Altered Microglial Plasticity in the Periaqueductal Grey of Pre-Symptomatic Mecp2-Heterozygous Mice Following Early-Life Stress

Rett syndrome (RTT), a severe neurodevelopmental disorder primarily affecting girls, is commonly caused by MECP2 loss-of-function mutations. Key symptoms include motor impairments, typical hand stereotypies and intellectual disability. Moreover, although not thoroughly studied, anxiety, heightened stress sensitivity, and aberrant pain perception are also an important component of the RTT phenotype. Emerging evidence suggests that early-life stress (ELS) worsens Mecp2-related phenotypic alterations in mice. Microglia, the resident immune cells within the central nervous system, play a critical role in RTT pathophysiology, yet the combined impact of ELS and Mecp2 deficiency on microglia has not been studied. Previously, we observed reduced activation of the periaqueductal grey (PAG, a cerebral structure involved in pain modulation, autonomic control, and defensive behaviours) in Mecp2-heterozygous (Mecp2-het) mice after thermal stimulation. Here, we investigated the impact of ELS on microglia morphology in the PAG under Mecp2 deficiency. To this end, we analysed microglia in the PAG of presymptomatic Mecp2-het mice previously subjected to maternal separation (MS) as a model of ELS, alongside corresponding control animals. Brain sections were immunolabelled for IBA1, a pan-microglial marker. Microglial cells within the PAG were evaluated for expression levels, morphological characteristics, and fractal properties. While global PAG analyses showed minimal differences, subdivision-specific analyses revealed significant microglial alterations. These findings suggest that ELS exacerbates Mecp2-related neurodevelopmental deficits, impairing microglia in a region-specific manner. Our data points to a microglial failure to morphologically adapt, rather than overt structural loss, in the PAG that may underlie some of the neurological dysfunctions observed in RTT.