In this study we have established methods for visualization and tracking of the dopamine transporter (DAT) in cultured dopaminergic neurons in real time using a fluorescent cocaine analogue JHC 1-64 and confocal fluorescence microscopy. The initial binding experiments in HEK 293 cells stably expressing EGFP-DAT revealed that JHC1-64 specifically labeled surface expressed EGFP-DAT and thus that the compound did not cross the membrane of intact HEK 293 cells. Upon treating the HEK 293 cells with the Protein Kinase C activator PMA, the surface expressed and JHC 1-64 labeled EGFP-DAT was internalized, corroborating the usefulness of this cocaine analogue as a tool for monitoring DAT trafficking. In the cultured neurons JHC 1-64 labeled the surface of almost the entire dopaminergic neurons including the cell body, although not as strongly as some of the neuronal extensions. This labeling by JHC 1-64 was prevented by excess concentrations of dopamine, cocaine, mazindol, or RTI-55, whereas the norepinephrine and/or serotonin transporter specific inhibitors desmethylimipramine and citalopram did not affect fluorescent labeling of the neurons. This strongly supports that JHC 1-64 specifically labels endogenously surface expressed DAT. The specific labeling of dopaminergic neurons was further supported by immunocytochemistry studies indicating that JHC 1-64 selectively labeled tyrosine hydroxylase positive neurons. Interestingly, immunostaining for the synaptic marker, VMAT2, revealed that JHC 1-64 labeling was clearly not restricted to synaptic sites. Altogether, the data provide the first direct visualization of the DAT in living neurons and support an unexpected uniform and widespread expression of the transporter in cultured neurons.
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Society for Neuroscience 35th annual meeting, 2005