Prolonged Exposure to Milk Caseins at Early Developmental Stage Increases Depressive-Like Behaviour and Induces Brain-Region Specific Neuroadaptations: Implications of Gut-Brain Axis

Nutrition during the postnatal period can influence brain development and play an important role in the progression of neuropsychiatric disorders such as depression. Milk is the main source of nutrition for infants during early postnatal development. Casein, a major protein in milk, releases casomorphines with opioid activity upon digestion. These casomorphines have been shown to cross the blood brain barrier1 and therefore may influence the development of the opioid system. Indeed, it has been shown that weaning rat pups on postnatal day 21 (PND21, the normal age of weaning) compared to late weaning (PND25) influences the developmental expression of µ and δopioid receptors (MOPr and DOPr respectively) and this has been shown to be dependent on the loss of dietary casein2. As both MOPr and DOPr have been shown to play protective rolesin anxiety and depression3, it ishypothesised that prolonged exposure to milk caseins, beyond the normal age of weaning may influence the development of these opioid receptors, resulting in depressive like behaviour. Furthermore considering the growing body of evidence implicating gut microbiota in neurobehavioural development, we sought to investigate the impact of a casein rich diet beyond the normal age of weaning on microbial populations in the gut.

Male Wistar Albino rats were divided into 3 groups: (n=8/group): 1) Rats provided with no milk (controls) 2) Rats provided with casein rich (CR) milk (20% casein) 3) Rats provided with casein free (CF) milk. All groups were provided with their respective diets from PND 21 through to PND 25. On PND25, depressive-like behaviour was tested using the forced-swim test (FST) after which Urine, brains and gut contents were collected for analysis. Quantitative receptor autoradiography of DOPr and MOPr were performed using 7nM [3 H]Delt-1 and 4nM [3 H]DAMGO respectively in the brains of the aforementioned rats. NMR metabonomic analysis was performed on the urine samples and Fluorescent In Situ Hybridization(FISH) analysis on the gut contents collected.

On PND 25 the group of pups provided with CR milk displayed a depressive phenotype as indicated by increased immobility times in the FST (p<0.05 one-way ANOVA followed by Bonferronipost-hoc test). MOPr binding was decreased in a number of regions including the amygdala in CR animals compared to CF, andin the deep layer of somatosensory cortex of CR treated animals compared to CF and controls(p<0.01 and p<0.05 respectivelyone-way ANOVAfollowed by LSD post-hoc test). Interestingly a significant decrease in DOPrdensity was observed in the deep layer of the somatosensory cortex in CF animals vs. CR and controls (p<0.05 one-way ANOVA followed by LSD post-hoc test). Metabonomic analysis of urine samples revealed 6 differently expressed urinary metabolites between the two treatment groups some of which were gut microbial metabolites. FISH analysis showed an increase in bacteria belonging to the Clostridum Histolyticum group in the caecum and colon of CR animals (p<0.001 and p<0.01 two way-ANOVA followed by Holm-sidakpost-hoc test respectively)

These provide the first evidence that prolonged exposure to milk and more specifically milk caseins in early life results in brain region specific alterations to the opioid system, which may be associated with the depressive-like phenotype observed in these rats. The differences in urinary metabolites and gut microbial populations indicate a possible gut-brain axis role in mediating the observed effects. The exact mechanisms underlying the observed depressive behaviour still remain to be determined.

1. Sun, Z., and Cade, J. R. (1999). “A Peptide Found in Schizophrenia and Autism Causes Behavioral Changes in Rats.” Autism, 3(1), 85-95.

2. Kitchen, et al. (1995) Development of delta-opioid receptor subtypes and the regulatory role of weaning: radioligand binding, autoradiography and in situ hybridization studies. Journal of Pharmacology and Experimental Therapeutics 275(3): 1597-1607.

3. Kieffer, B. L., and Pierre, E. L., (2013) "Opioid receptors: distinct roles in mood disorders"Trends Neurosci, 36(3), 195-206.