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Prenatal stress is a significant risk factor that can lead to neurobehavioral deficits in offspring. In the present study, we examined the effects of a probiotic mixture on anxiety, memory, and underlying possible molecular pathways in prenatally stressed rats. Male offspring exposed to chronic unpredictable stress (CUS) during fetal life were received either saline (CUS+SAL) or a probiotic mixture (CUS+PRO) for 30 days post-weaning. Non-stressed controls were also given either saline (CON+SAL) or probiotics (CON+PRO). The passive avoidance test and the elevated zero maze test were used to assess avoidance memory and anxiety-like behavior, respectively. In comparison to the CON+SAL controls, the CUS+SAL group exhibited significant anxiety-like behavior and impaired avoidance memory. At a molecular level, the behavioral impairments were accompanied by increased serum levels of the oxidant, MDA, and decreased serum levels of antioxidants, TAC, GSH, and SOD, upregulation of the hippocampal serotonin receptor Htr1a gene, while downregulation of microRNAs miR-26a and miR-320-3p, reduced BDNF, and increased Bax/Bcl-2 ratio apoptosis in the duodenum. Probiotics effectively mitigated these alterations. The intervention improved behavioral functions, normalized oxidative and antioxidative stress markers, and restored the expression of Htr1a and miR-320-3p to near-normal levels, while miR-26a expression remained unaffected by the treatment. It also enhanced the Bax/Bcl-2 ratio and increased BDNF content. Interestingly, unstressed control rats were unresponsive to the probiotic treatment. Conclusively, probiotic supplementation sufficiently alleviates the adverse effects of fetal life stress, possibly by affecting the gut-brain axis, highlighting the importance of beneficial bacteria in neurobehavioral development and maintenance. Show less