Activity dependent regulation of BDNF and NGF mRNAs in the rat hippocampus is mediated by non-NMDA glutamate receptors.

The mRNAs of nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) exhibit a similar, though not identical, regional and cellular distribution in the rodent brain. In situ hybridization experiments have shown that BDNF, like NGF, is predominantly expressed by neurons. The neuronal localization of the mRNAs of these two neurotrophic molecules raised the question as to whether neuronal activity might be involved in the regulation of their synthesis. After we had demonstrated that depolarization with high potassium (50 mM) resulted in an increase in the levels of both BDNF and NGF mRNAs in cultures of hippocampal neurons, we investigated the effect of a large number of transmitter substances. Kainic acid, a glutamate receptor agonist, was by far the most effective in increasing BDNF and NGF mRNA levels in the neurons, but neither N-methyl-D-aspartic acid (NMDA) nor inhibitors of the NMDA glutamate receptors had any effect. However, the kainic acid mediated increase was blocked by antagonists of non-NMDA receptors. Kainic acid also elevated levels of BDNF and NGF mRNAs in rat hippocampus and cortex in vivo. These results suggest that the synthesis of these two neurotrophic factors in the brain is regulated by neuronal activity via non-NMDA glutamate receptors.     

NMDA and Non-NMDA Receptor-Mediated Release of [3H]GABA from Granule Cell Dendrites of Rat Olfactory Bulb

Abstract: We have studied the effect of glutamate and the glutamatergic agonists N-methyl-d -aspartate (NMDA), kainate, and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) on [³H]GABA release from the external plexiform layer of the olfactory bulb. The GABA uptake blocker nipecotic acid significantly increased the basal [³H]GABA release and the release evoked by a high K⁺ concentration, glutamate, and kainate. The glutamate uptake blocker pyrrolidine-2,4-dicarboxylate (2,4-PDC) inhibited by 50% the glutamate-induced [³H]GABA release with no change in the basal GABA release. The glutamatergic agonists NMDA, kainate, and AMPA also induced a significant [³H]GABA release. The presence of glycine and the absence of Mg²⁺ have no potentiating effect on NMDA-stimulated release; however, when the tissue was previously depolarized with a high K⁺ concentration, a significant increase in the NMDA response was observed that was potentiated by glycine and inhibited by the NMDA receptor antagonist 2-amino-5-phosphonoheptanoic acid (AP-7). The kainate and AMPA effects were antagonized by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) but not by AP-7. The glutamate effect was also inhibited by CNQX but not by the NMDA antagonist 2-amino-5-phosphonopentanoic acid (AP-5); nevertheless, in the presence of glycine, [³H]GABA release evoked by glutamate was potentiated, and this response was significantly antagonized by AP-5. Tetrodotoxin inhibited glutamate- and kainate-stimulated [³H]GABA release but not the NMDA-stimulated release. The present results show that in the external plexiform layer of the olfactory bulb, glutamate is stimulating GABA release through a presynaptic, receptor-mediated mechanism as a mixed agonist on NMDA and non-NMDA receptors; glutamate is apparently also able to induce GABA release through heteroexchange.     

Presynaptic Glutamate Receptors Regulate Noradrenaline Release from Isolated Nerve Terminals

The wide-ranging neuronal actions of excitatory amino acids, such as glutamate, are thought to be mediated mainly by postsynaptic N-methyl-D-aspartate (NMDA) and non-NMDA receptors. We now report the existence of presynaptic glutamate receptors in isolated nerve terminals (synaptosomes) prepared from hippocampus, olfactory bulb, and cerebral cortex. Activation of these receptors by NMDA or non-NMDA agonists, in a concentration-dependent manner, resulted in Ca(2+)-dependent release of noradrenaline from vesicular transmitter stores. The NMDA-stimulated release was potentiated by glycine and was blocked by Mg2+ and selective NMDA antagonists. In contrast, release stimulated by selective non-NMDA agonists was blocked by 6-cyano-7-nitroquinoxaline-2,3- dione, but not by Mg2+ or NMDA antagonists. Our data suggest that the presynaptic glutamate receptors can be classified pharmacologically as both the NMDA and non-NMDA types. These receptors, localized on nerve terminals of the locus ceruleus noradrenergic neurons, may play an important role in interactions between noradrenaline and glutamate.     

Characterization of glutamate toxicity in cultured rat cerebellar granule neurons at reduced temperature

We have defined conditions whereby glutamate becomes toxic to isolated cerebellar granule neurons in a physiologic salt solution (pH 7.4). In the presence of a physiologic Mg⁺⁺ concentration, acute glutamate excitotoxicity manifests only when the temperature was reduced from 37°C to 22°C. In contrast to glutamate, N-methyl-D-aspartate (NMDA) was non-toxic at either temperature at concentrations as high as 1 mM. Glycine strongly potentiated both the potency and efficacy of glutamate but revealed only a modest NMDA response. The non-NMDA receptor antagonist, 6-cyano-7-nitroquinoxalinedione (CNQX), potently protected against glutamate challenge, although the contribution of antagonism at strychnine-insensitive glycine sites could not be excluded. To further characterize the non-NMDA receptor contribution to the excitotoxic response, the promiscuity of glutamate interaction with ionotropic receptors was simulated by exposing neurons to NMDA in the presence of non-NMDA receptor agonists. NMDA toxicity was potentiated four- to sevenfold when non-NMDA receptors were coactivated by a subtoxic concentration of AMPA, kainate, or domoate. These results suggest that non-NMDA receptor activation participates in the mechanism of acute glutamate toxicity by producing neuronal depolarization (via sodium influx), which in turn promotes the release of the voltage-dependent magnesium blockade of NMDA receptor ion channels. © 1997 John Wiley & Sons, Inc.     

Glutamate-induced cobalt uptake elicited by kainate receptors in rat taste bud cells

Glutamate-induced cobalt uptake reveals non-N-methyl-D-aspartate (non-NMDA) glutamate receptors (GluRs) in rat taste bud cells. However, it is not known which type of non-NMDA glutamate receptors is involved. We used a cobalt staining technique combined with pharmacological tests for kainate or alpha-amino-3-hydroxy-5-methyl-isoxazole-propionic acid (AMPA) receptors and/or immunohistochemistry against subunits of GluRs to examine the presence of non-NMDA receptors in rat foliate tastebud cells. Cobalt uptake into taste cells was elicited by treating taste buds with glutamate, kainate or SYM 2081, a kainate receptor agonist. Treating taste buds with AMPA or fluorowillardiine did not stimulate significant cobalt uptake. Moreover, 6-cyano-7-nitro-quinoxaline-2, 3-dione significantly reduced cobalt staining elicited by glutamate or kainate receptor agonists, but SYM 2206, an AMPA receptor antagonist, did not. Immunohistochemistry against subunits of GluRs reveals GluR6 and KA1-like immunoreactivity. Moreover, most glutamate-induced cobalt-stained cells showed GluR6 and KA1-like immunoreactivity. These results suggest that glutamate-induced cobalt uptake in taste cells occurs mainly via kainate type GluRs.     

Endogenous Interleukin-1β in Neuropathic Rats Enhances Glutamate Release from the Primary Afferents in the Spinal Dorsal Horn through Coupling with Presynaptic N-Methyl-d-aspartic Acid Receptors

Excessive activation of glutamate receptors and overproduction of proinflammatory cytokines, including interleukin-1β (IL-1β) in the spinal dorsal horn, are key mechanisms underlying the development and maintenance of neuropathic pain. In this study, we investigated the mechanisms by which endogenous IL-1β alters glutamatergic synaptic transmission in the spinal dorsal horn in rats with neuropathic pain induced by ligation of the L5 spinal nerve. We demonstrated that endogenous IL-1β in neuropathic rats enhances glutamate release from the primary afferent terminals and non-NMDA glutamate receptor activities in postsynaptic neurons in the spinal dorsal horn. Myeloid differentiation primary response protein 88 (MyD88) is a mediator used by IL-1β to enhance non-NMDA glutamate receptor activities in postsynaptic neurons in the spinal dorsal horn. Presynaptic NMDA receptors are effector receptors used by the endogenous IL-1β to enhance glutamate release from the primary afferents in neuropathic rats. This is further supported by the fact that NMDA currents recorded from small neurons in the dorsal root ganglion of normal rats are potentiated by exogenous IL-1β. Furthermore, we provided evidence that functional coupling between IL-1β receptors and presynaptic NMDA receptors at the primary afferent terminals is mediated by the neutral sphingomyelinase/ceramide signaling pathway. Hence, functional coupling between IL-1β receptors and presynaptic NMDA receptors at the primary afferent terminals is a crucial mechanism leading to enhanced glutamate release and activation of non-NMDA receptors in the spinal dorsal horn neurons in neuropathic pain conditions. Interruption of such functional coupling could be an effective approach for the treatment of neuropathic pain.     

Neuropharmacological analysis of synaptic transmission in the Lorenzinian ampulla of the skate Raja clavata

Summary Dissected ampullae of Lorenzini of the skate (Raja clavata) were studied with the aim of determining the synaptic transmitter between electroreceptor cell and afferent fibre. Resting activity and stimulus-evoked activity in response to electrical pulses were recorded in single afferent units at constant perfusion with normal and test solutions containing different putative neurotransmitters. Presynaptic transmitter release was blocked by Mg²⁺ (up to 50 mM) to investigate the effects of the test substances upon the postsynaptic membrane. l-Glutamate (l-GLU) and l-aspartate (l-ASP), both at concentrations between 10^-7 and 10^-3 M, enlarged strongly resting and stimulus-evoked discharge frequency in the afferent fibre. If transmission was blocked by high Mg²⁺, resting discharge frequency could be restored by l-GLU or l-ASP. The glutamate agonists quisqualate (10^-8–10⁵ M) and N-methyl-D-aspartate (10^-5–10^-3 M) enlarged spontaneous activity in the afferent fiber. The same was found for kainic acid (10^-9–10^-5 M). Taurine at concentrations between 10^-5 and 10^-3 M caused a concentration-dependent decrease in afferent activity. The same was found for gammaaminobutyric acid (GABA; 10^-5–10^-4 M), and for the catecholamines adrenaline and noradrenaline, both in concentrations between 10^-5 and 10^-3 M. Serotonine (10^-5–10^-3 M) and dopamine (10^-5-10^-3 M) had no effect on resting or evoked activity in the Lorenzinian ampulla afferents. Acetylcholine (ACh; 10^-4 M) enlarged discharge frequency in those units with initial rates lower than 22–25 Hz, but diminished discharge frequency in fibres with initial activity higher than 25 Hz. When synaptic transmission was blocked by high Mg²⁺ solution, perfusion with additional ACh did not restore resting activity in the afferent fibre. The results suggest that the most probable transmitter in the afferent synapse of the ampullae of Lorenzini is l-GLU or l-ASP, or a substance of similar nature.Abbreviations ACh acetylcholine - GABA gamma aminobutyric acid - KA kainic acid - l-ASP l-aspartate - l-GLU l-glutamate - NMDA N-methyl-D-aspartate - Q quisqualate - n.s. normal solution     

Localization and interaction of N-methyl-D-aspartate and non-N-methyl-D-aspartate receptors of lamprey spinal neurons.

Small volumes of N-Methyl-D-Aspartate (NMDA) and non-NMDA excitatory amino acid receptor agonists were applied to localized regions of the dendritic trees of lamprey spinal neurons along their medial-lateral axis to obtain a spatial map of glutamate receptor distribution. Voltage clamp and frequency domain methods were used to obtain quantitative kinetic data of the voltage dependent ionic channels located both on the soma and on highly branched dendritic membranes. Pressure pulses of NMDA applied to the most peripheral regions of the dendritic tree elicited large somatic impedance increases, indicating that the most peripheral dendrites are well supplied with NMDA receptors. Experiments done with kainate did not elicit somatic responses to agonist applications on peripheral dendrites. The data obtained are consistent with the hypothesis that the activation of NMDA receptors by exogenous glutamate is significantly modified by the simultaneous activation of non-NMDA receptors, which shunts the NMDA response. The non-NMDA shunting hypothesis was tested by a combined application of kainate and NMDA to mimic the action of glutamate showing that the shunting effect of non-NMDA receptor activation virtually abolished the marked voltage dependency typical of NMDA receptor activation. These data were interpreted with a compartmental neuronal model having both NMDA and non-NMDA receptors.     

Involvement of γ-Aminobutyric Acid and N-Methyl-D-Aspartate Receptors in the Inhibitory Effect of Ethanol on Pentylenetetrazole-Induced c-fos Expression in Rat Brain

The expression of c-fos mRNA in rat brain was induced by intraperitoneal administration of pentylenetetrazole (PTZ) and picrotoxin, which act on the picrotoxin binding site of the gamma-aminobutyric acid-benzodiazepine (GABA-BZ) receptor complex, by N-methyl-D-aspartate (NMDA) and kainic acid, agonists of different classes of glutamate receptors and by caffeine, an antagonist of adenosine receptors. The actions of PTZ and picrotoxin but not that of NMDA were blocked by ethanol and the NMDA-receptor antagonist, MK-801. Ro 15-4513 partially reversed the inhibitory effect of ethanol on PTZ-induced c-fos mRNA synthesis. Acute ethanol administration blocked the actions of PTZ and NMDA without affecting the response to kainic acid or caffeine. Taken together, these data suggest that ethanol blocks c-fos gene activation by noncompetitive antagonists of the GABA-BZ receptor via actions on both the NMDA and GABA receptors.     

Crystal structures of the glutamate receptor ion channel GluK3 and GluK5 amino-terminal domains

Ionotropic glutamate receptors (iGluRs) mediate the majority of fast excitatory synaptic neurotransmission in the central nervous system. The selective assembly of iGluRs into AMPA, kainate, and N-methyl-d-aspartic acid (NMDA) receptor subtypes is regulated by their extracellular amino-terminal domains (ATDs). Kainate receptors are further classified into low-affinity receptor families (GluK1-GluK3) and high-affinity receptor families (GluK4-GluK5) based on their affinity for the neurotoxin kainic acid. These two families share a 42% sequence identity for the intact receptor but only a 27% sequence identity at the level of ATD. We have determined for the first time the high-resolution crystal structures of GluK3 and GluK5 ATDs, both of which crystallize as dimers but with a strikingly different dimer assembly at the R1 interface. By contrast, for both GluK3 and GluK5, the R2 domain dimer assembly is similar to those reported previously for other non-NMDA iGluRs. This observation is consistent with the reports that GluK4-GluK5 cannot form functional homomeric ion channels and require obligate coassembly with GluK1-GluK3. Our analysis also reveals that the relative orientation of domains R1 and R2 in individual non-NMDA receptor ATDs varies by up to 10°, in contrast to the 50° difference reported for the NMDA receptor GluN2B subunit. This restricted domain movement in non-NMDA receptor ATDs seems to result both from extensive intramolecular contacts between domain R1 and domain R2 and from their assembly as dimers, which interact at both R1 and R2 domains. Our results provide the first insights into the structure and function of GluK4-GluK5, the least understood family of iGluRs.     

Role of non-NMDA receptors in vasopressin and oxytocin release from rat hypothalamo-neurohypophysial explants

Glutamate is recognized as a prominent excitatory transmitter in the supraoptic nucleus (SON) and is involved in transmission of osmoregulatory information from the osmoreceptors to the vasopressin (VP) and oxytocin (OT) neurons. Explants of the hypothalamo-neurohypophysial system were utilized to characterize the roles of the non-N-methyl-D-aspartate (NMDA) glutamate receptor subtypes (non-NMDA-Rs), kainic acid receptors (KA-Rs), and aminopropionic acid receptors (AMPA-Rs) and to evaluate the interdependence of NMDA-Rs and non-NMDA-Rs in eliciting hormone release. Although both KA and AMPA increased hormone release, a specific agonist of the KA-Rs, SYM-2081, was not effective. This combined with the finding that cyclothiazide, an agent that inhibits the desensitization of AMPA-Rs, increased the VP response to both KA and AMPA indicates that the increase in hormone release induced by the non-NMDA agonists is mediated via AMPA-Rs, rather than KA-Rs. Inhibition of osmotically stimulated VP and OT release by a specific AMPA-R antagonist indicated that AMPA-Rs are essential for mediating osmotically stimulated hormone release. NMDA-stimulated VP but not OT release was prevented by blockade of non-NMDA-Rs, but AMPA-stimulated VP/OT release was not prevented by NMDA-R blockade.     

Sulphur-Containing Amino Acids Modulate Noradrenaline Release from Hippocampal Slices

Abstract: The l - and d -enantiomers of the sulphur-containing amino acids (SAAs)—homocysteate, homocysteine sulphinate, cysteate, cysteine sulphinate, and S-sulphocysteine—stimulated [³H]noradrenaline release from rat hippocampal slices in a concentration-dependent manner. The relative potencies of the l -isomers (EC₅₀ values of 1.05–1.96 mM) were of similar order to that of glutamate (1.56 mM), which was 10-fold lower than that of NMDA (0.15 mM), whereas the d -isomers exhibited a wider range of potencies (0.75 to >5 mM). All stimulatory effects of the SAAs were significantly inhibited by the voltage-sensitive Na⁺ channel blocker tetrodotoxin (55–71%) and completely blocked by addition of Mg²⁺ or Co²⁺ to the incubation medium. All SAA-evoked responses were concentration-dependently antagonized by the selective NMDA receptor antagonist d -(?)-2-amino-5-phosphonopentanoic acid (IC₅₀ values of 3.2–49.5 µM). 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA receptor antagonist, at 100 µM inhibited the [³H]noradrenaline release induced by glutamate and NMDA (65 and 76%, respectively) and by all SAAs studied (65–85%), whereas 10 µM CNQX only inhibited the effects of S-sulpho-l -cysteine and l - and d -homocysteate (33, 32, and 44%, respectively). However, the more selective AMPA/kainic acid receptor antagonist 6-nitro-7-sulphamoylbenzo(f)quinoxaline-2,3-dione (100 µM), which did not antagonize the [³H]noradrenaline release induced by glutamate and NMDA, reduced only the S-sulpho-l -cysteine-evoked response (25%). Thus, the stimulation of Ca²⁺-dependent[³H]noradrenaline release from hippocampal slices elicited by the majority of the SAAs appears to be mediated by the NMDA receptor.     

Synaptic transmission in the pineal eye of young Xenopus laevis tadpoles: a role for NMDA and non-NMDA glutamate and non-glutaminergic receptors?

The pineal eye of Xenopus laevis tadpoles is directly photosensitive. A sudden reduction in light intensity produces a burst of activity in the pineal ganglion cells, which is closely followed by the onset of swimming. In this paper I present the results of experiments on the effects of agonists and antagonists of candidate pineal transmitters on ganglion cell activity. I found that NMDA and non-NMDA excitatory amino acid (EAA) agonists increased pineal activity, indicating the presence of both types of receptor. Kynurenic acid reduced activity, thus confirming that the photoreceptor transmitter is an EAA. Under physiological conditions, CNQX blocked activity almost completely whilst AP5 had little effect. In Mg²⁺-free saline CNQX had a considerably smaller effect, but joint application of CNQX and AP5 blocked almost all activity; therefore, the NMDA receptors are subject to blockage by Mg²⁺. Although GABA_A and ACh receptors appear to be present, no evidence was found for GABA or ACh as pineal transmitters. In addition, 5-HT had no effect on pineal activity. The main pineal transmitter is an EAA acting on ganglion cells through both NMDA and non-NMDA receptors. Other receptors are present but appear to have no role in controlling pineal activity at this stage. Accepted: 1 March 1997     

Parabrachial nucleus induces suppression of baroreflex bradycardia by the release of glutamate in the rostral ventrolateral medulla of the rat

The involvement of glutamatergic neurotransmission in the rostral ventrolateral medulla (RVLM) in the suppression of baroreflex bradycardia by the parabrachial nucleus (PBN) was investigated. Repeated electrical activation of the PBN increased the concentration of glutamate in the dialysate collected from the RVLM. The same stimulation also suppressed baroreflex bradycardia in response to transient hypertension evoked by phenylephrine (5 microg/kg, intravenously). Microinfusion of L-glutamate (10, 50 or 100 microM) via the microdialysis probe into the RVLM dose-dependently elicited a significant inhibition of baroreflex bradycardia that paralleled the concentration and time course of the PBN-elicited elevation in extracellular glutamate in the RVLM. The suppression of baroreflex bradycardia elicited by microinjection of L-glutamate (1 nmol) into the RVLM was appreciably reversed by coinjection of the NMDA receptor antagonist, dizocilpine (500 pmol), or the non-NMDA receptor antagonist, 6-cyano-7-nitroquinoxaline-2, 3-dione (50 pmol). These results suggest that an increase in the extracellular concentration of glutamate and activation of both NMDA and non-NMDA receptors in the RVLM may mediate the suppression of baroreflex bradycardia by activation of the PBN.     

Kainic acid binding to membranes of striatal neurons.

The specific binding of [³H]-kainic acid to membrane fragments of rat striatum was examined. The specific binding was found to be saturable and of high affinity. The dissociation constant was about 71 nM, while the apparent maximal number of receptor sites was 254 fmoles/mg protein. [³H]-Kainic acid binding was effectively competed by both unlabeled kainic acid and glutamate, Lesions of the striatum by stereotaxic injection of 5 nmoles of kainic acid reduced the density of [³H]-kainic acid binding sites by half, without affecting their affinity. Lesions of the cortico-striatal afferents, however, did not affect the binding of [³H]-kainic acid, although sodium-dependent glutamate uptake was reduced by 30%. It is concluded that [³H]-kainic acid binds to a population of receptors localized on neurons of the caudate-putamen.     

Actions of excitatory amino acids on mesencephalic trigeminal neurons

Mesencephalic trigeminal (MeV) neurons are primary sensory neurons of which the cell soma is located within the brainstem, and is associated with synaptic contacts. In previous studies it has been reported that these cells are resistant to kainic acid excitotoxicity, and have little or no responsiveness to exogenously applied glutamate or selective agonists. In an in vitro slice preparation with intracellular recording, we have found that these cells respond to pressure-applied glutamate, N-methyl-D-aspartic acid (NMDA), kainate (KA), and (R,S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). The kainate and AMPA responses appear to be mediated by different receptors, at least in part, since they exhibit differing sensitivity to an AMPA receptor selective antagonist. The agonists generally evoke larger responses than glutamate and exhibit a long-duration desensitization requiring approximately 10 min for full recovery. Some cross-desensitization between the glutamate agonists is also observed. Mesencephalic trigeminal neurons exhibit high-frequency oscillatory activity during depolarizations that approach threshold potentials, and these could combine with transmitter-induced depolarizations to enhance the excitability of these cells. Previous reports of nonsensitivity to glutamate and to kainate excitotoxicity are attributable to relatively small responses, and to the desensitization expressed by these neurons.     

Extracellular Striatal Dopamine and Glutamate After Decortication and Kainate Receptor Stimulation, as Measured by Microdialysis

Abstract: Disruption of corticostriatal glutamate input in the striatum decreased significantly extracellular striatal glutamate and dopamine levels. Local administration of 300 µ M concentration of excitatory receptor agonist kainic acid increased significantly extracellular striatal dopamine in intact freely moving rats. These findings support the hypothesis that glutamate exerts a tonic facilitatory effect on striatal dopamine release. The effect of kainic acid on extracellular striatal glutamate concentration in intact rats was a biphasic increase. The first glutamate increase can be explained by stimulation of presynaptic kainate receptors present on corticostriatal glutamatergic nerve terminals; the second increase is probably the result of a continuous interaction of the different striatal neurotransmitters after disturbance of their balance. Release of dopamine and glutamate was modulated differently in the intact striatum and in the striatum deprived of corticostriatal input. Dopamine release in the denervated striatum after kainate receptor stimulation was significantly lower than in intact striatum, confirming the so-called cooperativity between glutamate and kainic acid. Loss of presynaptic kainate receptors on the glutamatergic nerve terminals after decortication resulted in a loss of effect of kainic acid on glutamate release in denervated striatum. Aspartate showed no significant changes in this study.     

Ethanol sensitivity of NMDA receptors

NMDA receptors are ionotropic glutamate receptors assembled of subunits of the NR1 and of the NR2 family (NR2A–NR2D). The subunit diversity largely affects the pharmacological properties of NMDA receptors and, hence, gives rise to receptor heterogeneity. As an overall result of studies on recombinant and native NMDA receptors, ethanol inhibits the function of receptors containing the subunits NR2A and/or NR2B to a greater extent than those containing NR2C or NR2D. For example, in rat cultured mesencephalic neurons, NR2C expression was developmentally increased, whereas expression of NR2A and NR2B was decreased. These changes coincided with a developmental loss of sensitivity of NMDA responses to ethanol and ifenprodil, a non-competitive NMDA receptor antagonist that shows selectivity for NR2B-containing receptors. Also in rat locus coeruleus neurons, the low ethanol sensitivity of somatic NMDA receptors could be explained by a prominent expression of NR2C. The inhibitory site of action for ethanol on the NMDA receptor is not yet known. Patch–clamp studies suggest a target site exposed to or only accessible from the extracellular environment. Apparently, amino acid residue Phe⁶³⁹, located in the TM3 domain of NR1, plays a crucial role in the inhibition of NMDA receptor function by ethanol. Since this phenylalanine site is common to all NMDA and non-NMDA receptor (AMPA/kainate receptor) subunits, this observation is consistent with accumulating evidence for a similar ethanol sensitivity of a variety of NMDA and non-NMDA receptors, but it cannot explain the differences in ethanol sensitivity observed with different NR2 subunits.     

Effect of the Non-NMDA Receptor Antagonist GYKI 52466 on the Microdialysate and Tissue Concentrations of Amino Acids Following Transient Forebrain Ischaemia

Abstract: The effect of the non-N-methyl-D-aspartate (non-NMDA) receptor antagonist 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride (GYKI 52466) on ischaemia-induced changes in the microdialysate and tissue concentrations of glutamate, aspartate, and γ-aminobutyric acid (GABA) was studied in rats. Twenty minutes of four-vessel occlusion resulted in a transient increase in microdialysate levels of glutamate, aspartate, and GABA in striatum, cortex, and hippocampus. Administration of GYKI 52466 (10 mg/kg bolus + 10 mg/kg/60 min intravenously starting 20 min before onset of ischaemia) inhibited ischaemia-induced increases in microdialysate glutamate and GABA in striatum without affecting the increases in hippocampus or cortex. Twenty minutes of four-vessel occlusion resulted in immediate small decreases and larger delayed (72 h) decreases in tissue levels of glutamate and aspartate. Transient increases in tissue levels of GABA were shown in all three structures at the end of the ischaemic period. At 72 h, after the ischaemic period, significantly reduced GABA levels were observed in striatum and hippocampus. GYKI 52466, given under identical conditions as above, augmented the ischaemia-induced decrease in striatal tissue levels of glutamate and aspartate, without significantly affecting the decreases in hippocampus and cortex. Twenty minutes of ischaemia resulted in a large increase in microdialysate dopamine in striatum. GYKI 52466 failed to inhibit this increase. Kainic acid (500 μM infused through the probe for 20 min) caused increases in microdialysate glutamate and aspartate in the striatum. GYKI 52466 (10 mg/ kg bolus + 10 mg/kg/60 min) completely inhibited the kainic acid-induced glutamate release. In conclusion, the action of the non-NMDA antagonist, GYKI 52466, in the striatum is different from that in the cortex and hippocampus. The inhibition by GYKI 52466 of ischaemia-induced and kainate-induced increases in microdialysate glutamate concentration in the striatum may be related to the neuroprotection provided by GYKI 52466 in this region.