Selective changes in AMPA receptors in rabbit cerebellum following classical conditioning of the eyelid-nictitating membrane response

α-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors are critically involved in several forms of synaptic plasticity proposed to be neural substrates for learning and memory, e.g., long-term potentiation and long-term depression (LTD). The present study was designed to determine changes in cerebellar AMPA receptors following classical conditioning of the eyeblink-nictitating membrane response (NMR) in the rabbit. Quantitative autoradiography was used to assess changes in ligand binding properties of cerebellar AMPA receptors following NMR conditioning elicited by pairing electrical stimulation of the pontine nuclei with an airpuff to the eye. [³H]AMPA and [³H]-6-cyano-7-nitroquinoxaline-2,3-dion (CNQX) binding were determined following preincubation of frozen–thawed brain tissue sections at 0 or 35°C. With 0°C preincubation, no significant differences in [³H]AMPA binding to cerebellar AMPA receptors were seen between any of the experimental groups tested. In contrast, preincubation at 35°C revealed significant decreases in [³H]AMPA binding to the trained side of the cerebellar cortex resulting from paired presentations of the conditioned and the unconditioned stimuli, while unpaired presentations of the stimuli resulted in no significant effect. With 35°C preincubation, there were no significant differences in [³H]CNQX binding between any of the experimental groups and no significant differences in [³H]AMPA binding in the untrained side of the cerebellum. These results indicate that NMR conditioning is associated with a selective modification of AMPA-receptor properties in brain structures involved in the storage of the associative memory. Furthermore, they support the hypothesis that cerebellar LTD, resulting from decreased synaptic efficacy at parallel fiber–Purkinje cell synapses mediated by a change in AMPA-receptor properties, is a form of synaptic plasticity that supports this type of learning.     

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 alpha-amino-3-hydroxy-5-methyl-4 isoxazole propionic 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.     

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.     

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.     

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.     

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.     

Decreased density, but not number, of N-methyl-D-aspartate, glycine and phencyclidine binding sites in hippocampus of senescent rats

Aging is associated with reduced rates of kindling and spatial learning. Blockade of N-methyl-D-aspartate (NMDA) receptors in young animals produces effects similar to those of aging. These findings raise the possibility that age-dependent reductions in neuronal plasticity are the consequence of decreased NMDA receptor-mediated neurotransmission. Conceivably this reduction could be due to an alteration in the NMDA receptor itself. To test this idea we quantified ligand binding to 3 distinct sites on the NMDA receptor/channel complex in hippocampal membranes prepared from 3- and 24-month-old Fischer-344 rats. The binding parameters of the NMDA, glycine and non-competitive antagonist (A.K.A. phencyclidine) sites on the NMDA receptor/channel complex were examined using [3H]3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid ([3H]CPP), [3H]glycine and [3H]N-(1-[2-thienyl]cyclohexyl)3,4-piperidine ([3H]TCP), respectively. Aging produced no change in the affinity or stoichiometry of the binding sites. Aging was associated with a 30% reduction in the density of each of the 3 binding sites (when expressed as sites/mg membrane protein). However, this reduction in receptor density was the consequence of increased protein content in the hippocampus of aged animals, not a reduction in the number of binding sites. These findings suggest that a selective alteration in the NMDA receptor/channel complex itself does not account for the age-dependent reductions in neuronal plasticity.     

Long-term potentiation is associated with increased [3H]AMPA binding in rat hippocampus.

The location and nature of the changes underlying long-term potentiation (LTP) remain controversial issues. In this study, we tested the possibility that changes in binding properties of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA/quisqualate and N-methyl-D-aspartate (NMDA) subtype of glutamate receptors are associated with LTP. LTP was elicited in vivo by stimulation of the perforant pathway in anesthetized rats. One hour following stimulation the animals were sacrificed. We performed quantitative ligand binding autoradiography on frozen brain sections using [3H]AMPA and [3H]N-(1-(2-thienyl)cyclohexyl)-3,4-piperidine ([3H]TCP) to label the AMPA/quisqualate and the NMDA receptors, respectively. No changes in [3H]TCP binding were detected in any of the treatment groups. However, increases in [3H]AMPA binding were observed only in animals that exhibited LTP. These increases were bilateral and present in several subfields of the hippocampus and cortical areas. Administration of the NMDA receptor antagonist, ketamine, prior to tetanic stimulation prevented both the increase in binding and the induction of LTP. These results suggest that changes in the characteristics of AMPA/quisqualate receptors are a biochemical correlate of LTP.     

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.     

Selective increase of AMPA binding to the AMPA/quisqualate receptor in the hippocampus in response to acute stress.

The binding properties of ligands specific for two subclasses of glutamate receptors were studied by quantitative autoradiography after one hour of acute immobilization/shock stress. [3H]N-(1-(2-thienyl)cyclohexyl)-3,4-piperidine (TCP) and [3H]alpha-amino-3-methylisoxazole-4-propionic acid (AMPA) were used to visualize the N-methyl-D-aspartate receptor and the AMPA/quisqualate receptor types, respectively. While no change was observed in the binding properties of the [3H]TCP, [3H]AMPA binding was significantly increased in several areas of the hippocampus of acutely stressed rats relative to naive controls.     

Quinoxaline derivatives are high-affinity antagonists of the NMDA receptor-associated glycine sites

Membranes from rat telencephalon contain strychnine-insensitive glycine binding sites associated with NMDA receptors. Three quinoxaline derivatives, among them the high-affinity AMPA receptor antagonists CNQX and DNQX, were found to inhibit [3H]glycine binding to these sites with micromolar affinities. Binding of these compounds to the glycine site also inhibited glutamate-stimulated association and dissociation of [3H]TCP. This suggests that these AMPA antagonists, like the structurally related compound kynurenate, act as glycine site antagonists.     

Opposite effects of phospholipase A2 on [3H]AMPA binding in adult and neonatal membranes.

In the present study, we compared the effect of phospholipase A2 (PLA2) treatment of synaptic membranes from adult and neonatal rats on the characteristics of [3H]AMPA binding sites. Whereas PLA2 treatment of membranes from adult rats produces an increased affinity for [3H]AMPA binding, the same treatment in neonatal rats results in a decrease in the maximal number of binding sites. Since activation of PLA2 has been proposed to play a critical role in the formation of long-term potentiation (LTP), possibly mediated through a modification of the AMPA receptors, the results strengthen the hypothesis that PLA2-induced modification of [3H]AMPA binding sites is an important component of synaptic plasticity.     

Ionotropic glutamate receptor expression in human spinal cord during first trimester development

Quantitative receptor autoradiography and immunoblotting were used to study the expression and distribution of AMPA, kainate and NMDA receptors in first trimester human spinal cord obtained from elective abortions ranging from 4 to 11.5 weeks of gestational age. Spinal cord tissue sections were processed for receptor autoradiography with the ligands [3H]AMPA, [3H]kainate and [3H]MK-801 and the optical density was measured separately in a dorsal region (alar plate) and ventral region (basal plate) of the autoradiographs. Binding sites for all three ligands were demonstrated already at 4-5.5 weeks of gestation and increased continuously during the first trimester both in the dorsal and ventral regions. [3H]AMPA binding to both high- and low-affinity sites increased from undetectable levels to about 35 and 400 fmol/mg tissue, respectively, during this period. A temporal difference in the distribution of [3H]AMPA binding sites was observed. The early homogeneous pattern of [3H]AMPA binding in both alar and basal plates had changed to a heterogeneous pattern at 11 weeks of gestation with the highest density of [3H]AMPA binding sites in the superficial layers of the immature dorsal horn. [3H]kainate and [3H]MK-801 binding sites were densely and homogeneously distributed already at 4 weeks, and steadily increased six- and two-fold, respectively, to about 100 fmol/mg tissue at 11.5 weeks of gestation. Immunoreactive bands corresponding to the NMDA receptor subunits NR1, NR2A, NR2B, NR2C and NR2D were demonstrated by immunoblotting at the earliest between 4.5 and 7 weeks and increasing concentrations were seen up to 11 weeks of gestation. These results suggest that AMPA, kainate and NMDA receptors are expressed in the human spinal cord early in embryogenesis.     

Quantitative autoradiographic localization of NMDA, quisqualate and PCP receptors in the frog tectum

An organizing role for the N-methyl-D-aspartate (NMDA) receptor/channel has been suggested in the development of the retinotectal projection in Rana pipiens. The regional distributions of NMDA, phencyclidine (PCP) and quisqualic acid (QA) receptors were quantified using in vitro autoradiography in the tectum of normal and surgically produced 3-eyed juvenile frogs. NMDA and QA receptor binding was highest in the pretectum. Of the tectal layers, the superficial retinotectal synaptic zone, layer 9, had the highest amount of NMDA and QA receptor binding. Moderate binding was observed in layer 5, with little binding in the cellular layer 6. No specific [3H]N-(1-[2-thienyl]cyclohexyl) piperidine ([3H]TCP) binding was observed in any of the tectal regions.     

AMPA receptor modulation in previously frozen mouse brain sections: opposite effects of calcium in the cortex and hippocampus

Various forms of synaptic plasticity in the brain have been proposed to result from modifications in the properties of glutamate receptors by calcium-dependent mechanisms. In the present study, changes in glutamate receptors elicited by calcium treatment of previously frozen mouse brain sections were evaluated by qualitative as well as quantitative analysis of tritiated ligand binding to both alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and N-methyl-D-aspartate (NMDA) glutamate receptor subtypes. Quantitative analysis revealed that 3H-AMPA binding was reduced in a dose-dependent manner by calcium in the cerebral cortex and striatum formations. However, an opposite change in AMPA receptor properties was observed in the hippocampus, as calcium generated an increase of AMPA binding in all hippocampal fields. Analysis of the saturation kinetics of 3H-AMPA binding showed that the calcium-induced augmentation of AMPA binding in the stratum radiatum of the CA1 region was due to an alteration in the maximal number of sites, while the reduction of binding elicited by calcium in the cortex appeared to be due to modified AMPA receptor affinity. Calcium-induced downregulation of AMPA receptor affinity in the cortex and striatum was affected by baicalein, a selective inhibitor of the lipoxygenase pathways of arachidonic acid metabolism, whereas the same inhibitor did not modify calcium-mediated upregulation of receptor number in the CA1 region of the hippocampus. On the other hand, the effect of calcium appeared to be specific for the AMPA receptor, as the same treatment did not affect glutamate binding to the NMDA glutamate receptor subtype. Our results suggest the possibility that, depending on the brain regions, calcium ions may generate opposite modulation of AMPA receptor properties. Because the regulation of AMPA receptors by calcium-dependent enzymes has been implicated in synaptic plasticity, our results suggest that regional variations in the effect of calcium on AMPA binding account for differential plasticity at glutamatergic synapses.     

Postnatal development of the excitatory amino acid system in visual cortex of the rat. Changes in ligand binding to NMDA, quisqualate and kainate receptors

The postnatal development of the ligand binding to N-methyl-D-aspartate (NMDA), quisqualate and kainate receptor sites was examined in whole homogenates of the visual cortex of rats, aged 2-360 days. As selective ligands, [3H]CPP (3-(2-carboxypyperazine-4-yl)-propyl-1-phosphonic acid, [3H]AMPA (RS-alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid) and [3H]KA (kainic acid) were used, respectively. The binding of CPP was low in newborns, rapidly increased from the second postnatal week, reached its maximum between weeks 2 and 3, then slowly declined up to the age of 1 year. In contrast, the binding of AMPA and kainate was high perinatally, increased rapidly up to day 6 after birth to reach an early maximum value, then gradually decreased to adult values which were attained at an age of 3-4 weeks. These age-related changes were derived from alterations in the density of binding sites, which, in the case of AMPA, was accompanied by an increase in binding affinity. The results, compared with the developmental time-course of excitatory synapses, indicate that, in the immature cerebral cortex, NMDA receptors may be primarily involved in synaptic transmission, whereas quisqualate and kainate receptors may play some other (e.g. trophic) roles.     

Density and distribution of excitatory amino acid receptors in the developing human fetal brain: A quantitative autoradiographic study

The binding of [alpha-3H]amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) to quisqualate receptors, [3H]kainate (KA) to KA receptors, and L-[3H]glutamate to N-methyl-D-aspartate (NMDA) receptors was determined by quantitative autoradiography in brains obtained from twelve aborted human fetuses ranging from 16.5 to 26 weeks of gestational age. Among the three receptor subtypes, specific binding to AMPA was the highest, followed by NMDA and KA, respectively, in all age groups. Receptor binding was already apparent by 16.5 weeks in the hippocampus, thalamus, and subthalamic nucleus, rose sharply by 20-21.5 weeks, and subsequently declined to their lowest levels by 24-26 weeks. Anatomically distinct binding patterns for each of the three major excitatory amino acid (EAA) receptor subtypes were well established by 20-21.5 weeks. Within the hippocampus, AMPA was localized primarily in the stratum pyramidale, NMDA in the stratum radiatum, and KA in the molecular layer of the dentate gyrus and in the stratum lucidum of the CA3 region. The cerebral cortex showed dense labeling of AMPA in the outer layers, whereas KA binding was more prominent within the inner layers. The putamen and globus pallidus also showed relatively dense receptor binding in all age groups. The sharp rise in receptor density at 20-21.5 weeks of age suggests involvement of EAA pathways in developmental plasticity, including reorganization of neuronal processes or synapses, during this period of development. Developmental changes in the density and distribution of EAA receptors, as shown in this study, may also provide insight into shifts in the localization of age-dependent selective vulnerability within the developing human fetal brain.     

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.     

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.