NOVEL METHODS FOR VISUALIZING NEURON-SPECIFIC SYNAPSES

描述(摘自申请者的摘要)：哺乳动物的大脑 由数十亿个神经元组成。这些高度专业化的细胞中的每一个 据估计，即使不是数千，也有数百个突触连接到 其他神经元。该网络惊人的复杂性与 人类头脑中非凡的智力和创造力。只有一个 这些电路中的一部分是在个人层面上定义的 然而，细胞。而且我们甚至不太可能开始理解 这些复杂行为的起源直到更详细的布线 脊椎动物大脑的示意图是已知的。这项建议的目的是 开发微观方法来揭示特定物种之间的突触 神经元。我们已经选择在一个 具有简单的、明确定义的神经系统的模式生物。在线虫中 线虫神经系统恰好由302个神经元组成。这个 这些神经元的形态和突触连接已经被 由连续切片电子显微镜定义。因此，我们有一个完整的 整个线虫神经系统的电路图。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 在光学显微镜下观察完好的动物。因为我们将采用的方法 发展将消除对EM重建的需要，我们的方法应该 极大地促进了识别扰乱人类基因组的特定突变 这个简单的神经系统的接线图，从而导致了克隆 控制突触专一性的基因。此外，预计 我们将开发的方法可以在未来用于 定义其他更复杂的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 光学显微镜中的这些连接。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及其表达的神经元； 2)克隆基因调控区以驱动这些标记的表达 特定神经元中的蛋白质；3)共聚焦和多光子激发 显微镜下分辨这些标记的神经元突起及其突触 关系。

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.