To validate the formation of synaptic circuits in this system by different subtypes of iNs, we generated excitatory, inhibitory and DA neurons using different sets of transcription factors. Since glial cells are important for synapse formation32,33, we cultured mouse glial cells in the different compartments of the chamber system. We then initially seeded excitatory (glutamatergic) neurons into the outer chamber and DA neurons into the central chamber (Fig. 2c). The cultures were maintained in a humidified environment and the medium was changed every 2–3 days. The outer chamber solution was originally kept at a slightly higher fluid level compared with the central chamber to create hydrostatic pressure to direct the extension of axons from the side chambers through the microchannels. In subsequent experiments, we found that excitatory projections from the side chambers dominated axonal progression through the channels in spite of equal fluid levels, and so the medium height was thereafter maintained equally between chambers. After 4–7 days of culture, massive extended neuronal processes were observed to pass through the microchannels connecting the outer and the central chamber (Fig. 2c)
