Casein Intake Post Weaning Modulates Gut Microbial, Metabolic and Behavioral Profiles in Rats

The postnatal period is critical for brain and behavioral development and it is sensitive to environmental stimuli, such as nutrition. Prevention of weaning from maternal milk was previously shown to result in stress-induced immobility during the forced swim test (FST), indicative of depressive-like behavior, accompanied by neurochemical, gut microbial and metabolic alterations in rats. Separately, weaning was also found to act as a developmental trigger for a population of opioid receptor subtypes, an effect dependent on loss of dietary casein. In the present study, we explore the impact of exposure to the casein component of milk, and its two major genetic variants A1 and A2 β-casein, beyond weaning age. Rats exposed to casein-rich and casein-free milk, as well as to commercialized milk, containing a mixture of A1 and A2 β-casein, or exclusively A2 β-casein milk, from postnatal day (PND) 21 to PND25 were assessed for immobility behavior during the FST. Casein-rich and A1 β-casein containing milk but not casein-free and A2 β-casein milk increased stress-induced immobility compared to weaned control animals. This was concomitant with an increased abundance of bacteria from the Clostridium histolyticum group in the caecum and colon of the casein-rich group, region specific alterations of µ-opioid and oxytocin receptors in the brain, as well as modifications in urinary biochemical profiles. The A1 β-casein group additionally displayed a strong association between altered gut microbial metabolites in urine and brain metabolites. Our findings suggest that exposure to milk casein, in particular to A1 β-casein, beyond weaning age enhances stress-induced immobility during the FST, promoting a depressive-like phenotype, via a possible gut-brain axis dependent mechanism.