Since cultures are heterogeneous (Fig. 1C), we wished to evaluate larger sample sizes. Therefore, we used calcium imaging to assess spontaneous and glutamate-stimulated firing. We selected four cell lines for these studies (AF: 233 and 246, and UN: 420 and 472) and used the viral-transduced, genetically-encoded calcium indicator GCaMP6f as a proxy for neuronal activity [46]. Example images show consistent expression in all cell lines (Fig. 4A). Cultures were treated with repeated pulses of 10 μM glutamate (each followed by wash-out with ACSF), and pulses of 50 μM glutamate to elicit transient increases in fluorescence, indicating receptor-induced excitability, followed by pulses of 18 mM KCl, indicating cellular excitability (Fig. 4B–D). Neurons from both UN and AF groups had heterogeneous spiking patterns, with >50% of the neurons remaining inactive during the baseline period or after stimulation with glutamate or KCl. This is consistent with scRNAseq results indicating that only ~23% of the iN cells express genetic markers for neuronal activity (Fig. 1C). Increased spontaneous and induced spiking (>1–2 spikes) was apparent in the AF group when viewing individual cell responses in
