ANALYSIS OF DOPAMINE NERVE TERMINALS BY FLOW CYTOMETRY

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Dopamine (DA) neurons have been the focus of intensive interest because of their possible involvement in psychiatric and neurological disorders. However, the extent to which the inherent properties of DA neurons can be characterized is limited by the cellular complexity of most currently available experimental preparations. We propose to develop methods for the analysis and isolation of DA nerve terminals (synaptosomes) based upon flow cytometry. First, striatal synaptosomes will be permeabilized by fixation to enable penetration by immunoglobulins. Selective labeling of DA synaptosomes will be accomplished by indirect immunofluorescence techniques usng a monoclonal antibody directed against tyrosine hydroxylase (TH) and a fluorescein- labeled secondary antibody. DA and non-DA nerve terminals will then be separated by fluorescence-activated cell sorting. Control experiments will determine whether synaptosomes labeled and sorted using this method are enriched for TH content and whether labeling is decreased in response to lesions of nigrostriatal DA terminals. Pure populations of DA nerve terminals prepared in this manner will be used for protein analysis by 2 dimensional gel electrophoresis to determine whether discete proteins are enriched or depleted relative to non-DA terminals. Isolated DA terminals can also be examined, using flow cytometric and/or Western blot techniques, for the presence of particular proteins which have been proposed to be involved in the regulation of DA activity. Specifically, we will investigate the presence of guanine nucleotide regulatory proteins (G proteins) in purified DA nerve terminals. G proteins are known to play a coupling role in many receptor-effector systems in brain, and are thought to link DA receptor occupation to the generation of several second messengers. Flow cytometry can determine in a quantitative manner whether specific G proteins are associated with DA nerve terminals or with subpopulations of DA terminals. These data could provide the basis for rational models of the involvement of G proteins in the function of presynaptic DA receptors.