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A series of Special Issues of Neuropharmacology celebrates the 40th anniversary of a seminal review on excitatory amino acid (EAA) receptors by two pioneers of the field - Dick Evans and Jeff Watkins. Brought together in the Department of Pharmacology at the University of Bristol in the 1970s, they forged a partnership that, through the synthetic chemistry prowess of Jeff Watkins, which provided novel agonists and antagonists for EAA receptors for Dick Evans's deft experimental studies, generated enormous insight into the multitude of actions of EAAs in the nervous system. Among many achievements from this time was not just the naming of the N-methyl-d-aspartate (NMDA) receptor, but also the demonstration of its antagonism by magnesium ions. Here, Dick and Jeff reflect upon those early halcyon days of EAA research, which, as these six 1 Special Issues of Neuropharmacology demonstrate, is very much alive and kicking. Bruno G. Frenguelli, Editor-in-Chief, Neuropharmacology., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Studies of 5-HT-glutamate interactions suggest that activation of brain 5-HT(2A) receptors leads to an AMPA receptor-mediated induction of the immediate early (activity-dependent) gene, Arc (Arg3.1). In this respect, noradrenaline-glutamate interactions are poorly characterised. Here we investigated the influence on regional brain Arc gene expression of selective blockade of α(2)-adrenoceptors in rats. Several complementary techniques were used: qPCR (mRNA, discrete tissue punches), in situ hybridisation (mRNA, sections) and immunocytochemistry. The α(2)-adrenoceptor antagonist, RX 821002, dose-dependently and time-dependently (maximal effect 2 h) increased Arc mRNA levels as demonstrated both by qPCR and in situ hybridisation. The α(2)-adrenoceptor antagonist, atipamezole, also increased Arc mRNA in in situ hybridisation studies. Changes in Arc mRNA after RX 821002 were of similar magnitude in punches and intact tissue sections and region-specific, with effects being most pronounced in parietal cortex and caudate putamen, less robust in frontal cortex, and not detectable in hippocampal sub-regions. Both qPCR and in situ hybridisation studies demonstrated that RX 821002-induced Arc mRNA was blocked by the AMPA antagonist, GYKI 52466. Pretreatment with the NMDA antagonist MK 801 also prevented RX 821002-induced Arc mRNA, as did the mGluR5 antagonist MPEP, whilst the mGluR2/3 antagonist, LY341495, had no effect. Finally, immunocytochemical studies showed that RX 821002 increased Arc-immunoreactivity in cells in close apposition to α(2)-adrenoceptor-positive processes. Thus, employing three complementary techniques, these observations demonstrate that blockade of α(2)-adrenoceptors triggers brain expression of the immediate early gene, Arc, and that this effect involves the recruitment of AMPA, NMDA and mGluR5 but not mGluR2/3 glutamatergic receptors., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The neurobiological relationships between epilepsy and depression are receiving increased experimental attention. A key role for limbic monoamines in depression has been established and we recently showed the importance of hippocampal monoamines in limbic seizure control. We here studied whether anticonvulsant compounds are antidepressant and can elevate hippocampal dopamine (DA) or serotonin (5-HT) levels determined by in vivo microdialysis in rats. We used assessment of seizure severity in the focal pilocarpine model, antidepressant-like activity within the rat forced swim and the mouse tail suspension tests, and locomotor activity in an open field as behavioural tests. We studied the tricyclic antidepressant imipramine, the selective 5-HT reuptake inhibitor citalopram and the selective DA reuptake blocker GBR-12909. These compounds with combined antidepressant-anticonvulsant properties all directly enhanced extracellular hippocampal DA or 5-HT levels. Since glutamate-mediated hyperexcitability in temporal lobe regions seems to be involved in disturbed emotional behaviour, we next investigated possible antidepressant effects and hippocampal DA or 5-HT modulations exerted by selective ionotropic and metabotropic glutamate receptor ligands with anticonvulsant properties. Combined anticonvulsant-antidepressant activities of the NMDA antagonist MK-801 and the mGluR group I antagonists (AIDA, MPEP) were also associated with locally elicited increases in hippocampal DA and/or 5-HT levels. This study highlights that the hippocampus is an important site of action of combined anticonvulsant-antidepressant and monoamine enhancing effects.

Isoflurane, propofol and ketamine are representative general anesthetics with distinct molecular mechanisms of action that have neuroprotective properties in models of excitotoxic ischemic damage. We characterized the effects of these agents on neuronal glutamate and dopamine signaling by profiling drug-induced changes in brain intracellular protein phosphorylation in vivo to test the hypothesis that they affect common downstream effectors. Anesthetic-treated and control mice were killed instantly by focused microwave irradiation, frontal cortex and striatum were removed, and the phosphorylation profile of specific neuronal signaling proteins was analyzed by immunoblotting with a panel of phospho-specific antibodies. At anesthetic doses that produced loss of righting reflex, isoflurane, propofol, and ketamine all reduced phosphorylation of the activating residue T183 of ERK2 (but not of ERK1); S897 of the NR1 NMDA receptor subunit; and S831 (but not S845) of the GluR1 AMPA receptor subunit in cerebral cortex. At sub-anesthetic doses, these drugs only reduced phosphorylation of ERK2. Isoflurane and ketamine also reduced phosphorylation of spinophilin at S94, but oppositely regulated phosphorylation of presynaptic (tyrosine hydroxylase) and postsynaptic (DARPP-32) markers of dopaminergic neurotransmission in striatum. These data reveal both shared and agent-specific actions of CNS depressant drugs on critical intracellular protein phosphorylation signaling pathways that integrate multiple second messenger systems. Reduced phosphorylation of ionotropic glutamate receptors by all three anesthetics indicates depression of normal glutamatergic synaptic transmission and reduced potential excitotoxicity. This novel approach indicates a role for phosphorylation-mediated down-regulation of glutamatergic synaptic transmission by general anesthetics and identifies specific in vivo targets for focused evaluation of anesthetic mechanisms.

Hydrogen sulfide (H(2)S) is a cytotoxic gas recently proposed as a novel neuromodulator. Endogenous levels of H(2)S in the brain range between 50 and 160 microM and perturbed H(2)S synthesis has been reported in the brains from stroke, Alzheimer's disease and Down syndrome patients. Recently, in immature non-glutamate receptor expressing mouse cortical neurons H(2)S was shown to inhibit cell death exhibited by high concentrations of glutamate whereas H(2)S was not cytotoxic. Due to the reported role of H(2)S in facilitating LTP through NMDA receptors we examined the effects of H(2)S on glutamate receptor functioning using mature cortical neurons expressing functional glutamate receptor subtypes. Addition of 100 microM glutamate exhibited extensive cell death which was exacerbated by co-incubation with < or = 200 microM of the H(2)S donor sodium hydrosulfide (NaHS). At <200 microM NaHS induced apoptosis whereas >200 microM NaHS induced necrosis. Cell death was inhibited by pharmacological glutamate receptor antagonists MK801 and APV (NMDA receptor antagonists), and CNQX (kainate and AMPA receptor antagonist) but not kynurenate (broad spectrum glutamate receptor antagonist), GYKI52466 (more selective AMPA receptor antagonist) and CYZ (AMPA receptor potentiator). Although markers of apoptosis were observed, we did not detect caspase activation either by Western blotting or fluorescence assays and caspase inhibitors did not prevent cell death. Rather, H(2)S induced calpain activation and lysosomal membrane destabilization; processes inhibited by preferential antagonists of NMDA and kainate receptors. These data suggest that H(2)S induced neuronal death through ionotropic glutamate receptors, which recruits apoptosis to ensure cellular demise and employs calpains and lysosomal rupture. This study provides novel insights into cell death observed in neurodegenerative diseases involving glutamate receptor activation and perturbed H(2)S synthesis.

We have previously shown that pyramidal neurons engaged in cortico-cortical connectivity in limbic cortex are vulnerable to denervation lesions, i.e. relay pyramidal neurons in layer II of piriform cortex undergo transsynaptic apoptosis after lesions interrupting their inputs from the olfactory bulb (bulbotomies). At least one trigger of this transsynaptic degenerative phenomenon is the activation of inhibitory interneurons in layer I, which are induced to upregulate neuronal nitric oxide synthase (nNOS) and release NO. Thus, we have demonstrated that cortical interneurons play an essential role in transducing injury to apoptotic signaling that selectively targets pyramidal neurons. In the present study, we confirm the role of nNOS with pharmacological inhibition of a significant approximately 30% of transsynaptic apoptosis with the selective nNOS inhibitor BRNI at optimal doses. Outcomes were studied both at the histological and molecular level using DNA blots. We also show that the first-generation competitive non-NMDA (AMPA) antagonist NBQX ameliorates transsynaptic apoptosis by the same margin of difference as BRNI and it also reduces nNOS activation as indicated by a significant decrease in NADPH diaphorase histochemical activity in layer I of piriform cortex. Our findings confirm the role of nNOS activation/NO release in transsynaptic apoptosis and show that glutamatergic agonism at AMPA sites also plays a significant role. In addition, our data suggest that AMPA agonism may occur upstream to nNOS upregulation in inhibitory interneurons of layer I. In concert, our findings indicate that transsynaptic neuronal degeneration in limbic cortex involves complex AMPA-glutamatergic and nitrinergic signaling events. An AMPA-mediated upregulation of nNOS and release of NO by inhibitory interneurons may play a prominent role in this type of injury.

Willardiine derivatives with an N3-benzyl substituent bearing an acidic group have been synthesized with the aim of producing selective antagonists for GLUK5-containing kainate receptors. UBP296 was found to be a potent and selective antagonist of native GLUK5-containing kainate receptors in the spinal cord, with activity residing in the S enantiomer (UBP302). In cells expressing human kainate receptor subunits, UBP296 selectively depressed glutamate-induced calcium influx in cells containing GLUK5 in homomeric or heteromeric forms. In radioligand displacement binding studies, the willardiine analogues displaced [3H]kainate binding with IC50 values >100 microM at rat GLUK6, GLUK2 or GLUK6/GLUK2. An explanation of the GLUK5 selectivity of UBP296 was obtained using homology models of the antagonist bound forms of GLUK5 and GLUK6. In rat hippocampal slices, UBP296 reversibly blocked ATPA-induced depressions of synaptic transmission at concentrations subthreshold for affecting AMPA receptor-mediated synaptic transmission directly. UBP296 also completely blocked the induction of mossy fibre LTP, in medium containing 2 mM (but not 4 mM) Ca2+. These data provide further evidence for a role for GLUK5-containing kainate receptors in mossy fibre LTP. In conclusion, UBP296 is the most potent and selective antagonist of GLUK5-containing kainate receptors so far described.

During an incident of toxic mussel poisoning, the epileptogenic excitotoxin domoic acid (DOM) was associated with lasting neurological deficits mainly in older patients (), suggesting supersensitivity to excitotoxins is a feature of brain aging. Here, hippocampal slices from young (3 months) and aged (26-29 months) Sprague Dawley rats were assessed by CA1 field potential analysis before and after preconditioning with DOM. In naïve slices from young animals, DOM produced initial hyperexcitability followed by significant dose-dependent reductions in population spike amplitude during prolonged application. Following toxin washout, only small changes in neuronal activity were evident during a second application of DOM, suggesting that a resistance to the effects of DOM occurs in hippocampal slices which have undergone prior exposure to DOM. This inducible tolerance was not antagonized by the NMDA receptor blockers APV or MK-801, nor was it diminished by the group I, II or III mGluR blockers AIDA, CPPG and EGLU. Likewise, neither the AMPA/KA blocker CNQX nor the VSCC blocker nifedipine were effective in blocking tolerance induction in young slices. Field potential analysis revealed significant age-related reductions in CA1 EPSP strength, population spike amplitude and paired-pulse inhibition, but aged slices did not differ in sensitivity to DOM relative to young. However, aged CA1 failed to exhibit any tolerance to DOM following preconditioning, suggesting that a loss of inducible neuroprotective mechanisms may account for increased sensitivity to excitotoxins during aging.

Drimanial, a new sesquiterpene isolated from the barks of the plant Drimys winteri (Winteraceae), given systemically, intraplantarly, or by spinal or supraspinal routes, produced pronounced antinociception against both phases of formalin-induced licking. The systemic injection of drimanial also inhibited, in a graded manner, the pain-related behaviours induced by intraplantar or intrathecal (i.t.) administration of glutamate. Moreover, drimanial also caused marked inhibition of the nociception induced by i.t. administration of a metabotropic glutamate agonist (1S,3R)-ACPD, without affecting nociceptive responses induced by ionotropic agonists (NMDA, kainate, AMPA) or by substance P. The antinociception caused by drimanial was not influenced by naloxone, nor did it interfere with the motor coordination of animals in the rota-rod test. Furthermore, drimanial caused graded inhibition of [(3)H]glutamate binding in cerebral cortical membranes from mice, with an IC(50) value of 4.39 micro M. Together, these results provide strong evidence indicating that the sesquiterpene drimanial produces antinociception in mice at peripheral, spinal and supraspinal sites. An interaction with metabotropic glutamate receptors seems to contribute to the mechanisms underlying its antinociceptive action.

Two days following one-week administration of the benzodiazepine, flurazepam (FZP), rats exhibit anticonvulsant tolerance in vivo, while reduced GABA(A) receptor-mediated inhibition and enhanced EPSP amplitude are present in CA1 pyramidal neurons in vitro. AMPA receptor (AMPAR)-mediated synaptic transmission in FZP-treated rats was examined using electrophysiological techniques in in vitro hippocampal slices. In CA1 pyramidal neurons from FZP-treated rats, the miniature excitatory postsynaptic current (mEPSC) amplitude was significantly increased (33%) without change in frequency, rise time or decay time. Moreover, mEPSC amplitude was not elevated in dentate granule neurons following 1-week FZP treatment or in CA1 pyramidal neurons following acute desalkyl-FZP treatment. Regulation of AMPAR number was assessed by quantitative autoradiography with the AMPAR antagonist, [(3)H]Ro48-8587. Specific binding was significantly increased in stratum pyramidale of hippocampal areas CA1 and CA2 and in proximal dendritic fields of CA1 pyramidal neurons. Regulation of AMPAR subunit proteins was examined using immunological techniques. Neither abundance nor distribution of GluR1-3 subunit proteins was different in the CA1 region following FZP treatment. These findings suggest that enhanced AMPAR currents, mediated at least in part by increased AMPAR number, may contribute to BZ anticonvulsant tolerance. Furthermore, these studies suggest an interaction between GABAergic and glutamatergic systems in the CA1 region which may provide novel therapeutic strategies for restoring BZ effectiveness.

Neurotrophins jointly exert various functions in the nervous system, including neuronal differentiation, survival, and regulation of synaptic plasticity. However, the functional interactions of neurotrophins or mechanisms through which neurotrophins regulate each other are still not clear. In the present study, brain-derived neurotrophic factor (BDNF) mRNA expression is induced by neurotrophin-4/5 (NT-4/5) and by BDNF itself in neocortical neurons. K252a, a specific tyrosine kinase (Trk) inhibitor, completely suppresses BDNF- and NT-4/5-enhanced BDNF mRNA expression. NT-4/5 significantly augments BDNF protein production, which is also reversed by K252a. When neurons are incubated with neurotrophin-3 (NT-3) or nerve growth factor (NGF), there are no significant changes in BDNF mRNA or protein expression. Interestingly, the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor blocker 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) or the N-methyl-D-aspartate (NMDA) receptor blocker AP-5 completely suppresses NT-4/5-enhanced BDNF protein production, while tetrodotoxin (TTX) only suppresses NT-4/5-enhanced BDNF production by 50%. Additionally, the mitogen activated protein (MAP) kinase inhibitor PD98059 enhances BDNF-induced glutamate receptor-1 (GluR1) protein expression, but a phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 strongly reduces BDNF-induced GluR1 protein expression. Taken together, glutamate receptors are important for the regulation of BDNF expression by neurotrophins, and MAP and PI3K kinases differentially modulate AMPA receptor expression in the cortical neurons.

Since group II metabotropic glutamate (mGlu) receptors are a potential target for the amelioration of neuronal injury, we evaluated the ability of group II mGlu receptor agonists to attenuate toxicity induced by various insults in cortical, striatal and cerebellar granular (CGCs) pure neuronal cultures. The three cultures, when maintained under serum-free, anti-oxidant rich conditions for up to 13 days in vitro (div) were shown by immunocytochemistry to contain a maximum of 2-7% glia. At 6, 9 and 13 div a graded pattern of injury to cortical and striatal cultures was achieved with either hydrogen peroxide (60-110 microM), staurosporine (1 microM), N-methyl-D-aspartate (NMDA, 70 microM), alpha-amino-3-hydroxy-methylisoxazole-4-propionate (AMPA, 100 microM) or kainate (100 microM) over either 4, 24 or 48 h. CGCs were similarly exposed to low K(+) (5.4 mM KCl). Cell viability was examined via phase-contrast microscopy and assessed by a 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide assay. Treatment with group II mGlu receptor agonists (1-300 microM), 2R,4R-4-aminopyrrolidine-2,4-dicarboxylate ((2R,4R)-APDC), (2S,1'S,2'S)-2-(carboxycyclopropyl)glycine (L-CCG-I), (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV) and N-acetylaspartylglutamate (NAAG) failed to attenuate the toxicity. Pretreatment of cultures with the agonists and treatment following acute insult also failed to attenuate toxicity. Further investigations demonstrated the presence of second messenger activation whereby (2R,4R)-APDC reduced forskolin-stimulated production of cAMP in each culture. Thus, despite receptor coupling to intracellular signaling cascades, and regardless of culture development, agonist concentration, extent and mode of injury, group II mGlu receptor agonists were unable to protect against injury induced in cortical, striatal and cerebellar granular pure neuronal cultures. This result is in contrast to mixed cultures of neurones and glia and implies an important role for glia in the neuroprotective effects of group II mGlu receptor agonists.

The subthalamic nucleus (STN) is a key nucleus in the basal ganglia motor circuit that provides the major glutamatergic excitatory input to the basal ganglia output nuclei. The STN plays an important role in the normal motor function, as well as in pathological conditions such as Parkinson's disease. Development of a complete understanding of the role of the STN in motor control will require a detailed understanding of the mechanisms involved in the regulation of excitatory and inhibitory synaptic transmission in this nucleus. Here, we report that activation of groups I or III metabotropic glutamate (mGlu) receptors, but not group II, causes a depression of excitatory transmission in the STN. In contrast, mGlu receptor activation has no effect on the inhibitory transmission in this nucleus. Further characterization of the group I mGlu receptor-induced effect on EPSCs suggests that this response is mediated by mGlu1 and not mGlu5. Further, paired pulse studies suggest that both the mGlu1 receptor and the group III mGlu receptor-mediated effects are due to a presynaptic mechanism. If these receptors are involved in endogenous synaptic transmission in the STN, these results raise the exciting possibility that selective agents targeting mGlu receptors may provide a novel approach for the treatment of motor disorders involving the STN.

The effect of histamine on the main input to the striatum - the corticostriatal pathway - was studied using electrophysiological techniques in brain slices from rats and mice. Field potentials (FPs) were recorded in the striatum following stimulation at the border of the striatum and the cortex. Bath application of histamine caused a pronounced and long-lasting depression of FPs in rat slices with an IC(50) of 1.6 microM and a maximal depression of around 40%. In mouse slices histamine also depressed FPs, but to a lesser extent and more transiently. Further experiments in rat slices showed that histamine H(3) receptors were responsible for this depression since the selective H(3) receptor agonist R-alpha-methylhistamine (1 microM) mimicked the action of histamine whilst the selective H(3) receptor antagonist, thioperamide (10 microM) blocked the depression caused by histamine application. The histaminergic depression was probably not mediated indirectly through interneurons since blockade of GABA(A), GABA(B), nicotinic and muscarinic receptors or nitric oxide synthase did not prevent the histamine effect. Intracellular recordings from medium spiny neurons in the striatum revealed that histamine did not affect postsynaptic membrane properties but increased paired-pulse facilitation of excitatory synaptic responses indicating a presynaptic locus of action.

We have previously demonstrated that repeated brief increases in extracellular K(+) (K(+)(o)) induce a hyperexcitability in CA1 pyramidal cells that persists for a long time after the final application of K(+) [Neurosci. Lett. 223 (1997) 177; Epilepsy Research (2000) 75]. This epileptiform activity, which was associated with a lasting excitatory postsynaptic potential (EPSP)-spike potentiation, presented some of the characteristic features of traditional in vivo kindling. We have also found that Ca(2+) influx through L-type voltage-sensitive Ca(2+) channels is essential for the development of both in vitro kindling and EPSP-spike potentiation. The aims of this study were to investigate the involvement of ionotropic glutamate receptors, especially those of the NMDA subtype, and the requirement for Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in these phenomena. Field EPSPs with presynaptic fibre volleys from the stratum radiatum, and population spikes from the stratum pyramidale, were recorded in the CA1 area of rat hippocampal slices in response to electrical stimulation of the Schaffer collateral/commissural fibres. Repeated (three episodes) brief (30 s) increases in extracellular K(+) induced a sustained decrease in the threshold for development of evoked epileptiform discharges (i.e. an in vitro kindling-like state) and a lasting potentiation of the EPSP-spike transfer in CA1 pyramidal neurons (EPSP-spike potentiation). The selective antagonist of NMDA receptors, APV (50 microM), blocked the EPSP-spike potentiation, depressed the induction phase of the in vitro kindling-like state, and blocked the maintenance phase of this state. In contrast to APV, the blockade of AMPA/kainate receptors by CNQX (10 microM) had no effect. Like APV, KN62 (3 microM), a selective membrane permeable inhibitor of CaMKII, blocked the EPSP-spike potentiation and the maintenance phase of the in vitro kindling-like state. Our previous and present results therefore demonstrate that Ca(2+) influxes through L-type voltage-dependent-and NMDA receptor-dependent-Ca(2+) channels contribute differentially to the development of an in vitro kindling-like state, and both induce EPSP-spike potentiation in CA1 hippocampal pyramidal cells in response to repeated brief increases in K(+)(o). It is suggested that these effects of intracellular Ca(2+) on the maintenance phase of the in vitro kindling-like state and EPSP-spike potentiation are mediated by CaMKII-dependent mechanisms.

Activation of presynaptic nicotinic acetylcholine receptors (nAChRs) can enhance the release of glutamate from synapses in hippocampal slices and cultures. In hippocampal cultures making autaptic connections, rapid application of a high concentration of nicotine activated presynaptic, postsynaptic, and somatic nAChRs, which consequently enhanced the amplitude of evoked excitatory postsynaptic currents (eEPSCs) mediated by glutamate receptors. The increased eEPSC amplitudes arose from enhanced glutamate release caused by presynaptic nAChRs (Radcliffe and Dani, 1998, Journal of Neuroscience 18, 7075). The same whole-cell nicotine applications that enhanced non-NMDA eEPSCs often decreased the NMDA-receptor component of the eEPSCs. Furthermore, whole-cell activation of nAChRs by nicotine selectively reduced the amplitude of the whole-cell NMDA-receptor currents without affecting the non-NMDA receptor currents. The inhibition by nicotine was prevented by the alpha7-specific antagonist, methyllycaconitine, and required the presence of extracellular Ca(2+). The calmodulin antagonist fluphenazine prevented inhibition of the NMDA-receptor current by nAChR activity, suggesting that a Ca(2+)-calmodulin-dependent process mediated the effect of nicotine. Our results indicate that activation of nAChRs can modulate glutamatergic synapses in several ways. Presynaptic nAChR activity enhances synaptic transmission by increasing transmitter release. Additionally, somatic or postsynaptic nAChRs can initiate a Ca(2+) signal that can act via calmodulin to reduce the responsiveness of NMDA receptors.

In order to better understand the mechanism(s) of action of carbamazepine (CBZ), we studied its effects on the increase in [Ca2+]i and [Na+]i stimulated by glutamate ionotropic receptor agonists, in cultured rat hippocampal neurons, as followed by indo- or SBFI fluorescence, respectively. CBZ inhibited the increase in [Ca2+]i stimulated either by glutamate, kainate, alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA), or N-methyl-D-aspartate (NMDA), in a concentration-dependent manner. In order to discriminate the effects of CBZ on the activation of glutamate receptors from possible effects on Ca2+ channels, we determined the inhibitory effects of Ca2+ channel blockers on [Ca2+]i changes in the absence or in the presence of CBZ. The presence of 1 microM nitrendipine, 0.5 microM omega-conotoxin GVIA (omega-CgTx GVIA), or of both blockers, inhibited the kainate-stimulated increase in [Ca2+]i by 51.6, 32.9 or 68.7%, respectively. In the presence of both 100 microM CBZ and nitrendipine, the inhibition was similar (54.1%) to that obtained with nitrendipine alone, but in the presence of both CBZ and omega-CgTx GVIA, the inhibition was greater (54%) than that caused by omega-CgTx GVIA alone. However, CBZ did not inhibit the increase in [Na+]i stimulated by the glutamate receptor agonists, but inhibited the increase in [Na+]i due to veratridine. Tetrodotoxin, or MK-801, did not inhibit the influx of Na+ stimulated by kainate, indicating that Na+ influx occurs mainly through the glutamate ionotropic non-NMDA receptors. Moreover, LY 303070, a specific AMPA receptor antagonist, inhibited the [Na+]i response to kainate or AMPA by about 70 or 80%, respectively, suggesting that AMPA receptors are mainly involved. Taken together, the results suggest that CBZ inhibits L-type Ca2+ channels and Na+ channels, but does not inhibit activation of glutamate ionotropic receptors.

Both nitric oxide (NO) and glutamate in the brain stem nuclei are involved in central cardiovascular regulation. In the present study, we investigated possible functional interactions between NO and glutamate in the modulation of cardiovascular function in the nucleus tractus solitarii (NTS) of anesthetized rats. In Sprague-Dawley rats, intra-NTS unilateral microinjections of L-glutamate (0.1 nmol/60 nl) and its ionotropic agonists NMDA (5 pmol) and AMPA (2 pmol) resulted in significant decreases in mean blood pressure (MBP) and heart rate (HR). The cardiovascular effects of L-glutamate, NMDA and AMPA were significantly blocked by prior administration of the neuronal NO synthase (nNOS) inhibitor, 7-nitroindazole (7-NI, 0.5 nmol), or by the soluble guanylyl cyclase (sGC) inhibitor, 1H-[1.2.4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ, 0.03-1 pmol). Conversely, a depressor and bradycardic effect was elicited by microinjection of either the NO precursor L-arginine (10 nmol) or the NO donor sodium nitroprusside (SNP, 0.2 nmol) into the NTS. Prior administration of the NMDA receptor antagonists MK-801 (0.1-1 nmol) and APV (0.1-4 nmol) significantly attenuated these effects of L-arginine. Similarly, cardiovascular responses to L-arginine in the NTS were inhibited by pre-injections with the non-NMDA receptor antagonists CNQX (10-330 pmol) and NBQX (2-10 pmol). Furthermore, APV (4 nmol) and CNQX (330 pmol) attenuated the depressor and bradycardic effects of SNP, respectively. This study demonstrates that baroreflex-like responses to microinjections of L-glutamate and its ionotropic agonists into the NTS involve synthesis of NO and activation of sGC. Reciprocally, central cardiovascular effects of NO also depend on responsive ionotropic glutamate receptors.

Although GABA (gamma-aminobutyric acid) is the major inhibitory neurotransmitter in the brain, intense activation of GABA receptors can cause excitation under certain conditions. In the superficial layers of the guinea-pig superior colliculus (SC) slice the excitatory action of GABA (< or = 3 mM) is dominant and sufficient to induce a robust and novel form of long-term potentiation, termed LTPG, of evoked field excitatory postsynaptic potentials (fEPSPs). This action of GABA could neither be mimicked by GABA-A nor -B agonists which were found to suppress synaptic transmission. Additionally, LTPG was not inhibited by the GABA-A receptor antagonist bicuculline while the GABA-C receptor antagonist imidazol-4-acetic acid prevented LTPG. Glutamatergic synaptic transmission was found to be required, as LTPG was partially use-dependent and did not emerge when glutamate receptors of the non-NMDA type were blocked during GABA application. Moreover, LTPG declined to baseline values in the presence of the NMDA antagonist D,L-2-amino-5-phosphonovaleric acid (APV). In addition, the L-type calcium channel blocker nifedipine inhibited the induction of LTPG. It is suggested that activation of excitatory GABA non-A, non-B receptors can lead to LTP in the SC, which may be of major importance for plastic events since the content of GABA and GABA receptors are particularly high in this brain area.

We have investigated the influence of the nitric oxide synthase (NOS) substrate, NG-hydroxy-L-arginine (H-ARG) on dopamine (DA) and glutamate (GLU) efflux in vivo using concentric microdialysis probes implanted in the anterior-medial striatum of chloral hydrate-anesthetized rats. Intrastriatal infusion of H-ARG (100 microM, 200 microM, or 1 mM for 120 min) increased DA efflux in a dose-dependent fashion. The facilitatory effect of H-ARG (1 mM) on DA efflux was abolished following pretreatment (80 min) with the constitutive NOS inhibitor 7-nitroindazole (7-NI, 10 microM) but unaffected by L-NG(1-iminoethyl) lysine (100 microM) infusion. As both H-ARG (1 mM) and the NO-generator (+/-)-S-nitroso-N-acetylpenicillamine (1 mM) were observed to increase GLU efflux concurrently with the effect on DA efflux, we evaluated the potential intermediary role of GLU in NO-facilitated DA efflux using ionotropic GLU receptor antagonists. Local infusion of dizocilpine maleate (10 microM) or (+/-)-2-amino-3-[3-(carboxymethoxy)-5-methyl-isoxazol-4-yl] propionic acid (100 microM), attenuated the H-ARG (1 mM)-induced elevation of extracellular DA levels. Conversely, similar treatment with the kainate receptor antagonist d-gamma-glutamyl-aminomethanesulfonic acid did not alter H-ARG-induced DA efflux. To evaluate the regulatory influence of striatal NO on NMDA receptor activation, NMDA (100 microM) was co-perfused with either H-ARG (2 mM) or 7-NI (10 microM). While co-perfusion with 7-NI potentiated NMDA-induced DA efflux, similar treatment with H-ARG (2 mM) abolished the effect. These results demonstrate that endogenous NO production, stimulated via H-ARG-dependent activation of type 1 NOS, enhances striatal DA efflux via an increase in glutamatergic tone on ionotropic GLU-receptors. At higher levels of NOS activation (following H-ARG (2 mM) or NMDA infusion), NO may block glutamatergic neurotransmission via inhibition of NMDA receptor function.

Monitoring of extracellular cGMP during intracerebral microdialysis in freely moving rats permits the study of the functional changes occurring in the glutamate receptor/nitric oxide (NO) synthase/guanylyl cyclase pathway and the relationship of these changes to animal behaviour. When infused into the rat hippocampus in Mg2+-free medium, cyclothiazide, a blocker of desensitization of the AMPA-preferring receptor, increased cGMP levels. The effect of cyclothiazide (300 microM) was abolished by the NO synthase inhibitor L-NARG (100 microM) or the soluble guanylyl cyclase inhibitor ODQ (100 microM). During cyclothiazide infusion the animals displayed a pre-convulsive behaviour characterized by frequent "wet dog shakes" (WDS). Neither L-NARG nor ODQ decreased the WDS episodes. Both cGMP and WDS responses elicited by cyclothiazide were prevented by blocking NMDA receptor function with the glutamate site antagonist CGS 19755 (100 microM), the channel antagonist MK-801 (30 microM) or Mg2+ ions (1 mM). The AMPA/kainate receptor antagonists DNQX (100 microM) and NBQX (100 microM) abolished the WDS episodes but could not inhibit the cyclothiazide-evoked cGMP response. DNQX or NBQX (but not MK-801) elevated, on their own, extracellular cGMP levels. The cGMP response elicited by the antagonists appears to be due to prevention of a glutamate-dependent inhibitory GABAergic tone, since infusion of bicuculline (50 microM) caused a strong cGMP response. The results suggest that (a) AMPA/kainate receptors linked to the NO/cGMP pathway in the hippocampus (but not NMDA receptors) are tonically activated and kept in a desensitized state by endogenous glutamate; (b) blockade of AMPA/kainate receptor desensitization by cyclothiazide leads to endogenous activation of NMDA receptors; (c) the hippocampal NO/cGMP system is under a GABAergic inhibitory tone driven by non-NMDA ionotropic receptors; (d) the pre-convulsive episodes observed depend on hippocampal NMDA receptor activation but not on NO and cGMP production.

Intrahippocampal perfusion of bicuculline (50 microM) in Mg2+-free medium caused elevation of extracellular cGMP and epileptic-like behaviour. Both effects were partially prevented by blocking NMDA receptors with MK-801 or Mg2+ ions. Similarly, the GABA(B) receptor antagonists CGP52432 (0.1-30 microM) and CGP35348 (0.3-1 mM) evoked increases of extracellular cGMP. CGP52432 also elicited behavioural responses ranging from wet dog shakes to convulsions. MK-801 or Mg2+ ions reduced the effects of CGP52432. Local application of muscimol (100-300 microM) or (-)baclofen (300 microM) caused inhibition of extracellular cGMP. Administration of the AMPA/kainate receptor antagonist NBQX (100 microM) caused cGMP elevation which was almost abolished by co-perfusion of muscimol and (-)baclofen. In the presence of physiological Mg2+, perfusion of AMPA (30 microM) failed to affect cGMP levels, although rats displayed wet dog shakes episodes. When AMPA was co-perfused with low concentrations of bicuculline or CGP52432, cGMP elevations were observed in 60% of the rats. Addition of both antagonists to AMPA resulted in 85% of rats displaying a cGMP response. To conclude: (a) extracellular hippocampal cGMP is controlled by inhibitory GABA(A) and GABA(B) receptors tonically activated through GABAergic interneurons receiving AMPA/kainate-mediated glutamatergic inputs; (b) the GABAergic receptors are not endogenously saturated and can be further stimulated by exogenous agonists; (c) blockade of the GABA-mediated inhibition causes increase of cGMP and epileptic-like behaviour, due largely to endogenous activation of NMDA receptors; (d) reproducible cGMP responses to AMPA can be observed when the inhibitory GABAergic inputs to the NO/guanylyl cyclase system are blocked, confirming the previously proposed existence of AMPA/kainate receptors able to increase the nucleotide synthesis.

Light responses of retinal ON bipolar cells are mediated by metabotropic glutamate receptors selectively activated by L-2-amino-4-phosphonobutyric acid (L-AP4). Antagonists to L-AP4 receptors in ON bipolar cells have not previously been identified. This study examines the electrophysiological effects of (S)-2-amino-2-methyl-4-phosphonobutanoic acid (MAP4), (RS)-4-4-chloro-3,5-dihydroxyphenylglycine (CDHPG) and (RS)-3,4,5-trihydroxyphenylglycine (THPG), at L-AP4 receptors in ON bipolar cells of the amphibian retina. Unlike its actions in spinal cord, in retinal ON bipolar cells MAP4 is a weak agonist which exhibits no detectable antagonism to L-AP4. On the other hand, CDHPG exhibits a mixture of agonist and antagonist properties. Addition of Co2+ and oxygenation of CDHPG turns the solution brown and enhances antagonist effects, suggesting that the antagonism reflects actions of a breakdown product of CDHPG. Although THPG did not prove to be this breakdown product, it also has electrophysiological effects consistent with an L-AP4 receptor antagonist. The results suggest that THPG and breakdown products of CDHPG may be antagonists to L-AP4 receptors in retinal ON bipolar cells, although the possibility that these compounds antagonize effects of L-AP4 by acting at some site in the transduction pathway of L-AP4 receptors cannot yet be excluded.

We have studied the postnatal development of GABAA receptor alpha 1 and alpha 6 subunits expressed by primary cultures of cerebellar granule cells originating from 2-day-old (postnatal day 2, P2) and 10-day-old (P10) rat neonates. At these ages, the granule cells are at distinct stages of cerebellar development. In both cases, GABAA receptor alpha 1 and alpha 6 subunit-like immunoreactivities were detected, and displayed temporal expression profiles that were correlated with the maturity of the cerebella from which the cultured granule cells were derived. Using two different specificity anti-alpha 1 subunit-specific antibodies, immunoreactive species with M(r) 53,000 Da and 54,000 Da were detected by immunoblotting. The lower 53,000-Da band co-migrated with the alpha 1 subunit-like immunoreactivity detected in GABAA receptors purified from adult rat forebrain by benzodiazepine affinity chromatography. This 53,000-Da alpha 1 subunit-like immunoreactive species was detected at day 1 in vitro (1 DIV) in P10 cultures and 3-5 DIV in P2 cultures. The GABAA receptor alpha 6 subunit-like immunoreactivity (58,000 Da) was not detected until 5-7 DIV in P10 and 9-11 DIV in P2-derived cultures. The appearance of alpha 6 subunit-like immunoreactivity was paralleled by an up-regulation of alpha 1 subunit expression and a concomitant increase in diazepam-insensitive (DZ-IS) [3H]Ro 15-4513 binding activity, a pharmacological characteristic of alpha 6 and alpha 1 alpha 6-subunit-containing GABAA receptors (Pollard et al. J. Biol. Chem., 270, 21,285-21,290, (1995)). Antagonism of both non-NMDA and NMDA subtypes of ionotropic glutamate receptors did not significantly affect the developmental profile, the level of GABAA receptor alpha 6 subunit or the total DZ-IS or DZ-S [3H]Ro 15-4513 binding activities expressed by these neurons. These results provide further evidence that the expression of specific GABAA receptor subunit genes is subject to differential regulation. Furthermore, developmental expression of the GABAA receptor alpha 6 subunit gene by these neurons is either a preprogrammed event or is initiated by an environmental cue that is received early in granule cell development, and it is not a result of afferent activation of ionotropic glutamate receptors.

The neurotoxic effects of 4-quinolones alone and in combination with certain non-steroidal anti-inflammatory drugs (NSAIDs) may be related to an interaction at GABAA and/or ionotropic glutamate receptors. In the present study, the effects of the fluoroquinolone, ciprofloxacin, alone and in combination with the NSAID, biphenyl acetic acid (BPAA), were examined on GABAA-, NMDA-, AMPA-, and kainate-evoked current responses recorded from cultured rat hippocampal neurones, using the whole cell patch clamp technique. GABA-evoked currents were reversibly inhibited by bicuculline (3 microM) and ciprofloxacin (100 microM) to 11 +/- 5 and 38 +/- 7% of control, respectively. BPAA (100 microM) had little affect on the GABA current (the response was 82 +/- 4% of control) but enhanced the inhibitory potency of ciprofloxacin by approx. 3000-fold. The antagonist effects of ciprofloxacin (30 microM) and ciprofloxacin (0.03 microM) together with BPAA (100 microM) on the GABA-evoked current were not voltage-dependent. Whole cell currents evoked by NMDA, AMPA or kainate were little influenced by ciprofloxacin (100 microM), BPAA (100 microM), or ciprofloxacin plus BPAA (both at 100 microM); the responses being > or = 90% of control in all cases. These data suggest that the proconvulsant effects of quinolones when combined with BPAA may be related to antagonism of central GABAA receptors but not to an interaction at ionotropic glutamate receptors.

Metabotropic glutamate receptors (mGluRs) form a receptor family that consists of diverse receptor subtypes; now, numbering 8--exclusive of splice variants. (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) has been suggested to be a selective agonist for the mGluRs. We have recently reported that, in rat dorsolateral septal nucleus (DLSN) neurones, a 1S,3R-ACPD-preferring inward current (ACPDi) persists in pertussis toxin-treated rats. We now report that this ACPDi-current: (1) persists in DLSN neurones dialyzed with a stable analog of GTP, namely, GTP gamma S; (2) exhibits a negative slope region with inward rectification in its I-V relationship; (3) persists in neurones superfused with tetrodotoxin or low calcium solutions; (4) is dependent upon both sodium and calcium ions; and (5) is independent of a reduction in temperature. Furthermore, pharmacological data suggest that this current may be activated by a unique type of excitatory amino acid (EAA) receptor, i.e. a receptor which prefers "metabotropic" EAA agonists and is insensitive to AP5 or CNQX. Activation by ACPD of inward currents associated with a conductance increase have also been reported at cultured mouse cerebellar Purkinje neurones; in slices of rat hippocampal CA1 neurones and slice cultures of hippocampal CA3 neurones. We suggest that this ACPDi current may play an important role within the CNS in the induction of long-term potentiation and other neurological processes; processes attributed previously to currents associated with NMDA receptor activation.

As demonstrated recently, mGluRs are involved in some forms of learning. We thus investigated the effect of tADA (trans-azetidine-2,4-dicarboxylic acid) applied intracerebroventricularly prior to learning a spatial alternation paradigm. Compared to controls, tADA treated animals were amnesic when tested for retention 24 hr after training. Effects of state-dependency were excluded. These data and our earlier work indicate that both mGluR agonists and antagonists can have memory-disrupting effects.

Striatal slices, preincubated with [3H]dopamine and [14C]choline, were superfused continuously and subjected to electrical field stimulation (3 Hz) and perfused with amino acid analogues or 4-amino pyridine (4-AP). The released radioactivity was used to monitor release of the neurotransmitters dopamine (DA) and acetylcholine (ACh). Glutamate, NMDA (in the absence of Mg2+), AMPA, kainic acid, domoate and 4-AP all induced DA and ACh release in a concentration-dependent manner. The DA and ACh release induced by NMDA (15 microM) and glutamate (1 mM) was essentially abolished by Mg2+ (1.15 mM), whereas release induced by AMPA (100 microM), kainic acid (100 microM) or 4-AP (30 microM) was not reduced. Tetrodotoxin (1 microM) essentially abolished the effects of NMDA, markedly reduced the effects of glutamate, AMPA and 4-AP, whereas the effect of kainic acid was only modestly affected. MK-801 (30 nM) reduced NMDA-induced DA release by some 70% and ACh release by 30%. MK-801 reduced 4-AP-induced DA release by 40% but not ACh release. CNQX in a concentration (10 microM) that scarcely affected NMDA-induced ACh release, but blocked that induced by AMPA, kainic acid or domoate, reduced the ACh release induced by 4-AP. In summary, DA and ACh release from rat striatum can be stimulated by activation of NMDA and non-NMDA glutamate receptors, and this mechanism is activated by the potassium channel blocker 4-AP.

Acromelic acid, a kainate derivative of natural origin, markedly increased intracellular Ca2+ concentration ([Ca2+]i) in cultured rat spinal neurons in a concentration dependent manner; the half effective concentration (EC50) was 1.3 microM. Acromelic acid was more potent in increasing [Ca2+]i than any other glutamate receptor agonists tested, and the rank order of the activity was as follows: acromelic acid > alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) > kainate > N-methyl-D-aspartate (NMDA) > L-glutamate. Acromelic acid did not increase the [Ca2+]i in a Ca(2+-)free medium. 2,3-Dihydroxy-9-nitro-7-sulfamoylbenzo(F)quinoxaline (NBQX) completely inhibited the [Ca2+]i increase induced by acromelic acid. These results suggest that the [Ca2+]i increase was not through Ca2+ mobilization from intracellular stores but due to Ca2+ influx mediated by the activation of non-NMDA receptors. Acromelic acid increased the [Ca2+]i in rat hippocampal neurons as well; however, the EC50 (6 microM) was considerably higher than that in spinal neurons. The marked increase of [Ca2+]i in cultured spinal neurons would explain, at least in part, the earlier findings that systemic administration of acromelic acid causes selective degeneration confined to lower spinal interneurons.

The pharmacology and cellular mechanism by which metabotropic glutamate receptor (mGluR) activation modulates cAMP formation was studied in cross-chopped hippocampal slices from neonatal (7 day old) rats. The selective mGluR agonist 1S,3R-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD), and other non-selective mGluR agonists produced concentration-related stimulation of basal cAMP formation in this tissue. The relative agonist potency order was 1S,3R-ACPD = quisqualate > ibotenate >> 1R,3S-ACPD. 1S,3R-ACPD stimulated cAMP accumulation was antagonized in a stereoselective manner by L-2-amino-3-phosphonopropionate (L-AP3), but not by higher chain homologues such as L-2-amino-4-phosphonobutyrate (L-AP4) and 2-amino-5-phosphonopentanoate (AP5). 1S,3R-ACPD-enhanced cAMP formation was greatly inhibited by incubation with adenosine deaminase. In the adult rat hippocampus, 1S,3R-ACPD did not appreciably increase basal cAMP, but inhibited forskolin-stimulated cAMP formation, and this effect was observed with or without adenosine deaminase. In the presence of the adenosine receptor antagonist and cAMP phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX), 1S,3R-ACPD did not enhance cAMP formation in the neonatal hippocampus, but inhibited forskolin-stimulated cAMP (like in the adult tissue). These results demonstrate that mGluRs that increase cAMP in the neonatal hippocampus have a unique pharmacology when compared to mGluRs that decrease cAMP accumulation and increase phosphoinositide hydrolysis. 1S,3R-ACPD stimulation of cAMP in the neonatal rat hippocampal slice involves potentiation of responses to endogenous adenosine. Negatively coupled cAMP linked mGluRs are also present in the neonatal tissue, but are masked by the predominance of the positively coupled mGluR cAMP response.

The Ca(2+)-fraction of the ion current flowing through glutamate receptor channels activated either by glutamate or by AMPA was determined in forebrain neurons of the rat medial septum. By combining whole-cell patch-clamp and fura-2 fluorometric measurements we found that, at negative membrane potentials and at an extracellular free Ca(2+)-concentration of 1.6 mM, the Ca(2+)-fraction of the current activated by glutamate is 5.7%. A pharmacological analysis of responses produced by ionophoretically-released glutamate demonstrated a large contribution of NMDA-receptors but a small contribution of AMPA/kainate receptors to these responses. Interestingly, also AMPA-mediated currents were associated with significant changes in Ca(2+)-sensitive fluorescence. The fractional Ca2+ current of AMPA-induced responses was 1.2 +/- 0.4% (n = 5).

Repeated administration of the beta-carboline FG 7142 to mice leads to the development of kindled convulsions. In order to investigate a role for glutamatergic mechanisms in the processes underlying FG 7142 kindling, the N-methyl-D-aspartate (NMDA) antagonist, 2-amino-7-phosphono-heptanoic acid (AP7; 25 nmol), was administered intracerebroventricularly (i.c.v.) daily before administration of FG 7142 (40 mg/kg, i.p.). Under these conditions, kindling to FG 7142 did not occur. Administration of two antagonists at non-NMDA excitatory amino acid receptors, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and gamma-D-glutamylaminomethylsulphonic acid (gamma-D-GAMS; both 25 nmol) did not prevent the development of seizures; these doses were, however, adequate and selective in protecting against seizures induced by respectively quisqualic and kainic acids given by i.c.v. The susceptibility of mice kindled with FG 7142 to seizures induced by NMDA, or kainate or quisqualate was similar in mice which had shown 5 kindled seizures to that seen in drug-naive mice; mice which had shown 10 kindled seizures showed a decreased sensitivity to NMDA-induced convulsions (ED50 was increased from 0.24 to 0.31 nmol). No changes were seen in the convulsant thresholds of either NMDA or non-NMDA agonists. These observations suggest that although NMDA receptors appear to be involved in the processes underlying FG 7142 kindling, such kindling is not necessarily associated with an increased sensitivity of glutamate receptors, and in animals which have convulsed, a decreased sensitivity to NMDA agonists occurs.

The transient activation of the N-methyl-D-aspartate (NMDA) receptor system by high frequency (tetanic) stimulation results in a rapidly developing and long-lasting potentiation of synaptic transmission in the CA1 region of the hippocampus. This potentiation can be divided into an early decremental component, known as short-term potentiation (STP), and a more slowly developing persistent phase, termed long-term potentiation (LTP). Here we describe how activation of metabotropic glutamate receptors (mGluRs), by aminocyclopentane-1S,3R-dicarboxylic acid (1S,3R-ACPD), can induce the same stable form of LTP, but without the STP component. 1S,3R-ACPD-induced LTP does not require electrical stimulation during its induction, but is dependent on an intact connection between the CA3 and CA1 regions of the hippocampus. 1S,3R-ACPD-induced LTP circumvents the need for the activation of NMDA receptors and is likely to involve both the stimulation of protein kinase C (PKC) and the release of Ca2+ from intracellular stores.

With the aid of microiontophoretic techniques the action of caroverine, a quinoxaline-derivative, was tested on the receptor-linked depolarisation of the subsynaptic membrane of cochlear afferents. This membrane can be depolarised by the afferent transmitter agonist glutamate, mediated by NMDA and non-NMDA receptors and by acetylcholine, one of the different transmitter substances, released physiologically on axodendritic efferent synapses. Caroverine antagonized the membrane response to glutamate in an enduring but reversible manner. In contrast, the drug exhibited no effect on the depolarising action of acetylcholine. Therefore, the pharmacological profile of caroverine corresponded to the action of selective glutamate receptor antagonists. Since glutamate is likely to be the major mediator of neurotoxicity in the central nervous system, the selective glutamate-antagonism of caroverine is of particular interest, due to its putative neuroprotective competence. Caroverine is currently available clinically in some countries as a spasmolytic drug. Following these results it is proposed to test the drug for clinical efficacy in putatively glutamate-induced, excitotoxic disorders of the brain.