Age-associated microglial heterogeneity includes emergence of mobile microglial states.

Microglia maintain neuronal homeostasis through dynamic surveillance strategies that depend on their functional state. In the healthy brain, highly ramified microglia monitor neuronal integrity via motile processes and transient soma contacts. Aging is associated with reduced process motility and diminished expression of homeostatic markers, raising the question of how microglial surveillance adapts to these changes. Here, we used ex vivo time-lapse imaging of acute cortical slices from young and aged mice to characterize age-dependent alterations in microglial behavior. We found that, unlike microglia in young animals, a subset of aged microglia exhibited pronounced somatic mobility and loss of territorial confinement, resembling migratory behaviors previously described in neurodegenerative conditions. Unsupervised clustering analyses revealed increased heterogeneity in aged microglia, including the emergence of distinct high-mobility subpopulations absent in young brains. Despite these dynamic changes, aged microglia largely retained ramified morphology and did not show increased neuronal envelopment. In parallel, microglia from aged mice displayed prolonged intracellular Ca2+ bursts across all subpopulations, indicating a global shift in functional state. These sustained calcium signals resembled those observed in disease-associated microglia, suggesting that aging induces a primed, partially activated phenotype. Consistent with this interpretation, we observed a trend toward reduced P2Y12 expression in aged microglia. Together, our findings demonstrate that microglia adapt to aging by shifting from process-based surveillance toward increased soma-mediated mobility, accompanied by sustained Ca2+ signaling. This adaptive response may compensate for declining process dynamics while reflecting a transition toward a pro-inflammatory, disease-associated state.