We confirmed that the D2 receptor antagonist sulpiride blocked HFS-LTD (103% ± 8%; p < 0.05 compared to control; Figure 4A). Interestingly, sulpiride was also able to inhibit LFS-LTD (88% ± 4%; p < 0.05 compared to control; Figure 4B), indicating that D2 receptors act on eCB-LTD at or upstream of Gq. Adenosine A2A receptors are also highly expressed in indirect-pathway MSNs, where they influence eCB signaling and act in opposition to D2 receptors (Shen et al.,

2008 and Tozzi et al., 2007). Therefore, we tested whether activation of A2A receptors BVD-523 manufacturer could block HFS- or LFS-LTD. The A2A receptor agonist CGS21680 blocked both HFS- and LFS-LTD (102% ± 7%; p < 0.05 compared to control for HFS-LTD and 90% ± 12%; p < 0.05 compared to control for LFS-LTD; Figures 4C and 4D). Thus, like D2 receptors, A2A receptors appear to be acting at or upstream of Gq to modulate both forms of eCB-LTD in indirect-pathway MSNs. We confirmed these results in two different BAC transgenic mouse strains (Drd2-EGFP, target EGFP-positive MSNs; Drd1a-Tmt, target Tmt-negative MSNs), indicating that D2/A2A regulation is robust across multiple mouse lines (Figure S2C). We next considered Carfilzomib how D2 and A2A receptors modulate eCB mobilization

and LTD. Because regulation of eCB biosynthetic pathways by cAMP/PKA signaling is not well established, we first tested whether D2 receptors act to promote eCB-LTD through a reduction in cAMP levels or PKA activation. In this and subsequent experiments, we utilized HFS-LTD to examine the mechanisms regulating eCB-LTD, because this form of LTD remains a standard in the field. To examine whether inhibition of

cAMP production alone is sufficient to enable eCB-LTD induction, even in the presence of a D2 receptor antagonist, we used a membrane-impermeable adenylyl cyclase inhibitor, ddATP, and a membrane-impermeable inhibitor of PKA, PKI, which were added to our intracellular recording solution. The membrane-impermeability Electron transport chain of these drugs limited their effects to the recorded postsynaptic MSN, which allowed us to rule out effects on cAMP/PKA-dependent processes in the presynaptic terminal or in neighboring MSNs or interneurons. With either ddATP or PKI in our intracellular recording solution, we were able to elicit LTD in the presence of sulpiride (69% ± 9% with sulpiride and ddATP; 71% ± 10% with sulpiride and PKI; both p < 0.05 compared to LTD in sulpiride alone; Figure 5A). In contrast to the action of D2 receptors, A2A receptors are Gs-coupled receptors, and we therefore hypothesized that activation of A2A receptors blocks LTD by increasing cAMP/PKA signaling. In support of this hypothesis, we found that reducing cAMP/PKA activity by including either ddATP or PKI in the intracellular recording solution allowed LTD to occur in the presence of A2A agonist CGS21680 (61% ± 4% with CGS21680 and ddATP; 65% ± 7% with CGS21680 and PKI; both p < 0.