Sensing and manipulating neuromodulatory signaling in vivo

体内传感和操纵神经调节信号

基本信息

批准号：
10650681
负责人：
Haining Zhong
金额：
$ 256.58万
依托单位：
OREGON HEALTH & SCIENCE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-01 至 2026-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10650681
关键词：
Acetylcholine Acute Affect Animal Behavior Animals Behavior Benign Biological Assay Brain Cell physiology Cells Characteristics Clustered Regularly Interspaced Short Palindromic Repeats Communities Complement Cyclic AMP Cyclic AMP-Dependent Protein Kinases Defect Development Dissection Dopamine Event Exhibits Fluorescence Resonance Energy Transfer Functional disorder Future G-Protein-Coupled Receptors Goals Habitats Heterogeneity Image In Vitro Individual Kinetics Light Mammalian Cell Measurement Mediating Mediator Methods Microdialysis Monitor Mus Neuromodulator Neurons Noise Norepinephrine Outcome Oxygen Pathway interactions Performance Phosphotransferases Physiological Protein Kinase C Reagent Reporting Research Resolution Serotonin Side Signal Pathway Signal Transduction Synaptic Transmission Synaptic plasticity Testing Time Variant Viral Vector Virus Visualization Western Blotting awake cell type design experience experimental study extracellular high throughput screening improved in vivo in vivo imaging interdisciplinary approach neuronal excitability neuropsychiatric disorder neuroregulation novel optogenetics overexpression prototype rational design receptor response screening sensor side effect spatiotemporal subcellular targeting success temporal measurement tool voltage

项目摘要

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.

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，无法对大脑功能的强大控制。它调节兴奋，突触可塑性，和神经元功能的其他方面。神经调节中的缺陷与许多神经精神疾病。神经调节剂通过调节细胞内信号传导发挥其功能通过相应的G蛋白偶联受体（GPCR）的事件。虽然在体内审问细胞外神经调节剂已开始成为可能，神经调节剂对亚细胞信号传导和神经元功能是细胞类型特异性的。监视特定于细胞类型的神经调节性亚细胞信号事件的结果仍然很困难。也缺乏与体内高临时分辨率对抗神经调节信号事件的实用工具，建立信号事件与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，我们建议开发新的遗传编码传感器来检查具有理解神经调节信号传导的单神经元分辨率的体内活动途径：蛋白激酶C（PKC）途径。虽然原型遗传编码的PKC传感器基于Förster共振能量转移（FRET）已用于体外实验由于较低的挑战，因此在体内应用很难体内成像条件。基于我们以前成功地为开发传感器的经验营地和蛋白质激酶A（PKA）体内成像途径，我们将采用多管齐下的方法表征和改进体内成像的PKC传感器。此外，我们将开发小说 PKA和PKC途径的基因编码执行器，它们仅在它们时有效被蓝光刺激。我们将验证这些工具的效用以监视或操纵行为过程中清醒小鼠的神经调节活动。成功的工具将包装到病毒向量在体内易于介绍，并将被传播到研究界。如果成功，我们的努力将为研究社区提供以前无法实现的能力对大脑中神经调节信号传导活动进行大规模监测和操纵细胞和电路水平。这种量化和操纵神经调节信号传导的能力将补充细胞外神经调节剂和神经元电动活动的测量增强我们对动物行为基本脑功能的理解。