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.     

Effects ofl-cysteine-sulphinate andl-aspartate, mixed excitatory amino acid agonists, on the membrane potential of cat caudate neurons

Responses evoked byl-cysteine-sulphinate (l-CSA) andl-aspartate (l-Asp) were recorded with intracellular electrodes from caudate neurons in halothane anesthetized cats.l-CSA andl-Asp were applied microiontophoretically to caudate cells and their effects on membrane and action potentials, as well as on cortically evoked synaptic potentials were evaluated.l-CSA andl-Asp induced depolarizations accompanied by regular firing resembling kainate (KA)- or quisqualate (QUIS)-induced excitation patterns (type 1) in 82% and 72% of the recorded neurons, respectively, and a mixed pattern consisting of aN-methyl-d-aspartate (NMDA)-like excitation (type 2) followed by a regular type 1 pattern in the remaining cells. In about a quarter of the cells the effects ofl-CSA andl-Asp, but not those of KA or QUIS, were partially antagonized by 2-amino-7-phosphonoheptanoate (AP-7), a specific NMDA receptor antagonist. Kynurenate, a broad spectrum excitatory amino acid antagonist, blocked responses elicited by eitherl-CSA or QUIS. The actions ofl-CSA andl-Asp on the firing pattern and membrane potential of cat caudate neurons in situ provide evidence in favor of their mixed agonist nature with respect to NMDA and non-NMDA excitatory amino acid receptors.     

Receptor types mediating the excitatory actions of exogenous L-aspartate and L-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 and L-aspartate and also by gamma-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 and L-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 microM) were selectively abolished by (+/-)2-amino-5-phosphonopentanoic acid (APP; 100 microM) while depolarizations evoked by L-glutamate and L-aspartate (both at 10 mM) were only antagonized by 21 +/- 2 (n = 12) and 36 +/- 3 (n = 12) percent respectively (means +/- S.E.M.). gamma-D-Glutamylglycine (gamma-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 microM), gamma-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 and L-aspartate evoked depolarizations were only reduced by 51 +/- 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 by L-aspartate and L-glutamate.     

Action of 3-((±)-2-car☐ypiperazin-4-yl)-propyl-1-phosphonic aci (CPP): a new and highly potent antagonist of N-methyl-d-aspartate receptors in the hippocampus

A new compound, 3-((±)-2-car☐ypiperazin-4-yl)-propyl-1-phosphonic acid (CPP), has been evaluated as an excitatory amino acid receptor antagonist using electrophysiological assays and radioligand binding. In autoradiographic preparations, CPP reduces l-[³H]glutama binding in regions of the hippocampus rich in N-methyl-d-aspartate (NMDA) receptors, but not in regions richin kainate sites. In isolated membrane fraction preparations, CPP displaces l-[³H]glutamate binding to NMDA sites, but does not compete with the binding of selective kainate or quisqualate site ligands. CPP potently reduces depolarizations produced by application of NMDA but not depolarizations produced by quisqualate or kainate. Its order of potency against excitatory amino acid-induced responses in the hippocampus is NMDA > homocysteate > aspartate > glutamate > quisqualate. CPP has no efect on lateral perforant path responses or on inhibition of these responses by 2-amino-4-phosphonobutyrate. Finally, at doses that do not affect Schaffer collateral synpatic transmission, CPP reversibly blocks the induction of long-term potentiation of Schaffer synaptic responses. This new compounds is, therefore, a higly selective brain NMDA receptor blocker, and the most potent such by nearly an order of magnitude.     

Utilization of the resolvedl-isomer of 2-amino-6-phosphonohexanoic acid (l-AP6) as a selective agonist for a quisqualate-sensitized site in hippocampal CA1 pyramidal neurons

Brief exposure of rat hippocampal slices to quisqualic acid (QUIS) sensitizes neurons to depolarization by the α-amino-ω-phosphonate excitatory amino acid (EAA) analogues AP4, AP5 and AP6. These phosphonates interact with a novel QUIS-sensitized site. Whereasl-AP4 andd-AP5 cross-react with other EAA receptors,dl-AP6 has been shown to be relatively selective for the QUIS-sensitized site. This specificity ofdl-AP6, in conjuction with the apparent preference of this site forl-isomers, suggested that the hitherto unavailablel-isomer of AP6 would be a potent and specific agonist. We report the resolution of thed- andl-enantiomers of AP6 by fractional crystallization of thel-lysine salt ofdl-AP6. We also report the pharmacological responses of kainate / AMPA, NMDA, lateral perforant pathl-AP4 receptors and the CA1 QUIS-sensitized site tod- andl-AP6, and compare these responses to thed- andl-isomers of AP3, AP4, AP5 and AP7. Thed-isomers of AP4, AP5 and AP6 were 5-, 3- and 14-fold less potent for the QUIS-sensitized site than their respectivel-isomers. Whilel-AP4 andl-AP5 cross-reacted with NMDA andl-AP4 receptors,l-AP6 was found to be highly potent and specific for the QUIS-sensitized site (IC₅₀ = 40 μM). Its IC₅₀ values for kainate / AMPA, NMDA andl-AP4 receptors were > 10, 3 and 0.8 mM, respectively. As with AP4 and AP5, sensitization tol-AP6 was reversed byl-α-aminoadipate.     

N-methyld-aspartate acts as an antagonist of the photoreceptor transmitter in the carp retina

Glutamate, aspartate, and their agonists, kainate, quisqualate, cysteine sulfinate and N-methyl-d-aspartate (NMDA), were applied to the isolated carp retina while recording from horizontal cells. All these agents, except NMDA, depolarized horizontal cells membrane and reduced responses to light, thus mimicking the effect of the endogenous photorecepto transmitter. Application of NMDA, on the other hand, caused a membrane hyperpolarization of horizontal cells in the dark, an effect different from its depolarizing effect as observed elsewhere in the central nervous system. NMDA also reduced or blocked the light responses of these cells as well as the depolarizing responses to applications of glutamate, aspartate or kainate. Effects of NMDA on the spectral properties of the horizontal cell responses were identical to the effects of the acidic amino acid receptor antagonists α-methyl glutamate, and α-amino adipate. Thus, NMDA appears to act as a weak antagonist to the photoreceptor transmitter, whose receptors on the horizontal cell membrane interact with a glutamate-like substance but appear atypical of glutamate receptors described elsewhere in the brain.     

Inhibition of phosphoinositide hydrolysis by the novel neurotoxinβ-N-oxalyl-l-α, β-diaminopropionic acid (l-BOAA)

Inhibition by a recently isolated neurotoxic amino acid, β-N-oxalyl-l-α, β-diaminopropionic acid, (l-BOAA), of stimulated phosphoinositide hydrolysis was studied in rat brain cerebral cortical slices.l-BOAA inhibited the norepinephrine-stimulated response but did not affect hydrolysis induced by 55 mM K⁺, carbachol in the presence of 20 mM K⁺. The inhibition was concentration-dependent with anIC₅₀ of 300 μM. This inhibition was insensitive to the excitatory amino acid antagonists, γ-glutamylglycine, glutamic acid diethyl ether, CNQX, AP-4, AP-7, or kynurenate. Thus, we propose that thel-BOAA-mediated inhibition of the norepinephrine-stimulated response was due to an interaction at a novel site, which may also be sensitive to quisqualate (see discussion). The mechanism of the inhibition is still unknown but was not prevented by inhibition of phospholipase A₂ or polyamine synthesis and it was not affected by blockade of chloride channels. However, the presence of 20 mM K⁺ completely blocked the inhibitory effect ofl-BOAA on norepinephrine-stimulated phosphoinositide hydrolysis.     

Evidence forN-methyl-d-aspartic acid receptor-mediated modulation of the commissural input to central vestibular neurons of the frog

We have investigated the role ofN-methyl-d-asparte (NMDA) receptors in the excitatory synaptic transmission to central vestibular neurons in the isolated superfused brainstem of the frog. In superfusate containing 1 mM Mg²⁺ field potentials in the vestibular nuclei evoked by electrical stimulation of either the ipsi- or the contralateral VIIIth nerve were not affected by bath-appliedd-2-amino-5-phosphonovaleric acid (D-APV, 25–50 μM), a selective NMDA antagonist. In a low Mg²⁺ solution postsynaptic field potential components were larger than control but still unaffected by D-APV. Ipsi- and contralaterally evoked excitatory postsynaptic potentials (EPSPs) differed in their shape parameters as well as their pharmacological sensitivity. Ipsilaterally evoked EPSPs were not affected by D-APV and had a rise time that was faster than that of contralaterally evoked EPSPs. The peak amplitude of the latter was reduced by D-APV (25–50 μM) to about 65% of the control value in the presence of 1 mM Mg²⁺. During bath application of NMDA (100 μM) an increased input resistance and repetitive de- and hyperpolarizing membrane potential shifts were observed. Similar events were observed during a reduction of the Mg²⁺ concentration. Bath application of NMDA (0.1–1 μM) resulted in an enhanced size of the recorded EPSPs. Dendritic and somatic EPSPs were stimulated on a computer with the assumption of a constant NMDA receptor activation and a pulse-like non-NMDA receptor activation. The results of these stimulations are consistent with the hypothesis that the efficacy of non-NMDA-mediated vestibular commissural synaptic transmission is modulated through tonically activated NMDA receptors.     

High extracellular glycine does not potentiate N-methyl-

Auditory nerve fibers transmit signals from the cochlea to the 3 regions of the cochlear nuclear complex, the anteroventral (AVCN), posteroventral, and dorsal cochlear nucleus in the brainstem. It has been suggested that the amino acids l-aspartate and l-glutamate might serve as a neurotransmitter in auditory nerve fibers^{6–10,13,17–20}. The sensitivity of postsynaptic cells in the cochlear nuclei to these amino acids has been tested by iontophoretic techniques^4,9,10. One difficulty with these experiments is that responses were recorded only extracellularly. A second difficulty is that the concentrations needed to affect cells could not be determined. To avoid these difficulties a brain slice preparation was used to test the sensitivity of cells in the AVCN to bath applied l-glutamate and l-aspartate at concentrations ranging from 10⁻⁵ to 10⁻² M. All cells that were tested in the cochlear nuclear complex were insensitive at all concentrations used; the resting potentials and the input resistances remained unchanged and the synaptic responses to electrical stimulation of the auditory nerve were not desensitized. All cells that were tested in the hippocampus, however, depolarized in the presence of 10⁻⁴ M l-glutamate and l-aspartate. The synaptic responses to electrical stimulation of the auditory nerve were not blocked by d-α-aminoadipate, an amino acid which has been shown to block excitation of cells in the cochlear nuclei by auditory nerve fibers¹⁰. The results are not consistent with l-glutamate and l-aspartate serving as neurotransmitters in the AVCN.     

Cells in the anteroventral cochlear nucleus are insensitive to l-glutamate and l-aspartate; excitatory synaptic responses are not blocked by d-α-aminoadipate

Auditory nerve fibers transmit signals from the cochlea to the 3 regions of the cochlear nuclear complex, the anteroventral (AVCN), posteroventral, and dorsal cochlear nucleus in the brainstem. It has been suggested that the amino acids l-aspartate and l-glutamate might serve as a neurotransmitter in auditory nerve fibers^{6–10,13,17–20}. The sensitivity of postsynaptic cells in the cochlear nuclei to these amino acids has been tested by iontophoretic techniques^4,9,10. One difficulty with these experiments is that responses were recorded only extracellularly. A second difficulty is that the concentrations needed to affect cells could not be determined. To avoid these difficulties a brain slice preparation was used to test the sensitivity of cells in the AVCN to bath applied l-glutamate and l-aspartate at concentrations ranging from 10⁻⁵ to 10⁻² M. All cells that were tested in the cochlear nuclear complex were insensitive at all concentrations used; the resting potentials and the input resistances remained unchanged and the synaptic responses to electrical stimulation of the auditory nerve were not desensitized. All cells that were tested in the hippocampus, however, depolarized in the presence of 10⁻⁴ M l-glutamate and l-aspartate. The synaptic responses to electrical stimulation of the auditory nerve were not blocked by d-α-aminoadipate, an amino acid which has been shown to block excitation of cells in the cochlear nuclei by auditory nerve fibers¹⁰. The results are not consistent with l-glutamate and l-aspartate serving as neurotransmitters in the AVCN.     

Dilatation of cerebral arterioles in response to N-methyl-d-aspartate: role of CGRP and acetylcholine

The purpose of these experiments was to examine mechanisms by which N-methyl-d-aspartate (NMDA) produces nitric oxide-dependent vasodilatation in brain. Some nitrovasodilators appear to dilate cerebral arterioles, in part, by release of calcitonin gene-related peptide (CGRP) from trigeminal fibers. The first goal of this study was to examine the hypothesis that dilatation of cerebral arterioles in response to NMDA is mediated by activation of receptors for CGRP. Diameters of cerebral arterioles were measured using a closed cranial window in anesthetized rabbits. Topical CGRP (1 and 10 nM) dilated cerebral arterioles by 30 ± 9 (mean±S.E.M.) and 72 ± 9%, respectively, from a control diameter of 94 ± 7 μm. This response was inhibited almost completely by the CGRP antagonist CGRP(8–37) (0.5 μM). NMDA (100 and 300 μM) dilated cerebral arterioles by 14 ± 5and38 ± 7% in the absence and 20 ± 5%and30 ± 6% in the presence, respectively, of CGRP(8–37). Neurons may release acetylcholine in response to activation with NMDA. The second goal of the present study was to examine the hypothesis that dilatation of cerebral arterioles in response to NMDA is mediated by acetylcholine. Topical atropine (2 μg/ml) completely inhibited dilatation of cerebral arterioles in response to acetylcholine, but had no effect on vasodilatation in response to NMDA. Thus, vasodilatation of cerebral arterioles in response to NMDA does not appear to be dependent on activation of receptors for CGRP or acetylcholine.     

(±)-β-Parachlorophenylglutamate selectively enhances the depolarizing response to l-homocysteic acid in neocortical neurons of the rat: evidence for a specific uptake system

The effect of (±)-β-parachlorophenylglutamate (CP) on depolarizations induced by iontophoretically applied l-glutamate, l-aspartate, l-homocysteate (l-HCA) and d-HCA was investigated in neurons of the rat neocortex in vitro. CP, a reported blocker of amino acid uptake, strongly enhanced l-HCA responses to the other amino acids remained little affected. This action was observed irrespective of whether CP was administered iontophoretically or pneumatically from micropipettes. CP (5 mM) administered alone had no effect on membrane potential. These findings suggest the existence of a specific uptake system for l-HCA providing further evidence in favour of a possible function of l-HCA as an endogenous ligand for the N-methyl-d-aspartate receptor in the rat neocortex.     

Novel recognition site forl-quisqualate sensitizes neurons to depolarization byl-2-amino-4-phosphonobutanoate (l-AP4)

Brief exposure of rat hippocampal slices tol-quisqualate sensitizes pyramidal neurons to depolarization byl-2-amino-4-phosphonobutanoate (l-AP4). We report here experiments designed to clarify the duration, pharmacology, mechanism, and pathway specificity of this ‘QUIS-effect’. The quisqualate-induced sensitization tol-AP4 decreases only 3-fold over a 4 h period. No compound besides quisqualate has been found to induce the QUIS-effect, including quisqualate analogues, potent excitatory amino acid agonists,l-glutamate,l-aspartate, and compounds known to stimulus second messenger systems in hippocampal slices. Of 43 compounds assayed here, only 5 are able to block the induction of the QUIS-effect. Although these blockers are also potent ligands at a chloride-dependent glutamate uptake site, the marked difference in rank ordering of compounds for QUIS-effect blockade and uptake site potency suggests that the QUIS-effect is not induced through this uptake site. The QUIS-effect can be induced in the CA1 region, the medial perforant path, and the lateral olfactory tract of the rat, and in the guinea pig CA1. It cannot be induced in thel-AP4-sensitive rat lateral perforant path (LPP), suggesting that the receptors forl-AP4 in the LPP may be distinct from those that are sensitized by quisqualate in the other pathways.     

l-Glutamate evoked release of GABA from cultured avian retina cells does not require glutamate receptor activation

γ-Aminobutyric acid (GABA) and l-glutamate are the major inhibitory and excitatory transmitters in the central nervous system. Recent evidence has indicated that l-glutamate may stimulate GABA release by a novel exchange mechanism (Nascimento and De Mello, J. Neurochem., 1985, 45: 1820–1827). Here we provide strong support for this hypothesis by showing that the l-glutamate-evoked release of [³H]GABA from cultured avian retina cells is not dependent on the activation of excitatory amino acid receptors. Retina cells were found to incorporate [³H]GABA into a pool that was released when cultures were treated with l-glutamate (100 μM). This release was unaffected when calcium ions were removed, but was prevented when NaCl was replaced by LiCl. d-Aspartate, which in tracer experiments was shown to be taken into cells by the same carrier as l-glutamate, was also able to evoke release of [³H]GABA, with the same requirement for NaCl. In addition, l-glutamate and d-aspartate uptake by retina cells was inhibited in more then 80% when the uptake was measured in the presence of LiCl. As opposed to GABA, the release of acetylcholine (ACh) promoted by l-glutamate showed characteristics of classical mechanisms of neurotransmitter release. Glutamate-induced efflux of ACh was Ca²⁺-dependent and was not affected when NaCl was replaced by LiCl. Also, d-aspartate was ineffective in eliciting the release of ACh. Even at high concentrations, antagonists of excitatory amino acid receptors were unable to diminish the glutamate-evoked release of [³H]GABA. The antagonists tested were: 2-amino-5-phosphonovalerate and d-α-aminoadipate, which are relatively selective for N-methyl-d-aspartate receptors; l-glutamate diethyl ester, which selectively blocks quisqualate sites, and cis-2,3-piperidine dicarboxylate, w which non-selectively antagonizes all types of excitatory amino acid receptors. The data show that excitatory amino acid receptors are not involved in the Na-dependent l-glutamate-evoked release of [³H]GABA and support the concept that a glutamate-GABA exchange mechanism operates in the central nervous system. Since glutamate and GABA exert antagonistic effects on the electrophysiology of nerve cells, this mechanism might be important in regulating neuronal excitability.     

Effects of d-aspartate on excitatory amino acid-induced release of [H]GABA from goldfish retina

The rate of release of [³H]GABA from isolated intact goldfish retinas was studied. Release of [³H]GABA is markedly stimulated by the inclusion in the incubation medium of the photoreceptor neurotransmitter candidates l-glutamate (l-Glu) and l-aspartate (l-Asp), and the glutamate analogs, kainate and quisqualate. At micromolar concentrations, kainate and quisqualate are effective releasers of [³H]GABA, whereas millimolar concentrations of l-Glu and l-Asp are required to release comparable amounts of [³H]GABA. The d-isomers of aspartate (d-Asp) and glutamate (d-Glu) are able to release [³H]GABA, but only when applied at high concentrations (3–30 mM). In the presence of 5 mM d-Asp, the effect of l-Glu in releasing [³H]GABA was markedly potentiated. This dose-response curve of l-Glu was shifted to the left in the presence of d-Asp, although the maximal amount of release was unchanged. d-Asp at 5 mM only slightly increased the GABA release induced by quisqualate, and it did not increase the GABA release induced by kainate. Finally, low concentrations of l-Asp were potentiated by d-Asp, but higher concentrations of l-Asp (3–10 mM) were clearly inhibited by this agent. This biphasic effect of d-Asp on l-Asp-induced release of [³H]GABA is a possible explanation for previously conflicting reports of d-Asp's effect on l-Asp action^2,8,29. Our data suggest that d-Asp has both pre- and postsynaptic sites of action.     

Kinetic analysis ofl-leucine transport across the blood-brain barrier

Unidirectionall-leucine influx across cerebral capillaries was measured at different concentrations with an in situ rat brain perfusion technique, which has been several advantages over presently-used methods such as the Brain Uptake Index (BUI) technique. The maximal influx rate (V_max) and half-saturation concentration (K_m) equaled1.07 ± 0.02 × 10^3- μmol·s⁻·⁻¹and0.026 *+- 0.002 μmol·ml⁻¹, respectively, for the saturable component, and the constant for non-saturable equaled6.8 ± 1.4 × 10⁻⁵s⁻¹. These values differ by 3–4-fold from respective values obtained with the BUI technique.     

Effect ofd- andl-α-aminoadipate on the efflux ofl-aspartate,l-glutamate and γ-aminobutyrate from superfused rat brain slices

d-α-Aminoadipate (d-AA) andl-α-aminoadipate (l-AA) were found to significantly reduce spontaneous efflux of [¹⁴C]l-aspartate from preloaded rat brain slices. Onlyd-AA significantly reduced spontaneous efflux of [¹⁴C]l-glutamate and [³H]γ-aminobutyric acid (GABA);l-AA reduced but not significantly the efflux of these 2 labeled amino acids.d-AA reduced K⁺-stimulated release of [¹⁴C]l-aspartate and [^14]C]l-glutamate significantly, andl-AA that of [³H]GABA significantly. Since bothd-AA andl-AA inhibit the uptake ofl-aspartate,l-glutamate and GABA, their effects on the efflux of these amino acids are more specific. These results also suggest that it is unlikely that the depressant effect ofd-AA, and the excitant effect ofl-AA on neurons when applied locally by iontophoresis are secondary to the accelerated or decelerated release of more specific transmitter amino acids from neighboring cells.     

Role of nitric oxide, adenosine,N-methyl-d-aspartate receptors, and neuronal activation in hypoxia-induced pial arteriolar dilation in rats

In this study, we tested the hypothesis that nitric oxide (NO) and adenosine (ADO) are the principal mediators of severe hypoxia-induced vasodilation. In addition, we examined whether activation ofN-methyl-d-aspartate (NMDA) receptors and/or perivascular nerves plays a role. A closed cranial window and intravital microscopy system was used to monitor diameter changes in pial arterioles ( 40 μm) in anesthetized rats. The relative contributions of ADO, NMDA, NO, and neuronal activation to hypoxic cerebrovasodilation were assessed using the blockers 8-sulfophenyltheophylline (8-SPT), MK-801, nitro-l-arginine methylester (LNAME), and tetrodotoxin (TTX). Two experimental series were studied. In the first, we tested the effects of NOS inhibition, via topical L-NAME (1 mM), on moderate (PaO₂ ≈ 46 mmHg)then severe (PaO₂ ≈ 34 mmHg) hypoxia-induced dilation. To confirm that L-NAME was affecting specifically NO-dependent responses, we also examined, in each experiment, the vasodilatory responses to topical applications of NOS-dependent (adenosine diphosphate (ADP); acetylcholine (ACh)(and -independent (sodium nitroprusside (SNP)) agents, in the presence of L-NAME or, in controls, the presence of D-NAME or no added analogue. In the second series, topical suffusions of ADP, ADO, and NMDA were sequentially applied, followed by 5 min exposure to severe hypoxia (PaO, ≈ 32 mmHg). Following return to normoxia, a suffusion of either 8-SPT (10 μM), MK-801 (10 μM), TTX (1 μM), or 8-SPT + MK-801 was initiated (or, in controls, application of a drug-free suffusate was maintained), and the above sequence repeated. In control, TTX, and 8-SPT + MK-801 experiments, baseline conditions were then restored and hypercapnia (PaCO₂ = 70–85 mmHg) was imposed. In the series 1 control groups, moderate and severe hypoxia elicited ≈ 20% and 35–40% increases in diameter, respectively. L-NAME attenuated ADP- and ACh-induced dilations, did not alter the arteriolar responses to SNP or moderate hypoxia, but prevented further dilation upon imposition of severe hypoxia. This suggested that 45–50% of the severe hypoxia response was NO-dependent. In series 2, 8-SPT blocked the adenosine response and reduced severe hypoxia-induced dilation by 46%. MK-801 predictably blocked NMDA-induced relaxation and reduced the hypoxic response by 42%. When combined, 8-SPT and MK-801 affected hypoxic vasodilation additively. After TTX, the ADP and ADO responses were normal, but NMDA and hypoxia responses were completely blocked. Hypercapnia-induced dilation was unaffected by TTX or 8-SPT + MK-801. The results imply that severe hypoxia-induced release of NO and ADO, and the accompanying pial arteriolar dilation, are wholly dependent on the capacity to generate action potentials in perivascular nerves. The similarity of the L-NAME and MK-801 effects on hypoxic cerebrovasodilation suggests that the NO-dependency, to a large degree, derives from NMDA receptor activation.     

Inhibitory effects of hypoxia and adenosine on N-methyl-d-aspartate-induced pial arteriolar dilation in piglets

Our previous studies have indicated that oxygen radicals, produced during reoxygenation following short-term arterial hypoxia, lead to sustained suppression of cerebral arteriolar responses to N-methyl-

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-aspartate (NMDA). However, whether arteriolar dilator responses to NMDA are reduced during arterial hypoxia has never been examined. In this study, we determined whether hypoxia or hypoxia-related metabolites such as adenosine or nitric oxide (NO) will reduce NMDA-induced arteriolar dilation. We have also determined the location of NMDA receptor- and brain nitric oxide synthase (bNOS)-positive neurons in the cerebral cortex. In anesthetized piglets, pial arteriolar diameters were determined using intravital microscopy. Baseline arteriolar diameters were 100 μm. Topical application of NMDA at concentrations of 10⁻⁵, 5×10⁻⁵ and 10⁻⁴ M resulted in dose-dependent vasodilation (9±2, 18±2 and 29±2% above baseline, respectively, n=21). Administration of theophylline (20 mg/kg, i.v.) had no effect on NMDA-dependent vasodilation, but it did block dilation to hypoxia (inhalation of 8.5% O₂). In theophylline-treated animals, NMDA responses were completely abolished during hypoxia (28±2 vs. 2±1%, respectively to 10⁻⁴ M, n=7) while sodium nitroprusside (SNP, 10⁻⁴ M) still dilated pial arterioles normally. NMDA-induced vasodilation was not modified after application and removal of adenosine (10⁻⁴ M; n=5) or SNP (10⁻⁵ M; n=4), or when SNP (10⁻⁷ M) was coapplied with NMDA (n=6). Conversely, coapplication of adenosine (10⁻⁶ M) attenuated NMDA responses (31±5 vs. 20±3%, n=7). We also found that NMDA receptor- and bNOS-containing neurons were located predominantly in layers II/III of the cortex. Proximity of these neurons to the cortical surface is consistent with diffusion of NO to pial arterioles as the mechanism of dilation to NMDA. We conclude that NMDA-induced cerebral arteriolar dilation is inhibited by hypoxia alone and by exogenous adenosine, but not by NO.     

A quantitative estimate of the contribution made by various receptor categories to the depolarizations evoked by some excitatory amino acids in the olfactory cortex

A study has been undertaken to assess the percentage contributions made by N-methyl-D-aspartate (NMDA), kainate and quisqualate receptors to the composite depolarizations evoked by L-cysteate, L-cysteinesulphinate, L-homocysteate and S-sulpho-L-cysteine in the rat olfactory cortex slice. The percentage contribution made by NMDA receptors, which was quantified by measuring the reduction in agonist responses in the presence of the highly selective NMDA receptor antagonist 2-amino-5-phosphonopentanoate (0.1 mM), was: L-homocysteate, 73%; S-sulpho-L-cysteine, 65%; L-cysteate, 42% and L-cysteinesulphinate, 30%. Responses mediated by NMDA, kainate and quisqualate receptors were abolished by a 'desensitization' procedure involving repeated application of a mixture containing high concentrations of the selective agonists followed by perfusion of the non-selective receptor antagonist cis-2,3-piperidine dicarboxylate (5 mM). Following this procedure, responses to L-homocysteate and S-sulpho-L-cysteine were almost abolished and simple calculation gave the contribution of kainate plus quisqualate receptors to the agonist responses as: L-cysteinesulphinate, 46%; L-cysteate, 34%; S-sulpho-L-cysteine, 28% and L-homocysteate, 23%. However, approximately 24% of the composite depolarizations evoked by L-cysteate and L-cysteinesulphinate was mediated by a mechanism not involving NMDA, kainate or quisqualate receptors, neither did it reflect possible electrogenic uptake of the amino acids nor an interaction with 2-amino-4-phosphonobutyrate receptors. It is suggested that this fraction of the depolarizations evoked by L-cysteate and L-cysteinesulphinate might be due to a non-receptor-mediated release of K+ or, perhaps, to activation of an as yet unidentified receptor category.