The dopamine transporter: immunochemical characterization and localization in brain

Abstract

Antibodies specific for the dopamine transporter (DAT) was developed and characterized by immunoblot analysis, immunoprecipitation, and immunocytochemistry, and used for immunolocalization of transporter protein in rat brain at the light microscopic level. Antibodies targeting the N-terminus, the second extracellular loop, and the C- terminus were generated from fusion proteins containing amino acid sequences from these respective regions. Immunoblot analysis demonstrated that N-terminus and loop antibodies were specific for expressed cloned DAT, recognized transporter protein in rat and human striatal membranes, and were sensitive to preabsorption with excess homologous fusion protein. Immunoprecipitation studies demonstrated that anti-DAT antisera recognized solubilized, radiolabeled DAT protein in a concentration-dependent manner. DAT immunocytochemistry with these antibodies were also sensitive to preabsorption with fusion protein and to lesions of dopaminergic mesostriatal and mesocorticolimbic pathways. Regional distribution of DAT coincided with established dopaminergic innervation of several regions, including ventral mesencephalon, medial forebrain bundle, and dorsal and ventral striatum. However, certain mismatches between immunocytochemical distributions of DAT and tyrosine hydroxylase were apparent, indicating that dopaminergic systems are heterogeneous and may use independent mechanisms for the regulation of dopamine levels in brain. The generation of specific DAT antibodies will permit further characterization of the cellular and subcellular localization of DAT protein, and of dopaminergic circuits in neurological and psychiatric disorders.

Tyrosine Hydroxylase- and Dopamine Transporter-Immunoreactive Axons in the Primate Cerebellum Evidence for a Lobular- and Laminar-Specific Dopamine Innervation

“…Indeed, recent studies suggest that DAT is the critical element regulating the actions of DA (Jaber et al 1997;Kuhar 1998). Thus, DAT immunoreactivity would be expected to label DA-containing axons selectively, a view supported by multiple lines of evidence in other brain regions of both rodents and primates (Ciliax et al 1995(Ciliax et al , 1999Freed et al 1995;Lewis and Sesack 1997;Sesack et al 1998). For example, the DAT antibody used in this study robustly labels the DA-containing neurons of the primate mesencephalon, but does not label any neurons that utilize other monoamines (Ciliax et al 1999;Lewis and Sesack 1997).…”

“…Because TH is expressed in all catecholamine-synthesizing neurons, we also evaluated the distribution of axons immunoreactive for DAT, a protein found only in DA neurons (Ciliax et al 1995;Freed et al 1995;Lewis and Sesack 1997;Sesack et al 1998). In all animals examined, axons labeled by the DAT antibody were present only in the cerebellar vermis (Fig.…”

“…Consequently, in this study, we used immunocytochemical techniques to address the following questions. First, does the cerebellum of macaque monkeys contain axons immunoreactive for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, and for the DA membrane transporter (DAT), a specific marker of DA axons (Ciliax et al 1995;Freed et al 1995;Lewis and Sesack 1997;Sesack et al 1998)? Second, if so, do the distributions of TH-and DAT-positive axons differ from that of axons immunoreactive for dopamine-␤ -hydroxylase (DBH), the synthesizing enzyme for noradrenaline?…”

Clozapine Normalizes Prefrontal Cortex Dopamine Transmission in Monkeys Subchronically Exposed to Phencyclidine

“…In the dorsolateral prefrontal cortex a decrease in DA level contributed significantly to the increase in HVA/DA ratio elicited by clozapine. In the mesoprefrontal DA system, where the recapture mechanism is inefficient (Ciliax et al, 1995;Sesack et al, 1998;Lewis et al, 2001) and transmitter homeostasis is more dependent on synthesis (Galloway et al, 1986), increases in functional activity can be reflected by a decrease in transmitter as well as an increase in metabolite level (Bean and Roth, 1991). It is not entirely clear why clozapine, but not typical antipsychotic drugs such as haloperidol, has a preferential action on cortical DA systems compared to the striatal DA systems.…”

The promiscuity of the dopamine transporter: Implications for the kinetic analysis of [3H]serotonin uptake in rat hippocampal and striatal synaptosomes

“…One explanation for this disparity may be divergent transporter expression in midbrain as compared to hippocampus, most notably those transporters capable of transporting [ 3 H]5-HT. The midbrain ventral tegmental area and substantia nigra both display significant DAT expression (Mennicken et al, 1992, Ciliax et al, 1995, Freed et al, 1995, Hersch et al, 1997 and, as such, the reported kinetic analyses of 5-HT uptake into midbrain synaptosomes may reflect uptake via multiple transporters and not uptake specifically via SERT. In a more recent study, Samuvel and colleagues (2005) reported K m and V max values for 5-HT uptake into midbrain synaptosomes using 0.1 μM fluoxetine to define nonspecific uptake.…”