title: Brain Region-Specific Trafficking of the Dopamine Transporter.
abstract: The dopamine (DA) transporter (DAT) controls dopaminergic neurotransmission by removing extracellular DA. Although DA reuptake is proposed to be regulated by DAT traffic to and from the cell surface, the membrane trafficking system involved in the endocytic cycling of DAT in the intact mammalian brain has not been characterized. Hence, we performed immunolabeling and quantitative analysis of the subcellular and regional distribution of DAT using the transgenic knock-in mouse expressing hemagglutinin (HA) epitope-tagged DAT (HA-DAT) and by using a combination of electron microscopy and a novel method for immunofluorescence labeling of HA-DAT in acute sagittal brain slices. Both approaches demonstrated that, in midbrain somatodendritic regions, HA-DAT was present in the plasma membrane, endoplasmic reticulum, and Golgi complex, with a small fraction in early and recycling endosomes and an even smaller fraction in late endosomes and lysosomes. In the striatum and in axonal tracts between the midbrain and striatum, HA-DAT was detected predominantly in the plasma membrane, and quantitative analysis revealed increased DAT density in striatal compared with midbrain plasma membranes. Endosomes were strikingly rare and lysosomes were absent in striatal axons, in which there was little intracellular HA-DAT. Acute administration of amphetamine in vivo (60 min) or to slices ex vivo (10-60 min) did not result in detectable changes in DAT distribution. Altogether, these data provide evidence for regional differences in DAT plasma membrane targeting and retention and suggest a surprisingly low level of endocytic trafficking of DAT in the striatum along with limited DAT endocytic activity in somatodendritic areas.
  
  
  CONCLUSIONS
  The dopamine transporter (DAT) is the key regulator of the dopamine neurotransmission in the CNS. In the present study, we developed a new approach for studying DAT localization and dynamics in intact neurons in acute sagittal brain slices from the knock-in mouse expressing epitope-tagged DAT. For the first time, the fluorescence imaging analysis of DAT was combined with the immunogold labeling of DAT and quantitative electron microscopy. In contrast to numerous studies of DAT trafficking in heterologous expression systems and dissociated cultured neurons, studies in intact neurons revealed a surprisingly low amount of endocytic trafficking of DAT at steady state and after acute amphetamine treatment and suggested that non-vesicular transport could be the main mechanism establishing DAT distribution within the dopaminergic neuron.
authors.affiliation: Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, and.
authors.forename: Ethan R
authors.initials: ER
authors.lastname: Block
authors.affiliation: Department of Neuroscience, University of Pittsburgh Dietrich School of Arts and Sciences, Pittsburgh, Pennsylvania 15260.
authors.forename: Jacob
authors.initials: J
authors.lastname: Nuttle
authors.affiliation: Department of Neuroscience, University of Pittsburgh Dietrich School of Arts and Sciences, Pittsburgh, Pennsylvania 15260.
authors.forename: Judith Joyce
authors.initials: JJ
authors.lastname: Balcita-Pedicino
authors.affiliation: Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, and.
authors.forename: John
authors.initials: J
authors.lastname: Caltagarone
authors.affiliation: Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, and.
authors.forename: Simon C
authors.identifier: http://orcid.org/0000-0003-4092-1552
authors.initials: SC
authors.lastname: Watkins
authors.affiliation: Department of Neuroscience, University of Pittsburgh Dietrich School of Arts and Sciences, Pittsburgh, Pennsylvania 15260 sesack@pitt.edu sorkin@pitt.edu.
authors.forename: Susan R
authors.initials: SR
authors.lastname: Sesack
authors.affiliation: Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, and sesack@pitt.edu sorkin@pitt.edu.
authors.forename: Alexander
authors.initials: A
authors.lastname: Sorkin
mesh_terms: D000661:Amphetamine / Q000494:pharmacology; D000818:Animals; D001369:Axons / Q000737:chemistry / Q000648:ultrastructure; D002451:Cell Compartmentation; D002478:Cells, Cultured; D003342:Corpus Striatum / Q000378:metabolism / Q000473:pathology; D004298:Dopamine / Q000378:metabolism*; D050483:Dopamine Plasma Membrane Transport Proteins / Q000378:metabolism*; D059290:Dopaminergic Neurons / Q000378:metabolism; D004705:Endocytosis; D005260:Female; D055879:Gene Knock-In Techniques; D057809:HEK293 Cells; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D021381:Protein Transport; D051381:Rats; D013347:Subcellular Fractions
chemical_list: D050483:Dopamine Plasma Membrane Transport Proteins; D000661:Amphetamine; D004298:Dopamine
keywords: dopamine; electron microscopy; endocytosis; transporter