The influence of sex and neonatal stress on medullary microglia in rat pups.

Neonatal stress has wide-ranging consequences for the developing brain, including the medullary cardiorespiratory network. In rat pups, the reflexive cardiorespiratory inhibition triggered by the presence of liquids near the larynx is augmented by neonatal maternal separation (NMS), especially in males. Sex-specific enhancement of synaptic connectivity by NMS might explain this cardiorespiratory dysfunction. Microglia influence the formation, maturation, activity and elimination of developing synapses, but their role in the wiring of medullary networks is unknown. Owing to their sensitivity to sex hormones and stress hormones, microglial dysfunction could contribute to the abnormal cardiorespiratory phenotype observed in NMS pups. Here, we first used ionized calcium-binding adapter molecule-1 (Iba-1) immunolabelling to compare the density and morphology of microglia in the medulla of male versus female rat pups (14-15 days old) that were either undisturbed or subjected to NMS (3 h day-1 ; postnatal days 3-12). Neonatal maternal separation augmented the density of Iba-1+ cells (caudal region of the NTS), increased the size of the soma and reduced the arborization area (especially in the dorsal motor nucleus of the vagus). Sex-based differences were not observed. Given that the actions of microglia are regulated by neuronal fractalkine (CX3 CL1 ), we then used western blot analysis to compare the expression of CX3 CL1 and its microglial receptor (CX3 CR1 ) in medullary homogenates from control and NMS pups. Although CX3 CR1 expression was 59% greater in males versus females, NMS had no effect on CX3 CL1 /CX3 CR1 signalling. Given that an amoeboid morphology reflects an immature phenotype in developing microglia, NMS could interfere with synaptic pruning via a different mechanism.