No description available.

Dentate microcircuitry is thought to be involved in filtering, integrating, and relaying extrinsic hippocampal inputs to the hippocampus proper, which contributes to memory formation and retrieval. The axons of granule cells are called mossy fibres (MFs), and contain multiple terminal types that form characteristic synaptic connections with their postsynaptic targets. This diversity of presynaptic release sites that exists on the same MF provides an extremely interesting axonal type to study the organizing principles of presynaptic release regulation. A remarkable set of neurotransmitters and receptors present at the MF synaptic complex allow diverse computational modification of information from the dentate gyrus to the hippocampus. There are several types of glutamate receptors expressed at MF, such as group II/III mGluRs and kainate receptors (KARs). Presynaptic KARs modulate transmission at MF-CA3 pyramidal cell synapses; however, it is not known whether presynaptic KARs affect other synapses made by MFs. The aim of the first part of this thesis was to establish the principles of synapse-specific actions of presynaptic KARs in MFs. Combining electrophysiology and calcium imaging, this study provides compelling evidence that presynaptic KARs and Ca²⁺ stores can be activated by glutamate release from a single action potential in a single MF axon. This contributes to short-term, use-dependent facilitation of presynaptic Ca²⁺ entry and glutamate release exclusively at MF-CA3 pyramidal cell synaps, but not at other MF synapses, on hilar mossy cells or interneurons. Thus, our findings indicate that the presynaptic KARs, coupled with intracellular stores, exist in a synapse-specific autoreceptor mechanism. Activation of KARs strengthened MF-CA3 pyramidal cell synapses by increasing the Ca²⁺ influx at giant boutons, which might also contribute to the KAR-dependent hyper-excitability of the MF circuitry related to the mechanisms of temporal lobe epilepsy (TLE). This makes KARs good potential targets for therapies in CNS disorders such as epilepsy and other neurological and psychiatric disorders. The second part of this thesis was to explore the actions on the hippocampus of purified antibodies from a limbic encephalitis (LE) patient. LE is a CNS disease characterized by subacute onset of memory loss and temporal lobe seizures. The serum of these patients strongly labels MFs apparently co-localizing with the VGKC. The patients improve with immunotherapies that reduce the VGKC antibody levels in the serum, thus, strongly suggesting that these antibodies cause the condition. We found that LE serum IgGs enhance CA3 pyramidal cell excitability by blocking α-DTX sensitive VGKCs, which results in the increased release of glutamate. This, in turn, strengthens and desynchronizes MF and CA3 pyramidal cells synaptic transmission. However, these effects were occluded by α-DTX, a Kv1.1, Kv1.2 and Kv1.6 antagonist which, when applied alone, mimicked the action of the LE IgG, suggesting that they may share similar mechanisms of action. In contrast serum taken from healthy control patients had no significant effect under same recording conditions. Thus, this study provides the first evidence that the LE IgG functionally affects VGKC containing Kv1.1, Kv1.2 and/or Kv1.6 at both presynaptic MF axon terminals as well as the postsynaptic somatodendritic domain of CA3 pyramidal cells. Whatever defines the exact nature of LE IgG action, our results suggest that drugs acting specifically as openers of VGKC might help to protect the hippocampus from immune-mediated damage. In conclusion my data is consistent with the increasingly documented idea that MFs play a critical role in regulating the excitability of the hippocampal circuits and the dysfunction of MF transmission profoundly impairs hippocampal function.

<p>Long-term plasticity, the long-lasting, activity-dependent change in synaptic efficacy, is a fundamental property of the nervous system. Presynaptic forms of long-term plasticity are widely expressed throughout the brain, having been described in regions such as the cortex, cerebellum, hippocampus, thalamus, amygdala and striatum. Presynaptic long-term potentiation (LTP) is associated with an increase in presynaptic release probability, but further evidence of the cellular basis for the change in release probability is not known. At the molecular level, presynaptic LTP is known to require protein kinase A, the synaptic vesicle protein, Rab3A, and the active zone protein, RIM1alpha. RIM1alpha, a presynaptic scaffold protein, binds to many molecules with known functions at different stages of the neurotransmitter release process and the synaptic vesicle cycle. Understanding which interactions of RIM1alpha mediate presynaptic LTP would shed light on the molecular and cellular mechanisms for presynaptic long-term plasticity.</p><p>Here I developed a novel platform to achieve robust acute genetic</p><p>manipulation of presynaptic proteins at hippocampal mossy fiber synapses, where presynaptic LTP is expressed. With this platform, I perform structure-function analysis of RIM1alpha in presynaptic LTP. I find that RIM1alpha phosphorylation by PKA at serine 413 is not required for mossy fiber LTP, nor does RIM1alpha-Rab3A interation. These findings suggest that RIM1alpha, Rab3A and PKA signaling, instead of functioning synergistically, may represent separate requirements for presynaptic long-term plasticity. I then tested whether Munc13-1, a priming protein, is an effector for RIM1alpha in presynaptic LTP and provide the first evidence for the involvement of Munc13-1 in presynaptic long-term synaptic plasticity. I further demonstrate that the interaction between RIM1alpha and Munc13-1 is required for this plasticity. These results further our understanding of the molecular mechanisms of presynaptic plasticity and suggest that modulation of vesicle priming may provide the cellular substrate for expression of LTP at mossy fiber synapses.</p> / Dissertation

No description available.

Kindling epileptogenesis has been associated with a number of different forms of neuroplasticity in the hippocampus, including mossy fibre sprouting and an increase in both intracellular calcium and zinc. The purpose of this thesis was to determine whether interfering with the influx of calcium via the voltage gated calcium channels would interfere with kindling- induced plasticity. Both kindled and control rats were injected with either 5 or 25mg/kg of the L-type voltage gated calcium channel blocker, nifedipine, or a control vehicle, DMSO (dimethylsulfoxide). The kindled groups received a kindling stimulation twice a day for 11 days. It was revealed that both doses of nifedipine significantly increased afterdischarge duration (p<0.001) and furthermore, both doses of nifedipine were capable of significantly interfering with the rate of kindling (p<0.001). Three weeks following the last kindling stimulation, rats were perfused and brain tissue was processed according to the Timm method. The density of Timm granules, an indication of the level of intracellular zinc in the mossy fibre pathway, was quantified. The results of this analysis revealed that 25mg/kg of nifedipine is capable of significantly reducing the amount of intracellular zinc in both the IML (p<0.04) and the CA3 (p<0.01) region of the mossy fibre pathway, regardless of whether the rats had received kindling stimulations or not. These results provide support for the notion that nifedipine (5 or 25mg/kg) is an effective anticonvulsant agent. These results also suggest that, at a sufficient dose (25mg/kg), nifedipine can reduce the amount of intracellular zinc in the mossy fibre pathway in both kindled and non-kindled animals, suggesting that nifedipine may be a useful therapeutic agent for pathologies that have been associated with zinc-induced neurotoxicity. / Thesis / Master of Science (MSc)

L'hippocampe est une région du cerveau située dans le lobe temporal médian. Avec d'autres structures limbiques, l'hippocampe est impliqué dans des processus d'apprentissage et de mémorisation et possède un rôle crucial dans le traitement spatial de l'information. Les synapses de l'hippocampe formées entre les fibres moussues (fm) originaires du gyrus denté et les neurones pyramidaux de CA3 ont reçu une attention particulière, compte tenu de la position stratégique occupée par le gyrus denté à l'entrée de l'hippocampe. En outre les synapses fm- CA3 sont distinctes de la plupart des autres synapses excitatrices du système nerveux central par leurs propriétés morphologiques et physiologiques uniques. Cela soulève la question de savoir si ces propriétés uniques reflètent aussi un rôle fonctionnel unique dans le traitement de l'information effectué par cette synapse au sein du microcircuit de l'hippocampe. Malheureusement nous ne savons que peu de choses sur la façon dont les cellules granulaires modulent l'activité des neurones de CA3 dans le réseau intact in vivo (Henze et al, 2002 ; Hagena et Manahan - Vaughan, 2010, 2011). Le manque d'information est dû au fait que la manipulation classique des circuits neuronaux par des approches électriques, pharmacologiques et génétiques manque de précisions spatiale et temporelle in vivo. L'utilisation de la stimulation extracellulaire de fibres moussues peut conduire à l'activation polysynaptique de cellules pyramidales de CA3, qui peuvent ensuite contaminer les réponses enregistrées. Par ailleurs, l'utilisation de critères trop conservateurs peut conduire à l'exclusion des réponses provenant des fibres moussues «purs» aux propriétés méconnues (Henze et al., 2000). Toutefois, le développement récent et rapide de l’optogénétique dans les neurosciences a fourni de nouveaux outils offrant une sélectivité spatiale élevée (activation optique spécifique de la cellule), et une grande précision temporelle (à l'échelle de la milliseconde), permettant la dissection et l'étude des circuits neuronaux in vivo. L'objectif de ma thèse était de mieux comprendre les mécanismes et les conséquences physiologiques de la plasticité synaptique à court terme se produisant à la synapse formée entre les fibres moussues et les neurones pyramidaux de CA3 dans le cerveau de souris intact. La présente thèse se compose de deux parties principales. Dans la première partie, j'ai exploré de nouveaux outils optogénétiques dans le but de contrôler l'activité des cellules granulaires à l’aide d’impulsions de lumière. La stimulation optogénétique repose sur l'activation du canal ionique channelrhodopsin - 2 - lumière fermée ( ChR2 ) par une lumière bleue et induit des potentiels d'action sur une large gamme de fréquences de stimulation. J'ai aussi observé que la stimulation optique peut être utilisée pour déclencher la plasticité à court terme au niveau des synapses fm-CA3.Dans la deuxième partie j'ai affiné la méthodologie de stimulation optogénétique in vivo pour la caractérisation non invasive du fonctionnement synaptique des synapses fm- CA3. La fiabilité de la stimulation optogénétique d'une population neuronale génétiquement ciblée ainsi que la résolution d'une seule cellule obtenue en utilisant des enregistrements de cellules entières sont des étapes importantes vers une meilleure compréhension du rôle fonctionnel des fibres moussues dans le réseau de l'hippocampe in vivo. / The hippocampus is a brain region located in the medial temporal lobe. Along with other limbic structures, the hippocampus is involved in learning and memory processes and has a crucial role in spatial information processing. Within the hippocampus synapses made between mossy fibers (mf) originating from the dentate gyrus and CA3 pyramidal neurons have received particular attention, given the strategic position occupied by the dentate gyrus at the entrance of the hippocampus. Moreover mf-CA3 synapses are distinct from most of other excitatory synapses in the central nervous system for their unusual morphological and physiological properties. This raises the question if these unique properties reflect a unique functional role in information processing carried out by this synapse within the microcircuit of the hippocampus. Unfortunately very little is known on how granule cells modulate the activity of CA3 neurons in the intact network in vivo (Henze et al., 2002; Hagena and Manahan-Vaughan, 2010, 2011). The paucity of information is due to the fact that classical manipulation of neuronal circuits using electrical, pharmacological and genetic approaches lack spatial and temporal precision in vivo. The use of bulk extracellular stimulation may lead to polysynaptic activation of CA3 pyramidal cells, which can subsequently contaminate putative mossy fibers synaptic responses measured in CA3 pyramidal cells. The use of overly conservative criteria on the other side may lead to the exclusion of “pure” mossy fibers responses with unexpected properties (Henze et al., 2000).However the recent and fast growth of optogenetics in neuroscience has provided new tools with high spatial selectivity (cell specific optical activation) and temporal precision (at the millisecond scale), allowing the dissection and investigation of neuronal circuits in vivo. The aim of my thesis was to gain insight into the mechanisms and the physiological consequences of short-term synaptic plasticity occurring at mossy fibers to CA3 pyramidal neurons synapses in the intact mouse brain. The present thesis consists of two main parts. In the first part I explored new optogenetic tools to control the activity of granule cells with pulses of light. Optogenetic stimulation, which relies on the activation of the light-gated ion channel channelrhodopsin-2 (ChR2) by blue light reliably induced action potentials over a wide range of frequencies of stimulation. I also found that optical stimulation can be used to trigger short term plasticity at mf-CA3 synapses. In the second part I refined optogenetic stimulation methodology in vivo for non-invasive characterization of synaptic functioning of the mf-CA3 synapses. The reliability of optogenetic stimulation of a genetically targeted neuronal population together with the single cell resolution obtained using whole-cell recordings are important steps towards a better understanding of the functional role of the mossy fibers in the hippocampal network in vivo.

Posttraumatic epilepsy is a common consequence of brain trauma. However, little is known about how long-term changes in local excitatory and inhibitory synaptic networks contribute to epilepsy after closed-head brain injury. This study adapted a widely used model of experimental brain injury as a mouse model of posttraumatic epilepsy. Behavioral seizure activity and alterations in synaptic circuitry in the dentate gyrus were examined in mice after experimental cortical contusion brain injury. Spontaneous behavioral seizures were observed in 20% of mice after moderate injury and 36-40% of mice weeks after severe injury. In the dentate gyrus, most mice displayed regionally localized mossy fiber reorganization ipsilateral to the injury that was absent in control mice or sections contralateral to the injury. Extracellular field and whole-cell patch clamp recordings were performed in acute brain slice preparations of the dentate gyrus. Dentate granule cells displayed spontaneous and evoked activity that was consistent with network synchronization and the formation of recurrent excitatory network only in slices that had posttraumatic mossy fiber sprouting. The excitability of surviving hilar GABAergic interneurons, which provide important feedback inhibition to granule cells, was examined at similar time points. Cell-attached and whole-cell voltage-clamp recordings revealed increased spontaneous and glutamate photostimulation-evoked excitatory input to hilar GABA neurons ipsilateral to the injury, versus control and contralateral slices. Despite increased excitatory synaptic input to interneurons, whole-cell voltage-clamp recordings revealed a reduction in inhibitory synaptic input to granule cells. These findings suggest that there are alterations in excitatory and inhibitory circuits in mice with posttraumatic mossy fiber sprouting and seizures after cortical contusion head injury.

No description available.

Les synapses se forment selon plusieurs étapes comprenant la stabilisation des contacts nouvellement formés et leur maturation. Ces différentes étapes dépendent d’une mise en place coordonnée entre la terminaison pré- et postsynaptique. Les protéines composant la présynapse et les récepteurs ionotropiques du glutamate ont des rôles clés dans ces processus. Lors de ma thèse, je me suis intéressé à l’implication de la protéine présynaptique Bassoon lors de la maturation des synapses glutamatergiques entre les fibres moussues et les cellules pyramidales de CA3 dans l’hippocampe. Cette synapse constitue un modèle attractif pour l’étude de la maturation synaptique car elle suit des étapes de maturation morphologique et fonctionnelle bien définies. Bassoon est une des premières protéines se mettant en place au niveau des contacts synaptiques nouvellement formés. Par des approches électrophysiologiques, nous avons montré que la protéine Bassoon était importante pour l’organisation du site de libération de neurotransmetteur durant les deux premières semaines de vie post-natale chez la souris.Les récepteurs kaïnate jouent un rôle important dans la régulation de l’activité de réseau au cours du développement post-natal. Cependant l’impact de l’activation de ces récepteurs sur la maturation synaptique est peu connu. J’ai pu mettre en évidence un délai dans la maturation fonctionnelle de la synapse fibre moussue/cellule pyramidale de CA3 chez les souris déficientes pour la sous-unité GluK2 des récepteurs kaïnate (GluK2-/-). Afin de comprendre si ce délai de maturation fonctionnelle est corrélé à un retard dans la maturation morphologique de cette synapse, nous avons mis en place des infections de lentivirus codant pour une protéine membranaire fluorescente (YFP) chez le souriceau nouveau-né (P1-P2). A l’aide de microscopie confocale et de reconstruction en 3D, nous avons ainsi pu décrire la maturation morphologique de la synapse fibre moussue/cellule pyramidale de CA3. Cela m’a également permis de corréler la maturation fonctionnelle à la maturation morphologique et mes résultats montrent également un retard dans la mise en place des synapses chez les souris GluK2-/-. L’ensemble de cette étude révèle l’importance de l’activité synaptique et de la coordination entre mise en place de la pré- et de la postsynapse au cours de la maturation synaptique. / The formation of synapses follows different steps including synaptogenesis and maturation. These different steps depend on coordinated pre- and post-synaptic assembly. Pre-synaptic proteins and ionotropic glutamate receptors play a central role in these processes. During my thesis, I have been interested in the implication of the presynaptic protein Bassoon in the maturation of the hippocampal mossy fiber to CA3 pyramidal cell glutamatergic synapses. This synapse constitutes an attractive model for the study of synaptic maturation because it follows several steps of defined morphological and functional maturation. Bassoon in one of the first protein present at newly formed synaptic contacts. By electrophysiological approaches, we showed that Bassoon is important for the organization of the active zone during the first two postnatal weeks.Kainate receptors play an important role in the regulation of network activity during postnatal development. However, the impact of kainate receptors activation on synaptic maturation is less known. I showed a delay in functional maturation of mossy fiber synapses in mice deficient for the GluK2 subunit of kainate receptors (GluK2-/-). To know if this delay is correlated to morphological alterations of this synapse, we setup in vivo lentiviral infections of membrane fluorescent protein (YFP) in mouse pups (P1-P2). Using confocal microscopy and 3D reconstruction, we described the morphological maturation of mossy fiber synapses. We were able to correlate functional and morphological maturation and our results also showed an impairment in the formation of mossy fiber synapses in GluK2-/-. Together, these data reveal the importance of synaptic activity and of the coordination of pre- and post-synaptic assembly during synaptic maturation.

No description available.