N-methyl-D-aspartate, quisqualate and kainate receptors are all involved in transmission of photic stimulation in the suprachiasmatic nucleus in rats.

In order to clarify the neuronal transmission mechanism of photic stimulation in the suprachiasmatic nucleus (SCN), the effects of agonists and antagonists for excitatory amino acid receptors on N-acetyltransferase (NAT) activity in the pineal gland were observed following the microinjection of drugs into both sides of the nuclei. N-Methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate, and kainate (which are selective agonists for three different subtypes, i.e. NMDA, quisqualate and kainate receptors, respectively) significantly decreased NAT activity similarly to the suppressive effect of light. Moreover, compared with a control group, all the groups pretreated with a selective competitive antagonist for NMDA receptor (D-2-amino-5-phosphonovalerate or 3-((+-)-2-carboxypiperazine-4-yl)-propyl-1-phosphonate), or a selective non-competitive antagonist for non-NMDA receptors (Joro spider toxin-3 or 1-naphthylacetyl spermine) partially blocked the suppressive effect of photic stimulation on NAT activity. These results suggest that NMDA, quisqualate and kainate receptors are all involved in mediating photic stimulation in the SCN.     

A grease-gap method for studying the excitatory amino acid pharmacology of CA1 hippocampal pyramidal cells

A grease-gap method for studying the pharmacology of CA1 hippocampal pyramidal cells was developed with use of rat hippocampal slices that included only area CA1 and the retrohippocampal area. These slices were transferred to a two-compartment superfusion chamber and the pyramidal cell bodies in area CA1 were separated from their axons in the subiculum with a grease barrier. The CA1 pyramidal cells were depolarized relative to their axons by superfusion with N-methyl-D-aspartate (NMDA), (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), kainate and L-glutamate. NMDA was unusually potent in the CA1-subiculum slice compared to other preparations. The NMDA receptor antagonists D(-)-2-amino-5-phosphonovalerate (D-AP5), phencyclidine and Mg2+ shifted the NMDA dose-response curve to the right in a parallel manner. Similarly, the quisqualate receptor antagonist pentobarbitone shifted the AMPA dose-response curve to the right. Schild plots for these antagonists had slopes insignificantly different from 1. These results are consistent with the presence of a substantial NMDA receptor reserve on CA1 pyramidal cells. They are also in line with the high density of excitatory amino acid receptors on CA1 hippocampal pyramidal cells and with the known pharmacological properties of these receptors. Grease-gap studies on the CA1-subiculum slice fill the need for a means of obtaining quantitative pharmacological data on CA1 pyramidal cells.     

Serotonin Differentially Modulates Responses Mediated by Specific Excitatory Amino Acid Receptors in the Rat Locus Coeruleus

Microiontophoretic application of selective agonists for the three major excitatory amino acid receptors, N -methyl- d -aspartate (NMDA), quisqualate and kainate, increased the discharge rate of noradrenergic locus coeruleus (LC) neurons in vivo. NMDA activation was selectively attenuated by iontophoretic application of 2-amino-5-phosphonopentanoate (AP5), an antagonist at NMDA receptors, whereas kainate- and quisqualate-evoked responses were attenuated by both NMDA and non-NMDA antagonists iontophoresis. NMDA- and quisqualate-evoked responses were significantly decreased by co-iontophoresis of serotonin (5-HT). When the NMDA receptor-mediated component of the response to kainate was blocked with AP5 iontophoresis, 5-HT increased the response of LC neurons to kainate. These results revealed that 5-HT differentially modulates the responsiveness of LC neurons to excitatory amino acids, depending on the receptor subtypes responsible for the neuronal activation.     

Acidic Amino Acids and Self-stimulation of the Prefrontal Cortex in the Rat: A Pharmacological Study

The effects of intraventricular and intracortical microinjections of acidic amino acid antagonists on self-stimulation (SS) of the medial prefrontal cortex (MPC) were investigated. Self-stimulation was measured by depressing a lever in a standard chamber. Spontaneous motor activity of the animal and SS of the contralateral non-injected MPC were used as control for non-specific effects of the drugs. Intraventricular microinjections of gamma-d-glutamylglycine (DGG), an antagonist of NMDA, kainate and quisqualate receptors, or 2-amino-5-phosphonovalerate (AP-5), a specific antagonist of NMDA receptors, produced a dose-related decrease of SS in the MPC. Spontaneous motor activity of the animal was not significantly affected. Unilateral microinjections into the medial prefrontal cortex of DGG or AP-5 produced a decrease of SS in the ipsilateral side while no effects were found on the contralateral MPC. On the contrary, intraventricular microinjections of gamma-d-glutamyltaurine (Glu-tau), an antagonist with more relative affinity for kainate and quisqualate receptors, produced a dose-related decrease of both self-stimulation and spontaneous motor activity of the rats. Moreover, intracortical microinjections of Glu-tau had no effect on self-stimulation of this cortical area. These results suggest that acidic amino acids through NMDA, but not kainate or quisqualate, receptors could be part of the neurochemical substrate underlying SS of the MPC in the rat.     

Primary culture of identified neurons from the visual cortex of postnatal rats

We have examined the properties of neurons from the visual cortex of postnatal Long Evans rats in dissociated cell culture. Visual cortex from rat pups 1-15 d old was subjected to enzymatic and mechanical dissociation to yield a suspension of single cells. Neurons plated onto collagen or a feeder layer of astrocytes rapidly extended processes and survived for 4-10 weeks. Antisera to glutamic acid decarboxylase, choline acetyltransferase, and vasoactive intestinal polypeptide stained 22 +/- 2, 2.3 +/- 0.3, and 2.4 +/- 0.2% of all neurons, respectively, suggesting that different neuronal classes survived roughly in proportion to their number in vivo. In order to study a particular identified class of cortical neurons, we prelabeled cells in vivo by retrograde transport of a fluorescent tracer. Neurons in layer V of visual cortex that project to the superior colliculus were labeled after injecting fluorescent latex microspheres into the colliculus. Retrogradely labeled neurons were readily identified immediately after dissociation and throughout the period in vitro. After 2 weeks in culture, labeled cells exhibited many ultrastructural features characteristic of pyramidal neurons in vivo. Intracellular recording techniques were used to evaluate the response properties of labeled layer V neurons, as well as other, unlabeled neurons, to excitatory amino acid agonists and antagonists. Glutamate and aspartate--as well as the synthetic agonists N-methyl-D-aspartate (NMDA), kainate, and quisqualate--excited every cortical neuron tested. The antagonist 2-amino-5-phosphonovaleric acid had no effect on responses to quisqualate and kainate but completely blocked depolarizations due to NMDA and aspartate and reduced depolarizations elicited by low concentrations of glutamate. Kynurenic acid, piperidine dicarboxylic acid, and gamma-D-glutamylglycine antagonized responses to all 5 of the agonists. These results provide evidence that corticocollicular neurons in culture express both NMDA-type and non-NMDA receptors for excitatory amino acids.     

Presynaptic kainate and N-methyl-d-aspartate receptors regulate excitatory amino acid release in the olfactory cortex

The potassium-evoked release of endogenous aspartate, glutamate and GABA from olfactory cortex slices has been monitored. Release of aspartate alone is significantly reduced by N-methyl-d-aspartate (NMDA) whilst kainate significantly increases release of both aspartate and glutamate. These effects, which are blocked by appropriate receptor antagonists, suggest that presynaptic NMDA and kainate receptors regulate excitatory amino acid release in the olfactory cortex.     

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.     

Excitatory amino acid receptors on sustained retinal ganglion cells in the kitten during the critical period of development

Effects of iontophoretically applied excitatory amino acid analogues, kainate, quisqualate and N-methyl-D-aspartate (NMDA) and their receptor antagonists on the sustained class of retinal ganglion cells were studied in the optically intact eye of pentobarbitone-anaesthetized kittens (7-9 weeks of age). These results were compared with the effects obtained in adult cats. All 3 excitatory amino acid agonists had excitatory actions on the majority of On- and Off-sustained ganglion cells in the kitten but at higher current levels than those required for adult cells, suggesting all 3 types of receptors of weaker sensitivity are present on the kitten cells. Whilst the relative potency of kainate, quisqualate and NMDA was 15:3:1 in the adult cells, it was 5:2:1 in the kitten cells. As for other neurones in the CNS, an increase in the potency of kainate receptors and a decrease in that of NMDA receptors appear, therefore, to characterize the postnatal development of the excitatory amino acid receptors on the retinal ganglion cells. In accordance with the agonist results, a broadband receptor antagonist, kynurenate, powerfully antagonised responses of kitten cells as well as those of adult cells. The pure NMDA receptor antagonist, 3((+-)-2-carboxypiperazin-4-yl)propyl-1-phosphonate (CPP), however, only suppressed spontaneous firing of kitten cells. Furthermore, in kitten cells, the visually-driven firing was depressed while the level of firing was raised by these excitatory amino acid analogous, and a long period of inhibition of firing followed the agonist-induced excitation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cardiovascular changes induced by the local application of glutamate-related drugs in the rat nucleus tractus solitarii

The effects of the local application of drugs acting on glutamatergic receptors in the nucleus tractus solitarii (NTS) were investigated in anesthetized rats. Unilateral microinjection of agonists (L-glutamate, L-aspartate, N-methyl-D-aspartate (NMDA) and quisqualate) produced a dose-dependent hypotension and bradycardia. The effects of NMDA were prevented by low doses of the selective NMDA-receptor antagonist, 2-amino-5-phosphonovalerate (2-APV), or by the mixed NMDA/kainate antagonist, gamma-D-glutamylglycine. The response to all agonists and the bradycardia which was elicited in response to the intravenous administration of phenylephrine (vagal reflex response) could be prevented by the local microinjection of the glutamate antagonists kynurenic acid (3 nmol) and 2-APV (10 nmol) into the NTS. The present data suggest that in the NTS, NMDA and quisqualate receptors are implicated in blood pressure reflex regulation.     

Baclofen: Effects on evoked field potentials and amino acid neurotransmitter release in the rat olfactory cortex slice

A study has been made of the in vitro effects of (+/-)- and (-)-baclofen on the evoked field potentials and release of endogenous amino acid neurotransmitter candidates (aspartate, glutamate, GABA and possibly taurine) which accompany electrical stimulation of the excitatory input to the olfactory cortex slice, the lateral olfactory tract. Baclofen appears to reduce the excitatory input to the GABA-utilizing inhibitory interneurones; this action was manifest as a drug-induced abolition of the field potential known as the P-wave (IC50 for (-)-baclofen, 1.7 +/- 0.4 microM) together with a simultaneous reduction in the synaptically evoked release of aspartase and glutamate from the cut surface of slices. Both these actions of baclofen exhibited concentration dependence and stereospecificity and were not antagonized by picrotoxin (25 microM) thereby suggesting that they are directly related. The consequence of this action of baclofen was the abolition of GABA-mediated presynaptic and postsynaptic inhibition together with their respective field potential correlates, the late N- and I-waves. (+/-)-Baclofen (5 and 25 microM) also inhibited the potassium-evoked release of aspartate and glutamate from small cubes of tissue but, except at a high concentration (1 mM), had no effect on GABA release. Baclofen (up to 1 mM) did not affect transmission either at the lateral olfactory tract-superficial pyramidal cell synapse, a site where aspartate is the likely neurotransmitter, or at the superficial pyramidal cell collateral-deep pyramidal cell excitatory synapse. It is proposed that: (i) the actions of baclofen on the olfactory cortex are the result of inhibition of aspartate and glutamate release, probably from deep pyramidal cell collaterals; and (ii) not all neurones utilizing excitatory amino acids as their neurotransmitters are subject to the inhibitory action of baclofen.     

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.     

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.     

Pharmacology of glutamate neurotoxicity in cortical cell culture: attenuation by NMDA antagonists

The antagonist pharmacology of glutamate neurotoxicity was quantitatively examined in murine cortical cell cultures. Addition of 1-3 mM DL-2-amino-5-phosphonovalerate (APV), or its active isomer D-APV, acutely to the exposure solution selectively blocked the neuroexcitation and neuronal cell selectively blocked the neuroexcitation and neuronal cell loss produced by N-methyl-D-aspartate (NMDA), with relatively little effect on that produced by either kainate or quisqualate. As expected, this selective NMDA receptor blockade only partially reduced the neuroexcitation or acute neuronal swelling produced by the broad-spectrum agonist glutamate; surprisingly, however, this blockade was sufficient to reduce glutamate-induced neuronal cell loss markedly. Lower concentrations of APV or D-APV had much less protective effect, suggesting that the blockade of a large number of NMDA receptors was required to acutely antagonize glutamate neurotoxicity. This requirement may be caused by the amplification of small amounts of acute glutamate-induced injury by subsequent release of endogenous NMDA agonists from injured neurons, as the "late" addition of 10-1000 microM APV or D-APV (after termination of glutamate exposure) also reduced resultant neuronal damage. If APV or D-APV were present both during and after glutamate exposure, a summation dose-protection relationship was obtained, showing substantial protective efficacy at low micromolar antagonist concentrations. Screening of several other excitatory amino acid antagonists confirmed that the ability to antagonize glutamate neurotoxicity might correlate with ability to block NMDA-induced neuroexcitation: The reported NMDA antagonists ketamine and DL-2-amino-7-phosphono-heptanoate, as well as the broad-spectrum antagonist kynurenate, were all found to attenuate glutamate neurotoxicity substantially; whereas gamma-D-glutamylaminomethyl sulfonate and L-glutamate diethyl ester, compounds reported to block predominantly quisqualate or kainate receptors, did not affect glutamate neurotoxicity. The present study suggests that glutamate neurotoxicity may be predominantly mediated by the activation of the NMDA subclass of glutamate receptors--occurring both directly, during exposure to exogenous compound, and indirectly, due to the subsequent release of endogenous NMDA agonists. Given other studies linking NMDA receptors to channels with unusually high calcium permeability, this suggestion is consistent with previous data showing that glutamate neurotoxicity depends heavily on extracellular calcium.     

Electrical activity of the reticular formation during aversive and appetitive conditioning in rats

An investigation has been made of the effects of noradrenaline on excitatory transmission at the lateral olfactory tract (LOT)-superficial pyramidal cell synapse of the rat olfactory cortex slice by measuring the effects of bath-applied noradrenaline on the amplitudes and latencies of the field potentials evoked on LOT stimulation. Low concentrations of noradrenaline (0.1-5 microM) facilitate transmission whereas higher doses (20-250 microM) depress transmission. Both these effects were completely blocked by non-selective alpha- and beta-adrenoceptor antagonists, by 2-amino-5-phosphonovaleric acid (an antagonist of excitatory amino acid receptors of the N-methyl-D-aspartate type) and by the methylxanthine theophylline. The depressant effects of noradrenaline were mimicked by bath application of GABA or adenosine and specifically antagonized by bicuculline and picrotoxin. In parallel experiments, noradrenaline (100 microM) significantly increased the potassium-evoked release of endogenous aspartate, glutamate and GABA, proposed transmitters of the olfactory cortex, although the effect on GABA release was specifically antagonized by 2-amino-5-phosphonovaleric acid. Noradrenaline (100 microM) also significantly increased the potassium-evoked release of D-[3H]aspartate, an effect antagonized by a number of alpha- and beta-adrenoceptor antagonists. It is concluded that at low concentrations, noradrenaline facilitates transmission at the LOT-superficial pyramidal cell synapse by increasing excitatory amino acid neurotransmitter release. This effect is mediated by both alpha- and beta-adrenoceptors although the primary site of release is unknown. At higher concentrations of noradrenaline, the increased levels of excitatory transmitters release sufficient endogenous GABA (and possibly adenosine) to cause an overall depression of transmission. These conclusions are supported by the results of a series of experiments in which the effects of noradrenaline on stimulus input-evoked field potential output relationships were assessed. It is not possible to exclude additional direct effects of noradrenaline on membrane excitability.     

Excitatory amino acid-induced release of 3H-GABA from cultured mouse cerebral cortex interneurons

A newly developed continuous superfusion model was used for studies of 3H-GABA release from cultured mouse cerebral cortex neurons. It was found that a series of excitatory amino acids (EAAs) representing all receptor subtypes evoked Ca2+- dependent release of 3H-GABA from the neurons. Quisqualate was the most potent agonist tested, with an EC50 value of 75 nM. L-Glutamate, N-methyl-D-aspartate (NMDA), and kainate showed EC50 values of 12, 16 and 29 microM, respectively. The EAA-evoked 3H-GABA release could be blocked by a series of EAA antagonists. The highly selective NMDA antagonist D-2-amino-5-phosphonovaleric acid (D-APV) was found to block NMDA responses, whereas the nonselective antagonists cis-2,3-piperidine dicarboxylic acid (PDA) and gamma-D-glutamyl-aminomethyl sulphonic acid (GAMS) blocked responses to all agonists. NMDA responses were found to be sensitive to Mg+ blockade. EAA- as well as potassium-induced 3H-GABA release from the neurons could be detected as early as day 5 in culture. However, during the culture period up to 12 d, the responses to K+, quisqualate, and NMDA were increased. The ontogenetic development of binding sites for quisqualate, kainate, and NMDA in mouse cortex was studied using the radioligands 3H-alpha-amino-3-hydroxy-5-methyl-4-isoxasole propionate (3H-AMPA), 3H-kainate, and 3H-L-glutamate, respectively. The development of binding sites for the different EAA-receptor subtypes showed a good correlation with the development of neuronal 3H-GABA release evoked by the excitatory amino acids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antagonists of synaptic and amino acid excitation of neurones in the cat spinal cord

In the spinal cord of the anaesthetized cat microelectrophoretically administered (+/-)-cis-2,3-piperidine dicarboxylate (2,3-PDA), (+/-)-cis-2,5-piperidine dicarboxylate (2,5-PDA), gamma-D-glutamylglycine (gamma DGG), beta-D-aspartyl-beta-alanine (beta DAA), (+/-)-2-amino-4-phosphonobutyrate (2-APB), (+/-)-2-amino-5-phosphonovalerate (2-APV) and (+/-)-2-amino-7-phosphonoheptanoate (2-APH) were assessed as antagonists of chemical excitation of dorsal horn interneurones and Renshaw cells by N-methyl-D-aspartate (NMDA), L-aspartate, quisqualate (QUIS), kainate and L-glutamate, and of monosynaptic and polysynaptic excitation by impulses in primary afferent fibres of muscle and cutaneous origin. Whereas polysynaptic excitation of interneurones was readily and reversibly depressed by 2-APV, 2-APH, beta DAA, gamma DGG and 2,3-PDA, all of which also reduced excitation by NMDA (and L-aspartate) more than that by QUIS (and L-glutamate), no selective antagonism of monosynaptic excitation could be demonstrated. In particular, 2,3-PDA, which depressed excitation by kainate to a greater extent than that by either QUIS or NMDA, appeared to have no effect on monosynaptic excitation. The results support the involvement of L-aspartate as the transmitter of some spinal excitatory interneurones, but none of the antagonists tested were considered suitable for assessing the role of L-glutamate as the transmitter of some spinal primary afferent fibres.     

CNQX and DNQX block non-NMDA synaptic transmission but not NMDA-evoked locomotion in lamprey spinal cord

The motor pattern underlying locomotion in the lamprey is activated and maintained by excitatory amino acid neurotransmission. The quinoxalinediones 6,7-dinitroquinoxaline-2,3-dione (DNQX) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) are potent and selective antagonists of non-N-methyl-D-aspartate (NMDA) receptors in the mammalian central nervous system. In the lamprey, these compounds are now shown to block fast excitatory synaptic potentials elicited in neurones of the spinal ventral horn. They selectively antagonise responses to the application of selective kainate and quisqualate receptor agonists (kainate and alpha-amino-3-hydroxy-5-methyl-4-isoxalone (AMPA)) but do not influence NMDA receptor-mediated responses. Additionally, it is shown that the activation of NMDA receptors is sufficient to elicit and maintain fictive locomotion after blockade of non-NMDA receptors with either DNQX or CNQX. Conversely, activation of quisqualate receptors with AMPA, but not quisqualate leads to fictive locomotion with properties much like that activated by kainate.     

The role of putative excitatory amino acid neurotransmitters in the initiation of locomotion in the lamprey spinal cord. I. The effects of excitatory amino acid antagonists

The activation of N-methyl-D-aspartate (NMDA) and kainate receptors will evoke fictive locomotion in the appropriate motor pattern for locomotion in the isolated lamprey spinal cord, but not a selective activation of quisqualate receptors. The present experiments test whether the initiation of locomotion in response to sensory stimulation depends on these types of receptors. An in vitro preparation of the lamprey spinal cord with part of its tailfin left innervated has been used. In this preparation a sequence of fictive locomotion (i.e. alternating bursts in the segmental ventral roots with a rostrocaudal phase lag) can be elicited by continual sensory stimulation of the tailfin. The effects of excitatory amino acid antagonists were studied by recordings from ventral roots (extracellularly) and motoneurones (intracellularly). It was found that the strong initial bursts of each swimming sequence induced by sensory stimulation were depressed by combined NMDA/kainate antagonists (cis-2,3-piperidine dicarboxylate (PDA) and gamma-D-glutamylglycine (gamma-DGG] whereas the less intense burst activity, occurring particularly towards the end of each swimming sequence, was depressed by a selective NMDA antagonist, 2-amino-5-phosphonovalerate (2-APV). This condition could be mimicked in an isolated spinal cord preparation by an application of L-glutamate; the low-level fictive locomotion induced by low doses of L-Glu (less than 100 microM) was depressed by a NMDA antagonist (2-APV), and, if higher doses were applied, the activity was only depressed by PDA/gamma-DGG. The mode and time course of the depression (by excitatory amino acid antagonists) of fictive locomotion, induced by sensory stimulation, shows that the putative excitatory amino acid neurotransmitter directly or indirectly acts at the pattern generating circuitry within the spinal cord.     

Pharmacological characterization of the glutamate receptor in cultured astrocytes

Cultured astrocytes from neonatal rat cerebral hemispheres are depolarized by the excitatory neurotransmitter glutamate. In this study we have used selective agonists of different neuronal glutamate receptor subtypes, namely, the N-methyl-D-aspartate (NMDA), kainate, and quisqualate type, to characterize pharmacologically the glutamate receptor in astrocytes. The agonists of the neuronal quisqualate receptor, alpha-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionic acid (AMPA) and quisqualate, depolarized the membrane. Kainate, an agonist of the neuronal kainate receptor, depolarized astrocytes more effectively than quisqualate. Combined application of kainate and quisqualate depolarized astrocytes to a level which was intermediate to that evoked by quisqualate and kainate individually. Agonists activating the neuronal NMDA receptor, namely NMDA and quinolinate, were ineffective. Application of NMDA did not alter the membrane potential even in combination with glycine or in Mg2+-free solution, conditions under which neuronal NMDA receptor activation is facilitated. The nonselective agonists L-cysteate, L-homocysteate, and beta-N-oxalylamino-L-alanine (BOAA) mimicked the effect of glutamate. Dihydrokainate, a blocker of glutamate uptake, did not, and several antagonists of neuronal glutamate receptors only slightly affect the glutamate response. These findings suggest that astrocytes express one type of glutamate receptor which is activated by both kainate and quisqualate, lending further support to the notion that cultured astrocytes express excitatory amino acid receptors which have some pharmacological similarities to their neuronal counterparts.     

Unmyelinated cutaneous afferent neurons activate two types of excitatory amino acid receptor in the spinal cord of Xenopus laevis embryos

The trunk and tail skin of Xenopus laevis embryos near the time of hatching is innervated by the mechanoreceptive free nerve endings of Rohon-Beard neurons, a homogeneous class of cutaneous primary afferent fibers. Rohon-Beard neurons have cell bodies and axons in the dorsal spinal cord, where they monosynaptically excite a population of dorsolaterally situated interneurons (Clarke and Roberts, 1984). EPSPs can be recorded in these dorsolateral interneurons following electrical stimulation of the unmyelinated neurites of Rohon-Beard neurons in the skin. The EPSPs are dual component, consisting of separate fast and slow potentials that are usually evoked synchronously and that closely resemble those described previously in Xenopus and lamprey motoneurons (Dale and Roberts, 1985; Dale and Grillner, 1986). The excitation of dorsolateral interneurons by Rohon-Beard neurons is reduced by the bath application of excitatory amino acid antagonists. Kynurenic acid suppresses both the fast and slow components of the EPSPs, while both (+/-)-2-amino-5-phosphonovaleric acid (APV) and 1 mM magnesium reduce the slow component but have little or no effect on the peak amplitude of the EPSPs. These data suggest that Rohon-Beard neurons release an excitatory amino acid neurotransmitter, which acts simultaneously at both N-methyl-D-aspartate (NMDA) and non-NMDA receptor types. This is the first direct demonstration of dual-component excitatory amino acid-mediated synaptic transmission from cutaneous primary afferent neurons in the vertebrate spinal cord. The bath application of the agonists NMDA, kainate, or quisqualate in salines containing 1 microM TTX depolarized the interneurons and reduced their input resistance, which suggests that the interneurons possess all 3 types of excitatory amino acid receptor. Kynurenic acid strongly inhibits responses to NMDA and kainate, but is relatively less effective against the larger responses of quisqualate in this system.