A slow intravenous infusion of N-methyl-DL-aspartate as a seizure model in the mouse.

A seizure model involving slow i.v. infusion of the excitatory amino acid N-methyl-DL-aspartate (NMDLA) in the mouse is described. It allows determination of the threshold doses of NMDLA required to elicit clonic and tonic seizures in individual mice. The NMDA receptor antagonists MK-801, CPP, ifenprodil and 7-chlorokynurenic acid (7-CLK), and diazepam dose-dependently increased the dose of NMDLA required to elicit a tonic seizure. CPP, 7-CLK and diazepam also increased the dose of NMDLA inducing clonic seizures. In contrast, ifenprodil at doses which antagonised tonic seizures had no effect on clonic seizures. The glycine and polyamine modulatory site agonists, D-serine and spermidine respectively, dose-dependently reduced the dose of NMDLA required to induce clonic and tonic seizures. The NMDLA infusion model appears to be more sensitive than the classical bolus injection test and can detect both anticonvulsant and proconvulsant actions mediated by the NMDA receptor complex.     

Postnatal development of the chemosensitivity of rat cerebellar Purkinje cells to excitatory amino acids. An in vitro study

In vitro sagittal slices of immature rat cerebellum were used to study the development of the sensitivity of Purkinje cells (PCs) to L-aspartate (L-Asp), L-glutamate (L-Glu) and related derivatives. As early as postnatal day 0 all PCs already displayed clear excitatory responses to short iontophoretic applications of L-Asp, L-Glu and quisqualate while in the same conditions no effect of N-methyl-D,L-aspartate (NMDLA) was detected. By postnatal day 5, i.e. after the onset of the synaptogenesis, the sensitivity of PCs to L-Asp, L-Glu and quisqualate significantly increased up to values similar to those recorded in adult rat cerebellum and surprisingly nearly all (87%) the recorded cells now also displayed excitatory responses to NMDLA. Although this sensitivity of PCs to NMDLA was significantly lower than that observed with the other drugs, it persisted until the end of the first postnatal month when the adult type of connectivity is already well established but at this stage only 30 per cent of the tested cells were still sensitive to the agonist. During this period, excitatory responses elicited by NMDLA were selectively antagonized by 2-amino-5-phosphonovalerate (2-APV), suggesting that during postnatal development, NMDA receptor types are transiently expressed on PCs membranes since in the adult, NMDLA no longer had an excitatory effect. Instead, this drug now exerted a preferential antagonistic action on the excitatory response elicited by L-Asp. Also in the adult, no major changes occurred in the sensitivity of PCs to L-Asp, L-Glu and quisqualate when these drugs were ejected at a dendritic site whereas, when ejected at the somatic level, the sensitivity of the cell appeared 2-3 times lower.     

Stimulus-coupled release of amino acids from cerebellar granule cells in culture

Cerebellar cultures greatly enriched in excitatory granule neurones were depolarized by exposure to either elevated K⁺ or veratrine. Stimulation of the release of not only Glu, but also of certain amino acids, including Gly, Ala and Ser, was observed. The effect was specific, as depolarization did not induced the release of all the estimated amino acids or of lactate dehydrogenase. In comparison with the characteristics of the evoked release of Glu, those of the responsive neutral amino acids were similar in terms of Ca²⁺-dependence, but differences were also noted. Thus, upon stimulation, the relative rise was smaller than for Glu and the degree of depolarization causing maximal release was lower. The questions of whether stimulus-coupled release of the non-transmitter amino acids from granule cells may play a neuromodulatory role in the cerebellum is discussed.     

Effects of bath-applied excitatory amino acids and their analogs on spinal interneurons of the lamprey

Depolarizations, conductance increases and time courses of the responses to bath application of glutamate, aspartate, DL-homocysteate, N-methyl-DL-aspartate (NMDLA), quisqualate and kainate were determined in interneurons of the isolated spinal cord of the lamprey, one of the most primitive vertebrates. Conductance increases produced by these excitants in perfusate containing tetrodotoxin (0.5 microgram/ml), 4-aminopyridine (1 mM) and without Ca2+ were very small in comparison with those produced by glycine or GABA. NMDLA-induced depolarizations were associated with conductance decreases and rhythmic oscillations in membrane potentials in this perfusate. Quisqualate was strongest among these amino acids in producing depolarizations and conductance increases. Responses induced by analogs were slower than those produced by glutamate and aspartate. Phylogenetic distribution of N-methyl-D-aspartate receptors on neurons and muscles is discussed.     

Release of endogenous excitatory amino acids from ON-type bipolar cells isolated from the goldfish retina.

Excitatory amino acids are presumed to be the transmitter of retinal bipolar cells. However, one of the essential criteria for the identification of the transmitter, its release from the cells upon depolarization, has not been demonstrated. This article examines the release of endogenous excitatory amino acids from bipolar cells and correlates this release with the influx of Ca2+. Bipolar cells with a large, bulblike axon terminal (ON-type cells with mixed inputs from rods and cones) were enzymatically isolated from the goldfish retina. Horizontal cells dissociated from the catfish retina were used as a probe of excitatory amino acids, because these cells can detect submicromolar concentrations of L-glutamate with high selectivity. An isolated bipolar cell was closely apposed to a dissociated horizontal cell, and each cell was voltage clamped by a patch pipette in the whole-cell clamp configuration. When the bipolar cell was depolarized from -60 mV to a potential more positive than -40 mV using a 500-msec voltage pulse, an outward current (greater than 20 pA) was recorded from the apposed horizontal cell, which was maintained at +40 mV. The reversal potential of the current induced by the substance released from bipolar cells (Irs) was close to 0 mV and was almost identical to the responses evoked with ionophoretically applied L-glutamate. Both reversal potentials were shifted to the same, more negative value when the external Na+ was replaced with choline. Furthermore, the Irs was suppressed reversibly by the application of kynurenic acid, a glutamate antagonist. When the Ca current (ICa) of the bipolar cell was blocked by Cd2+, the Irs also disappeared. The peak amplitude of Irs was closely related to that of the ICa. We conclude that mixed rod/cone ON-type bipolar cells of the goldfish retina release an endogenous excitatory amino acid or a closely related compound in a Ca(2+)-dependent manner.     

Peripheral sensory stimulation and the release of transmitter amino acids in vivo from specific regions of cerebral cortex

The effects of sensory stimulation on the release of amino acids from sensorimotor and visual cortex have been studied using a superfusion technique. Electrical stimulation of the brachial plexus contralateral to the superfusion cannula increased significantly the release of glutamate and glutamine from the sensorimotor cortex of anaesthetized rats. No clear effect was observed with the other amino acids. Stimulation of the ipsilateral plexus had no effect on glutamate and glutamine release. In unanaesthetized animals stimulation of the contralateral brachial plexus raised the levels of all the amino acids in sensorimotor cortex superfusate. Weak photic stimulation of the eyes of dark-adapted rats increased glutamate release from the visual cortex but caused no significant change in the release of other amino acids. All evoked increases in amino acids release were reversible at the cessation of the stimuli.     

Sustained extracellular potentials in the cat spinal cord during the microiontophoretic application of excitatory amino acids

Sustained negative potentials were recorded in the ventral horn of the cat spinal cord during current balanced extracellular iontophoresis of excitatoyr amino acids. The potentials (referred to a distant indifferent electrode) were measured by an extracellular microelectrode. These focal potentials (FPs) were evoked by DL-homocysteate, L-glutamate, N-methyl-D-aspartate and kainate. These FPs are not an artifact of extracellular microiontophoresis. Their time course is correlated with the depolarization of spinal motoneurones by excitatory amino acids. During iontophoresis of kainate, FPs can be as large as 50 mV and can be recorded for up to 1 mm from the site of drug application. The FP and depolarization caused by kainate were usually irreversible. The depolarization of motoneurones evoked by excitatory amino acids is very much larger when recorded as a 'transmembrane potential' (i.e. the potential of an intracellular electrode minus the potential of a local extracellular electrode) rather than as a 'classical' intracellular potential (i.e. referred to a distant reference electrode). Possible mechanisms for the generation of the FP are discussed. It is suggested that FP may be recorded routinely during microiontophoretic studies employing extracellular recording of neuronal activity. The application of the FP as a measure of cell depolarization during pharmacological studies of excitatory amino acids and agents that block their action is discussed.     

The release of endogenous amino acids into the vitreous of the intact eye of the albino rat: effect of light, potassium, and ouabain

The vitreal space of the intact eye of albino rats was perfused in vivo. The concentration of several endogenous amino acids in the vitreal effluent was measured by the [³H]microdansylation procedure. GABA was never detected despite a sensitivity of the method of 0.5 pmol.

In contrast to previous results obtained in pigmented rats, photic stimulation with flashing white light did not alter the release of glycine or any of the other amino acids. Potassium (60 mM) and ouabain (0.1 mM) evoked a specific release of glycine. The potassium-evoked release was blocked by magnesium suggesting a neuronal site of origin of glycine. Ouabain-evoked release was not blocked by magnesium.

The results were contrasted with experiments on radiolabeled amino acid release from retinas preloaded and superfused in vitro, a condition in which glial localization of exogenous amino acids predominates.     

Neuropharmacological actions of kynurenic and quinolinic acids on horizontal cells of the isolated fish retina

We have studied the effects of naturally occurring metabolites of tryptophan, kynurenic and quinolinic acids, on the electrophysiological responses of retinal horizontal cells in the fish (Rutilus rutilus, the roach). Quinolinic acid usually hyperpolarizes the cells and reduces their light evoked responses (S-potentials) but on occasion, it causes a slight depolarization of the membrane potential. These actions are similar to those found with N-methyl-d-aspartate (NMDA) and our results are consistent with the proposal that it acts at NMDA binding sites. Kynurenic acid (1mM) invariably hyperpolarizes horizontal cells to their potassium Nernst equilibrium potential and, more significantly, blocks the depolarizing actions exerted on them by excitatory amino acids, such as kainic and quisqualic acids. We show that this action persists in the presence of the synaptic blocker, cobalt chloride, and thus is not mediated by chemical synaptic activity. Kynurenic acid does not reverse depolarization of horizontal cells by dopamine or γ-aminobutyric acid, thus its inhibitory effects are selective to the actions of excitatory amino acids. Neither xanthurenic acid, a close structural analogue of kynurenic acid, nor quinolinic acid are effective in blocking depolarizations by excitatory aino acids.     

Excitatory amino acid-evoked membrane currents and excitatory synaptic transmission in lamprey reticulospinal neurons

The characteristics of excitatory amino acid-evoked currents and of excitatory synaptic events have been examined in lamprey Müller neurons using voltage clamp and current clamp recording techniques. Application of glutamate evoked depolarizations associated with a decrease in input resistance. The reversal potential of the responses was -35 mV. Under voltage clamp conditions, a series of excitatory amino acid agonists evoked inward currents associated with little or no increase in baseline current noise. The order of potency of the excitatory amino acid agonists was quisqualate greater than kainate greater than glutamate greater than aspartate, while N-methyl-D-aspartic acid (NMDA) was inactive. Inward currents evoked by glutamate, as well as by kainate and quisqualate were attenuated reversibly by 1 mM kynurenic acid (KYN). In contrast, glutamate-evoked currents were not affected by 100 microM D(-)-2-amino-5-phosphonovaleric acid (APV), a selective NMDA antagonist. Spontaneously occurring and stimulus-evoked excitatory postsynaptic events were antagonized reversibly by 1 mM KYN. At this concentration, KYN had no effect on membrane potential, input resistance, or excitability of the cells. In contrast, excitatory postsynaptic currents were unaffected by APV. It is concluded that both glutamate responses and excitatory synaptic transmission in lamprey Müller neurons are mediated by non-NMDA-type receptors and that these receptors are associated with ionic channels with a low elementary conductance. The combined pharmacological and biophysical characteristics of these responses are therefore different from those previously reported in other preparations. Spontaneous (but not stimulus-evoked) inhibitory synaptic events in Müller neurons were blocked reversibly by 1 mM KYN but not by 100 microM APV, suggesting that excitation of interneurons inhibitory to Müller cells was also mediated by non-NMDA receptors.