To understand whether the decreased synaptic release in response to DAMGO is compounded by decreased intrinsic excitability, we examined the effect of DAMGO on induced action potential (AP) firing in N40 and D40 iN cells. Although no significant differences of AP firing rate, amplitudes and thresholds were observed between N40 (A10 clone) and D40 (C12 clone) (Supplemental Fig. 3), we observed that 10 ¼M DAMGO induced D40 versus N40 iN cells to fire significantly fewer APs (Fig. 4A–B) with no effect on AP amplitude and firing threshold (Fig. 4C–D). This is supported by an immediate and more robust decrease in spontaneous AP firing frequency following DAMGO application in D40 versus N40 iN cells (Fig. 4E), an effect which is sustained over the course of several minutes (Fig. 4F). This sustained decrease in AP frequency is paralleled by a rapid hyperpolarization of N40 and D40 iN cells (Fig. 4G). This effect was found to be significantly more robust in D40 versus N40 iN cells in the first minute following DAMGO application. The immediate drop in both AP firing frequency and membrane
