NOVEL METHODS FOR VISUALIZING NEURON-SPECIFIC SYNAPSES

描述（改编自申请人的摘要）：哺乳动物的大脑是 由数十亿个神经元组成。每一个高度特化的细胞 据估计，即使没有数千个，也有数百个突触连接， 其他神经元这个网络惊人的复杂性与 人类头脑中非凡的智力和创造力。只有 这些电路的一部分已被定义在个人的水平， 然而，细胞。我们似乎不太可能开始理解 这些复杂行为的起源，直到一个更详细的布线 脊椎动物大脑的示意图是已知的。说这一点是为 来开发显微镜下的方法， 神经元我们选择在一个 具有简单、明确的神经系统的模式生物。在线虫体内 C.神经系统由302个神经元组成。的 这些神经元中的每一个的形态和突触连接已经被 通过连续切片电子显微镜确定。因此，我们有一个完整的 电路图为整个C。线虫的神经系统我们的目标是 测试观察这些神经元及其特定突触的可行性 在光学显微镜下完整的动物。因为我们所使用的方法 发展将满足EM重建的需要，我们的方法应该 极大地促进了识别干扰细胞的特定突变的努力。 这一简单神经系统的线路图，从而导致克隆 控制突触特异性的基因。此外预计 我们将开发的方法在未来可以用来 定义其他更复杂神经系统的详细神经解剖学。我们 将开发可用于观察特定子集的方法， 在光学显微镜下观察这些连接。我们的战略利用了 以下工具：1）绿色荧光蛋白（GFP）和表位标签， 标记突触特异性蛋白质和它们在其中表达的神经元; 2)克隆基因调控区，以驱动这些标记的 特定神经元中的蛋白质; 3）共聚焦和多光子激发 显微镜来分辨这些标记的神经元过程及其突触 连接.

DESCRIPTION (Adapted from applicant's abstract): The mammalian brain is composed of billions of neurons. Each of these highly specialized cells is estimated to make hundreds, if not thousands, of synaptic connections to other neurons. The staggering complexity of this network parallels the remarkable intellectual and creative abilities of the human mind. Only a fraction of these circuits have been defined at the level of individual cells, however. And it seems unlikely that we can even begin to understand the origins of these complex behaviors until a much more detailed wiring diagram of the vertebrate brain is known. The purpose of this proposal is to develop microscopic methods that can reveal synapses between specific neurons. We have chosen to test the reliability of these approaches in a model organism with a simple, well defined nervous system. In the nematode C. elegans the nervous system is composed of precisely 302 neurons. The morphology and synaptic connectivity of each of these neurons has been defined by serial section electron microscopy. Thus, we have a complete circuit diagram for the entire C. elegans nervous system. Our goal is to test the feasibility of observing these neurons and their specific synapses in intact animals in the light microscope. Because the methods that we will develop would obviate the need for EM reconstruction, our approaches should greatly facilitate efforts to identify specific mutations that perturb the wiring diagram of this simple nervous system and thereby lead to the cloning of the genes that control synaptic specificity. In addition, it is expected that the methods that we will develop can be utilized in the future to define the detailed neuroanatomy of other, more complex nervous systems. We will develop methods that can be utilized to observe specific subsets of these connections in the light microscope. Our strategy exploits the following tools: 1) green fluorescent protein (GFP) and epitope tags to label synapse-specific proteins and the neurons in which they are expressed; 2) cloned gene regulatory regions to drive expression of these marked proteins in specific neurons; 3) confocal and multiphoton excitation microscopy to resolve these labeled neuronal processes and their synaptic connections.