化学シナプスにおいて、シナプス小胞内に蓄えられた神経伝達物質の開口放出によってシナプス伝達が起こる。開口放出後、シナプス小胞はエンドサイトーシスによって細胞内へ回収されて再利用される。膜容量測定法とpHイメージングを併用して、開口放出とエンドサイトーシスに伴う小胞膜および小胞タンパク質の動態を同時測定した。Ca2+-カルモジュリン-Munc13シグナリングが小胞タンパク質の回収に関与していることが示唆された。 / Neurotransmitter is released at synapses by fusion of synaptic vesicles with the plasma membrane. To sustain synaptic transmission, membranes and vesicular proteins has to be retrieved for reuse. I have combined capacitance measurements and pH-imaging via a pH-sensitive vesicular protein marker, and compared the retrieval kinetics of membranes and synaptotagmin2 (Syt2) at the calyx of Held presynaptic terminal. Data in this thesis identifies a novel mechanism of stimulus- and Ca2+-dependent regulation of coordinated endocytosis of synaptic membranes and Syt2. / 博士(理学) / Doctor of Philosophy in Science / 同志社大学 / Doshisha University

Synaptic vesicle recycling is the reuse of synaptic membrane material and proteins after vesicles have been exocytosed at the pre-synaptic terminal of a neuronal synapse. The discovery of the mechanisms by which recycling operates is a subject of active research. Within small mammalian central nervous system nerve terminals, two studied mechanisms of recovery are clathrin-mediated endocytosis and activity-dependent bulk endocytosis. Research into the comparative kinetics and mechanisms underlying these endocytosis mechanisms commonly involves time-series fluorescence microscopy of in vitro cultures. Synaptic proteins are tagged with fluorescent markers, or the synaptic vesicles are labelled with fluorescent dye. The change in fluorescence levels of individual synapses over time in response to stimuli is used to understand synaptic activity. The image analysis of these time-series images frequently requires substantial manual effort to extract the changing synaptic fluorescence intensity levels over time. This work focusses on two closely interlinked areas, the development of improved automated image analysis tools to facilitate the analysis of microscopy image data, and computational simulations to leverage the data obtained from these experiments to gain mechanistic insight into the underlying processes involved in synaptic vesicle recycling. The imaged properties of synapses within the time-series images are characterised, in terms of synapse movement during the course of an experiment. This characterisation highlights the properties which risk adding error to the extracted fluorescence intensity data, as analysis generally requires segmentation of regions of interest with fixed size and location. Where possible, protocols to optimise the manual selection of synapses in the image are suggested. The manual selection of synapses within time-series images is a common but time consuming and difficult task. It requires considerable skill on the part of the researcher to select synapses from noisy images without introducing error or bias. Automated tools for either general image segmentation or for segmentation of synapse-like puncta do exist, but have mixed results when applied to time-series experiments. This work introduces the use of knowledge of the experiment protocol into the segmentation process. The selection of synapses as they respond to known stimuli is compared against other current segmentation methods, and tools to perform this segmentation are provided. This use of synapse activity improves the quality of the segmented set of synapses over existing segmentation tools. Finally, this work builds a number of computational models, to allow published individual data points to be aggregated into a coherent view of overall synaptic vesicle recycling. The first is FM-Sim, a stochastic hybrid model of overall synapse recycling as is expected to occur during the course of an experiment. This closed system model handles the processes of exocytosis and endocytosis. It uses Bayesian inference to fit model parameters to experimental data. In particular, it uses the experimental protocol to separate the mechanisms and rates that may contribute to the observed experimental data. The second is a mathematical model of one aspect of synaptic vesicle recycling of particular interest - homoeostasis of plasma membrane integrity on the presynaptic terminal. This model provides bounds on efficiency of the studied endocytosis mechanisms at recovery of plasma membrane area during and after neuronal stimulus. Both the image analysis and the computational simulations demonstrated in this work provide useful tools and insights into current research of synaptic vesicle recycling and the role of activity-dependent bulk endocytosis. In particular, the utility of adding time-dependent experimental protocol knowledge to both the image analysis tools and the computational simulations is shown.

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Les vésicules synaptiques (VSs) sont essentielles pour la neurotransmission. Les recherches actuelles se focalisent sur la caractérisation de leur contenu en neurotransmetteurs, leur cinétique de libération, leur distribution et leur mobilité. Les VS ne sont pas présentes exclusievement en paquet dans les boutons présynaptiques mais sont echangées de façon dynamique avec le reste de l’axone dans un super-contingent (super-pool). Notre laboratoire a précédement montré que le transporteur vésiculaire de glutamate de type 1 (VGLUT1) jouerait un rôle dans la régulation du super-pool. Mon projet de thèse se focalise sur la mobilité des VS dans les axones. En premier lieu, j’ai généré une souris gain de fonction VGLUT1mEos2 afin d'étudier la mobilité des VSs et de mieux caractériser le super-pool. Ensuite j’ai engagé une étude des relation entre la structure de VGLUT1 et ses fonctions afin d’identifier les signatures moléculaires responsable de la régulation de la taille du super-pool. J’ai identifié le second motif poly-proline à l’extremité C-terminale de VGLUT1 comme étant nécessaire et suffisante pour induire une diminution de la taille du super-pool des VSs. Pour conclure mes travaux de thèse ont contribué à la compréhension du rôle de VGLUT1 dans la régulation de la mobilité des VSs et à fournir les outils nécessaires pour de futures investigations concernant la physiologie du super-pool. / Synaptic vesicles (SVs) are essential for neurotransmission, and more efforts are needed for better understanding their neurotransmitter content, release kinetics, distribution and mobility. SVs are not only clustered in presynaptic boutons, but also dynamically shared among multiple en passant presynaptic boutons, a phenomenon named SV super‐pool. Previous work from our laboratory suggested that the Vesicular GLUtamate Transporter 1 (VGLUT1) may play a role in regulating SV super-­pool size beyond loading glutamate into SV. My Ph.D project is focused on SVs mobility in axons. Firstly, I generated a VGLUT1mEos2 knock-in (KI) mouse line, which provides extended possibilities to study the SV trafficking and characterize SV super‐pool. Secondly, I engaged in a thorough VGLUT1 structure‐function analysis. I identified that VGLUT1 tends to cluster SVs in the presynaptic boutons and reduce SVs exchange with the super‐pool via the second poly‐proline motif of its C­‐terminus. Overall, my Ph.D work contributes to the knowledge of the role of VGLUT1 in regulating SVs mobility and provides new tools for the further investigations on SV super-­pool physiology.

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