Kainate Binding Sites Research Articles

Localization of striatal excitatory amino acid binding site subtypes to striatonigral projection neurons

Quantitative autoradiography was used to examine the cellular localization of excitatory amino acid binding sites in the striatum following selective lesion of striatonigral projection neurons. Degeneration of striatonigral neurons was induced unilaterally by injection of the suicide transport toxin, volkensin, into the left substantia nigra. Twelve days following nigral volkensin injection there was a reduction of all excitatory amino acid binding site subtypes in the striatum ipsilateral to the injected nigra. The reduction in N- methyl- d-aspartate (NMDA) binding sites was significantly greater than the loss of d,l-α-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA), kainate and metabotropic binding. These results indicate that there are NMDA, AMPA, metabotropic and kainate binding sites on striatonigral projection neurons and suggest that the NMDA subtype may be selectively enriched on striatonigral neurons.

Identification of the Serum Complex Which Induces Cerebellar Granule Cell In Vitro Differentiation and Resistance to Excitatory Amino Acids.

The protein complex promoting in vitro terminal differentiation of cerebellar granule cells has been isolated from rabbit serum. We designate the complex the neurite outgrowth and adhesion complex (NOAC). The apparent molecular weight, evaluated by gel filtration, is 80 - 100 kDa. Rat cerebellar granule cells cultured in NOAC exhibit much lower glial cell contamination and survive, in their differentiated state, much longer than in 10% foetal calf serum. While they bind tetanus toxin, express specific antigens such as synapsin I, synaptophisin and A2B5, and release [3H]d-aspartate in a fashion similar to that shown by cells cultured in foetal calf serum, they show a 60% reduction in the total number of kainate binding sites. Excitatory amino acid (EAA)-triggered and depolarization-stimulated calcium influx, measured in the presence of different agonists, is 50 - 80% lower in NOAC-cultured cells. NOAC cells are resistant to excitotoxic stimuli carried by EAAs or by depolarizing treatments with 50 mM KCl or 6 microM veratridine. The marked resistance of NOAC-cultured neurons to EAAs can be attributed to decreased calcium entry through EAA-coupled and voltage-gated calcium channels and possibly to other, as yet unidentified, phenotypic properties of these cells. These findings demonstrate that rabbit serum contains one or more polypeptide(s) endowed with the properties of promoting in vitro survival and differentiation of rat cerebellar granule cells and of conferring an EAA-resistant phenotype.

Characterization of l-glutamate and kainate binding sites in the brain of a freshwater fish, Telapilia monsanbica

[ 3H]Kainate and l-[ 3H]glutamate binding sites in a rich source of kainate binding sites, fish brain, have been thoroughly analysed here for the purpose of studying the correlation between kainate binding sites and l-glutamate receptors in vertebrate CNS. The brain of a freshwater fish, Telapilia monsanbica, was found to contain three types of kainate binding sites: Type 1 sites ( K d = 1050 ± 380μM, B max = 4 ± 4pmol/mg), Type 2 sites ( K d = 133 ± 20nM, B max = 190 ± 20pmol/mg), and Type 3 sites ( K d = 23 ± 15nM, B max = 28 ± 19pmol/mg). The dissociation constants of l-glutamate to Type 1, 2 and 3 sites were, respectively, 0.28 ± 0.04, 5.5 ± 0.2 and 137 ± 28μM. Pharmacological characterization of these binding sites showed that Type 1 and 2 sites, respectively, corresponded to N-methyl- d-aspartate-subtype l-glutamate receptors and Non-N-methyl- d-aspartate l-glutamate receptors. Autoradiographic studies showed that Type 1 and 2 sites were distributed widely in fish brain, indicating the involvement of l-glutamate receptors in various brain functions. Type 3 sites, on the other hand, were relatively insensitive to most endogenous amino acids and were only found in the molecular layer of cerebellum and torus longitudinalis. Type 3 sites possibly representing a distinctive class of receptor has been suggested by the results.

The interaction of a kainate receptor from goldfish brain with a pertussis toxin-sensitive GTP binding protein.

Kainate receptors are present in high concentrations in goldfish brain (Henley and Oswald, 1988a and b; Ziegra et al., 1990), possibly in neuronal and glial cells. In a number of systems, the kainate receptor has been assumed to be an integral ion channel (Watkins and Evans, 1981); but, for some kainate receptors, ion channel activity has not been demonstrated (Wada et al., 1989). This study presents evidence that a portion of the [3H]kainate-binding sites in goldfish brain is sensitive to guanine nucleotides, with a loss of high affinity binding in the presence of nonhydrolyzable GTP analogs. Pertussis toxin pretreatment of membranes causes a loss of high affinity [3H]kainate binding and of the guanine nucleotide-sensitive binding. Pertussis toxin catalyzes the specific [32P]ADP-ribosylation of a 40-kDa substrate in a kainate-sensitive manner. In addition, incorporation of [alpha-32P]GTP-gamma-azidoanilide by photoaffinity labeling was enhanced in the presence of kainate. These results indicate that a subpopulation of [3H]kainate-binding sites in goldfish brain may be coupled to G proteins.

Alterations in spinal cord excitatory amino acid receptors in amyotrophic lateral sclerosis patients

Excitatory amino acids (EAA) have been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). We have analyzed the distribution of the N-methyl- d-aspartate (NMDA) 1-(1-(2-thienyl)-cyclohexyl) piperidine (TCP), kainate and α-amino-3-hydroxy-5-methyl-4 isoxazole proprionic acid (AMPA) quisqualate subtypes of EAA receptors using quantitative receptor autoradiography in the cervical and thoracic spinal cords of patients who have died with ALS, and of controls. We observed that in control spinal cords [ 3H]TCP/NMDA binding sites were located both in the ventral and dorsal horns with the highest densities being situated in lamina II. [ 3H]AMPA and [ 3H]kainate binding sites were present almost exclusively in the substantia gelatinosa of the dorsal horn. In ALS, the distribution of these 3 types of receptors was unchanged, but [ 3H]TCP/NMDA binding was decreased both in the dorsal and ventral horns. [ 3H]kainate binding was possibly decreased in substantia gelatinosa, of ALS cords. However, the limited sample size available for [ 3H]kainate binding did not permit statistical analysis. [ 3H]AMPA binding sites were unaltered in ALS. These results indicate that there is a preferential reduction in NMDA receptors in ALS. We suggest that should an excitotoxic mechanism be involved in the pathogenesis of ALS, then NMDA receptors may be the target of this effect.

Coupling of a purified goldfish brain kainate receptor with a pertussis toxin-sensitive G protein.

Goldfish brain has a high density of [3H]kainate-binding sites, a subpopulation of which appears to be coupled to a pertussis toxin-sensitive G protein. We show here that a purified kainate receptor preparation reconstituted into phospholipid vesicles exhibits guanine nucleotide-sensitive high-affinity [3H]kainate binding. Pertussis toxin treatment abolishes the guanine nucleotide-sensitive portion of the [3H]kainate binding, and kainate promotes [3H]guanosine 5'-[beta,gamma-imido]triphosphate binding and [gamma-32P]GTP hydrolysis. Guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]) decreases the apparent Stokes radius of the soluble purified receptor preparation, consistent with dissociation of the kainate receptor-G protein complexes. The affinity-purified preparations contain proteins of 45, 41, and 35 kDa. The 45- and 41-kDa proteins crossreact with antibodies against the kainate receptor cloned from frog brain. The 35-kDa protein is recognized by an antiserum (SW) directed against the beta subunit of G proteins. When kainate receptors are purified in the presence of GTP[gamma S], the 35-kDa protein is no longer present. Also, [3H]kainate affinity is decreased and is no longer guanine nucleotide sensitive. Upon reconstitution with purified G proteins, high-affinity guanine nucleotide-sensitive binding and kainate-stimulated GTPase activity can be restored. These observations indicate that a kainate receptor from goldfish brain functionally interacts with a pertussis toxin-sensitive G protein.

Alterations in [ 3H]kainate and N-methyl- d-aspartate-sensitive l-[ 3H]-glutamate binding in the rat hippocampal formation following fimbria-fornix lesions

Following lesions of the fimbria-fornix, there is a time-dependent increase in interictal spikes and seizure susceptibility. This may result from sprouting of local excitatory and inhibitory circuits in response to the loss of subcortical and commissural innervation of the hippocampal formation. We used receptor autoradiography to examine the density of N-methyl- d-aspartate (NMDA)-sensitive l-[ 3H]glutamate and [ 3H]kainate (KA) binding sites in the hippocampal formation at 5 days, 3 months, and 1 year following bilateral aspiration lesions of the fimbria-fornix. At 5 days postlesion, the CA3 and CA1 strata radiatum and oriens displayed a decrease (20–42%, P < 0.01) in NMDA-sensitive l-[ 3H]glutamate binding. The initial decrease was followed by a moderate recovery at later time points but was still evident at 1 year postlesion. This may reflect a lesion-induced turnover of synaptic complexes, down-regulation of postsynaptic receptors, or loss of presynaptic receptors. Five days following fimbria-fornix lesion there was also a decrease (13–15%, P < 0.05) in [ 3H]KA binding in CA3 strata radiatum and pyramidale. However, at 3 months postlesion KA receptor density was elevated by 29–33% ( P < 0.01) in the outer molecular layer of the dentate gyrus with no significant change in binding to the inner molecular layer. By 1 year postlesion, the density of [ 3H]KA binding sites was not significantly different from that observed in control animals of the same age. The increase in KA receptor density in the outer molecular layer 3 months after fimbria-fornix lesion may reflect sprouting of the perforant path input or mossy fibers to this region and contribute to the increase in interictal spikes and seizure susceptibility.

Cloning of a putative glutamate receptor: A low affinity kainate-binding subunit

Kainate, a glutamate receptor agonist, is a potent neuroexcitatory agent that produces epileptiform activity and selective neuronal degeneration. Binding studies using neuronal membrane homogenates or brain sections have identified sites having either high or low affinity for [ 3H]kainate. Here we report the cloning of a gene, GluR7, with approximately 75% sequence identity with the previously cloned GIuR5 and GIuR6 subunit genes. Transcripts of the GIuR7 gene are evident in brain areas that bind [ 3H]kainate and are susceptible to kainateinduced neurotoxicity. We have performed ligand binding studies with membranes of transfected HeLa cells expressing GIuR6 or GIuR7 subunits. Our data show that the GIuR6 and GIuR7 subunits have a rank order of agonist affinity (domoate > kainate ≫ l-glutamate, quisqualate ≫ AMPA, NMDA) and a dissociation constant for kainate (95 and 77 nM, respectively) characteristic of the low affinity kainate-binding sites described in the brain.

Effects of acromelic acid A on the binding of [3H]‐kainic acid and [3H]‐AMPA to rat brain synaptic plasma membranes

1. The ability of acromelic acid A to inhibit [3H]-kainic acid and [3H]-(RS)-alpha-amino-3-hydroxy-5-methyloxazole-4-propionic acid ([3H]-AMPA) binding to rat brain synaptic plasma membranes was investigated by equilibrium radioligand binding assay. 2. Kinetic analysis of [3H]-kainic acid binding demonstrated the existence of two kainate binding sites in this tissue preparation and yielded equilibrium dissociation constants for [3H]-kainic acid of KD = 0.4 nM and KD = 20.8 nM. 3. Kainic acid and domoic acid both appeared to displace [3H]-kainic acid from a single binding site with equilibrium binding constants of KD = 19.4 nM and Ki = 14.5 nM respectively. Acromelic acid A exhibited a biphasic inhibition of [3H]-kainic acid binding to synaptic membranes with binding affinities of Ki = 15.1 nM and Ki = 1.49 microM. 4. In the absence of chaotropic ions, the order of potency of inhibition of [3H]-AMPA binding was acromelic acid A (Ki = 26 nM) greater than AMPA (KD = 184 nM) greater than domoic acid (Ki = 499 nM). 5. The inclusion of 100 mM thiocynanate ion in the [3H]-AMPA binding assay resulted in a change in the order of potency to: AMPA (KD = 160 nM) greater than acromelic acid A (Ki = 289 nM) greater than domoic acid (Ki = 9.02 microM). 6. These results show that acromelic acid A distinguishes two kainate binding sites in rat brain synaptic plasma membranes and in addition, that in the absence of chaotropic ions, acromelic acid A is the most potent displacer of [3H]-AMPA binding yet described.

Excitatory and inhibitory amino acid neurotransmitter binding sites in the cerebellar cortex of the pigeon ( Columba livia)

We used receptor autoradiography to determine the distribution of excitatory and inhibitory amino acid neurotransmitter binding sites in the cerebellar cortex of the pigeon ( Columba livia). α-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid, kainate and metabotropic binding sites had highest levels in the molecular layer. N-methyl- d-aspartate binding sites, assayed with both [ 3H]glutamate under selective conditions and with [ 3H]glycine binding to the associated strychnine-insensitive glycine site, had highest levels in the granule cell layer. There was little specific binding of the non-competitive N-methyl- d-aspartate antagonist, [ 3H]MK-801. The level of gamma-aminobutyric acid (GABA)-A binding sites was higher than GABA-B binding sites in both molecular and granule cell layers with the highest level of GABA-A sites in the granule cell layer. The highest level of GABA-B binding sites was in the molecular layer. [ 3H]Flunitrazepam binding levels were approximately the same in both molecular and granule cell layers. With the exception of kainate binding sites, the distribution of binding sites was identical to that seen in the cerebellar cortex of mammals. Our results support the concept that the chemoarchitecture of the cerebellar cortex has been conserved in the course of vertebrate evolution.

Excitatory and inhibitory amino acid binding sites in human dentate nucleus

Autoradiography of excitatory and inhibitory amino acid binding sites in human dentate nuclei indicated virtually no binding to N-methyl- d-aspartate (NMDA) or γ-aminobutyric acid B (GABA B) binding sites, and a low density of kainate binding sites. α-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid, metabotropic-quisqualate, benzodiazepine, and γ-aminobutyric acid A (GABA A) binding sites were present in moderate abundance. Our NMDA results differ from those found previously in rodents. GABA A receptors are probably the primary mediators of inhibitory neurotransmission and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid and metabotropic-quisqualate receptors are probably the primary mediators of excitatory neurotransmission within the human deep cerebellar nuclei.

The interaction of a kainate receptor from goldfish brain with a pertussis toxin-sensitive GTP-binding protein

Kainate receptors are present in high concentrations in goldfish brain (Henley and Oswald, 1988a and b; Ziegra et al., 1990), possibly in neuronal and glial cells. In a number of systems, the kainate receptor has been assumed to be an integral ion channel (Watkins and Evans, 1981); but, for some kainate receptors, ion channel activity has not been demonstrated (Wada et al., 1989). This study presents evidence that a portion of the [3H]kainate-binding sites in goldfish brain is sensitive to guanine nucleotides, with a loss of high affinity binding in the presence of nonhydrolyzable GTP analogs. Pertussis toxin pretreatment of membranes causes a loss of high affinity [3H]kainate binding and of the guanine nucleotide-sensitive binding. Pertussis toxin catalyzes the specific [32P]ADP-ribosylation of a 40-kDa substrate in a kainate-sensitive manner. In addition, incorporation of [alpha-32P]GTP-gamma-azidoanilide by photoaffinity labeling was enhanced in the presence of kainate. These results indicate that a subpopulation of [3H]kainate-binding sites in goldfish brain may be coupled to G proteins.

Alterations in Cortical [3H]Kainate and α‐[3H]Amino‐3‐Hydroxy‐5‐Methyl‐4‐Isoxazolepropionic Acid Binding in a Spontaneous Canine Model of Chronic Hepatic Encephalopathy

Excitatory amino acid receptor binding parameters were investigated in a spontaneous dog model of chronic hepatic encephalopathy. L-[3H]Glutamate, (+)-[3H]-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-im ine maleate ([3H]MK-801), [3H]kainate, and alpha-[3H]-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ([3H]AMPA) binding experiments were performed using crude cerebrocortical synaptosomal membrane preparations from dogs with congenital portosystemic encephalopathy (PSE) and control dogs. There was no change in the affinity or density of L-[3H]-glutamate or [3H]MK-801 binding sites in dogs with congenital PSE compared with control dogs. However, in the PSE dogs there was a significant reduction in the density of [3H]kainate binding sites compared with control dogs and abolition of the low-affinity [3H]AMPA binding site. The relative binding capacity of PSE synaptosomal membranes for [3H]kainate and [3H]AMPA was expressed as the ratio Bmax/KD. There was a significant inverse correlation between the Bmax/KD ratio for [3H]AMPA binding and the worst grade of encephalopathy experienced by each dog. These results suggest that there is a significant perturbation of cerebrocortical non-N-methyl-D-aspartate receptor binding in dogs with congenital PSE which may have relevance to the pathogenesis of hepatic encephalopathy.

Identification and localization of a kainate binding protein in the frog inner ear by electron microscopy immunocytochemistry.

A kainate binding protein (KBP) was studied in Rana pipiens inner ear using monoclonal and polyclonal antibodies against affinity purified KBP from frog brain. The KBP identified and analyzed in inner ear tissue homogenates, with one- and two-dimensional immunoblots, was similar to the affinity purified KBP and to the antibody-identified frog brain KBP. As brain KBP, inner ear KBP had 5 main components in the molecular weight dimension, centered at Mr = 48,000; however, inner ear KBP had a greater abundance of the higher molecular weight components. Light and electron microscopy observations showed KBP immunostaining at two locations: (1) in the dendrites of the eight nerve afferent fibers contacting sensory hair cells, with the postsynaptic density being more intensely stained; and (2) on the cytoplasmic membrane of fibroblasts present in the inner ear connective tissue which displayed intense immunostaining. The presence of kainate (KA) binding sites in the inner ear was assessed using in vitro receptor autoradiography. [3H]KA binding sites were found in connective tissue areas confirming the immunocytochemistry results. The postsynaptic localization of the KBP in afferent endings, strongly supports it as being a component of the KA receptor complex. However, its presence on fibroblasts situated in the inner ear connective tissue makes its function hypothetical. The dual presence of the KBP on non-neuronal cells as well as at postsynaptic membrane sites suggests the existence of a family of proteins involved in KA binding and KA receptors with a complex organization.

Selective alterations in glutamate receptor subtypes after unilateral orbital enucleation

Glutamate is the major excitatory neurotransmitter in the rat visual system. Using quantitative autoradiography the effect of unilateral orbital enucleation on [ 3H]kainate, [ 3H]α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid ([ 3H]AMPA) and [ 3H]glutamate binding to kainate, quisqualate and NMDA receptors respectively has been examined within anatomical components of the visual pathway at 4 time points up to 20 days post-lesion. The time course for the degeneration of retinal projection fibres was assessed in a separate group of animals by quantifying [ 3H]cyclohexyladenosine ([ 3H]CHA) binding to presynaptic adenosine A 1 receptors. Over the first 5 days after orbital enucleation, there were no significant alterations in glutamate or adenosine A 1 receptor binding in visual structures of the visually deprived hemisphere. However, at 10 days post-lesion [ 3H]AMPA binding was significantly reduced (30%) in the visually deprived superior colliculus but unaltered in other visual structures. At this time point there was also a significant reduction (50%) in [ 3H]CHA binding in the visually deprived superior colliculus but not in other retino-recipient nuclei. There were similar changes in [ 3H]AMPA and [ 3H]CHA binding at 20 days post-enucleation. [ 3H]Kainate binding was significantly increased in the visually deprived superior colliculus only at 20 days post-enucleation. Saturation analysis of [ 3H]kainate and [ 3H]AMPA binding at this time point indicated a selective increase in the b max value for the high affinity [ 3H]kainate binding site and a concomitant decrease in the b max value for the high affinity [ 3H]AMPA binding site in the visually deprived superior colliculus. There were, however, no significant alterations in [ 3H]AMPA or [ 3H]kainate binding in other primary projection areas or in secondary visual areas (e.g. visual cortex) at any time point. NMDA sensitive [ 3H]glutamate binding was unaltered in the visually deprived hemisphere up to 20 days post-enucleation. These results suggest an upregulation of kainate receptors in the visually deprived superior colliculus after orbital enucleation and a loss of presynaptic quisqualate receptors on degenerating retinal fibres. The plastic alterations in kainate receptors in the superior colliculus are supportive of electrophysiological data suggesting a physiological role for these sites in mediating excitatory postsynaptic potentials in tectal neurons.

Cerebellar [3H]kainate and [3H]ampa binding in dogs with congenital portosystemic encephalopathy

Abstract Previous studies have indicated that kainate and AMPA receptors are altered in cerebral cortex of dogs with chronic hepatic encephalopathy (HE). To ascertain whether receptors in dog cerebellum are similarly altered in HE [3H]kainate and [3H]α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) binding assays were performed on crude synaptosomal membranes prepared from cerebellar tissue from dogs with congenital portosystemic encephalopathy (PSE) and control dogs. There was no pathophysiologically relevant difference in the affinity or density of kainate or AMPA binding sites in PSE cerebellar tissue compared with control dogs. The failure to demonstrate alterations in these binding parameters in cerebellar tissue was expected as clinical signs of HE reflect cortical rather than cerebellar dysfunction.

Effects of neonatal γ-ray irradiation on rat hippocampus—II. Development of excitatory amino acid binding sites

In the rat, neonatal irradiation produces a destruction of dentate granule cells and prevents the development of the mossy fibre-CA3 pyramidal cell synapse. The developmental increase of high affinity kainate binding sites in the stratum lucidum was reduced on the irradiated side as compared with the control side. This suggests that a proportion of high affinity kainate binding sites is associated with mossy fibres. In contrast, the developmental profile of N-methyl- d-aspartate binding sites, which are associated with associational and commissural synapses in CA3, was not affected by irradiation. The role that afferent fibres may play in the development of pyramidal cells is discussed in connection with the modulatory effects of glutamate receptors on the development of neurons.

Isolation, localization, and cloning of a kainic acid binding protein from frog brain.

Excitatory amino acids (EAA) are major neurotransmitters in the vertebrate central nervous system. EAA receptors have been divided into three major subtypes on the basis of electrophysiological and ligand binding studies: N-methyl-D-aspartate, kainate, and quisqualate receptors. To understand their molecular properties, we undertook a project aimed at isolation and cloning of these receptor subtypes. We purified a kainate binding protein (KBP) from frog brain, in which kainate binding sites are about fortyfold more abundant than in rat brain, using domoic acid affinity chromatography, and made monoclonal and polyclonal antibodies to the purified protein. These antibodies immunoprecipitate the frog KBP but not KBPs from other species. Immunocytochemical analyses show that KBP has a synaptic and extrasynaptic localization in frog optic tectum, with most labeling being extrasynaptic. The cDNA encoding frog brain KBP was isolated by screening a frog brain cDNA library with oligonucleotide probes that were based on the amino acid sequence of the purified protein. The deduced amino acid sequence of the KBP has a hydrophobic profile similar to those of other ligand-gated ion channel subunits, such as the nicotinic acetylcholine receptor, the GABAA receptor, and the glycine receptor. Frog brain KBP is very similar (36% amino acid identity to the carboxyl half) to rat brain kainate receptor, suggesting that these two proteins evolved from a common ancestor. The function of KBP in frog brain remains a major question. Preliminary results showed that Xenopus laevis oocytes injected with KBP RNA did not produce a detectable electrophysiological response when perfused with kainate. These results suggest that additional subunits may be required to form a functional receptor or that KBP is not functionally related to a neurotransmitter receptor.

Molecular Cloning and Functional Expression of Glutamate Receptor Subunit Genes

Three closely related genes, GluR1, GluR2, and GluR3, encode receptor subunits for the excitatory neurotransmitter glutamate. The proteins encoded by the individual genes form homomeric ion channels in Xenopus oocytes that are sensitive to glutamatergic agonists such as kainate and quisqualate but not to N-methyl-D-aspartate, indicating that binding sites for kainate and quisqualate exist on single receptor polypeptides. In addition, kainate-evoked conductances are potentiated in oocytes expressing two or more of the cloned receptor subunits. Electrophysiological responses obtained with certain subunit combinations show agonist profiles and current-voltage relations that are similar to those obtained in vivo. Finally, in situ hybridization histochemistry reveals that these genes are transcribed in shared neuroanatomical loci. Thus, as with gamma-aminobutyric acid, glycine, and nicotinic acetylcholine receptors, native kainate-quisqualate-sensitive glutamate receptors form a family of heteromeric proteins.

Autoradiographic distribution of binding sites for the non-NMDA receptor antagonist CNQX in chick brain

The anatomical distribution of binding sites for the non-N- methyl- d-aspartate (non-NMDA) glutamate receptor antagonist [ 3H]6-cyano-7-nitroquinoxaline-2,3-dione ([ 3H]CNQX) in 1-day-old chick brain was investigated. Specific [ 3H]CNQX binding sites were widely distributed but were particularly densely localised in the molecular layer of the cerebellum. In the midbrain, the binding was comparatively low, except for relatively high levels in the nucleus isthmi of the optic lobe. The distribution of [ 3H]CNQX binding was markedly different to that of the NMDA receptor ligand [ 3H]MK-801. Overall, the localisation of [ 3H]CNQX binding was similar to the combined distributions of [ 3H]AMPA and [ 3H]kainate binding sites. Kainate inhibited [ 3H]CNQX binding throughout the brain. AMPA also inhibited [ 3H]CNQX binding in the fore- and midbrain, but the dense binding in the molecular layer of the cerebellum was notable in being AMPA-insensitive.