We next explored the functional properties of hSS. We found that 7 days of exposure to the neurosteroid and GABAA receptor agonist allopregnanolone (AlloP) combined with a 3-day exposure to retinoic acid (RA), significantly increased the frequency of spontaneous calcium spikes (Extended Data Fig. 3a–c). Notably, exposure to AlloP with or without RA did not alter subpallial fate, neurotransmitter identity or the GABAergic subtypes in hSS (Extended Data Fig. 3d–l). As a result, these two conditions were used for subsequent experiments. Considering the presence of spontaneous calcium activity and astrocytes (Fig. 1j), we investigated synaptogenesis in hSS using array tomography (AT). We found expression of the presynaptic protein synapsin-1 (SYN1) and the vesicular GABA transporter VGAT (Fig. 1k). Lastly, we used whole-cell patch clamping to record from neurons in sections of hSS and found that ~75% of neurons generated action potentials in response to depolarization (Fig. 1l). At the same time, ~60% of neurons exhibit spontaneous inhibitory postsynaptic currents (IPSCs) that reverse in direction around the chloride reversal potential and are abolished by the GABAA receptor antagonist gabazine (10 μM) (Fig. 1m, n; in contrast to synaptic currents in hCS, as shown in Extended Data Fig. 4).
