Stimulatory effects of centrally injected kainate and N-methyl-D-aspartate on gastric acid secretion in anesthetized rats

The effects of N-methyl-D-aspartate (NMDA), kainate and (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA), ionotropic glutamate agonists, on gastric acid secretion were investigated in the continuously perfused stomach of anesthetized rats. The lateral ventricular (LV) injection of kainate (0.01-1 microg) or NMDA (0.3-3 microg) dose-dependently stimulated gastric acid secretion. AMPA (3-10 microg) also stimulated gastric acid secretion but the effect was very weak. Repeated injections of kainate (0.1 microg) or NMDA (1 microg), at least twice, stimulated gastric acid secretion to a similar degree. The effect of kainate (0.1 microg) was blocked by the kainate receptor antagonists, 6-cyano-7-nitroquinoxaline-2,3-dione disodium (3 microg, LV) and D-gamma-glutamylaminomethanesulfonic acid (30 microg, LV), but not by NMDA receptor antagonists. The effect of NMDA (10 microg) was blocked by (+/-)-3-(2-carboxypiperazin-4-yl)-1-propylphosphonic acid (10 microg, LV), a competitive NMDA receptor antagonist, and (+)-5-methyl-10,11-dihydro-5H-dibenzocyclo-hepten-5,10-imine hydrogen maleate (10 microg, LV), a non-competitive NMDA receptor antagonist, but not by kainate receptor antagonists. Moreover, the gastric acid secretion stimulated by kainate and NMDA were completely blocked by systemic atropine injection (1 mg/kg, i.v.) and vagotomy. These findings suggest that kainate and NMDA receptor mechanisms are independently involved in the central nervous system to control gastric acid secretion through vagus cholinergic activation.     

Excitatory amino acid receptor subtype agonists induce feeding in the nucleus accumbens shell in rats: opioid antagonist actions and interactions with μ-opioid agonists

Administration of μ-opioid receptor subtype agonists into the nucleus accumbens shell elicits feeding which is dependent upon the normal function of μ-, δ- and κ-opioid receptors, D₁ dopamine receptors and GABA_B receptors in the nucleus accumbens shell for its full expression. Whereas the AMPA antagonist, DNQX administered into the nucleus accumbens shell elicits a transient, though intense feeding response, feeding is elicited by excitatory amino acid agonists administered into the lateral hypothalamus. The present study examined whether excitatory amino acid agonists elicited feeding following administration into the nucleus accumbens shell of rats, whether such feeding responses were altered by opioid antagonist pretreatment, and whether such feeding responses interacted with feeding elicited by μ-opioid agonists. Both AMPA (0.25–0.5 μg) and NMDA (1 μg) in the nucleus accumbens shell significantly and dose-dependently increased food intake over 4 h. Both feeding responses were blocked by naltrexone pretreatment in the nucleus accumbens shell. The μ-opioid agonist, [D-Ala²,NMe-Phe⁴,Gly-ol⁵]-enkephalin in the nucleus accumbens shell significantly increased food intake which was significantly enhanced by AMPA cotreatment. This enhanced feeding response was in turn blocked by pretreatment with either general or μ-selective opioid antagonists. In contrast, cotreatment of NMDA and the μ-opioid agonist in the nucleus accumbens shell elicited feeding which was significantly less than that elicited by either treatment alone. These data indicate the presence of important interactions between excitatory amino acid receptors and μ-opioid receptors in the nucleus accumbens shell in mediating feeding responses in nondeprived, ad libitum-fed rats.     

Receptor types mediating the excitatory actions of exogenousl-aspartate andl-glutamate in rat olfactory cortex

Changes in potential between the pial and cut surfaces of rat olfactory cortex slices evoked by N-methyl-d-aspartate (NMDA), quisqualate, kainate,l-glutamate andl-aspartate and also by γ-aminobutyric acid (GABA) have been monitored using extracellular electrodes. All agonists produced a pial-negative potential response when superfused onto the pial surface, GABA,l-aspartate andl-glutamate being less potent than the others. Repeated applications of NMDA, but not of the other agonists, led to a progressive reduction in response to approximately 30% of the initial depolarization. The responses to NMDA (100 μM) were selectively abolished by(±)2-amino-5-phosphonopentanoic acid (APP; 100 μM) while depolarizations evoked byl-glutamate andl-aspartate (both at 10 mM) were only antagonized by21 ± 2 (n = 12) and36 ± 3 (n = 12) percent respectively (means ± S.E.M.). γ-d-Glutamylglycine (γ-DGG; 1 mM) and(±)cis-2,3-piperidine dicarboxylate (cis-PDA; 2 and 5 mM), in addition to antagonizing responses to NMDA, also partially blocked quisqualate- and kainate-evoked depolarizations. When a mixture of APP (100 μM), γ-DGG (1 mM) and cis-PDA (5 mM) was applied to preparations, although NMDA receptors were completely blocked and responses to both quisqualate and kainate antagonized by approximately 80%,l-glutamate andl-aspartate evoked depolarizations were only reduced by51 ± 7 (n = 4) and 49 ± 4 (n = 4) percent respectively (means ± S.E.M.). The results are discussed in terms of the contributions made by NMDA, quisqualate and kainate receptors to the composite responses evoked byl-aspartate andl-glutamate.     

Epileptiform bursts elicited in CA3 hippocampal neurons by a variety of convulsants are not blocked by N-methyl-d-aspartate antagonists

Intracellular and extracellular recordings from CA₃ hippocampal neurons in vitro were used to study the ability of several NMDA (N-methyl-d-aspartate) receptor antagonists to suppress epileptiform bursts induced by NMDA and convulsants not thought to act at NMDA receptors. The antagonists, APV (d-2-amino-5-phosphonovalerate), AP-7 (d,l-2-amino-7-phosphonoheptanoate) and CPP (d,l-3-[(±)-2-car☐ypiperazin-4-yl-]-propyl-1-phosphonic acid), blocked the spontaneous and evoked bursts induced by NMDA. CPP, but not APV or AP-7, prevented the development of bursts induced by Mg-free medium. The NMDA antagonists failed to block bursting induced by kainate, 7 mM K⁺, mast cell degranulating peptide, anoxia or spontaneous bursting. In some cases the NMDA antagonists induced spontaneous bursts or enhanced burst frequency, a proconvulsant effect. It is concluded that activation of NMDA receptors is sufficient but not necessary for burst generation in the CA₃ region.     

NS-257, a novel competitive AMPA receptor antagonist, interacts with kainate and NMDA receptors

In this study, we examined the effects of a novel, water-soluble, putative competitive AMPA receptor antagonist, 1,2,3,6,7, 8-hexahydro-3-(hydroxyimino)-N,N,7-trimethyl-2-oxobenzo[2,1- b:3, 4-c']dipyrrole-5-sulfonamide (NS-257) on AMPA, kainate and NMDA receptors using the two-electrode voltage-clamp technique in Xenopus oocytes. All glutamate receptor subtypes were inhibited by NS-257 in a voltage-independent way. When kainate was applied to oocytes injected with total mouse brain mRNA, mainly AMPA receptors were activated. The antagonistic effects of NS-257 on these kainate-induced currents were concentration-dependent and competitive. In the same way, NS-257 blocked kainate-induced currents recorded from oocytes expressing homomeric GluR-1 receptors. In our experiments higher concentrations (>1 microM) of NS-257 also produced inhibitory effects on kainate and to a lesser extent on NMDA receptor function as indicated by recordings from GluR-6 or NR-1b/2A cRNA injected oocytes. While NMDA receptor function was inhibited in a competitive fashion, kainate responses recorded from homomeric GluR-6 receptors were blocked in a mixed competitive-noncompetitive manner. This mixed antagonistic action of NS-257 might have been caused by preincubating oocytes with concanavalin A, which blocks desensitization of kainate receptors. Although NS-257 appeared to be a less potent AMPA receptor antagonist then other known antagonists like NBQX, its main advantage over all other reported compounds so far is its higher aqueous solubility which still represents the major weakness of the other AMPA receptor antagonists, especially for clinical use.     

Biphasic modulatory action of the selective sigma receptor ligand SR 31742A on N-methyl- -aspartate-induced neuronal responses in the frontal cortex

The technique of intracellular recording was used to assess the effect of SR 31742A, a selective sigma receptor ligand, on N-methyl- -aspartate (NMDA) and (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor-mediated responses in pyramidal cells of the rat medial prefrontal cortex in vitro brain slice preparations. Bath application of SR 31742A produced a biphasic effect on NMDA responses: SR 31742A facilitated and inhibited NMDA-induced inward current at low (0.01, 0.05 and 0.1 μM) and higher (0.5, 1 and 10 μM) concentrations, respectively. The potentiating effect reached the peak (366%) at 0.1 μM, with an estimated EC₅₀ value of 23 nM. Correspondingly, SR 31742A also produced a similar biphasic modulatory action on excitatory postsynaptic potentials or currents (EPSPs/EPSCs) evoked by electrical stimulation of the forceps minor. In contrast, SR 31742A produced a modest potentiation of AMPA responses at the concentrations from 0.01 to 1 μM. The potentiating action of SR 31742A on NMDA-receptor mediated neurotransmission may account for, at least partially, its antipsychotic and cognitive-enhancing potential, whereas the inhibitory action on NMDA responses at higher concentrations may be related to the purported neuroprotective action of sigma receptor ligands.     

Extracellular reduced glutathione increases neuronal vulnerability to combined chemical hypoxia and glucose deprivation

In addition to its intracellular antioxidant role, reduced glutathione (GSH) is released by CNS cells and may mediate or modulate excitatory neurotransmission. Although extracellular GSH levels rise in the ischemic cortex, its effect on the viability of energy-compromised neurons has not been defined. In this study, we tested the hypothesis that exogenous GSH would increase the vulnerability of cultured cortical neurons to azide-induced chemical hypoxia combined with glucose deprivation. Thirty minutes azide exposure in a glucose-free buffer was tolerated by most neurons, with release of less than 10% of neuronal LDH over the subsequent 21–25 h. Concomitant treatment with 10–100 μM GSH increased cell death in a concentration-dependent fashion, to 71.6±5.1% of neurons at 100 μM; GSH alone was nontoxic. Injury was blocked by the selective N-methyl- -aspartate (NMDA) antagonist MK-801 but not by the AMPA/kainate antagonist NBQX. The sulfhydryl reducing agent mercaptoethanol (10–100 μM) mimicked the action of GSH; however, the zinc chelator ethylenediaminetetraacetic acid (EDTA) was ineffective. Two GSH analogues that lack a sulfhydryl group, S-hexylglutathione (SHG) and oxidized glutathione (GSSG), were inactive per se but attenuated the effect of both GSH and mercaptoethanol. These results suggest that micromolar concentrations of GSH enhance neuronal loss due to energy depletion by altering the extracellular redox state, resulting in increased NMDA receptor activation.     

Long-term potentiation in the hippocampus involves activation of N-methyl-D-aspartate receptors

A series of ω-phosphono-α-car☐ylic acids were tested as antagonists of excitatory amino acid depolarizations and long-term potentiation (LTP) in region CA1 of rat hippocampal slices. The 5- and 7-phosphono compounds (±AP5and±AP7) blocked N-methyl-D-aspartate (NMDA) depolarizations and prevented the induction of LTP of the synaptic field potential and population spike components of the Schaffer collateral response.±AP5and±AP7 did not reduce kainate or quisqualate depolarizations and did not affect unpoten synaptic response amplitude.±AP5, ±AP6and±AP8 did not block amino acid excitant responses or LTP.These results demonstrate that NMDA receptors present in hippocampal region CA1 are not necessary for normal synaptic transmission, but are involved in the initiation of long-term synaptic plasticity.     

Characterization of the glutamatergic system for induction and maintenance of allodynia

Glutamate is the main excitatory neurotransmitter in the central nervous system and has been shown to be involved in spinal nociceptive processing. We previously demonstrated that intrathecal (i.t.) administration of prostaglandin (PG) E₂ and PGF_2α induced touch-evoked pain (allodynia) through the glutamatergic system by different mechanisms. In the present study, we characterized glutamate receptor subtypes and glutamate transporters involved in induction and maintenance of PGE₂- and PGF_2α-evoked allodynia. In addition to PGE₂ and PGF_2α, N-methyl- -aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), but not kainate, induced allodynia. PGE₂- and NMDA-induced allodynia were observed in NMDA receptor 4 (NR2D) subunit knockout (GluR4(−/−)) mice, but not in 1 (NR2A) subunit knockout (GluR1(−/−)) mice. Conversely, PGF_2α- and AMPA-induced allodynia were observed in GluR1(−/−) mice, but not in GluR4(−/−) mice. The induction of allodynia by PGE₂ and NMDA was abolished by the NMDA receptor 2 (NR2B) antagonist CP-101,606 and neonatal capsaicin treatment. PGF_2α- and AMPA-induced allodynia were not affected by CP-101,606 and by neonatal capsaicin treatment. On the other hand, the glutamate transporter blocker -threo-β-benzyloxyaspartate ( -TBOA) blocked all the allodynia induced by PGE₂, PGF_2α, NMDA, and AMPA. These results demonstrate that there are two pathways for induction of allodynia mediated by the glutamatergic system and suggest that the glutamate transporter is essential for the induction and maintenance of allodynia.     

Enhancement of contextual fear-conditioning by putative (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor modulators and N-methyl-

The possible existence of N-methyl-d-aspartate (NMDA) and non-NMDA receptors on electrophysiologically identified nondopamine neurones in the ventral tegmental area (VTA) was tested in rat midbrain slice preparations. NMDA, kainate (KA), and AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) depolarized the membrane potential of nondopamine neurons in a dose-dependent manner. The NMDA effect was blocked by the selective NMDA receptor antagonist, CGS 19755 (cis-4-phosphonomethyl-2-piperidine carboxylate), but not by the non-NMDA receptor antagonist, NBOX [2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline]. In contrast, the effects of KA and AMPA were antagonized by NBOX, but not by CGS 19755. The rank order potency of the three agonists was AMPA > KA > NMDA, with thresholds of 0.1, 0.3, and 3 μM, respectively. These results provide clear electrophysiological evidence that nondopamine neurons in the ventral tegmental area possess both NMDA and non-NMDA receptors.     

NMDA, kainate, and AMPA depolarize nondopamine neurons in the rat ventral tegmentum

The possible existence of N-methyl-d-aspartate (NMDA) and non-NMDA receptors on electrophysiologically identified nondopamine neurones in the ventral tegmental area (VTA) was tested in rat midbrain slice preparations. NMDA, kainate (KA), and AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) depolarized the membrane potential of nondopamine neurons in a dose-dependent manner. The NMDA effect was blocked by the selective NMDA receptor antagonist, CGS 19755 (cis-4-phosphonomethyl-2-piperidine carboxylate), but not by the non-NMDA receptor antagonist, NBOX [2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline]. In contrast, the effects of KA and AMPA were antagonized by NBOX, but not by CGS 19755. The rank order potency of the three agonists was AMPA > KA > NMDA, with thresholds of 0.1, 0.3, and 3 μM, respectively. These results provide clear electrophysiological evidence that nondopamine neurons in the ventral tegmental area possess both NMDA and non-NMDA receptors.     

Interactions between excitatory amino acids and tachykinins in the rat spinal dorsal horn

Whole-cell patch-clamp technique of freshly isolated rat spinal dorsal horn (DH) neurons, intracellular recording from DH neurons in a slice preparation, and high performance liquid chromatography with fluorimetric detection of release of endogenous glutamate and aspartate from spinal cord slice following activation of primary afferent fibers were employed to investigate interactions between excitatory amino acids (EAA) and tachykinins [substance P (SP) and neurokinin A (NKA)]. Potentiation of N-methyl-D-aspartate (NMDA)-, quisqualate (QA)- and α-amino 3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-, but not kainate-induced currents by SP and NKA was found. Spantide II, a claimed novel nonselective tachykinin antagonist, effectively blocked the SP (2 nM )-induced potentiation of the responses of DH neurons to NMDA. In the presence of glycine (0.1 μM), the SP-evoked increase of the NMDA-induced current was prevented. However, 7-chlorokynurenic acid (2 μM), a competitive antagonist at the glycine allosteric site of the NMDA receptor, led to the reestablishment of the SP effect. Brief high frequency electrical stimulation of primary afferent fibers produced a longlasting potentiation of presumed monosynaptic and polysynaptic excitatory postsynaptic potentials and sustained enhanced release of endogenous glutamate (218.3± 66.1 %) and aspartate (286.3 ± 58.0%). Possible functional implications of the observed phenomena are discussed in relation to transmission and integration of sensory information, including pain.     

Pretreatment with SR48692 has different effects on central neurotensin-induced gastric mucosal defense and inhibition of gastric acid secretion in rats

Neurotensin is a tridecapeptide present in the brain and gastrointestinal tract. Administration of neurotensin into the brain results in responses in the gastrointestinal tract, suggesting a role for neurotensin in the interrelationships that comprise the brain–gut axis. Intracerebroventricular (i.c.v.) administration of neurotensin protects the gastric mucosa against injury caused by cold water restraint (CWR) and also inhibits gastrin-stimulated gastric acid secretion. The hypothesis tested was that these two actions of neurotensin are mediated via its high-affinity receptor. Rats were given neurotensin (60 μg, i.c.v.) prior to CWR or pylorus ligation after pretreatment with SR48692, a nonpeptide antagonist of the high-affinity neurotensin receptor (0.25 or 2.5 μg, i.c.v., or 10, 100, or 500 μg kg⁻¹, i.p.). Neurotensin reduced cold water restraint (CWR)-induced gastric mucosal injury and inhibited gastrin-stimulated acid secretion. Pretreatment with SR48692 (2.5 μg, i.c.v., or 100 μg kg⁻¹, i.p.) prior to CWR blocked neurotensin's protection of the gastric mucosa against injury. In contrast, pretreatment with 2.5 μg SR48692, i.c.v., did not block neurotensin-induced inhibition of acid secretion, whereas 500 μg kg⁻¹, i.p., partially blocked the inhibition. SR48692 (2.5 μg, i.c.v.) inhibited acid secretion, suggesting that SR48692 has agonist activity in this system. These results suggest that central neurotensin protects the gastric mucosa against CWR-induced injury via its high-affinity receptor. The receptor that mediates central neurotensin-induced inhibition of gastric acid secretion does not appear to be the high-affinity receptor since the neurotensin receptor antagonist SR48692, when given i.c.v., had agonist activity, inhibiting stimulated acid secretion. High-affinity neurotensin receptors in the periphery appear to play a role in inhibition of stimulated gastric acid secretion.     

Intracerebroventricular neuropeptide Y increases gastric acid secretion by decreasing tonic adrenergic inhibition of acid in dogs

Neuropeptide Y (NPY) has been shown to increase basal gastric acid secretion in dogs. We examined the hypothesis that NPY might increase gastric acid secretion by interaction with central catecholaminergic control of acid secretion in dogs. Studies were performed in awake canines with gastric fistulas and cerebroventricular guides which allowed injection into the lateral cerebral ventricle. Intracerebroventricular (i.c.v.) injection of yohimbine (5 μg/kg) increased acid secretion compared to control (yohimbine: 9.1 ± 3.3mmol//h;control: 1.8 ± 1.0mmol/2h, P < 0.05), whereas prazosin and propranolol (both 5 μg/kg i.c.v.) had no effect, suggesting that there is tonic central α₂-adrenergic inhibition of acid secretion. NPY_13–36 significantly increased acid secretion compared to control (NPY_13–36 1000pmol/kg i.c.v.: 5.6 ± 1.9mmol/2h;control: 1.3 ± 0.8mmol/2h, P < 0.05), whereas [Leu³¹, Pro³⁴]-NPY had no effect, suggesting that the central effect of NPY is mediated at a Y₂, probably pre-synaptic receptor. Finally, i.c.v. desmethylimipramine (DMI) inhibited the acid response to i.c.v. NPY when injected before but not after NPY (i.c.v. DMI then i.c.v. NPY: control,15.2 ± 6.6mmol/2h;DMI, 3.5 ± 1.2mmol/2h, P< 0.05; i.c.v. NPY followed by i.c.v. DMI: control,8.9 ± 4.0mmol/2h;DMI, 9.9 ± 2.9mmol/2h, P > 0.05). This suggests that NPY acts by decreasing noradrenaline release. These findings are compatible with the hypothesis that i.c.v. NPY increases acid secretion by decreasing tonic central adrenergic inhibition of acid by decreasing release of noradrenaline at pre-synaptic level.     

Selective protection against AMPA- and kainate-evoked neurotoxicity by (3S,4aR,6R,8aR)-6-[2-(1(2)H-tetrazole-5-yl)ethyl]decahydroisoquinoline-3-carboxylic acid (LY293558) and its racemate (LY215490)

Summary Glutamate receptor-mediated excitotoxicity is linked to the activation of multiple receptors including those activated by -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), N-methyl-D-aspartate (NMDA), and kainate. In this study, the novel glutamate receptor antagonist, as its active isomer (3S,4aR,6R,8aR)-6-[2-(1(2)H-tetrazole-5-yl)ethyl]-decahydroisoquinoline-3-carboxylic acid ((–)LY293558) and it's ± racemate (LY215490), was examined for neuroprotectant effects against excitotoxic injury in vitro and in vivo. This agent selectively protected against AMPA and kainate injury in cultured primary rat hippocampal neurons, an in vivo rat striatal neurotoxicity model, and against agonist-evoked seizures in mice. Thus, (3S,4aR,6R,8aR)-6-[2-(1(2)H-tetrazole-5-yl)ethyl]decahydroisguinoline-3-carboxylic acid represents a novel receptor selective and potent systemically active AMPA/kainate receptor antagonist for exploring neuroprotection via non-NMDA receptor mechanisms.     

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- -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 μM of the NMDA receptor antagonist (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801), while 20 μM 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 μM ACPD together with 10 μM 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 μM NMDA only. Adding the 300 μM 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 μM 2-methyl-6-(phenylethynyl)pyridine (MPEP) (mGluR5 antagonist) during the co-exposure, while a corresponding addition of 10 or 100 μM 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.     

Glutamate and N-Methyl-D-Aspartate Stimulate Rat Hypothalamic Corticotropin-Releasing Factor Secretion in vitro

It is known that in vivo excitatory amino acids (EAA) stimulate the hypothalamo-pituitary-adrenal axis. However their site of action is not fully understood. We investigated the possibility of a direct action of EAA on the secretion of the major adrenocorticotropin hormone (ACTH) secretagogue: corticotropin-releasing factor (CRF) from incubated rat hypothalamic slices. N-methyl-D-aspartic acid (NMDA) or L-glutamate (1×10^?7 to 1×10^?3 M) stimulated in a dose-dependent fashion CRF release. The maximal effect was obtained at a concentration of 1×10^?4 M for both drugs. The IC₅₀ was 1.3×10^?5 M and 3.3×10^?5 M for NMDA and L-glutamate, respectively. Incubation with 2.5×10^?4 M D-2-amino-5-phosphonovalerate (a NMDA receptor antagonist) or 2-amino-4-phosphonobutyrate (a metabotropic receptor antagonist) was without significant effect on basal CRF secretion and completely blocked the increase in CRF release induced by 5×10^?5 M NMDA or L-glutamate, respectively. Incubation with 1×10^?4 M kainate or 0.5×10^?4 M AMPA did not change basal CRF secretion. Incubation with 2×10^?4 M γ-D-glutamylglycine (a specific antagonist of kainate and AMPA receptor) had no effect under basal conditions or during exposure to kainate or AMPA. Our data demonstrate that EAA could stimulate directly CRF secretion, by an action through NMDA and metabotropic receptors, but not kainate or AMPA receptors. These findings may be relevant to the regulation of the hypothalamo-pituitary adrenal axis, both under basal conditions and during exposure to stress.     

Localization of striatal excitatory amino acid binding site subtypes to striatonigral projection neurons

Quantitative autoradiography was used to examine the cellular localization of excitatory amino acid binding sites in the striatum following selective lesion of striatonigral projection neurons. Degeneration of striatonigral neurons was induced unilaterally by injection of the suicide transport toxin, volkensin, into the left substantia nigra. Twelve days following nigral volkensin injection there was a reduction of all excitatory amino acid binding site subtypes in the striatum ipsilateral to the injected nigra. The reduction in (NMDA) binding sites was significantly greater than the loss of -α-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA), kainate and metabotropic binding. These results indicate that there are NMDA, AMPA, metabotropic and kainate binding sites on striatonigral projection neurons and suggest that the NMDA subtype may be selectively enriched on striatonigral neurons.     

A quantitative description of excitatory amino acid neurotransmitter responses on cultured embryonic Xenopus spinal neurons

We have performed a quantitative analysis of excitatory amino acid neurotransmitter receptors on cultured embryonic Xenopus spinal neurons using the whole-cell patch-clamp technique. Neuroblasts and underlying mesodermal cells isolated from spinal regions of neural plate-stage embryos were placed into dissociated cell culture, and responses were studied soon after the appearance of neurites on embryonic neurons. Glutamate (Glu) receptors were separated into two general classes based on responses to the characteristic agonists quisqualate (Quis), kainate (Ka) and N-methyl-d-aspartate (NMDA); these were NMDA receptors (those activated by NMDA) and non-NMDA receptors (those activated by Ka and Quis). Half-maximal responses to Glu and other agonists on NMDA and non-NMDA receptors were determined from Hill analysis of dose response relations. The order of sensitivities observed was: Glu_NMDA(ED₅₀ = 5.1 μM) >Glu_non-NMDA(ED₅₀ = 28 μM), and for Glu receptor agonists, Quis (ED₅₀ = 1.5 μM) >NMDA(ED₅₀ = 41 μM) >Ka(ED₅₀ = 58 μM). The order of response amplitudes recorded at concentrations near the appropriate ED₅₀s was Glu_NMDA > Glu_non-NMDA, and Ka > NMDA > Quis. A 10-fold decrease in external [Na⁺] shifted the reversal potentials for Glu_non-NMDA, Ka, and Quis to more negative voltages. Increasing external [Ca²⁺] shifted the reversal potential for NMDA responses to more positive potentials, an observation consistent with Ca²⁺ permeation of the embryonic NMDA-activated channel. NMDA-evoked currents could not be recorded in nominally glycine (Gly)-free media. Addition of Gly to external solutions potentiated NMDA responses (ED₅₀ = 644nM). NMDA responses were blocked by dl-2-amino-5-phosphonovaleric acid (APV;ED₅₀ = 1.9 μM) and by Mg²⁺ at negative potentials. In their sensitivities to agonists and antagonists, and ionic dependences, amino acid neurotransmitter responses on embryonic Xenopus neurons closely resembled those previously observed for mature Xenopus and mammalian central neurons. The Glu_NMDA receptors present on these immature neurons were sufficiently sensitive to be activated by endogenous concentrations of extracellular Glu, suggesting a possible role for receptor activation in modulating early neural development.     

Sympathetic preganglionic neurones in neonatal rat spinal cord in vitro: electrophysiological characteristics and the effects of selective excitatory amino acid receptor agonists

Intracellular recordings were made from 52 lateral horn neurones in thin slices of neonatal rat thoracolumbar spinal cord. Of these neurones 12 were spontaneously active and the remainder silent. A number of these cells could be activated antidromically by stimulation of ventral roots. The conduction velocity of the antidromic potential was estimated to be 0.9–2 m/s which is within the range reported for axons of sympathetic preganglionic neurones (SPNs). The membrane properties of antidromically identified SPNs were similar to other lateral horn neurones included in this study and comparable to those reported for SPNs by others. Spontaneous burst firing was recorded in 3 neurones and activity in a further 5 neurones was characterized by the discharge of an action potential followed by an afterhyperpolarization potential (AHP) of peak amplitude 3–13 mV and duration 0.5–4 s. The AHP had an initial fast component (fAHP) which was sensitive to the potassium channel blocker tetraethylammonium (TEA), and a second slower component (sAHP) which was both sensitive to extracellular calcium and TEA. The effects of the selective excitatory amino acid receptor agonists N-methyl-d-aspartate (NMDA), kainate and quisqualate were investigated by superfusion of the agonists, at known concentrations (100 nM to 100 μM). These agonists induced concentration-dependent depolarizations which were primarily associated with a reduction in neuronal input resistance. NMDA-induced depolarizations were potentiated in the absence of magnesium. In a number of neurones NMDA, kainate and quisqualate (1–50 μM) induced, in addition to a depolarizing response, the discharge of small (1.5–6.5 mV), brief (7–20 ms) IPSPs which were reversed just below resting membrane potential at −64 mV. These results suggest that both NMDA and non-NMDA receptors may have an important role in the excitation of SPNs and of inhibitory interneurones presynaptic to SPNs.