Evidence that excitatory amino acid receptors within the temporomandibular joint region are involved in the reflex activation of the jaw muscles

We have previously shown that injection of the inflammatory irritant and small-fiber excitant mustard oil (MO) into the temporomandibular joint (TMJ) region can reflexively induce a prolonged increase in the activity of both digastric and masseter muscles in rats. It is possible that peripheral excitatory amino acid (EAA) receptors play a role in this effect, because MO-evoked increases in jaw muscle activity are attenuated by preapplication of the noncompetitive NMDA receptor antagonist MK-801 into the TMJ region. In the present study the EAA receptor agonists glutamate, NMDA, kainate, and AMPA were applied locally to the TMJ region. Jaw muscle responses similar to those evoked by MO application to the TMJ region were achieved with glutamate, NMDA, AMPA, and kainate. Repeated application of glutamate, NMDA, or AMPA at intervals of 30 min evoked responses in the ipsilateral jaw muscles that were of comparable magnitude. Co-application of the NMDA receptor antagonist DL-2-amino-5-phosphonovalerate (0.5 micromol) significantly reduced the magnitude of the glutamate- and NMDA-evoked ipsilateral jaw muscle responses without affecting responses evoked by AMPA. In contrast, co-application of the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (1 nmol) significantly reduced the magnitude of the glutamate- and AMPA-evoked ipsilateral jaw muscle responses without affecting responses evoked by NMDA. This evidence suggests that both NMDA and non-NMDA EAA receptor types are located within the TMJ region and may contribute to jaw muscle activity that can be reflexively evoked from the TMJ region.     

Cobalt accumulation in neurons expressing ionotropic excitatory amino acid receptors in young rat spinal cord: morphology and distribution

Excitatory amino acids (EAA) acting on N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and kainate receptors play an important role in synaptic transmission in the spinal cord. Quantitative autoradiography and physiological experiments suggest that NMDA receptors are localized mainly in lamina II while kainate and AMPA receptors are found on both dorsal and ventral horn neurons. However the cell types expressing EAA receptors and their laminar distribution is not known. We have used a cobalt uptake method to study the morphology and distribution of spinal cord neurons expressing AMPA, kainate, or NMDA excitatory amino acid receptors in the lumbar enlargement of the rat spinal cord. The technique involved superfusion of hemisected spinal cords of 14 day-old rat pups in vitro with excitatory amino acid receptor ligands in the presence of CoCl2. Cobalt has been shown to enter cells through ligand-gated ion channels in place of Ca2+. Cells which accumulated cobalt ions following activation by ionotropic excitatory amino acid receptors were visualized histochemically. The cobalt uptake generated receptor-specific labeling of cells, as the NMDA receptor antagonist D-(-)-2-amino-(5)-phosphonovaleric acid (D-AP-5) (20 microM) blocked the NMDA, but not kainate-induced cobalt uptake. The kainate-induced cobalt labeling was reduced by the non-selective excitatory amino acid receptor antagonist kynurenic acid (4 mM). Passive opening of the voltage-gated Ca(2+)-channels by KCl (50 mM) did not result in cobalt uptake, indicating that cobalt enters the cells through ligand-gated Ca(2+)-channels. AMPA (500 microM), kainate (500 microM), or NMDA (500 microM) each induced cobalt uptake with characteristic patterns and distributions of neuronal staining. Overall, kainate induced cobalt uptake in the greatest number of neuronal staining. Overall, kainate induced cobalt uptake in the greatest number of neuronal perikarya while NMDA-induced uptake was the lowest. AMPA and kainate, but not NMDA superfusion, resulted in cobalt labeling of glial cells. Our results show that the cobalt uptake technique is a useful way to study the morphology and distribution of cells expressing receptors with ligand-gated Ca2+ channels.     

Domoic acid neurotoxicity in cultured cerebellar granule neurons is mediated predominantly by NMDA receptors that are activated as a consequence of excitatory amino acid release

The participation of NMDA and non-NMDA receptors in domoic acid-induced neurotoxicity was investigated in cultured rat cerebellar granule cells (CGCs). Neurons were exposed to 300 microM L-glutamate or 10 microM domoate for 2 h in physiologic buffer at 22 degrees C followed by a 22-h incubation in 37 degrees C conditioned growth media. Excitotoxic injury was monitored as a function of time by measurement of lactate dehydrogenase (LDH) activity in both the exposure buffer and the conditioned media. Glutamate and domoate evoked, respectively, 50 and 65% of the total 24-h increment in LDH efflux after 2 h. Hyperosmolar conditions prevented this early response but did not significantly alter the extent of neuronal injury observed at 24 h. The competitive NMDA receptor antagonist D(-)-2-amino-5-phosphonopentanoic acid and the non-NMDA receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX) reduced glutamate-induced LDH efflux totals by 73 and 27%, respectively, whereas, together, these glutamate receptor antagonists completely prevented neuronal injury. Domoate toxicity was reduced 65-77% when CGCs were treated with competitive and noncompetitive NMDA receptor antagonists. Unlike the effect on glutamate toxicity, NBQX completely prevented domoate-mediated injury. HPLC analysis of the exposure buffer revealed that domoate stimulates the release of excitatory amino acids (EAAs) and adenosine from neurons. Domoate-stimulated EAA release occurred almost exclusively through mechanisms related to cell swelling and reversal of the glutamate transporter. Thus, whereas glutamate-induced injury is mediated primarily through NMDA receptors, the full extent of neurodegeneration is produced by the coactivation of both NMDA and non-NMDA receptors. Domoate-induced neuronal injury is also mediated primarily through NMDA receptors, which are activated secondarily as a consequence of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate receptor-mediated stimulation of EAA efflux.     

Enhancement of the N-methyl-D-aspartate response in spinal dorsal horn neurons by cAMP-dependent protein kinase

Glutamate-gated ion channels mediate excitatory synaptic transmission in the central nervous system and are involved in synaptic plasticity, neuronal development and excitotoxicity (5,24). These ionotropic glutamate receptors were classified according to their preferred agonists as AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid), KA (kainate), and NMDA (N-methyl-D-aspartate) receptors [Trends Pharmacol. Sci., 11 (1990) 25-33]. The present study of NMDA receptor channels expressed in acutely isolated spinal dorsal horn (DH) neurons of young rat reveals that they are subject to modulation through the adenylate cyclase cascade. Whole-cell voltage-clamp recording mode was used to examine the effect of adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase (PKA) on the responses of DH neurons to NMDA. Whole-cell current response to NMDA was enhanced by 8 Br-cAMP, a membrane permeant analog of cAMP or by intracellular application of cAMP or catalytic subunit of PKA.     

Gender-specific factors in the utilization of medical services during adolescence

Properties of receptors for glutamate, consisting of kainate, alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and N-methyl-D-aspartate (NMDA), and gamma-aminobutyric acid (GABA) in the rat trigeminal ganglion (TG) neurons were studied by means of the whole-cell patch-clamp technique. 1. All TG neurons (diameter 20 approximately 40 microns) responded to GABA, but none of them responded to AMPA, NMDA, and glycine. TG neurons responding to kainate were smaller than 25 microns and desensitized by prolonged agonist exposure. 2. On the basis of the dose-response curves, EC50 and Hill coefficient were found to be 13. 7 microM and 1.2 for kainate, while those for GABA were 28.6 microM and 2.0, respectively. 3. Most kainate receptors in the TG neurons showed linear current-voltage (I-V) relations, though some showed inward rectification. The "Instantaneous" I-V relationship for GABA was linear, where as the "steady-state" I-V relationship produced by ramp changes in potential showed outward rectification in some neurons. 4. The number of TG neurons responding to kainate increased until the 8th postnatal day, while all the recorded neurons responded to GABA in postnatal days. The results indicated that there are kainate and GABAA receptors in the TG neurons and suggested that they are composed of various subunits.     

Role of desensitization and subunit expression for kainate receptor-mediated neurotoxicity in murine neocortical cultures

The neurotoxic actions of kainate and domoate were studied in cultured murine neocortical neurons at various days in culture and found to be developmentally regulated involving three components of neurotoxicity: (1) toxicity via indirect activation of N-methyl-D-aspartate (NMDA) receptors, (2) toxicity mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors, and (3) toxicity that can be mediated by kainate receptors when desensitization of the receptors is blocked. The indirect action at NMDA receptors was discovered because (5R, 10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-im ine (MK-801), an NMDA receptor antagonist, was able to block part of the toxicity. The activation of NMDA receptors is most likely a secondary effect resulting from glutamate release upon kainate or domoate stimulation. 1-(4-Aminophenyl)-3-methylcarbamyl-4-methyl-3,4-dihydro-7,8-ethyle nedioxy-5H-2,3-benzodiazepine (GYKI 53655), a selective AMPA receptor antagonist, abolished the remaining toxicity. These results indicated that kainate- and domoate-mediated toxicity involves both the NMDA and the AMPA receptors. Pretreatment of the cultures with concanavalin A to prevent desensitization of kainate receptors led to an increased neurotoxicity upon stimulation with kainate or domoate. In neurons cultured for 12 days in vitro a small but significant neurotoxic effect was observed when stimulated with agonist in the presence of MK-801 and GYKI 53655. This indicates that the toxicity is produced by kainate receptors in mature cultures. Examining the subunit expression of the kainate receptor subunits GluR6/7 and KA2 did, however, not reveal any major change during development of the cultures.     

Interactions between metabotropic and ionotropic glutamate receptor agonists in the rat spinal cord in vivo

Microelectrophoretic application of the non-selective metabotropic glutamate receptor (mGluR) agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid [(1S,3R)-ACPD] and the group I selective mGluR agonist (RS)-3,5-dihydroxyphenylglycine [(RS)-3,5-DHPG] potentiated the responses of rat spinal neurones to the cyclically-ejected ionotropic excitatory amino acid (EAA) agonists NMDA, AMPA and kainate in vivo. Potentiation was not selective between the three ionotropic responses and was paralleled by an enhancement of background activity in spontaneously active cells. "Correcting" spike count data for this increase in background activity showed that the EAA responses were not potentiated beyond the apparent enhancement of cell excitability. Neither mGluR agonist produced potentiation when NMDA/AMPA cycling was superimposed on background discharge held constant with kainate. It is concluded that potentiation produced by both (1S,3R)-ACPD and (RS)-3,5-DHPG is secondary to an enhancement of cell excitability rather than being due to a specific interaction with ionotropic EAA receptors. The mechanism of excitability enhancement cannot be determined by extracellular recording, but group I mGluRs are most likely to be responsible.     

Rapid Ca2+ entry through Ca2+-permeable AMPA/Kainate channels triggers marked intracellular Ca2+ rises and consequent oxygen radical production

The widespread neuronal injury that results after brief activation of highly Ca2+-permeable NMDA channels may, in large part, reflect mitochondrial Ca2+ overload and the consequent production of injurious oxygen radicals. In contrast, AMPA/kainate receptor activation generally causes slower toxicity, and most studies have not found evidence of comparable oxygen radical production. Subsets of central neurons, composed mainly of GABAergic inhibitory interneurons, express AMPA/kainate channels that are directly permeable to Ca2+ ions. Microfluorometric techniques were performed by using the oxidation-sensitive dye hydroethidine (HEt) to determine whether the relatively rapid Ca2+ flux through AMPA/kainate channels expressed on GABAergic neurons results in oxygen radical production comparable to that triggered by NMDA. Consistent with previous studies, NMDA exposures triggered increases in fluorescence in most cultured cortical neurons, whereas high K+ (50 mM) exposures (causing depolarization-induced Ca2+ influx through voltage-sensitive Ca2+ channels) caused little fluorescence change. In contrast, kainate exposure caused fluorescence increases in a distinct subpopulation of neurons; immunostaining for glutamate decarboxylase revealed the responding neurons to constitute mainly the GABAergic population. The effect of NMDA, kainate, and high K+ exposures on oxygen radical production paralleled the effect of these exposures on intracellular Ca2+ levels when they were monitored with the low-affinity Ca2+-sensitive dye fura-2FF, but not with the high-affinity dye fura-2. Inhibition of mitochondrial electron transport with CN- or rotenone almost completely blocked kainate-triggered oxygen radical production. Furthermore, antioxidants attenuated neuronal injury resulting from brief exposures of NMDA or kainate. Thus, as with NMDA receptor activation, rapid Ca2+ influx through Ca2+-permeable AMPA/kainate channels also may result in mitochondrial Ca2+ overload and consequent injurious oxygen radical production.     

Selective hypothermic perfusion of canine brain

A method for selective brain cooling by profound hemodilution with cold Ringer's lactate solution was previously reported in 1992. We recently modified this technique by combining it with an ultrafiltration and rewarming circuit between the left jugular vein and the inferior vena cava. We used 12 beagle dogs to study the efficacy of selective cerebral hypothermia induced by this modified technique. The brain temperature decreased to 28 degrees C within 5.4 +/- 2.7 minutes and to 20 degrees C within 15.5 +/- 9.4 minutes. The lowest brain and rectal temperatures were 17.0 +/- 1.8 degrees C and 32.1 +/- 2.2 degrees C, respectively. All animals survived in good condition without evidence of neurological deficits until they were killed at 10 weeks. Histological examination of the brains with 2,3,5-triphenyltetrazolim chloride demonstrated no evidence of ischemic lesions, and even in the hippocampus, there was no evidence of ischemic neuronal damage.     

Upregulation of Calbindin-D-28k immunoreactivity by excitatory amino acids

Excessive or prolonged exposure to excitatory amino acids (EAA) are thought to be neurotoxic by altering calcium homeostasis. A protective role of Calbindin-D-28 k (Calbindin) has been postulated due to its capacity to buffer calcium. Calbindin is highly expressed in the Purkinje cells (PCs), of the cerebellar cortex. Changes of the Calbindin immunoreactivity (IR) by the EAA has been here investigated in cerebellar slices maintained in vitro. It was found that at low temperature, PCs are very slightly immunoreactive and therefore the experiments were done at 22 degrees C. The results show that Calbindin-IR increases in PCs exposed to the neurotoxic agonists, Kainic acid (KA) and AMPA as well as to glutamate (Glu), the endogenous EAA. The increase is very rapid and slowly reversible; is induced by excitatory and excitotoxic concentrations of the agonists; is independent of the calcium influx. While KA- and AMPA-induced Calbindin-IR is blocked by CNQX, the KA/AMPA receptor antagonist, Glu-induced Calbindin-IR is only slightly decreased by CNQX and AP5, the NMDA receptor antagonist. It is concluded that Calbindin-containing neurons can increase their calcium buffering capacity in response to EAA binding to specific receptors, the response being independent of, but concomitant to calcium influx.     

Distinct roles for sodium, chloride, and calcium in excitotoxic dendritic injury and recovery

The postsynaptic neuronal dendrite is selectively vulnerable to hypoxic-ischemic brain injury and glutamate receptor overactivation. We explored the glutamate receptor pharmacology and ionic basis of rapid, reversible alterations in dendritic shape which occur in cultured neurons exposed to glutamate. Dendrite morphology was assessed with the fluorescent membrane tracer, DiI, or immunofluorescence labeling of the somatodendritic protein, MAP2. Cortical cultures derived from 15-day-old mouse embryos underwent segmental dendritic beading when exposed to NMDA, AMPA, or kainate, but not to metabotropic glutamate receptor agonists. Varicosity formation in response to NMDA or kainate application was substantially attenuated in reduced sodium buffer (substituted with N-methyl-D-glucamine). Furthermore, veratridine-induced sodium entry mimicked excitotoxic alterations in dendrites and additionally caused varicosity formation in axons. Solutions deficient in chloride (substituted with Na methylsulfate) and antagonists of chloride-permeable GABA/glycine receptors reduced NMDA- or kainate-induced varicosity formation. An increase in dendrite volume was observed as varicosities formed, and varicosity formation was attenuated in sucrose-supplemented hypertonic media. Despite marked structural changes affecting virtually all neurons, dendrite shape returned to normal within 2 h of terminating glutamate receptor agonist application. Neurons exposed to kainate recovered more rapidly than those exposed to NMDA, and neurons exposed to NMDA in calcium-free buffer recovered more rapidly than cells treated with NMDA in normal buffer. While sodium, chloride, and water entry contribute to excitotoxic dendritic injury acutely, calcium entry through NMDA receptors results in lasting structural changes in damaged dendrites.     

Multiple classes of the oligodendrocyte lineage are highly vulnerable to excitotoxicity

We have recently shown that galactocerebroside (Gal-C)-expressing oligodendrocytes are highly vulnerable to (AMPA)/kainate receptor-mediated death. Here we examined the vulnerability of cells at different developmental stages of the oligodendrocyte lineage to AMPA/kainate receptor-mediated excitotoxicity. Oligodendrocyte precursor cells, pre-oligodendrocytes and mature oligodendrocytes were killed by 24 h exposures to low concentrations of kainate (30-100 microM). Death was attenuated by the AMPA/kainate receptor antagonist 6-nitro-7-sulfamoylbenzo(f)quinoxaline-2,3-dione (NBQX). The high vulnerability of oligodendrocytes and their precursors to AMPA/kainate receptor excitotoxicity may represent an important mechanism of white matter damage resulting from trauma or ischemia in the perinatal and adult central nervous system (CNS).     

Glutamate receptor-mediated calcium entry in neurons derived from P19 embryonal carcinoma cells

We have examined the control of calcium elevation by glutamate in neurons derived from the mouse P19 embryonal carcinoma cell line. Following transient exposure to retinoic acid, P19 cells differentiate into neurons that express both NMDA and non-NMDA glutamate receptor subtypes. Fluorescence videomicroscopy using the indicator fura-2 revealed concentration-dependent elevation in cytosolic calcium levels with exposure to NMDA or kainate. Replacement of extracellular sodium with N-methylglucamine significantly reduced the action of kainate. Exposure to high K+ medium also elicited an elevation of cytosolic calcium in P19 cells, which was partially inhibited by the calcium channel antagonist nimodipine. These experiments suggest that the elevation in calcium produced by kainate involves the activation of voltage-gated calcium channels as a consequence of membrane depolarization, in contrast to direct calcium entry through NMDA receptor channels. Whole-cell recordings revealed that P19 NMDA receptors were highly permeable to calcium (PCa/PNa = 5.6 +/- 0.2). In most cells, channels gated by kainate displayed low permeability to calcium; the median permeability ratio, PCa/PNa, was 0.053 (range 0.045 to 0.132). Activation of peak currents by NMDA, glycine, and kainate was half-maximal at 24 microM, 240 nM, and 81 microM, respectively. In addition, cadmium-sensitive currents through voltage-gated calcium channels were recorded in P19 cells bathed in barium/TEA chloride. Staining with antibodies directed against AMPA receptor subunits revealed wide-spread immunoreactivity for anti-GluR-B/C and anti-GluR-B/D. About half of the P19 cells were stained with antibodies selective for GluR-D but there was little or no immunoreactivity for the GluR-A subunit.     

Ibudilast prevents oligodendroglial excitotoxicity

Previously we have demonstrated that cells of oligodendroglial lineage express non-N-methyl-D-aspartate (NMDA) glutamate receptor (GluR) genes and are damaged by kainate induced Ca2+ influx via non NMDA GluR channels of the alpha-amino-3-hydroxy-5-methyl 4 isoxazole propionate (AMPA) type, representing oligodendroglial excitotoxicity. We here present the finding that ibudilast, which is used clinically for treat patients with asthma and cerebrovascular diseases, prevents oligodendroglia excitotoxicity. The oligodendrocyte like cells (OLC), differentiated from the CG-4 cell line established from rat oligodendrocyte-type 2 astrocyte (O-2A) progenitor cells, were exposed to 2 mM kainate for 24 h and cell death was evaluated by measuring activity of lactate dehydrogenase (LDH) released into the culture medium. Kainate induced cell death was prevented by 10 to 100 microM ibudilast, which increased intracellular cAMP. A 45Ca2+ influx study revealed that ibudilast attenuated kainate-induced Ca2+ influx. Inhibition of kainate-induced Ca2+ influx by ibudilast was decreased by H-89, a protein kinase A (PKA) inhibitor, but increased by okadaic acid, an inhibitor of phosphatase 1 and 2A. Therefore, we concluded that ibudilast prevented oligodendroglial excitotoxicity by a PKA-dependent phosphorylation process resulting in decreased kainate-induced Ca2+ influx.     

Selective loss of [3H]kainic acid and [3H]AMPA binding in layer VI of frontal cortex in Huntington's disease

Excitatory amino acid neurotoxicity has been proposed to cause the neostriatal neuronal degeneration of Huntington's disease (HD); N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), and kainate receptors have been hypothesized to play important roles in this process. We have recently reported a loss of neurons in layer VI of the cerebral cortex in HD. Using quantitative autoradiographic methods, we have now measured NMDA, AMPA, and kainate receptor binding in the frontal cerebral cortex of the brains of controls and individuals with HD. We find no change in NMDA receptor binding but a selective decrease in kainate and AMPA receptor binding in layer VI. These data suggest that cerebral cortical neurons possessing kainate or AMPA receptors may be selectively vulnerable in individuals with HD.     

AMPA and NMDA receptors expressed by differentiating Xenopus spinal neurons

N-methyl--aspartate (NMDA) receptors are often the first ionotropic glutamate receptors expressed at early stages of development and appear to influence neuronal differentiation by mediating Ca2+ influx. Although less well studied, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors also can generate Ca2+ elevations and may have developmental roles. We document the presence of AMPA and NMDA class receptors and the absence of kainate class receptors with whole cell voltage-clamp recordings from Xenopus embryonic spinal neurons differentiated in vitro. Reversal potential measurements indicate that AMPA receptors are permeable to Ca2+ both in differentiated neurons and at the time they first are expressed. The PCa/Pmonocation of 1.9 is close to that of cloned Ca2+-permeable AMPA receptors expressed in heterologous systems. Ca2+ imaging reveals that Ca2+ elevations are elicited by AMPA or NMDA in the absence of Mg2+. The amplitudes and durations of these agonist-induced Ca2+ elevations are similar to those of spontaneous Ca2+ transients known to act as differentiation signals in these cells. Two sources of Ca2+ amplify AMPA- and NMDA-induced Ca2+ elevations. Activation of voltage-gated Ca2+ channels by AMPA- or NMDA-mediated depolarization contributes approximately 15 or 30% of cytosolic Ca2+ elevations, respectively. Activation of either class of receptor produces elevations of Ca2+ that elicit further release of Ca2+ from thapsigargin-sensitive but ryanodine-insensitive stores, contributing an additional approximately 30% of Ca2+ elevations. Voltage-clamp recordings and Ca2+ imaging both show that these spinal neurons express functional AMPA receptors soon after neurite initiation and before expression of NMDA receptors. The Ca2+ permeability of AMPA receptors, their ability to generate significant elevations of [Ca2+]i, and their appearance before synapse formation position them to play roles in neural development. Spontaneous release of agonists from growth cones is detected with glutamate receptors in outside-out patches, suggesting that spinal neurons are early, nonsynaptic sources of glutamate that can influence neuronal differentiation in vivo.     

The hydroxyl radical scavenger Nicaraven inhibits glutamate release after spinal injury in rats

Neuronal degeneration after trauma is mediated in part by release of excitatory amino acids (EAAs) and oxygen free radicals (OFR). We evaluated the effect of i.v. treatment with a hydroxyl radical scavenger ((+/-)-N,N'-propylenedinicotinamide; AVS) and spinal hypothermia (33 degrees C) on spinal CSF glutamate release after spinal trauma. In a control group, spinal compression evoked at 10 min a significant increase (5-fold) in glutamate which declined over 4 h (2.1-fold). AVS treatment attenuated glutamate release but had no additive effect. These data suggest that this compound can be effective in modulating spinal excitotoxicity resulting from increased OFR synthesis and corresponding potentiation of EAA release.     

Kainate excitotoxicity is mediated by AMPA- but not kainate-preferring receptors in embryonic rat hippocampal cultures

We investigated kainate-induced excitotoxicity in embryonic rat hippocampal cells cultured in a chemically defined medium. Treatment with kainate for 24 h resulted in neuronal death, as assessed by the release of lactate dehydrogenase into the culture media. This neurotoxic effect was kainate dose- and culture age-dependent. EC50 of kainate was 127 +/- 11 microM. 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo (f)quinoxaline (NBQX) completely blocked the toxicity, while MK801, an N-methyl-D-aspartate (NMDA) receptor antagonist, also blocked it but not completely. Furthermore, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) attenuated the kainate injury, while the selective and noncompetitive AMPA-preferring receptor antagonist 1-(4-aminophenyl)-4-methyl-7, 8-methylenedioxy-5H-2,3-benzo-diazepine (GYKI 52466) blocked it completely. Concanavalin A (ConA), which potentiates the response to kainate at kainate-preferring receptors, had little effect on kainate toxicity. Further, AMPA alone induced little toxicity, but produced remarkable toxicity when cyclothazide was used to block the desensitization of AMPA-preferring receptors. These results indicate that kainate excitotoxicity in hippocampal cultures is mediated by AMPA- but not kainate-preferring receptors, and that it involves NMDA-receptor-mediated toxicity. The non-desensitizing response at AMPA-preferring receptors may play an important role in kainate-induced excitotoxicity.     

Chondroitin sulfate proteoglycans protect cultured rat's cortical and hippocampal neurons from delayed cell death induced by excitatory amino acids

Protective effects of chondroitin sulfate proteoglycans (CSPGs) from rat's brain against delayed cell death induced by excitatory amino acids were examined in cultured neurons of the rat. CSPGs reduced delayed neuronal death induced by 10 min exposure to glutamate at a concentration between 100 microM and 1 mM when lactate dehydrogenase activity of culture medium was assayed 24 h after the exposure. CSPGs also protected neuronal death induced by 200 microM N-methyl-D-aspartate (NMDA), kainate or 100 microM alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA). CSPGs reduced death of cortical and hippocampal neurons even when they were administered at 2 h, but not 6 and 12 h, after the exposure to glutamate. These results indicate that CSPGs may have a neuroprotective action against acute noxious conditions in the brain.