Dissertationen zum Thema „Balance locale Excitation/Inhibition“

En psychologie, le contrôle inhibiteur est un mécanisme cognitif qui permet de stopper une ré-ponse motrice, émotionnelle ou cognitive non adaptée pour la réalisation d’un but désiré. Au niveaucérébral, le contrôle inhibiteur est associé au fonctionnement en réseau de régions cérébrales, quipeut être mesuré à partir du signal BOLD en IRMf. Des régions de contrôle préfrontales abaissentl’activité BOLD de régions cibles. L’IRMf permet de mesurer de manière indirecte l’activité desneurones. Comment peut-on alors inférer à partir de données d’IRMf des propriétés excitatriceset inhibitrices (E/I) neurales au sein de régions cérébrales impliquées dans une tâche de contrôleinhibiteur ?Nous partons d’un modèle biophysique non linéaire, hiérarchique qui décrit les évolutions tempo-relles des activités neurales excitatrice et inhibitrice par région (Naskar et al., 2021). Ces varia-tions d’activité produisent des changements BOLD dans chaque région cérébrale. L’analyse de cemodèle nous permet de : 1) identifier des paramètres neuraux de la balance E/I ; 2) montrer quel’augmentation d’activité BOLD d’une région de contrôle ne permet pas d’abaisser l’activité BOLDd’une région cible parce que les régions sont connectées par leurs neurones excitateurs uniquement ;3) proposer une nouvelle architecture de connectivité pour le permettre ; 4) étudier comment labaisse d’activité de la région cible dépend de la balance E/I dans la cible. Nous proposons alorsune nouvelle procédure d’inversion. Nous en vérifions la fiabilité avec des simulations, avant deprésenter une preuve de concept sur les données d’un sujet pendant une tâche de Think/NoThink,un paradigme d’étude du contrôle inhibiteur des intrusions mnésiques (Mary et al., 2020)
In psychology, inhibitory control is a cognitive mechanism that stops a motor, emotional orcognitive response from achieving a desired goal. At cerebral level, inhibitory control is associatedwith a network of brain regions, whose function may be measured using BOLD signals from fMRI.Prefrontal control regions lower the BOLD activity of target regions. fMRI provides an indirectmeasure of the activity of neurons. How can we then infer from fMRI data, neural excitatory andinhibitory (E/I) properties of brain regions involved in an inhibitory control task ?We start with a non-linear biophysical model that describes by region the temporal evolutionof neural excitatory and inhibitory activities (Naskar et al., 2021). These variations in activityproduce BOLD changes in each brain region. Analysis of this model enables us to : 1) identifyneural parameters of the E/I balance ; 2) show that increasing the BOLD activity of a controlregion does not lower the BOLD activity of a target region, since these regions are connected bytheir excitatory neurons only ; 3) propose a new connectivity architecture to enable this ; 4) studyhow the lowering of activity in the target region depends on the E/I balance in the target region.We then propose a new inversion procedure. We check its reliability through simulations, beforepresenting a proof-of-concept using real data from a subject during a Think/No-Think task, aparadigm used for studying the inhibitory control of memory intrusions (Mary et al., 2020)

The medial prefrontal cortex (mPFC} and hippocampus (HPC} are central to executive control, spatial learning and working memory. In order to enable behavioral control, the function of the mPFC and HPC is tuned by complex interplay between excitatory (glutamatergic) and inhibitory (GABAergic) neurotransmitter systems. This thesis has employed lesions, pharmacological and optogenetic methodologies to investigate how the relationship between excitation and inhibition within the adult mouse mPFC and HPC affects cognition, using a battery of touchscreen-based operant assays: the automated spatial array task (ASAT), Spatial Reversal (SR) and Visual Discrimination (VD). Behavioral testing following excitotoxic lesions showed that the HPC was strongly implicated in the performance of both spatial tests (ASAT and SR), while removal of the mPFC had only marginal effects on learning with several trends that did not reach significance. Additionally, in VD, effects were only present as trends towards an involvement of the mPFC in formations of new stimulus-reward relationships. Interdependent processing spanning the mPFC and HPC while not directly assessed can be considered likely to explain complex changes in task performance. Using the described assays helped validate their application to test mPFC and HPC function in mice.

Les travaux de l'équipe sont centrés sur la régulation de l'excitabilité et de la plasticité des réseaux neuronaux. La modulation de l'intégration synaptique est étudiée au niveau cortical où il s'agit de déterminer quels sont les acteurs de la plasticité homéostatique et comment l'action spécifique de certains types de récepteurs (cholinergique, sérotoninergique) peut moduler la balance excitation/inhibition déterminée dans des neurones pyramidaux de couche 5 qui génèrent les signaux de sortie du cortex visuel.

The generation of complex behaviors often requires the coordinated activity of diverse sets of neural circuits in the brain. Activation of neuronal circuits drives behavior. Inappropriate signaling can contribute to cognitive disorders such as epilepsy, Parkinson’s, and addiction (Nordberg et al., 1992; Quik and McIntosh, 2006; Steinlein et al., 2012). The molecular mechanisms by which the activity of neural circuits is coordinated remain unclear. What are the molecules that regulate the timing of neural circuit activation and how is signaling between various neural circuits achieved? While much work has attempted to address these points, answers to these questions have been difficult to ascertain, in part owing to the diversity of molecules involved and the complex connectivity patterns of neural circuits in the mammalian brain. My thesis work addresses these questions in the context of the nervous system of an invertebrate model organism, the nematode Caenorhabditis elegans. The locomotory circuit contains two subsets of motor neurons, excitatory and inhibitory, and the body wall muscle. Dyadic synapses from excitatory neurons coordinate the simultaneous activation of inhibitory neurons and body wall muscle. Here I identify a distinct class of ionotropic acetylcholine receptors (ACR-12R) that are expressed in GABA neurons and contain the subunit ACR-12. ACR-12R localize to synapses of GABA neurons and facilitate consistent body bend amplitude across consecutive body bends. ACR-12Rs regulate GABA neuron activity under conditions of elevated ACh release. This is in contrast to the diffuse and modulatory role of ACR-12 containing receptors expressed in cholinergic motor neurons (ACR-2R) (Barbagallo et al., 2010; Jospin et al., 2009). Additionally, I show transgenic animals expressing ACR-12 with a mutation in the second transmembrane domain [ACR-12(V/S)] results in spontaneous contractions. Unexpectedly, I found expression of ACR-12 (V/S) results in the preferential toxicity of GABA neurons. Interestingly loss of presynaptic GABA neurons did not have any obvious effects on inhibitory NMJ receptor localization. Together, my thesis work demonstrates the diverse roles of nicotinic acetylcholine receptors (nAChRs) in the regulation of neuronal activity that underlies nematode movement. The findings presented here are broadly applicable to the mechanisms of cholinergic signaling in vertebrate models.

The balance of synaptic excitation and inhibition plays a very important role in maintaining the function of central nervous system (CNS) and the imbalance is involved in neurologic diseases such as autism and epilepsy. PSD95 and gephyrin have been studied as scaffolding proteins, having critical functional and structural roles in excitatory and inhibitory synapses, respectively. In the thesis, I have attempted to develop small systemically applicable peptides that can reversibly knock down PSD95 or gephyrin in vitro and in vivo, using the novel peptide-mediated technology recently developed in our lab, as tools for modulating the balance of synaptic excitation and inhibition. The efficacy of the peptides knocking down respective targeted proteins was tested by immunoblots after the cultured neurons were treated with the peptides for the desired time at various concentrations. I found that peptides that target either PSD95 or gephyrin showed toxicity to the neurons in a dose and time dependent manner utilizing the LDH assay. The toxicity may also contribute to the reduction of protein levels. Using one of the peptides, TAT-NR2B9C-CMA that targets PSD95 as example, I systemically investigated the causes of the toxicity and tested several strategies to reduce the toxicity while keeping the efficacy of the protein knockdown. I found that while multiple treatments at low dose could not successfully separate the cell death and knockdown effect, treatment at high doses with shortened durations appeared partially effective in reducing the toxicity and maintaining knockdown efficacy. However, this protocol may not be applicable in vivo. I next modified the intrinsic properties of peptides by shortening CMA targeting motifs and/or adding a linker between the binding sequences and CMA targeting motif. I found that while both strategies could decrease the toxicity with varied degree, peptides with short CMA targeting motif kept the knockdown efficacy. Taken together, my study demonstrated the effective strategies to reduce the toxicity of the peptides one can consider in the process of developing novel protein knockdown peptides as novel research tools and therapeutic reagents.
Medicine, Faculty of
Graduate

One prominent feature of brain computation is the excitation inhibition balance (E/I balance) that represents one of the main homeostatic functions of the brain. Its aim is to maintain the neural circuits in a narrow and safe range of action. Within this range, the brain network can receive and analyze sensory inputs and produce a modulated output, proportional to the stimuli intensity. Any imbalance in this equilibrium leads to abnormal responses to external stimuli and results in pathological behavior. Indeed, neurological pathologies known for featuring a deep alteration of the E‐I balance are epilepsy and autism, which often occur together in the same patient. Several human genetic syndromes caused by alterations of genes involved in neural development feature signs like autism and epilepsy. Thus, they represent important cases for studying and understanding the role of these single altered genes in the development and regulation of the brain balance. In return, we hope that this knowledge of these genes and more generally of human brain network can be useful in treating the patients affected by these conditions and can help us improve their quality of life. In my work, I studied the regulation of the E/I balance in mouse models of neurological diseases from three different points of view. In the first set of experiments, I studied the E/I balance in a focal model of epileptiform activity. This model is produced by the local application of bicuculline to the mouse cortex. Bicuculline is a competitive GABAA receptor antagonist that, when applied, leads to the development of persistent and periodic interictal spikes at the injection site, while activity appears to be normal in nearby areas that are not reached by bicuculline. In our experiments, we showed that, even in the apparently normal area, there is a disruption of cortical computation. Specifically, the disruption occurs whenever an interictal spike is generated in the epileptic focus. This can have important impact on our understanding of epilepsy and of its treatment since interictal spikes are a common feature not only of epileptic patients, but can also appear in non‐epileptic subject, apparently without any consequence. From our results, we concluded that interictal activity can actually interfere with brain operation not only in the center of the epileptiform activity, but also in the connected areas, where the E/I balance is not directly disrupted. These results provide an example of the fact that apparently non‐symptomatic interictal spikes can affect brain computation. The second experimental model that I studied is a mouse model for a specific human genetic disease: the Phelan‐McDermid syndrome. This is a developmental disease, caused by a genomic deletion at site 22q13. The main suspect for causing the disease is one gene, Shank3, which encodes for a scaffold protein localized in the post‐synaptic density of glutamatergic synapses. In this model, we studied the computation of visual stimuli and we found an alteration of the contrast‐response curve. This is a defining relationship of visual processing: it is the transfer function that converts the visual input into a neural output. This means that to each intensity of visual stimulation corresponds a certain intensity of the neural response, of the visual cortex. We determined that, in Shank3 mutant mice, this curve was altered and showed an increased response to less intense stimuli and showed also a poor modulation of responses to high‐contrast stimulations. An interpretation of this can be that these mice are more sensitive to low‐contrast stimuli, but completely lose the ability of telling apart different high‐contrast stimuli from each other. Therefore, the Phelan McDermid mouse becomes “blinded” by weak stimulations as if they were seeing strong stimulus. Finally, we studied the behavior of the chloride ion in a drug‐induced epileptic seizure model. Chloride ion is of pivotal importance in neurons were the activation of ionotropic GABA and glycine receptors, which increase chloride membrane conductance in response to GABA or glycine release respectively. The intracellular concentration of chloride ions decides what is the effect of GABA release. Traditionally, ionotropic GABA receptors activation was thought to be inhibitory only, but the excitatory or inhibitory nature of these receptors is determined by the intracellular concentration of chloride ions. This concentration in normal adult neurons is thought to be around 5 mM: at this concentration, the effect of the activation of GABA receptors is an inhibition of the postsynaptic element. We investigated if the chloride concentration can be varied under extreme pathologic conditions as during epileptic seizures in a drug induced mouse model. In these animals, the epileptic seizures were produced by local administration of 4‐aminopyridine (4‐AP), a potassium channel antagonist. The effect of 4‐AP is to cause accumulation of chloride ions in neurons and this suggests that, in epileptic crisis, the role of inhibitory neurons can actually favor excitation.

The role of oxytocin (OXT) in controlling social behavior suggests a link to neuropsychiatric conditions in which social behavior behavior is aberrant or even absent, such as autism. Mice lacking the OXT receptor (Oxtr-/-) display an autistic-like phenotype, including deficits in social interaction, impaired cognitive flexibility (murine correlates of autism core symptoms), increased aggression and susceptibility to seizure (common co-occurring conditions). The deficit in cognitive flexibility is particularly interesting, because it is present in few animal model of autism. For this reason we decided to investigate its underlying neurobiological and molecular mechanisms. First, we compared Oxtr+/+and Oxtr-/- neuronal morphology and spine remodeling following a cognitive behavioral test. Interestingly, we highlighted, in the Oxtr-/- mice, an enhanced connectivity and overuse of the dorsolateral striatum, possibly arising from an hippocampal dysfunction, and we proposed it as substrate for habit-like symptoms and cognitive rigidity. Second, we investigated, at the molecular level, possible sources of this hippocampal dysfunction. In particular, we analyzed Oxtr-/- hippocampal neurons for the expression of proteins involved in the setting and maintenance of excitatio-inhibition (E-I) balance. We found an upregulation of several inwardly-rectifying K+ channels (belonging to Kir2 and Kir3 families), which could alter membrane excitability, and a lack of the physiological upregulation of the chloride transporter KCC2 during development, that may lead to aberrant GABAergic signaling in mature neurons. These data give important indications that the E-I balance is altered at multiple levels in Oxtr-/- hippocampal neurons, as an altered ratio between Glutamatergic and GABAergic synapses was also previously observed in these cultures. These observations are particularly intriguing, because an E-I imbalance has been frequently associated with several neurodevelopmental disorders such as autism. Third, we disclosed an OXTR-mediated pathway modulating KCC2 expression that may restore a correct E-I balance in hippocampal neurons. All this information could be useful to understand the pathophysiology of cognitive rigidity and to develop new therapies addressing specific symptoms of autism.

Le traitement de l'information sensorielle par le cortex cérébral requiert l'activation harmonieuse de micro-circuits neuronaux excitateurs et inhibiteurs interconnectés, ciblant les neurones pyramidaux de couche 5. Ces derniers élaborent les signaux de sortie corticaux et reçoivent un ratio de 20% d'excitation (E) et 80% d'inhibition (I). La dérégulation de cette balance E-I ou du système sérotoninergique conduit à des neuropathologies telles la dépression et la schizophrénie mais les interrelations entre la sérotonine et la balance E-I sont inconnues. Nous avons montré que la 5-HT endogène module la balance E-I en fonction du type de récepteur 5-HT recruté (1A, 2A, 3, 4, 7) et de sa localisation spécifique dans la colonne corticale. Ces données électrophysiologiques constituent la première évidence pour une action modulatrice fine de la sérotonine corticale sur la balance E-I et révèle la ségrégation fonctionnelle des récepteurs 5-HT dans les réseaux de neurones sensoriels.

Lorsqu'on effectue une tâche, les circuits neuronaux doivent représenter et manipuler des stimuli continus à l'aide de potentiels d'action discrets. On suppose communément que les neurones représentent les quantités continues à l'aide de leur fréquence de décharge et ceci indépendamment les un des autres. Cependant, un tel codage indépendant est inefficace puisqu'il exige la génération d'un très grand nombre de potentiels d'action pour atteindre un certain niveau de précision. Dans ces travaux, on montre que les neurones d'un réseau récurrent peuvent apprendre - à l'aide d'une règle de plasticité locale - à coordonner leurs potentiels d'actions afin de représenter l'information avec une très haute précision tout en déchargeant de façon minimale. La règle d'apprentissage qui agit sur les connexions récurrentes, conduit à un codage efficace en imposant au niveau de chaque neurone un équilibre précis entre excitation et inhibition. Cet équilibre est un phénomène fréquemment observer dans le cerveau et c'est un principe central de notre théorie. On dérive également deux autres règles d'apprentissages biologiquement plausibles qui permettent respectivement au réseau de s'adapter aux statistiques de ses entrées et d'effectuer des transformations complexes et dynamiques sur elles. Finalement, dans ces réseaux, le stochasticité du temps de décharge d'un neurone n'est pas la signature d'un bruit mais au contraire de précision et d'efficacité. Le caractère aléatoire du temps de décharge résulte de la dégénérescence de la représentation. Ceci constitue donc une interprétation radicalement différente et nouvelle de l'irrégularité trouvée dans des trains de potentiels d'actions
When performing a task, neural circuits must represent and manipulate continuous stimuli using discrete action potentials. It is commonly assumed that neurons represent continuous quantities with their firing rate and this independently from one another. However, such independent coding is very inefficient because it requires the generation of a large number of action potentials in order to achieve a certain level of accuracy. We show that neurons in a spiking recurrent network can learn - using a local plasticity rule - to coordinate their action potentials in order to represent information with high accuracy while discharging minimally. The learning rule that acts on recurrent connections leads to such an efficient coding by imposing a precise balance between excitation and inhibition at the level of each neuron. This balance is a frequently observed phenomenon in the brain and is central in our work. We also derive two biologically plausible learning rules that respectively allows the network to adapt to the statistics of its inputs and to perform complex and dynamic transformations on them. Finally, in these networks, the stochasticity of the spike timing is not a signature of noise but rather of precision and efficiency. In fact, the random nature of the spike times results from the degeneracy of the representation. This constitutes a new and a radically different interpretation of the irregularity found in spike trains

La plasticité homéostatique est un processus qui consiste à réguler l'efficacité globale des entrées synaptiques (excitatrices et inhibitrices) sur un neurone afin d'empêcher des modifications trop importantes de son niveau d'activité. Afin de caractériser les mécanismes à l'origine de ce processus, la balance Excitation/Inhibition des neurones pyramidaux de couche 5 du cortex visuel a été estimée. Elle est composée de 20 % d'excitation et de 80 % d'inhibition. A l'aide de protocoles de stimulation induisant des changements à long terme de l'efficacité des entrées synaptiques, les phénomènes de potentiation homéostatique et de dépression homéostatique ont été mis en évidence. L'induction de ces phénomènes, qui requiert l'activation de récepteurs NMDA et d'un signal NO, est sous le contrôle des systèmes inhibiteurs GABAergique et glycinergique. La récurrence entre signaux excitateurs et inhibiteurs apparaît comme l'élément clé de la régulation de l'activité neuronale.

The computations performed by the brain ultimately rely on the functional connectivity between neurons embedded in complex networks. It is well known that the neuronal connections, the synapses, are plastic, i.e. the contribution of each presynaptic neuron to the firing of a postsynaptic neuron can be independently adjusted. The modulation of effective synaptic strength can occur on time scales that range from tens or hundreds of milliseconds, to tens of minutes or hours, to days, and may involve pre- and/or post-synaptic modifications. The collection of these mechanisms is generally believed to underlie learning and memory and, hence, it is fundamental to understand their consequences in the behavior of neurons.(...)

L’intégrité des synapses neuronales est primordiale pour le développement et le maintien des capacités cognitives. Des mutations dans des gènes codant pour des protéines synaptiques ont été trouvées chez des patients atteints de déficience intellectuelle (DI), qui est une maladie neurodéveloppementale ayant des conséquences sur les fonctions intellectuelles et adaptatives. Ce travail de thèse porte sur l’étude de l’un de ces gènes, IL1RAPL1, dont les mutations sont responsables d’une forme non-syndromique de DI liée au chromosome X, et sur le rôle de la protéine IL1RAPL1 dans la formation et le fonctionnement des synapses. IL1RAPL1 est une protéine trans-membranaire qui est localisée dans les synapses excitatrices où elle interagit avec les protéines post-synaptiques PSD-95, RhoGAP2 et Mcf2l. De plus, IL1RAPL1 interagit en trans- avec une protéine phosphatase présynaptique, PTPd, via son domaine extracellulaire. Nous avons étudié les conséquences fonctionnelles de deux nouvelles mutations qui affectent le domaine extracellulaire d’IL1RAPL1 chez des patients présentant une DI. Ces mutations conduisent soit à une diminution de l’expression de la protéine, soit à une réduction de l’interaction avec PTPd affectant ainsi la capacité d’IL1RAPL1 à induire la formation de synapses excitatrices. En absence d’IL1RAPL1, le nombre ou la fonction des synapses excitatrices est diminué, ce qui mène à un déséquilibre entre les transmissions synaptiques excitatrice et inhibitrice dans des régions spécifiques du cerveau. Dans le cas particulier de l’amygdale latérale, nous avons montré que ce déséquilibre conduit à des défauts de mémoire associative chez la souris déficiente en Il1rapl1. L’ensemble des résultats qui font partie de ce travail montre que l’interaction IL1RAPL1/PTPd est essentielle pour la formation des synapses et suggère que les déficits cognitifs des patients avec une mutation dans il1rapl1 proviennent du déséquilibre de la balance excitation/ inhibition. Ces observations ouvrent des perspectives thérapeutiques visant à rétablir cette balance dans les réseaux neuronaux affectés
Preserving the integrity of neuronal synapses is important for the development and maintenance of cognitive capacities. Mutations on a growing number of genes coding for synaptic proteins are associated with intellectual disability (ID), a neurodevelopmental disease characterized by deficits in adaptive and intellectual functions. The present work is dedicated to the study of one of those genes, IL1RAPL1, and the role of its encoding protein in synapse formation and function. IL1RAPL1 is a trans-membrane protein that is localized at excitatory synapses, where it interacts with the postsynaptic proteins PSD-95, RhoGAP2 and Mcf2l. Moreover, the extracellular domain of IL1RAPL1 interacts trans-synaptically with the presynaptic phosphatase PTPd. We studied the functional consequences of two novel mutations identified in ID patients affecting this IL1RAPL1 domain. Those mutations lead either to a decrease of the protein expression or of its interaction with PTPd, affecting in both cases the IL1RAPL1-mediated excitatory synapse formation. In the absence of IL1RAPL1, the number or function of excitatory synapses is perturbed, leading to an imbalance of excitatory and inhibitory synaptic transmissions in specific brain circuits. In particular, we showed that this imbalance in the lateral amygdala results in associative memory deficits in mice lacking Il1rapl1. Altogether, the results included in this work show that IL1RAPL1/PTPd interaction is essential for synapse formation and suggest that the cognitive deficits in ID patients with mutations on IL1RAPL1 result from the imbalance of the excitatory and inhibitory transmission. These observations open therapeutic perspectives aiming to reestablish this balance in the affected neuronal circuits

In cortical circuits, neural responses are highly variable during both spontaneous and evoked activity. Nonetheless, coherent structures, such as propagating waves, can form at the population level. In this thesis, we provide a unified account of these seemingly contrasting dynamics by investigating spiking neural circuits that incorporate two essential features of cortical circuits: distance-dependent connectivity and the balance of excitation and inhibition. We show that propagating waves with complex dynamics only emerge when the neural circuits are balanced. These waves sweep past neurons, to which they provide highly synchronized synaptic inputs. We thus reconcile two major views of irregular neurodynamics, namely, the balanced state view and the synchronized input view. The propagating waves also provide a mechanism for double stochasticity of firing activity, and non-Gaussian dynamics of membrane potential. By applying a localized input to our balanced networks, we show that, as observed in experimental studies, a weak stimulus evokes a wave pattern propagating along lateral connections, whereas a strong stimulus triggers a localized pattern. We further identify the mechanisms underlying such response patterns, and show that their collective dynamics account for a range of recent experimental observations regarding cortical response properties. Such observations include the stimulus-evoked shift of cortical states from synchrony to asynchrony, and a decline in neural variability at stimulus onset. Furthermore, we extend previous theoretical studies of temporal chaos in balanced networks by showing that spatiotemporal chaos occurs in our network. This spatiotemporal chaos is characterized by the Lyapunov spectrum, and indicates that there are great fluctuations across space and time. By calculating finite-time Lyapunov vectors, we show that the spiking fronts of propagating wave patterns provide a mechanism for such spatiotemporal chaos.

Selon l'OMS, les troubles dépressifs majeurs (TDM) seront la 2ème cause d'incapacité dans le monde en 2020 et deviendront la 1ère en 2030. Les antidépresseurs classiques ont des effets thérapeutiques retardés et de nombreux patients sont résistants. La kétamine, antagoniste du récepteur N-methyl-D-aspartate (R-NMDA) du L-glutamate, possède un effet antidépresseur rapide chez les patients résistants à un traitement classique. Le mécanisme de cette activité étonnante n'est pas bien compris. En couplant la microdialyse intracérébrale à un test comportemental prédictif d'une activité antidépressive dans un modèle de souris BALB/cJ de phénotype anxieux, nous montrons que cette activité de la kétamine dépend de la balance excitation-inhibition entre les systèmes glutamate/R-NMDA et R-AMPA, GABA/R-GABAA, sérotonine du circuit cortex préfrontal/noyau du raphé. Nos résultats suggèrent également que ce serait la combinaison [kétamine-(2R,6R)-hydroxynorkétamine, son principal métabolite cérébral] qui porterait l'effet antidépresseur. Mes travaux de thèse contribuent à une meilleure compréhension de l'effet rapide antidépresseur de la kétamine
According to the WHO, major depressive disorder (MDD) will be the second leading cause of disability in the world in 2020 and will become the first in 2030. Conventional antidepressant drugs have delayed therapeutic effects and many patients are resistant. Ketamine, an N-methyl-D-aspartate (NMDA-R) receptor antagonist of L-glutamate, exerts a rapid antidepressant effect in patients who are resistant to standard therapy. The mechanism of this amazing activity is not well understood. By coupling intracerebral microdialysis to a predictive behavioral test of antidepressant activity in a BALB/cJ mouse model with an anxious phenotype, we show that this ketamine activity is dependent on the excitation-inhibition balance between glutamate/NMDA-R and AMPA-R, GABA/GABAA-R, serotonin systems in the prefrontal cortex/raphe nucleus circuit. Our results also suggest that it would be the combination [ketamine-(2R,6R)-hydroxynorketamine, its main brain metabolite] that would carry the antidepressant effect. My thesis work pave the way for the development of new fast-acting antidepressant drugs

Les désordres intellectuels (DI) comprennent une collection hétérogène de désordresneurodéveloppementaux qui émergent pendant l’enfance. Ils ont une incidence de 1 à 3% dansla population et sont associés avec des déficits dans les fonctions mentales et adaptives. Denombreuses mutations ont été identifiées dans des gènes codant pour des protéines quiremplissent des fonctions biologiques très diverses dans le cerveau. Parmi ces protéines,certaines sont enrichies à la synapse, supposant que les déficits cognitifs associés aux DIpourraient être reliés à des déficits synaptiques. L’objectif scientifique de notre équipe et decomprendre le rôle de certaines protéines dans la fonction synaptique et la cognition enutilisant des souris génétiquement modifiées portant des mutations dans le gènecorrespondant. Je me suis concentré sur Il1rapl1, un gène codant pour la protéine Interleukinreceptor-accessory-protein-like-1. Des mutations ou micro-délétions dans ce gène sont liés audéveloppement de DI chez l’homme. Dans les neurones, Il1rapl1 code pour une protéinetransmembranaire qui serait impliquée dans la formation et/ou la stabilisation de synapsesexcitatrices. Les conséquences de l’absence d’IL1RAPL1 à des niveaux plus intégrés restaientpeu étudiées lors du début de ma thèse. J’ai utilisé une souris déficiente pour IL1RAPL1 (KO) afinde comprendre le lien entre les déficits comportementaux et la fonction synaptique. Pour cela,j’ai soumis des souris KO à des taches comportementales de peur conditionnée. J’ai ensuiteutilisé une combinaison d’approches in vitro, ex vivo et in vivo afin de caractériser la fonctionsynaptique dans les circuits neuronaux dédiés : l’amygdale latérale et basolatérale. Desenregistrements electrophysiologiques ont montré une dérégulation de la balance entre latransmission inhibitrice et excitatrice (I/E) dans l’amygdale de souris Il1rapl1 KO, causant ainsides déficits dans la capacité d’acquérir et d’exprimer la mémoire de peur conditionnée. Lacorrection de ce déficit synaptique in vivo par pharmacologie ou par optogénétique a permis derestaurer le comportement chez les souris KO
Intellectual disability (ID) comprises a highly heterogeneous collection of neurodevelopmentaldisorders that arise during childhood. They have an incidence of 1-3% in the population withimpairments in mental and adaptive functions. While the etiologies of IDs are thought to bevery heterogeneous, a significant proportion of ID has genetic origins. Mutations in single IDgenes lead to dysfunctions in proteins that fulfill highly different biological functions in thebrain. Interestingly, ID-related proteins are often found enriched at synapses, suggesting thatcognitive impairments defining ID could be related to alterations of synaptic function. The maingoal of our research team is to understand the role of ID-related proteins in synaptic functionand cognition using mouse models bearing gene mutations associated to ID in humans. Myresearch focused on the study of Il1rapl1, a gene coding for the Interleukin-receptor-accessoryprotein-like-1 protein. Micro-deletions or point mutations in this gene are directly linked to thedevelopment of ID and autism spectrum disorder in humans. In neurons, Il1rapl1 encodes atrans-membrane protein and several in vitro experiments point to its important role in thedifferentiation and formation/stabilization of excitatory synapses trough interactions withpresynaptic, trans-synaptic or postsynaptic partners. However, the consequences of Il1rapl1deficiency at more integrated levels remains poorly understood. The principal objective of mythesis is to explore the link between synaptic deficits and behavioral impairments in Il1rapl1-deficient mice. To achieve that, wild-type and mutant animals were first submitted to fearlearning tasks. I then used a combination of in vivo, ex vivo and in vitro functional essays tocharacterize synaptic functions in behaviorally relevant neuronal circuits. Ultimately, ourworking hypothesis were challenged in vivo by pharmacological and optogenetic approaches tonormalize behavioral deficits in Il1rapl1 KO mice. Altogether my work demonstrates thatInhibitory/Excitatory imbalances associated with the absence of Il1rapl1 impaired both thecapacity to form new memories as well as the expression of previously formed memories

De nouvelles preuves suggèrent que les dysfonctionnements des circuits sous-jacents aux symptômes et aux déficits cognitifs des maladies psychiatriques pourraient être causés par une altération des paramètres d'équilibre d’excitation/inhibition (E/I). Cependant, les preuves physiologiques directes de cette hypothèse à partir de données électrophysiologiques et de neuro-imagerie non invasives sont jusqu'à présent rares. Pour apporter un soutien supplémentaire à l’hypothèse de l’équilibre E/I, la présente étude a appliqué une approche avancée de microscopie holographique numérique (MHN) pour examiner la dynamique des systèmes excitateurs/inhibiteurs suite à une stimulation glutamatergique dans des réseaux de neurones à différents stades de maturation neuronale. Cette approche fournissant une mesure approximative très précise des variations de mouvement de l’eau dans les cellules permet d’étudier certains processus physiologiques, tels que ceux reliés à l’activité neuronale. Cette étude a ainsi permis d’améliorer les connaissances sur la dynamique de la réponse neuronale induite par le glutamate, notamment en la caractérisant dans des cultures de neurones corticaux primaires de rats postnataux. L’activation des neurones engendrée par le glutamate, le principal neurotransmetteur excitateur, a révélé des changements plus ou moins persistants de la morphologie et des propriétés intracellulaires des neurones. De plus, les différentes réponses obtenues indiquent que le glutamate engendre des mécanismes d’activation et des processus de régulation du volume neuronal distincts d’un neurone à l’autre, probablement dépendant de l’état d’excitabilité de ce dernier qui résulte de l’interaction complexe des neurones inhibiteurs et excitateurs. Ainsi, la régulation de l’équilibre E/I de réseaux neuronaux pourrait potentiellement être reflétée par la proportion des différentes réponses de phase induites lors de stimulation de réseaux neuronaux au glutamate.
De nouvelles preuves suggèrent que les dysfonctionnements des circuits sous-jacents aux symptômes et aux déficits cognitifs des maladies psychiatriques pourraient être causés par une altération des paramètres d'équilibre d’excitation/inhibition (E/I). Cependant, les preuves physiologiques directes de cette hypothèse à partir de données électrophysiologiques et de neuro-imagerie non invasives sont jusqu'à présent rares. Pour apporter un soutien supplémentaire à l’hypothèse de l’équilibre E/I, la présente étude a appliqué une approche avancée de microscopie holographique numérique (MHN) pour examiner la dynamique des systèmes excitateurs/inhibiteurs suite à une stimulation glutamatergique dans des réseaux de neurones à différents stades de maturation neuronale. Cette approche fournissant une mesure approximative très précise des variations de mouvement de l’eau dans les cellules permet d’étudier certains processus physiologiques, tels que ceux reliés à l’activité neuronale. Cette étude a ainsi permis d’améliorer les connaissances sur la dynamique de la réponse neuronale induite par le glutamate, notamment en la caractérisant dans des cultures de neurones corticaux primaires de rats postnataux. L’activation des neurones engendrée par le glutamate, le principal neurotransmetteur excitateur, a révélé des changements plus ou moins persistants de la morphologie et des propriétés intracellulaires des neurones. De plus, les différentes réponses obtenues indiquent que le glutamate engendre des mécanismes d’activation et des processus de régulation du volume neuronal distincts d’un neurone à l’autre, probablement dépendant de l’état d’excitabilité de ce dernier qui résulte de l’interaction complexe des neurones inhibiteurs et excitateurs. Ainsi, la régulation de l’équilibre E/I de réseaux neuronaux pourrait potentiellement être reflétée par la proportion des différentes réponses de phase induites lors de stimulation de réseaux neuronaux au glutamate.
New evidences suggest that circuit dysfunctions underlying symptoms and cognitive deficits of psychiatric disorders may be caused by impaired excitation/inhibition equilibrium parameters (E/I). However, direct physiological evidences supporting this hypothesis from non-invasive electrophysiological and neuroimaging remain scarce. To provide additional support concerning the E/I balance hypothesis, this study uses an advanced digital holographic microscopy (DHM) approach to explore the dynamics of excitatory/inhibitory systems following glutamatergic stimulation in neural networks at different stages of neuronal maturation. This approach provides a very accurate approximate measurement of the water movement variations in cells allowing to study certain specific physiological processes, such as those related to neuronal activity. This study improves the knowledge regarding the dynamics of the glutamate-induced neuronal response, especially by characterizing it in cultures of primary cortical neurons of postnatal rats. The activation of neurons induced by glutamate, which is the main excitatory neurotransmitter, revealed more or less permanent changes in the morphology and intracellular properties of neurons. Moreover, the various responses obtained indicate that glutamate generates different neuronal activation mechanisms and neuronal volume regulation processes from a neuron to another, probably depending to the excitability state of the neuron that results from the complex interaction of inhibitory and excitatory neurons. Thus, the E/I balance regulation of neural networks could potentially be reflected by the proportion of different phase responses induced during glutamate neural network stimulation.
New evidences suggest that circuit dysfunctions underlying symptoms and cognitive deficits of psychiatric disorders may be caused by impaired excitation/inhibition equilibrium parameters (E/I). However, direct physiological evidences supporting this hypothesis from non-invasive electrophysiological and neuroimaging remain scarce. To provide additional support concerning the E/I balance hypothesis, this study uses an advanced digital holographic microscopy (DHM) approach to explore the dynamics of excitatory/inhibitory systems following glutamatergic stimulation in neural networks at different stages of neuronal maturation. This approach provides a very accurate approximate measurement of the water movement variations in cells allowing to study certain specific physiological processes, such as those related to neuronal activity. This study improves the knowledge regarding the dynamics of the glutamate-induced neuronal response, especially by characterizing it in cultures of primary cortical neurons of postnatal rats. The activation of neurons induced by glutamate, which is the main excitatory neurotransmitter, revealed more or less permanent changes in the morphology and intracellular properties of neurons. Moreover, the various responses obtained indicate that glutamate generates different neuronal activation mechanisms and neuronal volume regulation processes from a neuron to another, probably depending to the excitability state of the neuron that results from the complex interaction of inhibitory and excitatory neurons. Thus, the E/I balance regulation of neural networks could potentially be reflected by the proportion of different phase responses induced during glutamate neural network stimulation.

Le cortex est une structure qui supporte de nombreux processus tels que perception sensorielle, processus cognitifs et mémorisation. Il fonctionne grâce à une association de neurones excitateurs (E) et inhibiteurs (I) interconnectés de façon récurrente par des synapses dynamiques qui ciblent les neurones pyramidaux de couche 5 (NPy5) élaborant les signaux de sortie du cortex. Cette organisation neuronale est régulée par un équilibre entre E et I. La dérégulation des réseaux neuronaux peut conduire à des pathologies telles que la dépression ou la schizophrénie. Le fonctionnement du cortex est modulé entre autres par la sérotonine, la dopamine, la D-sérine et la glycine. Ce travail de thèse porte sur l’effet des interactions entre neuromodulateurs via les récepteurs 5-HT1A, D1, D2, NMDA et GlyR sur la balance et la plasticité synaptique de E et I dans le cortex. Mes données électrophysiologiques montrent que l’interaction entre les récepteurs 5-HT1A et D1 limite l’induction de la LTD, tandis que l’interaction entre les récepteurs 5-HT1A et D2, grâce à un carrefour commun de signalisation GSK3β, favorise l’induction de la LTD. Je montre dans le cortex visuel de rat que la D-sérine est nécessaire à l’induction de la LTP et que les GlyR ont un rôle de « shunt » le long de la dendrite des NPy5, ce qui entraîne un basculement d’une LTP en « LTD-like » apparente
The cortex is crucial for processes such as sensory perception, cognition and memory. Cortical organization is based on neuronal networks composed of excitatory (E) and inhibitory (I) neurons which target layer 5 pyramidal neurons. Dysfunctions of such networks result in psychiatric pathologies including major depression and schizophrenia. Regulations of cortical activity also involve neuromodulators such as serotonin, dopamine, D-serine and glycine. The current body of work decipher the interactions of the effects of 5-HT1A-, D1-, D2-, NMDA- and Glycine-receptors activation on the E-I balance and synaptic plasticity. The electrophysiological data that I have generated in the prefrontal cortex show that concomitant activation of 5-HT1A- and D1-receptors downregulates the induction of LTD whilst 5-HT1A coupled to D2-receptors activation promotes LTD induction, via a common modulation of GSK3β. I also collected data from the visual cortex, showing that D-serine is the co-agonist NMDA-receptor in this brain region and is, as such, required for LTP-induction. Glycine was instead found to act on dendritic Glycine-receptors, resulting in a shunt, which altered dendritic integration and thus turned LTP to a LTD-like effect at the somatic level

L'épilepsie est une maladie neurologique qui touche plus de 50 millions de personnes dans le monde. Elle se caractérise par des crises récurrentes dues à la surexcitation synchrone et spontanée de populations neuronales du cerveau. Les crises sont de nature très variable et les symptômes dépendent de la zone du cerveau touchée et de son étendue. Le terme «troubles épileptiques» est par conséquent préféré. Ceux-ci peuvent avoir de nombreuses causes, soient génétiques (par exemple, le syndrome de Dravet, une épilepsie infantile rare, provoquée dans 80% des cas par la mutation hétérozygote du gène SCN1A), soient environnementales (par exemple, après un empoisonnement aux organophosphorés, des composés présents dans les pesticides et les agents de guerre neurotoxiques). Dans les deux cas, les traitements actuels ne permettent pas un contrôle optimal des crises. Une meilleure compréhension de la physiopathologie de ces différentes formes d'épilepsie est donc nécessaire pour trouver de nouvelles cibles thérapeutiques et de nouveaux anticonvulsivants. Les cellules microgliales, les macrophages résidents du cerveau ont de nombreuses fonctions qui varient en fonction de la maturité du cerveau. Les microglies sont les gardiennes de l'homéostasie cérébrale, assurant en permanence le bon fonctionnement des neurones. Ce sont des cellules immunitaires capables de moduler leur activité en fonction des dangers qu'elles détectent. De plus, elles ont un rôle particulier dans la plasticité synaptique et la modulation de l'excitabilité neuronale. Ces différents rôles ont suscité de nombreuses hypothèses sur l'implication de ces cellules dans la physiopathologie des troubles épileptiques. Pour certaines, les microglies sont nocives pour l'excitabilité des neurones, par leur activation et la sécrétion chronique de cytokines pro-inflammatoires. Pour d'autres, elles ont un rôle bénéfique, la microglie tamponnant l'hyperexcitabilité neuronale et diminuant ainsi la fréquence des crises. L'objectif de mon travail de thèse était d'étudier les mécanismes de l'épileptogenèse impliquant les cellules microgliales afin d'identifier de nouvelles cibles thérapeutiques. J'ai développé deux modèles d'épilepsie chez le poisson zèbre, un modèle génétique du syndrome de Dravet et un modèle d'empoisonnement aux organophosphorés. Ceux-ci m'ont permis d'étudier les modifications du système nerveux central au cours de l'épileptogenèse. J'ai ainsi montré un déséquilibre de la balance excitateur/inhibiteur vers l'excitation qui pourrait déclencher des crises d'épilepsie. En utilisant le modèle de Dravet, j'ai également caractérisé les changements morphologiques, comportementaux et moléculaires des cellules microgliales après des crises. Ces travaux améliorent notre compréhension des conséquences des crises d'épilepsie dans le cerveau et contribuent à ouvrir la voie à la découverte de nouvelles cibles thérapeutiques pour traiter différentes formes d'épilepsie
Epilepsy is a neurological disease affecting some 50 million people worldwide. It is characterized by recurrent seizures due to the synchronous and spontaneous overexcitation of neuronal populations in the brain. Seizures vary widely in nature, and symptoms dependon the area of the brain affected and its extent. The term ‘epileptic disorders’ is accordingly preferred. These can have many causes, including both genetic (e.g. Dravet syndrome, a rare infantile epilepsy caused in 80% of cases by the heterozygous mutation of the SCN1A gene), and environmental (e.g. after poisoning with organophosphates, compounds present in pesticides and neurotoxic warfare agents). Whether for Dravet syndrome or organophosphate poisoning, current treatments do not enable optimal control of seizures. A better understanding of the pathophysiology of these different forms of epilepsy is thus needed to find new therapeutic targets and new anticonvulsants. Microglial cells are the resident macrophages in the brain. These cells have many functions, which can vary depending on the maturity of the brain. The microglia are the guardians of cerebral homeostasis, continuously ensuring the proper functioning of neurons. They are immune cells able to modulate their activity according to the dangers they detect. In addition, microglia have a special role in synaptic plasticity and the modulation of neuronal excitability. These different roles have prompted numerous hypotheses on the involvement of these cells in the pathophysiology of epileptic disorders. In some, microglia are harmful for the excitability of neurons, through their activation and the chronic secretion of proinflammatory cytokines. Others lend them a beneficial role, with microglia buffering neuronal hyperexcitability and thus decreasing the frequency of seizures. The objective of my PhD work was to study the mechanisms of epileptogenesis involving microglial cells in order to identify new therapeutic targets. I developed two models of epilepsy in zebrafish, a genetic model of Dravet syndrome and a model of organophosphate poisoning. These enabled me to study the modifications of the central nervous system during epileptogenesis. I specifically demonstrated an excitatory/inhibitory imbalance toward excitation that could trigger epileptic seizures. Using the Dravet model, I also successfully characterized the morphological, behavioral and molecular changes of microglial cells after seizures. This work improves our understanding of the consequences of epileptic seizures in the brain and helps pave the way for the discovery of new therapeutic targets to treat different forms of epilepsy

La décharge des cellules de Purkinje (CP), neurone de sortie du cortex cérébelleux, joue un rôle majeur dans le contrôle moteur. Les CP reçoivent des entrées excitatrices provenant des cellules des grains (CG), lesquelles génèrent également une inhibition antérograde sur les CP via l’activation d’interneurones de la couche moléculaire (IN). Lors de ma thèse, j’ai étudié l’influence simultanée de la balance excitation-inhibition (E/I) et des plasticités à court terme aux synapses CG-IN-CP sur la décharge des CP, par des techniques d’électrophysiologie, d’optogénétique et de simulation. Ces travaux démontrent l’existence d’une hétérogénéité d’E/I dans le cortex cérébelleux ainsi qu’une grande diversité de modulation des CP en réponse à la stimulation de CG. Le nombre de stimulation des CG influence fortement la direction et l’intensité de la modulation observée. Enfin, la combinaison de plasticités à court terme et d’E/I génère dans la décharge des CP des motifs de réponses complexes mais reproductibles, ayant sans doute un rôle essentiel dans l’encodage sensoriel
Rate and temporal coding in Purkinje cells (PC), the sole output of the cerebellar cortex, play a major role in motor control. PC receives excitatory inputs from granule cells (GC) which also provide feedforward inhibition on PC through the activation of molecular layer interneurons (MLI). In this thesis, I studied the influence of the combined action of excitation/inhibition (E/I) balance and short-term plasticity of GC-MLI-PC synapses on PC discharge, by using electrophysiological recordings, optogenetic stimulation and modelling. This work demonstrates that E/I balances are not equalized in the cerebellar cortex and showed a wide distribution of PC discharge modulation in response to GC inputs, from an increase to a shut down of the discharge. The number of stims in GC bursts strongly controls the strength and sign of PC modulation. Lastly, the interplay between short-term plasticity and E/I balance implements complex but reproducible output patterns of PC responses to GC inputs that should play a key role in stimulus encoding by the cerebellar cortex

In this work, we propose an approach that allows to explore the potential pathophysiological mechanisms (at neuronal population level) of ictogenesis by combining clinical intracranial electroencephalographic (iEEG) recordings with a neural mass model. IEEG recordings from temporal lobe epilepsy (TLE) patients around seizure onset were investigated. Physiologically meaningful parameters (average synaptic gains of the excitatory, slow and fast inhibitory population, Ae, B and G) were identified during interictal to ictal transition. We analyzed the temporal evolution of four ratios, i.e. Ae/G, Ae/B, Ae/(B + G), and B/G. The excitation/inhibition ratio increased around seizure onset and decreased before seizure offset, suggesting the disturbance and restoration of balance between excitation and inhibition around seizure onset and before seizure offset, respectively. Moreover, the slow inhibition may have an earlier effect on the breakdown of excitation/inhibition balance. Results confirm the decrease in excitation/inhibition ratio upon seizure termination in human temporal lobe epilepsy, as revealed by optogenetic approaches both in vivo in animal models and in vitro. We further explored the distribution of the average synaptic gains in parameter space and their temporal evolution, i.e. the path through the model parameter space, in TLE patients. Results showed that the synaptic gain values located roughly on a plane before seizure onset, dispersed during ictal and returned when the seizure terminated. Cluster analysis was performed on seizure paths and demonstrated consistency in synaptic gain evolution across different seizures from individual patients. Furthermore, two patient groups were identified, each one corresponding to a specific synaptic gain evolution in the parameter space during a seizure. Results were validated by a bootstrapping approach based on comparison with random paths. The differences in the path revealed variations in EEG dynamics for patients despite showing an identical seizure onset pattern. Our approach may have the potential to classify the epileptic patients into subgroups based on different mechanisms revealed by subtle changes in synaptic gains and further enable more robust decisions regarding treatment strategy. The increase of excitation/inhibition ratios, i.e. Ae/G, Ae/B and Ae/(B+G), around seizure onset makes them potential cues for seizure detection. We explored the feasibility of a model based seizure detection algorithm. A simple thresholding method was employed. We evaluated the algorithm against the manual scoring of a human expert on iEEG samples from patients suffering from different types of epilepsy. Results suggest that Ae/(B+G), i.e. excitation/(slow + fast inhibition) ratio, allowed the best performance and that the algorithm best suited TLE patients. Leave-one-out cross-validation showed that the algorithm achieved 94.74% sensitivity for TLE patients. The median false positive rate was 0.16 per hour, and median detection delay was -1.0 s. Of interest, the values of the threshold determined by leave-one-out cross-validation for TLE patients were quite constant, suggesting a general excitation/inhibition balance baseline in background iEEG among TLE patients. Such a model-based seizure detection approach is of clinical interest and could also achieve good performance for other types of epilepsy provided that more appropriate model, i.e. better describe epileptic EEG waveforms for other types of epilepsy, is implemented. Altogether, this thesis contributes to the field of epilepsy research from two perspectives. Scientifically, it gives new insights into the mechanisms underlying interictal to ictal transition, and facilitates better understanding of epileptic seizures. Clinically, it provides a tool for reviewing EEG data in a more efficient and objective manner and offers an opportunity for on-demand therapeutic devices.
Doctorat en Sciences de l'ingénieur et technologie
info:eu-repo/semantics/nonPublished

Des variants pathogènes du gène GLRA2 codant pour la sous-unité α2 du récepteur glycinergique ont été récemment impliqués dans le trouble du spectre autistique (TSA) et la déficience intellectuelle. Notre groupe a précédemment montré que les souris mâles déficientes en Glra2 (Glra2–/Y) présentent un déficit de mémoire lors du test de reconnaissance d’objets (TRO) et une altération de la plasticité synaptique du cortex préfrontal (CPF), une région fortement impliquée dans le TSA. Par ailleurs, des études développementales des souris Glra2–/Y ont décrit un défaut de migration des interneurones et une perte de neurones de projection corticaux associée à une microcéphalie. Dans ce projet, nous avons recherché les altérations cellulaires et fonctionnelles qui sous-tendent les défauts comportementaux et synaptiques des souris Glra2–/Y, en se focalisant sur le CPF. Contrairement à ce qui a été rapporté précédemment, les souris Glra2–/Y ne sont pas microcéphales et ne présentent ni perte globale de neurones excitateurs ou inhibiteurs, ni perte de sous-populations d’interneurones à parvalbumine, calrétinine ou cholécystokinine dans le CPF ou le cortex somatosensoriel. Cependant, le nombre d'interneurones corticaux à somatostatine est augmenté chez les souris Glra2–/Y. Ces résultats démontrent un rôle plus subtil de Glra2 dans le développement du cortex que ce qui avait été suggéré auparavant, cohérent avec le phénotype des patients mâles porteurs de mutations dans GLRA2. Au moyen d’approches anatomiques et électrophysiologiques, nous avons également mis en évidence chez les souris Glra2–/Y des altérations caractéristiques des troubles neurodéveloppementaux, retrouvées dans d'autres modèles murins du TSA. Dans le CPF, ces souris mutantes présentent une diminution du nombre de synapses inhibitrices, une augmentation de la densité des épines et de la complexité dendritique des neurones pyramidaux, et une augmentation de l’activité synaptique excitatrice des neurones pyramidaux, sans effet sur la transmission synaptique inhibitrice. L'ensemble de ces résultats révèle l’existence d’un déséquilibre de la balance excitation/inhibition dans le cortex des souris Glra2–/Y. Afin d'identifier les régions cérébrales impliquées dans le déficit cognitif des souris Glra2–/Y, nous avons quantifié les neurones exprimant c-Fos, un marqueur d’activation neurale, après le TRO. Nous avons mis en évidence une hypoactivation sélective du CPF infralimbique rostral chez les souris Glra2–/Y après cette tache. Par colocalisation de c-Fos avec des marqueurs neuronaux, nous avons montré que cette hypoactivation était due à un déficit d’activation des neurones glutamatergiques. Pour évaluer plus finement l'activité neuronale dans le CPF des souris Glra2–/Y lors de l'apprentissage, nous avons mesuré l’activité des neurones glutamatergiques du cortex infralimbique par photométrie de fibre à l’aide d’un senseur calcique, pendant le TRO. Chez les souris sauvages, l'exposition répétée à des objets pendant la phase d'entraînement induit une réduction progressive de l'activité calcique, alors que cette atténuation est absente chez les souris Glra2–/Y, renforçant l’implication d’une altération de l'activité des neurones excitateurs dans le déficit cognitif de ces souris. En outre, malgré l'absence de déficits sociaux apparents, la réponse calcique des neurones glutamatergiques à la nouveauté sociale est atténuée chez les souris Glra2–/Y. Dans leur ensemble, ces résultats montrent que des altérations subtiles des circuits préfrontaux chez les souris Glra2–/Y engendrent une altération de la balance excitation/inhibition et un dysfonctionnement des neurones glutamatergiques dans le CPF lors du TRO, entraînant un déficit de mémoire. Ils suggèrent que la sous-unité α2 est cruciale pour le développement normal du CPF, et que des défauts des circuits préfrontaux pourraient sous-tendre le dysfonctionnement neurocognitif observé chez les patients présentant une perte de GLRA2
Pathogenic variants in the GLRA2 gene, which encodes the glycine receptor α2 subunit, have been recently implicated as a novel cause of autism spectrum disorder (ASD) and intellectual disability. Our group previously showed that Glra2-deficient male (Glra2 /Y) mice display impaired learning and memory in the novel object recognition (NOR) task and altered synaptic plasticity in the prefrontal cortex (PFC), a region consistently implicated in ASD. In addition, developmental studies in mice expressing the same Glra2 mutation reported deficits in interneuron migration and loss of cortical projection neurons associated with microcephaly. In this project, we investigated the cellular and functional alterations underlying the behavioural and synaptic defects of Glra2 /Y mice, focusing on the PFC. In contrast with previous reports, Glra2 /Y mice were not microcephalic and neuronal quantification showed no loss of either glutamatergic neurons or interneurons, including parvalbumin, calretinin and cholecystokinin interneuron subpopulations in the PFC or the somatosensory cortex. However, the number of cortical somatostatin interneurons was increased in these regions in mutant mice. These findings imply that Glra2 plays a more subtle role in neocortical development and assembly than previously suggested and are consistent with the phenotype of male patients with pathogenic GLRA2 variants, who are not microcephalic and have normal brain imaging. We also show that Glra2 /Y mice exhibit many of the hallmarks of neurodevelopmental brain dysfunction observed in other rodent models of ASD. In the adult PFC, Glra2 /Y mice show a decreased number of inhibitory synapses and increased spine density and dendritic complexity of pyramidal neurons, whilst young mice (P14-P21) have increased excitatory synaptic inputs to prefrontal pyramidal neurons, with no effect on inhibitory synaptic transmission. Taken together, these findings point to excitatory hyperconnectivity in the PFC of Glra2 /Y mice, and suggest an imbalance of excitatory and inhibitory neurotransmission in these mutant mice. To identify which brain regions are associated with the recognition memory deficit observed in Glra2 /Y mice, we quantified c-Fos expression as a marker of neuronal activation following NOR. We found that the rostral infralimbic PFC was hypoactivated in Glra2 /Y mice following this task, whilst other brain regions quantified showed similar levels of c-Fos expression compared to wild-type mice. c-Fos colocalization with neuronal markers revealed that the hypoactivation of the PFC was driven by impaired activation of glutamatergic neurons following the task. To further assess neuronal activity in the PFC in Glra2 /Y mice during cognition, we recorded calcium transients from infralimbic glutamatergic neurons using in vivo fiber photometry during NOR, and compared them with the calcium response induced by social interaction with a novel mouse. In wild-type animals, repeated exposure to objects during the training phase of the NOR task caused a progressive reduction in calcium-dependent neuronal activity during exploration. This attenuation of the calcium signals was absent from Glra2 /Y mice, further implicating an impairment of prefrontal glutamatergic activity in the NOR deficit observed in this model. In addition, despite a lack of apparent social deficits, Glra2 /Y mice exhibited an attenuated glutamatergic calcium response to novel social stimuli in the PFC. Overall, these findings show that subtle alterations in prefrontal circuit organization and physiology in Glra2 /Y mice result in altered inhibitory/excitatory balance and an aberrant response of prefrontal glutamatergic neurons during recognition memory leading to impaired task performance. These results suggest that the glycine receptor α2 subunit is crucial for normal PFC development, and that defects in prefrontal circuits may underlie the neurocognitive dysfunction observed in patients lacking GLRA2

Tese de Doutoramento em Biologia Experimental e Biomedicina apresentada ao Instituto de Investigação Interdisciplinar da Universidade de Coimbra.
Excitation-inhibition (E-I) balance plays an important role in information processing, neuroplasticity and pathologic conditions. Evidence of E-I imbalance has been reported in a wide array of neuropsychiatric disorders such as autism spectrum disorder (ASD), schizophrenia, neurofibromatosis type 1, depression and attention-deficit/hyperactivity disorder. Of particular interest to us was the currently available evidence of cortical dysfunction in type 2 diabetes patients. Type 2 diabetes mellitus patients are known to have decreased cognitive ability before they develop any evidence of microvascular or macrovascular disease. This could be explained by impaired circuitry and/or E-I balance of certain networks in type 2 diabetes mellitus. It has been reported that patients with type 2 diabetes mellitus have increased levels of GABA in the occipital region in addition to evidence of blood-brain barrier disintegration in such patients. Because of this, type 2 DM was chosen as a disease model to study the possible impacts of the disease on GABAergic system in the occipital region and how that correlate with visual performance. The study of the GABAergic system and its dysfunction in epilepsy is gaining attention for several reasons. First, while GABA is mainly an inhibitory neurotransmitter in the brain it has been shown that it can act as an excitatory neurotransmitter on immature neurons. Second, there is growing evidence of increased GABA concentration in the epileptogenic zone in patients with drug-resistant epilepsy in vivo and ex vivo. Finally, the generation of pathologic and physiologic high frequency oscillations is expected to be related to and maintained by inhibitory postsynaptic potentials that are mediated by GABAA receptors. The second part of this thesis focuses on the study of the GABAergic system in epilepsy patients with drug-resistant disease and how it is related to physiologic or pathologic gamma activity. We chose a cohort of type 2 diabetes mellitus patients who have early diabetic retinopathy. The goal was to assess occipital cortical GABA as a predictor of visual performance in type 2 diabetes mellitus patients. GABA was measured by proton magnetic resonance spectroscopy from the occipital region, and visual performance was assessed in three domains (chromatic, achromatic and speed discrimination). We found for the first-time evidence of achromatic and speed discrimination abnormalities in type 2 diabetes mellitus patients as compared to healthy subjects. Moreover, we reported for the first time a positive correlation between occipital GABA and achromatic/speed discrimination thresholds (higher thresholds mean worse performance). Occipital GABA at baseline was also predictive of visual performance one year later, suggesting that modulating occipital GABA could have a long-term impact on visual performance. The second study to be included in the scope of this thesis focused on BBB permeability in type 2 diabetic patients and its relation to visual performance. The previously mentioned cohort of type 2 DM patients who had evidence of GABAergic dysfunction were included in this subsequent study of BBB integrity. In summary, we showed a relationship between BBB leakage and blood-retinal-barrier leakage, with patients with BRB leakage having higher BBB permeability. Moreover, we showed for the first time that metabolic control is correlated with BBB permeability (poor metabolic control is associated with impaired BBB integrity). Finally, we found that BBB permeability is predictive of visual acuity at baseline, one year and two years later in type 2 diabetics with established BRB leakage. We then moved to study the GABAergic system in a different disease model. In patients with drug resistant epilepsy, we are offered a unique opportunity where we can indirectly measure the function of the GABAergic system by measuring gamma activity with intracranial electroencephalography (EEG). The first study in this disease model focused on physiologic high frequency activity where we tested the relationship between functional topography of high gamma activity and perceptual decision-making. In summary, we found three distinct regional fingerprints of high frequency activity (HFA) in our cohort: a) Lower gamma frequency patterns dominated the anterior semantic ventral object processing, b) low gamma frequency patterns that involve dorsoventral integrating networks, and c) early sensory posterior patterns in the 60 to 250 Hz range. In summary, we show that accurate object recognition/perceptual decision-making is associated with low-gamma frequency activity that has a specific spatiotemporal signature. The second study that belonged to the disease model of drug resistant epilepsy focused on evidence of GABAergic dysfunction in epilepsy and how the modulation of the GABAergic system in the epileptogenic zone affects epileptogenecity and the GABAergic system in other reference brain region (the occipital region). We hypothesized that c-tDCS (cathodal transcranial direct current stimulation) which has an antiepileptic effect would modulate the neurotransmitters responsible for the abnormal and complex local synchrony and abnormal rhythmic activity seen in epilepsy. This is the first study to test for the impact of c-tDCS on physiologic and pathologic gamma activity and to measure GABA, glutamate and glutathione from the epileptogenic zone and occipital region simultaneously after c-tDCS in patients with drug resistant epilepsy. C-tDCS decreased the number of interictal discharges per minute. This was associated with a decrease in GABA concentration in the occipital and epileptogenic zones. We also found that cathodal tDCS stimulation of the epileptogenic zone suppressed grating evoked low gamma activity in the epileptogenic zone and increased it in the distant parieto-occipital regions. In summary, this study provided a window into the mechanism of action of c-tDCS as an antiepileptic and its effects on the GABAergic system and neural oscillatory patterning. In summary, we show that E-I balance is maintained across the different neural networks in a given time frame and alterations in this balance is linked to cognitive impairment and visual performance in type 2 DM, and epileptogenesis in epilepsy patients. Our results also suggest that GABAergic dysfunction in the epileptogenic zone is more than a consequence of epileptogenesis, and could be epileptogenic per se.
O equilíbrio entre excitação e inibição tem um papel importante no processamento de informação, na neuroplasticidade e em certas condições patológicas. Um desequilíbrio entre excitação e inibição tem sido referido em várias condições neuropsiquiátricas, tais como na perturbação do espectro do autismo, esquizofrenia, neurofibromatose de tipo 1, depressão e perturbação de hiperatividade e défice de atenção. A evidência desta disfunção cortical também em pessoas com diabetes mellitus tipo 2 revelou-se de particular interesse para nós. Sabe-se que pessoas com diabetes mellitus tipo 2 apresentam uma habilidade cognitiva diminuída antes até do aparecimento de doença micro ou macrovascular. Isto poderá ser explicado por alterações nos circuitos e/ou desequilíbrio entre excitação e inibição em certas redes neuronais. Estudos mostram que pessoas com diabetes mellitus tipo 2 têm concentrações de GABA aumentadas na região occipital, para além da evidência de disfunção da barreira hematoencefálica. Por estes motivos, a diabetes mellitus tipo 2 foi escolhida como modelo para o estudo do impacto da doença no sistema GABAérgico na região occipital e de como isso se relaciona com o desempenho em testes visuais. O estudo do sistema GABAérgico e a sua disfunção na epilepsia tem ganho atenção por vários motivos. Primeiro, apesar do GABA funcionar como neurotransmissor inibitório, tem sido mostrado que este funciona como neurotransmissor excitatório em neurónios imaturos. Segundo, há cada vez maior evidência da concentração aumentada de GABA na zona epileptogénica em pessoas com epilepsia refratária, sugerida por estudos in vivo e ex vivo. Por último, é expectável que as oscilações de alta frequência, quer de origem patológica quer fisiológica, se formem e sejam mantidas por potenciais pós-sinápticos mediados por recetores GABAA. A segunda parte desta tese refere-se ao estudo do sistema GABAérgico em pessoas com epilepsia refratária e como este se relaciona com a atividade fisiológica e patológica de frequências gamma. Neste trabalho foi incluído um grupo de participantes com diabetes mellitus tipo 2 e com retinopatia diabética. O objetivo era avaliar a concentração de GABA no córtex occipital como preditor do desempenho em testes visuais por estes participantes. A concentração de GABA na região occipital foi medida usando a técnica de espectroscopia por ressonância magnética nuclear e o desempenho em testes visuais foi avaliado em três áreas (visão cromática, acromática e discriminação de velocidade). Os resultados mostraram, pela primeira vez, evidência de diferenças na visão acromática e na discriminação de velocidade em participantes com diabetes mellitus tipo 2 quando comparados com participantes saudáveis. Além disso, foi encontrada pela primeira vez uma correlação positiva entre os níveis de GABA no córtex occipital e os limiares de visão acromática e discriminação de velocidade (maiores limiares significam pior desempenho). Os valores de GABA na região occipital também foram preditivos do desemprenho nos testes visuais quer na primeira avaliação, quer um ano depois, sugerindo que a modulação dos níveis de GABA no córtex occipital pode ter um impacto a longo termo no desempenho visual. O segundo trabalho realizado no âmbito desta tese refere-se ao estudo da permeabilidade da barreira hematoencefálica em pessoas com diabetes mellitus tipo 2 e a sua relação com o desempenho nos testes visuais. Os participantes com diabetes e com evidência de disfunção GABAérgica anteriormente referidos foram incluídos no estudo seguinte acerca da integridade da barreira hematoencefálica. Em suma, os resultados mostraram uma relação entre a integridade da barreira hematoencefálica e a integridade da barreira hemato-retiniana, sendo que participantes com maior ruptura da barreira hemato-retiniana apresentavam maior permeabilidade da barreira hematoencefálica. Além disso, os resultados mostraram pela primeira vez que o controlo metabólico está correlacionado com a permeabilidade da barreira hematoencefálica (pior o controlo metabólico associado a diminuição da integridade da barreira). Por último, em participantes com diabetes tipo 2 e ruptura da barreira hemato-retiniana, os resultados mostraram que a permeabilidade da barreira hematoencefálica é preditiva da acuidade visual quer no primeiro teste quer um e dois anos mais tarde em. Em seguida, o sistema GABAérgico foi estudado tendo outra doença como modelo. A função do sistema GABAérgico pode ser avaliada, de forma indireta, a partir da medição da atividade gamma usando eletroencefalografia intracraniana em pessoas com epilepsia refratária. No primeiro trabalho em que usámos a epilepsia refratária como modelo, estudou-se a atividade fisiológica de alta frequência, testando a relação entre a topografia funcional da atividade gamma alta e tomada de decisão percetual. Em suma, encontraram-se três padrões locais distintos dessa atividade de alta frequência neste grupo de participantes: a) domínio de padrões de frequência gamma baixa no processamento semântico em áreas anteriores e no processamento de objetos em áreas ventrais; b) padrões de frequência gamma baixa envolvendo redes dorsoventrais de integração de informação; c) padrões se surgimento inicial em áreas posteriores nas frequências de 60 a 250 Hz. Em suma, os resultados revelam que o reconhecimento de objetos de forma precisa e a tomada de decisão percetual estão associados a frequências gamma baixas com determinadas características espaciotemporais. O segundo trabalho usando a epilepsia refratária como modelo foi estudada a disfunção GABAérgica na epilepsia e como a modulação do sistema GABAérgico na zona epileptogénica afeta a epileptogenicidade e o sistema GABAérgico noutras áreas de referência (a região occipital). Considerando o efeito antiepilético da c-tDCS (estimulação catódica transcraniana por corrente direta), foi colocada a hipótese de que esta estimulação iria modelar os níveis de neurotransmissores responsáveis pela anormal e complexa sincronia local e pela atividade rítmica anormal comum na epilepsia. Este foi o primeiro trabalho a avaliar o impacto da c-tDCS na atividade gamma fisiológica e patológica e a medir GABA, glutamato e glutationa na zona epileptogénica e na região occipital depois da c-tDCS em participantes com epilepsia refratária. A estimulação c-tDCS diminuiu o número de descargas interictais por minuto. Esta redução revelou-se associada a uma diminuição da concentração de GABA na região occipital e na zona epileptogénica. Os resultados mostraram que a estimulação c-tDCS da zona epileptogénica cancelou a atividade gamma baixa tipicamente evocada por estímulos visuais em grelha na zona epileptogénica e aumentou essa atividade em regiões parieto-occipitais mais distantes. Em suma, este trabalho abre uma janela sobre os mecanismos de ação da estimulação c-tDCS como antiepilético e os seus efeitos no sistema GABAérgico e nos padrões de oscilações neuronais. Em síntese, os trabalhos mostram que o equilíbrio entre excitação e inibição é mantido por interação de diferentes redes neuronais numa dada janela temporal e as alterações desse equilíbrio estão associadas a dificuldades cognitivas e ao desempenho em testes visuais em pessoas com diabetes tipo 2 e à epileptogénese em pessoas com epilepsia. Os nossos resultados também sugerem que a disfunção GABAérgica na zona epileptogénica é mais do que uma consequência da epileptogénese, e poderá ser epileptogénica por si.

La déficience intellectuelle affecte de 1 à 3% de la population mondiale, ce qui en fait le trouble cognitif le plus commun de l’enfance. Notre groupe à découvert que des mutations dans le gène SYNGAP1 sont une cause fréquente de déficience intellectuelle non-syndromique, qui compte pour 1-3% de l’ensemble des cas. À titre d’exemple, le syndrome du X fragile, qui est la cause monogénique la plus fréquente de déficience intellectuelle, compte pour environ 2% des cas. Plusieurs patients affectés au niveau de SYNGAP1 présentent également des symptômes de l’autisme et d’une forme d’épilepsie. Notre groupe a également montré que SYNGAP1 cause la déficience intellectuelle par un mécanisme d’haploinsuffisance. SYNGAP1 code pour une protéine exprimée exclusivement dans le cerveau qui interagit avec la sous-unité GluN2B des récepteurs glutamatergique de type NMDA (NMDAR). SYNGAP1 possède une activité activatrice de Ras-GTPase qui régule négativement Ras au niveau des synapses excitatrices. Les souris hétérozygotes pour Syngap1 (souris Syngap1+/-) présentent des anomalies de comportement et des déficits cognitifs, ce qui en fait un bon modèle d’étude. Plusieurs études rapportent que l’haploinsuffisance de Syngap1 affecte le développement cérébral en perturbant l’activité et la plasticité des neurones excitateurs. Le déséquilibre excitation/inhibition est une théorie émergente de l’origine de la déficience intellectuelle et de l’autisme. Cependant, plusieurs groupes y compris le nôtre ont rapporté que Syngap1 est également exprimé dans au moins une sous-population d’interneurones GABAergiques. Notre hypothèse était donc que l’haploinsuffisance de Syngap1 dans les interneurones contribuerait en partie aux déficits cognitifs et au déséquilibre d’excitation/inhibition observés chez les souris Syngap1+/-. Pour tester cette hypothèse, nous avons généré un modèle de souris transgéniques dont l’expression de Syngap1 a été diminuée uniquement dans les interneurones dérivés des éminences ganglionnaires médianes qui expriment le facteur de transcription Nkx2.1 (souris Tg(Nkx2,1-Cre);Syngap1). Nous avons observé une diminution des courants postsynaptiques inhibiteurs miniatures (mIPSCs) au niveau des cellules pyramidales des couches 2/3 du cortex somatosensoriel primaire (S1) et dans le CA1 de l’hippocampe des souris Tg(Nkx2,1-Cre);Syngap1. Ces résultats supportent donc l’hypothèse selon laquelle la perte de Syngap1 dans les interneurones contribue au déséquilibre d’excitation/inhibition. De manière intéressante, nous avons également observé que les courants postsynaptiques excitateurs miniatures (mEPSCs) étaient augmentés dans le cortex S1, mais diminués dans le CA1 de l’hippocampe. Par la suite, nous avons testé si les mécanismes de plasticité synaptique qui sous-tendraient l’apprentissage étaient affectés par l’haploinsuffisance de Syngap1 dans les interneurones. Nous avons pu montrer que la potentialisation à long terme (LTP) NMDAR-dépendante était diminuée chez les souris Tg(Nkx2,1-Cre);Syngap1, sans que la dépression à long terme (LTD) NMDAR-dépendante soit affectée. Nous avons également montré que l’application d’un bloqueur des récepteurs GABAA renversait en partie le déficit de LTP rapporté chez les souris Syngap1+/-, suggérant qu’un déficit de désinhibition serait présent chez ces souris. L’ensemble de ces résultats supporte un rôle de Syngap1 dans les interneurones qui contribue aux déficits observés chez les souris affectées par l’haploinsuffisance de Syngap1.
Intellectual disability affects 1-3% of the world population, which make it the most common cognitive disorder of childhood. Our group discovered that mutation in the SYNGAP1 gene was a frequent cause of non-syndromic intellectual disability, accounting for 1-3% of the cases. For example, the fragile X syndrome, which is the most common monogenic cause of intellectual disability, accounts for 2% of all cases. Some patients affected by SYNGAP1 also showed autism spectrum disorder and epileptic seizures. Our group also showed that mutations in SYNGAP1 caused intellectual disability by an haploinsufficiency mechanism. SYNGAP1 codes for a protein expressed only in the brain which interacts with the GluN2B subunit of NMDA glutamatergic receptors (NMDAR). SYNGAP1 possesses a Ras-GAP activating activity which negatively regulates Ras at excitatory synapses. Heterozygote mice for Syngap1 (Syngap1+/- mice) show behaviour abnormalities and learning deficits, which makes them a good model of intellectual disability. Some studies showed that Syngap1 affects the brain development by perturbing the activity and plasticity of excitatory neurons. The excitatory/inhibitory imbalance is an emerging theory of the origin of intellectual disability and autism. However, some groups including ours, showed that Syngap1 is expressed in at least a subpopulation of GABAergic interneurons. Therefore, our hypothesis was that Syngap1 happloinsufficiency in interneurons contributes in part to the cognitive deficits and excitation/inhibition imbalance observed in Syngap1+/- mice. To test this hypothesis, we generated a transgenic mouse model where Syngap1 expression was decreased only in GABAergic interneurons derived from the medial ganglionic eminence, which expresses the transcription factor Nkx2.1 (Tg(Nkx2,1-Cre);Syngap1 mouse). We showed that miniature inhibitory postsynaptic currents (mIPSCs) were decreased in pyramidal cells in layers 2/3 in primary somatosensory cortex (S1) and in CA1 region of the hippocampus of Tg(Nkx2,1-Cre);Syngap1 mice. Those results suggest that Syngap1 haploinsufficiency in GABAergic interneurons contributes in part to the excitation/inhibition imbalance observed in Syngap1+/- mice. Interestingly, we also observed that miniature excitatory postsynaptic currents (mEPSCs) were increased in cortex S1 but decreased in CA1 region of the hippocampus. We further tested whether synaptic plasticity mechanisms that are thought to underlie learning and memory were affected by Syngap1 haploinsufficiency in GABAergic interneurons. We showed that NMDAR-dependent long-term potentiation (LTP) but not NMDAR-dependent long-term depression (LTD) was decreased in Tg(Nkx2,1-Cre);Syngap1 mice. We also showed that GABAA receptor blockade rescued in part the LTP deficit in Syngap1+/- mice, suggesting that a disinhibition deficit is present in these mice. Altogether, the results support a functional role of Syngap1 in GABAergic interneurons, which may in turn contributes to the deficit observed in Syngap1+/- mice.