Glycine, glycinamide and D-serine act as positive modulators of signal transduction at the N-methyl-D-aspartate (NMDA) receptor in vivo: differential effects on mouse cerebellar cyclic guanosine monophosphate levels

Direct intracerebellar (icb) administration of glycine, glycinamide and D-serine produced time- and dose-dependent changes in mouse cerebellar cGMP levels, indicating a modulation of ongoing neuronal activity through the NMDA receptor complex. Intracerebroventricular administration of glycinamide also produced a time-dependent change in cGMP levels, indicating a central mechanism of action. The icb dose-response data indicated a unimolecular interaction for these compounds. D-serine-, glycine-, and glycinamide-mediated increases in cGMP levels were reversed by the competitive NMDA antagonist, CPP and the NMDA-associated glycine receptor antagonist, HA-966, indicating mediation via the NMDA receptor complex. Glycine and D-serine were less effective than glycinamide at increasing cerebellar cGMP levels. In contrast, L- and D-serinamide did not affect cGMP levels. These results indicate that glycine receptor is not saturated under physiological conditions and also suggest possible existence of multiple glycine pools.     

N-methyl-D-aspartate exposure blocks glutamate toxicity in cultured cerebellar granule cells.

Exposure of cultured cerebellar granule cells to glutamate results in a concentration-dependent (EC50 = 22.7 +/- 0.4 microM) and delayed (24-72 hr) neurotoxicity, which is blocked by the specific N-methyl-D-aspartate (NMDA) receptor antagonists 2-amino-5-phosphovalerate and MK-801 but is unaffected by the non-NMDA receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione and 6,7-dinitroquinoxaline-2,3-dione. Although glutamate toxicity in these cells is mediated by the NMDA subtype of glutamate receptor, pretreatment of cerebellar granule cells with subtoxic concentrations of NMDA markedly antagonizes the neurotoxic actions of glutamate, with an IC50 of 55 +/- 4 microM. The neuroprotective effect of NMDA requires a preincubation time of approximately 120 min to be fully manifested and does not require the presence of NMDA during glutamate exposure. These data demonstrate that NMDA receptors mediate both neurotoxicity and neuroprotection in cerebellar granule cells. Among four glutamate receptor agonists tested (NMDA, quisqualate, ibotenate, and kainate), only NMDA was able to provide a robust neuroprotection against glutamate toxicity. Quisqualate was neither neurotoxic nor neuroprotective, whereas ibotenate, which was nontoxic by itself, induced a small degree of neuroprotection. In contrast, kainate, which was neurotoxic to cerebellar granule cells, also provided considerable neuroprotection against glutamate toxicity. Because preincubation of cerebellar granule cells with NMDA fails to alter NMDA receptor-mediated phosphoinositide hydrolysis or the specific binding of [3H]MK-801 to NMDA receptors, it appears that the neuroprotective effects of NMDA are not due to NMDA receptor desensitization.     

Glycine and D-serine increase the affinity of N-methyl-D-aspartate sensitive glutamate binding sites in rat brain synaptic membranes

In previously frozen and extensively washed brain membranes [3H]glutamate binds to a single population of sites characteristic of the NMDA-sensitive glutamate receptor subtype. This binding cannot be displaced by glycine and D-serine, but actually is enhanced by these amino acids in a dose-dependent manner. Glycine and D-serine increase the affinity of glutamate binding without changing the density of binding sites. These results delineate glycine as an allosteric modulator of the recognition site for the NMDA-sensitive glutamate receptor.     

Pertussis toxin inhibits signal transduction at a specific metabolotropic glutamate receptor in primary cultures of cerebellar granule cells

In primary cultures of cerebellar granule cells, glutamate receptors have been classified into metabolotropic (GP1 and GP2) and ionotropic (GC1 and GC2). The GP1 and GC1 receptors are negatively modulated by magnesium and noncompetitively inhibited by phencyclidine; GP2 and GC2 receptors are insensitive to inhibition by magnesium and phencyclidine (Costa, Fadda, Kozikowski, Nicoletti and Wroblewski, 1988). Exposure of cultured cerebellar granule cells to pertussis toxin (PTX, 1 microgram/ml for 14-16 hr) reduced the stimulation of the hydrolysis of inositol phospholipids (PI) by the GP2 receptor agonists, glutamate and quisqualate in the presence of magnesium, but did not inhibit the stimulation of the hydrolysis of PI by GP1 receptor agonists. The stimulation of the hydrolysis of PI by the muscarinic cholinergic receptor agonist, carbamylcholine, remained unchanged after pretreatment with pertussis toxin. In membranes prepared from cerebellar granule cells in primary culture, the addition of guanosine 5'-0-(3-thiotriphosphate) (GTP-gamma-s), a nonhydrolyzable analogue of GTP, enhanced the hydrolysis of PI and reduced the Bmax of quisqualate-displaceable binding of [3H]glutamate. These results indicate that, in primary cultures of cerebellar granule cells, a specific class of metabolotropic glutamate receptors (the GP2 receptor) is coupled with the hydrolysis of PI through a pertussis toxin-sensitive GTP-binding protein.     

Membrane depolarization and the expression of glutamate receptors in cerebellar granule cells.

The functional expression of glutamate receptors in cerebellar granule cells in culture was examined using the whole-cell patch clamp technique. All the cells tested responded with an inward current to superfusion with glutamate, kainate, or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate. Neurones cultured in a medium containing an elevated K+ concentration (25 mM) responded to N-methyl-D-aspartate (NMDA) provided they were simultaneously exposed to glycine (Gly). In contrast, NMDA/Gly-evoked currents were virtually undetectable in cells grown in medium containing 'low' K+ (10 mM), suggesting that only the NMDA-preferring class of glutamate receptors are subject to up-regulation by K(+)-induced chronic membrane depolarization.     

Appearance of highly sensitive cells to L-type Ca antagonist in cultured cerebellar granule cells.

We studied intracellular Ca2+ ([Ca2+]i) kinetics within single cells using and image-analyzing system. We prepared a culture dish with a bottom made of quartz (non-fluorescent glass) for reducing the background fluorescence during [Ca2+]i measurement. Furthermore, we changed the concentration of the poly-L-lysine solution which had been used to make cells adhere to the dish bottom from the conventional 5 micrograms/ml to 15 micrograms/ml. This modification improved cell viability, prevented colony formation, and therefore, provided favorable cell samples for detection within single cells. Then, we investigated 25 mM KCl-evoked changes in the [Ca2+]i level of rat cerebellar granule cells. We found that sensitivity to KCl was high in some cells but low in others. In a study using the highly sensitive cells, nicardipine, nimodipine and nifedipine were active even at nanomolar or picomolar levels, although their affinities to the L-type Ca ion channel have been reported to act only at micromolar levels.     

Characterization of metabotropic glutamate receptor-stimulated phosphoinositide hydrolysis in rat cultured cerebellar granule cells.

1. The pharmacology of excitatory amino acid (EAA)-stimulated phosphoinositide (PI) hydrolysis, monitored via [3H]-inositol monophosphate accumulation, was investigated in primary cultures of rat cerebellar granule cells. 2. EAA-stimulated PI hydrolysis peaked after 4-5 days in vitro and subsequently declined. 3. The agonist order of potency was found to be (EC50): L-quisqualic acid (Quis) (2 microM) >> L-glutamate (50 microM) > (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid ((1S,3R)-ACPD) (102 microM). L-Glutamate (Emax = 873% of basal activity) elicited the largest stimulation of PI hydrolysis, whereas Quis (Emax = 603%) and (1S,3R)-ACPD (Emax = 306%) produced somewhat lower stimulations. 4. Several phenylglycine derivatives were found to be active in inhibiting 2 microM Quis-stimulated PI hydrolysis, in order of potency (IC50): (S)-4-carboxy-3-hydroxyphenylglycine (41 microM) > or = (S)-4-carboxyphenylglycine (51 microM) >> (+)-alpha-methyl-4-carboxyphenylglycine (243 microM). 5. Cultured cerebellar granule cells of the rat appear to have Group I mGluR pharmacology similar to that reported for cloned mGluR1 and provide an ideal system for investigating novel mGluR1 ligands in a native environment.     

Recent advances in positive allosteric modulators of metabotropic glutamate receptors

Positive allosteric modulators of metabotropic glutamate receptors (mGluRs) are the subject of intensive research due to their emerging therapeutic potential for a range of psychiatric and neurological disorders such as pain, anxiety, cognition, Parkinson's disease and schizophrenia. Positive allosteric modulators, which are small molecules capable of enhancing agonist-mediated receptor activity while possessing no intrinsic agonist activity, have recently been described for group I (mGluR1 and mGluR5), group II (mGluR2) and group III (mGluR4) mGluRs. Relative to classical mGluR agonists, these molecules offer improved selectivity versus other mGluRs and chemical tractability, and may reduce the liability of receptor desensitization.     

Modulation of calcium uptake and D-aspartate release by GABAB receptors in cultured cerebellar granule cells

(-)Baclofen, a GABAB receptor agonist, and GABA attenuated by 60% the high K+-evoked 45Ca2+ uptake into cultured cerebellar granule cells with an EC50 of 110 +/- 18 nM and 2.4 +/- 0.2 microM, respectively. The attenuation by baclofen of 45Ca2+ uptake was stereospecific and the effect of GABA was unaffected by bicuculline. Moreover, muscimol, a GABAA receptor agonist did not affect the K+-evoked 45Ca2+ uptake. (-)Baclofen and GABA also decreased the K+-evoked and calcium-dependent release of preloaded [3H]D-aspartate from granule cells; however, their potency and efficacy appeared to be less than those for inhibiting the 45Ca2+ uptake. (+)Baclofen and muscimol failed to change this K+-evoked release. The release of [3H]D-aspartate induced by the calcium ionophore A23187 was unaffected by (-)-baclofen. The K+-evoked release of [3H]D-aspartate was effectively inhibited by nimodipine, a voltage sensitive calcium channel blocker. The results suggest that GABAB receptor in cultured cerebellar granule cells plays a crucial role in modulating the uptake of calcium and release of the excitatory transmitter. Moreover, these two effects mediated by GABAB receptor activation may be casually related.     

Activation of N-methyl-D-aspartate-sensitive glutamate receptors stimulates arachidonic acid release in primary cultures of cerebellar granule cells

In cultured granule cells prelabeled with [3H]arachidonate the activation of excitatory amino acid receptors by various agonists results in a dose-dependent stimulation of [3H]arachidonic acid release. Glutamate and aspartate were the most potent agonists, whereas N-methyl-D-aspartate, kainate and quisqualate were less potent. Other neurotransmitter receptor agonists--GABA, baclofen and norepinephrine--were inactive, while carbachol induced only a slight effect. Since the transmitter-mediated release of [3H]arachidonate was blocked by phencyclidine, a selective inhibitor of NMDA-sensitive glutamate receptors, it can be inferred that the effects of all other receptor agonists were indirectly mediated via the release of glutamate from granule cells. Aspartate-evoked release was Ca2+-dependent and was abolished by the glutamate receptor inhibitors: Mg2+ ions and 2-amino-5-phosphonovalerate. The inhibitors of phospholipase A2, quinacrine and p-bromophenacyl bromide, decreased the release of [3H]arachidonate in a dose-related manner.     

Serotonergic, adrenergic and histaminergic receptors coupled to phospholipase C in cultured cerebellar granule cells of rats

Phosphatidylinositol (Ptdlns) turnover has been studied in the primary culture of granule cells dissociated from the cerebellum of postnatal rat. Addition of serotonin (5-hydroxytryptamine, 5-HT) caused an increase (300-600% of control) of [3H]inositol monophosphate (IP1) accumulation in the presence of LiCl in cells prelabeled with [3H]myo-inositol. The EC50 and saturation concentrations of 5-HT were about 0.1 and 10 microM respectively. Some nonselective 5-HT receptor agonists, MK212, 5-methoxytryptamine, tryptamine and quipazine, were capable of stimulating IP1 accumulation; a selective 5-HT1A receptor agonist, 8-OH-DPAT, was ineffective. The 5-HT-induced response was potently blocked by several 5-HT2 receptor antagonists such as ketanserin, mianserin, spiroperidol and pyzotyline. The 5-HT-induced accumulation was dependent on the culturing time of granule cells with the maximal response seen in an 8-day culture. Norepinephrine (NE) also promoted an increased IP1 accumulation (300% of the control) with an EC50 of about 1 microM, while prazosin inhibited this NE-induced response with a Ki of about 0.2 nM. The responses induced by NE and 5-HT appeared to be additive. In addition, histamine in a dose-dependent manner enhanced the accumulation by about 100%; this histamine effect was blocked by triprolidine, an H1 receptor antagonist, but not by cimetidine, an H2 receptor antagonist. These results suggest that 5-HT, NE and histamine could be part of the neurotransmitter substances present in mossy fibers or other afferent nerve endings which innervate granule cells in vivo and that Ptdlns turnover regulated by their selective receptors on granule cells may play a role in modulating the excitatory function of these neurons.     

Gamma-aminobutyric acid and benzodiazepine receptors in cultured cerebellar granule cells: effects of taurine and its lipophilic derivatives

The effects of taurine and some lipophilic derivatives of taurine on binding to GABA and benzodiazepine receptors were studied in intact cerebellar granule cells. The phenylsuccinylimido derivatives of taurine appeared to increase the binding of muscimol in micromolar concentrations, while taurine decreased it slightly. Only minor changes were seen in the basal binding of flunitrazepam, whereas stimulation of the binding by GABA was strongly reduced by piperidino, benzamido and phenyl-succinylimidotaurine with taurine itself again showing only a weak effect. Diphenylhydantoin, which bears structural resemblance to the phenylsuccinylimido group, had a strong effect on the stimulated binding of flunitrazepam and it also slightly reduced the basal level of binding. Thus, it seems possible that the effects of the phenylsuccinylimido derivatives of taurine on the binding of flunitrazepam were due to this chemical structure and not to the taurine-like core of the molecules. The phthalimido derivative of taurine, taltrimide, which has been tested in clinical trials with epileptic patients, did not show any activity in the binding studies.     

Effect of metabotropic glutamate receptor agonists and signal transduction modulators on feeding by a caterpillar

Feeding in codling moth caterpillars was induced by the general glutamate receptor activator monosodium glutamate (MSG) and by three different mGluR agonists known to specifically stimulate different classes of vertebrate metabotropic glutamate receptors, including: (1S,3R)-ACPD, which stimulates group I mGluRs (2R,4R)-APDC, which stimulates group II mGluRs and L-AP4, which stimulates some group III mGluRs. Experiments exposing larvae to combinations of specific mGluR agonists and specific signal transduction modulators suggest that each tested mGluR uses a different signaling pathway. First, feeding stimulatory effects of (1S,3R)-ACPD were abolished by phospholipase C inhibitor, U 73122, but remained unaffected by adenylate cyclase activator, NKH 477, or phosphodiesterase inhibitor, Rolipram. Second, (2R,4R)-APDC induced feeding in presence of U 73122 or Rolipram, but lost its feeding stimulatory effects in presence of NKH 477. Finally, L-AP4 did not induce feeding in presence of Rolipram, but maintained its feeding stimulatory effects in presence of U 73122 or NKH 477. The activity of the general glutamate receptor activator MSG was abolished by NKH 477, and Rolipram. U 73122 did not affect MSG-stimulated feeding. These results suggest that transduction of MSG taste in the codling moth caterpillar relies mostly on cAMP-dependent signaling pathways.     

Chronic exposure of cerebellar granule cells to ethanol results in increased N-methyl-D-aspartate receptor function.

In primary cultures of cerebellar granule cells, activation of the N-methyl-D-aspartate (NMDA) receptor leads to Ca2+ influx. Previous work showed that this response is selectively inhibited by acute exposure to low concentrations of ethanol. The present results demonstrate that the response to NMDA (measured as an increase in intracellular Ca2+ concentration, using fura-2 fluorescence) is significantly enhanced after chronic in vitro exposure of the cells to ethanol (100 mM for 2-4 days; 20 mM for 3 or more days). This enhancement is consistent with an increased number of NMDA receptors, with no change in receptor properties. Specifically, there was no change in the EC50 values for NMDA and glycine or in the magnitude of inhibition of the NMDA response by competitive or uncompetitive antagonists. There was also no change in the ability of acute ethanol to inhibit the NMDA response after chronic exposure of the cells to ethanol. Furthermore, chronic ethanol exposure did not alter depolarization-dependent increases in intracellular Ca2+ observed after exposure of the cells to 30 mM KCl. The data suggest that chronic ethanol exposure produces a selective up-regulation of NMDA receptor function. In the intact animal, such a change may be associated with particular symptoms of ethanol withdrawal, i.e., withdrawal seizures.     

Selected amino acids, dipeptides and arylalkylamine derivatives do not act as allosteric modulators at GABAB receptors

Based on recent reports describing enhancing actions of arylalkylamines (fendiline [N-(3,3-diphenylpropyl)-alpha-methylbenzylamine] and prenylamine [N-(3,3-diphenylpropyl)-alpha-methylphenethylamine]), amino acids (L-phenylalanine, L-leucine and L-isoleucine), and dipeptides (L-Phe-Phe and L-Phe-Leu) on baclofen-induced responses in cortical slices, we have examined whether these compounds might act as positive allosteric modulators at GABA(B) receptors. Unlike the previously described allosteric GABA(B) receptor modulator CGP7930 (2,6-Di-tert-butyl-4-(3-hydroxy-2,2-dimethyl-propyl)-phenol), these compounds did not enhance GABA(B) receptor-mediated guanosine 5'-O-(3-thiotriphosphate) [GTP(gamma)35S] binding in native or recombinant cell membrane preparations. Similarly, in a competition binding assay using the antagonist radioligand [3H]CGP62349, CGP7930, but not the other compounds, enhanced the affinities of gamma-aminobutyric acid (GABA) for native GABA(B) receptors from rat brain cortex. Finally, in a cellular assay (Ca(2+) signaling in a recombinant cell line), CGP7930 was again the only compound found to enhance the GABA response. It is concluded that the arylalkylamines, amino acids and dipeptides tested do not act as allosteric modulators at native and recombinant GABA(B) receptors.     

The antipsychotic-like effects of positive allosteric modulators of metabotropic glutamate mGlu4 receptors in rodents

Background and Purpose

Because agonists at metabotropic glutamate receptors exert beneficial effects in schizophrenia, we have assessed the actions of Lu AF21934 and Lu AF32615, two chemically distinct, selective and brain-penetrant positive allosteric modulators (PAMs) of the mGlu₄ receptor, in several tests reflecting positive, negative and cognitive symptoms of schizophrenia in rodents.

Experimental Approach

Hyperactivity induced by MK-801 or amphetamine and head twitches induced by 2,5-dimethoxy-4-iodoamphetamine (DOI) in mice were used as models for positive symptoms. Disruption of social interaction and spatial delayed alternation tests induced by MK-801 in rats were used as models for negative and cognitive symptoms of schizophrenia, respectively.

Key Results

Lu AF21934 (0.1–5 mg·kg⁻¹) and Lu AF32615 (2–10 mg·kg⁻¹) dose-dependently inhibited hyperactivity induced by MK-801 or amphetamine. They also antagonized head twitches and increased frequency of spontaneous excitatory postsynaptic currents (EPSCs) in brain slices, induced by DOI. In mice lacking the mGlu₄ receptor (mGlu₄^−/−) mice, Lu AF21934 did not antagonize DOI-induced head twitches. MK-801-induced disruption in the social interaction test was decreased by Lu AF21934 at 0.5 mg·kg⁻¹ and by Lu AF32615 at 10 mg·kg⁻¹. In the delayed spatial alternation test, Lu AF21934 was active at 1 and 2 mg·kg⁻¹, while Lu AF32615 was active at 10 mg·kg⁻¹.

Conclusions and Implications

We propose that activation by PAMs of the mGlu₄ receptor is a promising approach to the discovery of novel antipsychotic drugs.     

Characterisation of kainate receptor mediated whole-cell currents in rat cultured cerebellar granule cells

Whole-cell voltage clamp recordings have been used to identify and characterise inward currents mediated by native kainate receptors in rat cultured cerebellar granule cells. While the selective AMPA receptor antagonist GYKI 53655 (50 microM) completely abolished inward currents evoked by AMPA (10-100 microM) in the presence of cyclothiazide (100 microM), kainate evoked currents in cells pretreated with concanavalin A (Con A) always showed a component (35-140 pA, n = 13) resistant to blockade. The majority (73+/-7%, n = 5) of GYKI 53655-resistant kainate-evoked inward currents remained in the presence of 100 microM AMPA. However, these currents were reversibly blocked by the competitive AMPA/kainate receptor antagonist NBQX (100 microM). (2S, 4R)-4-methylglutamate (SYM 2081, 10 microM) evoked inward currents in Con A treated cells (15-60 pA, n = 7), which were resistant to complete blockade by GYKI 53655 (50 microM) but antagonised by NBQX (100 microM). Kainate-evoked responses in the presence of GYKI 53655 (50 microM) had linear or slightly outwardly rectifying current-voltage (I-V) relationships in all cells examined (n = 5) and were resistant to blockade by Joro spider toxin (JsTx, 1 microM; n = 5). These results provide evidence that rat cultured cerebellar granule cells express functional kainate receptors made up of subunits which are edited at the Q/R site, and that SYM 2081 is an agonist at these native kainate receptors with a greater selectivity than kainate itself.     

Beta-amyloid-induced apoptosis of cerebellar granule cells and cortical neurons: exacerbation by selective inhibition of group I metabotropic glutamate receptors.

Administration of beta-amyloid fragment 25-35 (Abeta25-35) to cultured rat cerebellar granule cells (CGC) or cortical neurons caused cell death that was characterized by morphological and nuclear changes consistent with apoptosis. Inhibition of NMDA receptors produced a mild exacerbation of Abeta25-35 toxicity in cortical neurons; a similar effect was induced by AMPA/kainate receptor inhibition in CGC. Selective activation of group I metabotropic glutamate receptors (mGluR) by dihyroxyphenylglycine (DHPG) had no effect on Abeta25-35-induced apoptosis in either cell type, and was unaffected by blockade of ionotropic glutamate receptors. In contrast, selective inhibition of group I mGluR by (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA) exacerbated Abeta toxicity in cortical neurons, whereas this treatment was without effect on CGC. However, AIDA significantly increased Abeta-induced apoptosis in CGC in the presence of either NMDA or AMPA/kainate receptor inhibition; blockade of both ionotropic glutamate receptor classes further increased the exacerbation of apoptosis following treatment with AIDA. These findings suggest that Abeta25-35-induced neuronal injury leads to activation of group I mGluR, which attenuates the resulting apoptosis.