To characterize excitatory and inhibitory neuron identity in the cultures, we analyzed dendritic, axonal and synaptic marker expression by immunocytochemistry. At day 49, the neuronal cultures (direct contact) showed an abundant network of dendrites and axons as shown by dendritic marker MAP2 and axonal marker Smi312 (Fig 3A). To study presence of GABAergic neurons, we quantified the number of neurons expressing the GABA-producing enzyme GAD65/67 (Fig 3A–3C) as a fraction of the MAP2-positive cells (0.22 ± 0.5; Fig 3D). We also quantified the number of synaptophysin1 puncta per neuronal soma, which accounted to 298±74 pre-synapses per MAP2 dendritic soma (Fig 3E). We further studied the presence of GABAergic presynaptic terminals by co-localization of pre-synaptic protein synaptophysin1 puncta with vesicular GABA transporter (VGAT) puncta (Fig 3F–3H). The fraction of all VGAT puncta to synaptophysin1 puncta on MAP2-positive dendrites was 0.53 ± 0.01 (Fig 3I) and the number of VGAT synapses per dendritic soma (MAP2) was 103±18 (Fig 3J). Glutamatergic neurons in the cultures were identified by co-localization of presynaptic vesicular glutamate transporter 1 (VGLUT1) with synaptophysin1 puncta, and by presence of
