Early-life tryptophan exposure drives long-term behavioral and gut–brain changes in a neurodevelopmental model

Prenatal environmental factors critically shape brain development and may influence vulnerability to neurodevelopmental disorders. Tryptophan (Trp), the dietary precursor of serotonin (5-HT), is a key regulator of neurodevelopmental processes and also plays an important role in microbiota–gut–brain signaling. Alterations in serotonergic pathways and gut microbiota have both been implicated in autism spectrum disorder (ASD). However, the long-term consequences of increased prenatal Trp availability remain insufficiently understood, particularly in individuals with an underlying genetic vulnerability.

In this study, we investigated whether prenatal Trp supplementation induces persistent behavioral, neurochemical, and microbiota alterations in Nf1^+/− mice, a genetic model that recapitulates core ASD-relevant phenotypes. Pregnant dams were fed either a control or Trp-enriched diet from preconception through gestation and lactation. Offspring, including Nf1^+/− mice and wild-type (WT) littermates, were evaluated longitudinally from early postnatal development to the juvenile stage.

Behavioral assessments included early developmental milestones, ultrasonic vocalizations (USVs), anxiety-like behavior, sociability, and repetitive-like behaviors. Trp and 5-HT levels were quantified in cortical, hypothalamic, plasma, and intestinal tissues using high-performance liquid chromatography. Gut microbiota composition was analyzed using 16S rRNA sequencing. Microbial diversity and community structure were assessed using ecological metrics and permutational multivariate analysis of variance, and correlation analyses were performed to examine microbiota–brain–behavior relationships.

Prenatal Trp supplementation selectively influenced early development, accelerating acquisition of the surface righting reflex and reducing neonatal USV output, suggesting altered sensorimotor maturation and early-life emotional reactivity. In juvenile mice, effects were domain-specific. In Nf1^+/− offspring, Trp supplementation rescued social preference deficits and reduced repetitive digging behavior, indicating improvements in core ASD-relevant domains. In contrast, prenatal Trp increased anxiety-like behavior, particularly in WT mice, highlighting divergent effects across behavioral domains and developmental trajectories, and suggesting differential circuit-level sensitivity.

Neurochemical analyses revealed persistent and region-specific central adaptations. Prenatal Trp reduced cortical Trp levels in both genotypes and decreased hypothalamic 5-HT concentrations, while no significant changes were detected in plasma or gut compartments. These findings suggest long-term developmental reprogramming of central Trp–5-HT metabolism rather than sustained systemic alterations, potentially involving compensatory metabolic or transport mechanisms.

Microbiota profiling demonstrated increased alpha diversity and significant shifts in community composition in Nf1^+/− offspring exposed to prenatal Trp. Taxonomic changes included reduced abundance of Bacteroidaceae and increased Lactobacillaceae, taxa implicated in Trp metabolism and gut–brain communication. Correlation analyses further revealed genotype-dependent microbiota–brain–behavior interactions, with more integrated association networks observed in Nf1^+/− mice compared to WT animals.

Overall, prenatal Trp supplementation induces long-lasting and domain-specific effects across behavioral, neurochemical, and microbiota levels. While improving social and repetitive phenotypes relevant to ASD, it simultaneously promotes anxiety-like behavior, highlighting both the therapeutic potential and potential risks of early serotonergic modulation. These findings support a role for maternal nutrition in shaping neurodevelopment through microbiota–gut–brain mechanisms in a genotype-dependent manner and across multiple interacting biological systems.

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