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Visualising neuronal activity in cerebellar Purkinje cells

小脑浦肯野细胞神经元活动的可视化

基本信息

批准号：
BB/E001246/1
负责人：
Nicholas Hartell
金额：
$ 44.44万
依托单位：
University of Leicester
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2007
资助国家：
英国
起止时间：
2007 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FE001246%2F1
关键词：
Visualising neuronal activity cerebellar Purkinje

项目摘要

The term synapse refers to the specialised structures that allow excitable cells within the central nervous system to communicate with one another. The properties of synapses differ between cells and between parts of the brain and they can adapt over short or longer terms to modulate the strength and pattern of information transmission. Longer term changes in the strength of transmission are thought to provide a storage mechanism for learning and the process of learning may, in turn, help to sculpt patterns of information flow between networks of cells and between different structures in the brain. Understanding how synapses work, and how they can be modified, is fundamental to our understanding how the brain works and this, in turn, is an essential starting point for repairing brain function when it is damaged through injury or disease. In this proposal, we aim to generate strains of mice that have been genetically modified to express proteins that are fluorescent. These fluorescent proteins can be visualised microscopically and they alter their properties under different pH environments. By attaching these artificial proteins to natural protein structures that are involved in cell signalling and plasticity, we intend to develop methods that allow the real time visualisation of aspects of synaptic transmission, plasticity and communication in living brain cells. In the cerebellum, part of the brain necessary for the execution of skilled movement, information is transmitted from granule cells to Purkinje cells. Purkinje cells provide the sole output from this part of the brain and they are largely responsible for processing the information that enters the cerebellum. Activity within Purkinje cells triggers substantial increases in intracellular calcium, a chemical essential for cell signalling and plasticity. Calcium increases are accompanied by an acidification of the cell. By incorporating a fluorescent protein based pH sensor selectively into Purkinje cells, we aim to generate mice in which the activity of Purkinje cells can be directly visualised. We will then use brain slices prepared from these mice to evaluate how different patterns of neuronal input to the cerebellum are processed and passed on within this model network. Communication at a synapse requires the release of a chemical transmitter that diffuses across the synaptic space between the two cells and acts on a receptor present in the post-synaptic membrane to produce a response. Long-term changes in the strength of signalling between cells are thought to arise, in many cases, by either an increase or a decrease in the number of receptors present in the post-synaptic membrane. The movement of a receptor from the synaptic cleft to the inside of the cell (down-regulation) or vice verse (up-regulation), is accompanied by a sharp change in pH from the alkaline extracellular surface to the acidic inside of a transport vesicle. By tagging specific receptors expressed by Purkinje cells with a fluorescent protein pH sensor, we aim to develop mice in which we can directly visualise the movement of receptors to and from the membrane under conditions thought to produce learning. Brain slices derived from these mice will be used to examine the input conditions that produce changes in the number of receptors at a synapse and hence the strength of synaptic transmission within this part of the central nervous system. These mice will provide valuable tools to the research community and if successful, provide proof of concept for the development of other probes with uses in other parts of the brain.

术语突触指的是允许中枢神经系统内的兴奋细胞彼此通信的专门结构。突触的特性在细胞之间和大脑的不同部分之间是不同的，它们可以在短期或长期内适应，以调节信息传输的强度和模式。传输强度的长期变化被认为是为学习提供了一种存储机制，而学习过程反过来又有助于塑造细胞网络之间和大脑不同结构之间的信息流模式。了解突触如何工作，以及它们如何被修改，是我们理解大脑如何工作的基础，反过来，这也是修复因受伤或疾病而受损的大脑功能的重要起点。在这项提议中，我们的目标是产生经过遗传修饰的小鼠品系，以表达荧光蛋白质。这些荧光蛋白可以在显微镜下观察到，并且它们在不同的pH环境下改变其特性。通过将这些人工蛋白质连接到参与细胞信号传导和可塑性的天然蛋白质结构上，我们打算开发出能够真实的实时可视化活脑细胞中突触传递、可塑性和通信方面的方法。在小脑中，执行熟练运动所必需的大脑部分，信息从颗粒细胞传递到浦肯野细胞。浦肯野细胞提供了大脑这一部分的唯一输出，它们主要负责处理进入小脑的信息。浦肯野细胞内的活动触发细胞内钙的大量增加，这是细胞信号传导和可塑性所必需的化学物质。钙的增加伴随着细胞的酸化。通过将基于荧光蛋白的pH传感器选择性地结合到浦肯野细胞中，我们的目标是产生浦肯野细胞的活性可以直接可视化的小鼠。然后，我们将使用从这些小鼠制备的脑切片来评估输入小脑的不同神经元模式如何在这个模型网络中处理和传递。在突触处的通信需要释放一种化学递质，该化学递质扩散穿过两个细胞之间的突触空间，并作用于突触后膜中存在的受体以产生响应。在许多情况下，细胞间信号强度的长期变化被认为是由于突触后膜中受体数量的增加或减少而引起的。受体从突触间隙向细胞内部的移动（下调）或反之亦然（上调），伴随着pH从碱性细胞外表面到运输囊泡内部的酸性的急剧变化。通过用荧光蛋白pH传感器标记浦肯野细胞表达的特异性受体，我们的目标是开发小鼠，在这种小鼠中，我们可以在被认为产生学习的条件下直接观察受体往返膜的运动。来自这些小鼠的脑切片将用于检查产生突触处受体数量变化的输入条件，从而检查中枢神经系统这一部分内突触传递的强度。这些小鼠将为研究界提供有价值的工具，如果成功，将为开发用于大脑其他部位的其他探针提供概念证明。