Visualising neuronal activity in cerebellar Purkinje cells

术语突触是指允许中枢神经系统内的可兴奋细胞相互通信的特殊结构。突触的特性在不同的细胞和大脑的不同部分之间不同，它们可以在短时间或长时间内适应，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为学习提供了一种存储机制，学习过程反过来可能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。了解突触是如何工作的，以及它们如何被修改，是我们理解大脑如何工作的基础，而这反过来又是修复因损伤或疾病而受损的大脑功能的重要起点。在这项提案中，我们的目标是培育出能够表达荧光蛋白的转基因小鼠品系。这些荧光蛋白可以在显微镜下观察到，它们在不同的pH环境下会改变它们的性质。通过将这些人造蛋白质附着到与细胞信号传递和可塑性有关的天然蛋白质结构上，我们打算开发一种方法，允许实时可视化活的脑细胞中突触传递、可塑性和交流的各个方面。在小脑中，信息从颗粒细胞传递到浦肯野细胞，小脑是执行熟练运动所必需的大脑部分。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幅增加，这是细胞信号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传感器选择性地整合到Purkinje细胞中，我们的目标是培育出可以直接看到Purkinje细胞活动的小鼠。然后，我们将使用从这些小鼠准备的脑片来评估不同模式的神经元输入小脑是如何在这个模型网络中处理和传递的。在突触上的通信需要释放一种化学递质，这种化学递质扩散到两个细胞之间的突触空间，并作用于突触后膜中的受体以产生反应。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存在的受体数量的增加或减少而出现的。受体从突触裂隙移动到细胞内部(下调)或反之亦然(上调)，伴随着pH的急剧变化，从细胞外的碱性表面到运输小泡内部的酸性。通过用荧光蛋白pH传感器标记Purkinje细胞表达的特定受体，我们的目标是培育小鼠，在这种小鼠中，我们可以直接可视化受体在被认为产生学习的条件下进出膜的运动。来自这些小鼠的脑片将被用来检查输入条件，这些条件会导致突触上受体数量的变化，从而改变中枢神经系统这一部分突触传递的强度。这些小鼠将为研究界提供宝贵的工具，如果成功，将为开发其他用于大脑其他部分的探针提供概念证明。

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.