Pharmacological characterization of the glutamate receptor in cultured astrocytes

Cultured astrocytes from neonatal rat cerebral hemispheres are depolarized by the excitatory neurotransmitter glutamate. In this study we have used selective agonists of different neuronal glutamate receptor subtypes, namely, the N-methyl-D-aspartate (NMDA), kainate, and quisqualate type, to characterize pharmacologically the glutamate receptor in astrocytes. The agonists of the neuronal quisqualate receptor, alpha-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionic acid (AMPA) and quisqualate, depolarized the membrane. Kainate, an agonist of the neuronal kainate receptor, depolarized astrocytes more effectively than quisqualate. Combined application of kainate and quisqualate depolarized astrocytes to a level which was intermediate to that evoked by quisqualate and kainate individually. Agonists activating the neuronal NMDA receptor, namely NMDA and quinolinate, were ineffective. Application of NMDA did not alter the membrane potential even in combination with glycine or in Mg2+-free solution, conditions under which neuronal NMDA receptor activation is facilitated. The nonselective agonists L-cysteate, L-homocysteate, and beta-N-oxalylamino-L-alanine (BOAA) mimicked the effect of glutamate. Dihydrokainate, a blocker of glutamate uptake, did not, and several antagonists of neuronal glutamate receptors only slightly affect the glutamate response. These findings suggest that astrocytes express one type of glutamate receptor which is activated by both kainate and quisqualate, lending further support to the notion that cultured astrocytes express excitatory amino acid receptors which have some pharmacological similarities to their neuronal counterparts.     

Activation of NMDA receptors protects against glutamate neurotoxicity in the retina: evidence for the involvement of neurotrophins

Activation of glutamate receptors has been implicated in excitotoxicity. Here, we have investigated whether subtoxic concentrations of glutamate can modulate neuronal death in the developing retina. Explants of rat retinas were pre-incubated with glutamate, N-methyl-d-aspartate (NMDA), kainate, quisqualate or trans-1-amino-1,3-cyclopentanedicarboxylic acid (t-ACPD) for 18 h. Then, glutamate (6 mM) was added to the explants for an additional 6 h. Glutamate-induced degeneration was restricted to the emerging inner nuclear layer. Pre-incubation with glutamate, NMDA, or both, reduced glutamate-induced neuronal death and protected against neuronal death induced by irradiation (2 Gy). The NMDA receptor antagonists, 2-amino-5-phosphonovaleric acid (d-APV; 30 microM) or 5-methyl-10,11-dihydro-5H-dibenzocyclohepten-5,10-imine hydrogen maleate (MK-801; 30 microM), prevented glutamate-induced neuroprotection. To investigate whether this neuroprotection was mediated by neurotrophins, we incubated retinal explants with either brain-derived neurotrophic factor or neurotrophin-4. Both treatments resulted in partial protection against glutamate-induced neurotoxicity. Furthermore, NMDA mediated neuroprotection was totally reversed when a soluble form of the specific tyrosine kinase receptor B was simultaneously added to the explants. Our results suggest that activation of NMDA receptors may control neuronal death in the retina during development. This modulation seems to depend, at least in part, on the release of neurotrophins within the retina.     

Lack of correlation between glutamate-induced depletion of ATP and neuronal death in primary cultures of cerebellum

The aim of this work was to identify, using primary cultures of cerebellar neurons, the receptors involved in glutamate-induced depletion of ATP and to assess whether there is a correlation between glutamate-induced ATP depletion and neuronal death. Glutamate induced a rapid depletion of ATP (40% decrease at 5 min). After 60 min incubation with 1 mM glutamate ATP content decreased by 60–70%. Similar effects were induced by glutamate, NMDA and kainate while quisqualate, AMPA or trans-ACPD did not affect significantly ATP content. The EC₅₀ were 6, 25 and 30 μM for glutamate, NMDA and kainate, respectively. DNQX and AP-5, competitive antagonists of kainate and NMDA receptors, respectively, prevented in a dose-dependent manner the glutamate-induced depletion of ATP. These results indicate that glutamate-induced depletion of ATP is mediated by activation of kainate and NMDA receptors. Glutamate-induced neuronal death was prevented by MK-801, calphostin C, H7, carnitine, nitroarginine and W7. However, only MK-801 and W7 prevented glutamate-induced depletion of ATP, while calphostin C, H7, carnitine and nitroarginine did not. This indicates that there is not a direct correlation between ATP depletion and neuronal death.     

Repeated 4-aminopyridine induced seizures diminish the efficacy of glutamatergic transmission in the neocortex

Systemic administration of the potassium channel blocker 4-aminopyridine (4-AP) elicits acute convulsions. Synchronized tonic–clonic activity develops during the first hour after the treatment. However, subsequent chronic spontaneous seizures do not appear which suggests changes in neuronal excitability. The aim of our present work was to evaluate alterations in the glutamatergic transmission in the somatosensory cortex of rats following daily, brief convulsions elicited by 4-AP treatment. Changes in general neuronal excitability and pharmacological sensitivity of glutamate receptors were tested in ex vivo electrophysiological experiments on brain slices. In parallel studies quantitative changes in subunit composition of glutamate receptors were determined with immunohistoblot technique, together with the analysis of kainate induced Co²⁺ uptake. The results of our coordinated electrophysiological, receptor-pharmacological and histoblot studies demonstrated that repeated, daily, short convulsions resulted in a significant decrease of the general excitability of the somatosensory cortex together with changes in ionotropic glutamate receptor subunits. The relative inhibitory effect of the AMPA receptor antagonist, however, did not change. The NMDA receptor antagonist exerted somewhat stronger effect in the slices from convulsing animals. 4-AP pretreatment resulted in the attenuation of kainate induced Co²⁺ uptake, which suggests either reduction in non-NMDA receptors numbers or reduction in their Ca²⁺ permeability. Repeated seizures decreased GluR1–4 AMPA receptor subunit levels in all cortical layers with a relaitve increase in GluR1 subunits. While the principle NR1 NMDA receptor subunit showed no significant change, the staining density of NR2A subunit increased. These changes in ionotropic glutamate receptors are consistent with reduced excitability at glutamatergic synapses following repeated 4-AP induced seizures.     

Glutamate receptors communicate with Na+/K+-ATPase in rat cerebellum granule cells

Two glutamate receptor agonists, NMDA (N-methyl-d-aspartic acid) and ACPD (cis-(1S/3R)-1-aminocyclopentane-1,3-dicarboxylic acid), induce the reactive oxygen species (ROS) production in rat cerebellum granule cells, whereas the third one, 3-HPG (3-hydroxyphenylglycine), decreases this parameter. The simultaneous presence of 3-HPG, together with NMDA or ACPD, prevents the generation of ROS by neuronal cells. A similar effect of these ligands on Na⁺/K⁺-ATPase can be demonstrated: NMDA and ACPD inhibited the enzyme activity, but 3-HPG activated Na⁺/K⁺-ATPase and prevented its inhibition by NMDA or ACPD. In terms of current classification, NMDA is an agonist of ionotropic glutamate receptors of the so-called NMDA class, whereas ACPD and 3-HPG belong to metabotropic agonists, the former primarily being an activator of metabotropic glutamate receptors (mGluRs) of groups 2 and 3, and the latter, that of mGluRs of groups 1 and 5. Thus, the data presented illustrate the existence of diverse mechanisms of the cross talk between Na⁺/K⁺-ATPase and different glutamate receptors, as well as that between glutamate receptors of different classes.     

Peripheral type benzodiazepine binding sites are a sensitive indirect index of neuronal damage

The effects of excitotoxic lesions on the neuronal marker enzymes choline acetyltransferase and glutamate decarboxylase and on the levels of 'peripheral type' benzodiazepine binding sites (PTBBS) (a putative glial marker) have been compared to see whether PTBBS provide a suitable if indirect quantitative index of neuronal damage. Intrastriatal injection of excitotoxic compounds provoked a dose-dependent increase in the levels of PTBBS. The potency order was the following: kainate greater than AMPA greater than N-methyl-D-aspartate (NMDA) greater than quisqualate. The maximal increases in this parameter were 400, 470, 320 and 210% for kainate (12 nmol), AMPA (100 nmol), NMDA (500 nmol) and quisqualate (250 nmol), respectively. 2-Amino-5-phosphonovalerate (100 nmol)--an antagonist of the NMDA receptor subtype--completely blocked the increase in PTBBS induced by NMDA (250 nmol), but was without effect against the other excitotoxins. Increases in binding levels were in general mirrored by a decrease in choline acetyltransferase and glutamate decarboxylase activity. However, PTBBS were a more sensitive indirect index of neuronal damage than neuronal enzymes because the alterations in binding were statistically significant at doses of excitotoxins lower than those causing a loss of marker enzymes. It is concluded that PTBBS are a suitable and sensitive means of detecting discrete neurotoxic changes and that its measurement will help in the study of other pathological and experimental models.     

Neurons derived from embryonic stem (ES) cells resemble normal neurons in their vulnerability to excitotoxic death

We determined whether embryonic stem (ES) cells could provide a model system for examining neuronal death mediated by glutamate receptors. Although limited evidence indicates that normal neurons can be derived from mouse ES cells, there have been no studies examining pathophysiological responses in mouse ES cell systems. Mouse ES cells, induced down a neural lineage by retinoic acid (RA), were found to have enhanced long-term survival when plated onto a layer of cultured mouse cortical glial cells. In these conditions, the ES cells differentiated into neural cells that appeared normal morphologically and displayed normal features of immunoreactivity when tested for neuron-specific elements. Varying the culture medium generated cultures of mixed neuronal/glial cells or enriched in oligodendrocytes. These cultures were viable for at least four weeks. Real-time PCR analysis of N-methyl-D-aspartate (NMDA) receptor subunits revealed an appropriate age-in-vitro dependent pattern of expression. Neurons derived from ES cells were vulnerable to death induced by a 24-h exposure to the selective glutamate receptor agonists NMDA, kainate, and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). This vulnerability to agonist-induced death increased with age in vitro, and related closely to expression of receptor subunits, as it does in cultured primary neurons. Experiments with selective receptor antagonists showed that glutamate receptors mediated the NMDA- and kainate-induced death. Neuronal differentiated ES cells therefore exhibited an excitotoxic response resembling that displayed by central nervous system (CNS) neurons. Thus, ES cells, which are very amenable to genetic manipulation, provide a valid system for studying glutamate receptor-mediated toxicity at the molecular level.     

Reduced mitochondrial manganese-superoxide dismutase activity exacerbates glutamate toxicity in cultured mouse cortical neurons

Studies of neuronal injury and death after cerebral ischemia and various neurodegenerative diseases have increasingly focused on the interactions between mitochondrial function, reactive oxygen species (ROS) production and glutamate neurotoxicity. Recent findings suggest that increased mitochondrial ROS production precedes neuronal death after glutamate treatment. It is hypothesized that under pathological conditions when mitochondrial function is compromised, extracellular glutamate may exacerbate neuronal injury. In the present study, we focus on the relationship between mitochondrial superoxide production and glutamate neurotoxicity in cultured cortical neurons with normal or reduced levels of manganese-superoxide dismutase (MnSOD) activity. Our results demonstrate that neurons with reduced MnSOD activity are significantly more sensitive to transient exposure to extracellular glutamate. The increased sensitivity of cultured cortical neurons with reduced MnSOD activity is characteristically subject only to treatment by glutamate but not to other glutamate receptor agonists, such as N-methyl- -aspartate, kainate and quisqualate. We suggest that the reduced MnSOD activity in neurons may exacerbate glutamate neurotoxicity via a mechanism independent of receptor activation.     

Neuroprotective effects of MK-801 in vivo: selectivity and evidence for delayed degeneration mediated by NMDA receptor activation

The ability of the noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 to prevent neuronal degeneration in the rat striatum and hippocampus caused by intracerebral injection of excitotoxins has been examined. Excitotoxic damage was assessed after 7 d, using histological and biochemical [choline acetyltransferase (ChAT) glutamate decarboxylase (GAD)] measurements. Systemically administered MK-801 was found to protect against neurodegeneration caused by NMDA (200 nmol) and the naturally occurring NMDA receptor agonist quinolinate (120-600 nmol) but not against that induced by kainate (5 nmol) or alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA; 50 nmol), indicating a selectivity for NMDA receptor-mediated neuronal loss. Neurotoxicity caused by NMDA (200 nmol) or quinolinate (200 nmol) was prevented by MK-801 (1-10 mg/kg, i.p.) administered in a single dose after excitotoxin injection. In the striatum, significant protection of cholinergic neurons (assessed by ChAT measurements) was observed when MK-801 was given up to 5 hr after injection of NMDA or quinolinate, whereas protection of GABAergic neurons (assessed by GAD measurements) was obtained up to 2 hr. The results suggest that GABAergic neurons degenerate more rapidly than cholinergic neurons. The competitive NMDA receptor antagonist 3-[(+/-)-2-carboxypiperazin-4-yl]-propyl-1-phosphonate (100 mg/kg, i.p.) gave partial protection of striatal neurons when administered 1 hr after quinolinate injection. In the rat hippocampus, administration of 10 mg/kg MK-801 i.p. 1 hr after quinolinate injection caused almost complete protection of pyramidal and granule neurons, whereas the degeneration of CA3/CA4 pyramidal neurons caused by kainate injection was unaffected. These observations indicate that neurons in rat striatum and hippocampus do not die as an immediate consequence of exposure to high concentrations of NMDA agonists but that a delayed process is involved that requires NMDA receptor activation. In this respect, intracerebral injections of NMDA agonists may mimic the pathological changes that are thought to occur in the brain following periods of cerebral ischemia, where delayed neuronal degeneration occurs.     

Enhancement of NMDA receptor-mediated neurotoxicity in the hippocampal slice by depolarization and ischemia

Evidence from animal stroke models suggests that the proximate cause of neuronal degeneration after ischemia is massive release of glutamate and activation of NMDA receptors. However, in the physiologic presence of oxygen and glucose in the rat hippocampal slice preparation, the neurotoxicity of glutamate, as measured by inhibition of protein synthesis, requires high concentrations and is not prevented by glutamate receptor antagonists. Thus, the NMDA receptor-mediated neurotoxic effects of extracellular glutamate accumulation during ischemia might depend on additional factors, such as neuronal depolarization. In the experiments reported here, slices were exposed to glutamate in a medium intended to mimic the ionic conditions found during ischemia, high potassium (128 mM) and low sodium (26 mM). This depolarizing medium itself inhibited protein synthesis in a manner which was partially mediated by NMDA receptor activation, since it was significantly reversed by the noncompetitive NMDA antagonist, MK-801. Furthermore, the effect of glutamate under depolarizing conditions was also significantly decreased by MK-801, suggesting that glutamate was acting at NMDA receptors. Thus, depolarization appears to enhance the sensitivity of neurons to toxic NMDA receptor activation by glutamate. Under conditions that mimic ischemia, hypoxia plus hypoglycemia, a similar protective effect of NMDA receptor antagonists was observed. Depolarization and ischemia both appeared to attenuate the neurotoxicity of non-NMDA receptor agonists. It appears that under conditions of normal glucose and oxygen, high concentrations of bath applied glutamate inhibit protein synthesis at sites other than the NMDA receptor. However, when the Na+ gradient is decreased, as occurs during ischemia, glutamate's NMDA effects predominate. These findings suggest that ionic shifts may play a central role in permitting NMDA receptor-mediated ischemic neuronal damage.     

In vivo, the direct and seizure-induced neuronal cytotoxicity of kainate and AMPA is modified by the non-competitive antagonist, GYKI 52466

The 2,3-benzodiazepine GYKI 52466, administered intracerebrally or systemically, was assessed for its ability to protect against the neuronal death in the brain caused by intra-hippocampal injections of the non-N-methyl-D-aspartate (NMDA) receptor agonists, kainate and L-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA). In contrast to a previous report, a low intra-hippocampal dose of GYKI 52466 (25 nmol) did not protect against kainate toxicity. In order to achieve higher doses of GYKI 52466, solubilization in 2-hydroxypropyl-beta-cyclodextrin was used, and limited protection against AMPA, but not kainate toxicity was found. There was a commensurate reduction in seizure-related neuronal loss in the limbic regions of the brain. When diazepam was used to prevent seizures, GYKI 52466 had no effect on hippocampal neuronal loss caused by the direct toxicity of AMPA and kainate on hippocampal neurons. Systemic administration of GYKI 52466 had only a minimal effect on preventing neuronal death caused by AMPA. In vivo, GYKI 52466 is only weakly effective as a neuroprotective agent.     

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.     

Modulation of cell death by mouse genotype: differential vulnerability to excitatory amino acid-induced lesions

Previously we have demonstrated that mature inbred strains of mice differ significantly in their response to kainate-induced cell death. While both C57BL/6 and FVB/N mice exhibit similar seizure activity in response to kainate, only C57BL/6 mice can be characterized as resistant to kainate-induced cell death. To examine further the molecular pharmacological basis for this strain difference in hippocampal sensitivity, we assessed the ability of the ionotropic glutamate receptor agonists, kainic acid (KA), N-methyl-D-aspartate (NMDA), ibotenic acid (IBO), and quinolinic acid (QUIN), to promote excitotoxic damage. We examined seizure-related behavior and subsequent neurotoxicity in C57BL/6 and FVB/N mice following intrahippocampal administration of the kainate receptor agonist, KA, the NMDA receptor agonists NMDA or QUIN, or the NMDA and metabotropic glutamate receptor agonist, IBO. The time course and extent of cell death in mice were evaluated using Nissl and selective silver stains, and Fluoro-Jade, a fluorescent marker for dying neurons. In the present study, FVB/N mice were exquisitely sensitive to injection of KA at all doses, while susceptibility in C57BL/6 mice was dose dependent. In contrast, while hippocampal damage was present in both strains at all doses of QUIN, the extent of cell damage was significantly less in C57BL/6 mice at low doses (30 and 60 mM). Similarly, IBO administration resulted in differences in the extent of cell death when administered at the highest dose (126 mM). No strain-dependent differences in cell loss were observed following NMDA lesions. These results provide further evidence that susceptibility to excitotoxin-induced cell death is highly strain dependent and is kainate and NMDA receptor dependent.     

Inhibition of α2/α3 sodium pump isoforms potentiates glutamate neurotoxicity

Excessive stimulation of neurons by glutamic acid initiates a destructive cascade of ion fluxes, cellular swelling, and death. Homeostatic mechanisms which rectify these disturbances depend largely upon transmembrane ion gradients maintained by Na⁺, K⁺-ATPase (NaP). We proposed that the neurotoxicity of glutamate is enhanced when the NaP capacity is exceeded, and therefore, that the degree of neuronal death varies inversely with endogenous NaP activity. To test this concept, we directly reduced NaP activity in cultured rat telencephalic cells using either the specific inhibitor ouabain, or dcAMP, and assessed whether these treatments increased glutamate-induced neuronal death. Since rodent NaP catalytic subunits possess both low (α1) and high (α2/α3) affinity for ouabain, we were able to inhibit selectively the α2 (principally glial) and α3 (neuronal) catalytic subunits without affecting the α1 isoform. Brief exposures (5–60 min) to high ouabain concentrations (1–10 mM), which blocks the activity of all three catalytic subunits, killed differentiated neurons but spared glia. In contrast, differential inhibition of the α2/α3 isoforms (by 1 μM ouabain) was not of itself toxic, but produced a supersensitivity to glutamate. [³H]Ouabain binding studies confirmed that the glutamate neurotoxicity observed varied inversely with the degree of NaP inhibition. Further, this relationship was not absolutely dependent upon ouabain, since reduction in α2/α3 pump activity induced by dcAMP also amplified glutamate toxicity. We conclude glutamate excitotoxicity. Since the distribution of NaP is highly heterogenous in the nervous system, with similar cell types varying greatly in isoform expression, constitutive levels of this isoenzyme could constitute a major factor in the survival of stimulated neurons. Further, factors which directly affect pump activity, such as activation of protein kinase A, may modulate excitotoxicity.     

Stimulatory effects of centrally injected kainate and N-methyl- -aspartate on gastric acid secretion in anesthetized rats

The effects of N-methyl- -aspartate (NMDA), kainate and (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA), ionotropic glutamate agonists, on gastric acid secretion were investigated in the continuously perfused stomach of anesthetized rats. The lateral ventricular (LV) injection of kainate (0.01–1 μg) or NMDA (0.3–3 μg) dose-dependently stimulated gastric acid secretion. AMPA (3–10 μg) also stimulated gastric acid secretion but the effect was very weak. Repeated injections of kainate (0.1 μg) or NMDA (1 μg), at least twice, stimulated gastric acid secretion to a similar degree. The effect of kainate (0.1 μg) was blocked by the kainate receptor antagonists, 6-cyano-7-nitroquinoxaline-2,3-dione disodium (3 μg, LV) and -γ-glutamylaminomethanesulfonic acid (30 μg, LV), but not by NMDA receptor antagonists. The effect of NMDA (10 μg) was blocked by (±)-3-(2-carboxypiperazin-4-yl)-1-propylphosphonic acid (10 μg, LV), a competitive NMDA receptor antagonist, and (+)-5-methyl-10,11-dihydro-5H-dibenzocyclo-hepten-5,10-imine hydrogen maleate (10 μg, LV), a non-competitive NMDA receptor antagonist, but not by kainate receptor antagonists. Moreover, the gastric acid secretion stimulated by kainate and NMDA were completely blocked by systemic atropine injection (1 mg/kg, i.v.) and vagotomy. These findings suggest that kainate and NMDA receptor mechanisms are independently involved in the central nervous system to control gastric acid secretion through vagus cholinergic activation.     

Crucial role of kainate receptors in mediating striatal kainate injection-induced decrease in acetylcholine M1 receptor binding in rat forebrain

We investigated the roles of kainate-, α-amino-3-hydroxy-5-methylisoxazol-4-propionate (AMPA)- and N-methyl- -aspartate (NMDA)-receptors in mediating striatal kainate injection-induced decrease in the binding of acetylcholine M₁ receptors in rat forebrain. After unilateral intrastriatal injection of kainate (4 nmol), the bindings of [³H]kainate (10 nM), [³H]MK-801 (4 nM) and [³H]pirenzepine (4 nM) to the rat ipsilateral forebrain membranes declined, reaching the lowest on day 2 to 4 and recovering on day 8. Saturation binding studies, performed on day 2 post-injection, showed that kainate (1, 2, 4 nmol) dose-dependently decreased B_max and K_d of the three ligands. (+)-5-Methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801), a selective NMDA receptor channel blocker, antagonised (from a dose of 4 nmol) kainate-induced decreases in the bindings of [³H]kainate (up to 20%), [³H]MK-801 (up to 90%) and [³H]pirenzepine (up to 70%). In contrast, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a selective non-NMDA receptor antagonist, almost completely abolished (from a dose of 12 nmol) kainate-induced decreases in the bindings of all the three ligands (up to 95–98%). Cyclothiazide, a selective potentiator that enhances AMPA receptor-mediated responses, significantly enhanced (from a dose of 4 nmol) kainate-induced decrease in the binding of [³H]kainate but not that of [³H]pirenzepine or [³H]MK-801. In summary, these results indicate that striatal kainate injection-induced decrease in the binding of acetylcholine M₁ receptors in rat forebrain is dependent on activation of kainate receptors and, to a certain extent, a consequent involvement of NMDA receptors. These and previous studies provide some evidence showing that kainate receptors might play a crucial role in regulating excitatory amino acids (EAA)-modulated cholinergic neurotransmission in the central nervous system (CNS).     

Idebenone attenuates neuronal degeneration induced by intrastriatal injection of excitotoxins

Previous studies with the N18-RE-105 neuronal-like cell line and primary cortical cultures demonstrate that glutamate can produce a calcium-dependent, delayed form of neuronal degeneration that results from its competitive inhibition of cystine transport, which leads to cellular glutathione depletion and death by oxidative stress. Idebenone, a centrally active antioxidant used to treat multiinfarct dementia, protects cells from this form of glutamate-induced cytotoxicity in vitro. In the present study, we have examined the effects of systemic treatment with idebenone on the neurotoxic consequences of intrastriatal injection of kainic acid, quisqualic acid, or quinolinic acid, an NMDA receptor agonist, on neuronal degeneration. Striatal damage was assessed by quantitative neurochemistry with measurement of choline acetyltransferase activity and glutamate decarboxylase activity, by histochemical analysis for acetylcholinesterase and NADPH diaphorase staining and by behavioral assessment of circling produced by systemic apomorphine treatment 10 days after the unilateral lesion. The results indicate that treatment with idebenone provides significant protection against the neuronal degeneration induced by intrastriatal injection of kainic acid and quisqualic acid, but not the NMDA receptor agonist, quinolinic acid. The results suggest that oxidative stress may contribute to the proximate cause of neuronal degeneration induced by quisqualate and by kainate receptor agonists and that the mechanisms of neuronal degeneration caused by quisqualate/kainate receptor agonists differ from those associated with NMDA receptor agonists.     

NMDA receptor activition by residual glutamate in glutamine preparations: a cautionary note regarding weak NMDA receptor agonists

During an investigation of excitatory amino acids on cultured embryonic Xenopus neurons, we observed that commercial preparations of glutamine had weal agonist activity on NMDA-type glutamate receptors. Threshold responses were observed at 100 μM glutamine, and the dose-response relation did not show inflection or saturation at concentrations of up to 10 mM. However, NMDA receptor activation induced by glutamine probably represented activity of residual glutamate because: (a) recrystallization of glutamine reduced residual glutamate levels (measured by HPLC analysis) and NMDA receptor activation by comparable amounts; and (b) glutamate at concentrations close to those predicted to be present in glutamine preparations elicited currents of similar amplitudes. Our data indicate that residual glutamate at levels of less than 0.05% are sufficient to confound studies of weak NMDA receptor agonists.     

Failure of glycine site NMDA receptor antagonists to protect againstl-2-chloropropionic acid-induced neurotoxicity highlights the uniqueness of cerebellar NMDA receptors

Cultured cerebellar granule cells and cerebellar slices from neonatal rats have been widely used to examine the biochemistry of excitatory amino acid-induced cell death mediated in part by the activation of NMDA receptors. However, the NMDA subunit stoichiometry, producing functional NMDA receptors is different in cultured granule cells, neonatal and adult rat cerebellum as compared to the NMDA receptors in forebrain regions. We have used thel-2-chloropropionic acid (l-CPA) (750 mg/kg) model of NMDA-medialed selective cerebellar granule cell necrosis in vivo to examine the role of the glycine binding site and possible effect of the NR2C subunit (which is largely expressed only in the cerebellum) on granule cell necrosis. The abilities of various NMDA receptor antagonists were examined in vivo to determine the relative contribution of both glutamate and glycine sites involved in thel-CPA-induced neurotoxicity. The potent neuroprotective, non-competitive NMDA receptor antagonist dizocilpine (MK-801) was compared with glutamate and glycine site NMDA antagonists. We have examined a number of markers for thel-CPA-induced granule cell necrosis. Thel-CPA-induced reduction in cerebellar aspartate and glutamate concentrations were used as markers of granule cell necrosis. We also measured the cerebellar water content and sodium concentrations as measures of thel-CPA-induced cerebellar edema that accompanies the granule cell necrosis. Finally the ability of the NMDA antagonists to attenuate thel-CPA-induced reductions in body weight gain and the prevention of the loss in hindlimb function using a behavioral measure of hindlimb retraction were examined. The potent glutamate antagonists, CPP and CGP40116 and dizocilpine prevented thel-CPA-induced locomotor dysfunction and granule cell necrosis as measured by their ability to preventl-CPA-induced reductions in aspartate and glutamate concentrations. CPR CGP40116 and dizocilpine also prevented the appearance of cerebellar edema followingl-CPA administration. In addition, dizocilpine, CPP and CGP40116 were able to partially prevent thel-CPA-induced loss in body weight over the 48 h experimental period. In contrast, none of the glycine partial agonists or antagonists, namely (±)HA-966,d-cycloserine, MDL-29951, DPCQ, MNQX or L-701 252 were able to prevent thel-CPA-induced loss in body weight,l-CPA-induced granule cell necrosis and behavioral disturbances when administered to rats. None of the NMDA antagonists had any effect on the cerebellar neurochemistry when injected alone or had any effect on animal behavior except for dizocilpine, CPP, CGP401 l6 and (±)HA-966 which resulted in a transient sedation for between three and five hours immediately following their administration. In conclusion, we demonstrate that NMDA open channel blockade and glutamate antagonists can provide full neuroprotection against thel-CPA-induced granule cell necrosis. The failure of the glycine partial agonists and antagonists to provide any neuroprotection againstl-CPA-induced neurotoxicity in the cerebellum contrasts with their neuroprotective efficacy in other animal models of excitatory amino acid-induced cell death in forebrain regions in vivo. We therefore suggest that the glycine site plays a lesser role in modulating NMDA receptor function in the cerebellum and may explain why cells expressing NMDA receptors composed of NR1/NR2C subunits are particularly resistant to excitatory amino acid-induced neurotoxicity.     

Effects of MK-801 on glutamate-induced swelling of astrocytes in primary cell culture

The effects of glutamate and its agonists and antagonists on the swelling of primary astrocytes were studied. Glutamate (Glu), aspartate (Asp), homocysteate (HCA), and quisqualate (Quis) at 1 mM concentration caused a significant increase in astrocytic swelling as measured by the 3-0-methyl-[14C]-glucose, whereas kainate (KA), N-methyl-D-aspartate (NMDA), and receptor antagonists 2-amino-5-phosphonovaleric acid (APV), 2-amino-7-phosphonohepatanoic acid (APH), and kynurenic acid (Kynu) were not effective. This glial swelling was time-dependent since 1-hr or greater incubations with Glu or its agonists were needed to produce such an effect. Preincubation of glutamate or NMDA receptor anatogonists including Kynu, APH, and APV failed to ameliorate the Glu effects. However, MK-801, a noncompetitive NMDA antagonist, when added to the Glu-incubated astrocytes significantly reduced Glu-induced astrocytic swelling. MK-801 was also effective in reducing the astrocytic swelling induced by agonists including Asp, Quis, and HCA, suggesting that those agonists may share similar mechanisms of Glu in inducing astrocytic swelling. Since the cultured astrocytes lack the NMDA receptors, our data suggest that the observed beneficial effects of MK-801 on excitotoxin-induced swelling of astrocytes may be mediated by mechanisms other than NMDA receptors.