Low-affinity Kainate Receptor Research Articles

The Structure of a High-Affinity Kainate Receptor: GluK4 Ligand-Binding Domain Crystallized with Kainate

Ionotropic glutamate receptors play a key role in fast neurotransmission in the CNS and have been linked to several neurological diseases and disorders. Onesubfamily is the kainate receptors, which are grouped into low-affinity (GluK1-3) and high-affinity (GluK4-5) receptors based on their affinity for kainate. Although structures of the ligand-binding domain (LBD) of all low-affinity kainate receptors have been reported, no structures of the high-affinity receptor subunits are available. Here, we present theX-ray structure of GluK4-LBD with kainate at 2.05Å resolution, together with thermofluor and radiolabel binding affinity data. Whereas binding-site residues in GluK4 are most similar to the AMPA receptor subfamily, the domain closure and D1-D2 interlobe contacts induced by kainate are similar to the low-affinity kainate receptor GluK1. These observations provide a likely explanation for the high binding affinity of kainate at GluK4-LBD.

Mechanisms of bi-directional modulation of thalamocortical transmission in barrel cortex by presynaptic kainate receptors

Presynaptic kainate receptors play an important role in synaptic transmission and short-term plasticity to profoundly regulate network activity in many parts of the mammalian brain. In primary sensory neocortex, where short-term synaptic plasticity is important for receptive field structure and information processing, kainate receptors are highly expressed and regulate thalamocortical inputs, particularly during development. However, the mechanisms of the kainate receptor-dependent presynaptic regulation of thalamocortical transmission are unclear. We therefore investigated this issue using electrophysiology in neonatal thalamocortical slices of barrel cortex combined with pharmacology and biochemical analyses. We show that presynaptic kainate receptors can both facilitate or depress synaptic transmission depending on the extent of their activation. This bi-directional regulation is mediated in part by kainate receptors that directly influence thalamocortical axonal excitability, but also likely involves receptors acting at thalamocortical terminals to regulate transmitter release. The efficacy of kainate in regulating thalamocortical transmission is low compared to that reported for other inputs. Consistent with this low efficacy, our biochemical analyses indicate that the presynaptic kainate receptors regulating neonatal thalamocortical inputs likely lack the high kainate affinity GluK4 and 5 subunits. Thus thalamocortical transmission can be bi-directionally regulated by low affinity kainate receptors through two mechanisms. Such presynaptic regulation provides a potentially powerful mechanism to influence sensory processing during development of barrel cortex.

Structural Investigation of the 7-Chloro-3-hydroxy-1H-quinazoline-2,4-dione Scaffold to Obtain AMPA and Kainate Receptor Selective Antagonists. Synthesis, Pharmacological, and Molecular Modeling Studies

In this paper, the study of new 7-chloro-3-hydroxy-1H-quinazoline-2,4-dione derivatives, designed as AMPA and kainate (KA) receptor antagonists, is reported. Some derivatives bear different carboxy-containing alkyl chains on the 3-hydroxy group, while various heterocyclic rings or amide moieties are present at the 6-position of other compounds. Binding data at Gly/NMDA, AMPA, and high-affinity KA receptors showed that the presence of the free 3-hydroxy group is of paramount importance for a good affinity at all three investigated receptors, while introduction of some 6-heterocyclic moieties yielded AMPA-selective antagonists. The most significant result was the finding of the 6-(2-carboxybenzoylamino)-3-hydroxy-1H-quinazolin-2,4-dione 12, which possesses good affinity for high-affinity and low-affinity KA receptors (Ki=0.62 microM and 1.6 microM, respectively), as well as good selectivity. To rationalize the trend of affinities of the reported derivatives, an intensive molecular modeling study was carried out by docking compounds to models of the Gly/NMDA, AMPA, and KA receptors.

Glutamate receptor agonist kainate enhances primary dendrite number and length from immature mouse cortical neurons in vitro

Glutamate is an important regulator of dendrite development that may inhibit, (during ischemic injury), or facilitate (during early development) dendrite growth. Previous studies have reported mainly on the N-methyl-D-aspartate (NMDA) receptor-mediated dendrite growth-promoting effect of glutamate. In this study, we examined how the non-NMDA receptor agonist kainate influenced dendrite growth. E18 mouse cortical neurons were grown for 3 days in vitro and immunolabeled with anti-microtubule-associated protein 2 (MAP2) and anti-neurofilament (NF-H), to identify dendrites and axons, respectively. Exposure of cortical neurons to kainate increased dendrite growth without affecting neuron survival. This effect was dose-dependent, reversible and blocked by the alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate (AMPA)/kainate receptor antagonist NBQX and the low-affinity kainate receptor antagonist NS-102, but not by the AMPA receptor antagonist CFM-2. In addition, the NMDA receptor antagonist MK-801 had no effect on kainate-induced dendrite growth. Immunolabeling and Western blot analysis of kainate receptors using antibodies against the GluR6 and KA2 subunits, demonstrated that the immature cortical neurons used in this study express kainate receptor proteins. These results suggest that kainate-induced non-NMDA receptor activation promotes dendrite growth, and in particular primary dendrite number and length, from immature cortical neurons in vitro, and that kainate receptors may be directly involved in this process. Furthermore, these data support the possibility that like NMDA receptors, kainate receptor activation may also contribute to early neurite growth from cortical neurons in vitro.

Presynaptic low- and high-affinity kainate receptors in nociceptive spinal afferents

Presynaptic ionotropic glutamate receptors are increasingly attributed a role in the modulation of sensory input at the first synapse of dorsal root ganglion (DRG) neurons in the spinal dorsal horn. Central terminals of DRG neurons express AMPA and NMDA receptors whose activation modulates the release of glutamate, the main transmitter at these synapses. Previous work, with an antibody that recognizes all low-affinity kainate receptor subunits (GluR5, 6, 7), provided microscopic evidence of presynaptic kainate receptors in unidentified primary afferent terminals in superficial laminae of the spinal dorsal horn (Hwang SJ, Pagliardini S, Rustioni A, Valtschanoff JG. Presynaptic kainate receptors in primary afferents to the superficial laminae of the rat spinal cord. J Comp Neurol 2001; 436: pp. 275–289). We show here that, although all such subunits may be expressed in these terminals, GluR5 is the subunit most readily detectable at presynaptic sites in sections processed for immunocytochemistry. We also show that the high-affinity kainate receptor subunits KA1 and KA2 are expressed in central terminals of DRG neurons and are co-expressed with low-affinity receptor subunits in the same terminals. Quantitative data show that kainate-expressing DRG neurons are about six times more likely to express the P2X 3 subunit of the purinergic receptor than to express substance P. Thus, nociceptive afferents that express presynaptic kainate receptors are predominantly non-peptidergic, suggesting a role for these receptors in the modulation of neuropathic rather than inflammatory pain.

Differential oxidative stress in oligodendrocytes and neurons after excitotoxic insults and protection by natural polyphenols

Oligodendrocytes are vulnerable to overactivation of both their AMPA receptors and their high- and low-affinity kainate receptors. Depending on the intensity of the insult and the type of receptor activated, excitotoxic oligodendrocyte death mediated by these receptors has different characteristics. One important consequence at a cellular level is the ensuing oxidative stress, related to Ca2+-dependent alterations in mitochondrial functioning. We observed that oxidative stress associated with selective AMPA receptor activation is much higher than that associated with the selective activation of high- and low-affinity kainate receptors. Moreover, excitotoxic insults generate more intense oxidative stress in oligodendrocytes than in cortical neurons, though similar alterations in [Ca2+]i and mitochondrial potential were observed in both cell types. Nanomolar concentrations of mangiferin and morin, two natural polyphenols with antioxidant properties, partially protect oligodendrocytes as well as cortical neurons from mild, but not intense, insults mediated by AMPA receptors. In addition to presenting oxygen radical scavenging activity, mangiferin and morin attenuate the intracellular Ca2+ overload subsequent to the activation of AMPA receptors, a mechanism that may contribute to their protective properties. The inclusion of these antioxidant agents in therapeutic strategies for the treatment of diseases in which oligodendrocyte as well as neuron loss occurs may prove to be beneficial.

Caspase-Dependent and Caspase-Independent Oligodendrocyte Death Mediated by AMPA and Kainate Receptors

Oligodendrocytes are vulnerable to excitotoxic signals mediated by AMPA receptors and by high- and low-affinity kainate receptors. Here we investigated the nature of the cell death triggered by activation of these receptors in primary cultures of oligodendrocytes from the rat optic nerve. Activation of AMPA receptors at both submaximal and maximal concentrations of the agonist induced massive calcium entry, mitochondrial depolarization, and a rise in the level of reactive oxygen species that correlated with a decrease in the levels of reduced glutathione. In addition, excitotoxicity initiated by submaximal, but not maximal, activation of AMPA receptors was prevented by caspase-3 blockade and by the concomitant blockade of caspases 8 and 9. In turn, maximal activation of high- or low-affinity kainate receptors induced mitochondrial events and toxicity levels similar to those observed with submaximal activation of AMPA receptors. In contrast to AMPA receptor-mediated insults, calcineurin inhibition or caspase-9 blockade was sufficient to prevent cell death triggered by both types of kainate receptors. Consistent with these results, prolonged glutamate receptor activation in freshly isolated optic nerves caused selective activation of caspase-3 and chromatin condensation in oligodendrocytes. Overall, the evidence presented here indicates that oligodendrocyte death by excitotoxicity is mediated by caspase-dependent and -independent mechanisms.

Ca2+ Influx through AMPA or Kainate Receptors Alone Is Sufficient to Initiate Excitotoxicity in Cultured Oligodendrocytes

Oligodendrocytes are vulnerable to excitotoxic insults mediated by AMPA receptors and by low and high affinity kainate receptors, a feature that is dependent on Ca2+ influx. In the current study, we have analyzed the intracellular concentration of calcium [Ca2+]i as well as the entry routes of this cation, upon activation of these receptors. Selective activation of either receptor type resulted in a substantial increase (up to fivefold) of [Ca2+]i, an effect which was totally abolished by the non-NMDA receptor antagonist CNQX or by removing Ca2+ from the culture medium. Blockade of voltage-gated Ca2+ channels with La3+ or nifedipine, reduced the amplitude of the Ca2+ current triggered by AMPA receptor activation by ∼65%, but not that initiated by low and high affinity kainate receptors. In contrast, KB-R7943, an inhibitor of the plasma membrane Na+–Ca2+ exchanger, solely attenuated the rise in [Ca2+]i by ∼25% due to activation of low affinity kainate receptors. However, oligodendroglial death by glutamate receptor overactivation was largely unaffected in the presence of La3+ or KB-R7943. These findings indicate that Ca2+ influx via AMPA and kainate receptors alone is sufficient to initiate cell death in oligodendrocytes, which does not require the entry of calcium via other routes such as voltage-activated calcium channels or the plasma membrane Na+–Ca2+ exchanger.

Regional mapping of low-affinity kainate receptors in mouse brain using [ 3H](2 S,4 R)-4-methylglutamate autoradiography

Recent data indicate that (2 S,4 R)-4-methylglutamate is a selective agonist for low affinity (GluR5 and GluR6) kainate receptor subunits. In the present study, we have employed [ 3H](2 S,4 R)-4-methylglutamate to examine low affinity kainate receptor distribution in mouse brain. [ 3H](2 S,4 R)-4-Methylglutamate labelled a single site in murine cerebrocortical membranes ( K d=9.9±2.7 nM, B max=296.3±27.1 fmol mg protein −1). The binding of 8 nM [ 3H](2 S,4 R)-4-methylglutamate was displaced by several non-NMDA receptor ligands ( K i±S.E.M.): domoate (1.1±0.2 nM)>kainate (7.1±1.1 nM)≫ l-glutamate (187.6±31.9 nM)≫( S)-α-amino-3-hydroxy-5-methyl-4-isoazolepropionic acid (AMPA) (>50 μM). [ 3H](2 S,4 R)-4-Methylglutamate autoradiography revealed a widespread regional distribution of low affinity kainate receptors. Highest binding densities occurred within deep layers of the cerebral cortex, olfactory bulb, basolateral amygdala and hippocampal CA3 subregion. Moderate labelling was also evident in the nucleus accumbens, dentate gyrus, caudate putamen, hypothalamus and cerebellar granule cell layer. These data show that [ 3H](2 S,4 R)-4-methylglutamate is a useful radioligand for selectively labelling low affinity kainate receptors.

Excitotoxic profiles of novel, low-affinity kainate receptor agonists in primary cultures of murine cerebellar granule cells

The involvement of low-affinity kainate (KA) receptors in neuronal injury was investigated by employing a variety of agonists active at GluR5–7. Their excitotoxic profiles were determined in primary cultures of cerebellar granule cells, which abundantly expressed low-affinity KA receptors, and in the absence of any AMPA receptor-mediated neurotoxicity. Neurotoxicity induced by these compounds was analysed by phase contrast microscopy, a cell viability assay, the TUNEL technique (apoptosis), and by employing propidium iodide (PI; necrosis). All agonists induced concentration-dependent neurotoxicity, with rank order (EC 50 values; μM): ( S)-iodowillardiine (IW) 0.2>(2 S,4 R)-4-methylglutamate (4-MG) 36>(2 S,4 R,6 E)-2-amino-4-carboxy-7-(2-naphthyl)hept-6-enoic acid (LY339434) 46>KA 74>( RS)-2-amino-3-(hydroxy-5- tert-butylisoxazol-4yl)propanoic acid (ATPA) 88. IW exposure resulted in apoptosis at lower concentrations (<30 μM) and necrosis at higher concentrations, both of which were attenuated by CNQX (50 μM), but not MK-801 (10 μM). ATPA-mediated neurotoxicity was purely apoptotic and was attenuated by the non-NMDA receptor antagonists. Both IW and ATPA induced injury with the morphological characteristics of apoptosis shown by the presence of TUNEL-positive neurones. LY339434-mediated neuronal injury was only attenuated by MK-801 and was necrotic in nature. Similarly, 4-MG (>30 μM) exposure caused necrosis that was partially attenuated by MK-801 (10 μM) and CNQX (50 μM). The patterns of neurotoxicity possessed a complex pharmacological profile, demonstrated an apoptotic–necrotic continuum and were inconsistent with past findings, further outlining the importance of characterizing novel compounds at native receptors. ATPA and to a lesser extent IW appear to be suitable drugs for low-affinity KA receptors. Since toxicity-mediated by low-affinity KA receptors seem likely to contribute to neurodegenerative conditions, our study importantly examines the excitotoxic profile of these novel agonists.

(3)H](2S,4R)-4-Methylglutamate: a novel ligand for the characterization of glutamate transporters.

[(3)H](2S,4R)-4-Methylglutamate ([(3)H]4MG), used previously as a ligand for low-affinity kainate receptors, was employed to establish a binding assay for glutamate transporters (GluTs), as 4MG has also been shown to have affinity for the glial GluTs, GLT1 and GLAST. In rat brain membrane homogenates in the presence of Na(+) ions at 4 degrees C, specific binding of [(3)H]4MG was rapid and saturable (t(1/2) approximately 15 min), representing > 90% of total binding. Dissociation of [(3)H]4MG occurred in a biphasic manner, however, saturation studies and Scatchard analysis indicated a single site of binding (n(H) = 0.85) and a K(d) of 6.2 +/- 0.8 microM with a B(max) of 111.8 +/- 23.8 pmol/mg protein. Specific binding of [(3)H]4MG was Na(+)-dependent and inhibited by K(+) and HCO(3-). Pharmacological inhibition with compounds acting at GluTs revealed that Glu, D- and L-aspartate, L-serine-O-sulfate and Ltrans-pyrrolidine-2,4-dicarboxylate fully displaced specific binding. Drugs having preferential affinity for GLT1, kainate, dihydrokainate and Lthreo-3-methylglutamate, all inhibited approximately 40% of specific binding. The inhibition pattern of L-serine-O-sulfate in the presence of a saturating concentration of dihydrokainate was suggestive of [(3)H]4MG also labelling GLAST. 6-Cyano-7-nitroquinoxaline, a kainate receptor antagonist, and a range of Glu receptor agonists and antagonists failed to significantly inhibit [(3)H]4MG binding. The pharmacological profile of binding of [(3)H]4MG resembled that found for [(3)H]D-aspartate, a ligand specific for GluTs, reinforcing the hypothesis that [(3)H]4MG was labelling GluTs in this assay. Together, these data illustrate the development of an efficient, economic binding assay that is suitable for the characterization of different subtypes of GLuTs.

Maturational Aspects of Epilepsy Mechanisms and Consequences for the Immature Brain

Maturational Aspects of Epilepsy Mechanisms and Consequences for the Immature Brain

Excitotoxic profile of LY339434, a GluR5 agonist, in cultured murine cortical neurons

The neurotoxic profile of (2 S,4 R,6E)-2-amino-4-carboxy-7-(2-naphthyl)hept-6-enoic acid (LY339434), a low-affinity kainate receptor subtype 5 (GluR5) agonist at recombinant human glutamate receptors, was evaluated to investigate the involvement of GluR5 in excitotoxic neuronal death. Murine cortical neurons were exposed to treatments for 24 h and assessed by a cell viability assay and phase-contrast microscopy. LY339434 (1–1000 μM) caused a concentration-dependent decrease in cell viability (EC 50=11.4±1.2 μM) that was only attenuated by (5 R,10 S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[ a, d]cyclohepten-5,10-imine (MK-801, 10 μM), but not by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 50 μM) or 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI 52466, 20 μM). Labeling with nucleic acid binding dyes revealed that LY339434 induced few apoptotic-like characteristics. These findings indicate that in cultured murine cortical neurons, LY339434 acts predominantly through N-methyl- d-aspartate (NMDA) receptors rather than GluR5 to effect neuronal death that is rapid and involves predominantly necrosis rather than morphological apoptosis.

Low-affinity kainate receptor agonists induce insult-dependent apoptosis and necrosis in cultured murine cortical neurons

Overstimulation of ionotropic glutamate receptors leads to excitotoxic neuronal death, which has been implicated in the neurodegeneration of neurological diseases. The present study examined the role of putative low-affinity kainate receptor subtype (GluR5–7) agonists in excitotoxicity in cultured murine cortical neurons. The concentration-dependent decrease in cell viability induced by the agonists kainate (1–1,000 μM) and (RS)-2-amino-3-(hydroxy-5-tert-butylisoxazol-4-yl) propanoic acid (ATPA; 1–1,000 μM) was only attenuated by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 μM) and 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI 52466; 20 μM). (S)-5-iodowillardiine (1–1,000 μM)-induced toxicity was attenuated by CNQX (20 μM), GYKI 52466 (20 μM) and MK-801 (10 μM); however, (2S,4R)-4-methylglutamate (1–120 μM)-induced toxicity was not attenuated by the antagonists. None of the agonists possessed selective actions at GluR5–7. Morphological observations (phase-contrast and fluorescence microscopy) revealed that the agonists induced two distinct patterns of neuronal injury. After 24 hr of treatment, low concentrations of agonists (1–30 μM) produced cellular shrinkage and nuclear granulation consistent with slow, apoptotic-like neuronal death. Pyknotic labeling with the DNA binding dye Sytox green confirmed these apoptotic characteristics, which significantly decreased with increasing concentrations. After 4 hr, increasing concentrations of agonists (100–1,000 μM) induced cellular swelling, with subsequent extracellular debris; labeling with propidium iodide revealed isolated nuclei consistent with the increased involvement of rapid necrosis. Thus, all putative GluR5–7 agonists produced excitotoxicity across a necrotic-apoptotic continuum in murine cortical neuron cultures. J. Neurosci. Res. 59:788–796, 2000 © 2000 Wiley-Liss, Inc.

Low-affinity kainate receptor agonists induce insult-dependent apoptosis and necrosis in cultured murine cortical neurons.

Overstimulation of ionotropic glutamate receptors leads to excitotoxic neuronal death, which has been implicated in the neurodegeneration of neurological diseases. The present study examined the role of putative low-affinity kainate receptor subtype (GluR5-7) agonists in excitotoxicity in cultured murine cortical neurons. The concentration-dependent decrease in cell viability induced by the agonists kainate (1-1,000 microM) and (RS)-2-amino-3-(hydroxy-5-tert-butylisoxazol-4-yl) propanoic acid (ATPA; 1-1,000 microM) was only attenuated by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 microM) and 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI 52466; 20 microM). (S)-5-iodowillardiine (1-1,000 microM)-induced toxicity was attenuated by CNQX (20 microM), GYKI 52466 (20 microM) and MK-801 (10 microM); however, (2S, 4R)-4-methylglutamate (1-120 microM)-induced toxicity was not attenuated by the antagonists. None of the agonists possessed selective actions at GluR5-7. Morphological observations (phase-contrast and fluorescence microscopy) revealed that the agonists induced two distinct patterns of neuronal injury. After 24 hr of treatment, low concentrations of agonists (1-30 microM) produced cellular shrinkage and nuclear granulation consistent with slow, apoptotic-like neuronal death. Pyknotic labeling with the DNA binding dye Sytox green confirmed these apoptotic characteristics, which significantly decreased with increasing concentrations. After 4 hr, increasing concentrations of agonists (100-1,000 microM) induced cellular swelling, with subsequent extracellular debris; labeling with propidium iodide revealed isolated nuclei consistent with the increased involvement of rapid necrosis. Thus, all putative GluR5-7 agonists produced excitotoxicity across a necrotic-apoptotic continuum in murine cortical neuron cultures.

Expression and localization of ionotropic glutamate receptor subunits in the goldfish retina--an in situ hybridization and immunocytochemical study.

The expression and distribution of AMPA, kainate and NMDA glutamate receptor subunits was studied in the goldfish retina. For the immunocytochemical localization of the AMPA receptor antisera against GluR2, GluR2/3 and GluR4 were used, and for in situ hybridization rat specific probes for GluR1 and GluR2 and goldfish specific probes for GluR3 and GluR4 were used. The localization of the low affinity kainate receptor and NMDA receptor was studied using antisera against GluR5-7 and NR1. All AMPA receptor subtypes were demonstrated to be present in the goldfish retina both by immunocytochemistry and in situ hybridization. In situ hybridization revealed expression of all AMPA receptors subunit at the inner border of the INL. Only GluR3 was also strongly expressed in the outer border of the INL. Some of the ganglion cells displayed a strong signal for GluR1, GluR3 and GluR4. GluR1-immunoreactivity was present in subsets of bipolar, amacrine, and ganglion cells. GluR2 and GluR2/3-immunoreactivity was mainly localized in the outer plexiform layer. GluR2 and GluR2/3-immunoreactivity are associated with the photoreceptor synaptic terminals. GluR4-immunoreactivity is present on Müller cells in the inner retina and on dendrites of bipolar cells in the OPL, whereas GluR5-7-immunoreactivity was prominently present on horizontal cell axon terminals. Finally, NR1-immunoreactivity was confined to amacrine cells, the inner plexiform layer and ganglion cells. This study shows that there is a strong heterogeneity of glutamate receptor subunit expression in the various layers of the retina. Of the AMPA receptor subunits GluR3 seems to be expressed the most widely in all layers with strong glutamatergic synaptic interactions whereas all the other subunits seem to have a more restricted expressed pattern.

Reduced editing of low-affinity kainate receptor subunits in optic nerve glial cells

We have analyzed the degree of editing of adult optic nerve mRNAs encoding the low-affinity kainate receptor subunits, GluR5 and GluR6, the two major constituents of native receptors in this family. To this end, we used reverse transcription (RT) followed by polymerase chain reaction (PCR), and subsequent cloning and sequencing of the amplified fragments. Our results revealed that the GluR5 subunit is unedited at the Q/R site of cismembrane domain 2 (M2), whereas the GluR6 subunit is edited to a low extent at this site. These findings are in contrast to those reported by others using mRNAs from the adult brain in which GluR5 and GluR6 are edited at the Q/R site of M2 to a larger extent. In addition, we found that the adenosine deaminases, DRADA, RED1 and RED2, which edit ionotropic glutamate receptors in the brain, are expressed in the adult optic nerve and in oligodendrocytes, the major cell type in this structure. It thus appears that editing of kainate receptors in optic nerve cells is not limited by the availability of editing enzymes but rather by other, as yet unidentified factors. Overall, reduced editing of kainate receptor subunits in glial cells may result in different functional responses of the native receptors present in these cells with respect to those in neurons.

Excitotoxic injury profiles of low-affinity kainate receptor agonists in cortical neuronal cultures

Neurotoxic profiles of putative agonists for low-affinity kainate subtypes of l-glutamate receptors (GluR5-7) were determined in cultured cortical neurones. Rank order of neurotoxic potency (μM): (S)-5-iodowillardiine (9)≈(2 S,4 R,6 E)-2-amino-4-carboxy-7-(2-naphthyl)hept-6-enoic acid (LY339434, 11)>(2 S,4 R)-4-methylglutamate (33)>kainate (100)>( RS)-2-amino-3-(hydroxy-5- tert-butylisoxazol-4-yl)propanoic acid (ATPA, 360). Using ionotropic glutamate receptor antagonists, neurotoxicity induced by kainate, ATPA and ( S)-5-iodowillardiine appeared to involve a GluR5-7 component, unlike LY339434 and (2 S,4 R)-4-methylglutamate. These putative GluR5-7 agonists exhibited complex excitotoxic profiles highlighting the importance of studying native glutamate receptors.

Regional distribution of low affinity kainate receptors in brain of Macaca fascicularis determined by autoradiography using [ 3H](2 S,4 R)-4-methylglutamate

Binding of [ 3H](2 S,4 R)-4-methylglutamate, a novel low affinity kainate receptor agonist, was studied in brain sections of a Macaca fascicularis monkey. In cerebellar sections, [ 3H](2 S,4 R)-4-methylglutamate bound to a single population of sites ( K D=20 nM) and was inhibited by various glutamate receptor ligands: kainate>6-cyano-7-nitroquinoxaline-2,3-dione> l-glutamate>>AMPA. ( S)-5-Iodowillardiine and ( RS)-2-amino-3-(3-hydroxy-5- tert-butylisoxazol-4-yl)propanoic acid (ATPA), drugs selective for the GluR5 subunit, displaced 50% and 40% of binding, respectively. Autoradiography revealed topographic binding of [ 3H](2 S,4 R)-4-methylglutamate. Binding in cortex was highest in layer 5 and restricted to CA2/3 in hippocampus. Levels of binding were high in septum and hypothalamus. Moderate densities of binding were found in caudate-putamen, cerebellar granular layer, nucleus tractus solitarius, cuneate nucleus and area postrema. Binding in spinal cord was concentrated in dorsal horn. [ 3H](2 S,4 R)-4-Methylglutamate shows differential binding throughout primate brain and is a valuable new ligand for low affinity kainate receptors.

Low-affinity kainate receptors and long-lasting depression of NMDA-receptor-mediated currents in rat superficial dorsal horn.

In an in vitro spinal cord slice preparation whole cell electrophysiological recordings of rat superficial dorsal horn neurons responding differentially to glutamate (Glu) and N-methyl-D-aspartate (NMDA) were investigated systematically for the role of kainate (KA) receptors in modulating their activity. In these neurons, coapplication of Glu and NMDA, as well as application of Glu immediately before NMDA, induced long- and short-lasting depressions of NMDA-induced currents as well as depression of NMDA-receptor-mediated excitatory postsynaptic currents. KA applied before NMDA mimicked Glu-induced attenuating effects. Furthermore, the low-affinity KA receptor antagonist 5-nitro-6,7,8,9- tetrahydrobenzo[G]indole-2,3-dione-3-oxime potentiated Glu-induced NMDA-receptor-mediated currents in neurons responding differentially to Glu and NMDA. These results provide evidence for a novel mechanism, which may relate to classical long-term depression, involving low-affinity KA receptors in long-lasting modulation of NMDA-receptor-mediated currents. This implies a physiological role of KA receptors in long-term modulation of sensory transmission in the superficial dorsal horn of rat spinal cord.