PAMs of DOR could potentiate the inhibition by endogenously released opioids. We explored this possibility using the DOR PAM BMS-986187, which shows 100-fold selectivity for DOR over MOR and increases efficacy, potency and affinity of orthosteric ligands45. BMS-986187 (1 μM) significantly enhanced the inhibition induced by submaximal, exogenous ME (100 nM, Fig. 4c,e), indicating PAM activity at this synapse, which could be reversed to baseline levels by naloxone (6.3±2.6% inhibition; P>0.05 naloxone versus baseline, Tukey's post hoc comparisons, Fig. 4c,e). BMS-986187 is known to have agonist properties at higher concentrations45 and BMS-986187 alone induced a small but significant decrease in eEPSC amplitude (Fig. 4d,e; P<0.05, paired t-test). In these experiments, we used single synaptic stimuli (0.06 Hz) to minimize release of endogenous opioids. However, this BMS-986187 inhibition was reversed by naloxone (Fig. 4d,e) and we found that direct agonism by BMS-986187, resulting in β-arrestin recruitment and inhibition of cAMP production in CHO-OPRD1 cells, was insensitive to naloxone (Supplementary Fig. 1). Therefore, the naloxone sensitive inhibition of the BLA-Im synapse by BMS-986187 alone is not due to intrinsic agonist activity of BMS-986187, rather it is likely due to positive allosteric modulation of endogenously released opioids signalling at DORs. Although we know that PAMs can increase responses to exogenous opioids in cell lines45, this is the first instance in which increases in endogenous opioid signalling have been observed (Fig. 4d,e). To further investigate PAM activity on endogenous opioid signalling, we tested the effects of BMS-986187 on our previous treatment paradigms. When slices were preincubated with BMS-986187 (>45 min) and peptidases were inhibited, the naloxone-induced increase was significantly greater than peptidase inhibitors alone in response to the low stimulus (Fig. 4f,g), but had no further effect when using the moderate stimulus (CTL: 36.0±6.5% increase, n=8; BMS-986187: 36.8±5.9% increase n=5, P>0.05, unpaired t-test). Since naloxone produced comparable increases in eEPSC irrespective of stimulation intensity when in the presence of BMS-986187, this may indicate saturation of endogenously released opioid regulation at this synapse.