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

摘要 神经递质规格是定义神经回路身份和功能的关键步骤，因为它 帮助确定哪些神经元相互交流。令人惊讶的是，神经递质的规格是 既不是互斥的，也不是不可变的。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，并且不同神经递质的相对丰度可以被修改， 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，但对神经回路功能有深远的影响， 行为不幸的是，对神经递质可塑性的机制知之甚少。这 知识上的差距使得很难确定神经递质可塑性对正常 生理学和涉及神经回路功能障碍的精神障碍。为了克服这一障碍， 果蝇作为一个模型系统，我们开创了一个组合（1）核糖体分析，以确定所有 神经递质相关的转录物在小群体的神经元和（2）RNA酶为基础的定量 转录物在这些相同神经元中的分布，其可以同时被报告基因靶向， 免疫组织化学鉴定。在本提案中，我们将采用这些极其敏感的技术， 识别昼夜节律中各种觉醒调节神经元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 身份被神经元活动的变化所改变，例如在睡眠/觉醒周期期间预期的那些，以及 这种可塑性通过突触传递被细胞自主调节的程度。最后，我们将 使用RNAscope测量用于标记神经递质系统的常用驱动程序的选择性。 因此，我们的研究将为研究中枢神经系统引入一种新的、强大的技术组合。 系统的苍蝇，他们将首次定义的完整的神经递质使用的每一个 一个被充分研究的行为回路的细胞。我们的研究还将为确定如何 神经递质的身份可以通过生理和病理生理事件来调节，以改变神经递质的功能。 电路功能和最终行为。从长远来看，这种机制有望有助于我们的 了解神经系统疾病背后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.