Amphetamine exposure during embryogenesis changes expression and function of the dopamine transporter in Caenorhabditis elegans offspring

Abstract

The dopamine transporter (DAT) is a transmembrane protein that regulates dopamine (DA) neurotransmission by binding to and moving DA from the synaptic cleft back into the neurons. Besides moving DA and other endogenous monoamines, DAT is also a neuronal carrier for exogenous compounds such as the psychostimulant amphetamine (Amph), and several studies have shown that Amph-induced behaviors require a functional DAT. Here, we demonstrate that exposure to Amph during early development causes behavioral, functional, and epigenetic modifications at the Caenorhabditis elegans DAT gene homolog, dat-1, in C. elegans offspring. Specifically, we show that, while embryos exposed to Amph generate adults that produce offspring with no obvious behavioral alterations, both adults and offspring exhibit an increased behavioral response when challenged with Amph. Our functional studies suggest that a decrease in DAT-1 expression underlies the increased behavioral response to Amph seen in offspring. Moreover, our epigenetic data suggest that histone methylation is a mechanism utilized by Amph to maintain changes in DAT-1 expression in offspring. Taken together, our data reveal that Amph, by altering the epigenetic landscape of DAT, propagates long-lasting functional and behavioral changes in offspring.

Keywords: C. elegans; amphetamine; dopamine; dopamine transporter; histone methylation.

Figure-1.

Behavioral experiments in adult and offspring wildtype and dat-1 ko worms. (A) experimental design to examine behavioral, physiological, and functional outcomes of embryonic amphetamine (Amph) treatments. Wildtype and dat-1 ko embryos were exposed to 0.5 mM Amph for 15 hours. Then SWIP values were measured in adults (B) and offspring (C) challenged for 5 minutes with 0.5 mM Amph or vehicle solution. Numbers on top of each bar indicate the number of trials (N). A minimum of 210 (A) and 198 (B) worms were tested in each experimental group. Statistical analysis was performed using 2way-ANOVA Tukey’s multiple comparisons with Interaction Factor: F(3, 95) = 9.49 and F(3, 94) = 9.56 for F0 and F,1 respectively and Treatment Factor: F(3, 95) = 31 and F(3, 94) = 8.1 for F0 and F1 generations, respectively.

Figure-2.

Chromatin Immune Precipitation (ChIP) assays. Offspring originated from parents exposed to 0.5 mM amphetamine (Amph) (red bars) or control solution (blue bars) during embryogenesis were processed to obtain chromatin, and immune precipitated using the H3K4me3 (A), H3K36me3 (B) and H3K9me2 (C) antibodies. Gray bars indicate the assay background as samples were incubated with the non-specific IgG. Each graph shows an average of 3 separate experiments (N=3), wherein each group was tested in triplicate. Statistical analysis was performed using 2way-ANOVA Tukey’s multiple comparisons (*p = 0.01) with Interaction Factor: F(8, 30) = 2 and Treatment Factor: F(2, 30) = 30.2.

Figure-3.

mRNA quantification and [³H]-DA uptake assays. (A) Adult animals from embryos exposed to control solution (open blue bars) or 0.5 mM amphetamine (Amph) (red bars) were treated with 0.5 mM Amph or vehicle (200 mM sucrose) for 10 minutes. Then each group of animals was processed for RT-PCR. The number of replications (N) for each group was 12. (B) Cells from offspring embryos were incubated with 5nM [³H]-DA alone or with 10 μM imipramine to measure DA uptake. The number of replications (N) is indicated in each bar. Statistical analysis was performed using 2way-ANOVA (A) or 1way-ANOVA (B) Tukey’s multiple comparisons tests.