Microglial synaptic pruning on axon initial segment spines of dentate granule cells: Sexually dimorphic effects of early‐life stress and consequences for adult fear response

Axon initial segments (AIS) of dentate granule cells in the hippocampus exhibit prominent spines (AISS) during early development that are associated with microglial contacts. In the present study, we investigated whether developmental changes in AISS could be modified by early‐life stress (ELS), specifically neonatal maternal separation (MS), through stress hormones and microglial activation and examined the potential behavioural consequences. We examined AISS at postnatal day (PND)5, 15 and 50, using Golgi‐Cox staining and anatomical analysis. Neurone‐microglial interaction was assessed using antibodies against ankyrin‐G, PSD‐95 and Iba1, for AIS, AISS and microglia visualisation, respectively, in normally reared and neonatal maternally separated male and female rats. We observed a higher density of AISS in ELS rats at both PND15 and PND50 compared to controls. Effects were more pronounced in females than males. AIS‐associated microglia in ELS rats showed a hyper‐ramified morphology and less co‐localisation with PSD‐95 compared to controls at PND15. ELS‐associated alteration in microglial morphology and synaptic pruning was mimicked by treatment of acute hippocampal slices of normally reared rats with vasopressin. ELS rats exhibited increased freezing behaviour during auditory fear memory testing, which was more pronounced in female subjects and corresponded with increased Fos expression in dorsal and ventral dentate granule cells. Thus, microglial synaptic pruning in dentate AIS of hippocampus is influenced by ELS, with demonstrable sex bias regarding its anatomical characteristics and subsequent fear‐induced defensive behaviours. Axon initial segment (AIS) spines (AISS) of dentate gyrus granule cells receiving glutamatergic inputs are pruned during development by resident microglial cells, as measured by microglial engulfment of PSD‐95. AISS pruning declines with ageing, and this decline is slowed by early‐life stress in the form of maternal separation (MS), and can be modified in dentate granule cells in brain slices ex vivo by the stress‐associated neuropeptide vasopressin (decreased pruning). AISS pruning and later‐life responses to stress (predator sound) are both affected by MS, and there is a clear sexual bias in both behavioural response and microglial AISS engulfment responses to MS, in favour of greater female stress reactivity.