Dissociated postnatal (P0-2) rat hippocampal neurons were prepared as described previously (Aakalu et al., 2001). The PDMS replica molded microfluidic chambers were placed onto cover glass coated with poly-D-lysine (354210; BD Biosciences), then neurons were plated into the chambers (Taylor et al., 2005; Taylor et al., 2006; Taylor et al., 2003) at a density of 5×106 neurons per ml with 5 μl of cell suspension added to each well. During the incubation of the cultures, the withdrawal port was sealed to prevent evaporation until tubing was added for perfusion. All wells were filled with Neurobasal A/B27/Glutamax medium (150 μl for 8 mm wells; 100 μl for 6 mm wells). We waited one day before filling the perfusion channel to allow the neurons to attach sufficiently within the somatic compartments. To visualize potential contact between the separate neuron populations, we infected neurons with Sindbis viral vectors encoding either GFP or RFP. For isolating infections to one compartment, virus was added to 50 μl taken from the selected compartment, gently mixed, and then added back into the same compartment. Media within the
