Determining the ultrastructural differences between dually and singly innervated dendritic spines and their changes following glutamate excitotoxicity using Cryo-Electron Tomography

项目总结 谷氨酸兴奋毒性导致多种年龄相关神经病的神经细胞凋亡和坏死 中风和阿尔茨海默氏症等疾病。大剂量谷氨酸刺激神经元引起迅速的 树突棘(DS)的丧失，即从树突上发芽的膜性突起。DS对于学习和学习至关重要 记忆，但导致这种损失的结构变化仍然知之甚少，因为它们的体积很小。向上 到10%的DS由抑制性突触(DIDs)双重支配，并被发现比单一DIDs更稳定 仅含兴奋性突触的神经支配的DS(SID)。一种称为分隔化的过程也是 被认为是DS稳定性的关键，通过成熟的大头和窄颈的脊椎来限制分子 以及扩散进入和流出树枝晶的离子。最近的证据表明，肌动蛋白在体内存在扩散障碍 DS颈和头颈部交界处可能是分区的关键，但这仍然很差。 明白了。兴奋后钙内流导致肌动蛋白网络重构，从而驱动DS的形态 变化。DDS上的抑制性突触被发现抑制兴奋性突触后电位和 钙离子内流，且86%以上含有脊器。基于这些发现，我假设 在谷氨酸兴奋毒性后，DIDS保持了比SID更稳定的DS结构。为了调查我 将使用高分辨率冷冻电子断层扫描和相关荧光来比较DDS和 并阐明谷氨酸兴奋毒性后导致DS丢失的结构变化。目标1将 确定正常情况下DIDs和SIDs肌动蛋白网络的超微结构差异。目标 2将确定谷氨酸后DDS和SID之间发生的超微结构变化 兴奋性毒性。如果成功，这个项目将确定存在哪些高分辨率的结构性差异 DIDS和SID之间的关系以及兴奋性休克后DIDS是否更稳定。这也将是 为兴奋性毒性的研究人员提供高分辨率结构证据，解释为什么抑制性突触 预防神经元损伤的治疗靶点。

PROJECT SUMMARY Glutamate excitotoxicity causes neuronal apoptosis and necrosis in a myriad of age-associated neurologic conditions such as stroke and Alzheimer’s disease. High dose glutamate stimulation of neurons causes a rapid loss of dendritic spines (DS), membranous protrusions that bud off dendrites. DS are critical for learning and memory, but the structural changes that result in this loss remain poorly understood due to their small size. Up to 10% of DS are dually innervated with inhibitory synapses (DiDS) and found to be more stable than singly innervated DS (SiDS) containing only an excitatory synapse. A process termed compartmentalization is also considered key to DS stability, whereby mature spines with large heads and narrow necks restrict molecules and ions from diffusion into and out of the dendrite. Recent evidence suggests an actin diffusion barrier within the DS neck and head-neck junction could be key to compartmentalization, but this remains poorly understood. Following excitation calcium influx causes actin network remodeling that drives DS morphologic change. Inhibitory synapses on DiDS have been found to dampen excitatory post synaptic potentials and calcium influx, and upwards of 86% contain a spine apparatus. Based on these findings, I hypothesize following glutamate excitotoxicity DiDS maintain a more stable DS structure than SiDS. To investigate I will use high resolution cryo-electron tomography paired with correlative fluorescence to compare DiDS and SiDS and elucidate structural changes that result in DS loss following glutamate excitotoxicity. Aim 1 will determine the ultrastructural differences between DiDS and SiDS actin networks under normal conditions. Aim 2 will determine the ultrastructural changes that occur between DiDS and SiDS following glutamate excitotoxicity. If successful, this project would determine what high resolution structural differences exist between DiDS and SiDS and whether DiDS are more stable following excitotoxic shock. This would also provide investigators of excitotoxicity high resolution structural evidence for why inhibitory synapses could be therapeutic targets to prevent neuronal injury.