RT Journal Article
SR Electronic
T1 The dopamine transporter: immunochemical characterization and localization in brain
JF The Journal of Neuroscience
JO J. Neurosci.
FD Society for Neuroscience
SP 1714
OP 1723
DO 10.1523/JNEUROSCI.15-03-01714.1995
VO 15
IS 3
A1 BJ Ciliax
A1 C Heilman
A1 LL Demchyshyn
A1 ZB Pristupa
A1 E Ince
A1 SM Hersch
A1 HB Niznik
A1 AI Levey
YR 1995
UL http://www.jneurosci.org/content/15/3/1714.abstract
AB 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.