Amphetamine Exposure During Embryogenesis Leads To Long-Term And Transgenerational Increase In Behavioral Response And Decrease In Dopamine Uptake

Amphetamine (AMPH) is widely prescribed for the treatment of ADHD and a highly abused substance in society, yet little is known about the long-term effects of the drug. Here, we used Caenorhabditis elegans (C. elegans) to establish a model for the long-term and transgenerational effects of AMPH exposure on behavior. Furthermore, experiments were conducted to explore the molecular mechanisms of AMPH that were altered by embryonic AMPH exposure. C. elegans have a well characterized behavioral response to AMPH known as Swimming Induced Paralysis (SWIP). For the SWIP test, animals are placed in fluid, which normally induces a thrashing behavior. However, in the presence AMPH, the animals display a time- and dose-dependent paralysis. AMPH increases the levels of dopamine in the synapse by causing reverse transport through the protein known as the dopamine transporter (DAT), and the SWIP behavior has been shown to be dependent on dopaminergic transmission. We exposed embryos to either control solution alone (M9 solution) or 500μM AMPH dissolved in control solution for 15 hours. 4 days later the SWIP test was performed on young adult animals, revealing that animals previously exposed to AMPH as embryos displayed a higher response to AMPH. The progeny of both groups were tested for SWIP as well. Interestingly, the progeny of the animals exposed to AMPH as embryos showed a higher SWIP response with respect to the progeny of control animals, demonstrating that AMPH had both a long-term and transgenerational effect on the animals. Because the SWIP behavior was previously shown to be dependent on dopaminergic transmission, we performed DA uptake assays using primary cell cultures made from F1 generation animals to investigate alterations in DATs ability to uptake dopamine. Results from the uptake assays showed that primary cultures made from the progeny of animals exposed to AMPH as embryos had reduced ability to uptake DA with respect to control cultures. To further investigate the reduced uptake ability following AMPH exposure, a human neuroblastoma cell line (SH-SY5Y) was exposed to 15 hour of AMPH, and 5 days later, a DA uptake assay using a concentration response of DA was carried out. Results showed that the cells had a reduced Vmax with no change to Km, suggesting a reduced amount of DAT in the cells. We investigated changes in histone methylation as a mechanism for the long-term and transgenerational effect observed. Histones are proteins, which DNA wraps around to form the nucleosome, and methylation changes on histones can modify the binding of DNA to histones leading to a change in gene expression. Western blots of whole animal protein revealed a decreased level of histone 3 lysine 4 trimethylation (H3K4me3) in the F1 generation of AMPH exposed animals. Additionally, a reduction in the enzymes responsible for H3K4me2 methylation and H3K4me3 demethylation was observed in F1 progeny of AMPH exposed animals. Suggesting that AMPH exposure during embryogenesis alters methylation of specific histone markers. Taken together, these experiments show that in C. elegans, AMPH exposure causes a long-term and transgenerational alteration in behavioral response to AMPH, which correlates to alterations in DAT uptake ability