Sex-dependent and region-specific morphological adaptations in astrocytes following early-life stress

Astrocytes are structurally heterogeneous, with cell morphology providing a valuable readout of experi-ence-dependent plasticity. Despite this, single-cell reconstruction including complete cellular territories remains challenging to compare across brain regions and experimental groups. We used unilateral retro-orbital delivery of PHP.eB-GfaABC1D-eGFP as a practical method for sparse astrocyte labeling and ap-plied it to evaluate regional, sex, and early-life-stress comparisons in the mouse amygdala. Systemic vi-rus delivery to broadly target astrocytes across the brain produced eGFP-positive cells throughout cortex, hippocampus, hypothalamus, and amygdala with spatially isolated cells suitable for intensity projection, three-dimensional surface reconstruction, filament tracing, and Sholl analysis. In naive females, we report relatively homogenous morphological features between lateral amygdala and basal amygdala. In naive males, basal amygdala astrocytes had greater volume and a lower surface-area-to-volume ratio, while surface area, soma diameter, territory, and arborization measures did not differ significantly. Interestingly, early-life stress produced sex-specific morphological responses in the basal amygdala. Females showed increased perimeter, area, and Feret diameter without significant changes in three-dimensional volume or resolved filament measures. Males instead showed reduced surface area and volume, with no significant changes in projected territorial dimensions, soma diameter or resolved filament measures. S100β-positive cell density was unchanged in the displayed regional and stress comparisons. These findings es-tablish systemic sparse eGFP labeling as an efficient route to single-cell astrocyte morphometry and re-veal a sex-dependent and region-specific dissociation between territorial expansion and three-dimensional volumetric contraction after early-life stress.