To confirm that the effect was due to CNQX and not mechanical disruption of the cells from reagent dispensing, we added 10 μl HEPES (vehicle) as a negative control. When vehicle-only was added, the burst calcium spikes likely driven by the excitatory neurons were largely unaffected (Fig. 5D). This experiment indicates that synapses were formed between the excitatory neurons in one compartment and inhibitory neurons in the connected compartment, and that calcium signals can be recorded as an indicator of functional network activity.
