Doxycycline, which is used to induce expression of Tet-On vectors, was added to the Neurobasal media with Y-compound on day 1 at a concentration of 2 μg ml−1 and maintained in media throughout all experiments. On day 2, puromycin (1 μg ml−1) was added for selection. Inhibitory neurons were also selected with hygromycin (25 ng ml−1), since a hygromycin resistance was built into the Dlx2 vector. Concentrations of puromycin and hygromycin were occasionally increased, as needed, to more aggressively select for iNs against surviving iPS cells. Additional lentivirus was added on day 3, as applicable: vectors carrying DNA for GCaMP6f (200 μl ml−1 in Neurobasal media) were added, as needed, for calcium imaging studies on day 3, lentiviral vectors for hM3Dq-mCherry for DREADDs stimulation of excitatory neurons, and neuroligin-3 were added to the excitatory neuron cultures at 100 μl ml−1 in Neurobasal media on day 3. Induced neurons were maintained in Neurobasal media with puromycin and doxycycline until plating.
