Dynamic regulation of synaptic Ca2+ channel organization

项目摘要 突触前Ca 2+驱动数万亿个化学突触的神经传递， 神经系统中的交流。通过电压门控Ca 2+通道的Ca 2+内流引起局部Ca 2+内流， 胞内Ca 2+，其结合突触囊泡融合机制上的Ca 2+传感器，导致囊泡融合 以及神经递质在称为活动区的突触专门区域的释放。丰度和 Ca 2+通道的精确定位对突触功能有着深远的影响，然而， Ca 2+通道的组织和突触功能一直难以研究。果蝇的神经肌肉 连接（NMJ）提供了一个有吸引力的模型，研究这个问题，在内源性突触，让我们 比较两种相关运动神经元钙通道的组成、组织和动态调节 不同亚型的神经递质释放特性非常不同。在目标1中，我将研究Ca 2+的作用 通道辅助亚基和纳米级组织在建立突触特异性释放特性中的作用 使用CRISPR基因编辑，超分辨率成像和电子显微镜。 突触必须是可靠的，但也有可塑性，以适应动态环境的反应。突触前 稳态增强（PHP）是维持有效神经通讯的保守机制 在动态范围内通过突触释放概率的增加。我们实验室以前的工作 显示诱导PHP的操作导致电压门控Ca 2+通道快速募集， 区域，但新的频道如何被贩运到活跃的区域并在活跃的区域进行组织仍然是未知的。在目标2中，我将 使用遗传学和药理学来研究Ca 2+通道运输的细胞机制， 插入膜中，并聚集以促进稳态期间释放性质的变化， 可塑性。 这项研究将揭示钙离子通道是如何差异和动态调节，以实现和 保持突触特异性释放特性，并推进我们对神经系统中通信的理解。 系统 通过完成这些目标，申请人将获得细胞神经生物学方面的科学和技术专长， 神经遗传学、CRISPR基因编辑、超分辨率成像和电生理学。通过 全面的培训计划，该奖学金将支持申请人的专业发展， 实验设计和数据分析;书面和口头科学交流;有效和包容 指导。成功完成研究和培训目标得到了互动和 布朗大学和神经科学研究生课程的支持性制度环境，并将 为申请人迈向独立科学事业的下一步做好准备。

PROJECT SUMMARY Presynaptic Ca2+ drives neurotransmission at the trillions of chemical synapses that mediate most communication in the nervous system. Ca2+ influx through voltage-gated Ca2+ channels raises localized intracellular Ca2+, which binds to Ca2+ sensors on synaptic vesicle fusion machinery, resulting in vesicle fusion and release of neurotransmitter at specialized areas of synapses called active zones. The abundance and precise location of Ca2+ channels have a profound impact on synapse function, yet the relationship between Ca2+ channel organization and synaptic function has been difficult to study. The Drosophila neuromuscular junction (NMJ) provides an attractive model for studying this question at endogenous synapses by allowing us to compare Ca2+ channel composition, organization and dynamic regulation at two related motor neuron subtypes with very different neurotransmitter release properties. In Aim 1, I will investigate the role of Ca2+ channel auxiliary subunits and nanoscale organization in establishing synapse-specific release properties using CRISPR gene editing, super-resolution imaging, and electron microscopy. Synapses must be reliable, but also malleable to adapt their responses to a dynamic environment. Presynaptic homeostatic potentiation (PHP) is a conserved mechanism for maintaining effective neural communication within a dynamic range through an increase in synaptic probability of release. Previous work from our lab has shown that manipulations to induce PHP result in a rapid recruitment of voltage-gated Ca2+ channels to active zones, but how new channels are trafficked to and organized at active zones remains unknown. In Aim 2, I will use genetics and pharmacology to investigate the cellular mechanisms by which Ca2+ channels are trafficked, inserted in the membrane, and clustered to facilitate changes in release properties during homeostatic plasticity. This research will reveal how Ca2+ channels are differentially and dynamically regulated to achieve and maintain synapse-specific release properties, and advance our understanding of communication in the nervous system. By completing these aims, the applicant will gain scientific and technical expertise in cellular neurobiology, neurogenetics, CRISPR gene editing, super-resolution imaging, and electrophysiology. Through a comprehensive training plan, this fellowship will support the professional development of the applicant in robust experimental design and data analysis; written and oral scientific communication; and effective and inclusive mentoring. Successful completion of the research and training goals are fully supported by the interactive and supportive institutional environment of Brown University and in the Neuroscience Graduate Program, and will prepare the applicant for the next steps towards an independent scientific career.