Sex-dependent effects of early-life microbiota depletion on behaviour, neuroimmune function and neuronal development

Background: Numerous studies have emphasised the importance of the gut microbiota during early life and its role in modulating neurodevelopment and behaviour [1]. While, epidemiological studies have shown that early life antibiotic-induced microbial disruption can increase an individual’s risk of developing immune and metabolic diseases, preclinical studies have shown that long-term antibiotic-induced microbial disruption in early life can have enduring effects on brain physiology and behaviour [2]. However, these studies have not investigated the impact of targeted antibiotic-induced microbiota depletion during critical developmental windows and how this may be related to neurodevelopmental outcomes.

Aim: The main aim of this study was to investigate the long-term effects of antibiotic-induced microbiota depletion on sensitive periods of brain and behavioural development.

Methods: To identify critical windows of microbial influence on neurodevelopmental outcomes, an antibiotic cocktail (n = 8-12; ampicillin 200mg/kg, vancomycin 100mg/kg, imipenem 50 mg/kg, gentamicin 200mg/kg) or saline solution was orally administered to NIH Swiss mice either during postnatal (P2-9), pre-weaning (P12-18) or post-weaning (P21-27) developmental periods. Following treatment, behavioural outcomes and sexual dimorphic effects were assessed in offspring during early-life (P5-11; maternal care, communicative behaviour, and social attachment learning), adolescence (P40-45) and adulthood (P70-80) (anxiety, locomotor activity, spatial and episodic memory, depression, sociability, and social novelty). Nonparametric data were analysed by Kruskal–Wallis post hoc Dunn’s test, parametric data by Two-way ANOVA and post hoc Fishers LSD. Data are shown as mean ± SEM. Statistical significance was set at p < 0.05.

Results: Our results demonstrate that early life microbiota depletion has enduring effects into adolescence on the developing caecal microbiome. Principle component analysis (PCA) of beta-diversity demonstrated a clear separation between antibiotic-treated and saline-treated mice. Several bacterial genera were also significantly affected by early life microbiota disruption. Microbial disruption also induced changes in microbially derived gut-metabolic (GMMs) and gut-brain (GBMs) modules - functional pathways that encode the metabolism of neuroactive molecules. Furthermore, our results indicate that early-life antibiotic-induced microbiota depletion has subtle sex-and treatment timing effects on, circulating immune cells, and neurophysiology (including altered myelin-related gene expression in the prefrontal cortex and malformed microglia in the basolateral amygdala). We also observed an effect of sex and timing of treatment on behavioural outcomes in adolescence and adulthood, whereby microbial disruption in the PreWean timepoint was associated with increased anxiety-like behaviour. No significant effects of microbial disruption were observed in depressive-like, or memory related-behaviours.

Conclusion: In this study we provide evidence that early-life antibiotic-induced microbiota depletion perturbs the developing gut microbiome. Moreover, microbial depletion has subtle effects on anxiety-like behaviour, circulating immune cells and CNS molecular markers in a sex and time-dependent manner. Overall, this study highlights the vulnerability of the gut microbiota during critical windows of development and the subtle effects that microbiota-targeted perturbations can have on brain physiology and behaviour. Indeed, further research is required to understand the mechanistic underpinnings of these results and to determine if alterations in microbiota composition in early-life increases an individual’s susceptibility to neurodevelopmental or neuropsychiatric disorders.