Early-life adversity (ELA) consists of exposure to stressful situations during critical developmental periods and constitutes a major risk factor for poor mental health. Despite this, only around 30% of individuals exposed to ELA go on to develop persistent vulnerability, which often emerges later in life as anxiety disorders ¹. Microglia are immune cells involved in modulating brain maturation and neurogenesis and have been associated with perturbed developmental trajectories and negative mental health outcomes following ELA ². Yet, it remains unclear whether vulnerability and resilience to ELA are linked to distinct microglial phenotypes.

Here, we tested the hypothesis that vulnerability trajectories are associated with striking alterations in microglial morphology following exposure to ELA, using a translational ELA mouse model, the Limited Bedding and Nesting (LBN) ³ paradigm. C57BL/6J mice were subjected to LBN between postnatal days 2 and 9 (PND2-9). In adulthood (PND90), we assessed anxiety-like behaviour in male and female offspring using the elevated plus maze, open field, and light-dark box tests, as well as responses to a panicogenic stimulus (20% CO₂) ³. To stratify individuals as vulnerable or resilient in adulthood, we applied a Z-score approach ⁴, a statistical method that standardises behavioural outcomes across tests. Microglia morphology was analysed in the hippocampus, a brain region that plays a central role in the regulation of anxiety responses, using Iba-1 immunofluorescence and confocal microscopy ⁵. Student’s t-test was used for statistical analysis, with a significance level set at p < 0.05. This study was approved by the Animal Ethics Committee of the University of São Paulo (protocol: 1200/2023).

We observed anxiety- and panic-like responses in the open field (p < 0.05) and CO₂ exposure (p < 0.05) tests only in ELA-male offspring compared to Control, whereas no differences were detected in the elevated plus maze or light-dark box tests for both sexes. Following Z-score stratification, 29% of male and 17% of female mice were classified as vulnerable after ELA exposure. In the hippocampus, vulnerable ELA males exhibited a 24.9% reduction in microglial perimeter (p < 0.05) and a 54.1% increase in soma size (p < 0.05), while vulnerable females showed a 62.7% reduction in microglial perimeter (p < 0.05), compared with their respective ELA-resilient and Control groups. Notably, correlation analyses revealed a strong association (R² = 0.68; p < 0.01) between higher Z-score indices and reduced microglial perimeter, suggesting that this parameter may be indicative of vulnerability following ELA.

Together, these findings indicate that ELA differentially shapes microglial phenotypes in vulnerable and resilient individuals of both sexes, and that these alterations are associated with increased susceptibility to anxiety responses in adulthood.