Immunogold Localization of the Dopamine Transporter: An Ultrastructural Study of the Rat Ventral Tegmental Area

Abstract

The dopamine transporter (DAT) plays an important role in the plasmalemmal reuptake of dopamine and, thus, in the termination of normal dopaminergic neurotransmission. DAT is also a major binding site for cocaine and other stimulants, the psychoactive effects of which are associated primarily with the inhibition of dopamine reuptake within mesocorticolimbic dopaminergic neurons. We used electron microscopy with an anti-peptide antiserum directed against the N-terminal domain of DAT to determine the subcellular localization of this transporter in the rat ventral tegmental area (VTA), the region that contains the cell bodies and dendrites of these dopaminergic neurons. We show that in the VTA, almost 95% of the DAT immunogold-labeled profiles are neuronal perikarya and dendrites, and the remainder are unmyelinated axons. Within perikarya and large proximal dendrites, almost all of the DAT immunogold particles are associated with intracellular membranes, including saccules of Golgi and cytoplasmic tubulovesicles. In contrast, within medium-to smalldiameter dendrites and unmyelinated axons, most of the DAT gold particles are located on plasma membranes. In dually labeled tissue, peroxidase reaction product for the catecholaminesynthesizing enzyme tyrosine hydroxylase is present in DATimmunoreactive profiles. These findings suggest that intermediate and distal dendrites are both the primary sites of dopamine reuptake and the principal targets of cocaine and related psychostimulants within dopaminergic neurons in the VTA.

The somatodendritic release of dopamine in the ventral tegmental area and its regulation by afferent transmitter systems

“…This makes the somatodendritic uptake process in midbrain less efficient than that in terminal fields [67]. Autoradiographic and immunocytochemical studies have evidenced the existence of dopamine transporter in the VTA [77 -80], localized almost exclusively to somata and dendrites of dopaminergic neurons [81]. In accordance with the distribution of the protein, dopamine transporter mRNA is also expressed, albeit moderately, in the VTA [82 -86].…”

Three Ubiquitin Conjugation Sites in the Amino Terminus of the Dopamine Transporter Mediate Protein Kinase C–dependent Endocytosis of the Transporter

“…DAT is synthesized in the soma of DA neurons located in the substantia nigra and ventral tegmental area and trafficked to axonal processes projecting mainly to the dorsal striatum and nucleus accumbens, where it resides in the plasma membrane near the active zones of dopaminergic synapses (Nirenberg et al, 1997a(Nirenberg et al, , 1997b. The intensity and duration of dopaminergic neurotransmission depends on the level of the surface expression of DAT, which is regulated by trafficking of DAT between the plasma membrane and intracellular compartments.…”

“…Recently, a three-dimensional (3-D) structure of the homologous bacterial leucine transporter from Aquifex aeolicus (LeuT Aa ) has been solved at 1.65-Å resolution, providing a valuable framework for further studies of the structure and transport mechanisms of the family of Na ϩ /Cl Ϫ -dependent neurotransmitter transporters (Yamashita et al, 2005). This structure confirmed the predicted topology of SLC6 family transporters; however, it did not provide structural information about the amino-and carboxyl-termini of the transporters.DAT is synthesized in the soma of DA neurons located in the substantia nigra and ventral tegmental area and trafficked to axonal processes projecting mainly to the dorsal striatum and nucleus accumbens, where it resides in the plasma membrane near the active zones of dopaminergic synapses (Nirenberg et al, 1997a(Nirenberg et al, , 1997b. The intensity and duration of dopaminergic neurotransmission depends on the level of the surface expression of DAT, which is regulated by trafficking of DAT between the plasma membrane and intracellular compartments.…”

Multiple Molecular Determinants in the Carboxyl Terminus Regulate Dopamine Transporter Export from Endoplasmic Reticulum

“…The maximal surface expression of DAT is detected in synapses located along the axonal processes in striatum and in distal regions of dendrites in the ventral tegmental area (6,7). This specific targeting and the normal activity of DAT in neurons require efficient export of newly synthesized transporters from the ER, sorting to transport carriers in the Golgi complex, and delivery and retention of the transporters at the presynaptic functional sites.…”