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

Abstract

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.

Keywords: fear; optogenetics; prefrontal cortex; sex differences.

Figure 1.. Platform-mediated avoidance (PMA) under social conditions increases freezing and decreases pressing during the intertrial interval (ITI) compared to PMA under solitary conditions.

A. Schematic of PMA under social (left, purple, n=42) and solitary (blue, n=59) conditions. B. Percentage of time spent on the platform during the tone, C. Number of shocks avoided per day, D. Percentage of time freezing during the tone, E. Number of darting bouts during the tone, and F. Number of lever presses during the ITI. There was no significant difference in time on platform (z = −1.252, p=0.211) or number of shocks avoided (z=−0.552, p=0.581). There was a significant increase in freezing (z=4.068, p<0.001) and a significant decrease in darting (z = −4.786, p < 0.001) and ITI pressing (z=−6.034, p<0.001) in rats trained under social compared to solitary conditions. Data reported are main effects of the regression models (see Supplementary tables S1–S5). Data are shown across 10 days of training (trials shown in blocks of 3) and as mean ± SEM; ***p<0.001.

Figure 2.. PMA under social conditions reduces behavioral sex differences that are observed during PMA under solitary conditions.

A. Percentage of time on platform during the tone, B. Number of shocks avoided and shock reactivity (inset), as measured by the maximum velocity of each rat during Tone 1 on the first day of training, C. Percentage of time freezing during the tone, D. Number of darting bouts during the tone, and E. Number of lever presses during the ITI in female (n=21, salmon) and male (n=21, teal) rats trained under social conditions. During social partner PMA training, females pressed significantly less than males (z=−3.112, p=0.002), but there were no sex differences in time on platform (z=1.308, p=0.191), number of shocks avoided (z=1.590, p=0.112), freezing (z=−0.673, p=0.501), or darting (z=−0.886, p=0.375). F. Percentage of time on platform during the tone, G. Number of shocks avoided and shock reactivity (inset), H. Percentage of time freezing during the tone, I. Number of darting bouts during the tone, and J. Number of presses during the ITI in female (n=27, salmon) and male (n=32, teal) rats trained under solitary conditions. During solitary PMA training, females spent significantly more time on the platform (z=3.174, p=0.002), avoided significantly more shocks (z=2.949, p=0.003), and pressed significantly less (z=−3.044, p=0.002) compared to males. 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.

Figure 3.. Avoidance and freezing increase in the absence of a social partner.

A. Schematic of PMA under social conditions with a Trained Partner (n=20, purple) or another Learner Rat (n=22, yellow). B. Percentage of time on platform during the tone, C. Number of shocks avoided, D. Percentage of time freezing during the tone, E. Number of darting bouts during the tone, and F. Number of lever presses during the ITI across the 10 days of training (left), and on Day 11 in the absence of the partner (right). During social partner PMA training, Learner Rats with a Trained Partner darted significantly more compared to those trained with another Learner Rat (z=2.700, p=0.007). There were no differences in avoidance (time on platform: z=−1.548, p=0.122); number of shocks avoided: z=0.391, p=0.695), freezing (z=−1.168, p=0.243), or pressing (z=0.218, p=0.827) On Day 11 in the absence of the partner, Learner Rats trained with either partner type spent significantly more time on platform (with Trained Partner: z = −31.104, p < 0.001; with Learner Rat: z = −7.669, p < 0.001); avoided more shocks (with Trained Partner: z = −3.455, p < 0.001; with Learner Rat: z = −2.519, p = 0.012), and showed significantly increased freezing when trained with a Trained Partner (z = −3.921, p < 0.001). Learner Rats with a Learner Rat Partner showed significantly more darting on Day 11 compared to Day 10 (z = −10.019, p < 0.001), and those with a Trained Partner trended in the same direction (z = −1.953, p = 0.051). Data reported are main effects of the regression models (see Supplementary tables S6–S10). Day 10 vs. Day 11 comparisons are from Tukey’s post-hoc tests on the regression models. Data are shown in blocks of 3 for each day, mean ± SEM. *p<0.05, **p<0.01.

Figure 4.. Photoinactivation of ACC projection neurons impairs avoidance under social conditions regardless of partner presence.

A. Schematic of virus infusion, location of min/max expression of AAV, followed by avoidance training and tests. At Test w/ partner and w/o partner (Days 11/12), 532nm light was delivered to ACC during the entire 30-second tone presentation (Tone 1). B. Percentage of time on platform at Training (Day 10, Tone 1) and Test (Days 11/12, Tone 1 with laser ON and Tone 2 with laser OFF) for ArchT-eYFP (n=14, orange) and eYFP control rats (n=13, grey) when the partner was present. Avoidance decreased in ArchT-eYFP rats (repeated measures ANOVA, main effect of trial (F_(2,38)=13.88, p<0.001) and interaction between trial and AAV (F_(2,38)=3.70, p=0.034); post hoc Tukey test revealed a significant decrease between Tone 1 of Day 10 vs. Day 11 in ArchT-eYFP (p=0.002), but not in eYFP controls (p=0.478)). C. Latency of avoidance for each rat during Test w/ partner (Tone 1 at Test; Male ArchT, light orange, Female ArchT, dark orange; Male eYFP control, light grey; Female eYFP control, dark grey). D. Percentage of time on platform in 3 s bins (Tone 1 at Test) revealed no effect of photoinactivating ACC neurons in the presence of the partner (repeated measures ANOVA, post hoc Tukey, F_(9,126)= 0.891, p=0.535). E. Percentage of freezing (left), suppression of bar pressing (middle), and number of darting bouts (right) during the Tone+Laser trial. F. Percentage time on platform at Training (Day 10, Tone 1) and Test (Day 11, Tone 1 with laser ON and Tone 2 with laser OFF) for ArchT-eYFP (n=14, orange) and eYFP control rats (n=14, grey) when the partner was absent. Avoidance decreased in ArchT-eYFP rats (repeated measures ANOVA, main effect of trial (F_(2,40)=21.79, p<.001) and interaction between trial and AAV (F(2,40)=4.21, p=.022); post hoc Tukey test revealed a significant decrease between Tone 1 of Day 10 vs. Day 11 in ArchT-eYFP (p<0.001), but also in eYFP controls (p=0.008)). G. Latency of avoidance for each rat during Test w/o partner (Tone 1 at Test; Male ArchT, light orange, Female ArchT, dark orange; Male eYFP control, light grey; Female eYFP control, dark grey). H. Percentage of time on platform in 3 s bins (Tone 1 at Test) revealed a significant reduction in avoidance in ArchT-eYFP rats compared to eYFP controls in the absence of the partner (repeated measures ANOVA, post hoc Tukey, F(_9,135)=3.09, p=0.002). I. Percentage of freezing (left), suppression of bar pressing (middle), and number of darting bouts (right) during the Tone+Laser trial revealed a trend toward less freezing in ArchT-eYFP rats (t₍₂₀₎=2.036, p=0.0552) and significantly lower suppression of bar pressing in ArchT-eYFP rats (t₍₂₀₎=3.225, p=0.0042). All data are shown as mean ± SEM; #p<0.06, *p<0.05, **p<0.01, ***p<0.001.

Figure 5.. Photoinactivation of ACC projection neurons delays avoidance expression in male rats but blocks avoidance expression in female rats under solitary conditions.

A. Schematic of virus infusion, location of min/max expression of AAV, followed by avoidance training and test. At Test, 532nm light was delivered to ACC during the entire 30-second tone presentation (Tone 1). B. Percentage of time on platform at Training (Day 10, Tone 1) and Test (Day 11, Tone 1 with laser ON and Tone 2 with laser OFF) for ArchT-eYFP (n=22, left, orange) and eYFP control rats (n=26, right, grey). Avoidance was impaired in ArchT-eYFP rats (repeated measures ANOVA, main effect of trial (F_(2,70)=18.49, p<0.001); post hoc Tukey test revealed a significant decrease between Tone 1 of Day 10 vs. Day 11 in ArchT-eYFP (p<0.001), but also in eYFP controls (p=0.004)). C. Latency of avoidance for each rat (Tone 1 at Test). There was no significant difference between ArchT-eYFP and eYFP control rats (t₍₄₁₎=0.815, p=0.420). D. Percentage of time on platform in 3 s bins (Tone 1 at Test) revealed no significant differences between groups (repeated measures ANOVA, post hoc Tukey, F_(9,279)=1.25, p=0.265). E. Percentage of time on platform in 3 s bins (Tone 1 at Test) revealed a significant delay in avoidance when silencing ArchT-eYFP neurons in male rats (light orange) while blocking avoidance in female rats (dark orange; repeated measures ANOVA F_(9,162)=3.76, p<0.001, post hoc Tukeys, all p’s<0.001 15–30 sec). F. Percentage of freezing (left), suppression of bar pressing (middle), and number of darting bouts (right) during the Tone+Laser trial. Data shown as mean ± SEM;*p<0.05; **p<0.01; ***p<0.001.