Prior to stimulation, OSI-744 clinical trial the majority of surface-labeled FD1R immunoreactivity was concentrated at the cell periphery, whereas endogenous ACV was detected both peripherally and associated with internal structures (Figure 8A, top). Surface-labeled D1 receptors moved to endocytic membrane structures within 2 min after agonist addition and a number of these colocalized with ACV (Figure 8A, bottom). The fraction of D1

receptor immunoreactive structures that also contained ACV is quantified in Figure 8B. We used the same approach to look at the subcellular localization D1 receptors in relationship to Gαs/olf proteins. Prior to stimulation, Gαs/olf immunoreactivity localized both peripherally and in association with internal structures, whereas D1 receptors showed a peripheral distribution consistent with plasma membrane localization (Figure 8C, top). Following

acute receptor activation, D1 receptors redistributed to endocytic vesicles and Gαs/olf immunoreactivity colocalized with a significant fraction these structures (Figures 8C, bottom, and 8D). Examination of this distribution at higher magnification MK-1775 supplier suggested that both downstream transduction proteins localize to subdomains of D1 receptor-containing early endocytic membranes (insets in Figures 8A and 8C). To our knowledge, the present results provide the first analysis of the relationship between D1 receptor trafficking and signaling in neurons, and on a time scale approaching that of physiological dopaminergic neurotransmission. Our results demonstrate that D1 receptors enter the endocytic pathway within ∼1 min after activation by either DA or synthetic agonist and that receptor-mediated accumulation of cellular cAMP occurs with overlapping kinetics. They also establish a causal relationship whereby D1 receptor endocytosis augments acute dopaminergic signaling. heptaminol We demonstrate that recycling

is not required for this response and provide evidence that the endocytosis-dependent signal is generated from an early endosomal membrane, thus distinguishing the present results from endocytosis-dependent resensitization observed for several other GPCRs. Further, our results show that the endocytosis-dependent component of the D1 receptor-mediated signal is functionally relevant as it is required to increase AP firing in a native brain slice preparation. Previous studies of D1 receptor-mediated signaling effects, measured over longer time intervals (≥30 min), have suggested that endocytosis either inhibits (Jackson et al., 2002 and Zhang et al., 2007) or has no effect on dopaminergic signaling (Gardner et al., 2001).