Endocytic down-regulation of the striatal dopamine transporter by amphetamine in sensitized mice in sex-dependent manner

Dopamine transporter (DAT) controls dopamine signaling in the brain through the reuptake of synaptically released dopamine. DAT is a target of abused psychostimulants such as amphetamine (Amph). Acute Amph is proposed to cause transient DAT endocytosis which among other Amph effects on dopaminergic neurons elevates extracellular dopamine. However, the effects of repeated Amph abuse, leading to behavioral sensitization and drug addiction, on DAT traffic are unknown. Hence, we developed a 14-day Amph-sensitization protocol in knock-in mice expressing HA-epitope tagged DAT (HA-DAT) and investigated effects of Amph challenge on HA-DAT in sensitized animals. Amph challenge resulted in the highest locomotor activity on day 14 in both sexes, which was however sustained for 1 hour in male but not female mice. Strikingly, significant (by 30-60%) reduction in the amount of the HA-DAT protein in striatum was observed in response to Amph challenge of sensitized males but not females. Amph reduced Vmax of dopamine transport in striatal synaptosomes of males without changing Km values. Consistently, immunofluorescence microscopy revealed a significant increase of HA-DAT co-localization with the endosomal protein VPS35 only in males. Amph-induced HA-DAT down-regulation in the striatum of sensitized mice was blocked by chloroquine, vacuolin-1 (inhibitor of PIKfive kinase), and inhibitor of Rho-associated kinases (ROCK1/2), indicative of the involvement of endocytic trafficking in DAT down-regulation. Interestingly, HA-DAT protein down-regulation was observed in nucleus accumbens and not in dorsal striatum. We propose that Amph challenge in sensitized mice leads to ROCK-dependent endocytosis and post-endocytic traffic of DAT in a brain-region-specific and sex-dependent manner.