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

项目总结 哺乳动物的大脑非常活跃，能够迅速调整其功能状态以应对变化 在环境中。例如，当显著事件发生时，大脑进入增强记忆的模式 队形。这样的大脑状态变化发生得太快，不可能是由于解剖结构的重新连接。相反，他们是 被认为是由神经调节剂(NMS)和神经肽(NPs)的作用产生的。与小分子不同 NMS，如乙酰胆碱和单胺类，NPs通常不作为主要的神经递质释放 来自专门的神经元，它们在释放后不会被回收。相反，大多数神经元都会合成和 除了谷氨酸和GABA等快速递质外，还可以释放NPs，从而控制多肽的清除 通过扩散和蛋白质分解。尽管长期以来一直被用作解剖学标记，但我们对NP信号的理解是 只是草率而已。对肽能通讯的细胞密码的洞察现在才从大型- 揭示多肽及其受体跨细胞分布的规模转录图谱研究 类型。这些研究揭示了NP受体对在不同细胞类型之间的不同解剖分布 神经回路中NPs作为跨细胞通讯的重要介体。然而，功能性的 使用现有的工具研究NP信号的意义是极其困难的，如果不是不可能的话，这些工具还不能 揭示NP信号在体内的时间和位置，或NP信号对神经回路的影响 活动。因此，需要新的技术来实现准确定位目标的功能增减研究 在行为动物中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 传播用于NP时空动力学函数分析的新一代光学工具包 在行为过程中发出信号。我们的工具包包括：1)可快速传递NPs(或药物)的光敏剂 靶NP受体)高时空精确度的作用部位；2)遗传编码的NP 报告NPs何时释放以及它们作用的时间和空间尺度的传感器：3)新的光学元件 以及遗传方法，用于细胞和区域特定记录和操纵NP动作，使用这些 同时在哺乳动物大脑中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 确定NP信号对体内电路调制的必要性和充分性。我们会 确定NP释放的背景和位置，随后NP受体激活的时空模式， 以及这对神经元生理和行为的影响。我们将积极将这些工具包分发给 神经科学界。在不同的大脑区域和物种中的广泛应用将揭示这种动态 NPs对控制大脑回路和可塑性的贡献。这一知识将提供构建块和 为完善理论和开发神经和神经精神障碍的新疗法铺平道路。

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.