Multiplexed RNAscope imaging of gene expression in targeted cells across the fly brain

摘要 神经递质规范是定义神经回路特性和功能的关键一步，因为它 帮助确定哪些神经元可以相互通信。令人惊讶的是，神经递质规格是 既不相互排斥也不一成不变。也就是说，一些神经元释放多种类型的神经递质 具有不同的生理功能，不同神经递质的相对丰度可以改变 基于环境条件和神经元放电模式。这些特征赋予神经元一种 功能可塑性经常被忽视，但可以对神经回路功能产生深远影响，从而 行为。不幸的是，人们对神经递质可塑性的机制知之甚少。这 知识的空白使得很难确定神经递质的可塑性对正常的贡献 生理学和涉及神经回路功能障碍的精神障碍。要克服这一障碍，请使用 作为果蝇的模式系统，我们开创了(1)核糖体图谱的组合，以识别所有 小群神经元中神经递质相关的转录本和(2)基于RNAScope的定量 转录分布在这些相同的神经元上，这可以同时成为记者的目标，因此 经免疫组织化学鉴定。在这份提案中，我们将使用这些极其敏感的技术来 辨别昼夜节律中不同觉醒调节神经元所使用的不同神经递质 时钟网络和控制睡眠的神经元。我们还将使用RNAScope来测量神经递质 同一性被神经元活动的变化所改变，例如在睡眠/清醒周期中预期的变化，以及 这种可塑性在多大程度上是由细胞通过突触传递自主调节的。最后，我们将 使用RNAScope测量神经递质系统标记常用驱动器的选择性。 因此，我们的研究将为研究中枢神经引入一种新的、强大的技术组合 苍蝇的系统，他们将首次定义每个人使用的全部神经递质 一个研究得很好的行为回路的细胞。我们的研究也将为确定如何 神经递质的特性可以通过生理和病理生理事件来调节，从而改变神经 电路功能和最终行为。从长远来看，这样的机制有望为我们的 了解神经紊乱的神经回路功能障碍。

Abstract Neurotransmitter specification is a crucial step in defining neural circuit identity and function because it helps establish which neurons communicate with each other. Surprisingly, neurotransmitter specification is neither mutually exclusive nor immutable. That is, some neurons release multiple types of neurotransmitters with different physiological functions, and the relative abundance of different neurotransmitters can be modified based on environmental conditions and neuronal firing patterns. These features endow neurons with a type of functional plasticity that is often overlooked but can have profound effects on neural circuit function and thus on behavior. Unfortunately the mechanisms responsible for neurotransmitter plasticity are poorly understood. This gap in knowledge makes it very difficult to determine the contribution of neurotransmitter plasticity to normal physiology and to psychiatric disorders involving neural circuit dysfunction. To overcome this obstacle using fruit flies as a model system we have pioneered a combination of (1) ribosome profiling to identify all neurotransmitter-associated transcripts in small groups of neurons and (2) RNAscope-based quantification of transcript distribution across those same neurons, which can be simultaneously targeted by reporters and thus identified by immunohistochemistry. In this proposal we will employ these exquisitely sensitive techniques to identify the different neurotransmitters that are used by various arousal-regulating neurons in the circadian clock network and by neurons that control sleep. We will also use RNAscope to measure how neurotransmitter identity is modified by changes in neuronal activity, such as those expected during the sleep/wake cycle, and the extent to which this plasticity is cell-autonomously regulated through synaptic transmission. Lastly, we will use RNAscope to measure the selectivity of commonly used drivers for labeling of neurotransmitter systems. Our studies will thus introduce a new, powerful combination of techniques for studying the central nervous system of the fly, and they will define for the first time the full complement of neurotransmitters used by each cell of a well-studied behavioral circuit. Our studies will also provide a foundation for determining how neurotransmitter identity can be modulated by physiological and pathophysiological events to alter neural circuit function and ultimately behavior. In the long-term such mechanisms are expected to contribute to our understanding of neural circuit dysfunctions underlying neurological disorders.