Effect and mechanism of father's companionship on defensive attack behavior of adult male offspring mice

Abstract

Sociocultural changes in recent decades have promoted fathers' involvement in childcare, which is crucial for the brain and behavioral plasticity of offspring. The study elucidates the effect and mechanism of father's companionship on defensive attack behavior of adult male offspring mice. The study comprises a father companionship group, where offspring cohabited with sire and dam until weaning, and a control group, where offspring cohabited solely with the dam until weaning. The study shows that father's companionship increases defensive attack behavior of adult offspring. Additionally, the metabolite L-aspartic acid is upregulated in the father's companionship group compared to the control group in male offspring, and intracerebroventricular micro-injection of L-aspartic acid confirms its impact on defensive attack behavior. C-Fos immunohistochemistry reveals that c-Fos expression in lateral periaqueductal gray (LPAG) is activated. Subsequently, micro-injection of L-aspartic acid into LPAG increases defensive attack behavior. Additionally, 16S rRNA sequencing reveals a higher abundance of Bilophila and a lower abundance of Bifidobacterium in the father companionship group, which correlates with L-aspartic acid levels, suggesting a gut-brain axis mechanism for the effect of father companionship on defensive attack behavior.

Fig. 1. Father companionship increased the defensive attack behavior of offspring.

a Experimental design. In the father companionship group, the sire lived with the dam and offspring from conception through weaning. In the control group, only the dam lived with offspring during this period. b The effect of father’s companionship on the latency of biting (two-way ANOVA, effects of father companionship: F _{(1, 68)} = 2.185, P = 0.144, η2 = 0.031; effect of sex: F _{(1, 68)} = 1.766, P = 0.188, η2 = 0.025; interaction effect: F _{(1, 68)} = 0.236, P = 0.629, η2 = 0.003). c Father’s companionship increased the frequency of biting (two-way ANOVA, effects of father companionship: F _{(1, 68)} = 4.370, P = 0.040, η2 = 0.060; effect of sex: F _{(1, 68)} = 0.493, P = 0.485, η2 = 0.007; interaction effect: F _{(1, 68)} = 1.238, P = 0.270, η2  = 0.018). (d) Father’s companionship increased the biting duration (two-way ANOVA, effects of father companionship: F _{(1, 68)} = 5.331, P = 0.024, η2 = 0.073; effect of sex: F _{(1, 68)}= 0.241, P = 0.625, η2 = 0.004; interaction effect: F _{(1, 68)} = 0.803, P = 0.374, η2 = 0.012). n = 17-19 per group. Values are shown as mean ± standard error. FC father companionship, PD pregnancy day, PND postnatal day. * P <0.05.

Fig. 2. Father companionship changed the serum metabolomic profile of male offspring.

a Expression abundance of quality control for samples. b OPLS-DA plot between the father companionship and control group. c Volcano plot showing the differential metabolites between groups. d Bubble diagram of the KEGG enrichment pathway for significantly regulated differential metabolites. e The Lollipop map shows differences in up-regulated and down-regulated metabolites in the father companionship group compared with the control group. f The abundance of L-aspartic acid was elevated in the father’s companionship group compared to the control group (t₍₁₀₎ = 4.380, P = 0.001, t test). n = 6 per group. g The abundance of 3-sulfinoalanine was reduced in the father’s companionship group compared to the control group (t₍₁₀₎ = 9.374, P < 0.001, t test). n = 6 per group. h–j Correlation between the abundance of L-aspartic acid and defensive attack behavior. L-aspartic acid was positively associated with the frequency of biting (r = 0.706, P = 0.013). k–m Correlation between the abundance of 3-sulfinoalanine and defensive attack behavior. 3-sulfinoalanine was negatively associated with the frequency of biting (r = –0.617, P = 0.037). n = 12 (6 in the intervention group and 6 in the control group). FC father companionship, KSAHI Kaposi sarcoma-associated herpesvirus infection. PAC-DAP (2-Phenylacetyl) (2 R)-2,5-diaminopentanoate. * P < 0.05; **P  < 0.01; ***P < 0.001.

Fig. 3. Intracerebroventricular injection of L-aspartic acid increased defensive attack behavior of male offspring.

a The experimental design of intracerebroventricular injection of L-aspartic acid and defensive attack behavior test. b–d Latency of biting (one-way ANOVA, F _{(2, 21)} = 1.250, P = 0.307), frequency of biting (one-way ANOVA, F _{(2, 21)} = 2.275, P = 0.127), and biting duration (one-way ANOVA, F _{(2, 21)} = 4.069, P = 0.032) after intracerebroventricular injection of L-aspartic acid. Mice treated with 0.2 μmol/μL L-aspartic acid for 3 μL exhibited longer biting duration compared to the saline control group (Fisher’s LSD post-test, p = 0.011). n = 8 per group. e The experimental design of intracerebroventricular injection of 3-sulfinoalanine and defensive attack behavior test. f–h Latency of biting (t ₍₁₄₎ = 0.238, P = 0.815, t test), Frequency of biting (t ₍₁₄₎ = 0.646, P = 0.529, t test), and biting duration (t ₍₁₄₎ = 0.226, P = 0.825, t test) after intracerebroventricular injection of 3-sulfinoalanine. n = 8 per group. Values are shown as mean ± standard error. * P < 0.05.

Fig. 4. L-aspartic acid injections into the LPAG or administered intraperitoneally increased defensive attack behavior of male offspring.

a–e c-Fos expression in the LPAG was significantly higher in the father’s companionship group than in the control group (t ₍₁₆₎ = 2.366, P = 0.031, t test), whereas no significant difference was observed in the DMPAG (t ₍₁₆₎ = 0.346, P = 0.734, t test), DLPAG (t ₍₁₆₎ = 0.809, P = 0.430, t test), and VLPAG (t ₍₁₆₎ = 0.759, P = 0.459, t test); (3 mice per group were analyzed with 3 sections per mouse). f The experimental design of injection of L-aspartic acid in LPAG and defensive attack behavior test. g–i Latency of biting (one-way ANOVA, F _{(2, 23)} = 0.164, P = 0.850), frequency of biting (one-way ANOVA, F _{(2, 23)} = 1.042, P = 0.369), and biting duration (one-way ANOVA, F _{(2, 23)} = 3.988, P = 0.033) after injection of L-aspartic acid in LPAG. Mice treated with 1 μmol/μL L-aspartic acid for 0.2 μL exhibited longer biting duration compared to the saline control group (Fisher’s LSD post-test, P = 0.010). n = 8–9 per group. j The experimental design of intraperitoneal injection of L-aspartic acid and defensive attack behavior test. k–m The latency of biting (one-way ANOVA, F _{(3, 33)} = 1.582, P = 0.212), frequency of biting (one-way ANOVA, F _{(3, 33)} = 1.266, P = 0.302), and biting duration (one-way ANOVA, F _{(3, 33)} = 4.176, P = 0.013) after intraperitoneal injection of L-aspartic acid. Mice treated with 100 mg/kg (Fisher’s LSD post-test, P = 0.045) or 200 mg/kg (Fisher’s LSD post-test, P = 0.025) L-aspartic acid exhibited longer biting duration compared to the saline control group. n = 8–10 per group. Values are shown as mean ± standard error. DLPAG dorsolateral periaqueductal gray, DMPAG Dorsomedial periaqueductal gray. FC father companionship, LPAG lateral periaqueductal gray, VLPAG ventrolateral periaqueductal gray. PND, postnatal day. Scale bar, 200 µm. * P < 0.05.

Fig. 5. Father companionship changed the gut microbiota of male offspring.

a The ACE index, Chao1 index, Shannon index, and PD-whole-tree index indicate that father’s companionship and control groups did not show differences in α-diversity. b PCoA plot indicated the difference in β-diversity among male mice. c Relative abundance of gut microbial community at the genus level. d Linear discriminant analysis (LDA) effect size (LEfSe) analysis for taxonomic composition gut microbiota. e–g Correlation between the relative abundance of Bilophila and defensive attack behavior. The relative abundance of Bilophila was positively associated with the frequency of biting (r = 0.757, P = 0.006). h–j Correlation between the relative abundance of Bifidobacterium and defensive attack behavior. The relative abundance of Bifidobacterium was negatively associated with the frequency of biting (r = -0.690, P = 0.016). FC Father companionship.

Fig. 6. Correlation analysis of serum metabolomics and gut bacteria among male offspring.

a Heatmap showing the correlation of serum metabolomics and gut bacteria. b Network showing correlation of serum metabolomics and gut bacteria. The relative abundance of Bilophila was positively associated with L-aspartic acid, while the relative abundance of Bifidobacterium was negatively associated with L-aspartic acid. SSANYPA, (2S)-2-[(2S)-2-Aminopropanoyl]-naphthalen-2-ylamino] pentanedioic acid; ACAD,1-[(5-Amino-5-carboxypentyl) amino]-1-deoxyfructose.