Although we were able to identify potential regions of contact between axons and dendrites, it was unclear if bona fide synapses form within the microfluidic chambers. To answer this question, we first used immunocytochemistry to test for and visualize the position of presynaptic puncta (using an anti-bassoon antibody) relative to dendrites (using an anti-MAP2 antibody) (Figure 2C). Bassoon-positive puncta were clustered along the MAP2-positive dendrites, whereas axons in dendrite-free areas of the microgrooves did not contain such bassoon clustering. To detect functional synapses, whole-cell voltage clamp recordings of miniature synaptic currents were obtained from neurons inhabiting one of the compartments. Many synaptic events were visible, indicating abundant synaptic activity in the chambers. Recordings were obtained from neurons cultured up to 42 days. The glutamate receptor antagonists, NBQX and APV were added locally during some recordings, resulting in the immediate elimination of the miniature excitatory postsynaptic currents (Figure 2D). These results show that glutamatergic synapses are functional within the microfluidic chambers. Furthermore, we identified both glutamatergic and inhibitory neurons within the chambers, showing that the cultures mature normally and possess a mix of excitatory and inhibitory neurotransmitters (Figure S1).
