Medullary lateral tegmental field neurons influence the timing and pattern of phrenic nerve activity in cats.

In an effort to characterize the role of the medullary lateral tegmental field (LTF) in regulating respiration, we tested the effects of selective blockade of excitatory (EAA) and inhibitory amino acid (IAA) receptors in this region on phrenic nerve activity (PNA) of vagus-intact and vagotomized cats anesthetized with dial-urethane. We found distinct patterns of changes in central respiratory rate, duration of inspiratory and expiratory phases of PNA (Ti and Te, respectively), and I-burst amplitude after selective blockade of EAA and IAA receptors in the LTF. First, blockade of N-methyl-D-aspartate (NMDA) receptors significantly (P < 0.05) decreased central respiratory rate primarily by increasing Ti but did not alter I-burst amplitude. Second, blockade of non-NMDA receptors significantly reduced I-burst amplitude without affecting central respiratory rate. Third, blockade of GABAA receptors significantly decreased central respiratory rate by increasing Te and significantly reduced I-burst amplitude. Fourth, blockade of glycine receptors significantly decreased central respiratory rate by causing proportional increases in Ti and Te and significantly reduced I-burst amplitude. These changes in PNA were markedly different from those produced by blockade of EAA or IAA receptors in the pre-Bötzinger complex. We propose that a proper balance of excitatory and inhibitory inputs to several functionally distinct pools of LTF neurons is essential for maintaining the normal pattern of PNA in anesthetized cats.

General anaesthetic action at transmitter-gated inhibitory amino acid receptors.

Research within the past decade has provided compelling evidence that anaesthetics can act directly as allosteric modulators of transmitter-gated ion channels. Recent comparative studies of the effects of general anaesthetics across a structurally homologous family of inhibitory amino acid receptors that includes mammalian GABAA, glycine and Drosophila RDL GABA receptors have provided new insights into the structural basis of anaesthetic action at transmitter-gated channels. In this article, the differential effects of general anaesthetics across inhibitory amino acid receptors and the potential relevance of such actions to general anaesthesia will be discussed.

Compensatory physiological responses to chronic blockade of amino acid receptors during early development in spontaneously active organotypic cerebral cortex explants cultured in vitro.

Paired organotypic explants from rat occipital cortex were cultured for up to three weeks in the presence of selective blockers of amino acid receptor blockers, during which period spontaneous action potential generation was monitored electrophysiologically. In contrast to isolated explants (Corner, M.A., van Pelt, J., Wolters, P.S., Baker, R.E.and Nuytinck, R.H. (2002) Physiological e.ects of sustained blockade of excitatory synaptic transmission on spontaneously active developing neuronal networks--an inquiry into the reciprocal linkage between intrinsic biorhythms and neuroplasticity in early ontogeny. Neurosci. Biobehav. Rev., 26: 127-185), which upregulated their initially depressed spontaneous bursting activity only under conditions of N-methyl D-aspartate (NMDA) receptor blockade, cross-innervated co-cultures showed a large degree of functional recovery even when combined NMDA and AMPA receptor blockade was carried out. This compensatory activity could be eliminated by acute addition of a selective kainate receptor blocker to the medium. When kainate along with AMPA and NMDA receptor mediated activity was chronically suppressed, however, considerable functional recovery--in the form of recurrent burst discharges--took place gradually over a period of three weeks in vitro. These spontaneous bursts disappeared rapidly upon treatment with the muscarinic receptor blocker, atropine, but continuous low-level firing emerged at the same time. Similar "tonic" background activity was induced in control cultures as well, but without any noticeable reduction in burst discharges. Co-cultured neocortex explants, in which cyto-morphological maturation proceeds to a far greater degree than in isolated explants (Baker, R.E.and van Pelt, J. (1997) Co-cultured but not isolated cortical explants display normal dendritic development: a longterm quantitative study. Dev. Brain Res., 98: 21-27) are evidently capable of an astonishing degree of functional compensation for loss of excitatory synaptic drive during development. It could be shown, furthermore, that such homeostatic responses are not mediated largely by a weakening of inhibitory mechanisms in the absence of spontaneous firing. Chronic inhibitory synaptic blockade, on the other hand, led to intensified bursting activity which gradually normalized over a 3-week culture period. The cellular basis for this reversal of the disinhibited state, as well as for the residual neuronal firing even after cholinergic mechanisms have been largely eliminated, is at present unknown. The degree to which immature cortical networks attempt to compensate for altered levels of physiological activity, as documented in the present report, is another indication of how important such activity can be for normal development (see Corner, M.A., van Pelt, J., Wolters, P.S., Baker, R.E. and Nuytinck, R.H. (2002) Physiological e.ects of sustained blockade of excitatory synaptic transmission on spontaneously active developing neuronal networks-an inquiry into the reciprocal linkage between intrinsic biorhythms and neuroplasticity in early ontogeny. Neurosci. Biobehav. Rev., 26: 127-185).. At the same time, the large variations in overall firing levels and "macro-scale" temporal patterns from culture to culture within a given series, despite all attempts at identical preparation of the explants, can only mean that the "set-points" for such regulation are themselves subject to unknown ontogenetic factors which, apparently, are nonuniformly distributed even within a restricted region of the neocortex. On the other hand, it was striking to note that, regardless of age or treatment, an unexpected degree of consistency in temporal patterning existed at "mini-" and "micro-" time-scales (viz., EEG delta and beta frequency ranges, respectively) even when network bursting tendencies became greatly reduced in favor of tonic firing.

Cobalt accumulation in neurons expressing ionotropic excitatory amino acid receptors in young rat spinal cord: morphology and distribution.

Excitatory amino acids (EAA) acting on N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and kainate receptors play an important role in synaptic transmission in the spinal cord. Quantitative autoradiography and physiological experiments suggest that NMDA receptors are localized mainly in lamina II while kainate and AMPA receptors are found on both dorsal and ventral horn neurons. However the cell types expressing EAA receptors and their laminar distribution is not known. We have used a cobalt uptake method to study the morphology and distribution of spinal cord neurons expressing AMPA, kainate, or NMDA excitatory amino acid receptors in the lumbar enlargement of the rat spinal cord. The technique involved superfusion of hemisected spinal cords of 14 day-old rat pups in vitro with excitatory amino acid receptor ligands in the presence of CoCl2. Cobalt has been shown to enter cells through ligand-gated ion channels in place of Ca2+. Cells which accumulated cobalt ions following activation by ionotropic excitatory amino acid receptors were visualized histochemically. The cobalt uptake generated receptor-specific labeling of cells, as the NMDA receptor antagonist D-(-)-2-amino-(5)-phosphonovaleric acid (D-AP-5) (20 microM) blocked the NMDA, but not kainate-induced cobalt uptake. The kainate-induced cobalt labeling was reduced by the non-selective excitatory amino acid receptor antagonist kynurenic acid (4 mM). Passive opening of the voltage-gated Ca(2+)-channels by KCl (50 mM) did not result in cobalt uptake, indicating that cobalt enters the cells through ligand-gated Ca(2+)-channels. AMPA (500 microM), kainate (500 microM), or NMDA (500 microM) each induced cobalt uptake with characteristic patterns and distributions of neuronal staining. Overall, kainate induced cobalt uptake in the greatest number of neuronal staining. Overall, kainate induced cobalt uptake in the greatest number of neuronal perikarya while NMDA-induced uptake was the lowest. AMPA and kainate, but not NMDA superfusion, resulted in cobalt labeling of glial cells. Our results show that the cobalt uptake technique is a useful way to study the morphology and distribution of cells expressing receptors with ligand-gated Ca2+ channels.

Antinociceptive effects of NMDA and non-NMDA receptor antagonists in the tail flick test in mice.

Inhibition of spinal glutamate receptors induces antinociceptive effects in numerous animal models of pain. The present study compares the effects of intrathecally administered N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptor antagonists on nociceptive responses in the tail flick test. Potency of antagonists at NMDA and alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors was first measured by electrical assays in Xenopus oocytes expressing rat cerebral cortex poly(A)+ RNA. Subsequently, Swiss Webster mice were injected intrathecally with the antagonists and tested for antinociception. The drugs tested were: NBQX and GYKI-52466, selective AMPA receptor antagonists, ketamine, MK-801, R(+) HA-966 and ACEA-0762, selective NMDA receptor antagonists, and ACEA-1031, ACEA-1328 and ACEA-0593, NMDA receptor antagonists that also show inhibition of non-NMDA receptors. Selective NMDA receptor antagonists induced essentially no antinociceptive effects in the tail flick test. Antinociceptive activity generally correlated with inhibition of AMPA receptors. The exception was the non-competitive AMPA receptor antagonist GYKI-52466, which was unexpectedly weak. This may be due to inadequate dosing, because the compound has limited solubility, or may be due to differences in the non-NMDA receptor subtype-selectivity profile of GYKI-52466 as compared to competitive antagonists such as NBQX. Overall, our results suggest that inhibition of spinal non-NMDA receptors is the primary, and necessary, mechanism of antinociception by these drugs in the tail flick test in mice.

Effects of excitatory amino acid receptor antagonists on a capsaicin-evoked nociceptive reflex: a comparison with morphine, clonidine and baclofen.

The rat isolated spinal cord-tail preparation has been employed to examine the effects of several antinociceptive drugs and excitatory amino acid (EAA) receptor antagonists on nociceptive reflexes (recorded in ventral roots) stimulated by peripheral application of capsaicin (CAP). Non-nociceptive monosynaptic and polysynaptic dorsal root-evoked ventral root potentials (DR-VRPs) were also examined. Morphine (0.01-3 microM) and clonidine (0.03-1 microM) inhibited CAP-stimulated activity, but not the non-nociceptive dorsal root-evoked monosynaptic reflex (MSR) or polysynaptic (PSR) activity. These effects were antagonized by naloxone and efaroxan, respectively. The AMPA/KA receptor antagonists CNQX (0.1-100 microM) and DNQX (0.1-30 microM) blocked nociceptive activity and were 4-fold selective for CAP-evoked potentials compared to the monosynaptic reflex. Kynurenate (1-300 microM), DL-AP-4 (3-300 microM), L-AP-4 (3-300 microM), and the GABAB receptor agonist baclofen (0.1-10 microM), inhibited all evoked potentials with relatively little selectivity between nociceptive and non-nociceptive responses. NMDA receptor antagonism by AP-5 (100 microM) reduced nociceptive and non-nociceptive potentials by a maximum of 30-33%. These data indicate that AMPA/KA receptor-mediated synapses are involved in acute spinal nociceptive transmission and suggest that AMPA/KA receptor subtypes could provide novel analgesic targets.

Centrally administered non-NMDA but not NMDA receptor antagonists block peripheral knee joint inflammation.

An experimental arthritis of the knee joint resulted in limping, guarding, and an increased response to heat stimuli (heat hyperalgesia). Spinal administration of the non-N-methyl-D-aspartate (non-NMDA) antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), significantly reduced the degree of peripheral inflammation, thermal and behavioral manifestations of arthritis. NMDA antagonists had no effect on the inflammation but did prevent the development of the heat hyperalgesia. Thus, central non-NMDA receptors play a major role in the development of peripheral inflammation while both non-NMDA and NMDA receptors are involved in the development of heat hyperalgesia.

Characterization of glutamate efflux from preoptic area synaptosomes.

Treatment of ovariectomized rats in vivo with ovarian steroids has been found to influence the efflux of glutamate and gamma-aminobutyric acid from preoptic area synaptosomes incubated in vitro. Since these studies indicated a possible role of the glutamate carrier in steroid-modulated release of amino acids, the present studies examined the characteristics of efflux of glutamate and of the carrier system for glutamate in synaptosomes of the preoptic area derived from ovariectomized hormone-treated rats. The efflux of [3H]glutamate from preoptic area synaptosomes, was induced by glutamate and by the glutamate carrier agonist, D-aspartate; the putative glutamate carrier antagonist dihydrokainate failed to block this efflux. Dihydrokainate inhibited the uptake of glutamate but it was less effective than D-aspartate. The excitatory amino acid receptor agonists, N-methyl-D-aspartate and kainate were without effect while quisqualate modestly stimulated the efflux of [3H]glutamate. Efflux of [3H]glutamate, induced by glutamate itself or by D-aspartate was not blocked by the excitatory amino acid receptor antagonists, D-2-amino-5-phosphonovaleric acid, 6,7-dinitroquinoxaline-2,3-dione or kynurenate. Glutamate-induced efflux of [3H]glutamate did not require external Ca2+. Glutamate altered neither the basal nor the potassium-induced increases in the intrasynaptosomal concentration of Ca2+ as measured by the fura-2 method. Glutamate-induced efflux of [3H]glutamate was blocked by the putative chloride channel antagonist, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. It is concluded that the glutamate-induced efflux of [3H]glutamate in synaptosomes of the preoptic area is a carrier-mediated process that does not require activation of receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

GABAA receptor-mediated excitation of nociceptive afferents in the rat isolated spinal cord-tail preparation.

Algogens such as capsaicin, bradykinin, acetylcholine, 5-hydroxytryptamine and potassium ions applied to exposed tail skin of the rat isolated spinal cord-tail preparation evoke a ventral root response consisting of depolarization and spiking activity. L-glutamate and kainate also evoke similar reflexes. All these compounds evoke depolarization of afferent axons or dorsal root ganglion cells. Since GABA depolarizes unmyelinated afferent fibers, the ability of GABA receptor agonists to activate cutaneous nociceptive afferents has been examined. GABA superfused over exposed tail skin evoked a ventral root reflex essentially identical to that produced by capsaicin (3 microM). The EC50 was 27 microM. Muscimol, 3-aminopropane sulphonate, isoguvacine and beta-alanine had effects comparable to GABA, with EC50 values of 9.6, 26, 56 and 870 microM respectively. Baclofen (100 microM) or glycine (10 mM) had no effect. Bicuculline applied to the tail competitively antagonized GABA (Schild slope = -1.03) with a pA2 of 5.8. Spinal application of 1 microM morphine blocked the actions of GABA and capsaicin. These data indicate that GABAA receptors can depolarize and excite nociceptive afferents. GABA could be involved in nociception by contributing to firing of C-fibres, or by analogy to presynaptic inhibition in the spinal cord, may act to decrease neuropeptide transmitter release in cutaneous tissue.

Excitatory amino acid receptor mediation of sensory inputs to functionally identified dorsal horn neurons in cat spinal cord.

As excitatory amino acid receptors have been implicated in nociceptive sensory transmission, the principal objective of the present study was to investigate the effects of various excitatory amino acid antagonists on naturally evoked responses in spinal dorsal horn neurons. Extracellular single unit activity was recorded from functionally identified, spinal dorsal horn neurons in unanesthetized, decerebrated cats and in alpha-chloralose-anesthetized cats. The tests included iontophoretic application of the N-methyl-D-aspartate (NMDA) receptor antagonist 2-amino-5-phosphonovaleric acid (APV), the non-N-methyl-D-aspartate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and kynurenate, and also the intravenous administration of the N-methyl-D-aspartate receptor antagonist, ketamine. In addition, attempts were made to determine the effects on these neurons of iontophoretic application of the excitatory amino acid agonists, L-glutamate, N-methyl-D-aspartate, quisqualate, (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and domoate. Marked differences were noted in the actions of agonists and antagonists between the responses observed in the unanesthetized, decerebrated and the anesthetized animals. In decerebrated cats, responses to hair afferent stimulation were blocked by kynurenate, 6-cyano-7-nitroquinoxaline-2,3-dione and 2-amino-5-phosphonovaleric acid. Responses to noxious thermal stimulation were attenuated by 2-amino-5-phosphonovaleric acid and in one unit also by ketamine. Neither 6-cyano-7-nitroquinoxaline-2,3-dione nor kynurenate affected the responses to noxious thermal stimulation. The proportion of cells responding to the agonists were: N-methyl-D-aspartate 24/27 (89%), quisqualate 12/13 (92%) and domoate 6/7 (86%). In chloralose-anesthetized cats, responses to hair afferent stimulation were blocked by 6-cyano-7-nitroquinoxaline-2,3-dione and kynurenate but not by 2-amino-5-phosphonovaleric acid. Responses to noxious thermal stimulation were not affected by any of these antagonists, while the response to non-noxious thermal stimulation was blocked by 2-amino-5-phosphonovaleric acid, ketamine and kynurenate in the one neuron studied. The proportion of cells excited by the agonists differed from those observed in decerebrated cats: N-methyl-D-aspartate 9/32 (28%), quisqualate 50/54 (93%), (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate 19/23 (83%) and domoate 17/38 (45%). Application of the putative endogenous excitatory amino acid precursor N-acetyl-aspartyl-glutamate (NAAG) did not elicit a response in any of the neurons studied.(ABSTRACT TRUNCATED AT 400 WORDS)

Excitatory amino acids (EAAs) stimulate phosphatidylinositol turnover in adult rat striatal slices: interaction between NMDA and EAA metabotropic receptors.

The effect of excitatory amino acids (EAAs) on phosphatidylinositol (PI) turnover in adult rat striatal slices was investigated. Quisqualic acid (QA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainic acid (KA), ibotenic acid (IBO) and N-methyl-D-aspartic acid (NMDA) maximally increased inositol phosphate (IP) formation at 10 microM while trans-1-amino-cyclopentane-1,3-dicarboxylic acid (ACPD) was maximally effective at 100 microM. The NMDA channel blocker dizolcipine (MK-801) counteracted the effect of NMDA 10 microM and IBO 10 microM while it potentiated that of IBO 100 microM and IBO 1000 microM. Conversely, the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) prevented the effect of AMPA and KA and reduced that of QA (all at 10 microM). Lowering extracellular Ca2+ concentrations ([Ca2+]0) differentially affected the PI response to EAAs. The ACPD 30 microM effect was unchanged at low [Ca2+]0 (but abolished when EGTA 2 mM was added), while that of ACPD 100 microM was halved in 0.1 mM and almost abolished in a nominally free Ca2+ medium. NMDA 10 microM and AMPA 10 microM were ineffective at low [Ca2+]0 while NMDA 100 microM, ineffective in a 1.2 mM Ca2+ medium, strongly stimulated IP formation in 0.1 mM Ca2+ but not in a nominally free Ca2+ medium. The effect of NMDA on EAA metabotropic receptor agonist stimulated PI turnover was also studied. NMDA 10 microM potentiated the effect of ACPD 30 microM. This positive cooperation persisted at low [Ca2+]0 but not in the presence of EGTA. Conversely, NMDA 100 microM prevented the effect of ACPD 100 microM. This negative interference was reversed when Ca2+ was omitted from the medium. This study shows that in the adult rat striatum both EAA metabotropic and ionotropic receptor activation increases IP formation. A positive and negative interaction between NMDA and metabotropic receptor activation was also found to regulate PI turnover. The role of [Ca2+]0 in subserving the PI response to EAAs was made evident.

Excitatory amino acid receptor antagonists decrease hypoxia induced increase in extracellular dopamine in striatum of newborn piglets.

The present study tested the hypothesis that the increase in extracellular striatal dopamine during hypoxia is least partly associated with activation of N-methyl-D-aspartate (NMDA) and/or non-NMDA excitatory amino acid receptors. Studies were performed in anesthetized and mechanically ventilated 2-3 days old piglets. Hypoxic insult was induced by decreasing the oxygen fraction in inspired gas (FiO2) from 22 to 7% for 1 h, followed by 1 h reoxygenation at 22%. Cortical oxygen pressure was measured optically by oxygen dependent quenching of phosphorescence, and extracellular striatal dopamine was measured using in vivo microdialysis. The microdialysis probes were perfused with Ringer solution +/- 50 microM (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) or 50 microM 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX). One hour of hypoxia decreased the cortical oxygen pressure from 46 +/- 3 Torr to 10 +/- 1.8 Torr. In striatum perfused with Ringer, statistically significant increase in extracellular dopamine, to 1050 +/- 310% of control, was observed after 20 min of hypoxia. By 40 min of hypoxia the extracellular level of dopamine increased to 4730 +/- 900% of control; by the end of the hypoxic period the values increased to 18,451 +/- 1670% of control. The presence of MK-801 in the perfusate significantly decreased the levels of extracellular dopamine during hypoxia. At 20, 40 and 60 min of hypoxia extracellular level of dopamine increased to 278 +/- 94% of control, 1530 +/- 339% of control and 14,709 +/- 1095 of control, respectively. The presence of NBQX caused a statistically significant decrease, by about 30%, in the extracellular dopamine compared to control, only at the end of the hypoxic period. It can be concluded that in striatum of newborn piglets, the excitatory NMDA receptors but not the non-NMDA receptors may be modulating the changes in extracellular levels of dopamine. The NMDA receptor antagonist, MK-801, may exert part of its reported neuroprotective effect to hypoxic stress in striatum by decreasing the levels of extracellular dopamine.

Subthalamic nucleus modulates burst firing of nigral dopamine neurones via NMDA receptors.

The role of the subthalamic nucleus in the burst firing of dopamine neurones of the substantia nigra was investigated using extracellular single unit recordings combined with pressure or iontophoretic micro-injections in anaesthetized rats. Inhibition of subthalamic neurones by pressure injection of gamma-aminobutyric acid (GABA) regularized the burst firing pattern in eight out of 17 dopamine neurones. Bicuculline injection near subthalamic neurones increased their firing rate and increased burst discharge in a subpopulation of dopamine neurones tested (34 out of 102). The increase was depressed by iontophoresis of the N-methyl-D-aspartate (NMDA) antagonist (+-)2-amino,5-phosphonopentanoic acid (AP-5), but not of the non-NMDA antagonist, 6-cyano,7-nitroquinoxaline-2,3-dione (CNQX). These data suggest that the subthalamic nucleus promotes burst discharge in a subpopulation of substantia nigra dopamine neurones via NMDA receptors.

Differential effects of NMDA and non-NMDA receptor antagonists on spinal cutaneous vs muscular nociception in the cat.

To investigate the role of glutamate receptor subtypes in spinal transmission of nociceptive cutaneous and muscular afferents, nociceptive responses of WDR neurones in laminae IV-VI of the dorsal horn produced by noxious cutaneous and muscular stimulation were tested in the cat. With microelectrophoretic administration of the NMDA receptor antagonists, 2-amino-5-phosphonovalerate (APV) and ketamine, cutaneous nociceptive responses were preferentially reduced by more than 50%. In contrast, the non-selective excitatory amino acid receptor antagonist kynurenate (Kyn) markedly reduced both cutaneous and muscular nociceptive responses. The difference between APV- and Kyn-induced reductions of muscular nociceptive responses is statistically significant (chi 2-test, p < 0.01). The results suggest that NMDA and non-NMDA receptors preferentially mediate transmission of nociceptive information originating in skin and muscle, respectively.

NMDA receptors mediate neuronal burst firing in rat somatosensory cortex in vivo.

Two patterns of neuronal firing, bursting and regular spiking, are observed in the somatosensory cortex of anesthetized rats. The effects of excitatory amino acid receptor agonists and antagonists on these discharge types have been examined in vivo, in order to assess their involvement in the generation of such firing patterns. The analysis of interspike interval histograms indicates that N-methyl-D-aspartate (NMDA) receptors are involved in the generation of bursting responses while non-NMDA receptors mediate the regular spiking pattern of firing in individual neurons. It is also suggested that a basal level of ongoing neuronal excitation via the activation of non-NMDA receptors is required for bursting to be evoked through NMDA receptors.

Dextrorphan inhibits the release of excitatory amino acids during spinal cord ischemia.

The release of excitatory amino acids, particularly glutamate, into the extracellular space plays a causal role in irreversible neuronal damage after central nervous system ischemia. Dextrorphan, a noncompetitive N-methyl-D-aspartate receptor antagonist, has been shown to provide significant protection against cerebral damage after focal ischemia. We investigated the changes in extracellular neurotransmitter amino acid concentrations using in vivo microdialysis in a swine model of spinal cord ischemia. After lumbar laminectomies were performed, all animals underwent left thoracotomy and right atrial-femoral cardiopulmonary bypass with additional aortic arch perfusion. Microdialysis probes were then inserted stereotactically into the lumbar spinal cord. The probes were perfused with artificial cerebrospinal fluid and 15-minute samples were assayed using high-performance liquid chromatography. Group 1 animals (n = 9) underwent aortic clamping distal to the left subclavian and proximal to the renal arteries for 60 minutes. Group 2 animals (n = 7) were treated with dextrorphan before application of aortic clamps, and during aortic occlusion and reperfusion. Five amino acids were studied, including two excitatory neurotransmitters (glutamate and aspartate) and three putative inhibitory neurotransmitters (glycine, gamma-amino-butyric acid, and serine). Somatosensory-evoked potentials and motor-evoked potentials were monitored. Glutamate exhibited a threefold increase in extracellular concentration during normothermic ischemia compared with baseline values and remained elevated until 60 minutes after reperfusion. In animals treated with dextrorphan, glutamate concentrations decreased to one-third of baseline levels before aortic clamping and remained unchanged during ischemia and reperfusion. There was early loss of somatosensory-evoked potentials and motor-evoked potentials during ischemia in group 1 animals.(ABSTRACT TRUNCATED AT 250 WORDS)

NMDA and non-NMDA receptor antagonists protect against excitotoxic injury in the rat spinal cord.

The neuroprotective properties of the N-methyl-D-aspartate (NMDA) antagonist dizocilpine (MK-801) and the non-NMDA antagonists 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo[f]quinoxaline (NBQX) and alpha-methyl-4-carboxyphenylglycine (MCPG) were evaluated against neuronal injury produced by the intraspinal injection of NMDA and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA). Forty-nine animals were divided into eight groups in order to evaluate the effects of different drug combinations: (a) NMDA; (b) NMDA + MCPG; (c) NMDA + NBQX; (d) NMDA + MK-801; (e) AMPA; (f) AMPA + MCPG; (g) AMPA + MK-801; and (h) AMPA + NBQX. Drugs were microinjected into spinal segments T12-L3 through a micropipette attached to a Hamilton microliter syringe. Spinal cords were evaluated after a survival period of 48 h at which time NMDA and AMPA were found to produce morphological changes over the concentration ranges of 125-500 mM and 75-500 microM, respectively. Neuronal loss following injections of NMDA + MK-801 or AMPA + NBQX was significantly less than that following injections of NMDA or AMPA alone. By contrast, neuronal loss following co-injections of NMDA or AMPA with inappropriate antagonists, i.e., NMDA + NBQX/MCPG or AMPA + MCPG/MK-801, was not significantly different from that produced by NMDA or AMPA. The results suggest that elevations in spinal levels of glutamate followed by prolonged activation of NMDA and AMPA receptor subtypes initiate an excitotoxic cascade resulting in neuronal injury. Blockade of NMDA and AMPA effects by MK-801 and NBQX respectively confirms the well documented neuroprotective effects of these drugs and lends support to the potential importance of NMDA and especially AMPA receptor antagonists as therapeutic agents in the treatment of acute spinal cord injury.

NMDA and non-NMDA antagonists infused into the nucleus reticularis pontis caudalis depress the acoustic startle reflex.

The neural pathway that mediates the acoustic startle reflex has been proposed; however, the pharmacology underlying this reflex is less well known. The present study examined the role of excitatory amino acid receptors at the level of the nucleus reticularis pontis caudalis, a brainstem nucleus obligatory for the whole body startle reflex and implicated as the locus where extrinsic systems such as the amygdala may act to modulate acoustic startle. Twenty-nine rats, chronically implanted with bilateral cannulae aimed at the nucleus reticularis pontis caudalis, were tested to assess the effects of gamma-D-glutamylglycine (DGG), DL-2-amino-5-phosphonopentanoic acid (AP5), and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) on the amplitude of the acoustic startle reflex. Local infusion of each of the 3 compounds significantly reduced startle amplitude by as much as 70-80%. AP5 and CNQX attenuated startle over a dose range which indicated that the reticularis pontis caudalis may be much more sensitive to these compounds than other nuclei along the primary startle pathway. These results suggest that, at the level of the nucleus reticularis pontis caudalis, an excitatory amino acid neurotransmitter may mediate acoustic startle, and that both NMDA and non-NMDA receptor subtypes may be important for the expression of the acoustic startle reflex.

Long duration ventral root potentials in the neonatal rat spinal cord in vitro; the effects of ionotropic and metabotropic excitatory amino acid receptor antagonists.

Long duration, primary afferent evoked ventral root potentials (VRP's) have been recorded in vitro from hemisected spinal cords prepared from 8-12-day-old rat pups. Single shock stimulation of a dorsal root at stimulus strengths sufficient to recruit C/group IV afferent fibres evoked a long duration (11.9 +/- 1.2 s) ipsilateral VRP in all preparations. This long duration VRP consisted of two components, (i) a slow wave, time to peak 137.0 +/- 5.1 ms, the amplitude of which was reduced to 8.7% of mean control value in the presence of the N-methyl-D-aspartate (NMDA) antagonist D-AP5 (40 microM), (ii) a prolonged wave with a time to peak of 2.0 +/- 0.2 s which was partially resistant to D-AP5 (40 microM). Both the slow and the prolonged waves were unaffected following superfusion with the metabotropic excitatory amino acid (EAA) receptor antagonist L-AP3 (100-200 microM). Low frequency (1-10 Hz) repetitive stimulation (20 s duration) of high threshold dorsal root afferents evoked a temporal summation of synaptic activity which generated a progressively depolarizing VRP. This cumulative VRP was graded with frequency of stimulation (0.89 +/- 0.13 to 1.25 +/- 0.19 mV). The cumulative VRP was followed by a post-stimulus depolarization which outlasted the period of repetitive stimulation by tens of seconds (47.6 +/- 8.4 to 91.2 +/- 19.9 s). In the presence of AP5 the amplitude of the cumulative VRP was depressed to 54.5 +/- 11.5% of control values when low frequency (1.0 Hz) stimulation was used. The proportion of the cumulative VRP resistant to D-AP5 increased as the frequency of stimulation was increased to 10 Hz. The decay time of the post-stimulus depolarization was unaffected by AP5. Neither the amplitude nor the post-stimulus depolarization of the cumulative VRP was affected by 200 microM L-AP3. It is suggested that both an AP5 sensitive and AP5 insensitive potential contribute to the long duration VRP evoked in the neonatal rat spinal cord following single shock high threshold afferent stimulation. Moreover, the AP5 insensitive prolonged depolarization is manifest following sustained low frequency stimuli and higher frequency inputs.

NMDA and non-NMDA receptors on rat supraoptic nucleus neurons activated monosynaptically by olfactory afferents.

The recently discovered efferent projections from the main and accessory olfactory bulbs to the supraoptic nucleus (SON) were further investigated. Intracellular electrophysiological methods were used to determine (a) if these projections are monosynaptic, (b) which excitatory amino acid (EAA) receptor subtypes mediate responses to activation of these pathways and (c) whether the same receptor subtypes mediate responses of phasically firing (vasopressin) and continuously firing (putative oxytocin) neurons. Recordings were made from SON neurons in large explants and 500 microns thick horizontal slices, containing 2-6 mm of the piriform cortex and lateral olfactory tract (LOT). This allowed recording of synaptic responses to selective stimulation of the LOT. EPSPs in SON neurons faithfully followed stimulus frequencies of 50-100 Hz, indicating that these inputs were monosynaptic. Stimulus-evoked EPSPs were blocked by the non-specific EAA antagonist, kynurenate. Perifusion of the slice with Mg(2+)-free medium revealed the presence of NMDA receptors in addition to the non-NMDA receptors on both phasically and continuously firing cells, indeed, on all cells tested. Partial blockade of these EPSPs in Mg(2+)-free medium could be achieved with either the NMDA antagonist, AP5, or the non-NMDA antagonist, CNQX or NBQX. Full blockade of the stimulus-evoked EPSPs was effected by adding both types of antagonists to the medium, although spontaneous EPSPs were still observed in several cells. These results are consistent with prior studies showing both receptor subtypes in the SON. This is the first demonstration that afferent stimulation activates both subtypes in the same SON neuron regardless of its peptide content.