Pharmacology of glutamate receptor antagonists in the kindling model of epilepsy

It is widely accepted that excitatory amino acid transmitters such as glutamate are involved in the initiation of seizures and their propagation. Most attention has been directed to synapses using NMDA receptors, but more recent evidence indicates potential roles for ionotropic non-NMDA (AMPA/kainate) and metabotropic glutamate receptors as well. Based on the role of glutamate in the development and expression of seizures, antagonism of glutamate receptors has long been thought to provide a rational strategy in the search for new, effective anticonvulsant drugs. Furthermore, because glutamate receptor antagonists, particularly those acting on NMDA receptors, protect effectively in the induction of kindling, it was suggested that they may have utility in epilepsy prophylaxis, for example, after head trauma. However, first clinical trials with competitive and uncompetitive NMDA receptor antagonists in patients with partial (focal) seizures, showed that these drugs lack convincing anticonvulsant activity but induce severe neurotoxic adverse effects in doses which were well tolerated in healthy volunteers. Interestingly, the only animal model which predicted the unfavorable clinical activity of competitive NMDA antagonists in patients with chronic epilepsy was the kindling model of temporal lobe epilepsy, indicating that this model should be used in the search for more effective and less toxic glutamate receptor antagonists. In this review, results from a large series of experiments on different categories of glutamate receptor antagonists in fully kindled rats are summarized and discussed. NMDA antagonists, irrespective whether they are competitive, high- or low-affinity uncompetitive, glycine site or polyamine site antagonists, do not counteract focal seizure activity and only weakly, if at all, attenuate propagation to secondarily generalized seizures in this model, indicating that once kindling is established, NMDA receptors are not critical for the expression of fully kindled seizures. In contrast, ionotropic non-NMDA receptor antagonists exert potent anticonvulsant effects on both initiation and propagation of kindled seizures. This effect can be markedly potentiated by combination with low doses of NMDA antagonists, suggesting that an optimal treatment of focal and secondarily generalized seizures may require combined use of both non-NMDA and NMDA antagonists. Given the promising results obtained with novel AMPA/kainate antagonists and glycine/NMDA partial agonists in the kindling model, the hope for soon having potentially useful glutamate antagonists for use in epileptic patients is increasing.     

The synaptic activation of the GluR5 subtype of kainate receptor in area CA3 of the rat hippocampus

Two new compounds (LY293558 and LY294486), that antagonize homomeric human GluR5 receptors, were examined against responses mediated by kainate receptors in the CA3 region of rat hippocampal slices. Both compounds (applied at a concentration of 10 microM) antagonized reversibly currents induced by 200 nM kainate. They also antagonized reversibly the synaptic activation of kainate receptors, evoked by high-frequency stimulation of mossy fibres, in the presence of NMDA and AMPA receptor antagonists. These results show that GluR5 subunits are likely to contribute to a kainate receptor on CA3 neurones that mediates responses to both kainate and synaptically-released L-glutamate.     

Actions of kainate and AMPA selective glutamate receptor ligands on nociceptive processing in the spinal cord

Kainate receptors expressing the GluR5 subunit of glutamate receptor are present at high levels on small diameter primary afferent neurones that are considered to mediate nociceptive inputs. This suggests that GluR5 selective ligands could be novel analgesic agents. The role of kainate receptors on C fibre primary afferents has therefore been probed using three compounds that are selective for homomeric GluR5 receptors. The agonist, ATPA, and the antagonists, LY294486 and LY382884, have been tested in four models of nociception: responses evoked by noxious stimulation of the periphery have been recorded electrophysiologically (1) from hemisected spinal cords from neonatal rats in vitro, (2) from single motor units in adult rats in vivo, (3) from dorsal horn neurones in adult rats in vivo, and (4) in hotplate tests with conscious mice. In some protocols comparisons were made with the AMPA selective antagonist GYKI 53655. The agonist ATPA reduced nociceptive reflexes in vitro, but failed to have effects in vivo. In all tests, the GluR5 antagonists reduced nociceptive responses but only at doses that also affected responses to exogenous AMPA. The AMPA antagonist reduced nociceptive responses at doses causing relatively greater reductions of responses to exogenous AMPA. The results indicate that GluR5 selective ligands do reduce spinal nociceptive responses, but they are not strongly analgesic under these conditions of acute nociception.     

Differential production of IL-10 by T cells and monocytes of HIV-infected individuals: association of IL-10 production with CD28-mediated immune responsiveness

The present study determines the proportions of unmyelinated cutaneous axons at the dermal-epidermal junction in glabrous skin and of myelinated and unmyelinated axons in the sural and medial plantar nerves that immunostain for subunits of the ionotropic glutamate receptors. Approximately 20% of the unmyelinated cutaneous axon profiles at the dermal-epidermal junction immunostain for either N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), or kainate receptor subunits. These findings are consistent with previous observations that NMDA and non-NMDA antagonists ameliorate nociceptive behaviors that result from noxious peripheral stimulation. In the sural nerve, where the large majority of myelinated fibers are sensory, approximately half of the myelinated axon profiles immunostain for the NMDA receptor 1 (R1) subunit, 28% immunostain for the glutamate receptor 1 (GluR1) AMPA subunit, and 11% for the GluR5,6,7 kainate subunits. Even higher proportions immunostain for these receptors in the medial plantar nerve, a mixed sensory and motor nerve. In the sural nerve, 20% of the unmyelinated axon profiles immunostain for NMDAR1 and only 7% label for GluR1 or GluR5,6,7. Because the sural nerve innervates hairy skin, these data suggest that glutamate will activate a higher proportion of unmyelinated axons in glabrous skin than in hairy skin. Measurements of fiber diameters indicate that all sizes of myelinated axon profiles, including Adelta and Abeta, are positively labeled for the ionotropic receptors. The presence of glutamate receptors on large-diameter myelinated axons suggests that these mechanosensitive receptors, presumably transducing touch and pressure, may also respond to local glutamate and thus be chemosensitive.     

The glutamate receptor/NO/cyclic GMP pathway in the hippocampus of freely moving rats: modulation by cyclothiazide, interaction with GABA and the behavioural consequences

Monitoring of extracellular cGMP during intracerebral microdialysis in freely moving rats permits the study of the functional changes occurring in the glutamate receptor/nitric oxide (NO) synthase/guanylyl cyclase pathway and the relationship of these changes to animal behaviour. When infused into the rat hippocampus in Mg2+-free medium, cyclothiazide, a blocker of desensitization of the AMPA-preferring receptor, increased cGMP levels. The effect of cyclothiazide (300 microM) was abolished by the NO synthase inhibitor L-NARG (100 microM) or the soluble guanylyl cyclase inhibitor ODQ (100 microM). During cyclothiazide infusion the animals displayed a pre-convulsive behaviour characterized by frequent "wet dog shakes" (WDS). Neither L-NARG nor ODQ decreased the WDS episodes. Both cGMP and WDS responses elicited by cyclothiazide were prevented by blocking NMDA receptor function with the glutamate site antagonist CGS 19755 (100 microM), the channel antagonist MK-801 (30 microM) or Mg2+ ions (1 mM). The AMPA/kainate receptor antagonists DNQX (100 microM) and NBQX (100 microM) abolished the WDS episodes but could not inhibit the cyclothiazide-evoked cGMP response. DNQX or NBQX (but not MK-801) elevated, on their own, extracellular cGMP levels. The cGMP response elicited by the antagonists appears to be due to prevention of a glutamate-dependent inhibitory GABAergic tone, since infusion of bicuculline (50 microM) caused a strong cGMP response. The results suggest that (a) AMPA/kainate receptors linked to the NO/cGMP pathway in the hippocampus (but not NMDA receptors) are tonically activated and kept in a desensitized state by endogenous glutamate; (b) blockade of AMPA/kainate receptor desensitization by cyclothiazide leads to endogenous activation of NMDA receptors; (c) the hippocampal NO/cGMP system is under a GABAergic inhibitory tone driven by non-NMDA ionotropic receptors; (d) the pre-convulsive episodes observed depend on hippocampal NMDA receptor activation but not on NO and cGMP production.     

Pharmacological characterization of the human ionotropic glutamate receptor subtype GluR3 stably expressed in mammalian cells

We have cloned the human ionotropic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor GluR3 flip splice variant (hGluR3i) and developed a stable cell line expressing this receptor in HEK293 cells. Electrophysiological recordings demonstrated that glutamate-evoked currents desensitize rapidly, with a mean desensitization time constant of 5.4 ms. Robust glutamate-evoked increases in intracellular Ca++ ([Ca++]i) were observed in the presence of cyclothiazide, which attenuated receptor desensitization. [Ca++]i measurements were used to perform a detailed pharmacological characterization of hGluR3i with reference agonists and antagonists. The results of these studies showed that kainate and domoate were not fully efficacious agonists relative to glutamate. The binding affinities of agonists and competitive antagonists were determined in a [3H]AMPA competition binding assay. There was a good correlation between the functional data and the binding affinities obtained for competitive antagonists. However, the binding affinities of the agonists did not correlate with their functional EC50 values from [Ca++]i data, possibly because the binding assay predominantly measures the desensitized high-affinity state of the receptor. [3H]AMPA binding also was performed on membranes prepared from rat forebrain, and comparison of the data from HEK293 cells expressing hGluR3i and rat forebrain suggest that nearly all of the reference compounds show similar binding activities between the two membrane preparations, with the exception of fluoro-willardiine, kainate and 6-nitro-7-sulfamoylbenzo(f)quinoxaline-2-3-dione (NBQX). These data suggest that cells stably expressing recombinant hGluR3i represent pharmacologically valid experimental systems to study human AMPA receptors.     

Domoic acid neurotoxicity in cultured cerebellar granule neurons is mediated predominantly by NMDA receptors that are activated as a consequence of excitatory amino acid release

The participation of NMDA and non-NMDA receptors in domoic acid-induced neurotoxicity was investigated in cultured rat cerebellar granule cells (CGCs). Neurons were exposed to 300 microM L-glutamate or 10 microM domoate for 2 h in physiologic buffer at 22 degrees C followed by a 22-h incubation in 37 degrees C conditioned growth media. Excitotoxic injury was monitored as a function of time by measurement of lactate dehydrogenase (LDH) activity in both the exposure buffer and the conditioned media. Glutamate and domoate evoked, respectively, 50 and 65% of the total 24-h increment in LDH efflux after 2 h. Hyperosmolar conditions prevented this early response but did not significantly alter the extent of neuronal injury observed at 24 h. The competitive NMDA receptor antagonist D(-)-2-amino-5-phosphonopentanoic acid and the non-NMDA receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX) reduced glutamate-induced LDH efflux totals by 73 and 27%, respectively, whereas, together, these glutamate receptor antagonists completely prevented neuronal injury. Domoate toxicity was reduced 65-77% when CGCs were treated with competitive and noncompetitive NMDA receptor antagonists. Unlike the effect on glutamate toxicity, NBQX completely prevented domoate-mediated injury. HPLC analysis of the exposure buffer revealed that domoate stimulates the release of excitatory amino acids (EAAs) and adenosine from neurons. Domoate-stimulated EAA release occurred almost exclusively through mechanisms related to cell swelling and reversal of the glutamate transporter. Thus, whereas glutamate-induced injury is mediated primarily through NMDA receptors, the full extent of neurodegeneration is produced by the coactivation of both NMDA and non-NMDA receptors. Domoate-induced neuronal injury is also mediated primarily through NMDA receptors, which are activated secondarily as a consequence of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate receptor-mediated stimulation of EAA efflux.     

Pharmacological differentiation of kainate receptors on neonatal rat spinal motoneurones and dorsal roots

The objectives of this study, conducted on neonatal rat spinal cord and dorsal roots in vitro, were to characterise the actions of a range of willardiine analogues on GluR5-containing kainate receptors present in dorsal roots, to determine whether GluR5-containing receptors are also present on motoneurones, and to differentiate responses mediated by kainate receptors from those mediated by AMPA receptors on motoneurones. (S)-5-Trifluoromethyl-willardiine, (S)-5-iodowillardiine, (S)-5-iodo-6-azawillardiine and ATPA were found to be potent agonists of kainate receptors on dorsal roots (EC50 values 0.108 +/- 0.002, 0.127 +/- 0.010, 0.685 +/- 0.141 and 1.3 +/- 0.3 microM, respectively) being more potent but of lower efficacy than kainate (EC50 value 14.8 +/- 1.8 microM). (S)-5-Iodo-6-azawillardiine blocked kainate-induced depolarisations of the dorsal root, probably via its desensitising action. Kainate-induced responses of dorsal roots were weakly antagonised by (RS)-3,5-dicarboxyphenylglycine (DCPG) (apparent KD 1.5 +/- 0.4 mM). Kainate receptors containing GluR5 subunits do not appear to be present on motoneurones since (RS)-3,5-DCPG (1 mM) potentiated rather than antagonised kainate-induced depolarisations of motoneurones. Although (S)-5-iodowillardiine (a potent and selective agonist at GluR5-containing kainate receptors) depolarised motoneurones (EC50 value 5.8 +/- 0.6 microM), such depolarisations were antagonised by both (RS)-3,4- and (RS)-3,5-DCPG, which are selective AMPA receptor antagonists at motoneurones, showing a KD value of 73 microM (Schild slope, 0.96 +/- 0.09) and an apparent KD value of 123 +/- 38 microM, respectively. This accords with the previously reported activity of willardiine analogues at AMPA receptors. Since neither (RS)-3,4- nor (RS)-3,5-DCPG antagonised kainate-induced motoneuronal depolarisations but cyclothiazide enhanced and GYK153655 blocked these responses it is possible that a component of the kainate response may be mediated by a population of DCPG-insensitive AMPA receptors on motoneurones. However, it is also possible that a population of kainate receptors other than those containing GluR5 subunits, are responsible for these effects. The new compounds introduced in this study are likely to be useful tools for studying the physiological role of kainate receptors in CNS function.     

In-vitro characterization of YM872, a selective, potent and highly water-soluble alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor antagonist

The in-vitro pharmacological properties of (2,3-dioxo-7-(1H-imidazol-1-yl)-6-nitro-1,2,3,4-tetrahydro-1-quinoxal inyl)-acetic acid monohydrate, YM872, a novel and highly water-soluble alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-receptor antagonist were investigated. YM872 is highly water soluble (83 mg mL(-1) in Britton-Robinson buffer) compared with 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline (NBQX), 6-(1H-imidazol-1-yl)-7-nitro-2,3(1H,4H)-quinoxalinedione hydrochloride (YM90K) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). YM872 potently inhibits [3H]AMPA binding with a Ki (apparent equilibrium dissociation constant) value of 0.096 +/- 0.0024 microM. However, YM872 had very low affinity for other ionotropic glutamate receptors, as measured by competition with [3H]kainate (high-affinity kainate binding site, concentration resulting in half the maximum inhibition (IC50) = 4.6 +/- 0.14 microM), [3H]glutamate (N-methyl-D-aspartate (NMDA) receptor glutamate binding site, IC50 > 100 microM) and [3H]glycine (NMDA receptor glycine-binding site, IC50 > 100 microM). YM872 competitively antagonized kainate-induced currents in Xenopus laevis oocytes which express rat AMPA receptors, with a pA2 value of 6.97 +/- 0.01. In rat hippocampal primary cultures, YM872 blocked a 20-microM AMPA-induced increase of intracellular Ca2+ concentration with an IC50 value of 0.82 +/- 0.031 microM, and blocked 300-microM kainate-induced neurotoxicity with an IC50 value of 1.02 microM. These results show that YM872 is a potent and highly water-soluble AMPA antagonist with great potential for treatment of neurodegenerative disorders such as stroke.     

NMDA receptors of the vestibular nuclei neurones

Cloning and pharmacological studies have shown that glutamatergic receptors can be divided in two classes (refer to Table 1): ionotropic receptors including N-methyl-D-aspartate (NMDA) and non-NMDA subtypes, and the G-protein-coupled metabotropic receptors (glutamate metabotropic receptor). There are two types of non-NMDA receptors: the AMPA/low-affinity kainate receptor type (the AMPA receptors) activated by a specific agonist, the alpha-amino-3-hydroxy-5-methyl-4-iso-xalone propionate (AMPA), and the high affinity kainate receptors. The vestibular nuclei neurones are endowed with all these types of glutamatergic receptors, which fits well with the fact that various afferents, including the primary vestibular afferents, most probably use glutamate or aspartate as a neurotransmitter. This article is aimed at summarising several past studies of our group and some more recent data obtained in the in vitro whole-brain preparation concerning the NMDA receptors of the central vestibular neurones. In that process, we will detail also many valuable studies of other groups that had been devoted to the same topic.     

Synthesis of willardiine and 6-azawillardiine analogs: pharmacological characterization on cloned homomeric human AMPA and kainate receptor subtypes

Both willardiine and azawillardiine analogs (18-28) have been reported to be potent and selective agonists for either AMPA or kainate receptors. We report here the novel synthesis and pharmacological characterization of a range of willardiine (18-23) and 6-azawillardiine (24-28) analogs on cells individually expressing human homomeric hGluR1, hGluR2, hGluR4, or hGluR5 receptors. Reaction of the sodium salts of substituted uracils (7-12) or 6-azauracils (13-16) with (S)-3-[(tert-butoxycarbonyl)amino]oxetan-2-one (17) in dry DMF, subsequent deprotection in TFA, and purification by ion-exchange chromatography gave mainly the willardiine analog in which alkylation took place on N1 of the uracil ring. We have investigated the subtype selectivity of these compounds by examining their binding affinity for homomeric hGluR1, -2, -4, or -5 (and hGluR6 in the case of 5-iodowillardiine (22)). From this study we have demonstrated that 22 has high affinity for hGluR5 and, compared to kainate, displays excellent selectivity for this receptor over both the AMPA receptor subtypes and the homomeric kainate receptor, hGluR6. 5-Fluorowillardiine (19) has higher affinity than AMPA for both homomeric hGluR1 and hGluR2 and compared to AMPA displays greater selectivity for AMPA receptor subtypes over the kainate receptor, hGluR5. Some structural features required for optimal activity at homomeric AMPA or kainate receptor subtypes have also been identified. It would appear that quite large lipophilic substituents at the 5-position of the uracil ring not only are accommodated by hGluR5 receptors but also lead to enhanced affinity for these receptors. In contrast to this, for optimal binding affinity to hGluR1, -2, or -4, smaller, electron-withdrawing substituents are required. For optimal activity at hGluR4 receptors a 6-aza-substituted willardiine is favored. The subtype-selective compounds described here are likely to be useful tools to probe the distribution and the physiological roles of the various glutamate receptor subunits in the central nervous system.     

Pharmacological properties of homomeric and heteromeric GluR1o and GluR3o receptors

Homomeric and heteromeric alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunits GluR1o and GluR3o were expressed in Spodoptera frugiperda (Sf9) insect cells. Membranes containing the recombinant receptors showed a doublet of bands of the expected size (99-109 kDa) after western immunoblotting which was shifted to a single band upon deglycosylation. In (R,S)-[3H]AMPA binding experiments, high expression was seen (Bmax = 0.8-3.8 pmol/mg protein) along with high affinity binding to a single site (Kd, nM+/-S.D.): GluR1o, 32.5+/-2.7; GluR3o, 23.7+/-2.4; GluR1o + GluR3o, 18.1+/-2.9. The pharmacological profiles of these receptors resembled that of native rat brain AMPA receptors: AMPA analogues > L-glutamate > quinoxaline-2,3-diones > kainate. In the Xenopus oocyte expression system we had previously shown that the agonist (R,S)-2-amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionate (ACPA) exhibited an 11-fold selectivity for GluR3o vs. GluR1o. In this study, it was found that ACPA has 3-fold higher affinity at homomeric GluR3o and heteromeric receptors than at homomeric GluR1o, suggesting that its efficacy and/or desensitisation properties are different at GluR1o vs. GluR3o.     

Running fit and generalized tonic-clonic seizure are differently controlled by different subtype receptors in the brainstem

Rats neonatally treated with 0.02% propylthiouracil (PTU) through mother's milk showed a high incidence of audiogenic seizures after maturation. These audiogenic seizures were differently modified by MK-801 and NBQX; while intraperitoneal MK-801 equally inhibited running fit (RF) and generalized tonic-clonic seizure (GTCS), NBQX administered into cisterna ambiens significantly inhibited RF but not GTCS. The possible involvement of glutamate receptors in the inferior colliculus was further investigated using naive Sprague-Dawley rats injected with NMDA, AMPA or cyclothiazide, known as an inhibitor of desensitization of AMPA action. All drugs tested successfully induced RF followed by GTCS, resembling audiogenic seizures in PTU-treated rats. However, sound stimulation could augment AMPA-induced, but not NMDA-induced GTCS. Systemic administration with MK-801 potently blocked GTCS induced by AMPA/cyclothiazide, but the same drug failed to block RF after intracisternal injection with AMPA/cyclothiazide. Furthermore, intracisternal administration with NBQX significantly inhibited only RF induced by AMPA/cyclothiazide. The present study suggests that: 1) glutamate receptors in the brainstem, possible in the inferior colliculus, play a crucial role in audiogenic seizures, namely the initiation of RF and propagation into GTCS; and 2) the initiation mechanism is regulated by both NMDA and AMPA receptors, whereas propagation is mainly controlled by NMDA receptors.     

Desensitizing glutamate receptors shape excitatory synaptic inputs to tiger salamander retinal ganglion cells

AMPA/kainate (KA) receptors mediate a component of ganglion cell excitatory postsynaptic currents (EPSCs). We investigated whether desensitization at these receptors contribute to the shape of transient EPSCs in ON-OFF ganglion cells. Whole-cell, voltage-clamp recordings were made from ganglion cells in the retinal slice or in isolation. EPSCs were evoked by either stimulating the slice with light or puffing K+ at the outer plexiform layer (OPL). The AMPA/KA receptor-mediated component of the EPSCs was isolated by including NMDA receptor antagonists in the bath. Strychnine and picrotoxin blocked inhibitory inputs. In isolated ganglion cells, cyclothiazide (10 microM), which blocks desensitization in non-NMDA receptors, enhanced both the amplitude and the duration of currents evoked by puffs of AMPA or glutamate. EPSCs evoked by K(+)-puffs in the OPL were also enhanced by cyclothiazide (30 microM). When AMPA/KA receptors were blocked with NBQX (10 microM), no enhancement of the EPSCs by cyclothiazide was observed, indicating that cyclothiazide did not act presynaptically. Cyclothiazide also enhanced the amplitude and duration of both the ON and OFF light-evoked (L-) EPSCs recorded in ON-OFF ganglion cells. Current-voltage relationships showed the enhancement was not voltage dependent. When control and enhanced responses where normalized, it was observed that the rate of desensitization of both the ON and OFF L-EPSCs was decreased by cyclothiazide. Cyclothiazide selectively enhanced the AMPA/KA receptor-mediated component of ganglion cells EPSCs, suggesting that desensitization of AMPA/KA receptors shape transient L-EPSCs.     

Variable distributions of Ca(2+)-permeable and Ca(2+)-impermeable AMPA receptors on embryonic rat dorsal horn neurons

1. By measuring the apparent reversal potential (aErev) of kainate- and alpha-amino-3-hydroxyl-5-methyl-4-isoxazole propionic acid (AMPA)-evoked currents associated with changes in extracellular Ca2+ concentration ([Ca2+]e), we have been able to identify embryonic dorsal horn neurons grown in tissue culture that express Ca(2+)-permeable non-N-methyl-D-aspartic acid (NMDA) receptors. 2. The relative expression of Ca(2+)-permeable and Ca(2+)-impermeable non-NMDA receptors varies from cell to cell. This was evident from the range of a ErevS observed for kainate-evoked currents in a 0 mM [Na+]e, 10 mM [Ca2+]e bath. Under these conditions, aErev ranged from -96 to -21 mV, suggesting that the percentage of the non-NMDA receptors on each neuron that are Ca(2+)-permeable is variable. 3. To determine the extent to which the variability in aErev is due to variable receptor expression rather than experimental variability, we compared the effects of changes in [Ca2+]e on kainate-evoked currents and NMDA-evoked currents on the same cells. Assuming that all of the NMDA receptors on each neuron have a similar Ca2+ permeability, this approach provides an index of the sensitivity of our assay system. The reversal potential of NMDA-evoked currents in 10 mM [Ca2+]e ranged from -30 to -7 mV, whereas on the same population of neurons, the aErev of kainate-evoked currents ranged from -92 to -40 mV. 4. The rectification properties of the non-NMDA currents were generally linear or outwardly rectifying in normal bath solution. When the PCa/PCs ratio in 0 mM [Na+]e, 10 mM [Ca2+]e bath solution was assessed as a function of the rectification index in standard bath, a poor correlation was found between Ca2+ permeability and the rectification index. 5. The aErev of kainate-evoked currents was similar to that of cyclothiazide-enhanced AMPA-evoked currents observed on the same cells (-66.5 +/- 18.4 and -64.0 +/- 13.9 mV, mean +/- SD, respectively). This suggests that kainate is primarily activating the AMPA receptor and that the majority of non-NMDA receptors on embryonic dorsal horn neurons in culture are high-affinity AMPA receptors. 6. Immunocytochemical evidence suggests that the AMPA receptor subunits GluR1-4 are expressed to a variable degree from cell to cell in our cultures. We found evidence for low levels of expression of the kainate receptor subunits GluR5-7. The immunocytochemical observations support the physiological data indicating that much of the kainate-evoked current recorded in our experiments can be accounted for by kainate activation of AMPA receptors.(ABSTRACT TRUNCATED AT 400 WORDS)     

Comparative antagonism of kainate-activated kainate and AMPA receptors in hippocampal neurons

Native kainate receptors expressed by cultured hippocampal cells were studied in the whole-cell configuration of the patch-clamp technique by using a fast perfusion system. About 80% of the neurons expressed kainate receptors independently of the time in culture (0-4 days), which coincided with the number of cells immunoreactive for a monoclonal antibody against the GluR5/6/7 subunits. Three types of cells were considered: neurons in which the rapid application of kainate induced a rapidly desensitizing current, cells in which kainate induced a more slowly rising, non-desensitizing, response and those in which a mixture of both responses was apparent. Steady responses induced by 300 microM kainate were inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) in a dose-dependent manner (IC50 = 0.92 microM). CNQX was less potent in blocking transient kainate-induced responses (IC50 = 6.1 microM). Responses to kainate, whether steady or transient, were also inhibited by NS102, showing poor selectivity for the transient response (IC50 = 4.1 and 2.2 microM respectively). The new alpha-amino-3-hydroxy-5-methyl-4-isoxazole (AMPA) receptor antagonist NS394 was very potent in inhibiting steady kainate-induced currents (IC50 = 0.45 microM), but was even more effective in preventing peak responses (IC50 = 0.13 microM). In contrast, cyclothiazide did not affect transient kainate-induced responses but did potentiate current induced by activation of AMPA receptors by AMPA or kainate. These results demonstrate the lack of complete selectivity amongst some available competitive antagonists for AMPA and kainate receptors, and indicate that kainate receptors expressed by hippocampal cells lack the cyclothiazide modulatory site present at AMPA receptors. In addition, the present data support the idea that low-affinity kainate binding sites in the brain correspond to receptor channels selectively activated by kainate.     

Anticoagulants in pregnancy: fetal effects

The effects of an agonist (S-AMPA, i.c.v.), as well as competitive (CNQX, NBQX, DNQX, i.p.), and noncompetitive antagonists (GYKI 52466, i.p.) at the AMPA/kainate receptors were examined in the open field and the Vogel tests of anxiety. It was found that both kinds of antagonists inhibited rat exploratory behavior in a dose-dependent manner, at the dose range exhibiting a clear-cut tendency to decrease rat locomotor activity. They appeared inactive in the Vogel test over an examined dose-range. S-AMPA, whereas not changing in a significant way rat behavior in the open field, significantly enhanced the suppressive influence of a shock on drinking in the Vogel test. The drug administered at the dose of 2 micrograms/5 microliters, i.c.v., revealed also a tendency to decrease the motor activity followed by prodromal symptoms of epileptic-like activity in some subjects. It is concluded that AMPA/kainate receptors probably are not directly involved in the control of rat emotional behavior. Thus, their primary role as putative neuroprotective and anticonvulsant agents is indirectly confirmed.     

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.