Comparison of nociceptive effects produced by intrathecal administration of mGluR agonists

The present study examined the mGluR subtypes involved in (1S, 3R)-ACPD-induced spontaneous nociceptive behaviours (SNB) by administering the following selective agonists by the intrathecal (i.t.) route: (RS)-DHPG, trans-ADA (Group I; mGluR1/5 and mGluR5, respectively), (1S, 3S)-ACPD, (2R, 4R)-APDC (Group II), and L-AP4 (Group III). (RS)-DHPG administration induced SNB that were of significantly greater intensity and longer duration than those induced by an equal dose of (1S, 3R)-ACPD. No other agonists produced SNB, except (1S, 3S)-ACPD, which may be attributable to a nonselective action at mGluR1. Intrathecal treatment with the mGluR antagonist (+)-MCPG or the NMDA antagonist D-AP5 prior to (RS)-DHPG administration dose-dependently reduced SNB. It is suggested that a possible interaction between NMDA and mGluR1 is a critical event in the maintenance of persistent nociception.     

Metabotropic glutamate receptors in the rat nucleus accumbens

The effects of glutamate metabotropic receptors (mGluRs) on excitatory transmission in the nucleus accumbens were investigated using electrophysiological techniques in rat nucleus accumbens slices. The broad-spectrum mGluR agonist (1S,3R)-1-aminocyclopentyl-1,3-dicarboxylate, the mGluR group 2 selective agonists (S)-4-carboxy-3-hydroxyphenylglycine, (1S,3S)-ACPD) and (2S,1'S,2'S)-2-(2'-carboxycyclopropyl)glycine (L-CCG1), and the mGluR group 3 specific agonist L-2-amino-4-phosphonobutyrate (L-AP4) all reversibly inhibited evoked excitatory synaptic responses. The specific group 1 mGluR agonist (R,S)-3,5-dihydroxyphenylglycine [(R,S)-DHPG] did not depress transmission. Dose-response curves showed that the rank order of agonist potencies was: L-CCG1 > L-AP4 > (1S,3S)-ACPD. Group 2 and 3 mGluRs inhibited transmission via a presynaptic mechanism, as they increased paired-pulse facilitation, decreased the frequency of miniature excitatory postsynaptic currents and had no effect on their amplitude. The mGluRs did not inhibit transmitter release by reducing voltage-dependent Ca2+ currents through N- or P-type Ca2+ channels, as inhibition persisted in the presence of omega-conotoxin-GVIA or omega-Aga-IVA. The depression induced by mGluRs was not affected by specific antagonists of dopamine D1, GABA-B or adenosine A1 receptors, indicating direct effects. Finally, (R,S)-DHPG specifically blocked the postsynaptic afterhyperpolarization current (I(AHP)). Our results represent the first direct demonstration of functional mGluRs in the nucleus accumbens of the rat.     

Regulation of intrinsic and synaptic properties of neonatal rat trigeminal motoneurons by metabotropic glutamate receptors

We studied how metabotropic glutamate receptor (mGluR) activation modifies the synaptic and intrinsic membrane properties of neonatal rat trigeminal motoneurons using the broad-spectrum mGluR agonist (1S,3R)-1-amino-1,3-cyclopentane-dicarboxylic acid [(1S,3R)-ACPD], group I/II antagonist (+/-)-alpha-methyl-4-carboxy-phenylglycine (MCPG), and group III agonist L-2-amino-4-phosphonobutanoate (L-AP4). (1S,3R)-ACPD depressed excitatory transmission to trigeminal motoneurons presynaptically and postsynaptically via presynaptic inhibition and by reducing the currents carried by ionotropic glutamate receptors selective for AMPA. (1S,3R)-ACPD also depolarized trigeminal motoneurons and increased input resistance by suppressing a Ba2+-sensitive leakage K+ current. These effects were not mimicked by L-AP4 (100-200 microM). High-threshold Ca2+ currents were also suppressed by (1S,3R)-ACPD. Repetitive stimulation of excitatory premotoneurons mimicked the postsynaptic effects of (1S, 3R)-ACPD. The postsynaptic effects of (1S,3R)-ACPD and repetitive stimulation were both antagonized by MCPG, suggesting that mGluRs were similarly activated in both experiments. We conclude that mGluRs can be recruited endogenously by glutamatergic premotoneurons and that mGluR-mediated depression of excitatory transmission, combined with increased postsynaptic excitability, enhances the signal-to-noise ratio of oral-related synaptic input to trigeminal motoneurons during rhythmical jaw movements.     

Pharmacological characterization of metabotropic glutamate receptors coupled to phospholipase D in the rat hippocampus

1. Phospholipase D (PLD) is the key enzyme in a signal transduction pathway leading to the formation of the second messengers phosphatidic acid and diacylglycerol. In order to define the pharmacological profile of PLD-coupled metabotropic glutamate receptors (mGluRs), PLD activity was measured in slices of adult rat brain in the presence of mGluR agonists or antagonists. Activation of the phospholipase C (PLC) pathway by the same agents was also examined. 2. The mGluR-selective agonist (1S,3R)-l-aminocyclopentane-1,3-dicarboxylic acid [(1S,3R)-ACPD] induced a concentration-dependent (10-300 microM) activation of PLD in the hippocampus, neocortex, and striatum, but not in the cerebellum. The effect was particularly evident in hippocampal slices, which were thus used for all subsequent experiments. 3. The rank order of potencies for agonists stimulating the PLD response was: quisqualate > ibotenate > (2S,3S,4S)-alpha-(carboxycyclopropyl)-glycine > (1S,3R)-ACPD > L-cysteine sulphinic acid > L-aspartate > L-glutamate. L-(+)-2-Amino-4-phosphonobutyric acid and the ionotropic glutamate receptor agonists N-methyl-D-aspartate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, and kainate failed to activate PLD. (RS)-3,5-dihydroxyphenylglycine (100300 microM), an agonist of mGluRs of the first group, stimulated PLC but inhibited the PLD response elicited by 100 microM (1S,3R)-ACPD. 4. (+)-alpha-Methyl-4-carboxyphenylglycine (0.1-1 mM), a competitive antagonist of mGluRs of the first and second group, elicited a significant PLD response. L-(+)-2-Amino-3-phosphonopropionic acid (1 mM), an antagonist of mGluRs of the first group, inhibited the 100 microM (1S,3R)-ACPD-induced PLC response but produced a robust stimulation of PLD. 5. 12-O-Tetradecanoylphorbol 13-acetic acid and phorbol 12,13-dibutyrate (PDBu), activators of protein kinase C, at 1 microM had a stimulatory effect on mGluRs linked to PLD but depressed (1S,3R)-ACPD-induced phosphoinositide hydrolysis. The protein kinase C inhibitor, staurosporine (1 and 10 microM) reduced PLD activation induced by 1 microM PDBu but not by 100 microM (1S,3R)-ACPD. 6. Our results suggest that PLD-linked mGluRs in rat hippocampus may be distinct from any known mGluR subtype coupled to PLC or adenylyl cyclase. Moreover, they indicate that independent mGluRs coupled to the PLC and PLD pathways exist and that mGluR agonists can stimulate PLD through a PKC-independent mechanism.     

Roles of metabotropic glutamate receptor subtypes in modulation of pentylenetetrazole-induced seizure activity in mice

The anticonvulsant or proconvulsant properties of ligands at metabotropic glutamate receptors (mGluRs) were examined in a chemoconvulsant model using pentylenetetrazole (PTZ). Mice received mGluR ligands by intracerebroventricular (i.c.v.) infusion prior to a subcutaneous injection of PTZ and the latency to onset of tonic convulsions was recorded. The group I mGluR antagonist 1-aminoindan-1,5-dicarboxylic acid (AIDA) dose-dependently antagonised PTZ-induced seizures with a mean ED50 value of 465 nmol. In contrast, the selective group I mGluR agonist, (S)-3,5-dihydroxyphenylglycine [(S)-DHPG], was proconvulsive and decreased the PTZ-induced seizure latency (ED50=60 nmol i.c.v.). A selective agonist of group II mGluRs, (1S,3S)-1-aminocyclopentane dicarboxylic acid [(1S,3S)-ACPD], was proconvulsive but did not affect PTZ-induced seizure latency. Moreover, the proconvulsant effect of (IS,3S)-ACPD was not blocked by the mGluR2 antagonist, alpha-methylserine-O-phosphate monophenyl ester but was blocked by AIDA suggesting the involvement of group I mGluRs. (2S,1'S,2'S,3'R)-2-(2'-carboxy-3'-phenylcyclopropyl)glycine (PCCG-IV), which is a potent mGluR2 antagonist and a group III mGluR agonist at higher doses, increased the PTZ-induced seizure latency (ED50=51 nmol) and this effect was fully reversed by the group III mGluR antagonist, (S)-2-amino-2-methyl-4-phosphonobutanoic acid (MAP4). Similarly, the group III mGluR agonist 1-amino-3-(phosphonomethylene)cyclobutanecarboxylate (cyclobutylene-AP5) increased the PTZ-induced seizure latency (ED50=12 nmol) in a MAP4-sensitive manner. Collectively, these data suggest that mGluR ligands modulate PTZ-induced seizure activity in mice by either antagonizing group I mGluRs or activating group III mGluRs.     

Herpesvirus in atherosclerosis and thrombosis: etiologic agents or ubiquitous bystanders?

While it is well documented that the overactivation of ionotropic glutamate receptors leads to seizures and excitotoxic injury, little is known about the role of metabotropic glutamate receptors (mGluRs) in epileptogenesis and neuronal injury. Intracerebroventricular (i.c.v.) infusion of the group I mGluR specific agonist (R,S)-3,5-dihydroxyphenylglycine (3,5-DHPG) (1.5 micromol) to conscious rats produced severe and delayed seizures (onset at 4 hr) in 70% of the animals. The i.c.v. infusion of the group I mGluR non-selective agonist 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) (2 micromol) produced a similar rate of severe seizures, but with an early onset (0.6 hr). The analysis of motor activity showed that 3,5-DHPG elicited higher central stimulatory action than did 1S,3R-ACPD. Histopathological analysis of the hippocampus showed that 3,5-DHPG produced severe neuronal damage mainly in the CA1 pyramidal neurons and, to a lesser extent, in the CA3. Although 1S,3R-ACPD infusion also induced a slight injury of the CA1 and CA3 pyramidal neurons, damage was greater in the CA4 and dentate gyrus cells. In conclusion, the in vivo activation of group I mGluRs with the selective agonist 3,5-DHPG produces hyperexcitatory effects that lead to seizures and neuronal damage, these effects being more severe than those observed after infusion of the non-selective agonist 1S,3R-ACPD.     

Activation of metabotropic glutamate receptors induce differential effects on synaptic transmission in the dentate gyrus and CA1 of the hippocampus in the anaesthetized rat

Activation of ACPD-sensitive metabotropic receptors induced differential effects on synaptic transmission and the induction of LTP in CA1 and the dentate gyrus of the hippocampus i.c.v. injections of (1.S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid [(1S,3R)-ACPD] induced enduring potentiation of the fEPSP in CA1, which occluded tetanically induced LTP. In contrast, ACPD induced a dose-dependent biphasic effect on the fEPSP in the dentate gyrus, consisting of an initial short lasting potentiation, followed by enduring depression of the response, and blockade of LTP. These two effects are likely to be mediated by two different classes of the receptor as in the dentate gyrus the selective class I agonist, (RS)-3,5-dihydroxyphenylglycine (DHPG) induced sustained potentiation of the fEPSP, whereas the mixed mGluR2 agonist-mGluR1 antagonist, (S)-4-carboxy-3-hydrophenylglycine((S)-4C3H-PG) induced only depression. Increasing the concentration of calcium directly in the dentate gyrus prior to, and in conjunction with, injections of ACPD induced sustained potentiation rather than depression. The differential effects indicate that the second messenger cascades the subtypes of receptors are linked with, mediate different forms of synaptic plasticity within the hippocampus and have important implications for their role in learning.     

Endogenous activation of metabotropic glutamate receptors contributes to burst frequency regulation in the lamprey locomotor network

The effect of metabotropic glutamate receptor (mGluR) agonists and antagonists on the spinal cord network underlying locomotion in the lamprey has been analysed. The specific group I mGluR agonist (R,S)-3,5-dihydroxyphenylglycine (DHPG) and the broad-spectrum mGluR agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) both increased the burst frequency of N-methyl-D-aspartic acid (NMDA)-induced fictive locomotion and depolarized grey matter neurons. The burst frequency increase induced by the mGluR agonists was counteracted by the mGluR antagonists (+)-alpha-methyl-4-carboxyphenylglycine ((+)-MCPG), cyclopropan[b]chromen-1a-carboxylic acid ethylester (CPCCOEt) and (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA). Application of CPCCOEt alone reduced the locomotor burst frequency, indicating that mGluRs are endogenously activated during fictive locomotion. The mGluR antagonist CPCCOEt had no effect on NMDA-, or (S)-alpha-amino-3-hydroxy-5-methyl-4-isoazolepropionic acid (AMPA)-induced depolarizations. The mGluR agonists 1S,3R-ACPD and DHPG increased the amplitude of NMDA-induced depolarizations, a mechanism which could account for the increase in burst frequency. The group III mGluR agonist L-2-amino-4-phosphonobutyric acid reduced intraspinal synaptic transmission and burst frequency.     

Pharmacological differentiation of kainate receptors on neonatal rat spinal motoneurones and dorsal roots

The objectives of this study, conducted on neonatal rat spinal cord and dorsal roots in vitro, were to characterise the actions of a range of willardiine analogues on GluR5-containing kainate receptors present in dorsal roots, to determine whether GluR5-containing receptors are also present on motoneurones, and to differentiate responses mediated by kainate receptors from those mediated by AMPA receptors on motoneurones. (S)-5-Trifluoromethyl-willardiine, (S)-5-iodowillardiine, (S)-5-iodo-6-azawillardiine and ATPA were found to be potent agonists of kainate receptors on dorsal roots (EC50 values 0.108 +/- 0.002, 0.127 +/- 0.010, 0.685 +/- 0.141 and 1.3 +/- 0.3 microM, respectively) being more potent but of lower efficacy than kainate (EC50 value 14.8 +/- 1.8 microM). (S)-5-Iodo-6-azawillardiine blocked kainate-induced depolarisations of the dorsal root, probably via its desensitising action. Kainate-induced responses of dorsal roots were weakly antagonised by (RS)-3,5-dicarboxyphenylglycine (DCPG) (apparent KD 1.5 +/- 0.4 mM). Kainate receptors containing GluR5 subunits do not appear to be present on motoneurones since (RS)-3,5-DCPG (1 mM) potentiated rather than antagonised kainate-induced depolarisations of motoneurones. Although (S)-5-iodowillardiine (a potent and selective agonist at GluR5-containing kainate receptors) depolarised motoneurones (EC50 value 5.8 +/- 0.6 microM), such depolarisations were antagonised by both (RS)-3,4- and (RS)-3,5-DCPG, which are selective AMPA receptor antagonists at motoneurones, showing a KD value of 73 microM (Schild slope, 0.96 +/- 0.09) and an apparent KD value of 123 +/- 38 microM, respectively. This accords with the previously reported activity of willardiine analogues at AMPA receptors. Since neither (RS)-3,4- nor (RS)-3,5-DCPG antagonised kainate-induced motoneuronal depolarisations but cyclothiazide enhanced and GYK153655 blocked these responses it is possible that a component of the kainate response may be mediated by a population of DCPG-insensitive AMPA receptors on motoneurones. However, it is also possible that a population of kainate receptors other than those containing GluR5 subunits, are responsible for these effects. The new compounds introduced in this study are likely to be useful tools for studying the physiological role of kainate receptors in CNS function.     

Pharmacological characterization of 1-aminoindan-1,5-dicarboxylic acid, a potent mGluR1 antagonist

We examined the pharmacological profile of 1-aminoindan-1,5-dicarboxylic acid (AIDA), a rigid (carboxyphenyl)glycine derivative acting on metabotropic glutamate receptors (mGluRs). In cells transfected with mGluR1a, AIDA competitively antagonized the stimulatory responses of glutamate and (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid [(1S,3R)-ACPD] on phosphoinositide hydrolysis (pA2 = 4.21). In cells transfected with mGluR5a, AIDA displayed a much weaker antagonist effect. In transfected cells expressing mGluR2, AIDA (< or = 1 mM) did not affect the inhibition of forskolin-stimulated adenylate cyclase activity induced by (1S,3R)-ACPD, but at large concentrations, it displayed a modest agonist activity. In rat hippocampal or striatal slices, AIDA (0.1-1 mM) reduced the effects of (1S,3R)-ACPD on phospholipase C but not on adenylate cyclase responses, whereas (+)-alpha-methyl-4-carboxyphenylglycine (0.3-1 mM) was an antagonist on both transduction systems. In addition, AIDA (0.3-1 mM) had no effect on mGluRs coupled to phospholipase D, whereas (+)-alpha-methyl-4-carboxy-phenylglycine (0.5-1 mM) acted as an agonist with low intrinsic activity. In rat cortical slices, AIDA antagonized the stimulatory (mGluR1-mediated) effect of (1S,3R)-ACPD on the depolarization-induced outflow of D-[3H]aspartate, disclosing an inhibitory effect ascribable to (1S,3R)-ACPD activating mGluR2 and/or mGluR4. Finally, mice treated with AIDA (0.1-10 nmol i.c.v.) had an increased pain threshold and difficulties in initiating a normal ambulatory behavior. Taken together, these data suggest that AIDA is a potent, selective and competitive mGluR1 a antagonist.     

Carbachol can potentiate N-methyl-D-aspartate responses in the rat hippocampus by a staurosporine and thapsigargin-insensitive mechanism

Experiments were performed to investigate the mechanism underlying the potentiation of N-methyl-D-aspartate (NMDA) responses by carbachol (CCh) in the CA1 region of rat hippocampal slices. CCh (300 nM) potentiated responses to NMDA, but not to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), in a readily reversible manner. Potentiation occurred in slices treated with 200 nM tetrodotoxin and perfused with Mg(2+)-free medium. It also occurred in slices treated with either staurosporine (1 microM), which is a potent inhibitor of a variety of protein kinases including protein kinase C (PKC), or thapsigargin (10 microM), which depletes intracellular Ca2+ stores by preventing their refilling. However, CCh-induced potentiation was abolished in slices perfused with Ca(2+)-free medium. These data suggest that low concentrations of CCh can acutely potentiate NMDA responses in the hippocampus by a Ca(2+)-sensitive process that is probably independent of both the activation of PKC and the release of Ca2+ from intracellular stores. This mechanism is similar to that underlying the potentiation of NMDA responses by the metabotropic glutamate receptor (mGluR) agonist, aminocyclopentane-1S,3R-dicarboxylic acid (1S,3R-ACPD).     

NMDA receptor dependence of mGlu-mediated depression of synaptic transmission in the CA1 region of the rat hippocampus

1. The depression of synaptic transmission by the specific metabotropic glutamate receptor (mGlu) agonist (1S, 3R)-1-aminocyclopentane-1,3-dicarboxylate ((1S,3R)-ACPD) was investigated in area CA1 of the hippocampus of 4-10 week old rats, by use of grease-gap and intracellular recording techniques. 2. In the presence of 1 mM Mg2+, (1S,3R)-ACPD was a weak synaptic depressant. In contrast, in the absence of added Mg2+, (1S,3R)-ACPD was much more effective in depressing both the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) and N-methyl-D-aspartate (NMDA) receptor-mediated components of synaptic transmission. At 100 microM, (1S,3R)-ACPD depressed the slope of the field excitatory postsynaptic potential (e.p.s.p.) by 96 +/- 1% (mean +/- s.e.mean; n = 7) compared with 23 +/- 4% in 1 mM Mg(2+)-containing medium (n = 17). 3. The depressant action of 100 microM (1S,3R)-ACPD in Mg(2+)-free medium was reduced from 96 +/- 1 to 46 +/- 6% (n = 7) by the specific NMDA receptor antagonist (R)-2-amino-5-phosphonopentanoate (AP5; 100 microM). 4. Blocking both components of GABA receptor-mediated synaptic transmission with picrotoxin (50 microM) and CGP 55845A (1 microM) in the presence of 1 mM Mg2+ also enhanced the depressant action of (1S,3R)-ACPD (100 microM) from 29 +/- 5 to 67 +/- 6% (n = 6). 5. The actions of (1S,3R)-ACPD, recorded in Mg(2+)-free medium, were antagonized by the mGlu antagonist (+)-alpha-methyl-4-carboxyphenylglycine ((+)-MCPG). Thus, depressions induced by 30 microM (1S,3R)-ACPD were reversed from 48 +/- 4 to 8 +/- 6% (n = 4) by 1 mM (+)-MCPG. 6. In Mg(2+)-free medium, a group I mGlu agonist, (RS)-3, 5-dihydroxyphenylglycine (DHPG; 100 microM) depressed synaptic responses by 74 +/- 2% (n = 18). In contrast, neither the group II agonists ((2S,1'S,2'S)-2-(2'-carboxycyclopropyl)glycine; L-CCG-1; 10 microM; n = 4) and ((2S,1'R,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine; DCG-IV; 100 nM; n = 3) nor the group III agonist ((S)-2-amino-4-phosphonobutanoic acid; L-AP4; 10 microM; n = 4) had any effect. 7. The depolarizing action of (1S,3R)-ACPD, recorded intracellularly, was similar in the presence and absence of Mg(2+)-AP5 did not affect the (1S,3R)-ACPD-induced depolarization in Mg(2+)-free medium. Thus, 50 microM (1S,3R)-ACPD induced depolarizations of 9 +/- 3 mV (n = 5), 10 +/- 2 mV (n = 4) and 8 +/- 2 mV (n = 5) in the three respective conditions. 8. On resetting the membrane potential in the presence of 50 microM (1S,3R)-ACPD to its initial level, the e.p.s.p. amplitude was enhanced by 8 +/- 3% in 1 mM Mg2+ (n = 5) compared with a depression of 37 +/- 11% in the absence of Mg2+ (n = 4). Addition of AP5 prevented the (1S,3R)-ACPD-induced depression of the e.p.s.p. (depression of 4 +/- 5% (n = 5)). 9. It is concluded that activation by group 1 mGlu agonists results in a depression of excitatory synaptic transmission in an NMDA receptor-dependent manner.     

Light-regulated localization of the beta-subunit of Gq-type G-protein in the crayfish photoreceptors

The role of metabotropic (mGluRs) and N-methyl-D-aspartate (NMDA) glutamate receptors on 5-hydroxytryptamine (5-HT) release has been studied in rat periaqueductal gray (PAG) matter by using in vivo microdialysis. (1S,3R)-aminocyclopentane- 1,3-dicarboxylic acid [(IS,3R)-ACPD; 0.5 or 1 mM], a group I/group II mGluRs agonist, increased the dialysate 5-HT concentration. (2S)-alpha-ethylglutamic acid (EGlu; 1 mM), an antagonist of group II mGluRs, but not (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA; 1 mM), an antagonist of group I mGluRs, antagonized the 1S,3R-ACPD-induced effect. (S)-3,5-dihydroxyphenylglycine (DHPG; 0.5 and 1 mM), an agonist of group I mGluRs, did not modify dialysate 5-HT. (2S, 3S, 4S)-alpha-(carboxycyclopropyl)-glycine (L-CCG-I; 0.5 and I mM), an agonist of group II mGluRs, increased extracellular 5-HT. This effect was antagonized by EGlu. Similarly, L-serine-O-phosphate (L-SOP; 1 and 10 mM), an agonist of group III mGluRs, increased extracellular 5-HT and this effect was antagonized by (RS)-(alpha-methylserine O-phosphate (M-SOP; 1 mM), an antagonist of group III mGluRs. Out of the several N-methylD-aspartate concentrations used (NMDA; 10, 50, 100, 500 and 1000 microM) only the 50 microM infusion significantly decreased dialysate 5-HT. The GABA(A) receptor agonist, bicuculline (30 microM), increased 5-HT release on its own and antagonized the decrease caused by the opiate antagonist, naloxone (2 mM), as well as the increases caused by CCG-I or L-SOP. These data show that stimulation of PAG's group II/group II mGluRs increases 5-HT release, while stimulation of NMDA glutamate receptors may decrease it. We speculate that glutamate does not modulate 5-HT release in the PAG directly, but via activation of tonically active GABAergic interneurons.     

Synthesis of the four isomers of 4-aminopyrrolidine-2,4-dicarboxylate: identification of a potent, highly selective, and systemically-active agonist for metabotropic glutamate receptors negatively coupled to adenylate cyclase

The four isomers of 4-aminopyrrolidine-2,4-dicarboxylate (APDC) were prepared and evaluated for their effects at glutamate receptors in vitro. (2R,4R)-APDC (2a), an aza analog of the nonselective mGluR agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate (1S,3R)-ACPD, 1), was found to possess relatively high affinity for metabotropic glutamate receptors (mGluRs) (ACPD-sensitive [3H]glutamate binding IC50 = 6.49 +/- 1.21 microM) with no effects on radioligand binding to NMDA, AMPA, or kainate receptors up to 100 microM. None of the other APDC isomers showed significant mGluR binding affinity, indicating that this interaction is highly stereospecific. Both 1 and 2a were effective in decreasing forskolin-stimulated cAMP formation in the adult rat cerebral cortex (EC50 = 8.17 +/- 2.21 microM for 1; EC50 = 14.51 +/- 5.54 microM for 2a); however, while 1 was also effective in stimulating basal tritiated inositol monophosphate production in the neonatal rat cerebral cortex (EC50 = 27.7 +/- 5.2 microM), 2a (up to 100 microM) was ineffective in stimulating phosphoinositide hydrolysis in this tissue preparation, further supporting our previous observations that 2a is a highly selective agonist for mGluRs negatively coupled to adenylate cyclase. Microelectrophoretic application of either 1 or 2a to intact rat spinal neurons produced an augmentation of AMPA-induced excitation (95 +/- 10% increase for 1, 52 +/- 6% increase for 2a). Intracerebral injection of 1 (400 nmol) produced characteristic limbic seizures in mice which are not mimicked by 2a (200-1600 nmol, ic). However, the limbic seizures induced by 1 were blocked by systemically administered 2a in a dose-dependent manner (EC50 = 271 mg/kg, ip). It is concluded that (2R,4R)-APDC (2a) is a highly selective, systemically-active agonist of mGluRs negatively coupled to adenylate cyclase and that selective activation of these receptors in vivo can result in anticonvulsant effects.     

ERE environment- and cell type-specific transcriptional effects of estrogen in normal endometrial cells

Pregnenolone sulfate (PS) is an abundant neurosteroid that can potentiate or inhibit ligand gated ion channel activity and thereby alter neuronal excitability. Whereas PS is known to inhibit kainate and AMPA responses while potentiating NMDA responses, the dependence of modulation on receptor subunit composition remains to be determined. Toward this end, the effect of PS on recombinant kainate (GluR6), AMPA (GluR1 or GluR3), and NMDA (NR1(100)+NR2A) receptors was characterized electrophysiologically with respect to efficacy and potency of modulation. With Xenopus oocytes expressing GluR1, GluR3 or GluR6 receptors, PS reduces the efficacy of kainate without affecting its potency, indicative of a noncompetitive mechanism of action. Conversely, with oocytes expressing NR1(100)+NR2A subunits, PS enhances the efficacy of NMDA without affecting its potency. Whereas the modulatory efficacy, but not the potency, of PS is increased two-fold by co-injection of NR1(100)+NR2A cRNAs as compared with NR1(100) cRNA alone, there is little or no effect of the NR2A subunit on efficacy or potency of pregnanolone (or epipregnanolone) sulfate as an inhibitor of the NMDA response. This suggests that the NR2A subunit controls the efficacy of neurosteroid enhancement, but not inhibition, which is consistent with our previous finding that potentiating and inhibitory steroids act at distinct sites on the NMDA receptor. This represents a first step towards understanding the role of subunit composition in determining neurosteroid modulation of ionotropic glutamate receptor function.     

Developmental change of the potentiation of NMDA response by spermine

Developmental change in the potentiation of N-methyl-D-aspartate (NMDA) responses by spermine was investigated on the ventromedial hypothalamic neurones acutely dissociated from the rats aged between 5 and 21 days, using a nystatin perforated patch clamp recording in a whole cell mode. Spermine potentiated the NMDA response in a concentration dependent manner between 10(-5) M and 10(-5) M at all ages examined. This potentiation decreased significantly with age. On the other hand, spermine did not affect the kainate and AMPA responses at any age. This developmental change of the modulation of NMDA responses might influence to or be influenced by the behavioural and neuronal changes related to the VMH in the early postnatal life.     

Hyperalgesia and allodynia induced by intrathecal (RS)-dihydroxyphenylglycine in rats

To investigate the role of Group I mGluRs in allodynia and hyperalgesia, we examined the behavioural responses of rats to noxious and non-noxious mechanical and thermal stimuli following intrathecal (i.t.) treatment (25 nmol) with the selective mGluR1/5 agonist, (RS)-dihydroxyphenylglycine ((RS)-DHPG). (RS)-DHPG administration produced a persistent decrease in response latency on a 48 degrees C hotplate, a reduction in the 50% response threshold to von Frey hairs, and an increase in responses to a tail pinch. These data suggest that activation of spinal mGluR1/5 receptors plays a role in the development of persistent allodynia and hyperalgesia associated with tissue or nerve injury.     

Cobalt accumulation in neurons expressing ionotropic excitatory amino acid receptors in young rat spinal cord: morphology and distribution

Excitatory amino acids (EAA) acting on N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and kainate receptors play an important role in synaptic transmission in the spinal cord. Quantitative autoradiography and physiological experiments suggest that NMDA receptors are localized mainly in lamina II while kainate and AMPA receptors are found on both dorsal and ventral horn neurons. However the cell types expressing EAA receptors and their laminar distribution is not known. We have used a cobalt uptake method to study the morphology and distribution of spinal cord neurons expressing AMPA, kainate, or NMDA excitatory amino acid receptors in the lumbar enlargement of the rat spinal cord. The technique involved superfusion of hemisected spinal cords of 14 day-old rat pups in vitro with excitatory amino acid receptor ligands in the presence of CoCl2. Cobalt has been shown to enter cells through ligand-gated ion channels in place of Ca2+. Cells which accumulated cobalt ions following activation by ionotropic excitatory amino acid receptors were visualized histochemically. The cobalt uptake generated receptor-specific labeling of cells, as the NMDA receptor antagonist D-(-)-2-amino-(5)-phosphonovaleric acid (D-AP-5) (20 microM) blocked the NMDA, but not kainate-induced cobalt uptake. The kainate-induced cobalt labeling was reduced by the non-selective excitatory amino acid receptor antagonist kynurenic acid (4 mM). Passive opening of the voltage-gated Ca(2+)-channels by KCl (50 mM) did not result in cobalt uptake, indicating that cobalt enters the cells through ligand-gated Ca(2+)-channels. AMPA (500 microM), kainate (500 microM), or NMDA (500 microM) each induced cobalt uptake with characteristic patterns and distributions of neuronal staining. Overall, kainate induced cobalt uptake in the greatest number of neuronal staining. Overall, kainate induced cobalt uptake in the greatest number of neuronal perikarya while NMDA-induced uptake was the lowest. AMPA and kainate, but not NMDA superfusion, resulted in cobalt labeling of glial cells. Our results show that the cobalt uptake technique is a useful way to study the morphology and distribution of cells expressing receptors with ligand-gated Ca2+ channels.     

Metabotropic glutamate receptor-mediated inhibition and excitation of substantia nigra dopamine neurons

Microiontophoretic drug application and extracellular recording techniques were used to evaluate the effects of the selective metabotropic glutamate receptor (mGluR) agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate(1S,3R-ACPD) on dopamine (DA) neurons in the substantia nigra zona compacta (SNZC) of chloral hydrate-anesthetized rats. 1S,3R-ACPD had a biphasic effect on the firing rate of DA cells, initially decreasing, then increasing the firing rate. 1S,3R-ACPD also increased the burst-firing activity of DA neurons. Application of the ionotropic receptor (iGluR) agonists (R,S)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) or N-methyl-D-aspartate (NMDA) increased the firing rates of neurons which had responded to 1S,3R-ACPD, indicating that mGluRs and iGluRs reside on the same neurons. The initial inhibitory period was not antagonized by systemic haloperidol or iontophoretic bicuculline, indicating a lack of DA or gamma-amino-n-butyric acid (GABA) involvement in this effect. Combined application of the AMPA antagonist, 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX), and the NMDA antagonist, (I)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphoric acid (CPP), at currents which antagonized AMPA and NMDA, did not antagonize either the inhibitory or excitatory effects of 1S,3R-ACPD. Application of the metabotropic antagonist (S)-4-carboxy-phenylglycine antagonized both the inhibitory and excitatory effects of 1S,3R-ACPD. These results indicate that mGluRs may play a role in the modulation of dopaminergic activity in the SNZC.     

Metabotropic glutamate receptors modulate synaptic transmission in the perforant path: pharmacology and localization of two distinct receptors

Metabotropic glutamate receptors (mGluRs) have emerged as an interesting family of eight different receptor subtypes that can be divided into three groups according to their pharmacology and sequence similarity. In the present study, the specific mGluR agonists (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid ((1S,3R)-ACPD) and L(+)-2-amino-4-phosphonobutyric acid (L-AP4) depressed field excitatory postsynaptic potentials (fEPSPs) in the rat dentate gyrus evoked by perforant path stimulation in a concentration-dependent, rapid and reversible manner (EC50: L-AP4 5.9 +/- 1.6 microM, (1S,3R)-ACPD 80 +/- 34 microM). In a 'paired-pulse' stimulation protocol, the first fEPSP showed a stronger reduction, resulting in 'paired-pulse' facilitation. The effects of L-AP4 but not of (1S,3R)-ACPD could be antagonized by the group III mGluR antagonists (S)-2-amino-2-methyl-4-phosphonobutanoic acid (MAP4) and (RS)-alpha-methyl-4-phosphonophenylglycine (MPPG). Moreover, (1S,3R)-ACPD was still potently depressing fEPSPs after preperfusion of near saturating concentrations of L-AP4. Together, the results suggest that both substances act on different mGluRs. The effects of (1S,3R)-ACPD could not be further differentiated by selective group I or group II mGluR agonists. Although (2S,1'S,2'S)-2-carboxycyclopropylglycine (L-CCG-I) blocked fEPSPs at concentrations > 1 microM, these effects, as well as L-AP4 effects, were potently antagonized by MAP4. This suggests that mGluR8 might be responsible for the actions of L-AP4 and L-CCG-I. The two different mGluRs showed a distinct distribution when fEPSPs were recorded simultaneously in the outer and middle molecular layer (OML/MML): The L-AP4 sensitive receptor, possibly mGluR8, seems to be located in the OML while (1S,3R)-ACPD showed its main effect in the MML.