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.

项目概要神经调节，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强大的控制作用，它调节兴奋性、突触可塑性、以及神经功能的其他方面的缺陷与许多方面有关。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。通过其相应的 G 蛋白偶联受体 (GPCR) 进行体内研究。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功能是细胞类型特异性的。神经调节亚细胞信号传导事件的结果仍然很困难。在体内以高时间分辨率拮抗神经调节信号事件的实用工具，这是建立信号事件和神经元之间因果关系所必需的功能或动物行为。为了克服这些问题，我们建议开发新型基因编码传感器来检查尚未研究的神经调节信号传导在体内的单神经元分辨率活动途径：蛋白激酶 C (PKC) 途径，虽然是原型基因编码的 PKC 传感器。基于福斯特共振能量转移（FRET）的技术已用于体外实验，其由于在更具挑战性的条件下信噪比较低，体内应用一直很困难基于我们之前开发传感器的成功经验。 cAMP和蛋白激酶A（PKA）通路进行体内成像，我们将采用多管齐下的方法表征和改进用于体内成像的 PKC 传感器。 PKA 和 PKC 通路的基因编码执行器仅在它们有效时才有效我们将验证这些工具在监测或操纵方面的实用性。清醒小鼠行为过程中的神经调节活动将被包装成成功的工具。病毒载体易于引入体内，并将传播给研究界。如果成功的话，我们的努力将为研究界提供以前无法实现的能力对大脑中的神经调节信号活动进行大规模监测和操纵这种量化和操纵神经调节信号的能力将在细胞和电路水平上发挥作用。补充细胞外神经调节剂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。