interval. Then, a target (white square) was presented for at least 16 ms up to a maximum of 750 ms. Subjects were instructed to press a reaction button every time the target was presented regardless of the CS presented before. Pressing the reaction button following a CS+ while the target was visible always resulted in a win of 0.50€ (UCS). Directly following the target, feedback was presented for 2 s. The presentation time of the target was adjusted according to individual reaction times to ensure a similar reinforcement for all subjects (aim: 6.50€ for wins in 62% of CS+ trials). Individual mean reaction times and standard deviations used to calculate presentation times (win: M + 2 x SD ; loss: M − 2 x SD ) were determined from a practice session with different stimuli. If subjects won unplanned or did not win in scheduled reinforcement trials, the target presentation time was corrected online (subtracting or adding 20 ms to the presentation time, respectively) to ensure reinforcement as planned in future trials. CS+ trials that did not result in wins as planned or vice versa were adaptively repeated in scheduled CS+ trials with the according duration of target presentation.
#### Extinction
On the day after acquisition training, the extinction was conducted in the MRI scanner. Subjects were asked to freely recall the reinforced stimulus (“Which colored rectangle was followed by the chance to win a reward?”), and the collected data were excluded from this analysis, if they failed. The extinction consisted of 20 CS+ and 20 CS− trials. As before, subjects were instructed to always press the button when they saw the target but in contrast to the acquisition phase, this never resulted in winning money, regardless of how fast subjects reacted.
### Subjective ratings
Subjects completed ratings of the CS+ and CS− on three scales: arousal, valence, and UCS‐expectancy. Rating collection took place before and after acquisition training as well as after extinction training. Nine‐point self‐assessment manikin scales were used for the affective ratings of arousal and valence (Bradley & Lang, ), whereas UCS‐expectancy was rated in 10% steps from 0 to 100%. Ratings were analyzed in 2 (CS: CS+, CS−) × 3 (time: pre‐acquisition, post‐acquisition, post‐extinction) × 2 (gender: male, female) analyses of variance (ANOVA) using SPSS 23. Significant interactions were followed up with paired t ‐tests, which were corrected for multiple comparisons using Bonferroni‐correction ( α = 0.05).
### Skin conductance measuring
Skin conductance was measured during the acquisition and extinction training with reusable Ag/AgCl electrodes with 13/8 mm outer/inner diameter filled with isotonic (0.05 M NaCl) electrolyte medium placed proximal and distal on the hypothenar eminence on the non‐dominant left hand. Data were collected with a sampling rate of 1 kHz. For preprocessing and data analysis, Ledalab 3.4.4 was used (Benedek & Kaernbach, ). First, the data was downsampled to 100 Hz and smoothed with a 32 sample FWHM Gaussian kernel. As each picture was presented for 6 s, the time window from 1 to 6 s was defined as analysis window (entire interval response; Pineles, Orr, & Orr, ). The extracted response was defined as the largest difference between a maximum and the minimum that directly preceded it. The preceding minimum had to be within the analysis window for the response to be counted. Responses smaller than 0.01 μS were considered zero responses. All maximum responses were log(μS + 1) transformed to correct for violation of normal distribution of the data.
Mean SCRs for CS+ and CS− were calculated subsequently. Skin conductance data were analyzed in separate 2 (CS: CS+, CS−) × 2 (time: early phase, late phase) × 2 (gender: male, female) ANOVAs for acquisition and extinction training. Significant interactions were followed up with paired t ‐tests and corrected for multiple comparisons, using Bonferroni‐correction ( α = 0.05).
### Functional magnetic resonance imaging
All MRI images were acquired using a 3 Tesla whole‐body tomograph (Siemens Prisma, Siemens Healthineers, Erlangen) with a 64‐channel head coil. The structural images consisted of 176 T1‐weighted sagittal slices (slice thickness 0.9 mm; FoV = 240 mm; TR = 1.58 s; TE = 2.3 s). For the functional images, a total of 440 images was acquired with a T2*‐weighted gradient echo‐planar imaging (EPI) with 36 slices covering the whole brain (voxel size = 3 × 3 × 3.5 mm; gap = 0.77 mm; descending slice acquisition; TR = 2 s; TE = 30 ms; flip angle = 75; FoV = 192 × 192 mm; matrix size = 64 × 64; GRAPPA = 2; phase encoding direction: anterior–posterior). The field of view was positioned automatically relative to the AC‐PC line with an orientation of −30°. Preprocessing, first and second level analysis were conducted using SPM 12 (Wellcome Department of Cognitive Neurology, 2014) implemented in Matlab (The MathWorks Inc., 2012).
For preprocessing, all EPI images were coregistered to an EPI template, realigned, and unwarped using field maps recorded directly before the EPI images, slice time corrected, normalized to MNI standard space via segmentation of the structural T1‐image coregistered to a T1‐template and smoothed with a Gaussian kernel at 6 mm FWHM. Functional data were analyzed for outlying volumes using a distribution free approach for skewed data (Schweckendiek et al., ). For this approach, after realignment each volume is compared with the preceding and following volume in order to calculate deviation scores. Deviation scores are compared against a threshold calculated based on all collected data, to identify outliers. If more than 10% of volumes of a time series were marked as outliers, the time series was discarded from analysis. Each resulting outlying volume was later modeled within the general linear model as a separate regressor of no interest.
For the acquisition phase, the CS+, CS−, UCS+ (win feedback following a CS+), NoUCS+ (no win feedback following a CS+), and UCS− (no win feedback following a CS−) were modeled as regressors of interest. Although the UCS+ models the feedback that money was won after the CS+, NoUCS+ and UCS− model the feedback that no money is won after CS+ or CS−, respectively. The first CS+ and the first CS− were modeled separately as learning could not have taken place at that time. For extinction training, the regressors were similar to acquisition, excluding the UCS+ because the CS+ was no longer reinforced and not modeling the first CS separately.
CS regressors were split into an early (CS+ /CS− ) and a late phase (CS+ /CS− ) to enable clear differentiation between early and late effects (Kruse et al., ; Kruse et al., ; LaBar, Gatenby, Gore, LeDoux, & Phelps, ). Events were modeled as stick functions and were convolved with the canonical hemodynamic response function. Six movement parameters were entered as covariates alongside regressors of no interest for the identified outlying volumes. The time series was then filtered with a high pass filter (time constant = 128 s). For acquisition training, a CS+ − CS− contrast was calculated, while for the extinction training separate CS+ − CS− and CS+ − CS− contrasts were calculated. In addition to these main analyses, analyses were extended to include a (CS+ − CS− ) − (CS+ − CS− ) contrast for the extinction phase.
On the group level, one‐sample t ‐tests were performed for the first‐level contrasts to examine neural differences in appetitive conditioning and extinction. Region of interest (ROI) analyses on the voxel level were conducted using the small volume correction in SPM12 with p < 0.05 (FWE). The coordinates of peak voxels of the first study on neural correlates of appetitive extinction (Kruse et al., ) were used as centers of 6 mm‐spheres, which were used as ROIs for small volume correction. In addition, whole brain analyses were performed with p < 0.05 (FWE), k > 10 voxel for the extinction training and, exploratively, for a two‐sample t ‐test comparing male to female subjects. As no whole brain results emerged for the test for gender differences, we do not further include this analysis in the Results section.
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.
## RESULTS
### Subjective ratings
ANOVA of subjective ratings of valence, arousal, and UCS‐expectancy (Table ) revealed main effects of CS, time, and a CS × time interaction (see Table for detailed statistics). There was neither a significant main effect of gender, nor any significant gender × CS, gender × time, nor gender × CS × time interactions.
Mean (SD) subjective ratings of CS+ and CS−
Main effects and interaction effects from 2 (CS: CS+, CS−) × 3 (time: pre‐acquisition, post‐acquisition, post‐extinction) × 2 (gender: female, male) ANOVA for subjective ratings of arousal, valence, and UCS‐expectancy with F ‐value, p ‐value. ** p < 0.01; *** p < 0.001
### Skin conductance responses
For acquisition training, ANOVA of SCRs revealed a significant main effect of CS ( F [1, 74] = 36.92; p < 0.001). For detailed statistics, see Table . The main effect of CS was driven by significantly higher responses to the CS+ as compared to the CS− for the early phase ( t [75] = 5.33; p < 0.001), as well as the late phase ( t [75] = 5.29; p < 0.001). Similarly, for the extinction phase, we found a significant main effect of CS ( F [1, 74] = 10.90; p = 0.001) and time ( F [1, 74] = 26.63; p < 0.001). However, the main effect of CS was driven by significantly higher SCRs to the CS+ as compared to the CS− in the early phase ( t [75] = 2.55; p = 0.013), but not in the late phase ( t [75] = 1.55; p = 0.126). Neither during acquisition nor extinction there were any main effects or interactions qualified by gender (all p > 0.13).
Main effects and interaction effects from 2 (CS: CS+, CS−) × 2 (time: early phase, late phase) × 2 (gender: female, male) ANOVA for skin conductance responses during acquisition and extinction training with F ‐value, p ‐value. ** p < 0.01; *** p < 0.001
### Hemodynamic responses
#### Acquisition
Analysis of BOLD‐responses during the acquisition phase shows increased responses to the CS+ as compared to the CS− throughout key areas associated with acquisition of appetitive conditioning (see Table ).
Region of interest (ROI) activations during acquisition (CS+ − CS−). Localization, cluster size ( k ), and statistics (FWE‐corrected) of the peak voxel in the respective ROI
#### Extinction
Analysis of the early phase of extinction revealed increased BOLD contrast in dACC and hippocampus (see Table ). A whole brain analysis further revealed a significant differential BOLD response (CS+ − CS−) in the right frontal operculum ( k = 16; x = 36; y = 16; z = 10; Z = 5.26; p = 0.007). Crucially, during the late phase, increased differential BOLD (CS+ − CS−) emerged in the right amygdala and the left NAcc (see Figure ). Taken together, during both early and late phase, results from the first study on neural correlates of appetitive extinction were replicated with the exception of vACC and caudate nucleus during the early phase. To extend the previous results, activation during the late phase was compared to activation during the early phase. This revealed increased differential BOLD contrast (CS+ − CS−) in the right amygdala ( k = 102; x = 18; y = 2; z = −20; Z = 3.40; p = 0.026) during the late phase as compared to the early phase. In an exploratory analysis, we looked for significant activations in the contrast CS− − CS+, but found no significant results in neither phase. There were no significant differences between genders.
Region of interest (ROI) activations during the early and late extinction phase (CS+ − CS−). Localization, cluster size ( k ), and statistics (FWE‐corrected) of the peak voxel in the respective ROI
(left) Time course of contrast estimates (CS+ − CS−) at the location of the significant peak voxels during acquisition (blue) and late extinction (green) in the left NAcc (above) and right amygdala (below). The line plots show the contrast estimates for the location of each of the four peak voxels (acquisition/extinction peak in NAcc & acquisition/extinction peak in amygdala) for the early and late phase of acquisition as well as the early and late extinction phases. At the location of acquisition‐peak voxels, there is significant differential BOLD‐contrast during acquisition but a marked reduction of differential BOLD‐contrast during extinction. At the location of extinction‐peak voxels, there is differential BOLD‐contrast similar to that at the acquisition peak voxels during acquisition but discernible patterns of activation during extinction. (right) Significant activation in NAcc (above) and amygdala (below) during late extinction
To further discern amygdala and NAcc activation during acquisition and extinction learning, we extracted contrast estimates at the locations of the peak voxels identified during acquisition and the late phase of extinction for both acquisition and extinction (Figure ). For illustrative purposes, we created a new model also separating early and late acquisition phase. At the location of peak voxels showing greatest differentiation during acquisition training, there is a marked reduction in responding during extinction training. However, while at the location of peak voxels later showing greatest differentiation during late extinction, the pattern of activation at these locations seems similar during acquisition, responding at these locations increases during late extinction. In addition, at the location of the late extinction peak voxel in the amygdala, there is markedly reduced BOLD‐contrast during the early phase of extinction.
## DISCUSSION
The aim of the study was to examine neural correlates of appetitive extinction in humans. To this date, only one study with a small male‐only sample investigated the neural correlates of appetitive extinction (Kruse et al., ). The present study replicates and extends previous results in several ways. Subjects of all genders were included, as opposed to an all‐male sample. This independent sample consists of 76 instead of 21 subjects, and data were collected throughout the day instead of data collection solely in the afternoon. This change in protocol ensures that there is no time of day effects due to circadian rhythms (e.g., cortisol levels). In addition, we looked at the time‐course of responding for amygdala and NAcc substructures activated during acquisition or extinction training and tested for increased differential activations during the late phase of extinction as compared to the early phase. As in the first study, subjects acquired appetitive conditioning on the first day and returned 1 day later for extinction training. We will shortly discuss the results of the acquisition phase before focusing on the extinction phase.
### Acquisition training
Appetitive acquisition resulted in increased subjective ratings of arousal and valence to the CS+ as compared to ratings obtained directly before acquisition training and as compared to the CS−. In addition, UCS expectancy increased towards the CS+ as compared to pre‐acquisition ratings and compared to CS−. SCRs to the CS+ were significantly increased as compared to the CS− throughout acquisition training. fMRI showed significant differential BOLD contrast (CS+ − CS−) in the amygdala, the dorsal and ventral striatum, midbrain, dorsal ACC, and the OFC. Taken together, the results are in line with previous research on acquisition of appetitive conditioning (Andreatta & Pauli, ; Klucken, Kruse et al., ; Kruse et al., ; Tobler, Fletcher, Bullmore, & Schultz, ). In line with our findings, Chase et al. ( ) showed in their meta‐analysis that involvement of the amygdala is specific to acquisition of classical conditioning in contrast to tasks focusing instrumental learning. The NAcc has mainly been associated with acquiring the subjective CS/UCS‐association and contingency awareness (Klucken, Schweckendiek et al., ; Tapia León et al., ).
### Appetitive extinction
Extinction of appetitive conditioning was indicated by significant decreases in subjective ratings of valence and arousal toward the CS+ from post‐acquisition to post‐extinction and as compared to the CS−. UCS‐expectancy towards the CS+ was also reduced significantly from post‐acquisition to post‐extinction. However, even post‐extinction, it remained slightly but significantly higher than UCS‐expectancy following the CS−. Regarding SCRs, there were significant main effects of CS and time during extinction training. Post hoc tests revealed that the main effect of CS was mainly driven by significantly higher SCRs to the CS+ during the early phase of extinction. There was no significant difference between SCRs to the CS+ as compared to the CS− during the late phase. In general, this is in line results from the first study. There is a trend toward higher UCS‐expectancy to the CS+ following extinction training was visible as well, despite overall successful extinction learning.
### Early phase of appetitive extinction
Early extinction training was expected to be characterized by recall of the acquired CS/UCS association from the day before. This is in line with significantly increased SCRs following the CS+ as compared to the CS−. Regarding neural correlates, we were able to show differential activation of the dACC. The dACC is assumed to encode outcome expectancy and seems to be a neural correlate of the retrieval of a consolidated acquisition memory trace. Similarly, Ebrahimi et al. ( ) reported dACC activation during appetitive extinction in an exploratory analysis. In addition, we were able to show differential activation of the hippocampus, which might be a correlate of extinction learning being context specific (Bouton, ). Differential activation of caudate nucleus or vACC, that were shown to be activated during early appetitive extinction in the first study, did not replicate. This might be the case for a variety of reasons. First, nonsignificance can obviously not be interpreted as the absence of an effect. Higher in‐group variance in the sample of the present sample might have decreased the power to detect these effects more than the increased sample size increased power. Second, the previous study applied a more strict protocol, only collecting data in the afternoon to have constant baseline cortisol levels for a secondary research question (Kruse et al., ). As this is known to affect emotional learning, it is possible, that variation in involvement of vACC and caudate nucleus during early extinction is moderated by the time of day, for example, due to variation in baseline cortisol levels (Merz, Stark, Vaitl, Tabbert, & Wolf, ). While established for fear conditioning, future research should assess these factors in appetitive conditioning.
### Late phase of appetitive extinction
The late phase of extinction training was expected to be characterized by successful extinction of appetitive conditioning. Differences in SCRs between CS+ and CS− were no longer significant in this phase and subjective ratings collected directly after indicated significantly reduced ratings of valence and arousal toward the CS+. Crucially, as expected, we found differential activation (CS+ − CS−) of amygdala and NAcc during the late phase of extinction. This replicates the main finding of our previous study and generalizes the assumed involvement of amygdala and NAcc to a sample consisting of male and female subjects. In line with these findings, another study, investigating the appetitive extinction of drug cues in (mainly male) cocaine users on the same day as acquisition, also found increased activation of the striatum and the amygdala during appetitive extinction (Konova et al., ). Extending the previous results, we analyzed for potentially higher differential activation during the late phase as compared to the early phase. We found increased differential activation of the right amygdala during the late phase. Notably, we compared the time course of contrast estimates at the location of the significant peak voxels from acquisition of conditioning to activation at the locations of peak voxels from extinction of conditioning. At the location of peak voxels of the acquisition phase, there was a marked decrease of activation throughout the extinction phase. This is in line with the expected reduction of the acquired conditioned responses. In contrast, at the location of peak voxels of the late extinction phase, there was a differentiation of acquisition and extinction signaling with a markedly increased response during the late phase of extinction. This pattern suggests discernible substructures in amygdala and NAcc which differentiate to signal extinction learning. This is in line with neuronal recordings in animal studies also showing a subpopulation of neurons specifically active after successful extinction learning (Janak et al., ; Tye et al., ). Interestingly, particularly in the amygdala, at the location of the peak voxel identified in late extinction, there also was a marked decrease during early extinction. This level even lies below the mean level of activation in the peak voxel of acquisition. This unexpected finding suggests that the amygdala plays a specific role in both early and late extinction which needs to be studied in more detail. These exploratory, descriptive results suggest that future studies should try to focus on activation patterns within these regions using pattern analyses like representational similarity analysis (RSA; Jin, Zelano, Gottfried, & Mohanty, ), which the present design did not permit because the employed ITI was too short (Visser et al., ). This might allow to capture how subnuclei within amygdala and NAcc work in parallel during acquisition but show discernible patterns during extinction.
### Limitations
Despite the higher sample size, the higher number of subjects does not allow for a complete assessment of possible boundary conditions like the effect of the time of day on variation regarding extinction learning. Nevertheless, we were able to show that several neural correlates of appetitive extinction were robust and can be replicated with a less strict protocol in a sample consisting of male and female subjects, therefore increasing generalizability to the population. In addition, although we did not find sex differences regarding appetitive extinction, we were not able to include exact assessments of the female hormonal cycle, which is assumed to affect emotional learning, and the sample also includes women taking hormonal contraceptives (Merz, Kinner, & Wolf, ).
### Conclusion
In the light of the recent replicability crisis especially regarding fMRI research, it seems particularly important to investigate robustness and replicability. We set out to replicate previously reported neural correlates of appetitive extinction in an independent sample, while extending our findings to a sample consisting of subjects of all genders. Replication of the main finding, an involvement of NAcc and amygdala in the late phase of extinction, was successful. The absence of significant differences in neural activations between genders underscores the generalizability of the results. In addition, for the first time, it was possible to show subregions of amygdala and NAcc displaying separate acquisition and extinction coding signals which follow a similar course during acquisition but differentiate during extinction. As extinction of appetitive conditioning is regarded as a model for the treatment of psychiatric disorders like addiction, this allows to translate animal models that have built on the involvement of these areas in extinction learning and form the basis for pharmacological research, to humans.
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Generalized social anxiety disorder (GSAD) is associated with heightened limbic and prefrontal activation to negative social cues conveying threat (e.g. fearful faces), but less is known about brain response to negative non-threatening social stimuli. The neuropeptide oxytocin (Oxt) has been shown to attenuate (and normalize) fear-related brain activation and reactivity to emotionally negative cues. Here, we examined the effects of intranasal Oxt on cortical activation to non-threatening sad faces in patients with GSAD and matched controls (Con). In a double-blind placebo-controlled within-subjects design, the cortical activation to sad and happy (vs. neutral) faces was examined using functional magnetic resonance imaging following acute intranasal administration of 24 IU Oxt and placebo. Relative to the Con group, GSAD patients exhibited heightened activity to sad faces in the medial prefrontal cortex (mPFC/BA 10) extending into anterior cingulate cortex (ACC/BA 32). Oxt significantly reduced this heightened activation in the mPFC/ACC regions to levels similar to that of controls. These findings suggest that GSAD is associated with cortical hyperactivity to non-threatening negative but not positive social cues and that Oxt attenuates this exaggerated cortical activity. The modulation of cortical activity by Oxt highlights a broader mechanistic role of this neuropeptide in modulating socially negative cues in GSAD. |
Centrally acting monoamines have long been thought to be associated with component traits of behavior and emotion and are potential biological mediators of psychopathology. In this study we tested the hypothesis that centrally acting monoamines would be associated with measures of affective instability (i.e. affective intensity and affective lability) in healthy and personality disordered human subjects. In total, 57 adult subjects including 19 psychiatrically healthy volunteers and 38 personality disordered individuals were assessed for affective instability with the affective intensity measure (AIM) and the Affective Lability Scale (ALS). Samples of cerebrospinal fluid (CSF) were collected for assay of 5-hydroxyindoleacitic acid (5-HIAA), homovanillic acid (HVA) and 3-methoxy-4-hydroxy-phenylglycol (MHPG). CSF 5-HIAA concentration correlated directly with overall AIM score and, specifically, with the AIM Negative Intensity score, in all subjects and in personality disordered subjects. This result was not affected but the addition of aggression scores or life history of mood disorder to the model. Neither CSF HVA nor MHPG were found to uniquely correlate with either AIM or ALS measure. Higher Affective Intensity scores, Negative Intensity scores, specifically, are directly correlated with higher basal levels of CSF 5-HIAA. This relationship was independent of aggression, life history of mood disorder and general personality traits. |
Response to drug treatment of major depression is variable and biomarkers of response are needed. Cyclic AMP response element binding protein (CREB) is considered a key mediator of antidepressant drug effect. We studied CREB in T-lymphocytes as a potential predictor of response to a selective serotonin reuptake inhibitor (SSRI) in 69 Korean depressed patients. We determined total CREB (tCREB), phosphorylated CREB (pCREB) and CRE-DNA binding using immunoblot and electrophoretic mobility shift assays, at baseline and after 6 wk treatment. Thirty-four healthy controls were also studied. The rate of response was 36 of 69 cases (52%). Baseline levels of tCREB and pCREB were lower in the total depressed group compared to controls (p = 0.044 and p<0.001, respectively). Baseline tCREB values in responders were significantly reduced in comparison to non-responders and to controls. After 6 wk treatment, median values of change of all CREB measures were greater in responders (36) than in non-responders (33; p<0.001 for tCREB, p = 0.003 for pCREB, and p=0.072 for CRE-DNA binding). Similar but less robust changes in CREB variables distinguished remitters from non-remitters. The optimum value of baseline tCREB predicted response with a positive predicted value of 0.778 [21/27; 95% confidence intervals (CI) 0.621-0.935], negative predictive value of 0.643 (27/42; 95% CI 0.498-0.788) and accuracy of 0.695 (48/69; 95% CI 0.586-0.804). Patients with low baseline tCREB had a significantly greater rate of response (78%) than patients with high baseline tCREB (36%), p < 0.001. Moreover, the greatest changes in tCREB with treatment were observed in subjects who did respond. This preliminary study suggests that T-lymphocytic CREB biomarkers are reduced in depressed patients and may assist in the prediction of response to SSRI drugs in depression. |
## Background
The hippocampus is a region consistently implicated in schizophrenia and has been advanced as a therapeutic target for positive, negative, and cognitive deficits associated with the disease. Recently, we reported that the paraventricular nucleus of the thalamus (PVT) works in concert with the ventral hippocampus to regulate dopamine system function; however, the PVT has yet to be investigated as target for the treatment of the disease. Given the dense expression of orexin receptors in the thalamus, we believe these to be a possible target for pharmacological regulation of PVT activity.
## Methods
Here we used the methylazoxymethanol acetate (MAM) rodent model, which displays pathological alterations consistent with schizophrenia to determine whether orexin receptor blockade can restore ventral tegmental area dopamine system function. We measured dopamine neuron population activity, using in vivo electrophysiology, following administration of the dual orexin antagonist, TCS 1102 (both intraperitoneal and intracranial into the PVT in MAM- and saline-treated rats), and orexin A and B peptides (intracranial into the PVT in naïve rats).
## Results
Aberrant dopamine system function in MAM-treated rats was normalized by the systemic administration of TCS 1102. To investigate the potential site of action, the orexin peptides A and B were administered directly into the PVT, where they significantly increased ventral tegmental area dopamine neuron population activity in control rats. In addition, the direct administration of TCS 1102 into the PVT reproduced the beneficial effects seen with the systemic administration in MAM-treated rats.
## Conclusion
Taken together, these data suggest the orexin system may represent a novel site of therapeutic intervention for psychosis via an action in the PVT.
Significance Statement
Available pharmacological therapies for the treatment of schizophrenia are not always effective and produce undesirable side effects. For these reasons, patients with schizophrenia often discontinue antipsychotic treatments; thus, better medications are needed to adequately treat this devastating psychiatric disorder. Here, we demonstrate that the orexin system, via an action in the paraventricular nucleus of the thalamus, may represent a novel therapeutic target for the treatment of psychosis.
## Introduction
The ventral hippocampus (vHipp) and paraventricular nucleus of the thalamus (PVT) have been demonstrated to work in concert to regulate the activity of the mesolimbic dopamine system via a polysynaptic circuit involving the nucleus accumbens (NAc), ventral pallidum (VP), and ventral tegmental area (VTA) ( ; ). Individuals with schizophrenia display baseline hyperactivity in select hippocampal subregions, which is correlated with symptom severity ( , ; ). Similarly, rodent models used to study this disorder also display elevated baseline activity in the vHipp, which is directly responsible for an increase in dopamine neuron population activity (defined as the number of spontaneously active dopamine neurons) in the VTA ( ; ; ; , ). The PVT, much like the vHipp, can regulate VTA dopamine neuron population activity, without affecting average firing rate or bursting, via an indirect pathway involving the NAc and VP (see ) ( ). Further, activity in the vHipp is required for PVT-induced increases in dopamine neuron population activity, with the converse also being true ( ). Studies have demonstrated prominent glutamatergic projections from the PVT to the NAc ( ; ; ; ). Indeed, convergent inputs from the vHipp and PVT onto medium spiny neurons of the NAc work in concert to regulate VTA dopamine activity (see ) ( ). In addition, the PVT can regulate dopamine system function by direct modulation of dopamine terminals within the NAc ( ). These data suggest that the PVT may be a novel site of intervention for the treatment of psychosis in schizophrenia. Indeed, pharmacological or chemogenetic inactivation of the PVT can restore normal dopamine system function in different rodent models used to study schizophrenia ( ).
Schematic representation of the polysynaptic circuit involved in the regulation of dopamine neuron population activity, which includes the ventral hippocampus (vHipp), paraventricular nucleus of the thalamus (PVT), nucleus accumbens (NAc), ventral pallidum (VP), and ventral tegmental area (VTA). Under “normal” conditions, GABAergic projections from the VP provide a tonic suppression of VTA activity (left). Methylazoxymethanol acetate-treated rats model schizophrenia, whereby aberrant glutamatergic inputs to the NAc from the vHipp drive a downstream hyperfunction of the dopamine system (center). Aberrant dopamine neuron activity can be reversed by blocking orexin 1 and 2 receptors (OX R) in the PVT, resulting in a decreased glutamatergic transmission to the NAc (right). Adapted from ( ).
A unique characteristic of the PVT is the dense and robust peptidergic innervation, including those by the orexin/hypocretin (ORX) peptides, which are of particular importance to this study ( ). PVT neurons are dose dependently excited by orexin peptides A (OXA) and B (OXB) ( ). OXA and OXB are ligands of the G-protein–coupled receptors, orexin 1 and orexin 2, and display differential affinities such that OX R has a higher affinity for OXA (EC 30 nM) than OXB (EC 250 nM), whereas OX R is equally sensitive to OXA (EC 34 nM) and OXB (EC 60 nM). Activation of these receptors excites target neurons through various second messenger systems ( ) and can directly ( ) and indirectly ( ) modulate VTA dopamine system function. Additionally, orexin peptides produce dose-dependent increases in the firing rates of PVT neurons ( ), and these neurons can regulate dopamine levels in the NAc ( ). Dysfunction of the dopamine system has long been associated with positive symptoms of schizophrenia (i.e., psychosis) ( ; ; ; ). Taken together, we posit that targeting ORX receptors in the PVT can reverse the dysfunction present in the mesolimbic dopamine system commonly observed in preclinical models of the disease and, potentially, in individuals with schizophrenia. Here, we used a dual orexin receptor antagonist, TCS 1102, to evaluate whether this approach can normalize aberrant VTA dopamine neuron population activity in rats treated with methylazoxymethanol acetate (MAM), a gestational disruption that models some of the pathological alterations of schizophrenia ( ; ). This compound was specifically chosen because it blocks both OX R and OX R and has been shown to effectively block orexin-mediated behaviors for up to 4 hours ( ; ). Moreover, similar compounds are already approved by the FDA for the treatment of sleep disorders. Here, we provide evidence that targeting the ORX system may be a novel approach for the treatment of psychosis in schizophrenia.
## Materials and Methods
All experiments were performed in accordance with the guidelines outlined in the USPH Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of UT Health San Antonio.
### Animals
All experiments were performed on multiple litters of adult (>12 weeks) male MAM- and saline-treated rats, as previously described ( ; ). In brief, timed-pregnant female Sprague-Dawley rats were obtained from Envigo RMS Inc. (Indianapolis, IN) on gestational day (GD) 16. MAM (diluted in saline, 22 mg/kg i.p.) was administered on GD17, while control rats received injections of saline (1 mL/kg, i.p.). Pups were weaned on post-natal day 21 and housed with littermates in groups of 2–3 rats per cage. A separate set of experiments was performed on untreated adult male Sprague-Dawley rats obtained from Envigo.
### Drug Administration
For systemic drug administration, TCS 1102 (10 mg/kg; ; Ki values were 3.0 and 0.2 nM for OXR1 and OXR2, respectively) or vehicle (60% dimethyl sulfoxide in saline; 1 mL/kg) was administered i.p. (MAM- or saline-treated rats) 15 minutes prior to any electrophysiological recordings. Although the half-life of TCS 1102 is relatively short (approximately 20 minutes), behavioral responses are observed for up to 4 hours following administration ( ), which is consistent with the average time to complete VTA recordings for a single animal (2–3 hours). For unilateral intracranial drug administration, a 26-gauge guide cannula (Plastics One, Roanoke County, VA) was lowered into/immediately adjacent to the PVT (A/P −2.0 mm, M/L +0.4 mm from bregma and D/V −5.8 mm ventral of the brain surface) to avoid damage to the relatively small structure as previously reported ( ). An internal cannula (Plastics One), extending 1 mm past the end of the guide cannula, was used to deliver a 1-time injection of vehicle, peptides (OXA or OXB: 3 µg/750 nL; untreated SD rats), or TCS 1102 (1.5 µg/0.5 µL; MAM- or saline-treated rats) at a rate of approximately 0.5 μL/min at 10 minutes prior to any electrophysiological recording.
### Extracellular Dopamine Neuron Recordings
Male rats (approximately 300–450 g) were anesthetized with 8% chloral hydrate (400 mg/kg, i.p.), as this anesthetic does not significantly depress dopamine neuron activity ( ). Anesthesia was maintained by supplemental administration of chloral hydrate as required to maintain suppression of limb compression withdrawal reflex. Rats were positioned in a stereotaxic apparatus (Kopf, Tujunga, CA), and a core body temperature of 37°C was maintained by a thermostatically controlled heating pad (Kent Scientific, Torrington, CT). Extracellular glass microelectrodes (impedance: 6–14 MΩ) were lowered into the VTA (A/P ± 5.3 mm, M/L ± 0.6 mm from bregma and D/V −6.5 to −9.0 mm ventral of the brain surface) using a hydraulic micro-positioner (Kopf, Model 640) to measure dopamine neuron activity. Spontaneously active dopamine neurons were recorded for a period of 2–3 minutes and identified with open filter settings (low pass: 30 Hz; high pass: 30 kHz) using previously established electrophysiological criteria ( ). We measured 3 parameters of dopamine neuron activity: (1) population activity (defined as the number of spontaneously active dopamine neurons encountered while making 6–9 tracks or dorsal/ventral vertical passes), separated by 200 μm in a predetermined pattern to sample equivalent regions of the VTA); (2) basal firing rate; and (3) the proportion of action potentials occurring in bursts. At the cessation of all electrophysiological recordings, rats were rapidly decapitated.
### Analysis
Electrophysiological analysis of dopamine neuron activity was performed using commercially available computer software (LabChart version 7.1; ADInstruments Ltd., Chalgrove, Oxfordshire, UK) and plotted with Prism software (GraphPad Software Inc., San Diego, CA). Electrophysiological data was analyzed by 1-way ANOVA, 2-way ANOVA (strain × treatment) with post hoc comparisons performed using the Holm-Sidak method, or Kruskal-Wallis 1-way ANOVA on ranks with post hoc comparisons performed using Dunn’s method. Data are represented as the mean ± SEM unless otherwise stated, with n values representing the number of rats per group or neurons recorded per experimental group where indicated. Significance was determined at P < .05. All statistics were calculated using SigmaPlot (Systat Software, Chicago, IL).
### Materials
MAM was purchased from Midwest Research Institute (Kansas City, MO). TCS 1102 was purchased from Tocris (Cat. No. 3818; Minneapolis, MN). Orexin B was purchased from R &D systems (Catalog # 1457; Minneapolis, MN), and Orexin A was sourced from Cayman Chemicals (Item No. 15073; Ann Arbor, MI). Chloral hydrate was sourced from Sigma-Aldrich (St. Louis, MO). All other chemicals and reagents were of either analytical or laboratory grade and purchased from standard suppliers.
## Results
### TCS 1102, a Dual Orexin Antagonist, Reverses Aberrant VTA Dopamine Neuron Population Activity
We used TCS 1102 to examine whether blockade of orexin signaling could modulate VTA dopamine neuron activity in MAM-treated rats ( ). Similar to previous reports, SD control rats (n = 7 rats) displayed a population activity of 1.02 ± 0.04 cells per track, and MAM-treated rats (n = 8 rats) displayed a significantly higher population activity (1.77 ± 0.10 cells per track; 2-way ANOVA; F = 22.38; P < .001; F = 15.52; P < .001; F = 18.73; P < .001; Holm-Sidak post hoc; t = 6.79; P < .001). The i.p. administration of TCS 1102 had no effect on saline-treated rats (n = 6 rats; 1.05 ± 0.07 cells per track); however, it restored normal VTA dopamine system function in MAM-treated rats (n = 6 rats; 1.08 ± 0.08 cells per track; Holm-Sidak post hoc; t = 5.93; P < .001). Consistent with previous reports, saline-treated vehicle rats displayed an average firing rate of 3.97 ± 0.24 Hz (n = 45 neurons; ). The average firing rate of MAM-treated rats also treated with TCS 1102 (n = 38 neurons; 3.24 ± 0.31 Hz) displayed a significant decrease compared with control MAM-treated rats (n = 83 neurons; 4.52 ± 0.23 Hz; 2-way ANOVA; F = 8.53; P = .004; Holm-Sidak post hoc; t = 3.19; P = .002) and saline-treated rats administered TCS 1102 (n = 37 neurons; 4.46 ± 0.40 Hz; Holm-Sidak post hoc; t = 2.59; P = .01). No significant differences were observed in the average percent bursting between any of the groups ( ; saline-vehicle: n = 45 neurons; 36.12 ± 3.93% bursting; MAM-vehicle: n = 83 neurons; 38.97 ± 3.08% bursting; saline-TCS 1102: n = 37 neurons; 44.40 ± 4.77% bursting; MAM-TCS 1102: n = 38 neurons; 36.15 ± 4.56% bursting).
Administration of the dual orexin receptor antagonist, TCS 1102, restores normal dopamine system function in the MAM rodent model of schizophrenia (A). MAM-treated vehicle rats display a significant increase in ventral tegmental area (VTA) dopamine neuron activity compared with saline-treated vehicle rats. * P < .001. Further, TCS 1102 administration restores normal dopamine system function in MAM-treated rats. P = .01 denotes significance from MAM-treated vehicle rats. The average firing rate of VTA dopamine neurons is attenuated by TCS 1102 administration (B). * P = .002 denotes significance from MAM-treated vehicle rats. P = .01 denotes significance from saline-treated rats administered TCS 1102. No differences were observed in the average percent bursting between any groups (C). Representative dopamine recording and action potential from a saline-treated vehicle (D), MAM-treated vehicle (E), saline-treated TCS 1102 (F), and MAM-treated TCS 1102 (G) rats.
### Orexin Peptides A and B in the PVT Increase VTA Dopamine Neuron Population Activity
To determine whether orexin modulation of the PVT regulates VTA dopamine neurons, we unilaterally injected exogenous OXA and OXB peptides into the PVT ( ). Consistent with previous reports in Sprague-Dawley control rats, rats injected with vehicle displayed an average population activity of 1.01 ± 0.05 cells per track (n = 8 rats). Intra-PVT administration of the exogenous peptides, OXA (n = 5 rats; 1.95 ± 0.15 cells per track) or OXB (n = 6 rats; 1.92 ± 0.07 cells per track), caused a significant increase in population activity compared with control rats (1-way ANOVA; F = 42.69; P < .001; Holm-Sidak post hoc; OXA: t = 7.69; P < .001; OXB: t = 7.81; P < .001). No significant differences were observed in the average firing rate ( ; Kruskal-Wallis 1-way ANOVA on ranks; control: n = 56 neurons; 4.22 ± 0.27 Hz; OXA: n = 60 neurons; 4.45 ± 0.24 Hz; OXB: n = 65 neurons; 4.85 ± 0.31 Hz) or average percent bursting ( ; Kruskal-Wallis 1-way ANOVA on ranks; control: n = 56 neurons; 30.03 ± 3.27% bursting; OXA: n = 60 neurons; 32.26 ± 3.43% bursting; OXB: n = 65 neurons; 34.19 ± 3.23% bursting) between any of the groups.
Intracranial microinjection of exogenous orexin A (OXA) or orexin B (OXB) peptides into the paraventricular nucleus of the thalamus (PVT) causes a significant increase in ventral tegmental area (VTA) dopamine neuron activity (A). * P < .001 denotes significance from vehicle control. No differences were observed in the average firing rates (C) or average percent bursting between any groups (D). B depicts a representative brain slice (left) with a canula track indicated by an arrow in the PVT and corresponding schematic of the brain section (right). Representative dopamine recording and action potential from a control saline (E), OXA (F), and OXA (G) microinjected rats.
### TCS 1102 in the PVT Restores VTA Dopamine Neuron Population Activity
To determine whether orexin receptor blockade in the PVT could reverse aberrant VTA dopamine neuron population activity in MAM-treated rats, we unilaterally injected TCS 1102 or vehicle into the PVT of MAM- or saline-treated rats ( ). Consistent with previous reports in Sprague-Dawley control rats, saline-treated rats injected with vehicle displayed an average population activity of 1.01 ± 0.05 cells per track (n = 8 rats), and MAM-treated rats injected with vehicle displayed a significant increase in population activity (n = 6 rats; 1.67 ± 0.06 cells per track) compared with saline-treated controls (2-way ANOVA; F = 13.79; P = .001; F = 12.74; P = .002; F = 22.18; P < .001; Holm-Sidak post hoc; t = 6.49; P < .001). Intra-PVT administration of TCS 1102 caused a significant decrease in population activity in MAM-treated rats (n = 5 rats; 1.02 ± 0.06 cells per track) compared with MAM-treated control rats (Holm-Sidak post hoc; t = 5.69; P < .001). No changes in population activity were observed in saline-treated rats injected with TCS 1102 in the PVT (n = 5 rats; 1.10 ± 0.15 cells per track). Vehicle MAM-treated rats (n = 60 neurons; 5.27 ± 0.36 Hz; 49.27 ± 3.57% burst firing) displayed an elevated firing rate ( ) and bursting pattern ( ) compared with saline-treated vehicle control rats (n = 56 neurons; 4.22 ± 0.27 Hz; 2-way ANOVA; Holm-Sidak post hoc; t = 2.50; P = .01; 30.03 ± 3.27% burst firing; t = 3.90; P < .001) and MAM-treated rats injected with TCS 1102 in the PVT (n = 33 neurons; 3.94 ± 0.29 Hz; 2-way ANOVA; Holm-Sidak post hoc; t = 2.73; P = .007; 31.37 ± 4.54% burst firing; t = 3.11; P < .001). TCS 1102 injected in the PVT had no effect on saline-treated rats (n = 29 neurons; 3.65 ± 0.39 Hz; 36.11 ± 5.31% burst firing).
Intracranial microinjection of the dual orexin antagonist, TCS 1102, into the paraventricular nucleus of the thalamus (PVT), restored normal dopamine system function in MAM-treated rats (A). * P < .001 denotes significance from saline-treated vehicle. P < .001 denotes significance from MAM-treated vehicle. TCS 1102 administration attenuated the average firing rate (B) and average percent bursting (C) in MAM-treated vehicle rats. * P < .05 denotes significance from saline-treated vehicle. P < .05 denotes significance from MAM-treated vehicle. Representative dopamine recording and action potential from a saline-treated TCS 1102 (D) and MAM-treated TCS 1102 (E) rats.
## Discussion
The thalamus serves as a major point of convergence for various neuronal circuits and is composed of several nuclei, each with distinct afferent and efferent projections ( ; ). Moreover, thalamic abnormalities have been previously implicated in schizophrenia ( ). Specifically, a decrease in the thalamic volume and reduced gray matter has been reported in schizophrenia patients compared with healthy controls ( ; ; ; ; ; ). Furthermore, positron emission tomography studies performed in individuals with schizophrenia report abnormal activation of the thalamus during active auditory hallucinations ( ) and decreased thalamic blood flow ( ; ). However, the exact role of discrete thalamic nuclei in schizophrenia remains unclear, and it is not currently known whether the structural and functional alterations observed in this disease are a consequence of, or contribute to, the symptoms of schizophrenia ( ; ; ).
Of importance to this study is the PVT and its innervation of the NAc ( ; ; ; ). The PVT sends direct and indirect projections to dopamine neurons within the VTA ( ; ) and synapse on both medium spiny neurons and dopamine terminals within the NAc ( ; ). Thus, in addition to direct modulation of NAc neurons, the PVT can regulate presynaptic dopamine release ( ; ). Indeed, it has been reported that glutamate release from PVT terminals can act on ionotropic glutamate receptors to induce dopamine efflux in the NAc ( ). Thus, a decrease in PVT activity, by orexin receptor blockade, may decrease dopamine signaling multiple mechanisms, including (1) a direct effect on presynaptic release, and (2) an indirect effect on dopamine neuron activity in the VTA. We recently demonstrated that pharmacological (N-methyl-D-aspartate) activation of the PVT induces an increase in VTA dopamine neuron population activity without affecting the firing rate or bursting pattern of these neurons ( ), as those measures are altered by manipulations to other brain regions ( ; ). Indeed, this is consistent with previous studies examining the regulation of VTA dopamine neurons by the vHipp, in which activation of the vHipp produced selective increases in dopamine neuron population activity ( ). Interestingly, this PVT-induced increase in population activity is dependent on glutamatergic projections to the NAc, as chemogenetic activation of the PVT-NAc pathway produces a similar increase in dopamine neuron population activity ( ). Further, inactivation of the PVT was able to reverse aberrant dopamine neuron activity thought to contribute to psychosis in schizophrenia ( ).
The PVT receives a dense innervation from the ORX system ( ) and can directly (via direct innervation of dopaminergic neurons of the VTA ( ) or indirectly (via projections to the NAc ( ) modulate dopamine system function. Thus, we used the MAM rodent model, which displays neurophysiological and behavioral deficits consistent with schizophrenia to examine whether targeting the ORX system could reverse the mesolimbic dopamine system dysfunction, commonly observed in individuals with schizophrenia. As mentioned previously, numerous rodent models used to study the neurobiology of schizophrenia exhibit a significant increase in dopamine neuron population activity ( ; ; ; ; , , ). We observed a similar increase in MAM-treated vehicle rats, as they displayed a significant increase in VTA dopamine neuron population activity compared with saline-treated vehicle rats. Importantly, TCS 1102 administration was able to restore normal dopamine system function in MAM-treated rats, demonstrating that the ORX system may be a therapeutic target ( ). It should be noted that MAM-treated rats treated with TCS 1102 displayed a slight decrease in the average firing rate of VTA dopamine neurons compared with MAM-treated vehicle rats and saline-treated rats administered TCS 1102 ( ). Although we did observe this significant difference, it is likely that only subpopulations of dopamine neurons are affected. The ORX system has been demonstrated to facilitate dopamine neuron activity and fluctuations in ORX levels and can influence the excitability of VTA dopamine activity ( ). Thus, direct effects of the ORX antagonist on dopamine neurons are possible; however, the dramatic effects on population activity are likely attributable to indirect regulation.
Orexin peptides play a role in wakefulness and have been demonstrated to increase arousal and locomotor activity ( ; ). Although some antipsychotics have sedative effects on patients, this is often related to the dose and this sedative effect is not thought to contribute to the mechanism of action ( ; ). Indeed, other compounds that affect arousal, such as benzodiazepines, are not effective monotherapies ( ); therefore, it is likely that the results here are indicative of changes in the regulation of dopamine system function rather than nonselective changes in arousal. Further, it should be noted that the PVT has been shown to be activated after stressful or aversive events ( ; ). Interestingly, stress is also a known risk factor for a number of psychiatric conditions, including schizophrenia, suggesting that the PVT might be a potential site of convergence for stress-related psychiatric disorders ( ; ). To determine whether ORX system modulation could increase VTA dopamine neuron population activity via the PVT, we performed direct microinjections of the OXA or OXB peptides and recorded VTA dopamine neuron activity ( ). We evaluated the efficacy of OXA and OXB in modulating VTA dopamine neuron activity, as they differentially target OX R (binds OXA) and OX R (binds OXA and OXB), both of which are similarly expressed in the PVT ( ). Studies have reported activation of the dopamine system by orexins ( ) and that these peptides produce excitatory effects in the PVT ( ). Interestingly, infusion of either OXA or OXB into the PVT produced a significant increase in dopamine neuron population activity ( ), similar to what has been reported in rodent models used to study schizophrenia ( ; , , ). This increase was not observed in rodents that received vehicle infusions in the PVT, consistent with previous observations in control rats ( ; , , ). The ORX peptides were microinjected into or immediately adjacent to the PVT ( ) to avoid damage to the relatively small structure; thus, it is important to note that it is possible that other structures adjacent to the PVT may have been affected. Specifically, diffusion of the ORX peptides into the medial dorsal nucleus of the thalamus, immediately adjacent to the PVT, was likely; however, given that the density of ORX receptors in this region are very low, we do not believe this region to be modulated by OXR peptide infusion ( ). These data support previous chemogenetic studies targeting the PVT-NAc pathway ( ) and suggest that orexins can modulate mesolimbic dopamine neuron population activity via the PVT.
Lastly, to demonstrate that the effects of TCS 1102 on VTA dopamine neuron activity were indeed mediated by antagonizing orexin receptors, specifically in the PVT, we microinjected TCS 1102 into the PVT of MAM- and saline-treated rats and recorded the activity of VTA dopamine neurons. The significant increase in dopamine neuron population activity consistently observed MAM-treated rats was indeed reversed by local orexin receptor blockade in the PVT. This is consistent with a previous study that used chemogenetics to activate the PVT-NAc or the PVT-mPFC pathway and found that PVT-NAc activation was able to increase VTA dopamine neuron activity ( ). Thus, we posit that the effect observed in the current study is indeed specific to an action in the PVT. Further, it is likely that the OXR modulation occurs through an indirect pathway ( ) involving the PVT and the NAc-VP-VTA polysynaptic circuit. These data validate the action of OXR in modulating and restoring dopamine system function in a model with relevance to schizophrenia.
Collectively, these data suggest that targeting the ORX system could be beneficial in restoring normal dopamine system function and eliminating symptoms of psychosis linked to dysfunction of that system. Further, data collected in this study support the idea that the FDA-approved dual orexin receptor antagonist, Suvorexant, may also treat symptoms of psychosis in individuals with schizophrenia. Suvorexant, at high doses, is effective at treating insomnia ( ); however, lower, nonsedative doses could be effective at treating psychotic symptoms. Future studies will be aimed at observing the consequences of ORX modulation on behaviors associated with positive, negative, and cognitive symptom domains displayed by rodent models of schizophrenia. Indeed, activation of orexin neurons has been previously linked to improvements in working memory ( ; ; ) as well as symptoms often comorbid with schizophrenia, such as anxiety ( ; ), depression ( ), and sleep disorders ( ) (for orexin system review, see ). Further studies will explore the relative contribution of OX R and OX R separately, using an antagonist specific for each receptor. Therefore, this study provides evidence that the PVT is a potential site of intervention in schizophrenia and that targeting the ORX system may be a novel therapeutic approach for the treatment of psychosis.
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## Background
Conflicts are inherent to all groups and organizations, and most conflicts are resolved through mediation or negotiation. In conflict negotiation, predicting the preference information of opponents is of great significance to solve conflict problems and reduce negotiation costs. The key condition is that one obtains the preferences of other decision makers from cognitive psychology in order to take the initiative in conflict negotiation with multiple decision makers (DMS). In other words, the most important thing in conflict is to identify the intentions and preferences of other discourse markers from the perspective of psychological cognition. Mastering the preference ranking of opponents may lead to some results of conflict, which can help decision makers calmly face the tension in the process of conflict and predict the next strategy more accurately. For decision makers, how to adjust their emotions in the negotiation is particularly important.
## Research objects and methods
Under the framework of conflict resolution graph model (GMCR) of cognitive psychology, a method to obtain DM preference in multiple DM conflicts is constructed. Through reverse thinking, this method establishes three mathematical models: Nash, generalized sub rationality and sequence stability. These mathematical models can be used to obtain the minimum constraints of DM with unknown preferences. Achieving balance in conflict requires minimal constraints. This method allows other decision makers to obtain the preference ranking of their opponents on the premise that the conflict results are known. In turn, these preference rankings can balance known results. This study also used the questionnaire method to investigate the emotional micro behavior of each group in the negotiation process. This scale is used to measure the relationship between three independent variables: self accommodation, accommodation of others and the feeling of the degree to which others accommodate themselves. It includes 20 statements, with responses ranging from “almost always” (score 1) to “almost none” (score 5), with a total score between 20 (lowest accommodation) and 100 (highest accommodation).
## Results
The method was applied to the conflict analysis of water pollution in Lanzhou. There are three reasons for this conflict: Lanzhou Veolia Water Company, Sinopec Lanzhou branch and the local government. Firstly, the GMCR model of the above conflict is established. Then, the preferences of Lanzhou Veolia Water Company and Sinopec Lanzhou branch are analyzed. Finally, using the above mathematical model, they can obtain the preference ranking of their opponents - local governments, which makes them invincible in conflict negotiations. In addition, the theoretical results are consistent with the actual conflict situation. At the same time, the feasibility and effectiveness of this method are verified. The results showed that the scores of the four dimensions of emotion regulation in the first two groups were less than 2 points, and the difference was not statistically significant (P > 0.05). After 8 weeks of emotion regulation intervention, the average scores of initiative, negotiation psychological mastery, tension evaluation and conflict attitude in the intervention group were significantly higher than those in the control group (P < 0.01).
## Conclusion
The main contribution of this paper is to establish a mathematical model, which can be used to obtain the preference ranking of DM for ideal equilibrium. When obtaining the preference ranking of the main decision-makers, the mediator can guide the strategy choice of each decision-maker in the conflict and control the final result of the conflict. The results of this study provide a new and valuable perspective for conflict negotiation of multi discourse markers from the perspective of psychological cognition. This work can be extended to relative preferences or partially known preferences, because some DM preference information may be partially obtained. To sum up, through the comparative study of decision-makers' emotional behavior, this study found that emotional regulation can improve decision-makers' emotions in the negotiation process, not only solve conflicts more calmly, but also enable decision-makers to face difficulties and setbacks rationally, which is worthy of promotion.
## Acknowledgements
Supported by projects grant from Jiangsu Normal University (Grant No.19XSRX001), Philosophy and Social Science Research in Colleges and Universities in Jiangsu Province (Grant No.2019SJA0922), and Humanities and Social Sciences Fund Planning Project of the Ministry of Education (Grant No. 18YJA630128).
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Abstract
Objective : Clozapine is a potent antipsychotics agent commonly prescribed for patients with refractory schizophrenia. However, it is also related to many troublesome adverse effects. For example, the common genitourinary adverse effects such as enuresis and urinary incontinence. Although clozapine is known for high anticholinergic activity, there has been only one case report about clozapine-related urinary retention in the literature. The aim of this study is to report a case of with clozapine-induced urinary retention and to discuss potential mechanisms.
Methods : Case report.
Results: We report a 19-year-old male patient of refractory schizophrenia who developed acute urinary retention during treatment with clozapine 200 mg/day and haloperidol 10 mg/day. Urodynamic study suggested dysfunctional voiding. After a series of work-up, simplification of medications and dose adjustment, the urinary retention seemed to be resulted from clozapine. The total score of Adverse Drug Reaction Probability Scale is 7. The urinary retention didn’t respond to bethanechol (a cholinergic agent) and tamsulosin (a selective α1 receptor antagonist), and it resolved completely after discontinuation of clozapine while haloperidol 10 mg/day was kept.
Discussion and conclusions : Anticholinergic effect of clozapine has been suspected to contribute to impaired detrusor muscle contraction and therefore urinary retention, however, the urodynamic study in the case reported showed normal detrusor function during filling and voiding. Treatment with cholinergic agent didn’t improve urinary retention as well. This case report highlights that urinary retention can be an uncommon adverse effect of clozapine and may not be merely resulted from anticholinergic effect.
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Abstract
Objective : To explore tolerability, safety and treatment response of flexibly-dosed paliperidone palmitate (PP) in adult patients hospitalized with an exacerbation of schizophrenia.
Methods: International 6-week prospective open-label non-interventional study. Outcome parameters were changes in Brief Psychiatric Rating Scale (BPRS) total score, Clinical Global Impression-Severity Scale (CGI-S), Personal and Social Performance Scale (PSP), treatment satisfaction (Medication Satisfaction Questionnaire (MSQ)), Extrapyramidal Symptom Rating Scale (ESRS) scores and treatment-emergent adverse events (TEAEs) from baseline to last-observation-carried-forward endpoint.
Results: 367 patients (65.9% male, mean age (±SD) 39.8 ± 12.1 years, 85.8% paranoid schizophrenia) were documented. 91.6% of patients completed the 6-week observation period. Most frequent reasons for early discontinuation were loss to follow-up (2.7%) and withdrawal of consent (1.6%). The recommended PP initiation regimen (150 mg eq on day 1 and 100 mg eq on day 8, both in the deltoid muscle) was used in 88.8% of subjects. Mean time from hospital admission to initiation of PP was 9.4 ± 7.7 days. Mean baseline BPRS total score of 50.2 ± 13.6 improved by -6.5 ± 8.6 at day 8 and by -19.3 ± 12.6 from baseline to endpoint; 95% confidence interval [CI]-20.7;-18.0; both p<0.0001. At endpoint, 93.6% of patients were rated as improved in CGI-S. Functioning in PSP significantly improved from 49.4 ± 14.7 at baseline by 14.3 ± 12.4 at endpoint (95% CI 12.9;15.8, p<0.0001). Mean ESRS total score significantly decreased from 3.7 ± 5.9 at baseline to 2.0 ± 4.7 at endpoint (p<0.0001). 6.0% of patients were very or extremely satisfied with their previous antipsychotic medication at baseline, which increased to 46.1% with PP at endpoint. TEAEs reported in ≥2% of patients were tremor (2.5%) and schizophrenia (2.2%).
Conclusions: These data support results from previous randomized controlled and pragmatic studies that flexibly dosed paliperidone palmitate is well tolerated and associated with an early and clinically meaningful treatment response and functional improvement in patients hospitalized for an exacerbation of schizophrenia.
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Title: Ketamine and the pursuit of rapid-acting antidepressants for the treatment of depression and PTSD symptoms.
Abstract
The limitations of standard antidepressant treatments are well known, they work for too few patients and their beneficial effects emerge too slowly. APPROACH: The purpose of this presentation is to briefly describe the context leading to the discovery of the antidepressant effects of ketamine in humans, to characterize our current understanding of the profile of ketamine’s efficacy and ways that it is entering routine clinical treatment, to consider the management of risks associated with ketamine treatment, and to consider emerging alternatives to ketamine. Emerging studies also point to ketamine effectiveness in PTSD. CONCLUSIONS: The rapidity (onset within 24 hours) and magnitude (50%-75% clinical response rate, even in treatment-resistant depression populations) of the efficacy of ketamine suggest that it could be a new and important role in the treatment of mood disorders where clinical response is needed urgently or where there has been inadequate response to other treatments.
Disclosure: Dr. Krystal is a co-proponent of a use patent related to the intranasal administration of ketamine for the treatment of depression that has been licensed by Johnson and Johnson.
References
1. Berman RM, Cappiello A, Anand A, Oren DA, Heninger GR, Charney DS, Krystal JH. Antidepressant effects of ketamine in depressed patients. Biol Psychiatry. 2000 Feb 15;47(4):351–4. PubMed PMID: 10686270.
2. Duman RS, Aghajanian GK, Sanacora G, Krystal JH. Synaptic plasticity and depression: new insights from stress and rapid-acting antidepressants. Nat Med. 2016 Mar 3;22(3):238–49. doi: 10.1038/nm.4050. PubMed PMID: 26937618.
3. Krystal JH, Sanacora G, Duman RS. Rapid-acting glutamatergic antidepressants: the path to ketamine and beyond. Biol Psychiatry. 2013 Jun 15;73(12):1133–41. doi: 10.1016/j.biopsych.2013.03.026. Review. PubMed PMID: 23726151; PubMed Central PMCID: PMC3671489.
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Abstract
Objectives: Suicide attempters have impaired decision making and are at high risk of reattempt. Therefore it is important to refer them to psychiatric treatment. Especially, People with medical comorbidity are at higher risk of suicidal attempt and mortality. The aim of this study was to investigate the characteristics of suicidal attempters and to analyze the influence of the medical comorbidity on decision to receive psychiatric treatment after visit to an emergency department.
Methods: One hundred and thirty two patients, who visited the emergency room of a general hospital in Gyeonggi-do between January, 2012 and December, 2012 were enrolled as the subjects of this study. After reviewing each subject's medical records retrospectively, demographic and clinical factors were analyzed.
Results: Regardless of the engagement type, either via admission or outpatient clinic, the determinant factors of psychiatric treatment engagement were psychiatric diagnosis, employment status, previous psychiatric treatment history, and previous attempt history. Comparison of severity of medical comorbidity (Charlson Comorbidity Index) showed that suicide attempters who received psychiatric treatment via admission or refused the treatment tended to have higher level of medical comorbidity than who received psychiatric treatment via outpatient department.
Conclusions: Our findings showed that medical comorbidity of suicide attempters affected the decision to accept psychiatric treatment. All psychiatrists should evaluate the presence and the severity of medical comorbidity of the suicide attempters and consider implementing more intervention for the medically ill attempters who are willing to discharge against advice.
Key Words: Suicide attempter, Emergency room, Psychiatric consultation, Medical comorbidity
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Abstract
Background: Seizure threshold (ST) varies in patients that undergo electroconvulsive therapy (ECT). It is therefore necessary to titrate for optimal energy levels at the beginning of each treatment session. Titration for ST is crucial for achieving the best treatment results while minimizing the side effects. Most patients undergoing ECT take concomitant psychotropic medication, but little information is available on how those drugs affect ST.
Objective/hypothesis: To analyze the relationship between ST and psychotropic drugs in patients treated with ECT.
Methods: We examined clinical data from 43 patients receiving ECT. ST was titrated at each treatment session, and ECT was performed using a MECTA® device. We examined associations between ST and psychotropic drugs using multivariate correlation analyses. Data are presented as initial ST, the difference in ST between the first and 10th sessions (ΔST ), and the mean difference in ST between the first and last sessions (mean ΔST ).
Results: Multivariate regression analyses showed associations between initial ST and the total chlorpromazine-equivalent dose of antipsychotics (β = 0.363, p < 0.05). The total fluoxetine-equivalent dose of antidepressants was associated with ΔST (β = 0.486, p < 0.01) and mean ΔST (β = 0.472, p < 0.01).
Conclusions: Our study elucidated possible effects of psychotropic drugs on ST in patients undergoing ECT. We found that larger doses of antipsychotics are associated with higher initial ST, whereas higher doses of antidepressants are associated with shifts of ST during the course of treatment. Our findings provide a basis for creating safer and more efficient ECT protocols.
Keywords : Electroconvulsive therapy, seizure threshold, antipsychotics, antidepressants
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## Background
Increased pain sensitivity is observed following alcohol withdrawal, and attempts to alleviate this hyperalgesia can contribute to the cycle of addiction. The aim of this study was to determine if alcohol withdrawal-induced hyperalgesia was observed in a chronic ethanol exposure model and if this pain was affected by histone deacetylase inhibitors, thus revealing an epigenetic mechanism.
## Methods
Adult male Sprague Dawley rats received Lieber-DeCarli liquid control or ethanol (9% v/v) diet for 15 days. Mechanical sensitivity was measured with von Frey hair stimulation of the hindpaw during ethanol administration and 24- and 72-hour withdrawal.
## Results
Ethanol withdrawal produced severe and sustained mechanical hyperalgesia, an effect not observed in the control or ethanol-maintained groups. Furthermore, this hyperalgesia was attenuated by the histone deacetylase inhibitor, suberoylanilide hydroxamic acid treatment.
## Conclusions
Heightened pain sensitivity was observed following withdrawal from chronic ethanol exposure, and histone deacetylase inhibitors could be novel treatments for this alcohol withdrawal-induced hyperalgesia.
## Introduction
There is a complex relationship between alcohol and pain processing. A recent meta-analysis confirmed that acute alcohol is analgesic and that this effect is dose dependent ( ). However, an intoxicating blood alcohol content of approximately 0.08% (3–4 drinks) was required before an analgesic response was observed. In contrast, chronic alcohol use can result in peripheral neuropathy ( ), which may be due to the neurotoxic effects of alcohol and its metabolites ( ). Furthermore, withdrawal from chronic alcohol can also result in heightened pain sensitivity, which is resolved after several weeks to months of abstinence ( ). This alcohol withdrawal-induced hyperalgesia was also shown to correlate with negative emotional state ( ), and attempts to alleviate this heightened pain state could contribute to the cycle of addiction ( ; ). Furthermore, there are a number of overlapping circuits that are altered during drug dependence and chronic pain ( ), and adaptations within these shared pathways could mediate pain hypersensitivity following alcohol exposure and withdrawal ( ).
Chronic alcohol exposure can induce neuroplasticity that facilitates the development and maintenance of alcohol use disorders ( ). Epigenetic modifications are changes that occur at the structural level of DNA that alter the accessibility of the gene to the transcriptional machinery but do not alter the genetic code itself ( ). Epigenetic modulation of gene expression has emerged as a key regulator of the alcohol withdrawal state ( ). Alterations in the epigenome through DNA methylation or histone modifications are consistently observed following chronic alcohol exposure ( ; ). For example, withdrawal from chronic alcohol has been shown to result in increased histone deacetylase (HDAC) activity in the amygdala and heightened anxiety-like behaviors, which are attenuated by the HDAC inhibitor trichostatin A ( ). Furthermore, deficits in brain-derived neurotrophic factor expression and dendritic spine density in amygdaloid circuits in response to chronic alcohol and withdrawal are also attenuated by trichostatin A ( ; ), which is mechanistically consistent with the role of this brain region in regulation of negative affect ( ). Pain represents another behavioral phenotype of negative affect, which develops during withdrawal ( ; ; ; ; ) and can play a critical role in maintaining addictive behaviors ( ; ). To the best of our knowledge, the effects of HDAC inhibitor treatment on ethanol withdrawal-induced hyperalgesia has not been investigated.
The aim of this study was to develop an alcohol withdrawal-induced hyperalgesia animal model using the Lieber-DeCarli ethanol diet drinking paradigm to mimic human alcoholics. An additional goal was to further examine if this hyperalgesia was affected by treatment with the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA).
## Materials and Methods
### Chronic Ethanol Exposure Paradigm
All experiments were approved by the Institutional Animal Care and Use Committee of University of Illinois at Chicago and adhered to the NIH Guidelines for the Care and Use of Laboratory Animals. Animals were housed in a temperature-controlled room with a 12-hour-light/-dark cycle, with lights on at 7:00 and off at 7:00 and food and water were provided ad libitum. Adult male Sprague-Dawley rats (postnatal days 75–87 at start of treatment) were initially group housed and then separated during alcohol or control diet feeding. As described previously ( ), rats were offered 80 mL/d of the nutritionally complete Lieber-DeCarli liquid control diet (Lieber-DeCarli Diet 82; Bio-Serv, Frenchtown, NJ) for 3 days. Control groups continued with the control diet for 16 days, while ethanol groups were gradually introduced to ethanol (1.8% through 8.1% within 7 days) and maintained on 9% v/v ethanol diet for 15 days. Rats were pair-fed and liquid diet intake and body weights were closely monitored. Fresh diet was provided daily between 17 and 18 hours before the beginning of the dark cycle. One group of ethanol-diet fed rats was withdrawn for 24 to 72 hours (withdrawal group) and given the control diet. Another group of ethanol-diet fed rats was maintained on the ethanol diet during this time. Blood alcohol levels were measured using an Analox Alcohol Analyzer (Analox Instruments, Lunenberg, MA).
### Assessment of Sensory Sensitivity
Rats were habituated to the testing rack for 2 days for 20 minutes each prior to the first testing day. For all behavioral experiments, rats were counterbalanced into groups following the first test for mechanical sensitivity. The experimenter was blinded to the feeding paradigm and the drug condition tested. All rats were tested in a separate behavior room with low light (approximately 35–50 lux) and low noise, between 9 and 12 hours, roughly 15 to 18 hours after the placement of diet from the day before and 5 to 8 hours before the placement of fresh diet for that day. For hindpaw sensitivity, the threshold for responses to punctate mechanical stimuli (mechanical hyperalgesia) was tested according to the up-and-down method ( ) in the left hindpaw. The plantar surface of the hindpaw was stimulated with a series of 8 von Frey hair filaments (0.4–15 g). A response was defined as lifting, shaking, or licking of the paw on stimulation. The first filament tested was 4 g. In the absence of a response, a heavier filament (up) was tried, and in the presence of a response, a lighter filament (down) was tested. This pattern was followed for a maximum of 4 filaments following the first response ( ).
### Experimental Outline
For all experiments rats were tested the day before start of the ethanol or control diet (naïve), on the first day of ethanol exposure (titration), on days 1 and 8, 14 or 15 of 9% ethanol, and 24 and 72 hours withdrawal ( ).
Withdrawal from chronic ethanol exposure produces hyperalgesia. (A) Male rats were tested when naïve and during ethanol or control diet feeding, and mechanical responses to von Frey hair stimulation remained stable regardless of group or time. (B) Ethanol withdrawal produced a severe and sustained mechanical hyperalgesia compared with control and ethanol-maintained groups. *** P < .001 compared with the control group (2-way repeated-measures ANOVA, P < .001 for group; n = 8/group). Alcohol withdrawal increases pain sensitivity.
For the SAHA experiment, rats were injected daily with SAHA or vehicle at 24 hours withdrawal until 72 hours withdrawal. On test days, basal responses were determined before the treatment, and posttreatment responses were determined 2 hours post-drug administration. SAHA (Vorinostat, Selleck Chemicals, Houston, TX) solution was prepared by dissolving 62.5 mg SAHA in 0.2 mL dimethylsulfoxide, 4 mL of PEG300, 0.5 mL propylene glycol, 0.1 mL Tween‐80, and 5.2 mL normal saline were added to the solution sequentially and vortexed before each addition. The final concentration of SAHA was 6.25 mg/mL, 2% dimethylsulfoxide, 40% PEG300, 5% propylene glycol, and 1% Tween‐80 in saline. Control vehicle solution was the same solvents in the same concentrations in saline. The dose of SAHA was 50 mg/kg i.p.
### Statistical Analysis
Data are expressed as mean + SEM. All rats tested were included in the analysis. All statistical analyses were performed by SigmaStat, and graphs were generated using GraphPad Prism. Basal responses over time were analyzed using 2-way repeated-measures (RM) ANOVA with time and treatment as factors. For withdrawal responses, data were analyzed using 2-way RM ANOVA with Holm-Sidak post-hoc analysis.
## Results
### Alcohol Withdrawal Produces Severe Hyperalgesia
The mean ± SEM ethanol intake did not differ between ethanol and withdrawal groups (14.43 ± 0.3 and 14.75 ± 0.23 g/kg/d, respectively). The blood ethanol level in ethanol diet-maintained rats was 183 ± 22 mg%. These are similar levels to our previous publications ( ). In addition, there were no significant differences in the body weights (mean ± SEM) among the various groups (control, 305 ± 4.5 g; ethanol, 306 ± 5.6 g; withdrawal, 295 ± 3.9 g).
Mechanical responses were determined using von Frey hair stimulation of the plantar surface of the hindpaw. Naïve mechanical responses were taken when rats were still group housed and on the standard laboratory chow diet. In addition, measurements were taken during ethanol titration, and on days 1, 8, and 15 of 9% ethanol exposure. Ethanol diet did not significantly alter mechanical response to von Frey hair stimulation relative to liquid diet controls ( ). Data were analyzed using a 2-way RM ANOVA with group and time as factors. Further, there was no difference in responses in any group relative to naïve baselines; thus these results also show that singly housing the rats and switching to liquid diet did not significantly affect nociceptive responding. In contrast, ethanol withdrawal resulted in a significant decrease in mechanical thresholds, indicative of hyperalgesia, and this effect was observed at both 24 and 72 hours of withdrawal ( ). Data were analyzed using a 2-way RM ANOVA with group and time as factors, with a significant effect of group (F (2,21) = 12.357, P < .001).
### SAHA Treatment Attenuates Alcohol Withdrawal-Induced Hyperalgesia
To determine the effect of HDAC inhibitors on hyperalgesia induced by alcohol withdrawal, we tested the pan-HDAC inhibitor SAHA in a separate group of animals. The mean ± SEM ethanol intake between the ethanol, withdrawal-vehicle, and withdrawal-SAHA groups did not significantly differ from each other (ethanol = 11.89 ± 0.24; withdrawal-vehicle = 12.46 ± 0.73; withdrawal-SAHA = 11 ± 0.29 g/kg/d). As above, control or ethanol liquid diet did not affect baseline mechanical responses ( ). For 24 and 72 hours withdrawal, baseline and post-drug responses were analyzed using 2-way RM ANOVA with Holm-Sidak post-hoc analysis. Twenty-four hours after withdrawal, the withdrawn rats were significantly different from the ethanol control group [ ,F (2,21) = 3.917, P = .036]. Animals were then injected with vehicle or SAHA (50 mg/kg i.p.) and tested 2 hours later ( , post-drug). At this time point, a comparison between control-VEH/SAHA and withdrawal-VEH/SAHA groups revealed a significant group difference [F(3, 28) = 4.063, P = .016]. Rats were given a second injection of VEH/SAHA at 48 hours withdrawal, but they were not tested on this day. However, rats were tested prior to the 3rd injection at 72 hours withdrawal ( , baseline). After 2 injections, SAHA significantly inhibited hyperalgesia ( , baseline), an effect that was maintained after the third and final SAHA injection [ , post-drug; control-VEH/SAHA vs withdrawal-VEH/SAHA group effect F(3,27) = 6.769, P = .002]. These data clearly suggest that SAHA is an effective treatment for preventing development of pain during ethanol withdrawal after chronic ethanol exposure in rats.
Alcohol withdrawal-induced hyperalgesia is blocked by suberoylanilide hydroxamic acid (SAHA). (A) Mechanical responses remained stable throughout ethanol or control treatment. During the withdrawal period, control diet and withdrawal groups were injected daily with SAHA (50 mg/kg i.p.) or vehicle following ethanol withdrawal, while the ethanol-maintained group was concurrently injected with vehicle. (B) At 24-hour withdrawal, there was a significant difference between withdrawn animals and ethanol ( P < .05) and control groups ( P < .05). (C) Repeated injection of SAHA significantly reduced this hyperalgesia by 72-hour withdrawal; n = 8/group, ** P < .01, *** P < .001 compared with the control-VEH group (2-way repeated-measures ANOVA with Holm Sidak post-hoc analysis).
## Discussion
In this study we found that withdrawal from chronic ethanol exposure resulted in severe mechanical hyperalgesia, an effect that was maintained for at least 72 hours post-withdrawal. We also observed that this hyperalgesia was prevented by treatment with an HDAC inhibitor, suggesting that this heightened pain sensitivity is regulated epigenetically.
Increased pain sensitivity has been observed in alcoholics undergoing alcohol withdrawal or abstinence ( ). In addition, several studies have observed alcohol withdrawal-induced hyperalgesia in different animal models of alcohol exposure. Withdrawal following chronic intermittent exposure to ethanol vapor was shown by 2 separate groups to induce increased pain sensitivity during withdrawal periods ( ; ). Additionally, repeated cycles of exposure and withdrawal from the Lieber-DeCarli diet was also found to increase mechanical hyperalgesia ( ). In mice, withdrawal from voluntary alcohol consumption ( ; ), or forced exposure through oral gavage ( ) can also reliably produce a robust hyperalgesic response. In our study, rats were gradually introduced to ethanol over 7 days and then maintained at 9% for 15 days. Withdrawal from this paradigm resulted in severe mechanical hyperalgesia that was maintained for at least 72 hours post-withdrawal. Increased pain sensitivity during withdrawal could be a factor to the “dark side” of addiction, and a study in patients confirmed that alcohol withdrawal-induced hyperalgesia correlated with increased depression scores ( ).
In the paradigm used in this study, rats maintained mechanical responses similar to controls during ethanol exposure, and hyperalgesia was only observed in the withdrawal state. However, a previous study using the Lieber-DeCarli diet showed that chronic alcohol alone could result in painful peripheral neuropathy ( ). However in this latter study, rats were maintained on a liquid diet for 12 weeks, and significant hyperalgesia was only observed 4 weeks into the diet. Interestingly, in this model, females showed a similar time course to develop hyperalgesia but had increased pain sensitivity relative to males ( ), an effect that was estrogen dependent. In an ethanol vapor exposure model, decreased pain thresholds were observed only after 8 weeks of alcohol exposure ( ). Furthermore, peripheral neuropathy has been demonstrated in chronic alcoholics ( ). In our study, rats were maintained at 9.0% ethanol for only 15 days, and perhaps a longer duration of exposure would result in alcohol-induced hyperalgesia or neuropathy.
Epigenetic modifications mediated by acetylation or methylation of histones and DNA methylation have been identified as important factors regulating chronic alcohol use and withdrawal ( ; Berkel and ; ). In our study, alcohol withdrawal-induced hyperalgesia was blocked by the pan-HDAC inhibitor SAHA. We have previously observed that alcohol withdrawal also results in increased anxiety-related behaviors ( ; ) and that these effects are blocked by HDAC inhibition ( ; ). Furthermore, withdrawal from chronic alcohol is associated with decreased expression of BDNF, Arc, and NPY in the central nucleus of amygdala, and this process is correspondingly reversed by HDAC inhibition ( ; ). Alcohol withdrawal-induced hyperalgesia could be the result of similar changes in brain circuitry regulating pain processing and may be related to increased HDAC activity, changes in histone acetylation or methylation, and subsequent neuroplasticity. A recent study demonstrated that alcohol withdrawal-induced hyperalgesia could be regulated by disruption to circuits projecting from the central amygdala to the periaqueductal grey ( ). In addition, chronic alcohol exposure and withdrawal could also be due to alterations occurring in the dorsal root ganglia or lumbar spinal cord. Future studies will focus on identifying the epigenetic mechanisms that mediate ethanol withdrawal-induced hyperalgesia and where these changes occur. In addition, SAHA is a pan-HDAC inhibitor and can affect acetylation of both histones and other targets, such as tubulin ( ), which will also be explored in future studies.
Alcohol use disorder is a chronic relapsing condition characterized by cycles of alcohol use and withdrawal. A major characteristic of alcohol dependence is the need to consume alcohol not just for its rewarding effects but also to alleviate negative states associated with withdrawal ( ). The alcohol withdrawal-induced hyperalgesia we observed reflects the increased pain sensitivity observed in patients undergoing withdrawal ( ). HDAC inhibitors have been proposed as therapeutic targets for the treatment of emotional dysregulation associated with withdrawal ( ). Our behavioral and pharmacological findings indicate that hyperalgesia could contribute to the cycle of addiction and that HDAC inhibitors may also be effective at managing this component of the “dark side” of addiction.
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It had previously been shown that influences from two cortical areas, the anterior ectosylvian sulcus (AES) and the rostral lateral suprasylvian sulcus (rLS), play critical roles in rendering superior colliculus (SC) neurons capable of synthesizing their cross-modal inputs. The present studies examined the consequences of selectively eliminating these cortical influences on SC-mediated orientation responses to cross-modal stimuli. Cats were trained to orient to a low-intensity modality-specific cue (visual) in the presence or absence of a neutral cue from another modality (auditory). The visual target could appear at various locations within 45 degrees of the midline, and the stimulus effectiveness was varied to yield an average of correct orientation responses of approximately 45%. Response enhancement and depression were observed when the auditory cue was coupled with the target stimulus: A substantially enhanced probability in correct responses was evident when the cross-modal stimuli were spatially coincident, and a substantially decreased response probability was obtained when the stimuli were spatially disparate. Cryogenic blockade of either AES or rLS disrupted these behavioral effects, thereby eliminating the enhanced performance in response to spatially coincident cross-modal cues and degrading the depressed performance in response to spatially disparate cross-modal cues. These disruptive effects on targets contralateral to the deactivated cortex were restricted to multisensory interactive processes. Orientation to modality-specific targets was unchanged. Furthermore, the pattern of orientation errors was unaffected by cortical deactivation. These data bear striking similarities to the effects of AES and rLS deactivation on multisensory integration at the level of individual SC neurons. Presumably, eliminating the critical influences from AES or rLS cortex disrupts SC multisensory synthesis that, in turn, disables SC-mediated multisensory orientation behaviors. |
On the assumption that linguistic faculties reflect both lexical storage in the temporal cortex and combinatorial rules in the striatal circuits, several authors have shown that striatal-damaged patients are impaired with conjugation rules while retaining lexical knowledge of irregular verbs [Teichmann, M., Dupoux, E., Kouider, S., Brugières, P., Boissé, M. F., Baudic, S., Cesaro, P., Peschanski, M., & Bachoud-Lévi, A. C. (2005). The role of the striatum in rule application. The model of Huntington's disease at early stage. Brain, 128, 1155-1167; Ullman, M. T., Corkin, S., Coppola, M., Hickok, G., Growdon, J. H., Koroshetz, W. J., & Pinker, S. (1997). A neural dissociation within language: Evidence that the mental dictionary is part of declarative memory, and that grammatical rules are processed by the procedural system. Journal of Cognitive Neuroscience, 9, 266-276]. Yet, such impairment was documented only with explicit conjugation tasks in the production domain. Little is known about whether it generalizes to other language modalities such as perception and whether it refers to implicit language processing or rather to intentional rule operations through executive functions. We investigated these issues by assessing perceptive processing of conjugated verb forms in a model of striatal dysfunction, namely, in Huntington's Disease (HD) at early stages. Rule application and lexical processes were evaluated in an explicit task (acceptability judgments on verb and nonword forms) and in an implicit task (lexical decision on frequency-manipulated verb forms). HD patients were also assessed in executive functions, and striatal atrophy was evaluated with magnetic resonance imaging (bicaudate ratio). Results from both tasks showed that HD patients were selectively impaired for rule application but lexical abilities were spared. Bicaudate ratios correlated with rule scores on both tasks, whereas executive parameters only correlated with scores on the explicit task. We argue that the striatum has a core function in linguistic rule application generalizing to perceptive aspects of morphological operations and pertaining to implicit language processes. In addition, we suggest that the striatum may enclose computational circuits that underpin explicit manipulation of regularities. |
Adults seem to have greater difficulties than children in acquiring a second language (L2) because of the alleged "window of opportunity" around puberty. Postpuberty Japanese participants learned a new English rule with simplex sentences during one month of instruction, and then they were tested on "uninstructed complex sentences" as well as "instructed simplex sentences." The behavioral data show that they can acquire more knowledge than is instructed, suggesting the interweaving of nature (universal principles of grammar, UG) and nurture (instruction) in L2 acquisition. The comparison in the "uninstructed complex sentences" between post-instruction and pre-instruction using functional magnetic resonance imaging reveals a significant activation in Broca's area. Thus, this study provides new insight into Broca's area, where nature and nurture cooperate to produce L2 learners' rich linguistic knowledge. It also shows neural plasticity of adult L2 acquisition, arguing against a critical period hypothesis, at least in the domain of UG. |
The melodic contour of speech forms an important perceptual aspect of tonal and nontonal languages and an important limiting factor on the intelligibility of speech heard through a cochlear implant. Previous work exploring the neural correlates of speech comprehension identified a left-dominant pathway in the temporal lobes supporting the extraction of an intelligible linguistic message, whereas the right anterior temporal lobe showed an overall preference for signals clearly conveying dynamic pitch information [Johnsrude, I. S., Penhune, V. B., & Zatorre, R. J. Functional specificity in the right human auditory cortex for perceiving pitch direction. Brain, 123, 155-163, 2000; Scott, S. K., Blank, C. C., Rosen, S., & Wise, R. J. Identification of a pathway for intelligible speech in the left temporal lobe. Brain, 123, 2400-2406, 2000]. The current study combined modulations of overall intelligibility (through vocoding and spectral inversion) with a manipulation of pitch contour (normal vs. falling) to investigate the processing of spoken sentences in functional MRI. Our overall findings replicate and extend those of Scott et al. [Scott, S. K., Blank, C. C., Rosen, S., & Wise, R. J. Identification of a pathway for intelligible speech in the left temporal lobe. Brain, 123, 2400-2406, 2000], where greater sentence intelligibility was predominately associated with increased activity in the left STS, and the greatest response to normal sentence melody was found in right superior temporal gyrus. These data suggest a spatial distinction between brain areas associated with intelligibility and those involved in the processing of dynamic pitch information in speech. By including a set of complexity-matched unintelligible conditions created by spectral inversion, this is additionally the first study reporting a fully factorial exploration of spectrotemporal complexity and spectral inversion as they relate to the neural processing of speech intelligibility. Perhaps surprisingly, there was little evidence for an interaction between the two factors-we discuss the implications for the processing of sound and speech in the dorsolateral temporal lobes. |
Cognitive theories on reading propose that the characteristics of written stimuli determine how they are processed in the brain. However, whether the brain distinguishes between regular words, irregular words, and pseudowords already at an early stage of the reading process is still subject to debate. Here we used chronometric TMS to address this issue. During the first 140 msec of regular word, irregular word, and pseudoword reading, TMS was used to disrupt the function of the ventral occipitotemporal, posterior middle temporal, and supramarginal gyri, which are key areas involved in orthographic, semantic, and phonological processing, respectively. Early TMS stimulation delivered on posterior middle temporal and supramarginal gyri affected regular and irregular word, but not pseudoword, reading. In contrast, ventral occipitotemporal disruption affected both word and pseudoword reading. We thus found evidence for an early distinction between word and pseudoword processing in the semantic and phonological systems, but not in the orthographic system. |
Change blindness-the failure to detect changes in visual scenes-has often been interpreted as a result of impoverished visual information encoding or as a failure to compare the prechange and postchange scene. In the present electroencephalography study, we investigated whether semantic features of prechange and postchange information are processed unconsciously, even when observers are unaware that a change has occurred. We presented scenes composed of natural objects in which one object changed from one presentation to the next. Object changes were either semantically related (e.g., rail car changed to rail) or unrelated (e.g., rail car changed to sausage). Observers were first asked to detect whether any change had occurred and then to judge the semantic relation of the two objects involved in the change. We found a semantic mismatch ERP effect, that is, a more negative-going ERP for semantically unrelated compared to related changes, originating from a cortical network including the left middle temporal gyrus and occipital cortex and resembling the N400 effect, albeit at longer latencies. Importantly, this semantic mismatch effect persisted even when observers were unaware of the change and the semantic relationship of prechange and postchange object. This finding implies that change blindness does not preclude the encoding of the prechange and postchange objects' identities and possibly even the comparison of their semantic content. Thus, change blindness cannot be interpreted as resulting from impoverished or volatile visual representations or as a failure to process the prechange and postchange object. Instead, change detection appears to be limited at a later, postperceptual stage. |
Social attention when viewing natural social (compared with nonsocial) images has functional consequences on contextual memory in healthy human adults. In addition to attention affecting memory performance, strong evidence suggests that memory, in turn, affects attentional orienting. Here, we ask whether the effects of social processing on memory alter subsequent memory-guided attention orienting and corresponding anticipatory dynamics of 8-12 Hz alpha-band oscillations as measured with EEG. Eighteen young adults searched for targets in scenes that contained either social or nonsocial distracters and their memory precision tested. Subsequently, RT was measured as participants oriented to targets appearing in those scenes at either valid (previously learned) locations or invalid (different) locations. Memory precision was poorer for target locations in social scenes. In addition, distractor type moderated the validity effect during memory-guided attentional orienting, with a larger cost in RT when targets appeared at invalid (different) locations within scenes with social distractors. The poorer memory performance was also marked by reduced anticipatory dynamics of spatially lateralized 8-12 Hz alpha-band oscillations for scenes with social distractors. The functional consequences of a social attention bias therefore extend from memory to memory-guided attention orienting, a bidirectional chain that may further reinforce attentional biases. |
In natural vision, processing of spatial and nonspatial features occurs simultaneously; however, the two types of attention in charge of facilitating this processing have distinct mechanisms. Here, we tested the independence of spatial and feature-based attention at different stages of visual processing by examining color-based attentional selection while spatial attention was focused or divided. Human observers attended to one or two of four fields of randomly moving dots presented in both left and right visual hemifields. In the focused attention condition, the target stimulus was defined both by color and location, whereas in the divided attention condition stimuli of the target color had to be attended in both hemifields. Sustained attentional selection was measured by means of steady-state visual evoked potentials elicited by each of the frequency-tagged flickering dot fields. Additionally, target and distractor selection was assessed with ERPs to these stimuli. We found that spatial and color-based attention independently modulated the amplitude of steady-state visual evoked potentials, confirming independent top-down influences on early visual areas. In contrast, P3 amplitudes elicited only by targets and distractors of the attended color were subject to space-based enhancement, suggesting increasing integration of spatial and feature-based selection over the course of perceptual processing. |
The human visual system can only process a fraction of the information present in a typical visual scene, and selection is historically framed as the outcome of bottom-up and top-down control processes. In this study, we evaluated how a third factor, an individual's selection history, interacts with top-down control mechanisms during visual search. Participants in our task were assigned to one of two groups in which they developed a history of either shape or color selection in one task, while searching for a shape singleton in a second task. A voluntary task selection procedure allowed participants to choose which task they would perform on each trial, thereby maximizing their top-down preparation. We recorded EEG throughout and extracted lateralized ERP components that index target selection (N<sub>T</sub>) and distractor suppression (P<sub>D</sub>). Our results showed that selection history continued to guide attention during visual search, even when top-down control mechanisms were maximized with voluntary task selection. For participants with a history of color selection, the N<sub>T</sub> component elicited by a shape target was attenuated when accompanied by a color distractor, and the distractor itself elicited a larger P<sub>D</sub> component. In addition, task-switching results revealed that participants in the color group had larger, asymmetric switch costs implying increased competition between task sets. Our results support the notion that selection history is a significant factor in attention guidance, orienting the visual system reflexively to objects that contradict an individual's current goals-even when these goals are intrinsically selected and prepared ahead of time. |
The orienting response evoked by the appearance of a salient stimulus is modulated by arousal; however, neural underpinnings for the interplay between orienting and arousal are not well understood. The superior colliculus (SC), causally involved in multiple components of the orienting response including gaze and attention shifts, receives not only multisensory and cognitive inputs but also arousal-regulated inputs from various cortical and subcortical structures. To investigate the impact of moment-by-moment fluctuations in arousal on orienting saccade responses, we used microstimulation of the monkey SC to trigger saccade responses, and we used pupil size and velocity to index the level of arousal at stimulation onset because these measures correlate with changes in brain states and locus coeruleus activity. Saccades induced by SC microstimulation correlated with prestimulation pupil velocity, with higher pupil velocities on trials without evoked saccades than with evoked saccades. In contrast, prestimulation absolute pupil size did not correlate with saccade behavior. Moreover, pupil velocity correlated with evoked saccade latency and metrics. Together, our results demonstrated that small fluctuations in arousal, indexed by pupil velocity, can modulate the saccade response evoked by SC microstimulation in awake behaving monkeys. |
We examined the changes of two transcription factors, CREB and c-Jun, in dorsal root ganglia (DRG) after acute (8 or 48 hours) or chronic (10 days) cyclophosphamide (CYP)-induced cystitis. Results showed an increase in the number of p-CREB-immunoreactive (-IR) cells in the L1 and L2 DRG (5-7-fold; P < or = 0.05) as well as L6 and S1 DRG (2-4-fold; P < or = 0.05) after acute and chronic cystitis. The number of p-CREB-IR cells in the L4-L5 DRG was not altered with cystitis. The number of c-Jun-IR cells increased in the L1-L2 DRG (L1: 10-fold; L2: 8-fold; P < or = 0.05) only with chronic cystitis, although it increased in the L6-S1 DRG with CYP-induced cystitis of acute (2-3-fold; P < or = 0.05) and chronic (6-10-fold; P < or = 0.05) duration. After CYP treatment, the percentage of bladder afferent cells expressing p-CREB immunoreactivity (3-7-fold; P < or = 0.05) increased in L1, L2, L6, and S1 DRG. The increase occurred 8 hours post-CYP injection and was maintained with chronic cystitis. There were few c-Jun-IR cells in the bladder afferent population. These results demonstrate that CYP induces p-CREB and c-Jun expression in DRG in a time-dependent manner. However, c-Jun expression is not associated with bladder afferent neurons. Resiniferatoxin reduced CYP-induced up-regulation of p-CREB in DRG, suggesting that cystitis can reveal an altered CREB phosphorylation that may be mediated by capsaicin-sensitive bladder afferents. Colocalization of p-CREB and Trk receptor(s) showed that a subpopulation of p-CREB-IR cells expressed p-Trk with cystitis. These results suggest that up-regulation of p-CREB may be mediated by a neurotrophin/Trk signaling pathway. |
One of the key elements concerning our understanding of the organization of the mouse retina is the complete classification of the various types of bipolar cells. With the present study, we tried to contribute to this important issue. Unfortunately, most of the antibodies that stain specifically bipolar cells in the retina of other mammals hardly work for the retina of the mouse. We succeeded in overcoming this limitation by using a relatively novel technique based on the gene gun transfer of fluorescent dyes to cells. Hence, we were able to stain a considerable number of bipolar cells that could be characterized according to morphological and comparative criteria. We also performed a complete morphometric analysis of a subset of bipolar cells stained by anti-neurokinin-3 receptor antibodies. We found nine types of cone bipolar cells and one type of rod bipolar cell; these data are consistent with the findings of previous studies on the retinas of other mammals, such as rabbits, rats, and monkeys and with a recent study based on the mouse retina (Ghosh et al. [2004] J Comp Neurol 469:70-82). Our results also confirm the existence of a common structural similarity among mammalian retinas. It remains to be elucidated what is exactly the functional role of the various types of cone bipolar cells and what is the specific contribution they provide to the perception of a given visual stimulus. Most probably, each bipolar cell type constitutes a specialized channel for the computation of a selected component of the visual stimulus. More complex signal coding, involving the coordinated activity of various types of bipolar cells, could also be postulated, as it has been shown for ganglion cells (Meister [1996] Proc Natl Acad Sci U S A 93:609-614). |
Our previous study tracked the ontogeny of aminergic systems in zebrafish (Danio rerio). Here we use tyrosine hydroxylase (TH) and serotonin (5-hydroxytryptamine; 5-HT) immunoreactivity, in conjunction with retrograde and genetic labeling techniques, to provide a more refined examination of the potential synaptic contacts of aminergic systems. Our focus was on different levels of the sensorimotor circuit for escape, from sensory inputs, through identified descending pathways, to motor output. We observed 5-HT reactivity in close proximity to the collaterals of the Rohon-Beard sensory neurons in spinal cord. In the brainstem we found TH and 5-HT reactivity closely apposed to the dendritic processes of the nucleus of the medial longitudinal fascicle (nMLF), in addition to the ventral dendrites of the Mauthner neuron and its serial homologs MiD2cm and MiD3cm. Only TH reactivity was observed near the lateral dendrites of the Mauthner cell. TH and 5-HT reactivity were also positioned near the outputs of reticulospinal cells in spinal cord. Finally, both TH and 5-HT reactivity were detected close to the dendritic processes of primary and secondary spinal motor neurons. We also confirmed, using dual TH and 5-HT staining and retrograde labeling, that the sources of spinal aminergic reactivity include the posterior tuberculum (dopamine) and inferior raphe region (5-HT). Our data indicate that aminergic systems may interact at all levels of the sensorimotor pathways involved in escape. The identification of some of these likely sites of aminergic action will allow for directed studies of their functional roles using the powerful combination of techniques available in zebrafish. |
Mammalian retinas are innervated by histaminergic axons that originate from perikarya in the posterior hypothalamus. To identify the targets of these retinopetal axons, we localized histamine receptors (HR) in monkey and rat retinas by light and electron microscopy. In monkeys, puncta containing HR3 were found at the tips of ON-bipolar cell dendrites in cone pedicles and rod spherules, closer to the photoreceptors than the other neurotransmitter receptors. This is the first ultrastructural localization of any histamine receptor and the first direct evidence that HR3 is present on postsynaptic membranes in the central nervous system. In rat retinas, most HR1 were localized to dopaminergic amacrine cells. The differences in histamine receptor localization may reflect the differences in the activity patterns of the two species. |
The avian circadian system is composed of multiple inputs, oscillators, and outputs. Among its oscillators are the pineal gland, retinae, and a hypothalamic structure assumed to be homologous to the mammalian suprachiasmatic nucleus (SCN). Two structures have been suggested as this homolog -- the medial SCN (mSCN) and the visual SCN (vSCN). The present study employed biotin dextran amine (BDA) and cholera toxin B subunit (CTB) as anterograde and retrograde tracers to investigate the connectivity of the mSCN and vSCN in order to address this issue. Intravitreal injections of CTB were used to determine whether one or both of these structures receives afferent input from retinal ganglion cells. Both the vSCN and mSCN receive terminal retinal input, with the strongest input terminating in the vSCN. Precise iontophoretic injections of BDA and CTB in the mSCN and vSCN were used to identify efferents and afferents. The avian mSCN and vSCN collectively express more efferents and afferents than does the mammalian SCN. A subset of these connections matches the connections that have been established in rodent species. Individually, both the mSCN and vSCN are similar to the mammalian SCN in terms of their connections. Based on these data and other studies, we present a working model of the avian SCN that includes both the mSCN and vSCN as hypothalamic oscillators. We contend that both structures are involved in a suprachiasmatic complex that, as a functional group, may be homologous to the mammalian SCN. |
Olfactory and vomeronasal projections have been traditionally viewed as terminating in contiguous non-overlapping areas of the basal telencephalon. Original reports, however, described areas such as the anterior medial amygdala where both chemosensory afferents appeared to overlap. We addressed this issue by injecting dextran amines in the main or accessory olfactory bulbs of rats and the results were analyzed with light and electron microscopes. Simultaneous injections of different fluorescent dextran amines in the main and accessory olfactory bulbs were performed and the results were analyzed using confocal microscopy. Similar experiments with dextran amines in the olfactory bulbs plus FluoroGold in the bed nucleus of the stria terminalis indicate that neurons projecting through the stria terminalis could be integrating olfactory and vomeronasal inputs. Retrograde tracing experiments using FluoroGold or dextran amines confirm that areas of the rostral basal telencephalon receive inputs from both the main and accessory olfactory bulbs. While both inputs clearly converge in areas classically considered olfactory-recipient (nucleus of the lateral olfactory tract, anterior cortical amygdaloid nucleus, and cortex-amygdala transition zone) or vomeronasal-recipient (ventral anterior amygdala, bed nucleus of the accessory olfactory tract, and anteroventral medial amygdaloid nucleus), segregation is virtually complete at posterior levels such as the posteromedial and posterolateral cortical amygdalae. This provides evidence that areas so far considered receiving a single chemosensory modality are likely sites for convergent direct olfactory and vomeronasal inputs. Therefore, areas of the basal telencephalon should be reclassified as olfactory, vomeronasal, or mixed chemosensory structures, which could facilitate understanding of olfactory-vomeronasal interactions in functional studies. |
The ability to identify and respond to food is essential for survival, yet little is known about the neural substrates that regulate natural variation in food-related traits. The foraging (for) gene in Drosophila melanogaster encodes a cGMP-dependent protein kinase (PKG) and has been shown to function in food-related traits. To investigate the tissue distribution of FOR protein, we generated an antibody against a common region of the FOR isoforms. In the adult brain we localized FOR to neuronal clusters and projections including neurons that project to the central complex, a cluster within the dorsoposterior region of the brain hemispheres, a separate cluster medial to optic lobes and lateral to brain hemispheres, a broadly distributed frontal-brain cluster, axon bundles of the antennal nerve and of certain subesophageal-ganglion nerves, and the medulla optic lobe. These newly described tissue distribution patterns of FOR protein provide candidate neural clusters and brain regions for investigation of neural networks that govern foraging-related traits. To determine whether FOR has a behavioral function in neurons we expressed UAS-for in neurons using an elav-gal4 driver and measured the effect on adult sucrose responsiveness (SR), known to be higher in rovers than sitters, the two natural variants of foraging. We found that pan-neuronal expression of for caused an increase in the SR of sitters, demonstrating a neural function for PKG in this food-related behavior. |
The lanceolate sensory endings that form palisades around the hair follicle associate with networks of branched Schwann cells. To define the properties of these glial networks as possible conduits of Ca2+ signals, lanceolate endings isolated from rat vibrissae were observed by confocal microscopy while the signaling was locally activated by mechanical stimulation. Intercellular coupling by gap junctions was also assessed by a technique employing fluorescence recovery after photobleaching (FRAP) and by transmission electron microscopy (TEM). Results showed that the glial Ca2+ signals can spread among the arrays of lanceolates in two forms: rapid signals that originate in individual Schwann processes covering the lanceolate axon terminals around the locus of mechanical stimulation, and delayed ones that travel from the stimulation locus through cytoplasmic arborization of the primarily activated cell to the adjacent cell processes. The former signaling was suppressed by the antipurinergic agents suramin and apyrase, whereas the latter was sensitive to the gap junction blocker carbenoxolon. FRAP experiments and TEM observations corroborated the presence of gap junction communications between the Schwann processes of different cell origins. These findings show that, in the Schwann networks, purinergically induced Ca2+ signals and those dependent on gap junctions are propagated in their own spatiotemporal patterns to constitute two distinct forms of communication among the mechanoreceptor palisades. |
The retinal photoreceptor ribbon synapse is a chemical synapse structurally and functionally specialized for the tonic release of neurotransmitter. It is characterized by the presynaptic ribbon, an electron-dense organelle at the active zone covered by hundreds of synaptic vesicles. In conventional synapses, dense-core transport vesicles carrying a set of active zone proteins are implicated in early steps of synapse formation. In photoreceptor ribbon synapses, synaptic spheres are suggested to be involved in ribbon synapse assembly, but nothing is known about the molecular composition of these organelles. With light, electron, and stimulated emission depletion microscopy and immunocytochemistry, we investigated a series of presynaptic proteins during photoreceptor synaptogenesis. The cytomatrix proteins Bassoon, Piccolo, RIBEYE, and RIM1 appear early in synaptogenesis. They are transported in nonmembranous, electron-dense, spherical transport units, which we called precursor spheres, to the future presynaptic site. Other presynaptic proteins, i.e., Munc13, CAST1, RIM2, and an L-type Ca(2+) channel alpha1 subunit are not associated with the precursor spheres. They cluster directly at the active zone some time after the first set of cytomatrix proteins has arrived. By quantitative electron microscopy, we found an inverse correlation between the numbers of spheres and synaptic ribbons in the postnatally developing photoreceptor synaptic terminals. From these results, we suggest that the precursor spheres are the transport units for proteins of the photoreceptor ribbon compartment and are involved in the assembly of mature synaptic ribbons. |
In rats, whisking behavior is characterized by high-frequency synchronous movements and other stereotyped patterns of bilateral coordination that are rarely seen in the bilateral movements of the limbs. This suggests that the motor systems controlling whisker and limb movements must have qualitative or quantitative differences in their interhemispheric connections. To test this hypothesis, anterograde tracing methods were used to characterize the bilateral distribution of projections from the whisker and forepaw regions in the primary motor (MI) cortex. Unilateral tracer injections in the MI whisker or forepaw regions revealed robust projections to the corresponding MI cortical area in the contralateral hemisphere. Both MI regions project bilaterally to the neostriatum, but the corticostriatal projections from the whisker region are denser and more evenly distributed across both hemispheres than those from the MI forepaw region. The MI whisker region projects bilaterally to several nuclei in the thalamus, whereas the MI forepaw region projects almost exclusively to the ipsilateral thalamus. The MI whisker region sends dense projections to the contralateral claustrum, but those to the ipsilateral claustrum are less numerous. By contrast, the MI forepaw region sends few projections to the claustrum of either hemisphere. Bilateral deposits of different tracers in MI revealed overlapping projections to the neostriatum, thalamus, and claustrum when the whisker regions were injected, but not when the forepaw regions were injected. These results suggest that the bilateral coordination of the whiskers depends, in part, on MI projections to the contralateral neostriatum, thalamus, and claustrum. |
The central caudal nidopallium (NCC) is a large subdivision of the nidopallium in the pigeon brain, but its connectional anatomy is unknown. Here, we examined the connections of NCC by using tract-tracing methods. Injections of cholera toxin B-chain (CTB) in NCC labeled many neurons within NCC. Outside NCC, many labeled neurons were found in the dorsal intermediate mesopallium and medialmost part of the medial intermediate nidopallium, with a few in the intermediate (AI) and medial (AM) arcopallium. In the thalamus, labeled neurons were located in the subrotundal nucleus, the shell region of nucleus ovoidalis, and the caudal part of the dorsolateral posterior thalamic nucleus. Injections of biotinylated dextran amine (BDA) in NCC labeled many fibers running rostrocaudally within NCC. Some of these terminated in the dorsal intermediate mesopallium, but the size of the terminal field was smaller than the region of the dorsal intermediate mesopallium that provided the projection to NCC. NCC sent numerous efferents to AI and AM but few to the thalamus. In contrast, after CTB injections in the dorsal intermediate mesopallium, a few neurons were labeled in NCC, but, after BDA injections in the dorsal intermediate mesopallium, large numbers of labeled fibers were seen to project widely throughout NCC. These findings indicate that the flow of information is predominantly from the dorsal intermediate mesopallium to NCC and from there to the arcopallium (AI and AM). The arcopallial outflow to the medial hypothalamus could imply that NCC is involved in neuroendocrine and autonomic functions and is limbic in nature. |
Lepidopterans like the giant sphinx moth Manduca sexta are known for their conspicuous sexual dimorphism in the olfactory system, which is especially pronounced in the antennae and in the antennal lobe, the primary integration center of odor information. Even minute scents of female pheromone are detected by male moths, facilitated by a huge array of pheromone receptors on their antennae. The associated neuropilar areas in the antennal lobe, the glomeruli, are enlarged in males and organized in the form of the so-called macroglomerular complex (MGC). In this study we searched for anatomical sexual dimorphism more downstream in the olfactory pathway and in other neuropil areas in the central brain. Based on freshly eclosed animals, we created a volumetric female and male standard brain and compared 30 separate neuropilar regions. Additionally, we labeled 10 female glomeruli that were homologous to previously quantitatively described male glomeruli including the MGC. In summary, the neuropil volumes reveal an isometric sexual dimorphism in M. sexta brains. This proportional size difference between male and female brain neuropils masks an anisometric or disproportional dimorphism, which is restricted to the sex-related glomeruli of the antennal lobes and neither mirrored in other normal glomeruli nor in higher brain centers like the calyces of the mushroom bodies. Both the female and male 3D standard brain are also used for interspecies comparisons, and may serve as future volumetric reference in pharmacological and behavioral experiments especially regarding development and adult plasticity. J. Comp. Neurol. 517:210-225, 2009. (c) 2009 Wiley-Liss, Inc. |
Understanding the development of nociceptive circuits is important for the proper treatment of pain and administration of anesthesia to prenatal, newborn, and infant organisms. The spinothalamic tract (STT) is an integral pathway in the transmission of nociceptive information to the brain, yet the stage of development when axons from cells in the spinal cord reach the thalamus is unknown. Therefore, the retrograde tracer Fluoro-Gold was used to characterize the STT at several stages of development in the mouse, a species in which the STT was previously unexamined. One-week-old, 2-day-old and embryonic-day-18 mice did not differ from adults in the number or distribution of retrogradely labeled STT neurons. Approximately 3,500 neurons were retrogradely labeled from one side of the thalamus in each age group. Eighty percent of the labeled cells were located on the side of the spinal cord contralateral to the injection site. Sixty-three percent of all labeled cells were located within the cervical cord, 18% in thoracic cord, and 19% in the lumbosacral spinal cord. Retrogradely labeled cells significantly increased in diameter over the first postnatal week. Arborizations and boutons within the ventrobasal complex of the thalamus were observed after the anterograde tracer biotinylated dextran amine was injected into the neonatal spinal cord. These data indicate that, whereas neurons of the STT continue to increase in size during the postnatal period, their axons reach the thalamus before birth and possess some of the morphological features required for functionality. |
Tenascin-R is an extracellular matrix glycoprotein that is restricted to the central nervous system, where it acts as a multifunctional and versatile molecule. We report spatial and temporal distribution of tenascin-R in the developing human cerebral cortex for the first time. At 7.5 gestational weeks (GW), tenascin-R was expressed in a restricted area of the basal telencephalon. At 9.5 and 11 GW, it showed a unique double band expression pattern that delineated the boundaries of the future cortical plate. From 14 to 30 GW, tenascin-R labeling extended to the whole cortex from the deep layers toward the marginal zone with an inside-to-outside progression pattern reminiscent of neuronal migration. Moreover, tenascin-R labeling initially appeared in the form of thin, straight, or slightly tortuous intercellular processes directed toward the surface in parallel with the axis of neuronal migration. At the end of pregnancy and at adulthood, diffuse and homogeneous immunolabeling of the whole cortex thickness was observed. The striatum and thalamus were faintly positive for TNR as early as 14 GW, and this positivity intensified with brain maturation. At all developmental stages, the germinative zone, the corpus callosum, the anterior commissure, and the internal capsule appeared clearly negative for tenascin-R immunostaining whereas the adjacent parenchyma was immunopositive. Our results show that tenascin-R expression is tightly regulated in a spatiotemporal manner during brain development, especially cortical plate formation. Its pattern of expression suggests a role for tenascin-R in corticogenesis. |
Cephalopods have the largest and most complex nervous system of all invertebrates, and the brain-to-body weight ratio exceeds those of most fish and reptiles. The brain is composed of lobe units, the functions of which have been studied through surgical manipulation and electrical stimulation. However, how information is processed in each lobe for the animal to make a behavioral decision has rarely been investigated. To perform such functional analyses, it is necessary to precisely describe how brain lobes are spatially organized and mutually interconnected as a whole. We thus made three-dimensional digital brain atlases of both hatchling and juvenile pygmy squid, Idiosepius paradoxus. I. paradoxus is the smallest squid and has a brain small enough to scan as a whole region in the field-of-view of a low-magnification laser scan microscope objective. Precise analyses of the confocal images of the brains revealed one newly identified lobe and also that the relative volume of the vertical lobe system, the higher association center, in the pygmy squid represents the largest portion compared with the cephalopod species reported previously. In addition, principal component analyses of relative volumes of lobe complexes revealed that the organization of I. paradoxus brain is comparable to those of Decapodiformes species commonly used to analyze complex behaviors such as Sepia officinalis and Sepioteuthis sepioidea. These results suggest that the pygmy squid can be a good model to investigate the brain functions of coleoids utilizing physiological methods. J. Comp. Neurol. 524:2142-2157, 2016. © 2016 Wiley Periodicals, Inc. |
The habenula is an epithalamic structure differentiated into two nuclear complexes, medial (MHb) and lateral habenula (LHb). Recently, MHb together with its primary target, the interpeduncular nucleus (IP), have been identified as major players in mediating the aversive effects of nicotine. However, structures downstream of the MHb-IP axis, including the median (MnR) and caudal dorsal raphe nucleus (DRC), may contribute to the behavioral effects of nicotine. The afferent and efferent connections of the IP have hitherto not been systematically investigated with sensitive tracers. Thus, we placed injections of retrograde or anterograde tracers into different IP subdivisions or the MnR and additionally examined the transmitter phenotype of major IP and MnR afferents by combining retrograde tract tracing with immunofluorescence and in situ hybridization techniques. Besides receiving inputs from MHb and also LHb, we found that IP is reciprocally interconnected mainly with midline structures, including the MnR/DRC, nucleus incertus, supramammillary nucleus, septum, and laterodorsal tegmental nucleus. The bidirectional connections between IP and MnR proved to be primarily GABAergic. Regarding a possible topography of IP outputs, all IP subnuclei gave rise to descending projections, whereas major ascending projections, including focal projections to ventral hippocampus, ventrolateral septum, and LHb originated from the dorsocaudal IP. Our findings indicate that IP is closely associated to a distributed network of midline structures that modulate hippocampal theta activity and forms a node linking MHb and LHb with this network, and the hippocampus. Moreover, they support a cardinal role of GABAergic IP/MnR interconnections in the behavioral response to nicotine. |
The paraflocculus and the neighboring smaller flocculus form a remarkable protrusion in the ventrolateral aspect of the mouse cerebellum, in which the longitudinal compartments are conspicuously oriented perpendicularly to the sagittal plane. The developmental process of such anatomical arrangements in these lobules has not been fully clarified. Here, we used the genetic tractability of pcdh10-lacZ knock-in (OL-KO), IP <sub>3</sub> R1-nls-lacZ transgenic (1NM13) and Gpr26cre-Ai9-AldocV mice to track the development of compartments and examined local longitudinal orientation of Purkinje cells within the paraflocculus and flocculus. We observed a distinct pcdh10-positive (pcdh10+) compartment in the flocculus, whereas the paraflocculus and other lobules had a continuous paravermal pcdh10+ compartment, in the embryonic OL-KO cerebellum. During the first postnatal week, the parafloccular pcdh10+ compartment shifted laterally to the most lateral edge in the caudal part of the protruding paraflocculus. Although the most medial edge of the parafloccular pcdh10+ compartment remained in the nonprotruding part of the paraflocculus, it was disrupted from the originally continuous pcdh10+ compartment in the copula pyramidis. The local longitudinal orientation changed gradually along with the mediolateral extent of the copula pyramidis, almost becoming perpendicular to the sagittal plane in the laterally connected paraflocculus in the adult cerebellum. This rotational change in orientation was derived from the short U-shaped embryonic cerebellum, in which the surfaces of the flocculus and paraflocculus were oriented laterally. These results indicated that the peculiar compartmental organization of the paraflocculus originates from the embryonic common hemispheric compartmental organization and shaped by the significant reorganization process in the first postnatal week. |
The central complex (CX) in the insect brain is a higher order integration center that controls a number of behaviors, most prominently goal directed locomotion. The CX comprises the protocerebral bridge (PB), the upper division of the central body (CBU), the lower division of the central body (CBL), and the paired noduli (NO). Although spatial orientation has been extensively studied in honeybees at the behavioral level, most electrophysiological and anatomical analyses have been carried out in other insect species, leaving the morphology and physiology of neurons that constitute the CX in the honeybee mostly enigmatic. The goal of this study was to morphologically identify neuronal cell types of the CX in the honeybee Apis mellifera. By performing iontophoretic dye injections into the CX, we traced 16 subtypes of neuron that connect a subdivision of the CX with other regions in the bee's central brain, and eight subtypes that mainly interconnect different subdivisions of the CX. They establish extensive connections between the CX and the lateral complex, the superior protocerebrum and the posterior protocerebrum. Characterized neuron classes and subtypes are morphologically similar to those described in other insects, suggesting considerable conservation in the neural network relevant for orientation. |
Box jellyfish have an elaborate visual system and perform advanced visually guided behaviors. However, the rhopalial nervous system (RNS), believed to be the main visual processing center, only has 1000 neurons in each of the four eye carrying rhopalia. We have examined the detailed structure of the RNS of the box jellyfish Tripedalia cystophora, using immunolabeling with antibodies raised against four putative neuropeptides (T. cystophora RFamide, VWamide, RAamide, and FRamide). In the RNS, T. cystophora RF-, VW-, and RAamide antibodies stain sensory neurons, the pit eyes, the neuropil, and peptide-specific subpopulations of stalk-associated neurons and giant neurons. Furthermore, RFamide ir+ neurites are seen in the epidermal stalk nerve, whereas VWamide antibodies stain the gastrodermal stalk nerve. RFamide has the most widespread expression including in the ring and radial nerves, the pedalium nerve plexus, and the tentacular nerve net. RAamide is the putative neurotransmitter in the motor neurons of the subumbrellar nerve net, and VWamide is a potential marker for neuronal differentiation as it is found in subpopulations of undifferentiated cells both in the rhopalia and in the bell. The results from the FRamide antibodies were not included as only few cells were stained, and in an unreproducible way. Our studies show hitherto-unseen details of the nervous system of T. cystophora and allowed us to identify specific functional groups of neurons. This identification is important for understanding visual processing in the RNS and enables experimental work, directly addressing the role of the different neuropeptides in vision. |
We examined the number, distribution, and immunoreactivity of the infracortical white matter neuronal population, also termed white matter interstitial cells (WMICs), throughout the telencephalic white matter of an adult female chimpanzee. Staining for neuronal nuclear marker (NeuN) revealed WMICs throughout the infracortical white matter, these cells being most numerous and dense close to the inner border of cortical layer VI, decreasing significantly in density with depth in the white matter. Stereological analysis of NeuN-immunopositive cells revealed an estimate of approximately 137.2 million WMICs within the infracortical white matter of the chimpanzee brain studied. Immunostaining revealed subpopulations of WMICs containing neuronal nitric oxide synthase (nNOS, approximately 14.4 million in number), calretinin (CR, approximately 16.7 million), very few WMICs containing parvalbumin (PV), and no calbindin-immunopositive neurons. The nNOS, CR, and PV immunopositive WMICs, possibly all inhibitory neurons, represent approximately 22.6% of the total WMIC population. As the white matter is affected in many cognitive conditions, such as schizophrenia, autism, epilepsy, and also in neurodegenerative diseases, understanding these neurons across species is important for the translation of findings of neural dysfunction in animal models to humans. Furthermore, studies of WMICs in species such as apes provide a crucial phylogenetic context for understanding the evolution of these cell types in the human brain. |
<i>Objective.</i>Electro/Magnetoencephalography (EEG/MEG) source-space network analysis is increasingly recognized as a powerful tool for tracking fast electrophysiological brain dynamics. However, an objective and quantitative evaluation of pipeline steps is challenging due to the lack of realistic 'controlled' data. Here, our aim is two-folded: (a) provide a quantitative assessment of the advantages and limitations of the analyzed techniques and (b) introduce (and share) a complete framework that can be used to optimize the entire pipeline of EEG/MEG source connectivity.<i>Approach.</i>We used a human brain computational model containing both physiologically based cellular GABAergic and Glutamatergic circuits coupled through Diffusion Tensor Imaging, to generate high-density EEG recordings. We designed a scenario of successive gamma-band oscillations in distinct cortical areas to emulate a virtual picture-naming task. We identified fast time-varying network states and quantified the performance of the key steps involved in the pipeline: (a) inverse models to reconstruct cortical-level sources, (b) functional connectivity measures to compute statistical interdependency between regional signals, and (c) dimensionality reduction methods to derive dominant brain network states (BNS).<i>Main results.</i>Using a systematic evaluation of the different decomposition techniques, results show significant variability among tested algorithms in terms of spatial and temporal accuracy. We outlined the spatial precision, the temporal sensitivity, and the global accuracy of the extracted BNS relative to each method. Our findings suggest a good performance of weighted minimum norm estimate/ Phase Locking Value combination to elucidate the appropriate functional networks and ICA techniques to derive relevant dynamic BNS.<i>Significance.</i>We suggest using such brain models to go further in the evaluation of the different steps and parameters involved in the EEG/MEG source-space network analysis. This can reduce the empirical selection of inverse model, connectivity measure, and dimensionality reduction method as some of the methods can have a considerable impact on the results and interpretation. |
<i>Objective.</i>A large part of the cerebral cortex is dedicated to the processing of visual stimuli and there is still much to understand about such processing modalities and hierarchies. The main aim of the present study is to investigate the differences between directional visual stimuli (DS) and non-directional visual stimuli (n-DS) processing by time-frequency analysis of brain electroencephalographic activity during a visuo-motor task. Electroencephalography (EEG) data were divided into four regions of interest (ROIs) (frontal, central, parietal, occipital).<i>Approach.</i>The analysis of the visual stimuli processing was based on the combination of electroencephalographic recordings and time-frequency analysis. Event related spectral perturbations (ERSPs) were computed with spectrum analysis that allow to obtain the average time course of relative changes induced by the stimulus presentation in spontaneous EEG amplitude spectrum.<i>Main results.</i>Visual stimuli processing enhanced the same pattern of spectral modulation in all investigated ROIs with differences in amplitudes and timing. Additionally, statistically significant differences in occipital ROI between the DS and n-DS visual stimuli processing in theta, alpha and beta bands were found.<i>Significance.</i>These evidences suggest that ERSPs could be a useful tool to investigate the encoding of visual information in different brain regions. Because of their simplicity and their capability in the representation of brain activity, the ERSPs might be used as biomarkers of functional recovery for example in the rehabilitation of visual dysfunction and motor impairment following a stroke, as well as diagnostic tool of anomalies in brain functions in neurological diseases tailored to personalized treatments in clinical environment. |
We describe a spatio-temporal linear discriminator for single-trial classification of multi-channel electroencephalography (EEG). No prior information about the characteristics of the neural activity is required, i.e., the algorithm requires no knowledge about the timing and spatial distribution of the evoked responses. The algorithm finds a temporal delay/window onset time for each EEG channel and then spatially integrates the channels for each channel-specific onset time. The algorithm can be seen as learning discrimination trajectories defined within the space of EEG channels. We demonstrate the method for detecting auditory-evoked neural activity and discrimination of task difficulty in a complex visual-auditory environment. |
A method based on wavelet packet best basis decomposition (WPBBD) is investigated for the purpose of extracting features of electroencephalogram signals produced during motor imagery tasks in brain-computer interfaces. The method includes the following three steps. (1) Original signals are decomposed by wavelet packet transform (WPT) and a wavelet packet library can be formed. (2) The best basis for classification is selected from the library. (3) Subband energies included in the best basis are used as effective features. Three different motor imagery tasks are discriminated using the features. The WPBBD produces a 70.3% classification accuracy, which is 4.2% higher than that of the existing wavelet packet method. |
Hypothalamic-pituitary-adrenal (HPA) axis dysfunction has been implicated in the pathogenesis of addictive behaviour and especially in alcohol craving. The pro-opiomelanocortin gene (POMC), encoding a 241 amino acids stretching polypeptide hormone precursor, plays an important role in the regulation of the HPA, and is prone to epigenetic regulation due to promoter-related DNA methylation. Aim of the present study therefore was to investigate possible differences in promoter-related DNA methylation in patients suffering from alcohol dependence compared to healthy controls. We analysed the DNA methylation of the 5' promoter of the POMC gene that is embedded in a CpG island using bisulfite sequencing in 145 alcohol-dependent patients and 37 healthy controls taken from the Franconian Alcoholism Research Studies. We found only marginal, hence significant differences at single CpG sites between patients and controls. We identified a cluster of CpGs showing a significant association with alcohol craving in the patients group. These results implicate that epigenetic changes possibly due to alcohol intake may contribute to craving via promoting HPA-axis dysfunction. Further studies should more closely investigate the impact of these changes on the several derivatives of the POMC gene. |
Type B monoamine oxidase (MAO-B) is proposed to be involved in the pathogenesis of neurodegenerative disorders, such as Parkinson's disease, through oxidative stress and synthesis of neurotoxins. MAO-B inhibitors, rasagiline and selegiline [(-)deprenyl], protect neuronal cells by direct intervention in mitochondrial death signaling and induction of pro-survival Bcl-2 and neurotrophic factors. Recently, type A MAO (MAO-A) was found to mediate the induction of anti-apoptotic Bcl-2 by rasagiline, whereas MAO-A increases in neuronal death and also serves as a target of neurotoxins. These controversial results suggest that MAO-A may play a decisive role in neuronal survival and death. This paper reports that rasagiline and selegiline increased the mRNA, protein and catalytic activity of MAO-A in SH-SY5Y cells. Silencing MAO-A expression with small interfering (si)RNA suppressed rasagiline-dependent MAO-A expression, but MAO-B overexpression in SH-SY5Y cells did not affect, suggesting that MAO-A, not MAO-B, might be associated with MAO-A upregulation. Rasagiline reduced R1, a MAO-A specific repressor, but selegiline did not. Mithramycin-A, an inhibitor of Sp1 binding, and actinomycin-D, a transcriptional inhibitor, reduced the rasagiline-dependent upregulation of MAO-A mRNA, indicating that rasagiline induced MAO-A transcriptionally through R1-Sp1 pathway, whereas selegiline by another non-defined pathway. These results are discussed in relation to the role of MAO-A and these MAO-B inhibitors in neuronal death and neuroprotection. |
We evaluated the immunohistochemical intensities of α-synuclein, phosphorylated α-synuclein (p-syn), dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32), calbindin-D 28k, calpain-cleaved carboxy-terminal 150-kDa spectrin fragment, and tyrosine hydroxylase in multiple system atrophy (MSA). The caudate head, anterior putamen, posterior putamen, substantia nigra, pontine nucleus, and cerebellar cortex from six MSA brains, six age-matched disease control brains (amyotrophic lateral sclerosis), and five control brains were processed for immunostaining by standard methods. Immunostaining for α-synuclein, p-syn, or both was increased in all areas examined in oligodendrocytes in MSA. Immunostaining for DARPP-32 and calbindin-D 28k was most prominently decreased in the posterior putamen, where neuronal loss was most prominent. Immunostaining for DARPP-32 and calbindin-D 28k was also diminished in the anterior putamen and caudate head, where neuronal loss was less prominent or absent. Calbindin immunostaining was also decreased in the dorsal tier of the substantia nigra and cerebellar cortex. Loss of immunostaining for DARPP-32 and calbindin-D 28k compared with that of neurons indicates calcium toxicity and disturbance of the phosphorylated state of proteins as relatively early events in the pathogenesis of MSA. |
The psychostimulant, methylphenidate (MPD), is the first line treatment as a pharmacotherapy to treat behavioral disorders such as attention deficit hyperactivity disorder (ADHD). MPD is commonly misused in non-ADHD (normal) youth and young adults both as a recreational drug and for cognitive enhancing effects to improve their grades. MPD is known to act on the reward circuit; including the caudate nucleus (CN). The CN is comprised of medium spiny neurons containing largely dopamine (DA) D1 and D2 receptors. It has been widely shown that the DA system plays an important role in the response to MPD exposure. We investigated the role of both D1 and D2 DA receptors in the CN response to chronic MPD administration using specific D1 and D2 DA antagonist. Four groups of young adult, male SD rats were used: a saline (control) and three MPD dose groups (0.6, 2.5, and 10.0 mg/kg). The experiment lasted 11 consecutive days. Each MPD dose group was randomly divided into two subgroups to receive either a 0.4 mg/kg SCH-23390 selective D1 DA antagonist or a 0.3 mg/kg raclopride selective D2 DA antagonist prior to their final (repetitive) MPD rechallenge administration. It was observed that selective D1 DA antagonist (SCH-23390) given 30 min prior to the last MPD exposure at ED11 partially reduced or prevented the effect induced by MPD exposure in CN neuronal firing rates across all MPD doses. Selective D2 DA antagonist (raclopride) resulted in less obvious trends; some CN neuronal firing rates exhibited a slight increase in all MPD doses. |
Type A monoamine oxidase (MAOA) catabolizes monoamine transmitters, serotonin, norepinephrine and dopamine, and plays a major role in the onset, progression and therapy of neuropsychiatric disorders. In depressive disorders, increase in MAOA expression and decrease in brain levels of serotonin and norepinephrine are proposed as the major pathogenic factors. The functional polymorphism of MAOA gene and genes in serotonin signal pathway are associated with depression. This review presents recent advance in studies on the role of MAOA in major depressive disorder and related emotional disorders. MAOA and serotonin regulate the prenatal development and postnatal maintenance of brain architecture and neurocircuit, as shown by MAOA-deficient humans and MAO knockout animal models. Impaired neurogenesis in the mature hippocampus has been proposed as "adult neurogenesis" hypothesis of depression. MAOA modulates the sensitivity to stress in the stages of brain development and maturation, and the interaction of gene-environmental factors in the early stage regulates the onset of depressive behaviors in adulthood. Vice versa environmental factors affect MAOA expression by epigenetic regulation. MAO inhibitors not only restore compromised neurotransmitters, but also protect neurons from cell death in depression through induction of anti-apoptotic Bcl-2 and prosurvival neurotrophic factors, especially brain-derived neurotrophic factor, the deficiency of which is detected in depression. This review discusses novel role of MAOA and serotonin in the pathogenesis and therapy of depressive disorders. |
The glutamate hypothesis of schizophrenia is related to the proposed dysregulation of D-amino acid oxidase (DAO), DAO activator (DAOA)/G72, and Neuregulin 1 (NRG1) genes. Genetic studies have shown significant associations between DAO, DAOA, NRG1 single-nucleotide polymorphisms (SNPs), and schizophrenia. The systematic literature search yielded 6, 5, and 18 new studies for DAO, DAOA, and NRG1 published after 2011 and not included in the previous SchizophreniaGene (SZGene) meta-analysis. We conducted meta-analyses of 20, 23, and 48 case-control studies, respectively, to comprehensively evaluate the association of 8 DAO, 12 DAOA, and 14 NRG1 SNPs with schizophrenia. The updated meta-analyses resulted in the following findings: the C-allele of DAO rs4623951 was associated with schizophrenia across all pooled studies [Odds ratio (OR) = 0.88, 95% confidence interval (CI) = 0.79-0.98, p = 0.02, N = 3143]; however, no new reports could be included. The G-allele of DAOA rs778293 was associated with schizophrenia in Asian patients (OR = 1.17, 95% CI = 1.08-1.27, p = 0.00008, N = 6117), and the T-allele of DAOA rs3916971 was associated with schizophrenia across all studies (OR = 0.84, 95% CI = 0.73-0.96, p = 0.01, N = 1765). Again, for both SNPs, no new eligible studies were available. After adding new reports, the T-allele of NRG1 SNP8NRG241930 (rs62510682) across all studies (OR = 0.95, 95% CI = 0.91-0.997, p = 0.04, N = 22,898) and in Caucasian samples (OR = 0.95, 95% CI = 0.90-0.99, p = 0.03, N = 16,014), and the C-allele of NRG1 rs10503929 across all studies (OR = 0.89, 95% CI = 0.81-0.97, p = 0.01, N = 6844) and in Caucasian samples (OR = 0.89, 95% CI = 0.81-0.98, p = 0.01, N = 6414) were protective against schizophrenia. Our systematic meta-analysis is the most updated one for the association of DAO, DAOA, and NRG1 SNPs with schizophrenia. |
Professor Kurt Jellinger is well known for his seminal work on the neuropathology of age-associated neurodegenerative disorders, particularly Lewy body diseases. However, it is less well known that he also contributed important insights into the neuropathological features of several paediatric neurometabolic diseases, including Alpers-Huttenlocher syndrome, a syndrome of mitochondrial disease caused by POLG mutations, and infantile neuroaxonal dystrophy, a phenotype resulting from PLA2G6 mutations. Despite these rare diseases occurring in early life, they share many important pathological overlaps with age-associated Lewy body disease, particularly dysregulation of α-synuclein. In this review, we describe several neurometabolic diseases linked to Lewy body disease mechanisms, and discuss the wider context to pathological overlaps between neurometabolic and Lewy body diseases. In particular, we will focus on how understanding disease mechanisms in neurometabolic disorders with dysregulated α-synuclein may generate insights into predisposing factors for α-synuclein aggregation in idiopathic Lewy body diseases. |
Cell-based assays are a novel method to determine potency of botulinum toxin drugs. Manufacturers are working on their acquisition, development and implementation to reduce animal consumption during the manufacturing process. Potency labelling of botulinum toxin drugs differes principally between Ipsen and the other manufacturers. Reference to a uniform international standard would avoid this potentially dangerous situation. However, this has not been demanded by the registration authorities and has not been persued by the manufacturers for decades. |
The pathogenesis of HIV-induced neurological disorders is still incompletely understood. Since many aspects of this disease are difficult to explore in humans, animal models are necessary to fill the gaps in our knowledge. Based on the high concordance with the human system, the SIV-infection of macaques currently provides the best animal model to study pathogenesis, therapy and prevention of HIV-infection. In this review, important features of the CNS-infection in this model are outlined. Recent virological, immunological, neurophysiological and neurochemical findings obtained with this animal model are presented and key factors in the development of neurological disease are identified. |
Accumulating data confirm the usefulness of transcranial sonography (TCS) in the diagnosis of Parkinson’s disease. The relevance of basal ganglia abnormalities depicted by TCS in atypical parkinsonian syndromes still needs further assessment. In the present study, 20 patients with progressive supranuclear palsy (PSP) and 13 patients with corticobasal syndrome (CBS) were studied with the use of transcranial sonography. Echogenicity of the substantia nigra (SN) and lenticular nucleus (LN) were assessed. 0/20 patients with PSP and 8/12 (66.6 %) patients with CBS were characterized with SN hyperechogenicity. LN hyperechogenicity was observed in 9/20 patients diagnosed with PSP and 0/11 of CBS patients. The combination of SN isoechogenicity and LN hyperechogenicity reached 100 % sensitivity and positive predictive value for the diagnosis of PSP. The results of this study point out that CBS has to be taken into consideration when SN hyperechogenicity is depicted in a patient with parkinsonian syndrome. Normal echogenicity of the SN coexisting with LN hyperechogenicity practically excludes CBS.
## Introduction
In the wake of the increasing need for reliable preclinical diagnosis of Parkinson`s disease (PD), numerous diagnostic tools are under investigation. Neuroimaging studies are among the most promising. SN hyperechogenicity depicted by TCS is a sensitive marker for PD (Becker et al. ). Consecutive studies (Berg et al. ) confirmed the reliability of TCS as a supporting diagnostic tool in PD patients. Due to a more limited data the appearance of SN hyperechogenicity in atypical Parkinsonism (APS) is still a matter of controversy. According to available data (Walter et al. ; Behnke et al. ) 6–25 % of patients with multiple system atrophy (MSA) are characterized by SN hyperechogenicity, whereas in patients diagnosed with progressive supranuclear palsy (PSP) this echo feature is observed in 8–47 %. To date there is only one published study (Walter et al. ) describing TCS findings in merely 8 patients diagnosed with corticobasal syndrome (CBS). Walter et al. reported high incidence (ca. 90 %) of bilaterally marked SN hyperechogenicity in CBS patients. Due to discrepant data SN hyperechogenicity might be of diverse specificity in the differential diagnosis of parkinsonian syndromes when comparing different populations. Hyperechogenicity of the lenticular nucleus (LN) is a frequent (72–88 % in available literature) and valuable supporting finding (Walter et al. , ; Behnke et al. ) in APS but lacks specificity for particular disease entities. Due to clinical overlap and limited TCS data patients with tauopathies constitute a group of special interest. In the present study, SN and LN echogenicity assessment was applied to patients with two types of tauopathies—PSP and CBS—to evaluate the clinical usefulness of TCS as a supporting tool in the diagnosis of these conditions.
## Patients and methods
A total of 34 consecutive patients fulfilling diagnostic criteria for clinically probable PSP ( N = 20) and CBS ( N = 14) were recruited from in- and outpatient clinic (see Table for details). All patients gave written consent for the study. The study obtained IRB approval. TCS was performed using a phased-array ultrasound system with a 1–4 MHz transducer (MyLab 70XVision, Esaote, Italy). SN and LN echogenicity were assessed according to available guidelines (Walter et al. ). The examiner was blinded to final clinical diagnosis, but not separated from the patient during TCS examination. The examination was performed bilaterally through temporal acoustic window with penetration depth of 16 cm and dynamic range of 40–45 dB. At the midbrain plane butterfly-shaped midbrain was depicted. The echogenic area of the ipsilateral SN was manually encircled and automatically measured. As in previous studies, the cut-off value of ≥0.20 cm on at least one side defined SN hyperechogenicity and ≥0.25 cm - marked hyperechogenicity. By tilting the probe 10° in the upward direction the thalamus plane was visualized. At this level the contralateral lenticular nucleus was assessed qualitatively as iso- or hyperechogenic. The third ventricle width was not evaluated. Due to the authors opinion this measure, however undoubtedly valuable, might be as well obtained with the use of standard MRI imaging whereas basal ganglia echogenicity assessment brings new and unique diagnostic data unavailable with the use of other imaging modalities. Obtained images were assessed by two raters (M.S.-K. and K.S.) to increase the final reliability of measures; final picture analysis for each patient was performed jointly. The Chi-square test was employed to compare the results in both groups.
Demographic and clinical data of patients
## Results
All patients with PSP and 12 patients with CBS had sufficient acoustic temporal window. No significant difference in demographic data was observed ( p > 0.05). SN hyperechogenicity was observed in 8 out of 12 (66.6 %) patients diagnosed with CBS. In all these cases the criteria for marked hyperechogenicity (≥0.25 cm ) were fulfilled. In all cases observed hyperechogenicity was unilateral and located contralaterally to the more affected side. None of PSP patients fulfilled the criteria for SN hyperechogenicity. The median size of 0.3 cm for CBS and 0.1 cm for PSP was observed (Fig. ; p < 0.05). The larger echogenic side was taken into consideration in calculations. Assuming that the isoechogenic SN is characteristic for PSP the positive predictive value for PSP diagnosis reached 83.33 %, specificity 66.66 % and sensitivity 100 %. LN hyperechogenicity was depicted in 9 out of 20 patients with PSP and was not observed in patients diagnosed with CBS ( p < 0.05). The combination of SN isoechogenicity and LN hyperechogenicity reached 100 % sensitivity and positive predictive value as a characteristic feature of CBS patients.
Comparison of the area of echogenicity in the SN (cm ) in PSP and CBS patients ( p < 0.05)
## Discussion
The results of this study confirm the relatively high occurrence of SN hyperechogenicity in patients diagnosed with CBS distinguishing them from other patients with atypical parkinsonian syndromes. Walter et al. ( ) reported that 7/8 (88 %) patients with CBS had marked bilateral SN hyperechogenicity (in comparison to 66.6 % and asymmetric in this study). In both studies none of the PSP patients showed this feature. Lenticular nucleus hyperechogenicity in this study was observed only in patients with PSP, whereas Walter et al. noted this echo feature in both groups with comparative frequency. The dynamic range setting at the level of 40–45 dB used in this study might result in higher contrast of TCS images enabling clear interpretation, but at the cost of slightly lower incidence of SN hyperechogenicity in comparison to Walter et al. (45–50 dB used). Patients with CBS appear to have distinctive features in TCS examination. The fact that SN hyperechogenicity is a frequent finding in CBS patients has to be taken seriously into consideration, especially in the context of the specificity of this finding in PD patients. In a patient with newly diagnosed parkinsonian syndrome and SN hyperechogenicity, CBS has to be taken into account. Marked SN hyperechogenicity, when considered in a proper clinical context, might support the diagnosis of corticobasal syndrome. Due to frequent overlap of symptomatology in PSP and CBS transcranial sonography findings might be also of value in more advanced cases. The pathophysiology of observed changes is still at the centre of debate. SN hyperechogenicity in PD patients is considered to be a result of iron storage dysregulation (Zecca et al. ). The absolute increase of iron within a substantia nigra of PD patients raises concerns (Wypijewska et al. ). Thus, it can be argued that the progressive loss of dopaminergic neurons with simultaneous microglial activation might explain the change in tissue sonographic features (Berg et al. ; Sadowski et al. ). This mechanism might be universal for diverse neurodegenerative disorders with substantia nigra involvement. The observation of significant SN hyperechogenicity in CBS cases might be, at least theoretically related to the very intense tau pathology in corticobasal degeneration. One may also assume that the asymmetry of the pathology of the CBS patients creates an artifact that looks like SN hyperechogenicity. An analogous argument can be made for the lentiform nucleus isoechogenicity. Interpretation of LN hyperechogenicity observed in atypical parkinsonian syndromes meets with difficulties. There are few research groups applying this part of TCS examinations. A more subjective way of LN echogenicity assessment (qualitative analysis) is another source of potential difficulties. Thus, results are more ambiguous in comparison to SN echogenicity studies. Most probably LN hyperechogenicity is characteristic for atypical parkinsonian syndromes. No studies explaining the pathogenesis of LN hyperechogenicity in atypical parkinsonian syndromes were conducted so far. Due to high incidence of clinical–pathological mismatch in CBS patients (only 50 % of patients clinically presenting as corticobasal syndrome have corticobasal degeneration) neuropathological studies would be of key importance. Would the neurodegenerative process within the SN in PSP and CBS differ in a way affecting the sonographic imaging, will probably be explained in consecutive studies. Short period of observation and lack of pathological confirmation are obvious drawbacks of this study. Thus, the results are useful as justification for further in-depth studies. On the other hand, obtained results might be important for everyday clinical practice and broaden our knowledge of practical use of transcranial sonography in movement disorders.
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The pathophysiological mechanisms of cognitive and gait disturbances in subjects with normal-pressure hydrocephalus (NPH) are still unclear. Cholinergic and other neurotransmitter abnormalities have been reported in animal models of NPH. The objective of this study was to evaluate the short latency afferent inhibition (SAI), a transcranial magnetic stimulation protocol which gives the possibility to test an inhibitory cholinergic circuit in the human brain, in subjects with idiopathic NPH (iNPH). We applied SAI technique in twenty iNPH patients before ventricular shunt surgery. Besides SAI, also the resting motor threshold and the short intracortical inhibition to paired stimulation were assessed. A significant reduction of the SAI ( p = 0.016), associated with a less pronounced decrease of the resting motor threshold and the short latency intracortical inhibition to paired stimulation, were observed in patients with iNPH at baseline evaluation. We also found significant ( p < 0.001) correlations between SAI values and the gait function tests, as well as between SAI and the neuropsychological tests. These findings suggest that the impairment of cholinergic neurons markedly contributes to cognitive decline and gait impairment in subjects with iNPH.
## Introduction
Idiopathic normal-pressure hydrocephalus (iNPH) is characterized by the classic Adams triad of cognitive dysfunction, urinary incontinence, and gait impairment. Gait and balance disorders are the leading presentations, whereas cognitive decline and incontinence appear as the disease progresses (Williams and Relkin ). Improvement in walking performance following a large-volume lumbar puncture (CSF tap test) is the key selection criterion for ventricular shunt surgery and for predicting responsiveness to the shunt therapy. Despite the clinical importance of the symptoms, the pathophysiological mechanisms of iNPH cognitive and gait disturbances remain unclear and objective methods for its assessment are lacking.
In fact, the degree of ventriculomegaly does not predict the level of behavioral impairment in NPH (Del Bigio et al. ) nor does the regression of ventriculomegaly after shunting necessarily correspond to the degree of clinical improvement (Del Bigio et al. , ).
Disturbed cholinergic neurotransmission, together with delayed hippocampal neuronal death and accumulation of beta-amyloid may contribute to memory dysfunction (Kondziella et al. ).
On the other hand, cholinergic dysfunction was found to be an important contributor to gait dysfunction in subjects with Parkinson’s disease (PD) (Rochester et al. ), a condition that is similarly characterized by loss of balance, slowness and small steps, although NPH patients perform worse (Bugalho et al. ).
A transcranial magnetic stimulation (TMS) protocol, the short latency afferent inhibition (SAI), may give direct information about the function of some cholinergic pathways in the human motor cortex.
SAI is significantly reduced in Alzheimer’s disease (AD) (Di Lazzaro et al. , , ; Nardone et al. ) and can be increased by acetylcholinesterase inhibitors (Di Lazzaro et al. ).
We hypothesize that SAI assessment might allow the contribution of cholinergic dysfunction to cognitive decline and gait disturbances to be evaluated. Therefore, we aimed to evaluate in the present study SAI in a group of subjects with NPH, and its correlation with cognitive and gait impairments.
## Materials and methods
### Patients
Twenty patients with a clinical and neuroimaging diagnosis of iNPH were recruited consecutively for the study. Patients were included in the study if they met criteria for probable iNPH (Relkin et al. ) and were capable of cooperating sufficiently for neurophysiological testing. The diagnosis of iNPH was based on the presence of the following symptoms and signs: (1) gradually developing gait disturbance; (2) cognitive deterioration, urinary incontinence, or both; (3) progression of symptoms over time; (4) a CT/MR imaging study showing ventricular enlargement (Evans index > 0.3), with transependymal diffusion and no significant cortical atrophy; (5) normal CSF pressure at lumbar puncture; and (6) improvement of clinical symptoms after lumbar tap test or following continuous external lumbar CSF drainage over 2 days. Exclusion criteria were as follows: (1) any spinal cord lesion that might cause myelopathy or severe lumbar radiculopathy, as verified by neuroimaging and EMG evaluation; (2) history of head trauma with documented brain damage in the last year that might affect excitatory and inhibitory responses to TMS; (3) CNS disease that might explain the clinical symptoms; (4) history or evidence of conditions that might cause secondary hydrocephalus; (5) diabetic polyneuropathy; and (6) pacemaker, metallic implants, or any other contraindication to TMS as specified in the safety guidelines for that procedure (Rossi et al. ).
A total of 20 patients (8 women and 12 men; mean age 73.6 years) fulfilled inclusion and exclusion criteria and were enrolled in the study.
Twenty age-matched neurologically healthy subjects (9 women and 11 men; mean age 74.1 years) constituted the control group.
Global cognitive function was examined by means of the Mini-Mental Scale Examination (MMSE, Folstein et al. ). Learning and memory were evaluated by means of the Rey Auditory Verbal Learning Test (RAVLT), executive functions by means of the Digit Span and the Trail Making Test (TMT) B, psychomotor speed by means of the TMT A. For reference and description of these tests see Lezak et al. ( ).
In addition, gait abnormalities were quantified by the Timed Up and Go Test (TUG, Podsiadlo and Richardson ) and the total walking distance for the 6-min walk test (6MWT, ATS Committee on Proficiency Standards for Clinical Pulmonary Function Laboratories ).
Based on CT and MR imaging studies, ventricular enlargement was assessed using an Evans index defined as the maximum width of the frontal horns divided by the maximum inner width of the skull.
The demographic characteristics, as well as the clinical und neurophysiological findings, are shown in the Table .
Demographic characteristics, clinical and neurophysiological data of the patients with idiopathic normal-pressure hydrocephalus and the control subjects
Data are expressed as mean values ± SD
Y years, s seconds, TUG timed up and go test, 6MWT total walking distance for the 6-min walk test, MMSE mini-mental scale examination, RAVLT rey auditory verbal learning test, TMT trail making test, RMT the resting motor threshold, SICI the short latency intracortical inhibition, SAI short latency afferent inhibition
% of maximum stimulator output
% of test response
### Transcranial magnetic stimulation
TMS was performed using a High-power Magstim 200 magnetic stimulator (Magstim Co., Whitland, Dyfed, UK) connected to a Bistim module throughout all measurements. A figure-of-eight coil with external loop diameters of 9 cm was held over the motor cortex at the optimum scalp position to elicit motor responses in the first dorsal interosseous (FDI) muscle. The dominant hemisphere was selected for stimulating patients and healthy subjects. The induced current flows in a postero-anterior direction. Motor evoked potentials (MEPs) were recorded via two 9-mm diameter Ag–AgCl electrodes with the active electrode applied over the motor point of the muscle and the reference on the metacarpophalangeal joint of the index finger. MEPs were amplified and filtered (bandwidth 3–3000 Hz) by D150 amplifiers (Digitimer, Welwyn Garden City, Hertfordshire, UK).
We evaluated the following TMS parameters: the resting motor threshold (RMT), the short latency intracortical inhibition (SICI) to paired TMS, and the SAI.
RMT was defined as the minimum stimulus intensity that produced a minimal motor evoked response (about 50 µV in 50% of ten trials) at rest (Rossini et al. ).
SICI was studied using the technique of Kujirai et al. ( ). Two magnetic stimuli were given through the same stimulating coil over the motor cortex and the effect of the first (conditioning) stimulus on the second (test) stimulus was investigated. The intensity of the conditioning stimulus was set to 80% RMT; the second (test) shock intensity was adjusted to evoke a MEP in relaxed FDI with an amplitude of approximately 1 mV, peak-to-peak. The timing of the conditioning shock was altered in relation to the test shock. Inhibitory interstimulus intervals (ISIs) of 2, 3, and 5 ms were investigated. Ten stimuli were delivered at each ISI also for test stimulus and single MEP. For these recordings, muscle relaxation is very important and the subject was given audiovisual feedback at high gain to assist in maintaining complete relaxation. The presentation of conditioned and unconditioned trials was randomized. The amplitude of the conditioned EMG responses was expressed as the percentage of the amplitude of the test EMG responses. The amplitudes of the conditioned responses were averaged obtaining grand mean amplitudes of the three inhibitory ISIs.
SAI was studied using the technique of Tokimura et al. ( ). Conditioning stimuli were single pulses (200 ms) of electrical stimulation (with the cathode positioned proximally), applied through bipolar electrodes to the median nerve at the wrist. The intensity of the conditioning stimuli was set at just over motor threshold for evoking a visible twitch of the thenar muscles. The intensity of the test cortical magnetic shock was adjusted to evoke a muscle response in relaxed FDI with an amplitude of approximately 1 mV peak-to-peak. The conditioning stimulus to the peripheral nerve preceded the test magnetic cortical stimulus. ISIs were determined relative to the latency of the N20 component of the somatosensory evoked potential evoked by stimulation of the median nerve. In right-handed subjects, the active electrode for recording the N20 potential was attached 3 cm posterior to C3 (10–20 system), and the reference was 3 cm posterior to C4 (vice versa for left-handed subjects). Five hundred responses were averaged to identify the latency of N20 peak. ISIs from the latency of the N20 component plus 2 ms to the latency of the N20 component plus 8 ms were investigated in steps of 1 ms. Eight stimuli were delivered at each ISI also for test stimulus and single MEP. We calculated the average of the MEP obtained after cortical magnetic stimulation alone, and the average of the MEP obtained by conditioning cortical magnetic stimulus with a peripheral stimulus to the median nerve at the wrist at the seven different ISIs studied. The amplitude of the conditioned MEP was expressed as a percentage of the amplitude of the test MEP. The percentage inhibition of the conditioned responses at the seven different ISIs was averaged to obtain a grand mean. Subjects were given audio–visual feedback at high gain to assist in maintaining complete relaxation.
### Statistical analysis
Statistics were carried out using the software environment R (R Core Team ), including the package npmv which is available at and is based on the nonparametric multivariate inference methodology described by Bathke et al. ( ).
We included group (patients vs. controls) as a between-subject factor and as within-factors (repeated measures; response variables) age, RMT, SAI, SICI, TUG, 6MWT, MMSE, RAVLT, Digit Span, TMT-A, and TMT-B. Together with the test, we provide also relative effects. The relative effects can be understood as tendencies expressed as estimated probabilities.
We also evaluated the correlation between SAI and the gait function tests (TUG and 6MWT), as well as between SAI and the neuropsychological tests (MMSE, RAVLT, TMT A and B, Digit Span) for the two groups. Spearman correlation was used for this purpose. Because of 14 separate correlations, we used Bonferroni correction to interpret the results.
To control whether SAI correlates with age, we performed again a Spearman correlation for the whole group.
## Results
We found a significant main effect of group ( F (5.28,200.59) = 28.13; p < 0.001). The closed-loop post hoc test shows that the main effect of group is significant for SAI, 6MWT, MMSE, RAVLT, Digit Span, TMT A, and TMT B, but not for age, RMT, SICI, and TUG.
Figure shows the distributions of RMT, SICI, and SAI for the two groups.
The distributions of RMT, SICI, and SAI are shown for the two groups are shown. As the boxplots reveal, the distributions overlap largely for RMT and SICI, resulting in no significant difference, while there is minimal overlap for SAI, resulting in a significant difference between groups. MSO maximum stimulator output
Relative effects indicate that the probability that a randomly chosen participant from the patient group exhibits a larger SAI than a randomly chosen participant from the control group is high with 0.81, and for 6MWT it is null (Table ).
Relative effects for factor group
SAI short latency afferent inhibition, RMT the resting motor threshold, SICI the short latency intracortical inhibition, TUG timed up and go test, 6MWT total walking distance for the 6‐min walk test, MMSE mini-mental scale examination, RAVLT rey auditory verbal learning test, TMT trail making test
The correlations are shown in Table and illustrated in Fig. . All variables (TUG, 6MWT, RAVLT, Digit Span, TMT A and TMT B) correlated with SAI in patients, but not in control subjects. After Bonferroni correction for multiple conduction of the correlation test, these correlations were still significant ( p divided by 14 correlations: p < 0.003).
Correlations separately for the two groups
TUG timed up and go test, 6MWT total walking distance for the 6-min walk test, MMSE mini-mental scale examination, RAVLT rey auditory verbal learning test, TMT trail making test
Scatterplots of SAI vs. TUG, 6MWT, MMSE, RAVLT, digit span, TMT-A, and TMT-B. The line represents the regression line, fitted to the data. SAI correlates significantly with all gait and neuropsychological tests. MMSE mini-mental scale examination, RAVLT rey auditory verbal learning test, TMT trail making test, TUG timed up and go test, 6MWT total walking distance for the 6-min walk test
SAI did not correlate significantly with age (rho = 0.25; p = 0.12).
## Discussion
The main and novel finding of this study was a significant reduction of SAI, a surrogate measure of cholinergic activity. Also SICI and RMT were decreased, similar to that reported in a previous TMS study (Chistyakov et al. ), even if in our study the differences did not reach statistical significance. Methodological issues could account for this discrepancy. In particular, in the previous study single- and paired-pulse TMS have been performed over leg motor area.
The present findings are consistent with previous experimental studies. So far, the only valid animal model of NPH is the chronic adult kaolin hydrocephalus (Khan et al. ). Adult rats with chronic kaolin hydrocephalus show cognitive impairment such as decreased learning and spatial memory, as well as other symptoms including gait ataxia and bradykinesia comparable to NPH patients (Del Bigio et al. , , , ).
In the adult kaolin hydrocephalus complex neurotransmitter disturbances, including the cholinergic system (Tashiro et al. ; Egawa et al. ), have been described.
Progressive injury to cholinergic systems (Tashiro et al. , ; Egawa et al. ), in combination with the delayed neuronal death in hippocampus (Klinge et al. ), may contribute to hydrocephalic dementia. In particular, progressive hydrocephalus results in functional injuries of cholinergic and GABAergic neurons in the neostriatum and dopaminergic neurons in the substantia nigra compacta by mechanical distortion (Tashiro et al. ).
In another experimental study in Wistar neonatal rats, the number of cholinergic neostriatal neurons was significantly reduced at 2 and 4 weeks in the acute form, at 8 weeks in the subacute form of hydrocephalus (Ishizaki et al. ).
The impairment of spatial memory in kaolin-induced hydrocephalic rats is associated with dysfunction of the hippocampal cholinergic and noradrenergic systems (Egawa et al. ).
The disturbance in balance of these neurotransmitter systems in the basal ganglia may explain some motor functional disabilities in hydrocephalus.
On the other hand, pathological, neuroimaging, and clinical evidence suggest that degeneration of cholinergic systems may contribute to impaired balance and gait in PD (Mancini et al. ).
Interestingly, significant associations were found between SAI, gait speed, inhibition, age and postural instability and gait disorder score in subjects with PD (but not control subjects) (Rochester et al. ). Regression analysis showed that reduced SAI was an independent predictor of slower gait speed in participants with PD.
It should be noted that SAI did not correlate in our study with age, therefore, the abnormal SAI values in our iNPH patients cannot be related only to the deterioration of the cholinergic system seen in normal human cognitive aging (Young-Bernier et al. ; Di Lorenzo et al. ).
The major limitation of our preliminary study was that we cannot exclude a concomitant AD pathology, which is present (neuritic plaques) in over 40% of patients (Golomb et al. ). Notably, there is a relationship between amyloid pathology (elevated amyloid precursor protein-derived proteins) and iNPH (Jeppsson et al. ) as well as between SAI and CSF beta-amyloid levels in AD (Martorana et al. ). It has been hypothesized, that AD and NPH might represent the extremes in a cluster of disorders characterized by a continuum of CSF circulatory failure with subsequent neurodegeneration (Silverberg et al. ). Indeed, many NPH cases remain with severe cognitive and motor deficits after shunting, even when ventricular size decreases postoperatively (Vanneste ; Kondziella et al. ).
Interestingly, in a recent paper SAI has been correlated to gait disturbances also in AD patients (Schirinzi et al. ), shedding light on the role of cholinergic system in cognitive motor control (Pelosin et al. ). Several studies are thus highlighted the usefulness of SAI as a marker of gait in cognitive impaired people. Conversely, SAI has been correlated to cognitive functions (especially memory more than executive function) in a sample of cognitively unimpaired old and young subjects (Young-Bernier et al. ; Bonnì et al. ) but not in AD patients (Koch et al. ). It should also be considered that patients with iNPH show a specific pattern of impairment on tests sensitive to frontostriatal dysfunction, which is distinct from that exhibited by patients with mild AD (Iddon et al. ).
In conclusion, the present study provides neurophysiological evidence that disturbances in balance of cholinergic neurotransmitter system may partly explain dementia and motor functional disabilities in subjects with NPH. It would be of great interest to examine in future studies the impact of shunt surgery on these neurophysiological findings.
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Rehabilitation for isolated forms of dystonia, such as cervical or focal hand dystonia, is usually targeted towards the affected body part and focuses on sensorimotor control and motor retraining of affected muscles. Recent evidence, has revealed people who live with dystonia experience a range of functional and non-motor deficits that reduce engagement in daily activities and health-related quality of life, which should be addressed with therapeutic interventions. These findings support the need for a holistic approach to the rehabilitation of dystonia, where assessment and treatments involve non-motor signs and symptoms, and not just the dystonic body part. Most studies have investigated Cervical Dystonia, and in this population, it is evident there is reduced postural control and walking speed, high fear of falling and actual falls, visual compensation for the impaired neck posture, and a myriad of non-motor symptoms including pain, fatigue, sleep disorders and anxiety and depression. In other populations of dystonia, there is also emerging evidence of falls and reduced vision-related quality of life, along with the inability to participate in physical activity due to worsening of dystonic symptoms during or after exercise. A holistic approach to dystonia would support the management of a wide range of symptoms and signs, that if properly addressed could meaningfully reduce disability and improve quality of life in people living with dystonia.
## Introduction
Dystonia is a neurological movement disorder, where one or more body parts are affected by involuntary, sustained or intermittent muscle contractions causing abnormal postures, repetitive movements, tics or tremors (Albanese et al. ). Cervical dystonia (CD) is the most common form of idiopathic isolated dystonia affecting the head and neck (Dauer et al. ), with motor dysfunction and pain causing significant distress and disability (Pauw et al. ; Dool et al. ). The most common medical treatment for CD are regular botulinum toxin injections (BTX) (Ferreira et al. ), even though patients express limited satisfaction (Comella and Bhatia ). Rehabilitation by allied health professionals usually takes the form of exercises of the neck, to reduce activity in contracted muscles and enhance strength and function of their antagonist muscles (Boyce et al. ; Pauw et al. ), with limited success. Many studies have provided physiotherapy with and without BTX in small clinical trials with varying results (Counsell et al. ; Hu et al. ; Tassorelli et al. ; Dool et al. ). There have been different rehabilitation approaches for CD, (Prudente ; Delnooz et al. ), however, these interventions all pirmarily target the impaired musculature of the neck. While motor symptoms such as head tremor, abnormal head posture and jerks are among the most burdensome symptoms, there is emerging evidence of non-motor symptoms such as pain, fatigue, sleep and mood disorders that further limit daily life functions and negatively impact on quality of life (Smit et al. , , ). Recent studies showing deficits in balance, gait and function (Barr et al. ), indicate a more holistic approach to rehabilitation is needed (Batla et al. ). In the current review, relevant dysfunctions outside the dystonic body part/s and their importance for holistic rehabilitation are summarised.
## Physical function, gait and balance
People living with CD exhibit impairments in physical function; not surprising considering the vital role of proprioception, visual and vestibular feedback in maintaining upright posture and balance. Central nervous system (CNS) processing of sensory inputs from all three systems are impacted by the head turn posture. Consequently, assessment of postural control and gait function should be included in physical assessments of dystonia patients. Emerging evidence demonstrates gait deficits and slower walking speed in people with CD when spatiotemporal parameters are measured in a laboratory setting (Barr et al. ; Hoffland et al. ; Esposito et al. ). Ten people living with CD were compared to ten control adults across a range of gait kinematic measures assessed using an instrumented walkway (Barr ). The dystonia group on average displayed reduced step length and increased step time, and spent more time in double support (both feet on the ground), all of which are consequences of poor balance control. In support, the time to perform the timed-up-and-go (TUG) test, a common test of physical function involving gait speed, turning and balance, was increased in CD (Barr et al. ( )). The CD group demonstrated greater postural sway, as measured by centre of pressure (COP) path length using a force plate, and increased choice, but not simple stepping reaction time. The functional deficits may be related to the aberrant head posture, as reduced cervical range of motion (neck flexion) was significantly correlated to delayed simple and choice reaction times and increased postural sway, while limited cervical rotation was associated with slower TUG completion time (Fig. a–d). There were also strong correlations between functional measures, with a longer time to complete the TUG associated with greater delay in stepping reaction times (Fig. a). Longer time spend in double support and reduced time in single support during gait is a known strategy for enhancing stability during walking. Not surprisingly, there was correlation between these measures in people living with CD (Fig. b), indicating maintaining balance during gait may be an issue for some, slowing down walking speed. Finally, longer time spend in double support and reduced time in single support was also associated with a higher rhomboid quotient (RQ) (Fig. c). The RQ is a ratio of postural sway between eyes closed and eyes open, and as normal adults sway more with eyes closed, the RQ is usually between 1 and 2. However, CD patients had lower RQ’s than controls as they swayed more with their eyes open in comparison to eyes closed. The RQ in dystonia was also correlated with gait kinematics (Barr et al. ). Greater postural sway amplitude and velocity was also reported in people living with CD compared to control adults during sitting postures; particularly noticeable in those with head tremor and more impaired cervical sensorimotor control (Pauw et al. ). We recently investigated postural sway in ten people with CD and ten matched controls, using an accelerometer attached to the waist rather than the gold-standard force plate. We recorded root mean square (RMS) of the acceleration in medial–lateral and anterior–posterior directions during eyes open and eyes closed conditions while in tandem stance. We found greater acceleration in the medial–lateral but not anterior–posterior directions in the CD group, and this postural sway was increased in the presence of head tremor. Instability in the medial–lateral direction could be an important new finding, as it can differentiate fallers from non-fallers in community-dwelling older adults (Park et al. ), so has potential as a screening test for falls risk in dystonia. Current treatment of CD is focused on the cervical region, however, the evidence summarised highlights the value of adding physical function assessments, and postural control and/or stepping reaction exercises along with gait rehabilitation into the therapeutic management of dystonia.
Correlations between cervical range of motion and functional measures from the study by Barr et al. ( ). a Cervical flexion and simple foot reaction time (RT), demonstrating longer RT was associated with reduced cervical flexion range of motion. b Cervical flexion and choice foot reaction time, demonstrating longer RT for both feet (ipsilateral and contralateral to the head turn direction) was associated with reduced cervical flexion. c Cervical flexion and COP path length during force plate postural sway measures for eyes open and eyes closed conditions, demonstrating greater COP pathlength for both conditions were associated with reduced cervical flexion. D. Cervical rotation (contralateral to dystonic rotation) and timed-up-and-go (TUG) test, where longer TUG times were associated with reduced cervical rotation
Correlations between functional measures from the study by Barr et al. ( ). a TUG and choice stepping reaction time, where longer RT was associated with slower times to complete the TUG. b Walking speed and time spend in single and double support, where longer time in double support and shorter time in single support was associated with slower walking speed. c Time spend in single and double support and the rhomboid quotient (RQ), where longer time in double support and shorter time in single support was associated with a lower RQ. Lower RQ indicates greater sway with eyes closed relative to eyes open, hence this finding indicates those with more ‘normal’ RQ’s actually presented with the impaired gait pattern
## Falling and fear of falling
Impairments of gait, balance and vision may impact on people with dystonia in ways that could result in falls. Fear of falling is high in people with CD, and was first reported by Hoffland and colleagues (Hoffland et al. ) using the Activities-Specific Balance Confidence Scale (ABC), and by Barr and co-workers (Barr et al. ) using the Falls Self-Efficacy Scale-International (FES-I). We later conducted an international survey of a mixed dystonic population to further explore the falls experience, and found 39% of the 122 respondents reported falling over in the previous 6 months (Boyce et al. , ). Many of the fallers were living with isolated forms of dystonia such as CD, blepharospasm and focal hand dystonia, and not dystonia directly affecting the trunk and/or lower limbs. This suggests falling may be a consequence of the physical function impacts of dystonia, such as poor sensorimotor control, balance and gait function. In that study, fallers had significantly more fear of falling and poorer balance confidence than those who did not fall according to the ABC and FES-I scales (Boyce et al. , ). We then validated both scales in the dystonia population and determined cut off points indicating falls risk for each scale. The cut off points for the ABC scale was 71.3 out of 100 (Boyce et al. ), somewhat lower than scores reported in Parkinson’s disease (Hoehn and Yahr 3, ABC cut off = 81) (Bello-Haas et al. ) and Multiple Sclerosis (mean score = 79) (Cameron and Huisinga ), but similar to the falls cut-off score of 67 reported in the healthy elderly (Lajoie and Gallagher ), despite the relatively younger sample of dystonia participants that completed our survey (51.2 ± 21.1 years). Similarly, the FES-I cut off point for a fall in the dystonia population was 29.5 out of 64 which is close to that of 29.6 reported in a mixed neurological population (Jonasson et al. ) and within the range that defines “high risk of falls” (cut off score > 23) in the healthy elderly (Delbaere et al. ). From the current research, it appears that people with dystonia report less fear of falling and higher balance confidence than people with other progressive neurological diseases, but similar fear of falling and balance confidence to older healthy people. This points to the importance of assessing balance and falls risk in the dystonia population during rehabilitation, even in those with isolated forms affecting the neck, face, voice or hand, and not just people with truncal or lower limb dystonia.
There were a variety of reasons for falling reported by survey respondents (Boyce et al. , ). The most common reasons were related to losing balance when walking, turning, reaching or using stairs, as illustrated by the following quotations from study participants; “walking and just lost balance “; “loss of balance and my feet are turning inwards”; and “lost my balance standing and walking”. Falling after tripping was also common, and often related to restricted vision due to CD or blepharospasm (BLP), as per the participant quotes; “Tripped over a raised metal edging outside of a shopping centre”; “tripped when stepping out from a picnic shelter”; “tripping over carpet rugs”; and “I was walking over my farm and didn't see a small rabbit hole which my foot went into”. Falls were reported as occurring as a direct result of dystonic impairments, illustrated by the quotes of; “I often lose track of where I am on stairs and sometimes skip steps going down stairs and fall”; “shopping in stores, always bumping into displays, people and certain lighting set Dystonia off”; “both feet turned in and I was walking on my ankles which made me fall down”; and “had Blepharospasms lost balance, or something in the way I didn't see”. Falls may occur secondarily to impaired vison, balance or proprioception due to dystonia, or caused by dystonic impairments causing difficulty in scanning the environment for hazards to ambulation. Tripping over objects due to poor vision related to a fixed head posture in CD or eye closure in blepharospasm may also cause falls, rather than impaired balance per se. This is supported by our research showing reduced vision-related quality of life in dystonia (Bradnam et al. ), where participant quotes also spoke of tripping or falling due vision impairment secondary to dystonic postures (see below). We recently completed a prospective study in adults with CD, investigating functional balance and walking tests using scales validated in other neurological populations or healthy older people. Our preliminary analysis suggests balance and mobility may not be as impaired as previously indicated, as people with CD mostly performed well on the physical functional scales. Therefore, it is possible that the fear of falling previously reported (Boyce et al. ) may be more related to psychological rather than physical limitations. While this idea is speculative and requires further investigation, it has an important impact on the way people with dystonia are managed during rehabilitation following a fall. In these cases, improving balance confidence from a psychological perspective could be more influential than physical or exercise-based rehabilitation. Regardless, falls-related fear should be addressed from a multi-disciplinary perspective, and falls prevention in the most appropriate format based on robust physical and psychological assessment should be included in rehabilitation programs.
## Vision and function
People living with CD exhibit increased postural sway with their eyes open compared to control adults, indicating vision is not used to maintain centre of gravity within the base of support to the same degree as normal (Barr et al. ). This may arise due to the abnormal head posture in CD, meaning vision cannot be relied upon to provide reference points for spatial orientation and balance. Certainly, vision impairment as a consequence of dystonic postures is a major issue for many people living with CD. A survey of 42 mixed dystonia participants found reduced vision-related quality of life compared to normative data (Bradnam et al. ). People living with dystonia significantly differed on two domains of a vision-related quality of life questionnaire; ocular symptoms and role performance. The following participant quotes supported reduced vision-related quality of life in a powerful way; ‘blurriness, tired eyes, eyes not facing what I want to see due to twisted head—have to look out of the corner of my eye or not look at all’; ‘focusing difficulty, judgement of distance in regard to steps and narrow walkways. I become unbalanced easily’; ‘my field of vision is affected when walking by head pulling to right’; ‘the only difficulty I have is looking at things directly because my head turns. That is, I find I have to look at some things with my peripheral vision’; ‘because I need to assist my head to be straight forward with my hand is my main problem. When I do this I can see quite o.k.’. These previously unpublished quotes point to a relationship between head posture, vision and functional impairments, including balance, which may help to explain the incidence of falls and high fear of falling in the dystonia population outlined in the previous section. Neurological vision deficits should also be considered in CD, as we found one young female participant (out of ten) to have marked visuospatial neglect using a battery of computer-based and pencil and paper spatial neglect tests (Bradnam et al. ). Clinicians should be aware of the possibility their CD patients could experience neglect and screening for this should be included in a comprehensive assessment of dystonia. In this study, we used mobile eye-tracking to determine the direction of eye movements made by people with CD when navigating an indoor circuit and identifying visual targets. Participants made a significant number of eye movements away from the dystonic head turn direction, indicating compensatory behaviour arising from the abnormal head posture (Bradnam et al. ). What is unknown, and of potential important impact for navigation around real world environments, is whether such compensatory eye movements induce fatigue in the oculomotor system. A fatigue-inducing paradigm in healthy young adults impaired saccade velocity (speed of eye movement) due to central neural mechanisms (Connell et al. , , ). With relevance for individuals with CD, making compensatory eye movements may produce oculomotor fatigue with potential safety consequences when navigating fast-paced, real-life environments requiring visual compensation for a rotated neck, such as crossing a busy road. Clinicians and patients alike should be made aware of the potential for oculomotor fatigue secondary to repetitive eye movements in the contralateral direction to head turn so they can make the necessary compensations. We are currently investigating eye movement adaptation in people with CD and have found in our preliminary data from nine participants that normal adaptation occurs in the contralateral, but not ipsilateral, direction relative to the dystonic head turn. This may indicate the compensatory movements in the contralateral direction described by Bradnam et al. ( ), may provide adequate stimulation to the cerebellar structures responsible for saccade adaptation. This would not be the case for the ipsilateral direction as the eyes are rarely moved that way since the head is already turned. If this novel finding is maintained in a larger participant group it suggests eye movement training in the ipsilateral direction may be a therapeutic intervention to possibly normalise cerebellar-mediated saccade adaptation and oculomotor function. Vision impairment secondary to dystonic postures and its impact on physical function, visual compensation and oculomotor fatigue along with potential neurological impairments like spatial neglect should be considered important components of holistic rehabilitation of dystonia.
## Non-motor symptoms
Many people living with dystonia experience non-motor symptoms contributing to disability and reducing participation in daily activities (Smit et al. ; Stamelou et al. ; Torres and Rosales ), leading to the development of non-motor symptom scales for dystonia (Smit et al. ; Klingelhoefer et al. ). Non-motor symptoms featured strongly when people with CD were asked about their most burdensome symptoms (Smit et al. ) and the most prevalent experienced by people with idiopathic, isolated dystonia were pain, depression, anxiety, apathy, and impaired sleep (Smit et al. ; Novaretti et al. ). Other non-motor symptoms such as fatigue, catastrophizing, sensorimotor disturbances, olfactory and visual problems have also been noted amongst others, and also impact negatively on quality of life (Zetterberg et al. ). Non-motor symptoms are important when considering the overall management of dystonia as they play a significant role in quality of life (Smit et al. ; Torres and Rosales ; Tomic et al. ).
Pain is a prevalent and debilitating non-motor symptom in dystonia. In people with CD, pain is reported in 55–89% of people (Avenali et al. ). The prevalence of pain in other dystonia types is lower than CD, with studies reporting pain was a symptom in 30–40% of people with FHD or lower limb dystonia and only 3% in BLP primarily related to photophobia pain (Avenali et al. ). Despite being a common co-occurring symptom of dystonia, current knowledge of the mechanism of dystonic pain is incomplete and effective management strategies are largely insufficient (Avenali et al. ). Pain is very likely not only a consequence of having a sustained muscle contraction caused by dystonia but may primarily arise from abnormal neural processing. Better understanding how people with dystonia describe the pain they experience is the focus of our current research, which will be used to help inform appropriate therapeutic strategies. To date, pain is most commonly treated with BTX and medication (Marciniec et al. ; Siongco et al. ). Novel cerebellar neuromodulation techniques also show promise for reducing pain in CD when combined with physical therapy as an adjuvant to BTX injections (Bradnam et al. , ). Evidence for oral medications for management of pain in CD or other focal dystonia’s is lacking (Avenali et al. ).
Neuropsychiatric features also commonly coexist with dystonia, be the case focal, segmental or generalized, idiopathic or heredodegenerative dystonia’s (Torres and Rosales ). These broadly include depression, anxiety, personality disorder, obsessive–compulsive disorder (Stamelou et al. ), as well as reduced self-efficacy, catastrophizing, fear of movement, and stigma (Zetterberg et al. ). Neuropsychiatric comorbidities have been found to be significantly higher than controls and are prevalent in all forms of idiopathic dystonia, though the prevalence is particularly high in CD (up to 90%) (Avenali et al. ). Interestingly, psychiatric features such as depression and anxiety precede the onset of motor symptoms for many people, suggesting they may be a primary feature of dystonia (Fabbrini et al. ; Wenzel et al. ). However, given the debilitating experience of pain and many other symptoms that co-occur with dystonia it is likely that a proportion of patients experience depression as a secondary feature of living with their dystonic symptoms. Although the relationship between dystonia severity and the severity of depression is unclear, studies in CD or segmental and generalized dystonia patients show that mood does improve with positive treatment effects (Stamelou et al. ; Torres and Rosales ).
Fatigue appears to be a major issue and occurs independently to psychological factors and quality of sleep (Smit et al. ; Wagle Shukla et al. ). Fatigue emerged as a significant barrier to participation in exercise and physical activity in our own research (McCambridge et al. ), detailed below. Fatigue in dystonia is worthy of further exploration and holistic rehabilitation strategies should include fatigue management. Fatigue commonly is associated with sleep disturbances and disrupted sleep is also a non-motor symptom of dystonia, with an estimated prevalence between 44 and 70% in CD and BLP patients (Eichenseer et al. ; Paus et al. ). Studies on sleep in dystonia have found a reduction in sleep quality and efficiency, less time in REM (rapid eye movement) sleep, and more awakenings (Silvestri et al. ; Sforza et al. ). Impaired sleep quality or excessive daytime sleepiness is associated with depression and anxiety in dystonia (Smit et al. ; Wagle Shukla et al. ; Paus et al. ), however, the association between impaired sleep and severity of dystonia is mixed. Sleep quality is also not improved by BTX injection (Eichenseer et al. ), though this has not been specifically explored. Non-motor symptoms contribute significantly to the disability experience of those living with dystonia, and must be addressed to take a truly holistic approach to therapeutic management and rehabilitation (Torres and Rosales ).
## Exercise and physical activity
Exercise is not only important for cardiometabolic health in general, but for neurological populations, it also has the potential to improve neuroplasticity and provide therapeutic benefits. All the above-mentioned movement impairments and non-motor symptoms experienced, along with vision-related impairments, have implications for the amount of physical activity (PA) and exercise that people living with dystonia engage in. Several neurological populations (Parkinson’s disease, Multiple Sclerosis, Stroke) have been extensively investigated for benefits of PA on disease-specific signs such as fatigue, depression and pain, and general cardiovascular and musculoskeletal health (Latimer-Cheung, et al. , ; Motl et al. ). In these conditions, remaining active can attenuate disease progression and physical deconditioning, and maintain or improve cognitive function, and exercise guidelines have been published (Kim et al. ). However, in a systematic review that analysed the number of studies assessing physical activity in neurological populations, no studies in people living with dystonia were included (Block et al. ). There is little understanding of how PA and exercise engagement may affect physical and psychological health in people living with dystonia. Even less research has investigated the effects that sedentary behaviour (SB), an independent risk factor for poor health, has on health in dystonic populations. Exercise guidelines specific to this patient cohort are needed. We recently published the first investigation of this kind; an international survey to determine the amount of PA and SB engagement and reported barriers and facilitators to physical activity using questionnaires (McCambridge et al. ). Upon analysis of the 263 people who completed the questionnaires, people living with dystonia appeared to achieve the minimum recommendations for PA, by means of incidental activity during transport and domestic activities (McCambridge et al. ). However, on average, people reported spending in excess of 8 h per day in sedentary behaviour. Common barriers to engaging in PA that were identified were personal barriers, relating to physical impairments, and financial barriers and a lack of trained exercise specialists (McCambridge et al. ). The most reported dystonic symptom barriers were pain, fatigue and poor balance. Zetterberg and colleagues (Zetterberg et al. ) found that employment as well as self-efficacy for exercise had the greatest association with physical activity in their survey of over 350 people with cervical dystonia (Zetterberg et al. ). Furthermore, many people with dystonia do not exercise as it tends to aggravate a range of dystonic symptoms (McCambridge et al. ). However, our survey revealed that lower intensity exercise was less aggravating for dystonia symptoms than high-intensity exercise (Fig. ), a finding that has potential implications for prescribing exercise in practice. When asked what would help them be more active, symptom reduction was a common reply, as illustrated by the following participant quotes; I'm as physically active as symptoms permit on any given day. So only reduced symptoms could make me more active’ and ‘To be less tired from medications; to be spasm and pain free; to have better posture and balance for more than a few minutes at a time; to have more energy; to have daily enjoyable beneficial short sessions of exercises or gentle short walks with supportive people. Encouragement and inclusion are motivating for everyone’. Other quotes included; ’More energy, less social anxiety’, ‘Some way to reduce the tremor and muscle spasm’, ‘If I had better balance and movement in my neck’, Less pain, able to walk without pain’, ‘Increased motivation as I am so fatigued’ and ‘I am as physically active as I am capable of being without significantly aggravating my symptoms and pain’.
Physical activity and exercise and their impact on dystonia symptoms in survey respondents from the study by McCambridge et al. ( ) showing the proportion (percentage) of respondents that answered ‘better’ (green), ‘no change’ (blue) and ‘worse’ (red) for each activity. Obvious higher impact exercise tended to worsen dystonia, while low impact exercise may be beneficial, or at least not aggravating for around 2/3 of dystonia patients
People with dystonia face extensive barriers to physical activity and exercise engagement and more effective tailored interventions are needed to reap the benefits of activity for overall health and well-being. Future studies are critical as they have the potential to inform behaviour change addressing barriers in order to promote feasible and beneficial activity behaviours in people with dystonia. It is also important to understand motivational aspects of why people living with CD continue to exercise despite worsening of dystonic symptoms to inform a behavioural intervention with a self-management focus, and a study is currently underway for this.
## Discussion
Treatments for dystonia offer limited effectiveness and patient satisfaction. A reason for this could be that current therapies are too narrow focused and have not considered the wider impacts of dystonia on motor function as well as non-motor symptoms (Batla et al. ; Torres and Rosales ; Franco and Rosales ). In Fig. , we have depicted the motor and non-motor symptoms of dystonia on an ice-berg where the motor symptoms are often seen as the problem that needs to be addressed, but there are many more unseen problems that are significant contributors to a person’s level of disability and quality of life (Pauw et al. ; Dool et al. ; Smit et al. , ; Zetterberg et al. ; Tomic et al. ). Tremor and jerks, pain and fatigue, balance and gait, fear of falling, vision issues, poor sleep, anxiety and depression cause significant disability, negative impact on daily life, and reduce quality of life (Dool et al. ; Smit et al. , , ; Zetterberg et al. ; Tomic et al. ). Therapists should begin to consider and integrate all of these aspects into a person’s rehabilitation. Recognition and rehabilitation of the wider spectrum of dystonic signs and symptoms could be key to improving treatment outcomes, life participation, and overall quality of life for people living with dystonia.
Visualisation of the motor and non-motor symptoms of dystonia as an ice-berg with physical symptoms that can be seen, but non-motor symptoms that often go unseen
Without a curative treatment for dystonia, the focus for therapists must be on alleviating the symptoms which encompass more than the dystonic muscle contraction. To begin to provide a holistic treatment, therapists will need to perform assessments of these issues. Physical therapists should include gait and physical function and balance tests (e.g. TUG, Berg Balance scale), and clinical assessment of gait in their overall assessment of a patient. In addition, non-motor symptoms could be screened for using a validated non-motor symptom scale (Smit et al. ; Klingelhoefer et al. ). The Dystonia Non-Motor Symptoms Questionnaire (Smit et al. ) includes multiple domains, such as sleep, fatigue, emotional well-being, sensory symptoms, activities of daily living, autonomic symptoms and stigma, and could be easily implemented into routine clinical practice. The multitude of findings that people living with dystonia experience significant psychological issues, such as depression and anxiety, fear of falling and heightened feelings of stigma, strongly suggests that a multi-disciplinary approach to rehabilitation is needed. Self-efficacy and self-management strategies are vital for adherence to long-term rehabilitation programs. Self-management interventions are another component of treatment for dystonia that is currently under explored and should urgently be addressed.
Finally, an awareness of maintaining an overall healthy lifestyle is also needed for people with dystonia. In our self-reported PA research, we found that people were reporting adequate levels of PA, though they did not feel supported or guided with their exercise routines (McCambridge et al. ). We are currently following up this research with objective measurements, and we hope to better understand the health impacts of dystonia on obesity and other co-morbidities. Exercise guidelines developed specifically for dystonia, that are prescribed to the patient by a trained professional, are needed to reduce barriers to participation and facilitate dystonia patients to engage in PA without symptom exacerbation. Only after careful consideration of these issues can we understand the longer term benefits of exercise and PA on participation and quality of life in people living with dystonia.
While the practice of neurorehabilitation has its place in dystonia (Franco and Rosales ), a holistic rehabilitation approach is needed for managing the myriad of symptoms, both motor and non-motor, experienced by people living with isolated dystonia (Batla et al. ). Motor symptoms such sustained muscle contractions and abnormal postures, tremor and muscle jerks are often the main focus of treatment. However, pain, depression, stigma, impaired sleep, and fatigue are only a few of the debilitating non-motor symptoms that contribute to the lived experience of dystonia (Torres and Rosales ). Given that non-motor symptoms have been found to decrease health-related quality of life more so than motor symptoms (Smit et al. , , ; Torres and Rosales ; Zetterberg et al. ; Wagle Shukla et al. ), a multi-disciplinary approach to rehabilitation must be adopted. Future studies should continue to explore the wider impairments associated with dystonia, and the impact of a holistic rehabilitation approach using carefully designed multi-disciplinary clinical trials on patient-reported outcome measures.
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Synthesis of acetylcholine depends on the plasma membrane uptake of choline by a high affinity choline transporter (CHT1). Choline uptake is regulated by nerve impulses and trafficking of an intracellular pool of CHT1 to the plasma membrane may be important for this regulation. We have generated a hemagglutinin (HA) epitope tagged CHT1 to investigate the organelles involved with intracellular trafficking of this protein. Expression of CHT1-HA in HEK 293 cells establishes Na+-dependent, hemicholinium-3 sensitive high-affinity choline transport activity. Confocal microscopy reveals that CHT1-HA is found predominantly in intracellular organelles in three different cell lines. Importantly, CHT1-HA seems to be continuously cycling between the plasma membrane and endocytic organelles via a constitutive clathrin-mediated endocytic pathway. In a neuronal cell line, CHT1-HA colocalizes with the early endocytic marker green fluorescent protein (GFP)-Rab 5 and with two markers of synaptic-like vesicles, VAMP-myc and GFP-VAChT, suggesting that in cultured cells CHT1 is present mainly in organelles of endocytic origin. Subcellular fractionation and immunoisolation of organelles from rat brain indicate that CHT1 is present in synaptic vesicles. We propose that intracellular CHT1 can be recruited during stimulation to increase choline uptake in nerve terminals. |
There is now considerable knowledge concerning neuron death following necrotic insults, and it is believed that the generation of reactive oxygen species (ROS) and oxidative damage play a pivotal role in the neuron death. Prompted by this, we have generated herpes simplex virus-1 amplicon vectors over-expressing the genes for the antioxidant enzymes catalase (CAT) or glutathione peroxidase (GPX), both of which catalyze the degradation of hydrogen peroxide. Over-expression of each of these genes in primary hippocampal or cortical cultures resulted in increased enzymatic activity of the cognate protein. Moreover, each enzyme potently decreased the neurotoxicity induced by kainic acid, glutamate, sodium cyanide and oxygen/glucose deprivation. Finally, these protective effects were accompanied by parallel decreases in hydrogen peroxide accumulation and the extent of lipid peroxidation. These studies not only underline the key role played by ROS in the neurotoxicity of necrotic insults, but also suggest potential gene therapy approaches. |
In the present work, several experimental approaches were used to determine the presence of the glucagon-like peptide-1 receptor (GLP-1R) and the biological actions of its ligand in the human brain. In situ hybridization histochemistry revealed specific labelling for GLP-1 receptor mRNA in several brain areas. In addition, GLP-1R, glucose transporter isoform (GLUT-2) and glucokinase (GK) mRNAs were identified in the same cells, especially in areas of the hypothalamus involved in feeding behaviour. GLP-1R gene expression in the human brain gave rise to a protein of 56 kDa as determined by affinity cross-linking assays. Specific binding of 125I-GLP-1(7-36) amide to the GLP-1R was detected in several brain areas and was inhibited by unlabelled GLP-1(7-36) amide, exendin-4 and exendin (9-39). A further aim of this work was to evaluate cerebral-glucose metabolism in control subjects by positron emission tomography (PET), using 2-[F-18] deoxy-D-glucose (FDG). Statistical analysis of the PET studies revealed that the administration of GLP-1(7-36) amide significantly reduced (p < 0.001) cerebral glucose metabolism in hypothalamus and brainstem. Because FDG-6-phosphate is not a substrate for subsequent metabolic reactions, the lower activity observed in these areas after peptide administration may be due to reduction of the glucose transport and/or glucose phosphorylation, which should modulate the glucose sensing process in the GLUT-2- and GK-containing cells. |
Apoptosis may be initiated in neurons via mitochondrial release of the respiratory protein, cytochrome c. The mechanism of cytochrome c release has been studied extensively, but little is known about its dynamics. It has been claimed that release is all-or-none, however, this is not consistent with accumulating evidence of cytosolic mechanisms for 'buffering' cytochrome c. This study has attempted to model an underlying disease pathology, rather than inducing apoptosis directly. The model adopted was diminished activity of the mitochondrial respiratory chain complex I, a recognized feature of Parkinson's disease. Titration of rat brain mitochondrial respiratory function, with the specific complex I inhibitor rotenone, caused proportional release of cytochrome c from isolated synaptic and non-synaptic mitochondria. The mechanism of release was mediated, at least in part, by the mitochondrial outer membrane component Bak and voltage-dependent anion channel rather than non-specific membrane rupture. Furthermore, preliminary data were obtained demonstrating that in primary cortical neurons, titration with rotenone induced cytochrome c release that was subthreshold for the induction of apoptosis. Implications for the therapy of neurodegenerative diseases are discussed. |
In rat frontal cortex, extracellular levels of glutamate are raised by the anti-psychotic drug clozapine. We have recently shown that a significant reduction in the levels of the glutamate transporter GLT-1 may be one of the mechanisms responsible for this elevation. Here we studied whether GLT-1 down-regulation induced by chronic clozapine treatment is associated with changes in the expression of synaptophysin, synaptosome-associated protein of 25 kDa (SNAP-25) and vesicular glutamate transporter 1 (VGLUT1), three major presynaptic proteins involved in neurotransmitter release. Quantitative high-resolution confocal microscopy studies in vivo showed that GLT-1 down-regulation is closely associated with a significant increase in synaptophysin, but not SNAP-25 and VGLUT1, expression. This was confirmed in vitro studies, and in western blotting studies of synaptophysin, SNAP-25 and VGLUT1. In addition, our results show that, following clozapine treatment, synaptophysin expression increases in the very cortical regions in which GLT-1 expression is down-regulated. These findings suggest that part of the effects of clozapine may be exerted via an action on the presynaptic machinery involved in neurotransmitter release. |
Mammalian cells synthesize ceramide in the endoplasmic reticulum (ER) and convert this to sphingomyelin and complex glycosphingolipids on the inner, non-cytosolic surface of Golgi cisternae. From there, these lipids travel towards the outer, non-cytosolic surface of the plasma membrane and all membranes of the endocytic system, where they are eventually degraded. At the basis of the selective, anterograde traffic out of the Golgi lies the propensity of the sphingolipids to self-aggregate with cholesterol into microdomains termed 'lipid rafts'. At the plasma membrane surface these rafts are thought to function as the scaffold for various types of (glyco) signaling domains of different protein and lipid composition that can co-exist on one and the same cell. In the past decade, various unexpected findings on the sites where sphingolipid-mediated events occur have thrown a new light on the localization and transport mechanisms of sphingolipids. These findings are largely based on biochemical experiments. Further progress in the field is hampered by a lack of morphological techniques to localize lipids with nanometer resolution. In the present paper, we critically evaluate the published data and discuss techniques and potential improvements. |
Prion diseases are transmissible fatal neurodegenerative diseases of humans and animals, characterised by the presence of an abnormal isoform (scrapie prion protein; PrP(Sc)) of the endogenous cellular prion protein (PrP(C)). The pathological mechanisms at the basis of prion diseases remain elusive, although the accumulation of PrP(Sc) has been linked to neurodegeneration. Different genomic approaches have been applied to carry out large-scale expression analysis in prion-infected brains and cell lines, in order to define factors potentially involved in pathogenesis. However, the general lack of overlap between the genes found in these studies prompted us to carry an analysis of gene expression using an alternative approach. Specifically, in order to avoid the complexities of shifting gene expression in a heterogeneous cell population, we used a single clone of GT1 cells that was de novo infected with mouse prion-infected brain homogenate and then treated with quinacrine to clear PrP(Sc). By comparing the gene expression profiles of about 15 000 genes in quinacrine-cured and not cured prion-infected GT1 cells, we investigated the influence of the presence or the absence of PrP(Sc). By real-time PCR, we confirmed that the gene encoding for laminin was down-regulated as a consequence of the elimination of PrP(Sc) by the quinacrine treatment. Thus, we speculate that this protein could be a specific candidate for further analysis of its role in prion infection and pathogenesis. |
Neuropeptides in the stomatogastric ganglion (STG) and the brain of adult and late embryonic Homarus americanus were compared using a multi-faceted mass spectral strategy. Overall, 29 neuropeptides from 10 families were identified in the brain and/or the STG of the lobster. Many of these neuropeptides are reported for the first time in the embryonic lobster. Neuropeptide extraction followed by liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry enabled confident identification of 24 previously characterized peptides in the adult brain and 13 peptides in the embryonic brain. Two novel peptides (QDLDHVFLRFa and GPPSLRLRFa) were de novo sequenced. In addition, a comparison of adult to embryonic brains revealed the presence of an incompletely processed form of Cancer borealis tachykinin-related peptide 1a (CabTRP 1a, APSGFLGMRG) only in the embryonic brain. A comparison of adult to embryonic STGs revealed that QDLDHVFLRFa was present in the embryonic STG but absent in the adult STG, and CabTRP 1a exhibited the opposite trend. Relative quantification of neuropeptides in the STG revealed that three orcokinin family peptides (NFDEIDRSGFGF, NFDEIDRSGFGFV, and NFDEIDRSGFGFN), a B-type allatostatin (STNWSSLRSAWa), and an orcomyotropin-related peptide (FDAFTTGFGHS) exhibited higher signal intensities in the adult relative to the embryonic STG. RFamide (Arg-Phe-amide) family peptide (DTSTPALRLRFa), [Val(1)]SIFamide (VYRKPPFNGSIFa), and orcokinin-related peptide (VYGPRDIANLY) were more intense in the embryonic STG spectra than in the adult STG spectra. Collectively, this study expands our current knowledge of the H. americanus neuropeptidome and highlights some intriguing expression differences that occur during development. |
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