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通信。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，并被称为树突棘的微小膜性突起所覆盖。脊椎包含突触后密度和构成突触后突触后侧的相关机制，尽管在了解这些蛋白质的相互作用和生理学方面取得了重大进展，但许多问题仍然存在。光学技术的最新进展使得刺激个体突触和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单个突触的突触后离子通道的相互作用。这项工作的具体目标是详细了解在CA1锥体神经元树突上的单个突触上形成突触后反应的事件。特别是，这里提出的实验将解决两个主要问题。首先，谷氨酸受体激活下游的信号事件是什么，它们塑造了树突棘中的电位和钙离子积累？其次，近乎一致的突触活动和动作电位是否会影响CA1区锥体神经元脊椎中的钙信号？为了解决这些问题，我们将使用全细胞膜片钳生理学、双光子扫描激光显微镜(2PLSM)和双光子激光谷氨酸浸泡相结合的方法来刺激和记录单个树突棘中的事件。更广泛地说，这项提议旨在探索神经元之间交流的基础事件的基本方面。中枢神经系统神经元之间连接强度的变化被认为是学习和记忆的基础，了解树突中发生的复杂信号事件对于理解这些现象至关重要。此外，脊椎中蛋白质的许多突变与人类神经疾病有关，在许多神经疾病的小鼠模型中，脊椎的形态受到干扰。了解突触传递的特性和脊椎中的信号动力学对于解释阿尔茨海默氏症和帕金森氏症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.