a, Average traces of 50 trials where 72-dB tones were presented (red arrow) with and without laser activation of LC-GABA neurons for 1 mouse. The dashed box delineates the averaging window for calculating the tonic effect of VGAT-ChR2 activation (panels b–d). b, Pupil size at different tone intensities, with and without laser activation, for the example mouse in a using a 7-s averaging window after tone onset. c, Normalized suppression of tonic pupil response due to activation of LC-GABA neurons. Suppression was calculated by subtracting the response of laser-on from the laser-off trials and normalizing to maximum value (P = 0.0284 using one-way ANOVA, F 4,20 = 3.39; Tukey post-hoc test reveals that only the 66 and 71-dB conditions were significantly different). d, Comparison of laser to no laser trials for all trials regardless of tone intensity or frequency (***P = 2 × 10−149 using two-tailed unpaired t-test: t 1673 = 28.91). e, Average traces of 50 trials for the same mouse and condition as in a, showing the phasic post-tone-onset response. To compare response amplitudes, the traces were normalized to a baseline of 0.5 s preceding the tone onset. The dashed box delineates the averaging window for calculating the phasic effect of VGAT-ChR2 activation (panels f–h). f, Pupil size at different tone intensities, with and without laser activation, for the same example mouse using a 1.5-s averaging window after tone onset. g. Normalized suppression of phasic pupil response to tone intensity due to activation of LC-GABA neurons (P = 0.00023 using one-way ANOVA, F 4,20 = 9.13). h, Comparison of laser to no laser trials for all trials regardless of tone intensity or frequency (***P = 4 × 10−34 using two-tailed unpaired t-test: t 1673 = 12.46). i, Average normalized suppression of pupil dilation to tone pips produced by activation of LC-GABA in VGAT-YFP-ChR2 mice for each frequency tested. The P value is for significance of the correlation using one-way ANOVA.