Next, we aimed to further demonstrate the capacity for perturbing the in vitro neural circuitry function with different concentrations of small molecules or drugs. We applied a chemogenetic approach by expressing hM3Dq DREADDs in human neurons and applying the synthetic molecule clozapine-N-oxide (CNO), a synthetic agonist for hM3Dq.29 We infected excitatory neurons with hM3Dq and paired them within the circuit model with inhibitory neurons expressing GCaMP6, which were not infected with the hM3Dq lentivirus. Excitatory and inhibitory neurons were also cultured on coverslips for morphological analysis of their subtype identity (Fig. S2†). CNO (at varying concentrations), clozapine (at 500 nM), and vehicle (HEPES) were added to the system to measure network stimulation (Fig. 6). Calcium imaging was performed in the same manner as described above. Basal activity was recorded for 100 seconds, after which the CNO drug was added. An additional 2:45 minutes were then recorded to capture the effect of the drug. Excitatory neurons which were mCherry (and therefore hM3Dq) positive showed a strong response to 500 nM CNO addition (Fig. 6A and B) while mCherry-hM3Dq-negative excitatory neurons did not show any signs of signal change (Fig. 6C and D).
