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Genetically-encoded ACh sensors

基因编码的 ACh 传感器

基本信息

批准号：
10065020
负责人：
J. Julius Zhu
金额：
$ 35.33万
依托单位：
UNIVERSITY OF VIRGINIA
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-12-01 至 2022-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10065020
关键词：
Acetylcholine Address Advocate Alzheimer&apos s Disease Area Attention Behavior Biological Process Brain Brain Stem Brain region Cardiovascular Diseases Cell Communication Cell Nucleus Cells Cognition Complex Corpus striatum structure Coupling Cultured Cells Data Development Diabetes Mellitus Diffuse Epilepsy Family Fluorescence Resonance Energy Transfer Fright G-Protein-Coupled Receptors Green Fluorescent Proteins Habenula Health Heart Immune In Vitro Kinetics Link Liver Malignant Neoplasms Medial Mediating Mental Depression Mental disorders Monitor Motivation Movement Mus Muscarinic Acetylcholine Receptor Neuroglia Neurologic Neuromuscular Junction Neurons Neurotransmitters Noise Obesity Organ Pancreas Parkinson Disease Pathologic Perception Performance Peripheral Peripheral Nervous System Physiological Population Property Psyche structure Rattus Receptor Activation Regulation Rewards Role Schizophrenia Signal Transduction Site-Directed Mutagenesis Sleep Specificity Stress Suggestion System Techniques Tissues Translational Research Validation Work addiction awake basal forebrain base biological research cholinergic cholinergic neuron classical conditioning experimental study improved in vivo nervous system disorder real-time images screening sensor success tool transmission process user-friendly

项目摘要

PROJECT SUMMARY Acetylcholine (ACh) mediates cell-to-cell communication in the central and peripheral nervous systems, as well as non-neuronal systems. ACh released by neuronal and non-neuronal cells in these systems regulates complex brain functions, such as attention, perception, associative learning, and sleep/awake states, and various biological processes in other tissues and organs, including the heart, liver and pancreas. Dysregulation of cholinergic transmission is linked to a number of neurological diseases, including addiction, Alzheimer’s disease, epilepsy, schizophrenia, Parkinson’s disease and depression, as well as many other health problems, including cardiovascular diseases, obesity, diabetes, immune deficiency and cancer. Despite the significance of ACh in physiological and pathological conditions, the precise regulations and exact functional roles of cholinergic transmission in the majority of tissues and organs remain poorly understood, due primarily to the limitations of available tools for monitoring ACh. We recently initiated development of genetically-encoded G-protein-coupled receptor activation-based sensors for ACh (GACh) by coupling a circular permutated green fluorescent protein (cpGFP) with a muscarinic receptor. We are improving the sensors with large-scale site-directed mutagenesis and screening. Our preliminary data suggest that GACh sensors will have specificity, signal-to-noise ratio, kinetics and photostability suitable for real-time imaging of endogenous ACh signals. Here, I propose to complete the development and validation of GACh sensors following two specific aims: Aim 1 is to optimize and characterize GACh sensors. In our pilot work, we constructed a family of GACh sensors. We plan to use large-scale site-directed mutagenesis and screening to generate more GACh sensors with better performance (Aim 1a). Moreover, we will characterize the properties of GACh sensors in cultured cells and neurons (Aim 1b). We expect these experiments to optimize the specificity, signal-to-noise ratio, kinetics and photostability of GACh sensors. Aim 2 is to validate and utilize GACh sensors. In our preliminary study, we found that GACh sensors selectively detect exogenously applied ACh and endogenously released ACh. We will verify whether GACh sensors can be easily employed to detect ACh signals in various brain regions of both mice and rats (Aim 2a). Finally, we plan to explore the applications of GACh sensors in vitro and in vivo, and address a few fundamental questions about central cholinergic transmission (Aim 2b). We expect these experiments to testify the general applicability of GACh sensors in monitoring the dynamics of endogenous ACh signals and reveal some key features of cholinergic transmission.

项目总结