Differential entrainment of a social rhythm in adolescent mice

Abstract

Daily routines in animal activities range from sleep-wake cycles, to foraging bouts, to social interactions. Among animals living within groups, it is unclear whether the motivations that underlie social interactions respond to daily light-dark (LD) cycles or endogenous circadian rhythms. Employing two mouse strains (BALB/cJ [BALB] and C57BL/6J [B6]) with genetically based differences in social affect and circadian rhythms, we examined how social investigation (SI) is modulated by social deprivation and circadian factors. We found a genetic influence on SI that was moderated by the preceding duration of social deprivation, requiring 3-6 h of social isolation prior to testing. Following 6h of social deprivation, the SI responses of adolescent B6 mice were greater than those of BALB mice only when the isolation period was imposed during the dark phase of the LD cycle. When B6 mice were weaned into conditions of constant darkness, a novel, endogenous social rhythm emerged, which was characterized by two pronounced peaks of social responsiveness (relative to one peak under LD entrainment) that were separated by 12-h intervals. Irrespective of the lighting conditions during social isolation, the SI responses of adolescent BALB mice did not oscillate across the day. Similar strain-dependent patterns of sociability were evident within groups of mice that were left undisturbed in their home cage under LD entrainment or constant darkness. Overall, genetic influences on the social phenotypes of adolescent mice are thus moderated by an interaction between social deprivation and oscillations of an endogenous social rhythm that entrains to the LD cycle.

Figure 1. Social investigation responses plotted as a function of the genotype of test mice and the preceding social isolation period

(a) SI responses are depicted above the abscissa. The duration and LD conditions of the respective social isolation periods are represented graphically below the abscissa. Sample sizes for the BALB and B6 genotypes were 6-15 and 10-24 test mice/isolation duration, respectively. Each asterisk represents a significant post-hoc comparison between the 2 genotypes (Tukey's HSD test, P < 0.05). (b) Log-linear regression functions were used to summarize the relationship between SI and social isolation for each genotype. The shaded area bounding each regression line represents the 95% confidence interval of the respective fit. Inter-rater reliability, r_p = 0.94, d.f. = 275. All data are presented as the mean ± standard error.

Figure 2. Social investigation responses of adolescent mice plotted as function of genotype, isolation duration and test time

The SI responses of adolescent mice were evaluated at ZT 14 or ZT 22 following either 6 or 24 h of social isolation. Data for ZT 22 are re-plotted from Figure 1a. Sample sizes for ZT 14 were 18-24 and 12-22 mice/genotype for the 6 h- and 24 h-isolation periods, respectively. Sample sizes for ZT 22 were 14-21 and 15-24 mice/genotype for the 6-h and 24 h-isolation periods, respectively. Asterisks represent a significant (P<0.05) orthogonal contrast between the 2 genotypes. Inter-rater reliability, r_p = 0.88, d.f. = 147. All data are presented as the mean ± standard error.

Figure 3. Social investigation responses of adolescent mice maintained under different entrainment schedules and following circadian rhythm disruption

The SI responses of mice (a) entrained to a 14:10 h LD cycle (b) weaned into constant darkness and (c) maintained in constant darkness from birth. All SI tests were conducted at 6-h intervals following 6 h of social isolation. At each test time, respective sample sizes for the BALB and B6 strains were (a) 14-16 and 16-18 mice, (b) 13-16 and 16 mice, and (c) 6-8 and 10-12 mice. Each asterisk represents a significant (P<0.05) orthogonal contrast between the 2 genotypes. Inter-rater reliability for Figures 3a-c, r_p = 0.91, d.f. = 309. The SI responses of mice were also measured after a 6-h phase shift. Mice were entrained to a 14:10 h LD cycle and tested at (d) ZT 0/24 following 6 h of social isolation in the dark (‘Iso. dark’) or in the light (‘Phase advance’), and at (e) ZT 6 following 6 h of social isolation in the light (‘Iso. light’) or in the dark (‘Phase delay’). (f) Mice were maintained in constant darkness from weaning and tested at CT 0/24 following 6 h of social isolation in the dark (‘Iso. dark’) or in the light (‘Light pulse’). Respective sample sizes for BALB and B6 strains for each isolation condition were (d) 10 and 8 mice, (e) 6 and 8 mice and (f) 8 and 8 mice. Each asterisk represents a significant post-hoc comparison (Tukey's HSD test, P<0.05) between the 2 genotypes. Numeric symbols represent a significant difference (Tukey's HSD test, P<0.05) between B6 mice that underwent different LD conditions during social isolation. Inter-rater reliability for Figures 3d-f, r_p = 0.85, d.f. = 93. All data are presented as the mean ± standard error.

Figure 4. Comparisons of mouse activity patterns and social behaviors within their home cage environment

Behaviors of BALB mice (left panels) and B6 mice (right panels) maintained under a 14:10 h LD cycle (stippled landscapes) or under constant darkness (black landscapes) were compared. Behavioral measures include (a,b) behavioral quiescence, (c,d) ambulatory activity and (d,e) social behavior. The abscissa represents zeitgeber/circadian time. Samples sizes were 3-5 social groups/genotype for each zeitgeber condition. Measures of inter-rater reliability were high for behavioral quiescence (r_p = 0.95), ambulatory activity (r_p = 0.91) and social behavior (r_p = 0.82). Standard error bars (pooled across the entire 24-h cycle) are presented for each genotype and behavioral category at the top right of each panel.