Genetic tools for visualization of co-transmission in identified neurons

 描述(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取决于我们对行为回路进行的神经计算的理解。神经元之间化学交流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向其所有靶点释放一种类型的递质。然而，近年来，人们已经清楚地看到，脊椎动物和无脊椎动物参与成瘾、记忆形成、觅食行为和繁殖的神经回路中的关键神经元含有违反这一规则的神经元，释放出多种神经递质。在这项建议中，我们开发了基因工具，通过用荧光蛋白修改神经递质的囊泡转运体的内源遗传位点，以多种颜色定位单个神经元中特定神经递质释放位点的分布。由于标记的产生和周转依赖于控制内源蛋白质存在的过程，这使得对递质释放部位的准确和完整的计算成为可能。单细胞分辨率是通过这种策略的交叉版本获得的，在该策略中，分裂的荧光蛋白仅在特定细胞中重组。目前在任何系统中都没有能够提供这种级别分辨率的技术。这项技术将提供在复杂的情况下确定 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。它还将允许绘制神经递质释放的时间变化图，无论是发育变化还是可塑性引起的变化。这项技术最初是为了在果蝇身上使用而开发的，果蝇是一种模式生物，对于我们理解行为的遗传和回路基础非常重要，但作为一种一般策略，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.