Elucidation of Nanostructure and Function of Spontaneous GABAergic Transmission at the Inhibitory Synapse

项目摘要 在兴奋性突触处，异质分子布局产生精确的突触前和突触后神经元。 促进不同神经传递模式的结构。神经传递的空间分离 导致自发性谷氨酸释放的自主功能。的调查 兴奋性突触的结构/功能关系促进了疾病通路的发现 和精神疾病干预的目标。然而，异常的抑制性神经传递也是 与许多精神疾病有关，包括精神分裂症、抑郁症和焦虑症。尽管 抑制性神经传递在疾病中的重要性，很少有研究调查 纳米结构和抑制性突触功能之间的关系。我的初步数据显示 动作电位依赖性和自发性神经传递在中枢抑制区的分离 GABA能突触，但这种分离是如何实现的是未知的。很少有研究调查过 抑制结构/功能关系，由于有限的工具，允许选择性操纵 不同的抑制性神经传递模式。我已经生成了初步数据，一个新的小 分子药物，青蒿素，选择性失调抑制自发释放，因为它竞争性结合 在GABAAR-桥蛋白结合口袋中;提供了一种新的药理学工具， 这个提议。本课题将从两个方面研究GABA能突触，阐明突触后的 分离神经传递的结构，以及这种结构如何导致神经传递的自主功能。 稳态可塑性中自发GABA能传递在原代海马培养中，青蒿素 将被用作减少GABA能自发释放的工具，以研究这如何对应于 纳米结构和信号通路。核心假设是GABA能突触具有特定的后- 突触桥蛋白支架和GABAAR纳米结构促进自发性 神经传递目标#1将研究突触后结构如何隔离神经传递 使用超分辨率和电子显微镜来评估1A。桥蛋白成簇动力学和1b. GABAAR 亚基定位和聚类。目标#2将研究这种纳米结构的功能途径 通过描绘自发性GABA能神经传递在稳态可塑性中的作用来促进。 2a.首先将评估下游基因表达途径，特别是BDNF表达。2b.然后 自发性GABA能信号如何改变钙动力学以触发基因转录途径， 被描绘出来。本研究将阐明抑制蛋白的结构与功能之间的关系， 突触，最终提供了新的见解调节突触强度的基础 健康和疾病中的兴奋/抑制平衡。

PROJECT SUMMARY At the excitatory synapse, a heterogenous molecular layout creates a precise pre- and post-synaptic structure that facilitates different modes of neurotransmission. The spatial segregation of neurotransmission leads to the autonomous function of spontaneous glutamate release. The investigation into the structure/function relationship at the excitatory synapse has facilitated the discovery of disease pathways and targets for psychiatric disease intervention. However, aberrant inhibitory neurotransmission is also implicated in numerous psychiatric illnesses including schizophrenia, depression, and anxiety. Despite the fundamental importance of inhibitory neurotransmission in disease, few studies have investigated the relationship between nanostructure and function at the inhibitory synapse. My preliminary data suggests a segregation of action potential dependent and spontaneous neurotransmission at central inhibitory GABAergic synapses, but how this segregation is achieved is unknown. Few studies have investigated the inhibitory structure/function relationship due to the limited tools that allow for the selective manipulation of different modes of inhibitory neurotransmission. I have generated preliminary data that a novel small molecule drug, Artemisinin, selectively dysregulates inhibitory spontaneous release as it competitively binds in the GABAAR-gephyrin binding pocket; providing a new pharmacological tool that will be utilized throughout this proposal. This project will investigate the GABAergic synapse in two-fold by elucidating the post-synaptic structure that segregates neurotransmission and how this structure leads to an autonomous function of spontaneous GABAergic transmission in homeostatic plasticity. In primary hippocampal culture, Artemisinin will be used as a tool to decrease GABAergic spontaneous release to investigate how this corresponds to nanostructure and signaling pathways. The central hypothesis is GABAergic synapses have a specific post- synaptic gephyrin scaffold and GABAAR nanostructure that facilitates an autonomous role of spontaneous neurotransmission. Aim#1 will investigate how the post-synaptic structure segregates neurotransmission using super resolution and electron microscopy to assess 1a. gephyrin clustering dynamics and 1b. GABAAR subunit localization and clustering. Aim#2 will investigate the functional pathway this nanostructure facilitates by delineating the role of spontaneous GABAergic neurotransmission in homeostatic plasticity. 2a. First downstream gene expression pathways will be assessed, specifically BDNF expression. 2b. Then how spontaneous GABAergic signaling alters calcium dynamics to trigger gene transcription pathways will be delineated. This research will elucidate the relationship between structure and function at the inhibitory synapse, ultimately providing novel insight into the regulation of synaptic strength underlying excitatory/inhibitory balance in health and disease.