Supplementary Figure 11: Development and characterization of an inhibitory neuron-specific enhancer.

a, The inhibitory marker Gad67 co-localized with eGFP expression. Left: mI12b-eGFP; Right: anti-GAD67 immunostaining (Methods). Scale bars, 30 μm (n = 39 cells; representative data are shown). b, Correspondence of mI12b-eGFP fluorescence and Gad67 immunostaining in neurons that were positive for at least one of these reporters. Of n = 41 Gad67⁺ neurons, 39 were mI12b-eGFP⁺. Of n = 41 mI12b-eGFP⁺ neurons, 39 were also Gad67⁺. c, Top: Schematic showing patterned light stimulation onto an mI12b-eGFP⁺ neuron and voltage imaging from a nearby cell expressing QuasAr2. Middle: dark green: mI12b-eGFP, red: QuasAr2-dark Citrine, blue: DMD mask for patterned blue light stimulation. Right: IPSP was evoked by stimulation of the presynaptic cell expressing mI12b-EGFP. Scale bar, 10 μm. Two-trial average (n = 3 cells; representative data are shown). d, Optopatch measurements of spiking patterns and action potential waveforms revealed differences between mI12b-eGFP⁺ and mI12b-eGFP⁻ neurons consistent with differences between inhibitory versus excitatory neurons reported by patch-clamp measurements (Methods).^35,36 Blue: blue light stimulation of cells expressing both CheRiff and QuasAr2. Example traces show QuasAr2 fluorescence from a nominal excitatory neuron (orange, mI12b-EGFP⁻) and a nominal inhibitory neuron (green, mI12b-EGFP⁺). Middle: raster plots of spiking. Bottom: spike rate of nominal excitatory neurons (orange, mI12b-EGFP⁻) and inhibitory neurons (green, mI12b-EGFP⁺). e, Spiking rate and f, spiking waveform of cells not expressing mI12b (orange) and expressing mI12b (dark green). All shaded error bars and error bars, s.e.m. mI12b-eGFP⁺ neurons (putative interneurons) showed higher average evoked firing rates (13.6 ± 1.7 versus 6.7 ± 1 Hz, P = 0.001, two-sided two-sample t-test), lower probability of depolarization block under strong stimulus, narrower action potentials (6.9 ± 0.2 ms versus 9.4 ± 0.4 ms, P = 1 × 10⁻⁶, two-sided two-sample t-test) and larger after-hyperpolarization compared to simultaneously measured mI12b-eGFP⁻ cells (n = 36 mI12b-eGFP⁺ and 32 mI12b-eGFP⁻ neurons).