Determinants of Calcium Ion Influx into Dendritic Spines

描述（申请人提供）：哺乳动物中枢神经系统中的神经元通过突触相互沟通。接受兴奋性谷氨酸能输入的细胞的树突是高度复杂的结构，被称为树突棘的微小膜状突起所覆盖。棘包含突触后密度和突触后侧的相关机制，虽然在理解这些蛋白质的相互作用和生理学方面取得了重大进展，但仍存在许多问题。光学技术的最新进展使得刺激单个突触并同时在单个树突棘的精细空间尺度上可视化局部神经元活动成为可能。我们建议使用这些方法来研究突触后离子通道在单个突触中的相互作用。这项工作的具体目标是获得对CA1锥体神经元树突上单个突触突触后反应形成的事件的详细了解。特别地，这里提出的实验将解决两个主要问题。首先，树突棘中谷氨酸受体激活下游形成电位和钙离子积累的信号事件是什么？其次，CA1锥体神经元棘中的钙信号是否受到几乎一致的突触活动和动作电位的影响？为了解决这些问题，我们将结合使用全细胞膜片钳生理学，双光子扫描激光显微镜（2PLSM）和谷氨酸双光子激光释放来刺激和记录单个树突棘中的事件。更一般地说，这一提议旨在探索神经元之间通信事件的基本方面。中枢神经系统中神经元之间连接强度的变化被认为是学习和记忆的基础，理解发生在树突棘中的复杂信号事件对理解这些现象至关重要。此外，位于脊柱中的许多蛋白质突变与人类神经系统疾病有关，并且在许多神经系统疾病的小鼠模型中脊柱形态受到干扰。了解突触传递和脊髓信号动力学的特性对于解释阿尔茨海默病和帕金森病等神经退行性疾病的细胞机制至关重要。

DESCRIPTION (provided by applicant): Neurons in the mammalian central nervous system communicate with each other via synapses. The dendrites of cells which receive excitatory glutamatergic input are highly complex structures, and covered in tiny membranous protuberances called dendritic spines. Spines house the postsynaptic density and associated machinery that constitute the post-synaptic side of the synapse, and while significant progress has been made in understanding the interactions and physiology of these proteins, many questions remain. Recent advances in optical techniques have allowed for stimulating individual synapses and simultaneously visualizing local neuronal activity on the fine spatial scale of single dendritic spines. We propose to use these methods to investigate the interplay of postsynaptic ion channels at individual synapses. The specific goal of this work is to obtain a detailed understanding of the events that shape the postsynaptic responses at single synapses on the dendrites of CA1 pyramidal neurons. In particular, the experiments proposed here will address two main questions. First, what are the signaling events downstream of glutamate receptor activation that shape potentials and calcium ion accumulation in dendritic spines? Secondly, are calcium signals in spines of CA1 pyramidal neurons influenced by near-coincident synaptic activity and action potentials? To address these questions, we will use a combination of whole-cell patch clamp physiology, 2-photon scanning laser microscopy (2PLSM), and 2-photon laser uncaging of glutamate to stimulate and record events in individual dendritic spines. More generally, this proposal aims to explore fundamental aspects of the events that underlie communication between neurons. Changes in the strength of connections between neurons in the central nervous system are thought to underlie learning and memory, and understanding the complex signaling events that occur in dendritic spines is crucial to understanding these phenomena. Furthermore, many mutations in proteins located in spines are linked to human neurological disorders, and spine morphology is perturbed in many mouse models of neurological diseases. Understanding the properties of synaptic transmission and the signaling dynamics in spines is vital to explaining the cellular mechanisms of neurodegenerative disorders like Alzheimer's and Parkinson's diseases.