Molecular mechanisms of amphetamine actions in Caenorhabditis elegans

Abstract

Amphetamine (AMPH) poses a serious hazard to public health. Defining the molecular targets of AMPH is essential to developing treatments for psychostimulant abuse. AMPH elicits its behavioral effects primarily by increasing extracellular dopamine (DA) levels through the reversal of the DA transporter (DAT) cycle and, as a consequence, altering DA signaling. In Caenorhabditis elegans, an excess of synaptic DA results in a loss of motility in water, termed swimming-induced paralysis (SWIP). Here we demonstrate that AMPH produces SWIP in a time- and dose-dependent manner in wild-type (wt) animals but has a reduced ability to generate SWIP in DAT knock out worms (dat-1). To determine whether D1-like and/or D2-like receptors are involved in AMPH-induced SWIP, we performed experiments in DOP-1 and DOP-4, and DOP-2, and DOP-3 receptor knockout animals, respectively. AMPH administration resulted in a reduced ability to induce SWIP in animals lacking DOP-3, DOP-4, and DOP-2 receptors. In contrast, in worms lacking DOP-1 receptors, AMPH-induced SWIP occurred at wt levels. Using microamperometry on C. elegans DA neurons, we determined that in contrast to wt cells, AMPH failed to promote DA efflux in dat-1 DA neurons. These data suggest that DA efflux is critical to sustaining SWIP behavior by signaling through DOP-3, DOP-4, and DOP-2. In a double mutant lacking both DAT-1 and DOP-1 expression, we found no ability of AMPH to induce SWIP or DA efflux. This result supports the paradigm that DA efflux through C. elegans DAT is required for AMPH-induced behaviors and does not require DOP-1 signaling.

Fig. 1.

AMPH-induced SWIP requires DAT-1. A, AMPH treatment induces a time- and concentration-dependent increase in SWIP (solid symbols). In contrast, vehicle treatment (□) does not induce paralysis (*, p < 0.01, one-way analysis of variance). B, in dat-1 animals, AMPH treatment (100 μM at 4 min; ●) did not induce a significant increase in SWIP with respect to vehicle treated animals (□). Inset, ΔSWIP calculated after treatment for 4 min with 100 μM AMPH for wt (n = 15, 271 animals) and dat-1 animals (n = 8, 136 animals).

Fig. 2.

AMPH-induced SWIP is supported by CAT-2, CAT-1, DOP-2, DOP-3, and DOP-4 expression. AMPH-induced ΔSWIP was significantly (Student's t test) diminished in A animals lacking tyrosine hydroxylase (A; *, p < 0.01; n = 6, 121 animals), VMAT (B; *, p < 0.05; n = 11, 163 animals), DOP-2 (D; *, p < 0.05; n = 14, 206 animals), DOP-3 (E; *, p < 0.005; n = 8, 151 animals), and DOP-4 (F; *, p < 0.005; n = 4, 106 animals) receptors compared with wt animals (dotted line). In contrast, AMPH-induced SWIP (C) was not significantly reduced in worms lacking DOP-1 (n = 12; 228 animals). All data are expressed as a percentage of wt.

Fig. 3.

AMPH-induced SWIP is significantly reduced in dat-1/dop-1 double mutants. In dop-1 animals, AMPH induces a significant increase in SWIP (■) compared with vehicle-treated controls (□). A transporter/receptor double mutant, dat-1;dop-1, was generated. AMPH-induced SWIP was not significantly reduced in dat-1;dop-1 animals (▴, n = 4, 54 animals) with respect to vehicle treated controls (▵; n = 4, 59 animals). Inset, the ΔSWIP induced by 100 μM AMPH (4 min) in dop-1 animals was significantly different from that measured in dat-1;dop-1 double mutants (*, p ≤ 0.001, Student's t test, n = 4; 103 animals).

Fig. 4.

AMPH-induced DA efflux in C. elegans DA neurons. A, in DA neurons isolated from wt animals, 10 μM AMPH induced robust DA efflux (oxidation is represented as a positive current by convention) and inhibited by 10 μM mazindol. B, in contrast, in DA neurons from dat-1 animals, neither AMPH nor mazindol had any effect on DA efflux. C, DA neurons isolated from dat-1;dop-1 double mutants did not show DA effluxes after AMPH treatment. In addition, no changes were caused by mazindol to the basal amperometric current. D, in dop-1 DA neurons, perfusion of 10 μM AMPH induced a mazindol sensitive DA efflux.