Chronic pregabalin treatment protects against spreading depolarization and alters hippocampal synaptic characteristics in a model of familial hemiplegic migraine-type 1.

Familial hemiplegic migraine type-1 (FHM-1) is a form of migraine with aura caused by mutations in the P/Q-type (Cav2.1) voltage-gated calcium channel. Pregabalin, used clinically in the treatment of chronic pain and epilepsy, inhibits P/Q-type calcium channel activity and recent studies suggest that it may have potential for the treatment of migraine. Spreading Depolarization (SD) is a neurophysiological phenomenon that can occur during migraine with aura by propagating a wave of silenced neuronal function through cortex and sometimes subcortical brain structures. Here, utilizing an optogenetic stimulation technique optimized to allow for non-invasive initiation of cortical SD, we demonstrate that chronic pregabalin administration [12 mg/kg/day (s.c.)] in vivo increased the threshold for cortical spreading depolarization in transgenic mice harboring the clinically-relevant Cav2.1S218L mutation (S218L). In addition, chronic pregabalin treatment limited subcortical propagation of recurrent spreading depolarization events to the striatum and hippocampus in both wild-type and S218L mice. To examine contributing underlying mechanisms of action of chronic pregabalin, we performed whole-cell patch-clamp electrophysiology in CA1 neurons in ex vivo brain slices from mice treated with chronic pregabalin vs vehicle. In WT mice, chronic pregabalin produced a decrease in spontaneous excitatory postsynaptic current (sEPSC) amplitude with no effect on frequency. In contrast, in S218L mice chronic pregabalin produced an increase in sEPSC amplitude and decreased frequency. These electrophysiological findings suggest that in FHM-1 mice chronic pregabalin acts through both pre- and post-synaptic mechanisms in CA1 hippocampal neurons to elicit FHM-1 genotype-specific inhibitory action. The results highlight the potential of chronic pregabalin to limit recurrent SD to subcortical brain structures during pathophysiological events in both the genetically-normal and FHM-1 brain. The work further provides insights into FHM-1 pathophysiology and the potential for chronic pregabalin treatment to prevent SD in migraineurs.

Pannexin-1 opening in neuronal edema causes cell death but also leads to protection via increased microglia contacts.

Neuronal swelling during cytotoxic edema is triggered by Na+ and Cl- entry and is Ca2+ independent. However, the causes of neuronal death during swelling are unknown. Here, we investigate the role of large-conductance Pannexin-1 (Panx1) channels in neuronal death during cytotoxic edema. Panx1 channel inhibitors reduce and delay neuronal death in swelling triggered by voltage-gated Na+ entry with veratridine. Neuronal swelling causes downstream production of reactive oxygen species (ROS) that opens Panx1 channels. We confirm that ROS activates Panx1 currents with whole-cell electrophysiology and find scavenging ROS is neuroprotective. Panx1 opening and subsequent ATP release attract microglial processes to contact swelling neurons. Depleting microglia using the CSF1 receptor antagonist PLX3397 or blocking P2Y12 receptors exacerbates neuronal death, suggesting that the Panx1-ATP-dependent microglia contacts are neuroprotective. We conclude that cytotoxic edema triggers oxidative stress in neurons that opens Panx1 to trigger death but also initiates neuroprotective feedback mediated by microglia contacts.

Age-dependent gray matter demyelination is associated with leptomeningeal neutrophil accumulation.

People living with multiple sclerosis (MS) experience episodic CNS white matter lesions instigated by autoreactive T cells. With age, patients with MS show evidence of gray matter demyelination and experience devastating nonremitting symptomology. What drives progression is unclear and studying this has been hampered by the lack of suitable animal models. Here, we show that passive experimental autoimmune encephalomyelitis (EAE) induced by an adoptive transfer of young Th17 cells induced a nonremitting clinical phenotype that was associated with persistent leptomeningeal inflammation and cortical pathology in old, but not young, SJL/J mice. Although the quantity and quality of T cells did not differ in the brains of old versus young EAE mice, an increase in neutrophils and a decrease in B cells were observed in the brains of old mice. Neutrophils were also found in the leptomeninges of a subset of progressive MS patient brains that showed evidence of leptomeningeal inflammation and subpial cortical demyelination. Taken together, our data show that while Th17 cells initiate CNS inflammation, subsequent clinical symptoms and gray matter pathology are dictated by age and associated with other immune cells, such as neutrophils.

Hyperexcitable superior colliculus and fatal brainstem spreading depolarization in a model of Sudden Unexpected Death in Epilepsy.

Cardiorespiratory arrest and death in mouse models of sudden unexpected death in epilepsy occur when spreading depolarization is triggered by cortical seizures and then propagates to the brainstem. However, the critical brain regions and the specific changes required to allow spreading depolarization to propagate to the brainstem under the relatively rare circumstances leading to a fatal seizure are unknown. We previously found that following cortical seizure-inducing electrical stimulation, spreading depolarization could occur in both the superior and inferior colliculi in Cacna1aS218L mice, but was never observed in wild-type animals or following non-seizure-inducing stimuli in Cacna1aS218L mice. Here, we show that optogenetic stimulation of the superior/inferior colliculi in Cacna1aS218L mice induces severe seizures, and resulting spreading depolarization in the superior/inferior colliculi that propagates to the brainstem and correlates with the respiratory arrest followed by cardiac arrest. Further, we show that neurons of the superior colliculus in Cacna1aS218L mice exhibit hyperexcitable properties that we propose underlie a distinct susceptibility to spreading depolarization. Our data suggest that the susceptibility of the superior colliculus to elicit fatal spreading depolarization is a result of either genetic or seizure-related alterations within the superior colliculus that may involve changes to structure, connectivity and/or excitability.

Gut microbes shape microglia and cognitive function during malnutrition.

Fecal-oral contamination promotes malnutrition pathology. Lasting consequences of early life malnutrition include cognitive impairment, but the underlying pathology and influence of gut microbes remain largely unknown. Here, we utilize an established murine model combining malnutrition and iterative exposure to fecal commensals (MAL-BG). The MAL-BG model was analyzed in comparison to malnourished (MAL mice) and healthy (CON mice) controls. Malnourished mice display poor spatial memory and learning plasticity, as well as altered microglia, non-neuronal CNS cells that regulate neuroimmune responses and brain plasticity. Chronic fecal-oral exposures shaped microglial morphology and transcriptional profile, promoting phagocytic features in MAL-BG mice. Unexpectedly, these changes occurred independently from significant cytokine-induced inflammation or blood-brain barrier (BBB) disruption, key gut-brain pathways. Metabolomic profiling of the MAL-BG cortex revealed altered polyunsaturated fatty acid (PUFA) profiles and systemic lipoxidative stress. In contrast, supplementation with an ω3 PUFA/antioxidant-associated diet (PAO) mitigated cognitive deficits within the MAL-BG model. These findings provide valued insight into the malnourished gut microbiota-brain axis, highlighting PUFA metabolism as a potential therapeutic target.

Location Matters: Navigating Regional Heterogeneity of the Neurovascular Unit.

The neurovascular unit (NVU) of the brain is composed of multiple cell types that act synergistically to modify blood flow to locally match the energy demand of neural activity, as well as to maintain the integrity of the blood-brain barrier (BBB). It is becoming increasingly recognized that the functional specialization, as well as the cellular composition of the NVU varies spatially. This heterogeneity is encountered as variations in vascular and perivascular cells along the arteriole-capillary-venule axis, as well as through differences in NVU composition throughout anatomical regions of the brain. Given the wide variations in metabolic demands between brain regions, especially those of gray vs. white matter, the spatial heterogeneity of the NVU is critical to brain function. Here we review recent evidence demonstrating regional specialization of the NVU between brain regions, by focusing on the heterogeneity of its individual cellular components and briefly discussing novel approaches to investigate NVU diversity.

Gamma frequency activation of inhibitory neurons in the acute phase after stroke attenuates vascular and behavioral dysfunction.

Alterations in gamma oscillations occur in several neurological disorders, and the entrainment of gamma oscillations has been recently proposed as a treatment for neurodegenerative disease. Optogenetic stimulation enhances recovery in models of stroke when applied weeks after injury; however, the benefits of acute brain stimulation have not been investigated. Here, we report beneficial effects of gamma-frequency modulation in the acute phase, within 1 h, after stroke. Transgenic VGAT-ChR2 mice are subject to awake photothrombotic stroke in an area encompassing the forelimb sensory and motor cortex. Optogenetic stimulation at 40 Hz in the peri-infarct zone recovers neuronal activity 24 h after stroke in motor and parietal association areas, as well as blood flow over the first week after stroke. Stimulation significantly reduces lesion volume and improves motor function. Our results suggest that acute-phase modulation of cortical oscillatory dynamics may serve as a target for neuroprotection against stroke.

Immunometabolism in the Brain: How Metabolism Shapes Microglial Function.

Immune cells react to their environment by flexibly reprogramming intracellular metabolic pathways that subsequently alter immune function, in a process called immunometabolism. However, in the CNS, the impact of metabolic reprogramming on microglia, neuroinflammation, and subsequently on brain function is poorly understood. As brain-resident macrophages, microglia are the CNS immune effectors and share similarities with peripheral immune cells. New tools for studying immunometabolism now allow the analysis of bioenergetic regulation with cellular resolution and, as a result, have uncovered previously unappreciated roles for microglial immunometabolism in shaping neuroinflammation. This review highlights evidence that microglia metabolism adapts to changes in brain energy homeostasis and that metabolic reprogramming regulates microglial polarization, thereby impacting pathological inflammatory responses in the brain.

Microglial metabolic flexibility supports immune surveillance of the brain parenchyma.

Microglia are highly motile cells that continuously monitor the brain environment and respond to damage-associated cues. While glucose is the main energy substrate used by neurons in the brain, the nutrients metabolized by microglia to support surveillance of the parenchyma remain unexplored. Here, we use fluorescence lifetime imaging of intracellular NAD(P)H and time-lapse two-photon imaging of microglial dynamics in vivo and in situ, to show unique aspects of the microglial metabolic signature in the brain. Microglia are metabolically flexible and can rapidly adapt to consume glutamine as an alternative metabolic fuel in the absence of glucose. During insulin-induced hypoglycemia in vivo or in aglycemia in acute brain slices, glutaminolysis supports the maintenance of microglial process motility and damage-sensing functions. This metabolic shift sustains mitochondrial metabolism and requires mTOR-dependent signaling. This remarkable plasticity allows microglia to maintain their critical surveillance and phagocytic roles, even after brain neuroenergetic homeostasis is compromised.

Nanoscale Surveillance of the Brain by Microglia via cAMP-Regulated Filopodia.

Microglia, the brain's immune cells, maintain homeostasis and sense pathological changes by continuously surveying the parenchyma with highly motile large processes. Here, we demonstrate that microglia also use thin actin-dependent filopodia that allow fast nanoscale sensing within discrete regions. Filopodia are distinct from large processes by their size, speed, and regulation mechanism. Increasing cyclic AMP (cAMP) by activating norepinephrine Gs-coupled receptors, applying nitric oxide, or inhibiting phosphodiesterases rapidly increases filopodia but collapses large processes. Alternatively, Gi-coupled P2Y12 receptor activation collapses filopodia but triggers large processes extension with bulbous tips. Similar control of cytoskeletal dynamics and microglial morphology by cAMP is observed in ramified primary microglia, suggesting that filopodia are intrinsically generated sensing structures. Therefore, nanoscale surveillance of brain parenchyma by microglia requires localized cAMP increases that drive filopodia formation. Shifting intracellular cAMP levels controls the polarity of microglial responses to changes in brain homeostasis and alters the scale of immunosurveillance.

CaV 3.2 drives sustained burst-firing, which is critical for absence seizure propagation in reticular thalamic neurons.

OBJECTIVE: Genetic alterations have been identified in the CACNA1H gene, encoding the CaV 3.2 T-type calcium channel in patients with absence epilepsy, yet the precise mechanisms relating to seizure propagation and spike-wave-discharge (SWD) pacemaking remain unknown. Neurons of the thalamic reticular nucleus (TRN) express high levels of CaV 3.2 calcium channels, and we investigated whether a gain-of-function mutation in the Cacna1h gene in Genetic Absence Epilepsy Rats from Strasbourg (GAERS) contributes to seizure propagation and pacemaking in the TRN. METHODS: Pathophysiological contributions of CaV 3.2 calcium channels to burst firing and absence seizures were assessed in vitro using acute brain slice electrophysiology and quantitative real-time polymerase chain reaction (PCR) and in vivo using free-moving electrocorticography recordings. RESULTS: TRN neurons from GAERS display sustained oscillatory burst-firing that is both age- and frequency-dependent, occurring only in the frequencies overlapping with GAERS SWDs and correlating with the expression of a CaV 3.2 mutation-sensitive splice variant. In vivo knock-down of CaV 3.2 using direct thalamic injection of lipid nanoparticles containing CaV 3.2 dicer small interfering (Dsi) RNA normalized TRN burst-firing, and in free-moving GAERS significantly shortened seizures. SIGNIFICANCE: This supports a role for TRN CaV 3.2 T-type channels in propagating thalamocortical network seizures and setting the pacemaking frequency of SWDs.

3DMorph Automatic Analysis of Microglial Morphology in Three Dimensions from Ex Vivo and In Vivo Imaging.

Microglia are dynamic immune cells of the central nervous system, and their morphology is commonly used as a readout of cellular function. However, current morphological analysis techniques rely on either tracing of cells or two-dimensional projection analysis, which are time-consuming, subject to bias, and may ignore important three-dimensional (3D) information. Therefore, we have created 3DMorph, a MATLAB-based script that analyzes microglial morphology from 3D data. The program initially requires input of threshold levels, cell size expectations, and preferred methods of skeletonization. This makes 3DMorph easily scalable and adaptable to different imaging parameters or cell types. After these settings are defined, the program is completely automatic and can batch process files without user input. Output data includes cell volume, territorial volume, branch length, number of endpoints and branch points, and average distance between cells. We show that 3DMorph is accurate compared to manual tracing, with significantly decreased user input time. Importantly, 3DMorph is capable of processing in vivo microglial morphology, as well as other 3D branching cell types, from mouse cranial windows or acute hippocampal slices. Therefore, we present a novel, user-friendly, scalable, and semiautomatic method of analyzing cell morphology in 3 dimensions. This method should improve the accuracy of cell measurements, remove user bias between conditions, increase reproducibility between experimenters and labs, and reduce user input time. We provide this open source code on GitHub so that it is free and accessible to all investigators.

P2X receptor channels in chronic pain pathways.

Chronic pain is a highly prevalent debilitating condition for which treatment options remain limited for many patients. Ionotropic ATP signalling through excitatory and calcium-permeable P2X receptor channels is now rightfully considered as a critical player in pathological pain generation and maintenance; therefore, their selective targeting represents a therapeutic opportunity with promising yet untapped potential. Recent advances in the structural, functional and pharmacological characterization of rodent and human ATP-gated P2X receptor channels have shed brighter light on the role of specific subtypes in the pathophysiology of chronic inflammatory, neuropathic or cancer pain. Here, we will review the contribution of P2X3, P2X4 and P2X7 receptors to chronic pain and discuss the opportunities and challenges associated with the pharmacological manipulation of their function. LINKED ARTICLES: This article is part of a themed section on Recent Advances in Targeting Ion Channels to Treat Chronic Pain. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.12/issuetoc.

Microglial modulation of neuronal activity in the healthy brain.

Investigations on the role of microglia in the brain have traditionally been focused on their contributions to disease states. However, recent observations have now convincingly shown that microglia in the healthy brain are not passive bystanders, but instead play a critical role in both central nervous system development and homeostasis of synaptic circuits in the adult. Here, we review the various mechanisms by which microglia impact neuronal communication in the healthy adult brain, both by sensing nearby synaptic responses and by actively modulating neuronal function. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 78: 593-603, 2018.

The cost of communication in the brain.

Imaging ATP in axons reveals that they rely on glucose from the blood and lactate produced by glial cells as sources of energy.

Ca2+ transients in astrocyte fine processes occur via Ca2+ influx in the adult mouse hippocampus.

Astrocytes display complex morphologies with an array of fine extensions extending from the soma and the primary thick processes. Until the use of genetically encoded calcium indicators (GECIs) selectively expressed in astrocytes, Ca2+ signaling was only examined in soma and thick primary processes of astrocytes where Ca2+ -sensitive fluorescent dyes could be imaged. GECI imaging in astrocytes revealed a previously unsuspected pattern of spontaneous Ca2+ transients in fine processes that has not been observed without chronic expression of GECIs, raising potential concerns about the effects of GECI expression. Here, we perform two-photon imaging of Ca2+ transients in adult CA1 hippocampal astrocytes using a new single-cell patch-loading strategy to image Ca2+ -sensitive fluorescent dyes in the cytoplasm of fine processes. We observed that astrocyte fine processes exhibited a high frequency of spontaneous Ca2+ transients whereas astrocyte soma rarely showed spontaneous Ca2+ oscillations similar to previous reports using GECIs. We exploited this new approach to show these signals were independent of neuronal spiking, metabotropic glutamate receptor (mGluR) activity, TRPA1 channels, and L- or T-type voltage-gated calcium channels. Removal of extracellular Ca2+ almost completely and reversibly abolished the spontaneous signals while IP3 R2 KO mice also exhibited spontaneous and compartmentalized signals, suggesting they rely on influx of extracellular Ca2+ . The Ca2+ influx dependency of the spontaneous signals in patch-loaded astrocytes was also observed in astrocytes expressing GCaMP3, further highlighting the presence of Ca2+ influx pathways in astrocytes. The mechanisms underlying these localized Ca2+ signals are critical for understanding how astrocytes regulate important functions in the adult brain. GLIA 2016;64:2093-2103.

Post-translational regulation of P2X receptor channels: modulation by phospholipids.

P2X receptor channels mediate fast excitatory signaling by ATP and play major roles in sensory transduction, neuro-immune communication and inflammatory response. P2X receptors constitute a gene family of calcium-permeable ATP-gated cation channels therefore the regulation of P2X signaling is critical for both membrane potential and intracellular calcium homeostasis. Phosphoinositides (PIPn) are anionic signaling phospholipids that act as functional regulators of many types of ion channels. Direct PIPn binding was demonstrated for several ligand- or voltage-gated ion channels, however no generic motif emerged to accurately predict lipid-protein binding sites. This review presents what is currently known about the modulation of the different P2X subtypes by phospholipids and about critical determinants underlying their sensitivity to PIPn levels in the plasma membrane. All functional mammalian P2X subtypes tested, with the notable exception of P2X5, have been shown to be positively modulated by PIPn, i.e., homomeric P2X1, P2X2, P2X3, P2X4, and P2X7, as well as heteromeric P2X1/5 and P2X2/3 receptors. Based on various results reported on the aforementioned subtypes including mutagenesis of the prototypical PIPn-sensitive P2X4 and PIPn-insensitive P2X5 receptor subtypes, an increasing amount of functional, biochemical and structural evidence converges on the modulatory role of a short polybasic domain located in the proximal C-terminus of P2X subunits. This linear motif, semi-conserved in the P2X family, seems necessary and sufficient for encoding direct modulation of ATP-gated channels by PIPn. Furthermore, the physiological impact of the regulation of ionotropic purinergic responses by phospholipids on pain pathways was recently revealed in the context of native crosstalks between phospholipase C (PLC)-linked metabotropic receptors and P2X receptor channels in dorsal root ganglion sensory neurons and microglia.

Inhibition of P2X4 function by P2Y6 UDP receptors in microglia.

ATP-gated P2X4 receptor channels expressed in spinal microglia actively participate in central sensitization, making their functional regulation a key process in chronic pain pathologies. P2Y6 metabotropic Gq -coupled receptors, also expressed in microglia, are involved in the initial response to nerve injury, triggering phagocytosis upon activation by UDP. It has been reported recently that expression of both P2X4 and P2Y6 is upregulated in activated microglia following nerve injury. We show here, in resting as well as LPS-activated primary microglia, that P2Y6 decreases P2X4-mediated calcium entry and inhibits the dilation of P2X4 channels into a large-conductance pore measured with a YO-PRO-1 uptake assay. Furthermore, P2Y6 activation modulates the ATP-dependent migration of microglia, a process likely involved in their shift from migratory to phagocytic phenotype. Reconstituting the P2X4-P2Y6 interaction in recombinant systems shows that P2Y6 activation decreases P2X4 current amplitude, activation and desensitization rates, and reduces P2X4 channel permeability to the large cation NMDG(+) . Phospholipase C-mediated hydrolysis of the phosphoinositide PI(4,5)P2 , a necessary cofactor for P2X4 channel function, underlies this inhibitory crosstalk. As extracellular levels of both ATP and UDP are increased in the spinal cord following nerve injury, the control of P2X4 activity by P2Y6 might play a critical role in regulating neuropathic pain-inducing microglial responses.

Dual effects of daily FTY720 on human astrocytes in vitro: relevance for neuroinflammation.

BACKGROUND: FTY720 (fingolimod, Gilenya) is a daily oral therapy for multiple sclerosis that readily accesses the central nervous system (CNS). FTY720 is a structural analog to the sphingolipid sphingosine-1-phosphate (S1P) and is a cognate ligand for the S1P G-protein coupled receptors (S1PR). Studies in experimental autoimmune encephalomyelitis using mice with conditionally deleted S1P1R from astrocytes indicate that one beneficial effect of FTY720 in this model is via downregulating external receptors, which inhibits responses induced by the natural ligand. Another proposed effect of FTY720 on neuroinflammation is its ability to maintain persistent signaling in cells via internalized S1P1R resulting in functional responses that include suppressing intracellular calcium release. We used human fetal astrocytes to investigate potential dual inhibitory- and function-inducing effects of daily FTY720 on responses relevant to neuroinflammation. For the inhibitory effects, we used signaling and proliferation induced by the natural ligand S1P. For the function-inducing responses, we measured inhibition of intracellular calcium release stimulated by the proinflammatory cytokine, interleukin (IL)-1ß. METHODS: Astrocytes derived from human fetal CNS specimens and maintained in dissociated cultures were exposed to 100 nM of the biologically active form of FTY720 over a dosing regimen that ranged from a single exposure (with or without washout after 1 h) to daily exposures up to 5 days. Responses measured include: phosphorylation of extracellular-signal-regulated kinases (pERK1/2) by Western blotting, Ki-67 immunolabeling for cell proliferation, IL-1ß-induced calcium release by ratiometric fluorescence, and cytokine/chemokine (IL-6, CXCL10) secretions by ELISA. RESULTS: We observed that a single addition of FTY720 inhibited subsequent S1PR ligand-induced pERK1/2 signaling for >24 h. Daily FTY720 treatments (3-5 days) maintained this effect together with a loss of proliferative responses to the natural ligand S1P. Repeated FTY720 dosing concurrently maintained a functional cell response as measured by the inhibition of intracellular calcium release when stimulated by the cytokine IL-1ß. Recurrent FTY720 treatments did not inhibit serum- or IL-1ß-induced pERK1/2. The secretions of IL-6 and CXCL10 in response to IL-1ß were unaffected by FTY720 treatment(s). CONCLUSION: Our results indicate that daily FTY720 exposures may regulate specific neuroinflammatory responses by desensitizing astrocytes to external S1PR stimuli while sustaining cellular influences that are independent of new surface S1PR activation.