To facilitate neuronal specific analysis in the absence of astrocytes, we grew neurons in indirect contact with astrocytes. These cultures manifested morphology of very well connected neurons at early (day 26; Fig 5A) and later differentiated stages (day 56; Fig 5B). At day 56, VGAT and synaptophysin1 (Fig 5C–5F) as well as VGLUT1 and HOMER1 (Fig 5G–5J) were expressed in a punctate pattern, suggesting the presence of GABAergic and glutamatergic synapses. Similar to the direct contact cultures, the indirect contact cultures were positive for the lineage identity markers CTIP2, SATB2, PROX1, MEIS2 and Calbindin at day 56 using immunocytochemistry (S3I–S3M Fig). Indeed, these neurons established GABAergic and glutamatergic synapses as measured by whole-cell patch clamp electrophysiological recordings of spontaneous release (Fig 5K and 5L). Furthermore, Ca2+ transients in indirect contact cultures demonstrated a functionally integrated network (Fig 5M and 5O) of mixed neurons (Fig 5N and 5P), which showed increased amplitude of response to an increasing number of stimuli (Fig 5Q). We also observed ROIs, which upon bicuculline addition showed either increased (20%) or decreased (55%) frequency of activity, and
