Early-life stress and adolescent circadian dysrhythmia drives unique behavioral and microbial profiles in rats

OBJECTIVES: Early life adversity and circadian disruptions are known to impact neurodevelopment and physiology. This study investigated the effects of maternal separation (MS), adolescent circadian dysrhythmia, and their combination (double hit) on anxiety-like behavior and gut microbiota composition in rats. METHODS: Rats were divided into four groups: CL (control group: normal early-life conditions with a standard light/dark cycle during adolescence), MS + N (maternal separation (MS) with a standard light/dark cycle (N=normal)) during adolescence), N + ALD (normal early-life conditions (N) with an altered light/dark cycle (ALD) during adolescence), and MS + ALD (combined exposure: MS with an altered light/dark cycle (ALD) during adolescence). Anxiety-like behavior and locomotor activity were assessed using the Open Field Test. Gut microbial diversity and taxonomic composition were analysed to identify microbial shifts across groups. RESULTS: Behavioral analysis indicated that the combined stress group (MSLD) spent significantly (p < 0.05) more time in the center of the arena compared to the CL, MS + N, and N + ALD groups, suggesting a compromise in risk assessment ability due to dual stress exposure. Microbiome profiling revealed that while a core microbiome was conserved, each stressor generated a unique taxonomic signature. The N + ALD group appeared as the most distinct outlier, characterized by the lowest number of unique features and a specific enrichment of the viral species of phylum Uroviricota. Conversely, the MS + ALD group was distinguished by an enrichment of Bacteroidota species, including Muribaculum intestinale and Phocaeicola vulgatus, while the MS + N group showed enrichment in Bacteroides acidifaciens. Mycobiome analysis showed that early-life stress was the primary driver of fungal restructuring, distinguishing maternal separation groups by the loss of Neocallimastix species and the competitive expansion of Piromyces finnis. While adolescent circadian disruption alone largely preserved the baseline mycobiome, the cumulative dual-hit stress (MS + ALD) generated a distinct dysbiotic profile evident by the unique proliferation of Anaeromyces robustus. CONCLUSIONS: In conclusion, the developmental timing of stress exposure drives distinct dysbiotic shifts. Specifically, adolescent circadian disruption selectively targets the virome, whereas early-life stress causes shift in the microbiome which endures a long-term foundation for adolescent psychiatric vulnerability. Notably, the cumulative effect of early life and adolescence stressors results in a unique microbial and behavioral profile, highlighting that the specific developmental window of exposure is a decisive factor in gut-brain axis dysfunction.