Vigorous Motor Activity inCaenorhabditis elegansRequires Efficient Clearance of Dopamine Mediated by Synaptic Localization of the Dopamine Transporter DAT-1

Abstract

The catecholamine dopamine (DA) functions as a powerful modulatory neurotransmitter in both invertebrates and vertebrates. As in man, DA neurons in the nematode Caenorhabditis elegans express a cocaine-sensitive transporter (DAT-1), presumably to regulate synaptic DA signaling and limit DA spillover to extrasynaptic sites, although evidence supporting this is currently lacking. In this report, we describe and validate a novel and readily quantifiable phenotype, swimming-induced paralysis (SWIP) that emerges in DAT-1-deficient nematodes when animals exert maximal physical activity in water. We verify the dependence of SWIP on DA biosynthesis, vesicular packaging, synaptic release, and on the DA receptor DOP-3. Using DAT-1 specific antibodies and GFP::DAT-1 fusions, we demonstrate a synaptic enrichment of DAT-1 that is achieved independently of synaptic targeting of the vesicular monoamine transporter (VMAT). Importantly, dat-1 deletions and point mutations that disrupt DA uptake in cultured C. elegans neurons and/or impact DAT-1 synaptic localization in vivo generate SWIP. SWIP assays, along with in vivo imaging of wild-type and mutant GFP::DAT-1 fusions identify a distal COOH terminal segment of the transporter as essential for efficient somatic export, synaptic localization and in vivo DA clearance. Our studies provide the first description of behavioral perturbations arising from altered trafficking of DATs in vivo in any organism and support a model whereby endogenous DA actions in C. elegans are tightly regulated by synaptic DAT-1.

Coexpressed D1- and D2-Like Dopamine Receptors Antagonistically Modulate Acetylcholine Release in Caenorhabditis elegans

“…Wild-type and control strains were tested for SWIP ( Figure 3). As previously reported, wild-type animals and dop-3 (vs106) mutants swam vigorously for .30 min when placed in water while dat-1(ok157) mutants became paralyzed within 10 min (McDonald et al 2007) (Figure 3B). The dop-3(vs106) mutation suppressed the SWIP phenotype of dat-1(ok157) mutants as dat-1(ok157); dop-3(vs106) double mutants swam freely like wild-type animals ( Figure 3B).…”

“…Therefore, we investigated the site of action of another, more robust, DAspecific locomotion behavior: swimming-induced paralysis. Wild-type animals placed in water swim vigorously for .30 min while animals with increased DA signaling caused by mutation of the DA reuptake transporter DAT-1 become paralyzed within 10 min (McDonald et al 2007) (Figure 3). Swimming-induced paralysis is a robust behavioral phenotype as .80% of wild-type animals continue to swim after 10 min while nearly 100% of dat-1(ok157) animals are paralyzed at this time.…”

“…Swimming-induced paralysis is a robust behavioral phenotype as .80% of wild-type animals continue to swim after 10 min while nearly 100% of dat-1(ok157) animals are paralyzed at this time. The excess DA present in dat-1(ok157) mutants acts through DOP-3 to cause SWIP as dat-1(ok157); dop-3(vs106) double mutants do not show SWIP (McDonald et al 2007) (Figure 3 and supporting information movies, File S1, File S2, and File S3 ).…”

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).…”