In addition to testing various CNO concentrations, we also screened the circuit response to clozapine, the metabolite of CNO. Gomez et al. recently demonstrated the differences between clozapine-N-oxide (CNO) and clozapine, a metabolite of CNO.30 They demonstrated, using both human embryonic kidney cells (HEK293) in vitro and mouse in vivo experiments, that clozapine has much greater reactivity and affinity for the hM3Dq receptor than CNO. Gomez et al. argue that CNO likely only reacts with hM3Dq at high concentrations due to its ability to convert to significant clozapine levels for binding hM3Dq. However, in our system, we only observed a minimal, non-significant, increase in activity when a high concentration (500 nM) of clozapine was added to our cultures compared to a large stimulation following an addition of 500 nM CNO. For the efficacy of CNO on activation of neuronal activity in neurons expressing hM3Dq, we were able to estimate a half maximal effective concentration (EC50) of approximately 30 nM based on our dose-response curve. This is within the same order of magnitude as the EC50 of 85 ± 17 nM
