Behavioral characterization of the persistent effect of adolescent social isolation

In short, we observed a general decrease in social recognition memory in both stress groups (EASI and LASI) regardless of sex. Both male and female LASI rats showed reduced social interactions. On the hotplate test, Male EASI and LASI rats demonstrated heightened thermal pain sensitivity, whereas the opposite was true for EASI females when compared to their sex-matched CTLs. We also observed some alterations to time spent in the center zone in male EASI rats and an increase total arm entries in female LASI rats.

Elevated plus maze

The ANOVA performed on stress group (CTL, EASI, LASI) and sex (Male, Female) examined the effects of time spent on the open arm time, time spent in the center zone and total arm entries on anxiety-like and locomotor activity. The ANOVA on time spent on the open arm did not reveal a significant interaction effect (F (1, 68) = 2.636, p = 0.079, ηp2 = 0.079) suggesting that ASI does not affect time spent on the open arm, regardless of sex. However, EASI males spent a lower percent of time in the center zone compared to CTL males (Fig. 3A), which may reflect an indirect measure of impulsive behavior [41]. Female LASI rats on the other hand showed higher general activity in the EPM compared to female CTL and EASI rats, as indicated by the total number of arm entries performed during the test.

Fig. 3

Behavioral performance in the elevated plus maze. (A) percent time spent in the center zone, (B) total arm entries in CTL (● control), early (■ EASI), and late (▲LASI) adolescent social isolation groups tested in adulthood (PD 90). Data are shown as individual data points with the mean ± SEM. *p < 0.5, **p < 0.01

The ANOVA for time spent in the center zone revealed an interaction between stress group and sex (F (1, 68) = 3.199, p = 0.047, ηp2 = 0.086) and a main effect of sex (F (1, 68) = 11.396, p = 0.001, ηp2 = 0.144). The stress group effect was not significant (F (1, 68) = 1.324, p = 0.273, ηp2 = 0.037). To further explore the interaction effect, pairwise comparisons were conducted. Among males there was a significant difference in percent time spent on the center zone between male CTL (M = 0.285, SD = 0.149) and male EASI rats (M = 0.188 SD = 0.077) (Fig. 3A). The overall model was significant, (F(1, 68) = 4.523, p = 0.001, ηp2 = 0.250), accounting for approximately 25% of the variance in time spent in the center zone.

The interaction effect between the stress group and sex for total number of arm entries was statistically significant (F (1, 68) = 5.390 p = 0.0004, ηp2 = 0.165) as was the main effect of sex (F (1, 68) = 13.395, p = 0.001, ηp2 = 0.144). Importantly, female LASI (M = 13.250, SD = 3.387) rats made more total entries compared to the other female groups; EASI (p = 0.009, M = 10.333, SD = 3.143), CTL (p = 0.027, M = 10.006, SD = 2.186) (Fig. 3B). Additionally, female LASI (M = 13.250, SD = 3.387) performed more total arm entries compared to male LASI (p < 0.00001, M = 8.090, SD = 1.868) rats (Fig. 3B). In general, female rats (M = 11.128, SD = 3.163) made more total arm entries than did male rats (p = 0.0004, M = 8.857, SD = 2.463). The overall model was significant, (F(1, 68) = 5.643, p = 0.001, ηp2 = 0.293), accounting for approximately 29.3% of the variance of total arm entries.The different estrous cycle phases did not influence anxiety-like behavior (data not shown). Summary statistics in Supplementary materials.

Open field

The ANOVA was conducted to investigate the effects of stress group (CTL, EASI, LASI) and sex (Male, Female) on total distance travelled on the OFT. We observed no long-lasting impact of ASI on the adult locomotor activity in the OFT (F (1, 68) = 0.101 p = 0.904, ηp2 = 0.410) but observed a sex effect (F (1, 68) = 51.395 p = 0.000001, ηp2 = 0.410). On average, female rats (M = 5653.987, SD = 1682.047) travelled a longer distance during the 30 min OFT compared to males (M = 3401.445, SD = 926.988) (Graph in Supplementary Materials). No effect was observed in the stress group (F (1, 74) = 0.705, p = 0.498, ηp2 = 0.019). The overall model was significant, (F (1, 68) = 10.990, p = 0.00001, ηp2 = 0.426), accounting for approximately 42.6% of the variance of total arm entries. Summary statistics in Supplementary materials.

Novel object recognition memory

NOR data was analyzed using stress group (CTL, EASI, LASI) and sex (Male, Female) ANOVA. Adult ASI rats did not differ in object recognition ability (F (1, 74) = 2.283, p = 0.109, ηp2 = 0.058). The ANOVA revealed no statistical differences between stress groups or sex on time spent investigating the two same objects during the acquisition phase (P1) (F (1, 74) = 0.911, p = 0.407, ηp2 = 0.024). In the test phase (P2) we first assessed if all rats preferences differed from chance, which was statistically significant (t (79) = 12.095, p = 0.000001; Hedge’s g = 11.115) implying all rats recognition ability differed from chance.

A non-significant interaction between stress group and sex was found on object discrimination ability during the test phase (P2) (F (1, 74) = 2.283, p = 0.109, ηp2 = 0.058). The main effect of stress group (F (1, 74) = 0.915, p = 0.405, ηp2 = 0.024) and sex (F (1, 74) = 2.946, p = 0.090, ηp2 = 0.038) was statistically non-significant. The overall model was also not statistically significant, (F (1, 74) = 1.695, p = 0.146, ηp2 = 0.426), indicating that the model as a whole did not explain a significant proportion of the variance of objection recognition ability. Summary statistics in Supplementary materials.

Social interaction test

The ANOVA was conducted to analyze the effects of stress group (CTL, EASI, LASI) and sex (Male, Female) on total social interaction time, anogenital and non-anogenital sniffing in a free social interaction test performed in open field arena. We did not observe stress group differences between males and females (F (1, 74) = 0.287, p = 0.752, ηp2 = 0.008). However, the analysis revealed a main effect of stress group, whereby LASI rats spent less time in social interactions bouts (based on social interactions initiated by the experimental rat) than CTL rats. This was largely driven by a reduction in interaction times across both sexes in both LASI groups.

The main effect of stress group was statistically significant (F (1, 74) = 9.036, p = 0.0003, ηp2 = 0.196). Pairwise comparisons revealed LASI (M = 33.708, SD = 14.827) rats spent less time in social investigation than EASI (p = 0.003, M = 53.541, SD = 14.911) and CTL (p = 0.0005, M = 54.781, SD = 24.874) rats (Fig. 4B). The overall model was statistically significant (F (1, 74) = 3.736, p = 0.005, ηp² = 0.202), suggesting that it accounts for 20.2% of the varianceof time spenting in social investigation during the social interaction test .

Fig. 4

Behavioral performance in the social interaction test. (A) Total social interaction time in the social interaction test in the CTL (control), early (EASI), and late (LASI) adolescent social isolation groups tested in adulthood (PD 96) (SIT) across stress group and sex and (B) total interaction time by stress group (C) number of non-anogenital sniffing bouts and (D) sex difference in non-anogenital sniffing bouts between male and female rats. Data are shown as individual data points with the mean ± SEM. *p < 0.5, **p < 0.01

The ANOVA on non-anogenital sniffing indicated difference in bouts between the stress groups (F (1, 74) = 5.483, p = 0.006, ηp2 = 0.129) and sex (F (1, 74) = 24.670, p = 0.000004, ηp2 = 0.250) independently. Pair-wise comparisons between stress groups revealed that LASI (M = 10.708, SD = 5.901) rats performed fewer non-anogenital social investigation bouts than CTLs (p = 0.013, M = 15.093, SD = 8.294) and EASI (p = 0.031, M = 11.166, SD = 3.726) rats (Fig. 4C). While, male rats (M = 15.800, SD = 7.660) on average performed more non-anogential social bouts than female rats (p = 0.000004, M = 9.400, SD = 3.506) (Fig. 4D). The overall model was significant, (F (1, 74) = 8.743, p = 0.000001, ηp2 = 0.371), accounting for approximately 37.1% of the variance of non-anogential sniffing bouts. We observed no effect of ASI on the frequency of anogenital sniffing bouts (F (1, 74) = 3.001, p = 0.056, ηp2 = 0.075). The overall model was not statistically significant (F (1, 74) = 1.334, p = 0.260, ηp² = 0.083), suggesting that it did not account for a significant proportion of the variance of the anogential sniffing bouts.

The ANOVA revealed an interaction effect of the stress group and sex on rearing behaviors (F (1, 74) = 4.341, p = 0.017, ηp2 = 0.105) and a main effect of the stress group (F (1, 74) = 8.887, p = 0.0003, ηp2 = 0.194). Further analysis of the interaction effect revealed that male EASI (M = 17.666, SD = 5.804) rats reared less than LASI (p = 0.00001, M = 39.416), SD = 11.212) and CTL (p = 0.012, M = 29.687, SD = 16.684) rats, and male LASI rats reared more than LASI female rats (p = 0.008, M = 27.500, SD = 6.142). The overall model was statistically significant (F (1, 74) = 5.939, p = 0.0001, ηp2 = 0.286) accounting for approximately 28.6% of the variance of rearing in the SIT. Summary statistics in supplementary materials.

Social recognition memory

The ANOVA was conducted on stress group (CTL, EASI, LASI) and sex (Male, Female) effects on investigation time (P1) and social recognition memory in the test phase (P2) based on investigations times between the novel and familiar conspecific. We found that in general ASI rats demonstrated impaired social recognition memory compared to CTL rats.

First, we assessed all rats’ preference against chance during the test phase (P2) before further analysis. To test this we performed a one sample t-test which was statistically significant (t (79) = 45.569, p = 0.000001; Hedge’s g = 5.046), implying all rats differed from chance level in their discrimination ability.

No differences were observed in the total time spent investigating both conspecifics during the test phase (P2) between stress groups independent of sex (F (1, 74) = 0.219, p = 0.804, ηp2 = 0.006). Neither of the main effect of stress group (F (1, 74) = 0.794, p = 0.456, ηp2 = 0.021) or sex (F (1, 74) = 0.058, p = 0.810, ηp2 = 0.021) were statistically significant. However, analysis of the social recognition memory discrimination ability revealed a main effect of stress group (F (1, 74) = 11.241, p = 0.00003, ηp2 = 0.241) (Fig. 5). Pairwise comparisons of the stress group variable revealed that EASI (p = 0.00002, M = 56.791, SD = 15.137) and LASI (p = 0.018, M = 62.632, SD = 9.524) rats showed impaired social recognition ability compared to CTL (M = 71.093, SD = 8.644) rats (Fig. 5, small inlet) regardless of sex. The overall model was statistically significant (F (1, 74) = 0.794, p = 0.456, ηp2 = 0.021) accounting for 27.65% of the variance in social recognition discrimination ability. Summary statistics in Supplementary materials.

Fig. 5

Behavioral performance on the social recognition memory test: The ability of rats to discriminate between social partners was indicated by a decrease in discrimination ability compared to CTL rats. The data are shown as individual data points with the mean ± SEM. *p < 0.5, **p < 0.01, ***p < 0.001. Main effect of stress group in small inlet (top right)

Hotplate test

The hotplate test data were analyzed using stress group (CTL, EASI, LASI) and sex (Male, Female) ANOVA. Compared to male CTL rats males EASI and LASI rats showed heightened thermal pain sensitivity. While female EASI rats in contrast demonstrated a reduction in thermal pain sensitivity in the hotplate test compared to female CTL rats. The interaction effects of stress group and sex (F (1, 74) = 11.843, p = 0.00003, ηp2 = 0.242) (Fig. 6) and the main effects of stress group (F (1, 74) = 3.380, p = 0.039, ηp2 = 0.084) and sex (F (1, 74) = 7.812, p = 0.007, ηp2 = 0.095) were statistically significant. Further exploration of the interaction effect revealed that both male EASI (p = 0.0005, M = 7.950, SD = 2.043) and LASI (p = 0.001, M = 7.950, SD = 2.043) rats demonstrated higher thermal pain sensitivity than male CTL rats. While, female EASI (M = 9.409, SD = 2.587) rats showed reduced thermal pain sensitivity compared to LASI (p = 0.031, M = 6.912, SD = 1.795) and CTL (p = 0.016, M = 6.862, SD = 1.074) rats. Male CTL (M = 9.418, SD = 3.134) rats demonstrated longer thermal latencies than female CTL (M = 7.641, SD = 2.148). We observed no differences in thermal pain sensitivity between estrous cycle phases captured immediately after the hotplate test. Summary statistics in Supplementary materials.

Fig. 6

Behavioral performance on the hotplate test (52.5 °C). Latency to react to thermal pain stimuli in the hotplate test compared with controls (CTL). Data are shown as individual data points with the mean ± SEM. *p < 0.5, **p < 0.01, ***p < 0.001

Molecular characterization of the OTR binding in adulthood following adolescent social isolation

Data were analyzed using stress group (CTL, EASI, LASI) and sex (Male, Female) ANOVA to determine differences in OTR bindings levels in the several brain regions involved in social and anxiety-like behaviors.

We observed sex-dependent effects of ASI on OTR binding in the CeA, PVN, and PVT (Figs. 7; ; 8). In the PVT, OTR binding increased in female LASI and EASI by 154% and 141% respectively compared to female CTL rats. While in male EASI rats showed a 47% decrease in OTR binding compared male controls. While in the PVN OTR binding in female EASI and LASI rats increased by 136 and 54%, respectively. Male EASI rats showed a 52% increase in OTR binding in the CeA compared to CTL males.

Fig. 7

OTR binding sites in the CeA, PVN and PVT in adult rats (PD90) representative autoradiograph and bregma coordinates for regions of interest. (A) OTR bindings sites in the CeA, PVN, and PVT measured by saturated [125I] OVTA receptor autoradiography (fmol/mg). Bar graphs show OTR binding sites in the (B) CeA (mean female CTL values = 0.705 ± 0.05, mean male CTL values = 0.663 ± 0.08), (C) PVN (mean female CTL values = 0.124 ± 0.01, mean male CTL values = 0.205 ± 0.01), (D) PVT (mean female CTL values = 0.124 ± 0.01, mean male CTL values = 0.239 ± 0.02) are defined as 0% and changes in binding density show increase and decrease from baseline. Data shown as µ ± SEM. *p < 0.5, **p < 0.01, ***p < 0.001. Statistical analysis was performed by region-wise one-way ANOVA. n = 4–8/group. Scale bar 1 mm. CeA; central amygdala, PVN; paraventricular nucleus of the hypothalamus, PVT; paraventricular nucleus of the hypothalamus, CTL; control, EASI; early adolescent social isolation, LASI; late adolescent social isolation

Fig. 8

Main findings from the ASI study. Summary of behavioral and oxytocin receptor binding findings in early adolescent social isolation and late social isolation male and female rats compared to controls. Minor effects in brackets

For the PVT, we observed an interaction effect between stress group and sex (F = 150.791 (1, 29), p = 0.00003, ηp2 = 0.521), and the main effect of stress group (F = 70.870 (1, 29), p = 0.002, ηp2 = 0.352). Pairwise comparisons revealed that both EASI (p = 0.00005) and LASI (p = 0.00002) females had higher OTR binding levels in the PVT compared to CTL females. While male EASI rats demonstrated significantly less OTR binding levels compared to their CTL (p = 0.003) and LASI (p = 0.002) male counterparts. Interestingly, we observed an opposing effect of EASI on OTR binding in the PVT, with OTR binding increasing in females and but decreasing in males following EASI (p = 0.00001) (Appendix 8). Pair-wise comparisons of the sex and stress group interaction effects revealed that CTL females had lower levels of OTR binding compared to CTL males (p = 0.007) in adulthood.

In the PVN the interaction between stress group and sex (F = 80.267 (1, 21), p = 0.002, ηp2 = 0.441) was significant. Here, female EASI rats showed significantly more OTR binding in the PVN compared to female CTL (p = 0.0004) and LASI (p = 0.004) rats. We observed similar opposing sex specific effects in EASI rats in the PVN as we did in the PVT, with EASI leading to an increase in OTR binding in females but an opposing decrease in males (p = 0.001).

Analysis of the CeA revealed a significant stress group and sex interaction (F = 30.519 (1, 21), p = 0.048, ηp2 = 0.251), and the main effects of stress group (F = 30.904 (1, 21), p = 0.036, ηp2 = 0.271) and sex (F = 60.771 (1, 21), p = 0.016, ηp2 = 0.244). Pair-wise comparisons on the interaction effect demonstrated that female EASI had higher OTR binding levels compared to female LASI rats (p = 0.018) (Appendix 8). Additionally, OTR binding levels were higher in male EASI rats compared to male CTL rats (p = 0.016). We also observed sex differences between males LASI rats and female LASI (p = 0.002) rats were the former had significantly higher OTR binding levels. Post-hoc analysis for the stress group revealed statistically significant differences between the stress groups, with EASI rats demonstrating the highest OTR binding levels compared to CTL (p = 0.026) and LASI (p = 0.024). All statistical values are shown by sex for clarity but were analyzed together. Summary statistics in Supplementary Materials.

Figure 7 OTR binding sites in the CeA, PVN and PVT in adult rats (PD90) representative autoradiograph and bregma coordinates for regions of interest. (A) OTR bindings sites in the CeA, PVN, and PVT measured by saturated [125I] OVTA receptor autoradiography (fmol/mg). Bar graphs show OTR binding sites in the CeA (mean female CTL values = 0.705 ± 0.05, mean male CTL values = 0.663 ± 0.08), PVN (mean female CTL values = 0.124 ± 0.01, mean male CTL values = 0.205 ± 0.01), PVT (mean female CTL values = 0.124 ± 0.01, mean male CTL values = 0.239 ± 0.02) are defined as 0% and changes in binding density show increase and decrease from baseline. Data shown as µ ± SEM. *p < 0.5, **p < 0.01, ***p < 0.001. Statistical analysis was performed by region-wise one-way ANOVA. n = 4–8/group. Scale bar 1 mm. CeA; central amygdala, PVN; paraventricular nucleus of the hypothalamus, PVT; paraventricular nucleus of the hypothalamus, CTL; control, EASI; early adolescent social isolation, LASI; late adolescent social isolation.

Sex differences independent of adolescent social isolation

Additionally, we performed an analysis comparing male CTL rats to female CTL in order to identify sex difference independent of ASI. Across all tests, notable sex differences were observed in the EPM, OFT, SIT, and hotplate test, independent of ASI. The receptor autoradiography results revealed sex differences in the PVT between CTL males and females.

In the EPM male rats spent significantly more time in the central zone of the EPM compared to female CTL rats (F (27) = 11.414, p = 0.002, ηp2 = 0.297). On the other hand, female CTL rats made a significantly greater number of total arm entries (F (27) = 26.773, p = 0.00001, ηp2 = 0.472).

In the SIT, male rats engaged in more non-anogenital sniffing bouts compared to females (F (30) = 10.496, p = 0.003, ηp2 = 0.257), interestingly no significant sex differences were observed in the total time spent in social investigation (F (30) = 0.011, p = 0.917, ηp2 = 0.00). Additionally, no sex differences were noted in the SRM. In the hotplate test, males demonstrated a significantly higher thermal pain threshold on the hotplate test compared to females(F (30) = 40.387, p = 0.000001, ηp2 = 0.574).

The ANOVA on sex (male vs. female) conducted for the receptor autoradiography data revealed a general sex difference in OTR binding within the PVT (F (29) = 30.421, p = 0.075, ηp2 = 0.106), with males showing higher baseline levels of OTR binding compared to females (Appendix 8, Supplementary Materials).