Considering the functional maturity of the cortical neurons and the presence of astrocytes, we decided to investigate synaptogenesis within ~180-day-old hCSs. We used array tomography to visualize individual synapses27 within the dense neuropil of the hCSs (n = 5) and antibodies against multiple pre- and postsynaptic proteins to identify individual synapses with a high degree of confidence. We found that the presynaptic protein synapsin-1 (SYN-1) and postsynaptic protein PSD-95 were expressed throughout the interior of the hCSs in both large and small puncta (Fig. 5a). Larger SYN-1 puncta were often found adjacent to PSD-95 puncta, indicating the presence of a synapse. To examine these larger puncta in further detail, we constructed ‘synaptograms’ consisting of a series of high-resolution sections through a single synapse where we could probe for potential colocalization of at least three independent synaptic markers. In many cases, we observed colocalization of the presynaptic proteins SYN-1 and the glutamate transporter VGLUT1 in close apposition to postsynaptic protein PSD-95, indicating the presence of a glutamatergic synapse (Fig. 5b). In some cases, the NMDA receptor subunit NR2B was also colocalized
