The functional role of the dopamine transporter (DAT) in central dopaminergic neurotransmission was assessed further by investigating the consequences on dopamine (DA) turn-over of up- and down-regulation of this protein induced by a non-viral gene transfer approach. For this purpose, expression plasmids containing the sense or antisense coding sequence of DAT complexed with the cationic polymer, polyethylenimine (PEI), were injected into the rat substantia nigra, the brain region containing the majority of DA cell bodies.
Before in vivo injection, the efficacies of the different DNA constructs were assessed by transfection studies into LLC-PK1 cells. Stereotaxic administration of the sense plasmid complexed to PEI induced, 3 days later, a significant increase in the immunoautoradiographic labelling by anti-DAT antibodies of the substantia nigra and various DA projection areas. These effects were associated with a significantly enhanced capacity of striatal synaptosomes to take up [3H]-DA and lasted up to 14 days postinjection. In contrast, 7 days after intranigral administration of the antisense plasmid complexed to PEI, we observed a significant decrease of DAT immunolabelling in the substantia nigra and [3H]-DA uptake by striatal synaptosomes.
Whereas DA turnover in the striatum was unaltered 3 days after intranigral administration of the sense plasmid, it was increased 7 days after intranigral administration of the antisense construct.
These data indicate that non-viral transfer of the sense or antisense coding sequence of DAT can be used as a novel approach to induce long-term changes in central DA neurotransmission.