IL PV neurons signal active avoidance

(A) Fiber photometry schematic. (B) GCaMP6f expression in IL PV neurons. Scale bar, 100 μm. (C) Active avoidance task schematic. The green box indicates presence of the tone, which lasts till animal crosses. (D) Example IL PV ΔF/F (red) and speed (black) during two successful avoidance trials. Vertical line indicates chamber crossing. Tone, light green. (E) IL PV ΔF/F during successful avoidance trials, data aligned to chamber crossing. White ticks: chamber crossing. Black ticks: tone onset. Same example mouse as D. (F) Example average IL PV ΔF/F (red) and speed (grey), aligned to chamber crossing (left) and movement initiation (right). Same example mouse as D. (G) Average IL PV ΔF/F before chamber crossing (pre, 4 to 2 seconds before cross) and during chamber crossing (peri, 1 second before to 1 second after crossing). **p < 0.01, paired t-test. Shaded regions indicate SEM.

IL PV neural activity reflects the avoidance movement, not the predictive tone

(A) Schematic of a modified version of avoidance task including 10% short tone trials (S), where the tone is always 1.5 seconds; 10% long tone trials (L), where the tone lasts until 1.5 seconds after successful avoidance; and 80% regular trials (R), where the tone terminates upon successful avoidance. (B-C) Average IL PV ΔF/F aligned to (B) chamber crossing and (C) tone offset. (D) Comparison between average IL PV calcium activity 0 to 0.1 seconds after avoidance chamber crossings during regular trials and long tone trials (ns = non-significant, paired t-test). (E) Comparison between average IL PV calcium activity 0 to 0.1 seconds after tone offsets during regular trials, short tone trials, and long tone trials (**p < 0.01, one-way repeated ANOVA). (F-G) Average avoidance kernel (F) and average tone offset kernel (G) across all animals calculated by a linear model. (H) Loss of predictive power (ΔR2) in a reduced model with shuffled avoidance or tone offset time points (ns = non-significant, *p<0.05, one-sample t test). Error bars and shaded regions indicate SEM.

IL PV neural activity does not reflect movement to obtain rewards.

(A) Reward approach task schematic. (B) Example IL PV ΔF/F (red) and speed (black) during two successful approach trials. Vertical line indicates chamber crossing. Tone, light green. (C) IL PV ΔF/F during successful approach trials, data aligned to chamber crossing. Black ticks: tone onset; white ticks: chamber crossing; magenta ticks: first licks. Same example mouse as B. (D) Example average IL PV ΔF/F (red) and speed (grey), aligned to chamber crossing (left). Same example mouse as B. (E) Average IL PV ΔF/F before chamber crossing (pre, 4 to 2 seconds before cross) and during chamber crossing (peri, 1 second before to 1 second after crossing). ns = non-significant, paired t-test. Shaded regions indicate SEM.

IL PV neural activity becomes correlated to movement after shock.

(A-B) Example IL PV ΔF/F (red) and speed (black) during (A) habituation prior to the first-ever shock exposure, and (B) the intertrial interval after avoidance training. (C-D) Evolution of cross-covariance between IL PV activity and speed over trials (upper panel, black) and corresponding average avoidance success rate across animals (bottom panel, cyan) (C) on the first day of avoidance training (N = 7 mice) and (D) during approach in well-trained mice (N = 6 mice, green shading marks the time in chamber before the task (pre-task)). Error bars indicate SEM.

Inhibiting IL PV neural activity delays avoidance.

(A) Optogenetic schematic. (B) NpHR-eYFP expression in IL PV neurons in a PV-Cre mouse. Scale bar: 200 μm. (C) Optogenetic inactivation schematic. Interleaved simulation blocks were introduced, with optogenetic inhibition of IL PV neurons 0.5 to 2.5 seconds after tone onset. (D) Speed of NpHR-expressing mice during avoidance with (red) and without (dark brown) illumination. (E) Speed of NpHR- and eYFP-expressing mice, averaged over the laser stimulation period, with and without illumination. (F) Distribution of crossing latencies during avoidance in NpHR-expressing animals with (red) and without illumination (brown). (G) Ratio of illuminated/non-illuminated chamber crossing probabilities. (H-K) Same as (D-G) for approach task. (L-M) Same as (D-E) but for OFT with dim light. (N-O) Same as (L-M) but for OFT with bright light. ns = nonsignificant, *p < 0.05, **p < 0.01; for (E), (I), (M), and (O), 2-way ANOVA interaction term; for (G) and (K), unpaired t-test. Shaded regions indicate SEM.

(A) Average control IL GFP PV ΔF/F (red) and speed (grey), aligned to chamber crossing in avoidance (N = 2 mice). (B) Average IL PV GCaMP6f ΔF/F before (pre, −2 to 0 seconds) and after (peri, 0 to 2 seconds) avoidance movement initiation. (C) IL PV GCaMP6f ΔF/F during successful avoidance trials, data aligned to tone onset. White ticks: tone onset. Black ticks: chamber crossing. (D) Example average IL PV GCaMP6f ΔF/F (red) and speed (grey), aligned to tone onsets. Same example mouse as C. (E) Example average IL PV GCaMP6f ΔF/F (red) and speed (grey) of long latency trials (avoidance latency longer than 3 seconds), aligned to tone onset. Same example mouse as C. (F) Average IL PV GCaMP6f ΔF/F before (pre, −4 to −2 seconds) and after (peri, 0 to 2 seconds) tone onset of long latency trials (avoidance latency longer than 3 seconds). (G) Correlation between maximum speed and peak IL PV GCaMP6f ΔF/F at avoidance. Each dot represents an avoidance trial, and trials from one animal are marked in the same color. (H) Distribution of slope of linear correlation between maximum speed and peak IL PV GCaMP6f ΔF/F at avoidance. One dot represents one animal. (I) Clustering of distribution of z score of maximum speed and z score of IL PV GCaMP6f ΔF/F at avoidance. (J-L) The same as (G-I), but the correlation was calculated between avoidance latency and peak IL PV GCaMP6f ΔF/F at avoidance. (M) Avoidance success rate over time. Success rate was calculated every 10 trials. (N) Peak IL PV GCaMP6f ΔF/F at avoidance (average ΔF/F −1 to 1 second around avoidance) every 10 trials over 2 days. ns = non-significant, **p < 0.01; for B and F, paired t-test; for H and K, one-sample t-test; for N, one-way ANOVA. Shaded regions and error bars indicate SEM.

(A) Average IL PV ΔF/F (red) and speed (grey), aligned to chamber crossing in approach from PV::GFP mice. (N=2 mice). (B) Average IL PV ΔF/F (red) and speed (grey), aligned to initiation of movement for reward approach (N = 6 mice). (C) Average IL PV ΔF/F before initiation of movement for reward approach (pre, 2 to 0 seconds before movement initiation) and after movement initiation (peri, 0 second to 2 seconds after movement initiation) (N = 6 mice, p=0.0382, paired t-test). (D) Schematic of a modified reward approach task with reward omissions. (E) Average IL PV ΔF/F (top) and speed (bottom) of rewarded trials, aligned to the first lick after chamber crossing (N=5 mice).

(A) OFT schematic. (B) Example IL PV GCaMP6f ΔF/F (red) and speed (black) during OFT under bright light (white shading) and dim light epochs (grey shading). (C) IL PV GCaMP6f ΔF/F during OFT, aligned to maximum speed of each movement epoch in OFT. White ticks: maximum speed of movement epoch. Same example mouse as B. (D) Example average IL PV GCaMP6f ΔF/F (red; purple) and speed (grey; dark blue), aligned to maximum speed of each movement epoch in OFT under bright lighting (red; grey) and under dim lighting (purple; dark blue). Same example mouse as B. (E) Average IL PV GCaMP6f ΔF/F before (pre, −4 to −2 seconds) and during (peri, −1 to 1 seconds) peak movement under bright light (red) and dim light (blue). (F) Average control IL PV GFP ΔF/F (red; purple) and speed (grey; dark blue), aligned to the maximum speed of each movement epoch in OFT under bright light (red; grey) and dim light (purple; dark blue) (N=2 mice). Shaded regions indicate SEM.

(A) Evolution of cross-covariance between movement speed and PV activity in habituation and inter-trial intervals on the first day of avoidance training (N=7 mice). (B) Evolution of cross-covariance between movement speed and PV activity in habituation and inter-trial intervals of well-trained animals during the approach task (N=6 mice).

(A) Speed of eYFP-expressing mice during avoidance with (red) and without (dark brown) illumination. (B) Distribution of crossing latencies during avoidance in eYFP-expressing animals with (red) and without illumination (brown). (C-D) Same as (A-B) for approach. (E) Freezing probabilities of NpHR-expressing animals during avoidance with (red) and without (dark brown) illumination. (F) Difference in freezing probabilities between with (on) and without (off) illumination in NpHR-expressing mice (red) and in eYFP-expression mice (grey). (G) Difference in freezing probabilities, averaged over the laser stimulation period, between with (on) and without (off) illumination in NpHR-expressing mice (red) and in eYFP-expression mice (grey). (H) Change in ratio of freezing duration/avoidance latency of NpHR-expressing and eYFP-expression mice during illumination compared to non-illumination. (I) Crossing latencies during avoidance in NpHR-expressing mice, grouped by relative position prior or within a stimulation block. (J) Average crossing latencies of all non-illuminated trials (off), 1st trials (1st stim) and 2nd-6th trials in illuminated blocks (2-6th stim). (K) Crossing latencies during avoidance in NpHR-expressing mice, grouped by relative position within or after a stimulation block. (L) Average crossing latencies of all illuminated trials (stim), 1st trials (1st off) and 2nd-6th trials (2-6th off) in non-illuminated blocks. (M) Post-crossing optogenetic inactivation schematic. (N) Speed of NpHR-expressing mice during avoidance with (red) and without (dark brown) illumination. (O) Same as (N), but the y-axis was plotted on a log scale. (P) Change in speed of NpHR-expressing and eYFP-expression mice during illumination (0 to 2 s from avoiding chamber crossing) compared to non-illumination trials. (Q) The latency of the first trials in stimulation blocks is compared to the latency of the second trials in stimulation blocks, and the latency of the first trials after stimulation blocks is compared to the latency of the second trials after stimulation blocks. ns = non-significant, *p < 0.05, **p < 0.01; for G and H, unpaired t-test; for J, L and Q, paired t-test; for P, 2-way ANOVA interaction term. Shaded regions and error bars indicate SEM.