Mapping dopamine neuron cotransmission by proximity detection

通过邻近检测绘制多巴胺神经元共传递

• 批准号: 8985749
• 负责人: STEPHEN RAYPORT
• 金额: $ 25.72万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8985749
• 关键词: Address; Affect; Area; Axon; Biological Assay; Brain; Cell membrane; Corpus striatum structure; Detection; Development; Disease; Disease model; Dopamine; Dopamine Receptor; Electron Microscopy; Elements; Epitopes; Fluorescence; Functional disorder; Genetic; Glutamates; Hybrids; Image; Imagery; In Situ Hybridization; Label; Ligation; Maps; Measurement; Measures; Membrane; Membrane Proteins; Mental disorders; Modification; Molecular; Mus; Neurons; Neurotransmitters; Parkinson Disease; Pharmaceutical Preparations; Phenotype; Population; Presynaptic Terminals; Proteins; Reading; Rhodopsin; Role; Schizophrenia; Signal Transduction; Site; Slice; Specificity; Synapses; Synaptic Vesicles; Technology; Time; Tissues; Transgenic Organisms; Vesicle; Wild Type Mouse; addiction; base; cholinergic neuron; clinical material; density; dopaminergic neuron; gamma-Aminobutyric Acid; neuropsychiatry; neurotransmitter release; noradrenergic; optogenetics; psychostimulant; public health relevance; synaptic function; tomography; vesicle-associated membrane protein; vesicular SNARE proteins; vesicular glutamate transporter 2; vesicular monoamine transporter 2

 DESCRIPTION (provided by applicant): Many brain neurons release a mix of transmitters, but it has been challenging to identify their synapses based on transmitter status. The application of proximity ligation assay (PLA) technology in optogenetic mice offers a way to address these issues comprehensively, enabling visualization of all synapses of an identified population of neurons and their transmitter status. PLA is a hybrid immunochemical and in situ hybridization approach in which two selected epitopes, which are within about 20 nm of each other, generate a discrete fluorescence signal. Synaptic vesicles at the active zone are a distinctive functional element of synapses, which are within 20 nm of the plasmalemma when poised for release. With PLA, we have recently visualized dopamine receptor oligomerization in striatal neurons and addressed the developmental trajectory of dopamine receptor colocalization. In this project, we will use PLA to visualize the proximity of synaptic markers in dopamine neurons to determine key measures of dopamine neuron synaptic function. The specific aims are to: <1> Visualize dopamine neuron synaptic release sites specifically, then visualize dopamine neuron release sites based on the transmitter released. This will be done in optogenetic mice conditionally expressing the exogenous membrane, axon-targeted protein ChR2-EYFP in dopamine neurons by detecting proximity of ChR2-EYFP in the plasmalemma to key synaptic vesicle proteins. <2> Correlate PLA measurements of dopamine neuron connectivity in target areas with functional connectivity to determine the functional readout of the PLA measurements. <3> Demonstrate the ability of PLA to identify synaptic release sites in tissue from wild type mice using proximity of native plasmalemma and vesicular membrane proteins. Stereology will be used for systematic image acquisition and mapping. The quantitative information obtained with PLA will enable asking how does synaptic connectivity change, with regional specificity, over development, and in disease models? PLA will further enable addressing questions such as, where in the brain do drugs impact most, over what time, and for how long? Once prototyped in the dopamine neurons, the PLA approaches developed in this project could be extended to the other major modulatory neuron groups. This will lay the groundwork for post-mortem studies in clinical material, and enable asking, questions such as, which synaptic connections are most affected in disease states, do treatments impact connectivity, are treatments inducing compensatory changes or reversing pathological changes?