Kainic acid responses and toxicity show pronounced Ca2+ dependence

Responses of pyramidal neurons to ionophoretic kainate, quisqualate and N-methyl aspartate were studied in a submerged rat piriform cortex slice as a function of Ca2+ and Mg2+ concentrations. The results suggest that the channel activated by kainate is unusually influenced by Ca2+, excitotoxicity is Ca2+-dependent and a function of Ca2+ concentration, and the excitotoxic actions of various amino acid agonists are correlated with the Ca2+ dependence of their responses.     

Kainic acid responses and toxicity show pronounced Ca dependence

Responses of pyramidal neurons to ionophoretic kainate, quisqualate and N-methyl aspartate were studied in a submerged rat piriform cortex slice as a function of Ca²⁺ and Mg²⁺ concentrations. The results suggest that (a) the channel activated by kainate is unusually influenced by Ca²⁺, (b) excitotoxicity is Ca²⁺-dependent and a function of Ca²⁺ concentration, and (c) the excitotoxic actions of various amino acid agonists are correlated with the Ca²⁺ dependence of their responses.     

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.     

Endogenous ligands for the quisqualate receptor: presence in rat brain cortex synaptic vesicles

The presence in highly purified rat brain cortex synaptic vesicles of endogenous ligands for rat brain quisqualate receptors was investigated. The vesicles were extracted, and their contents fractionated by high voltage electrophoresis. Endogenous ligands were detected by a radioreceptor assay in which such ligands competed with 50 nM -[³H]glutamate for binding to quisqualate receptors present in rat brain postsynaptic densities (PSDs). Binding of -[³H]glutamate to (NMDA) receptors, also present in PSDs, was blocked by 100 μM NMDA. We found that the endogenous ligands present in brain cortex synaptic vesicles for quisqualate receptors, were glutamate and aspartate, in a molar ratio of about two to one. The quisqualate receptor had an affinity 130-fold higher for glutamate (K_d 0.3 μM) than for aspartate, and the latter amino acid also showed a marked negative cooperativity for binding (Hill number 0.29, against 0.67 for glutamate). These findings suggest that glutamate is the natural transmitter that activates quisqualate receptors at some central excitatory synapses, and also that aspartate may be a classical transmitter, the receptor for which still remains to be shown.     

Pharmacological properties of gamma-aminobutyric acid-, glutamate-, and aspartate-induced depolarizations in cultured astrocytes

Differentiated glial fibrillary acidic protein-positive astrocytes in homogeneous cultures of early postnatal rat cerebral hemispheres respond by membrane depolarization to gamma-aminobutyric acid (GABA), glutamate, and aspartate with a threshold concentration of approximately 10(-5) M. The GABA-induced depolarization is antagonized by two blockers of the neuronal GABAA receptor, picrotoxin and bicuculline, but is not affected by the uptake blockers beta-alanine or nipecotic acid. An agonist of the GABAA receptor, muscimol, produces a dose-response curve similar to that of GABA, whereas the agonist of the GABAB receptor, baclofen, did not alter the membrane potential. When repetitive pulses of GABA are given to one cell, its responsiveness depends on the time interval between pulses. Within 30 sec after termination of the first pulse the cell remains unresponsive to the second pulse. With increased time intervals between the pulses, reactivity toward GABA recovers. Five minutes after the first pulse the cell regains 75% of its initial depolarization peak. Aspartate results in a depolarization similar in size and time course to that induced by glutamate. The glutamate agonists, quisqualate and ibotenate, and kainate are less potent than glutamate. N-Methyl-D-aspartate has no effect on the membrane potential of astrocytes. The pharmacological features of the glutamate response are therefore similar to those of the receptor mediating neuronal glutamate transport.     

Characterization of quisqualate receptor desensitization in cultured postnatal rat hippocampal neurons.

The quisqualate class of glutamate receptors is thought to play an important role in excitatory synaptic transmission, synaptic plasticity, and neuronal death. Since desensitization is a prominent feature of the responses mediated by this class of receptors, we have characterized the rapidly desensitizing quisqualate response in cultured postnatal rat hippocampal neurons using the whole-cell patch-clamp technique. Quisqualate and its structural analogs elicit a peak current that rapidly decays to a steady-state level. In contrast, currents induced by kainate, NMDA, and their structural analogs exhibit either no decay or a much slower decay. The biophysical and pharmacological properties of the peak and steady-state quisqualate currents indicate that both are mediated by an ionotropic quisqualate receptor. Quisqualate currents desensitized monoexponentially by approximately 70% with a time constant near 80 msec. Both the rate and percentage of desensitization showed slight voltage dependence and were concentration dependent, reaching maximal values at saturation. Additionally, the overlap of the dose-response curves for activation of the steady-state current and desensitization of the peak current by a conditioning dose suggests that the two processes are related. Furthermore, desensitizing quisqualate currents were observed when Ca2+, Mg2+, Na+, K+, and Cl- were removed from the extracellular solution or their concentrations greatly reduced. These results suggest that the decline in the response is not caused by a simple open channel block mechanism. Despite the lack of desensitization by kainate, our observations are consistent with the hypothesis that quisqualate and kainate act at a single receptor-channel complex. Kainate and quisqualate appeared to interact competitively when applied simultaneously and noncompetitively when quisqualate was applied first. In addition, saturating doses of quisqualate and kainate gave steady-state currents of equal amplitude in neurons treated with the lectin WGA, an inhibitor of quisqualate receptor desensitization.     

Altered excitatory amino acid function and morphology of the cerebellum of the spastic Han-Wistar rat.

A mutant strain of Han-Wistar rat carries an autosomal recessive gene producing spastic paresis which is characterized by ataxia, tremor and hind limb rigidity. Brains of affected rats and unaffected littermate controls were transected at the mesencephalon into rostral and caudal portions (the caudal portion contained the cerebellum and brainstem). Poly(A)+ mRNA was isolated from pooled rostral or caudal portions and injected into Xenopus oocytes. The oocytes were voltage-clamped and exposed to 1 mM L-glutamate, 500 microM kainate, 500 microM quisqualate, 200 microM N-methyl-D-aspartate (NMDA) or 1 mM gamma-aminobutyric acid (GABA). Oocytes injected with mRNA isolated from the caudal portions of the affected rat brains exhibited statistically significant increases in glutamate and kainate peak current responses compared to oocytes injected with mRNA from other brain samples. No differences were noted in the responses of the groups when exposed to quisqualate, NMDA or GABA. Cerebellar and brain stem mRNA were also isolated separately in different groups of mutants and unaffected littermates. Only oocytes injected with cerebellar mRNA from mutants displayed statistically significant increases in responses to glutamate and kainate. In parallel morphological studies changes in the cerebellum of mutants were also observed. These consisted of a loss of Purkinje cells and an asymmetrical disarrangement of the granule cell layer of cerebellar cortex. Taken together, the physiological and morphological results suggest that alterations in glutamate/kainate receptors in the cerebellum are phenotypic manifestations of the Han-Wistar mutation. The results are consistent with the hypothesis that this mutant rat might serve as a model of glutamate/kainate excitotoxicity in the brain.     

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.     

Barbiturates, alcohols and the CNS excitatory neurotransmission: Specific effects on the kainate and quisqualate receptors

The effects of barbiturates and straight-chain aliphatic alcohols on the responses of rat striatal neurons to excitatory amino acids have been investigated. The responses to N-methyl-D-aspartate, quisqualate, kainate, L-glutamate and L-aspartate were measured by the increase in 22Na+ efflux rate that they produce in brain slices. The responses to quisqualate and kainate, measured in the 22Na+ efflux assay, were found to be partially blocked by barbiturates whereas the responses to N-methyl-D-aspartate, glutamate and aspartate were not. The kainate and quisqualate-induced increases in 22Na+ efflux rate were much more readily blocked by the presence of aliphatic alcohols than were the responses to N-methyl-D-aspartate, glutamate and aspartate. These results strengthen the idea of the existence of 4 distinct receptors for excitatory amino acids in the rat striatum. They are consistent with the presence on the kainate and quisqualate receptors, but not on the N-methyl-D-aspartate and glutamate/aspartate receptors of a hydrophobic domain which would provide a site of interaction for barbiturates and alcohols. They suggest that receptors for excitatory amino acids can be targets for the actions of barbiturates and alcohols on the central nervous system, and may mediate some of the anesthetic and hypnotic effects of these drugs.     

The piperidine analogues prevent desensitization of glutamate receptors on crustacean muscle

The effect of the fourcis piperidine decarboxylate analogues on glutamate receptor desensitization was investigated. Intracellular recordings of the depolarization in response to ionophoretic application ofl-glutamate were made from the opener muscle of the Hermit crab (Eupagurus bernhardus). Following bath application of any of the piperidine dicarboxylates (1 h) the decline in the amplitude of successive glutamate potentials in response to a train of ionophoretic pulses was prevented. This result indicated that the piperidine dicarboxylates inhibited the development of glutamate receptor desensitization. This effect was reversed on washing. During a prolonged ionophoretic glutamate application (5 s) the depolarization declined due to the onset of desensitization. This decline in the potential no longer occurred after exposure to a piperidine dicarboxylate analogue (30–60 min). Similarly, after application of the piperidine dicarboxylates the shape of the bath-applied glutamate potential was transformed from a rapid depolarization followed by repolarization to a maintained depolarization. These effects of the piperidine dicarboxylates are consistent with a loss of glutamate receptor desensitization.     

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.     

Antibodies to glutamate and aspartate recognize non-endogenous ligands for excitatory amino acid receptors

Antisera raised against glutaraldehyde conjugates of glutamate (Glu) and aspartate (Asp) with hemocyanin proved highly specific for their respective unconjugated amino acid haptens when tested in immunocytochemical blocking experiments on sections of the rat spinal cord. In addition, immunocytochemical staining by the Glu antiserum was effectively blocked by quisqualate but not by kainate or N-methyl-D-aspartate (NMDA); staining with the Asp antiserum was effectively blocked by kainate, to a lesser extent by quisqualate, and was not affected by NMDA. These results may be explained by assuming that the specific binding regions of the antibodies tested share certain recognition characteristics with endogenous binding sites or receptors for excitatory amino acids and their agonists.     

Insulin-specific sensitization of cultured cerebrocortical neurons to glutamate excitotoxicity

The effect of insulin on the sensitivity of neurons to excitatory amino acid-induced cytotoxic cell death was examined in primary cultures of the rat cerebral cortex. Cells developed for two weeks in serum supplemented medium in the presence or absence of insulin, insulin-like growth factor or b-fibroblast growth factor. Excitotoxic cell death was induced by 1 mmol/l glutamate, N-methyl-D-aspartate, kainate or quisqualate. The vulnerability of cells was evaluated by the measurement of lactate dehydrogenase release due to cytotoxic injury. In contrast to the moderate evaluation of protein content by all the 3 growth factors, only insulin increased the vulnerability of cells to the neurotoxic effects of glutamate and of the 3 excitatory amino acid receptor agonists examined. Our results show that the induction of vulnerability in cortical cultures is a specific action of insulin and not a general effect of growth factors. Moreover, the increased vulnerability to N-methyl-D-aspartate, quisqualate and kainate suggests that the effect of insulin is exerted through intracellular mechanisms other than a selective induction of one subpopulation of excitatory amino acid receptors.     

AMPA, kainate, and quisqualate activate a common receptor-channel complex on embryonic chick motoneurons

The actions of the putative quisqualate-selective agonist DL-alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) were examined in identified embryonic chick motoneurons using gigaseal recording techniques and compared with properties of the selective non-NMDA excitatory amino acid agonists kainate and quisqualate. Pressure application of AMPA induces an inward going current when neurons are voltage-clamped at negative membrane potentials. The current-voltage relationship for this response is linear with reversal near 0 mV. Over the range of 1 microM-10 mM, the AMPA-induced current is dose-dependent with an ED50 of 40 microM. AMPA currents are insensitive to the selective NMDA receptor antagonist, 2-amino-5-phosphonovalerate, and the putative quisqualate selective blocker, glutamate diethyl ester, but are partially inhibited by kynurenic acid. In competition experiments, applications of saturating concentrations of AMPA and either kainate or quisqualate produce responses intermediate between the response to either agonist alone, indicating commonality in the mechanism of these agents. Applications of AMPA with the NMDA-selective agonist aspartate give an additive response. Analysis of current fluctuations indicates that AMPA, quisqualate, and kainate gate a channel with a primary conductance near 20 pS. Differences in maximal macroscopic current evoked by saturating concentrations of AMPA, kainate, and quisqualate cannot be explained by differences in mean channel open time as the most efficacious agonist, kainate, has the shortest channel open time (AMPA = 5.9 +/- 0.4 msec, kainate = 2.7 +/- 0.1 msec, quisqualate = 5.0 +/- 0.5 msec). Rather, kainate induces a greater frequency of channel opening. This finding contrasts with results obtained at the nicotinic ACh receptor, where the most efficacious agonists have the longest mean channel open time. Our results suggest that AMPA acts at the same receptor-channel complex as kainate and quisqualate on chick motoneurons and support the hypothesis that only 2 classes of excitatory amino acid receptor complexes exist in this preparation.     

Chloride channels gated by extrajunctional glutamate receptors (H-receptors) on locust leg muscle

Outside-out patches of extrasynaptic membrane were isolated from leg muscles of locusts. L-Glutamate and its agonists were applied to such patches either continuously or in rapidly switched pulses. When the pipette contained a high chloride concentration, 2.5 x 10(-5) M glutamate triggered single-channel currents (gated by H-receptors) with a conductance of 25 pS which were carried by chloride, in addition to cationic channels (gated by D-receptors). For the chloride channels, the distribution of channel open times had components of about 2 and 12 ms. Pulses of higher glutamate concentrations elicited many superimposed channel openings, and the approximately saturating concentration of 10(-3) M glutamate opened 100-200 channels simultaneously. When the pipette contained low chloride, channel conductance was reduced, and the current voltage relation was shifted towards the now negative chloride equilibrium potential. H-Receptor-gated chloride channels were activated by glutamate, ibotenate and aspartate, but not by GABA, quisqualate, kainate, N-methyl-D-aspartate and carbachol. The currents declined in the continued presence of agonist showing a time constant of desensitization greater than 1 s. Recovery from desensitization after removal of the agonist was tested with double pulses and was found to have a time constant of about 300 ms.     

Glutamate, kainate and quisqualate enhance GABA-dependent chloride uptake in cortex

Several lines of evidence indicate a possible interaction between the major inhibitory and excitatory cortical neurotransmitters, GABA and glutamate. To assess the neurochemical basis for such an interaction, we examined the effects of glutamate and several analogs on GABA-dependent chloride uptake in a mouse cortical synaptoneurosome preparation. L-Glutamate and the specific receptor subtype ligands kainate and quisqualate led to a small but significant enhancement in chloride uptake in the presence, but not the absence, of the GABA analog muscimol (5 microM). Enhancement was seen at excitatory amino acid (EAA) concentrations of 2-10 microM, but not at higher concentrations. D-Glutamate, NMDA, the NMDA-related antagonists APV and MK801, and the kainate/quisqualate antagonist CNQX, had no effect on chloride uptake. However, CNQX (50 microM) but not APV (50 microM) blocked the increase in chloride uptake due to kainate or quisqualate (10 microM). In addition, depolarization of synaptoneurosomes using high potassium (40 mM KC1) or ouabain pretreatment (5 microM) blocked the effects of kainate and quisqualate. Glutamate, kainate, and quisqualate had no effect on binding at the benzodiazepine, TBPS, or GABA sites on the GABAA receptor complex.     

Fade of the response to prolonged glutamate application in the rat hippocampal slice

The effect of prolonged glutamate (GLU) application was examined on 60 CA1 pyramidal neurons in the in vitro rat hippocampal slice preparation. Continuous application of L-GLU, either by bath perfusion (0.5-2 mM) of the slices or iontophoresis (200 mM) into the dendritic region of the neurons, elicited a transient depolarization which faded to a mean of 53% of the initial peak amplitude despite continued exposure to the agonist. Membrane depolarization to aspartate (ASP) and the d-isomer of GLU also faded with time. In contrast, the depolarizing response to the excitatory amino acid agonists N-methyl-D,L-aspartate (NMA), quisqualate (QUIS), and kainate (KA) did not fade significantly during continuous application. The fade of the GLU depolarization was not affected by the NMDA antagonist D-2-amino-5-phosphonovalerate (APV) or by blocking synaptic transmission with tetrodotoxin. At the time of maximum fade of the GLU depolarization, there was no change in input resistance or GLU reversal potential. In addition, fade of the response was not a consequence of changes in extracellular potassium concentration, GLU uptake mechanisms, or the electrogenic pump. The most likely explanation for fade is postsynaptic receptor desensitization.     

N-methyl D-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 photoreceptor 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 alpha-methyl glutamate, and alpha-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.     

AMPA receptor desensitization as a determinant of vulnerability to focally evoked status epilepticus

Within the area tempestas (AT) in the anterior piriform cortex, unilateral microinfusions of GABA receptor antagonists and glutamate receptor agonists trigger brief episodic limbic seizures. In the present study, we document a synergistic effect of coinfusing bicuculline (GABAA receptor antagonist) with either carbachol (muscarinic receptor agonist) or cyclothiazide (inhibitor of AMPA receptor desensitization) but not with glutamate receptor agonists (AMPA, NMDA or kainate) in the rat AT. In particular, coadministration of bicuculline (118 pmol) with either carbachol (328 pmol) or cyclothiazide (1.2 nmol) triggered continuous self-sustaining seizures (status epilepticus; SE). Cyclothiazide alone did not evoke seizures. Although blockade of NMDA receptors with AP-7 (100 or 500 pmol) prevented episodic seizures evoked by carbachol or bicuculline alone, it was without effect on the continuous seizures evoked by combined treatments. NMDA-insensitive self-sustaining seizures were also evoked by the combination of AMPA and cyclothiazide. Regardless of the mechanism by which SE was evoked, it was prevented only by an AMPA receptor antagonist, NBQX, thus reinforcing the crucial role of AMPA receptors in the transition to SE. Further evidence for AMPA receptor regulation of seizure severity came from the overexpression of the GluR1 AMPA receptor subunit in AT. This resulted in substantially increased severity of bicuculline-evoked seizures that was reversed by focal application of NBQX. Thus, desensitization of AMPA receptors appears to limit the duration and severity of seizure activity, and a failure of this mechanism, or an overabundance of slowly desensitizing AMPA receptors, predisposes to severe and prolonged seizures.     

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.