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

项目摘要 谷氨酸兴奋性毒性可导致大量与年龄相关的神经系统疾病中的神经元凋亡和坏死 中风和老年痴呆症等疾病。高剂量谷氨酸刺激神经元引起快速的 树突棘（DS）缺失，从树突上出芽的膜状突起。DS对学习至关重要， 记忆，但导致这种损失的结构变化仍然知之甚少，由于它们的小尺寸。起来 到10%的DS与抑制性突触（DiDS）双重支配，并且发现比单独支配更稳定。 受神经支配的DS（SiDS）仅含有兴奋性突触。一个被称为划分的过程也是 被认为是DS稳定性的关键，因此具有大头部和窄颈部的成熟棘限制分子 以及离子从枝晶扩散进出。最近的证据表明，肌动蛋白扩散屏障内 DS颈和头颈连接可能是区室化的关键，但这仍然很差 明白兴奋后钙离子内流引起肌动蛋白网络重塑，驱动DS形态学改变， 变化已发现对DiDS的抑制性突触抑制兴奋性突触后电位， 钙内流，86%以上含有脊椎器官。基于这些发现，我假设 在谷氨酸兴奋毒性之后，DiDS比SiDS保持更稳定的DS结构。为了调查我 将使用高分辨率冷冻电子断层扫描与相关荧光配对来比较DiDS和 SiDS和阐明结构变化，导致DS损失后谷氨酸兴奋性毒性。目标1将 确定正常条件下DiDS和SiDS肌动蛋白网络之间的超微结构差异。目的 2将确定谷氨酸后DiDS和SiDS之间发生的超微结构变化 兴奋性毒性如果成功，该项目将确定存在哪些高分辨率结构差异 DiDS和SiDS之间的关系以及DiDS在兴奋性休克后是否更稳定。这也将 为兴奋性毒性研究者提供了高分辨率的结构证据，说明为什么抑制性突触可以 治疗靶点以防止神经元损伤。

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.