Neonatal immune challenge influences the microbiota and behaviour in a sexually dimorphic manner

• There is comorbidity between anxiety disorders and gastrointestinal disorders, with both linked to adverse early life events. • Using a rodent model of neonatal immune activation found sex-specific changes in behaviour and gut microbiome . • Observed microbial associations with behaviour following neonatal immune activation. • Sex-specific alterations in predicted functional pathways of neurotransmitters . • The importance of sex in determining the impact of neonatal immune activation on anxiety behaviours and the gut microbiome. There is comorbidity between anxiety disorders and gastrointestinal disorders, with both linked to adverse early life events. The microbiome gut-brain-axis, a bidirectional communication system, is plastic throughout the neonatal period and is a possible mediator of this relationship. Here, we used a well-established neonatal rodent immune activation model to investigate the long-term effect of neonatal lipopolysaccharide (LPS) exposure on adult behaviour and the relationship to microbiome composition. Wistar rats were injected with LPS (0.05 mg/kg) or saline (equivolume) on postnatal days 3 and 5. In adulthood, behavioural tests were performed to assess anxiety-like behaviour, and microbiota sequencing was performed on stool samples. There were distinctly different behavioural phenotypes for LPS-exposed males and females. LPS-exposed males displayed typical anxiety-like behaviours with significantly decreased social interaction (F (1,22) = 7.576, p = 0.009) and increased defecation relative to saline controls (F (1,23) = 8.623, p = 0.005). LPS-exposed females displayed a different behavioural phenotype with significantly increased social interaction (F (1,22) = 6.094, p = 0.018), and exploration (F (1,24) = 6.359, p = 0.015), compared to saline controls. With respect to microbiota profiling data, Bacteroidota was significantly increased for LPS-exposed females (F (1,14) = 4.931p = 0.035) and Proteobacteria was decreased for LPS-exposed rats of both sexes versus controls (F (1,30) = 4.923p = 0.035). Furthermore, alterations in predicted functional pathways for neurotransmitters in faeces were observed with a decrease in the relative abundance of D-glutamine and D-glutamate metabolism in LPS exposed females compared to control females ( p < 0.05). This suggests that neonatal immune activation alters both later life behaviour and adult gut microbiota in sex-specific ways. These findings highlight the importance of sex in determining the impact of neonatal immune activation on social behaviour and the gut microbiota.