Metabotropic Receptor Subtype Research Articles

Altered spatial learning and memory in mice lacking the mGluR4 subtype of metabotropic glutamate receptor.

Altered spatial learning and memory in mice lacking the mGluR4 subtype of metabotropic glutamate receptor.

Metabotropic glutamate receptors regulate N-methyl-D-aspartate-mediated synaptic transmission in nucleus accumbens.

We recorded intracellularly from core nucleus accumbens (NAcc) neurons in brain slices to study the regulation by metabotropic glutamate receptors (mGluRs) of pharmacologically isolated N-methyl--aspartate-mediated excitatory postsynaptic currents (NMDA-EPSCs). Monosynaptic NMDA-EPSCs, evoked by local stimulation, were isolated by superfusion of the non-NMDA and gamma-aminobutyric acid-A (GABAA) receptor antagonists, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 microM) and bicuculline (15 microM), respectively. Trans-1-aminocyclopentane-1,3-decarboxylic acid (trans-ACPD; 50 microM), a nonspecific group 1 and 2 mGluR agonist, had no effect on resting membrane potential (RMP) or input resistance of NAcc neurons. However, it consistently decreased NMDA-EPSC areas (time integrals) dose dependently (1-100 microM; EC50 = 8 microM) and reversibly. The specific group 1 mGluR agonists quisqualate (1-4 microM) and (RS)-3, 5-dihydroxyphenylglycine (DHPG; 100 microM) did not mimic the trans-ACPD effect on NMDA-EPSCs, nor did exposure of the slice to the group 1 mGluR antagonist (+)-2-amino-3-phosphonopropionic acid (-AP3, 0.4 mM) inhibit the trans-ACPD effect. The putative mGluR1 and mGluR2 antagonist (+)-alpha-methyl-4-carboxyphenylglycine (MCPG) at 0.5 mM failed to antagonize trans-ACPD effects but at 1 mM blocked them. Both the group 2 mGluR agonist (2S,3S, 4S)-alpha-(carboxycyclopropyl)-glycine (-CCG-I, 2 microM) and the group 3 mGluR specific agonist (+)-2-amino-4-phosphonobutyric acid (-AP4, 20 microM) attenuated NMDA-EPSC areas; the effect of -AP4 was blocked by the group 3 antagonist (S)-2-amino-2-methyl-4-phosphonobutanoic acid (MAP4; 0.5 mM). Exogenously applied NMDA, in the presence of tetrodotoxin to prevent presynaptic effects, induced inward currents that were decreased by 20 microM -AP4 but not by 10 microM trans-ACPD. These findings suggest that NMDA receptor-mediated neurotransmission in NAcc is under dual inhibitory regulation by group 2 and 3 metabotropic receptor subtypes: -AP4-sensitive receptors located postsynaptically and those sensitive to trans-ACPD located presynaptically.

Metabotropic glutamate receptor subtype-1α (mGluR1α) immunoreactivity in ependymal cells of the rat caudal medulla oblongata and spinal cord

Immunohistochemical study of the distribution of glutamate receptor subunits 1–4 (GluR1–4), NMDA receptor subunit 1 and metabotropic glutamate receptor subtypes 1–3 (mGluR1–3) in the ependymal cells of the caudal medulla oblongata and upper thoracic spinal cord was carried out. The results showed that ependymal cells and tanycytes expressed only the metabotropic receptor subtype, mGluR1α. Some of the mGluR1α-positive long basal processes of the tanycytes reached the pia mater; some made contact with capillaries. It was suggested that the activity of these mGluR1a-positive tanycytes may be regulated by the glutamate in both the CSF and blood. The presence of many mGluR1α positive fibres in the area postrema suggests that mGluR1α subtype in this region may be involved in cardiovascular regulation.

Ontogenetic and Pharmacological Studies on Metabotropic Glutamate Receptors Coupled to Phospholipase D Activation

The present study was aimed at characterizing the metabotropic receptor subtype which is involved in the activation of phospholipase D (PLD) by glutamate in rat hippocampal slices. We first observed that the ontogenetic profile of glutamate-induced hydrolysis of phosphoinositides and of phosphatidylcholine was strikingly similar. Both pathways were significantly activated by glutamate in tissue taken from 3-, 8- and 15-day old rats, but not in adult rats. PLD activation was strongest in slices taken from 8-day old rats. At this age, quisqualate had a higher potency for PLD activation ( ec 50: 0.6 μM) than 1S,3R-ACPD ( ec 50: 16 μM) and DHPG, a specific activator of group I mGluR, was a full agonist at PLD activation ( ec 50: 3.5 μM) indicating an involvement of a group I mGluR (mGluR1 and 5). MCPG and AIDA, two pulative antagonists at mGluR1 receptors, caused a small but (in the case of MCPG) significant inhibition. DCG-IV, an activator of group II mGluR, was a weak partial agonist at PLD activation ( ec 50: 22 nM) while l-AP 4, an activator at group III mGluR, was totally inactive. Likewise, forskolin, a stimulant of cyclic AMP formation, was inactive either alone, or in combination with glutamatergic agonists. Pretreatment of the slices with pertussis toxin did not affect PLD activation. In summary, the glutamate-mediated activation of hippocampal PLD, which occurs transiently during postnatal development, is mediated by a group I mGluR, possibly involving mGluR5. © 1997 Elsevier Science Ltd. All rights reserved.

Excitatory amino acid receptors coupled to the phosphoinositide pathway in Bergmann glia.

Glutamate (L-glu) receptors coupled to phosphoinositide hydrolysis in primary cultures of Bergmann cells from chick cerebellum were characterized biochemically and pharmacologically. Both ionotropic and metabotropic receptor agonists stimulated [3H] inositol phosphates accumulation in the following order of potency: QA > NMDA > L-glu > KA approximately QA > AMPA > > t-ACPD. QA showed a biphasic dose-response curve (EC50 = 0.07 and 53 microM), suggesting interaction with two populations of receptors; L-glu was the most efficient agonist. Stimulation by NMDA was blocked by CPP, AP5 and MK-801; that by AMPA and KA was inhibited 100% by CNQX and DNQX, whereas the effect of QA was decreased both by CNQX and the metabotropic antagonist 4-CPG. Stimulation of PIP2 hydrolysis induced by metabotropic L-glu receptor agonist t-ACPD was blocked by 4-CPG but was only moderately inhibited by MCPG. EAA-induced [3H]IPs accumulation was dependent on external Ca2+ and was not affected by nifedipine verapamil, or dantrolene; thapsigargin increased the effect. Results suggest that EAA activate the PI pathway in Bergmann glia through ionotropic (NMDA and AMPA/KA) as well as metabotropic receptor subtypes (t-ACPD) which could act jointly influencing neurotransmission at the parallel fiber-Purkinje cell synapses in the cerebellum.

Impaired Cerebellar Synaptic Plasticity and Motor Performance in Mice Lacking the mGluR4 Subtype of Metabotropic Glutamate Receptor

The application of the glutamate analog L-2-amino-4-phosphonobutyric acid (L-AP4) to neurons produces a suppression of synaptic transmission. Although L-AP4 is a selective ligand at a subset of metabotropic glutamate receptors (mGluRs), the precise physiological role of the L-AP4-activated mGluRs remains primarily unknown. To provide a better understanding of the function of L-AP4 receptors, we have generated and studied knockout (KO) mice lacking the mGluR4 subtype of mGluR that displays high affinity for L-AP4. The mGluR4 mutant mice displayed normal spontaneous motor activity and were unimpaired on the bar cross test, indicating that disruption of the mGluR4 gene did not cause gross motor abnormalities, impairments of novelty-induced exploratory behaviors, or alterations in fine motor coordination. However, the mutant mice were deficient on the rotating rod motor-learning test, suggesting that mGluR4 KO mice may have an impaired ability to learn complex motor tasks. Patch-clamp and extracellular field recordings from Purkinje cells in cerebellar slices demonstrated that L-AP4 had no effect on synaptic responses in the mutant mice, whereas in the wild-type mice 100 microM L-AP4 produced a 23% depression of synaptic responses with an EC50 of 2.5 microM. An analysis of presynaptic short-term synaptic plasticity at the parallel fiber-->Purkinje cell synapse demonstrated that paired-pulse facilitation and post-tetanic potentiation were impaired in the mutant mice. In contrast, long-term depression (LTD) was not impaired. These results indicate that an important function of mGluR4 is to provide a presynaptic mechanism for maintaining synaptic efficacy during repetitive activation. The data also suggest that the presence of mGluR4 at the parallel fiber-->Purkinje cell synapse is required for maintaining normal motor function.

Impaired cerebellar synaptic plasticity and motor performance in mice lacking the mGluR4 subtype of metabotropic glutamate receptor

Impaired cerebellar synaptic plasticity and motor performance in mice lacking the mGluR4 subtype of metabotropic glutamate receptor

Glutamate induces c-fos proto-oncogene expression and inhibits proliferation in oligodendrocyte progenitors: receptor characterization.

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

Excitatory amino acid-, except 1S,3R-ACPD, induced transient high stimulation of phosphoinositide metabolism during hippocampal neuron development

Rat hippocampal neurons in culture extended their neurites until day 5 in vitro (DIV). Then, the mean neuritic length slightly decreased. Excitatory amino acid (EAA)-elicited inositol phosphate (IP) formation increased from 0.5 to 2 DIV, reached a plateau between 2 and 4–5 DIV, and then gradually decreased until 10 DIV. This decrease was likely not due to neuronal death. This developmental pattern was observed for N-methyl-d-aspartate, kainate, glutamate, ibotenate and quisqualate (QA). Interestingly, the 1S,3R-aminocyclopentane dicarboxylate (1S,3R-ACPD) response slightly increased during neuronal culture development. At 3 DIV, the ionotropic antagonists 6,7-dinitro-quinoxalin-2,3-dion and d-2-amino-5-phosphonopentanoate efficiently blocked N-methyl-d-aspartate and kainate-elicited IP formation, and partially inhibited glutamate and ibotenate responses. QA and 1S,3R-ACPD responses were not affected, suggesting a metabotropic action for these two compounds. Furthermore, QA and 1S,3R-ACPD potencies significantly increased between 3 and 10 DIV.The transient high activity periods induced by EAA, except for 1S,3R-ACPD, are not observed for norepinephrine, carbachol and potassium chloride responses.Taken together, these data suggest that: (i) QA and 1S,3R-ACPD can act on two different glutamate metabotropic receptors subtypes during development; and (ii) the EAA-induced transient peaks of IP stimulation, which are specific with respect to other neuroactive substances profiles, could be involved in the development of hippocampal neurons. Indeed, these transient high activities take plase when the neuritic length regularly increases in vitro.

Metabotropic glutamate receptors and visual cortical synaptic plasticity.

Two forms of use-dependent synaptic plasticity, called long-term potentiation (LTP) and long-term depression (LTD), can be elicited in the visual cortex following different paradigms of electrophysiological stimulation. These neurobiological phenomena often are considered as necessary components of models for the alteration in function of the nervous system that must occur at some level for the establishment and (or) maintenance of memory engrams, for learning processes, or for the consolidation of active neural connections and regression of inactive contacts in the developing brain. It has been postulated that for LTP and LTD to be produced in the hippocampus, activation of a particular subtype of excitatory amino acid receptor, the metabotropic receptor, is a critical requirement. Only recently has it become possible to test this hypothesis directly, as a new compound, (+/-)-alpha-methyl-4-carboxyphenylglycine (MCPG), has been introduced and the suggestion made that it selectively antagonizes the metabotropic receptor. This substance has been tested in the present study on responses recorded from slices of rat visual cortex and has been found both to block the activation of the metabotropic receptor and to interfere selectively with the form of synaptic plasticity called LTD. It thus appears from the experiments reported in this paper as though the metabotropic receptor subtype that is blocked by MCPG is required for the expression of LTD but not for the expression of LTP, in the visual cortex of adult rats.

Metabotropic glutamate receptors in the guinea-pig medial vestibular nucleus in vitro.

Despite the accumulation of a large body of evidence on the function of the kainate/alpha-amino-3-5-methyl-4-iso-xazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) excitatory amino acid (EAA) receptor subtypes within the vestibular nucleus, little is known of the metabotropic receptor subtype. The aim of the present study was to examine the response of guinea-pig medial vestibular nucleus (MVN) neurons in brain stem slices to the selective metabotropic receptor agonist, 1S,3R-amino-cyclopentyl-1,3-dicarboxylate (ACPD). Sixty percent (12/20) of MVN neurones responded to 1S,3R-ACPD at a concentration of 10(-6) M, compared with 40% (8/20) and 35% (7/20) of neurones at concentrations of 10(-8) and 10(-10) M. Both increases and decreases in firing rate were observed in different neurones: in general, the magnitude of the responses was large and the duration of the responses was long. Recordings from a slice which contained only the MVN confirmed that these responses were produced by the action of the metabotropic agonist within the MVN itself. These results demonstrate that many MVN neurones have functional metabotropic receptors.

Regional and developmental profile of modulation of hippocampal synaptic transmission and LTP by AP4-sensitive mGluRs in vivo

l-AP4 is an agonist at the presynaptic metabotropic receptor subtypes mGluR4, mGluR6 and mGluR7. In vitro, l-AP4 has been shown to reduce glutamate release and thereby suppress hippocampal excitatory transmission. Little data is available with regard to the actions of this compound in vivo. This study examined the effects of l-AP4 injected i.c.v. in the hippocampus of freely-moving rats on synaptic transmission and long-term potentiation (LTP). Two age groups were employed: 8-week-old and 12-week-old. Administration of l-AP4, 80 mM/5 μl, reduced evoked baseline responses in the dentate gyrus (DG) and CA1 of 8-week-old rats when compared with controls. This effect was blocked by MCPG. l-AP4, 40 mM/5 μl, also reduced baseline in DG but not CA1. l-AP4 (20, 40 or 80 mM/5 μl) had no effect on baseline in either DG or CA1 of 12-week-old animals. However, injection of l-AP4 (80 mM/5 μl) significantly reduced the amplitude of LTP induced by tetanization in CA1 and DG. This effect was blocked by MCPG (200 mM/5 μl). LTP reduction, tested in 12-week-old animals, also occurred with an l-AP4 concentation of 40 mM/5 μl in CA1 but not in DG. These data indicate that l-AP4 inhibits LTP in vivo with a variation in sensitivity to the drug occurring between regions. It is suggested that the response in CA1 is produced by mGluR7, and in DG by presynaptic mGluR4 present on perforant path neurons. These results offer in vivo physiological evidence for a variation in functional response and in developmental regulation of these subtypes, dependent on the region of the hippocampus where they are located.

The messenger RNAs encoding metabotropic glutamate receptor subtypes are expressed in different neuronal subpopulations of the rat suprachiasmatic nucleus

Glutamate is the principal transmitter of retinal projections to the rodent suprachiasmatic nucleus, a circadian clock synchronized with the light-dark cycle through the activation of glutamate receptors of the ionotropic type. In vitro, an intracellular mobilization of calcium can be induced by glutamate within cells of the suprachiasmatic nucleus maintained in a calcium-free medium, suggesting a participation of metabotropic glutamate receptors coupled to phospholipase C. Using in situ hybridization histochemistry, we examined the expression of messenger RNAs encoding the mGluR1 and mGluR5 subtypes of metabotropic glutamate receptors in the suprachiasmatic nucleus of the adult rat and during postnatal development. In the adult, mGluR1 was expressed in a small subset of neurons segregated caudally within the ventrolateral subdivision of the nucleus, while mGluR5 was mainly expressed in ventrolateral neurons within the middle third of the nucleus. Both subtypes were expressed in morphologically similar small cells, but mGluR5 was also solely expressed in a small population of larger neurons located at the dorsalmost aspect of the ventrolateral subdivision. In addition, with mGluR1 probe silver grain clusters exhibiting a grain density close but below the significant level were observed throughout the ventrolateral subdivision of the nucleus. At birth, mGluR1 and mGluR5 were similarly expressed throughout the caudal half of the nucleus. The expression of mGluR1 increased during early postnatal development and exhibited an adult pattern at postnatal day 21. The expression of mGluR5 increased from postnatal day 7 and reached the adult pattern at postnatal day 45. These observations suggest that each subtype of metabotropic glutamate receptor coupled to phospholipase C underlies specific roles within the rat suprachiasmatic nucleus during postnatal development and in the adult. In the adult, ionotropic and metabotropic receptors likely co-expressed within neuronal subsets located in the retinal terminal field may have interactive and/or additive effects on intracellular calcium concentration. Metabotropic receptors may thus participate in the mediation of photic information conveyed to a subset of neurons. During postnatal development, metabotropic receptors may play a role in the maturation of glutamatergic synapses associated with the retinal input.

Metabotropic glutamate receptor mGluR1 distribution and ultrastructural localization in hypothalamus

The metabotropic glutamate receptor mGluR1 is a G-protein-coupled glutamate receptor whose activation induces phosphotidylinositol hydrolysis and increases diacylglycerol and cytoplasmic calcium. By using affinity-purified antisera against a partial amino acid sequence of mGluR1 alpha, deduced from the nucleotide sequence of the cloned gene, the heterogeneous expression of this glutamate receptor was studied immunocytochemically with light and electron microscopy in the rat hypothalamus. Immunoreactivity was restricted to cell bodies and dendrites throughout many regions of the adult hypothalamus, including the preoptic area, anterior hypothalamus, suprachiasmatic nucleus, dorsomedial hypothalamus, and periventricular region. Strong immunolabeling was found in the lateral hypothalamus where immunoreactivity could be detected as early as embryonic day 18. Intense immunoreactivity was also found in the medial mammillary nuclei. In contrast to the strong labeling in many other regions, the neuroendocrine neurons of the arcuate, supraoptic, and paraventricular nuclei showed relatively little staining in adults. With light microscopy, immunoperoxidase labeling was found distributed in patches on the cytoplasmic side of the plasma membrane of immunoreactive neurons. When the same tissue was examined ultrastructurally, the patches were not restricted to synaptic specializations but were also found distributed on perikaryal and dendritic membranes sometimes associated with synapses and sometimes not. Some immunoreactive membranes showed no immunolabeling at the synaptic junction. When the tissue was strongly stained, labeling could be found in the cytoplasm of immunoreactive cells. No immunostaining was found on axons or presynaptic boutons. Together with other evidence showing a widespread expression of many different subtypes of both ionotropic and metabotropic receptors, these data support the hypothesis that glutamate may regulate hypothalamic cellular activity with a number of physiologically different mechanisms, and these mechanisms include second-messenger systems activated by G proteins.

Inhibition of sympathetic preganglionic neurons by spinal glycinergic interneurons

Intracellular and whole-cell patch-clamp recordings were obtained from sympathetic preganglionic neurons in rat spinal cord slices. Perfusion of selective ionotropic and metabotropic excitatory amino acid agonists induced depolarizing responses in all neurons. In approximately 20% of neurons the application of these agonists also evoked inhibitory postsynaptic potentials. The application of the ionotropic receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (5–40 μM) blocked the inhibitory postsynaptic potential discharges induced by ( S)-α/^uamino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (0.5–50μM) and N-methyl- d-aspartate (0.5–50μM), but failed to block the inhibitory postsynaptic potentials induced by quisqualate (0.5–50μM) and (1S,3R)-l-aminocyclopentane-1,3-dicarboxylic acid (10–200μM). Similar inhibitory postsynaptic potentials were seen to occur spontaneously or could be evoked by electrical stimulation of the dorsal horn. The application of tetrodotoxin blocked the spontaneous and evoked inhibitory postsynaptic potential, indicating that they result from activity-dependent release of neurotransmitter. Strychnine antagonized all inhibitory postsynaptic potentials suggesting that they were mediated via glycine receptors. The reversal potential of the inhibitory postsynaptic potentials was −65 mV for intracellular and −55 mV for whole-cell recordings. This latter value is close to the reversal potential for chloride, suggesting that the inhibitory postsynaptic potentials were mediated by a chloride conductance. Perfusion of glycine (0.1–1 mM) induced inhibitory hyperpolarizing responses in the majority of neurons. This hyperpolarizing response was associated with a reduction in neuronal input resistance, persisted in the presence of tetrodotoxin, was blocked by strychnine and reversed at -55mV. In some neurons, glycine induced a membrane depolarization and increased the rate of spontaneous action potential firing. This excitatory effect of glycine was blocked by tetrodotoxin, showed voltage dependency and was less sensitive to strychnine than the glycine-induced inhibitory response. We conclude from these data that spinal interneurons which synapse with sympathetic preganglionic neurons can be activated through multiple subtypes of excitatory amino acid receptor, including both ionotropic and metabotropic receptors. These interneurons release glycine to evoke inhibitory postsynaptic potentials which are mediated via a strychnine-sensitive glycine receptor coupled to a chloride conductance.

Functional expression of 2-amino-4-phosphonobutyrate (APB) receptors in Xenopus laevis oocytes by injection of poly(A) + RNA from quail brain

The glutamate analogue 2-amino-4-phosphonobutyrate (APB) is known to activate a subtype of metabotropic glutamate receptor in the central nervous system, including the retina. In the present study, APB receptors were studied using the Xenopus oocyte expression system. No endogenous APB sensitivity was detected in control oocytes. In contrast, microinjection of mRNA, extracted from quail brain, into Xenopus oocytes resulted in the functional expression of APB receptors after 3–5 days incubation. Application of 50 μM-1 mM APB to injected oocytes voltage clamped at a holding potential of −60 mV produced a sustained outward current which was associated with a significant decrease in membrane conductance; the reversal potential was around −11 mV. The response to APB was dose-dependent and non-desensitizing. This is the first demonstration of the expression of a conductance-decreasing receptor mechanism in Xenopus oocytes.

Changes in mRNA for metabotropic glutamate receptors after transient cerebral ischaemia.

Using a rat 4-vessel occlusion model of cerebral ischaemia we studied the changes in the mRNA level for the metabotropic receptor subtypes mGluR1 alpha, mGluR1 beta, mGluR2, mGluR3, mGluR4, and mGluR5 by means of in situ hybridization with oligonucleotides. After 24 hours of reperfusion the mRNA levels were significantly increased for mGluR2 and mGluR4 while it was significantly decreased for mGluR5. These results suggest that vulnerable neurones react to an increased extracellular glutamate concentration by differential regulation of the mRNA for metabotropic glutamate receptor subtypes which perhaps reflects the different pre- or postsynaptic location and different involvement in ischaemic neurodegeneration.

Modulation of cyclic AMP formation by putative metabotropic receptor agonists

1. The effects of various metabotropic glutamate receptor agonists on [3H]-cyclic AMP accumulation and phosphoinositide hydrolysis were investigated in guinea-pig cerebral cortical slices prelabelled with [3H]-adenine or [3H]-inositol. 2. 1-Aminocyclopentane-1S,3R-dicarboxylate (1S,3R-ACPD), L-2-amino-4-phosphonobutanoate (L-AP4) and (2S,3S,4S)-alpha-(carboxycyclopropyl)glycine (L-CCG-I), elicited concentration-dependent inhibitions of forskolin-stimulated [3H]-cyclic AMP accumulation, with IC50 values of 2.1 +/- 0.3, 71 +/- 17 and 0.2 +/- 0.1 microM respectively. 3. 1S,3R-ACPD and L-CCG-I increased the cyclic AMP responses to histamine H2 receptor stimulation with EC50 values of 7 +/- 2 microM and 19 +/- 2 microM respectively. 1S,3R-ACPD (EC50 values 17 +/- 2 microM) and L-CCG-I (EC50 value 15 +/- 3 microM) potentiated the cyclic AMP responses to the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA, 10 microM). This potentiating effect of L-CCG-I was reduced in the presence of a protein kinase C inhibitor, and also in the absence of extracellular calcium. In contrast, L-AP4 inhibited the NECA response in a concentration-dependent manner, with an IC50 value of 120 +/- 20 microM. 4. L-AP4 (at concentrations up to 1 mM) failed to stimulate phosphoinositide hydrolysis in guinea-pig cerebral cortical slices, but both 1S,3R-ACPD (EC50 value 35 +/- 6 microM) and L-CCG-I (approximately 160 microM) elicited concentration-dependent stimulations of phosphoinositide turnover. 5. These results confirm the existence of at least two distinct subtypes of metabotropic receptor in guinea-pig cortex. The data also substantiate previous studies indicating the importance of the agonist L-CCG-I as a pharmacological tool for distinguishing metabotropic receptor subtypes.

Molecular characterization of a novel retinal metabotropic glutamate receptor mGluR6 with a high agonist selectivity for L-2-amino-4-phosphonobutyrate

A cDNA clone for a new metabotropic glutamate receptor, termed mGluR6, was isolated from a rat retinal cDNA library by cross-hybridization with the previously isolated cDNA clone for a metabotropic glutamate receptor. The cloned mGluR6 subtype consists of 871 amino acid residues and exhibits a structural architecture common to the metabotropic receptor family, possessing a large extracellular domain preceding the seven putative membrane-spanning domains. mGluR6 shows the highest sequence similarity to mGluR4 among the metabotropic receptor subtypes and inhibits the forskolin-stimulated cyclic AMP accumulation in Chinese hamster ovary cells transfected with the cloned cDNA. mGluR6 potently reacts with L-2-amino-4-phosphonobutyrate (L-AP4) and L-serine-O-phosphate, and the potencies of these compounds are one order of magnitude greater than that of L-glutamate. Blot and in situ hybridization analyses indicated that mGluR6 mRNA is restrictedly expressed in the inner nuclear layer of the retina where ON-bipolar cells are distributed. The metabotropic receptor that responds strongly to L-AP4 and L-serine-O-phosphate in ON-bipolar cells is known to mediate glutamate synaptic transmission between photoreceptor cells and ON-bipolar cells. On the basis of the agonist selectivity of mGluR6 and its specific expression in retinal cells, the physiological role of this receptor subtype in the visual system is discussed.

Quisqualate resolves two distinct metabotropic [3H]glutamate binding sites.

Metabotropic receptor subtypes have been proposed based on pharmacological, signal transduction and cDNA sequence data. We assessed potential metabotropic binding site subtypes with in vitro quantitative [3H]glutamate autoradiography in adult rat brains in the presence of saturating concentrations of N-methyl-D-aspartate (NMDA) and (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate (AMPA). Quisqualate (QUIS) competition curves resolved two differentially distributed binding sites (KIhigh = 17 nM; KIlow = 62 microM). Trans-1-amino-cyclopentane- 1,3-dicarboxylic acid (t-ACPD) and 1S,3R-ACPD displaced [3H]glutamate binding both in the absence and presence of a quisqualate concentration (2.5 microM) that saturates the high affinity sites, suggesting that both sites are linked to metabotropic receptors. We conclude that two metabotropic binding sites with different distributions and pharmacological profiles can be detected with selective [3H]glutamate binding assays.