Activation of metabotropic glutamate receptor 5 is associated with effect of amphetamine on brain neurons

The role of metabotropic glutamate receptor 5 (mGluR5) was explored in mechanisms underlying the action of amphetamine (AMPH). The activity of mGluR5 was monitored by measuring the level of [3H]inositol monophosphates in brain neurons, in response to stimulation of 2-choloro-5-hydroxyphenylglycine (CHPG), a selective agonist of mGluR5. Treatment with 1 microM of AMPH for 1 h or 7 days increased the CHPG (1 mM, 30 min)-evoked phosphoinositide turnover by 46% or 92% and 26% or 84% in cultured cortical and hippocampal neurons, respectively, from that of CHPG-only treated cells. When AMPH was present during CHPG application post-1 h or 7 day AMPH incubation, the rate of phosphoinositide hydrolysis in cortical neurons became 121% or 142% higher than that treated with CHPG only. The postnatal day (P) 21 (juvenile) and P60 (adult) rats received three intraperitoneal injections of 5 mg/kg of AMPH or saline daily for 6 days. They were challenged on the eighth day with one dosage and sacrificed 3 h later. Reversible 3H-glutamate binding detected increases of 22-89% in the binding levels of cortex and hippocampus of both ages following the AMPH injections. Increases of 13-18% in the levels of mGluR5 mRNA were seen in the juvenile pyramidal neurons of hippocampal CA1-4, granular cells of dentate gyrus, and ventral thalamic nuclei, as shown by in situ hybridization. The AMPH-induced altered activity of mGluR5 is probably associated with changes in the expression of the glutamate receptors, including mGluR5. AMPH may modify the sensitivity of mGluR5 or interact with the receptor itself.     

L-glutamate binding sites of normal and atrophic human cerebellum

The binding kinetics, pharmacologic properties, ontogeny and localization of L-glutamate binding sites were studied in membrane preparations and sections of normal and olivopontocerebellar atrophy (OPCA) human cerebellum. One binding component was found with a Kd value in the order of 150 x 10(-9) M. No significant changes of Kd values were observed with age, whereas the highest Bmax value was observed at the age of 1 year. L-Aspartate, ibotenate, quisqualate and L-homocysteic acid were potent inhibitors of L-[3H]glutamate binding. Quantitative densitometric measurements indicated the presence of L-glutamate sites in both the molecular and granule cell layer. In OPCA cerebella a very significant decrease of L-[3H]glutamate specific binding (Bmax) was observed, whereas Kd values were found unchanged. The pharmacologic properties of L-[3H]glutamate binding sites of OPCA cerebellar tissues were similar to those of normal cerebellum. [3H]quinuclidinyl benzylate binding, expressed in fmol/mg protein, did not show significant differences between normal and OPCA cerebella.     

3H]AMPA binding to glutamate receptor subpopulations in rat brain

The glutamate analog (RS)-alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA), displaced 11% of the binding of L-[3H]glutamate to rat brain membranes, amounting to 22% of the specific binding displaceable by excess non-radioactive glutamate. AMPA-sensitive L-[3H]glutamate binding was additive with that displaced by kainic acid (1 microM) plus N-methyl-D-aspartate (10 microM) when low concentrations of non-radioactive AMPA (1 microM) were employed to determine non-specific background, but partially overlapped when higher concentration of AMPA (100 microM) were used. [3H]AMPA binding was 21% specific (displaceable by non-radioactive 0.1 mM AMPA) in sodium-, calcium- and chloride-free buffer, but increased to over 30% in the presence of 0.1 M chloride. AMPA-sensitive glutamate binding and AMPA binding were both stimulated dramatically by thiocyanate and by several other anions. [3H]AMPA binding activity was resistant to freezing and thawing, optimal at 0-4 degrees C, and detectable at slightly reduced levels by filtration assays and in tissue section autoradiography. AMPA showed a heterogeneous affinity in displacement of L-[3H]glutamate, and [3H]AMPA binding showed heterogeneity with respect to AMPA, quisqualate, and glutamic acid diethyl ester. Scatchard plots gave a best fit for two sites with Kd values of 28 and 500 nM and Bmax values of 200 and 1800 fmol/mg protein, respectively. [3H]AMPA was inhibited by quisqualate (IC50 = 60 nM), L-glutamate (2 microM), (RS)-3-hydroxy-4,5,6,7-tetrahydroisoxazolo-[5,4-c]-pyridine-7-carboxylic acid (7-HPCA, 5 microM), kainic acid (20 microM) and glutamic acid diethyl ester (21 microM) but insensitive to L-aspartate, ibotenic acid, N-methyl-D-aspartate, (RS)-2-amino-phosphonobutyric acid and (RS)-2-amino-phosphonovaleric acid. This is consistent with labeling of a quisqualate-specific subpopulation of glutamate receptors. The high affinity (28 nM) and intermediate affinity (0.5 microM) AMPA sites had similar pharmacological specificity and brain regional distribution as determined by autoradiography. The latter revealed high densities of [3H]AMPA binding in the superficial layers of the cerebral cortex; stratum pyramidale, stratum radiatum, and stratum oriens of the hippocampus; and stratum moleculare of the dentate gyrus. Within the cerebellum, higher densities of binding were observed in the molecular layer than in the granule cell layer. In many regions, [3H]AMPA binding had a similar distribution to that of L-[3H]glutamate binding displaced by AMPA (1 microM).(ABSTRACT TRUNCATED AT 400 WORDS)     

Direct radiolabeling by [3H]quisqualic acid of group I metabotropic glutamate receptor in rat brain synaptic membranes

[3H]Quisqualic acid (QA) was synthesized and used to label metabotropic glutamate receptor (mGluR) in rat brain synaptic membranes in the presence of three different ionotropic glutamate receptor agonists at respective saturating concentrations. Of several mGluR agonists tested, group I agonists were more potent in displacing [3H]QA binding than group II and group III agonists in the presence of the three ionotropic agonists. [3H]QA binding was markedly inhibited by guanine nucleotide analogues in a concentration-dependent manner at a concentration range of 10 nM to 1 mM. Scatchard analysis revealed that [3H]QA binding consisted of a single component with a K(d) of 50.9+/-5.3 nM and a B(max) of 431. 6+/-18.3 fmol/mg protein. These results suggest that [3H]QA indeed labels group I mGluR functionally coupled to GTP binding protein in rat brain synaptic membranes when determined under the experimental conditions employed.     

Effects of a metabotropic glutamate agonist, trans-ACPD, on cortical epileptiform activity.

The effect of the metabotropic glutamate receptor agonist trans-1-amino-1,3,cyclopentanedicarboxylic acid (trans-ACPD) on epileptiform activity induced in rat neocortical slices by exposure to Mg(2+)-free medium was examined. Trans-ACPD dose dependently (10-200 microM) decreased the frequency of spontaneous epileptiform events whilst increasing both the duration of afterpotentials and the number of afterbursts associated with single events. This effect on afterpotentials and afterbursting was particularly pronounced in 14-17 day-old rats and was blocked by the sigma ligand ditolyguanidine (DTG) 10 microM. The putative metabotropic glutamate receptor antagonist L-AP3 did not antagonise the actions of trans-ACPD. The results suggest a role for metabotropic glutamate receptors in epilepsy, possibly in the transition from interictal to ictal activity.     

A metabotropic glutamate receptor agonist does not mediate neuronal degeneration in cortical culture.

In light of the evidence that calcium plays a critical role in excitotoxic neuronal death, it has been speculated that the metabotropic glutamate receptor may also contribute to excitotoxic damage through the mobilization of Ca2+ from intracellular stores. In the present study we examined this possibility by studying the neurotoxicity of trans-1-amino-cyclopentyl-1,3-dicarboxylate (trans-ACPD), a selective agonist of the metabotropic glutamate receptor. Exposure of cortical neurons to 100 microM trans-ACPD substantially increased phosphoinositide hydrolysis and intraneuronal free calcium in the presence of CPP and CNQX. Despite the presence of functional metabotropic receptors on cultured neurons, however, exposure of cultures to as high as 1 mM trans-ACPD for 24 h failed to produce any morphological or chemical signs of neuronal damage. Furthermore, trans-ACPD did not potentiate submaximal neurotoxicity produced by other non-N-methyl-D-aspartate (NMDA) agonists, kainate and D,L-alpha-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionic acid (AMPA).     

Multiple Cl(-)-independent binding sites for the excitatory amino acids: glutamate, aspartate and cysteine sulfinate in rat brain membranes

As we have recently reported that Cl(-)-dependent glutamate (GLU) binding reflects GLU accumulation into membrane vesicles, the characteristics, kinetics and pharmacological specificities of L-[3H]glutamate (L-[3H]GLU) binding to crude rat brain synaptic membranes, were investigated in Cl(-)-free medium. L-[3H]GLU binding was systematically compared to that of L-[3H]cysteine sulfinate (L-[3H]CSA) and L-[3H]ASP), two other putative excitatory amino acids. A high affinity site was determined for each of these radioactive ligands (L-[3H]GLU: Kd = 0.14 microM, Bm = 3.4 pmol/mg protein; L-[3H]CSA: Kd = 0.07 microM, Bm = 2.2 pmol/mg protein; L-[3H]ASP: Kd = 5.8 microM, Bm = 31.2 pmol/mg protein). The pharmacological specificity of these Cl(-)-independent binding sites indicate the existence of at least 3 distinct high affinity sites, all different from the Cl(-)-dependent GLU binding 'site': one having a similar affinity for GLU and CSA, a second one preferring CSA, and a third one preferring ASP. Among the large quantity of structural analogs of the neuroexcitatory amino acids tested, only endogenous compounds (GLU, ASP and CSA) (except hydroxylamine-o-sulfate) were able to interact efficiently. No inhibition by classical agonists and antagonists (such as N-methyl-D-aspartate, quisqualate, kainate, 2-amino-4-phosphonobutyrate, or 2-amino-5-phosphonovalerate) was found. In addition to their high specificity, these Cl(-)-independent sites possess most other biochemical characteristics of receptor proteins.     

NAAG inhibits KCl-induced [(3)H]-GABA release via mGluR3, cAMP, PKA and L-type calcium conductance

The peptide neurotransmitter, N-acetylaspartylglutamate (NAAG), is a selective agonist at the type 3 metabotropic glutamate receptor (mGluR3) where it acts to decrease cAMP levels. Rat cortical interneurons express both NAAG and glutamic acid decarboxylase, as well as mGluR3 mRNA. In the presence of ionotropic glutamate receptor antagonists, both NAAG and the group II metabotropic glutamate receptor agonist, DCG-IV, reduced the calcium-dependent, KCl-induced [(3)H]-GABA release from rat cortical neurons by 35%. This release process was unaffected by tetrodotoxin. The group II antagonist, ethyl glutamate, reversed the effects of DCG-IV and NAAG. The mGluR3-selective antagonist, beta-N-acetylaspartylglutamate, reversed the effect of NAAG. While pretreatment of cortical neurons with forskolin alone did not significantly affect KCl-stimulated [(3)H]-GABA-release, forskolin abolished the inhibition of release produced by NAAG. The protein kinase A inhibitor, H-89, decreased [(3)H]-GABA release while NAAG produced no additional inhibition in the presence of H-89. In contrast, the protein kinase C inhibitor, Ro 31--8220, had no effect on KCl-stimulated release, nor did it affect the inhibition of release produced by NAAG. The L-type calcium channel blocker, nifedipine, also inhibited the release of [(3)H]-GABA and coapplication with NAAG resulted in no significant additional inhibition of release. These data support the hypothesis that the inhibition of KCl-stimulated [(3)H]-GABA release by NAAG is mediated via presynaptic mGluR3 on GABAergic cortical neurons and that this effect is obtained by decreasing cAMP with a consequent decrease in protein kinase A activity and L-type calcium channel conductance.     

Thiamine deficiency results in downregulation of the GLAST glutamate transporter in cultured astrocytes

Pyrithiamine-induced thiamine deficiency (TD) is a well-established model of Wernicke's encephalopathy in which a glutamate-mediated excitotoxic mechanism may play an important role in determining selective vulnerability. In order to examine this possibility, cultured astrocytes were exposed to TD and effects on glutamate transport and metabolic function were studied. TD led to decreases in cellular levels of thiamine and thiamine diphosphate (TDP) after 24 h of treatment and decreased activities of the TDP-dependent enzymes alpha-ketoglutarate dehydrogenase and transketolase after 4 and 7 days, respectively. TD treatment for 10 days led to a reversible decrease in the uptake of [(3)H]-D-aspartate, a nonmetabolizable analogue of glutamate. Kinetic analysis revealed that the uptake inhibition was caused by a 47% decrease in the V(max) for uptake of [(3)H]-D-aspartate, with no change in the K(m) value. Immunoblotting showed that this decrease in uptake was due to an 81% downregulation of the astrocyte-specific GLAST glutamate transporter. Loss of uptake activity and GLAST protein were blocked by treatment with the protein kinase C inhibitor H7, while exposure to DCG IV, a group II metabotropic glutamate receptor (mGluR) agonist, resulted in improvement of [(3)H]-D-aspartate uptake and a partial reversal of transporter downregulation. These results are consistent with our recent in vivo findings of a loss of astrocytic glutamate transporters in TD and provide evidence that TD conditions may increase phosphorylation of GLAST, contributing to its downregulation. In addition, manipulation of group II mGluR activity may provide an important strategy in the treatment of this disorder.     

Trans-ACPD-induced Ca2+ signals in cerebellar Purkinje cells.

A combination of intracellular recording and fluorometric measurements of cytosolic calcium [( Ca2+]i) was used to locate changes in [Ca2+]i induced by the specific metabotropic glutamate receptor (mGluR) agonist trans-D,L-1-amino-1,3-cyclopentanedicarboxylic acid (t-ACPD), in Purkinje cells of rat cerebellar slices. Under voltage-clamp conditions, application of t-ACPD (100 microM) induced an inward current accompanied by a large increase in [Ca2+]i located primarily in the soma but also, to a lesser degree, in restricted parts of the dendrites. In contrast, elevations of [Ca2+]i associated with calcium spikes were confined to the dendrites and inward currents of a similar amplitude induced by (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), an agonist of ionotropic glutamate receptors, did not raise [Ca2+]i.     

Quantitative autoradiographic study of L-glutamate binding sites in normal and atrophic human cerebellum.

In the present work the distribution of L-glutamate binding sites in the different layers of human cerebellum of normal individuals and of seven patients who died with olivopontocerebellar atrophy (OPCA) was examined with the technique of quantitative autoradiography. Specific L-[3H]glutamate binding was higher in the molecular than in the granule cell layer of normal cerebellar tissue. A significant decrease of L-[3H]glutamate specific binding was observed in the molecular layer of all OPCA tissues. In the granule cell layer L-[3H]glutamate binding was decreased only in two patients who suffered from late-onset sporadic OPCA and in one patient who suffered from a form of OPCA inherited in a dominant manner. Quisqualate-sensitive binding sites were the most abundant binding sites in the molecular layer of normal cerebella, whereas N-methyl-D-aspartic acid (NMDA)-sensitive binding sites were the most abundant type in the granule cell layer. A significant decrease of quisqualate-sensitive and an increase in NMDA-sensitive binding sites were observed in the molecular layer of OPCA cerebellar tissues. No significant changes were observed in the granule cell layer of these tissues.     

The metabotropic glutamate receptor agonist 1S,3R-ACPD stimulates and modulates NMDA receptor mediated excitotoxicity in organotypic hippocampal slice cultures

The potential toxic effects of the metabotropic glutamate receptor agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) and its interactions with the N-methyl-D-aspartate (NMDA) receptor were studied in hippocampal brain slice cultures, using densitometric measurements of the cellular uptake of propidium iodide (PI) to quantify neuronal degeneration. Cultures exposed to ACPD, showed a concentration (2-5 mM) and time (1-4 days) dependent increase in PI uptake in CA1, CA3 and dentate subfields after 24 h and 48 h of exposure, with CA1 pyramidal cells being most sensitive. The neurodegeneration induced by 2 mM ACPD was completely abolished by addition of 10 microM of the NMDA receptor antagonist (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801), while 20 microM of the 2-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)/kainic acid receptor antagonist 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX) had no effect. Co-exposing cultures to a subtoxic dose of 300 microM ACPD together with 10 microM NMDA, which at this dose is known to induce a fairly selective degeneration of CA1 pyramidal cells, significantly increased the PI uptake in both CA1 and CA3, compared to cultures exposed to 10 microM NMDA only. Adding the 300 microM ACPD as pretreatment for 30 min followed by a 30 min wash in normal medium before the ACPD/NMDA co-exposure, eliminated the potentiation of NMDA toxicity. The potentiation was also blocked by addition of 10 or 100 microM 2-methyl-6-(phenylethynyl)pyridine (MPEP) (mGluR5 antagonist) during the co-exposure, while a corresponding addition of 10 or 100 microM 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt) (mGluR1 antagonist) had no effect. We conclude that, stimulation of metabotropic glutamate receptors with ACPD at concentrations of 2 mM or higher induces a distinct subfield-related and time and concentration dependent pattern of hippocampal degeneration, and that ACPD at subtoxic concentrations modulates NMDA-induced excitotoxicity through the mGluR5 receptor in a time dependent way.     

Chloride-dependent binding sites for L-[3H]glutamate on dendrodendritic synaptosomal membranes of rat olfactory bulb

Dendrodendritic synapses occur between granule cell dendrites and secondary dendrites of mitral cells within the olfactory bulb and are attainable in a subcellular fraction (DDS). Since the mitral cells are thought to utilize an excitatory amino acid as a neurotransmitter, we determined the pharmacologic specificity of Na+-independent L-[3H]glutamate binding to fresh membranes of DDS in 50 mM Tris-HCl, pH 7.1. Binding of L-glutamate to membranes of DDS was specific, Cl(-)-dependent, and saturable. Scatchard plots were analyzed by nonlinear regression analyses using the computer program LIGAND, and the data was best-fitted to a one-site model with KD of 0.56 +/- 0.04 microM and an apparent Bmax of 48 +/- 5 pmol/mg protein. Hill plots also indicated the presence of one site and no cooperativity (nH = 0.99 +/- 0.03). However, the relative effectiveness of several compounds in inhibiting L-glutamate binding to membranes of DDS clearly demonstrated the presence of more than one site. Electrophysiological studies suggest that 2-amino-4-phosphonobutyrate (APB) is a potent antagonist of evoked responses elicited by stimulation of mitral cell axons and that quisqualate is a potent agonist; both of these compounds were highly effective inhibitors of L-glutamate binding to DDS membranes. APB displaced about 70% of the sites labeled with 200 nM L-glutamate with a KI of 1.6 microM, whereas quisqualate inhibition of L-glutamate binding yielded a line that was curvilinear in the Scatchard plot and was resolved into two sites of relatively high affinity (KI values of 0.02 and 0.65 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Trans-ACPD-induced phosphoinositide hydrolysis and modulation of hippocampal pyramidal cell excitability do not undergo parallel developmental regulation.

The selective metabotropic glutamate receptor agonist, trans-1-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD), stimulates phosphoinositide hydrolysis and elicits a number of electrophysiological responses in the hippocampus. If these effects are mediated by the same receptor subtype, they should undergo parallel developmental regulation. Therefore, we compared the phosphoinositide hydrolysis response and the electrophysiological responses to trans-ACPD at two different developmental stages. Trans-ACPD-stimulated phosphoinositide hydrolysis was significantly greater in hippocampal slices from immature (6-11-day-old) rats than from adults. In contrast, trans-ACPD elicited decreases in spike frequency adaptation and in the amplitude of the slow afterhyperpolarization in roughly equal percentages of immature and adult CA1 pyramidal cells. Similar results were obtained using the putative endogenous agonist, glutamate. These data support the hypothesis that certain electrophysiological effects of trans-ACPD are mediated by a metabotropic glutamate receptor that is distinct from the phosphoinositide hydrolysis-linked glutamate receptor.     

Interaction between beta-N-methylamino-L-alanine and excitatory amino acid receptors in brain slices and neuronal cultures.

beta-N-Methylamino-L-alanine (BMAA) stimulated the hydrolysis of polyphosphoinositides (PPI) in hippocampal slices prepared from 8-day old rats. The action of BMAA was antagonized by D,L-2-amino-3-phosphonopropionate (an antagonist of metabotropic receptors) and was largely reduced after lowering the concentration of bicarbonate ions from 25 to 1 mM. In cultured cerebellar neurons, stimulation of PPI hydrolysis by BMAA was mediated by the activation of both metabotropic and N-methyl-D-aspartate (NMDA) receptors. However, BMAA exhibited low activity as an NMDA receptor agonist, as reflected by its low efficacy in increasing cGMP formation in cultures incubated in the absence of extracellular Mg2+. A preferential interaction of BMAA with non-NMDA receptors was confirmed by binding studies on crude synaptic membranes from rat brain. Accordingly, BMAA was more potent in displacing specifically bound [3H]glutamate than 3-(2-carboxypiperazin-4-yl)[1,23H]propyl-1-phosphonic acid (CPP) (a selective NMDA receptor ligand). As expected, the affinity of BMAA for [3H]glutamate or [3H]CPP binding sites was greater in the presence of 25 mM bicarbonate. BMAA weakly displaced specifically bound [3H]glycine in the absence of bicarbonate and, in cultured neurons incubated with buffer containing 1 mM bicarbonate, mimicked glycine in reversing the inhibitory action of kynurenic acid on glutamate-stimulated 45Ca2+ influx. Taken collectively, these results suggest that BMAA acts as a mixed agonist of 'metabotropic' and NMDA receptors.     

Reduced glutamate binding in rat dorsal vagal complex after nodose ganglionectomy

Quantitative receptor autoradiography with L-[3H]glutamate was employed to examine the distribution and properties of glutamate binding sites in the rat brain 14 days after excision of the right nodose ganglion. Slide-mounted coronal sections of the brain showed reduced L-[3H]glutamate binding in the nucleus tractus solitarius/dorsal motor nucleus of the vagus in the ipsilateral relative to the sham-operated side. Densitometric and saturation analyses of binding data indicated a significant reduction in the density of glutamate binding sites (57% decrease relative to sham), while there was a significant increase in receptor affinity (40% greater than sham). Binding was unaltered in the inferior olivary complex. Glutamate receptors are likely to exist on synaptic nerve terminals of vagal afferent fibres within the nucleus tractus solitarius and on vagal preganglionic neurones within the dorsal motor nucleus of the vagus and/or their dendritic processes within the nucleus tractus solitarius. Additionally, our receptor autoradiographic studies provide evidence for L-glutamate being a transmitter of vagal afferent neurones.     

Microinjections of the metabotropic glutamate receptor agonist,trans-(±)-1-amino-cyclopentane-1,3-dicarboxylate (trans-ACPD) into the amygdala increase the acoustic startle response of rats

The present study examined the effect of intraamygdaloid application of the metabotropic glutamate receptor agonist trans-ACPD on the acoustic startle response. Trans-ACPD led to a disruption of between-session habituation which is normally seen after repeated testing after injections of the vehicle into the amygdala. More specifically, a statistically significant increase of the magnitude of the startle response was observed 4 h after injection of 30 nmol of trans-ACPD into the central amygdaloid nucleus. The present findings suggest a role for the metabotropic glutamate receptor in the amygdala in the enhancement of the acoustic startle response.     

Nitric oxide dependence of glutamate-mediated modulation at a vertebrate neuromuscular junction

Recent evidence has revealed a contribution of glutamate in the stereotyped cholinergic neuromuscular transmission. Indeed, receptors, transporters and glutamate itself are present at the neuromuscular junction (NMJ) while glutamate activation of metabotropic receptors (mGluRs) decreases synaptic transmission and mediates depression through presynaptic mechanisms. However, we have shown that the mGluRs are located postsynaptically, inconsistent with the presynaptic action of glutamate. In the present study, we tested whether nitric oxide (NO) serves as a retrograde messenger mediating the distant effect of glutamate. Glutamate or an mGluR agonist [trans-(1S,3R)-aminocyclopentanedicarboxylic acid (ACPD)] failed to reduce synaptic transmission in the presence of an NOS inhibitor (3Br7NINa, 3-bromo-7-nitroindazole sodium salt). Moreover, application of 3Br7NINa precluded the effect of the mGluR antagonist MCPG [(S)-alpha-methyl-4-carboxyphenylglycine] on high-frequency-induced synaptic depression. Iontophoretic injections of BAPTA [1,2-bis(2-aminophenoxy)ethane-N,N,N'-tetraacetic acid] in muscle fibres abolished the effect of trans-ACPD on synaptic transmission and blocked the mGluR component of depression, indicating the involvement of muscular calcium in mGluR-induced depression. Also, the use of this protocol unveiled a muscular calcium-dependent potentiating pathway dependent on cyclo-oxygenase activity. In addition, local application of trans-ACPD induced an increase in NO production by muscle fibres visualized with the indicator DAF-FM (4-amino-5-methylamino-2',7'-difluorofluorescein). This was prevented by 3Br7NINa or the iontophoretic injection of BAPTA. Moreover, motor nerve stimulation (50 Hz, 30 s) induced an increase in DAF-FM fluorescence that was abolished by 3Br7NINa and MCPG. Hence, the data suggest that the production of the retrograde molecule NO depends on the postsynaptic calcium-dependent activation of nitric oxide synthase following mGluRs stimulation and is essential for the glutamatergic modulation of synaptic efficacy and plasticity at the NMJ.     

Effects of amygdaloid kindling on NMDA receptor function and regulation

These studies were conducted to determine whether amygdaloid kindling results in the long-term alteration of NMDA receptors which could explain the persistent reduction in seizure threshold seen in this phenomenon. NMDA-induced [3H]norepinephrine (NE) release, NMDA-sensitive L-[3H]glutamate binding, and NMDA and glycine-enhanced [3H]TCP binding were measured in brain tissue from kindled rats and nonstimulated control rats 3 to 6 weeks after the last seizure. There was no difference in the ability of NMDA to induce [3H]NE release from kindled or control slices of amygdala or hippocampus. There was also no difference in the ability of phencyclidine (PCP) or Mg2+ to inhibit [3H]NE release induced by 100 microM NMDA. Equilibrium saturation experiments of NMDA-sensitive L-[3H]glutamate binding revealed no differences in KD or Bmax values between control and kindled cortex, amygdala, and hippocampus. The Ki values for NMDA displacement of L-[3H]glutamate binding also did not differ in kindled tissue. NMDA-enhanced [3H]TCP binding was similar in cortex, amygdala, and hippocampus of kindled and control tissues. Finally, glycine-enhanced [3H]TCP binding was not different in control or kindled tissues. These studies suggest that the NMDA recognition site and the modulation of the NMDA receptor/ion channel complex by magnesium, PCP, and glycine are not altered several weeks after the last seizure. Even though NMDA-mediated electrophysiological responses are reportedly enhanced in kindled tissue at that time, the mechanism(s) underlying the enhancement remains to be determined.     

Selective and reversible increase in the number of quisqualate-sensitive glutamate binding sites on hippocampal synaptic membranes after angular bundle kindling

The specific binding of L-[3H]glutamate to hippocampal synaptic membranes was examined in rats kindled by tetanic stimulation of the angular bundle. One day after the last of a minimum of 3 class 4 kindled seizures, the binding of L-[3H]glutamate to a quisqualate-sensitive site (GLU A) was about 40% greater than in electrode-implanted unstimulated controls. Saturation binding data indicated an increase in the maximum density of GLU A binding sites with no change in their affinity for L-glutamate. No such increase was detected 28 days after the last kindled seizure, although the animals were still kindled. Radioligand binding to a site that is much less sensitive to quisqualate (GLU B) was unaffected by kindling stimulation. These observations suggest that an increase in GLU A binding sites could be involved in the induction, but not in the maintenance, of the kindled state.