CNiFERs Cell-based Neurotransmitter Fluorescent Engineered Reporters

CNiFERs 基于细胞的神经递质荧光工程记者

基本信息

批准号：
8654321
负责人：
David Kleinfeld
金额：
$ 46.22万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-07-01 至 2016-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8654321
关键词：
Acetylcholine Address Animals Antipsychotic Agents Area Basal Nucleus of Meynert Beds Binding Biological Assay Blood Vessels Blood flow Brain Butyric Acids Cataloging Catalogs Cell Line Cells Chemicals Clozapine Cocaine Communication Contralateral Data Detection Dopamine Drug effect disorder Electric Stimulation Engineering Environment Family Fluorescence Fluorescence Resonance Energy Transfer G-Protein-Coupled Receptors GTP-Binding Proteins Generations Goals Image Implant Individual Interneurons Ipsilateral Lighting Maps Measurement Measures Mediating Mental disorders Microscopy Monitor Muscarinic Antagonists Neocortex Nervous system structure Neurons Neuropeptides Neurosecretory Systems Neurotransmitters Norepinephrine Optical reporter Optics Parietal Lobe Pathway interactions Pattern Peptides Pharmacologic Substance Pharmacology Physiological Play Property Proteins Rattus Reporter Resolution Rodent Role Schizophrenia Serotonin Signal Transduction Somatostatin Stimulus Technology Testing Time Transgenic Mice Vasoactive Intestinal Peptide Ventral Tegmental Area Work addiction base brain cell cholinergic design detector human CHRM1 protein hypocretin in vivo monoamine neocortical neuronal patterning neuropeptide Y neuroregulation neurotransmission new technology olanzapine optogenetics preference public health relevance receptor receptor coupling research study response sensor small molecule transmission process two-photon

项目摘要

DESCRIPTION (provided by applicant): We propose to advance a technology that, for the first time, will permit in vivo detection of any neurotransmitter that binds to a G-protein coupled receptor. This includes the monoamines, which play a fundamental role in neuromodulation and a biomedical role in addiction and mental disorders, and peptide transmitters, which are important for the neuroendocrine system and control of vascular tone and blood flow. At present, only limited in vivo assays are available to detect monoamines and peptide neurotransmission. This deficit is a major impediment to understanding normal signaling in the brain. To fill this gap of missing, we introduce Cell-based Neurotransmitter Fluorescent Engineered Reporters (CNiFERs) for the optical measurement of exogenous receptor activity in vivo. A CNiFER is a clonal cell-line that is engineered to express a specific metabotropic receptor that couples to G proteins and a genetically encoded FRET-based Ca2+ sensor that detects changes in intracellular [Ca2+]. Stimulation of the metabotropic receptor leads to elevations in cytosolic [Ca2+], providing a direct and rapid readout of local neurotransmitter activity. CNiFERs are acutely or chronically implanted and fluorescence measured with in vivo two-photon microscopy. This new technology will make it possible to map the spatial patterns of in vivo signaling with up to ~ 100 5M spatial precision and ~ 1 s temporal resolution.

描述（由申请人提供）：我们提议推进一项技术，该技术首次允许在体内检测任何与G蛋白偶联受体结合的神经递质。其中包括单胺，它们在神经调节中起着基本作用，在成瘾和精神疾病中起生物医学的作用以及肽发射器，这对于神经内分泌系统以及对血管张力和血液流动的控制至关重要。目前，只有有限的体内测定可检测单胺和肽神经传递。这种赤字是理解大脑正常信号传导的主要障碍。为了填补这一缺失的空白，我们引入了基于细胞的神经递质荧光工程记者（CNIFER），以在体内对外源受体活性的光学测量。 CNIFER是一种经过精心设计的克隆细胞系，可表达特定的代谢型受体，该受体将其伴随到G蛋白和基于遗传编码的基于FRET的CA2+传感器，可检测细胞内的变化[Ca2+]。刺激代谢受体会导致胞质[Ca2+]的升高，从而直接且快速地读出局部神经递质活性。 CNIFER是急性或慢性植入的，并用体内两光子显微镜测量荧光。这项新技术将使可以绘制体内信号的空间模式，并具有〜100 5m的空间精度和〜1 s的时间分辨率。