Subcellular localization and molecular topology of the dopamine transporter in the striatum and substantia nigra

Abstract

Plasma membrane transporters remove neurotransmitters from the extracellular 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 subcellular location of the dopamine transporter (DAT) by immunoperoxidase and immunogold electron microscopy, using monoclonal antibodies to both the N-terminus and the second extracellular loop. The two DAT epitopes were found on opposite faces of cellular and intracellular membranes, providing confirmation of the predicted molecular topology of DAT. In the striatum, DAT was localized in the plasma membrane of axons and terminals. Double immunocytochemistry demonstrated DAT colocalization with two other markers of nigrostriatal terminals, tyrosine hydroxylase and D2 dopamine receptors. The latter was thus demonstrated to be an autoreceptor. Labeled striatal terminals formed symmetrical synapses with spines, dendrites, and perikarya. DAT was not identified within any synaptic active zones, however, even using serial section analysis. These results suggest that striatal dopamine reuptake may occur outside of synaptic specializations once dopamine diffuses from the synaptic cleft. In the substantia nigra, DAT appears to be specifically transported into dendrites, where it can be found in smooth endoplasmic reticulum, plasma membrane, and pre- and postsynaptic active zones. These localizations suggest that DAT modulates the intracellular and extracellular dopamine levels of nigral dendrites. Within the perikarya of pars compacta neurons, DAT was localized primarily to rough and smooth endoplasmic reticulum, Golgi complex, and multivesicular bodies, identifying probable sites of synthesis, modification, transport, and degradation.

Redistribution of DAT/α-Synuclein Complexes Visualized by “In Situ” Proximity Ligation Assay in Transgenic Mice Modelling Early Parkinson's Disease

“…In the substantia nigra, the DAT can be specifically transported into dendrites. In particular, it is involved in the tuning of intracellular and extracellular DA levels in the somatodendritic compartment [49]. Therefore, our observations indicate that, whether α-synuclein aggregation may compromise DAT trafficking, this event may lead to alterations in somatodendritic DA release.…”

Striatal dopamine neurotransmission: Regulation of release and uptake

“…This anatomy suggests that it is necessary for DA to spillover from release sites to activate its receptors, and therefore that DA transmission is mainly extrasynaptic [423]. Consistent with this mode of action, DA uptake sites are rarely observed on the DA synaptic membrane and rather, may be distributed throughout the membrane of DA fibers [358,423,424]. …”

Trafficking of dopamine transporters in psychostimulant actions

“…2). Electron microscopy studies demonstrated the presence of DAT in the ER in the soma and proximal dendrites of DA neurons in VTA and SNc [13, 23]. …”

“…Electron microscopy analysis revealed the presence of DAT in the plasma membrane of axonal varicosities and between these varicosities in dSTR and NAc. Plasma membrane DAT was also observed in the distal dendrites, but not in the neuronal soma [13, 23]. Interestingly, in axonal varicosities DAT was not detected in the active zone of the synapses, but rather it was located extrasynaptically, including perisynaptically[23, 32].…”