Antibiotic perturbation of the gut microbiota in zebrafish larvae reveals a microbiota-gut-brain axis signalling pathway influencing behaviour and neurobiology

The microbiota-gut-brain axis (MGBA) enables bidirectional communication between intestinal microbes and the central nervous system, influencing host neurodevelopment, metabolism, and behaviour [1]. Early-life antibiotic exposure can profoundly disrupt the developing microbiome during critical developmental windows [2,3]; however, the specific neurochemical and metabolic mechanisms linking microbial community disruption to altered neurobiology and behaviour during development remain poorly defined.

This study aimed to (1) characterise the effects of antibiotic-induced gut microbiome perturbation on microbial community composition, (2) determine whether such perturbations alter anxiety-related behaviour and serotonergic gene expression, and (3) identify metabolic signatures in the brain and periphery associated with microbiome disruption during early neurodevelopment.

To study this, zebrafish larvae (3 dpf) were exposed to meropenem, vancomycin, or their combination for 48h. Gut microbial composition was profiled by 16S rRNA gene sequencing (V3–V4 region) with PICRUSt2-based functional prediction. Anxiety-like behaviour was assessed using the light-dark transition assay, with locomotor data analysed by two-way repeated-measures ANOVA with Tukey’s post hoc correction. Transcriptional changes in the brain serotonergic system were quantified by RT-qPCR. Targeted metabolomics (LC-MS/MS) was used to measure central and peripheral neurotransmitter levels, and untargeted metabolomic profiling was applied to brain and body extracts. Statistical analyses of microbiome data included alpha and beta diversity metrics, differential abundance testing, principal coordinates analysis, and permutational multivariate analysis of variance.

All antibiotic treatments markedly altered microbial community structure, with significant reductions in alpha diversity and distinct beta-diversity clustering by treatment. Functional prediction identified treatment-specific reductions in pathways for polyamine biosynthesis and amino acid metabolism. In the light-dark assay, antibiotic-treated larvae exhibited significantly attenuated dark-phase locomotor activity and impaired light habituation, indicating a specific effect on anxiety-related behaviour rather than general locomotor function. Behavioural changes were accompanied by significant downregulation of the serotonin biosynthetic enzyme tph1a and the serotonin reuptake transporter slc6a4a in the brain, as determined by RT-qPCR.

Targeted metabolomics revealed antibiotic-dependent reductions in central serotonin, glutamate, and GABA levels. Complementary untargeted profiling of head and body samples demonstrated widespread metabolic remodelling, including disruption of amino acid homeostasis with near-complete depletion of lysine across all treatment groups, implicating the microbiome in essential amino acid availability. Additional alterations included suppression of the arginine-polyamine axis, shifts in purine metabolism consistent with reduced anabolic capacity, disruption of membrane lipid turnover, changes in redox-associated metabolites, and loss of microbially derived biotin in the brain. The extent and nature of these changes varied by treatment. Meropenem induced the most extensive metabolic perturbations, vancomycin had more moderate effects, and combination treatment produced a distinct metabolic profile. Differences between head and body compartments further indicate that microbiome disruption shapes tissue-specific neurochemical and metabolic outcomes.

Taken together, our findings demonstrate that antibiotic-induced disruption of the developing gut microbiome leads to treatment-specific changes in behaviour, neurotransmitter signalling, and brain metabolism in zebrafish larvae. In addition, we identified specific metabolic and neurochemical pathways, including essential amino acid depletion and polyamine suppression, through which microbiota perturbation may influence host neurobiology during early neurodevelopment.

References

1. Cryan, J. F. et al. The Microbiota-Gut-Brain Axis. Physiol. Rev. 99, 1877–2013 (2019)
2. Phelps, D. et al. Microbial colonization is required for normal neurobehavioral development in zebrafish. Sci. Rep. 7, 11244 (2017)
3. Li, P. et al. The Function and the Affecting Factors of the Zebrafish Gut Microbiota. Front. Microbiol. 13, 903471 (2022)