Characterization of neuronal migration disorders in neocortical structures: quantitative receptor autoradiography of ionotropic glutamate, GABAA and GABAB receptors

Epileptiform activity was previously described [ Luhmann et al. (1998 ) Eur.J. Neurosci., 10, 3085–3094] in the neocortex of the adult rat following freeze lesioning of the newborn neocortex. After a survival time of 3 months, a small area of dysplastic cortex surrounded by histologically normal (exofocal) neocortex was observed. The dysplastic cortex is characterized by the formation of a small sulcus and a three- to four-layered architecture. Two questions are addressed here: (i) is the hyperexcitability associated with changes in binding to major excitatory and inhibitory transmitter receptors in the dysplastic cortex?; and (ii) do such changes also occur in the exofocal cortex? Alterations in binding to glutamatergic N-methyl-d -aspartate (NMDA), (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), kainate and GABA_A and GABA_B (γ-aminobutyric acid) receptors are demonstrated with quantitative in vitro receptor autoradiography by using the ligands [³H]MK-801, [³H]AMPA, [³H]kainate, [³H]muscimol and [³H]baclofen, respectively. In the dysplastic cortex, the binding to NMDA, AMPA and kainate receptors is significantly increased, whereas the binding to GABA_A and GABA_B receptors is reduced. Exofocal areas of the lesioned hemisphere show an imbalance between excitatory and inhibitory receptor binding with an up-regulation of the binding to AMPA and kainate, and a down-regulation to GABA_A receptors. The binding to GABA_B and NMDA receptors is not significantly changed in the exofocal areas. The imbalance between excitatory and inhibitory receptors may cause the hyperexcitability, as previously found in the identical experimental model, and may also induce epileptiform activity in the human cortex with migration disorders.     

Involvement of Metabotropic and lonotropic Glutamate Receptors in Inositol Polyphosphate Formation in Carp Retinal Slices

The contribution of ionotropic and metabotropic glutamate receptors to inositol polyphosphate accumulation in carp retinal slices was investigated using myo-[2-³H]inositol prelabelling. In the presence of the glutamate agonists quisqualate, (RS)-α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and trans-(±)-1-amino-1, 3-cyclopentane-dicarboxylic acid (t-ACPD), formation of [³H]inositol phosphate was significantly increased in a dose-dependent manner, with EC₅₀ values of 350 nM, 1.5 μM and 10 μM respectively. The complete AMPA-induced response and a large component of the quisqualate-induced response were inhibited in a competitive manner when the ionotropic antagonist 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX) was present. Furthermore, the remaining level of quisqualate-induced [³H]inositol phosphate formation closely matched that produced by ACPD alone, and coincubation of AMPA and ACPD showed additive effects, suggesting that the quisqualate-induced response resulted from coactivation of metabotropic and ionotropic glutamate receptors. The ionotropic component was partially reduced in the presence of cobalt, suggesting indirect effects resulting from synaptic interactions. We could exclude indirect effects through depolarization-induced release of other neurotransmitters. Only serotonin (EC₅₀ 1 μM) and carbachol (at a concentration of 1 mM) stimulated [³H]inositol phosphate formation, but their antagonists did not affect the quisqualate response and coactivation with quisqualate and serotonin or carbachol resulted in additive effects. The ionotropic component was completely suppressed when Ca²⁺ was omitted from the medium and cobalt was present. This makes it likely that the ionotropic component resulted from Ca²⁺ entry through AMPA-gated channels and subsequent Ca²⁺-dependent activation of phospholipase C.     

Regional differences in the enhancement by GABA of [H]zolpidem binding to ω1 sites in rat brain membranes and sections

The potential heterogeneity in the allosteric coupling between GABA and ω₁ binding sites within the native GABA_A receptor complex has been evaluated in the rat by measuring the enhancement by GABA of [³H]zolpidem binding to ω₁ site in membranes from three rat brain structures (neocortex, cerebellum and hippocampus) and brain sections. The maximal stimulatory effect of GABA was significantly higher (265 ± 47%) in cortical membranes than in cerebellar (165 ± 48%) or in hippocampal (118 ± 17%) membranes. These differences are not due either to the presence of different amounts of residual GABA in the membrane preparations or to the labeling, in presence of GABA, of binding sites other than ω₁ since: (1) the pharmacological properties of the [³H]zolpidem binding sites were similar in the three regions; (2) the degree of allosteric enhancement was unrelated to the relative proportion of ω₁ sites in each structure; and (3) GABA did not increase the B_max for [³H]zolpidem. Regional differences in the effect of 100 μM GABA on [³H]zolpidem binding were also observed by quantitative autoradiography. Regions where the strongest (3–4-fold) effects of GABA in [³H]zolpidem binding were observed included the substantia nigra, lateral geniculate body, olfactory tubercule and red nucleus. A large increase in [³H]zolpidem binding was also demonstrated in the cingulate and frontoparietal cortices with higher effects in deep (4.2-fold) rather than in superficial layers (3.3-fold). Heterogeneous subregional increases in [³H]zolpidem binding in the presence of GABA were quantified within the cerebellum, hippocampus and superior colliculus. In the cerebellum the effect of this neurotransmitter was larger in the molecular (3.8-fold) than in the granular (2.2-fold) layer. In the hippocampus the effect of GABA was also heterogeneous with larger increases in CA1 and CA2 fields (3.5-fold) than in CA3 field (2.2-fold) and dentate gyrus (2.5-fold). Finally in the deep layers of the superior colliculus GABA stimulation of [³H]zolpidem binding was greater than the superficial layer. In the other structures examined the GABA-induced increase in [³H]zolpidem binding was less than 3-fold. The smallest stimulations were quantified in the entorhinal cortex (2.1-fold), amygdala (2.4-fold) and nucleus accumbens (1.7-fold). These results suggest that [³H]zolpidem sites are associated to, at least, two GABA_A receptor subtypes that can be differentiated by their allosteric interaction between GABA and [³H]zolpidem sites.     

In ovo chronic neurosteroid treatment affects the function and allosteric interactions of GABAA receptor modulatory sites

We investigated the effects of in ovo chronic administration of the endogenous neurosteroid epipregnanolone (5β-pregnan-3β-ol-20-one) on the GABA_A receptor complex present in chick optic lobe synaptic membranes. Chronic epipregnanolone treatment failed to exert any effect on the chick optic lobe total protein content and wet weight at the different doses tested. [³H]Flunitrazepam control binding remained unaltered after neurosteroid exposure, however, the positive allosteric modulation of this ligand by 4 μM allopregnanolone was reduced in a dose-dependent manner by neurosteroid treatment. Embryo exposure to 30 μM epipregnanolone decreased allopregnanolone EC₅₀ and E_max values. Analyses of saturation binding isotherms disclosed that such administration had no effect on K_d and B_max values for [³H]flunitrazepam and [³H]GABA binding. [³H]GABA binding modulation disclosed an increase in allopregnanolone EC₅₀ value with a decrease in its E_max value. Diazepam EC₅₀ and E_max values were enhanced, while low affinity sodium pentobarbital EC₅₀ value was reduced by epipregnanolone treatment. The investigation of the GABA_A receptor function revealed that administration of this neurosteroid reduces the efficacy of GABA to induce ³⁶Cl⁻ influx into microsacs prepared from chick optic lobe. These results indicate that endogenous neurosteroid epipregnanolone chronically administered in ovo produces homologous uncoupling between steroid modulatory sites, and those corresponding to benzodiazepine and GABA receptors. Thus epipregnanolone is able to induce heterologous changes in the allosteric linkage between benzodiazepine and barbiturate modulatory sites, and the GABA receptor site. Taken jointly with results on epipregnanolone enhancing effects on [³H]flunitrazepam and [³H]GABA binding, in the context of its endogenous synthesis, our present findings support this neurosteroid as the endogenous modulator of GABA_A receptor sites and function during chick optic lobe development.     

Polyamine effects uponN-methyl-D-aspartate receptor functioning: differential alteration by glutamate and glycine site antagonists

Polyamines such as spermidine potentiate activation of theN-methyl-D-aspartate (NMDA)-type excitatory amino acid receptor. The goal of the present study was to investigate interactions between the putative polyamine binding site and previously described sites for glutamate and glycine. Binding of the high-potency PCP receptor ligand [³H]MK-801 to well-washed rat brain membranes was used as an in vitro probe of NMDA receptor activation. Spermidine concentration-response studies were performed in the absence and presence of both glutamate and glycine, with and withoutD-(−)-2-amino-5-phosphonovaleric acid (D(−)AP-5) or 7-chlorokynurenic acid (7Cl-KYN). Incubation in the presence of spermidine alone induced a 20.4-fold increase in [³H]MK-801 binding with an EC₅₀ value of 13.3 μM. The mean concentration of spermidine which induced maximal stimulation of binding was 130 μM (n = 10,S.E.M.= 24.66,range= 25–250 μM). Glutamate (10 μM) decreased the EC₅₀ value for spermidine-induced stimulation of [³H]MK-801 binding to 3.4 μM. Glycine (10 μM) did not significantly alter either maximum spermidine-induced [³H]MK-801 binding or the EC₅₀ value for spermidine-induced stimulation of [³H]MK-801 binding. Incubation in the presence of the specific glutamate antagonistD(−)AP-5 attenuated [³H]MK-801 binding in a glutamate-reversible fashion. The competitive glycine antagonist 7Cl-KYN decreased maximum spermidine-induced [³H]MK-801 binding in a glycine-reversible fashion. In addition, 7Cl-KYN increased the EC₅₀ value for spermidine-induced stimulation of [³H]MK-801 binding whileD(−)AP-5 was without effect. These findings suggest that glutamate and glycine regulate the polyamine binding site differentially. PCP-like agents induce a psychotomimetic state closely resembling schizophrenia by inhibiting NMDA receptor-mediated neurotransmission. The ability of polyamines to modulate NMDA receptor functioning suggests a potential site for pharmacological intervention.     

Species differences in the distribution of central 5-HT1 binding sites: a comparative autoradiographic study between rat and guinea pig

In this study, we compared the localization of central 5-HT₁ binding sites of rat and guinea pig. The 5-HT_1B sites were absent in the guinea pig brain. Good correlations were found between species in the regional distribution of 5-HT₁ sites labelled with [³H]5-HT(r = 0.73), 5-HT_1A sites labelled with [³H]8-OH-DPAT (r = 0.87), and 5-HT_1B versus 5-HT_1D sites labelled with [³H]5-HT in the presence of ipsapirone and DOI (r = 0.76). Despite the overall similarities, species differences were observed in many brain regions. The CA1/CA2 fields of the hippocampus and the dorsal subiculum displayed significantly more 5-HT_1A receptor binding in guinea pig than in rat. Conversely, the 5-HT_1A binding in dorsolateral septum, cingulate cortex and laminae IV-V of the neocortex, was more pronounced in rat. Areas almost exclusively containing 5-HT_1B or 5-HT_1D sites, such as the ventral pallidum, globus pallidus and substantia nigra, expressed markedly more [³H]5-HT binding in rat as compared to guinea pig, while the opposite occurred in claustrum, dorsal endopiriform nucleus, lateral geniculate nucleus, and superficial grey layer of the superior colliculus. The implications of the species differences are illustrated by the binding of [³H]eltoprazine. The distribution of [³H]eltoprazine binding sites showed a good correlation with that of the 5-HT_1B sites in rat (r = 0.89), and with that of the 5-HT_1A sites in guinea pig (r = 0.97). The data give rise to the possibility that differences in the presence and distribution of 5-HT₁ receptor sites are related to species differences in behavioral, neurochemical and physiological responses to drugs with 5-HT₁ receptor affinity.     

An autoradiographic study of [H]AMPA receptor binding and in situ hybridization of AMPA sensitive glutamate receptor A (GluR-A) subunits following morphine withdrawal in the rat brain

Chronic treatment with opioids is well known to result in the development of physical dependence. More recently, glutamatergic mechanisms have been implicated in expression of the withdrawal syndrome from opioids. To better examine glutamatergic involvement, an autoradiographic study of [³H]AMPA receptor binding and an assessment of in situ hybridization of AMPA sensitive glutamate receptor A (GluR-A) subunits in the rat brain were each performed 7 h after withdrawal from morphine infusion. Animals were rendered dependent by intracerebroventricular (i.c.v.) infusion of morphine (26 nmol/μl/h) via osmotic minipumps for 3 days. Brain sections of 14-μm thickness were incubated with 15 nM [³H]AMPA for quantitation of binding to the AMPA receptor. The probe for in situ hybridization was labeled at its 3′ end using terminal deoxynucleotidyl transferase and [³⁵S]dATP. The highest degree of [³H]AMPA binding was shown in the hippocampus. The extent of [³H]AMPA binding was increased significantly in the cortex areas (18–21%), caudate-putamen (20%), and hippocampus (7–9%) of rats following withdrawal from morphine. The highest levels of mRNA for GluR-A, flop and flip subunits, were found in the dentate gyrus and in the CA3 region of the hippocampus, respectively. The levels of mRNA for the flop form of GluR-A were decreased in the CA3 of hippocampus (8%) of the rat brain. The levels of mRNA for the flip form of GluR-A were increased in the parietal cortex (7%) and the entorhinal cortex (8%). Increases in the binding of [³H]AMPA to its receptor may play an important role during withdrawal from morphine dependence.     

Autoradiographic localization of 5-HT1A receptors in the post-mortem human brain using [H]WAY-100635 and [C]WAY-100635

The distribution of 5-HT_1A receptors was examined in the post-mortem human brain using whole hemisphere autoradiography and the selective 5-HT_1A receptor antagonist [³H]WAY-100635 ([O-methyl-³H]-N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl)cyclohexanecarboxamide trihydrochloride). The autoradiograms showed very dense binding to hippocampus, raphe nuclei and neocortex. The labeling in neocortex was slightly lower than in the hippocampus and was mainly at superficial layers, although a faintly labeled band could be seen in deeper neocortical layers. Other regions, such as the amygdala, septum and claustrum, showed low densities of [³H]WAY-100635 binding, reflecting low densities of 5-HT_1A receptors. The labeling was very low in basal ganglia, such as nucleus caudatus and putamen, in cerebellum or in structures of the brain stem except in the raphe nuclei. The labeling of human 5-HT_1A receptors with [³H]WAY-100635 was antagonized by the addition of the 5-HT_1A receptor ligands, 5-HT, buspirone, pindolol or 8-OH-DPAT (10 μM), leaving a very low background of non-specific binding. Saturation analysis of semiquantitative data from several human regions indicated that [³H]WAY-100635 has a K_d of approximately 2.5 nM. The selective labeling of 5-HT_1A receptors with [³H]WAY-100635 clearly show that this compound is useful for further studies of the human 5-HT_1A receptor subtype in vitro. [¹¹C]WAY-100635 is used for the characterization of 5-HT_1A receptors with positron emission tomography (PET). WAY-100635 was also radiolabeled with the short-lived positron-emitting radionuclide carbon-11 (t_1/2=20 min) and used for in vitro autoradiography on human whole hemisphere cryosections. [¹¹C]WAY-100635 gave images qualitatively similar to those of [³H]WAY-100635, although with a lower resolution. Thus, the hippocampal formation was densely labeled, with lower density in the neocortex. Buspirone, pindolol or 8-OH-DPAT (10 μM), blocked all binding of [¹¹C]WAY-100635. The in vitro autoradiography of the distribution of 5-HT_1A receptors obtained with radiolabeled WAY-100635 provide detailed qualitative and quantitative information on the distribution of 5-HT_1A-receptors in the human brain. Moreover, the studies give reference information for the interpretation of previous initial results at much lower resolution in humans with PET and [¹¹C]WAY-100635. These data provide a strong basis for expecting [¹¹C]WAY-100635 to behave as a highly selective radioligand in vivo.     

Quantitative autoradiography of binding sites for [H]AMPA, a structural analogue of glutamic acid

Binding sites for the potent glutamate agonist [³H]α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) were localized in rat brain frozen sections by quantitative autoradiography. Highest levels of binding were seen in stratum radiatum and stratum oriens of the CA1 hippocampal subfield and in the dorsal subiculum. Substantially less but still high amounts of [³H]AMPA binding occurred in other hippocampal subfields and in rostral forebrain structures. The heterogeneous nature of [³H]AMPA binding is discussed in relation to [³H]glutamate binding visualized by similar methods. From these data it is suggested that [³H]AMPA may label a particular subclass of the glutamate receptor population which exhibits a high affinity for quisqualic acid.     

In vitro binding of a radio‐labeled positive allosteric modulator for metabotropic glutamate receptor subtype 5

The positive allosteric modulator (PAM) binding site for metabotropic glutamate receptor subtype 5 (mGlu₅) lacks a readily available radio‐labeled tracer fordetailed structure‐activity studies. This communication describes a selective mGlu₅ compound, 7‐methyl‐2‐(4‐(pyridin‐2‐yloxy)benzyl)‐5‐(pyridin‐3‐yl)isoindolin‐1‐one (PBPyl) that binds with high affinity to human mGlu₅ and exhibits functional PAM activity. Analysis of PBPyl by FLIPR revealed an EC₅₀ of 87 nM with an 89% effect in transfected HEK293 cells and an EC₅₀ of 81 nM with a 42% effect in rat primary neurons. PBPyl exhibited 5‐fold higher functional selectivity for mGlu₅ in a full mGlu receptor panel. Unlabeled PBPyl was tested for specific binding using a liquid chromatography mass spectrometry (LC/MS/MS)‐based filtration binding assay and exhibited 40% specific binding in recombinant membranes, a value higher than any candidate compound tested. In competition binding studies with [³H]MPEP, the mGlu₅ receptor negative allosteric modulator (NAM), PBPyl exhibited a k _i value of 34 nM. PBPyl also displaced [³H]ABP688, a mGluR₅ receptor NAM, in tissue sections from mouse and rat brain using autoradiography. Areas of specific binding included the frontal cortex, striatum and nucleus accumbens. PBPyl was radiolabeled to a specific activity of 15 Ci/mmol and tested for specific binding in a filter plate format. In recombinant mGlu_5b membranes, [³H] PBPyl exhibited saturable binding with a K_d value of 18.6 nM. In competition binding experiments, [³H] PBPyl was displaced by high affinity mGlu₅ positive and negative modulators. Further tests showed that PBPyl displays less than optimal characteristics as an in vivo tool, including a high volume of distribution and ClogP, making it more suitable as an in vitro compound. However, as a first report of direct binding of an mGlu₅ receptor PAM, this study offers value toward the development of novel PET imaging agents for this important therapeutic target. Synapse, 2013. © 2012 Wiley Periodicals, Inc.     

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.     

The interaction of [3H]PY 108-068 and of [3H]PN 200-110 with calcium channel binding sites in rat brain

Summary The binding properties of the tritiated calcium channel antagonists PY 108-068 and PN 200-110 were investigated in membrane fractions from rat brainin vitro. Both ligands reversibly interact with one apparent population of stereoselective binding sites which have pharmacological properties described for calcium channel binding sites. In a calcium buffer enhancement of [³H]PY 108-068 binding is observed with an EC₅₀ at pCa 6.28 [³H]PN 200-110 binding is less sensitive to allosteric stimulation by diltiazem and to allosteric inhibition by verapamil and D 600 than [³H]PY 108-068 binding, suggesting that the former ligand may stabilize a high affinity configuration of the binding sites. Afteri.v. administration of [³H]PY 108-068in vivo binding to membranes is observed in brain and heart, which, in contrast to total tissue radioactivity is sensitive to inhibition by unlabelled (+) PN 200-110. These observations suggest that PY 108-068 can interact with its binding sites alsoin vivo. The results ofex vivo binding studies in brain and heart with [³H]PY 108-068 confirm and extend these observations. It could be shown that all investigated 1,4-dihydropyridines (PY 108-068, PN 200-110, nifedipine, Bay K 8644) after i.p. administration can readily enter brain and heart tissue.     

Distribution of [H]AMPA binding sites in rat brain as determined by quantitative autoradiography

Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) is a potent neuroexcitatory compound which acts at the quisqualate class of excitatory amino acid receptors. In this study we describe the pharmacological characteristics and anatomical distribution of [³H]AMPA binding sites in rat brain using quantitative autoradiography. These binding sites exhibit the appropriate pharmacological characteristics and are found in high concentrations in the hippocampus, cerebral cortex (especially layers I–III), induseum griseum, and dorsal lateral septum. Intermediate concentrations are found in the corpus striatum and deeper layers of cerebral cortex. Lower concentrations are found in the diencephalon, midbrain and brainstem. These results demonstrate that [³H]AMPA binding sites are found throughout the CNS and suggest brain regions which may use quisqualate receptors as glutamate neurotransmitter receptors.     

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.     

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.     

Analysis of NMDA Receptors in the Human Spinal Cord

NMDA receptors in postmortem human spinal cord were analyzed using [³H]MK-801 ligand binding and immunoblotting with NMDA receptor subunit-specific antibodies. The averageK_Dfor [³H]MK-801 binding was 1.77 nM with aB_maxof 0.103 pmol/mg. The EC₅₀for stimulation of [³H]MK-801 binding withl-glutamate was 0.34 μM. None of these parameters were affected by postmortem intervals up to 72 h. Immunoblotting of native NMDA receptors showed that NR1, NR2A, NR2C, and NR2D subunits could all be found in the human spinal cord of which NR1 was preferentially located to the dorsal half. Immunoprecipitation of solubilized receptors revealed that NR1, NR2C, and NR2D subunits coprecipitated with the NR2A subunit, indicating that native human spinal cord NMDA receptors are heteroligimeric receptors assembled by at least three different receptor subunits. These results provide a basis for the development of drugs selectively aimed at spinal cord NMDA receptors for the future treatment of spinal cord disorders.     

Potassium-induced long-term potentiation in area CA1 of the hippocampus involves phospholipase activation

Previous studies have shown that potassium-induced long-term potentiation (LTP) of the Schaffer collateral/commissural synapses in area CA1 of the hippocampus shares common properties with tetanus-induced LTP. In the present investigation, we performed electrophysiological and binding experiments on CA1 hippocampal slices to evaluate the location and nature of the changes underlying potassium-induced LTP. Paired-pulse facilitation, which represents an index of transmitter release, was markedly reduced by potassium-induced LTP. In addition, KC1-induced LTP was associated with an increase in ³H-AMPA ([³H]-amino-3-hydroxy-5-methylisoxazole-4-propionate) binding to CA1 synaptic membranes when measured 40 min after high-potassium exposure; however, no changes were detected in binding of an antagonist ([³H]-6-cyano-7-nitroquinoxaline-2,3-dione; ³H-CNQX) to AMPA receptors in slices expressing KC1-induced LTP. Administration of the phospholipase A₂ (PLA₂) inhibitor bromophenacyl bromide (BPB) prior to potassium application prevented LTP formation as well as the changes in paired-pulse facilitation and ³H-AMPA binding that characterized this type of potentiation. Taken together, these data indicate that potassium-in-duced LTP may be related to modifications in both pre-and postsynaptic properties and confirm the hypothesis that PLA₂ activation is an important mechanism in long-term changes of synaptic operation. © 1994 Wiley-Liss, Inc.     

Regulation of nitric oxide synthase activity in cortical slices by excitatory amino acids and calcium

Nitric oxide synthase (NOS) activity was determined in adult rat frontal cortex and hippocampus by measuring the conversion of L-[³H]arginine to L-[³H]citrulline. N-methyl-D-aspartate (NMDA), but not kainate or α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA), stimulated NOS activity. This effect was concentration dependent (EC₅₀ ≈ 30μM) and was inhibited by tetrodotoxin, EGTA, N^ω-nitro-L-arginine (NOARG), Mg²⁺, phencyclidine, and (cis)-4-phosphonomethyl-2-piperidine carboxylate (CGS 19755), but not by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). NOS activity was increased to an even greater extent by the calcium ionophores ionomycin and A23187 and by depolarization with 50 mM K⁺. Interestingly, neither caffeine nor 1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD), drugs that would be expected to increase intracellular Ca²⁺ concentration by release of Ca²⁺ from intracellular ryanodine- and inositol-1,4,5-trisphosphate-sensitive stores, respectively, had any significant effect on NOS activity. It is concluded that NOS can be activated by NMDA binding to a classic NMDA glutamate receptor subtype as well as by depolarization or other agents that increase the influx of extracellular Ca²⁺. The paradoxical lack of effect of caffeine, as well as the inhibitory effect of tetrodotoxin, are discussed. © 1994 Wiley-Liss, Inc.     

Autoradiographic localization of extrastriatal D2-dopamine receptors in the human brain using [125I]epidepride

Epidepride is a benzamide with high affinity for central D₂- and D₃-dopamine receptors. The anatomical distribution of [¹²⁵I]epidepride binding was examined by autoradiography, using postmortem human whole-hemisphere cryosections. The density of [¹²⁵I]epidepride binding sites was high in caudate nucleus and putamen. [¹²⁵I]epidepride also labeled receptors in extrastriatal region such as in the pallidum, some thalamic nuclei, the neocortex, and the substantia nigra. The neocortical binding was heterogeneously distributed. In most cortical regions, binding sites were located in superficial layers (I-II). However, in basal levels of the occipital cortex, [¹²⁵I]epidepride binding was located in a deeper layer, probably corresponding to layer V. Competition studies indicated that most of the [¹²⁵I]epidepride binding represented predominantly D₂-dopamine receptors, in striatal as well as in extrastriatal regions. The presence of extrastriatal D₂-dopamine receptor populations is of particular interest for research on schizophrenia and antipsychotic drug action. © 1996 Wiley-Liss, Inc.     

Interaction of Felbamate with [H]DCKA-Labeled Strychnine-Insensitive Glycine Receptors in Human Postmortem Brain

The dicarbamate felbamate has been shown to be capable of competing for the binding of 5,7-[³H]dichlorokynurenic acid ([³H]DCKA) to strychnine-insensitive glycine receptors in sections of human postmortem brain. The IC₅₀ for this interaction was 305.8 μM and the inhibition was complete at 1 mM. Autoradiographic localization of [³H]DCKA binding revealed many regions of human brain in which strychnine-insensitive glycine receptors are manifest. The specific binding in most of these areas was markedly reduced in the presence of 625 μM felbamate. In many regions, [³H]DCKA binding was reduced to background in the presence of felbamate, but some areas retained binding by as much as 41% (i.e., the CA2 region of the hippocampus). This is in contrast to the binding of [³H]DCKA in the presence of carbamazepine, phenytoin, or valproic acid. The binding of the glycine receptor antagonist was not affected by any of these latter agents to the same degree as felbamate. Strychnine-insensitive glycine receptors represent a site of action of felbamate in the human brain.