Additionally, since the device was designed to enable central chamber neurons to receive inputs from neurons in two distinct side chambers, we wanted to demonstrate the capacity of the device to facilitate formation of a three-way circuit. We infected excitatory neurons with either GFP or ChR2-tdTomato lentivirus prior to seeding, and placed them in adjacent side chambers. Non-labeled excitatory neurons were seeded in the central chamber. After 4 weeks, both GFP and tdTomato-labeled projections were observed to fill the microchannels (Fig. 4a). Interestingly, axons projected outward and turned to follow the circular edge of the device. Central chamber neurons located near the intersection between side chambers were found to have nearby projections from both GFP and tdTomato iNs. These neurons stained positive for synapsin, suggesting synapse formation between side chamber projections and central chamber iNs (Figs. 4b–d). These data suggest that axonal projections originating from neuronal bodies located in different outer compartments could form mature synapses onto the same neurons in the central chamber.
