Two-hit model of stress: Outcome-dependent effects of maternal separation and post-weaning isolation rearing on behaviour and BDNF in the prefrontal cortex

The two-hit model proposes that an adverse experience during an early sensitive period modifies vulnerability to a subsequent stressor. This study aimed to determine whether maternal separation during early postnatal life and isolation rearing from weaning produce cumulative or interactive effects or preferentially affect distinct behavioural and neurobiological domains in male and female rats. We hypothesised that the two developmental stressors would induce persistent but partly distinct alterations, reflecting the different developmental periods and maturational processes affected by each manipulation. Seventy-eight Wistar rat offspring (=39/sex) were exposed to maternal separation for 3 h/day from postnatal day 3 to postnatal day 8, while control littermates remained in the home cage with the dam. At weaning on postnatal day 21, rats from both early-life conditions were assigned to social rearing in same-sex groups or isolation rearing in individual cages until postnatal day 58. Behavioural testing began 30 days post-weaning, and brain-derived neurotrophic factor (BDNF) concentrations in the prefrontal cortex were measured at the end of the experiment. Maternal separation and isolation rearing independently reduced locomotor activity and increased anxiety-like behaviour, without consistent intensification following combined exposure. Isolation rearing reduced sucrose preference and altered relative sucrose intake in a sex- and prior experience-dependent manner. Maternal separation reduced BDNF in the prefrontal cortex, with an additional sex-dependent interaction between the two manipulations, whereas isolation rearing had no significant overall effect. Females were generally more active and displayed less anxiety-like behaviour, but sex did not define a uniform pattern of vulnerability. These findings support the hypothesis that maternal separation and post-weaning isolation rearing produce partly distinct, outcome-dependent profiles rather than simple cumulative impairment, with their consequences shaped by sex and the functional domain examined.