Supplementary Figure 7: Behavior during tasks involving unavoidable aversive air puff or tail shock and related photometry controls.

a, Percentage change in eye closure behavior for task involving unavoidable air puff, relative to pre-cue baseline period for reward cue (RC), neutral cue (NC), and aversive cue (AC-Un) trials. Error bars: s.e.m. across 6 mice. See Methods for details. b, Left: percentage change in eye closure during each cue period. Right: percentage change in eye closure after air puff delivery (n = 6, *** p = 0.0008, ** p = 0.0055, two-sided t-test against null distribution with mean of zero). Mean ± s.e.m. across 6 mice. c, Percentage change in eye closure for air puff experiments, relative to pre-cue baseline period from hungry trials. Note that persistent defensive eye closure increased with satiety. Note that VTA^DA→BA axon responses to unavoidable aversive outcome predicting cue (AC-Un) did not decrease with satiety, indicating that vision was not impaired in the contralateral eye receiving visual stimuli. Mean ± s.e.m., n = 6 mice. d, Percentage increase in pre-cue persistent eye closure during sated trials relative to hungry trials. (n = 6, * p = 0.011, two-sided t-test again null distribution with mean of zero). e, Mean running behavior for task involving unavoidable tail shock during second session of training for reward cue (RC), neutral cue (NC), and aversive cue (AC-Un) trials. Error bars: s.e.m. across 8 mice (1 session/mouse). f, Left: change in speed during cue period relative to pre-cue baseline (n = 8 mice, * p = 0.028, two-sided t-test again null distribution with mean of zero). Right: change in speed during tail shock delivery relative to pre-cue baseline (n = 8 mice, * p = 0.011, two-sided t-test again null distribution with mean of zero). Mean ± s.e.m. g, Left: mean cue responses in fiber photometry recordings from VTA^DA→BA axons (4^th and final day of air puff exposure) in hungry mice and sated mice. Error bars: s.e.m. across 6 mice. Note that response to AC-Un progressively decreased across days (see also Supplementary Fig. 6; we speculate that this may reflect a decrease in motivational salience of the cue as mice develop learned helplessness), but continues to increase in sated sessions. Right: mean VTA^DA→BA cue responses (first session of tail shock exposure) in hungry mice. Error bars: s.e.m. across 6 mice. Z: Z-score. Note that the same mice previously learned the AC-Un association with unavoidable air puff and that the AC-Un response is rapidly restored upon exposure to the same visual cue now predicting unavoidable tail shock. h, Top: example GCaMP6s fiber photometry recording from VTA^DA→BA axons showing mean response and single-trial responses to reward cue (RC; left, mean ± s.e.m., n = 56 trials from 1 mouse) and to cues predicting unavoidable air puff delivery (AC-Un; right, mean ± s.e.m., n = 32 trials from 1 mouse). Note that Ensure reward delivery or air puff delivery occur following the second vertical dashed line. Bottom: mean and single-trial photometry traces from GFP controls during reward cues (RC; left, mean ± s.e.m., n = 69 trials from 1 mouse) and during cues predicting unavoidable air puff delivery (AC-Un; right; mean ± s.e.m., n = 30 trials from 1 mouse) do not show the same transient events observed in GCaMP6s recordings. Images at right: example histological reconstructions of fiber placements over BA and fluorescence of VTA^DA→BA axons for GCaMP6s (top) and GFP (bottom) experiments.