In Vivo Imaging of the Microglial Landscape After Whole Brain Radiation Therapy.

PURPOSE: Whole brain radiation therapy (WBRT) is an important treatment for patients with multiple brain metastases, but can also cause cognitive deterioration. Microglia, the resident immune cells of the brain, promote a proinflammatory environment and likely contribute to cognitive decline after WBRT. To investigate the temporal dynamics of the microglial reaction in individual mice to WBRT, we developed a novel in vivo experimental model using cranial window implants and longitudinal imaging. METHODS AND MATERIALS: Chronic cranial windows were surgically implanted over the somatosensory cortex of transgenic Cx3cr1-enhanced green fluorescent protein (EGFP)/+ C57BL/6 mice, where microglia were fluorescently tagged with EGFP. Cx3cr1-EGFP/+ mice were also crossed with Thy1-YFP mice to fluorescently dual label microglia and subsets of neurons throughout the brain. Three weeks after window implantation and recovery, computed tomography image guided WBRT was delivered (single dose 10 Gy using two 5 Gy parallel-opposed lateral beams). Radiation dosing was confirmed using radiochromic film. Then, in vivo 2-photon microscopy was used to longitudinally image the microglial landscape and microglial motility at 7 days and 16 days after irradiation in the same mice. RESULTS: Film dosimetry confirmed the average delivered dose per beam at midpoint was accurate within 2%, with no attenuation from the window frame. By 7 days after WBRT, significant changes in the microglial landscape were seen, characterized by apparent loss of microglial cells (20%) and significant rearrangements of microglial location with time after irradiation (36% of cells not found in original location). CONCLUSIONS: Using longitudinal in vivo 2-photon imaging, this study demonstrated the feasibility of imaging microglia-neuron interactions and defining how microglia react to WBRT in the same mouse. Having demonstrated utility of the model, this experimental paradigm can be used to investigate the dynamic changes of many different brain cell types and their interactions after WBRT and uncover the underlying cellular mechanisms of WBRT-induced cognitive decline.

Loss of P2Y12 Has Behavioral Effects in the Adult Mouse.

While microglia have been established as critical mediators of synaptic plasticity, the molecular signals underlying this process are still being uncovered. Increasing evidence suggests that microglia utilize these signals in a temporally and regionally heterogeneous manner. Subsequently, it is necessary to understand the conditions under which different molecular signals are employed by microglia to mediate the physiological process of synaptic remodeling in development and adulthood. While the microglial purinergic receptor P2Y12 is required for ocular dominance plasticity, an adolescent form of experience-dependent plasticity, it remains unknown whether P2Y12 functions in other forms of plasticity at different developmental time points or in different brain regions. Using a combination of ex vivo characterization and behavioral testing, we examined how the loss of P2Y12 affects developmental processes and behavioral performance in adulthood in mice. We found P2Y12 was not required for an early form of plasticity in the developing visual thalamus and did not affect microglial migration into barrels in the developing somatosensory cortex. In adult mice, however, the loss of P2Y12 resulted in alterations in recognition and social memory, as well as anxiety-like behaviors, suggesting that while P2Y12 is not a universal regulator of synaptic plasticity, the loss of P2Y12 is sufficient to cause functional defects.

Phosphoinositide-3-Kinase γ Is Not a Predominant Regulator of ATP-Dependent Directed Microglial Process Motility or Experience-Dependent Ocular Dominance Plasticity.

Microglia are dynamic cells whose extensive interactions with neurons and glia during development allow them to regulate neuronal development and function. The microglial P2Y12 receptor is crucial for microglial responsiveness to extracellular ATP and mediates numerous microglial functions, including ATP-dependent directional motility, microglia-neuron interactions, and experience-dependent synaptic plasticity. However, little is known about the downstream signaling effectors that mediate these diverse actions of P2Y12. Phosphoinositide-3-kinase γ (PI3Kγ), a lipid kinase activated downstream of Gi-protein-coupled receptors such as P2Y12, could translate localized extracellular ATP signals into directed microglial action and serve as a broad effector of P2Y12-dependent signaling. Here, we used pharmacological and genetic methods to manipulate P2Y12 and PI3Kγ signaling to determine whether inhibiting PI3Kγ phenocopied the loss of P2Y12 signaling in mouse microglia. While pan-inhibition of all PI3K activity substantially affected P2Y12-dependent microglial responses, our results suggest that PI3Kγ specifically is only a minor part of the P2Y12 signaling pathway. PI3Kγ was not required to maintain homeostatic microglial morphology or their dynamic surveillance in vivo Further, PI3Kγ was not strictly required for P2Y12-dependent microglial responses ex vivo or in vivo, although we did observe subtle deficits in the recruitment of microglial process toward sources of ATP. Finally, PI3Kγ was not required for ocular dominance plasticity, a P2Y12-dependent form of experience-dependent synaptic plasticity that occurs in the developing visual cortex. Overall, our results demonstrate that PI3Kγ is not the major mediator of P2Y12 function in microglia, but may have a role in amplifying or fine-tuning the chemotactic response.

Author Correction: Noradrenergic signaling in the wakeful state inhibits microglial surveillance and synaptic plasticity in the mouse visual cortex.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Noradrenergic signaling in the wakeful state inhibits microglial surveillance and synaptic plasticity in the mouse visual cortex.

Microglia are the brain's resident innate immune cells and also have a role in synaptic plasticity. Microglial processes continuously survey the brain parenchyma, interact with synaptic elements and maintain tissue homeostasis. However, the mechanisms that control surveillance and its role in synaptic plasticity are poorly understood. Microglial dynamics in vivo have been primarily studied in anesthetized animals. Here we report that microglial surveillance and injury response are reduced in awake mice as compared to anesthetized mice, suggesting that arousal state modulates microglial function. Pharmacologic stimulation of ß2-adrenergic receptors recapitulated these observations and disrupted experience-dependent plasticity, and these effects required the presence of ß2-adrenergic receptors in microglia. These results indicate that microglial roles in surveillance and synaptic plasticity in the mouse brain are modulated by noradrenergic tone fluctuations between arousal states and emphasize the need to understand the effect of disruptions of adrenergic signaling in neurodevelopment and neuropathology.

Microglia-mediated synaptic elimination in neuronal development and disease.

It has recently become clear that microglia, the immune cells of the central nervous system, are far more active in the healthy brain than previously thought. Microglia facilitate many stages of brain development by shaping neuronal connectivity via synaptic elimination. Dysfunction of these same processes likely underlies a wide range of neurological and neurodevelopmental disorders.

Cerebellar microglia are dynamically unique and survey Purkinje neurons in vivo.

Microglia are the innate immune cells of the central nervous system and are also important participants in normal development and synaptic plasticity. Here, we demonstrate that the microglia of the mouse cerebellum represent a unique population compared to cortical microglia. Microglia are more sparsely distributed within the cerebellum and have a markedly less ramified morphology compared to their cortical counterparts. Using time-lapse in vivo imaging, we found that these differences in distribution and morphology ultimately lead to decreased parenchymal surveillance by cerebellar microglia. We also observed a novel form of somal motility in cerebellar microglia in vivo, which has not been described in cortical populations. We captured microglial interactions with Purkinje neurons in vivo. Cerebellar microglia interact dynamically with both the dendritic arbors and somas of Purkinje neurons. These findings suggest that cerebellar microglia are physiologically distinct from cortical populations and that these differences may ultimately alter how they could contribute to plasticity and disease processes in the cerebellum. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 78: 627-644, 2018.

Inhibitors of glutamate dehydrogenase block sodium-dependent glutamate uptake in rat brain membranes.

We recently found evidence for anatomic and physical linkages between the astroglial Na(+)-dependent glutamate transporters (GLT-1/EAAT2 and GLAST/EAAT1) and mitochondria. In these same studies, we found that the glutamate dehydrogenase (GDH) inhibitor, epigallocatechin-monogallate (EGCG), inhibits both glutamate oxidation and Na(+)-dependent glutamate uptake in astrocytes. In the present study, we extend this finding by exploring the effects of EGCG on Na(+)-dependent l-[(3)H]-glutamate (Glu) uptake in crude membranes (P2) prepared from rat brain cortex. In this preparation, uptake is almost exclusively mediated by GLT-1. EGCG inhibited l-[(3)H]-Glu uptake in cortical membranes with an IC50 value of 230 µM. We also studied the effects of two additional inhibitors of GDH, hexachlorophene (HCP) and bithionol (BTH). Both of these compounds also caused concentration-dependent inhibition of glutamate uptake in cortical membranes. Pre-incubating with HCP for up to 15 min had no greater effect than that observed with no pre-incubation, showing that the effects occur rapidly. HCP decreased the V max for glutamate uptake without changing the K m, consistent with a non-competitive mechanism of action. EGCG, HCP, and BTH also inhibited Na(+)-dependent transport of d-[(3)H]-aspartate (Asp), a non-metabolizable transporter substrate, and [(3)H]-γ-aminobutyric acid (GABA). In contrast to the forebrain, glutamate uptake in crude cerebellar membranes (P2) is likely mediated by GLAST (EAAT1). Therefore, the effects of these compounds were examined in cerebellar membranes. In this region, none of these compounds had any effect on uptake of either l-[(3)H]-Glu or d-[(3)H]-Asp, but they all inhibited [(3)H]-GABA uptake. Together these studies suggest that GDH is preferentially required for glutamate uptake in forebrain as compared to cerebellum, and GDH may be required for GABA uptake as well. They also provide further evidence for a functional linkage between glutamate transport and mitochondria.