Testing NMDA receptor block as a therapeutic strategy for reducing ischaemic damage to CNS white matter

Damage to oligodendrocytes caused by glutamate release contributes to mental or physical handicap in periventricular leukomalacia, spinal cord injury, multiple sclerosis, and stroke, and has been attributed to activation of AMPA/kainate receptors. However, glutamate also activates unusual NMDA receptors in oligodendrocytes, which can generate an ion influx even at the resting potential in a physiological [Mg2+]. Here, we show that the clinically licensed NMDA receptor antagonist memantine blocks oligodendrocyte NMDA receptors at concentrations achieved therapeutically. Simulated ischaemia released glutamate which activated NMDA receptors, as well as AMPA/kainate receptors, on mature and precursor oligodendrocytes. Although blocking AMPA/kainate receptors alone during ischaemia had no effect, combining memantine with an AMPA/kainate receptor blocker, or applying the NMDA blocker MK-801 alone, improved recovery of the action potential in myelinated axons after the ischaemia. These data suggest NMDA receptor blockers as a potentially useful treatment for some white matter diseases and define conditions under which these blockers may be useful therapeutically. Our results highlight the importance of developing new antagonists selective for oligodendrocyte NMDA receptors based on their difference in subunit structure from most neuronal NMDA receptors.     

AMPA receptors are the major mediators of excitotoxic death in mature oligodendrocytes

Myelination of axons is important for central nervous system function, but oligodendrocytes, which constitute CNS myelin, are vulnerable to excitotoxic injury and death. Although mature oligodendrocytes express functional alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA) and kainate-type glutamate receptors, the relative roles of these subtypes in excitotoxicity are not well understood. Using recently developed selective antagonists for subtypes of ionotropic non-NMDA receptors, we addressed this issue. By examining the pharmacological, biochemical, and morphologic features of kainite-induced excitotoxic death, we also determined whether it occurs by apoptosis, necrosis, or both. We conclude that when mature oligodendrocytes die after exposure to kainate: (1) AMPA receptors are the most important mediators, (2) kainate receptors play a smaller role, and (3) death occurs predominantly by necrosis, not apoptosis.     

AMPA and kainate receptors each mediate excitotoxicity in oligodendroglial cultures

Recent studies indicate that oligodendrocytes are vulnerable to excitotoxic insults mediated by glutamate receptors. The present study was carried out to characterize the type of glutamate receptors triggering cell death in optic nerve oligodendrocyte cultures. Acute activation of either AMPA or kainate receptors was toxic to oligodendrocytes, an effect that was prevented by CNQX. However, exposure to agonists of the NMDA and metabotropic glutamate receptors did not impair cell viability. Dose-response curves showed that toxicity was mediated by three distinct populations of receptors: an AMPA-type receptor and high- and low-affinity kainate-type receptors. Expression and immunocytochemical studies suggested that the glutamate receptor subunits give rise to the native receptors in each population. In all instances, Ca(2+) entry was a major determinant of glutamate receptor excitotoxicity. However, its influence varied for each receptor subtype. These results indicate that aberrantly enhanced activation of AMPA and/or kainate receptors may be involved in demyelinating diseases.     

Calretinin-Containing Neurons in Rat Cerebellar Granule Cell Cultures

Using an antiserum against calretinin, a calcium-binding protein, we discovered two distinct neuronal cell types that stain intensely in enriched cerebellar granule cells. One neuronal cell type resembles unipolar brush cells, whereas the other resembles Lugaro cells. During early culture times, these calretinin-positive neurons are most numerous but represent less than one percent of the total neuronal population. In cultured cells, calretinin mRNA levels peak at day three in vitro, followed by a rapid decline to undetectable levels by day six in vitro. However, calretinin-immunoreactive neurons are observed up to 29 days in vitro. Excitotoxic concentrations of glutamate receptor agonists failed to elicit an excitotoxic response on the intensely staining calretinin-positive neurons, whereas greater than 95% of the cerebellar granule cells were susceptible to the excitotoxic actions of the glutamate receptor agonists. To distinguish between the two possibilities that calretinin-positive neurons either do not express glutamate receptors or they are not susceptible to the excitotoxic effects of glutamate receptor agonists, we performed immunocytochemistry using glutamate receptor antibodies to detect the presence of receptor protein. We found that the AMPA/kainate glutamate receptor (GluR₂R₃) colocalized with calretinin, suggesting that calretinin-immunoreactive neurons express the AMPA/kainate receptor; cerebellar granule cells, which are known to express this receptor, were also immunoreactive for the GluR₂R₃ receptor.     

Glutamate induces rapid loss of axonal neurofilament proteins from cortical neurons in vitro

One of the primary hallmarks of glutamate excitotoxicity is degradation of the neuronal cytoskeleton. Using a tissue culture approach, we have investigated the relationship between excitotoxicity and cytoskeletal degradation within axons, with particular reference to the axon specific neurofilament proteins. Neurofilaments were rapidly lost from axons over a 24-h period in response to excitotoxic insult (as observed by immunocytochemistry and western blotting), while other axonal cytoskeletal markers (such as betaIII-tubulin) remained intact. Treatment with kainic acid and NMDA, or complementary experiments using the pharmacological glutamate receptors blockers CNQX (kainate/AMPA receptor antagonist) and MK-801 (NMDA receptor antagonist), demonstrated that neurofilament degeneration was mediated primarily by NMDA receptor activity. This work suggests that excitotoxicity triggers a progressive pathway of cytoskeletal degeneration within axons, initially characterised by the loss of neurofilament proteins.     

Glutamate inhibition in MS: the neuroprotective properties of riluzole

In addition to demyelination and damage to oligodendrocytes, axonal injury and neuronal cell death are dominating histopathological characteristics of multiple sclerosis (MS). Still little is known about the cause of the damage. Extracellular accumulation of glutamate contributes to excitotoxic injury of neurons and glial cells, suggesting that the maintenance of subtoxic extracellular glutamate levels may be crucial. Riluzole is a neuroprotective agent that inhibits the release of glutamate from nerve terminals and modulates glutamate, i.e., kainate and NMDA receptors. It inhibits excitotoxic injury in several experimental models of neurodegenerative disease. We performed a small run-in versus treatment MR-monitored pilot study in 16 primary progressive MS patients. The results suggest that riluzole reduces the rate of cervical cord atrophy and the development of T1 hypointense lesions on magnetic resonance imaging in primary progressive MS. The rate of brain atrophy was only slightly decreased. The results indicate an effect on mechanisms involving lesion evolution and axonal loss, but no clear effect on new lesion formation. However, the data suffer from several limitations and must be confirmed in future trials.     

Chronic exposure of kainate and NBQX changes AMPA toxicity in hippocampal slice cultures.

Receptor agonist/antagonist mediated modulation of the excitotoxic effect of AMPA was studied in organotypic hippocampal slice cultures. Treatment of developing cultures for 2 weeks with a subtoxic dose of 2 microM kainate reduced the toxicity of 3 microM AMPA, applied for 48 h with 24 h of recovery, as measured by cellular uptake of the fluorescent dye propidium iodide. In contrast long-term treatment with 0.3 microM of the AMPA/KA antagonist NBQX increased the susceptibility of the cultures to an even lower dose of 2 microM AMPA. The modulatory effects of long-term application of low doses of kainate and NBQX, have implications for the development and use of related drugs that aim to protect against glutamate receptor-mediated disturbances.     

Glutamate excitotoxicity--a mechanism for axonal damage and oligodendrocyte death in Multiple Sclerosis?

Glutamate excitotoxicity mediated by the AMPA/kainate-type of glutamate receptors is known not only to damage neurons but also the myelin-producing cell of the central nervous system (CNS), the oligodendrocyte. In Multiple Sclerosis (MS), myelin, oligodendrocytes and axons are lost or damaged as a result of an inflammatory attack on the CNS. Activated immune cells produce glutamate in large quantities by deamidating glutamine via glutaminase. Thus, we hypothesized that during inflammation in MS, glutamate excitotoxicity may contribute to the lesion. This was addressed by treating mice sensitized to develop acute experimental autoimmune encephalomyelitis (EAE) with an AMPA/kainate antagonist, NBQX. Treatment resulted in substantial amelioration of disease, increased oligodendrocyte survival and reduced axonal damage, as indicated by the levels of dephosphorylated neurofilament-H. Despite the clinical differences, NBQX-treatment had no effect on lesion size and did not reduce the degree of CNS inflammation. In addition, NBQX did not alter the proliferative activity of antigen-primed T cells in vitro, further indicating a lack of effect at the level of the immune system. In separate studies, infiltrating immune cells present in perivascular cuffs, commonly the site of entry for invading immune cells, were found to express glutaminase in abundance, supporting the production of glutamate in inflammatory lesions. Thus, glutamate excitotoxicity appears to be an important mechanism in autoimmune demyelination and its prevention with AMPA/kainate antagonists may prove to be an effective therapy for MS.     

Emergence of cellular markers and functional ionotropic glutamate receptors on tangentially dispersed cells in the developing mouse retina

Tangential cell dispersion in the retina is a spacing mechanism that establishes a regular mosaic organization among cell types and contributes to their final positioning. The present study has used the X-inactivation transgenic mouse expressing the lacZ reporter gene on one X chromosome. Due to X chromosome inactivation, 50% of early progenitor cells express beta-galactosidase (beta-Gal); therefore, all cells derived from a particular beta-Gal-expressing progenitor cell can be identified in labeled columns. The radial segregation of clonally related beta-Gal-positive and beta-Gal-negative cells can be used to determine whether single cells transgress a clonal boundary in the retina. We investigated the extent to which particular cell classes tangentially disperse by analyzing the placement of labeled cells expressing particular markers at several ages and quantifying their tangential displacement. Retinal neurons expressing cell markers at postnatal day (P) 1 have a greater degree of tangential dispersion compared with amacrine and bipolar cells at P5-6. We also studied whether there is a functional correlation with these dispersion patterns by investigating the emergence of functional ionotropic glutamate receptors. To determine the degree of functional glutamate receptor activation, agmatine (AGB) was used in combination with cell-specific labeling. AGB permeates functional glutamate receptor channels following activation with alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate or N-methyl-D-aspartate (NMDA). Within these receptor groups, high concentrations of AMPA, kainate, and NMDA are associated with a high degree of tangential dispersion in the adult. Developmentally, functional kainate and AMPA receptors were detected by P1 and were associated with tangentially dispersed cells. Functional NMDA receptors were not detected as early as kainate and AMPA receptors. These results indicate that cells generated early during development are more likely to disperse tangentially compared with those generated later in development. Therefore, functional AMPA and kainate receptors may play a critical role in tangentially displacing cell types.     

Reduced size of the dendritic tree does not protect Purkinje cells from excitotoxic death

Purkinje cell loss by excitotoxic damage is a typical finding in many cerebellar diseases. One important aspect of this high sensitivity of Purkinje cells to excitotoxic death might be the enormous size of their dendritic tree, with a high load of excitatory glutamate receptors. We have studied whether reduction in the size of the dendritic tree might confer resistance against excitotoxic death to Purkinje cells. We have grown Purkinje cells in organotypic cerebellar slice cultures under chronic activation of metabotropic glutamate receptors or of protein kinase C. Both treatments strongly reduced dendritic tree size. After this treatment, cells were exposed to the glutamate receptor agonist AMPA, which has a strong excitotoxic effect on Purkinje cells. We found that Purkinje cells with small dendritic trees were as sensitive to AMPA exposure as untreated control cells with large dendritic trees. Immunostaining against vesicular glutamate transporter 1 revealed that the small dendritic trees were densely covered by glutamatergic terminals. Our results indicate that the expansion of the dendritic tree and the total number of AMPA receptors per neuron do not play a major role in determining the susceptibility of Purkinje cells to excitotoxic death. © 2009 Wiley‐Liss, Inc.     

Excitotoxic mechanisms of ischemic injury in myelinated white matter.

Axonal injury and dysfunction in white matter (WM) are caused by many neurologic diseases including ischemia. We characterized ischemic injury and the role of glutamate-mediated excitotoxicity in a purely myelinated WM tract, the mouse optic nerve (MON). For the first time, excitotoxic WM injury was directly correlated with glutamate release. Oxygen and glucose deprivation (OGD) caused duration-dependent loss of axon function in optic nerves from young adult mice. Protection of axon function required blockade of both alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and kainate receptors, or removal of extracellular Ca(2+). Blockade of N-methyl-D-aspartate receptors did not preserve axon function. Curiously, even extended periods of direct exposure to glutamate or kainate or AMPA failed to induce axon dysfunction. Brief periods of OGD, however, caused glutamate receptor agonist exposure to become toxic, suggesting that ionic disruption enabled excitotoxic injury. Glutamate release, directly measured using quantitative high-performance liquid chromatography, occurred late during a 60-mins period of OGD and was due to reversal of the glutamate transporter. Brief periods of OGD (i.e., 15 mins) did not cause glutamate release and produced minimal injury. These results suggested that toxic glutamate accumulation during OGD followed the initial ionic changes mediating early loss of excitability. The onset of glutamate release was an important threshold event for irreversible ischemic injury. Regional differences appear to exist in the specific glutamate receptors that mediate WM ischemic injury. Therapy for ischemic WM injury must be designed accordingly.     

Topiramate protects against glutamate- and kainate-induced neurotoxicity in primary neuronal-astroglial cultures

Potential neuroprotective effects of the antiepileptic drug (AED) topiramate (TPM) were evaluated using primary neuronal-astroglial cultures or astroglial-enriched cultures from newborn rats exposed to excitotoxic concentrations of glutamate (Glu) or kainate. Neurons expressed functional Glu receptors of the NMDA and AMPA/kainate types as evaluated by immunocytochemistry and Ca(2+) imaging. When Glu (10 mM) was added to 9-10-day cultures incubated with the fluorescent dye calcein/AM for 5h, there was a marked cell loss in both culture types, but was more pronounced in the neuronal-astroglial cultures. When TPM (5-10 microM) was included in the medium together with Glu, the amount of surviving cells was significantly higher in the neuronal-astroglial cultures, but not in the astroglial-enriched cultures. Immuno-labeling of the cultures revealed an enhanced survival of MAP positive neuronal cells when TPM was included in the Glu containing medium. As TPM has a proven negative modulatory effect on kainate activated receptors, neuronal-astroglial cultures were further exposed to excitotoxic concentrations of kainate (100 microM) and analyzed immunohistochemically. Significantly more MAP positive neurons survived in the TPM containing medium and showed a morphology similar to untreated cells. Valproate and phenytoin were used as reference AEDs. In conclusion, our results demonstrate a protective effect of TPM upon neuronal cells in primary culture, exposed to excitotoxic levels of Glu or kainate.     

Agonist-stimulated cobalt uptake provides selective visualization of neurons expressing AMPA- or kainate-type glutamate receptors in the retina

Fast-acting excitatory neurotransmission in the retina is mediated primarily by glutamate, acting at alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) -selective and kainate-selective receptors. To localize these sites of action, cat retinas were stimulated with either AMPA or kainate and processed for histochemical visualization of cobalt uptake through calcium-permeable channels. Treatment with both agonists resulted in staining of A- and B-type horizontal cells and several types of OFF cone bipolar cells; there was no evidence for staining of ON cone bipolar cells or rod bipolar cells. The subpopulations of OFF cone bipolar cells differed in their responses with two distinct types that stained heavily with cobalt after exposure to AMPA and three different types that were preferentially labeled after exposure to kainate. Although many amacrine and ganglion cells appeared to respond to both agonists, AII amacrine cells were stained after stimulation by AMPA but not by kainate. The OFF cone bipolar cells that exhibit AMPA-stimulated cobalt uptake were found to have a high level of correspondence with cells that show immunocytochemical staining for the AMPA-selective glutamate receptor subunits GluR1 and GluR2/3. Similarly, the cone bipolar cells exhibiting kainate-stimulated cobalt uptake resemble those that are immunoreactive for the kainate subunit GluR5. The results indicate that, whereas many retinal neurons express both AMPA and kainate receptors, AII amacrine cells and subpopulations of OFF cone bipolar cells are limited to the expression of either AMPA or kainate receptors. This differential expression may contribute to the unique character of transmission by these cell types.     

A revised excitotoxic hypothesis of schizophrenia: therapeutic implications

A revision of an "excitotoxic hypothesis" of schizophrenia is summarized. The hypothesis suggests that in, at least, a subtype of patients with schizophrenia, progressive excitotoxic neuronal cell death in hippocampal and cortical areas occurs via "disinhibition" of glutamatergic projections to these areas. Patients who have excitotoxic damage would be expected to have poor outcomes characterized, perhaps, by anatomic evidence of progressive neurodegeneration, pronounced negative symptoms and cognitive deficits, and profound psychosocial deterioration. Disinhibited glutamatergic activity could result from inhibition of N-methyl-D-aspartate (NMDA) receptor-mediated neurotransmission and a consequent failure to stimulate inhibitory gamma-aminobutyric acid (GABA)-ergic interneurons, and/or anatomic degeneration of inhibitory GABAergic interneurons. The result of these hypothesized mechanisms is excessive stimulation of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate class of glutamate receptor complexes. In turn, this excessive stimulation of AMPA/kainate receptors could lead to disruption of ionic gradients, depletion of energy reserves expended in an attempt to restore and maintain the ionic disequilibrium across neuronal membranes, generation of reactive oxygen species, and cell death from apoptotic and other mechanisms. The postulated existence of disinhibited glutamatergic neurotransmission and the subsequent cascade of excitotoxic events resulting from NMDA receptor hypofunction (NRH), anatomic degeneration of inhibitory GABAergic interneurons, or a combination of the two has suggested a diverse variety of experimental therapeutic interventions for schizophrenia. These interventions include facilitation of NMDA receptor-mediated neurotransmission, potentiation of GABAergic neurotransmission, antagonism of AMPA/kainate receptors, and "quenching" of locally generated reactive oxygen species. In fact, several of these approaches have already been pursued or are proposed as part of a systematic clinical investigation of the revised excitotoxic hypothesis of schizophrenia.     

Differential expression of glutamate receptor subtypes in human brainstem sites involved in perinatal hypoxia-ischemia

This study delineates the development of N-methyl-D-aspartate (NMDA) and non-NMDA receptor binding in the human brainstem, particularly as it relates to issues of the trophic effects of glutamate, the glutamate-mediated ventilatory response to hypoxia, and regional excitotoxic vulnerability to perinatal hypoxia-ischemia. We used tissue autoradiography to map the development of binding to NMDA, alpha-amino-3-hydroxy-5-methyl-4-isoxazole-proprionate (AMPA), and kainate receptors in brainstem sites involved in the glutamate ventilatory response to hypoxia, as well as recognized sites vulnerable to perinatal hypoxia-ischemia. NMDA receptor/channel binding was virtually undetectable in all regions of the human fetal brainstem at midgestation, an unexpected finding given the trophic role for NMDA receptors in early central nervous system maturation in experimental animals. In contrast, non-NMDA (AMPA and kainate) receptor binding was markedly elevated in multiple nuclei at midgestation. Although NMDA binding increased between midgestation and early infancy to moderately high adult levels, AMPA binding dramatically fell over the same time period to low adult levels. High levels of kainate binding did not change significantly between midgestation and infancy, except for an elevation in the infant compared with fetal inferior olive; after infancy, kainate binding decreased to negligible adult levels. Our data further suggest a differential development of components of the NMDA receptor/channel complex. This baseline information is critical in considering glutaminergic mechanisms in human brainstem development, physiology, and pathology.     

Activated microglia initiate motor neuron injury by a nitric oxide and glutamate-mediated mechanism

Recent studies suggest that motor neuron (MN) death may be non-cell autonomous, with cell injury mediated by interactions involving non-neuronal cells, such as microglia and astrocytes. To help define these interactions, we used primary MN cultures to investigate the effects of microglia activated by lipopolysaccharide or IgG immune complexes from patients with amyotrophic lateral sclerosis. Following activation, microglia induced MN injury, which was prevented by a microglial iNOS inhibitor as well as by catalase or glutathione. Glutamate was also required since inhibition of the MN AMPA/kainate receptor by CNQX prevented the toxic effects of activated microglia. Peroxynitrite and glutamate were synergistic in producing MN injury. Their toxic effects were also blocked by CNQX and prevented by calcium removal from the media. The addition of astrocytes to cocultures of MN and activated microglia prevented MN injury by removing glutamate from the media. The protective effects could be reversed by inhibiting astrocytic glutamate transport with dihydrokainic acid or pretreating astrocytes with H2O2. Astrocytic glutamate uptake was also decreased by activated microglia or by added peroxynitrite. These data suggest that free radicals released from activated microglia may initiate MN injury by increasing the susceptibility of the MN AMPA/kainate receptor to the toxic effects of glutamate.     

Expressions of nitrotyrosine and TUNEL immunoreactivities in cultured rat spinal cord neurons after exposure to glutamate, nitric oxide, or peroxynitrite

Although excitotoxic and oxidative stress play important roles in spinal neuron death, the exact mechanism is not fully understood. We examined cell damage of primary culture of 11-day-old rat spinal cord by addition of glutamate, nitric oxide (NO) or peroxynitrite (PN) with detection of nitrotyrosine (NT) or terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL). With addition of glutamate, NOC18 (a slow NO releaser) or PN, immunoreactivity for NT became stronger in the cytoplasm of large motor neurons in the ventral horn at 6 to 48 hr and positive in the axons of the ventral horn at 24 to 48 hr. TUNEL positive nuclei were found in spinal large motor neurons from 24 hr, and the positive cell number greatly increased at 48 hr in contrast to the vehicle. Pretreatment of cultures with alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate receptor antagonist, NO-suppressing agent, and antioxidant protected the immunoreactivity for NT or TUNEL. The present results suggest that both excitotoxic and oxidative stress play an important role in the upregulation of NT nitration and the apoptotic pathway in cultured rat spinal neurons.     

Low concentrations of glutamate induce apoptosis in cultured neurons: implications for amyotrophic lateral sclerosis

Evidence is accumulating that excessive glutamate concentration in the extracellular space is neurotoxic and plays a role in amyotrophic lateral sclerosis (ALS). However, the published results on glutamate levels in cerebrospinal fluid (CSF) and on glutamate-mediated toxicity of CSF in ALS disease remain controversial. In this report, we studied CSF from patients with sporadic ALS and controls to determine glutamate concentrations, and then analyzed the neurotoxic effect of glutamate at the concentrations present in CSF from ALS patients on cultured cortical neuronal cells. Our study shows that glutamate, at the concentrations found in CSF from ALS patients (5.8 microM), diminished cell viability and increased apoptosis determined by the fluorescent DNA-binding dye Hoechst 33342 as well as by Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP Nick End-Labeling (TUNEL) reaction in cultured neuronal cells. However, glutamate concentrations as those found in CSF from controls (2.8 microM or below) did not induce any effect. Both significant glutamate-induced effects were inhibited in the presence of NBQX (2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline-2,3-dione), an alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate-sensitive glutamate receptor antagonist. These results demonstrate that AMPA/kainate receptors are involved in the glutamate-mediated neurotoxic effects on cultured neurons, according to reports that implicate these receptors in ALS disease. We conclude that the glutamate-mediated neuronal apoptosis through AMPA/kainate receptors could occur in ALS patients who have elevated CSF glutamate concentration.     

Quantitative autoradiographic distribution of glutamate receptors in the cervical segment of the spinal cord of the wobbler mouse

The reduction of glutamate content has been observed in the spinal cord of the wobbler mouse, a purported model of amyotrophic lateral sclerosis (ALS). To elucidate glutamate receptors in the wobbler spinal cord, we measured densities ofN-methyl-d-aspartate (NMDA), kainate, -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and metabotropic glutamate (mGlu) binding sites using in vitro autoradiography. In wobbler mice, NMDA, kainate, and AMPA binding sites were increased in the dorsal horn and kainate binding sites were also increased in the intermediate zone. However, mGlu binding was unchanged. These results disagree with those observed in ALS spinal cords, in which NMDA and kinate binding sites are decreased. The wobbler mouse may have the glutamate dysfunction, but in a different way from ALS.