SUBCELLULAR-LOCALIZATION AND MOLECULAR TOPOLOGY OF THE DOPAMINE TRANSPORTER IN THE STRIATUM AND SUBSTANTIA-NIGRA

Plasma membrane transporters remove neurotransmitters from the extrace llular space and have been postulated to terminate synaptic activity. Their specific roles in synaptic and nonsynaptic neurotransmission at a cellular level, however, remain unclear. We have determined the subc ellular location of the dopamine transporter (DAT) by immunoperoxidase and immunogold electron microscopy, using monoclonal antibodies to bo th the N-terminus and the second extracellular loop. The two DAT epito pes were found on opposite faces of cellular and intracellular membran es, providing confirmation of the predicted molecular topology of DAT. In the striatum, DAT was localized in the plasma membrane of axons an d terminals. Double immunocytochemistry demonstrated DAT colocalizatio n with two other markers of nigrostriatal terminals, tyrosine hydroxyl ase and D2 dopamine receptors. The latter was thus demonstrated to be an autoreceptor. Labeled striatal terminals formed symmetrical synapse s with spines, dendrites, and perikarya. DAT was not identified within any synaptic active zones, however, even using serial section analysi s. These results suggest that striatal dopamine reuptake may occur out side of synaptic specializations once dopamine diffuses from the synap tic cleft. In the substantia nigra, DAT appears to be specifically tra nsported into dendrites, where it can be found in smooth endoplasmic r eticulum, plasma membrane, and pre- and postsynaptic active zones. The se localizations suggest that DAT modulates the intracellular and extr acellular dopamine levels of nigral dendrites. Within the perikarya of pars compacta neurons, DAT was localized primarily to rough and smoot h endoplasmic reticulum, Golgi complex, and multivesicular bodies, ide ntifying probable sites of synthesis, modification, transport, and deg radation. (C) 1997 Wiley-Liss, Inc.