In vivo analysis of astroctye-neuron dynamics in circuit formation, function, and maintenance

项目总结 哺乳动物的大脑是由数十亿个神经元组成的，这些神经元通过特殊的化学连接进行交流。 叫做突触。单个神经元相互连接，形成正常学习所需的功能电路 和记忆，以及神经元电路的中断是患者经历的衰弱症状的基础 患有癫痫和精神分裂症等神经系统疾病。虽然适当的阵型和 神经元回路的维持对于高质量的人类生活是必不可少的，通过这个过程，给定的 神经元找到正确的突触对，以及这些突触是如何随着时间的推移而维持和修改的 人们对此知之甚少。我们实验室和其他实验室最近的工作已经确定了星形胶质细胞，这是最丰富的中枢神经系统 神经胶质细胞类型，作为突触发育的主要调节因子。星形胶质细胞既支持突触生成(例如，丢失 星形胶质细胞导致突触生成减少)以及反突触生成(例如，星形胶质细胞吞噬和修剪 突触)。星形胶质细胞在调节突触数量方面的这些重要功能表明，星形胶质细胞可能 调控更广泛的电路形成，尽管这一假说尚未得到充分研究。 行为相关回路中星形胶质细胞-神经元动力学的特征尚未被 进行，可能是因为它需要在体内操纵一对确定的突触耦合神经元 以及相关的星形胶质细胞。考虑到哺乳动物神经系统的巨大复杂性，这些类型 在哺乳动物身上进行实验还不可行。令人兴奋的是，现在可以在 果蝇神经系统由于星形胶质细胞操纵工具的最新发展 弗里曼实验室，以及在能源部实验室中控制幼虫运动的神经回路的鉴定。作为联合导师 在能源部和弗里曼实验室的博士后研究员，我将合并这些新工具，以拥有独特的 可视化和基因操作单个中央突触的能力，我将与之结合 靶向操纵相关星形胶质细胞以确定星形胶质细胞在突触形成中的作用， 维护和功能。对于所有的研究，我将使用最近识别的转基因品系，这些转基因品系被标记为 突触对：E2和SA1中间神经元之间的兴奋性胆碱能突触，以及抑制性突触， A31k中间神经元与RP2运动神经元之间的GABA能突触。星形胶质细胞将使用 抗Gat免疫荧光或在Alrm-GAL4作用下表达UAS-MYR：：天青素。 在我的第一个目标中，我将结合星形胶质细胞消融实验和突变分析来测试 功能性星形胶质细胞在兴奋性和抑制性突触的发展(形成)中。在我的第二个目标中，我 将使用光遗传策略来测量兴奋性和抑制性突触的活动(功能) 对星形胶质细胞功能变化的反应。最后，在我的第三个目标中，我将操纵神经元活动(通过 已定义的突触前神经元的结构性激活或沉默)，并检验神经元 活动影响星形胶质细胞的形态和功能。总而言之，这些实验将定义体内的 星形胶质细胞在兴奋性和抑制性突触的形成、功能和维持中的作用 与行为相关的感觉运动回路。

PROJECT SUMMARY The mammalian brain is formed by billions of neurons which communicate at specialized chemical junctions called synapses. Individual neurons connect to form functional circuits which are required for proper learning and memory, and disruption of neuronal circuitry underlies the debilitating symptoms experienced by patients suffering from neurological disorders such as epilepsy and schizophrenia. Although proper formation and maintenance of neuronal circuits is essential for a high quality of human life, the process by which a given neuron finds the correct synaptic pair, and how these synapses are maintained and modified over time is poorly understood. Recent work from our labs and others have identified astrocytes, the most abundant CNS glial cell type, as a major regulator of synaptic development. Astrocytes are both pro-synaptogenic (e.g. loss of astrocytes results in decreased synaptogenesis) as well as anti-synaptogenic (e.g. astrocytes engulf and prune synapses). These important functions of astrocytes in regulating synapse number suggest that astrocytes may regulate broader circuit formation, though this hypothesis has not been fully investigated. Characterization of astrocyte-neuron dynamics within a behaviorally-relevant circuit has not been performed, probably because it requires in vivo manipulation of a defined pair of synaptically-coupled neurons and the associated astrocytes. Given the enormous complexity of the mammalian nervous system, these types of experiments are not yet feasible in mammals. Excitingly, it is now possible to perform these studies in the Drosophila nervous system due to the recent development of tools for astrocyte manipulation from the Freeman lab, and identification of neural circuits governing larval locomotion in the Doe lab. As a co-mentored postdoctoral fellow within the Doe and Freeman laboratories, I will merge these new tools to have the unique ability to visualize and genetically manipulate individual central synapses, which I will couple with targeted manipulation of the associated astrocytes to define the role of astrocytes in synapse formation, maintenance, and function. For all studies, I will use recently identified transgenic lines that label defined synaptic pairs: the excitatory cholinergic synapses between E2 and SA1 interneurons, and the inhibitory, GABAergic synapses between A31k interneuron and RP2 motor neuron. Astrocytes will be visualized using anti-Gat immunofluorescence or expression of UAS-myr::Cerulean under alrm-GAL4. In my first aim, I will couple astrocyte ablation experiments with mutant analyses to test the necessity of functional astrocytes in the development (formation) of excitatory and inhibitory synapses. In my second aim, I will use an optogenetic strategy to measure the activity (function) of excitatory and inhibitory synapses in response to changes in astrocyte function. Finally, in my third aim, I will manipulate neuronal activity (through constitutive activation or silencing of defined pre-synaptic neurons) and test the hypothesis that neuronal activity influences both astrocyte morphology and function. In sum, these experiments will define the in vivo role of astrocytes in the formation, function, and maintenances of excitatory and inhibitory synapses within a behaviorally-relevant, sensorimotor circuit.