To verify that the LTD that occurs Raf inhibitor when cAMP/PKA signaling is inhibited is actually eCB-LTD and not another form of synaptic plasticity, we added the CB1 receptor antagonist AM251 to an extracellular recording solution, which already contained CGS21680 and patched cells with PKI included in the intracellular solution. The addition of AM251 to the extracellular solution blocked LTD (119% ± 16%; p < 0.05 compared to LTD with CGS21680 and PKI; Figure S2D), demonstrating that cAMP/PKA inhibition is allowing eCB-LTD to occur. To further test the hypothesis that increases in cAMP/PKA signaling are sufficient to block LTD, we tested
whether directly activating either adenylyl cyclase or PKA would Alpelisib block HFS-LTD when there were no drugs present in the external saline solution. To activate adenylyl cyclase we used the water-soluble (membrane-impermeable) forskolin analog NKH477. To activate PKA we used a membrane-impermeable PKA activator, Sp-8-OH-cAMPS. When either NKH477 or Sp-8-OH-cAMPS were included in
our intracellular recording solution, LTD was inhibited (78% ± 5% with NKH477; 89% ± 9% with Sp-8-OH-cAMPS; both p < 0.05 compared to control LTD; Figures 5C and 5D). From these experiments, we concluded that increased cAMP/PKA activity inhibits LTD. How does cAMP/PKA activity block LTD? Because D2 and A2A receptor drugs act on both LFS- and HFS-LTD, they likely act on a common target in both pathways: group I mGluRs or Gq. A particularly attractive candidate for such modulation is regulator of G protein signaling 4 (RGS4). RGS4 is a GTPase-activating protein expressed strongly in MSNs in the dorsolateral striatum, where it is associated with mGluR5 and PLCβ (Gold et al., 1997 and Schwendt and McGinty, 2007), its activity is increased by tuclazepam PKA phosphorylation (Huang et al., 2007), and it strongly inhibits signaling through Gq (Saugstad et al.,
1998). To test whether RGS4 is involved in the regulation of LTD by D2 and A2A receptors, we obtained RGS4−/− mice, crossed those mice into our D2-EGFP BAC transgenic line so that we could identify indirect-pathway MSNs, and applied the HFS-LTD induction protocol to indirect-pathway MSNs. In RGS4−/− mice, we observed significant HFS-LTD (66% ± 5%; Figure 6A). If RGS4 is responsible for the connection between D2 and A2A receptor signaling and Gq signaling, then LTD in RGS4−/− mice should occur even in the presence of the D2 antagonist sulpiride or the A2A agonist CGS21680. Indeed, LTD was readily observed in the presence of either drug in RGS4−/− mice (80% ± 6% in sulpiride; 64% ± 7% in CGS21680; Figure 6B). While genetic knockouts offer complete elimination of the gene product, they also may yield developmental and/or homeostatic changes. Therefore, we used a recently identified small-molecule inhibitor of RGS4, CCG-63802 (Blazer et al., 2010), to test whether acute inhibition of RGS4 could uncouple LTD from D2 receptors.