Ukpong B. Eyo

Neuronal Hyperactivity Recruits Microglial Processes via Neuronal NMDA Receptors and Microglial P2Y12 Receptors after Status Epilepticus

Microglia are highly dynamic immune cells of the CNS and their dynamism is proposed to be regulated by neuronal activities. However, the mechanisms underlying neuronal regulation of microglial dynamism have not been determined. Here, we found an increased number of microglial primary processes in the hippocampus during KA-induced seizure activity. Consistently, global glutamate induced robust microglial process extension toward neurons in both brain slices and in the intact brain in vivo. The mechanism of the glutamate-induced microglial process extension involves the activation of neuronal NMDA receptors, calcium influx, subsequent ATP release, and microglial response through P2Y12 receptors. Seizure-induced increases in microglial process numbers were also dependent on NMDA receptor activation. Finally, we found that P2Y12 KO mice exhibited reduced seizure-induced increases in microglial process numbers and worsened KA-induced seizure behaviors. Our results elucidate the molecular mechanisms underlying microglia–neuron communication that may be potentially neuroprotective in the epileptic brain.

Bidirectional microglia-neuron communication in the healthy brain.

Unlike other resident neural cells that are of neuroectodermal origin, microglia are resident neural cells of mesodermal origin. Traditionally recognized for their immune functions during disease, new roles are being attributed to these cells in the development and maintenance of the central nervous system (CNS) including specific communication with neurons. In this review, we highlight some of the recent findings on the bidirectional interaction between neurons and microglia. We discuss these interactions along two lines. First, we review data that suggest that microglial activity is modulated by neuronal signals, focusing on evidence that (i) neurons are capable of regulating microglial activation state and influence basal microglial activities; (ii) classic neurotransmitters affect microglial behavior; (iii) chemotactic signals attract microglia during acute neuronal injury. Next, we discuss some of the recent data on how microglia signal to neurons. Signaling mechanisms include (i) direct physical contact of microglial processes with neuronal elements; (ii) microglial regulation of neuronal synapse and circuit by fractalkine, complement, and DAP12 signaling. In addition, we discuss the use of microglial depletion strategies in studying the role of microglia in neuronal development and synaptic physiology. Deciphering the mechanisms of bidirectional microglial-neuronal communication provides novel insights in understanding microglial function in both the healthy and diseased brain.

Microglia: Key Elements in Neural Development, Plasticity, and Pathology

A century after Cajal identified a “third element” of the nervous system, many issues have been clarified about the identity and function of one of its major components, the microglia. Here, we review recent findings by microgliologists, highlighting results from imaging studies that are helping provide new views of microglial behavior and function. In vivo imaging in the intact adult rodent CNS has revolutionized our understanding of microglial behaviors in situ and has raised speculation about their function in the uninjured adult brain. Imaging studies in ex vivo mammalian tissue preparations and in intact model organisms including zebrafish are providing insights into microglial behaviors during brain development. These data suggest that microglia play important developmental roles in synapse remodeling, developmental apoptosis, phagocytic clearance, and angiogenesis. Because microglia also contribute to pathology, including neurodevelopmental and neurobehavioral disorders, ischemic injury, and neuropathic pain, promising new results raise the possibility of leveraging microglia for therapeutic roles. Finally, exciting recent work is addressing unanswered questions regarding the nature of microglial-neuronal communication. While it is now apparent that microglia play diverse roles in neural development, behavior, and pathology, future research using neuroimaging techniques will be essential to more fully exploit these intriguing cellular targets for effective therapeutic intervention applied to a variety of conditions.

Microglia and monocytes synergistically promote the transition from acute to chronic pain after nerve injury.

Microglia and peripheral monocytes contribute to hypersensitivity in rodent models of neuropathic pain. However, the precise respective function of microglia and peripheral monocytes has not been investigated in these models. To address this question, here we combined transgenic mice and pharmacological tools to specifically and temporally control the depletion of microglia and monocytes in a mouse model of spinal nerve transection (SNT). We found that although microglia and monocytes are required during the initiation of mechanical allodynia or thermal hyperalgesia, these cells may not be as important for the maintenance of hypersensitivity. Moreover, we demonstrated that either resident microglia or peripheral monocytes are sufficient in gating neuropathic pain after SNT. We propose that resident microglia and peripheral monocytes act synergistically to initiate hypersensitivity and promote the transition from acute to chronic pain after peripheral nerve injury.

Modulation of Microglial Process Convergence Toward Neuronal Dendrites by Extracellular Calcium

Extracellular calcium concentrations in the brain fluctuate during neuronal activities and may affect the behavior of brain cells. Microglia are highly dynamic immune cells of the brain. However, the effects of extracellular calcium concentrations on microglial dynamics have not been investigated. Here, we addressed this question in mouse brain slices and in vivo using two-photon microscopy. We serendipitously found that extracellular calcium reduction induced microglial processes to converge at distinct sites, a phenomenon we termed microglial process convergence (MPCs). Our studies revealed that MPCs target neuronal dendrites independent of neuronal action potential firing and is mediated by ATP release and microglial P2Y12 receptors. These results indicate that microglia monitor and interact with neurons during conditions of cerebral calcium reduction in the normal and diseased brain.

Microglia–Neuron Communication in Epilepsy

Epilepsy has remained a significant social concern and financial burden globally. Current therapeutic strategies are based primarily on neurocentric mechanisms that have not proven successful in at least a third of patients, raising the need for novel alternative and complementary approaches. Recent evidence implicates glial cells and neuroinflammation in the pathogenesis of epilepsy with the promise of targeting these cells to complement existing strategies. Specifically, microglial involvement, as a major inflammatory cell in the epileptic brain, has been poorly studied. In this review, we highlight microglial reaction to experimental seizures, discuss microglial control of neuronal activities, and propose the functions of microglia during acute epileptic phenotypes, delayed neurodegeneration, and aberrant neurogenesis. Future research that would help fill in the current gaps in our knowledge includes epilepsy‐induced alterations in basic microglial functions, neuro–microglial interactions during chronic epilepsy, and microglial contribution to developmental seizures. Studying the role of microglia in epilepsy could inform therapies to better alleviate the disease. GLIA 2016;65:5–18

“P2X7 Receptor Activation Regulates Microglial Cell Death During Oxygen-Glucose Deprivation”

Brain-resident microglia may promote tissue repair following stroke but, like other cells, they are vulnerable to ischemia. Here we identify mechanisms involved in microglial ischemic vulnerability. Using time-lapse imaging of cultured BV2 microglia, we show that simulated ischemia (oxygen-glucose deprivation; OGD) induces BV2 microglial cell death. Removal of extracellular Ca(2+) or application of Brilliant Blue G (BBG), a potent P2X7 receptor (P2X7R) antagonist, protected BV2 microglia from death. To validate and extend these in vitro findings, we assessed parenchymal microglia in freshly isolated hippocampal tissue slices from GFP-reporter mice (CX3CR1(GFP/+)). We confirmed that calcium removal or application of apyrase, an ATP-degrading enzyme, abolished OGD-induced microglial cell death in situ, consistent with involvement of ionotropic purinergic receptors. Indeed, whole cell recordings identified P2X7R-like currents in tissue microglia, and OGD-induced microglial cell death was inhibited by BBG. These pharmacological results were complemented by studies in tissue slices from P2X7R null mice, in which OGD-induced microglia cell death was reduced by nearly half. Together, these results indicate that stroke-like conditions induce calcium-dependent microglial cell death that is mediated in part by P2X7R. This is the first identification of a purinergic receptor regulating microglial survival in living brain tissues. From a therapeutic standpoint, these findings could help direct novel approaches to enhance microglial survival and function following stroke and other neuropathological conditions.

Microglial P2Y12 Receptors Regulate Microglial Activation and Surveillance during Neuropathic Pain

Microglial cells are critical in the pathogenesis of neuropathic pain and several microglial receptors have been proposed to mediate this process. Of these receptors, the P2Y12 receptor is a unique purinergic receptor that is exclusively expressed by microglia in the central nervous system (CNS). In this study, we set forth to investigate the role of P2Y12 receptors in microglial electrophysiological and morphological (static and dynamic) activation during spinal nerve transection (SNT)-induced neuropathic pain in mice. First, we found that a genetic deficiency of the P2Y12 receptor (P2Y12(-/-) mice) ameliorated pain hypersensitivities during the initiation phase of neuropathic pain. Next, we characterised both the electrophysiological and morphological properties of microglia in the superficial spinal cord dorsal horn following SNT injury. We show dramatic alterations including a peak at 3days post injury in microglial electrophysiology while high resolution two-photon imaging revealed significant changes of both static and dynamic microglial morphological properties by 7days post injury. Finally, in P2Y12(-/-) mice, these electrophysiological and morphological changes were ameliorated suggesting roles for P2Y12 receptors in SNT-induced microglial activation. Our results therefore indicate that P2Y12 receptors regulate microglial electrophysiological as well as static and dynamic microglial properties after peripheral nerve injury, suggesting that the microglial P2Y12 receptor could be a potential therapeutic target for the treatment of neuropathic pain.

Effects of oxygen-glucose deprivation on microglial mobility and viability in developing mouse hippocampal tissues.

As brain‐resident immune cells, microglia (MG) survey the brain parenchyma to maintain homeostasis during development and following injury. Research in perinatal stroke, a leading cause of lifelong disability, has implicated MG as targets for therapeutic intervention during stroke. Although MG responses are complex, work in developing rodents suggests that MG limit brain damage after stroke. However, little is known about how energy‐limiting conditions affect MG survival and mobility (motility and migration) in developing brain tissues. Here, we used confocal time‐lapse imaging to monitor MG viability and mobility during hypoxia or oxygen‐glucose deprivation (OGD) in hippocampal tissue slices derived from neonatal GFP‐reporter mice (CX3CR1GFP/+). We found that MG remain viable for at least 6 h of hypoxia but begin to die after 2 h of OGD, while both hypoxia and OGD reduce MG motility. Unexpectedly, some MG retain or recover motility during OGD and can engulf dead cells. Additionally, MG from younger neonates (P2–P3) are more resistant to OGD than those from older ones (P6–P7), indicating increasing vulnerability with developmental age. Finally, transient (2 h) OGD also increases MG death, and although motility is rapidly restored after transient OGD, it remains below control levels for many hours. Together, these results show that MG in neonatal mouse brain tissues are vulnerable to both transient and sustained OGD, and many MG die within hours after onset of OGD. Preventing MG death may, therefore, provide a strategy for promoting tissue restoration after stroke.

Microglial Hv1 proton channel promotes cuprizone-induced demyelination through oxidative damage.

NADPH oxidase (NOX)‐dependent reactive oxygen species (ROS) production in inflammatory cells including microglia plays an important role in demyelination and free radical‐mediated tissue injury in multiple sclerosis (MS). However, the mechanism underlying microglial ROS production and demyelination remains largely unknown. The voltage‐gated proton channel, Hv1, is selectively expressed in microglia and is required for NOX‐dependent ROS generation in the brain. In the present study, we sought to determine the role of microglial Hv1 proton channels in a mouse model of cuprizone‐induced demyelination, a model for MS. Following cuprizone exposure, wild‐type mice presented obvious demyelination, decreased myelin basic protein expression, loss of mature oligodendrocytes, and impaired motor coordination in comparison to mice on a normal chow diet. However, mice lacking Hv1 (Hv1−/−) are partially protected from demyelination and motor deficits compared with those in wild‐type mice. These rescued phenotypes in Hv1−/− mice in cuprizone‐induced demyelination is accompanied by reduced ROS production, ameliorated microglial activation, increased oligodendrocyte progenitor cell (NG2) proliferation, and increased number of mature oligodendrocytes. These results demonstrate that the Hv1 proton channel is required for cuprizone‐induced microglial oxidative damage and subsequent demyelination. Our study suggests that the microglial Hv1 proton channel is a unique target for controlling NOX‐dependent ROS production in the pathogenesis of MS.