Synaptic mechanisms of Hebbian and homeostatic plasticity driven by intrinsic activity in the developing hippocampus

The formation of synaptic connections in the brain is guided by genetic and activity-dependent mechanisms. The initial hard-wiring of the circuitry is followed by a phase during which connections are refined. During this process the genetic factors are less important and refinement is guided by electrical activity. However, the mechanisms underlying the activity-driven synaptic fine-tuning are still poorly understood. The features of electrical activity and the mechanisms of synaptic transmission differ in the developing networks as compared to those of the adult. Electrical activity in the developing networks comprises of intermittent, highly synchronous bursts of action potentials interleaved by more silent, asynchronous neuronal firing. In the hippocampus this immature-type electrical patternity coincides temporally with the intense synaptic reorganization. Moreover, there is a parallel, developmentally-regulated expression GluA4 subunit of AMPA-type ionotropic glutamate receptors in the hippocampal neurons. Despite frequent speculation on the relative importance of synchronous vs. asynchronous neuronal activity on the synaptic development in the brain, there have been no direct experiments to study this issue. In this thesis we have, for the first time, been able to experimentally dissect the roles of asynchronous vs. synchronous activity on synaptic refinement in the hippocampus. Specifically, we show that spontaneous synchronous activity is essential for the stabilization and maturation of immature CA3-CA1 synapses, and that network desynchronization leads to weakening of glutametergic transmission in the CA1 area. Plasticity changes caused by different endogenous activity patterns were strongly dependent on the synapse type (glutamatergic vs. GABAergic), the anatomical area (CA1 vs. CA3) and maturational stage of the neurons. In addition, the GluA4 was shown to be critical for both the Hebbian type and homeostatic plasticity mechanisms in developing glutamatergic synapses. In the absence of GluA4, the homeostatic regulation of the immature glutamatergic networks in response to manipulation of endogenous activity patterns was perturbed. Finally, GluA4 was shown to be necessary and sufficient for protein kinase A dependent long-term potentiation (LTP), typical of immature CA3-CA1 synapses. These data demonstrate the instrumental role of spontaneous synchronous activity and GluA4 AMPAR subunit expression in the formation and refinement of hippocampal synaptic networks.Aivojen hermosolujen välinen tiedonsiirto perustuu niiden välisiin synaptisiin yhteyksiin. Yksittäinen hermosolu voi olla synapsiyhteydessä useisiin satoihin tai tuhansiin muihin hermosoluihin. Osa näistä yhteyksistä on hermosolun toimintaa edistäviä (eksitoivia) ja osa estäviä (inhiboivia). Yksi aivojen keskeisimmistä ominaisuuksista on plastisuus, eli kyky muuttaa hermosolujen välisten synapsien määrää ja vahvuutta. Plastisuusmekanismit luovat molekulaarisen pohjan mm. oppimisen ja muistin solutason mekanismeille. Voimakas hermosolujen samanaikainen sähköinen aktiivisuus tai korkeataajuinen ärsytys johtaa yleensä kyseisten solujen välisen synapsiyhteyden vahvistumiseen (LTP, long-term potentiation), kun taas matalalla taajuudella toistuva ärsytys heikentää kyseisten solujen välisen kontaktin vahvuutta (LTD, long-term deprssion). Tätä ominaisuutta kutsutaan hebbiläiseksi plastisuudeksi. Yksittäisten synapsiyhteyksien liiallinen vahvistuminen tai heikkeneminen voi kuitenkin johtaa hermosolujen yliaktiivisuuteen tai totaaliseen hiljaisuuteen. Näitä ääripäitä välttääkseen aivot käyttävät ns. tasapainottavia eli homeostaattisia plastisuusmekanismeja, jotka muuttavat hermosolujen yhteyksiä niin, että yksittäisten synapsien vahvuuserot ja erojen sisältämä informaatio säilyvät. Sekä hebbiläiset että homeostaattiset plastisuusmekanismit ovat tärkeitä jo varhaiskehityksen aikana ensimmäisten hermosolujen yhteyksien muodostuessa. Aluksi synapsien muodostus on runsasta ja ensimmäisten yhteyksien muodostumista seuraa niiden testaus ja hienosäätö, jonka aikana tarpeelliset synapsit vahvistuvat ja tarpeettomat poistetaan. Tätä hienosäätöä ja siihen tarvittavia plastisuusmekanismeja ohjaa hermosolujen sähköinen aktiivisuus. Kaikkien nisäkäsaivojen varhaiskehitykselle on ominaista spontaani eli sisäsyntyinen sähköinen aktiivisuus. Tälle aktiivisuudelle on tyypillistä hermosolujen samanaikaisen (synkronisen) aktiivisuuden muodostamat hermoverkkoryöpyt, joilla on uskottu olevan tärkeä rooli synapsiyhteyksien hienosäädössä ja aivojen kehitykselle sopivan sähköisen aktiivisuustason ylläpidossa. Synaptiseen plastisuuteen liittyvien solutason mekanismien sekä hermoverkkoryöppyjen merkitys synaptisten kontaktien synnyssä varhaiskehityksen aikana on kuitenkin ollut tähän asti epäselvää. Tärkein aivojen viestien välitystä edistävä synapsissa vaikuttava välittäjäaine on glutamaatti. Tässä väitöskirjatyössä on ensimmäistä kertaa osoitettu, että sisäsyntyiset hermoverkkoryöpyt ohjaavat aivojen viestinvälitystä edistävien glutamaattivälitteisten synapsien kehitystä hippokampuksessa. Ilman synkronista aktiivisuutta glutamaattivälitteinen aktiivisuus heikkenee ja toimimattomien ns. hiljaisten synapsien määrä kasvaa. Glutamaatin vapautumisen aikaansaama viestinvälitys synapsissa perustuu sen vastaanottajasolun synapsin solukalvolla sijaitsevien reseptorimolekyylien aktivaatioon. Väitöskirjassa osoitettiin myös, että synapsien kehityksen solutason mekanismit riippuvat tietyn glutamaattireseptorin, 1-amino-3-hydroksi-5-metyyli-iso-oksatsoli-4-propionaatti (AMPA)-reseptorin, alayksikön, GluA4, ilmentymisestä. Tämän alayksikön ilmentyminen hippokampuksessa katoaa samaan aikaan sisäsyntyisen aktiivisuuden kanssa ja se korvataan muilla alayksiköillä aikuisissa aivoissa. GluA4:n ohimenevän ilmentymisen fysiologista merkitystä ei ole aikaisemmin tiedetty. Nykykäsityksen mukaan alttius monille hermostoperäisille sairauksille saattaa juontaa juurensa jo keskushermoston varhaiskehityksen aikaisista häiriöistä. Tässä väitöskirjassa tutkittua sisäsyntyistä spontaania aktiivisuutta havaitaan ihmissikiöillä viimeisen raskauskolmanneksen aikana. Tulosten perusteella voidaan olettaa, että jo pienet häiriöt aivojen spontaanissa aktiivisuudessa voivat aiheuttaa merkittäviä muutoksia hermosolujen synapsiyhteyksien muodostumisessa. Häiriö voi olla esimerkiksi alkoholin tai lääkeaineiden aiheuttama. Väitöskirjassa löydetyt synapsiyhteyksien kehitysmekanismit ja niiden muutokset auttavat ymmärtämään tiettyjen kehitysperäisten keskushermostosairauksien syntymekanismeja

Cholinergic Control of Cortical Circuit Activity

Cholinergic neurons of the basal forebrain send extensive projections to all regions of the neocortex and are critically involved in a diverse array of cognitive functions, including sensation, attention and learning. Cholinergic signaling also plays a crucial role in the moment-to-moment control of ongoing cortical state transitions that occur during periods of wakefulness. Yet, the underlying circuit mechanisms of synaptic cholinergic function in the neocortex remain unclear. Moreover, acetylcholine continues to be widely viewed as a slow and diffuse neuromodulator, despite the preponderance of in vivo evidence demonstrating rapid cholinergic function. In this study, we used a combination of optogenetics and in vitro electrophysiology to examine spatiotemporally precise control of cortical network activity by endogenous acetylcholine. We show that even brief activation of cholinergic afferents could powerfully suppress evoked cortical recurrent activity for several seconds. This suppression was reliant on the engagement of both nicotinic and muscarinic acetylcholine receptors. Nicotinic receptors mediated transient suppression by acting in the superficial cortical layers, while muscarinic receptors mediated prolonged suppression in layer 4. In agreement, we found nicotinic-mediated excitation of inhibitory neurons in the supragranular layers, and muscarinic-mediated hyperpolarization of excitatory cells in layer 4. Together, these findings present novel circuit mechanisms for fast and robust cholinergic signaling in neocortex

Caractérisation du potentiel de champ local dans le cortex préfrontal médian du rat durant le stress et la prise alimentaire

Le stress joue un rôle important dans le maintien de la qualité de vie quotidienne. Une exposition à une situation stressante peut causer divers désordres neuropsychiatriques du cerveau qui sont associés avec des problèmes liés au sommeil, à la dépression, à des problèmes digestifs et à des troubles de l'alimentation. Les traitements de ces troubles liés au stress sont très coûteux à travers le monde. De nos jours, des considérations importantes ont été soulevées afin de trouver des moyens appropriés pour la prévention plutôt que de dépenser ultérieurement plus de budget sur les traitements. De cette façon, l'étude et l'expérimentation sur les animaux des troubles liés au stress sont l'un des moyens les plus fiables pour atteindre une compréhension plus profonde des problèmes liés au stress. Ce projet visait à révéler la modulation des potentiels de champ locaux (LFP) lors de la consommation de sucrose dans deux conditions englobant la condition de contrôle non-stressante et celle stressante d'un choc électrique aiguë à la patte dans le cortex préfrontal médian (CPFm) du cerveau de rat. Le CPFm est une structure importante dans la réponse au stress et à l'anxiété par l'interaction avec l'axe hypothalamique-pituitaire surrénale (HPA). Les résultats de ce projet ont révélé que la plupart des coups de langue se sont produits dans les 15 premières minutes de l'accès à une solution de sucrose autant pour la condition contrôle non-stressante que pour la condition stressante. En outre, le stress aigu d'un choc à la patte affecte de manière significative la consommation horaire de sucrose en diminuant le volume de la consommation. Les résultats ont également révélé une présence importante du rythme thêta dans le CPFm pendant la condition de base et pendant l'ingestion de sucrose dans les deux conditions. De plus, les résultats ont montré une diminution de puissance des bandes delta et thêta lors des initiations de léchage du sucrose. Ce projet conduit à des informations détaillées sur les propriétés électrophysiologiques du cortex infra-limbique (IL) du CPFm en réponse à l'exposition à des conditions de stress et de l'apport d'une solution de sucrose. Ce projet permet également de mieux comprendre les mécanismes neurophysiologiques des neurones du CPFm en réponse à l'exposition à une condition stressante suivie d'apport de sucrose. Ce projet a également permis de confirmer les effets anorexigènes du stress et suggèrent également que la synchronisation neuronale dans le cortex IL peut jouer un rôle dans le comportement de léchage et sa désynchronisation pendant le léchage après une exposition à des conditions stressantes.Stress has been playing important role in maintaining daily life quality. Exposure to stressful situation may cause vast varieties of neuropsychiatric brain disorders associated with sleep related problems, depression, digestive problems and eating disorders. Treatments of such stress-related disorders are costly all across the world. Nowadays, significant consideration has been raised in order to find the appropriate ways for prevention rather than later spending more budgets on treatments. In this way, animals’ modeling and studying the stress-related disorders is one of the most reliable ways for deeper understanding the stress-related problems. This project aimed to reveal the modulation of local field potentials (LFPs) that carries important information about neuronal activities within very specific domains during sucrose consumption in two conditions encompassing non-stressful control condition and acutely foot shock stressed condition in the medial prefrontal cortex (mPFC) of the rat’s brain. The mPFC plays an important role in stress response and anxiety via its interaction with hypothalamic-pituitary adrenal (HPA) axis. The results of this project revealed that licks mostly occurred in the first 15-min of access to palatable sucrose solution in either non-stressful control or stressful conditions. Also, acute foot shock stress significantly affects the 1-h intake of sucrose by decreasing the volume of intake. It also revealed the mPFC prominent theta band oscillation during both baseline and sucrose ingestion in non-stressful and stressful conditions. Moreover, the results showed an increase in power of delta and theta oscillation bands on the licking initiation onsets. This project reveals detailed information on the electrophysiological properties of IL cortex of mPFC in response to exposure to stressful condition and intake of palatable sucrose solution. This project also helps to better understand the neurophysiological mechanisms of mPFC neurons in response to the exposure to stressful condition following by sucrose intake. This project also helped to confirm the anorectic effects of stress and also suggested that the neuronal synchronization in the IL cortex may play a role in licking behavior and showed desynchronization in the IL during licking after exposure to stressful conditions

Intrinsic and synaptic membrane properties of neurons in the thalamic reticular nucleus

Tableau d’honneur de la Faculté des études supérieures et postdoctorales, 2004-2005Le noyau réticulaire thalamique (RE) est une structure qui engendre des fuseaux, une oscillation bioélectrique de marque pendant les stades précoces du sommeil. De multiples propriétés neuronales, intrinsèques et synaptiques, sont impliquées dans la génération, la propagation, le maintien et la terminaison des ondes en fuseaux. D’un autre côté, ce rythme constitue un état spécial de l’activité du réseau qui est généré par le réseau lui-même et affecte les propriétés cellulaires du noyau RE. Cette étude se concentre sur ces sujets: comment les propriétés cellulaires et les propriétés du réseau sont inter-reliées et interagissent pour engendrer les ondes fuseaux dans les neurones du RE et leurs cibles, les neurones thalamocorticaux. La présente thèse fournit de nouvelles évidences montrant le rôle fondamental joué par les neurones du noyau RE dans la genèse des ondes en fuseaux, dû aux synapses chimiques établies par ces neurones. La propagation et la synchronisation de l’activité sont modulées par les synapses électriques entre les neurones réticulaires thalamiques, mais aussi par les composantes dépolarisantes secondaires des réponses synaptiques évoquées par le cortex. De plus, la forme générale et la terminaison des oscillations thalamiques sont probablement contrôlées en grande partie par les neurones du RE, lesquels expriment une conductance intrinsèque leurs procurant une membrane avec un comportement bistable. Finalement, les oscillations thalamiques en fuseaux sont aussi capables de moduler les propriétés membranaires et l’activité des neurones individuels du RE.The thalamic reticular nucleus (RE) is a key structure related to spindles, a hallmark bioelectrical oscillation during early stages of sleep. Multiple neuronal properties, both intrinsic and synaptic, are implicated in the generation, propagation, maintenance and termination of spindle waves. On the other hand, this rhythm constitutes a special state of network activity, which is generated within, and affects single-cell properties of the RE nucleus. This study is focused on these topics: how cellular and network properties are interrelated and interact to generate spindle waves in the pacemaking RE neurons and their targets, thalamocortical neurons. The present thesis provides new evidence showing the fundamental role played by the RE nucleus in the generation of spindle waves, due to chemical synapses established by its neurons. The propagation and synchronization of activity is modulated by electrical synapses between thalamic reticular neurons, but also by the secondary depolarizing component of cortically-evoked synaptic responses. Additionally, the general shaping and probably the termination of thalamic oscillations could be controlled to a great extent by RE neurons, which express an intrinsic conductance endowing them with membrane bistable behaviour. Finally, thalamic spindle oscillations are also able to modulate the membrane properties and activities of individual RE neurons

GABA signaling in the thalamus

Inhibition of neuronal activity in networks of the mammalian central nervous system is essential for all fundamental brain functions, ranging from perception, to consciousness, to action. Both exacerbation and diminution of inhibition dramatically affect our behavioral capacities, indicating that, in the healthy brain, strength and dynamics of inhibition must be precisely balanced. Inhibitory functions are primarily accomplished by neurons releasing the neurotransmitter GABA. According to their wide variety of functions, GABAergic neurons show a tremendous diversity in morphological, biochemical and functional characteristics. The combination of these diverse properties allows the brain to generate interneurons acting as, for examples, filters, co-incidence detectors or contrast enhancers. GABAergic signaling in thalamus plays an essential role in controlling sensory information flow from the periphery to the cortical processing centers, and in generating sleep-related neuronal rhythms. Surprisingly, however, the diversity of GABAergic neurons is remarkably limited in thalamic networks. Both functions mentioned have been tightly associated with two homogeneous groups of GABAergic neurons arising within thalamic nuclei or within the nucleus reticularis, a shell of inhibitory nuclei surrounding the dorsal thalamus. The results arising from the present thesis challenge the view that the diversity of GABAergic signaling in thalamus is comparatively limited and proposes that, to fully understand GABAergic signaling in thalamus, at least two additional aspects have to be considered. First, it shows that GABAergic signaling arising from the nucleus reticularis can have a profound effect on the synthesis of second messenger compounds that are important in the control of neuronal rhythmicities and in the statedependent control of gene expression. Second, it demonstrates the functional relevance of a previously undescribed extrathalamic and extrareticular inhibitory pathway that arises within the anterior pretectal nuclei, indicating that the architecture of GABAergic signaling in thalamus has to be complemented by a conceptually novel, powerful afferent pathway. The first part investigates the modulation of cAMP synthesis by GABA in thalamocortical neurons through the activation of the Gi-coupled GABAB receptors. GABAB receptors can provide two different cAMP signals in the neurons. First, GABAB receptor activation depresses the level of cAMP inside thalamocortical neurons. However, a large and long cAMP signal is observed when GABAB receptors are activated concomitantly with b-adrenergic receptors, which are Gscoupled receptors. In the presence of GABAB receptor agonists, the moderate cAMP increase produced by b-adrenergic receptor activation is transformed into a large synthesis of cAMP. Remarkably, the activation of the GABAB receptors at the synapses between reticular neurons and thalamocortical neurons also potentiates the effects of b-adrenergic receptors. Thus, GABAB receptors modulate cAMP signals at synapses that are important for the regulation of the state of arousal. The second part provides the first electrophysiological description of synaptic connections between the anterior pretectum group and the thalamic higher-order nuclei. Electric stimulation in the anterior pretectum group evoked inhibitory postsynaptic responses (IPS) in the thalamocortical neurons of the higher-order nuclei. We showed that the IPS responses were mediated via the GABAA receptors activated through monosynaptic connections between the APT and the higher-order nuclei. Functionally, the anterior pretectum modulated the discharge properties of the thalamocortical neurons, suggesting an important role of this nucleus in the dialogue between the thalamus and the cortex

Mechanisms of amyloid-beta cytotoxicity in hippocampal network function : rescue strategies in Alzheimer's disease

The origin and nature of cognitive processes are strongly associated with synchronous rhythmic activity in the brain. Gamma oscillations that span the frequency range of 30–80 Hz are particularly important for sensory perception, attention, learning, and memory. These oscillations occur intrinsically in brain regions, such as the hippocampus, that are directly linked to memory and disease. It has been reported that gamma and other rhythms are impaired in brain disorders such as Alzheimer’s disease, Parkinson’s disease, and schizophrenia; however, little is known about how these oscillations are affected. In the studies contained in this thesis, we investigated a possible involvement of toxic Amyloid-beta (Aβ) peptide associated with Alzheimer’s disease in degradation of gamma oscillations and the underlying cellular mechanismsin rodent hippocampi. We also aimed to prevent possible Aβ- induced effects by using specially designed molecular tools known to reduce toxicity associated with Aβ by interfering with its folding and aggregation steps. Using electrophysiological techniques to study thelocal field potentials and cellular properties in the CA3 region of the hippocampus, we found that Aβ in physiological concentrations acutely degrades pharmacologically- induced hippocampal gamma oscillations in vitro in a concentration- and time- dependent manner. The severity of degradation also increased with the amount of fibrillar Aβ present. We report that the underlying cause of degradation of gamma oscillations is Aβ-induced desynchronization of action potentials in pyramidal neurons and a shift in the equilibrium of excitatory-inhibitory synaptic transmission. Using specially designed molecular tools such as Aβ-binding ligands and molecular chaperones, we provide evidence that Aβ-induced effects on gamma oscillations, cellular firing, and synaptic dynamics can be prevented. We also show unpublished data on Aβ effects on parvalbumin-positive baskets cells or fast-spiking interneurons, in which Aβ causes an increase in firing rate during gamma oscillations. This is similar to what is observed in neighboring pyramidal neurons, suggesting a general mechanism behind the effect of Aβ. The studies in this thesis provide a correlative link between Aβ-induced effects on excitatory and inhibitory neurons in the hippocampus and extracellular gamma oscillations, and identify the Aβ aggregation state responsible for its toxicity. We demonstrate that strategies aimed at preventing peptide aggregation are able to prevent the toxic effects of Aβ on neurons and gamma oscillations. The studies have the potential to contribute to the design of future therapeutic interventions that are aimed at preserving neuronal oscillations in the brain to achieve cognitive benefits for patients

Focal Augmentation of Somatostatin Interneuron Function and Subsequent Circuit Effects in Developmentally Malformed, Epileptogenic Cortex

Drug-resistant epilepsy (DRE) is a common clinical sequela of developmental cortical malformations such as polymicrogyria. Unfortunately, much remains unknown about the aberrant GABA-mediated circuit alterations that underlie DRE\u27s onset and persistence in this context. To address this knowledge gap, we utilized the transcranial freeze lesion model in optogenetic mice lines (Somatostatin (SST)-Cre or Parvalbumin (PV)-Cre x floxed channelrhodopsin-2) to dissect features of the SST, PV, and pyramidal neuron microcircuit that are potentially associated with DRE. Investigations took place within developmental microgyria’s known pathological substrate, the adjoined and epileptogenic paramicrogyral region (PMR). As well, microcircuit relationships within the previously unexplored range of normal-appearing cortex beyond PMR’s terminus were also interrogated. We previously demonstrated SST interneuron output enhancement onto postsynaptic layer V pyramidal neurons of PMR. Dissertation studies elaborated on this SST-interneuron mediated effect through the utilization of ex vivo slice electrophysiology in conjunction with selective optical activation of either SST or PV interneurons. An ostensible mechanism was identified in the form of a novel structural schematic for SST interneurons of PMR whereby they exhibit wider reaching, within-layer arborization of axons within this pathological substrate. Also, within PMR, SST interneuron output was not enhanced onto postsynaptic layer V PV interneurons, indicating targeting specificity of the SST to pyramidal neuron effect. Moving beyond PMR, past its terminus, SST interneuron output onto layer V pyramidal cells was found to be equivalent to controls, indicating effect focality. Finally, a novel disinhibitory relationship was demonstrated beyond PMR’s terminus, wherein PV interneurons exhibited output enhancement onto postsynaptic layer V SST interneurons. This indicates a putative in vivo mechanism for the PMR-focality of the SST to pyramidal neuron output enhancement scheme. These novel discoveries will provide the field with more context as to the role SST and PV interneurons potentially play in the emergence and/or modulation of drug-resistant epilepsy in and outside the terminus of PMR

Rôle de deux groupes de vésicules dans la transmission synaptique

Les synapses formées par les fibres moussues (FM) sur les cellules principales de la région CA3 (FM-CA3) jouent un rôle crucial pour la formation de la mémoire spatiale dans l’hippocampe. Une caractéristique des FM est la grande quantité de zinc localisée avec le glutamate dans les vésicules synaptiques recyclées par la voie d’endocytose dépendante de l’AP3. En combinant l’imagerie calcique et l’électrophysiologie, nous avons étudié le rôle des vésicules contenant le zinc dans la neurotransmission aux synapses FM-CA3. Contrairement aux études précédentes, nous n’avons pas observé de rôle pour le zinc dans l’induction des vagues calciques. Nos expériences ont révélé que les vagues calciques sont dépendantes de l’activation des récepteurs métabotropiques et ionotropiques du glutamate. D’autre part, nos données indiquent que les vésicules dérivées de la voie dépendante de l’AP3 forment un groupe de vésicules possédant des propriétés spécifiques. Elles contribuent principalement au relâchement asynchrone du glutamate. Ainsi, les cellules principales du CA3 de souris n’exprimant pas la protéine AP3 avaient une probabilité inférieure de décharge et une réduction de la synchronie des potentiels d’action lors de la stimulation à fréquences physiologiques. Cette diminution de la synchronie n’était pas associée avec un changement des paramètres quantiques ou de la taille des groupes de vésicules. Ces résultats supportent l’hypothèse que deux groupes de vésicules sont présents dans le même bouton synaptique. Le premier groupe est composé de vésicules recyclées par la voie d’endocytose utilisant la clathrine et participe au relâchement synchrone du glutamate. Le second groupe est constitué de vésicules ayant été recyclées par la voie d’endocytose dépendante de l’AP3 et contribue au relâchement asynchrone du glutamate. Ces deux groupes de vésicules sont nécessaires pour l’encodage de l’information et pourraient être importants pour la formation de la mémoire. Ainsi, les décharges de courte durée à haute fréquence observées lorsque les animaux pénètrent dans les places fields pourraient causer le relâchement asynchrone de glutamate. Finalement, les résultats de mon projet de doctorat valident l’existence et l’importance de deux groupes de vésicules dans les MF qui sont recyclées par des voies d’endocytoses distinctes et relâchées durant différents types d’activités.Mossy fiber-CA3 pyramidal cell synapses play a crucial role in the hippocampal formation of spatial memories. These synaptic connections possess a number of unique features substantial for its role in the information processing and coding. One of these features is presence of zinc co-localized with glutamate within a subpopulation of synaptic vesicles recycling through AP3-dependent bulk endocytosis. Using Ca2+ imaging and electrophysiological recordings we investigated role of these zinc containing vesicles in the neurotransmission. In contrast to previous reports, we did not observe any significant role of vesicular zinc in the induction of large postsynaptic Ca2+ waves triggered by burst stimulation. Moreover, our experiments revealed that Ca2+ waves mediated by Ca2+ release from internal stores are dependent not only on the activation of metabotropic, but also ionotropic glutamate receptors. Nevertheless, subsequent experiments unveiled that the vesicles derived via AP3-dependent endocytosis primary contribute to the asynchronous, but not synchronous mode of glutamate release. Futhermore, knockout mice lacking adaptor protein AP3 had a reduced synchronization of postsynaptic action potentials and impaired information transfer; this was not associated with any changes in the synchronous release quantal parameters and vesicle pool size. These findings strongly support the idea that within a single presynaptic bouton two heterogeneous pools of releasable vesicles are present. One pool of readily releasable vesicles forms via clathrin mediated endocytosis and mainly participates in the synchronous release; a second pool forms through bulk endocytosis and primarily supplies asynchronous release. The existence of two specialized pools is essential for the information coding and transfer within hippocampus. It also might be important for hippocampal memory formation. In contrast to low firing rates at rest, dentate gyrus granule cells tend to fire high frequency bursts once an animal enters a place field. These burst activities, embedded in the lower gamma frequency, should be especially efficient in the triggering of substantial asynchronous glutamate release. Therefore, the results of my PhD project for the first time provide strong evidence for the presence and physiological importance of two vesicle pools with heterogeneous release and recycling properties via separate endocytic pathways within the same mossy fiber bouton