Oligodendrocytes from forebrain are highly vulnerable to AMPA/kainate receptor-mediated excitotoxicity.

Little is known of the molecular mechanisms that trigger oligodendrocyte death and demyelination in many acute central nervous system insults. Since oligodendrocytes express functional alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate-type glutamate receptors, we examined the possibility that oligodendrocyte death can be mediated by glutamate receptor overactivation. Oligodendrocytes in primary cultures from mouse forebrain were selectively killed by low concentrations of AMPA, kainate or glutamate, or by deprivation of oxygen and glucose. This toxicity could be blocked by the AMPA/kainate receptor antagonist 6-nitro-7-sulfamoylbenzo(f)quinoxaline-2,3-dione (NBQX). In vivo, differentiated oligodendrocytes in subcortical white matter expressed AMPA receptors and were selectively injured by microstereotaxic injection of AMPA but not NMDA. These data suggest that oligodendrocytes share with neurons a high vulnerability to AMPA/kainate receptor-mediated death, a mechanism that may contribute to white matter injury in CNS disease.

Traumatic neuronal injury in vitro is attenuated by NMDA antagonists.

Pure traumatic neuronal injury was modeled in dispersed neocortical cell cultures derived from fetal mice. A plastic stylet was used to tear the neuronal and glial cell layer; medium oxygen content, pH, and glucose remained unchanged. Adjacent to this local disruption, many neurons developed acute swelling and went on to degenerate over the next day, but glia were relatively spared. If the same mechanical insult was delivered in the presence of the N-methyl-D-aspartate (NMDA) antagonists dextrorphan or D-2-amino-5-phosphonovalerate, resultant neuronal degeneration was markedly reduced. The protective effect of these NMDA antagonists was concentration-dependent between 1 and 100 microM, with EC50 near 10 microM for both compounds. Present findings suggest that endogenous excitatory amino acids may participate significantly in the propagation of central neuronal cell loss in response to a purely mechanical insult.

Oxygen or glucose deprivation-induced neuronal injury in cortical cell cultures is reduced by tetanus toxin.

We examined glutamate-mediated neurotoxicity in cortical cell cultures pretreated with 1-5 micrograms/ml tetanus toxin to attenuate the Ca(2+)-dependent release of neurotransmitters. Efficacy of the tetanus toxin pretreatment was suggested by blockade of electrical burst activity induced by Mg2+ removal and by reduction of glutamate efflux induced by high K+. Tetanus toxin reduced neuronal injury produced by brief exposure to elevated extracellular K+ or to glutamate, situations in which release of endogenous excitatory neurotransmitter is likely to play a role. Furthermore, although glutamate efflux evoked by anoxic conditions may occur largely via Ca(2+)-independent transport, tetanus toxin attenuated both glutamate efflux and neuronal injury following combined oxygen and glucose deprivation. With prolonged exposure periods, the neuroprotective efficacy of tetanus toxin was comparable to that of NMDA receptor antagonists. Presynaptic inhibition of Ca(2+)-dependent glutamate release may be a valuable approach to attenuating hypoxic-ischemic brain injury.

Ampa/kainate receptor activation mediates hypoxic oligodendrocyte death and axonal injury in cerebral white matter.

We developed an in situ model to investigate the hypothesis that AMPA/kainate (AMPA/KA) receptor activation contributes to hypoxic-ischemic white matter injury in the adult brain. Acute coronal brain slices, including corpus callosum, were prepared from adult mice. After exposure to transient oxygen and glucose deprivation (OGD), white matter injury was assessed by electrophysiology and immunofluorescence for oligodendrocytes and axonal neurofilaments. White matter cellular components and the stimulus-evoked compound action potential (CAP) remained stable for 12 hr after preparation. OGD for 30 min resulted in an irreversible loss of the CAP as well as structural disruption of axons and subsequent loss of neurofilament immunofluorescence. OGD also caused widespread oligodendrocyte death, demonstrated by the loss of APC labeling and the gain of pyknotic nuclear morphology and propidium iodide labeling. Blockade of AMPA/KA receptors with 30 microm NBQX or the AMPA-selective antagonist 30 microm GYKI 52466 prevented OGD-induced oligodendrocyte death. Oligodendrocytes also were preserved by the removal of Ca(2+), but not by a blockade of voltage-gated Na(+) channels. The protective action of NBQX was still present in isolated corpus callosum slices. CAP areas and axonal structure were preserved by Ca(2+) removal and partially protected by a blockade of voltage-gated Na(+) channels. NBQX prevented OGD-induced CAP loss and preserved axonal structure. These observations highlight convergent pathways leading to hypoxic-ischemic damage of cerebral white matter. In accordance with previous suggestions, the activation of voltage-gated Na(+) channels contributes to axonal damage. Overactivation of glial AMPA/KA receptors leads to oligodendrocyte death and also plays an important role in structural and functional disruption of axons.

Dendritic spines lost during glutamate receptor activation reemerge at original sites of synaptic contact.

During cerebral ischemia, neurons undergo rapid alterations in dendritic structure consisting of focal swelling and spine loss. We used time-lapse microscopy to determine the fate of dendritic spines that disappeared after brief, sublethal hypoxic or excitotoxic exposures. Dendrite and spine morphology were assessed in cultured cortical neurons expressing yellow fluorescent protein or labeled with the fluorescent membrane tracer, DiI. Neurons exposed to NMDA, kainate, or oxygen-glucose deprivation underwent segmental dendritic beading and loss of approximately one-half of dendritic spines. Most spine loss was observed in regions of local dendritic swelling. Despite widespread loss, spines recovered within 2 hr after termination of agonist exposure or oxygen-glucose deprivation and remained stable over the subsequent 24 hr. Recovery was slower after NMDA than AMPA/kainate receptor activation. Time-lapse fluorescence imaging showed that the vast majority of spines reemerged in the same location from which they disappeared. In addition to spine recovery, elaboration of dendritic filopodia was observed in new locations along the dendritic shaft after dendrite recovery. Spine recovery did not depend on actin polymerization because it was not blocked by application of latrunculin-A, which eliminated filamentous actin staining in spines and blocked spine motility. Throughout spine loss and recovery, presynaptic and postsynaptic elements remained in physical proximity. These results suggest that elimination of dendritic spines is not necessarily associated with loss of synaptic contacts. Rapid reestablishment of dendritic spine synapses in surviving neurons may be a substrate for functional recovery after transient cerebral ischemia.

Amyloid-beta peptides are cytotoxic to oligodendrocytes.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive dementia. Amyloid-beta peptide (Abeta), a 39-43 amino acid peptide derived from beta-amyloid precursor protein, forms insoluble fibrillar aggregates that have been linked to neuronal and vascular degeneration in AD and cerebral amyloid angiopathy. Here we demonstrate that Abeta 1-40 and a truncated fragment, Abeta 25-35, induced death of oligodendrocytes (OLGs) in vitro in a dose-dependent manner with similar potencies. Abeta-induced OLG death was accompanied by nuclear DNA fragmentation, mitochondrial dysfunction, and cytoskeletal disintegration. Abeta activation of redox-sensitive transcription factors NF-kappaB and AP-1 and antioxidant prevention of Abeta-mediated OLG death suggest that oxidative injury contributes to Abeta cytotoxicity in OLGs. Recent demonstration of Abeta deposition and white matter abnormalities in AD implies a potential pathophysiological role for Abeta-mediated cytotoxicity of OLGs in this neurodegenerative disease.

Neuronal free calcium measurement using BTC/AM, a low affinity calcium indicator.

BTC is a low affinity calcium indicator (Kd approximately 7-26 microM) featuring many desirable properties for cellular calcium imaging, including long excitation wavelengths (400/485 nm), low sensitivity to Mg2+, and accuracy of ratiometric measurement [Iatridou H., Foukaraki E., Kuhn M.A., Marcus E.M., Haugland R.P., Katerinopoulos H.E. The development of a new family of intracellular calcium probes. Cell Calcium 1994; 15: 190-198]. To assess the usefulness of this indicator in cultured neurons, we examined properties of BTC and its acetoxymethyl ester, BTC/AM. BTC/AM had substantial calcium-independent fluorescence at all excitation wavelengths. BTC/AM was readily loaded into neurons and was rapidly hydrolysed. There was little dye compartmentalization, as assessed by digitonin lysis, Co2+ quenching of BTC fluorescence and by confocal microscopy. Despite adequate loading, BTC gradually became unresponsive to [Ca2+]i when cultures were examined under routine imaging conditions. This effect was a function of the cumulative fluorescence illumination and could be minimized by attenuating light intensity or duration. Ratio imaging after exposure of neuronal cultures to 1-50 microM ionomycin revealed distinct sensitivity ranges for BTC and Fura-2. BTC reported graded neuronal [Ca2+]i responses to glutamate receptor stimulation with N-methyl-D-aspartate in the range 10-50 microM, whereas Fura-2 did not distinguish between these stimuli. Under appropriate loading and illumination conditions, bath-loaded BTC/AM may be well suited for measurement of moderate to high calcium concentrations in cultured neurons.

Ionic selectivity of low-affinity ratiometric calcium indicators: mag-Fura-2, Fura-2FF and BTC.

Accurate measurement of elevated intracellular calcium levels requires indicators with low calcium affinity and high selectivity. We examined fluorescence spectral properties and ionic specificity of three low-affinity, ratiometric indicators structurally related to Fura-2: mag-Fura-2 (furaptra), Fura-2FF, and BTC. The indicators differed in respect to their excitation wavelengths, affinity for Ca2+ (Kd approximately 20 microM, 6 microM and 12 microM respectively) and selectivity over Mg2+ (Kd approximately 2 mM for mag-Fura-2, > 10 mM for Fura-2FF and BTC). Among the tested indicators, BTC was limited by a modest dynamic range upon Ca2+ binding, susceptibility to photodamage, and sensitivity to alterations in pH. All three indicators bound other metal ions including Zn2+, Cd2+ and Gd3+. Interestingly, only in the case of BTC were spectral differences apparent between Ca2+ and other metal ions. For example, the presence of Zn2+ increased BTC fluorescence 6-fold at the Ca2+ isosbestic point, suggesting that this dye may be used as a fluorescent Zn2+ indicator. Fura-2FF has high specificity, wide dynamic range, and low pH sensitivity, and is an optimal low-affinity Ca2+ indicator for most imaging applications. BTC may be useful if experimental conditions require visible wavelength excitation or sensitivity to other metal ions including Zn2+.

Calcium ionophores can induce either apoptosis or necrosis in cultured cortical neurons.

Cultured cortical neurons exposed for 24 h to low concentrations of the Ca2+ ionophores, ionomycin (250 nM) or A-23187 (100 nM), underwent apoptosis, accompanied by early degeneration of neurites, cell body shrinkage, chromatin condensation and internucleosomal DNA fragmentation. This death could be blocked by protein synthesis inhibitors, as well as by the growth factors brain-derived neurotrophic factor or insulin-like growth factor I. If the ionomycin concentration was increased to 1-3 microM, then neurons underwent necrosis, accompanied by early cell body swelling without DNA laddering, or sensitivity to cycloheximide or growth factors. Calcium imaging with Fura-2 suggested a possible basis for the differential effects of low and high concentrations of ionomycin. At low concentrations, ionomycin induced greater increases in intracellular Ca2+ concentration in neurites than in neuronal cell bodies, whereas at high concentrations, ionomycin produced large increases in intracellular Ca2+ concentration in both neurites and cell bodies. We hypothesize that the ability of low concentrations of Ca2+ ionophores to raise intracellular Ca2+ concentration preferentially in neurites caused early neurite degeneration, leading to loss of growth factor availability to the cell body and consequent apoptosis, whereas high concentrations of ionophores produced global cellular Ca2+ overload and consequent necrosis.

NMDA receptors mediate hypoxic spine loss in cultured neurons.

We examined the pharmacology of dendritic morphologic changes in cultured cortical neurons exposed to sublethal oxygen-glucose deprivation (OGD). Confocal analysis of DiI-labeled neurons demonstrated transient dendritic swelling and spine loss after OGD. These morphological changes were reproduced by direct application of NMDA, kainate, veratridine, ionomycyin, and gramicidin, but not KCl. Blockade of voltage-gated sodium or calcium channels did not prevent OGD-induced dendritic spine loss. In contrast, the NMDA receptor antagonist, MK-801, fully prevented these changes. An AMPA/kainate receptor antagonist, NBQX, had no effect by itself but reduced spine loss when added to MK-801. While alterations in dendrite morphology may be triggered by activation of disparate ion channels, rapid spine loss in hypoxic cortical neurons is mediated preferentially through activation of the NMDA subtype glutamate receptor.

Glucose deprivation neuronal injury in cortical culture.

Murine cortical cell cultures deprived of glucose for 6-8 h developed extensive neuronal degeneration, apparent both morphologically and by efflux of lactate dehydrogenase to the bathing medium. This neuronal damage could be substantially reduced by addition of D-2-amino-5-phosphonovalerate (D-APV), in a concentration-dependent (IC50 about 2 microM) and stereospecific (D-APV more potent than L-APV) fashion. A similar neuron-protective effect could also be obtained with several other NMDA antagonists, 2-amino-7-phosphonoheptanoate, phencyclidine, MK-801, ketamine, and (+)-SKF 10,047, as well as with the broad spectrum glutamine antagonist kynurenate. In contrast, little protection could be obtained with gamma-D-glutamylaminomethyl sulfonate and L-glutamate diethyl ester, compounds which have been reported to act primarily at non-NMDA receptors. These observations support the hypothesis that glucose deprivation-induced cortical neuronal injury is largely mediated by NMDA receptors, and suggest that cell culture methodology can be useful in the quantitative characterization of that injury.

Ketamine protects cultured neocortical neurons from hypoxic injury.

The general anesthetic ketamine, which has recently been reported to block the excitation of cortical neurons by N-methyl-D-aspartate (NMDA), was found to markedly reduce neuronal loss in murine neocortical cell cultures exposed to a hypoxic atmosphere or to cyanide. These observations may be relevant to attempts to find pharmacological means of minimizing hypoxic brain damage in the clinical setting.

Antagonism of non-NMDA receptors augments the neuroprotective effect of NMDA receptor blockade in cortical cultures subjected to prolonged deprivation of oxygen and glucose.

A 30-60 min period of oxygen and glucose deprivation induced widespread degeneration of cultured murine neocortical neurons. Neuronal degeneration could be blocked by adding the selective NMDA antagonist MK-801 to the bathing medium; however, if the deprivation period was prolonged to 90-105 min, the neuroprotective effect of MK-801 was overcome. The non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) at 1-100 microM concentrations also failed to protect neurons against this prolonged insult, but the combination of CNQX with either MK-801 or D-APV produced marked neuroprotection. This synergistic action of CNQX was not due to enhanced blockade of NMDA receptors, as it was not mimicked by combining MK-801 with D-APV or 7-chlorokynurenate. These observations support the idea that combined NMDA and non-NMDA receptor blockade may have value in ameliorating the neuronal loss associated with prolonged ischemic insults in vivo.

Dextromethorphan reduces neocortical ischemic neuronal damage in vivo.

The dextrorotatory morphinan dextromethorphan (DM), a clinically tested antagonist of the N-methyl-D-aspartate (NMDA) receptor-channel complex, was tested in an in vivo model of acute transient focal cerebral ischemia. Rabbits were randomly assigned to pretreatment with a 20 mg/kg i.v. bolus followed by 10 mg/kg/h of 0.4% DM in normal saline (NS), or with an equivalent volume of NS alone. They then underwent 1 h occlusion of the left internal carotid artery an anterior cerebral artery followed by 4 h of reperfusion. DM-treated animals showed a significant decrease in the percentage of severe neocortical ischemic neuronal damage (10.5%), as compared to NS-treated animals (49.6%).

High concentrations of naloxone attenuate N-methyl-D-aspartate receptor-mediated neurotoxicity.

(-)-Naloxone, 1 mM, partially reduced neuronal loss induced by exposure of murine cortical cell cultures to N-methyl-D-aspartate (NMDA) or quinolinate, but produced little or no attenuation of kainate or quisqualate neurotoxicity. Antagonism of NMDA neurotoxicity was (-)-naloxone concentration-dependent between 100 microM and 3 mM. (+)-Naloxone produced a slightly greater reduction of NMDA neurotoxicity, arguing against mediation by opioid receptors. Although this novel neuron-protective action of (-)-naloxone was weak, it may contribute to reported beneficial effects in CNS ischemia.

Hypoxic neuronal injury in vitro depends on extracellular glutamine.

Hypoxic neuronal injury (HNI) in cortical cell cultures was enhanced in a concentration-dependent fashion by the presence of 500 microM to 2 mM (EC50 about 500 microM) glutamine in the medium, concentrations approximating those normally present in cerebrospinal fluid (CSF). Regardless of the glutamine concentration, glutamate receptor antagonists 2-amino-5-phosphonovalerate or dextrorphan could substantially reduce HNI. Thus, the availability of extracellular glutamine could be a determinant of hypoxic neuronal injury in vivo, most likely reflecting its importance in the synthesis of the neurotransmitter excitotoxins glutamate and aspartate.

Adenosine reduces cortical neuronal injury induced by oxygen or glucose deprivation in vitro.

The endogenous neuromodulatory purine, adenosine, substantially attenuated neuronal degeneration when added to dissociated cortical cell cultures acutely deprived of either oxygen or glucose. The protective effect of adenosine, was concentration-dependent between 30 and 1000 microM (EC50 about 100 microM), and could be mimicked by the stable adenosine analogue N6-cyclohexyladenosine (10 microM). Unlike postsynaptic glutamate receptor antagonists, which also block these forms of neuronal injury, adenosine did not alter the neurotoxicity of exogenously applied glutamate.

Dextrorphan and dextromethorphan attenuate hypoxic injury in neuronal culture.

The dextrorotatory opioid derivatives, dextrorphan and dextromethorphan, can attenuate hypoxic injury in cortical cell cultures. This effect is concentration-dependent in the micromolar range, and not strongly stereospecific, as it can also be demonstrated with the levorotatory enantiomer of dextrorphan, levorphanol. The possibility that these clinically available compounds may have therapeutic utility in hypoxic or ischemic encephalopathy warrants further investigation.

Oligodendrocyte degeneration and recovery after focal cerebral ischemia.

The vulnerability of oligodendrocytes to ischemic injury may contribute to functional loss in diseases of central white matter. Immunocytochemical methods to identify oligodendrocyte injury in experimental models rely on epitope availability, and fail to discriminate structural changes in oligodendrocyte morphology. We previously described the use of a lentiviral vector (LV) carrying enhanced green fluorescent protein (eGFP) under the myelin basic protein (MBP) promoter for selective visualization of oligodendrocyte cell bodies and processes. In this study, we used LV-MBP-eGFP to label oligodendrocytes in rat cerebral white matter prior to transient focal cerebral ischemia, and examined oligodendrocyte injury 24 h, 48 h and 1 week post-reperfusion by quantifying cell survival and assaying the integrity of myelin processes. There was progressive loss of GFP+ oligodendrocytes in ischemic white matter at 24 and 48 h. Surviving GFP+ cells had non-pyknotic nuclear morphology and were terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-negative, but there was marked fragmentation of myelin processes as early as 24 h after stroke. One week after stroke, we observed a restoration of GFP+ oligodendrocytes in ischemic white matter, reflected both by cell counts and by structural integrity of myelin processes. Proliferating cells were not the main source of GFP+ oligodendrocytes, as revealed by bromodeoxyuridine (BrdU) incorporation. These observations identify novel transient structural changes in oligodendrocyte cell bodies and myelinating processes, which may have consequences for white matter function after stroke.