Differential effects of NMDA and AMPA/kainate receptor antagonists on superoxide production and MnSOD activity in rat brain following intrahippocampal injection

The involvement of NMDA and AMPA/kainate receptors in the induction of superoxide radical production in the rat brain was examined after injection of kainate, non-NMDA receptor agonist, kainate plus 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), selective AMPA/kainate receptor antagonist, or kainate plus 2-amino-5-phosphonopentanoic acid (APV), selective NMDA receptor antagonist. Competitive glutamate receptor antagonists were injected with kainate unilaterally into the CA3 region of the rat hippocampus. We investigated superoxide production and mitochondrial MnSOD activity after injection. The measurements took place at different times (5, 15 min, 2, 48 h and 7 days) in the ipsi- and contralateral hippocampus, forebrain cortex, striatum, and cerebellum homogenates. Used glutamate antagonists APV and CNQX both expressed sufficient neuroprotection in sense of decreasing superoxide production and increasing MnSOD levels, but with differential effect in mechanisms and time dynamics. Our findings suggest that NMDA and AMPA/kainate receptors are differentially involved in superoxide production. Following intrahippocampal antagonists injection they, also, interpose different neuroprotection effect on the induction of MnSOD activity in distinct brain regions affected by the injury, which are functionally connected via afferents and efferents. It suggests that MnSOD protects the cells in these regions from superoxide-induced damage and therefore may limit the retrograde and anterograde spread of neurotoxicity.     

Glutamate receptors mediate regulation of Na pump isoform activities in neurons

Rat cerebral neurons matured in culture were stimulated with glutamate analogues, and the K+ uptake activities of Na pump isoforms were measured. Ionotropic receptor agonists, kainate, AMPA, and NMDA, increased total K+ uptake activity via activation of the alpha2/alpha3 isoforms and an inhibition of the alpha1 isoform as reported previously for glutamate. The effects of kainate or AMPA were antagonized by CNQX and those of NMDA were by APV or MK-801. In contrast, metabotropic receptor agonist ACPD had no effects on the Na pump isoform activities. Glutamate transporter substrate, PDC, was effective, but NMDA receptor antagonists abolished the effects of PDC. These results suggest that the ionotropic glutamate receptors mediate the regulation of Na pump isoform activities in neurons.     

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.     

Interleukin-1beta promotes oligodendrocyte death through glutamate excitotoxicity

Glutamate excitotoxicity is implicated in the progressive loss of oligodendrocytes in multiple sclerosis, but how glutamate metabolism is dysregulated in the disease remains unclear. Because there is microglia activation in all stages of multiple sclerosis, we determined whether a microglia product, interleukin-1beta, could provide the mechanism for glutamate excitotoxicity. We found that whereas interleukin-1beta did not kill oligodendrocytes in pure culture, it produced apoptosis of oligodendrocytes in coculture with astrocytes and microglia. This requirement for a mixed glia environment suggests that interleukin-1beta impairs the well-described glutamate-buffering capacity of astrocytes. In support, antagonists at AMPA/kainate glutamate receptors, NBQX and CNQX, blocked the interleukin-1beta toxicity to oligodendrocytes. Another microglia/macrophage cytokine, tumor necrosis factor-alpha, also evoked apoptosis of oligodendrocytes in a mixed glia environment in an NBQX-blockable manner. These results provide a mechanistic link between the persistent and insidious microglia activation that is evident in all stages of multiple sclerosis, with the recent appreciation that glutamate excitotoxicity leads to the destruction of oligodendrocytes in the disease.     

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.     

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.     

Vascular endothelial growth factor increases the function of calcium-impermeable AMPA receptor GluA2 subunit in astrocytes via activation of protein kinase C signaling pathway

Astrocytic calcium signaling plays pivotal roles in the maintenance of neural functions and neurovascular coupling in the brain. Vascular endothelial growth factor (VEGF), an original biological substance of vessels, regulates the movement of calcium and potassium ions across neuronal membrane. In this study, we investigated whether and how VEGF regulates glutamate-induced calcium influx in astrocytes. We used cultured astrocytes combined with living cell imaging to detect the calcium influx induced by glutamate. We found that VEGF quickly inhibited the glutamate/hypoxia-induced calcium influx, which was blocked by an AMPA receptor antagonist CNQX, but not D-AP5 or UBP310, NMDA and kainate receptor antagonist, respectively. VEGF increased phosphorylation of PKCα and AMPA receptor subunit GluA2 in astrocytes, and these effects were diminished by SU1498 or calphostin C, a PKC inhibitor. With the pHluorin assay, we observed that VEGF significantly increased membrane insertion and expression of GluA2, but not GluA1, in astrocytes. Moreover, siRNA-produced knockdown of GluA2 expression in astrocytes reversed the inhibitory effect of VEGF on glutamate-induced calcium influx. Together, our results suggest that VEGF reduces glutamate-induced calcium influx in astrocytes via enhancing PKCα-mediated GluA2 phosphorylation, which in turn promotes the membrane insertion and expression of GluA2 and causes AMPA receptors to switch from calcium-permeable to calcium-impermeable receptors, thereby inhibiting astrocytic calcium influx. The present study reveals that excitatory neurotransmitter glutamate-mediated astrocytic calcium influx can be regulated by vascular biological factor via activation of AMPA receptor GluA2 subunit and uncovers a novel coupling mechanism between astrocytes and endothelial cells within the neurovascular unit.     

Tumor necrosis factor-alpha induces cFOS and strongly potentiates glutamate-mediated cell death in the rat spinal cord

Excitotoxic cell death due to glutamate release is important in the secondary injury following CNS trauma or ischemia. Proinflammatory cytokines also play a role. Both glutamate and tumor necrosis factor-alpha (TNF(alpha)) are released immediately after spinal cord injury. Neurophysiological studies show that TNF(alpha) can potentiate the effects of glutamatergic afferent input to produce hyperactivation of brain-stem sensory neurons. Therefore, we hypothesized that TNF(alpha) might act cooperatively with glutamate to affect cell death in the spinal cord as well. Nanoinjections of either TNF(alpha) (60 pg) or kainate (KA; 32 ng) alone into the thoracic gray resulted in almost no tissue damage or cell death 90 min after injection. However, the combination of TNF(alpha) plus KA at these same doses produced a large area of tissue necrosis and neuronal cell death, an effect which was blocked by the AMPA receptor antagonist CNQX (17 ng). These results suggest that secondary injury may involve potentiation of AMPA receptor-mediated excitatory cell death by TNF(alpha).     

Glutamate Induces c-fos Proto-oncogene Expression and Inhibits Proliferation in Oligodendrocyte Progenitors: Receptor Characterization

The effect of glutamate on c-fos expression in oligodendrocyte progenitors was investigated by Northern blot analysis. Glutamate caused rapid and transient induction. Both 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX) and 6,7-dinitroquinoxaline-2,3-dione (DNQX), two competitive non-NMDA ionotropic receptor antagonists, reduced glutamate-induced c-fos expression, whereas the NMDA antagonist MK-801 was ineffective. In addition, the glutamate receptor agonists (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid hydrobromide (AMPA) and kainate strongly induced c-fos. However, the metabotropic receptor agonist trans-(±)-1-amino-(1S,3R)-cyclopentanedicarboxylic acid (trans-(±)-ACPD) did not increase c-fos mRNA level and the antagonist L-(+)-2-amino-3-phosphonopropionic acid did not block glutamate-induced c-fos mRNA. These findings indicate that c-fos induction in oligodendrocyte progenitors is mediated through the AMPA/kainate receptors, while NMDA and metabotropic receptor subtypes are not involved. Chelation of extracellular calcium by EDTA prevented glutamate-induced c-fos expression. Similarly, the protein kinase C inhibitor 1-(5-isoquinoline-sulphonyl)-2-methylpiperazine dihydrochloride (H7) and down-regulation of protein kinase C by prolonged exposure to phorbol-12-myristate 13-acetate blocked c-fos induction. These results suggest that induction of c-fos through AMPA/kainate receptors is dependent on extracellular calcium influx and involves downstream activation of phorbol ester-sensitive protein kinase C. The effect of glutamate on oligodendrocyte progenitor proliferation was assessed by [³H]thymidine incorporation. Glutamate and the agonists kainate and AMPA, but not trans-(±)-ACPD, caused a dose-dependent decrease in [³H]thymidine incorporation. All these pharmacological agents were not toxic to oligodendrocyte progenitors. CNQX reversed the inhibitory effects produced by glutamate and the various agonists. These results suggest that glutamate may modulate the growth and differentiation of oligodendrocytes in the central nervous system.     

Non-NMDA receptor-mediated neurotoxicity in cortical culture

The neurotoxicity of 3 non-NMDA glutamate receptor agonists--kainate, alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA), and quisqualate--was investigated quantitatively in dissociated murine cortical cultures. Five minute exposure to 500 microM kainate, but not AMPA, produced widespread acute neuronal swelling. Kainate-induced swelling was resistant to 2-amino-5-phosphonovalerate (APV) or replacement of extracellular sodium with choline but attenuated by either kynurenate or low concentrations of quisqualate. Unlike NMDA agonists, kainate or AMPA did not produce much late neuronal loss after a 5 min exposure. In contrast, 5 min exposure to 500 microM quisqualate produced both acute neuronal swelling and widespread late neuronal degeneration. This acute swelling was blocked by APV or by replacement of extracellular sodium by choline, consistent with mediation by NMDA receptors; we speculate that high concentrations of quisqualate may directly activate NMDA receptors or induce the release of endogenous glutamate. Quisqualate-induced late neuronal degeneration may be due to another unexpected process: cellular quisqualate uptake and delayed release, converting brief addition into prolonged exposure. Hours after thorough washout of exogenously added quisqualate, micromolar concentrations could be detected in the bathing medium by high performance liquid chromatography. With lengthy exposure (20-24 hr), all 3 non-NMDA agonists were potent neurotoxins, able to destroy neurons with EC50's of about 20 microM for kainate, 4 microM for AMPA, and 1 microM for quisqualate. Kynurenate and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), but not APV or L-glutamate diethyl ester, were effective in attenuating the neuronal degeneration induced by these agonists. CNQX was about 3 times more selective than kynurenate against kainate-induced neuronal injury, but CNQX was still nearly equipotent with APV against NMDA-induced injury. Gamma-D-glutamylaminomethyl sulfonate exhibited partial antagonist specificity for AMPA-induced toxicity.     

Differential effects of NMDA and AMPA/kainate receptor antagonists on nitric oxide production in rat brain following intrahippocampal injection

Stimulation of glutamate receptors induces neuronal nitric oxide (NO) release, which in turn modulates glutamate transmission. The involvement of ionotropic glutamate NMDA and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainite (KA) receptors in the induction of NO production in the rat brain was examined after injection of kainate, non-NMDA receptor agonist, KA+6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), selective AMPA/KA receptor antagonist, or KA+2-amino-5-phosphonopentanoic acid (APV), selective NMDA receptor antagonist. Competitive glutamate receptor antagonists were injected with KA unilaterally into the CA3 region of the rat hippocampus. The accumulation of nitrite, the stable metabolite of NO, was measured by the Griess reaction at different times (5 min, 15 min, 2 h, 48 h and 7 days) in the ipsi- and contralateral hippocampus, forebrain cortex, striatum and cerebellum homogenates. The detected increase of NO production in distinct brain regions, which are functionally connected via afferents and efferents, suggests that these regions are affected by the injury. The effect of KA on nitrite production was blocked by the glutamate antagonists. Intrahippocampal KA+CNQX injection resulted in decrease of nitrite production, around control levels, in all tested brain structures. Significant decrease in nitrite levels was found only in comparison to KA-treated animals, i.e. the overall effect of selective AMPA/KA receptor antagonist was a decrease of KA-induced excitotoxicity. The accent effect of intrahippocampal KA+APV injection resulted, also, in decrease of nitrite production. However, this effect was detected after 5 min from the injection indicating the existence of an NMDA receptor-mediated component of basal nitrite production in physiological conditions and difference in mechanisms and time dynamics between CNQX and APV. The used antagonists showed same pattern in all tested brain structures. APV and CNQX both expressed sufficient neuroprotection in sense of reducing nitrite concentrations, but with differential effect in mechanisms and time dynamics. Our findings suggest that NMDA and AMPA/KA receptors are differentially involved in NO production.     

Differential intracellular calcium responses to glutamate in type 1 and type 2 cultured brain astrocytes.

Fluorescence image analysis using the calcium indicator fluo-3 was used to examine changes in [Ca2+]i induced by glutamate in mixed glia populations cultured from neonatal rat brains. [Ca2+]i responses were correlated with glia type by performing immunohistochemistry using markers specific for type 1 and type 2 astrocytes on the same cells used in the imaging experiments. Glutamate (30-500 microM) induced two markedly different [Ca2+]i responses in the two astrocyte types: the response in type 1 astrocytes consisted of an initial fast transient followed by varying degrees of oscillations, whereas the predominant response in type 2 astrocytes was a slow rise in [Ca2+]i to a more or less sustained and nonoscillatory level. In some type 2 astrocytes, an initial spikelike transient similar to that in type 1 astrocytes was observed; the overall size of the spike, however, was smaller than in type 1 astrocytes. Two agonists for the ionotropic glutamate receptor, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) and kainate, elicited a 6-cyano-7-dinitroquinoxaline-2,3-dione (CNQX)-sensitive, external Ca(2+)-dependent, sustained [Ca2+]i rise in type 2 but not type 1 astrocytes. The initial spike in type 2 astrocytes was less dependent on external Ca2+ and not blocked by CNQX. [Na+]i as measured by the Na(+)-fluorescence dye SBFI, was elevated by kainate in both astrocyte types, though the increase was larger in type 2 astrocytes. This increase was reduced by CNQX, suggesting this [Ca2+]i increase was mediated, at least in part, by ionotropic glutamate receptors. The results are discussed in terms of the relative distribution of two classes of glutamate receptors on these two astrocyte types: one, the ionotropic class, is linked directly to an ion channel, and the other, the metabotropic class, induces internal mobilization of Ca2+ via inositol phospholipid hydrolysis.     

Dual roles in feeding for AMPA/kainate receptors: receptor activation or inactivation within distinct hypothalamic regions elicits feeding behavior

We have previously shown that hypothalamic injections of glutamate, or agonists of its ionotropic receptors (iGluRs), elicit intense feeding responses in satiated rats [Brain Res. 613 (1993) 88, Brain Res. 630 (1993) 41]. While attempting to clarify the role of the AMPA and kainate (KA) receptor subtypes in glutamatergic feeding systems, we discovered that lateral hypothalamic (LH) injection of high doses of the competitive AMPA/KA receptor antagonist, NBQX (10 and 30 nmol), elicited a pronounced feeding response. We questioned whether this effect was due to inactivation of AMPA or possibly KA receptors. To determine whether other AMPA/KA antagonists can also elicit feeding, we tested whether injection of CNQX, another AMPA/KA receptor antagonist, also stimulates eating and whether these feeding stimulatory effects were due to antagonists' actions in the LH or in other hypothalamic sites. Here we report that NBQX and CNQX elicit feeding in a dose dependent manner and are most effective when injected into the perifornical hypothalamus (PFH), or into the paraventricular nucleus (PVN) and, to a lesser extent, into the LH of satiated rats. In contrast, AMPA was most effective in stimulating feeding when injected into the LH, confirming previous reports. These data suggest that either activation or inactivation of AMPA/KA receptors in distinct but overlapping hypothalamic sites may be sufficient to induce feeding behavior, indicating a broadened role for glutamate in hypothalamic feeding mechanisms.     

Simultaneous AMPA/kainate receptor blockade and dopamine D(2/3) receptor stimulation in the nucleus accumbens decreases brain stimulation reward in rats

Interactions between dopamine (DA) and glutamate (GLU) in the mesocorticolimbic pathway of the brain may influence motivation and reward. Previous work from this laboratory has demonstrated that alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate receptor blockade may potentiate decreases in exploratory motor activity induced by the DA D(2/3) receptor agonist 7-OH-DPAT in the nucleus accumbens septi (NAS). This study investigated the interaction of AMPA/kainate receptor antagonists CNQX or NBQX with 7-OH-DPAT on ventral tegmental area (VTA) brain stimulation reward (BSR). Effects of these compounds, alone and combined, were measured in male Sprague-Dawley rats stereotaxically implanted with a unilateral VTA electrode and bilateral guide cannulae in the NAS core or shell subregions. Rate-frequency analysis was used to assess BSR frequency thresholds and maximum response rates of rats trained to lever-press for reinforcing electrical stimulation. When given alone, CNQX (0.5 microg), NBQX (0.5 microg), or 7-OH-DPAT (5.0 microg) did not affect BSR frequency thresholds. Co-administration of CNQX or NBQX with 7-OH-DPAT synergistically increased BSR frequency thresholds, indicative of decreased reward. These data indicate that simultaneous AMPA/kainate receptor blockade and DA D(2/3) receptor stimulation in the NAS may act synergistically to inhibit motivated behaviours such as electrical brain self-stimulation.     

Characterization and regulation of a high affinity [3H]CNQX labelled AMPA receptor in rat neocortex.

We have characterized a high affinity site of the alpha-amino-3 hydroxy-5-methyl-isoxazole-4-propionate (AMPA) receptor in in vitro living slices of adult rat neocortex using [3H]CNQX, and AMPA antagonist. [3H]CNQX labelled multiple binding sites with a Bmax of a high affinity site of approximately 470 fmol/mg protein and an apparent Kd of 11.3 nM. The high affinity site of the AMPA receptor could be down-regulated (36%) by 2 h preincubations in quisqualate, an AMPA agonist. Increases in electrical activity induced by a combination of veratridine and glutamate also led to an average decrease of the high affinity AMPA receptor number of 17%. In addition, preincubations with muscimol, a GABAA agonist, as well as glutamate agonists kainate and N-methyl-D-aspartate (NMDA) led to an average increase in high affinity AMPA receptor number of 17%, 14%, and 37%, respectively. The present results show that a ligand-gated high affinity AMPA receptor can be regulated by agonist stimulation as well as changes in neural activity.     

The excitatory amino acid glutamate mediates reflexly increased tracheal blood flow and airway submucosal gland secretion

In six decerebrated and in eight alpha-chloralose anesthetized, paralyzed and mechanically ventilated beagle dogs, we have studied involvement of glutamate and glutamate receptors in transmission of excitatory inputs from the airway sensory receptors to the nucleus tractus solitarius and from this site to airway-related vagal preganglionic cells that regulate the tracheal circulation and the submucosal gland secretion. Stimulation of airway sensory fibers by lung deflation-induced reflex increase in tracheal blood flow and submucosal gland secretion. These responses were diminished by prior administration of AMPA/kainate receptor antagonist CNQX into the fourth ventricle (n=6). Furthermore, topical application or microinjection of AMPA/kainate receptor blockers, into the region of the ventrolateral medulla, where airway-related vagal preganglionic neurons are located, abolished the reflex changes in tracheal submucosal gland secretion (n=8); in these dogs mucosal blood flow was not measured). These findings indicate that reflex increase in tracheal blood flow and submucosal gland secretions are mediated mainly via release of glutamate and activation of the AMPA/kainate subtype of glutamate receptors.     

Hypoxic injury of isolated axons is independent of ionotropic glutamate receptors

Axonal injury in white matter is an important consequence of many acute neurological diseases including ischemia. A role for glutamate-mediated excitotoxicity is suggested by observations from in vitro and in situ models that AMPA/kainate blockers can reduce axonal injury. We assessed axonal vulnerability in primary murine neuronal cultures, with axons isolated from their cell bodies using a compartmented chamber design. Transient removal of oxygen and glucose in the axon compartment resulted in irreversible loss of axon length and neurofilament labeling. This injury was not prevented by addition of ionotropic glutamate receptor blockers and could not be reproduced by glutamate receptor agonists. However, hypoxic injury was prevented by blockade of voltage-gated sodium channels, inhibition of calpain and removal of extracellular calcium. These results suggest that isolated, unmyelinated axons are vulnerable to hypoxic injury which is mediated by influx of sodium and calcium but is independent of glutamate receptor activation.     

Selective and AMPA receptor-dependent astrocyte death following prolonged blockade of glutamate reuptake in rat cerebellar cultures

In this study we examined the effects of prolonged l-trans-pyrrolidine-2,4-dicarboxylate (PDC)-induced glutamate reuptake blockade on the viability of glial cells in cerebellar granule cell cultures. Immunofluorescence staining for the glial-specific intermediate filament protein, GFAP, revealed that the PDC- induced increase of extracellular glutamate concentration was accompanied by increased astrocyte death, while neurons and oligodendrocytes remained intact and viable. Astrocytic cell death was manifested as fragmentation of processes and cell bodies. The selective astrocyte death was completely prevented by the competitive alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate receptor antagonist, NBQX (10 microM), whereas MK-801 (10 microM), a noncompetitive blocker of N-methyl-D-aspartate receptors, gave only partial protection. Double staining for GFAP and the AMPA receptor subunits GluR2/3 showed that astrocytes had much higher immunoreactivity for GluR2/3 than neurons or oligodendrocytes, suggesting that the number of AMPA receptors is likely to be higher on astrocytes. Furthermore, we employed real-time RT-PCR to measure GluR1-4 subunit mRNA expression in control and PDC-exposed cultures. Following treatment with PDC, GluR1 and GluR4 mRNAs were reduced by 40% and GluR3 was reduced by 70% relative to control levels. In contrast, GluR2 expression was not affected by the PDC treatment, indicating that GluR3 was the dominant type of AMPA receptor subunit expressed on astrocytes. Our results show that astrocytes appear to be more vulnerable than neurons or oligodendrocytes to a gradual increase in the extracellular glutamate concentration, suggesting that astrocytes may be critically involved in the pathophysiology of slowly developing chronic neurodegenerative disorders.     

Ionotropic glutamate receptor modulation preferentially affects NMDA receptor expression in rat hippocampus

Electrophysiological data suggest that alterations in the function of one glutamate receptor subtype may affect the function of other subtypes. Further, previous studies have demonstrated that NMDA receptor antagonists affect NMDA and kainate receptor expression in rat hippocampus. In order to address the mutual regulation of NMDA, AMPA, and kainate receptor expression in rat hippocampus, we conducted two experiments examining the effects of NMDA and non-NMDA glutamate receptor modulators on NMDA, AMPA, and kainate receptor expression using in situ hybridization and receptor autoradiography. NMDA receptor expression was preferentially affected by systemic treatments, as all drugs significantly altered [(3)H]MK-801 binding, and several drugs increased [(3)H]ifenprodil binding. GYKI52466 and aniracetam treatments resulted in changes in both [(3)H]ifenprodil binding and NR2B mRNA levels, consistent with the association of this subunit and binding site in vitro. There were more modest effects on AMPA and kainate receptor expression, even by direct antagonists. Together, these data suggest that ionotropic glutamate receptors interact at the level of expression. These data also suggest that drug regimens targeting one ionotropic glutamate receptor subtype may indirectly affect other subtypes, potentially producing unwanted side effects.