A behavioral paradigm for investigating innate decision-making in mice.

(A) Schematic of the behavioral assay (3D and top-down views). (B) Top: arena occupancy patterns for five example mice in one session. Bottom: visit frequency and average duration for each visit. Colors mark the mouse’s identity. (C) Visit frequency and duration under exploration and looming conditions. Error bars represent the standard deviation. N = 46 mice, paired t-test. (D) The pipeline for behavioral classification. (E) Distribution of behavioral decisions across 3862 trials from 140 mice. (F) Left: distance to the safe zone for four decision types (10 example trials each). Red dashed lines mark the onset of each stimulus repetition; solid lines mark the end of the last repetition. Grey shade indicates the safe zone. Right: locomotion speed towards the safe zone for the same trials. (G) Distribution of the longest stationary time for “freezing” and “assessment+flight”. N= 224 and 1667, p < 0.001, Mann-Whitney-Wilcoxon test. (H) Distribution of latency to flee for “direct flight” and “assessment+flight”. N= 458 and 1667, p < 0.001, Mann-Whitney-Wilcoxon test. (I) Distribution of peak speed for “direct flight” and “assessment+flight”. N= 458 and 1667, p < 0.001, Mann-Whitney-Wilcoxon test. (J) Distribution of latency to hide in the safe zone for “direct flight” and “assessment+flight”. N= 455 and 1563, p < 0.001, Mann-Whitney-Wilcoxon test. (K) Distribution of behavioral states at the end of each trial for “direct flight”, “assessment+flight”, and “freezing”. (L) Distribution of fear recovery time for “direct flight”, “assessment+flight”, and “freezing”. N= 74, 458, and 205, p < 0.001, Kruskal-Wallis test and Dunn’s post-hoc test.

Mice learn quickly from experience.

(A) Distance to the safe zone (top) and locomotion speed (bottom) for the 1st, 2nd, 5th, 10th, and 20th trials of 19 mice. Dash lines mark the start of each stimulus, and solid lines mark the end of the stimulus. Positive speed is towards the safe zone. (B) Defensive probability decreases with the number of trials. (C) Summary of the decisions made by 19 mice for the first 20 trials. (D) Latency to flee increases with the number of trials. Gray shade indicates the standard error of the mean. N=19. (E) Hiding latency increases with the number of trials. N=19. (F) Peak fleeing speed varies with the number of trials. N=19.

Mice make economic decisions modulated by vigilance.

(A) Distance to the safe zone and locomotion speed towards the safe zone across trials in response to low- and high-contrast looming stimuli with different reward values. Dash lines indicate stimulus onset; solid lines mark stimulus offset. (B) Distribution of decision types for six experimental conditions. N = 40 trials from 4 mice (none, low), 50 trials from 5 mice (water, low), 50 trials from 5 mice (sucrose, low), 50 trials from 5 mice (none, high), 39 trials from 5 mice (water, high) and 30 trials from 5 mice (sucrose, high), Chi-squared test. (C-F) Distance fled, duration in the reward zone, latency to flee, and peak fleeing speed across all conditions. Two-way ANOVA with post hoc Tukey’s test. N = 40, 50, 50, 50, 39, 30 trials for C, D, F, and N = 27, 30, 19, 49, 39, 29 trials for E. (G) Water or sucrose consumption during exploration and looming experiments at low and high contrasts. N = 10, 10, 5, 5, 5, 5 trials. Mann-Whitney-Wilcoxon test. Whiskers indicate the minimum and maximum. Boxes indicate the first and third quartiles. (H) Number of up-attention action bouts before exposed to looming at the first and second trials. N = 4, 5, 5, 5, 5, 5 mice. Wilcoxon signed-rank test. (I) Experimental timeline investigating the influence of water reward on innate decision-making in the same mouse. (J) Distance to the safe zone and locomotion speed towards the safe zone across trials in response to low-contrast looming stimuli with and without water reward in an example mouse. Left, non-reward condition followed by reward condition; Right, reward condition followed by non-reward condition. (K) Decision types of nine mice across five trials. Gray squares indicate trials where the mouse did not enter the arena within 30 min. (L) Defensive probability in non-reward and reward conditions. N = 9 mice, paired t-test. (M) Distance fled, duration in reward zone, latency to flee, and peak speed in non-reward and reward conditions. N = 5 mice for latency to flee and 9 mice for other features, paired t-test, two-sided. For all panels: #p < 0.1, **p < 0.05, **p < 0.01, ***p < 0.001.

Influence of social hierarchy structure on innate decision-making.

(A) Schematic of the social hierarchy experiment. Top, experimental timeline. Each session lasted 2 hours per mouse pair during the pre-looming, looming, and post-looming phases. Bottom, schematic of the tube test. (B) Arena occupancy for an example pair of mice during the three sessions. (C) Visiting frequency and average duration per visit for dominant and subordinate mice during the pre-looming, looming, and post-looming sessions. N = 9 pairs, paired t-test. (D) Distance to the safe zone over seven days for an example pair. Looming stimuli were presented on days 4 and 5. (E) Percentage of time spent in the reward zone across days. Error bars represent SEM. N = 9 pairs, paired t-test. (F) Distance to the safe zone (left) and locomotion speed (right) for an example pair. (G) Behavioral decisions across the first 10 trials for 9 mouse pairs. (H) Pie charts showing decision distributions for dominant and subordinate mice. N = 90, 90 trials, Chi-squared test. (I) Violin plots comparing dominant and subordinate mice for: latency to flee (N = 90, 78 trials), distance fled, peak fleeing speed, and duration in the reward zone (N = 90, 90 trials) for dominant and subordinate mice. Mann-Whitney-Wilcoxon test. For all panels: **p < 0.05, **p < 0.01, ***p < 0.001.

The drift-diffusion leaky integrator model.

(A-F) Simulated evidence level for escape and predicted latencies to flee and decision types across six conditions. (G) Compare decision models based on threat intensity, reward value, and vigilance to experimental results. Color saturation indicates the likelihood of defensive decisions.

Quality control for behavioral classification model.

(A) Tracking accuracy of mouse nose and tail base in DeepLabCut. (B) Model performance evaluated by a confusion matrix on the test dataset.

Behavioral responses to looming stimuli presented at four distances from the safe zone.

(A) Distance to the safe zone. Red dashed lines mark the onset of each stimulus repetition; solid lines mark the end of the last repetition. Grey shade indicates the safe zone. N = 18 (70 cm), 19 (50 cm), 13 (30 cm), and 11 (10 cm) trials. (B) Distribution of escape directions.

Comparison of behavioral responses to looming stimuli before and after the tube test.

(A) Schematic timeline of the looming experiment before and after the tube test. (B) Behavioral decisions for dominant and subordinate mice before and after the tube test; Chi-squared test. (C) Violin plots showing latency to flee, distance fled, peak fleeing speed, and duration in the reward zone for dominant (top) and subordinate (bottom) mice before and after the tube test; Mann-Whitney-Wilcoxon test. *p<0.05.