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Novel fluorescent sensors for imaging neuromodulation

用于神经调节成像的新型荧光传感器

基本信息

批准号：
10201786
负责人：
Yang DAN
金额：
$ 100.29万
依托单位：
UNIVERSITY OF CALIFORNIA BERKELEY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-08-15 至 2023-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10201786
关键词：
Acetylcholine Acute Affinity Agonist Amino Acid Sequence Animal Behavior Animals Area Argipressin Axon Behavior Behavioral Behavioral Paradigm Binding Binding Sites Brain Cell physiology Chemicals Cholecystokinin Cognition Communities Corpus striatum structure Coupled Data Dimensions Disease Dopamine Eating Electrophysiology (science)Endocannabinoids Engineering Feedback Fiber Fluorescence G-Protein-Coupled Receptors Goals Health Human Hypothalamic structure Image In Vitro Kinetics Lead Learning Libraries Ligand Binding Ligands Lipids Mammalian Cell Measures Membrane Membrane Proteins Memory Methods Microdialysis Midbrain structure Mission Molecular Conformation Monitor Moods Mus Nervous system structure Neuromodulator Neurons Neuropeptides Oxytocin Pathologic Peptides Performance Photometry Physiological Play Preparation Process Public Health Receptor Activation Research Resolution Role Signal Transduction Signaling Molecule Site Sleep Sleep Wake Cycle Slice Social Behavior Specificity Technology Therapeutic United States National Institutes of Health Universities Validation Vasopressins Viral Yang addiction base cell type detection of nutrient enhanced green fluorescent protein experience experimental study imaging modality in vivo in vivo evaluation in vivo imaging in vivo monitoring lens millisecond monoamine motor learning nanomolar nervous system disorder neural circuit neuroregulation non-invasive monitor novel pain sensation preservation prototype real time monitoring receptor relating to nervous system response screening sensor sleep regulation tool transmission process two-photon

项目摘要

SUMMARY Animal behaviors are orchestrated by the sophisticated nervous system, which is dynamically regulated by neuromodulators including lipids and neuropeptides. Endocannabinoids (eCBs) are neurolipids exist broadly in the brain and regulate learning and memory, addiction, pain sensation, and food intake. Among neuropeptides, cholecystokinin (CCK) is involved in nutrient sensing, food intake, and sleep regulation, and oxytocin (OXT) and vasopressin (AVP) play important roles in various aspects of social behaviors. However, how and when lipid and neuropeptide transmission occur in the brain are largely unclear. Existing methods (e.g. microdialysis) that measures brain chemical content suffer from low temporal and spatial resolution. Additionally, since neurolipid and neuropeptide releases often require repetitive neuronal firing and can occur at both axonal and dendritic sites, activity of the neuromodulator- releasing neurons cannot reliably predict where and when neurolipids and neuropeptides are released. Here we propose to develop a set of new tools for long-term monitoring of neurolipids and neuropeptides. Our strategy taps into their natural receptors, human G protein-coupled receptors (GPCRs), which are coupled to GFP. In the presence of neurolipids or neuropeptides, these GPCR Activation-Based (GRAB) sensors transform ligand binding-induced conformational changes into rapid fluorescent signals. We aim to develop and optimize neurolipid and neuropeptide GRAB sensors with >500% fluorescence change (dF/F) and 10- nanomolar affinity in vitro and validate these novel tools in brain slices ex vivo and mouse behavioral paradigms in vivo. In Aim 1, we will develop GRAB sensors for endocannabinoids, CCK, vasopressin, and OXT by systematically varying key sites involved in ligand binding, conformational change, etc. In Aim 2, we will validate the performance of these sensors in brain slice following long-term expression using viral tools. In Aim 3, we will use three different imaging methods (fiber photometry, epifluorescence and 2-photon imaging coupled with GRIN lens) in different behavioral paradigms to test in vivo performance of the novel GRAB sensors in mice. Feedback from experiments in Aims 2-3 will guide iterative optimization in Aim 1. Successful completion of our proposal will yield a suite of powerful tools and technical approaches, which will greatly facilitate studies of neurolipids and neuropeptides under both physiological and pathological conditions, helping reveal disease mechanisms, providing therapeutic guidance, and eventually benefiting human health.

总结