Glutamate receptors and white matter stroke.

White matter (WM) damage during ischemia occurs at multiple sites including myelin, oligodendrocytes, astrocytes and axons. A major driver of WM demise is excitoxicity as a consequence of excessive glutamate release by vesicular and non-vesicular mechanisms from axons and glial cells. This results in over-activation of ionotropic glutamate receptors (GluRs) profusely expressed by all cell compartments in WM. Thus, blocking excitotoxicity in WM with selective antagonists of those receptors has a potential therapeutic value. The significance of WM GluR expression for WM stroke injury is the focus of this review, and we will examine the role of GluRs in injury to myelin, oligodendrocytes, astrocytes and the axon cylinder.

Vesicular glutamate release from central axons contributes to myelin damage.

The axon myelin sheath is prone to injury associated with N-methyl-D-aspartate (NMDA)-type glutamate receptor activation but the source of glutamate in this context is unknown. Myelin damage results in permanent action potential loss and severe functional deficit in the white matter of the CNS, for example in ischemic stroke. Here, we show that in rats and mice, ischemic conditions trigger activation of myelinic NMDA receptors incorporating GluN2C/D subunits following release of axonal vesicular glutamate into the peri-axonal space under the myelin sheath. Glial sources of glutamate such as reverse transport did not contribute significantly to this phenomenon. We demonstrate selective myelin uptake and retention of a GluN2C/D NMDA receptor negative allosteric modulator that shields myelin from ischemic injury. The findings potentially support a rational approach toward a low-impact prophylactic therapy to protect patients at risk of stroke and other forms of excitotoxic injury.

White matter tauopathy: Transient functional loss and novel myelin remodeling.

Early white matter (WM) changes are common in dementia and may contribute to functional decline. We here examine this phenomenon in an induced dementia model for the first time. We report a novel and selective form of myelin injury as the first manifestation of tauopathy in the adult central nervous system. Myelin pathology rapidly followed the induction of a P301 tau mutation associated with fronto-temporal dementia in humans (rTG4510 line). Damage involved focal disruption of the ad-axonal myelin lamella and internal oligodendrocyte tongue process, followed by myelin remodeling with features of re-myelination that included myelin thinning and internodal shortening. The evolution of the re-myelinated phenotype was complete in the molecular layer of the dentate gyrus after 1 month and in the optic nerve (ON) after 9 months of transgene induction and proceeded in the absence of actual demyelination, reactive glial changes or inflammatory response. The initial rapid myelin pathology was associated with loss of WM function and performance decline in a novel recognition test and both these effects largely reversed during the myelin re-modeling phase. The initial phase of myelin injury was accompanied by disruption of the vesicle population present in the axoplasm of hippocampal and ON axons. Axoplasmic vesicle release is significant for the regulation of myelin plasticity and disruption of this pathway may underlie the myelin damage and remodeling evoked by tauopathy. WM dysfunction early in tauopathy will disorder neural circuits, the current findings suggest this event may make a significant contribution to early clinical deficit in dementia.

An alternative surgical approach reduces variability following filament induction of experimental stroke in mice.

Animal models are essential for understanding the pathology of stroke and investigating potential treatments. However, in vivo stroke models are associated, particularly in mice, with high variability in lesion volume. We investigated whether a surgical refinement where reperfusion is not reliant on the Circle of Willis reduced outcome variability. Mice underwent 60 min of transient middle cerebral artery occlusion avoiding ligation of the external carotid artery. During reperfusion, the common carotid artery was either ligated (standard approach), or it was repaired to allow re-establishment of blood flow through the common carotid artery. All mice underwent MRI scanning for assessment of infarct volume, apparent diffusion coefficient and fractional anisotropy, along with terminal assessment of infarct volume by 2,3,5-triphenyltetrazolium chloride (TTC) staining. Repairing the common carotid artery following middle cerebral artery occlusion enhanced reperfusion (P<0.01) and reduced the variability seen in both total (histological analysis, P=0.008; T2-weighted MRI, P=0.015) and core (diffusion tensor MRI, P=0.043) lesion volume. Avoiding external carotid artery ligation may improve animal wellbeing, through reduced weight loss, while using an alternative surgical approach that enabled reperfusion through the common carotid artery decreased the variability in lesion volume seen within groups.

The Leukocentric Theory of Neurological Disorder: A Manifesto.

Approximately half of the human brain is composed of white matter (WM), a specialized tissue housing the axonal projection of neurons and their necessary supporting glial cells. Axons course long distances from their parent soma, have a delicate structure, large surface area and in many cases are dependent upon a uniquely close morphological arrangement with myelinating oligodendrocyte partners; all factors that may predispose them to injury and disease. WM damage is central to a range of well-characterized disorders including multiple sclerosis and spinal cord injury and is also makes a significant contribution to disorders often considered to be largely focused in gray matter; for example, in stroke where ~49% of injury by volume is located in WM. In addition, advances in brain imaging have revealed early, often prodromal, changes in WM structure in most forms of neurodegeneration including Alzheimer's, Huntingdon's and Parkinson's diseases as well as during normal cognitive decline and a variety of behavioral conditions. The significance of the early WM changes for the etiology of these diseases is largely unknown. Subtle, early changes in synaptic function may produce the prodromal WM changes evident in imaging, or WM and gray mater structures may undergo simultaneous reactions to the underlying disease process. However, there are rational mechanisms for the transmission of pathology from WM to gray matter and this article suggests an alternative hypothesis: that WM pathology precedes and to some extent is causal of synaptic dysfunction in many common neurological disorders. Neurological disorders that have their origin or their principle lesion in WM are here defined as "leukopathologies".

Plasmalemmal and mitochondrial Na(+) -Ca(2+) exchange in neuroglia.

In the absence of the electrical signaling for which neurons are so highly specialized, GLIA rely on the slow propagation of ionic signals to mediate network events such as Ca(2+) and Na(+) waves. Glia differ from neurons in another important way, they are replete with a high density of ionic-transport proteins that are essential for them to fulfil their basic functions as guardians of the intra and extra-cellular milieux. Both the signaling and the homeostatic properties of glial cells are therefore particularly dependent upon the regulation of the two principle physiological metal cations, Ca(2+) and Na(+) . For both ions, glia express high-affinity/low capacity ATP-fuelled pumps that can rapidly move small numbers of ions against an electro-chemical gradient. For both Ca(2+) and Na(+) regulation, a single transporter family, the Na(+) -Ca(2+) exchanger (NCX), is used to maintain cellular ion homeostasis over the longer term and under conditions of prolonged or acute ionic dysregulation in astrocytes, oligodendroglia and microglia. Our understanding of glial NCX, both plasmalemmal and mitochondrial, is undergoing the kind of transformation that our understanding of glial cells, in general, has undergone in recent decades. These exchange proteins are becoming increasingly recognized for their essential roles in intracellular homeostasis while their signaling functions are starting to come to light. This review summarizes these key aspects and highlights the many areas where work has yet to begin in this rapidly evolving field. GLIA 2016;64:1646-1654.

Phenotype overlap in glial cell populations: astroglia, oligodendroglia and NG-2(+) cells.

The extent to which NG-2(+) cells form a distinct population separate from astrocytes is central to understanding whether this important cell class is wholly an oligodendrocyte precursor cell (OPC) or has additional functions akin to those classically ascribed to astrocytes. Early immuno-staining studies indicate that NG-2(+) cells do not express the astrocyte marker GFAP, but orthogonal reconstructions of double-labeled confocal image stacks here reveal a significant degree of co-expression in individual cells within post-natal day 10 (P10) and adult rat optic nerve (RON) and rat cortex. Extensive scanning of various antibody/fixation/embedding approaches identified a protocol for selective post-embedded immuno-gold labeling. This first ultrastructural characterization of identified NG-2(+) cells revealed populations of both OPCs and astrocytes in P10 RON. NG-2(+) astrocytes had classic features including the presence of glial filaments but low levels of glial filament expression were also found in OPCs and myelinating oligodendrocytes. P0 RONs contained few OPCs but positively identified astrocytes were observed to ensheath pre-myelinated axons in a fashion previously described as a definitive marker of the oligodendrocyte lineage. Astrocyte ensheathment was also apparent in P10 RONs, was absent from developing nodes of Ranvier and was never associated with compact myelin. Astrocyte processes were also shown to encapsulate some oligodendrocyte somata. The data indicate that common criteria for delineating astrocytes and oligodendroglia are insufficiently robust and that astrocyte features ascribed to OPCs may arise from misidentification.

HCO3(-)-independent pH regulation in astrocytes in situ is dominated by V-ATPase.

The mechanisms of HCO3(-)-independent intracellular pH (pHi) regulation were examined in fibrous astrocytes within isolated neonatal rat optic nerve (RON) and in cultured cortical astrocytes. In agreement with previous studies, resting pHi in cultured astrocytes was 6.82 ± 0.06 and inhibition of the V-ATPase H(+) pump by Cl(-) removal or via the selective inhibitor bafilomycin had only a small effect upon resting pHi and recovery following an acid load. In contrast, resting pHi in RON astrocytes was 7.10 ± 0.04, significantly less acidic than that in cultured cells (p < 0.001), and responded to inhibition of V-ATPase with profound acidification to the 6.3-6.5 range. Fluorescent immuno-staining and immuno-gold labeling confirmed the presence V-ATPase in the cell membrane of RON astrocyte processes and somata. Using ammonia pulse recovery, pHi recovery in RON astrocyte was achieved largely via V-ATPase with sodium-proton exchange (NHE) playing a minor role. The findings indicate that astrocytes in a whole-mount preparation such as the optic nerve rely to a greater degree upon V-ATPase for HCO3(-)-independent pHi regulation than do cultured astrocytes, with important functional consequences for the regulation of pH in the CNS.

Ischemic tolerance in pre-myelinated white matter: the role of astrocyte glycogen in brain pathology.

In isolated white matter, ischemic tolerance changes dramatically in the period immediately before the onset of myelination. In the absence of an extrinsic energy source, postnatal day 0 to 2 (P0 to P2) white matter axons are here shown to maintain excitability for over twice as long as axons >P2, a differential that was dependent on glycogen metabolism. Prolonged withdrawal of extrinsic energy supply tended to spare axons in zones around astrocytes, which are shown to be the sole repository for glycogen particles in developing white matter. Analysis of mitochondrial volume fraction revealed that neither axons nor astrocytes had a low metabolic rate in neonatal white matter, while oligodendroglia at older ages had an elevated metabolism. The astrocyte population is established early in neural development, and exhibits reduced cell density as maturation progresses and white matter expands. The findings show that this event establishes the necessary conditions for ischemia sensitivity in white matter and indicates that astrocyte proximity may be significant for the survival of neuronal elements in conditions associated with compromised energy supply.

The dichotomy of memantine treatment for ischemic stroke: dose-dependent protective and detrimental effects.

Excitotoxicity is a major contributor to cell death during the acute phase of ischemic stroke but aggressive pharmacological targeting of excitotoxicity has failed clinically. Here we investigated whether pretreatment with low doses of memantine, within the range currently used and well tolerated for the treatment of Alzheimer's disease, produce a protective effect in stroke. A coculture preparation exposed to modeled ischemia showed cell death associated with rapid glutamate rises and cytotoxic Ca(2+) influx. Cell death was significantly enhanced in the presence of high memantine concentrations. However, low memantine concentrations significantly protected neurons and glia via excitotoxic cascade interruption. Mice were systemically administered a range of memantine doses (0.02, 0.2, 2, 10, and 20 mg/kg/day) starting 24 hours before 60 minutes reversible focal cerebral ischemia and continuing for a 48-hour recovery period. Low dose (0.2 mg/kg/day) memantine treatment significantly reduced lesion volume (by 30% to 50%) and improved behavioral outcomes in stroke lesions that had been separated into either small/striatal or large/striatocortical infarcts. However, higher doses of memantine (20 mg/kg/day) significantly increased injury. These results show that clinically established low doses of memantine should be considered for patients 'at risk' of stroke, while higher doses are contraindicated.

Premyelinated central axons express neurotoxic NMDA receptors: relevance to early developing white-matter injury.

Ischemic-type injury to developing white matter is associated with the significant clinical condition cerebral palsy and with the cognitive deficits associated with premature birth. Premyelinated axons are the major cellular component of fetal white matter and loss of axon function underlies the disability, but the cellular mechanisms producing ischemic injury to premyelinated axons have not previously been described. Injury was found to require longer periods of modelled ischemia than at latter developmental points. Ischemia produced initial hyperexcitability in axons followed by loss of function after Na(+) and Ca(2+) influx. N-methyl-D-aspartate- (NMDA) type glutamate receptor (GluR) agonists potentiated axon injury while antagonists were protective. The NMDA GluR obligatory Nr1 subunit colocalized with markers of small premyelinated axons and expression was found at focal regions of axon injury. Ischemic injury of glial cells present in early developing white matter was NMDA GluR independent. Axons in human postconception week 18 to 23 white matter had a uniform prediameter expansion phenotype and postembedded immuno-gold labelling showed Nr1 subunit expression on the membrane of these axons, demonstrating a shared key neuropathologic feature with the rodent model. Premyelinated central axons therefore express high levels of functional NMDA GluRs that confer sensitivity to ischemic injury.

White matter injury: Ischemic and nonischemic.

Ischemic pathologies of white matter (WM) include a large proportion of stroke and developmental lesions while multiple sclerosis (MS) is the archetype nonischemic pathology. Growing evidence suggests other important diseases including neurodegenerative and psychiatric disorders also involve a significant WM component. Axonal, oligodendroglial, and astroglial damage proceed via distinct mechanisms in ischemic WM and these mechanisms evolve dramatically with maturation. Axons may pass through four developmental stages where the pattern of membrane protein expression influences how the structure responds to ischemia; WM astrocytes pass through at least two and differ significantly in their ischemia tolerance from grey matter astrocytes; oligodendroglia pass through at least three, with the highly ischemia intolerant pre-oligodendrocyte (pre-Oli) stage linking the less sensitive precursor and mature phenotypes. Neurotransmitters play a central role in WM pathology at all ages. Glutamate excitotoxicity in WM has both necrotic and apoptotic components; the latter mediated by intracellular pathways which differ between receptor types. ATP excitotoxicity may be largely mediated by the P2X7 receptor and also has both necrotic and apoptotic components. Interplay between microglia and other cell types is a critical element in the injury process. A growing appreciation of the significance of WM injury for nonischemic neurological disorders is currently stimulating research into mechanisms; with curious similarities being found with those operating during ischemia. A good example is traumatic brain injury, where axonal pathology can proceed via almost identical pathways to those described during acute ischemia.

Neurotransmitter signaling in white matter.

White matter (WM) tracts are bundles of myelinated axons that provide for rapid communication throughout the CNS and integration in grey matter (GM). The main cells in myelinated tracts are oligodendrocytes and astrocytes, with small populations of microglia and oligodendrocyte precursor cells. The prominence of neurotransmitter signaling in WM, which largely exclude neuronal cell bodies, indicates it must have physiological functions other than neuron-to-neuron communication. A surprising aspect is the diversity of neurotransmitter signaling in WM, with evidence for glutamatergic, purinergic (ATP and adenosine), GABAergic, glycinergic, adrenergic, cholinergic, dopaminergic and serotonergic signaling, acting via a wide range of ionotropic and metabotropic receptors. Both axons and glia are potential sources of neurotransmitters and may express the respective receptors. The physiological functions of neurotransmitter signaling in WM are subject to debate, but glutamate and ATP-mediated signaling have been shown to evoke Ca(2+) signals in glia and modulate axonal conduction. Experimental findings support a model of neurotransmitters being released from axons during action potential propagation acting on glial receptors to regulate the homeostatic functions of astrocytes and myelination by oligodendrocytes. Astrocytes also release neurotransmitters, which act on axonal receptors to strengthen action potential propagation, maintaining signaling along potentially long axon tracts. The co-existence of multiple neurotransmitters in WM tracts suggests they may have diverse functions that are important for information processing. Furthermore, the neurotransmitter signaling phenomena described in WM most likely apply to myelinated axons of the cerebral cortex and GM areas, where they are doubtless important for higher cognitive function.

Central axons preparing to myelinate are highly sensitive [corrected] to ischemic injury.

OBJECTIVE: Developing central white matter is subject to ischemic-type injury during the period that precedes myelination. At this stage in maturation, central axons initiate a program of radial expansion and ion channel redistribution. Here we test the hypothesis that during radial expansion axons display heightened ischemic sensitivity, when clusters of Ca(2+) channels decorate future node of Ranvier sites. METHODS: Functionality and morphology of central axons and glia were examined during and after a period of modeled ischemia. Pathological changes in axons undergoing radial expansion were probed using electrophysiological, quantitative ultrastructural, and morphometric analysis in neonatal rodent optic nerve and periventricular white matter axons studied under modeled ischemia in vitro or after hypoxia-ischemia in vivo. RESULTS: Acute ischemic injury of central axons undergoing initial radial expansion was mediated by Ca(2+) influx through Ca(2+) channels expressed in axolemma clusters. This form of injury operated only in this axon population, which was more sensitive to injury than neighboring myelinated axons, smaller axons yet to initiate radial expansion, astrocytes, or oligodendroglia. A pharmacological strategy designed to protect both small and large diameter premyelinated axons proved 100% protective against acute ischemia studied under modeled ischemia in vitro or after hypoxia-ischemia in vivo. INTERPRETATION: Recent clinical data highlight the importance of axon pathology in developing white matter injury. The elevated susceptibility of early maturing axons to ischemic injury described here may significantly contribute to selective white matter pathology and places these axons alongside preoligodendrocytes as a potential primary target of both injury and therapeutics.

Novel morphological features of developing white matter pericytes and rapid scavenging of reactive oxygen species in the neighbouring endothelia.

Capillary endothelia and pericytes form a close morphological arrangement allowing pericytes to regulate capillary blood flow, in addition to contributing to vascular development and support. Vascular changes associated with oxidative stress are implicated in important pathologies in developing whiter matter, but little is known about the vascular unit in white matter of the appropriate age or how it responds to oxidative stress. We show that the ultrastructural arrangement of post-natal day 10 (P10) capillaries involves the apposition of pericyte somata to the capillary inner basement membrane and penetration of pericyte processes onto the abluminal surface where they form close connections with endothelial cells. Some pericytes have an unusual stellate morphology, extending processes radially from the vessel. Reactive oxygen species (ROS) were monitored with the ROS-sensitive dye 2',7'-dichlorofluorescin (DCF) in the endothelial cells. Exposure to exogenous ROS (100 µm H(2) O(2) or xanthine/xanthine oxidase), evoked an elevation in intracellular ROS that declined to baseline during the ongoing challenge. A second challenge failed to evoke an intracellular ROS rise unless the nerve was rested for > 4 h or exposed to very high levels of exogenous ROS. Exposure to a first ROS challenge prior to loading with DCF also prevented the intracellular ROS rise from a second challenge, proving that dye washout during exposure to ROS is not responsible for the loss of a second response. Perfusion with 30 µm extracellular Ca(2+) or the voltage-gated Ca(2+) antagonist diltiazem partially prevented this rapid scavenging of intracellular ROS, but blocking either catalase or glutathione peroxidase did not. The phenomenon was present over a range of post-natal ages and may contribute to the high ROS-tolerance of endothelial cells and act to limit the release of harmful ROS onto neighbouring pericytes.

Identification of four functional NR3B isoforms in developing white matter reveals unexpected diversity among glutamate receptors.

Functional neurotransmitter receptors are expressed in central white matter, where they mediate ischemic damage to glia and may be involved in cell-cell signalling. In this study, we analysed NMDA receptor NR1, NR2B-C and NR3A-B subunit expression in the brain and optic nerve by molecular cloning. In addition to the canonical forms of NR1 and NR2, four previously unknown NR3B variants, generated by alternative splicing, were identified. The variants encoded for isoforms with deletions of 8/15 amino acids in the N-terminal domain or 200/375 amino acids removing one or three transmembrane domains and part of the C-terminal domain, as compared with the previously characterized NR3B isoform. Co-expression of NR3B isoforms with NR1/NR2A-C modulated the amplitude and Mg(2+)-sensitivity of glutamate responses in a NR2 subunit-dependent fashion, with significant variations in the effects produced by different isoforms. These effects were not the result of reduced surface expression of the receptor complex since all NR3B isoforms reduced surface expression by a similar degree. These data reveal previously uncharacterized regulation of NMDA receptor function by alternative splicing of the NR3B subunit.

Glia as transmitter sources and sensors in health and disease.

Glial cells express a bewildering array of neurotransmitter receptors. To illustrate the complexity of expression, we have assayed non-glutamatergic neurotransmitter receptor mRNA in isolated rat optic nerve, a preparation devoid of neurons and neuronal synapses and from which relatively pure "glial" RNA can be isolated. Of the 44 receptor subunits examined which span the GABA-A, nicotinic, adreno- and glycine receptor families, over three quarters were robustly expressed in this mixed population of white matter glial cells, with several expressed at higher levels than found in control whole brain RNA. In addition to the complexity of glial receptor expression, numerous neurotransmitter release mechanisms have been identified. We have focused on glutamate release from astrocytes, which can occur via at least seven distinct pathways and which is implicated in excitotoxic injury and are neurons and glia. Recent findings suggest that non-glutamatergic receptors can also mediate acute glial injury are also discussed.

Glutamate receptor-mediated ischemic injury of premyelinated central axons.

OBJECTIVE: Ischemic injury of axons is a feature of periventricular leukomalacia, a pathological correlate of cerebral palsy. Recent evidence suggests that axons are damaged before they receive the first layer of compact myelin. Here we examine the cellular mechanisms underlying ischemic-type injury of premyelinated central axons. METHODS: Two-thirds of axons in the postnatal day 10 (P10) rat optic nerve are small premyelinated axons (<0.4microm in diameter), and one-third have undergone radial expansion in preparation for glial contact and the onset of myelination. Compound action potential recording and quantitative electron microscopy were used to examine the effect of modeled ischemia (oxygen-glucose deprivation) upon these two axon populations. Glutamate receptor (GluR) expression was investigated using polymerase chain reaction (PCR) and immunostaining approaches at the confocal light and ultrastructural levels. RESULTS: Oxygen-glucose deprivation produced action potential failure and focal breakdown of the axolemma of small premyelinated axons at sites of contact with oligodendrocyte processes, which were also disrupted. The resulting axon loss was Ca(2+)-dependent, Na(+)- and Cl(-)-independent, and required activation of N-methyl-D-aspartic acid (NMDA) and non-NMDA GluRs. NMDA receptor expression was localized to oligodendrocyte processes at sites of contact with premyelinated axons, in addition to expression within compact myelin. No periaxonal NMDA receptor expression was observed on oligodendrocyte processes ensheathing large premyelinated axons and no protective effect of GluR block was observed in these axons. INTERPRETATION: NMDA receptor-mediated injury to oligodendrocyte processes navigating along small premyelinated axons precedes damage to the underlying axon, a phenomena that is lost following radial expansion and subsequent oligodendrocyte ensheathment.