Do synapses form within the perfusion channel? The ability to target synapses made between cells located in pre- and post-synaptic compartment would be a considerable advancement for experimental manipulations of synapses. To visualize axons from neurons isolated within the presynaptic compartment, we infected neurons in this compartment with a GFP-virus (Figure 5). Many axons were labeled and extended into the microgrooves and through the perfusion channel (not shown). To examine whether there are active presynaptic terminals associated with these axons, we loaded presynaptic terminals within the perfusion channel with the styryl dye, FM5-95 (Figure 5C) by stimulating exo- and endocytosis. We perfused the dye while simultaneously depolarizing the neurons with a high KCl solution. After a couple of wash steps, multiple presynaptic terminals were loaded with the FM dye, which also co-localized with the GFP-labeled axon, showing that active presynaptic terminals are present within the perfusion channel. (Figure 5D).
