Sensing and manipulating neuromodulatory signaling in vivo

项目摘要 神经调节，例如由神经调节剂去甲肾上腺素、乙酰胆碱和 多巴胺对大脑功能有着强大的控制作用。它调节兴奋性，突触可塑性， 以及其他神经元功能的研究神经调节的缺陷与许多 神经精神疾病神经调质通过调节细胞内信号传导发挥其功能 事件通过其相应的G蛋白偶联受体（GPCR）。虽然在体内审讯 细胞外神经调质已经开始成为可能，神经调质的作用， 亚细胞信号传导和神经元功能是细胞类型特异性的。监测细胞类型特异性 神经调节亚细胞信号传导事件的结果仍然是困难的。也缺乏 用于在体内以高时间分辨率拮抗神经调节信号传导事件的实用工具， 这是建立信号事件和神经元之间的因果关系所必需的。 功能或动物行为。 为了克服这些问题，我们建议开发新的基因编码传感器， 活动，在体内与单神经元分辨率，一个研究不足的神经调节信号 蛋白激酶C（PKC）途径。虽然原型基因编码的PKC传感器 基于Förster共振能量转移（FRET）的方法已用于体外实验， 由于在更具有挑战性的环境中信噪比较低， 体内成像条件。基于我们以前在开发传感器方面的成功经验， cAMP和蛋白激酶A（PKA）通路的体内成像，我们将采用多管齐下的方法 以表征和改进用于体内成像的PKC传感器。此外，我们还将开发新的 PKA和PKC途径的基因编码执行器，只有当它们 受到蓝光的刺激。我们将验证这些工具在监视或操纵 在清醒的小鼠行为期间的神经调节活动。成功的工具将被打包到 病毒载体易于引入体内，并将传播给研究界。如果 如果成功，我们的努力将为研究界提供以前无法实现的能力， 进行大规模监测和操纵大脑中的神经调节信号活动， 细胞和电路水平。这种量化和操纵神经调节信号的能力将 补充细胞外神经调质和神经元电活动的测量， 增强我们对动物行为背后的大脑功能的理解。

PROJECT SUMMARY Neuromodulation, such as that mediated by the neuromodulators norepinephrine, acetylcholine, and dopamine, imposes powerful control over brain function. It regulates the excitability, synaptic plasticity, and other aspects of neuronal function. Defects in neuromodulation are associated with many neuropsychiatric diseases. Neuromodulators exert their functions by regulating intracellular signaling events via their corresponding G protein-coupled receptors (GPCRs). Although in vivo interrogation of extracellular neuromodulators has started to become possible, the effects of neuromodulators on subcellular signaling and neuronal function are cell type-specific. Monitoring the cell type-specific outcomes of neuromodulatory subcellular signaling events remains difficult. There is also a lack of practical tools for antagonizing neuromodulatory signaling events with high temporal resolution in vivo, which is required for establishing the causal relationship between the signaling events and neuronal functions or animal behavior. To overcome these problems, we propose to develop novel genetically encoded sensors for examining the activities, in vivo with single-neuron resolution, of an understudied neuromodulatory signaling pathway: the protein kinase C (PKC) pathway. Although prototypic genetically encoded PKC sensors based on Förster resonance energy transfer (FRET) have been used for experiments in vitro, their application in vivo has been difficult due to lower signal-to-noise ratios under the more challenging in vivo imaging conditions. Building on our previous successful experience in developing sensors for the cAMP and protein kinase A (PKA) pathway for in vivo imaging, we will employ a multi-pronged approach to characterize and improve PKC sensors for in vivo imaging. In addition, we will develop novel genetically encoded actuators for both the PKA and PKC pathways that are effective only when they are stimulated by blue light. We will validate the utility of these tools for monitoring or manipulating neuromodulatory activities in awake mice during behavior. The successful tools will be packaged into viral vectors for their easy introduction in vivo, and will be disseminated to the research community. If successful, our efforts will provide the research community with a previously unattainable ability to conduct large-scale monitoring and manipulation of neuromodulatory signaling activities in the brain at the cellular and circuit levels. This ability to quantify and manipulate neuromodulatory signaling will complement the measurements of extracellular neuromodulators and neuronal electric activities to enhance our understanding of brain function underlying animal behavior.