Epileptogenesis in vivo enhances the sensitivity of inhibitory presynaptic metabotropic glutamate receptors in basolateral amygdala neurons in vitro

Modulation of excitatory synaptic transmission by presynaptic metabotropic glutamate receptors (mGluRs) was examined in brain slices from control rats and rats with amygdala-kindled seizures. Using whole-cell voltage-clamp and current-clamp recordings, this study shows for the first time that in control and kindled basolateral amygdala neurons, two pharmacologically distinct presynaptic mGluRs mediate depression of synaptic transmission. Moreover, in kindled neurons, agonists at either group II- or group III-like mGluRs exhibit a 28- to 30-fold increase in potency and suppress synaptically evoked bursting. The group II mGluR agonist (2S,3S,4S)-2-(carboxycyclopropyl)glycine (L-CCG) dose-dependently depressed monosynaptic EPSCs evoked by stimulation in the lateral amygdala with EC50 values of 36 nM (control) and 1.2 nM (kindled neurons). The group III mGluR agonist L-2-amino-4-phosphonobutyrate (L-AP4) was less potent, with EC50 values of 297 nM (control) and 10.8 nM (kindled neurons). The effects of L-CCG and L-AP4 were fully reversible. Neither L-CCG (0.0001-10 microM) nor L-AP4 (0.001-50 microM) caused membrane currents or changes in the current-voltage relationship. The novel mGluR antagonists (2S,3S,4S)-2-methyl-2-(carboxycyclopropyl)-glycine (MCCG; 100 microM) and (S)-2-methyl-2-amino-4-phosphonobutyrate (MAP4; 100 microM) selectively reversed the inhibition by L-CCG and L-AP4 to 81.3 +/- 12% and 65.3 +/- 6.6% of predrug, respectively. MCCG and MAP4 (100-300 microM) themselves did not significantly affect synaptic transmission. The exquisite sensitivity of agonists in the kindling model of epilepsy and the lack of evidence for endogenous receptor activation suggest that presynaptic group II- and group III-like mGluRs might be useful targets for suppression of excessive synaptic activation in neurological disorders such as epilepsy.     

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.     

Glutamate metabotropic receptor agonists depress excitatory and inhibitory transmission on rat mesencephalic principal neurons

Intracellular and whole-cell patch-clamp recordings were used to evaluate the actions of different metabotropic glutamate receptor (mGluR) agonists on the synaptic inputs evoked on principal cells of the rat mesencephalon. Bath application of the group III mGluR agonists L-2-amino-4-phosphonobutyric acid (L-AP4) and L-serine-O-phosphonobutanoate (L-SOP) did not change the holding current of the cells held at resting potential (-60 mV) but produced a dose-dependent inhibition of the amplitude of the excitatory and inhibitory events. L-AP4 and L-SOP were more effective at inhibiting the excitatory postsynaptic currents (EPSCs) than the GABA(A) and GABA(B) inhibitory postsynaptic currents (IPSCs). The suppressing effects of L-AP4 and L-SOP were antagonized by (S)-2-amino-2-methyl-4-phosphonobutanoic acid (MAP-4) but not by +/- -alpha-methyl-4-carboxyphenylglycine (MCPG). Moreover, the group II agonist (2S,1'S,2'S)-(carboxycyclopropyl)glycine (L-CCG1) and the group I agonist (RS)-3,5-dihydrophenylglycine (3,5-DHPG) depressed in a dose-related manner the EPSC, the GABA(A) IPSC and the GABA(B) IPSC. The suppressing effect of the two mGluRs agonists was partially antagonized by MCPG but not by MAP-4. In addition, both L-CCG1 and 3,5-DHPG caused an inward shift of the holding current. To characterize the site of action of the metabotropic receptor agonists, experiments were performed to examine the amplitude and ratio of EPSC and GABA(A) IPSC pairs. The increase of the s2/s1 ratio caused by the agonists suggests that the location of the inhibitory mGluRs was presynaptic. These results indicate that the activation of presynaptic mGluRs controls the release of excitatory and inhibitory transmitters on presumed dopaminergic cells within the ventral mesencephalon.     

Modulation of adenosine-induced cAMP accumulation via metabotropic glutamate receptors in chick optic tectum

Changes on cyclic adenosine monophosphate (cAMP) levels in response to adenosine and glutamate and the subtype of glutamate receptors involved in this interaction were studied in slices of optic tectum from 3-day-old chicks. cAMP accumulation mediated by adenosine (100 microM) was abolished by 8-phenyltheophylline (15 microM). Glutamate and the glutamatergic agonists kainate or trans-D, L-1-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD) did not evoke cAMP accumulation. Glutamate blocked the adenosine response in a dose-dependent manner. At 100 microM, glutamate did not inhibit the effect of adenosine. The 1 mM and 10 mM doses of glutamate inhibited adenosine-induced cAMP accumulation by 55% and 100%, respectively. When glutamatergic antagonists were used, this inhibitory effect was not affected by 200 microM 6,7-dihydroxy-2,3,dinitroquinoxaline (DNQX), an ionotropic antagonist, and was partially antagonized by 1 mM (RS)-alpha-methyl-4-carboxyphenylglycine [(RS)M-CPG], a metabotropic antagonist, while 1 mM L-2-amino-3-phosphonopropionate (L-AP3) alone, another metabotropic antagonist, presented the same inhibitory effect of glutamate. Kainate (10 mM) and trans-ACPD (100 microM and 1 mM) partially blocked the adenosine response. This study indicates the involvement of metabotropic glutamate receptors in adenylate cyclase inhibition induced by glutamate and its agonists trans-ACPD and kainate.     

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 phosphoinositide turnover in neonatal rat cerebral cortex by group I- and II- selective metabotropic glutamate receptor agonists

1. The interactive effects of different metabotropic glutamate (mGlu) receptor subtypes to regulate phosphoinositide turnover have been studied in neonatal rat cerebral cortex and hippocampus by use of agonists and antagonists selective between group I and II mGlu receptors. 2, The group II-selective agonist 2R,4R-4-aminopyrrolidine-2,4-dicarboxylate (2R,4R-APDC; 100 microM) had no effect on basal total inositol phosphate ([3H]-InsPx) accumulation (in the presence of Li+) in myo-[3H]-inositol pre-labelled slices, but enhanced the maximal [3H]-InsPx response to the group I-selective agonist (S)-3,5-dihydroxyphenylglycine (DHPG) by about 100% in both hippocampus and cerebral cortex. In cerebral cortex the enhancing effect of 2R,4R-APDC occurred with respect to the maximal responsiveness and had no effect on EC50 values for DHPG (-log EC50 (M): control, 5.56+/-0.05; +2R,4R-APDC, 5.51+/-0.08). 2R,4R-APDC also caused a significant enhancement of the DHPG-stimulated inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) mass response over an initial 0-300 s time-course. 3. The enhancing effects of 2R,4R-APDC on DHPG-stimulated [3H]-InsPx accumulation were observed in both the presence and nominal absence of extracellular Ca2+, and irrespective of whether 2R,4R-APDC was added before, simultaneous with, or subsequent to DHPG. Furthermore, increasing the tissue cyclic AMP concentration up to 100 fold had no effect on DHPG-stimulated Ins(l,4,5)P3 accumulation in the absence or presence of 2R,4R-APDC. 4. 2R,4R-APDC and (2S, 1'R, 2'R, 3'R)-2-(2,3-dicarboxylcyclopropyl)glycine (DCG-IV), the latter agent in the presence of MK-801 to prevent activation of NMDA-receptors, each inhibited forskolin-stimulated cyclic AMP accumulation by about 50%, with respective EC50 values of 1.3 and 0.04 microM (-log EC 50 (M): 2R,4R-APDC, 5.87+/-0.09; DCG-IV, 7.38+/-0.05). In the presence of DHPG (30 microM), 2R,4R-APDC and DCG-IV also concentration-dependently increased [3H]-InsPx accumulation with respective EC50 values of 4.7 and 0.28 microM (-log EC50 (M): 2R,4R-APDC, 5.33+/-0.04; DCG-IV, 6.55+/-0.09) which were 3-7 fold rightward-shifted relative to the adenylyl cyclase inhibitory responses. 5. The group II-selective mGlu receptor antagonist LY307452 (30 microM) caused parallel rightward shifts in the concentration-effect curves for inhibition of forskolin-stimulated adenylyl cyclase, and enhancement of DHPG-stimulated [3H]-InsPx accumulation, by 2R,4R-APDC yielding similar equilibrium dissociation constants (KdS, 3.7+/-1.1 and 4.1+/-0.4 microM respectively) for each response. 6. The ability of 2R,4R-APDC to enhance receptor-mediated [3H]-InsPx accumulation appeared to be agonist-specific; thus although DHPG (100 microM) and the muscarinic cholinoceptor agonist carbachol (10 microM) stimulated similar [3H]-InsPx accumulations, only the response to the former agonist was enhanced by co-activation of group II mGlu receptors. 7. These data demonstrate that second messenger-generating phosphoinositide responses stimulated by group I mGlu receptors are positively modulated by co-activation of group II mGlu receptors in cerebral cortex and hippocampus. The data presented here are discussed with respect to the possible mechanisms which might mediate the modulatory activity, and the physiological and pathophysiological significance of such crosstalk between mGlu receptors.     

Excitation of rat subthalamic nucleus neurones in vitro by activation of a group I metabotropic glutamate receptor

The subthalamic nucleus (SThN) provides a glutamate mediated excitatory drive to several other component nuclei of the basal ganglia, thereby significantly influencing locomotion and control of voluntary movement. We have characterised functionally the metabotropic glutamate (mGlu) receptors in the SThN using extracellular single unit recording from rat midbrain slices. SThN neurones fired action potentials spontaneously at a rate of 10 Hz which was increased by the group I/II mGlu receptor agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate (1S,3 R-ACPD; 1-30 microM) and the group I selective agonist (S, R)-dihydroxyphenylglycine (DHPG; 1-30 microM). However, both the group II selective agonist (1S,1'R,2'R,3'R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV; 1 microM) and the group III selective agonist (S)-2-amino-4-phosphonobutanoic acid (L-AP4; 10 microM) were without effect, indicating that the excitation was mediated by a group I mGlu receptor. The excitation caused by DHPG (3 microM) was reversed by co-application of the mGlu receptor antagonist (+)-alpha-methyl-4-carboxyphenylglycine (MCPG; 500 microM). Thus a group I mGlu receptor mediates excitation of SThN neurones, and suggests a use for group I mGlu receptor ligands for treatment of both hypo- and hyperkinetic disorders of basal ganglia origin, such as Parkinson's disease and Huntington's disease.     

Active immunization using dendritic cells mixed with tumor cells inhibits the growth of primary breast cancer

The potential role of alpha2-adrenoceptors in modulating the activity of adenylyl cyclase in the rat striatum was examined. The selective alpha2-adrenoceptor agonist, UK14,304, produced a concentration-dependent inhibition of forskolin-stimulated accumulation of cAMP in striatal slices. The effect of UK14,304 was reversed by pre-incubation of striatal slices with the selective alpha2-adrenoceptor antagonist, RX821002. To determine whether alpha2C-adrenoceptors contribute to the alpha2-adrenoceptor-induced inhibition of forskolin-stimulated cAMP accumulation, an antisense oligodeoxynucleotide directed against alpha2C-adrenoceptor mRNA (alpha(2C)AS) or a random sequence (RS) was infused directly into the striatum. The ability of alpha(2C)AS to reduce the expression of alpha2C-adrenoceptors has been previously demonstrated. Alpha2C(AS) infusions did not reduce the ability of UK14,304 to inhibit forskolin-stimulated cAMP accumulation. Instead, alpha(2C)AS significantly enhanced forskolin-stimulated cAMP accumulation on the infusion side compared to the contralateral striatum. In contrast to the effects of alpha(2C)AS, infusions of RS had no effects on forskolin-stimulated cAMP accumulation or on the ability of UK14,304 to inhibit this effect. Incubation of striatal slices from untreated rats with RX821002 could mimic the ability of alpha(2C)AS infusion to enhance forskolin-stimulated cAMP accumulation, and did so in a concentration-dependent manner. Alpha2-adrenoceptors are negatively coupled to adenylyl cyclase in the rat striatum and alpha2C-adrenoceptors appear to be under tonic activation by an endogenous ligand in striatal slices.     

Modulation of excitatory synaptic transmission in locus coeruleus by multiple presynaptic metabotropic glutamate receptors

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.     

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.     

The group I mGlu receptor agonist DHPG induces a novel form of LTD in the CA1 region of the hippocampus

The group I specific metabotropic glutamate (mGlu) receptor agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) (100 microM, 10 min) induced long-term depression (LTD) of synaptic transmission in the CA1 region of adult rat hippocampal slices, measured using a grease-gap recording technique. In "normal" (1 mM Mg2+-containing) medium, LTD (measured 30 min after washout of DHPG) was small (13+/-3%), but LTD was enhanced if DHPG was applied when the tissue was made hyperexcitable, either by omitting Mg2+ from the perfusate (35+/-3%) or by adding the GABA(A) receptor antagonist picrotoxin (29+/-2%). The N-methyl-D-aspartate (NMDA) receptor antagonist AP5 (100 microM) substantially reduced the generation of DHPG-induced LTD in Mg2+-free medium, but had little effect on LTD induced in the presence of picrotoxin. In Mg2+-free medium, the threshold concentration of DHPG required to induce LTD was between 1 and 3 microM. Neither agonists specific for group II (100 nM DCG-IV or 1 microM LY354740) or group III (10 microM L-AP4) mGlu receptors or a combined group I and II agonist (30-100 microM (1S,3R)-ACPD) induced LTD. However, an agonist (1 mM CHPG) which activates mGlu5 but not mGlu1 receptors did induce LTD. Surprisingly, DHPG-induced LTD was reversed by mGlu receptor antagonists, applied hours after washout of DHPG. DHPG-induced LTD did not occlude with LTD induced by synaptic activation (1200 stimuli delivered at 2 Hz), in Mg2+-free medium. These data show that activation of group I mGlu receptors (probably mGlu5) can induce LTD and that this mGlu receptor-mediated LTD may, or may not, require activation of NMDA receptors, depending on the experimental conditions.     

Involvement of a cyclic-AMP pathway in group I metabotropic glutamate receptor responses in neonatal rat cortex

3,5-Dihydroxyphenylglycine (DHPG), (S)-3-hydroxyphenylglycine and (S)-4-carboxy-3-hydroxyphenylglycine (S-4C3HPG) stimulated phosphoinositide hydrolysis in neonatal rat cortical slices, but with lower maximal effect, in comparison with 2S,1'S,2'S-2-(2'-carboxycyclopropyl)glycine (L-CCG I) or (1S,3R)-1-aminocyclo-pentane-1,3-dicarboxylic acid (1S,3R-ACPD). DHPG, 1S,3R-ACPD, and S-4C3HPG also evoked a rapidly desensitizing increase in [Ca2+]i in cortical layers of neonatal brain slices. (R,S)-alpha-methyl-4-tetrazolyl-phenylglycine (MTPG), and (R,S)-alpha-methyl-4-phosphono-phenylglycine (MPPG) inhibited the increase of phosphoinositide hydrolysis elicited by 1S,3R-ACPD but not that by R,S-DHPG. In contrast, the selective group II receptor agonist (1S,2S,5R,6S)-2-amino-bicyclo-[3.1.0]-hexane-2,6-dicarboxylate (LY 354740) potentiated the response of R,S-DHPG. Finally, 8-(4-chlorophenylthio)-cAMP, a membrane permeant analogue of cAMP, reversed the stimulatory effect of 1S,3R-ACPD and S-4C3HPG on phosphoinositide hydrolysis and [Ca2+]i mobilization, without affecting the response induced by R,S-DHPG. These data suggest that, in neonatal rat cortex, the activation of group II metabotropic glutamate receptors potentiates the phosphoinositide hydrolysis and [Ca2+]i responses mediated by group I metabotropic glutamate receptors.     

Characterization of (2S,2'R,3'R)-2-(2',3'-[3H]-dicarboxycyclopropyl)glycine binding in rat brain

[(2S,2'R,3'R)-2-(2',3'-[3H]Dicarboxycyclopropyl)glycine ([3H]DCG IV) binding was characterized in vitro in rat brain cortex homogenates and rat brain sections. In cortex homogenates, the binding was saturable and the saturation isotherm indicated the presence of a single binding site with a K(D) value of 180 +/- 33 nM and a Bmax of 780 +/- 70 fmol/mg of protein. The nonspecific binding, measured using 100 microM LY354740, was <30%. NMDA, AMPA, kainate, L(-)-threo-3-hydroxyaspartic acid, and (S)-3,5-dihydroxyphenylglycine were all inactive in [3H]DCG IV binding up to 1 mM. However, several compounds inhibited [3H]DCG IV binding in a concentration-dependent manner with the following rank order of potency: LY341495 = LY354740 > DCG IV = (2S,1'S,2'S)-2-(2-carboxycyclopropyl)glycine > (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid > (2S,1'S,2'S)-2-methyl-2-(2-carboxycyclopropyl)glycine > L-glutamate = ibotenate > quisqualate > (RS)-alpha-methyl-4-phosphonophenylglycine = L(+)-2-amino-3-phosphonopropionic acid > (S)-alpha-methyl-4-carboxyphenylglycine > (2S)-alpha-ethylglutamic acid > L(+)-2-amino-4-phosphonobutyric acid. N-Acetyl-L-aspartyl-L-glutamic acid inhibited the binding in a biphasic manner with an IC50 of 0.2 microM for the high-affinity component. The binding was also affected by GTPgammaS, reducing agents, and CdCl2. In parasagittal sections of rat brain, a high density of specific binding was observed in the accessory olfactory bulb, cortical regions (layers 1, 3, and 4 > 2, 5, and 6), caudate putamen, molecular layers of the hippocampus and dentate gyrus, subiculum, presubiculum, retrosplenial cortex, anteroventral thalamic nuclei, and cerebellar granular layer, reflecting its preferential (perhaps not exclusive) affinity for pre- and postsynaptic metabotropic glutamate mGlu2 receptors. Thus, the pharmacology, tissue distribution, and sensitivity to GTPgammaS show that [3H]DCG IV binding is probably to group II metabotropic glutamate receptors in rat brain.     

The potent mGlu receptor antagonist LY341495 identifies roles for both cloned and novel mGlu receptors in hippocampal synaptic plasticity

Understanding the roles of metabotropic glutamate (mGlu) receptors has been severely hampered by the lack of potent antagonists. LY341495 (2S-2-amino-2-(1S,2S-2-carboxycyclopropyl-1-yl)-3-(xanth-9-y l)propanoic acid) has been shown to block group II mGlu receptors in low nanomolar concentrations (Kingston, A.E., Ornstein, P.L., Wright, R.A., Johnson, B.G., Mayne, N.G., Burnett, J.P., Belagaje, R., Wu, S., Schoepp, D.D., 1998. LY341495 is a nanomolar potent and selective antagonist at group II metabotropic glutamate receptors. Neuropharmacology 37, 1-12) but can be used in higher concentrations to block all hippocampal mGlu receptors, identified so far by molecular cloning (mGlu1-5,7,8). Here we have further characterised the mGlu receptor antagonist activity of LY341495 and have used this compound to investigate roles of mGlu receptors in hippocampal long-term potentiation (LTP) and long-term depression (LTD). LY341495 competitively antagonised DHPG-stimulated PI hydrolysis in AV12-664 cells expressing either human mGlu1 or mGlu5 receptors with Ki-values of 7.0 and 7.6 microM, respectively. When tested against 10 microM L-glutamate-stimulated Ca2+ mobilisation in rat mGlu5 expressing CHO cells, it produced substantial or complete block at a concentration of 100 microM. In rat hippocampal slices, LY341495 eliminated 30 microM DHPG-stimulated PI hydrolysis and 100 microM (1S,3R)-ACPD-inhibition of forskolin-stimulated cAMP formation at concentrations of 100 and 0.03 microM, respectively. In area CA1, it antagonised DHPG-mediated potentiation of NMDA-induced depolarisations and DHPG-induced long-lasting depression of AMPA receptor-mediated synaptic transmission. LY341495 also blocked NMDA receptor-independent depotentiation and setting of a molecular switch involved in the induction of LTP; effects which have previously been shown to be blocked by the mGlu receptor antagonist (S)-MCPG. These effects may therefore be due to activation of cloned mGlu receptors. In contrast, LY341495 did not affect NMDA receptor-dependent homosynaptic LTD; an effect which may therefore be independent of cloned mGlu receptors. Finally, LY341495 failed to antagonise NMDA receptor-dependent LTP and, in area CA3, NMDA receptor-independent, mossy fibre LTP. Since in the same inputs these forms of LTP were blocked by (S)-MCPG, a novel type of mGlu receptor may be involved in their induction.     

Homology-based gene prediction using neural nets

1 In this study we have determined the pharmacological profile of (S)-quisqualic acid, (S)-2-amino-4-phosphonobutyric acid ((S)-AP4) and their higher homologues (S)-homoquisqualic acid, (S)-2-amino-5-phosphonopentanoic acid ((S)-AP5), respectively, and (R)-AP5 at subtypes of metabotropic (S)-glutamic acid (mGlu) receptors expressed in Chinese hamster ovary cells. 2 (S)-Quisqualic acid was a potent mGlu1/mGlu5 agonist (EC50 values of 1.1 microM and 0.055 microM, respectively) showing no activity at mGlu2 and weak agonism at mGlu4 (EC50 approximately 1000 microM). 3 (S)-Homoquisqualic acid displayed competitive antagonism at mGlu1 (KB = 184 microM) and full agonism at mGlu5 (EC50 = 36 microM) and mGlu2 (EC50 = 23 microM), but was inactive at mGlu4. 4 (S)-AP4 was a potent and selective mGlu4 agonist (EC50 = 0.91 microM) being inactive at mGlu1, mGlu2 and mGlu5 both as agonist and antagonist. 5 (S)-AP5 displayed very weak agonist activity at mGlu4. At the mGlu2 receptor subtype (S)-AP5 acted as a competitive antagonist (KB = 205 microM), whereas the compound was inactive at mGlu, and mGlu5. (R)-AP5 was inactive at all mGlu receptor subtypes tested both as agonist and antagonist. 6 These studies demonstrate that incorporation of an additional carbon atom into the backbone of (S)-glutamic acid and its analogues, to give the corresponding homologues, and replacement of the terminal carboxyl groups by isosteric acidic groups have profound effects on the pharmacological profiles at mGlu receptor subtypes. Furthermore, (S)-homoquisqualic acid has been shown to be a potentially useful tool for differentiating mGlu1 and mGlu5.     

Metabotropic glutamate receptor antagonists, like GABA(B) antagonists, potentiate dorsal root-evoked excitatory synaptic transmission at neonatal rat spinal motoneurons in vitro

Recordings of whole-cell synaptic current responses elicited by electrical stimulation of dorsal roots were made from motoneurons, identified by antidromic invasion, in isolated spinal cord preparations from five- to eight-day-old Wistar rats. Supramaximal electrical stimulation of the dorsal root evoked complex excitatory postsynaptic currents with mean latencies (+/- S.E.M.) of 6.1 +/- 0.26 ms, peak amplitude of -650 +/- 47 pA and duration of 4.30 +/- 0.46 s (n=34). All phases of excitatory postsynaptic currents were potentiated to approximately 20% above control levels in the presence of the metabotropic glutamate receptor antagonists S-2-amino-2-methyl-4-phosphonobutanoate (MAP4; 200 microM; n=15) and 2S, 1'S,2'S-2-methyl-2-(carboxycyclopropyl)glycine (MCCG; 200 microM; n=9). A similar level of potentiation was produced by the GABA(B) receptor antagonist 3-N[1-(S)-(3,4-dichlorophenyl)ethyl]amino-2-(S)-hydroxypropyl-P-benzyl-p hosphinic acid (CGP55845; 200 nM; n=5). MAP4 (200 microM) produced a six-fold rightward shift in the concentration-effect plot for the depressant action of the metabotropic glutamate receptor agonist S-2-amino-4-phosphonobutanoate (L-AP4), whereas CGP55845 produced no significant change in the potency of L-AP4. MAP4 did not antagonize the depressant actions of baclofen (n=8), 1S,3S-1-aminocyclopentane-1,3-dicarboxylate (n=4) or 2-S,1'S,2'S-2-(carboxycyclopropyl)glycine (n=4). The metabotropic glutamate receptor antagonists produced no change in the holding current of any of the neurons, indicating that they had no significant postsynaptic excitatory actions. These results are the first to indicate a possible physiological role for metabotropic glutamate receptors in the spinal cord. Like GABA(B) receptors, they control glutamatergic synaptic transmission in the segmental spinal pathway to motoneurons. This is likely to be a presynaptic control mechanism.     

Heteroaryl analogues of AMPA. 2. Synthesis, absolute stereochemistry, photochemistry, and structure-activity relationships

We have previously shown that (S)-2-amino-3-(3-hydroxy-5-phenyl-4-isoxazolyl)propionic acid [(S)-APPA, 2] is a weak agonist at (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptors, specifically activated by (S)-AMPA (1), whereas (S)-2-amino-3-[3-hydroxy-5-(2-pyridyl)-4-isoxazolyl]propionic acid [(S)-2-Py-AMPA, 5] and (RS)-2-amino-3-[3-hydroxy-5-(2-thiazolyl)-4-isoxazolyl]propionic acid (4) are potent AMPA agonists. On the other hand, (R)-APPA (3) and (R)-2-Py-AMPA (6) have been shown to be weak AMPA antagonists. We now report the synthesis of 2-Py-AMPA (7a) and the isomeric compounds 3-Py-AMPA (7b) and 4-Py-AMPA (7c) as well as the 7a analogues, (RS)-2-amino-3-[3-hydroxy-5-(6-methyl-2-pyridyl)-4-isoxazolyl]p ropion ic acid (7d) and (RS)-2-amino-3-[3-hydroxy-5-(2-quinolinyl)-4-isoxazolyl]propionic acid (7e). Furthermore, (RS)-2-amino-3-[3-hydroxy-5-(2-furyl)-4-isoxazolyl]propionic acid (2-Fu-AMPA, 7f) and its 5-bromo-2-furyl derivative (7g) were synthesized, and (S)-2-Fu-AMPA (8) and (R)-2-Fu-AMPA (9) were prepared by semipreparative chiral HPLC resolution of 7f. HPLC analyses and circular dichroism spectroscopy indicated the absolute stereochemistry of 8 and 9 to be S and R, respectively. This was confirmed by an X-ray crystallographic analysis of 9.HCl. In receptor binding (IC50 values) and rat cortical wedge electrophysiological (EC50 values) studies, 7c (IC50 = 5.5 +/- 0.6 microM; EC50 = 96 +/- 5 microM) was shown to be markedly weaker than 7a (IC50 = 0.57 +/- 0.16 microM; EC50 = 7.4 +/- 0.2 microM) as an AMPA agonist, whereas 7b,d,e were inactive. The very potent AMPA agonist effect of 7f (IC50 = 0.15 +/- 0.03 microM; EC50 = 1.7 +/- 0. 2 microM) was shown to reside exclusively in 8 (IC50 = 0.11 +/- 0.01 microM; EC50 = 0.71 +/- 0.11 microM), whereas 9 did not interact significantly with AMPA receptors, either as an agonist or as an antagonist. 8 was shown to be photochemically active and is a potential photoaffinity label for the recognition site of the AMPA receptors. Compound 7g turned out to be a very weak AMPA receptor agonist (IC50 = 12 +/- 0.7 microM; EC50 = 160 +/- 15 microM). None of these new compounds showed detectable effects at N-methyl-d-aspartic acid (NMDA) or kainic acid receptors in vitro. The present studies have emphasized that the presence of a heteroatom in the 2-position of the heteroaryl 5-substituent greatly facilitates AMPA receptor agonist activity.     

Grand rounds: sports medicine

Experiments were undertaken to examine the influence of corticosterone on forskolin-stimulated cyclic AMP accumulation in rat cerebral cortical slices. Incubation in vitro of cerebral cortical slices with increasing concentrations of corticosterone (1 nM-100 microM) did not influence basal cyclic AMP response. Despite the lack of effect when used alone, corticosterone attenuated the effect of forskolin (10 microM) on cyclic AMP accumulation with IC50 = 24.1 +/- 5.1 microM. Corticosterone (10 microM) added to the incubation medium, for 10 min, reduced the cyclic AMP accumulation in response to increasing concentrations of forskolin (1 microM-100 microM), the concentration-response curve was shifted to the right by about one order of magnitude. In order to compare the in vitro effect of corticosterone with its effects in vivo in the next experiment the forskolin-induced cyclic AMP accumulation was measured in cerebral cortical slices from rats which were treated with corticosterone or vehicle. Similarly as in in vitro model, single dose of corticosterone (10 mg/kg sc) given to rats 2 h before sacrifice inhibited forskolin-stimulated cyclic AMP accumulation when compared with vehicle-treated control animals. In contrast, prolonged administration of corticosterone (10 mg/kg sc, twice daily for 4 and 7 days) increased forskolin-stimulated cyclic AMP accumulation in rat cerebral cortical slices when measured 2, 24 and 48 h after the administration of the last dose. These findings suggest that glucocorticoids exert multiple actions on the adenylate cyclase-coupled cyclic AMP generating system in the brain, with the ultimate effect being dependent upon amount and duration of exposure to these hormones.     

Molecular pharmacology of homologues of ibotenic acid at cloned metabotropic glutamic acid receptors

We have studied the effects of the enantiomers of 2-amino-3-(3-hydroxyisoxazol-5-yl)propionic acid (homoibotenic acid, HIBO) and analogues substituted with a methyl, bromo or butyl group in the four position of the ring at cloned metabotropic glutamate (mGlu) receptors expressed in Chinese hamster ovary (CHO) cells. In contrast to the parent compound ibotenic acid, which is a potent group I and II agonist, the (S)-forms of homoibotenic acid and its analogues are selective and potent group I antagonists whereas the (R)-forms are inactive both as agonists and antagonists at group I, II, and III mGlu receptors. Interestingly, (S)-homoibotenic acid and the analogues display equal potency at both mGlu1alpha and mGlu5a with Ki values in the range of 97 to 490 microM, (S)-homoibotenic acid and (S)-2-amino-3-(4-butyl-3-hydroxyisoxazol-5-yl)propionic acid [(S)-4-butylhomoibotenic acid] displaying the lowest and highest potency, respectively. The homoibotenic acid analogues thereby differ from mGlu receptor antagonists derived from phenylglycine such as (S)-4-carboxyphenylglycine which only antagonizes mGlu1alpha (Ki = 18 microM) showing no effect at mGlu5a (Ki > 300 microM).     

Molecular diagnosis and monitoring of acute myeloid leukemia

Using an in vitro static incubation system of adult male rat hypothalami, we have studied the effect of melatonin on the release of gonadotropin-releasing hormone (GnRH) and cyclic adenosine monophosphate (cAMP). Mediobasal hypothalamus (MBH) and preoptic area (POA) were incubated separately in Minimum Essential Medium (MEM) for 6 h. The release of GnRH was measured by radioimmuno-assay in the incubation medium sampled every 7.5 min. In the MBH and POA incubation medium, the mean amount of GnRH released was 8.9 +/- 1.1 and 3.4 +/- 0.6 pg GnRH/7.5 min, respectively (P < 0.01). The mean number of GnRH pulses under basal conditions was 2 +/- 0.3 per 2 h in the MBH and 1.6 +/- 0.3 per 2 h in the POA (P > 0.05). Melatonin (10(-8) M) did not alter the release of GnRH in the presence or absence of forskolin (10(-4) M). Melatonin, which was without effect on basal cAMP, inhibited forskolin-stimulated cAMP accumulation in the medium by 50% in the MBH and 40% in the POA. These results suggest that in our incubation system, melatonin does not modify GnRH release, but probably acts through the melatonin binding sites located in the hypothalamus to inhibit forskolin-stimulated cAMP.