Article Figures & Data
Figures
- Figure 1.
Experimental timeline and social stress. A, Experimental timeline. Rats underwent surgery for GCaMP6s expression and fiber implantation followed by 2 weeks rest prior to social stress. Rats were pre-exposed to the social preference test (SoPT) apparatus and then exposed to 5 consecutive days of social stress or control procedures. Within 10 d of the last stress, rats underwent two trials of a SoPT with simultaneous fiber photometry recordings. B, All rats in the social stress group experienced repeated attacks [average number of attacks per day and (C) total number of attacks]. D, Most rats displayed at least one episode of submission posture. E, Social stress reduced weight gain compared with control. Data shown as mean ± SEM. *p < 0.05, ***p < 0.001, ****p < 0.0001. Panel A created with BioRender.com.
- Figure 2.
Social stress altered social behavior. A, Experimental timeline and description of apparatus zones for Trial 1 (novel same-strain rat vs novel object) and Trial 2 (novel same-strain rat vs novel different-strain rat). The “proximity” zone consisted of the space immediately surrounding the cage (dotted line), and the “area” zone included the entire third of the apparatus (labelled). B, Left, During Trial 1, both social stress and control groups spent more time in the novel same-strain rat area than the novel object area or center; however, socially stressed rats spent more time in the novel same-strain rat area compared with controls. B, Right, During Trial 2, both groups spent more time in the different-strain rat area than the novel same-strain rat area or center, but there were no differences between stress and control groups. C, In Trial 1, socially stressed rats spent more time investigating in the novel same-strain rat proximity zone at the beginning of the test relative to control and (D) did not show differences in time spent in the novel object proximity zone. E, Socially stressed and control rats showed similar social preference over the course of Trial 1. F, In Trial 2, socially stressed rats avoided spending time in the novel same-strain rat proximity zone compared with controls. G, Socially stressed rats also avoided spending time in the novel different-strain rat proximity zone at the beginning of the test relative to controls. H, These results were also reflected in the preference index in Trial 2, where both groups initially spent more time with the novel different-strain rat than the novel same-strain rat, but control rats slowly regained a preference for the novel same-strain rat over time while stressed rats did not (significant condition × time interaction). I, Socially stressed and control rats showed a similar number of transitions between stimuli in Trial 1. J, In Trial 2, socially stressed rats showed significantly more transitions between stimuli than control rats. We measured the amount of time spent with each stimulus rat to see if a high rate of transitions was associated with shorter bout duration. K, In Trial 1, the average bout duration was similar between socially stressed and control rats while in both the novel same-strain rat proximity zone and the novel object proximity zone. L, In Trial 2, the average bout duration was similar between social stress and control groups in the novel same-strain rat proximity zone. However, socially stressed rats had shorter average bout durations while in the novel different-strain rat proximity zone compared with controls, suggesting that socially stressed rats show increased social behavior fragmentation in the presence of a novel threat. Data shown as mean ± SEM. *p < 0.05, **p < 0.01. Panel A created with BioRender.com. Panel B bars are superimposed, as the “proximity” zone is included within the “area” zone.
- Figure 3.
Histology and sample fiber photometry traces. A, Placements of GCaMP6s virus below fiber with A/P coordinates displayed as mm from the bregma based on a rat brain atlas (Paxinos and Watson, 2009). Placements from control rats marked in black and placements from socially stressed rats marked in blue. B, Sample fiber photometry recording in the posterior medial amygdala (MeAp) from a control rat in Trial 1 (novel same-strain rat vs novel object). Periods of social investigation are overlaid in blue and periods of object investigation are overlaid in gray. Darker shades represent closer exploration in the “proximity” zone rather than the “area” zone. Periods of time with no color overlay represent time spent in the center zone. C, Peri-event heat maps demonstrated that the MeAp had increased activity following entry (red line) to the novel same-strain rat proximity zone in Trial 1 in both control and socially stressed rats. D, The MeAp was also activated after entry to the novel different-strain rat proximity zone in both control and socially stressed rats, though the social stress group appeared to show higher MeAp activity than controls. E, Sample peri-event traces from one socially stressed rat are shown in inset. F, Representative images of GCaMP6s viral expression in control and (G) socially stressed rats.
- Figure 4.
Socially stressed rats showed stimulus-selective MeAp responses. A, In Trial 1 (novel same-strain rat vs novel object), both social stress and control rats showed higher posterior medial amygdala (MeAp) activity while in the novel same-strain rat proximity zone than the novel object proximity zone or center zone. B, In Trial 2 (novel same-strain rat vs novel different-strain rat), socially stressed rats showed lower MeAp activity while in the novel same-strain rat proximity zone and higher MeAp activity while in the novel different-strain rat proximity zone compared with controls. We calculated the relative MeAp ΔF/F signal for Trial 1 (same-strain–object) and Trial 2 (same-strain–different-strain). C, In Trial 1, relative MeAp activity was similar between social stress and control groups. D, In Trial 2, MeAp activity favored the novel same-strain rat in controls but favored the novel different-strain rat in socially stressed rats. We measured the amount of nose–nose contact between the experimental rat and the novel same-strain rat and novel different-strain rat during social preference tests. E, In Trial 1, the amount of nose–nose contact was similar between social stress and control groups. F, In Trial 2, socially stressed rats engaged in significantly less nose–nose contact with the novel same-strain rat but showed a similar amount of nose–nose contact with the novel different-strain rat, compared with controls. G, Posterior medial amygdala (MeAp) activity was significantly higher in both groups during nose–nose contact with the novel same-strain rat compared with MeAp activity while in the proximity zone in general during Trial 1. H, There was a trend toward higher MeAp activity while engaging in nose–nose contact with the novel same-strain rat compared with the novel different-strain rat; however, there were no differences between social stress and control groups in Trial 2. We illustrated the temporal pattern of MeAp activity in relation to the onset of several behaviors, including (I) nose–nose contact with the novel same-strain rat in Trial 1, (J) nose–nose contact with the novel same-strain rat in Trial 2, (K) nose–nose contact with the novel different-strain rat in Trial 2, and (L) startle events while in the novel different-strain rat proximity zone in Trial 2. Perievent MeAp activity was also quantified using the AUC of each event. M, In Trial 1, MeAp activity 0 to 5 s after the onset of nose–nose contact with the novel same-strain rat was similar in socially stressed and control groups. N, In Trial 2, MeAp activity 0 to 5 s after the onset of nose–nose contact with the novel same-strain rat was also similar in both groups. O, However, MeAp activity 0 to 5 s after the onset of nose–nose contact with the novel different-strain rat was higher in socially stressed rats compared with controls. P, MeAp activity 2.5 s before to 5 s after the onset of startle behaviors (red vertical line) near the novel different-strain rat was higher during events in socially stressed rats compared with controls. Data shown as mean ± SEM for each rat in A–L and mean ± SEM for each event in M–P. *p < 0.05, **p < 0.01, ***p < 0.001.
Tables
Data structure Type of test Power (confidence interval) a—average number of attacks/day 5.0 ± 0.22 Normal No test 95% CI [4.2, 5.8] b—average number of total attacks 24.6 ± 1.1 Normal No test 95% CI [21.0, 28.0] c—weight gained, main effect of condition (stress), F(1,98) = 11.6, p = 0.0010 Normal Two-way mixed-effects model ANOVA 95% CI of difference [1.2, 4.4] d—weight gained, Day 4, p = 0.022 Normal Holm–Sidak'sa multiple-comparisons test 95% CI [0.16, 6.0] e—weight gained, recording day, p < 0.0001 Normal Holm–Sidak's multiple-comparisons test 95% CI [4.2, 10.1] f—time spent in the novel same-strain rat or novel object area, condition × zone interaction, F(2,264) = 6.9, p = 0.0012 Normal Two-way mixed-effects model ANOVA 95% CI of difference [10.5, 106.2] g—time in the novel same-strain rat area, p = 0.011 Normal Holm-Sidak's multiple-comparisons test 95% CI [−61.9,−6.2] h—time spent in the novel same-strain rat or novel different-strain rat area, main effect of zone, F(2,210) = 74.1, p < 0.0001 Normal Two-way mixed-effects model ANOVA 95% CI of difference [48.1, 127.0] (to obtain CI of difference between same-strain and different strain, reran without center zone) i—time in the novel same-strain rat proximity zone over time, condition × time interaction (T1), F(4,352) = 2.7, p = 0.031 Normal Two-way mixed-effects model ANOVA 95% CI of difference [−16.7, −1.8] True CI interaction computation not possible because >2 (5) time points. As proxy, CI of interaction computed from data collapsed to T0-2 and T8-10 j—time in the novel same-strain rat proximity zone (T1), 0–2 min time point, p = 0.0067 Normal Holm–Sidak's multiple-comparisons test 95% CI [−18.0, −2.0] k—preference index over time (T1), main effect of time, F(4,350) = 4.8, p = 0.0009 Normal Two-way mixed-effects model ANOVA 95% CI of difference [14.9, 41.8] True CI main effect computation not possible because >2 (5) time points. As proxy, CI of interaction computed from data collapsed to T0-2 and T8-10 l—time in the novel same-strain rat proximity zone over time (T2), main effect of condition, F(1,70) = 5.5, p = 0.022 Normal Two-way mixed-effects model ANOVA 95% CI of difference [0.42, 5.3] m—time in the novel same-strain rat proximity zone (T2), 4–6 min time point, p = 0.048 Normal Holm–Sidak's multiple-comparisons test 95% CI [−0.13, 10.0] n—time in the novel same-strain rat proximity zone (T2), 6–8 min time point, p = 0.0073 Normal Holm–Sidak's multiple-comparisons test 95% CI [1.2, 11.4] o—time in the novel different-strain proximity zone over time, main effect of time, F(4,280) = 21.2, p < 0.0001 Normal Two-way mixed-effects model ANOVA 95% CI of difference [13.3, 20.4] True CI main effect computation not possible because >2 (5) time points. As proxy, CI of interaction computed from data collapsed to T0-2 and T8-10 p—time in the novel different-strain proximity zone over time, condition × time interaction, F(4,280) = 6.3, p < 0.0001 Normal Two-way mixed-effects model ANOVA 95% CI of difference [9.7, 23.9] True CI interaction computation not possible because >2 (5) time points. As proxy, CI of interaction computed from data collapsed to T0-2 and T8-10 q—time in the novel different-strain rat proximity zone, 0–2 min time point, p = 0.0038 Normal Holm–Sidak's multiple-comparisons test 95% CI [3.0, 22.0] r—preference index over time (T2), main effect of time, F(4,278) = 9.1, p < 0.0001 Normal Two-way mixed-effects model ANOVA 95% CI of difference [−59.2, −24.7] True CI main effect computation not possible because >2 (5) time points. As proxy, CI of interaction computed from data collapsed to T0-2 and T8-10 s—preference index over time (T2), condition × time interaction, F(4,278) = 3.0, p = 0.020 Normal Two-way mixed-effects model ANOVA 95% CI of difference [−79.4, 10.5] True CI interaction computation not possible because >2 (5) time points. As proxy, CI of interaction computed from data collapsed to T0-2 and T8-10 t—number of transitions between novel same-strain and novel object proximity zones, U = 905, p = 0.39 (n.s.) Non-normal Mann–Whitney U test Control 10.6 ± 0.7 transitions, 95% CI [9.2, 12.0]; stress 11.1 ± 0.7 transitions, 95% CI [9.7, 12.5]; sum of ranks [1,940, 2,155], n.s. u—bout duration in the novel same-strain rat proximity zone and novel object proximity zone, main effect of apparatus zone, F(1,88) = 27.5, p < 0.0001 Normal Two-way mixed-effects model ANOVA 95% CI of difference [−0.52 to −0.24] v—number of transitions between same-strain and different-strain rat proximity zones, U = 386, p = 0.0032 Non-normal Mann–Whitney U test Control 14.5 ± 1.2 transitions, 95% CI [12.2, 16.9]; stress: 20.1 ± 1.1 transitions, 95% CI [17.8, 22.3]; sum of ranks [947, 1,681] w—bout duration in the novel same-strain rat proximity zone and novel different-strain rat proximity zone, main effect of apparatus zone, F(1,70) = 20.2, p < 0.0001 Normal Two-way mixed-effects model ANOVA 95% CI of difference [0.26, 0.68] x—bout duration in the novel same-strain rat proximity zone and novel different-strain rat proximity zone, main effect of condition, F(1,70) = 11.0, p = 0.0015 Normal Two-way mixed-effects model ANOVA 95% CI of difference [0.14, 0.56] y—bout duration in the different-strain rat proximity zone, p = 0.0019 Normal Holm–Sidak's multiple-comparisons test 95% CI [0.17, 0.84] z—average MeAp ΔF/F while in the novel same-strain rat proximity zone compared with the novel object proximity zone, main effect of zone, F(1,19) = 55.5, p < 0.0001 Normal Two-way mixed-effects model ANOVA 95% CI of difference [−0.95, −0.53] A—average MeAp ΔF/F while in the novel same-strain proximity zone compared with novel different-strain rat proximity zone, condition × zone interaction, F(1,18) = 17.2, p = 0.0006 Normal Two-way mixed-effects model ANOVA 95% CI of difference [0.39, 1.2] B—average MeAp ΔF/F while in the novel different-strain rat proximity zone, p = 0.0031 Normal Holm–Sidak's multiple-comparisons test 95% CI of difference [−0.82, −0.15] C—average MeAp ΔF/F while in the novel same-strain rat proximity zone, p = 0.033 Normal Holm–Sidak's multiple-comparisons test 95% CI of difference [−0.016, 0.65] D—relative (same-strain–object) MeAp ΔF/F, t(20) = 0.53, p = 0.60 (n.s.) Normal Two-tailed unpaired t test Control, 0.77 ± 0.12, 95% CI [0.51, 1.0]; stress, 0.67 ± 0.16, 95% CI [0.32, 1.0]; 95% CI of difference [−0.51, 0.30], n.s. E—relative (same-strain–different-strain) MeAp ΔF/F, t(18) = 4.1, p = 0.0006 Normal Two-tailed unpaired t test Control: n = 10 rats, 0.47 ± 0.14, 95% CI [0.16, 0.79]; stress: n = 10 rats, (−0.33) ± 0.13, 95% CI [−0.63, −0.024]; CI of difference [−1.2, −0.39] F—amount of time in nose–nose interaction with the novel same-strain rat (T1), t(19) = 0.47, p = 0.64 (n.s.) Normal Two-tailed unpaired t test Control, 27.6 ± 2.9 s, 95% CI [21.2, 33.9]; stress, 25.2 ± 4.3 s, 95% CI [15.4, 34.9]; CI of difference [−13.1, 8.2], n.s. G—amount of time in nose–nose interaction with the novel same-strain rat (T2), t(19) = 2.9, p = 0.010 Normal Two-tailed unpaired t test Control, 15.0 ± 3.3 s, 95% CI [7.5, 22.4]; stress, 5.4 ± 1.1 s, 95% CI [2.9, 7.9]; CI of difference [−16.6, −2.6] H—amount of time in nose–nose interaction with the novel different-strain rat (T2), p = 0.24 (n.s.) Normal Two-tailed unpaired t test Control, 10.4 ± 1.7 s, 95% CI [6.6, 14.2]; stress, 13.6 ± 2.0 s, 95% CI [9.1, 18.2]; CI of difference [−2.4, 8.8], n.s. I—MeAp ΔF/F, novel same-strain rat, nose–nose contact and proximity zone (T1), main effect of location, F(2,38) = 76.7, p < 0.0001 Normal Two-way mixed-effects model ANOVA 95% CI of difference [−0.77, −0.46] J—MeAp ΔF/F, nose–nose contact novel same-strain rat and novel different-strain rat (T2), main effect of zone, F(1,38) = 3.7, p = 0.060 (n.s.) Normal Two-way mixed-effects model ANOVA 95% CI of difference [−0.016, 0.70], n.s. K—MeAp ΔF/F after initiating nose–nose contact with novel same-strain rat (T1), U = 1691, p = 0.60 (n.s.) Non-normal Mann–Whitney U test Control, 4.7 ± 0.6, 95% CI [3.4, 5.9]; stress, 4.3 ± 0.6, 95% CI [3.0, 5.5]; sum of ranks [3,973, 3,287], n.s. L—MeAp ΔF/F after initiating nose–nose contact with novel same-strain rat (T2), t(35) = 0.67, p = 0.51 (n.s.) Normal Two-tailed unpaired t test Control, 5.6 ± 0.8, 95% CI [4.1, 7.2]; stress, 6.7 ± 1.4, 95% CI [3.4, 9.9]; CI of difference [−2.1, 4.1], n.s. M—MeAp ΔF/F after initiating nose–nose contact with novel different-strain rat (T2), t(91) = 2.8, p = 0.0065 Normal Two-tailed unpaired t test Control, 1.9 ± 0.4, 95% CI [1.1, 2.8]; stress, 4.0 ± 0.6, 95% CI [2.8, 5.2]; CI of difference [0.59, 3.5] N—MeAp ΔF/F after initiation of defensive response (startle, T2), t(44) = 3.4, p = 0.0015 Normal Two-tailed unpaired t test Control, 1.1 ± 0.9, 95% CI [−0.8, 3.0]; stress, 5.6 ± 1.0, 95% CI [3.6, 7.6]; CI of difference [1.8, 7.1] ↵a T1 = Trial 1, T2 = Trial 2. Note: GraphPad Prism does not calculate CI for Holm–Sidak's multiple-comparisons test. Therefore, CI was calculated independently of Holm–Sidak's multiple-comparisons test.
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