Active avoidance recruits the anterior cingulate cortex regardless of social context in male and female rats

Actively avoiding danger is necessary for survival. Most research has focused on the behavioral and neurobiological processes when individuals avoid danger alone, under solitary conditions. Therefore, little is known about how social context affects active avoidance. Using a modified version of the platform-mediated avoidance task in rats, we investigated whether the presence of a social partner attenuates conditioned freezing and enhances avoidance learning compared to avoidance learned under solitary conditions. Rats spent a similar percentage of time avoiding during the tone under both conditions; however, rats trained under social conditions exhibited greater freezing during the tone as well as lower rates of darting and food seeking compared to solitary rats. Under solitary conditions, we observed higher levels of avoidance in females compared to males, which was not present in rats trained under social conditions. To gain greater mechanistic insight, we optogenetically inactivated glutamatergic projection neurons in the anterior cingulate cortex (ACC) following avoidance training. Photoinactivation of ACC neurons reduced expression of avoidance under social conditions both in the presence and absence of the partner. Under solitary conditions, photoinactivation of ACC delayed avoidance in males but blocked avoidance in females. Our findings suggest that avoidance is mediated by the ACC, regardless of social context, and may be dysfunctional in those suffering from trauma-related disorders. Furthermore, sex differences in prefrontal circuits mediating active avoidance warrant further investigation, given that females experience a higher risk of developing anxiety disorders.


Table S2 . Number of shocks avoided in social vs. solitary PMA Fixed
their interactions and random intercept effects of Animal and Stimulus Number predicting the proportion of time spent on the platform.All categorical variables are effect coded.Trial Number and Stimulus Number were both rescaled to range from 0 to 1 to improve model convergence.effects parameter estimates of the multilevel negative binomial regression with Group Type (social vs. solo), Sex, Trial Number (1-10), and their interactions and random intercept effects of Animal and Stimulus Number predicting the number of shocks avoided.All categorical variables are effect coded.

Table S3 . Freezing in social vs. solitary PMA Fixed
effects parameter estimates of the multilevel binomial logistic regression with Group Type (social vs. solitary), Sex, Trial Day, and their interactions and random intercept effects of Animal and Stimulus Number predicting the proportion of time spent freezing.All categorical variables are effect coded.Trial Number and Stimulus Number were both rescaled to range from 0 to 1 to improve model convergence.

Table S5 . ITI pressing in social vs. solitary PMA Fixed
effects parameter estimates of the multilevel negative binomial regression with Group Type (social vs. solitary), Sex, Trial Day, and their interactions and random intercept effects of Animal and Stimulus Number predicting the number of lever presses in each ITI.All categorical variables are effect coded.Trial Number and Stimulus Number were both rescaled to range from 0 to 1 to improve model convergence.

Table S6 . Time on platform in Learner Rats with Trained Partners or Learner Rats Fixed
effects parameter estimates of the multilevel binomial logistic regression with Partner Type (Trained Partner vs. Learner Rat), Sex, Trial Number (1-11), and their interactions and random intercept effects of Animal and Stimulus Number predicting the proportion of time spent on the platform.Model was run on only data from social rats, with 10 days of social partner PMA and Day 11 in the absence of the partner (11 days total).All categorical variables are effect coded.Trial Number and Stimulus Number were both rescaled to range from 0 to 1.1 to improve model convergence.

Table S7 . Number of shocks avoided in Trained partners and Learner rats
Fixed effects parameter estimates of the multilevel binomial logistic regression with Partner Type(Trained Partner vs. Learner Rat), Sex,, and their interactions and random intercept effects of Animal and Stimulus Number predicting the number of shocks avoided.Model was run on only data from social rats, with 10 days of partner PMA and Day 11 in the absence of the partner (11 days total).All categorical variables are effect coded.

Table S9 . Darting in Trained partners and Learner rats in social PMA Fixed
effects parameter estimates of the multilevel binomial logistic regression with Partner Type(Trained Partner vs. Learner Rat), Sex,, and their interactions and random intercept effects of Animal and Stimulus Number predicting the number tone-induced darting bouts.Model was run on only data from social rats, with 10 days of partner PMA and Day 11 in the absence of the partner (11 days total).All categorical variables are effect coded.

Table S10 . ITI pressing in Trained partners and Learner rats in social PMA Fixed
effects parameter estimates of the multilevel binomial logistic regression with Partner Type (Trained Partner vs. Learner Rat), Sex, Trial Number (1-11), and their interactions and random intercept effects of Animal and Stimulus Number predicting the number of lever presses in each ITI.Model was run on only data from social rats, with 10 days of partner PMA and Day 11 in the absence of the partner (11 days total).All categorical variables are effect coded.Trial Number and Stimulus Number were both rescaled to range from 0 to 1.1 to improve model convergence.

PMA under social conditions increases freezing and decreases pressing, regardless of sex. A.
Percentage of time on platform during the tone, B. Number of shocks avoided, C. Percentage of freezing during the tone, D. Number of darting bouts during the tone, and E. Number of presses during the ITI in females trained under social (n=21, dark red) or solitary (n=27, orange) conditions.F. Percentage of time on platform during the tone, G. Number of shocks avoided, H. Percentage of freezing during the tone, I. Number of darting bouts during the tone, and J. Number of presses during the ITI in males trained under social (n=21, dark blue) or solitary (n=32, light blue)

Figure 2. Acquisition of PMA under social and solitary conditions for optogenetic rat cohorts A.
).Data reported are post-hoc Tukey tests on the regression models.Data are shown across 10 days of training (trials shown in blocks of 3) and as mean ± SEM; **p<0.01;***p<0.001.Supplementary Percentage of time on platform during the tone, B. Number of shocks avoided, C. Percentage of freezing during the tone, and D. Number of lever presses during the ITI in ArchT-eYFP (n=14, orange) and eYFP controls (n=14, grey) during 10 days of PMA training under social conditions.There was a small but significant main effect of AAV in time on platform (repeated measures ANOVA, F(1,14)=6.04,p=0.028)where eYFP controls spent more time on the platform compared to ArchT-eYFP rats; however, no Tukey's post hoc comparisons were statistically significant for any specific day of PMA training.There were no significant differences in the number of shocks avoided (repeated measures ANOVA, F(1,14)=2.73,p=0.121), freezing (repeated measures ANOVA, F(1,14)=0.006,p=0.941), or number of ITI presses (repeated measures ANOVA, F(1,14)=0.624,p=0.443).E. Percentage of time on platform during the tone, F. Number of shocks avoided, G. Percentage of freezing during the tone, and H. Number of presses during the ITI in ArchT-eYFP (n=13, orange) and eYFP controls (n=20, grey) during 10 days of PMA training under solitary conditions.There were no significant differences in time on platform (repeated measures ANOVA, F(1,20)=0.447,p=0.511), number of shocks avoided (repeated measures ANOVA, F(1,20)=0.005,p=0.944), freezing (repeated measures ANOVA, F(1,20)=1.758,p=0.200), or number of ITI presses (repeated measures ANOVA, F(1,20)=0.960,p=0.339).Data shown across 10 days of training (trials shown in blocks of 3).Data are shown as mean ± SEM.