Glutamate release evoked by glutamate receptor agonists in cultured chick retina cells: Modulation by arachidonic acid

We studied the effect of ionotropic glutamate receptor agonists on the release of endogenous glutamate or of [³H]D -aspartate from reaggregate cultures (retinospheroids) or from monolayer cultures of chick retinal cells, respectively. Kainate increased the fluorescence ratio of the Na⁺ indicator SBFI and stimulated a dose-dependent release of glutamate in low (0.1 mM) Ca²⁺ medium, as measured using a fluorometric assay. Under the same experimental conditions, the release evoked by N-methyl-D -aspartate (NMDA; 400 μM) was about half of that evoked by the same kainate concentration; α-amino-3-hydroxy-5-methyl-4-isoxasolepropionic acid (AMPA; 400 μM) did not trigger a significant response. In the presence of 1 mM CaCl₂, all of the agonists increased the [Ca²⁺]_i, as determined with the fluorescence dye Indo-1, but the glutamate release evoked by NMDA and kainate was significantly lower than that measured in 0.1 mM CaCl₂ medium. Inhibition by Ca²⁺ of the kainate-stimulated release of glutamate was partially reversed by the phospholipase A₂ inhibitor oleiloxyethyl phosphorylcholine (OPC), suggesting that the effect was mediated by the release of arachidonic acid, which inhibits the glutamate carrier. Accordingly, kainate, NMDA, and AMPA stimulated a Ca²⁺-dependent release of [³H]arachidonic acid, and the direct addition of the exogenous fatty acid to the medium decreased the release of glutamate evoked by kainate in low (0.1 mM) CaCl₂ medium. In monolayer cultures, we showed that NMDA, kainate, and AMPA also stimulated the release of [³H]D -aspartate, but in this case release in the presence of 1 mM CaCl₂ was significantly higher than that evoked in media with no added Ca²⁺. The ranking order of efficacy for stimulation of Ca²⁺-dependent release of [³H]D -aspartate was NMDA ≪ kainate < AMPA. © 1996 Wiley-Liss, Inc.     

Exposure to N-methyl-D-aspartate increases release of arachidonic acid in primary cultures of rat hippocampal neurons and not in astrocytes

The release of [3H]arachidonic acid (ARA) was investigated from prelabelled primary cultures of hippocampal neurons and astroglial cells. The activation of N-methyl-D-aspartate (NMDA) subtype of glutamate receptors resulted in a dose-dependent stimulation of [3H]ARA release. The half maximal effect was obtained at about 15 microM NMDA, whereas the maximum concentration (50 microM NMDA) produced about a 2-fold increase in 7-day-old cultures. This elevation in [3H]ARA release was blocked in a dose-related manner by the NMDA receptor antagonist, 2-amino-5-phosphonovaleric acid (APV), and by Mg2+ which blocks NMDA receptor-linked Ca2+ ion channels. The removal of external Ca2+ inhibited NMDA-induced release, whereas treatment with calcimycin (A 23187, a Ca2+ ionophore) greatly increased the [3H]ARA release. The inhibitors of phospholipase A2, nordihydroguaiaretic acid and mepacrine, decreased the NMDA-dependent [3H]ARA release in a dose-related manner, maximum inhibition reaching to about 90% at high doses. Entry of Ca2+ brought about by opening the voltage-sensitive channels by high K+ had no effect on the release of [3H]ARA, indicating that NMDA gated channels are situated in a part of the neuron where Ca2+ entry through this route is more efficiently coupled to the activation of phospholipase A2. Treatment with NMDA had no significant effect on [3H]ARA release in hippocampal astroglial cells as opposed to neurons. This was not due to inability of astrocytes to release ARA, for ATP still evoked [3H]ARA release, and this was markedly inhibited by mepacrine. It is suggested that ARA act as both intracellular and intercellular messengers in the functioning of NMDA receptors in synaptic transmission and plasticity in the hippocampus.     

Nordihydroguaiaretic acid and RHC 80267 potentiate astroglial injury during combined glucose-oxygen deprivation

Membrane phospholipid degradation has been proposed to play a key role in hypoxic-ischemic brain injury. We tested the hypotheses that both nordihydroguaiaretic acid, a phospholipase A₂ and lipoxygenase inhibitor, and RHC 80267, a diacylglycerol lipase inhibitor, would decrease the release of [³H]arachidonic acid metabolites from prelabeled cultures of astroglia subjected to combined glucose-oxygen deprivation and that these inhibitors would also decrease astroglial injury during combined glucose-oxygen deprivation. Both nordi-hydroguaiaretic acid and RHC 80267 significantly inhibited the release of [³H]arachidonic acid metabolites during combined glucose-oxygen deprivation. This suggests that two separate enzymic pathways, the phospholipase A₂ pathway and the phospholipase C/diacylglycerol lipase pathway, contribute to the release of astroglial [³H]arachidonic acid metabolites during combined glucose-oxygen deprivation. However, both of these lipase inhibitors increased astroglial cell death during combined glucose-oxygen deprivation, probably due to inhibition of arachidonic acid release. We speculate that arachidonic acid release may be a mechanism of astroglial self-preservation during combined glucose-oxygen deprivation.     

Contribution of endogenously formed arachidonic acid in the presynaptic facilitatory effects of NMDA and carbachol on dopamine release in the mouse striatum

Arachidonic acid stimulated the release of [3H]-dopamine from striatal microdiscs in a concentration-dependent and partially calcium-dependent manner. Inhibitors of cytosolic and membrane-bound phospholipase A2 were used to determine whether endogenously formed arachidonic acid also contributes to the release of [3H]-DA (previously taken up in tissues or endogenously synthesized from [3H]-tyrosine) evoked by N-methyl-d-aspartate (NMDA) and carbachol alone or in combination. In the presence of magnesium, carbachol was found to remove the magnesium block of NMDA receptors and to facilitate the NMDA-evoked release of [3H]-DA from striatal microdiscs and synaptosomes. In addition, in the absence of magnesium, synergistic responses were induced by both agonists on microdiscs but not on synaptosomes. Responses induced by NMDA, carbachol or both agonists on microdiscs were reduced by phospholipase A2 inhibitors, the most striking effects being observed with mepacrine. Mepacrine was also shown to reduce the oxotremorine, but neither the nicotine- nor the potassium-evoked release of [3H]-DA. Tetrodotoxin decreased the release of [3H]-DA evoked by the co-application of NMDA and carbachol on microdiscs, but mepacrine still decreased this tetrodotoxin-resistant response. Similarly, mepacrine still decreased the release of [3H]-DA evoked by NMDA and carbachol on synaptosomes. Altogether, these results indicate that arachidonic acid which is formed in striatal neurons, and to a lesser extent in DA fibres, under stimulation of NMDA and muscarinic receptors, partially contributes to the presynaptic facilitation of DA release evoked by NMDA and carbachol.     

Endothelins activate phospholipase A2 in brain capillary endothelial cells

Addition of endothelin-1 to cultured rat brain capillary endothelial cells induced a 2.7-fold activation of phospholipase A₂, as evidenced from the release of [³H]arachidonic acid from prelabelled cells. Half maximum activation by endothelin-1 was observed at 1 nM. The action of endothelin-1 was not mimicked by low concentrations of endothelin-3 and it was largely suppressed by BQ-123, suggesting the involvement of an ETA receptor subtype. It is suggested that the activation of phospholipase A₂ by endothelins plays a role in the development of delayed cerebral vasopasm following subarachnoid hemorrhage.     

Induction of neurosteroid synthesis by NMDA receptors in isolated rat retina: a potential early event in excitotoxicity

Here we investigated the possible regulation of neurosteroidogenesis by N-methyl-d -aspartic acid (NMDA) receptor activation and addressed the hypothesis that neurosteroid synthesis may be involved in acute excitotoxicity. In the isolated retina, exposure to NMDA modified pregnenolone and pregnenolone sulphate formation. This effect was dose and time dependent, the synthesis being increased by relatively moderate NMDA doses (1–100 μm ) within 30 min exposure and reduced to its control value by 60 min or by raising drug concentrations. NMDA-stimulated neurosteroid synthesis was blocked by (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclo-hepten-5,10-imine hydrogen maleate (MK-801) and 3(2-carboxypiperazine-4-yl)propyl-1-phosphonic acid (CPP), depended on extracellular calcium and reproduced by glutamate. Lactate dehydrogenase (LDH) release and morphological analysis revealed that retinal cell viability was not significantly affected after 30 min exposure to 50 μm NMDA, but severe cell damage occurred by 60 min. When the GABA_A (γ-aminobutyric acid) receptor agonist muscimol (1–1000 μm ), known to activate retinal neurosteroidogenesis, was added together with NMDA, no additional increase in neurosteroid synthesis was observed, and NMDA-induced LDH release remained unchanged. However, exposure to a high concentration of muscimol alone (500 μm ) provoked a similar degree of toxicity to NMDA. By contrast, bicuculline abolished the increase in neurosteroidogenesis and LDH release. Similarly, pretreatment with R (+)-p-aminoglutethimide (AMG), an inhibitor of cholesterol side-chain cleavage cytochrome P450, attenuated acute retinal cell damage. The inhibitory nature of AMG on NMDA-stimulated neurosteroidogenesis was confirmed in the observation that drug treatment reduced pregnenolone content and did not affect the bindings of [³H] MK-801 and [³H] muscimol. The results demonstrate that NMDA receptors regulate neurosteroidogenesis through a transneuronal mechanism, which implies GABA_A receptor activation. The early NMDA-mediated stimulation of neurosteroid synthesis seems to play a critical role in acute excitotoxicity; consequently, its inhibition is likely to delay neuronal cell death.     

Role of arachidonic acid in the regulation of the NMDA-evoked release of acetylcholine in striatal compartments

The role of endogenously released arachidonic acid in the control of the NMDA (50 microM)-evoked release of [3H]-acetylcholine previously formed from [3H]-choline was investigated in striosome-enriched areas and in the matrix of the rat striatum using a microsuperfusion procedure in vitro. Experiments were performed with either mepacrine (0.2 microM) or bovine serum albumin (BSA, 0.02%) which inhibits phospholipase A2 activity or binds endogenously released arachidonic acid, respectively. Both treatments similarly reduce the NMDA-evoked release of [3H]-acetylcholine, this effect being more pronounced in striosomes than in the matrix. These reductions result from a facilitation of dopamine release, since they were not observed in the presence of (-)sulpiride, the D2 dopamine receptor antagonist. Moreover, the superfusion with BSA was shown to enhance the release of [3H]-dopamine (formed from [3H]-tyrosine), this effect being of larger amplitude in striosomes than in the matrix. In control conditions, due to the blockade of the presynaptic inhibitory effect of GABA on dopamine release, bicuculline (GABA(A) receptor antagonist) reduces the NMDA-evoked release of [3H]-acetylcholine in both striatal compartments. Bicuculline was no longer effective following superfusions with either mepacrine or BSA, suggesting that these treatments eliminate the GABAergic presynaptic inhibitory control on dopamine transmission and thus lead to the dopamine-mediated inhibition of [3H]-acetylcholine release. These results indicate that arachidonic acid endogenously formed under weak stimulation of NMDA receptors contributes to the regulation of the evoked release of [3H]-acetylcholine by facilitating GABAergic transmission and that this process is more important in striosomes than in the matrix.     

Nitric oxide responsible for NMDA receptor-evoked inhibition of arachidonic acid incorporation into lipids of brain membrane

The activation of the glutamatergic NMDA receptor has no effect on arachidonic acid release from cortical synaptoneurosomal lipids prelabeled with [1-¹⁴C]arachidonic acid ([¹⁴C]AA). However, activation of NMDA receptor leads to the reduction of AA incorporation into rat brain cortex synaptoneurosomal membrane phosphatidylinositol (PI). The competitive NMDA receptor antagonist, 2-amino-5-phosphovaleric acid (APV), completely eliminates the effect of NMDA on this process. More precise analysis of the sequence of events leading to NMDA-induced decrease of AA incorporation indicates that this process is significantly blocked by voltage-gated sodium and calcium channels inhibitors, such as tetrodotoxin (TTX) and ω-conotoxin (CTX), respectively. Then the antagonist of inositol trisphosphate receptor, TMB-8, totally abolishes the effect of NMDA on AA incorporation into PI. The lowering of AA incorporation evoked by NMDA is significantly diminished by nitric oxide (NO) synthase inhibitor,N ^G-nitro-l-arginine (NNLA). Further studies were carried out with NO donor(s) to explain the mechanism of NO action in the inhibition of AA incorporation into PI. Our results suggest the following sequence of events: opening of voltage-dependent sodium and calcium channels, subsequent activation of PI-4,5-bisphosphate-specific phospholipase C (PLC), elevation of inositol trisphosphate (IP₃)-sensitive calcium ions, stimulation of NO production and NO-mediated S-nitrosylation, or free radical effect on enzymes involved in AA incorporation. Our data suggest that NO-mediated events may be responsible for NMDA-evoked inhibition of AA incorporation into PI of synaptoneurosomal membrane.     

Excitatory amino acid-evoked release of [H]GABA from hippocampal neurons in primary culture

The novel glutamate antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) inhibited glutamate stimulated [³H]GABA release from cortical neurons in vitro. Kainate-induced release was blocked in a competitive fashion butN-methyl-d-aspartate (NMDA)-induced release was blocked non-competitively by CNQX. 7-Chlorokynurenate (7-CK) also inhibited NMDA evoked [³H]GABA release non-competitively, but had no effect on kainate induced release. The effects of both CNQX and 7-CK on NMDA-induced release were reversed by addition of exogenous glycine but the effects of CNQX on kainate-induced release were not altered by glycine. This suggests that both CNQX and 7-CK may interact with the glycine regulatory site of the NMDA receptor.     

Glutamate increases the [Ca]i but stimulates Ca-independent release of [H]GABA in cultured chick retina cells

The effect of glutamate of [Ca²⁺]_i and on [³H]γ-aminobutyric acid (GABA) release was studied on cultured chick embryonic retina cells. It was observed that glutamate (100 μM) increases the [Ca²⁺]_i by Ca²⁺ influx through Ca²⁺ channels sensitive to nitrendipine, but not to ω-conotoxin GVIA (ω-Cg Tx) (50%), and by other channels insensitive to either Ca²⁺ channel blocker. Mobilization of Ca²⁺ by glutamate required the presence of external Na⁺, suggesting that Na⁺ mobilization through the ionotropic glutamate receptors is necessary for the Ca²⁺ channels to open. The increase in [Ca²⁺]_i was not related to the release of [³H]GABA induced by glutamate, suggesting that the pathway for the entry of Ca²⁺ triggered by glutamate does not lead to exocytosis. In fact, the glutamate-induced release of [³H]GABA was significantly depressed by Ca_o²⁺, but it was dependent on Na_o⁺, just as was observed for the [³H]GABA release induced by veratridine (50 μM). The veratridine-induced release could be fully inhibited by TTX, but this toxin had no effect on the glutamate-induced [³H]GABA release. Both veratridine- and glutamate-induced [³H]GABA release were inhibited by 1-(2-(((diphenylmethylene)amino)oxy)ethyl)-1,2,5,6-tetrahydro-3-pyridine-carboxylic acid (NNC-711), a blocker of the GABA carrier. Blockade of the NMDA and non-NMDA glutamate receptors with MK-801 and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), respectively, almost completely blocked the release of [³H]GABA evoked by glutamate. Continuous depolarization with 50 mM K⁺ induced maximal release of [³H]GABA of about 1.5%, which is much smaller than the release evoked by glutamate under the same conditions (6.0–6.5%). Glycine (3 μM) stimulated [³H]GABA release induced by 50 mM K⁺, and this effect was blocked by MK-801, suggesting that the effect of K⁺ on [³H]GABA release was partially mediated through the NMDA receptor which probably was stimulated by glutamate released by K⁺ depolarization. We conclude that glutamate induces Ca²⁺-independent release of [³H]GABA through reversal of the GABA carrier due to Na⁺ entry through the NMDA and non-NMDA, TTX-insensitive, channels. Furthermore the GABA carrier seems to be inhibited by Ca²⁺ entering by the pathways open by glutamate. This Ca²⁺ does not lead to exocytosis, probably because the Ca²⁺ channels used are located at sites far from the active zones.     

Excitatory amino acid-evoked release of [3H]GABA from hippocampal neurons in primary culture

We investigated the release of gamma-[2,3-3H(N)]aminobutyric acid ([3H]GABA) from hippocampal neurons in primary cell culture. [3H]GABA release was stimulated by the excitatory amino acid neurotransmitter glutamate as well as by N-methyl-D-aspartate (NMDA) and kainate. Cell depolarization induced by raising [K+]o or by veratridine also stimulated [3H]GABA release. NMDA-induced release was completely blocked by 3-((+/-)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP+), Mg2+ and Zn2+ whereas the release induced by glutamate and kainate was much less susceptible to inhibition by these substances. Furthermore, removal of external Ca2+ inhibited NMDA-induced release, but not that induced by glutamate, kainate, veratridine or 50 mM K+. Removal of external Na+ reduced [3H]GABA release evoked by all stimuli, but to different extents. All of the excitatory amino acids tested increased [Ca2+]i within hippocampal neurons as assessed by fura-2 based microspectrofluorimetry. This increase in [Ca2+]i was completely dependent on the presence of external Ca2+. These results suggest that Ca2+-dependent and -independent forms of GABA release from hippocampal interneurons may occur. [3H]GABA release evoked by glutamate, kainate, veratridine or 50 mM K+, appeared to be mediated by the reversal of electrogenic, Na+-coupled GABA uptake. Release was inhibited by nipecotic acid, an inhibitor of the Na+-coupled GABA uptake system. However, release induced by NMDA may also include a Ca2+-dependent component.     

Role of Ca-independent phospholipase A2 and n−3 polyunsaturated fatty acid docosahexaenoic acid in prostanoid production in brain: perspectives for protection in neuroinflammation

Various diseases of the central nervous system are characterized by induction of inflammatory events, which involve formation of prostaglandins. Production of prostaglandins is regulated by activity of phospholipases A₂ and cyclooxygenases. These enzymes release the prostaglandin precursor, the n−6 polyunsaturated fatty acid, arachidonic acid and oxidize it into prostaglandin H₂. Docosahexaenoic acid, which belongs to the n−3 class of polyunsaturated fatty acids, was shown to reduce production of prostaglandins after in vivo and in vitro administration. Nevertheless, the fact that in brain tissue cellular phospholipids naturally have a uniquely high content of docosahexaenoic acid was ignored so far in studies of prostaglandin formation in brain tissue. We consider the following possibilities: docosahexaenoic acid might attenuate production of prostaglandins by direct inhibition of cyclooxygenases. Such inhibition was found with the isolated enzyme. Another possibility, which has been already shown is reduction of expression of inducible cyclooxygenase-2. Additionally, we propose that docosahexaenoic acid could influence intracellular Ca²⁺ signaling, which results in changes of activity of Ca²⁺-dependent phospholipase A₂, hence reducing the amount of arachidonic acid available for prostaglandin production. Astrocytes, the main type of glial cells in the brain control the release of arachidonic acid, docosahexaenoic acid and the formation of prostaglandins. Our recently obtained data revealed that the release of arachidonic and docosahexaenoic acids in astrocytes is controlled by different isoforms of phospholipase A₂, i.e. Ca²⁺-dependent phospholipase A₂ and Ca²⁺-independent phospholipase A₂, respectively. Moreover, the release of arachidonic and docosahexaenoic acids is differently regulated through Ca²⁺- and cAMP-dependent signal transduction pathways. Based on analysis of the current literature and our own data we put forward the hypothesis that Ca²⁺-independent phospholipase A₂ and docosahexaenoic acid are promising targets for treatment of inflammatory related disorders in brain. We suggest that Ca²⁺-independent phospholipase A₂ and docosahexaenoic acid might be crucially involved in brain-specific regulation of prostaglandins.     

Regulation of NMDA-stimulated [C]GABA and [H]acetylcholine release by striatal glutamate and dopamine receptors

Striatal function is heavily influenced by glutamatergic and dopaminergic afferent input. To ultimately better understand how the N-methyl- -aspartate (NMDA) antagonist, phencyclidine (PCP), alters striatal function, we sought to determine how NMDA receptor function is influenced by activation of other glutamatergic receptors and by dopaminergic receptors. To this end, we used NMDA-stimulated efflux of [¹⁴C]GABA and [³H]acetylcholine (ACh) from striatal slices to assess the influence of these receptors on NMDA function. NMDA-stimulated [¹⁴C]GABA release was more sensitive to NMDA and glycine antagonists than was [³H]ACh release, suggesting that different NMDA receptors regulate the release of these neurotransmitters. Furthermore, NMDA-stimulated [³H]ACh release was inhibited by a D₂ receptor mechanism whereas NMDA-stimulated [¹⁴C]GABA release was enhanced by D₁ receptor activation. NMDA and (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid hydrobromide (AMPA) interact additively to evoke [³H]ACh release, and synergistically to evoke [¹⁴C]GABA release. An additive effect of NMDA and kainate (KA) was found on [¹⁴C]GABA release, but NMDA and KA acted in a less than additive manner in evoking [³H]ACh release. KA-stimulated [³H]ACh release was largely blocked by NMDA antagonists, suggesting mediation through activation of NMDA receptors, probably secondary to KA-induced glutamate release. A selective group II metabotropic receptor agonist inhibited NMDA-stimulated [¹⁴C]GABA and [³H]ACh release. On the other hand, NMDA-stimulated [¹⁴C]GABA release was potentiated by activation of group I metabotropic receptors. Thus, in addition to the differential modulation by D₁- and D₂-like receptors, the release of striatal neurotransmitters by NMDA receptor activation depends on the extent to which the other glutamate receptors, both ionotropic and metabotropic, are activated.     

NMDA receptor activation induces glutamate release through nitric oxide synthesis in guinea pig dentate gyrus

We tested the hypothesis that the release of glutamate following activation of N-methyl-d-aspartate (NMDA) receptors is mediated by nitric oxide (NO) production, using slices of the guinea pig hippocampus. The NMDA-induced glutamate release from slices of dentate gyrus or CA1, which was both concentration-dependent and Ca²⁺-dependent, was also Mg²⁺-sensitive and abolished by MK-801, a selective non-competitive NMDA receptor antagonist. In dentate gyrus, the NMDA-induced glutamate release was inhibited non-significantly by tetrodotoxin, whereas the NO synthase (NOS) inhibitor N^G-nitro-l-arginine (l-NNA) blocked the NMDA-induced release of glutamate in a concentration-dependent manner, but not a high K⁺-evoked release of glutamate. In addition, the l-NNA blockade of NMDA-induced release of glutamate was recovered by pretreatment with l-arginine, the normal substrate for NOS. These results suggest that activation of NMDA receptors in dentate gyrus, as well as subsequent Ca²⁺ fluxes, is required for the neuronal glutamate release mediated by NO production. On the other hand, the NMDA-evoked glutamate release from CA1 region was tetrodotoxin-sensitive and was not inhibited by l-NNA, thereby suggesting that activation of NMDA receptors in CA1 results in increased glutamate release in an NO-independent manner. Taken together, the NMDA receptor-mediated neuronal release of glutamate from the guinea pig dentate gyrus likely involves the recruitment of NOS activity.     

Intraseptal administration of (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid induces immediate early gene expression in lateral septal neurons

Prior work has shown that activation of metabotropic glutamate receptors can induce burst firing and a form of NMDA receptor independent long term potentiation in lateral septal slice preparations. To study this phenomenon in vivo we used the expression of immediate early gene products as markers for increased neuronal activity following intraseptal injection of the metabotropic agonist 1S,3R-ACPD. Intraseptal injection of 1S, 3R ACPD induced the expression of Fos-like, Jun 13-like and Krox24-like immunoreactivity in lateral septal neurons in a dose-dependent fashion. Immediate early gene product expression peaked at 4 to 6 h post-injection and then declined to baseline. Immediate early gene expression was diminished by co-injection of -AP3 and was not elicited by intraseptal injection of -AP4, cysteine sulfinic acid or DHPG. Immediate early gene expression was not diminished by chronic lithium treatment but was diminished by chronic treatment with the phospholipase A₂ inhibitor quinacrine. Co-injection of the phospholipase AZ inhibitor NDGA partially suppressed the induction of immediate early gene expression. Metabotropic glutamate receptors regulate lateral septal neuron excitability in vivo and some of their effects may be mediated by activation of phospholipase A₂. Alternatively, arachidonic acid may play a permissive role it the effects of metabotropic glutamate receptors on lateral septal neurons.     

Development and regulation of excitatory amino acid receptors involved in the release of arachidonic acid in cultured hippocampal neural cells

Release of [3H]arachidonic acid mediated by excitatory amino acid (EAA) receptors was investigated from prelabelled primary cultures of hippocampal neurons and astroglial cells. Treatment with N-methyl-D-aspartate (NMDA), quisqualate (QA) and kainate resulted in age- and dose-dependent stimulation of [3H]arachidonic acid release. During development, the maximum response for NMDA was observed relatively earlier (at 7 days) than those for QA and kainate (at 14 days) in the hippocampal neuronal cultures. The half maximal effects were obtained at about 15 microM NMDA at all ages studied and about 0.5 microM QA at 14 and 20 days. At optimum concentrations NMDA- and QA-induced releases were additive. Unlike with neurons, treatment with all the 3 EAA receptor agonists, NMDA, QA and kainate, had no significant effect on [3H]arachidonate release in hippocampal astroglial cells. In cultured 14-day-old neurons, the increases in NMDA- and QA-mediated [3H]arachidonic acid release were completely blocked by the NMDA receptor antagonist, 2-amino-5-phosphonovaleric acid, and the ionotropic QA receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione, respectively. But the iontropic QA receptor agonist alpha-amino-3-hydroxy-5-methyl-isoxazole-4- propionic acid (AMPA) had no significant effect on [3H]arachidonate release, indicating that interaction between ionotropic QA and metabolotropic QA receptors may be essential for optimal QA-mediated arachidonic acid release. At physiological concentrations of Mg2+ (1.2 mM), AMPA was found to potentiate NMDA-induced release of [3H]arachidonic acid; the effect appeared to be related to a removal of Mg2+ blockade mediated by mild depolarisation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of MK-801, kainate,AMPA, and muscimol binding sites and the effect of dark rearing in rat visual cortex

We used quantitative autoradiography to determine whether the development of glutamate receptors correlates with the plastic period for monocular deprivation in rat visual cortex. To study glutamate receptors, we incubated sections of rat visual cortex with tritiated (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10imine maleate (MK-801), tritiated kainate, and tritiated amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA). [³H]MK-801 is a noncompetitive ligand for the N-methyl-D-aspartate (NMDA) receptor. [³H]kainate and [³H]AMPA are competitive ligands for non-NMDA receptors. To compare glutamate binding sites with a nonglutamate binding site, we studied [³H]muscimol, which binds to γ-aminobutyric acid (GABA)_A receptors. [³H]MK-801 binding was maximal at postnatal day 26 (P26) and decreased in adulthood. [³H]AMPA binding was maximal at P18. [³H]kainate binding and [³H]muscimol binding were not age dependent. Dark rearing partially prevented the age-dependent decrease in [³H]MK-801 binding but had no effect on [³H]kainate or [³H]AMPA binding. Dark rearing decreased muscimol binding in adult animals. These results suggest that NMDA receptors, but not other glutamate receptors or GABA_A receptors, are likely to be critical for developmental plasticity in rat visual cortex. J. Comp. Neurol. 383:73–81, 1997. © 1997 Wiley-Liss, Inc.     

Effect of phospholipase A2 on calcium transport in brain synaptosomes

Pretreatment of isolated brain synaptic endings with commercial phospholipase A₂ isolated from venom of Apis mellifera, followed by a BSA washing, selectively inhibited the depolarization-dependent portion of ⁴⁵Ca uptake. Phospholipase A₂ initially caused an increase of synaptosome respiration and subsequently inhibited their oxygen uptake, but this effect was completely abolished in BSA-containing media. The classical uncoupler of oxidative phosphorylation, DNP, produced release of ⁴⁵Ca or [³H]GABA from superfused synaptosomes previously loaded with radioactive calcium or GABA. The treatment of synaptosomes with phospholipase A₂ had no effect on the spontaneous efflux of ⁴⁵Ca or [³H]GABA. However, depolarization-dependent release of [³H]GABA from synaptosomes treated with phospholipase A₂ was significantly inhibited. We suggest that the inhibition of depolarization-dependent influx of ⁴⁵Ca into synaptosomes treated with phospholipase A₂ may be attributed to the lesion of the specific function of plasma membrane rather than to the impairment of the calcium-sequestrating function of intraterminal structures.     

Changes of GABAA receptor binding and subunit mRNA level in rat brain by infusion of subtoxic dose of MK-801

In the present study, we have investigated the effects of prolonged inhibition of NMDA receptor by infusion of subtoxic dose of MK-801 to examine the modulation of GABA_A receptor binding and GABA_A receptor subunit mRNA level in rat brain. It has been reported that NMDA-selective glutamate receptor stimulation alters GABA_A receptor pharmacology in cerebellar granule neurons in vitro by altering the levels of selective subunit. However, we have investigated the effect of NMDA antagonist, MK-801, on GABA_A receptor binding characteristics in discrete brain regions by using autoradiographic and in situ hybridization techniques. The GABA_A receptor bindings were analyzed by quantitative autoradiography using [³H]muscimol, [³H]flunitrazepam, and [³⁵S]TBPS in rat brain slices. Rats were infused with MK-801 (1 pmol/10 μl per h, i.c.v.) for 7 days, through pre-implanted cannula by osmotic minipumps (Alzet, model 2ML). The levels of [³H]muscimol binding were highly elevated in almost all of brain regions including cortex, caudate putamen, thalamus, hippocampus, and cerebellum. However, the [³H]flunitrazepam binding and [³⁵S]TBPS binding were increased only in specific regions; the former level was increased in parts of the cortex, thalamus, and hippocampus, while the latter binding sites were only slightly elevated in parts of thalamus. The levels of β2-subunit were elevated in the frontal cortex, thalamus, hippocampus, brainstem, and cerebellar granule layers while the levels of β3-subunit were significantly decreased in the cortex, hippocampus, and cerebellar granule layers in MK-801-infused rats. The levels of α6- and δ-subunits, which are highly localized in the cerebellum, were increased in the cerebellar granule layer after MK-801 treatment. These results show that the prolonged suppression of NMDA receptor function by MK-801-infusion strongly elevates [³H]muscimol binding throughout the brain, increases regional [³H]flunitrazepam and [³⁵S]TBPS binding, and alters GABA_A receptor subunit mRNA levels in different directions. The chronic MK-801 treatment has differential effect on various GABA_A receptor subunits, which suggests involvement of differential regulatory mechanisms in interaction of NMDA receptor with the GABA receptors.