It is important to note these targeted peptidases are non-selective metalloproteases. Therefore, blocking their activity has the potential to enhance the activity of other ‘off-target' endogenous signalling peptides (for example, substance P3940, neurokinin4142; neurotensin4243) that may act either in concert or opposition to endogenous opioid signalling. Consistent with this, while inhibiting peptidase activity with the peptidase inhibitor cocktail on average, reduced eEPSC amplitude in response to both low (12.9±5.4% inhibition, n=7) and moderate (32.1±6.2% inhibition, n=8) stimuli (for example, Fig. 3g,h), there was a high degree of variability, with either inhibition (12/18 neurons); no effect (3/18 neurons) or an increase observed (3/18 neurons). Thus, we defined endogenous opioid action as the naloxone-induced increase in eEPSC following peptidase inhibitor treatment (Fig. 3a). In addition, there was variability in the timing of the peptidase inhibitor response, which could reflect the time required to accumulate sufficient peptide or differences in restricting microarchitecture. Despite this variability in peptidase inhibitor response, naloxone significantly increased eEPSC amplitudes in all cells. Due to possible dominance of confounding ‘off-target' signalling peptides in cells where the peptidase inhibitors increased the eEPSC amplitude, these cells were excluded from further analysis of the endogenous opioid effect. In the remaining cells, naloxone induced a significant increase in eEPSC amplitude using both low (Fig. 3g,i) and moderate stimuli (Fig. 3h,i). These data indicate endogenous opioids are released during low-stimulus experiments but eEPSC amplitude remains unchanged due to rapid degradation of the peptide. Further, it shows that the actions of endogenously released opioids, released in response to the moderate stimulus, are limited by peptidase activity. Interestingly, when peptidases are inhibited, the first pulse of the moderate stimulus train was also increased by naloxone (40.2±4.8% increase, n=8, P<0.01 peptidase inhibitor versus naloxone, paired t-test). As this did not occur without peptidase inhibitors (10.7±6.4% increase, n=7, P>0.05 baseline versus naloxone, paired t-test), this suggests endogenously released opioids are broken down by peptidases during the 15 s inter-stimulus interval.