Genetic tools for visualization of co-transmission in identified neurons

 描述（由申请人提供）：理解大脑在正常和病理情况下如何产生行为的能力依赖于我们对行为电路进行的神经计算的理解。神经元之间化学通讯的性质是由释放到突触后靶点的特定神经递质决定的，多年来人们一直认为神经元释放一种类型的递质到所有的靶点。然而，近年来，人们已经清楚地发现，脊椎动物和无脊椎动物回路中涉及成瘾、记忆形成、进食行为和生殖的关键神经元含有违反这一规则的神经元，释放多种神经递质。在这个建议中，我们开发的遗传工具，允许映射特定的神经递质释放位点在单个神经元中的多种颜色的分布，通过修改内源性遗传基因位点的囊泡转运蛋白的神经递质与荧光蛋白。这允许准确和完整地计算递质释放位点，因为标记物的产生和周转依赖于控制内源性蛋白质存在的过程。单细胞分辨率通过这种策略的交叉版本获得，其中分裂的荧光蛋白仅在特定细胞中重建。在任何系统中，目前还没有技术可以提供这种级别的分辨率。这项技术将提供确定复杂 神经元释放几种化学物质，每种化学物质的空间分布及其与该神经元下游靶点的关系。它也将允许映射的时间变化，无论是发育或可塑性诱导，在神经递质释放。这项技术最初是为果蝇而开发的，果蝇是一种模式生物，对我们理解行为的遗传和电路基础非常重要，但作为一种通用策略，它也可以适用于哺乳动物的大脑。

 DESCRIPTION (provided by applicant): The ability to understand how brains generate behavior both in normal and pathological situations relies on our understanding of the neural computations carried out by behavioral circuits. The nature of chemical communication between neurons is determined by the specific neurotransmitters released onto postsynaptic targets and it was thought for many years that neurons released a single type of transmitter onto all their targets. In recent years, however, it has become clear that key neurons in vertebrate and invertebrate circuits involved in addiction, memory formation, feeding behavior and reproduction contain neurons that violate this rule, releasing multiple neurotransmitters. In this proposal we develop genetic tools to allow mapping of the distribution of specific neurotransmitter release sites in single neurons in multiple colors by modifying the endogenous genetic loci of vesicular transporters for neurotransmitter with fluorescent proteins. This allows both accurate and complete accounting of transmitter release sites since the marker's generation and turnover rely on processes that control the presence of the endogenous protein. Single cell resolution is obtained via an intersectional version of this strategy in which split fluorescent proteins become reconstituted only in specific cells. There is no current technique in any system which can provide this level of resolution. This technology will provide the ability to determine, in complex neurons releasing several chemical substances, the spatial distribution of each of the chemicals and its relationship to downstream targets of that neuron. It will also allow the mapping of temporal changes, either developmental or plasticity-induced, in neurotransmitter release. The technique is developed initially for use in Drosophila, a model organism which has been immensely important for our understanding of both the genetic and circuit basis of behavior, but as a general strategy can also be adapted for use in mammalian brain.