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Mécanismes moléculaires de la stabilisation synaptique des récepteurs du glutamate de type kaïnate dans les cellules pyramidales de CA3 / Molecular mechanisms for the synaptic stabilization of kainate receptors in CA3 pyramidal cellsFievre, Sabine 19 November 2015 (has links)
Les récepteurs ionotropiques du glutamate peuvent être compartimentés de manière très spécifique au niveau des différentes afférences synaptiques d’un neurone. Dans les neurones pyramidaux de CA3, les récepteurs de type kaïnate (rKA) post-synaptiques sont localisés à la synapse formée entre les fibres moussues et les cellules pyramidales de CA3 (synapse FM-CA3) mais ils sont totalement absents des autres afférences glutamatergiques sur ce même neurone. Nous avons cherché à comprendre les mécanismes moléculaires de cette compartimentation subcellulaire. En réalisant une cartographie fonctionnelle des récepteurs du glutamate par décageage focalisé de glutamate dans les cellules pyramidales de CA3, nous avons montré que les rKA présentent une localisation subcellulaire strictement confinée dans les excroissances épineuses, éléments post-synaptiques des synapses FM-CA3, et sont exclus des compartiments somato-dendritiques, contrairement aux récepteurs AMPA. Nous avons identifié une séquence du domaine C-terminal de GluK2a nécessaire pour la stabilisation des rKA. Cette séquence est responsable d’une interaction avec la protéine d’adhérence N-cadhérine. L’altération de la fonction de la N-cadhérine dans les cellules pyramidales de CA3 entraine une déstabilisation des rKA à la synapse FM-CA3. Ces travaux suggèrent que plusieurs mécanismes participent à la compartimentation des rKA à la synapse FMCA3 impliquant le recrutement et la stabilisation des rKA par les N-cadhérines. / Distinct subtypes of ionotropic glutamate receptors can be segregate to specific synaptic inputs in a given neuron. In CA3 pyramidal cells (PCs), kainate receptors (KARs) are present at mossy fiber (mf) synapses and absent from other glutamatergic inputs. The mechanisms for such a constrained subcellular segregation is not known. We have investigated the molecular determinants responsible for the subcellular segregation of KARs at mf-CA3 synapses. Using functional mapping of glutamate receptors by focal glutamate uncaging we show that KARs display a strictly confined expression on thorny excrescences, the postsynaptic elements of mf-CA3 synapses, being excluded from extrasynaptic somatodendritic compartments, at variance with AMPA receptors. We have identified a sequence in the GluK2a C-terminal domain necessary for restricted expression of KARs which is responsible for GluK2a interaction with N-Cadherin. Targeted deletion of N-Cadherin or overexpression of a dominant negative N-Cadherin in CA3 PCs greatly induce a destabilization of KARs at the mf-CA3 synapses. Our findings suggest that multiple mechanisms combine to control the compartmentalization of KARs at mf-CA3 synapses, including a stringent control of the amount of GluK2 subunit in CA3 PCs, a limited number of slots for KARs, and the recruitment/stabilization of KARs by N-Cadherins.
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Investigating the role of the hippocampal formation in episodic and spatial memoryStevenson, Cassie Hayley January 2011 (has links)
This thesis aims to explore the two dominant functional roles of the hippocampal formation, in the relational encoding of episodic memory and the neural representation of allocentric space, using a combination of pharmaceutical manipulations and single-unit recording techniques in rodents. The first part of this thesis focuses on episodic-like memory, defined by the original episodic memory triad: ‘what-where-when’ (Tulving 1972), which enables the behavioural aspects of episodic memory to be tested in non-human animals. Permanent neurotoxic lesions of the hippocampus and it’s subregions were induced to assess their role in a putative episodic-like memory task developed by Eacott and Norman (2004). In view of the difficulties encountered in successfully demonstrating the temporal component of episodic-like memory in rats, this task tested integrated memory for ‘what-where-which’, where the temporal component (when) was replaced with another event specifier: context (on ‘which’ occasion). Disruption of the hippocampal circuitry led to a specific impairment in the integration of all three event components, whereas the associative recognition of any combination of these features in isolation was left intact. These results confirm the hippocampal dependence of this episodic-like memory task and further reveals the necessity of both CA3 and CA1, hypothetically due to the underlying autoassociative role of CA3 with CA1 functioning as the vital output pathway for this associated information and/or as a mismatch detector. There has been much debate over the inclusion of the temporal component and sceptics may argue that any such interpretations of task-dependence on episodic-like memory processing are invalid considering the requirement for temporal processing is absent. Due to the proposal that a temporal framework necessarily provides the foundation on which episodic memories are built, the second chapter focuses on the development of a suitable protocol in which integrated memory for the original ‘what-where-when’ episodic memory triad can be reliably tested. The other main function attributed to the hippocampus was brought to light by the fascinating revelation that it’s neurons selectively fire in different regions of an environment, termed ‘place cells’ (O’Keefe and Dostrovsky 1971). From the numerous publications resulting from this discovery it has emerged that place cells not only respond to the spatial features of the environment but are also sensitive to a multitude of non-spatial features. These characteristics support the logical assumption that the primary firing patterns of the hippocampus should underlie it’s main purported roles, leading to speculations that they reflect episodic memory processes. The second part of this thesis aims to examine the relationship between hippocampal cells and behaviour by extending the work of Ainge et al. (2007a), in which a subset of hippocampal place cells were found to encode both current and intended destination in a double Y-maze ‘win-stay’ task. The development of these ‘goal-sensitive’ cells were initially investigated during the learning phase of this task. An exciting pattern of results showed a strong positive correlation between the emergence of goal-sensitive firing and behavioural performance on the task, tempting speculations that these firing patterns may underlie spatial learning and future planning, necessary to support performance. To ensure these firing patterns were not a mere reflection of greater experience on the maze, a second study was conducted in which the task demands changed over set periods of days. A significant increase in the proportion of cells demonstrating goal-sensitive firing was revealed when the protocol shifted to incorporate the spatial memory demands of the ‘win-stay’ task, with all other parameters of the protocol remaining constant. These results support the theory that goal-sensitive firing patterns are specifically related to the learning and memory demands of the spatial task, not a result of increased exploration of the maze. The last of this series of studies assessed hippocampal-dependence of this task and revealed that bilateral hippocampal lesions induced an impairment in spatial ‘win-stay’ performance. Collectively, these experiments demonstrate that goal-sensitive firing of hippocampal cells emerge in line with behavioural performance in a hippocampal-dependent task and the emergence of these firing patterns are specific to the learning and memory demands of a spatial ‘win-stay’ protocol. The functional role of the hippocampus in allocentric spatial processing may thus underpin it’s function in episodic memory and potentially in the imagining and planning of future events, whereby the hippocampus provides a ‘space’ in which retrieved information can be integrated in a coherent context to support the fluent and flexible use of information. This hippocampal function would necessarily require visual information to be accessed, concerning the arrangement of landmarks and cues within the environment, in association with information regarding internal orientation and direction and this leads to the question assessed in the final part of this thesis of where this integration occurs. Based on anatomical evidence and the current literature, the postsubiculum, an input structure to the hippocampus, emerged as a potential site for the convergence of sensory cues into the internally generated head direction cell and place cell networks to enable hippocampal-dependent spatial processing. Thus, the effects of temporary pharmacological blockade of AMPARs and NMDARs in the postsubiculum were assessed on the encoding of spatial memory in an object recognition paradigm. The impairment revealed in the ability to recognise novel object-place configurations demonstrates a key role for NMDAR-dependent plasticity within the postsubiculum itself in the formation of allocentric spatial memory. In summary, the experimental results reported in this thesis further elucidate the critical role the hippocampal formation plays in spatial and episodic memory by combining evidence from cellular physiology and neuroanatomy to the behaving animal and extends these findings to discuss a more general role for the hippocampus in imagining both past and future events, in order to successfully navigate, learn and enable past experience to influence our intended future plans and decisions.
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Envelhecimento, declínio cognitivo e plasticidade astroglial em ca3TOKUHASHI, Tatyana Pereira 18 November 2011 (has links)
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Previous issue date: 2011 / Poucos estudos tem se dedicado a investigar em detalhe possíveis relações entre o declínio cognitivo associado ao envelhecimento e a plasticidade astroglial no hipocampo. No presente trabalho investigamos possíveis relações entre o desempenho em testes de memória de reconhecimento de objeto e o numero e a distribuição laminar dos astrocitos em CA3 em modelo murino. Para isso empregamos camundongos fêmeas adultas da linhagem C57Bl6 de 6 (n = 7) e 20 meses (n = 5) de idade, mantidos em gaiolas padrão desde o nascimento,comparando seus desempenhos em tarefas hipocampo-dependentes para reconhecimento da forma, do lugar e do momento em que os objetos selecionados lhes foram expostos. Apos os testes comportamentais todos os animais foram per fundidos com fixadores aldeidicos e tiveram seus cérebros removidos e processados para imunomarcação empregando anticorpo seletivo para detecção da proteína acida fibrilar dos astrocitos (GFAP). Para evitar possível viés amostral empregamos o fracionador óptico, um método estereológico que não e afetado pelo processamento histologico. Os resultados nos testes comportamentais isolados e integrados (memória episódica) revelaram que o envelhecimento compromete significativamente (teste T bi-caudal, p<0.05) o reconhecimento da forma, do lugar e do momento em que objetos selecionados são apresentados aos sujeitos. As análises
estereologicas das estimativas do numero de astrocítos revelaram que o envelhecimento afetou a distribuicao laminar com aumento na proporção relativa daqueles na camada piramidal de CA3 dorsal e ventral e reducao no lacunoso molecular de CA3 dorsal. O número total de astrocitos em consequência apresentou significativa reorganização na distribuição laminar em função da idade com os animais senis mostrando redução no percentual daqueles localizados na camada oriens. Nenhuma diferenca significativa foi encontrada entre os volumes das camadas de CA3 sugerindo que as mudanças induzidas pelo envelhecimento alteram diretamente a plasticidade astroglial em CA3. Finalmente os estudos de correlação linear entre as estimativas do numero dos astrocitos da camada piramidal e os testes comportamentais demonstraram correlação inversa com os piores desempenhos estando associados a um maior número de astrocitos naquela camada. Evidencias diretas adicionais dessa correlação com os astrocitos alterados em CA3 e possíveis mecanismos moleculares para explicar o declinio cognitivo associado ao envelhecimento permanecem por ser investigados. / A few studies investigated in detail possible relationships between aging cognitive decline and hippocampal astroglial plasticity. In the present report we investigated in murine model possible relationships between performances in object recognition tests and the astrocytes laminar distribution in CA3. To do so, young (6 months old, n = 7) and old (20 months old, n= 5) C57Bl6 mice, were maintained in standard cages and assessed in object recognition hippocampal-dependent tasks. Isolated or integrated (episodic-like memory) tests were applied and revealed that object identity (What?), place (Where?) and time (When?), were impaired in old subjects, whereas in young mice only spatial memory was impaired. After behavioral tests all subjects were sacrificed and perfused with aldehyde fixatives had their
brains removed and processed for glial fibrillary acid protein (GFAP) immunohistochemistry, a selective marker for astrocytes. To avoid sample bias we used the optical fractionator, a stereological method that is no affected by histological procedures. The results on behavioral isolated or integrated tests revealed that aging significantly impairs object, spatial and time recognition (two-tail t-test, p<0.05). As compared to young subjects, old mice showed laminar changes in the astrocytes distribution with proportional increase of the astrocytes number in the pyramidal layer of dorsal and ventral CA3 and a reduction in the lacunosum molecular layer of dorsal CA3. Coherently, the total number of CA3 astrocytes showed significant
reorganization of its laminar distribution as a function of age with reduction of its numbers in the stratum oriens. No significant differences were detected in the mean values of laminar volumes suggesting that aging induced changes directly affected astroglial plasticity in CA3. Finally, a linear inverse correlation was found between the estimations of pyramidal cell layer astrocytes and performances in the behavioral tasks. Further direct evidences of this correlation with altered CA3 astrocytes and possible molecular mechanisms to explain aging cognitive decline remains to be investigated.
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The presynaptic protein Mover buffers synaptic plasticity at the hippocampal mossy fiber synapseViotti, Julio Santos 21 November 2017 (has links)
No description available.
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Direito ou esquerdo? Avaliação da lateralização funcional do hipocampo dorsal na modulação da memória de reconhecimento e espacial de ratos Wistar /Pimentel, Gabrielle Araujo January 2020 (has links)
Orientador: Luiz Henrique Florindo / Resumo: Atualmente é bem estabelecido que o hipocampo (HPC) possui um papel importante nos processamentos de aprendizagem e memória. No entanto, existem controvérsias sobre as funções das sub-regiões do Corno de Amon (CA) do HPC propriamente dito e em relação à lateralização funcional dessa estrutura. A partir disso, a função do hipocampo dorsal (HPCd) na memória de reconhecimento e espacial (recente e remota), foi analisada através da inativação direita, esquerda ou bilateral da área CA3. Foram utilizados 37 ratos Wistar distribuídos em quatro grupos: grupo GVe (n=8), que receberam injeção bilateral de tampão fosfato-salina (PBS - veículo) na região CA3 do HPCd; grupo HPCd-D (n=9), que receberam injeção do lesionador ácido ibotênico (IBO) na região do hemisfério direito; grupo HPCd-E (n=10), que receberam injeção de IBO na região do hemisfério esquerdo; e grupo HPCd-BI (n=10), que receberam injeção bilateral de IBO na região de ambos HPCd. Os animais foram submetidos ao labirinto aquático de Morris (LAM), teste de reconhecimento de objetos (TRO) e labirinto em T forçado. Os dados foram submetidos ao teste de homogeneidade de Shapiro-Wilk, seguido de análise de variância (ANOVA), e pelo teste de Tukey para dados paramétricos, ou pelo teste de KruskalWallis seguido de teste de Dunn para dados não paramétricos. Foi admitido nível de significância para p<0,05. Nenhum dos animais apresentou comprometimento para realização de comportamentos exploratórios. Não houve diferenças entre os ani... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Currently the hippocampus (HPC) has a well established role in learning and memory processing. However, there are controversies about the functions of sub-regions of the Corno de Amon (CA) of the HPC itself and in relation to the functional lateralization of this structure. From this, the function of the dorsal hippocampus (dHPC) in the recognition and spatial memory (recent and remote), was analyzed through the right, left or bilateral inactivation of the CA3 area. Thirty seven Wistar rats were distributed in four groups: group GVe (n = 8), with animals received bilateral injection of phosphate-saline buffer (PBS - vehicle) in the CA3 region of dHPC; group dHPC-R (n = 9), with animals received an injection of the ibotenic acid injury (IBO) in the right hemisphere region; group dHPC-L (n = 10), with animals received an IBO injection in the left hemisphere region; and group dHPC-BI, with animals received bilateral IBO injection in the region of both HPCd. The animals were submitted to the Morris water maze (MWM), object recognition test (ORT) and forced T maze. Data were by first analyzed by Shapiro-Wilk’s homogeneity test, followed by analysis of variance (ANOVA), and by Tukey’s post-test for parametric data, or by Kruskal-Wallis’s test followed by Dunn's post-test for nonparametric data. Significance level was admitted for p <0.05. None of the animals was compromised to perform exploratory behaviors. There were no differences between the animals in relation to the allocentri... (Complete abstract click electronic access below) / Mestre
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DORSAL HIPPOCAMPUS INFUSIONS OF CNQX INTO THE DENTATE GYRUS DISRUPT EXPRESSION OF TRACE FEAR CONDITIONINGPierson, Jamie L. 12 December 2012 (has links)
No description available.
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Morpho-functional impact of Vangl2 on hippocampus development / Impact morpho-fonctionnel de Vangl2 sur le développement de l’hippocampeDos Santos Carvalho, Steve Francois 30 November 2016 (has links)
La Polarité Cellulaire Planaire (PCP) est une voie de signalisation originellement identifiée chez les invertébrés pour son rôle dans l’établissement d’une asymétrie cellulaire perpendiculaire à l’axe apico‐basal. Elle définit une polarité dans le plan d’un épithélium et coordonne cette polarité dans tout l'épithélium. L'activation de la voie PCP conduit à une réorganisation ducyto squelette en passant par une modulation des zones d'adhésion, régulant ainsi la forme et les mouvements des cellules. La voie de signalisation de la PCP est conservée tout au long de l'évolution jusqu'au mammifères, et contrôle la morphogénèse de divers tissus dont les tissus épithéliaux et mésenchymateux, ainsi que pour les tissues cardiaques, osseux, pulmonaire ou encore rénaux, mais aussi le système nerveux pour n'en citer que quelques‐uns.Afin d'identifier le rôle de vangl2, un des gènes centraux de la PCP, dans la mise en place de la circuiterie hippocampale, nous avons créé un modèle murin où vangl2 est supprimé de façon conditionnelle (cKO) dans le télencéphale à des stades précoces de l’embryogénèse. J’ai d'abord montré que Vangl2 est enrichi dans les neurones immatures de la zone sous granulaire du DG, ainsi que dans l’arborisation des neurites (axones et dendrites) des cellules granulaires (CG) du gyrus denté (DG) de l’hippocampe. Ainsi, Vangl2 est enrichi dans le stratum lucidum (sl), une région dense en contacts synaptiques entre le DG et le CA3. Dans cette région a lieu une synapse très particulière entre l'axone des CG, la fibre moussue (Mf) qui forme des boutons géants (MfB) et les excroissances épineuse (TE) issues de la partie proximale des dendrites apicaux. L'analyse structurale et ultra structurale de ces épines démontre que l'élargissement et la complexification de la synapse MfB/TE est bloquée dans nos mutants, alors que les zones actives (PSD) des épines sont présentes, mais réorganisées. De façon intéressante,dans une zone plus distale des dendrites des neurones du CA3 (sl), les épines sont, elles, plus grosses, suggérant un remodelage complexe du réseau en l'absence de vangl2. Enfin, j’ai pu montrer que ces défauts morphologiques étaient corrélés à des problèmes de mémoire complexe (mémoire déclarative) qui dépendent de l’hippocampe mais aussi du cortex. Cette étude montre pour la première fois l’importance du signal PCP dans maturation in vivo d’un circuit hippocampique spécifique ainsi que ces conséquences cognitives. D'autres résultats in vitro montrent que la suppression de vangl2 augmente la vitesse de déplacement des cônes de croissance sur des substrats de N‐cadhérine. J’ai utilisé la microscopie en super résolution spt‐PALM‐TIRF pour montrer que cette augmentation de croissance est inversement proportionnelle à la vitesse du flux rétrograde d’actine. Des expériences de FRAP permettent de suggérer que les molécules de N‐cadhérine engagées dans des interactions hémophiliques (adhésion) est plus importante dans les mutants vangl2 Je propose que Vangl2 contrôle le recyclage et la stabilité des protéines N‐cadhérine dans les sites d’adhésion afin de réguler localement les dynamiques d’actine et par conséquent la croissance neuronale. / Planar Cell Polarity (PCP) is a signaling pathway originally known for its role in the establishment of cellular asymmetry perpendicular to the apico‐basal axis, in the plane of an epithelium. PCPsignaling has been shown to be crucial for many tissue patterning, including epithelial and mesenchymal tissue, but also cardiac, lung, bone, or kidney tissues, to cite a few. PCP signaling controls the regulation of cellular movement via the control of adhesion turnover and cytoskeleton reorganization. Vangl2 is one of the most upstream core PCP proteins that has been implicated in the recent years in various neuronal mechanisms, such as axonal guidance, dendrite morphogenesis or synaptogenesis. However, most of these studies rely on acute downregulation of the gene in vitro or in the use of a mouse presenting a spontaneous mutation of this gene, called Loop‐tail (Vangl2Lp) which causes the death of the embryo at birth. Moreover, the Vangl2Lp form of this protein has been described has a dominant‐negative form, making it difficult to untangle the molecular mechanism leading to the many phenotypes (included neuronal ones) reported inhomozygotes Looptail mice. To bypass this problem we created a conditional knockout (cKO) mouse in which vangl2 is deleted in the telencephalon during early embryogenesis. First, I analyzed the profile of expression of the protein during the first 3 weeks after birth, and I show that Vangl2 is specifically targeted to the arborization of granular cells (GC) of the dentate gyrus (DG) of the hippocampus, and excluded from cell bodies. Also, the protein was highly enriched in immature neurons of the subgranular zone of the DG, and in the stratum lucidum, a region of high‐density contacts between the GC and the CA3. In this region, a special type of synapse is formed: the Mossy Fiber Bouton (MfB) / Thorny Excrescence (TE) synapse. These synapses are bigger and more complex than conventional synapses. I then performed a structural and ultrastructural analysis of the DG/CA3 circuit in the Vangl2 cKO mice in order to understand the role of Vangl2 in the hippocampus maturation. For this, I used stereotaxic mice infection viruses, and Serial block face scanning electron microscopy (SBFsEM) with 3D reconstruction. Results show that in cKO mice, Mfs fasciculation is mildly impacted, and that the enlargement and complexification of the MfB/TE synapse is arrested, with TEs almost absent. I was able to link these morphological abnormalities to deficits in complex hippocampal‐dependent learning tasks. This work demonstrates for the first time the importance of PCP signaling for the in vivo maturation of a specific hippocampal circuit and its specific cognitive consequences. Next, I attempted to identify the functional consequences of vangl2 deletion on young hippocampal neuron maturation. My results confirm that Vangl2 is expressed in young hippocampal neurons and that the deletion of the gene affected neurite outgrowth on Ncadherin substrate. I used spt‐PALM‐TIRF super‐resolution microscopy to show that this increased neurite outgrowth was inversely proportional to a decrease in actin retrograde flowand to a decrease in the number of directed actin trajectories. These results strongly suggest that N‐cadherin adhesions are affected by Vangl2 deletion. FRAP experiments demonstratedthat in Vangl2 cKO neurons the recovery of N‐cadherin molecules engaged in homophilicbindings (adhesion) was decreased, suggesting that the turnover of N‐cadherin involved inadhesion is reduced. Altogether, I propose that Vangl2 controls the turnover/stability of Ncadherin proteins at adhesion sites to regulate local actin dynamics and consequently neuronal outgrowth
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NEUROPROTECTIVE EFFECTS OF POSTINJURY LITHIUM TREATMENT: DETERMINING THE OPTIMAL DOSING PARADIGM AND ASSESSING POTENTIAL MECHANISMS OF ACTIONEakin, Katharine 10 May 2010 (has links)
Traumatic brain injury (TBI) has a dramatic impact on our society in terms of mortality, morbidity, and inherently high financial costs. Formidable research efforts are being addressed to the identification of neuroprotective agents capable of ameliorating the neurological outcome after TBI. Preclinical studies have recently demonstrated lithium to be a promising neuroprotective agent for both acute ischemic brain injury and chronic neurodegenerative disease. In light of these encouraging data, we designed a lateral fluid-percussion injury (FPI) study aimed at investigating the role of early post-traumatic administration of lithium as a strategy for reducing TBI-induced motor and cognitive deficits. The optimal dose of this agent and the time window for its administration have been determined on the basis of data derived from the assessment of motor and cognitive functioning in experimental animals, as well as from the stereological quantification of neuronal survival (PID 7) within the CA3 and hilar regions of the hippocampus ipsilateral to the FPI. In addition, we attempted to elucidate the mechanisms underlying the neuroprotective properties of this drug via western blot analysis of levels of the pro-apoptotic marker caspase-3 (PID 1, 7) and two neuroplasticity markers, growth associated protein-43 (GAP-43) and brain-derived neurotrophic factor (BDNF) (PID 1, 7, 21). Our findings indicate that low-dose lithium chloride (0.125 or 0.25 mmol/kg), given either 30 min or 8 hr after lateral FPI significantly ameliorates injury-induced cognitive and motor impairment. Specifically, cell survival in the CA3 region of the hippocampus of the injured lithium-treated animals (but not in the hilus) was significantly increased compared to injured vehicle-treated animals. Western blot analyses revealed a significant increase in GAP-43 levels on PID 7 in injured animals when treated with lithium, indicating a possible mechanism for lithium-induced neuroprotection. In contrast, BDNF levels were relatively unchanged until PID 21, and caspase-3 activation was not observed at all, suggesting that these proteins play less significant roles in the observed neuroprotective effects of lithium treatment after lateral FPI. Early administration of lithium, within 8 hours after TBI, holds promise as an effective therapy to ameliorate postinjury neurobehavioral deficits and warrants further investigation in clinical TBI studies.
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Molecular mechanisms of presynaptic plasticity and function in the mammalian brainWeyrer, Christopher January 2018 (has links)
Synaptic plasticity describes efficacy changes in synaptic transmission and ranges in duration from tens to hundreds of milliseconds (short-term), to hours and days (long-term). Short-term plasticity plays crucial roles in synaptic computation, information processing, learning, working and short-term memory as well as its dysfunction in psychiatric and neurodegenerative diseases. The main aim of my PhD thesis was to determine the molecular mechanisms of different forms of presynaptic plasticity. Short-term facilitation increases neurotransmitter release in response to a high-frequency pair (paired-pulse facilitation; PPF) or train (train facilitation; TF) of presynaptic stimuli. Synaptotagmin 7 (Syt7) has been shown to act as residual calcium (Ca$_{res}$) sensor for PPF and TF at various synapses. Syt7 also seems to be involved in recovery from depression, whereas its role in neurotransmission remains controversial. My aim was to express Syt7 in a synapse where it is not normally found and determine how it affects short-term synaptic plasticity. Immunohistochemistry indicated that Syt7 is not localized to cerebellar climbing fibers (CFs). Wild-type (WT) and Syt7 knockout (KO) recordings at CF to Purkinje cell (CF-PC) synapses established that at near-physiological external calcium (Ca$_{ext}$) levels both genotypes displayed similar recovery from paired-pulse depression. In low Ca$_{ext}$,WT CF-PC synapses showed robust PPF, which turned out to be independent of Syt7. All my experiments strongly suggested that WT CFs do not express native Syt7, but display low Ca$_{ext}$ CF-PC PPF and TF. Thus, channelrhodopsin-2 and Syt7 were bicistronically expressed via AAV9 virus in CFs. This ectopic Syt7 expression in CFs led to big increases in low-Ca$_{ext}$ CF-PC facilitation, more than doubling PPF and more than tripling TF. While overexpression of Syt7 might turn out to have an effect on the initial release probability (pr), the observed CF-PC facilitation increase still critically depended on presynaptic Syt7 expression. And when comparing only cells in a defined EPSC1 amplitude range, the Syt7-induced increase in low-Ca$_{ext}$ PPF could not be accounted for by changes in initial pr, suggesting a general role for Syt7 as calcium sensor for facilitation. Another form of short-term plasticity, post-tetanic potentiation (PTP), is believed to be mediated presynaptically by calcium-dependent protein kinase C (PKC) isoforms that phosphorylate Munc18-1 proteins. It is unknown how generally applicable this mechanism is throughout the brain and if other proteins might be able to modulate PTP. Combining genetic (PKCαβy triple knockout [TKO] and Munc18-1SA knock-in [Munc18 KI] mice, in which Munc18- 1 cannot get phosphorylated) with pharmacological tools (PKC inhibitor GF109203), helped us show that PTP at the cerebellar parallel fiber to Purkinje cell (PF-PC) synapse seems to depend on PKCs but seems mostly independent of Munc18-1 phosphorylation. In addition, compared to WT animals, genetic elimination of presynaptic active zone protein Liprin-α3 led to similar PF-PC PTP and paired-pulse ratios (PPRs). At the hippocampal CA3-CA1 synapse previous pharmacological studies suggested that PKC mediates PTP. A genetic approach helped to show that calcium dependent PKCs do not seem to be required for CA3-CA1 PTP. Pharmacologically inhibiting protein kinase A as well as genetically eliminating Syt7 also had no effect on CA3-CA1 PTP. In addition, Ca IM-AA mutant mice, in which Ca$_{v}$2.1 channels have a mutated IQ-like motif (IM) so that it cannot get bound by calcium sensor proteins any more, not only displayed regular PTP, but also normal PPF and TF at CA3-CA1 synapses. In conclusion, my PhD thesis helped further characterize different forms of presynaptic plasticity, underlined that short-term synaptic plasticity can be achieved through diverse mechanisms across the Mammalian brain and supported a potentially general role for synaptotagmin 7 acting as residual calcium sensor for facilitation.
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