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. Author manuscript; available in PMC: 2019 Apr 4.
Published in final edited form as: Nature. 2019 Jan 16;565(7741):645–649. doi: 10.1038/s41586-018-0866-8

Extended Data Fig. 9 |. Activation of feeding- or social-excited cells does not alter whisker activity or pupil diameter and activation of NSNF neurons does not affect licking.

Extended Data Fig. 9 |

a, Representative whisker (orange) tracking from an example GCaMP6m + bReaChES mouse. b, c, Two-photon spiral stimulation of 20 feeding-or social-excited cells per animal does not significantly alter whisker activity. Animals are able to detect noise produced from the spiral-stimulation galvanometer mirrors during both no stimulation (laser shutter closed) and stimulation (laser shutter open) sessions, as demonstrated by a significant increase in classified whisking events during the spiral scanning period (b; unstimulated, 0 to 5 s versus –5 to 0 s: n = 5 mice, interaction F2,8 = 68.34, P = 0.02; and stimulated 0 to 5 s versus −–5 to 0 s: n = 5 mice, interaction F2,8 = 68.34, P = 0.002, two-way ANOVA with repeated measures); this sensory response was not disrupted by spiral stimulation of feeding (b; n = 5 mice, interaction F2,8 = 2.31, P > 0.99, two-way ANOVA with repeated measures) or social (c; n = 6 mice, interaction F2,10 = 0.61, P > 0.99, two-way ANOVA with repeated measures) cells. d, Representative pupil-diameter tracking (purple) from an example GCaMP6m + bReaChES mouse. e, f, Pupil diameter is not significantly affected by stimulation of 20 feeding (e; n = 4 mice, F2,6 = 0.17, P = 0.85, one-way ANOVA with repeated measures) or social (f; n = 4 mice, F2,6 = 0.05, P = 0.95, oneway ANOVA with repeated measures) cells. g, Cell masks of neurons that do not display significant responses to either feeding or social stimuli (NSNF cells; orange) and spiral-stimulation targets (red) from a GCaMP6m + bReaChES mouse. Image from n = 1 representative mouse; this experiment was repeated in six mice with similar results. Scale bar, 100 μm. h, Normalized mean Ca2+ activity of NSNF neurons across 20 trials of 5-s stimulation. n = 20 stimulation-targeted NSNF cells. i, Single-cell stimulation of NSNF neurons did not significantly alter licking for caloric rewards in GCaMP6m + bReaChES mice when compared to baseline sessions. n = 20 NSNF-stimulated cells per mouse, n = 6 mice; interaction F2,10 = 1.76, P = 0.51, two-way ANOVA with repeated measures. j, Average cumulative lick rate (30-s bins) across 10-min baseline and NSNF cell stimulation sessions in GCaMP6m + bReaChES mice. n = 6 mice. n.s., non-significant (P > 0.05).