Two distinct quisqualate receptor systems are present on striatal neurons

At concentrations at which it did not alter spontaneous release, quisqualate (QUIS) induced a dose-dependent (EC50, 0.5 microM) potentiation of KCl- or veratrine-evoked release of [3H]GABA from striatal neurons in primary culture. QUIS potentiation of KCl-evoked [3H]GABA release was mimicked by the selective agonist alpha-amino-3-hydroxy-5-methylisoxazole-propionic acid (AMPA), glutamate and kainate, and was blocked by kynurenic acid and gamma-D-glutamylglycine. QUIS also induced a dose-dependent (EC50, 0.2 microM) augmentation of [3H]inositol monophosphate production in striatal neurons. This action of QUIS was mimicked by glutamate, but not by AMPA nor by kainate. Furthermore, none of the antagonists tested (kynurenic acid, gamma-D-glutamylglycine, glutamic acid diethyl ester, and 4-aminophosphonobutanoic acid) could block QUIS-induced elevations in [3H]inositol monophosphate production. The results of the present study suggest that two QUIS receptor systems, distinguished on the basis of their pharmacological properties, may subserve specific roles in the regulation of striatal neuron function by excitatory amino acids.     

Development of excitatory amino acid induced cytotoxicity in cultured neurons

The neurotoxicity of the excitatory amino acids (EAAs) L-glutamate (L-glu), L-aspartate (L-asp), N-methyl-D-aspartate (NMDA), kainate (KA), quisqualate (QA) and RS-alpha-amino-3-hydroxy-5-methyl-4-isoxazolopropionate (AMPA) was followed as a function of development in primary cultures of cerebral cortex neurons and cerebellar granule cells. These two types of neurons express, respectively, glutamate receptor subtypes with sensitivity to all of these excitatory amino acids or only to glutamate and aspartate. None of the EAAs were toxic in cerebral cortex neurons at 2 days in culture, whereas at culture day 4 the neurons became sensitive to glutamate, at day 5 to KA followed by sensitivity to QA at day 6, and finally to NMDA, L-asp and AMPA at day 7. The rank order of potency of the EAAs was in cerebral cortex neurons cultured for 12 days: L-asp (ED50 = 0.5 microM) = L-glu (ED50 = 1 microM) greater than AMPA (ED50 = 10 microM) greater than NMDA (ED50 = 65 microM) greater than QA = KA (ED50 = 100 microM). Cerebellar granule cells were insensitive to all of the EAAs at 3 and 5 days in culture but at day 8 the cells became sensitive to toxicity induced by L-glu (ED50 = 70 microM) and L-asp (ED50 = 30 microM). In order to determine ED50 values for L-asp and L-glu accurately, media in these experiments also contained 500 microM of the glutamate uptake inhibitor L-aspartate-beta-hydroxamate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glutamate receptors in striatum and substantia nigra: Effects of medial forebrain bundle lesions

We examined NMDA-sensitive [³H]glutamate, [³H]AMPA, [³H]kainate and metabotropic-sensitive [³H]glutamate binding sites in neostriatum and substantia nigra pars reticulata (SNr) in rats after unilateral 6-hydroxydopamine lesions of the medial forebrain bundle. One week after the lesion, NMDA, AMPA, kainate and metabotropic receptors were decreased in the ipsilateral neostriatum, whereas at three months NMDA receptors were increased while AMPA, kainate and metabotropic receptors were not changed. In the SNr at one week, only AMPA and metabotropic receptors were significantly decreased whereas three months after the lesion NMDA, AMPA and kainate binding sites were decreased. The early decrease of excitatory amino acid receptors in the striatum is likely to reflect degeneration of dopaminergic fibers, suggesting that specific subpopulations of excitatory amino acid binding sites are located on dopaminergic terminals.     

Autoradiographical analysis of excitatory amino acid binding sites in rat hippocampus during the development of hippocampal kindling

Binding sites for excitatory amino acids have been determined by autoradiographical procedures in the rat hippocampus and striatum during hippocampal kindling. The binding sites measured were the N-methyl-D-aspartate (NMDA)-sensitive sites for L-[3H]glutamate and [3H]MK-801 sites (transmitter recognition site and ion channel of the NMDA receptor, respectively), [3H]alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) sites (quisqualate receptor), [3H]kainate sites (kainate receptor) and NMDA-insensitive sites for L-[3H]glutamate. In general, little change was apparent in the hippocampus or striatum for any of these binding sites when assessed 48 h after attaining stages 1/2, 3 or 5 of kindling. These results suggest that hippocampal kindling does not bring about a change in the excitatory amino acid receptor binding sites examined, and that the appearance of an NMDA receptor-mediated component to synaptic responses in the hippocampus produced by kindling, cannot be explained on this basis.     

3H]AMPA binding to glutamate receptor subpopulations in rat brain

The glutamate analog (RS)-alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA), displaced 11% of the binding of L-[3H]glutamate to rat brain membranes, amounting to 22% of the specific binding displaceable by excess non-radioactive glutamate. AMPA-sensitive L-[3H]glutamate binding was additive with that displaced by kainic acid (1 microM) plus N-methyl-D-aspartate (10 microM) when low concentrations of non-radioactive AMPA (1 microM) were employed to determine non-specific background, but partially overlapped when higher concentration of AMPA (100 microM) were used. [3H]AMPA binding was 21% specific (displaceable by non-radioactive 0.1 mM AMPA) in sodium-, calcium- and chloride-free buffer, but increased to over 30% in the presence of 0.1 M chloride. AMPA-sensitive glutamate binding and AMPA binding were both stimulated dramatically by thiocyanate and by several other anions. [3H]AMPA binding activity was resistant to freezing and thawing, optimal at 0-4 degrees C, and detectable at slightly reduced levels by filtration assays and in tissue section autoradiography. AMPA showed a heterogeneous affinity in displacement of L-[3H]glutamate, and [3H]AMPA binding showed heterogeneity with respect to AMPA, quisqualate, and glutamic acid diethyl ester. Scatchard plots gave a best fit for two sites with Kd values of 28 and 500 nM and Bmax values of 200 and 1800 fmol/mg protein, respectively. [3H]AMPA was inhibited by quisqualate (IC50 = 60 nM), L-glutamate (2 microM), (RS)-3-hydroxy-4,5,6,7-tetrahydroisoxazolo-[5,4-c]-pyridine-7-carboxylic acid (7-HPCA, 5 microM), kainic acid (20 microM) and glutamic acid diethyl ester (21 microM) but insensitive to L-aspartate, ibotenic acid, N-methyl-D-aspartate, (RS)-2-amino-phosphonobutyric acid and (RS)-2-amino-phosphonovaleric acid. This is consistent with labeling of a quisqualate-specific subpopulation of glutamate receptors. The high affinity (28 nM) and intermediate affinity (0.5 microM) AMPA sites had similar pharmacological specificity and brain regional distribution as determined by autoradiography. The latter revealed high densities of [3H]AMPA binding in the superficial layers of the cerebral cortex; stratum pyramidale, stratum radiatum, and stratum oriens of the hippocampus; and stratum moleculare of the dentate gyrus. Within the cerebellum, higher densities of binding were observed in the molecular layer than in the granule cell layer. In many regions, [3H]AMPA binding had a similar distribution to that of L-[3H]glutamate binding displaced by AMPA (1 microM).(ABSTRACT TRUNCATED AT 400 WORDS)     

Evidence that [3H]glutamate binding sites are masked by biologically relevant endogenous factor on cell membranes of frog spinal cord.

We identified the possible endogenous factor effective to modulate the binding of [3H]-labeled excitatory amino acid agonists and antagonists in the 100,000 x g supernatant of Triton X-100 (0.01%)-treated cell membranes from frog spinal cords. The factor inhibited the binding of [3H]glutamate to Triton X-100-treated cell membranes, to which the binding capacity of [3H]glutamate increased much more than that to intact cell membranes. The binding capacities of [3H]AMPA (an AMPA type agonist) and [3H]CPP (an NMDA type antagonist) to cell membranes remained low by Triton treatment, but they were enhanced significantly by the addition of the factor. The effect of the factor on the [3H]kainate binding was hardly observable. The factor may provide key information on receptor structures and the classification of receptor types concerning excitatory amino acids in the mammalian central nervous system.     

L-[3H]Glutamate binds to kainate-, NMDA- and AMPA-sensitive binding sites: an autoradiographic analysis

The anatomical distribution of L-[3H]glutamate binding sites was determined in the presence of various glutamate analogues using quantitative autoradiography. The binding of L-[3H]glutamate is accounted for by the presence of 3 distinct binding sites when measured in the absence of Ca2+, Cl- and Na+ ions. The anatomical distribution and pharmacological specificity of these binding sites correspond to that reported for the 3 excitatory amino acid binding sites selectively labelled by D-[3H]2-amino-5-phosphonopentanoate (D-[3H]AP5), [3H]kainate ([3H]KA) and [3H] alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ([3H]AMPA) which are thought to be selective ligands for the N-methyl-D-aspartate (NMDA), KA and quisqualate (QA) receptors, respectively.     

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]-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 A₁ receptors. Over the first 5 days after orbital enucleation, there were no significant alterations in glutamate or adenosine A₁ 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.     

Localization and Quantitative Autoradiography of Glutamatergic Ligand Binding Sites in Chick Brain

The anatomical localization of glutamate receptor subtype-selective ligand binding sites was investigated in 1-day-old chick brain using quantitative autoradiography. Under the conditions used, the regional distributions of [3H]glutamate, [3H]AMPA (a selective quisqualate receptor ligand) and [3H]kainate binding sites are manifestly different. [3H]l-glutamate binding is densely localized in the telencephalon, particularly in the neostriatum (2.8 pmol/mg protein). In addition, [3H]l-glutamate labels the thalamus, the nucleus mesencephalicus lateralis pars dorsalis, the superficial layers of the optic tectum and the molecular layer of the cerebellum. [3H]AMPA binding sites are most densely localized in the hippocampus (0.90 pmol/mg protein), with an otherwise relatively uniform distribution of binding within the telencephalon. [3H]AMPA also labels the striatum griseum et fibrosum superficiale of the optic tectum and the molecular layer of the cerebellum. [3H]Kainate binding sites are extremely densely packed in the molecular layer of the cerebellum (10 pmol/mg protein). Other regions of [3H]kainate binding include the hyperstriatum and the thalamus. The binding of the NMDA receptor channel blocker [3H]MK-801 is increased in the presence of 1 mM l-glutamate. [3H]MK-801 binding is generally widespread in the telencephalon but is notably absent from the ectostriatum. No evidence of [3H]MK-801 binding sites was detected in the cerebellum, even in the presence of 1 mM l-glutamate. The relatively high densities and the well-defined localizations of the glutamate receptor subtype binding sites suggest that chick brain provides a useful system for the further study of excitatory amino acid receptors.     

Excitatory amino acid receptor regulation after subthalamic nucleus lesions in the rat

The subthalamic nucleus plays a pivotal role in the regulation of basal ganglia output. Recent electrophysiologic, lesion and immunocytochemical studies suggest that the subthalamic nucleus uses an excitatory amino acid as a neurotransmitter. After complete ablation of the subthalamic nucleus, we have examined the NMDA, AMPA, kainate and metabotropic subtypes of excitatory amino acid receptors in two major subthalamic projection areas (globus pallidus and substantia nigra pars reticulata) with quantitative autoradiography. Two weeks after ablation, binding sites for [³H]AMPA and [³H]kainate increased in substantia nigra pars reticulata ipsilateral to the lesion. In globus pallidus on the lesioned side, [³H]glutamate binding to the NMDA recognition site decreased. The results suggest that glutamate receptors regulate after interruption of subthalamic nucleus output.     

Development and regulation of excitatory amino acid receptors involved in the release of arachidonic acid in cultured hippocampal neural cells

Release of [3H]arachidonic acid mediated by excitatory amino acid (EAA) receptors was investigated from prelabelled primary cultures of hippocampal neurons and astroglial cells. Treatment with N-methyl-D-aspartate (NMDA), quisqualate (QA) and kainate resulted in age- and dose-dependent stimulation of [3H]arachidonic acid release. During development, the maximum response for NMDA was observed relatively earlier (at 7 days) than those for QA and kainate (at 14 days) in the hippocampal neuronal cultures. The half maximal effects were obtained at about 15 microM NMDA at all ages studied and about 0.5 microM QA at 14 and 20 days. At optimum concentrations NMDA- and QA-induced releases were additive. Unlike with neurons, treatment with all the 3 EAA receptor agonists, NMDA, QA and kainate, had no significant effect on [3H]arachidonate release in hippocampal astroglial cells. In cultured 14-day-old neurons, the increases in NMDA- and QA-mediated [3H]arachidonic acid release were completely blocked by the NMDA receptor antagonist, 2-amino-5-phosphonovaleric acid, and the ionotropic QA receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione, respectively. But the iontropic QA receptor agonist alpha-amino-3-hydroxy-5-methyl-isoxazole-4- propionic acid (AMPA) had no significant effect on [3H]arachidonate release, indicating that interaction between ionotropic QA and metabolotropic QA receptors may be essential for optimal QA-mediated arachidonic acid release. At physiological concentrations of Mg2+ (1.2 mM), AMPA was found to potentiate NMDA-induced release of [3H]arachidonic acid; the effect appeared to be related to a removal of Mg2+ blockade mediated by mild depolarisation.(ABSTRACT TRUNCATED AT 250 WORDS)     

The expression of excitatory amino acid binding sites during neuritogenesis in the developing rat cerebellum

The present study has examined excitatory amino acid transmitter binding sites as measured autoradiographically in cryostat sections prepared from developing rat cerebella during the period of granule cell neuritogenesis. The external germinal layer (EGL) and molecular layer (ML), which during development contain granule cells at early stages of axon growth, contained only low levels of NMDA-displaceable L-[3H]glutamate binding sites. Similarly, [3H]glycine binding to the NMDA receptor linked binding site was not enriched in the EGL. Radioligand binding to the NMDA receptor was always greater in the granular layer (GL) than in the ML. The developmental increases in NMDA-displaceable L-[3H]glutamate and in [3H]glycine binding to the GL were similar but NMDA displaceable L-[3H]glutamate binding density increased before [3H]glycine binding sites. Glycine increased NMDA-displaceable L-[3H]glutamate binding only in the adult cerebellum. These results suggest that NMDA stimulation of neuritogenesis in granule cell cultures may reflect stimulation of dendritogenesis in the developing glomerulus rather than a stimulation of axon growth in the EGL. Also, NMDA receptors may be present in an immature form during cerebellar development and have different properties to the adult receptor. Binding sites for [3H]kainate and [3H]AMPA were present in both the GL and ML and increased during development. At all times the amount of binding sites for [3H]kainate were highest in the GL whereas those for [3H]alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate were highest in the ML.     

Crucial role of kainate receptors in mediating striatal kainate injection-induced decrease in acetylcholine M(1) receptor binding in rat forebrain

We investigated the roles of kainate-, alpha-amino-3-hydroxy-5-methylisoxazol-4-propionate (AMPA)- and N-methyl-D-aspartate (NMDA)-receptors in mediating striatal kainate injection-induced decrease in the binding of acetylcholine M(1) receptors in rat forebrain. After unilateral intrastriatal injection of kainate (4 nmol), the bindings of [3H]kainate (10 nM), [3H]MK-801 (4 nM) and [3H]pirenzepine (4 nM) to the rat ipsilateral forebrain membranes declined, reaching the lowest on day 2 to 4 and recovering on day 8. Saturation binding studies, performed on day 2 post-injection, showed that kainate (1, 2, 4 nmol) dose-dependently decreased B(max) and K(d) of the three ligands. (+)-5-Methyl-10, 11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801), a selective NMDA receptor channel blocker, antagonised (from a dose of 4 nmol) kainate-induced decreases in the bindings of [3H]kainate (up to approximately 20%), [3H]MK-801 (up to approximately 90%) and [3H]pirenzepine (up to approximately 70%). In contrast, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a selective non-NMDA receptor antagonist, almost completely abolished (from a dose of 12 nmol) kainate-induced decreases in the bindings of all the three ligands (up to approximately 95-98%). Cyclothiazide, a selective potentiator that enhances AMPA receptor-mediated responses, significantly enhanced (from a dose of 4 nmol) kainate-induced decrease in the binding of [3H]kainate but not that of [3H]pirenzepine or [3H]MK-801. In summary, these results indicate that striatal kainate injection-induced decrease in the binding of acetylcholine M(1) receptors in rat forebrain is dependent on activation of kainate receptors and, to a certain extent, a consequent involvement of NMDA receptors. These and previous studies provide some evidence showing that kainate receptors might play a crucial role in regulating excitatory amino acids (EAA)-modulated cholinergic neurotransmission in the central nervous system (CNS).     

Laminar distribution of MK-801, kainate,AMPA, and muscimol binding sites in cat visual cortex: A developmental study

We used quantitative autoradiography to determine whether the development of glutamate receptors correlates with the sensitive period for monocular deprivation in the visual cortex. To study glutamate receptors, we incubated sections of cat visual cortex with tritiated (+)-5-methyl-10, 11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10imine-maleate (MK-801), tritiated kainate, and tritiated amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA). [³H]MK-801 is a noncompetitive ligand for the N-methyl-D-aspartate (NMDA) receptor. [³H]kainate and [³H]AMPA are competitive ligands for non-NMDA receptors. We used [³H]muscimol, which binds to GABA_A receptors, so that we would have one control ligand that binds to a nonglutamate receptor. When all layers were combined, the results confirmed our previous studies with homogenate binding. [³H]MK-801 and [³H]kainate binding were significantly greater at 42 days than at earlier or later times. [³H]AMPA and [³H]muscimol binding did not show such a peak. This suggests that MK-801 and kainate binding sites are more likely to be involved in plasticity than are AMPA and muscimol binding sites. In layers 2/3, MK-801 had the greatest age-dependent changes; in layers 5 and 6, kainate binding changed most with age. This suggests that the mechanisms of plasticity may vary with cortical layer. © 1996 Wiley-Liss, Inc.     

Multiple Cl(-)-independent binding sites for the excitatory amino acids: glutamate, aspartate and cysteine sulfinate in rat brain membranes

As we have recently reported that Cl(-)-dependent glutamate (GLU) binding reflects GLU accumulation into membrane vesicles, the characteristics, kinetics and pharmacological specificities of L-[3H]glutamate (L-[3H]GLU) binding to crude rat brain synaptic membranes, were investigated in Cl(-)-free medium. L-[3H]GLU binding was systematically compared to that of L-[3H]cysteine sulfinate (L-[3H]CSA) and L-[3H]ASP), two other putative excitatory amino acids. A high affinity site was determined for each of these radioactive ligands (L-[3H]GLU: Kd = 0.14 microM, Bm = 3.4 pmol/mg protein; L-[3H]CSA: Kd = 0.07 microM, Bm = 2.2 pmol/mg protein; L-[3H]ASP: Kd = 5.8 microM, Bm = 31.2 pmol/mg protein). The pharmacological specificity of these Cl(-)-independent binding sites indicate the existence of at least 3 distinct high affinity sites, all different from the Cl(-)-dependent GLU binding 'site': one having a similar affinity for GLU and CSA, a second one preferring CSA, and a third one preferring ASP. Among the large quantity of structural analogs of the neuroexcitatory amino acids tested, only endogenous compounds (GLU, ASP and CSA) (except hydroxylamine-o-sulfate) were able to interact efficiently. No inhibition by classical agonists and antagonists (such as N-methyl-D-aspartate, quisqualate, kainate, 2-amino-4-phosphonobutyrate, or 2-amino-5-phosphonovalerate) was found. In addition to their high specificity, these Cl(-)-independent sites possess most other biochemical characteristics of receptor proteins.     

Autoradiographic characterization of the non-N-methyl-D-aspartate binding sites in human cerebellum using the antagonist [3H]6-cyano-7-nitroquinoxaline-2,3-dione

Using quantitative autoradiography, we have characterized the binding properties of the non-N-methyl-D-aspartate (NMDA) glutamate receptor antagonist [³H]6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) in adult human cerebellum. Saturation experiments revealed [³H]CNQX binding to a single class of sites with similar affinity in the molecular and granule cell layer (K_d = 89.0 ± 6.4 and 83.3 ± 9.9nM, respectively). The maximum number of [³H]CNQX binding sites was much higher in the molecular compared to the granule cell layer (B_max = 16.2 ± 1.1 and 2.8 ± 0.5 pmol/mg protein, respectively). Inhibition experiments were performed in order to examine the pharmacological profile of [³H]CNQX binding in the molecular layer. [³H]CNQX labeled sites with high affinity for both non-NMDA agonists, (RS)-α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and kainate. Dose-response curves for inhibition of [³H]CNQX by AMPA and Kainate were biphasic. The potency of AMPA for displacement of [³H]CNQX binding (K_i © 1994 Wiley-Liss, Inc.:2.8 ± 0.8 nM and 12.5 ± 0.8 μM) was 4- to 6-fold greater than the corresponding potency of kainate (Kⁱ:18.1 ± 5.7 nm and 48.7 ± 9.3 μM). In conclusion, the pharmacological analysis of [³H]CNQX binding in the human cerebellar molecular layer reflects the existence of multiple binding sites of the non-NMDA receptor that have different affinities for both AMPA and kainate. © 1994 Wiley-Liss, Inc.     

Excitatory amino acids and neuronal plasticity: modulation of AMPA receptors as a novel substrate for the action of nootropic drugs.

The nootropic drug, aniracetam, behaves as a positive modulator of AMPA-sensitive glutamate receptors in a variety of systems, including intact brain tissue, amphibian oocytes injected with rat brain mRNA, and cultured neurons. In electrophysiological studies, aniracetam both increases the peak amplitude and reduces the rate of decay of the ion current generated by AMPA or quisqualate. In cultured neurons, aniracetam (as well as oxiracetam and piracetam) enhances the stimulation of 45Ca2+ influx produced by AMPA but not that produced by kainate or NMDA. In addition, aniracetam (as other nootropic drugs) increases the maximal density of low affinity binding sites for [3H]AMPA in crude synaptic membranes. Positive modulation of AMPA receptors by aniracetam provides a novel molecular substrate which explains the clinical efficacy of nootropic drugs as memory and cognition enhancers.     

The binding of acidic amino acids to snail, Helix aspersa, periosophagic ring membranes reveals a single high-affinity glutamate/kainate site

The characterization of specific acidic amino acid binding sites to snail, Helix aspersa, ganglia membranes has been assayed using tritiated glutamate (L-[3H]Glu), aspartate (L-[3H]Asp), cysteine sulfinate (L-[3H]CSA) and kainate. At 2 degrees C, only L-[3H]Glu and [3H]kainate specific binding could be measured using a filtration procedure to separate bound from free ligand. The analysis of L-[3H]Glu specific binding reveals the presence of one class of high-affinity binding sites with Kd = 0.12 microM and Bmax = 30 pmol/mg protein. This L-[3H]Glu binding was specific, reversible and saturable. The order of potency of different substances, agonists or antagonists of the rat brain excitatory amino acid receptors, has been determined. Kainate was the best displacing agent, followed by ibotenate = L-Glu greater than L-alpha-aminoadipate (L-alpha-AA) greater than homocysteate (HCA). Using 10 nM [3H]kainate, a single class of binding site was detected. Its pharmacological properties indicate that it is likely identical to the L-[3H]Glu binding site. This L-Glu-kainate site possesses most of the properties expected for a specific receptor. However, whereas L-[3H]Glu binding could be detected on purified neuronal membranes, the major component of specifically bound L-[3H]Glu appeared to be located on the sheaths surrounding neuronal cell bodies. These findings suggest that Glu or another endogenous acidic amino acid may function as a transmitter at neuromuscular junctions in Helix periesophagic ring, acting at a receptor distinct from those on nerve cells.     

Development of MK-801, kainate,AMPA, and muscimol binding sites and the effect of dark rearing in rat visual cortex

We used quantitative autoradiography to determine whether the development of glutamate receptors correlates with the plastic period for monocular deprivation in rat visual cortex. To study glutamate receptors, we incubated sections of rat visual cortex with tritiated (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10imine maleate (MK-801), tritiated kainate, and tritiated amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA). [³H]MK-801 is a noncompetitive ligand for the N-methyl-D-aspartate (NMDA) receptor. [³H]kainate and [³H]AMPA are competitive ligands for non-NMDA receptors. To compare glutamate binding sites with a nonglutamate binding site, we studied [³H]muscimol, which binds to γ-aminobutyric acid (GABA)_A receptors. [³H]MK-801 binding was maximal at postnatal day 26 (P26) and decreased in adulthood. [³H]AMPA binding was maximal at P18. [³H]kainate binding and [³H]muscimol binding were not age dependent. Dark rearing partially prevented the age-dependent decrease in [³H]MK-801 binding but had no effect on [³H]kainate or [³H]AMPA binding. Dark rearing decreased muscimol binding in adult animals. These results suggest that NMDA receptors, but not other glutamate receptors or GABA_A receptors, are likely to be critical for developmental plasticity in rat visual cortex. J. Comp. Neurol. 383:73–81, 1997. © 1997 Wiley-Liss, Inc.