Dissertations / Theses on the topic 'Inhibitory synapse'

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, June 2018.
Cataloged from PDF version of thesis. Page 75 blank.
Includes bibliographical references (pages 66-74).
Structural plasticity is one of the physical manifestations of circuit rewiring in the brain. Once thought to be relegated solely to developmental time periods, we now know that even in the mature brain inhibitory or excitatory connections can be made and broken, modifying the information flow within a circuit by enabling or removing specific information channels. However, the properties of inhibitory and excitatory synapse dynamics are not well understood. To address this issue, we utilized triple-color two photon microscopy to examine inhibitory and excitatory synapses across time with daily imaging. We found that the majority of dynamic spines at these intervals lacked a mature excitatory synapse as indicated by the absence of PSD-95. Inhibitory synapses were also highly dynamic during daily imaging, much more so than expected from previous results imaging at longer intervals, especially those located on spines which also contain an excitatory synapse. Surprisingly, we found that many inhibitory synapses, on the dendritic shaft and on spines, were also repeatedly removed and then reformed again at the same locations on the dendritic arbor. These recurrent inhibitory dynamic events at persistent locations represent a novel role for synapse dynamics, modulating local excitatory activity via their addition or removal. The rate of inhibitory synapse turnover was also modified by experience, as shown through their responses following monocular deprivation. We further sought to investigate these events on even shorter time scales by developing a dual color labeling strategy in combination with a newly developed line scanning temporal focusing two photon microscope, enabling imaging of the entire dendritic arbor and its inhibitory synapses in just a few minutes. This system allows for examination of synapse dynamics on the hourly time scale in vivo and can be expanded to study other molecular events that occur too fast for conventional two photon imaging.
by Kalen P. Berry.
Ph. D.

Recent innovations in live-cell imaging have demonstrated that the synapse undergoes constant remodelling and reorganisation. One well characterised aspect of this process is the lateral mobility of neurotransmitter gated receptors, which enables their dynamic exchange between synaptic and extrasynaptic populations. Regulation of this process, primarily via transient receptor-scaffold interactions, determines receptor number at the synapse and thus directly shapes the strength of synaptic neurotransmission. Neuroligins (NLs) are trans-synaptic proteins that project across the synaptic cleft and bind to partners positioned on the presynaptic side, physically fusing the two synapses in close apposition. Further to this function, the various NL isoforms (NL1-4) are essential regulators of the composition of the postsynaptic density. NL2 is primarily localised to inhibitory synapses, where it influences synaptic activity through interactions with, among others, gephyrin and collybistin. In contrast to neurotransmitter receptors, the membrane dynamics of the NL proteins are poorly understood. Thus, the aim of this thesis is to uncover the mechanisms underlying NL2 mobility on the membrane surface. Novel features of NL2 expression in non-neuronal cells were harnessed to create innovative systems in which the role of specific synaptic mechanisms was studied in isolation. Single particle tracking of NL2 with quantum dots in hippocampal neurons confirmed that endogenous NL2 exhibits transient confinement at inhibitory synapses, in line with previous findings on receptors. To uncover the underlying mechanisms, a number of important NL2 mutants were designed. Collectively, these experiments suggested that NL2 transient confinement primarily depends on intracellular interactions, specifically via phosphorylation and the PDZ and gephyrin binding domains, rather than trans-synaptic signalling mechanisms. Thus, the work described in this thesis contributes to the concept of the dynamic synapse and attributes a non-static membrane profile to NL2, which may ultimately influence synaptic remodelling and plasticity events.

Neuronal activity and subsequent calcium influx activates a signaling cascade that causes transcription factors in the nucleus to rapidly induce an early-response program of gene expression. This early-response program is composed of transcriptional regulators that in turn induce transcription of late-response genes, which are enriched for regulators of synaptic development and plasticity that act locally at the synapse.

The proper functioning of the brain and central nervous system (CNS) requires the precise formation of synapses between neurons. The two main neurotransmitter systems for fast synaptic communication in the CNS are excitatory glutamate and inhibitory gamma-aminobutyric acid. A growing body of evidence has begun to uncover several shared and divergent rules for the establishment of each of these two types of synapses. At the molecular level, a number of key proteins have been shown to be involved in the initial formation and subsequent development of synaptic connection, including cell adhesion molecules (CAMs). Among the CAMs, neurexins and neuroligins are important synaptogenic proteins that act trans-synaptically to organize synapses: binding of axonal beta-neurexins by neuroligins is sufficient to cause development of a presynaptic specialization at that site, while binding of dendritic neuroligin-1 or neuroligin-2 by beta-neurexins is sufficient to cause development of postsynaptic excitatory or inhibitory specializations, respectively. In Chapter 2, we explore the role of alpha-neurexins in synapse organization. We find alpha-neurexins are able to specifically induce the formation of inhibitory synapses, presumably through clustering of postsynaptic neuroligin-2. Moreover, we find that the expression of various splice variants of alpha- and beta-neurexins is regulated both during development and by activity, suggesting a physiological role for alternative splicing in the modulation of synapse assembly. At the cellular level, it is now clear from live imaging studies that synapses and their formation are highly dynamic processes. A number of studies have established the temporal recruitment of pre- and postsynaptic components to nascent synapses and how synapse formation can influence neuron growth. However, these studies have focused on excitatory synapses. In Chapter 3, we explore the cellular mechanisms of inhibitory synapse formation and modulation. We find that entire synapses are highly mobile and can undergo dynamic structural modulation. New synapses are formed by gradual accumulation of components from diffuse cytoplasmic pools, with a significant contribution of presynaptic vesicles from previously recycling sites. These results provide new insights into the mechanisms of inhibitory synapse formation and how it is both similar and different from excitatory synapse formation.

Le récepteur ionotrope de l'acide γ-aminobutyrique (GABAAR), perméable aux ions chlorures, est le principal récepteur neurotransmetteur médiateur de l'inhibition dans le cerveau des mammifères. La transmission GABAergique est soumise à une régulation complexe et multifactorielle. Non seulement façonnée par le cycle d'ouverture du GABAAR, qui dicte le passage entre ses conformations au repos, ouverte et désensibilisée, mais aussi par l'homéostasie des ions chlorure, laquelle détermine la polarité et l'efficacité de la transmission GABAergique, la transmission GABAergique repose aussi fortement sur le nombre de GABAARs présents à la membrane postsynaptique, localisée face aux sites présynaptiques de libération de GABA. Le nombre de ces récepteurs aux synapses est rapidement régulé par un mécanisme de "diffusion-capture", dans lequel les GABAARs alternent entre une diffusion rapide à la membrane plasmique extrasynaptique et un ralentissement suivi d'un confinement aux synapses. De fait, ce confinement et cette agrégation synaptique sont médiés par l'interaction entre les GABAARs et leur principale protéine d'échafaudage synaptique, la géphyrine. La régulation de la diffusion latérale des récepteurs est considérée comme le principal mécanisme d'ajustement du nombre de récepteurs aux synapses en réponse à la demande synaptique. De plus, l'activité neuronale régule cette diffusion latérale des GABAARs, notamment en contrôlant la liaison des récepteurs à la géphyrine par la modulation de la phosphorylation des récepteurs et/ou de la géphyrine en aval des cascades de kinases, influençant ainsi la conformation de ces protéines. Au cours de ma thèse, j'ai étudié la régulation dynamique des synapses GABAergiques dans l'hippocampe à travers la phosphorylation de la géphyrine et la conformation des récepteurs, en utilisant notamment des techniques de microscopie optique de pointe, telles que le suivi de particules individuelles (SPT), la microscopie de reconstruction optique stochastique (STORM) et la microscopie de localisation photo-activée (PALM), en recourant à des stratégies pharmacologiques et de mutagenèse dirigée in vitro et in vivo. Plus précisément, mes recherches suggèrent que l'organisation synaptique des GABAARs et de la géphyrine dans l'hippocampe est régulée dynamiquement et est médiée, entre autres, par la phosphorylation de la géphyrine via la voie de signalisation sensible au chlorure WNK/SPAK/OSR1, une cascade de kinases précédemment liée à l'homéostasie des ions chlorures et à la transmission inhibitrice. D'autre part, mes résultats indiquent que l'état de conformation des GABAARs impacte leur régulation dynamique et leur organisation au niveau de la synapse. En somme, mes travaux doctoraux apportent de nouvelles perspectives sur la régulation dynamique de l'organisation et de la fonction des synapses GABAergiques dans l'hippocampe mature des modèles murins
The chloride ion permeant ionotropic γ-aminobutyric acid receptor (GABAAR) is the principal neurotransmitter receptor mediating inhibition in the mammalian brain. The GABAergic GABAergic transmission is subjected to a complex and multifactorial regulation. Shaped by the gating cycle of GABAAR, which dictates the switch between their resting, open, and desensitized conformation and by chloride homeostasis which dictates the polarity and efficacy of the GABAergic transmission, the GABAergic transmission also relies heavily on the number of GABAARs present in the postsynaptic membrane opposite to presynaptic GABA-releasing sites. The number of GABAARs at synapses is rapidly regulated by a “diffusion-capture” mechanism wherein receptors alternate between rapid diffusion into the extrasynaptic plasma membrane and slowing down and confinement to the synapses. This confinement and synaptic aggregation are mediated by the interaction between GABAARs and their primary scaffolding protein at the synapse, gephyrin. Regulation of receptor lateral diffusion is considered the first mechanism for adjusting the number of receptors at synapses in response to synaptic demand. Neuronal activity regulates the lateral diffusion of GABAARs, particularly by controlling receptor binding to gephyrin through the modulation of receptor and/or gephyrin phosphorylation downstream of kinase cascades, which subsequently influences the conformation of these proteins. During my PhD, I have investigated the dynamic regulation of GABAergic synapses in the hippocampus through the lens of gephyrin phosphorylation and receptor conformation, using state of the art optical microscopy techniques, such as Single Particle Tracking (SPT), STochastic Optical Reconstruction Microscopy (STORM) or Photo-Activated Localization Microscopy (PALM), and relying on pharmacological and directed mutagenesis strategies in vitro and in vivo. More specifically, my research suggests that GABAergic synapses in the hippocampus are dynamically regulated, with modulation of GABAAR and gephyrin synaptic organization mediated by gephyrin phosphorylation through the chloride-sensitive WNK/SPAK/OSR1 signaling pathway, a kinase cascade previously linked to chloride homeostasis and inhibitory transmission. Additionally, my findings indicate that the conformation state of GABAARs impacts their dynamic regulation and organization at the synapse. Overall, my doctoral work provides new insights into the dynamic regulation of GABAergic synapses organization and function in the mature hippocampus of murine models

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

La synapse est une structure macromoléculaire dont les composants sont renouvelés en permanence alors que l’assemblage est quasi-stable. A l’échelle mésoscopique, les récepteurs aux neurotransmetteurs (RN) sont accumulés dans le compartiment post-synaptique (PSD). Cette accumulation résulte de la diffusion latérale des RNs dans la membrane neuronale et de leurs immobilisations transitoires dans la PSD. Les protéines d’échafaudage (PE) localisées sous la membrane post-synaptique constituent des sites de capture en interagissant avec les RNs. Mon travail de thèse s’inscrit dans le cadre d’une collaboration avec des chimistes et des physiciens afin de comprendre les paramètres impliqués dans le processus de diffusion-capture. Nous nous sommes intéressés au cas de la capture du récepteur de la glycine (RGly) par les agrégats de PEs des synapses inhibitrices, les géphyrines. Nous avons étudié l’impact de la liaison RGly-géphyrine sur le processus de diffusion-capture sous deux aspects. Le premier est lié à la nature bimodale de liaison du RGly. Le second aborde l’impact des phosphorylations de la boucle M3-M4 de la sous-unité β du RGly sur la liaison avec la géphyrine.Mon travail de thèse montre qu’il est maintenant possible, en utilisant des approches de microscopie super-résolutive, de quantifier les aspects thermodynamiques des interactions moléculaires dans les cellules vivantes
The synapse is a macromolecular structure whose components are constantly renewed while the assembly remains quasi-stable. At the mesoscopic level, neurotransmitter receptors (RNs) accumulate in the post-synaptic compartment (PSD). This accumulation is the result of the lateral diffusion of RNs in the neuronal membrane and transient immobilization within the PSD. This mechanism, called diffusion-trapping has been highlighted by single-molecule-tracking techniques. Scaffold proteins (PE) are localized under the post-synaptic membrane. These proteins form trapping-sites by interacting with RNs. Through an interdisciplinary approach in collaboration with chemists and physicists, the aim of my doctoral research was to understand the parameters that are involved in diffusion-trapping mechanisms. We especially focused on glycine receptor (RGly) trapping by PE clusters at inhibitory synapses, namely the scaffold protein gephyrin. The gephyrin- interaction motif of the GlyR is located within the cytoplasmic domain of the β-subunit of the receptor, the so-called β-loop. Two aspects of the impact of RGly-gephyrin binding on diffusion-trapping were studied. The first was to identify the source of the RGly-gephyrin bimodal binding. The second one addressed the regulation of gephyrin binding by phosphorylation of the GlyR βLoop.My research thus shows that it is now possible to quantify thermodynamic aspects of molecular interactions in living cells using high-density single-molecule-tracking

La microglie désigne l'ensemble des macrophages résidents du système nerveux central (SNC). Longtemps considérées comme étant actives uniquement en conditions pathologiques, les cellules microgliales sont pourtant essentielles à l'activité physiologique du SNC. En particulier, pendant la formation du SNC, elles régulent apoptose et survie neuronales, et interagissent directement avec les synapses en les éliminant, en promouvant leur formation ou en régulant leur activité. Toutefois, les mécanismes microgliaux impliqués dans la mise en place et la maturation fonctionnelle des circuits corticaux pendant le développement ne sont pas intégralement élucidés. Afin de mieux comprendre le rôle de la microglie dans le développement cortical, nous avons utilisé le système des champs de tonneaux du cortex somatosensoriel de la souris, et combiné des manipulations in vivo avec des approches électrophysiologiques, optogénétique, pharmacologique et histologique sur tranches de cerveaux de souris génétiquement modifiées. Dans une première étude, nous nous sommes intéressés aux conséquences de l'arrivée de la microglie à proximité des zones de terminaison des fibres thalamiques (les centres des tonneaux) dans le cortex somatosensoriel au cours de la première semaine postnatale sur les propriétés fonctionnelles des synapses thalamocorticales et de l'inhibition disynaptique associée (inhibition antérograde ou feedforward). Nos résultats montrent qu'une déplétion de la microglie pendant la première semaine postnatale entraîne un retard de maturation fonctionnelle de la connexion thalamocorticale excitatrice monosynaptique et de l'inhibition feedforward disynaptique au niveau des cellules principales excitatrices de la couche 4 (CP) entre les 10ème et 12ème jours postnataux (P10-12). Nous avons ensuite testé si le facteur neurotrophique BDNF (brain-derived neurotrophic factor) pouvait être la molécule microgliale impliquée dans la maturation de ces synapses corticales en utilisant une approche transgénique (lignée CX3CR1+/CreERT2;BDNFlox/lox). Nos enregistrements indiquent que l'absence de BDNF microglial entraîne aussi un déficit de maturation fonctionnelle des connexions excitatrices monosynaptiques et inhibitrices disynaptiques thalamocorticales entre P10-12. Nous avons donc identifié un facteur microglial clé dans la maturation des synapses corticales, et nos enregistrements chez le jeune adulte suggèrent que la suppression de BDNF microglial pendant la première semaine postnatale altère la synapse thalamocorticale excitatrice sur le long terme. Dans une deuxième étude, nous avons examiné les conséquences de perturbations de la microglie au cours du développement embryonnaire sur la mise en place des réseaux corticaux. L'induction d'une activation immunitaire maternelle (MIA) par injection de lipopolysaccharide (LPS) bactérien ou la déplétion de la microglie aux stades embryonnaires modifie la répartition laminaire des interneurones inhibiteurs exprimant la parvalbumine (PV+) _cellules responsables de l'inhibition feedforward_ dans le cortex jusqu'à P20. Nos données fonctionnelles ont révélé que ces protocoles de MIA et de déplétion induisent une augmentation de l'inhibition périsomatique des CP à P20, ainsi qu'une exubérance horizontale de l'inhibition soutenue par les interneurones PV+. Cette inhibition exacerbée ne perdure pas et nos enregistrements chez l'adulte indiquent au contraire un affaiblissement des synapses inhibitrices entre les interneurones PV+ et les CP. Nous postulons donc que les cellules microgliales sont le chaînon manquant entre des stimulations immunitaires, telles qu'elles peuvent se produire durant une inflammation pendant la grossesse, et l'augmentation du risque de développer des pathologies neurodéveloppementales. Ainsi, nos résultats mettent en exergue le rôle crucial de la microglie dans le développement des réseaux neuronaux corticaux pendant la période périnatale
Microglial cells are a population of specialized macrophages residing in the CNS only. They have long been studied solely under pathological contexts and were thought to be active only upon homeostatic disturbance following a brain lesion. However, over the last decade, they have been increasingly recognized to be essential players in the physiological functioning of the CNS. Specifically, during the CNS formation, microglia has been shown to regulate apoptosis and neuronal survival. They are also able to directly interact with synapses, by eliminating supernumerary and inappropriate connections, by promoting synapse formation or by regulating their activity. However, mechanisms by which microglia influence wiring and functional maturation of cortical are not fully understood. To better assess the role of microglia in cortical development, we used the barrel field as a model of neuronal development and we combined in vivo manipulations together with electrophysiology, optogenetics, pharmacologic and histologic approaches on brain slices of genetically-engineered mice. We first explored the consequences of microglia entry near the terminals of thalamic afferents (center of the barrels) in the primary somatosensory cortex during the first postnatal week on functional properties of thalamocortical synapses and associated disynaptic feedforward inhibition. By selectively depleting microglia at early postnatal days by intracerebral injections of clodronate-encapsulated liposomes, we show that microglia absence during the first postnatal week delays the functional maturation of both monosynaptic thalamocortical synapse and feedforward inhibition of layer 4 principal cells of the barrel cortex (PC) up to the 10th and 12th postnatal days (P10-12). To identify the mechanism underlying this process, we used the CX3CR1+/CreERT2; BDNFlox/lox mouse line allowing the conditional deletion of microglial BDNF during the first postnatal week. Our recordings indicate that the absence of microglial BDNF, as well as early microglia depletion, leads to a deficit in the functional maturation of both monosynaptic excitatory and disynaptic inhibitory thalamocortical connexions between P10-12. We therefore identified a microglial key factor in the maturation of cortical synapses. Our recordings in the young adult suggest that early microglial BDNF deletion has a long-term effect on thalamocortical excitatory synapses. In a second study, we investigated the consequences of microglia dysfunction during embryonic development on cortical networks wiring. Maternal immune activation (MIA) triggered by bacterial lipopolysaccharide (LPS) injection modifies the laminar repartition of parvalbumin-expressing inhibitory interneurons (PV+), key actors in neuropsychiatric disease, in the cortex until P20. Our functional data revealed that these MIA and depletion protocols lead to an increase of layer 4 PC perisomatic inhibition at P20, as well as a horizontal exuberance of cortical inhibition supported by PV+ interneurons. This increased inhibition does not last within development as suggested by our recordings in the adult. On the opposite, it seems that MIA and early microglia depletion result in weaker inhibitory synapses at P60. To conclude, we postulate that microglial cells are the missing link between maternal immune challenge and à higher risk of having neurodevelopmental pathologies like autism or schizophrenia. Our results highlight the crucial role of microglial cells in neuronal network development during perinatal period

Diazepam is an allosteric modulator of GABAA receptors which potentiates GABAA receptor activity resulting in enhanced inhibitory synaptic transmission. Diazepam is used to treat anxiety, insomnia and seizures, however, its use is limited due to the development of tolerance. Here I show that prolonged treatment of cortical neurones with diazepam triggers endocytosis and subsequent downregulation of cell-surface GABAA receptors. Using pharmacological reagents, I have demonstrated that diazepam triggers PLC-dependent release of calcium from the endoplasmic reticulum which activates the phosphatase calcineurin resulting in dephosphorylation of the γ2 subunit of GABAA receptors and their endocytosis. This was elucidated using a combination of biochemical and cell biological approaches. In addition, I have developed HEK293 cell lines stably expressing various subtypes of GABAA receptors to investigate further diazepam and isoguvacine-dependent regulation of GABAA receptors. The same calcium-dependent signalling pathway that regulates cell-surface stability of GABAA receptors in neurones was found to operate in HEK293 cells. Subsequently, I focused on a key component of this signalling pathway; PLCδ1. Using biochemical techniques I have demonstrated that PLCδ1 binds directly to the GABAA receptor β3 subunit at two independent sites. This binding was confirmed by coimmunoprecipitation of PLCδ1 and GABAA receptors from cortical neuronal lysates. Interestingly, upon diazepam treatments, PLCδ1 was shown to dissociate from GABAA receptors, thus leading to mobilisation of calcium from the intracellular stores and activation of calcineurin. To assess how changes in cell-surface expression of GABAA receptors affect the stability of GABAergic synapses, I characterised the size and number of post-synaptic GABAA receptor clusters and the number of presynaptic GABA-releasing terminals following chronic diazepam treatment. I observed a reduction in the size and number of post-synaptic GABAA receptor clusters and a reduction in the number of GABA-releasing terminals. These data are consistent with the loss of cell-surface GABAA receptors following long-term treatments of cortical neurones with diazepam. These changes correlated with an increase in the expression of the early apoptosis marker, cleaved-caspase 3, in glutamatergic neurones suggesting indirect cytotoxic effects of diazepam treatments. The loss of inhibitory GABAergic synapses following chronic diazepam treatment may contribute to the well-known development of tolerance to these clinically important therapies for stress- and anxiety- related neurological disorders.

Synapsins are the most abundant family of neuro-specific phosphoproteins associated with the cytoplasmic surface of the synaptic vesicle membrane. These proteins actively regulate synaptic transmission at the level of the presynaptic terminal by controlling the storage and mobilization of synaptic vesicles within a reserve pool. However, it is hypothesized that synapsins could be involved in other stages of synaptic vesicle dynamics such as trafficking, docking, fusion with the plasma membrane and consequent recycling. Synapsin I (SynI) in particular is expressed two isoforms (Ia and Ib) at the presynaptic compartment of all neurons in the adult brain. Several studies suggest that SynI is also involved in axon elongation and synaptic vesicle fusion kinetics. In human, nonsense and missense mutations of SYN1 gene are related to several diseases such as epilepsy and autism spectrum disorder; in fact, SynI knockout (KO) mice show an epileptic and autism-like phenotype. To carry out its functions, SynI requires to bind ATP in a Ca2+-dependent manner thanks to the coordination of a glutamate residue (E373). As ATP binding regulates SynI oligomerization and SV clustering, we analyzed the effect of E373K mutation on neurotransmitter release and short-term plasticity in excitatory and inhibitory synapses. We coupled electrophysiology (patch-clamp recordings) with electron microscopy in primary SynI KO hippocampal neurons in which either the human wild type or the E373K mutant SynI were re-introduced by infection with lentiviral vectors. Our data indicate that E373K mutation affects predominantly excitatory synapses. The frequency of miniature excitatory postsynaptic currents (mEPSCs) was enhanced, without changes in the amplitude and in the number of excitatory synapses. The increment of mEPSCs frequency was totally abolished after acute injection of BAPTA-AM (a specific Ca2+ chelator), suggesting a possible alteration of Ca2+ homeostasis at the presynaptic terminal. Excitatory E373K-Syn I neurons showed reduced evoked EPSC amplitude attributable to a reduction of the readily releasable pool (RRP), while, on the contrary, inhibitory E373K-Syn I neurons did not show any difference both in miniature, evoked IPSC amplitude and RRP size. While no effects in the dynamics and steady state of depression were detected, both excitatory and inhibitory E373K-Syn I neurons failed to recover after stimulation with long high-frequency trains. No mutation-induced changes were observed in network firing/bursting activity as determined with multi-electrode extracellular recordings. Our data suggest that the Ca2+-dependent regulation of ATP-binding to SynI plays important roles in spontaneous and evoked neurotransmitter release that differentially affect the strength of excitatory and inhibitory transmission.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2016.
This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Cataloged from student-submitted PDF version of thesis. "February 2016."
Includes bibliographical references (pages 113-123).
Structural plasticity, the rewiring of synaptic connections, occurs not only during development, but is prevalent in the adult brain and likely represents the physical correlate of learning and memory. Removal or addition of excitatory and inhibitory synaptic inputs onto a neuron can affect their relative influence on excitation in specific dendritic segments, and ultimately regulate neuronal firing. However, the structural dynamics of excitatory and inhibitory synapses in vivo, and their relation to each other, is not well understood. To gain insight into synaptic remodeling in the adult brain in vivo, we used dual- and triple- color two-photon imaging to track the dynamics of all inhibitory and excitatory synapses onto a given neuron in the cerebral cortex at different timescales. By studying synaptic changes over 4-day or 24-hour intervals we were able to determine that inhibitory synapses are remarkably dynamic in vivo. We found that Inhibitory synapses occur not only on the dendritic shaft, but also a significant fraction is present on dendritic spines, alongside an excitatory synapse. Inhibitory synapses on these dually innervated spines are remarkably dynamic and in stark contrast to the stability of excitatory synapses on the same spines. Many of the inhibitory synapses on dendritic spines repeatedly disappear and reappear in the same location. These reversible structural dynamics indicate a fundamentally new role for inhibitory synaptic remodeling - flexible, input-specific modulation of stable excitatory connections. To determine whether synapse dynamics are regulated by experience-dependent plasticity, we performed monocular deprivation, finding that an ocular dominance shift reduces inhibitory synaptic lifetime and increases recurrence. To investigate the molecular mechanism of rapid inhibitory synapse appearance and removal, I am currently testing molecular interventions that influence the clustering of gephyrin, a scaffolding molecule that anchors inhibitory receptors at postsynaptic sites.
by Katherine L. Villa.
Ph. D.

The cerebellum is vitally important for motor learning and this behaviour is heavily reliant on the plasticity of excitatory and inhibitory synaptic transmission. Over the past two decades, numerous forms of synaptic plasticity within the cerebellum have been described, particularly affecting the principle output inhibitory neuron, the Purkinje cell (PC). In this study, I have focused on the mechanism underlying the phenomenon of rebound potentiation (RP), which is a long-lasting enhancement of inhibitory transmission at interneuron-PC synapses. RP is triggered by strong climbing fibre induced depolarization of postsynaptic PCs. Subsequent Ca^{2+} influx via voltage-dependent calcium channels, and Ca^{2+} release from intracellular stores, synergise to activate Ca^{2+} dependent kinase pathways resulting in a persistent enhancement of synaptic γ aminobutryic acid type-A receptor (GABA_AR) mediated current on PCs. The types of RP that are induced can be classified as early and late RP. The induction of RP critically depends on both Ca^{2+}/Calmodulin-dependent protein kinase II (CaMKII) and protein tyrosine kinase (PTK) activation, as selective inhibitors (CaMKIINtide and genistein) blocked the RP of miniature inhibitory postsynaptic currents (mIPSCs). These kinases were found to work through inter-dependent pathways with PTK acting dowmtream of CaMKII. Furthermore, CaMKII activity is required for both the induction and the maintenance of RP. In PCs, the majority of GABA_ARs are comprised of α1β2γ2 subunits. The α1 subunit was essential for mediating the phasic inhibition observed in PCs; whereas, the β2 subunit-containing receptors underlied the large amplitude, fast rise time mIPSCs and were also critically important for the induction of RP. Thirdly, tyrosine phosphorylation of the γ2 subunit was found to determine the direction of plasticity, converting RP to a new phenomenon of rebound depression. Finally, by using inhibitors of SNARE-based exocytosis pathways, we determined that GABA_A receptor insertion is the underlying mechanism of RP. In conclusion, RP is a phosphorylation-dependent subunit-specific plasticity of GABA_ARs, which is manifest by postsynaptic GABA_A receptor insertion. As such, it may be an important contributory factor to motor learning in the cerebellum.

For years, GABAergic synapses were considered poorly plastic. However, an increasing body of evidence demonstrated that, similarly to excitatory synapses, inhibitory synapses formed by interneurons onto pyramidal cells can be modulated in response to neuronal activity. The spiking output of a neuron is determined by the opposite but concomitant action of excitation and inhibition. In this view, it is crucial to understand how plasticity at excitatory and inhibitory synapses is coordinated. In this thesis, I will describe two different types of plasticity interplay between excitation and inhibition, in space and time domains. In our previous work (Petrini et al., 2014) we characterized the induction and the expression of postsynaptic inhibitory long term potentiation (iLTP) following the administration of NMDA and CNQX (chemical protocol of induction of iLTP). Here we induced postsynaptic iLTP by administering electrophysiological stimulations intended to better mimic physiological neuronal activity. We observed that the delivery to the postsynaptic neuron of a train of action potentials at frequency of 2Hz, (that we define here low frequency stimulation, LFS) potentiated the amplitude of the inhibitory postsynaptic currents (iPSCs) up to 30 minutes. This particular form of iLTP depended on moderate calcium increase in the postsynaptic neuron and the activation of the Calcium calmodulin kinase II (CaMKII). The concomitant investigation of excitatory transmission revealed the depression of excitatory postsynaptic currents (ePSCs) amplitude thus indicating that LFS induced excitatory (LTD). In order to further study the interaction between excitatory and inhibitory synaptic plasticity, we paired the protocol of iLTP together with simultaneous photorelease of glutamate on a single spine. This particular type of Hebbian stimulation induced LTP at the photostimulated spine (Lee et al., 2009; Matsuzaki et al., 2001, 2004) and LTD on the other spines. In contrast, we observed that the GABAergic synapses located distant from the potentiated spine showed an iLTP while the ones located in a range of 3 micron from the potentiated spine were depressed. Such “inversion of plasticity” was promoted by a massive influx of calcium and the activation of calpain, a protease involved in the cleavage of the gephyrin. In the second part of this thesis, I will show a new type of structural inhibitory short term depression plasticity (siSTD) that occurs during the induction of the plasticity protocol, before the expression of iLTP. In particular, the depolarization of the postsynaptic neuron induced a transient depression of inhibitory synapses that was dependent on the fast activation of the protease calpain. We propose that such “early” calpain activation pathway competes with that of the CaMKII thus defining the extent of long-term inhibitory plasticity.

Tableau d'honneur de la FÉSP
Tableau d'honneur de la FÉSP
La signalisation calcique dendritique joue un rôle important dans la régulation de mécanismes neuronaux, tels que la plasticité synaptique et l’intégration de l’information transmise. Bien compris chez les neurones principaux, ce processus de régulation est moins étudié chez les divers types d’interneurones GABAergiques qui modulent l’acquisition et l’envoi de signaux neuronaux. Chez les interneurones à décharge rapide, un type d’interneurone commun dans les circuits corticaux, il a été démontré qu’il y a absence de rétropropagation des potentiels d’action dans les dendrites distales (Hu et al., 2010). Cette découverte a des implications fonctionnelles, car la rétropropagation des potentiels d’action est un signal important pour l’induction des formes de plasticité synaptique hebbiennes. Par contre, il a été suggéré que l’activité dendritique locale pourrait compenser pour l’absence de rétropropagation des potentiels d’action. En conséquence, ce travail porte sur l’étude des évènements calciques dans les dendrites distales des interneurones à décharge rapide. Nous avons cherché à déterminer s’il est possible de générer ces signaux calciques par stimulation dendritique locale, à étudier les mécanismes responsables de ces signaux et à déterminer si ces signaux jouent un rôle dans la régulation de la plasticité synaptique à ces synapses. Pour atteindre ces objectifs, nous avons utilisé une combinaison de méthodes électrophysiologiqes (patch-clamp en mode cellule entière), d’imagerie calcique deux-photons et de modélisation computationnelle. Nous avons pu établir qu’il est possible de générer des évènements calciques postsynaptiques supralinéaires dans les synapses excitatrices étudiées par stimulation électrique locale. Ces signaux sont médiés par l’influx calcique provenant de l’activation des récepteurs AMPA perméables au Ca2+, qui déclenche à son tour le relâchement de Ca2+ par les récepteurs ryanodine présents sur réserves calciques intracellulaires. Ces signaux comprennent aussi une contribution calcique mineure des récepteurs NMDA, et ils restent locaux (pas de propagation dans l’arbre dendritique). De plus, nous avons déterminé que ces évènements calciques supralinéaires produisent un revirement de la plasticité synaptique, car ils induisent la dépression à long-terme dans les synapses étudiées, alors que les signaux calciques de basse amplitude induisent la potentiation à long-terme. Nous avons aussi examiné si ces évènements calciques supralinéaires étaient générés de façon équivalente dans les dendrites apicales et basales, qui reçoivent des synapses de différentes sources. Nous avons observé que les signaux des dendrites apicales avaient une plus grande amplitude et étaient associés à un plus haut niveau de dépolarisation. À partir de la modélisation, nous avons pu prédire le nombre de synapses nécessaires à la génération de ces signaux et la contribution potentielle des mécanismes d’extrusion du Ca2+. Finalement, nous avons étudié la spécificité cellulaire des mécanismes d’intégration dendritique en combinant l’imagerie calcique et la modélisation dans un type différent d’interneurone, les interneurones spécifiques aux interneurones type III. En conclusion, nous avons prouvé qu’il existe dans certains interneurones des mécanismes alternatifs, médiés par des hausses de Ca2+ locales, permettant la régulation de la plasticité aux synapses excitatrices.
La signalisation calcique dendritique joue un rôle important dans la régulation de mécanismes neuronaux, tels que la plasticité synaptique et l’intégration de l’information transmise. Bien compris chez les neurones principaux, ce processus de régulation est moins étudié chez les divers types d’interneurones GABAergiques qui modulent l’acquisition et l’envoi de signaux neuronaux. Chez les interneurones à décharge rapide, un type d’interneurone commun dans les circuits corticaux, il a été démontré qu’il y a absence de rétropropagation des potentiels d’action dans les dendrites distales (Hu et al., 2010). Cette découverte a des implications fonctionnelles, car la rétropropagation des potentiels d’action est un signal important pour l’induction des formes de plasticité synaptique hebbiennes. Par contre, il a été suggéré que l’activité dendritique locale pourrait compenser pour l’absence de rétropropagation des potentiels d’action. En conséquence, ce travail porte sur l’étude des évènements calciques dans les dendrites distales des interneurones à décharge rapide. Nous avons cherché à déterminer s’il est possible de générer ces signaux calciques par stimulation dendritique locale, à étudier les mécanismes responsables de ces signaux et à déterminer si ces signaux jouent un rôle dans la régulation de la plasticité synaptique à ces synapses. Pour atteindre ces objectifs, nous avons utilisé une combinaison de méthodes électrophysiologiqes (patch-clamp en mode cellule entière), d’imagerie calcique deux-photons et de modélisation computationnelle. Nous avons pu établir qu’il est possible de générer des évènements calciques postsynaptiques supralinéaires dans les synapses excitatrices étudiées par stimulation électrique locale. Ces signaux sont médiés par l’influx calcique provenant de l’activation des récepteurs AMPA perméables au Ca2+, qui déclenche à son tour le relâchement de Ca2+ par les récepteurs ryanodine présents sur réserves calciques intracellulaires. Ces signaux comprennent aussi une contribution calcique mineure des récepteurs NMDA, et ils restent locaux (pas de propagation dans l’arbre dendritique). De plus, nous avons déterminé que ces évènements calciques supralinéaires produisent un revirement de la plasticité synaptique, car ils induisent la dépression à long-terme dans les synapses étudiées, alors que les signaux calciques de basse amplitude induisent la potentiation à long-terme. Nous avons aussi examiné si ces évènements calciques supralinéaires étaient générés de façon équivalente dans les dendrites apicales et basales, qui reçoivent des synapses de différentes sources. Nous avons observé que les signaux des dendrites apicales avaient une plus grande amplitude et étaient associés à un plus haut niveau de dépolarisation. À partir de la modélisation, nous avons pu prédire le nombre de synapses nécessaires à la génération de ces signaux et la contribution potentielle des mécanismes d’extrusion du Ca2+. Finalement, nous avons étudié la spécificité cellulaire des mécanismes d’intégration dendritique en combinant l’imagerie calcique et la modélisation dans un type différent d’interneurone, les interneurones spécifiques aux interneurones type III. En conclusion, nous avons prouvé qu’il existe dans certains interneurones des mécanismes alternatifs, médiés par des hausses de Ca2+ locales, permettant la régulation de la plasticité aux synapses excitatrices.
Dendritic Ca2+ signaling plays an important role in the regulation of neuronal processes, such as synaptic plasticity and input integration. Well-studied in principal neurons, this form of regulation is not well understood in the various types of GABAergic interneurons that modulate activity in neuronal networks. In fastspiking (FS) interneurons, a common interneuron type in cortical circuits, it has been shown that there is a lack of action potential (AP) backpropagation in distal dendrites (Hu et al., 2010). This discovery has functional implications, AP backpropagation is an important signal for the induction of Hebbian forms of synaptic plasticity. However, it has been suggested that local dendritic activity could compensate for the absence of AP backpropagation. Consequently, this work focuses on the study of Ca2+ transients in distal dendrites of FS interneurons. We sought to determine whether it is possible to generate supralinear Ca2+ transients through local dendritic stimulation, to study the mechanisms responsible for those transients and to determine whether those signals play a role in the regulation of synaptic plasticity at those synapses. To reach those objectives, we used a combination of electrophysiological methods (whole-cell patch-clamp recordings), two-photon Ca2+ imaging and of computational modeling. We were able to establish that supralinear postsynaptic Ca2+ transients can be generated through local electrical stimulation of excitatory synapses in distal dendrites. These Ca2+ transients were mediated by Ca2+ influx from the activation of Ca2+-permeable AMPA receptors, which triggers Ca2+ release through ryanodine receptors present on intracellular Ca2+ stores (Ca2+-induced Ca2+ release). These Ca2+ signals also contain a minor contribution from NMDA receptors, and stay localized (no significant propagation in the dendritic arbor). In addition, we determined that these supralinear Ca2+ signals constitute a switch in the expression of synaptic plasticity, as they induce long-term depression in local synapses, while low-amplitude Ca2+ signals induced synaptic long-term potentiation. We also examined whether these supralinear Ca2+ transients were generated in both apical and basal dendrites, which receive synaptic contacts from different sources (Schaffer collaterals vs local collaterals). We observed that Ca2+ transients in apical dendrites had a higher amplitude and were associated with a higher level of somatic depolarization. We were also able to predict, through computational modeling, the number of synapses necessary to the generation of those signals and the potential contribution of Ca2+ extrusion mechanisms. Finally, we studied the cell-specificity of dendritic integration mechanisms by combining Ca2+ imaging and modeling in a different interneuron type, interneuron-specific interneurons type III. In conclusion, we were able to prove that certain interneurons possess alternative mechanisms, mediated through local Ca2+ transients, that allow for the regulation of plasticity at excitatory synapses.
Dendritic Ca2+ signaling plays an important role in the regulation of neuronal processes, such as synaptic plasticity and input integration. Well-studied in principal neurons, this form of regulation is not well understood in the various types of GABAergic interneurons that modulate activity in neuronal networks. In fastspiking (FS) interneurons, a common interneuron type in cortical circuits, it has been shown that there is a lack of action potential (AP) backpropagation in distal dendrites (Hu et al., 2010). This discovery has functional implications, AP backpropagation is an important signal for the induction of Hebbian forms of synaptic plasticity. However, it has been suggested that local dendritic activity could compensate for the absence of AP backpropagation. Consequently, this work focuses on the study of Ca2+ transients in distal dendrites of FS interneurons. We sought to determine whether it is possible to generate supralinear Ca2+ transients through local dendritic stimulation, to study the mechanisms responsible for those transients and to determine whether those signals play a role in the regulation of synaptic plasticity at those synapses. To reach those objectives, we used a combination of electrophysiological methods (whole-cell patch-clamp recordings), two-photon Ca2+ imaging and of computational modeling. We were able to establish that supralinear postsynaptic Ca2+ transients can be generated through local electrical stimulation of excitatory synapses in distal dendrites. These Ca2+ transients were mediated by Ca2+ influx from the activation of Ca2+-permeable AMPA receptors, which triggers Ca2+ release through ryanodine receptors present on intracellular Ca2+ stores (Ca2+-induced Ca2+ release). These Ca2+ signals also contain a minor contribution from NMDA receptors, and stay localized (no significant propagation in the dendritic arbor). In addition, we determined that these supralinear Ca2+ signals constitute a switch in the expression of synaptic plasticity, as they induce long-term depression in local synapses, while low-amplitude Ca2+ signals induced synaptic long-term potentiation. We also examined whether these supralinear Ca2+ transients were generated in both apical and basal dendrites, which receive synaptic contacts from different sources (Schaffer collaterals vs local collaterals). We observed that Ca2+ transients in apical dendrites had a higher amplitude and were associated with a higher level of somatic depolarization. We were also able to predict, through computational modeling, the number of synapses necessary to the generation of those signals and the potential contribution of Ca2+ extrusion mechanisms. Finally, we studied the cell-specificity of dendritic integration mechanisms by combining Ca2+ imaging and modeling in a different interneuron type, interneuron-specific interneurons type III. In conclusion, we were able to prove that certain interneurons possess alternative mechanisms, mediated through local Ca2+ transients, that allow for the regulation of plasticity at excitatory synapses.

L'hippocampe est une région du cerveau importante pour la formation de mémoire. Des études récentes ont montré que la zone CA2 de l'hippocampe, longtemps ignorée, joue un rôle clef dans certaines formes de mémoire et notamment dans la mémoire sociale. De plus, des études post-mortem ont révélé des altérations spécifiques à la région CA2 chez les patients schizophrènes. Cependant, l’implication des neurones de CA2 dans les circuits de l'hippocampe reste peu connu, tant dans des conditions physiologiques que pathologiques. En combinant pharmacologie, génétique et électrophysiologie sur tranches d’hippocampe de souris, nous avons étudié comment les neurones pyramidaux (NP) CA2 sont recrutés dans les circuits hippocampiques après des changements d’inhibition et comment le recrutement des NP CA2 pourrait moduler l’information sortant de l'hippocampe. D’autre part, nous avons examiné les altérations fonctionnelles de la zone CA2 dans le modèle murin Df(16)A+/- de la microdélétion 22q11.2, le facteur génétique de risque de schizophrénie le plus élevé. Dans la région CA2 de l’hippocampe, les synapses inhibitrices contrôle les afférences des collatérales de Schaeffer (CS) et expriment une dépression à long-terme (DLTi) unique qui dépendant des récepteurs delta-opioïdes (RDO). Contrairement aux synapses CS-CA1, les synapses excitatrices CS-CA2 n’expriment pas de potentialisation à long-terme après application des protocoles d'induction. Cependant, nous avons constaté que différents types d'activités induisent une augmentation durable de l’amplitude des potentiels post-synaptiques (PPS) évoqués aussi bien par une stimulation des CS que des afférences distales des NP CA2, et ceci via une modulation de la balance excitation/inhibition. Nous avons démontré que ces augmentations du rapport excitation/inhibition sont les conséquences directes de la DLTi RDO-dépendante. De plus, la DLTi permet le recrutement des NP CA2 par les NP CA3 alors qu’une inhibition intacte empêche complètement leur activation en réponse aux stimulations des CS. Par ailleurs, le recrutement des pyramides de CA2 par les CS après disinhibition activité-dépendante ajoute une composante polysynaptique (SC-CA2-CA1) au PPS monosynaptique (SC-CA1) dans les NP CA1 et augmente leur activité. De plus, l’inactivation des interneurones exprimant la parvalbumine à l’aide d’outils pharmacogénétiques, a montré que ces cellules inhibitrices contrôlent fortement l'amplitude du PPS et l’activité des NP CA2 en réponse à la stimulation des CS et qu’elles sont nécessaires à l'augmentation RDO-dépendante du rapport excitation/inhibition entre CA3 et CA2. Enfin, l'étude de la zone CA2 chez les souris Df(16)A+/- a révélé plusieurs modifications dépendantes de l'âge dont une réduction de l'inhibition, une altération de la plasticité du rapport excitation/inhibition entre CA3 et CA2 et une hyperpolarisation NP CA2. Ces modifications cellulaires peuvent expliquer les déficiences de mémoire sociale que nous observons chez les souris Df(16)A+/- adultes. L’ensemble de nos études a permis de mettre en évidence le rôle des neurones CA2 dans les circuits de l'hippocampe. Enfin pour conclure, nous postulons que le recrutement des neurones CA2 dans les réseaux neuronaux sous-tend des aspects particuliers de la fonction de l'hippocampe
The hippocampus is a region of critical importance for memory formation. Recent studies have shown that the long-overlooked hippocampal region CA2 plays a role in certain forms of memory, including social recognition. Furthermore, post-mortem studies of schizophrenic patients have revealed specific changes in area CA2. As yet, the role of CA2 neurons in the hippocampal circuitry remains poorly understood under both normal physiological and pathological conditions. By combining pharmacology, mouse genetics and electrophysiology, we investigated how CA2 pyramidal neurons (PNs) could be recruited in hippocampal circuits in mice hippocampal slices following an activity-dependent change in the strength of their inhibitory inputs. We further investigated how subsequent recruitment of CA2 PNs could modulate hippocampal output. Moreover, we examined the functional alterations of area CA2 in the Df(16)A+/- mouse model of the 22q11.2 microdeletion, a spontaneous chromosomal deletion that is the highest known genetic risk factor for developing schizophrenia. In area CA2, inhibitory synapses exert a powerful control of Schaffer collateral (SC) inputs and undergo a unique long-term depression (iLTD) mediated by delta-opioid receptor (DOR) activation. Unlike SC-CA1 synapses, SC-CA2 excitatory synapses fail to express long-term potentiation after classical induction protocols. However, we found that different patterns of activity persistently increase both the SC and the distal input net excitatory drive onto CA2 PNs via a modulation of the balance between excitation and inhibition. We demonstrated that increases in the excitatory/inhibitory ratio are direct consequences of the DOR-mediated iLTD. Interestingly, we found that the inhibition in area CA2 completely preventing CA3 PNs to activate CA2 PNs, and following iLTD, SC stimulation allows CA2 PNs to fire action potentials. Moreover, the recruitment of CA2 PNs by SC intra-hippocampal inputs after their activity-dependent disinhibition adds a delayed SC-CA2-CA1 response to the SC-CA1 monosynaptic post-synaptic potential (PSP) in CA1 and increases CA1 PN activity. Furthermore, pharmaco-genetic silencing of parvalbumin-expressing interneurons revealed that these inhibitory cells control the PSP amplitude and the firing of CA2 PNs in response to SC stimulation and are necessary for the DOR-mediated increase in excitatory/inhibitory balance between CA3 and CA2. Finally, we found several age-dependent alterations in area CA2 in Df(16)A+/- mouse model of the 22q11.2 microdeletion. These included a reduction in inhibition, an impaired activity-dependent modulation of the excitatory drive between CA3 and CA2 and a more hyperpolarized CA2 PN resting potential. These cellular disruptions may provide a potential mechanism for the social memory impairment that we observe in Df(16)A+/- adult mice. Altogether, our studies highlight the role of CA2 neurons in hippocampal circuitry. To conclude, we postulate that the recruitment of CA2 neurons in neuronal networks underlies key aspects of hippocampal function

Ma thèse est consacrée à l’étude des réseaux neuronaux spinaux impliqués dans la motricité chez l’Homme est comprend deux chapitres. Des travaux récents effectués sur la moelle épinière du rat ont mis en évidence qu’au cours du développement chez les mammifères, les synapses GABAergiques et glycinergiques sont tout d’abord excitatrices avant de devenir inhibitrices et qu’une section de la moelle épinière ne permet pas cette transformation. Cette transition développementale semble due à l’action d’un transporteur transmembranaire (KCC2) au cours de développement qui diminue après section de la moelle épinière. La diminution de l’expression du KCC2 dépolarise l’action du GABA et de la glycine, ce qui conduit donc à une réduction de l'efficacité de synapse inhibitrice. Le but de ce projet est d’explorer si chez l’Homme une section traumatique de la moelle épinière qui prive les neurones inhibiteurs de leur contrôle suprasegmentaire a pour conséquence de modifier leur comportement synaptique, voire de les ramener à un fonctionnement « immature », c’est-à-dire de transformer des synapses inhibitrices en synapses facilitatrices. Pour tester cette hypothèse, nous avons étudié l’effet sur des synapses inhibitrices de la moelle épinière d’une prise per os de furosémide, un antagoniste de KCC2, et comparé ses effets chez des sujets sains et chez des patients porteurs d’une section de la moelle épinière. L’étude sur les sujets sains suggère que le furosémide (40 mg) a pour effet une réduction du fonctionnement des synapses inhibitrices. Cet effet du furosémide sur les synapses inhibitrices semble être réduit chez des patients. Les résultats obtenus chez les sujets sains indiquent que furosémide administré per os à des dose largement utilisé en clinique humain modifie sélectivement le fonctionnement des synapses inhibitrices et permet donc de disposer d’un mesure non-invasive de fonctionnement intrinsèque de la synapse inhibitrice. Les résultats préliminaires obtenus chez les patients porteurs d’une section de la moelle épinière suggèrent une réduction de l’efficacité de synapses inhibitrices qui probablement contribue à la spasticité. La stimulation électrique transcrânienne de courant continu encore appelée « transcranial direct current stimulation (tDCS) » par les anglo-saxons, a connu un essor considérable et constitue aujourd’hui une technique de référence pour moduler l’excitabilité du cortex chez l’Homme. En 2009, Roche et al. ont montré dans notre laboratoire, que la tDCS anodale appliquée sur l’hémisphère contralateral pouvait également modifier l’excitabilité des réseaux neuronaux spinaux (i.e. l’inhibition réciproque au niveau du poignet) enregistrée sur le côté dominant chez les sujets sains. L’existence de projection corticale ipsilatérale sur les réseaux neuronaux spinaux de la moelle épinière et leurs éventuelles modifications après lésion cortico-sous-corticale reste très controversée. Dans ce projet, nous avons testé les effets de la tDCS ipsi- et contralarérale du cortex non-lésé sur l’inhibition réciproque chez des patients AVC. La tDCS ipsilatérale n’induit pas de modifications de l’inhibition réciproque chez les sujets sains. Des résultats similaires enregistrés sur le membre supérieur lésé ont été observés chez des patients AVC, mais ces résultats mériteraient d’être confortés avec un plus grand nombre de sujets. La tDCS contralatérale chez les sujets sains n’induit pas de modifications de l’inhibition réciproque enregistrées sur le membre supérieur non-dominant. Ce résultat est différent de celui observé sur le membre supérieur dominant par Roche et al. (2009). Ce contrôle asymétrique sur l'inhibition réciproque est argument en faveur de l'hypothèse que l'inhibition inter-hémisphérique (IHI) entre les deux cortex moteurs est asymétrique. L’IHI à partir de l'hémisphère «dominant» est probablement plus importante
My thesis is devoted to the study of the spinal circuitry involved in motor functions using non-invasive electrophysiological methods in humans. It comprises two research projects.Studies in animals have shown that during neural development, GABAergic and glycinergic neurons are first excitatory, and then become inhibitory during maturation. This developmental transition is mainly due to the activation of co-transporter KCC2 at the mature state. A down-regulation of KCC2 was reported after spinal cord transection in the rat that leads to the depolarising (excitatory) action of GABA and glycine and thus results in a reduction of inhibitory synaptic efficiency. The aim of this project was to explore if spinal cord injury (SCI) in human reverses the pattern of GABAergic and glycinergic neurons back towards the immature state (primarily excitatory). To test this hypothesis, we studied the effects of furosemide (a KCC2 antagonist) on spinal inhibitory synaptic function, and compared the results obtained in healthy subjects and SCI patients. Results in healthy subjects suggest that furosemide (40 mg, orally-administrated) induces a reduction of inhibitory synapse functions. This effect of furosemide on inhibitory synapses seems to be reduced in SCI patients. Our results suggest that furosemide has the potential to test functions of inhibitory synapses in humans. The difference of furosemide effects on spinal inhibitory synapse excitability in healthy subjects and SCI patients favours the hypothesis of a decrease in inhibitory neuronal activity induced by down-regulation of KCC2 after SCI in humans that likely contributes to spasticity. Transcranial direct current stimulation (tDCS) has emerged as a method for exploring cortex excitability in humans. Roche et al. (2009) have shown in our laboratory that using anodal tDCS over contralateral motor cortex can also induce changes in spinal network excitability (i.e. reciprocal inhibition between forearm muscles) in the dominant limb in healthy subjects. It is unknown whether motor activity from the unaffected cerebral hemisphere could be employed after semi-brain damage in patients with hemiplegia. Moreover, little is known about the non-affected limb if it always functions like 'normal' after unilateral stroke. In this project, the ipsi- and contralateral corticospinal controls on reciprocal inhibition between forearm muscles were explored using anodal tDCS applied over the unaffected motor cortex of stroke patients and then compared to the results obtained in healthy subjects. Ipsilateral tDCS induces no change in reciprocal inhibition in healthy subjects. Similar results recorded on the affected upper limb are observed in stoke patients. However a larger number of patients is required to confirm the results. Contralateral anodal tDCS in healthy subjects shows no changes of reciprocal inhibition recorded in the non-dominant upper limb. This result is different from that observed in the dominant upper limb by Roche et al. (2009). This asymmetrical control on reciprocal inhibition would favour the hypothesis that the inter-hemispheric inhibition (IHI) between both motor cortices is asymmetric, with prominent IHI projections originating in the “dominant” left hemisphere. Contralateral anodal tDCS of the unaffected motor cortex induces a strong decrease in reciprocal inhibition in non-affected upper limb in stoke patients.This is different from that observed in both dominant and non-dominant upper limb in healthy subjects suggesting that the pathophysiological changes after unilateral stroke would probably not occur only on the hemiparesis side, but may also the non-affected side. A larger number of patients is still required to confirm the results

Missense mutations in the human CACNA1A gene, which encodes the pore-forming a1 subunit of the CaV2.1 (P/Q-type) Ca2+ channel, cause familial hemiplegic migraine type 1 (FHM1), a rare subtype of migraine with aura (Ophoff et al., 1996). Apart from the characteristic hemiparesis, the headache, autonomic and aura symptoms of typical attacks of FHM1 are similar to those of the common forms of migraine with aura (Pietrobon, 2007; Pietrobon and Striessnig, 2003). CaV2.1 channels are located in presynaptic terminals and somatodendritic membranes throughout the brain, where they play a dominant role in controlling neurotransmitter release. FHM1 mutations produce gain of function of human recombinant CaV2.1 channels, mainly due to a shift of channel activation to more negative voltages and an increase of the open probability and single channel influx over a broad voltage range (Hans et al., 1999; Tottene et al., 2002, 2005; Pietrobon, unpublished data). Accordingly, homozygous knock-in (KI) mice carrying the R192Q FHM1 mutation show an increased P/Q-type Ca2+ current density in cerebellar granule cells and cortical pyramidal cells (Tottene et al., 2009; van den Maagdenberg et al., 2004). Interestingly, both the induction and the propagation of cortical spreading depression (CSD: the phenomenon underlying migraine aura, and a possible trigger of migraine headache) are facilitated in homozygous R192Q (RQ/RQ) FHM1 KI mice in vivo (Pietrobon, 2005, 2007; van den Maagdenberg et al., 2004). To investigate the mechanisms underlying the facilitation of CSD, in my laboratory we have recently studied excitatory cortical synaptic transmission in neuronal microcultures (this was part of my PhD project) and at connected pairs of pyramidal cells and multipolar fast-spiking (FS) interneurons in acute thalamocortical slices of RQ/RQ KI mice (Tottene et al., 2009). We found gain of function of excitatory neurotransmission due to increased action potential (AP)-evoked Ca2+ influx through presynaptic P/Q-type Ca2+ channels and increased probability of glutamate release at pyramidal cell synapses. Using an in vitro model of CSD, we provided direct evidence of a causative link between enhanced glutamate release at pyramidal cell synapses and facilitation of experimental CSD in RQ/RQ KI mice. In striking contrast, inhibitory neurotransmission at connected pairs of multipolar FS interneurons and pyramidal cells in thalamocortical slices of RQ/RQ KI mice was unaltered, despite being initiated by P/Q-type Ca2+ channels (Tottene et al., 2009). The synapse-specific effect of FHM1 mutations supports the view of migraine as an episodic disorder of brain excitability, with disruption of the excitation-inhibition balance and hyperactivity of cortical circuits in response to specific migraine triggers as the basis for episodic vulnerability to CSD ignition in migraine. In patients, the R192Q mutation causes typical FHM attacks; in contrast, the S218L mutation causes a dramatic hemiplegic migraine syndrome that is associated with seizures, coma and severe cerebral oedema often triggered by mild head trauma (Kors et al., 2001). In comparison with R192Q, mutation S218L produces a larger shift of activation of recombinant human CaV2.1 channels towards more negative membrane potentials and, accordingly, a larger gain of function of neuronal Ca2+ influx at low voltages and a larger facilitation of CSD induction and propagation in homozygous S218L (SL/SL) FHM1 KI mice compared to RQ/RQ KI mice in vivo (Tottene et al., 2002, 2005; van den Maagdenberg et al., 2004, 2010). Moreover, the effect of the S218L mutation on the neuronal CaV2.1 Ca2+ current and facilitation of CSD is allele dosage-dependent. To investigate the mechanisms underlying the greater facilitation of CSD in SL/SL KI mice compared to RQ/RQ KI mice, I studied glutamatergic synaptic transmission in cortical pyramidal cells grown on glial microislands from heterozygous S218L (SL/WT) KI mice. I found gain of function of cortical excitatory neurotransmission due to increased action potential-evoked Ca2+ influx through presynaptic P/Q-type Ca2+ channels and increased probability of glutamate release at cortical pyramidal cell synapses of SL/WT KI mice. In fact, in single cortical pyramidal cells forming autapses from mutant mice the amplitude of the evoked excitatory postsynaptic current (EPSC) and the contribution of P/Q-type Ca2+ channels to synaptic transmission were both increased. Moreover, saturation of the EPSC occurred at lower Ca2+ concentration and the paired pulse ratio (PPR) was decreased. The changes in EPSC amplitude, Ca2+ dependence of the EPSC and PPR in SL/WT KI mice were quantitatively similar to those measured in RQ/RQ KI mice. Therefore, the S218L mutation produces a larger increase in presynaptic Ca2+ influx and glutamate release at cortical pyramidal cell synapses than the mild R192Q FHM1 mutation. Given our evidence of a causative link between enhanced glutamate release and CSD facilitation (Tottene et al, 2009), this may explain the greater susceptibility to CSD induced by the S218L mutation, and possibly its dramatic clinical phenotype. I also found that cortical excitatory transmission in both FHM1 KI mice was less susceptible to presynaptic inhibition by activation of G protein-coupled GABAB receptors. In fact, the fraction of the EPSC inhibited by the GABAB receptor agonist baclofen was lower in SL/WT KI and RQ/RQ KI mice than in wild-type (WT) mice. As a consequence, excitatory neurotransmission was further facilitated in the presence of baclofen. Heterozygous S218L KI and homozygous R192Q KI mice showed a similar reduction in presynaptic inhibition. The data suggest that hyperactivity of cortical circuits due to both enhanced CaV2.1-dependent glutamate release and reduced presynaptic inhibition of glutamate release during G protein-coupled neuromodulation may render the cortex of FHM patients vulnerable to CSD ignition in response to migraine triggers. Another aim of my PhD project was to investigate the mechanism underlying the different effect of the R192Q FHM1 mutation on excitatory and inhibitory cortical synaptic transmission found in Tottene et al. (2009). I investigated inhibitory autaptic neurotransmission in single cortical multipolar fast-spiking interneurons grown on glial microislands from WT and RQ/RQ KI mice. The average amplitude of the AP-evoked inhibitory postsynaptic current (IPSC) was similar in multipolar interneurons of WT and RQ/RQ KI mice, despite a dominant role of P/Q Ca2+ channels in controlling GABA release at these synapses. I found that AP-evoked Ca2+ influx nearly saturates the presynaptic Ca2+ sensor at multipolar interneuron autapses in WT mice. However, the unaltered IPSC amplitude in RQ/RQ KI mice is not due to saturation of the Ca2+ sensor, because a similar IPSC at WT and RQ/RQ KI autapses was found also at low external Ca2+ concentrations. Indeed, I found a similar Ca2+ dependence of the IPSC at WT and RQ/RQ KI multipolar interneuron autapses. These findings suggest that the unaltered cortical inhibitory neurotransmission in RQ/RQ KI mice is largely due to the lack of significant increase of action potential-evoked Ca2+ influx through mutant presynaptic P/Q-type Ca2+ channels at multipolar interneuron synapse, possibly as a consequence of the short action potential and/or the expression of a splice variant of the CaV2.1 a1 subunit little affected by the R192Q mutation in FS interneurons. The unaltered AP-evoked Ca2+ influx through mutant presynaptic CaV2.1 channels at multipolar FS interneuron synapses is probably a common effect of all FHM1 mutations, as I also found unaltered inhibitory synaptic transmission at multipolar FS interneuron autapses in heterozygous S218L KI mice.
L’emicrania emiplegica familiare di tipo 1 (FHM1), un raro sottotipo di emicrania con aura, è causata da mutazioni missense nel gene umano CACNA1A che codifica per la subunità α1 dei canali del calcio CaV2.1 (tipo P/Q) (Ophoff et al., 1996). Il mal di testa e i sintomi neurologici dell’aura che caratterizzano i tipici attacchi di FHM1 sono simili a quelli delle forme comuni di emicrania, eccetto per il sintomo dell’emiparesi (Pietrobon, 2007; Pietrobon and Striessnig, 2003). I canali CaV2.1 sono espressi nei terminali presinaptici e nelle membrane somatodendritiche di tutti i neuroni del cervello, dove svolgono un ruolo fondamentale nel controllo del rilascio di neurotrasmettitore. Le mutazioni FHM1 determinano un guadagno di funzione della corrente Ca2+ dei canali ricombinanti umani CaV2.1; in particolare causano un aumento dell’influsso di Ca2+ a livello di singolo canale in un ampio intervallo di potenziali vicini alla soglia di attivazione del canale, dovuto a un’aumentata probabilità d’apertura del canale, causata per lo più dallo spostamento della curva di attivazione del canale verso potenziali più negativi (Hans et al., 1999; Tottene et al., 2002, 2005; Pietrobon, unpublished data). In accordo con tali risultati, i topi omozigoti knock-in (KI) recanti la mutazione FHM1 R192Q (RQ/RQ) presentano, in granuli di cervelletto e in cellule piramidali corticali, un aumento della densità di corrente Ca2+ di tipo P/Q (Tottene et al., 2009; van den Maagdenberg et al., 2004). Questi topi mostrano, inoltre, una facilitazione dell’induzione e propagazione, in vivo, della cortical spreading depression (CSD: il fenomeno neurologico che causa l’aura e il possibile trigger del mal di testa emicranico) (Pietrobon, 2005, 2007; van den Maagdenberg et al., 2004). Per capire i meccanismi che determinano la facilitazione della CSD nei topi KI, in laboratorio abbiamo recentemente studiato la trasmissione sinaptica eccitatoria in neuroni corticali in microcoltura e in neuroni piramidali e interneuroni multipolari fast-spiking (FS) connessi tra loro sinapticamente in fettine acute talamo-corticali di topi KI RQ/RQ (Tottene et al., 2009). Lo studio della neurotrasmissione in microcolture di neuroni corticali ha rappresentato parte del mio progetto di Dottorato. I risultati hanno dimostrato un guadagno di funzione della neurotrasmissione eccitatoria dovuto a un aumentato influsso di Ca2+ evocato da potenziale d’azione, attraverso i canali del Ca2+ presinaptici di tipo P/Q, e a un’aumentata probabilità di rilascio di glutammato alle sinapsi delle cellule piramidali. Inoltre, usando un modello in vitro di CSD, abbiamo dimostrato una correlazione causale tra l’aumentato rilascio di glutammato alle sinapsi delle cellule piramidali e la facilitazione sperimentale della CSD nei topi KI RQ/RQ. Abbiamo costatato che la neurotrasmissione inibitoria alle sinapsi tra gli interneuroni multipolari FS e le cellule piramidali nelle fettine talamo-corticali di topi KI RQ/RQ era invece inalterata, sebbene la trasmissione sinaptica fosse controllata dai canali del Ca2+ di tipo P/Q (Tottene et al., 2009). Il suddetto effetto sinapsi-specifico delle mutazioni FHM1 consolida la visione dell’emicrania come un disordine episodico dell’eccitabilità cerebrale. Infatti, la distruzione del bilancio tra eccitazione e inibizione e la conseguente iperattività dei circuiti neuronali possono costituire, in risposta a specifici triggers, la causa degli episodi di suscettibilità all’innesco della CSD nell’emicrania. Mentre nei pazienti la mutazione R192Q causa tipici attacchi di FHM, la mutazione S218L causa una grave sindrome di emicrania emiplegica associata a convulsioni epilettiche, coma e grave edema cerebrale, dovuti spesso a traumi alla testa di lieve entità (Kors et al., 2001). La mutazione S218L, rispetto alla mutazione R192Q, determina un maggior spostamento verso potenziali più negativi della curva di attivazione dei canali umani ricombinanti CaV2.1 (Tottene et al., 2002, 2005). Inoltre, in accordo con il suddetto effetto, determina un maggior guadagno di funzione dell’influsso di Ca2+ nei neuroni a potenziali negativi e una maggiore facilitazione dell’induzione e propagazione della CSD in vivo nei topi omozigoti FHM1 KI S218L (SL/SL) rispetto ai topi KI RQ/RQ (van den Maagdenberg et al., 2004, 2010). L’effetto della mutazione S218L sulla corrente CaV2.1 Ca2+ neuronale e sulla facilitazione dell’induzione e propagazione della CSD risulta essere dipendente dal dosaggio allelico. Al fine di investigare i meccanismi che determinano la maggior facilitazione della CSD in topi KI SL/SL rispetto ai topi KI RQ/RQ, ho studiato la trasmissione sinaptica glutamatergica in neuroni piramidali corticali di topi eterozigoti KI S218L (SL/WT) cresciuti su microisole di cellule gliali in modo da formare sinapsi su se stessi (autapsi). Ho trovato un guadagno di funzione della neurotrasmissione corticale eccitatoria dovuto a un aumentato influsso di Ca2+ evocato da potenziale d’azione, attraverso i canali del Ca2+ presinaptici di tipo P/Q e a un’aumentata probabilità di rilascio di glutammato alle sinapsi delle cellule piramidali corticali dei topi KI SL/WT. Infatti, l’ampiezza della corrente postsinaptica eccitatoria evocata (EPSC) e il contributo dei canali Ca2+ di tipo P/Q alla trasmissione sinaptica erano entrambi aumentati alle sinapsi dei neuroni piramidali corticali in microcoltura. Inoltre ho dimostrato che la saturazione del sensore del Ca2+ avveniva a minor concentrazioni esterne di Ca2+ e che la paired pulse ratio (PPR) era diminuita. I cambiamenti nell’ampiezza dell’EPSC, nella Ca2+ dipendenza dell’EPSC e nella PPR, trovati nei topi KI SL/WT erano quantitativamente simili a quelli rilevati nei topi KI RQ/RQ. La mutazione S218L determina quindi un aumento maggiore dell’influsso di Ca2+ presinaptico e del rilascio di glutammato alle sinapsi corticali delle cellule piramidali rispetto alla mutazione R192Q che causa un fenotipo lieve di FHM1. Vista la correlazione causale tra l’aumentato rilascio di glutammato e la facilitazione sperimentale della CSD (Tottene et al., 2009), questo risultato potrebbe spiegare la maggior suscettibilità alla CSD indotta dalla mutazione S218L e il suo fenotipo grave. I dati hanno anche dimostrato che la trasmissione corticale eccitatoria in entrambi i topi FHM1 KI era meno suscettibile all’inibizione presinaptica conseguente all’attivazione dei recettori GABAB accoppiati a proteine G. Infatti, la frazione dell’EPSC inibito dal baclofen, agonista dei recettori GABAB, era minore nei topi KI SL/WT e KI RQ/RQ rispetto ai topi selvatici (WT) e, conseguentemente, la neurotrasmissione eccitatoria era ulteriormente facilitata in presenza di modulazione. I topi eterozigoti KI S218L e omozigoti KI R192Q presentavano una riduzione dell’inibizione presinaptica simile. I dati suggeriscono che l’iperattività dei circuiti corticali dovuta sia all’aumentato rilascio di glutammato dipendente dai canali CaV2.1, sia alla ridotta inibizione presinaptica del rilascio di glutammato durante neuromodulazione (attraverso l’attivazione di proteine G) possono rendere la corteccia dei pazienti FHM vulnerabile all’innesco della CSD in risposta a triggers emicranici. Un altro scopo del mio progetto di Dottorato consisteva nel determinare il meccanismo che causa l’effetto diverso della mutazione FHM1 R192Q sulla trasmissione sinaptica corticale eccitatoria e inibitoria, come descritto in Tottene et al. (2009). Ho studiato la neurotrasmissione inibitoria autaptica in singoli interneuroni corticali multipolari fast-spiking cresciuti su microisole di cellule gliali da topi WT e topi KI RQ/RQ. L’ampiezza media della corrente postsinaptica inibitoria (IPSC) evocata da potenziale d’azione era simile negli interneuroni multipolari dei topi WT e KI RQ/RQ, nonostante l’importante ruolo svolto dai canali Ca2+ di tipo P/Q nel controllo del rilascio di GABA a queste sinapsi. Ho trovato che il sensore del Ca2+ presinaptico, alle autapsi degli interneuroni multipolari nei topi WT, viene quasi saturato dall’influsso di Ca2+ evocato da potenziale d’azione. La mancanza del guadagno di funzione della neurotrasmissione inibitoria nei topi RQ/RQ KI non è comunque causata dalla saturazione del sensore Ca2+, poiché l’ampiezza dell’IPSC in interneuroni di topi WT e KI RQ/RQ era simile anche a basse concentrazioni di Ca2+ esterno. Ho costatato, infatti, una simile dipendenza dell’IPSC dalle concentrazioni esterne di Ca2+ alle autapsi degli interneuroni multipolari WT e KI RQ/RQ. Tali risultati suggeriscono che l’inalterata neurotrasmissione corticale inibitoria nei topi KI RQ/RQ sia dovuta soprattutto ad un aumento non significativo dell’influsso di Ca2+, evocato da potenziale d’azione attraverso i canali presinaptici CaV2.1 alle sinapsi degli interneuroni multipolari fast-spiking. Una possibile causa potrebbe essere la forma più corta del potenziale d’azione degli interneuroni FS e/o l’espressione, in questi interneuroni, di una variante di splicing della subunità α1 CaV2.1 poco influenzata dalla mutazione R192Q. L’inalterato influsso di Ca2+ evocato da potenziale d’azione attraverso i canali mutati presinaptici CaV2.1, alle sinapsi degli interneuroni multipolari FS, è probabilmente un effetto comune a tutte le mutazioni FHM1, poiché ho trovato inalterata anche la trasmissione sinaptica inibitoria alle autapsi degli interneuroni corticali multipolari FS nei topi eterozigoti KI S218L.

Différentes études ont montré que des altérations du gène codant pour la Cadhérine-13 (CDH13) sont impliquées dans de nombreux troubles neurologiques (autisme (TSA), troubles de déficit de l’attention avec ou sans hyperactivité, déficiences intellectuelles, maladies psychiatriques). La CDH13 est un membre atypique de la superfamille des cadhérines, car elle est dépourvue de domaine transmembranaire et intracellulaire et, contrairement aux autres cadhérines, elle est fixée à la membrane par une ancre glycosylphosphatidylinositol (GPI). Il a été démontré que la CDH13 joue un rôle dans le guidage et la croissance des axones ainsi que dans la régulation de l'apoptose au cours du développement cérébral. Les souris KO de la CDH13 montrent une augmentation des courants synaptiques inhibiteurs ainsi que des déficits d'apprentissage et de mémoire. Dans le cervelet, la CDH13 est exprimée uniquement dans un sous type de neurones : les cellules de Golgi. La délétion de la CDH13 spécifiquement dans les cellules de Golgi entraine des déficits comportementaux rappelant les TSA chez les souris. Nous avons donc cherché à déterminer les mécanismes moléculaires et cellulaires de la CDH13 dans le développement du cervelet et plus particulièrement dans la mise en place des synapses des cellules de Golgi. En utilisant des souris mutantes constitutives ou conditionnelles pour la CDH13 et différentes approches de biologie cellulaire, de biochimie et d’immunohistochimie in vitro et in vivo, nous avons caractérisé les mécanismes d’action de la CDH13 dans la synaptogénèse des cellules de Golgi. Nous avons montré que la CDH13 est localisée à la présynapse inhibitrice des cellules d e Golgi au sein des glomérules et que la suppression de son expression entraine une diminution de la densité des marqueurs synaptiques dans les glomérules. La CDH13 est donc nécessaire pour un développement normal de la synapse glomérulaire et est impliquée dans la mise en place du circuit cérébelleux. Nous avons identifié que le partenaire trans-synaptique de la CDH13 était la protéine Neuroligine-2 (NLG2), un élément crucial pour la différenciation postsynaptique des synapses inhibitrices. Enfin, nous avons montré que l’expression de la CDH13 était suffisante pour induire la différenciation synaptique via son interaction avec NLG2. Nos résultats démontrent ainsi le rôle clé de la CDH13 dans la synaptogénèse du glomérule au niveau du cervelet et identifient un nouvel acteur moléculaire présynaptique dans la mise en place d es synapses inhibitrices
Various studies have shown that alterations in the gene coding for Cadherin-13 (CDH13) are involved in many neurological disorders (autism (ASD), attention deficit disorders with or without hyperactivity, intellectual disabilities, psychiatric diseases). CDH13 is an atypical member of the cadherin superfamily. It lacks a transmembrane and intracellular domain and, unlike other cadherins, is attached to the membrane by a glycosylphosphatidylinositol (GPI) anchor. CDH13 plays several roles in axon guidance and growth and in the regulation of apoptosis during brain development. In the CDH13 KO mice, an increase in inhibitory synaptic currents and deficits in learning and memory were observed. In the cerebellum, CDH13 expression is restricted to the inhibitory Golgi cells. Deletion of CDH13 specifically in Golgi cells leads to ASD-like behavioral deficits in mice. Therefore, we sought to determine the molecular and cellular mechanisms of CDH13 in the formation of Golgi cell synapses during cerebellar development. Using constitutive or conditional mutant mice for CDH13, together with cell biology, biochemistry and immunohistochemistry approaches, both in vitro and in vivo, we characterized the mechanisms of action of CDH13 in Golgi cell synaptogenesis. We show that CDH13 is localized at the presynaptic sites of Golgi cells within glomeruli. In CDH13 knock-out mice, we observe a decrease in the density of synaptic markers at the pre and postsynaptic sites in glomeruli, demonstrating that CDH13 is required for normal glomerular synapse formation during cerebellar circuit development. In addition, we show that CDH13 trans-synaptically interacts with NLG2A, a splicing isoform of NLG2 that is crucial for the postsynaptic differentiation of inhibitory synapses. Finally, we demonstrate that CDH13 expression is sufficient to induce synaptic differentiation via its interaction with NLG2. Altogether, our results identify a new presynaptic molecular pl ayer in the establishment of inhibitory synapses and characterize the cellular and molecular mechanisms of glomerular synapse formation in the cerebellum

The brain network is a multiscale hierarchical organization from neurons and local circuits to macroscopic brain areas. The precise synaptic transmission at each synapse is therefore crucial for neural communication and the generation of orchestrated behaviors. Activation of presynaptic voltage-gated calcium channels (CaV2) initiates synaptic vesicle release and plays a key role in neurotransmission. In this dissertation, I have aimed to uncover how CaV2 activity affects synaptic transmission, circuit function and behavioral outcomes using Caenorhabditis elegans as a model. The C. elegans genome encodes an ensemble of highly conserved neurotransmission machinery, providing an opportunity to study the molecular mechanisms of synaptic function in a powerful genetic system. I identified a novel gain of function CaV2α1 mutation that causes CaV2 channels to activate at a lower membrane potential and slow the inactivation. Cell-specific expression of these gain-of-function CaV2 channels is sufficient to hyper-activate neurons of interest, offering a way to study their roles in a given circuit. CaV2(gf) mutants display behavioral hyperactivity and an excitation-dominant synaptic transmission. Imbalanced excitation and inhibition of the nervous system have been associated with several neurological disorders, including Familial Hemiplegic Migraine type 1 (FHM1) which is caused by gain- of-function mutations in the human CaV2.1α1 gene. I showed that animals carrying C. elegans CaV2α1 transgenes with corresponding human FHM1 mutations recapitulate the hyperactive behavioral phenotype exhibited by CaV2(gf) mutants, strongly suggesting the molecular function of CaV2 channels is highly conserved from C. elegans to human. Through performing a genome-wide forward genetic screen looking for CaV2α(gf) suppressors, we isolated new alleles of genes that required for CaV2 trafficking, localization and function. These regulators include subunits of CaV2 channel complex, components of synaptic and dense core vesicle release machinery as well as predicted extracellular proteins. Taken together, this work advances the understanding of CaV2 malfunction at both cellular and circuit levels, and provides a genetically amenable model for neurological disorders associated with excitation-inhibition imbalance. Additionally, through identifying regulators of CaV2, this research provides new avenues for understanding the CaV2 channel mediated neurotransmission and potential pharmacological targets for the treatments of calcium channelopathies.

The brain network is a multiscale hierarchical organization from neurons and local circuits to macroscopic brain areas. The precise synaptic transmission at each synapse is therefore crucial for neural communication and the generation of orchestrated behaviors. Activation of presynaptic voltage-gated calcium channels (CaV2) initiates synaptic vesicle release and plays a key role in neurotransmission. In this dissertation, I have aimed to uncover how CaV2 activity affects synaptic transmission, circuit function and behavioral outcomes using Caenorhabditis elegans as a model. The C. elegans genome encodes an ensemble of highly conserved neurotransmission machinery, providing an opportunity to study the molecular mechanisms of synaptic function in a powerful genetic system. I identified a novel gain of function CaV2α1 mutation that causes CaV2 channels to activate at a lower membrane potential and slow the inactivation. Cell-specific expression of these gain-of-function CaV2 channels is sufficient to hyper-activate neurons of interest, offering a way to study their roles in a given circuit. CaV2(gf) mutants display behavioral hyperactivity and an excitation-dominant synaptic transmission. Imbalanced excitation and inhibition of the nervous system have been associated with several neurological disorders, including Familial Hemiplegic Migraine type 1 (FHM1) which is caused by gain- of-function mutations in the human CaV2.1α1 gene. I showed that animals carrying C. elegans CaV2α1 transgenes with corresponding human FHM1 mutations recapitulate the hyperactive behavioral phenotype exhibited by CaV2(gf) mutants, strongly suggesting the molecular function of CaV2 channels is highly conserved from C. elegans to human. Through performing a genome-wide forward genetic screen looking for CaV2α(gf) suppressors, we isolated new alleles of genes that required for CaV2 trafficking, localization and function. These regulators include subunits of CaV2 channel complex, components of synaptic and dense core vesicle release machinery as well as predicted extracellular proteins. Taken together, this work advances the understanding of CaV2 malfunction at both cellular and circuit levels, and provides a genetically amenable model for neurological disorders associated with excitation-inhibition imbalance. Additionally, through identifying regulators of CaV2, this research provides new avenues for understanding the CaV2 channel mediated neurotransmission and potential pharmacological targets for the treatments of calcium channelopathies.

Thesis (Ph.D.)--Medical University of Ohio, 2006.
"In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Medical Sciences." Major advisor: Elizabeth I. Tietz. Includes abstract. Document formatted into pages: iv, 234 p. Title from title page of PDF document. Bibliography: pages 86-95,126-135,167-174,190-232.

La microscopie optique stochastique de reconstruction (STORM) contourne la limite de diffraction en enregistrant des signaux monomoléculaires spatialement et temporellement séparés, atteignant une résolution de ~10-40 nm. Dans mon étude, j'ai développé une stratégie d'imagerie et d'analyse de données dSTORM bicolore afin d'étudier l'ultrastructure des synapses inhibitrices mixtes. Mes résultats ont montré que les GlyRs, les GABAARs, la géphyrine et RIM1/2 présentent une organisation intra-synaptique hétérogène et forment des domaines sous-synaptiques (SSDs). Les GlyR et les GABAAR ne sont pas complètement mélangés, mais peuvent occuper des espaces différents à la densité post-synaptique (PSD). De plus, les SSD de géphyrine postsynaptique sont alignées avec les SSD de RIM1/2 pré-synaptiques, formant des nanocolonnes trans-synaptiques. Au cours d'une activité neuronale élevée par traitement 4-AP, la corrélation spatiale entre les GlyRs, les GABAARs et la géphyrine a augmentée au PSD. De plus, la corrélation spatiale des GlyRs et RIM1/2 a également augmenté, tandis que celle des GABAARs et RIM1/2 n'a pas changé. Le nombre de SSD par synapse pour ces protéines synaptiques n'est pas modifié par 4-AP. Cette étude fourni un nouvel angle de compréhension des mécanismes sous-jacents à la co-transmission GABAergique/glycinergique
Stochastic optical reconstruction microscopy (STORM) bypasses the diffraction limit by recording spatially and temporally separated single molecule signals, achieving a resolution of ~10-40 nm. In my study, I have developed a two-color dSTORM imaging and data analysis strategy, in order to investigate the ultrastructure of mixed inhibitory synapses. My results show that GlyRs, GABAARs, gephyrin and RIM1/2 exhibit a heterogeneous intra-synaptic organization and form sub-synaptic domains (SSDs). GlyRs and GABAARs were not fully intermingled, but sometimes occupied different spaces at the post-synaptic density (PSD). In addition, post-synaptic gephyrin SSDs were aligned with pre-synaptic RIM1/2 SSDs, forming trans-synaptic nanocolumns. During elevated neuronal activity by 4-AP treatment, the spatial correlation between GlyRs, GABAARs and gephyrin was increased at the PSD. Moreover, the spatial correlation of GlyRs and RIM1/2 was also increased, while that of GABAARs and RIM1/2 did not change. The number of SSDs per synapse for these synaptic proteins was not changed by 4-AP. My study thus provides a new angle for understanding the mechanisms underlying GABAergic/glycinergic co-transmission

Dans les cornes dorsales (CD), la transmission synaptique inhibitrice joue un rôle clef dans le traitement des informations nociceptives. Cette inhibition peut subir des changements plastiques menant à des symptômes d’hyperalgésie et d’allodynie liés aux douleurs neuropathiques. Dans les CD, les récepteurs NMDA sont recrutés suite à une lésion nerveuse, bien que leur rôle dans les phénomènes de plasticités de la synapse excitatrice soit bien étudié, leur implication dans la plasticité de l’inhibition spinale reste peu connue. Mon projet de thèse a visé à déterminer l’effet de l’activation des récepteurs NMDA sur l’inhibition synaptique spinale en condition normale et en condition de douleur neuropathique. Pour cela nous utilisons des approches d’électrophysiologies sur tranches aiguës de moelle épinière de souris adultes. Les résultats obtenus ont permis d'améliorer la compréhension des mécanismes de modulation et plasticité de l’inhibition au sein du réseau nociceptif spinal
In the dorsal horn (DH) of the spinal cord, inhibitory synaptic transmission plays a key role in the processing of nociceptive information. This inhibition can display plastic changes linked with hyperalgesia and allodynia associated with neuropathic pain. In the DH, NMDA receptors are recruited following a nerve injury. Although their role in plastic phenomenon is well established, little is known about their involvement in spinal inhibition plasticity. My research project aims at studying the effect of NMDA receptor activation on spinal synaptic inhibition in a normal state and during neuropathic pain. To do so we used an electrophysiological approach on acute spinal cord slices of adult mice. Results obtained allow a better understanding of the mechanism underlying the modulation and plasticity of inhibitory transmission within the spinal nociceptive network

Dissertação de Mestrado em Biologia Celular e Molecular apresentada à Faculdade de Ciências e Tecnologia
A remodelação das estruturas sinápticas, dependente do tipo de estímulos que recebem, é o mecanismo molecular responsável pela plasticidade dos circuitos neuronais – um processo que se julga estar na base da aprendizagem e da memória. O processamento de informação e a plasticidade dos circuitos no sistema nervoso central dependem do equilíbrio entre a função excitatória e a função inibitória. O estabelecimento de uma correta transmissão glutamatérgica (excitatória) e GABAérgica (inibitória) é essencial para o controlo do equilíbrio entre excitação e inibição e para o funcionamento normal dos circuitos neuronais; a perda deste equilíbrio está geralmente associada ao desenvolvimento de neuro-psiquiátricos. As GTPases são uma família de proteínas associadas com a regulação do citoesqueleto de actina, e que têm um papel relevante no desenvolvimento e plasticidade da sinapse. As GTPases apresentam um ciclo de activação (quando ligadas a GTP) e inactivação (quando ligadas a GDP) que é regulado pelos factores de troca de nucleotídeos de guanina (GEFs) e pelas proteínas activadoras de GTPase (GAPs), respectivamente. Apesar de existirem dezenas de GEFs e GAPs no cérebro (um número superior ao de GTPases), a função da maioria destas proteínas ainda não foi descrita. Tipicamente, as proteínas reguladoras de GTPases possuem vários domínios proteicos, que lhes conferem um papel importante como integradores de sinalização intracelular. Uma vez que as GTPases estão envolvidas em vários processos do desenvolvimento neuronal, como por exemplo, a migração neuronal, a formação da árvore dendrítica e o desenvolvimento sináptico – quer excitatório quer inibitório, a sua regulação é de extrema importância para o normal desenvolvimento dos circuitos neuronais e normal função cognitiva. De facto, distúrbios na sinalização pelas GTPases podem causar defeitos sinápticos que originam défices cognitivos. Para além disso, mutações em genes que codificam proteínas reguladoras e sinalizadores das GTPases já foram extensamente associadas a défices cognitivos e outros distúrbios comportamentais. Neste estudo, focamo-nos na caracterização da função neuronal da proteína ARHGAP8, uma nova proteína potenciadora da actividade de GTPases de Rho-GTPases. Resultados preliminares do nosso grupo indicam que a proteína ARHGAP8 está presente nas densidades pós-sinápticas das sinapses excitatórias e que esta GAP pode estar envolvida na regulação deste tipo de sinapses. Tendo em consideração esta hipótese, foram realizadas experiências com o objectivo de testar os efeitos da sobre-expressão de ARHGAP8 na transmissão sináptica mediada por receptores AMPA. Os nossos resultados demonstraram que a sobre-expressão de ARHGAP8 causa uma diminuição na frequência e amplitude de correntes excitatórias pós sinápticas miniatura, o que indica que a proteína ARHGAP8 regula negativamente a transmissão sináptica excitatória mediada pelo receptor AMPA. Para além disto, caracterizámos a presença desta proteína nas sinapses inibitórias. Os nossos resultados indicam que a proteína ARHGAP8 está presente nas sinapses inibitórias e que regula a acumulação de marcadores sinápticos inibitórios. Estes resultados sugerem que a proteína ARHGAP8 coordena o desenvolvimento de ambos os tipos de sinapses (excitatórias e inibitórias). Mais experiencias são necessárias de forma a desvendar os mecanismos através dos quais a proteína ARHGAP8 regula a transmissão sináptica medida por receptores AMPA e a composição da sinapse inibitória, bem como, para avaliar se a proteína endógena está envolvida na regulação de sinapses excitatórias e inibitórias.
The activity-dependent modifications of synaptic strength are the molecular mechanism that underlie circuit plasticity, the molecular device for learning and memory. However, maintaining proper balance of excitation and inhibition (E/I balance) is critical for information processing and plasticity in the central nervous system (CNS). Correct excitatory glutamatergic transmission and inhibitory GABAergic signalling are essential for tight control of E/I balance and normal neural circuit function, and disruption of E/I often underlies the development of neuropsychiatric disorders. As key regulators of the actin cytoskeleton, Rho-family GTPases play a critical role in synapse development and plasticity. They shuttle between an active GTP-bound form and an inactive GDP-bound form. Their activation and inactivation cycle is under the regulation of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs), respectively. Even though dozens of GEFs and GAPs have been detected in the brain (outnumbering Rho GTPases), the function of most of them has not been elucidated. Rho-regulatory proteins typically comprise multiple signalling domains, playing an important role as key signalling integrators and scaffolds. Given that Rho GTPases regulate a myriad of neurodevelopmental processes including neuronal migration, dendritic arborization and synaptogenesis, their precise regulation is important for circuitry development and normal cognitive function. In fact, aberrant Rho GTPase signalling can cause synaptic defects that can ultimately lead to cognitive impairments. Furthermore, mutations in genes encoding regulators and effectors of the Rho GTPase family have already been associated with intellectual disability (ID) and other neurodevelopmental disorders.Here, we focus on the characterization of a novel Rho-GTPase activating protein, ARHGAP8, in the brain. Preliminary data from our group showed that ARHGAP8 is present at the post-synaptic densities of excitatory synapses in an NMDA receptor-dependent way, and that this Rho-GAP might be involved in the regulation of excitatory synapses. Considering this hypothesis, further studies were conducted testing the functional effects of overexpressing ARHGAP8 in AMPA receptor-mediated transmission. Our data show that overexpression of ARHGAP8 decreases the amplitude and frequency of miniature excitatory post-synaptic currents, indicating that ARHGAP8 downregulates AMPA receptor-mediated excitatory synaptic transmission. Furthermore, we characterized the presence of this protein in inhibitory synapses to further extend our knowledge of its role in neurons. Our results indicate that ARHGAP8 is present in inhibitory synapses, and regulates the synaptic accumulation of inhibitory synapse markers. Collectively, these observations suggest that ARHGAP8 coordinates the development of excitatory and inhibitory synapses. Further investigation should be done in order to unravel the mechanisms through which ARHGAP8 modulates AMPAR-mediated synaptic transmission and inhibitory synapse composition, and to evaluate if endogenous ARHGAP8 is involved in the regulation of both excitatory and inhibitory synapses.
Outro - FCT: PTDC/SAU-NMC/4888/2014 e UID/NEU/045S39/2013 Programa Mais Centro: CENTRO-07-ST24-FED ER-002002, 002006, 002008

During nervous system development, thousands of distinct neuronal cell types are generated and assembled into highly precise circuits. The proper wiring of these circuits requires that developing neurons recognize their appropriate synaptic partners. Analysis of a vertebrate spinal circuit that controls motor behavior reveals distinct synaptic connections of two types of inhibitory interneurons, a ventral V1 class that synapses with motor neurons and a dorsal dI4 class that selectively synapses with proprioceptive sensory neuron terminals that are located on or in close proximity to motor neurons. What are the molecular and cellular programs that instruct this remarkable synaptic specificity? Are only subsets of these interneurons capable of integrating into this circuit, or do all neurons within the same class behave similarly? The ability to answer such questions, however, is hampered both by the complexity of the spinal cord, where many different neuronal cell types can be found synapsing in the same area; as well as by the challenge of obtaining enough neurons of a particular subtype for analysis. Meanwhile, pluripotent stem cells have emerged as powerful tools for studying neural development, particularly because they can be differentiated to produce large amounts of diverse neuronal populations. Mouse embryonic stem cell-derived neurons can thus be used in a simplified in vitro system to study the development of specific neuronal cell types as well the interactions between defined cell types in a controlled environment. Using stem cell-derived neurons, I investigated how the V1 and dI4 cardinal spinal classes differentiate into molecularly distinct subtypes and acquire cell type-specific functional properties, including synaptic connectivity. In Chapter Two, I describe the production of lineage-based reporter stem cell lines and optimized differentiation protocols for generating V1 and dI4 INs from mouse embryonic stem cells, including confirming that they have molecular and functional characteristics of their in vivo counterparts. In Chapter Three, I show that a well-known V1 interneuron subtype, the Renshaw cell, which mediates recurrent inhibition of motor neurons, can be efficiently generated from stem cell differentiation. Importantly, manipulation of the Notch signaling pathway in V1 progenitors impinges on V1 subtype differentiation and greatly enhances the generation of Renshaw cells. I further show that sustained retinoic acid signaling is critical for the specific development of the Renshaw cell subtype, suggesting that interneuron progenitor domain diversification may also be regulated by spatially-restricted cues during embryonic development. In Chapter Four, using a series of transplantation, rabies virus-based transsynaptic tracing, and optogenetics combined with whole-cell patch-clamp recording approaches, I demonstrate that stem cell-derived Renshaw cells exhibit significant differences in physiology and connectivity compared to other V1 subpopulations, suggesting that synaptic specificity of the Renshaw cell-motor neuron circuit can be modeled and studied in a simplified in vitro co-culture preparation. Finally, in Chapter Five, I describe ongoing investigations into molecular mechanisms of dI4 interneuron subtype diversification, as well as approaches to studying their synaptic specificity with proprioceptive sensory neurons. Overall, my results suggest that our stem cell-cell based system is well-positioned to probe the functional diversity of molecularly-defined cell types. This work represents a novel use of embryonic stem cell-derived neurons for studying inhibitory spinal circuit assembly and will contribute to further understanding of neural circuit formation and function during normal development and potentially in diseased states.

One of the central questions of neuroscience research has been how the cellular and molecular components of the brain give rise to complex behaviours. Three major breakthroughs from the past sixty years have made the study of learning and memory central to our understanding of how the brain works. First, the psychologist Donald Hebb proposed that information storage in the brain could occur through the strengthening of the connections between neurons if the strengthening were restricted to neurons that were co-active (Hebb, 1949). Second, Milner and Scoville (1957) showed that the hippocampus is required for the acquisition of new long-term memories for consciously accessible, or declarative, information. Third, Bliss and Lømo (1973) demonstrated that the synapses between neurons in the dentate gyrus of the hippocampus could indeed be potentiated in an activity-dependent manner. Long-term potentiation (LTP) of the glutamatergic synapses in area CA1, the primary output of the hippocampus, has since become the leading model of synaptic plasticity due to its dependence on NMDA receptors (NMDARs), required for spatial and temporal learning in intact animals, and its robust pathway specificity. Using whole-cell recording in hippocampal slices from adult rats, I find that the efficacy of synaptic transmission from CA3 to CA1 can in fact be enhanced without the induction of classic LTP at the glutamatergic inputs. Taking care not to directly stimulate inhibitory fibers, I show that the induction of GABAergic plasticity at feedforward inhibitory inputs in CA1 results in the reduced shunting of excitatory currents, producing a long-term increase in the amplitude of Schaffer collateral-mediated postsynaptic potentials which is dependent on NMDAR activation and is pathway specific. The sharing of these fundamental properties with classic LTP suggests the possibility of a previously unrecognized target for therapeutic intervention in disorders linked to memory deficits, as well as a potentially overlooked site of LTP expression in other areas of the brain.

Temporal lobe epilepsy (TLE) is among the most common forms of epilepsy in adults. A significant proportion of patients experience drug-resistant seizures associated with hippocampal sclerosis (HS), in which there is extensive cell loss in the hippocampal cornu ammonis 1 (CA1) and cornu ammonis 3 (CA3) subfields. The dentate gyrus (DG) and cornu ammonis 2 (CA2) subfield are more resilient to neurodegeneration, and a prior report found that CA2 neurons in tissue from TLE patients show interictal-like firing and receive aberrant perisomatic excitatory synapses from DG granule cell (GC) mossy fibers (Wittner et al. Brain. 2009;132:3032–3046). Furthermore, findings from a collaborative study in the laboratory of Dr. Helen Scharfman demonstrated that chronic chemogenetic inhibition of CA2 pyramidal neurons (PNs) in vivo significantly reduced the frequency of spontaneous recurring convulsive seizures in epileptic mice. I therefore explored the hypothesis that pathophysiological changes to CA2 PN excitability or synaptic connectivity may be associated with chronic epilepsy by examining CA2 properties in a mouse model of TLE.Pilocarpine-induced status epilepticus in mice leads to a pattern of hippocampal sclerosis-like neurodegeneration and recurring spontaneous seizures, and thus recapitulates key features of TLE. I performed whole-cell electrophysiological recordings from PNs in acute hippocampal slices from pilocarpine (PILO)-treated mice in the chronic phase of epilepsy as well as age-matched controls. In some experiments I used Cre-expressing mouse lines to selectively express a light-activated excitatory channel in CA2 PNs or DG GCs. I also performed immunohistochemistry to examine CA2 interneuron (IN) populations following PILO-induced status epilepticus. I found that in healthy tissue CA2 PNs, like those in CA3, both directly excited other CA2 PNs via a recurrent CA2-CA2 PN circuit and indirectly inhibited other CA2 PNs by recruiting local INs. The CA2 and CA3 subfields also form reciprocal excitatory and feedforward inhibitory circuits. These recurrent and reciprocal circuits constitute an auto-associative network in which INs crucially control CA2/CA3 population excitability. DG GC mossy fibers made direct but relatively weak excitatory synapses onto CA2 PNs. Following PILO-induced status epilepticus, feedforward inhibition is diminished in the DG GC mossy fiber circuit to CA2, in the CA2/CA3 recurrent network, and in the forward-projecting circuit from CA2 PNs to CA1. I found a modest decrease in the density of parvalbumin-immunopositive INs and a profound decrease of cholecystokinin-immunopositive IN density, combined with degradation of the pyramidal neuron-associated perisomatic perineuronal net, which together may contribute to this inhibitory disruption. DG GC mossy fiber excitatory input to CA2 PNs is strengthened, along with CA2 PN excitatory input to CA1 PNs. Finally, in hippocampal slices from PILO-treated mice I found an increase in CA2 PN input resistance and thus elevated intrinsic excitability, leading to a higher firing rate upon direct current injection. The combined effect of these changes may drive the emergence of epileptiform synchronization in the CA2 network and facilitate the propagation of seizure activity from the DG and entorhinal cortex directly to CA1 via the CA2-centered disynaptic (EC LII --> CA2 --> CA1) and alternate trisynaptic circuits (EC LII --> DG --> CA2 --> CA1).

Calretinin (CR) and parvalbumin (PV) neurons are inhibitory interneurons (INs) that play important roles in the modulation of excitatory pyramidal neurons. They are found in many species are and throughout the neocortex. However, their characteristics vary between species and brain region. The aim of this study was to compare the density, distribution, and inhibitory signaling of CR and PV neurons in monkey primary visual cortex (V1), monkey lateral prefrontal cortex (LPFC), mouse V1 and mouse frontal cortex (FC). Coronal brain slices from each of the species and brain regions were stained using immunohistochemistry and then the slices were scanned using high-resolution confocal imaging. High resolution image stacks were used to count the somata of CR and PV. The vesicular gamma aminobutyric acid (GABA) transporter (VGAT), CR and PV particles were analyzed to quantify these inhibitory markers in monkey V1, LPFC, and mouse V1 and FC. There were significant differences in the laminar distribution of CR and PV neurons in that CR neurons were concentrated in L2/3 and PV neurons were concentrated in L2-5. In L2/3, Monkey V1 had more CR neurons than did monkey LPFC. Furthermore, there were a greater number of PV neurons in monkey and mouse V1 compared to monkey LPFC and mouse FC. In L2/3, monkey V1 had the highest number of PV neurons. In L5, there significantly greater PV neurons in mouse V1 compared to monkey V1. There was significantly higher density of CR neurons in the upper middle layers of Monkey V1 compared to mouse V1 and monkey LPFC compared to mouse FC. The upper middle layers of monkey V1 had significantly higher density of PV neurons compared to monkey LPFC and mouse V1. There was significantly higher density of VGAT particles in monkey V1 and LPFC compared to mouse V1 and FC, which indicates more inhibitory synapses. There were significantly more VGAT+ boutons colocalized with PV+ boutons than CR+ boutons. Finally, discriminant analysis and hierarchical cluster analysis show that species is the largest separating factor between monkey V1, LPFC and mouse V1 and FC. Mouse V1 and FC are very similar, and monkey V1 and LPFC are dissimilar from one another. This data, united with comparative data on pyramidal neurons, demonstrates that neurons have differences between species, and monkeys have more regional specialization than mice.

No
The ability of neurons to modulate synaptic strength underpins synaptic plasticity, learning and memory, and adaptation to sensory experience. Despite the importance of synaptic adaptation in directing, reinforcing, and revising the behavioral response to environmental influences, the cellular and molecular mechanisms underlying synaptic adaptation are far from clear. Brain-derived neurotrophic factor (BDNF) is a prime initiator of structural and functional synaptic adaptation. However, the signaling cascade activated by BDNF to initiate these adaptive changes has not been elucidated. We have previously shown that BDNF activates mitogen- and stress-activated kinase 1 (MSK1), which regulates gene transcription via the phosphorylation of both CREB and histone H3. Using mice with a kinase-dead knock-in mutation of MSK1, we now show that MSK1 is necessary for the upregulation of synaptic strength in response to environmental enrichment in vivo. Furthermore, neurons from MSK1 kinase-dead mice failed to show scaling of synaptic transmission in response to activity deprivation in vitro, a deficit that could be rescued by reintroduction of wild-type MSK1. We also show that MSK1 forms part of a BDNF- and MAPK-dependent signaling cascade required for homeostatic synaptic scaling, which likely resides in the ability of MSK1 to regulate cell surface GluA1 expression via the induction of Arc/Arg3.1. These results demonstrate that MSK1 is an integral part of a signaling pathway that underlies the adaptive response to synaptic and environmental experience. MSK1 may thus act as a key homeostat in the activity- and experience-dependent regulation of synaptic strength.