Search

We have previously shown that the release of central neurotransmitters can be modulated by the activation of Group I and Group II subtypes of G-protein-linked metabotropic glutamate (mGlu) receptors. To date, however, very little is known about the regulation of serotonergic neurotransmission by these receptor subtypes. In the present study, we have utilized in vivo intracerebral microdialysis to elucidate the roles of Group I and Group II mGlu receptors in the regulation of neuronal 5-hydroxytryptamine (5-HT) release in the frontal cortex of conscious, freely moving rats. Dialysate 5-HT was of neuronal origin with basal release showing strong calcium dependency and tetrodotoxin sensitivity and marked elevation following K(+)-induced depolarization. The broad-spectrum mGlu receptor agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid [(1S,3R)-ACPD; 1-3 mM] did not significantly modify basal cerebrocortical 5-HT release. Similarly, the Group I mGlu receptor-specific agonist (RS)-3,5-dihydroxyphenylglycine [(RS)-3,5-DHPG; 1-3 mM] showed no marked effect on cortical dialysate 5-HT levels. To eliminate the possibility that these findings were the result of receptor desensitization, the effects of lower concentrations of (RS)-DHPG (100-300 microM) and shorter ligand exposure time (15 min) were also evaluated. Dialysate 5-HT levels remained unmodified by these manipulations. In comparison, the Group II mGlu receptor agonist, (2S,1'S,2'S)-2-(carboxycyclopropyl)glycine (L-CCG-1; 500 microM), evoked a marked facilitation of release (approximately 150% of basal) which was fully reversed by the Group I/II antagonist, (S)-alpha-methyl-4-carboxyphenylglycine [(S)-MCPG; 3 mM]. The modulatory action of L-CCG-1 showed a bell-shaped concentration-response relationship. (S)-MCPG (3 mM) and the potent and selective mGlu(5) receptor antagonist, 2-methyl-6-(phenylethynyl)pyridine (MPEP; 100 microM), when given alone, did not significantly modify 5-HT levels.The current data provide strong evidence to suggest that while the release of neuronal 5-HT in the rat frontal cortex is not subject to regulation by facilitatory Group I mGlu receptors, it may be positively modulated by activation of Group II mGlu receptors. Taken together with data from other studies, the present investigation lends emphasis to the notion that neuromodulation by mGlu receptors is a region-specific phenomenon and also proposes that the heterogeneous distribution of these receptors is neurone-specific in its complexity. The failure of (S)-MCPG alone to modify cortical 5-HT release suggests that Group II mGlu receptors do not tonically modulate serotonergic neurotransmission in the cerebral cortex but this does not preclude an important functional role for these receptors during pathological conditions when endogenous neurotransmitter levels become excessively elevated. The strategic development of new subtype-specific mGlu receptor ligands may provide novel therapeutic agents for the treatment of a range of neurological and psychiatric disorders., (Copyright 2003 IBRO)

The effects of metabotropic glutamate receptor (mGluR) activation on non-dopamine (putative GABAergic) neurons and inhibitory synaptic transmission in the ventral tegmental area were examined using intracellular recordings from rat midbrain slices. Perfusion of (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD; agonist for group I and II mGluRs), but not L-amino-4-phosphonobutyric acid (L-AP4; agonist for group III mGluRs), produced membrane depolarization (current clamp) and inward current (voltage clamp) in non-dopamine neurons. The t-ACPD-induced depolarization was concentration-dependent (concentration producing 50% maximal depolarization [EC(50)]=6.1+/-2.5 microM), and was blocked by the antagonist (+/-)-alpha-methyl-4-carboxyphenylglycine, but not by tetrodotoxin and ionotropic glutamate-receptor antagonists. The t-ACPD-evoked responses were mimicked comparably by selective group I mGluR agonist (RS)-3,5-dihydroxyphenylglycine (DHPG). Furthermore, the DHPG-induced depolarization in non-dopamine neurons was greatly reduced by mGluR1-specific antagonist 7(hydroxyimino)cyclopropachromen-1a-carboxylate ethyl ester. When recorded in dopamine neurons, the frequency of spontaneous GABA(A) receptor-mediated inhibitory postsynaptic potentials was increased by t-ACPD but not L-AP4. However, the amplitude of evoked inhibitory postsynaptic currents in dopamine neurons was reduced by all three group mGluR agonists. These results reveal a dual modulation of mGLuR activation on inhibitory transmission in midbrain ventral tegmental area: enhancing putative GABAergic neuronal excitability and thus potentiating tonic inhibitory synaptic transmission while reducing evoked synaptic transmission at inhibitory terminals.

Deep dorsal horn neurons present plateau properties involved in non-linear integration of nociceptive inputs, in the windup of the discharge, and in the expression of long-lasting afterdischarges. In vitro experiments using intracellular recordings in a slice preparation of the rat spinal cord have established that they are supported in part by voltage-dependent calcium currents, and positively modulated by metabotropic glutamate receptor activation. In the present study, whole-cell patch-clamp recordings in acutely isolated soma of dorsal horn neurons (n=48) were used to analyse the voltage-dependent calcium currents involved.Deep dorsal horn neurons expressed both inactivating and non-inactivating calcium currents with Ca(2+) or Ba(2+) used as a charge carrier. The non-inactivating component activated at intermediate threshold (-55mV), and was blocked mostly by nifedipine (61+/-6%). Although voltage-dependent facilitation of whole-cell calcium currents could be obtained by prepulses to +100mV, repetitive depolarization at potentials compatible with the plateau (-45mV and -10mV) failed to induce facilitation of calcium currents. No direct modulation of somatic calcium currents by application of (S)-3,5-dihydroxyphenylglycine, a selective group I metabotropic glutamate receptor agonist and 1S,3R-1-amino-1,3-cyclopentanedicarboxylic acid, a group I and II metabotropic glutamate receptor agonist, was found, while application of the metabotropic GABA(B) receptor agonist baclofen induced a significant decrease in calcium currents.Thus, the present voltage-clamp study shows that rat deep dorsal horn neurons express a non-inactivating, nifedipine sensitive, intermediate threshold (-55mV) calcium current which could provide the depolarizing drive to generate plateau potentials near threshold. Our results also indicate that calcium currents are not sensitized following repetitive stimulation, and not modulated by metabotropic glutamate receptor activation. They provide, however, the first evidence for a direct modulation of voltage-gated calcium channels in dorsal horn neurons by GABA(B) receptor activation, which may contribute to the mechanism of baclofen's antinociceptive activity.

We studied N-methyl-D-aspartate-induced cell death in organotypic hippocampal slices from seven-day-old Wistar rat pups cultured for 12-14 days in a medium containing no added glutamate. Propidium iodide fluorescence intensity was used as an indicator of cell death measured with the help of confocal microscopy. Exposure of slices for 2h to L-glutamate (1-500 microM) prior to the N-methyl-D-aspartate challenge significantly reduced N-methyl-D-aspartate-induced cell death. Glutamate at 10 and 500 microM concentrations was highly protective against N-methyl-D-aspartate-induced cell death, but was less protective at the 1 microM concentration. The protection was not blocked by the Na(+) channel blocker tetrodotoxin (1 microM), the N-methyl-D-aspartate receptor antagonist D-2-amino-5-phosphonopentanoic acid (20 microM) or the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (20 microM). 1S, 3R-1-Aminocyclopentane-trans-1,3-dicarboxylic acid, an agonist at metabotropic glutamate receptor types 1, 2/3 and 5, was protective at 100 microM but not at 50 microM. In contrast, the ionotropic glutamate receptor agonist aspartate (250 microM) facilitated N-methyl-D-aspartate toxicity. Treatment of slices with the protein kinase C inhibitor staurosporine (0.2 microM) or antisense oligonucleotide (10nM, 72 h) that selectively inhibits metabotropic glutamate receptor type 5 synthesis significantly reduced glutamate protection. These results suggest that ambient glutamate may reduce nerve cell susceptibility to injury caused by excessive N-methyl-D-aspartate receptor activation by acting at metabotropic glutamate receptors linked to protein kinase C.

Maintenance of long-term potentiation in perforant path-granule cell synapses is associated with an increase in glutamate release, which we have suggested relies on an interaction between arachidonic acid and the metabotropic glutamate receptor agonist, trans-1-amino-cyclopentyl-1,3-dicarboxylate (ACPD). Evidence suggests that this interaction is dependent on stimulation of tyrosine kinase, which phosphorylates and activates phospholipase Cgamma. In this study, we demonstrate that arachidonic acid and ACPD stimulate tyrosine phosphorylation of a protein of about 40,000 mol. wt and further analysis, using a specific antibody, suggested that this may be extracellular signal-regulated kinase, one member of the family of mitogen-activated protein kinases. Activity of extracellular signal-regulated kinase was increased by arachidonic acid and ACPD in vitro, but it was also increased by induction of long-term potentiation in perforant path-granule cell synapses. A role for extracellular signal-regulated kinase in long-term potentiation was supported by the observation that expression of long-term potentiation, as well as the associated increases in endogenous glutamate release and extracellular signal-regulated kinase activation, were inhibited by pretreatment with the mitogen-activated protein kinase inhibitor, PD98059, while PD98059 pretreatment inhibited the interaction between arachidonic acid and ACPD on glutamate release. An age-related decrease in extracellular signal-regulated kinase activity was observed in the dentate gyrus, and there was no evidence of increased extracellular signal-regulated kinase activity or endogenous glutamate release in tissue prepared from aged rats in which long-term potentiation was compromised. The evidence is consistent with the view that increased activation of extracellular signal-regulated kinase plays a role in long-term potentiation, and that activation of this kinase relies on the interaction between arachidonic acid and ACPD.

It has been previously reported that block of high-frequency stimulation-induced long-term potentiation of synaptic transmission in the hippocampus does not necessarily lead to impairment of spatial learning. Here we show that (1S,3S)-1-aminocyclopentane-1,3-dicarboxylic acid, an agonist at group II metabotropic glutamate receptors, completely blocked long-term potentiation induced by a theta-burst type of stimulation protocol (five pulses at 75 Hz per train, 200 ms inter-train interval) in the CA1 region in vivo. The drug did not significantly affect synaptic responses during each train whereas inter-train facilitation of excitatory postsynaptic potentials was slightly reduced. It also produced a large reduction in paired-pulse facilitation (50 ms inter-stimulus interval), possibly indicating that an increase in inhibition might be involved in the block of long-term potentiation. The drug dose used (5 microliters of a 10 mM solution i.c.v.) was half the dose which inhibited high-frequency stimulation-induced long-term potentiation in earlier experiments but which did not prevent learning of spatial tasks. We conclude that long-term potentiation induced by a more physiological stimulation protocol which uses theta-like inter-train intervals does not appear to accurately model the synaptic changes which are believed to occur during learning either.

The transition from sleep to waking is associated with the abolition of spindle waves and the appearance of tonic activity in thalamocortical neurons and thalamic reticular/perigeniculate GABAergic cells. We tested the possibility that changes such as these may arise through modulation of the leak potassium current, IKL, by examining the effects of neurotransmitters known to modulate this current on spindle wave generation in the ferret geniculate slice maintained in vitro. Local application of agents that reduce IKL in thalamocortical neurons, including acetylcholine, DL-muscarine chloride and the glutamate metabotropic receptor agonist 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD), to spontaneously spindling thalamocortical neurons resulted in a 5-10 mV membrane depolarization and the abolition of spindle waves. Local application of 1S,3R-ACPD and cholecystokinin-8-sulfate, both of which reduce IKL, to GABAergic neurons of the perigeniculate nucleus resulted in a 10-20 mV membrane depolarization, appearance of tonic discharge and the abolition of spindle wave generation. Local application of 1S,3R-ACPD and cholecystokinin to the perigeniculate nucleus while recording from thalamocortical neurons resulted in the abolition of spindle wave-associated inhibitory postsynaptic potentials and the occurrence of a continuous barrage of smaller amplitude inhibitory postsynaptic potentials, presumably in response to depolarization and tonic discharge of perigeniculate neurons. These results indicate that modulation of IKL in thalamocortical neurons and perigeniculate neurons is capable of abolishing the generation of spindle waves in thalamic networks. Through the modulation of IKL, ascending and descending activating systems may control the state of the thalamus such that the transition from slow wave sleep to waking is associated with the abolition of slow, synchronized rhythms and the facilitation of a state that is conducive to sensory receptor field analysis, arousal and perception.

We have reported previously that a selective metabotropic glutamate receptor agonist, (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD), caused two primary postsynaptic membrane changes, namely, a slow membrane depolarization, and burst firing in rat dorsolateral septal nucleus neurons. In addition, (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid also potentiates a slow after depolarization in rat dorsolateral septal nucleus neurons. We now report that, among all the postsynaptic membrane changes induced by (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid, only the burst firing was selectively blocked by pertussis toxin pretreatment. Thus, aminocyclopentane-1,3-dicarboxylic acid induced burst firing was mediated by a metabotropic receptor coupled to a pertussis toxin-sensitive GTP-binding protein, while the other induced cellular responses may be mediated by metabotropic glutamate receptors insensitive to pertussis toxin. We further characterized this receptor pharmacologically. This metabotropic receptor is activated by several metabotropic glutamate receptor agonists, but is insensitive to L-glutamate or L-aspartate. On the basis of its agonist activity profile, particularly the ineffectiveness of glutamate as an agonist, we have tentatively assigned the name aminocyclopentane-1,3-dicarboxylic acid metabotropic receptor, to this native, pertussis toxin-sensitive metabotropic receptor in the dorsolateral septal nucleus. Furthermore, this receptor is coupled to protein kinase C, probably via a phospholipase C independent pathway.

To investigate the electrophysiological effects of the stimulation of the metabotropic excitatory amino acid receptors, we applied trans-1-amino-cyclopentane-1,3-dicarboxylate, an agonist of this type of receptors, on presumed rat dopamine cells intracellularly recorded in vitro. Trans-1-amino-cyclopentane-1,3-dicarboxylate (3-30 microM, t-ACPD) caused a sustained increase of the spontaneous firing rate and a depolarization. When the membrane potential was held at about the resting level (-50, -60 mV), by the single-electrode voltage-clamp technique, t-ACPD induced an inward current. In 57% of the tested cells the inward current was associated with a decrease of the apparent input conductance. In the remaining cells no obvious changes in membrane conductance were observed. The active form of t-ACPD, (1S,3R)-1-amino-cyclopentane-1,3-dicarboxylate [3-50 microM, (1S,3R)-ACPD] also produced a reversible inward current on the dopaminergic cells and this was antagonized by (S)-4-carboxy-3-hydroxyphenylglycine (300 microM), a selective antagonist of the (1S,3R)-ACPD-induced depolarization on central neurons. The (1S,3R)-ACPD-induced inward current was not antagonized by L-2-amino-3-phosphonopropionic acid (100 microM), an antagonist of the t-ACPD-induced activation of inositide synthesis. 6-cyano-7-nitroquinoxaline-2,3-dione (10 microM), an alfa-amino-3-hydroxy-5- methyl-isoxazole propionic acid/kainate antagonist, DL-amino-5-phosphonopentanoic acid (30 microM), an N-methyl-D-aspartate antagonist, and scopolamine (10 microM), a muscarinic antagonist, did not significantly affect the actions of t-ACPD. A block of synaptic transmission obtained by applying tetrodotoxin failed to prevent the action of t-ACPD.(ABSTRACT TRUNCATED AT 250 WORDS)

The possible roles of G-protein coupled metabotropic glutamate receptors in central nervous function are currently the focus of intensive investigation. The complexity of effects produced by agonists at these receptors probably reflects the activity of a range of sub-types. The metabotropic glutamate receptors first described are linked to phospholipase C, mediating phosphoinositide hydrolysis and release of Ca2+ from intracellular stores. A substance generally considered to be a selective agonist for these receptors is (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD). This substance not only stimulates phosphoinositide hydrolysis, but also inhibits cyclic AMP formation. A family of metabotropic glutamate receptors, incorporating both phospholipase C- and adenylcyclase-linked sub-types has been cloned. Various effects of metabotropic glutamate receptor agonists on membrane ion fluxes and synaptic events have been reported, including neuronal depolarization and/or excitation, hyperpolarization, inhibition of Ca(2+)-dependent and voltage-gated K+ currents, potentiation of N-methyl-D-aspartate-induced responses, depression of synaptic excitation and either induction or augmentation of long-term potentiation. To clarify the role of metabotropic glutamate receptors in central nervous activity and to aid the characterization of the various receptor types that may be involved, a range of highly selective agonists and antagonists is required. To date, currently available antagonists such as L-2-amino-3-phosphonopropionate and L-aspartic acid-beta-hydroxamate appear to be unselective and insufficiently potent. We report here the actions of three phenylglycine derivatives, the particular agonist and/or antagonist properties of which may help to elucidate the roles of metabotropic glutamate receptors in central nervous activity.(ABSTRACT TRUNCATED AT 250 WORDS)

The slow (<1 Hz) rhythm is an electroencephalogram hallmark of resting sleep. In thalamocortical neurons this rhythm correlates with a slow (<1 Hz) oscillation comprising recurring UP and DOWN membrane potential states. Recently, we showed that metabotropic glutamate receptor activation brings about an intrinsic slow oscillation in thalamocortical neurons of the cat dorsal lateral geniculate nucleus in vitro which is identical to that observed in vivo. The aim of this study was to further assess the properties of this oscillation and compare them with those observed in thalamocortical neurons of three other thalamic nuclei in the cat (ventrobasal complex, medial geniculate body; ventral lateral nucleus) and two thalamic nuclei in rats and mice (lateral geniculate nucleus and ventrobasal complex). Slow oscillations were evident in all of these additional structures and shared several basic properties including, i) the stereotypical, rhythmic alternation between distinct UP and DOWN states with the UP state always commencing with a low-threshold Ca2+ potential, and ii) an inverse relationship between frequency and injected current so that slow oscillations always increase in frequency with hyperpolarization, often culminating in delta (delta) activity at approximately 1-4 Hz. However, beyond these common properties there were important differences in expression between different nuclei. Most notably, 44% of slow oscillations in the cat lateral geniculate nucleus possessed UP states that comprised sustained tonic firing and/or high-threshold bursting. In contrast, slow oscillations in cat ventrobasal complex, medial geniculate body and ventral lateral nucleus thalamocortical neurons exhibited such UP states in only 16%, 11% and 10% of cases, respectively, whereas slow oscillations in the lateral geniculate nucleus and ventrobasal complex of rats and mice did so in <12% of cases. Thus, the slow oscillation is a common feature of thalamocortical neurons that displays clear species- and nuclei-related differences. The potential functional significance of these results is discussed.

Following the hypothesis of the "signal-to-noise" ratio we examined whether changes in the activity of group-I metabotropic glutamate (mGlu) receptors in the hippocampus are associated with a condition that specifically enhances the learning capacity in rats. As a model, we used rats that had been nursed by mothers drinking a solution of corticosterone (13.5 mg of daily intake of corticosterone hemisuccinate) during the lactation period. These rats were prone to learn, as indicated by a better performance in a passive avoidance test. Stimulation of polyphosphoinositide (PI) hydrolysis by the mGlu receptor agonist, 1S,3R-1-amino-cyclopentan-1,3-dicarboxylic acid (1S,3R-ACPD), was attenuated in hippocampal slices prepared from corticosterone-nursed male and female rats at 30 or 60 days of postnatal life, an age at which an increased learning capacity could be demonstrated. This effect was specific because the PI response to carbamylcholine was unchanged. A reduced PI hydrolysis in corticosterone-nursed rats was also observed when group-I mGlu receptors (i.e. mGlu1 and -5 receptors) were selectively activated using 3,5-dihydroxyphenylglycine or 1S,3R-APCD combined with the selective group-II mGlu receptor antagonist, 2S-2-amino-2-(1S,2S-2-carboxycyclopropan-1-yl)-3-(xanth-9-yl)propionate. Western blot analysis showed a selective reduction in the expression of mGlu1a receptor protein in the hippocampus of corticosterone-nursed rats, whereas expression of mGlu5 and mGlu2/3 receptors was unchanged. The reduction in mGlu-receptor mediated PI hydrolysis in the hippocampus may contribute to the greater learning capacity of corticosterone-nursed rats by reducing the background noise over which a specific signal must be superimposed during learning. This hypothesis was supported by the evidence that mGlu-receptor stimulated PI hydrolysis was amplified in hippocampal slices from rats subjected to a passive avoidance learning paradigm, and that this amplification was greater in slices from corticosterone-nursed rats of both sexes.

The modulation of spontaneous miniature GABAergic inhibitory postsynaptic currents (mIPSC) by the metabotropic glutamate receptors was investigated in the mechanically dissociated rat nucleus basalis of Meynert neurons using the conventional whole-cell patch recording configuration. An application of (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (tACPD) reversibly reduced the frequency of mIPSC without affecting the current amplitude distribution. The application of K+ channel blockers such as 4-aminopyridine, Cs+, Ba2+ or tetraethylammonium increased the mIPSC frequency, but failed to inhibit the tACPD action on mIPSC. Although the removal of Ca2+ from the extracellular solution reduced the mIPSC frequency, the inhibitory effect of tACPD on mIPSC was unaltered. These results suggested that neither voltage-dependent K+ or Ca2+ channels are involved in the inhibitory effect of tACPD on mIPSC frequency. Forskolin, an activator of adenylate cyclase, facilitated the mIPSC frequency in a concentration-dependent manner and inhibited the tACPD-induced suppression of mIPSC frequency. 8-Br-cAMP, a membrane permeable analog of cAMP, also prevented the inhibitory action of tACPD. However, Sp-cAMP, an activator of protein kinase A, could not prevent the inhibitory action of tACPD. L-CCG-I and (2R,4R)-APDC, group II mGluR agonists, mimicked the tACPD action on mIPSC frequency, but L-AP4, a group III mGluR agonist, had no such effect. MCCG, a group II mGluR antagonist, fully blocked the tACPD action. It was concluded that the activation of group II mGluR on the GABAergic presynaptic nerve terminals projecting to the rat nucleus basalis of Meynert neurons therefore inhibits the GABA release by reducing the activity of the cAMP-dependent pathway.

The cyclobutylglycine (+/-)-2-amino-2-(3-cis and trans-carboxycyclobutyl-3-(9-thioxanthyl)propionic acid) (LY393053) has been identified as a functionally potent metabotropic glutamate receptor antagonist. It is most potent on the two group I metabotropic glutamate receptors, 1alpha and 5alpha, with IC50 values of 1.0+/-0.4 microM and 1.6+/-1.4 microM, respectively. In this study, LY393053 has also been evaluated electrophysiologically on native group I metabotropic glutamate receptors in an in vitro spinal cord preparation as well as behaviourally, in a mouse model of visceral pain. LY393053 dose-dependently antagonised group I agonist, (RS)-3, 5-dihydroxyphenylglycine, or a broad-spectrum agonist (1S,3R)-amino-1,3-cyclopentanedicarboxylic acid-induced depolarisation of spinal motoneurons. The apparent Kd values were estimated to be 0.3 microM against (RS)-3, 5-dihydroxyphenylglycine-induced depolarisation and 0.5 microM against (1S,3R)-amino-1,3-cyclopentanedicarboxylic acid-induced depolarisation, respectively. On the other hand, the dorsal root-ventral root potential elicited at 8 x threshold was depressed by LY393053 with IC50 values of 9.0+/-0.7 microM and 12.7+/-1.7 microM on monosynaptic and polysynaptic responses, respectively. When investigated using the mouse acetic acid writhing test, LY393053 showed significant analgesic effects at doses of 1-10 mg/kg intraperitoneally. An ED50 value of 6.0 mg/kg was obtained in this test. By revealing a potent effect of LY393053 in antagonising the native group I metabotropic receptor-mediated responses in the spinal cord in rodents, and an antinociceptive efficacy in a mouse visceral pain model, these results, therefore, provide additional evidence in support of the analgesic potential of metabotropic glutamate receptor antagonists.

Depolarization-induced transmitter release in synaptosomes prepared from the hippocampus of aged rats is decreased compared with release from young animals. Although the underlying cause of this deficit is not known, some evidence suggests that increased membrane rigidity may contribute to these age-related synaptic changes. One possible consequence of the decreased transmitter release in the hippocampus of aged rats is a reduced ability to sustain long-term potentiation in perforant path-granule cell synapses, a pathway in which maintenance of long-term potentiation and increased glutamate release have been coupled. The observation that there is an age-dependent impairment in long-term potentiation is consistent with this view. If the age-related deficits in release and long-term potentiation are a consequence of increased membrane rigidity, it must be predicted that any manoeuvre which reverses membrane rigidity should reverse these functional deficits. In the present study, we investigated the effect of dietary manipulation of aged rats with omega-3 fatty acids on synaptic function. The data obtained indicate that an eight-week modified feeding schedule reversed the age-related impairments in long-term potentiation and depolarization-induced glutamate transmitter release. We also report that the concentrations of both docosahexanoic acid and arachidonic acid, two main polyunsaturated fatty acids in neuronal membranes, were decreased in the hippocampus of aged rats, and were restored by dietary manipulation. The data are consistent with the hypothesis that these deficits results from a change in membrane composition.

Numerous studies have suggested that excitatory projections from the ventral hippocampus to the nucleus accumbens modulate locomotor activity in rats. Furthermore, the ability of ventral hippocampal neurons to alter locomotor activity may involve the dense dopaminergic innervation found in the nucleus accumbens. The purpose of this study was to: (i) more fully characterize the locomotor effects of acute alterations in ventral hippocampal activity; (ii) ascertain the influence of dopamine agonists and antagonists on locomotor changes produced by altered ventral hippocampal activity; and (iii) use immediate early gene induction to determine whether dopamine antagonists alter the response of nucleus accumbens neurons to ventral hippocampal stimulation. By comparing a variety of excitatory amino acid agonists, it was found that ventral hippocampal infusion of N-methyl-D-aspartate elevated locomotor activity in a subconvulsive manner, while other excitatory amino acid receptor agonists did not. Inactivation of the ventral hippocampus achieved by lidocaine infusion did not suppress ongoing locomotor activity, nor did it affect amphetamine-induced increases in locomotor activity. Increases in locomotor activity induced by ventral hippocampal N-methyl-D-aspartate infusion were blocked by systemic administration of haloperidol (a D2 receptor antagonist), SCH-23390 (a D1 receptor antagonist) or reserpine. Cellular expression of the protein product of the immediate early gene, c-fos, was dramatically increased in the nucleus accumbens shell after ventral hippocampal N-methyl-D-aspartate infusion, and haloperidol, SCH-23390 and reserpine attenuated this effect. These results suggest that the increases, but not decreases, in ventral hippocampal activity have a measurable effect on ongoing rates of locomotion, and that this effect requires both D1 and D2 receptors. Moreover, the studies of Fos expression suggest that dopamine receptor antagonists attenuate neuronal responses to ventral hippocampal stimulation within the nucleus accumbens, a brain region important in the generation and maintenance of locomotor activity.

In this study, we investigated the possible contribution of presynaptic group 1 metabotropic glutamate receptor activation to changes in synaptic efficacy by means of analysis of glutamate release in hippocampal synaptosomes. Data were interpreted in the context of group 1 metabotropic glutamate receptor involvement in synaptic plasticity in the CA1 region of freely moving rats. In synaptosomes, 3,5-dihydroxyphenylglycine enhanced diacylglycerol formation and facilitated vesicular Ca(2+)-dependent glutamate release, whereas trans-azetidine-2,4-dicarboxylic acid had no effect on these processes. Trans-azetidine-2,4-dicarboxylic acid enhanced glutamate release, but in a Ca(2+)-independent manner. This effect was mimicked by the L-glutamate uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid. (R,S)-alpha-Methyl-4-carboxyphenylglycine blocked the effects of 3,5-dihydroxyphenylglycine, but not trans-azetidine-2,4-dicarboxylic acid in synaptosomes. Short-term potentiation (100 Hz, three bursts of 10 stimuli, 0.1 ms stimulus duration, 10 s interburst interval) was induced in the CA1 region in vivo. The metabotropic glutamate receptor agonist 1S,3R-aminocyclopentane-2,3-dicarboxylic acid, or the group 1 metabotropic glutamate receptor agonists, 3,5-dihydroxyphenylglycine and trans-azetidine-2,4-dicarboxylic acid, dose-dependently facilitated short-term potentiation into long-term potentiation, which lasted > 24 h. The facilitation was inhibited by the metabotropic glutamate receptor antagonist, (R,S)-alpha-methyl-4-carboxyphenylglycine, and the group 1 metabotropic glutamate receptor antagonist, (S)-4-carboxy-phenylglycine, but not by the group 2 metabotropic glutamate receptor antagonist, (R,S)-alpha-methylserine-O-phosphate monophenyl ester. L-Trans-pyrrolidine-2,4-dicarboxylic acid dose-dependently facilitated short-term potentiation into long-term potentiation, which lasted < 4 h. These data suggest that activation of group 1 metabotropic glutamate receptors results in presynaptic modulation of glutamate release. This effect may contribute to group 1 metabotropic glutamate modulation of the expression of long-term potentiation in vivo.

The effects of the metabotropic glutamate receptor agonists against apoptosis induced by oxygen-glucose deprivation or colchicine were studied in the primary cultures of mature cerebellar granule cells. Oxygen-glucose deprivation (90 min) or addition of colchicine (1 microM) resulted in neuronal damage as revealed by Trypan Blue assay 12 h later. Further analysis demonstrated that the cells exposed to oxygen-glucose deprivation or colchicine exhibit typical features of apoptosis: internucleosomal DNA fragmentation, condensation and fragmentation of chromatin and typical DNA ladder on agarose gel electrophoresis. Metabotropic glutamate receptor agonist, (1S,3R)-1-aminocycloheptane-trans-1,3-dicarboxylic acid, acting at group I and II receptors, and selective agonist, (2S,1'R,2R',3R')-2(2,3-dicarboxycyclopropyl)glycine, acting at group II receptors, added to cells recovering from oxygen-glucose deprivation exerted neuroprotective action against oxygen-glucose deprivation-induced apoptosis. Similar neuroprotective effects of metabotropic glutamate receptor agonists were observed against colchicine-induced apoptosis. The results thereby provide evidence that metabotropic glutamate receptor agonists have therapeutic potential in the treatment of pathologies associated with increased neuronal apoptosis. The selective protein kinase C inhibitor bisindolylmaleimide (100 nM) abolished the neuroprotective action of (1S,3R)-1-aminocycloheptane-trans-1,3-dicarboxylic acid, whereas the activator of adenylyl cyclase forskolin (10 microM) abolished the neuroprotective action of (2S,1'R,2R',3R')-2(2,3-dicarboxycyclopropyl)glycine (30 microM) against colchicine-induced apoptosis. It is concluded that both phosphoinositide hydrolysis with consequent activation of protein kinase C and inhibition of adenylyl cyclase seem to contribute to the neuroprotective action of metabotropic glutamate receptor agonists against neuronal apoptosis in the primary culture of cerebellar granule cells.

The perirhinal cortex is crucially involved in various forms of learning and memory. Decrements in neuronal responsiveness occur in the perirhinal cortex with stimulus repetition during visual recognition performance. However, very little is known concerning the underlying mechanisms of synaptic transmission and plasticity in this cortical region. In this study, we provide evidence demonstrating the presence of functional group I, II and III metabotropic glutamate receptors in the rat perirhinal cortex in vitro. Furthermore, the results demonstrate long-lasting synaptic depression in the perirhinal cortex. Extracellular synaptic responses were recorded from superficial layers of the perirhinal cortex directly below the rhinal sulcus, in response to electrical stimuli delivered in the superficial or intermediate layers to the entorhinal or temporal cortex sides of the rhinal sulcus. Evoked synaptic potentials were depressed during bath perfusion of each of the following: the broad-spectrum metabotropic glutamate receptor agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid, the selective group I agonist (R,S)-3,5-dihydroxyphenylglycine, the group II agonist (2S,1'R,2'R,3'R)-(2',3'-dicarboxycyclopropyl)glycine and the group III agonist (S)-2-amino-4-phosphonobutanoate. Furthermore, there was a long-lasting depression of synaptic transmission following washout of (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid, (R,S)-3,5-dihydroxyphenylglycine or (2S,1'R,2'R,3'R)-(2',3'-dicarboxy-cyclopropyl)glycine. Activation of group III metabotropic glutamate receptors by (S)-2-amino-4-phosphonobutanoate did not result in long-lasting changes in synaptic transmission. Thus, the pharmacological activation of metabotropic glutamate receptors can produce short- or long-term changes in synaptic transmission in the perirhinal cortex. It is possible therefore, that metabotropic glutamate receptors are involved in the decrement in neuronal responsiveness associated with visual recognition in the perirhinal cortex.

In rat brainstem slices, we investigated the possible role of metabotropic glutamate receptors in modulating the synaptic transmission within the medial vestibular nuclei, under basal and plasticity inducing conditions. We analysed the effect of the metabotropic glutamate receptor antagonist (R,S)-alpha-methyl-4-carboxyphenylglycine on the amplitude of the field potentials and latency of unitary potentials evoked in the ventral portion of the medial vestibular nuclei by primary vestibular afferent stimulation, and on the induction and maintenance of long-term potentiation, after high-frequency stimulation. Two effects were observed, consisting of a slight increase of the field potentials and reduction of unit latency during the drug infusion, and a further long-lasting development of these modifications after the drug wash-out. The long-term effect depended on N-methyl-D-aspartate receptor activation, as D,L-2-amino-5-phosphonopentanoic acid prevented its development. We suggest that (R,S)-alpha-methyl-4carboxyphenylglycine enhances the vestibular responses and induces N-methyl-D-aspartate-dependent long-term potentiation by increasing glutamate release, through the block of presynaptic metabotropic glutamate receptors which actively inhibit it. The block of these receptors was indirectly supported by the fact that the agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid reduced the vestibular responses and blocked the induction of long-term potentiation by high-frequency stimulation. The simultaneous block of metabotropic glutamate receptors facilitating synaptic plasticity, impedes the full expression of the long-term effect throughout the (R,S)-alpha-methyl-4-carboxyphenylglycine infusion. The involvement of such a facilitatory mechanism in the potentiation is supported by its reversible reduction following a second (R,S)-alpha-methyl-4-carboxyphenylglycine infusion. The drug also reduced the expression of potentiation induced by high-frequency stimulation. Conversely the electrical long-term potentiation was still induced, but it was occluded by the previous drug potentiation. We conclude that metabotropic glutamate receptors play a dual functional role in the medial vestibular nuclei, consisting in the inhibition of glutamate release under basal conditions, and the facilitation of N-methyl-D-aspartate-dependent plasticity phenomena.

Activation of metabotropic glutamate receptors is known to elicit a rise in intracellular Ca2+ and the present study was undertaken to see whether they also modulate the intracellular pH (pHi) of neurons and glia. Measurements of the pHi of neurons and astrocytes were made with the ratiometric fluorescent dye 2',7'-biscarboxyethyl-5,6-carboxyfluorescein. In the absence of bicarbonate, stimulation with the specific metabotropic glutamate receptor agonist 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid caused a fall in pHi in both astrocytes and neurons. In the presence of bicarbonate, stimulation with 25 microM 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid elicited a rise in pHi in the astrocytes, while the neurons responded with a small acidification. The astrocytic alkalinization could also be elicited by the specific group I metabotropic glutamate receptor agonist (S)-3-hydroxyphenylglycine but not by the group II agonist (2S,1'S,2'S)-(2-carboxycyclopropyl)glycine or by the group III agonist L(+)-2-amino-4-phosphonobutyric acid. The alkalinization of glial cells could be reduced by preloading the cells with BAPTA, but not by removal of extracellular Ca2+. Depolarization of the astrocytes with potassium elicited a small alkalinization, but stimulation with 100 microM 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid in high potassium medium elicited a further alkalinization. It is concluded that activation of group I metabotropic glutamate receptors leads to an alkalinization of astrocytes by a process that involves an elevation of intracellular Ca2+. The pHi changes that follow activation of the metabotropic glutamate receptors may play a role in initiation of glial proliferation following cerebral injury.

Previous work has shown that responses of thalamic neurons in vivo to the metabotropic glutamate receptor agonists 1S,3R-aminocyclopentane-1,3-dicarboxylate and S-3,5-dihydroxyphenylglycine can be reduced by a variety of phenylglycine antagonists. Responses of thalamic neurons to noxious thermal somatosensory stimuli were reduced in parallel by these antagonists, indicating that these responses are mediated by Group I metabotropic glutamate receptors (i.e. metabotropic glutamate receptor-1 and/or metabotropic glutamate receptor-5), which are known to be linked to phosphoinositol phosphate hydrolysis. The recent development of S-2-methyl-4-carboxyphenylglycine as an antagonist which is highly selective for metabotropic glutamate receptor-1 compared to metabotropic glutamate receptor-5 on human receptors expressed in AV-12 cells, now offers the possibility of discriminating between these two receptor subtypes in order to distinguish which is involved in thalamic responses. We have made recordings from single somatosensory neurons in the thalamus of the rat, and find that S-2-methyl-4-carboxy-phenylglycine is able to reduce responses of neurons to 1S,3R-aminocyclopentane-1,3-dicarboxylate, S-3,5-dihydroxyphenylglycine, and noxious stimuli without significant effect on responses to either N-methyl-D-aspartate or (+/-)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate. These results suggest that excitatory responses of thalamic neurons to 1S,3R-aminocyclopentane-1,3-dicarboxylate and S-3,5-dihydroxyphenylglycine may be mediated by metabotropic glutamate receptor-1. Furthermore, the reduction of nociceptive responses by S-2-methyl-4-carboxy-phenylglycine indicates that metabotropic glutamate receptor-1 is involved in thalamic nociceptive processing and that such antagonists may have analgesic properties.

The role of metabotropic glutamate receptors in the processing of somatosensory information was studied in dorsal horn neurons of the rat spinal cord. Activation of metabotropic glutamate receptors by local iontophoresis of (1S,3R)-1-amino-cyclopentane-1,3-dicarboxylic acid resulted in an increased response of dorsal horn neurons to ionotropic glutamate receptor agonists (N-methyl-D-aspartate and kainic acid) applied by iontophoresis. Greater amounts of 1S,3R-1-amino-cyclopentane-1,3-dicarboxylic acid, ejected at high iontophoresis currents, directly excited dorsal horn neurons. Application of (1S,3R)-1-amino-cyclopentane-1,3-dicarboxylic acid also led to a significant increase in responses to innocuous (brush, pressure) but not in responses to noxious (pinch, squeeze) mechanical stimulation. The excitatory effects of (1S,3R)-1-amino-cyclopentane-1,3-dicarboxylic acid were selectively blocked by (S)-4-carboxy-3-hydroxyphenyl-glycine, an antinociceptive phenylglycine derivative which is a selective group 1 metabotropic glutamate receptor antagonist, confirming the involvement of these receptors. In wide dynamic range neurons, wind-up, the progressive potentiation of C-fibre-evoked responses during a train of stimuli, was increased by iontophoretic application of (1S,3R)-1-amino-cyclopentane-1,3-dicarboxylic acid or decreased by iontophoresis of (S)-4-carboxy-3-hydroxyphenyl-glycine without significant change in the C-fibre input. The results suggest an interaction between metabotropic and ionotropic glutamate receptors in spinal dorsal horn neurons. Metabotropic glutamate receptors proved to be involved in the frequency-dependent potentiation of C-fibre responses possibly via modulation of ionotropic glutamate receptors. The long-lasting effects of (1S,3R)-1-amino-cyclopentane-1,3-dicarboxylic acid on wind-up and on responses to peripheral mechanical stimuli strongly support the view that metabotropic glutamate receptors in these neurons may play a significant role in spinal synaptic plasticity, and therefore, may contribute to the central sensitization during mechanical hyperalgesia.

Synchronous neuronal activity that resembles interictal epileptiform discharges occurs in hippocampal slices if there is an imbalance of inhibitory and excitatory synaptic activity. Antagonists of the GABAA receptor and agonists of the ionotropic glutamate receptors are convulsants that produce epileptiform discharges in hippocampal slices. We evaluated the effects of activation of the metabotropic class of glutamate receptors on epileptiform activity produced by convulsants. The metabotropic glutamate agonist (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (ACPD, 30-100 microM) accelerated the rate of interictal epileptiform discharges produced by either bicuculline methiodide or 4-aminopyridine and had minimal effects on discharges produced by high [K+]o. The increase in rate was associated with a significant decrease in the amplitude and duration of the afterhyperpolarization that follows the paroxysmal depolarizing shift, the intracellular correlate of the interictal epileptiform discharge. A modest increase in input resistance (approximately 10%) accompanied the rate increase. beta-adrenergic or muscarinic agonists, neurotransmitters that also decrease the afterhyperpolarization, acted synergistically with ACPD (100 microM) to increase the control rate of bicuculline-induced interictal discharges by more than eight-fold. Antagonists of beta-adrenergic or muscarinic receptors reduced, but did not block, the acceleration of bicuculline-induced discharge rate produced by 30 microM ACPD. The results show that metabotropic glutamate receptors enhance the rate of interictal epileptiform discharges produced by bicuculline or 4-aminopyridine. ACPD had no effect on interictal epileptiform activity induced by high [K+]o, a finding that may indicate that in high [K+]o conditions the metabotropic receptor is activated or that the effects of high [K+]o already reduced the effect of depolarizing currents that are enhanced by ACPD. The acceleration in interictal discharge rate was associated with a reduction in the afterhyperpolarization that follows the paroxysmal depolarizing shift and this action appears to be important in determining the synchronization of neurons and the rate of interictal epileptiform discharges. Furthermore, interaction between mGluR activation and either muscarinic or beta-adrenergic activation may be important for seizure generation.

Sucrose gap recordings from the dorsal roots of isolated, hemisected frog spinal cords were used to determine the effects of metabotropic L-glutamate receptor activation on primary afferent terminals by (+/-)-1-amino-trans-1,3-cyclopentane-dicarboxylic acid (t-ACPD). Dorsal root potentials evoked by ventral root volleys were significantly reduced by t-ACPD (30 microM), as were GABA- and muscimol-induced afferent terminal depolarizations. The effects of t-ACPD on GABA-depolarizations depended upon activation of group I metabotropic glutamate receptors, i.e. the effects were blocked by the group I/II antagonist (RS)-alpha-methyl-4-carboxyphenylglycine, but not by the group II antagonist alpha-methyl-(2S,3S,4S)-alpha-(carboxycyclopropyl)-glycine or the group III antagonist alpha-methyl-(S)-2-amino-4-phosphonobutyrate and were mimicked by the group I agonist 3,5-dihydroxyphenylglycine but were not mimicked by the group III agonist (S)-2-amino-4-phosphonobutyrate. Increasing the intracellular concentration of 3'-5'-cyclic adenosine monophosphate with 8-bromo-cAMP, forskolin, and 3-isobutyl-1-methylxanthine significantly reduced GABA depolarizations, but the protein kinase inhibitors Rp-adenosine 3,5-cyclic monophosphothioate triethylamine and N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide did not alter t-ACPD's depression of GABA depolarizations. The actions of t-ACPD on GABA depolarizations were neither mimicked nor blocked by phorbol-12-myristate 13-acetate, thapsigargin, staurosporine, or arachidonic acid, presumptive indications that the effects of t-ACPD did not involve phosphoinositide hydrolysis, the release of Ca2+ from intracellular stores, or the formation of arachidonate. t-ACPD's effects on GABA depolarizations were blocked by 20 mM Mg2+, the broad spectrum L-glutamate antagonist kynurenate, and the selective N-methyl-D-aspartate antagonist D(-)-2-amino-5-phosphonovaleric acid, but not by the non-N-methyl-D-aspartate antagonist 6-cyano-7-nitroquinoxaline-2,3-dione. Low concentrations of N-methyl-D-aspartate (10 microM) mimicked the effect of t-ACPD on GABA responses. These results suggest that t-ACPD's depression of GABA depolarizations involves an indirect, three-stage mechanism that includes activation of Group I metabotropic glutamate receptors on interneurons and/or on afferent terminals, the release of L-glutamate from the latter structures, and the activation of N-methyl-D-aspartate receptors on primary afferent terminals. The depression of GABA depolarizations caused by the release of L-glutamate from afferent terminal and/or interneurons leads to a block of presynaptic inhibition (produced in the frog spinal cord by GABA) resulting in a positive feed-forward amplification of reflex transmission.

Metabotropic glutamate receptors have been implicated in modulation of synaptic transmission in many different systems. This study reports the effects of selective activation of metabotropic glutamate receptors on synaptic transmission in intracellularly recorded locus coeruleus neurons in brain slice preparations. Perfusion of either L-2-amino-4-phosphonobutyric acid (L-AP4; 0.1-500 microM) or (+/-)-1-aminocyclopentane-trans-1,3,dicarboxylic acid (t-ACPD; 0.1-500 microM) caused a depression of excitatory postsynaptic potentials in a dose-dependent fashion to about 70% inhibition. Both agonists exerted their effects at relatively low concentrations with estimated EC50s of 2.6 microM and 11.5 microM for L-AP4 and t-ACPD, respectively. This inhibition was not observed with the potent group I metabotropic glutamate receptor agonist (RS)-3,5-dihydroxyphenylglycine (DHPG; 100 microM). Conversely, (R)-4-carboxy-3-hydroxyphenyl-glycine (4C-3H-PG), a group I antagonist/group II agonist, and 2R,4R-4-aminopyrrolidine-2,4-dicarboxylate (APDC), a novel and specific group II agonist, also caused an inhibition of excitatory postsynaptic potentials. Both t-ACPD and L-AP4 produced an increase in paired-pulse facilitation, and failed to change the locus coeruleus response to focally applied glutamate, indicating a presynaptic locus of action. The L-AP4 inhibition was antagonized by (S)-amino-2-methyl-4-phosphonobutanoic acid (MAP4: group III antagonist) but not by (RS)-alpha-methyl-4-carboxyphenylglycine [(RS)-MCPG; mixed antagonist], suggesting that this agonist acts through a type 4 metabotropic glutamate receptor. Conversely, t-ACPD was antagonized by MCPG and by ethyl glutamate (group II antagonist), but not by aminoindan dicarboxylic acid (AIDA; group I antagonist) or MAP4, suggesting that this agonist acts on a type 2 or 3 metabotropic glutamate receptor. Taken together, these results suggest that two pharmacologically distinct presynaptic metabotropic glutamate receptors function in an additive fashion to inhibit excitatory synaptic transmission in locus coeruleus neurons. These receptors may be involved in a feedback mechanism and as such may function as autoreceptors for excitatory amino acids.

The functional relevance of presynaptic glutamate receptors in controlling presynaptic Ca2+ influx and thereby transmitter release is unknown. To test if presynaptic Ca2+ entry in the hippocampus is controlled by glutamate autoreceptors, we created a hippocampal slice preparation for investigation of presynaptic Ca2+ signals with Ca(2+)-sensitive microelectrodes after lesioning of neurons by glucose deprivation or kainate. Stratum radiatum and alveus stimulation-induced postsynaptic field potential components were irreversibly abolished in areas CA1 and CA3 of lesioned slices, whereas stratum radiatum stimulation still evoked afferent volleys. Repetitive stimulation of the stratum radiatum still induced decreases in extracellular Ca2+ concentration. Repetitive stimulation of the alveus no longer induced decreases in extracellular Ca2+ concentration, suggesting complete damage of pyramidal cells. The stratum radiatum stimulation-induced decreases in extracellular Ca2+ concentration in lesioned slices were comparable to those elicited during application of the glutamate antagonists 6-cyano-7-nitroquinoxaline-2,3-dione and L-2-amino-5-phosphonovalerate. In lesioned slices the stimulus-induced presynaptic Ca2+ influx was reversibly reduced by kainate. RS-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), N-methyl-D-aspartate and glutamate without effects on afferent volleys. The kainate and N-methyl-D-aspartate effects on presynaptic Ca2+ signals were partly sensitive to 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(f)quinoxaline and L-2-amino-5-phosphonovalerate, respectively, while the AMPA effects were not significantly affected by 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(f)quinoxaline, suggesting involvement of a novel glutamate receptor subtype. The involvement of a novel glutamate receptor subtype was supported by our findings that ionotropic glutamate receptor agonists also reduce presynaptic Ca2+ influx under conditions of blocked synaptic transmission by 6-cyano-7-nitroquinoxaline-2,3-dione and L-2-amino-5-phosphonovalerate. 1-Aminocyclopentane-trans-1,3-dicarboxylic acid had no significant effect on presynaptic Ca2+ entry. Also, the presynaptic Ca2+ influx was not influenced by the glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione, 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(f)quinoxaline and L-2-amino-5-phosphonovalerate when applied alone. Low kainate concentrations (5 microM) reduced presynaptic Ca2+ signals in area CA3 but not in area CA1, demonstrating the higher affinity of presynaptic kainate receptors on mossy fibre terminals.

High frequency synchronized neuronal activity plays an important role in spatiotemporal processing of sensory inputs and possibly cognitive function. This activity, in the so called beta and gamma electroencephalographic frequency range is found in cortical areas and in the hippocampus. Here we show that activation of metabotropic glutamate receptors by the agonist 1-aminocyclopentane-(1S,3R)-dicarboxylate evokes synchronous 20 Hz rhythmic activity in rat hippocampal slices. Quisqualate was more potent than 1-aminocyclopentane-(1S,3R)-dicarboxylate which suggests involvement of either the metabotropic glutamate receptor subtypes I, V or both. The 20 Hz rhythmic activity was different from the theta-rhythmic effect induced by the cholinergic muscarinic receptor agonist carbachol which showed a dominant frequency of 9.0 +/- 0.5 Hz. The 20 Hz rhythmic activity is correlated with an increased firing rate of pyramidal neurons. The 20 Hz rhythmic activity was reduced by the GABA(A) receptor antagonist bicuculline, thus interneuron activity plays a major role in the 20 Hz oscillations. The 20 Hz rhythmic activity induced by activation of metabotropic glutamate receptors represents a new aspect of synchronized activity in the hippocampus and may be involved in the binding and representation of converging sensory inputs.

The extent to which glutamate receptors in the nucleus accumbens and ventral tegmental area regulate feeding-evoked increases in dopamine release in the nucleus accumbens was determined using in vivo brain microdialysis in the rat. In some animals a second dialysis probe was implanted in the ventral tegmental area ipsilateral to the nucleus accumbens probe. The feeding protocol involved access to standard rat chow after 18 h of food deprivation. Under these conditions rats began eating approximately 30 s after the introduction of food and consumed 7-8 g, resulting in a 50% increase in dopamine release. Application of the glutamate receptor antagonist kynurenate (1 mM) in the nucleus accumbens potentiated the feeding-evoked increase in dopamine release by 80%. Application of the metabotropic glutamate receptor agonist trans-1S,3R-1-amino-1,3-cyclopentanedicarboxylic acid (100 microM) in the nucleus accumbens blocked the feeding-evoked increase in dopamine release. Application of a combination of the ionotropic glutamate receptor antagonists 2-amino-5-phosphopentanoic acid (200 microM) and 6-cyano-7-nitroquinoxaline-2,3-dione (50 microM) through the dialysis probe in the ventral tegmental area reduced basal dopamine output in the nucleus accumbens by 20% and markedly attenuated (by 70%) the effect of feeding on dopamine release. None of the treatments affected the latency to eat or the volume of food consumed. These results indicate that glutamatergic afferents to the ventral tegmental area mediate feeding-induced increases in dopamine release in the nucleus accumbens. In contrast, at physiological concentrations, glutamate in the nucleus accumbens appears to decrease dopamine release via actions on ionotropic and metabotropic receptors.

Intracellular recordings were obtained from neocortical brain slices of adult rats maintained in vitro. The effect of metabotropic glutamate receptor activation on spike frequency adaptation in regular spiking layer II and III neurons was determined. Putative metabotropic glutamate receptor agonists and antagonists, as well as inhibitors of intracellular signaling systems, were tested. Activation of metabotropic glutamate receptors by bath applied (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate (1S,3R-ACPD; 50-200 microM) reduced the first interspike interval and increased action potential frequency at all current intensities. This effect was not blocked by ionotropic glutamate receptor antagonists. Under these recording conditions, quisqualate (1-10 microM) similarly reduced spike frequency adaptation. Neither 1R,3S-ACPD, L-2-carboxycyclopropylglycine-I nor the putative presynaptic metabotropic glutamate receptor agonist, L-2-amino-4-phosphonobutyrate, mimicked the effects of 1S,3R-ACPD or quisqualate. Bath application of the putative metabotropic glutamate receptor antagonist, alpha-methyl-4-carboxyphenylglycine, competitively antagonized the excitatory actions of 1S,3R-ACPD. Another putative antagonist, L-2-amino-3-phosphonopropionate, failed to antagonize the reduction in spike frequency adaptation. Intracellular injection of guanosine-5'-O-(2-thiodiphosphate), a non-hydrolysable analog of GTP, inhibited the postsynaptic metabotropic glutamate receptor-mediated effects. However, the depression of synaptic transmission by 1S,3R-ACPD was not antagonized by this compound. The decrease in spike frequency adaptation by 1S,3R-ACPD was not prevented by prior exposure to the non-specific protein kinase inhibitors H-7 or H-8 (10 microM), the protein kinase A inhibitor H-89 (0.25 microM) or the protein kinase C inhibitor staurosporine (0.10 microM). These data suggest that the metabotropic glutamate receptor-mediated reduction in spike adaptation requires the activation of specific G-protein-coupled metabotropic glutamate receptor subtypes located on postsynaptic sites. The increase in neuronal excitability observed in the adult neocortex may be mediated either by an unidentified G-protein-coupled second messenger or via a membrane-delimited G-protein action.

Extracellular application of glutamate (100 microM) increased the spontaneous secretion of acetylcholine, as well as the amplitude and decay time of miniature endplate potentials at developing neuromuscular synapses in Xenopus tadpoles. Kainate, quisqualate and N-methyl-D-aspartate (100 microM each) increased miniature endplate potential frequency by 26-, 13- and four-fold, respectively. The rank order of efficacy at 100 microM was kainate > quisqualate > N-methyl-D-aspartate > glutamate. The effect of kainate on miniature endplate potential frequency was inhibited by 6-cyano-2,3-dihydroxy-7-nitroquinoxaline (20 microM), but not by (+/-)-2-amino-5-phosphonovalerate (20 microM). Treatment with the voltage-dependent Ca2+ channel blockers verapamil (10 microM), Cd2+ (100 microM) or omega-conotoxin (1 microM) inhibited the potentiating action of kainate on miniature endplate potential frequency. On the other hand, 1S,3R-1-aminocyclopentane-1,3-dicarboxylate (300 microM), a glutamate metabotropic receptor agonist, inhibited the spontaneous acetylcholine release, which was antagonized by the application of 2-amino-3-phosphonopropionate (500 microM). The potentiating effect of glutamate receptor agonists on the miniature endplate potential frequency declined or disappeared in older Xenopus tadpoles. Quisqualate (100 microM) and N-methyl-D-aspartate (100 microM) but not kainate (30 microM) increased the amplitude and decay time of miniature endplate potential, whereas 1S, 3R-1-aminocyclopentane-1, 3-dicarboxylate (300 microM) only increased the decay time of miniature endplate potentials. These results suggest that there are kainate/quisqualate and N-methyl-D-aspartate receptors existing in the motor nerve terminals of younger Xenopus tadpoles and the activation of these receptors potentiates spontaneous acetylcholine release through increasing Ca2+ influx. Our data suggest that the presynaptic glutamate receptors on cholinergic terminals may be involved in feedback regulation of acetylcholine secretion at earlier embryonic stages.

The neurophysiological effects of ionophoretic application of the metabotropic glutamate receptor agonist (1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid on the firing rate of single suprachiasmatic nucleus cells was studied in a hypothalamic slice preparation. In addition, the effects of the phenylglycine derivative (RS)-alpha-methyl-4-carboxyphenylglycine, a selective metabotropic glutamate receptor antagonist, on responses evoked by (1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid and N-methyl-D-aspartate were assessed. (1S,3R)-1-Amino-1,3-cyclopentanedicarboxylic acid elicited current-dependent increases in neuronal activity in 65% of all suprachiasmatic nucleus cells studied, while N-methyl-D-aspartate activated 93% of the same cells. Cells in the ventrolateral suprachiasmatic nucleus were more sensitive to (1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid application (82% of cells activated) than those in the dorsomedial suprachiasmatic nucleus (28% of cells activated). In addition, responses evoked by (1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid in the ventrolateral suprachiasmatic nucleus had a rapid onset and a prolonged recovery from agonist application, whereas responses elicited in the dorsomedial suprachiasmatic nucleus were slower in onset and recovered more quickly from agonist application. Co-application of (RS)-alpha-methyl-4-carboxyphenylglycine selectivity attenuated (1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid-evoked responses but had no effect on N-methyl-D-aspartate-evoked activity in the same cells. These results indicate that (1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid application activates single cells in the hamster suprachiasmatic nucleus and these responses are selectively attenuated by (RS)-alpha-methyl-4-carboxyphenylglycine. Cells in the ventrolateral suprachiasmatic nucleus are more sensitive to (1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid application than those in the dorsomedial suprachiasmatic nucleus. These results are the first demonstration that metabotropic glutamate receptors can modulate spontaneous neuronal activity within the suprachiasmatic nucleus, the predominant mammalian circadian pacemaker.

We have examined the role of metabotropic glutamate receptor activation in regulating neurotrophin messenger RNA levels in the brain with the use of the selective agonist (1S,3R)-1-aminocy-clopentane-1,3-dicarboxylic acid. Intracerebroventricular injection of (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid into adult adult rats resulted in increased expression of nerve growth factor and brain-derived neurotrophic factor messenger RNA in the hippocampal and pyriform cortex and decreased levels of neurotrophin-3 messenger RNA in the hippocampal dentate gyrus granule cell layer. C-fos messenger RNA levels were also increased throughout hippocampal and cortical subfields following (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid administration. (1S,3R)-1-Aminocyclopentane-1,3-dicarboxylic acid-induced changes in messenger RNA levels occurred without behavioral seizures, yet these changes were similar in magnitude and time course to early changes in neurotrophin and c-fos messenger RNA levels observed following recurrent limbic seizures. In contrast quisqualate, a potent agonist of metabotropic as well as ionotropic kainate/alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors, was only capable of inducing increased expression of brain-derived neurotrophic factor messenger RNA at doses which produced recurrent motor seizures, and both effects were completely inhibited by the non-N-methyl-D-aspartate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione. Neurotrophin messenger RNA changes induced by (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid were also partially susceptible to 6-cyano-7-nitroquinoxaline-2,3-dione antagonism, as well as the specific N-methyl-D-aspartate receptor antagonist (+)-5-methyl-10,11-dihydroxy-5H-dibenzo(a,d)-cyclohepten-5,10- iminedizoleipine. These results suggest that (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid-sensitive metabotropic glutamate receptors can dramatically increase the expression of neurotrophin and c-fos messenger RNAs in rat forebrain without producing significant behavioral trauma and that these influences may involve ionotropic glutamate receptors in certain brain regions.

Hippocampal slices were transiently exposed to an oxygen- and glucose-free environment which causes a pronounced drop of both ATP and creatine phosphate, an anoxic depolarization, and an incomplete recovery of synaptically evoked population spike in the CA1 region after 1 h (48.5 +/- 3.6% of baseline values). This recovery could be markedly enhanced by the application of N-methyl-D-aspartate receptor antagonists. To examine the influence of metabotropic glutamate receptors on neuronal recovery from hypoxia/hypoglycemia, we applied various antagonists and agonists of the metabotropic glutamate receptors to the bath during the interval from 20 min before to 10 after hypoxia/hypoglycemia. The metabotropic glutamate receptor antagonists (+)-alpha-methyl-4-carboxyphenylglycine and L-2-3- amino-phosphonopropionic acid were both able to enhance the population spike recovery significantly. However, the mixed metabotropic glutamate receptor agonist 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid also exhibited a protective effect on population spike recovery, leaving the anoxic depolarization and N-methyl-D-aspartate responses during the hypoxia/hypoglycemia untouched. With the help of more subtype-specific agonists, we found that an activation of phospholipase C coupled (class 1) metabotropic glutamate receptors prior to hypoxia/hypoglycemia may be responsible for the protective effect seen with 1S, 3R-1-aminocyclopentane-1,3-dicarboxylic acid, because the specific class 1 metabotropic glutamate receptor agonist trans-azetidine-2,4-dicarboxylic acid appeared to be highly protective, but only if it was applied 20 min before the hypoxia/hypoglycemia. An activation of class 2 metabotropic glutamate receptors by (2S,1'R,2'R,3'R)-2-(2,3-dicarboxycyclopropyl)glycine, which inhibits adenylyl cyclase activity, led to a marked deterioration of the population spike recovery and even to a total prevention of the protective effect of the N-methyl-D-aspartate agonist D-2-amino-5-phosphonopentanoic acid. Our data suggest that prior activation of class 1 metabotropic glutamate receptors is beneficial, while their activation during hypoxia/hypoglycemia is detrimental. Furthermore, the activation of class 2 metabotropic glutamate receptors decreases the recovery from hypoxia/hypoglycemia.

We have addressed the role of presynaptic metabotropic glutamate receptors in the control of glutamate release from cerebrocortical nerve terminals. The metabotropic glutamate receptor agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid enhances the release evoked by a submaximal depolarization in the presence of low concentrations of arachidonic acid and in a staurosporine-sensitive manner. In contrast, (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid and L(+)-2-amino-4-phosphonobutyrate inhibit the release evoked by a maximal depolarization, in the absence of arachidonic acid and by a staurosporine-insensitive mechanism. Interestingly, the effects of the metabotropic glutamate receptors that inhibit glutamate release are only observed in the nerve terminals from young rats (one to three weeks), while the facilitatory effects are better seen in latter developmental stages (three to four weeks) and adult (two to three months) rats, coinciding with the development of the maximal capacity of glutamate uptake. These results indicate the existence of important developmental changes in the presynaptic control of glutamate release.

Prior work has shown that intrastriatal injection of the metabotropic glutamate receptor agonist 1S,3R-ACPD results in pronounced contralateral rotation, and the basis for this effect is thought to be increased activity of dopaminergic nigrostriatal neurons. We tested this hypothesis by determining the expression of Fos-like immunoreactivity after intrastriatal injection of 1S,3R-ACPD. Intense Fos-like immunoreactivity was noted in the globus pallidus, entopeduncular nucleus, subthalamic nucleus and substantia nigra pars reticulata. Ablation of the subthalamic nucleus 10 days prior to intrastriatal injection of 1S,3R-ACPD abolished rotational behaviour but not Fos-like immunoreactivity in the globus pallidus, entopeduncular nucleus and substantia nigra. Intrasubthalamic injection of 1S,3R-ACPD produced marked contralateral rotation and a pattern of Fos-like immunoreactivity similar to that seen after intrastriatal 1S,3R-ACPD injection. These results suggest that stimulation of striatal metabotropic glutamate receptors inhibits striatal projection neuron activity, while stimulation of subthalamic metabotropic glutamate receptors increases subthalamic nucleus activity. Increased subthalamic nucleus activity is necessary and sufficient for the expression of rotational behavior. These results also suggest that metabotropic glutamate receptor antagonists may be useful in the treatment of Parkinson's disease.

Glutamate induced an increase in cell volume within one minute and evoked cytosolic Ca2+ transients in type 1 astroglial cells in primary culture obtained from the cerebral cortex of newborn rat. Even the metabotropic glutamate receptor agonists (1S,3R)-1-aminocyclopentane- 1,3-dicarboxylic acid (1S-3R-ACPD) and L(+)-2-amino-4 phosphonobutyric acid (L-AP4) induced a cell swelling with ACPD inducing a parallel Ca2+ transient while L-AP4 did not. A new method was used where rapid changes in relative cell volume could be followed at the single cell level. Relative volume changes in cultured single astroglial cells were examined by microspectrofluorimetry after loading the cells with the highly fluorescent intracellular probe fura-2/AM. At its isosbestic point, 358 nm, fura-2 is ion-insensitive and the fluorescent signals emitted are related only to the intracellular dye concentration. By varying the excitation wavelengths, changes in intracellular Ca2+ transients could be recorded simultaneously with the relative volume variations of the individual cells. Thus, as rapid changes in cell volume were followed, the results from this method could be of physiological significance. Glutamate-induced cell swelling was blocked by BaCl2 and by tetraethylammonium, suggesting that K+ channels are operative in glutamate-induced cell swelling. Furthermore, the glutamate-induced swelling was blocked by the Na+; K+, and 2Cl- co-transport inhibitor furosemide. The glutamate-induced swelling was partially blocked by pertussis toxin and partially blocked also by the glutamate carrier-blocker dihydroaspartate. When the ionotropic glutamate receptor alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid was blocked with the antagonist 2,3-dihydroxy-6-nitro-7- sulfamoyl-benzo(F)quinoxaline, glutamate still induced a swelling, suggesting that this receptor was not directly involved in the glutamate-induced volume increase. Even in situations of blocked or partially blocked swelling, intracellular Ca2+ transients could be obtained. Furthermore, the glutamate-induced swelling was evoked even in low extracellular Ca2+ concentrations. Our data suggest that glutamate-induced rapid swelling is a complex process at the molecular level. One hypothetical mechanism might be that glutamate interacts with metabotropic glutamate receptors and induces a release of Ca2+ from internal stores. Furthermore glutamate interacts with K+ channels, and probably at least one co-transporter and the sodium-dependent high-affinity uptake glutamate carrier, resulting in cell swelling.

The functional roles played by metabotropic glutamate receptors in the neostriatum is just beginning to be examined. One possibility, raised by previous studies, is that metabotropic glutamate receptors act to modulate responses mediated by ionotropic glutamate receptors. In the present study, we examined this possibility in a neostriatal brain slice preparation using intracellular recording and iontophoretic techniques. We found that the iontophoretic application of the metabotropic glutamate receptor agonist 1-amino-cyclopentane-1,3-dicarboxylic acid markedly attenuated both the amplitude and duration of excitatory responses induced by the iontophoretic application of N-methyl-D-aspartate. These inhibitory effects were stereo-selective and relatively long-lasting. The metabotropic glutamate receptor antagonist 2-amino-3-phosphonopropionic acid applied either iontophoretically or in the bath prevented the inhibitory effects of 1-amino-cyclopentane-1,3-dicarboxylic acid. The inhibitory action of 1-amino-cyclopentane-1,3-dicarboxylic acid was specific to N-methyl-D-aspartate, as 1-amino-cyclopentane-1,3-dicarboxylic acid had no consistent action on the responses evoked by the iontophoretic application of glutamate, amino-3-hydroxy-5-methyl-4-isoazolepropionic acid or quisqualate. Bath application of 1-amino-cyclopentane-1,3-dicarboxylic acid inhibited the evoked depolarizing postsynaptic potentials recorded in neostriatal cells. Thus, activation of metabotropic glutamate receptors may play an important role in modulating N-methyl-D-aspartate receptor function in neostriatal neurons.

A novel autoradiographic method to identify individual neurons responding to neurotransmitter stimulation with increased phosphoinositide turnover is described. When phosphoinositide-coupled receptors are activated, phosphatidylinositol 4,5-bisphosphate is hydrolysed by phospholipase C generating the two second messengers, inositol 1,4,5-trisphosphate and diacylglycerol. During prolonged receptor stimulation, both second messengers are actively recycled to maintain the effective intracellular levels of agonist-sensitive phosphoinositides. Lithium ions inhibit this recycling pathway by blocking the recovery of free inositol from inositol 1,4,5-trisphosphate thus leading to the accumulation of phosphatidyl cytidine monophosphate, a membrane bound molecule which is the activated precursor of the synthesis of phosphoinositides. Therefore, addition of excess myo-inositol reverts the effects of lithium inhibition. Thus, taking advantage of this fact and using [3H]cytidine as precursor, phosphatidyl [3H]cytidine monophosphate accumulation was induced in rat neocortical and hippocampal slices after muscarinic or metabotropic glutamate receptor stimulation. The labelled slices were then fixed, dehydrated and embedded in Durcupan resin. Semithin sections (1 micron thick) were cut and exposed to autoradiographic emulsion for several weeks. Biochemical analysis of the incorporation of [3H]cytidine into the chloroform extracted (containing lipids) and the alkali-solubilized (containing nucleic acids and proteins) fractions were carried out in parallel with morphological studies. The stimulation of both receptor types induced labelling of neurons in neocortex and hippocampus. In labelled cells silver grains were characteristically observed over the cytoplasm surrounding the nucleus and main dendritic processes. The anatomical location and distribution of labelled cells as well as the levels of response obtained in both brain regions studied, was found to be receptor specific. Inclusion of 30 mM myo-inositol in the incubation media reversed completely both the accumulation of phosphatidyl [3H]cytidine monophosphate and the labelling of cells, thus demonstrating that the label detected autoradiographically corresponds to phosphatidyl [3H]cytidine monophosphate. It is concluded that the method is sensitive and specific, allowing identification of individual neurons in both neocortical and hippocampal slices and after stimulation of both muscarinic and metabotropic glutamate receptor subtypes. The method may open a new means to study the phosphoinositide second messenger signalling pathway and the cells in which it takes place.

The aim of the described experiments was to use immunohistochemistry to visualize the release of GABA from specific retinal amacrine cells following ischaemia and to establish the involvement of defined glutamatergic receptors. In initial experiments, rabbit retinas were exposed in vitro to excitatory amino acid agonists alone or in combination with a putative antagonist, or in physiological solution lacking oxygen and glucose, or in solution containing potassium cyanide for 45 min at 37 degrees C. The nature of the GABA immunoreactivity was then examined by immunohistochemistry. In other in vitro experiments, retinas were first allowed to accumulate exogenous serotonin before exposing the tissues to the combinations as described. These tissues were then processed immunohistochemically for the localization of serotonin. In yet other experiments, the intraocular pressure of a rabbit's eye was raised to about 110 mmHg for 60 min and a reperfusion time of 45 min allowed before dissecting the retina and processing for the localization of GABA immunoreactivity. The other eye served as a control. Of the excitatory amino acid agonists tested, only N-methyl-D-aspartate, kainate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid caused a change in the GABA immunoreactivity. The N-methyl-D-aspartate effect was specifically antagonized by dizocilpine maleate, dextromethorphan and memantine, and was characterized by a reduction in the number of GABA-immunoreactive perikarya. The GABA "staining" in the inner plexiform layer also appeared as four clear bands. The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid- and kainate-induced effects were both antagonized by 6-cyano-2,3-dihydroxy-7-nitroquinoxaline-2,3-dione and partially by kynurenic acid at the concentrations used. Here, the amount of GABA-positive perikarya was greatly reduced and three immunoreactive bands appeared in the inner plexiform layer. However, for low concentrations of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid four GABA-immunoreactive bands could be identified in the inner plexiform layer. The normal GABA immunoreactivity of the inner plexiform layer also appeared to be in defined bands in retinas which received an ischaemic insult either by reducing the availability of glucose and oxygen, exposing the tissue to potassium cyanide or raising the intraocular pressure of an eye. In these cases the number of GABA-positive perikarya was also reduced. Only alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and kainate of the excitatory amino acid agonists tested caused a release of serotonin and this process was antagonized by 6-cyano-2,3-dihydroxy-7-nitroquinoxaline-2,3-dione and partially by kynurenic acid.(ABSTRACT TRUNCATED AT 400 WORDS)

The stimulatory effect of excitatory sulphur-containing amino acids on inositol phosphate formation was investigated in primary cultures of cerebellar granule cells. L-Cysteine sulphinate (CSA), L-cysteate (CA), L-homocysteine sulphinate (HCSA), L-homocysteate (HCA) and S-sulpho-L-cysteine (SSC) dose-dependently stimulated the formation of [3H]inositol phosphates exhibiting EC50 values in the range 60-200 microM and maximal effects of six- to 17-fold that of basal [3H]inositol phosphate levels. Endogenous L-glutamate spontaneously released into the extracellular medium or following exposure of cells to HCSA, HCA or SSC did not contribute significantly to formation of [3H]inositol phosphates, whereas 10% of the total [3H]inositol phosphates accumulated following exposure to CSA and CA was due to released L-glutamate. The selective N-methyl-D-aspartate receptor antagonist, D,L-2-amino-5-phosphonopentanoic acid (APV, 500 microM) attenuated by 20% (HCSA) to between 80 and 100% (CSA, CA, SSC, HCA) the formation of [3H]inositol phosphates induced by 1 mM sulphur-containing amino acids. When, however, HCSA was used at 100 microM (a concentration near to its EC50 for phosphoinositide hydrolysis), APV inhibited induced responses by 70%. Sulphur-containing amino acid-stimulated [3H]inositol phosphate formation was unaffected by the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 microM). Inhibition of sulphur-containing amino acid-stimulated [3H]inositol phosphate formation by co-administration of APV and CNQX was similar to that obtained in the presence of APV alone. CSA-, CA-, SSC- and HCA-stimulated [3H]inositol phosphate formation was markedly reduced by removal of Ca2+ from the extracellular medium whereas that stimulated by HCSA was less affected. A similar inhibitory profile was observed when the levels of sulphur-containing amino acid-induced increases in intracellular free calcium ([Ca2+]i) were measured in the presence of 500 microM APV; 1 mM HCSA-induced responses being inhibited by only 30% whereas responses to the remaining sulphur-containing amino acid (also at 1 mM) were inhibited by > 45%. When the sulphur-containing amino acids were used at concentrations approximating their EC50 values for phosphoinositide hydrolysis, APV inhibited the induced increases in [Ca2+]i by 70-100%. HCA and SSC co-administered with the less efficacious but selective metabotropic glutamate receptor agonist, (+-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD) at maximally effective concentrations (1 mM) of each agonist stimulated [3H]inositol phosphate formation in an additive manner.(ABSTRACT TRUNCATED AT 400 WORDS)

Slices of hippocampal area CA1 in the rat were employed to test the hypothesis that the activation of metabotropic glutamate receptors during tetanization is necessary for the late maintenance of long-term potentiation. If the metabotropic glutamate receptor antagonist L-2-amino-3-phosphonopropionate was present during tetanization, post-tetanic and early long-term potentiation of the population spike as well as field excitatory postsynaptic potential developed almost normally. However, 100 min after tetanization, long-term potentiation of the field excitatory postsynaptic potential decreased in an irreversible manner. The same concentration of D-2-amino-3-phosphonopropionate was ineffective. If L-2-amino-3-phosphonopropionate was applied 120 min after tetanization, it did not influence long-term potentiation. The presence of the metabotropic glutamate receptor agonist trans-D,L-1-aminocyclopentane-1,3-dicarboxylic acid during tetanization weakly enhanced the slope of field excitatory postsynaptic potential long-term potentiation. The influence of L-2-amino-3-phosphonopropionate and D,L-1-aminocyclopentane-1,3-dicarboxylic acid on ionotropic glutamate receptors was studied using whole-cell voltage-clamp and pressure application techniques. No effect of L-2-amino-3-phosphonopropionate on either early or late components of excitatory postsynaptic currents could be detected at the concentration used to block long-term potentiation. It is therefore unlikely that the effect of L-2-amino-3-phosphonopropionate on long-term potentiation is due to an interaction with N-methyl-D-aspartate receptors or alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors. However, bath-applied 1S,3R-D,L-1-aminocyclopentane-1,3-dicarboxylic acid facilitated the N-methyl-D-aspartate-induced depolarization in response to N-methyl-D-aspartate pressure application in a reversible manner. These data suggest that besides the involvement of N-methyl-D-aspartate receptors the activation of a 2-amino-3-phosphonopropionate-sensitive metabotropic glutamate receptors during or immediately after tetanization is necessary for subsequent mechanisms responsible for the maintenance of long-term potentiation. A link between metabotropic glutamate receptors and protein kinase C activation during long-term potentiation is discussed considering the similar time course of long-term potentiation blockade after application of L-2-amino-3-phosphonopropionate and protein kinase C inhibitors.