Next generation all-optical toolkits for functional analysis of neuropeptide dynamics in neural circuits

项目摘要 哺乳动物的大脑是非常动态的，可以迅速调整其功能状态，以应对变化 在环境中。例如，当一个显著的事件发生时，大脑会进入一种增强记忆的模式 阵这样的大脑状态变化发生得太快，不可能是由于解剖学上的重新布线。取而代之的是 被认为是由神经调质（NM）和神经肽（NP）的作用引起的。不像小分子 NMs，如乙酰胆碱和单胺，NPs通常不作为主要神经递质释放 它们来自专门的神经元，释放后不会再循环。相反，大多数神经元合成， 除了快速递质如谷氨酸和GABA外，还释放NP， 通过扩散和蛋白水解。虽然长期以来被用作解剖学标记，但我们对NP信号传导的理解是 只是粗略的。对肽能通讯的细胞密码的深入了解现在才出现在大规模的研究中， 大规模的转录谱研究揭示了肽及其受体在细胞内的分布， 类型这些研究揭示了NP-受体对在细胞类型中的分化解剖分布， 平衡NPs作为神经回路中跨细胞通信的重要介质。然而，功能 NP信号的重要性是非常困难的，如果不是不可能的话，使用目前的工具来研究， 揭示体内NP信号的时间和位置，或NP信号对神经回路的影响 活动因此，需要新的技术来进行功能获得和功能丧失的研究， 行为动物中NP活动的正常位置和时间。 为了克服这些技术障碍，我们组建了一个多学科团队来开发、验证、应用和 传播下一代光学工具包，用于NP时空动力学的功能分析 行为中的信号。我们的工具包包括：1）光活化剂，以快速提供纳米粒子（或药物， 靶向NP受体）以高时空精确性到达其作用位点; 2）遗传编码NP 传感器来报告NP何时被释放以及它们在什么时间和空间尺度上起作用：3）新的光学 和遗传方法的细胞和区域特异性记录和操纵的NP行动，使用这些 同时在哺乳动物大脑的多个位置进行探测。 将这些方法与行为动物的功能研究相结合，我们的目标是建立范式， 确定NP信号传导用于体内电路调制的必要性和充分性。我们将 确定NP释放的背景和位置，随后NP受体激活的时空模式， 以及这对神经生理和行为的影响。我们将积极向 神经科学社区在各种大脑区域和物种中的广泛应用将揭示 NPs对控制大脑回路和可塑性的贡献。这些知识将提供积木， 为完善理论和开发神经和神经精神疾病的新疗法铺平道路。

Project summary The mammalian brain is remarkably dynamic and can quickly adjust its functional state in response to changes in the environment. For example, when a salient event occurs, the brain enters a mode that enhances memory formation. Such brain state changes occur too rapidly to be due to anatomical rewiring. Instead, they are thought to arise from the action of neuromodulators (NMs) and neuropeptides (NPs). Unlike small-molecule NMs, such as acetylcholine and monoamines, NPs are not generally released as the major neurotransmitter from specialized neurons and they are not recycled after release. Instead most neurons synthesize and release NPs in addition to fast transmitters such as glutamate and GABA, and peptide clearance is governed by diffusion and proteolysis. Although long utilized as anatomical markers, our understanding of NP signaling is only cursory. Insights into the cellular code of peptidergic communication are only now emerging from large- scale transcriptional profiling studies that reveal the distribution of peptides and their receptors across cell types. These have revealed a differentiated anatomic distribution of NP-receptor pairs across cell types that poise NPs as important mediators of trans-cellular communication in neural circuits. However, the functional significance of NP signaling is extremely difficult, if not impossible, to study using current tools, which do not reveal the timing and location of NP signaling in vivo, or the consequences of NP signaling on neural circuit activity. Thus, new technologies are needed to enable gain- and loss-of-function studies that precisely target the normal location and timing of NP activity in behaving animals. To overcome these technical barriers, we assembled a multi-disciplinary team to develop, validate, apply, and disseminate next-generation optical toolkits for functional analysis of the spatiotemporal dynamics of NP signaling during behavior. Our toolkits include: 1) photoactivatable agents to rapidly deliver NPs (or drugs that target NP receptors) to their sites of action with high spatiotemporal precision; 2) genetically-encoded NP sensors to report when NPs are released and over what temporal and spatial scales they act: 3) new optical and genetic approaches for cell- and region-specific recording and manipulation of NP action using these probes at multiple sites in the mammalian brain simultaneously. Combining these methods with functional studies in behaving animals, we aim to establish paradigms for determining the necessity and sufficiency of NP signaling for the modulation of circuits in vivo. We will determine the context and location of NP release, the ensuing spatiotemporal pattern of NP receptor activation, and the effects this has on neuronal physiology and behavior. We will actively disseminate these toolkits to the neuroscience community. Broad applications in various brain regions and species will reveal the dynamic contribution of NPs to the control of brain circuits and plasticity. This knowledge will provide building blocks and pave the ways to refine theory and develop novel therapeutics for neurological and neuropsychiatric disorders.