Because the mature hippocampal granule neurons in our system were almost exclusively glutamatergic, we employed pharmacological perturbation during calcium imaging to further assess the dependency of the network activity on glutamate-mediated synaptic transmission. Application of APV, an antagonist of NMDA receptor (NMDAR), and CNQX, an antagonist of AMPA receptor, reduced the neuronal network activity, as indicated by a decrease in the proportion of signaling neurons as well as their calcium transient frequency. Importantly, we observed a greater sensitivity of the networks to APV at the 3-week time point, whereas CNQX exerted a greater effect at the 6-week time point (Figures 4C–4F). This observation is consistent with previous work on synaptic maturation of hippocampal granule neurons that indicated a transition from an NDMA-receptor to an AMPA-receptor-mediated excitatory transmission as the granule neurons mature and integrate into the existing circuitry (Tashiro et al., 2006). Finally, studies have also shown a transition in hippocampal granule neurons from a GABA-excitatory to a GABA-inhibitory state during early maturation as a result of changes in intracellular Cl− concentration due to the upregulation of the KCC2 channel
