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.     

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.     

Reversible kinase and phosphatase regulation of brain amino acid receptors in postnatal development.

In a previous study [Shaw, C., Pasqualotto, B. and Lanius, R.A., Mol. Neuropharmacol., in press] we have shown that phosphorylation and dephosphorylation actions of protein kinase and alkaline phosphatase lead to decreases or increases in the number of GABAA and AMPA receptors in adult rat neocortex. Using the same in vitro cortical slice preparation, we have now examined the role of these enzymes in regulating GABAA and AMPA receptors at different stages of postnatal development. GABAA receptors were labelled with [3H]SR95531 [Shaw, C. and Scarth, B.A., Mol. Brain Res., 11 (1991) 273-282]; AMPA receptors were labelled with [3H]CNQX [Lanius, R.A. and Shaw, C., Mol. Brain Res., 15 (1992) 256-262]. At postnatal day 14, GABAA receptors showed a decrease in binding in response to alkaline phosphatase treatment as opposed to an increase in binding observed in response to protein kinase treatment. Similar effects were observed for AMPA receptors at 20 days of age. The direction of regulation following the enzyme treatments were opposite to those observed in the adult cortex for both receptor populations. These fundamental changes in the enzymatic nature of regulation for such key inhibitory and excitatory receptor populations in cortex may signal an important role for age-dependent kinases and phosphatases in the events leading to modifications in neuronal function during postnatal development.     

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.     

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.     

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.     

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.     

Differential ontogenic development of three receptors comprising the NMDA receptor/channel complex in the rat hippocampus

The postnatal development of the three receptor binding sites that constitute the N-methyl-D-aspartate (NMDA) receptor channel/complex was examined in six hippocampal regions of rats using quantitative receptor autoradiography. NMDA-sensitive [3H]-glutamate binding, strychnine-insensitive [3H]glycine binding, and [3H]N-(1-[2-thienyl]cyclohexyl)-3,4-piperidine [( 3H]TCP) binding were measured to examine the ontogeny of NMDA recognition sites, glycine modulatory sites, and PCP receptors, respectively. NMDA-sensitive [3H]glutamate binding transiently exceeded adult levels by 50 to 120% in all regions examined, with peak densities generally occurring between postnatal days (PND) 10 and 28. Stratum radiatum CA1 binding increased slowly from 49 to 61% of the adult value between PND 1 and 7, after which, binding rapidly rose to 151% of adult values at PND 14, remained elevated through PND 28, and then decreased to adult levels. The ontogenic profile of NMDA recognition site binding was similar in other hippocampal regions, although the initial age of maximal binding and the period of stabilization varied. The ontogenic profiles of glycine modulatory site binding and PCP receptor binding were very similar to each other. Development was delayed, however, with respect to NMDA recognition site binding. The rapid development of binding observed between PND 7 and 14 with NMDA receptors in stratum radiatum CA1 was contrasted by a much slower increase in glycine and PCP receptor binding. Furthermore, maximal glycine and PCP receptor binding densities were not reached until PND 28 and were lower than NMDA recognition site binding densities. The observed developmental patterns of binding to each of the receptor components of the NMDA receptor channel/complex are consistent with postnatal changes in cytoarchitecture, synaptogenesis, afferent lamination, and functional development of the hippocampus. However, the relative overexpression of NMDA recognition sites with respect to glycine and PCP receptors between PND 7 and 21 suggests that there is differential expression of these binding sites during development.     

Characterization and regulation of a high affinity [3H]CNQX labelled AMPA receptor in rat neocortex.

We have characterized a high affinity site of the alpha-amino-3 hydroxy-5-methyl-isoxazole-4-propionate (AMPA) receptor in in vitro living slices of adult rat neocortex using [3H]CNQX, and AMPA antagonist. [3H]CNQX labelled multiple binding sites with a Bmax of a high affinity site of approximately 470 fmol/mg protein and an apparent Kd of 11.3 nM. The high affinity site of the AMPA receptor could be down-regulated (36%) by 2 h preincubations in quisqualate, an AMPA agonist. Increases in electrical activity induced by a combination of veratridine and glutamate also led to an average decrease of the high affinity AMPA receptor number of 17%. In addition, preincubations with muscimol, a GABAA agonist, as well as glutamate agonists kainate and N-methyl-D-aspartate (NMDA) led to an average increase in high affinity AMPA receptor number of 17%, 14%, and 37%, respectively. The present results show that a ligand-gated high affinity AMPA receptor can be regulated by agonist stimulation as well as changes in neural activity.     

Development of glutamate binding sites and their regulation by calcium in rat hippocampus

The postnasal development of the Na-independent [3H]glutamate binding sites, which exhibit some characteristics of postsynaptic glutamate receptors, has been studied in rat hippocampal membranes. The amount of binding sites (expressed in pmol/hippocampus) represents 4% of the adult level at postnatal day (PND) 4, increases very rapidly until PND 9, and then increases at a slower rate reaching 80% of the adult value at PND 23. In contrast, the density of binding sites (expressed in pmol/mg protein) exhibits a maximum at PND 9 and slowly decreases to reach the adult value at PND 23. These changes seen to be only quantitative since the affinity (about 450nM) and Hill coefficient (about 1.0) of these binding sites remain constant throughout development. Calcium ions have been shown to markedly stimulate [3H]glutamate binding in adult hippocampal membranes. This effect appears on PND 9--10 and increases rapidly until PND 16 when it is similar to that seen in the adult rat. We also determined the minimum age at which long-term potentiation (LTP) of synaptic transmission could be detected in the CA1 field of hippocampal slice preparations following repetitive electrical stimulation of the Schaffer-commissural pathways. LTP was only rarely detected at PND 8 whereas it could be reliably obtained after PND 9. These results indicate that the postnatal development of Na-independent glutamate binding sites closely parallels synapse formation in the hippocampus, further supporting the idea that the binding sites are associated with a physiological receptor. They also show that the appearance of the stimulatory effect of calcium on glutamate binding occurs at a time when several forms of synaptic plasticity appear in the hippocampus. In particular the correlation of the development of LTP with the calcium-stimulation of glutamate binding suggests that these phenomena have similar cellular mechanisms.     

Muscarinic cholinergic receptors in human infant forebrain: [3H]quinuclidinyl benzilate binding in homogenates and quantitative autoradiography in sections

The ontogeny of muscarinic receptors in human brain was studied by comparing [3H]quinuclidinyl benzilate [( 3H]QNB) binding in postmortem tissue from infants 1 week to 3 months of age with binding in adult specimens. Saturation analysis with [3H]QNB and displacement studies with muscarinic antagonists and agonists in tissue homogenates demonstrated that binding sites in the infants' forebrain regions were present in adult or higher than adult concentrations (Bmax). Binding affinity (Kd) and pharmacological characteristics were nearly identical at the two ages. Quantitative receptor autoradiography demonstrated more [3H]QNB binding in the gray matter of infants than adults and revealed a marked difference between the two ages in the laminar distribution of binding sites in neocortex. In contrast to the adult pattern with higher binding in superficial layers 1-3 than in layers 4-6, the distribution in the immature cortex was inverted. These results suggest that muscarinic receptors in infants resemble closely those in mature brain. However, the topography of receptors in the immature neocortex is distinct and they are redistributed in a gradient from inside outward during postnatal development.     

AMPA receptor potentiation by acetylcholinesterase is age-dependently upregulated at synaptogenesis sites of the rat brain

We have used radioligand binding to synaptic membranes from distinct rat brain regions and quantitative autoradiography to investigate the postnatal evolution of acetylcholinesterase (AChE)-evoked up-regulation of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors in CNS areas undergoing synaptogenesis. Incubation of synaptosomal membranes or brain sections with purified AChE caused a developmentally modulated enhancement in the binding of [3H]-(S)-AMPA and the specific AMPA receptor ligand [3H]-(S)-5-fluorowillardiine, but did not modify binding to kainate neither N-methyl-D-aspartate receptors. In all postnatal ages investigated (4, 7, 14, 20, 27, 40 days-old and adult rats), AChE effect on binding was concentration-dependent and blocked by propidium, BW 284c51, diisopropylfluorophosphonate and eserine, therefore requiring indemnity of both peripheral and active sites of the enzyme. AChE-mediated enhancement of [3H]-fluorowillardiine binding was measurable in all major CNS areas, but displayed remarkable anatomical selectivity and developmental regulation. Autoradiograph densitometry exhibited distinct temporal profiles and peaks of treated/control binding ratios for different cortices, cortical layers, and nuclei. Within the parietal, occipital and temporal neocortices, hippocampal CA1 field and cerebellum, AChE-potentiated binding ratios peaked in chronological correspondence with synaptogenesis periods of the respective AMPA-receptor containing targets. This modulation of AMPA receptors by AChE is a molecular mechanism able to transduce localized neural activity into durable modifications of synaptic molecular structure and function. It might also contribute to AChE-mediated neurotoxicity, as postulated in Alzheimer's disease and other CNS disorders.     

Postnatal development of detergent-insoluble properties of NMDA and AMPA receptor subunits in the rat brain synaptic membrane.

We investigated the developmental changes of detergent-insoluble characteristics of NMDA and AMPA receptor subunits in the synaptic membranes prepared from the rat cerebral cortex. At postnatal day (PND) 1, the majority of NMDAR1 and NMDAR2B subunits of NMDA receptors in the synaptic membranes were insoluble to the treatment of 1% Triton X-100. The detergent-insoluble properties of both subunits were not significantly changed during postnatal development. At PND 1, about 45% of GluR1 and 10% of GluR2/3 subunits of AMPA receptors in the synaptic membrane were insoluble to Triton X-100, whereas 70% of GluR1 and 56% of GluR2/3 subunits were insoluble at PND 22. These findings indicate that the postsynaptic clustering of NMDA and AMPA receptors during development seems to be differentially regulated in vivo.     

Molecular pathways mediating activation by kainate of mitogen-activated protein kinase in oligodendrocyte progenitors

Oligodendroglial cells express ionotropic glutamate receptors of alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid hydrobromide (AMPA) and kainate (KA) subtypes. Recently, we reported that AMPA receptor agonists increased 45Ca2+ uptake and phospholipase C (PLC) activity. To further elucidate the intracellular signaling mechanisms, we examined the effects of AMPA and KA on mitogen-activated protein kinase (MAPK). KA caused a time- and concentration-dependent increase in MAPK activity (predominantly the p42mapk or ERK2) and the effect was blocked by 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX), a competitive AMPA/KA receptor antagonist. Furthermore, the noncompetitive antagonists of AMPA receptor GYKI 52466 and LY 303070 prevented the actions of the agonists, indicating that the effect of KA on MAPK activation is mediated through AMPA receptors in oligodendrocyte progenitors. Chelation of extracellular Ca2+ by EDTA or inhibition of PLC with U73122 abolished MAPK activation by KA. In addition, KA-stimulated MAPK activation was reduced by the protein kinase C (PKC) inhibitors, H7 and bisindolylmaleimide, as well as downregulation of PKC by prolonged exposure to phorbol esters. The involvement of PKC in the signal transduction pathways was further supported by the ability of KA to induce translocation of PKC measured by [3H]PDBu binding. Interestingly, a wortmannin-sensitive phosphatidylinositol 3-kinase and a pertussis toxin (PTX)-sensitive G protein form part of the molecular pathways mediating MAPK activation by AMPA receptor. A specific inhibitor of MAPK kinase, PD 098059, blocked MAPK activation and reduced KA-induced c-fos gene expression. All together, these results indicate that MAPK is implicated in the transmission of AMPA signaling to the nucleus and requires extracellular Ca2+, and PLC/PKC activation.     

Inhibition of N6-[3H]cyclohexyladenosine binding by carbamazepine

The mechanism of action of carbamazepine (CBZ) (Tegretol), despite widespread use in the management of partial and tonic-clonic seizures in adults, is not completely understood. In animals, adenosine and adenosine analogues have anticonvulsant effects that may be due to interactions with central A1 adenosine receptors. CBZ (at therapeutically relevant concentrations) inhibits the binding of agonists and antagonists to brain A1 adenosine receptors, but whether as an agonist/antagonist is not clear. The adenosine agonist, N6-[3H]cyclohexyladenosine ([3H]CHA), binds to membranes from rat cortex and hippocampus at two nanomolar binding sites or states. To clarify the actions of carbamazepine at the A1 adenosine receptor, its inhibitory actions were compared with those of known adenosine agonists and xanthine antagonists using 0.1 nM[3H]CHA, in which almost all binding is to the higher affinity state, or 10 nM [3H]CHA, in which there is a substantial contribution of binding from both states. The ratios of the IC50 values (concentration that inhibits specific binding by 50%) at 10 nM [3H]CHA to the IC50 values at 0.1 nM [3H]CHA were 18-31 for the agonists and 4-10 for the xanthine antagonists. CBZ had a ratio of 3. The inhibitory effects of GTP on [3H]CHA binding were less in the presence of the adenosine agonist, 2-chloroadenosine than were inhibitory effects in the presence of the xanthine antagonist theophylline or CBZ in both cortex and hippocampus. These in vitro studies indicate that CBZ is an antagonist at A1 adenosine receptors in cerebral cortical and hippocampal membranes from rat brain. Agonist activity at A1 adenosine receptors would have been compatible with the sedative anticonvulsant effects of CBZ, but these data do not support a role of the anticonvulsant action of carbamazepine on A1 adenosine receptors in cerebral cortex or hippocampus.     

Synchronized overproduction of AMPA,kainate, and NMDA glutamate receptors during human spinal cord development

Quantitative receptor autoradiography was used to map the distribution in the developing human spinal cord of the three types of ionotropic glutamate receptors. N-methyl-D-Aspartate (NMDA) receptors were labeled with [³H]glutamate, kainic acid (KA) receptors were labeled with [³H]KA, and α-amino-3-hydroxy-5-methyl-4-isoxazole proprionate (AMPA) receptors were labeled with [³H]AMPA. In the adult, labeling of all three receptor subtypes is largely restricted to the substantia gelatinosa (SG) in the dorsal horn, with very low level labeling elsewhere in the spinal gray matter. In marked distinction, in late fetal life, high level ligand binding is seen throughout the spinal gray matter. In early postnatal life, binding sites diminish in all regions, but least so in the SG, until the adult pattern emerges. Thus a coordinated transient high level of ionotropic glutamate receptor expression occurs within the developing spinal cord. Saturation analysis of ligand binding shows that the affinity of [³H]KA and [³H]AMPA binding is not developmentally regulated. In contrast, the affinity of [³H]glutamate binding to the NMDA receptor in the fetal ventral horn is three-fold greater than in the adult ventral horn. Thus, in addition to quantitative changes in glutamate receptor expression, qualitative changes occur in the expression of NMDA receptors during development. The distinct glutamate receptor phenotype of fetal and early postnatal spinal cord cells suggests that alterations in the excitable properties of these cells plays an important role in activity-dependent development and in susceptibility to excitotoxic injury. J. Comp. Neurol. 384:200-210, 1997. © 1997 Wiley-Liss, Inc.     

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.     

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).     

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.