Our data with the MGL inhibitor MAFP indicate that DSI is likely mediated by 2-AG, however, MAFP also inhibits degradation of another eCB, anandamide. To confirm that DSI is mediated by 2-AG in the BLA, we applied the DAGL inhibitor THL in combination with MAFP. In the presence of MAFP, bath application of the DAGL inhibitor THL 10 µM for >30 min before experiments completely blocked DSI (Figure 6a). Two-way ANOVA showed a significant effect of THL (p < 0.0001), time (p < 0.05), and a significant interaction (p < 0.01). Summary data indicate that THL blocked both maximal DSI (MAFP-vehicle 66.3 ± 5.4% N = 8 vs MAFP + THL 106.4 ± 7.8% N = 9, p < 0.001; Figure 6b) and late DSI (MAFP-vehicle 74.7 ± 3.6% N = 8 vs MAFP-THL 104.7 ± 5.8% N = 9, p < 0.01; Figure 6b). These data indicate that the ability of MAFP to prolong DSI requires DAGL activity, thus indicating DSI is mediated by 2-AG in the BLA.
