Vigorous Motor Activity in Caenorhabditis elegans Requires Efficient Clearance of Dopamine Mediated by Synaptic Localization of the Dopamine Transporter DAT-1

摘要

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.