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.Dysfunction in DA signaling has been implicated in multiple pathologies, including schizophrenia, attention-deficit/ hyperactivity disorder, Parkinson's disease, and AMPH abuse. Although it is well known that dopaminergic pathways are involved in cocaine and AMPH abuse, a comprehensive understanding of the key players coordinating AMPH behaviors is still missing. Upon exocytotic release of DA into the synapse, DATs coordinate the spatial and temporal action of DA by actively clearing the amine from the extracellular space. Although diffusion and enzymatic degradation may partially govern the synaptic concentration of DA, knockout studies have established DAT function as the primary mechanism controlling extracellular DA levels. DAT is the major molecular target responsible for the reward properties and abuse potential of several psychostimulants, including cocaine, methamphetamine, and AMPH. As a substrate of DAT, AMPH elevates extracellular DA levels by antagonizing DA clearance and stimulating DAT-mediated DA efflux. The subsequent increase in dopaminergic signaling mediated by DA receptors leads to secondary plasticity that mediates addiction. Whereas multiple lines of evidence implicate DA receptors in the mechanism of action of abused psychostimulants such as AMPH (Hiroi and White, 1991;Vallone et al., 2000;Ball et al., 2003), the degree to which different receptors support AMPH-induced behaviors has not yet been complet...

Amphetamine Activates an Amine-gated Chloride Channel to Generate Behavioral Effects in Caenorhabditis elegans

“…In each SWIP trial, 8 to 16 age synchronized larva-4 animals were placed in 40 l of vehicle (200 mM sucrose) with or without Amph (NIDA, Research Triangle Institute) or tyramine (Sigma-Aldrich) in a single well of a Pyrex spot plate (Thermo Fisher Scientific, Waltham, MA). 200 mM sucrose was used as vehicle solution instead of water (21,22) because of the discrepancy of data obtained by changing type of water. Experiments including tyramine (Sigma) were performed by including 500 M of ascorbic acid to prevent tyramine oxidation.…”

“…We named this behavior swimming-induced paralysis (SWIP) (22). WT animals treated for 4 min with 0.1 mM Amph exhibited SWIP, whereas DAT KO animals (dat-1) showed a basal level of SWIP, which could not be further enhanced by exposure to 0.1 mM Amph (21). In this study, we examined the SWIP phenotype using higher (0.3-1 mM) but still physiologically relevant concentrations of Amph (24) for longer treatment periods (10 min).…”

“…Transporter Knock-out Animals-Previously, we reported that Amphinduced DA release through the DAT inhibits C. elegans swimming (21). We named this behavior swimming-induced paralysis (SWIP) (22).…”

Chronic nicotine exposure augments gustatory plasticity inCaenorhabditis elegans:involvement of dopamine signaling

“…18) Cocaine and amphetamines are inhibitors of monoamine transporters, and acute treatment with these drugs changes locomotor activity in nematodes. 19,20) Dual effects of nicotine on locomotion speed, which *Corresponding author. Email: matsuura@iwate-u.ac.jp are dependent on differences in its dosage and treatment duration, have also been revealed.…”

“…elegans is also a model organism in studies to evaluate the effects of some drugs such as ethanol, 17,18) cocaine, 19) amphetamines, 20) and nicotine. [21][22][23] Ethanol administration interferes with gustatory plasticity in nematodes, i.e.…”