Dopamine (DA) transporter (DAT) functions at the surface of dopaminergic neurons to clear extracellular DA. DAT surface levels are regulated by endocytosis. However, the endosome-lysosome system is not well characterized in dopaminergic neurons and the endocytic trafficking of endogenous DAT is poorly studied. Hence we analyzed the distribution of endocytic compartments and DAT localization in cultured rat embryonic and postnatal neurons using fluorescence microscopy. Early Rab5 and EEA.1 containing endosomes were mostly found in somatodendritic regions of neurons, whereas endosomes containing recycling markers were primarily found in axons. In axons, DAT was located mainly in recycling endosomes and plasma membrane whereas in cell bodies and dendrites DAT was detected in early, late and recycling endosomal compartments. Subcellular fractionation of adult rat striatal synaptosomes demonstrated that DAT is enriched in fractions containing plasma membrane and recycling endosomes. This pattern of DAT distribution was not altered upon activation of protein kinase C in postnatal DA neurons. Altogether, our data suggest that axonal DAT mainly shuttles between the plasma membrane and recycling endosomes, whereas in the somatodendritic region of neurons DAT traffics through all conventional endosomal pathways.
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