Chronic amphetamine exposure causes long‐term effects on dopamine uptake

Abstract

The dopamine transporter (DAT) is a plasma membrane transporter that clears released dopamine from the synaptic cleft; therefore it is an important regulator of extracellular levels of dopamine. DAT is also the main molecular target of many drugs of abuse, most notably the psychostimulants cocaine and amphetamine. Amphetamine (AMPH) is a substrate of DAT and competes for reuptake with dopamine. Several groups have shown that acute AMPH treatments alter the function and the number of DAT on the cell membrane. However, no data is available regarding the long‐term effects of AMPH on DAT activity. Data collected in our laboratory suggest that parental AMPH exposure affects dopamine uptake in C. elegans dopaminergic neurons isolated from progeny. These results led us to hypothesize that reduced dopamine reuptake in subsequent generations, as a result of parental AMPH exposure, is caused by down‐regulation of DAT. In order to overcome the complexity of multicellular organisms and the challenges of primary cell isolation and culture, where very few cells differentiate into dopaminergic neurons, we investigated the long‐term effects caused by AMPH in simplified systems. Initial experiments were carried out in LLC‐PK1 cells, stably transfected with human DAT. Cells were pre‐exposed to AMPH for 15 hours and uptake assays, carried out after cells had doubled, showed a significant decrease in dopamine uptake compared to control (untreated cells). These data suggest that changes caused by AMPH were maintained after one cell cycle. We repeated the same experiment using the human neuroblastoma SH‐SY5Y cells which can be differentiated into authentic dopaminergic neurons. Prior to the uptake assay one group of undifferentiated SH‐SY5Y cells was exposed to AMPH for 15 hours and the other group was treated with control solution. The cells were then allowed to grow and cross generations before they were treated with Retinoic Acid (RA) to induce differentiation. Uptake assays carried out 5 days after RA initiated differentiation revealed a significant decrease in dopamine uptake in AMPH treated groups with respect to control. These results suggest that parental AMPH pretreatment down regulates the expression or activity of DAT. We are currently investigating whether chronic exposure to AMPH in progenitor cells alters DAT expression in daughter cells.Support or Funding InformationCOBRE P20 GM104360