Dopamine (DA) is a neurotransmitter used throughout phylogeny to modulate circuits controlling movement, attention, reward and cognition. Alterations in DA signaling in man have been implicated in Parkinson's Disease, Attention-Deficit Hyperactivity Disorder (ADHD), addiction and schizophrenia. DA signaling is tightly controlled by a presynaptic, DA transporter (DAT) that is a major target for addictive psychostimulants such as cocaine and amphetamine (AMPH), as well as agents used in the treatment of ADHD. Our recent identification of multiple, functional alleles of human DAT in ADHD and Bipolar disorder subjects adds translational significance to our efforts to decipher the presynaptic mechanisms that control DA release and inactivation. To date, these mechanisms have largely been studies through pharmacological and genetic manipulation of genes identified two decades or more ago. In the current application, we capitalize on a robust, forward genetic approach to identify and characterize novel presynaptic regulators of DA signaling using the powerful model system Caenorhabditis elegans. Over the past 15 years, the Blakely lab has developed skills in the C. elegans model with a focus on DAT and DA signaling, initiated by the identification of the C. elegans DAT gene (T23G5.5, dat-1). The present effort arises from our discovery of a simple DA and dat-1 dependent phenotype termed Swimming-Induced Paralysis (SWIP). Whereas wildtype animals thrash in water at ~1 Hz for up to 30 minutes, dat-1 (ok157) (DAT-deficient) animals paralyze in 3-5 minutes. This phenotype is dependent on DA synthesis, vesicular DA packaging, and DA release and is overcome by DAT activity. We request support to advance our screen to identify and characterize genes acting presynaptically to regulate DA release and reuptake, localize their mode of contribution to DA signaling, assess their impact on the actions of AMPH, and initiate an analysis of conserved vertebrate homologs. Together our efforts provide an opportunity to identify novel and conserved regulators of DA signaling that would be extremely difficult to elucidate in vertebrate models. PUBLIC HEALTH RELEVANCE: DAT proteins are major presynaptic regulators of DA signaling and are targets of therapeutic and addictive psychostimulants. To identify novel presynaptic mechanisms that control DA release and reuptake, we implement a novel, forward genetic approach in the model system C. elegans. As all known regulators of DA signaling in mammals are conserved in C. elegans, our efforts have a high probability of yielding fundamental insights that underlie potential contributors to human, DA-dependent disorders as well as novel targets for medication development.