Presynaptic Gq-coupled receptors drive biphasic dopamine transporter trafficking that modulates dopamine clearance and motor function

Abstract

AbstractExtracellular dopamine (DA) is temporally constrained by the presynaptic DA transporter (DAT). Decades of work stemming from primarily in vitro systems support that DAT is dynamically regulated by endocytic trafficking. However, despite its pivotal role in DA neurotransmission, DAT regulation in situ is poorly understood, and it is unknown whether regulated DAT trafficking impacts dopaminergic signaling and/or DA-dependent behaviors. Here, we leveraged chemogenetics and conditional gene silencing in mice to probe the endogenous signaling mechanisms that regulate DAT trafficking, and their impact on DA signaling and DA-dependent behaviors. Activating presynaptic Gq-coupled receptors, either hM3Dq or mGluR5, drove rapid DAT membrane insertion followed by endocytic retrieval, with region-specific differences in ventral and dorsal striata. Gq-stimulated DAT membrane insertion required DRD2 autoreceptors and intact Vps35+ retromer complex, whereas subsequent DAT retrieval required PKC activation and the neuronal GTPase, Rit2. Ex vivo voltammetry in striatal slices revealed that DAT trafficking impacts DA clearance. Conditional mGluR5 silencing in DA neurons demonstrated that presynaptic mGluR5 is specifically required for biphasic DAT trafficking, and its loss increased baseline DAT membrane presentation. Locomotor studies revealed that DAergic mGluR5 silencing abolished motor learning, and selective DAT inhibition rescued this motor deficit, consistent with a causal role for DAT trafficking dysfunction in motor performance. These studies report that striatal presynaptic DAT trafficking is complex, multimodal, and region-specific, and identify cell autonomous mechanisms required for DAT trafficking. Importantly, the findings establish a link between regulated DAT trafficking, DA clearance, and motor function.Significance StatementDA transmission is central to motivation, learning, and motor function, and mechanisms that impact DA signaling exert profound control over these behaviors. The DAT is pivotal in constraining the DA signal, but little is known about how DAT is regulated in DA terminals, or whether DAT regulation may impact DA-dependent behaviors. Using a variety of complementary genetic approaches in mice, this study provides the first evidence that regulated DAT trafficking plays a strong contributory role in DA signaling and DA-dependent behaviors.