Dynamic regulation of synaptic Ca2+ channel organization

项目总结 突触前钙离子在数万亿个化学突触上驱动神经传递，这些突触在 神经系统中的交流。通过电压门控钙离子通道的钙离子内流提高了局部性 细胞内钙离子，它与突触小泡融合机制上的钙传感器结合，导致小泡融合 并在突触的特定区域释放神经递质，称为活动区。富足和 钙通道的准确定位对突触功能有深远的影响，但两者之间的关系 钙离子通道的组织和突触功能一直很难研究。果蝇的神经肌肉 连接(NMJ)为研究内源性突触的这一问题提供了一个有吸引力的模型，它允许我们 两种相关运动神经元钙离子通道组成、组织及动态调节的比较 具有非常不同的神经递质释放特性的亚型。在目标1中，我将研究钙离子的作用 建立突触特异性释放特性中的通道辅助亚单位和纳米级组织 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，但也具有可塑性，以适应动态环境的反应。突触前 内稳态增强(Php)是维持有效神经通讯的一种保守机制 在一个动态范围内，通过增加突触释放的概率。我们实验室以前的工作是 结果显示，诱导PHP的操作导致电压门控钙通道迅速重新激活 但是，新的频道如何被传输到活动区并在活动区组织起来仍然是未知的。在《目标2》中，我会 利用遗传学和药理学来研究钙离子通道的细胞机制， 插入到膜中，并聚集以促进在动态平衡期间释放特性的变化 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的，以实现和 维持突触特有的释放特性，促进我们对神经中枢交流的理解 系统。 通过完成这些目标，申请者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。通过一个 全面的培训计划，该奖学金将支持申请者在稳健的专业发展 实验设计和数据分析；书面和口头科学交流；有效和包容 辅导。成功完成研究和培训目标得到了互动和培训的充分支持 布朗大学和神经科学研究生项目的支持性制度环境，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.