Next, we sought to determine the functionality of the circuit formed between the two chambers. To do so, we paired excitatory neurons with inhibitory neurons in each well. Both neuronal populations were infected with GCaMP6f, which enables visualization of changes in intracellular free calcium levels via fluorescence intensity as a metric for cellular activity/neuronal firing. To use calcium imaging as a high-throughput readout, it is convenient to have access to a semi-/fully-automated system such as the IN Cell Analyzer for data collection. To capture both chambers in the excitatory-inhibitory circuit, we used 2048 × 2048 images to capture the 500 μm width of the microchannels and approximately an additional 200 μm on either side of the wall. This provided a large enough number of cells within the field of view to simultaneously record network activity populations of human neurons. Baseline activity was captured for 2:12 minutes (1 frame captured every 0.11 seconds), after which the AMPA receptor blocker CNQX was added to the chamber at a final concentration of 20 μM to suppress excitatory synaptic currents and an additional 3:51
