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.

PurposeMicroglia and Müller cells are prominent participants in retinal responses to injury and disease that shape eventual tissue adaptation or damage. This investigation examined how microglia and Müller cells interact with each other following initial microglial activation.MethodsMouse Müller cells were cultured alone, or co-cultured with activated or unactivated retinal microglia, and their morphological, molecular, and functional responses were evaluated. Müller cell-feedback signaling to microglia was studied using Müller cell-conditioned media. Corroborative in vivo analyses of retinal microglia-Müller cell interactions in the mouse retina were also performed.ResultsOur results demonstrate that Müller cells exposed to activated microglia, relative to those cultured alone or with unactivated microglia, exhibit marked alterations in cell morphology and gene expression that differed from those seen in chronic gliosis. These Müller cells demonstrated in vitro (1) an upregulation of growth factors such as GDNF and LIF, and provide neuroprotection to photoreceptor cells, (2) increased pro-inflammatory factor production, which in turn increased microglial activation in a positive feedback loop, and (3) upregulated chemokine and adhesion protein expression, which allowed Müller cells to attract and adhere to microglia. In vivo activation of microglia by intravitreal injection of lipopolysaccharide (LPS) also induced increased Müller cell-microglia adhesion, indicating that activated microglia may translocate intraretinally in a radial direction using Müller cell processes as an adhesive scaffold.ConclusionOur findings demonstrate that activated microglia are able to influence Müller cells directly, and initiate a program of bidirectional microglia-Müller cell signaling that can mediate adaptive responses within the retina following injury. In the acute aftermath following initial microglia activation, Müller cell responses may serve to augment initial inflammatory responses across retinal lamina and to guide the intraretinal mobilization of migratory microglia using chemotactic cues and adhesive cell contacts. Understanding adaptive microglia-Müller cell interactions in injury responses can help discover therapeutic cellular targets for intervention in retinal disease.

Chronic retinal inflammation in the form of activated microglia and macrophages are implicated in the etiology of neurodegenerative diseases of the retina, including age-related macular degeneration, diabetic retinopathy, and glaucoma. However, molecular biomarkers and targeted therapies for immune cell activation in these disorders are currently lacking. To address this, we investigated the involvement and role of translocator protein (TSPO), a biomarker of microglial and astrocyte gliosis in brain degeneration, in the context of retinal inflammation. Here, we find that TSPO is acutely and specifically upregulated in retinal microglia in separate mouse models of retinal inflammation and injury. Concomitantly, its endogenous ligand, diazepam-binding inhibitor (DBI), is upregulated in the macroglia of the mouse retina such as astrocytes and Müller cells. In addition, we discover that TSPO-mediated signaling in microglia via DBI-derived ligands negatively regulates features of microglial activation, including reactive oxygen species production, TNF-␣ expression and secretion, and microglial proliferation. The inducibility and effects of DBI-TSPO signaling in the retina reveal a mechanism of coordinated macroglia-microglia interactions, the function of which is to limit the magnitude of inflammatory responses after their initiation, facilitating a return to baseline quiescence. Our results indicate that TSPO is a promising molecular marker for imaging inflammatory cell activation in the retina and highlight DBI-TSPO signaling as a potential target for immodulatory therapies.