SummaryMicroglia, the primary resident immune cells of the central nervous system (CNS), exhibit dynamic behavior involving rapid process motility and cellular migration that is thought to underlie key functions of immune surveillance and tissue repair. Although age-related changes in microglial activation have been implicated in the pathogenesis of neurodegenerative diseases of aging, how dynamic behavior in microglia is influenced by aging is not fully understood. In this study, we employed live imaging of retinal microglia in situ to compare microglial morphology and behavioral dynamics in young and aged animals. We found that aged microglia in the resting state have significantly smaller and less branched dendritic arbors, and also slower process motilities, which probably compromise their ability to survey and interact with their environment continuously. We also found that dynamic microglial responses to injury were age-dependent. While young microglia responded to extracellular ATP, an injury-associated signal, by increasing their motility and becoming more ramified, aged microglia exhibited a contrary response, becoming less dynamic and ramified. In response to laser-induced focal tissue injury, aged microglia demonstrated slower acute responses with lower rates of process motility and cellular migration compared with young microglia. Interestingly, the longer term response of disaggregation from the injury site was retarded in aged microglia, indicating that senescent microglial responses, while slower to initiate, are more sustained. Together, these altered features of microglial behavior at rest and following injury reveal an agedependent dysregulation of immune response in the CNS that may illuminate microglial contributions to agerelated neuroinflammatory degeneration.

PurposeMicroglia represent the primary resident immune cells in the CNS, and have been implicated in the pathology of neurodegenerative diseases. Under basal or “resting” conditions, microglia possess ramified morphologies and exhibit dynamic surveying movements in their processes. Despite the prominence of this phenomenon, the function and regulation of microglial morphology and dynamic behavior are incompletely understood. We investigate here whether and how neurotransmission regulates “resting” microglial morphology and behavior.MethodsWe employed an ex vivo mouse retinal explant system in which endogenous neurotransmission and dynamic microglial behavior are present. We utilized live-cell time-lapse confocal imaging to study the morphology and behavior of GFP-labeled retinal microglia in response to neurotransmitter agonists and antagonists. Patch clamp electrophysiology and immunohistochemical localization of glutamate receptors were also used to investigate direct-versus-indirect effects of neurotransmission by microglia.ResultsRetinal microglial morphology and dynamic behavior were not cell-autonomously regulated but are instead modulated by endogenous neurotransmission. Morphological parameters and process motility were differentially regulated by different modes of neurotransmission and were increased by ionotropic glutamatergic neurotransmission and decreased by ionotropic GABAergic neurotransmission. These neurotransmitter influences on retinal microglia were however unlikely to be directly mediated; local applications of neurotransmitters were unable to elicit electrical responses on microglia patch-clamp recordings and ionotropic glutamatergic receptors were not located on microglial cell bodies or processes by immunofluorescent labeling. Instead, these influences were mediated indirectly via extracellular ATP, released in response to glutamatergic neurotransmission through probenecid-sensitive pannexin hemichannels.ConclusionsOur results demonstrate that neurotransmission plays an endogenous role in regulating the morphology and behavior of “resting” microglia in the retina. These findings illustrate a mode of constitutive signaling between the neural and immune compartments of the CNS through which immune cells may be regulated in concert with levels of neural activity.

Ongoing research is warranted because A. vera may represent a new therapeutic class of medications for OSSN treatment.