Induced GABAergic neurons for human neurological disease modeling. (a) Collybistin expression is reduced by shRNA knockdown (hairpin numbers 1–5) compared with control (Ctrl) in iN cells as measured by qRT-PCR (mean ± s.e.m.). (b) Representative traces of postsynaptic currents induced by exogenous application of GABA (1 mM). (c,d) Cumulative (cum.) plots (i) and summary graphs (ii) show reduction in average peak amplitude (c) and total charge-transfer (d) of GABA puff-induced IPSCs in human Ngn2-iN cells subjected to collybistin knockdown. (e) Patch–clamp configuration for postsynaptic recordings performed on day 28–30 human neurons expressing collybistin shRNAs. Collybistin KD iN cells (EGFP positive) were cocultured with Ascl1, Dlx2, Myt1l-generated iN cells (EGFP negative, black arrowheads). Rec, recording electrode. Scale bars, 15 μm. (f) Sample traces of GABAAR- mediated spontaneous IPSCs recorded from control (top) or collybistin shRNA no. 4 (Sh# 4)-infected neurons (bottom). (g) Cumulative plots (left) and average graphs (right) representing mean ± s.e.m. values of sIPSC amplitude (amp, i) and event frequency (freq, ii), recorded from control versus collybistin shRNA no. 4 – infected neurons in (e). Numbers inside bars indicate total number of independent batches (for batch-wise comparisons) or total number of cells recorded/number of batches. Two-tailed, unpaired Student’s t-test; ***, P < 0.005; **, P < 0.01; *, P < 0.05; n.s., not significant. P > 0.05 was used for all comparisons except batch-wise comparisons, where paired t-tests were performed. For cumulative plots, circles represent average values recorded from individual cells.
