Autoradiographic comparison of the distribution of [H]MK801 and [H]CNQX in the human cerebellum during development and aging

The autoradiographic distribution of (NMDA) and -a-amino-3-hydroxyl-5-methyl-4-isoxazoleproprionic acid/quisqualate (AMPA/QUIS) receptors was determined in cerebellum obtained at autopsy from 37 human individuals, aged from 24 weeks gestation to 95 years. [³H]MK801 was used to label the NMDA receptor and [³H]CNQX to label the AMPA/QUIS receptor. AMPA/QUIS receptors were concentrated in the cerebellar molecular layer, and NMDA receptors in the granular layer. Significant (3- to 4-fold) increases in binding were seen for both ligands from the fetal to neonatal periods in the molecular layer (CNQX) and in both molecular and granular layers (MK801). MK801 binding in the molecular layer continued to increase with age up to the tenth decade and together with binding in the granular layer, increased 2-fold between 10–40 years. The Purkinje cell layer was negative for MK801 binding until the 6–7th decade when it became positive. [³H]CNQX binding in the molecular layer increased significantly with age between the fetal period and the tenth decade, whereas in the granular layer binding increased from neonate to 40 years, but then decreased significantly from 60 years to the tenth decade. Lamination of the molecular and granular layers was absent during the fetal period and appeared with both ligands during the neonatal period. These marked differences in age-related expression of ligand binding sites in the granular layer during development and aging are of potential significance in relation both to selective vulnerability to ischaemia, and synaptic plasticity and remodelling related to neuronal loss in senescence.     

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.     

Postnatal changes in AMPA receptor regulation by phospholipase A2 treatment of synaptic membranes: temporally differential effects on agonist and antagonist binding.

Previous results have indicated that phospholipase A2 (PLA2) treatment of telencephalic membranes produced opposite effects on [3H]amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) binding in neonatal and adult rats. In the present study, we compared the effects of PLA2 treatment of telencephalic membranes on the binding characteristics of agonists and antagonists of the AMPA receptors in the developing rat brain. Whereas PLA2 treatment of telencephalic membranes from postnatal day (PND) 5 and 10 animals produced an important decrease in [3H]AMPA binding, the same treatment performed on PND 20, 25 and adult membranes resulted in a marked increase in [3H]AMPA binding; the shift from decreased to increased [3H]AMPA binding occurred at about PND 15. In contrast to [3H]AMPA binding, [3H]6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) binding was substantially reduced following PLA2 treatment at PND 5, 10 and 20, and effect due to a decrease in the maximal number of [3H]CNQX binding sites. In adult membranes, the effect of PLA2 treatment on [3H]CNQX binding was markedly reduced when compared to neonatal membranes. Pretreatment of synaptic membranes with PCMBS (a sulfhydryl reagent) increased [3H]AMPA binding in both young (PND 10) and adult telencephalic membranes, without significantly changing [3H]CNQX binding. The various effects of PLA2 treatment on agonist and antagonist binding did not appear to be due to major differences in the pharmacological properties of the AMPA receptors at different ages. The present results indicate that the characteristics of the binding sites for agonists and antagonists of the AMPA receptors are differentially modulated by the lipid environment during the postnatal period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glycine-induced changes in synaptic efficacy in hippocampal slices involve changes in AMPA receptors

Brief applications of high glycine concentrations to hippocampal slices have been shown to produce long-lasting changes in synaptic efficacy. In the present study, we show that glycine application transiently and reversibly increases the amplitude and prolongs the duration of synaptic potentials mediated by (NMDA) receptors. The long-lasting changes in synaptic potentials mediated by AMPA receptors are correlated with changes in the binding of [³H]α-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid ([³H]AMPA) to membranes prepared from glycine-treated slices. The changes in binding properties of AMPA receptors in adult slices are due to an increase in affinity of the agonist for the receptor. Furthermore, glycine-induced increases in [³H]AMPA binding and in synaptic potentials in adult hippocampal slices are markedly reduced in the presence of low extracellular calcium or of the phospholipase inhibitor bromophenacylbromide. Finally, glycine-induced potentiation of synaptic potentials is associated with an increased potency of the glutamate receptor antagonist, 6,7-dinitroquinoxaline (DNQX), to inhibit synaptic potentials. The results indicate that glycine-induced changes in synaptic efficacy are likely triggered by the activation of NMDA receptors and expressed by changes in the properties of AMPA receptors. As similar events underly long-term potentiation (LTP), this phenomenon might provide important clues to elucidate the molecular mechanisms involved in LTP maintenance.     

Classical conditioning selectively increases AMPA receptor binding in rabbit hippocampus.

The NMDA and AMPA receptors have been shown to play critical roles in various forms of synaptic plasticity (learning and memory, long-term potentiation). The present study investigated the involvement of these two receptors in a well-characterized classical conditioning paradigm. Following classical conditioning of the rabbit nictitating membrane the binding properties of these two subclasses of excitatory amino acid transmitter receptors were analyzed in dorsal hippocampi by quantitative autoradiography. [3H] TCP and [3H] AMPA were used to identify the NMDA and AMPA receptors, respectively. The binding of [3H]TCP to the NMDA receptor remained unchanged in all the experimental groups tested. Paired presentations of the conditioned and unconditioned stimuli resulted in increased [3H] AMPA binding to the AMPA receptor in several subfields of the hippocampus, while unpaired presentations had no significant effects. The increase in binding was due to an increased affinity of the low-affinity component of the AMPA receptor. The results support the hypothesis that changes in glutamate receptors participate in the synaptic plasticity involved in certain forms of learning.     

Comparative studies on binding of 3 different ligands to theN-methyl-d-aspartate recognition domain in brain synaptic membranes treated with Triton X-100

Treatment with a low concentration of Triton X-100 almost tripled the binding of [³H]d,l-(E)-2-amino-4-propyl-5-phosphono-3-pentenoic acid (CGP 39653), a novel competetive antagonist at anN-methyl-d-aspartate (NMDA)-sensitive subclass of brain excitatory amino acid receptors, in synaptic membranes of the rat brain. The binding linearly increased with increasing protein concentrations of up to 0.4 mg/ml and also increased in proportion to incubation time with a plateau within 60 min after the initiation of incubation at 2°C in Triton-treated membranes. Elevation of incubation temperature from 2°C to 30°C resulted in a marked decrease in the binding at equilibrium by 80%, and a maximal level was obtained within 1 min after the initiation of incubation at 30°C with a gradual decline of up to 10 min. Bound [³H]CGP 39653 was rapidly dissociated by the addition of excess unlabeledl-glutamic acid (Glu), and the time required to attain complete dissociation was 60 min at 2°C and 1 min at 30°C, respectively. Among several agonists and antagonists tested, Glu was the most potent displacer of [³H]CGP 39653 binding with progressively less potent displacement byd-2-amino-5-phosphonovaleric,(±)-3-(2-carboxypiperain-4-yl)propyl-1-phosphonic (CPP),d-2-amino-7-phosphonoheptanoic,N-methyl-d-aspartic andN-methyl-l-aspartic acids. Agonists at the glycine (Gly) domain on the NMDA receptor ionophore complex invariably inhibited the binding of [³H]CGP 39653 without virtually affecting the binding of [³H]CPP or [³H]Glu in a manner that was sensitive to antagonism by 4 different antagonists at the Gly domain. In contrast, the Gly antagonist 1-hydroxy-3-aminopyrrolidone-2 markedly potentiated [³H]CPP binding with the binding of both [³H]CGP 39653 and [³H]Glu being unaltered. These results suggest that [³H]CGP 39653 may predominantly label an antagonist-preferring form of the NMDA domain in a state different from that favorable to labeling by [³H]CPP.     

Clathrin-coated vesicles from bovine brain contain uncoupled GABAA receptors

Clathrin-coated vesicles are thought to be a vehicle for the sequestration of GABA_A receptors. For coated vesicles from bovine cerebrum, we examined the binding properties of [³H]muscimol, a GABA_A-specific agonist, [³H]flunitrazepam, a benzodiazepine agonist, and [³⁵S]t-butylbiocyclophosphorthionate (TBPS), a ligand for GABA_A receptor channels. Under standard conditions, the binding level of [³H]muscimol, [³H]flunitrazepam, and [³⁵S]TBPS to coated vesicles represented 12.3±1.8%, 7.9±1%, and 10.2±1.8%, respectively, of that in crude synaptic membranes. Coated vesicles showed a single [³H]flunitrazepam binding site with a K_D value (12 nM) which was 9-fold that for synaptic membranes. The allosteric coupling between binding sites was measured by the addition of GABA to [³H]flunitrazepam and [³⁵S]TBPS binding assays. For [³H]flunitrazepam binding to synaptic membranes, GABA gave an EC₅₀=2.0 μM and at saturation (100 μM) an enhancement of 122%. This stimulation was completely blocked by the GABA antagonist SR95531. In contrast, neither GABA nor SR95531 had a significant effect on [³H]flunitrazepam binding to CCVs, indicating that the allosteric interaction between GABA and benzodiazepine binding sites is abolished. Likewise, GABA displaced nearly all of the [³⁵S]TBPS binding to synaptic membranes but had no effect on binding to coated vesicles, indicating that coupling between the GABA binding sites and chloride channel is also impaired. Thus GABA_A receptors appear to be uncoupled during normal intracellular trafficking via coated vesicles. The presence of major GABA_A receptor subunits on these particles was verified by quantitative immunoblotting. Relative to the levels in synaptic membranes, CCVs contained 110±14% and 29.5±3.8%, respectively, of the immunoreactivity for GABA_A receptor β2 and α1 subunits. Thus, in comparison to GABA_A receptors on synaptic membranes, those on CCVs have a reduced α1/β2-subunit ratio. It may be suggested that a selective decline in the content of α1 subunits in coated vesicles could in part account for GABA_A receptor uncoupling.     

Effect of phosphatidylserine on the binding properties of glutamate receptors in brain sections from adult and neonatal rats

The effects of phosphatidylserine (PS) on the binding properties of the AMPA (-amino-3-hydroxy-5-methylisoxazolepropionic acid) and NMDA ( N-methyl-d-aspartate) subtypes of glutamate receptors were analyzed by quantitative autoradiography of [³H]AMPA, [³H]6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and [³H]glutamate binding on at brain tissue sections. Preincubation of brain sections with PS produced an increase in [³H]AMPA binding without modifying the binding properties of [3H]CNQX, an antagonist of AMPA receptors. This effect of PS appeared to be specific for the AMPA subtype of glutamate receptors as the same treatment did not modify [³H]glutamate binding to the NMDA receptors. Furthermore, the PS-induced increase in [3H]AMPA binding was different in various brain structures, being larger in the molecular layer of the cerebellum and almost absent in the striatum. Preincubation with calcium also augmented [³H]AMPA binding, and the lack of additivity of the effects of calcium and PS on [³H]AMPA binding strongly suggests that both treatments share a common mechanism(s) for producing increased agonist binding. Finally, the effect of PS on AMPA receptor properties was markedly reduced in rat brain sections prepared from neonatal rats at a developmental stage that is normally characterized by the absence of LTP expression in certain brain regions. The present data are consistent with the hypothesis that alteration in the lipid composition of synaptic membranes may be an important mechanism for regulating AMPA receptor properties. which could be involved in producing long-lasting changes in synaptic operation.     

β-adrenoceptor stimulation-induced increase in the number of α2-adrenoceptors of cerebral cortical membranes in rats

Effects of pre-treatment of synaptic membranes with β-adrenoceptor agonists and cholera toxin on [³H]clonidine and [³H]yohimbine binding were examined in rat cerebral cortex. Pre-incubation of cerebral cortical membranes with isoproterenol (10 or 200 μM) or dobutamine (1, 10 or 200 μM) at 37 °C for 40 min caused a significant elevation of specific [³H]clonidine binding but treatment with salbutamol (10 or 200 μM) did not. Scatchard analysis showed that 200 μM isoproterenol treatment resulted in a significant elevation of high affinity component of [³H]clonidine binding which was significantly decreased by the addition of 10μM GTP. A significant elevation in high affinity [³H]clonidine binding was observed by treatment with 100 μg/ml cholera toxin, while a significant decrease in low affinity one was by the treatment. Specific [³H]yohimbine binding was also elevated by 10 or 200 μM isoproterenol treatment. It is suggested that stimulation of β-receptors, presumably β₁-subtype could elevate the number of agonist and antagonist binding sites in α₂-receptors in synaptic membranes by partially mediated by stimulatory and/or inhibitory GTP binding regulatory proteins.     

Characterization of Na-dependent binding sites of [H]glutamate in synaptic membranes from rat brain

Some biochemical characteristics of Na⁺-dependent binding of [³H]l-glutamic acid (Glu) were studied using crude synaptic membrane preparations from the rat brain as compared with Na⁺-independent binding. In vitro addition of sodium chloride (1–100 mM) exhibited a significant enhancement of [³H]Glu binding to synaptic membranes in a concentration-dependent manner independent of the incubation temperature employed (2 or 30 °C). In contrast, sodium acetate elicited a concentration-dependent augmentation of the binding at 2 °C to a significantly greater extent than that found at 30 °C. It was found that the binding found in the presence of 100 mM sodium acetate reached its maximal value within 10 min of incubation followed by a rapid decline up to 60 min at 30 °C, while gradually increasing up to 60 min at 2 °C. The Na⁺-independent basal binding was significantly activated by the alteration of incubation temperature from 2 to 30 °C and reached equilibrium within 10 min of incubation at both incubation temperatures. The Na⁺-dependent binding was more promptly attenuated by the addition of excess of non-radiactive Glu (1 mM) at 30 °C than that at 2 °C, whereas the Na⁺-independent binding was greatly suppressed by the addition at 2 °C in comparison with that at 30 °C. Quisqualic acid induced a considerably less-potent inhibition of the Na⁺-dependent binding than that of the Na⁺-independent binding. Neither N-methyl-l-aspartic acid nor kainic acid had such a significant effect on each binding.d-Aspartic acid exerted a significant elimination of the Na⁺-dependent binding in a concentration-dependent manner without significantly affecting the Na⁺-independent binding. In vitro addition of sodium acetate (1–100 mM) exhibited a concentration-dependent suppression of [³H]Glu binding in the presence ofd-aspartate. Pretreatment of synaptic membranes with various sulphhydryl reagents such asp-chloromercuribenzoic acid (PCMB), N-ethylmaleimide (NEM) and5, 5′-dithio-bis-(2-nitrobenzoic acid) (DTNB) invariably resulted in a significant deterioration of the Na⁺-independent binding in a concentration-dependent manner, whereas the Na⁺-dependent binding was significantly abolished exclusively by 1 mM PCMB. These results suggest that the Na⁺-dependent binding sites may be distinctly different from the Na⁺-independent binding sites in nature and the former sites may be closely related to the Na⁺-dependent sites of Glu uptake with a high affinity for this putative neurotransmitter.     

Effect of Temperature and Calcium on the Binding Properties of the AMPA Receptor in Frozen Rat Brain Sections

The elucidation of the mechanisms regulating the properties of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) subtype of glutamate receptors is important for understanding glutamatergic transmission. Here we report that qualitative as well as quantitative analysis of tritiated ligand binding to the AMPA receptor on thin frozen rat brain tissue sections reveals the existence of several mechanisms regulating the binding properties of AMPA receptors. Preincubation of tissue sections at 35 degrees C results in a decreased amount of [3H]AMPA binding as compared to that measured following preincubation at 0 degrees C. The decrease in binding appears to be mainly localized to cell bodies as evaluated by autoradiography, and could be due to proteolysis. Preincubation with calcium at 35 degrees C produces increased levels of [3H]AMPA binding. The effect of calcium is mimicked by manganese and to a lesser extent by magnesium; it is concentration-dependent with a 50% effective concentration for calcium of approximately 150 microM, time-dependent and temperature-dependent. The calcium-induced increase in [3H]AMPA binding is different among various brain structures, being larger in area CA1 of the hippocampus and in the superficial layers of the cerebral cortex. The effect of calcium is partly reduced by preincubation with the calpain inhibitor leupeptin and potentiated by preincubation with purified calpain II. The calcium-induced increase in [3H]AMPA binding is associated with a decrease in the binding of an antagonist of AMPA receptors, [3H]6-nitro-7-cyanoquinoxaline-2,3-dione. The results indicate that the binding properties of the AMPA receptor are rapidly regulated by calcium-dependent processes, and possibly by calcium-dependent proteases. They suggest that modulation of the binding properties involves changes in the configuration of the receptor, producing opposite changes in the affinities of the receptor for agonists and antagonists. Finally, these results strengthen the hypothesis that changes in the properties of AMPA receptors might underlie various forms of synaptic plasticity.     

Crucial role of kainate receptors in mediating striatal kainate injection-induced decrease in acetylcholine M1 receptor binding in rat forebrain

We investigated the roles of kainate-, α-amino-3-hydroxy-5-methylisoxazol-4-propionate (AMPA)- and N-methyl- -aspartate (NMDA)-receptors in mediating striatal kainate injection-induced decrease in the binding of acetylcholine M₁ 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 20%), [³H]MK-801 (up to 90%) and [³H]pirenzepine (up to 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 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 M₁ 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).     

Differential pharmacological profile of striatal and cerebellar dopamine receptors labeled by [3H]quinpirole: Identification of a discrete population of putative D3 receptors

We have previously identified [³H]quinpirole-labeled dopamine receptors in the molecular layer of cerebellar lobule 10 which have a D₂-like pharmacological profile, are guanine nucleotide-insensitive, and are juxtaposed to putative D₃ receptor mRNA. This study compares the pharmacological profiles of [³H]quinpirole-labeled dopamine receptors in striatum and cerebellar lobule 10 using quantitative autoradiography. Dopaminergic compounds inhibited the specific binding of [³H]quinpirole in the caudate/putamen with the following rank order of potencies: spiperone > haloperidol ≥ (+)butaclamol ≥ quinpirole ≥ 7-OH-DPAT ≥ bromocriptine > clozapine > (-)sulpiride. In cerebellar lobule 10, a somewhat different rank order of potencies was observed: 7-OH-DPAT > quinpirole ≥ bromocriptine > spiperone > (+)butaclamol > haloperidol > clozapine > (-)sulpiride. Quinpirole possessed equal affinity for [³H]quinpirole-labeled receptors in the caudate/putamen and cerebellum. 7-OH-DPAT exhibited 5-fold greater affinity for cerebellar receptors than those in the caudate/putamen. Spiperone, haloperidol, (+)butaclamol, and clozapine were more potent in competing for [³H]quinpirole binding at striatal dopamine receptors than cerebellar receptors by 83-, 59-, 11-, and 6-fold, respectively. The relative potencies of these compounds at striatal and cerebellar dopamine receptors are generally similar to the differential affinities reported at D₂ and D₃ dopamine receptors expressed in CHO cells, respectively. These data provide additional evidence that the dopamine receptors observed in cerebellar lobule 10 represent a discrete population of putative D₃ receptors. © Wiley-Liss, Inc.     

Binding sites for the glutamate-analogue [H]AMPA in cultured rat brainstem and spinal cord

By means of light microscopic autoradiography, binding sites for the glutamate-analogue [ [³H]AMPA [(RS)-α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid] were observed on the soma and processes of many brainstem and spinal neurons but not on glial cells. Unlabeled AMPA, glutamate or the glutamate antagonist glutamic acid diethyl ester at high concentrations inhibited or reduced binding of [³H]AMPA. Our data indicate that binding sites for [³H]AMPA exhibit characteristics expected for glutamate receptors.     

Rapid Effects of Kainate Administration on α-Amino-3-hydroxy-5-methyl-4-isoxazole Propionic Acid (AMPA) Receptor Properties in Rat Hippocampus

We investigated changes in AMPA receptor properties in rat hippocampus 5 h after systemic kainate administration. Quantitative [³H]AMPA autoradiography and Western blot analysis of receptor subunits GluR1-3 in different subcellular fractions were used to evaluate possible alterations in binding characteristics and immunological properties of the receptors in synaptic and nonsynaptic fractions. Both ligand-binding and Western blots revealed significant changes in binding and immunological properties of nonsynaptic receptors but relatively smaller changes in synaptic receptors 5 h after kainate administration. GluR2/3 showed a greater relative change in the synaptic receptor population compared to GluR1, suggesting either a shift in subunit composition of AMPA receptors or the formation of a synaptic subpopulation of AMPA receptors with truncated C-terminal domain of GluR1 subunits. The effects of kainic acid were blocked by cycloheximide treatment indicating that the changes were due at least in part to increased synthesis of AMPA receptor subunits. The results indicate that excessive synaptic activity produces rapid changes in both synaptic and nonsynaptic AMPA receptor properties.     

Phospholipase A2-induced changes in AMPA receptor: an autoradiographic study.

The expression of long-term potentiation and learning of a classical conditioning task increase [3H]-AMPA binding in hippocampus. Phospholipase A2 (PLA2) has been proposed to underly these changes, as PLA2 treatment of membrane preparations increases the affinity of AMPA receptors for agonists. We demonstrate here that preincubation of thin (10 microns) frozen rat brain sections with exogenous PLA2 and calcium at physiological temperature changes the binding properties of AMPA receptors. Quantitative autoradiography reveals that PLA2-treatment produces a differential increase in [3H]-AMPA binding across brain regions. The same treatment also decreases the binding of an antagonist ([3H]-CNQX) throughout the brain. We propose that PLA2 treatment results in a modification of the AMPA receptors which is regionally specific, probably due to different AMPA receptor subunit compositions.     

Sialic acid residues indirectly modulate the binding properties of AMPA-type glutamate receptors

Manipulations that disrupt the extracellular interactions of neural cell adhesion molecules (NCAMs) block the formation of stable long-term potentiation (LTP) but do not reverse already established potentiation. Several studies have implicated a change in AMPA-type glutamate receptors as being responsible for the expression of LTP but there are no evident links between NCAMs and the receptors. NCAMs are major carriers of sialic acid residues in the brain and removal of these with neuraminidase markedly affects the binding properties of the adhesion molecules. Therefore, the present study tested if neuraminidase treatment produces a change in AMPA receptors. Preincubation of cortical membranes with the enzyme for 15 min at 37°C caused a 5% reduction in the apparent sizes of NCAMs 140 and 180 but had no detectable influence on the sizes of various glutamate receptor subunits. The same treatment resulted in a 20±1% increase in the binding of [³H]AMPA with no apparent effect on binding to NMDA-type glutamate receptors or to high affinity kainate receptors. In membranes from the hippocampus, neuraminidase induced a 30±2% increase in binding which Scatchard analyses showed to be due to an increase in receptor affinity. Finally, neuraminidase had no effect on either the binding properties of solubilized AMPA receptors or on AMPA receptors stably expressed in a non-neuronal cell line. These results: (i) demonstrate that modulation of the extracellular environment can influence the binding properties of AMPA receptors, (ii) indicate that sialic acid residues in the extracellular compartment of synapses exert a significant and indirect influence on AMPA receptors and, (iii) suggest a route whereby NCAMs and LTP could be linked.     

Direct radiolabeling by [H]quisqualic acid of group I metabotropic glutamate receptor in rat brain synaptic membranes

[³H]Quisqualic acid (QA) was synthesized and used to label metabotropic glutamate receptor (mGluR) in rat brain synaptic membranes in the presence of three different ionotropic glutamate receptor agonists at respective saturating concentrations. Of several mGluR agonists tested, group I agonists were more potent in displacing [³H]QA binding than group II and group III agonists in the presence of the three ionotropic agonists. [³H]QA binding was markedly inhibited by guanine nucleotide analogues in a concentration-dependent manner at a concentration range of 10 nM to 1 mM. Scatchard analysis revealed that [³H]QA binding consisted of a single component with a K_d of 50.9±5.3 nM and a B_max of 431.6±18.3 fmol/mg protein. These results suggest that [³H]QA indeed labels group I mGluR functionally coupled to GTP binding protein in rat brain synaptic membranes when determined under the experimental conditions employed.     

[H]-dynophin A (1–8): Degradation profile and binding characteristics in brain homogenates

[³H]-Dynorphin A (1–8) was degraded in brain homogenates at 25° and even at 0°C. The peptidase inhibitors bestatin and captopril almost completely protected[³H]-dynorphin A (1–8) from degradation at 0°C but had only little effect on binding at this temperature. At 25°C, the binding of [³H]-dynorphin A (1–8) was markedly improved by addition of bestatin, captopril and L-leucyl-L-arginine, which afforded some, but not complete protection from degradation. The results of saturation binding assays at 25°C in the presence of the peptidase inhibitors were variable. However, it was found from saturation binding assays at 0°C that the maximum binding capacity for [³H]-dynorphin A (1–8) at the κ-site is similar to that of [³H]-(−)-bremazocine and [³H]-dynorphin A (1–9).