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6-Nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione (NBQX) is a competitive antagonist selective for alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors. Here we report the pharmacological characteristics and anatomical distribution of [3H]NBQX binding to rat brain. The association rate of [3H]NBQX to rat cerebrocortical membranes was rapid, with peak binding occurring within 10 min at 0 degree C. The off-rate was also rapid, with near-complete dissociation of the radioligand within 5 min of addition of 1 mM unlabelled L-glutamate. [3H]NBQX bound to a single class of sites with KD and Bmax values of 47 nM and 2.6 pmol mg-1 of protein, respectively. The rank order of inhibition of [3H]NBQX binding by AMPA receptor ligands was NBQX > > 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) > or = (S)-5-fluorowillardiine > or = AMPA > > L-glutamate. The chaotrope KSCN had no effect on the IC50 value of unlabelled NBQX displacement of [3H]NBQX binding. The kainate receptor-selective ligands NS102 and kainate were only very weak displacers. It is interesting that NBQX and CNQX displaced significantly more [3H]NBQX than any of the agonists tested. Autoradiographic analysis of the binding of [3H]NBQX to coronal sections showed a distribution compatible with that of [3H]AMPA binding. These data indicate that [3H]NBQX provides a useful novel tool to characterise the antagonist binding properties of AMPA receptors.

Neurofibrillary tangle (NFT)-rich brain samples from patients with progressive supranuclear palsy (PSP) or Alzheimer's disease (AD) were probed with a large panel of anti-tau antibodies to compare the species of tau present in PSP and AD NFTs by immunohistochemistry and Western blot methods. These antibodies have been shown to recognize phosphate-independent or -dependent epitopes that extend from the amino to the carboxy terminal domains of normal brain tau and the abnormal tau in the paired helical filaments (PHFs) of AD NFTs (PHFtau). The immunohistochemical studies showed that all of the tau epitopes detected in brainstem PSP NFTs also were found in hippocampal AD NFTs and vice versa. While Western blots demonstrated 2 PHFtau-like immunobands in PSP brainstem, a triplet of PHFtau proteins were seen in the AD and PSP hippocampus. Despite differences in the distribution, ultrastructure and immunoblot profile of NFTs in PSP and AD, the same constellation of tau epitopes is present in the abnormal tau proteins in PSP and AD NFTs. Thus, the generation of abnormal tau proteins in PSP (PSPtau) and AD (PHFtau) may have similar adverse biological consequences in both diseases.

Guanyl nucleotides inhibit the binding of the AMPA receptor agonists [3H]fluorowillardiine and [3H]AMPA and the competitive antagonist [3H]CNQX to rat brain cerebrocortical membranes. The rank order of inhibition for each of the radioligands tested was GTP > GDP > GMP. The nucleotides CTP and ATP showed no effect. GTP inhibition was unaffected by the presence or absence of NaCl and MgCl2. Pre-treatment of the membranes with GTP, and its removal before addition of radioligand, did not inhibit binding. Quantitative autoradiography demonstrated that GTP inhibition occurred throughout the brain. These results are consistent with guanyl nucleotides acting at an extracellular site present on all AMPA receptor subunits, occupation of which inhibits agonist and antagonist binding.

Goldfish brain is a widely used model system for the study of the mechanisms involved in neuronal regeneration and synaptic plasticity. Because of the proposed role of glutamate receptors in these processes we have investigated the anatomical localisations of [3H]AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate), [3H]kainate, [3H]CNQX (6-cyano-7-nitroquinoxaline-2,3-dione) and [3H]L-glutamate binding sites in horizontal and sagittal sections. Binding sites for [3H]L-glutamate were the most widespread and both NMDA (N-methyl-D-aspartate) and non-NMDA sensitive components were detected. The density of [3H]kainate binding was very high in the cerebellum compared to other regions and in comparison with the other radioligands used. Conversely, relatively low amounts of [3H]AMPA binding were present with the telencephalon being the most densely labelled structure. [3H]CNQX binding was most densely localised in the tectum with the cerebellum also possessing high binding. In addition, there was a small population of [3H]CNQX binding sites located in the telencephalon and lobus vagi that appeared insensitive to AMPA and kainate.

Radiation inactivation analysis was used to estimate the target size of a putative glutamate receptor subtype in goldfish brain. A simple, linear inactivation curve was obtained. The calculated molecular size of the [3H]kainate binding site was 33.8 kDa. The results presented here are comparable to the molecular masses determined for putative glutamate receptors in other lower vertebrates but are markedly different from the sizes of the corresponding glutamate receptor subtypes in mammalian central nervous system.

In the Xenopus central nervous system the binding sites for [3H]kainate and [3H]alpha-amino-3-hydroxy-5-methylisoxazolepropionate [( 3H]AMPA) are highly localised and the distributions of both ligands are largely coincident. The telencephalon was the most strongly labelled area with a relatively uniform distribution of binding sites. In addition, the infundibulum and the cerebellum were also heavily labelled. Areas containing a lower density of binding sites included the septum, the thalamus and the optic lobes. [3H]Kainate binding was potently inhibited by 1 microM AMPA in the presence of 0.1 M KSCN and [3H]AMPA binding was blocked by 1 microM kainate. These data are consistent with the hypothesis that kainate and AMPA bind to the same site on a single protein entity and that this unitary AMPA/KA binding protein may constitute the predominant type of excitatory amino acid receptor in the Xenopus brain.

[3H]Kainate binding to membrane homogenates and detergent extracts prepared from Xenopus central nervous system was evaluated in 50 mM Tris-citrate buffer, pH 7.0. In membrane fragment preparations, [3H]kainate bound with a KD of 54.4 nM to a large number of sites (Bmax = 27.8 pmol/mg of protein). Up to 80% of the total number of membrane-bound binding sites were solubilised using the nonionic detergent n-octyl-beta-D-glucopyranoside. Values for the KD of [3H]kainate for solubilised binding sites were 46.0 nM and 53.6 nM derived from equilibrium and kinetic binding experiments, respectively. Competitive binding studies revealed that a variety of ligands had similar Ki values in both membranes and solubilised extracts, with domoate and kainate being the most potent inhibitors of [3H]kainate binding. The dissociation rate of [3H]kainate from solubilised binding sites was 0.022 min-1. The binding component migrated in sucrose density gradients in a single 8.6S peak. These results demonstrate that the kainate receptor in Xenopus central nervous system, although similar to the [3H]kainate binding site from goldfish brain, differs in a number of important respects. In particular, the slower dissociation rate and higher affinity of [3H]kainate suggest that Xenopus provides the most convenient model system yet investigated for biochemical analysis of kainate receptors.

Monoclonal antibodies raised against the nicotinic acetylcholine receptor of Electrophorus electricus electroplaque have been used as probes to characterize putative nicotinic acetylcholine receptors in goldfish brain. One monoclonal antibody (mAb), mAb 47, recognized a protein which binds both (-)-[3H]nicotine and 125I-alpha-bungarotoxin with high affinity. Another monoclonal antibody (mAb 172) recognized a protein which binds (-)-[3H]nicotine but not 125I-alpha-bungarotoxin. Both antibodies precipitated a protein(s) (biosynthetically labeled with [35S]methionine) in the absence, but not in the presence, of excess purified nicotinic acetylcholine receptor from Torpedo nobiliana. The dilution of mAb 47 that precipitated half of the maximum amount of 125I-alpha-bungarotoxin binding protein was the same as that which precipitated half of the maximum amount of (-)-[3H]nicotine binding activity. When used in combination, the two antibodies precipitated more (-)-[3H]nicotine radioactivity than either antibody alone. The (-)-[3H]nicotine and 125I-alpha-bungarotoxin binding component-mAb complexes were characterized by sucrose density centrifugation. In the presence of either mAb 172 or 47, the (-)-[3H] nicotine binding component migrated further into the gradient, but only mAb 47 shifted the 125I-alpha-bungarotoxin peak. Incubation of solubilized brain extract with alpha-bungarotoxin-coupled Sepharose reduced the amount of (-)-[3H]nicotine radioactivity precipitated by mAb 47 but not by mAb 172. These data suggest that the antibodies may recognize distinct subtypes of (-)-nicotine binding sites in goldfish brain, one subtype which binds both 125I-alpha-bungarotoxin and (-)-[3H]nicotine and a second subtype which binds only (-)-[3H] nicotine.

The kainate (KA) and the quisqualate (QUIS) receptors that activate cation channels in the central nervous system have previously been defined as two of the major glutamate receptor types. In amphibian brain, an exceptionally rich source of these sites, they can be coextracted by octylglucoside and shown to behave as one entity in all analyses made in solution. When partly purified by lectin affinity, ion-exchange chromatography or by sucrose gradient centrifugation, the two activities comigrate in a 1:1 ratio. When the QUIS component is bound to an immobilized specific QUIS agonist, the KA component is extracted in parallel with it. There are equivalent numbers of the QUIS and KA sites and the two sites show a single affinity series for the binding of glutamatergic agonists. We deduce that KA or QUIS select different conformations of a single KA/QUIS receptor binding site, leading thus to the different channel-opening events that have been reported for these two agonists.