(fluorescence curve area of post-CNO)/(area of pre-CNO) ratio, determined in the analysis time windows over all conditions (Fig. 7C). While there was variation in the area ratio between wells of the same condition, the CNO concentrations of 500 nM and 100 nM showed significantly (p < 0.001) higher total bursting activity following CNO addition compared to hM3Dq negative and vehicle controls in both the excitatory and inhibitory neuronal chambers (Videos S2–S4†). Other CNO concentrations show a trend of effect that did not reach statistical significance. Clozapine at 500 nM did show an increase in area but was not significantly different from the vehicle control (HEPES only). Importantly, high concentrations of CNO showed significant differences from both vehicle-treated hM3Dq-positive cells and 500 nM CNO-treated hM3Dq-negative cells (Fig. 7C). We generated a dose response curve for both excitatory and inhibitory neurons within the device (Fig. 7D). hM3Dq-negative inhibitory neurons showed a similar increase in activity with increasing CNO concentration, indicating the presence of excitatory input via synaptic connections across the microchannels. Higher CNO concentrations also showed significant differences compared to vehicle and hM3Dq-negative cells in inhibitory neurons (Fig. 7C and D). This experiment indicates that functionality of neurons within a defined neurocircuitry
