Programming Healthy Neurodevelopment Through the Gut–Brain Axis: From Early-Life Preclinical Probiotic Interventions to Clinical Metabolomics

Neurodevelopment is increasingly recognized as being shaped by dynamic interactions within the microbiota–gut–brain axis. This thesis investigates the gut–brain axis in both preclinical models and a human cohort, focusing on how microbial modulation and metabolites impact neurodevelopmental outcomes. Mouse models were used to investigate how maternal probiotic exposure during critical perinatal periods programs long-term neurobehavioral and molecular trajectories.

Prenatal supplementation with a multispecies probiotic (Ecologic® Panda; gestational day 6 to birth) produced persistent, sex- and age-dependent effects on behavior, microbiota composition and gene expression in both brain and colon of mice. Juvenile males and females showed reduced anxiety-like behavior. In adulthood, only males maintained reduced anxiety, whereas females displayed greater anxiety and increased exploratory behavior. Expression of tight-junction genes that maintain blood-brain barrier (BBB) integrity were increased differently in adult and juvenile prefrontal cortex (PFC) of male offspring. Expression of the anti-inflammatory cytokine interleukin-10 (Il10) increased in juveniles of both sexes but persisted only in adult males. The oxytocin receptor gene Oxtr expression was higher in juvenile females and adult males, while the microglial receptor Trem2 increased only in adult females. The peptidoglycan transporter PepT1 (Slc15a1) was consistently upregulated in juvenile and adult males but not females. Colon tissue showed similar sex- and age-specific changes in tight-junction and mucin proteins. 16S rRNA sequencing revealed persistent alterations in beta diversity with enrichment of anti-inflammatory genera such as Eubacterium xylanophilum and Ruminococcus in males and Lachnospiraceae A2 and Faecalibaculum in females, together with reduced Dubosiella in females.

Perinatal exposure to Limosilactobacillus reuteri (L. reuteri) was similarly investigated (gestational day 6 to postnatal day 7), reshaping social behavior and producing sex- and region-specific molecular signatures in the PFC and striatum. Adult males showed enduring improvements in sociability and social recognition, while females exhibited impaired social recognition and only a minor trend toward reduced anxiety. Both sexes displayed increased Oxtr expression, yet behavioral changes emerged mostly in males, indicating that receptor upregulation alone is insufficient for social improvement. Male PFC also showed elevated microglial markers and myelin-related genes, while females displayed slight alterations without corresponding behavioral changes. In the striatum, Il10 and the peptidoglycan transporter Slc46a2 were upregulated in males, whereas in females Slc46a2, Slc46a3, myelin oligodendrocyte glycoprotein (Mog) and the synaptic marker synaptophysin (Syp) were upregulated. Gut microbiota profiling showed sex-dependent beta diversity shifts, with increased relative abundance in males of Lachnospiraceae_NK4A136, ASF356 and Anaerotruncus, taxa associated with short-chain fatty acid (SCFA) production. Females showed increased relative abundance of A2 and the Eubacterium xylanophilum group, together with reductions in Roseburia, Oscillibacter and Blautia, also linked to SCFA metabolism.

Transcriptomics analysis comparing hippocampus and hypothalamus revealed broad and enduring signatures in both probiotics, highlighting shared neuroimmune pathways alongside strain- and sex-specific molecular profiles. The multispecies formulation produced broader transcriptional regulation, affecting genes involved in neuroplasticity, immune signaling, myelination and peptidoglycan transport, while L. reuteri produced more selective region- and sex-specific changes. Both interventions converged on Il10, Oxtr, Trem2 and bacterial peptidoglycan transporters, highlighting shared neuroimmune pathways through which maternal microbiota influences offspring brain development. Across studies, female offspring generally displayed more selective changes in both behavioral and transcriptional outcomes.

Given the growing evidence that early-life microbial metabolites influence neurodevelopment, we performed metabolomic profiling in the ELICIT birth cohort, a longitudinal study of mother–infant dyads in rural Tanzania investigating how early environmental and microbial exposures shape growth and cognition. Microbial- and host-derived metabolites, including choline, lactate and dimethylamine, alongside vitamins were associated with infant growth and cognitive performance. Plasma lipids, bile acids and amino acids showed additional sex-dependent metabolic signatures linked to neurodevelopmental outcomes. These findings highlight multiple candidate biomarkers of early neurodevelopment.

Together, these studies demonstrate that maternal probiotic supplementation durably reprograms offspring neurobehavioral and molecular trajectories through the microbiota–gut–brain axis. By integrating mechanistic insights from preclinical models with human metabolomics, this thesis identifies convergent microbiota, immune, and metabolic pathways that may guide the development of precision microbiota-targeted strategies to promote healthy brain development.