There have been numerous studies 6, 8–15 examining the functional consequences of MOR N40D on receptor activation in overexpression models and in knock-in mice harboring MOR N40D, but no functional or electrophysiological analyses on cultured neurons have been conducted, specifically not in a human neuronal context. To gauge whether N40 and D40 iN cells respond differently to MOR activation, we used a MOR-specific agonist DAMGO ([D-Ala2, N-MePhe4, Gly-ol]-enkephalin) to study its role on modulating synaptic release. In both N40 and D40 iN cells, DAMGO suppressed sIPSCs in a dose-dependent manner (Fig. 1J). However, the suppression of sIPSC frequency was more robust in D40 iN cells compared to N40 iN cells in multiple repeated experiments and multiple iPS cell lines (Fig. 1K–L), with no difference in sIPSC amplitude by genotype. To confirm that the observation is not due to a residual effect of prolonged agonist exposure, we applied a single concentration of 10 ¼M DAMGO (Fig. 1M–N) and similarly found that D40 iN cells respond more robustly to MOR activation compared to N40 iN cells, illustrating genotype-dependent regulation of MOR signaling.
