Fig. 2. Effects of prior “2-hit” stress with familiar (FAM) versus novel (NOV) context on anxiety-related behaviors during abstinence.

Rats were tested across 3 weeks in the following order beginning after at least 1 week of abstinence: elevated plus-maze, novelty-induced hypophagia, bottle-brush irritability, and then in counter-balanced order, by CLAMS and social investigation, and then acoustic startle and finally fear overgeneralization. (a) Timeline illustrating behavioral tests relative to two-bottle choice limited access. (b) Diagram of fear overgeneralization testing. Stress rats received a second footshock in a familiar context (Ctx A) or novel (Ctx B) box. Ninety-four days later, latency to cross to the dark compartment of a distinct, novel box (Ctx C) was measured. (c) FAM males showed longer latencies (P=0.009, planned comparisons). (d) Both FAM males (P=0.019, within-sex planned comparisons) and NOV males (P=0.049, within-sex planned comparison) spent significantly less times in the closed arms of the elevated plus-maze. There was no interaction with Sex (F_1,42=1.376, P<0.264) and a trend for a Sex main effect (F_1,41=2.455, P=0.098). (e) We did not observe any significant effects of Stress or Sex on the number of entries to the closed arms (all P’s>0.05). (f) NOV and FAM males did not consume any pellets in novelty-induced hypophagia testing, producing maximum and significantly greater intake latencies (P’s≤0.008, planned comparisons). (g) A main effect of Stress (F_2,41=4.309, P=0.02), reflected reduced food intake in NOV and FAM rats (P’s≤0.037, Dunnett’s t-test) during novelty-induced hypophagia testing. Planned comparisons suggest males (P’s≤0.011) rather than females (P’s≥0.3) drive the effect. (h). In bottle-brush testing, there was a significant main effect of stress (F_2,42=4.324, P=0.02) wherein FAM rats showed increased startle compared with controls (P=0.034, Dunnett’s t-test). Within-sex planned comparisons showed that the effect was driven by females (P=0.045) vs. males (P=0.168). (i) Stress reduced the longest bout of sleep in females (NOV, P=0.028; FAM, P=0.068; planned comparisons) during the first 11-hr of the light phase of a Comprehensive Lab Animal Monitoring System testing. (j) For social investigation/avoidance, NOV rats spent less time in the zone containing soiled bedding from unfamiliar same-sex conspecifics (P=0.019, Dunnett’s t-test; significant main effect of Stress, F_2,41=3.695, P=0.033). (k) There was also a main effect of Stress (F_2,41=3.329, P=0.046) on percentage of time in the bedding zone (interaction bedding zone time/(interaction zone time+acclimation zone time)*100), and NOV rats favored the unsoiled bedding (P=0.05, Dunnett’s t-test). *P<0.05, **P=0.01 within-sex planned comparisons, ^#P<0.05, Dunnett’s t-test, ^@P=0.05, Dunnett’s t-test, ^‡P<0.05, main effect of Sex. n=8 rats/group, except NOV males where n=7 as one NOV male was removed before behavior studies due to illness. All data shown as mean±SEM. (l-q) Acoustic startle response was also assessed. (l) The very first trial of acoustic startle testing was considered separately, as is commonly done, and did not yield significant Stress (F_2,41=0.867, P=0.428) or interaction effects (F_1,41=1.046, P=0.361) in two-way ANOVA. Females showed an overall greater response than males (F_1,41=7.914, P=0.007, main effect of Sex). (m) During the first block of 120-dB stimuli, NOV females (P=0.045, within-sex planned comparisons) and FAM females (P=0.001, within-sex planned comparisons), but not males of either stress group (P’s>0.6) exhibited significantly increased startle responses (Stress*Sex interaction: F_2,41=4.126, P=0.023, two-way ANOVA). Females still had higher responses (Sex: F_1,41=18.457, P<0.001), with no main effect of Stress (F_2,41=2.345, P=0.109). (n) Groups did not differ in their responses to the final block of 120-dB stimuli (all P’s>0.05, two-way ANOVA with within-sex planned comparisons). (o) There were no Stress or interaction effects on prepulse inhibition (all P’s>0.05, two-way ANOVA with within-sex planned comparisons), but females showed a lower prepulsed response (Sex: F_1,41=11.992, P=0.001). (p) Whereas NOV and FAM males also showed normal responses to less intense acoustic stimuli (all within-sex planned comparisons P’s>0.05), (q) within-sex planned comparisons showed that NOV females also exhibited exaggerated startle to 85-dB (P=0.005) and 100-dB stimuli (P=0.003), yielding Stress*Sex interactions (85-dB: F_2,41=3.326, P=0.046; 100-dB: F_2,41=5.740, P=0.006). *P<0.05, **P=0.001, within-sex planned comparison, ^#P<0.05, Dunnett’s t-test, ^@P<0.07, Dunnett’s t-test, ^‡P<0.05, main effect of Sex. n=8 rats /group, except NOV males where n=7. All data are shown as mean±SEM.