Excitatory amino acid receptors in the human cerebral cortex: a quantitative autoradiographic study comparing the distributions of [3H]TCP, [3H]glycine, L-[3H]glutamate, [3H]AMPA and [3H]kainic acid binding sites

The excitatory amino acids are probably the major neurotransmitters in the cerebral cortex, and they act through at least three receptors: the N-methyl-D-aspartate, the quisqualate and the kainic acid receptors. Under the appropriate conditions, [3H]1-(1-(2-thienyl)-cyclohexyl)piperidine [( 3H]TCP), [3H]glycine and L-[3H]glutamate label different sites on the N-methyl-D-aspartate receptor, [3H]-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid [( 3H]AMPA) labels the quisqualate receptor and [3H]kainic acid the kainic acid receptor. The anatomical localizations of these binding sites were studied in sections of blocks removed from the cerebral cortices of eight post-mortem human brains. The results showed that, in the human cerebral cortex, [3H]TCP, [3H]glycine and L-[3H]glutamate binding sites had congruent distributions, with [3H]AMPA binding sites showing a similar distribution. In the hippocampus, these four ligands had high binding site densities in the CA1 region and the dentate gyrus molecular layer. With the exception of the striate cortex, in the neocortex, a tri-laminar pattern was seen consisting of a high density across laminae I-III, a layer of low density corresponding to the region of lamina IV, and a band of moderate density across laminae V and VI, except for [3H]AMPA where the middle zone of low density was usually wider. [3H]Kainic acid showed a binding pattern which was generally complementary to that of the other four ligands. There were low levels of [3H]kainic acid binding sites in the CA1 region of the hippocampus with higher levels in the CA3 region, the hilus, and the inner third of the dentate gyrus molecular layer. In the neocortex there was a band of high density corresponding to laminae V and VI, with a thin band of moderate binding corresponding to lamina I and the outer region of lamina II. An exception was the motor cortex where the highest level of [3H]kainic acid binding was in laminae I and II. The high degree of congruence between the binding patterns of [3H]TCP, [3H]glycine and L-[3H]glutamate (using conditions appropriate for the N-methyl-D-aspartate receptor) supports data indicating that these ligands bind to different regions of the same receptor complex. The similar distribution of [3H]AMPA binding sites, with the exception of the striate cortex, supports observations made in rodents that N-methyl-D-aspartate receptors and quisqualate receptors have similar distributions and perform different but related functions in excitatory transmission.(ABSTRACT TRUNCATED AT 400 WORDS)     

In vivo binding of [3H]d-N-allylnormetazocine and [3H]haloperidol to sigma receptors in the mouse brain

Specific binding to sigma sites has been demonstrated and characterized in vitro using [3H]d-N-allylnormetazocine ([3H]d-NANM) and [3H]haloperidol ([3H]HAL) as ligands. As an extension of these experiments, we examined the regional in vivo specific binding of [3H]d-NANM and [3H]HAL in the mouse brain. Specific in vivo sigma binding was seen with both ligands; average estimates of specific binding across brain regions were 54 per cent and 56 per cent of total brain radioactivity, using [3H]d-NANM and [3H]HAL, respectively. Both ligands showed high levels of specific binding in the cerebellum, medulla-pons and midbrain, and lowest levels in the hippocampus. Estimated average [3H]d-NANM binding to phencyclidine (PCP) receptors across seven brain regions was only 13 per cent of total brain radioactivity, and showed a more uniform regional distribution than sigma binding. While the distributions of in vivo specific binding of [3H]d-NANM and [3H]HAL to sigma sites were comparable to findings obtained in vitro, the present estimates of in vivo [3H]d-NANM binding to PCP sites did not resemble the distribution of PCP receptors found in vitro. The results suggest that radiolabelled d-NANM and HAL may be useful for imaging sigma binding sites in vivo.     

Cerebellar excitatory amino acid binding sites in normal, granuloprival, and Purkinje cell-deficient mice.

Using quantitative autoradiography, the cellular localization and characterization of cerebellar excitatory amino acid binding sites in normal, Purkinje cell-deficient and granuloprival (granule cell-deficient) mouse cerebella were investigated. In the molecular layer of normal mouse cerebellum, the quisqualate subtype of excitatory amino acid receptor (assayed by [3H](RS)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate, quisqualate-sensitive L-[3H]glutamate, and [3H]6-cyano-7-nitroquinoxaline-2,3-dione binding) predominated. In the granule cell layer of the cerebellum, N-methyl-D-aspartate-sensitive L-[3H]glutamate and [3H]glycine binding sites were predominant. In the molecular layer of Purkinje cell-deficient mutant mice, [3H](RS)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate binding sites and [3H]6-cyano-7-nitro-quinoxaline-2,3-dione binding were reduced to 24% (P less than 0.01) and 36% (P less than 0.001) of control, respectively, while quisqualate-sensitive [3H]glutamate binding sites were reduced to 54% of control (P less than 0.01). N-Methyl-D-aspartate-sensitive [3H]glutamate and [3H]glycine binding were unchanged. In the granule cell layer of these mouse cerebella, there was no change in excitatory amino acid receptor binding. In the molecular layer of granuloprival mouse cerebella, [3H](RS)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate binding was increased to 205% of control (P less than 0.01), [3H]6-cyano-7-nitro-quinoxaline-2,3-dione binding was increased to 136% of control (P less than 0.02), and quisqualate-sensitive [3H]glutamate binding was increased to 152% of control (P less than 0.01). N-Methyl-D-aspartate-sensitive [3H]glutamate and [3H]glycine binding were unchanged. In areas of granule cell depletion N-methyl-D-aspartate-sensitive [3H]glutamate and [3H]glycine binding were reduced to 68% (P less than 0.01) and 59% (P less than 0.01) of control, respectively. In the granule cell layer, binding to quisqualate receptors was not significantly different from binding in controls with any of the ligands tested. These results suggest that three different receptor assays: [3H](RS)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate, quisqualate-sensitive L-[3H]glutamate, and [3H]6-cyano-7-nitro-quinoxaline-2,3-dione binding can be used to demonstrate that quisqualate receptor specific binding sites are located on Purkinje cell dendrites in the molecular layer of cerebellum, and that these binding sites apparently up-regulate in response to granule cell ablation and Purkinje cell deafferentation.     

L-[3H]glutamate labels the metabotropic excitatory amino acid receptor in rodent brain

A quantitative autoradiographic assay for a novel L-[3H]glutamate binding site in rodent brain has been developed. Binding to this site was distinguished by its high affinity for quisqualate (QA), ibotenate, glutamate and trans-1-amino-cyclopentyl-1,3-dicarboxylic acid (trans-ACPD), but low affinity for [RS]-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate and N-methyl-D-aspartate (NMDA). 'AMPA-insensitive, QA-sensitive [3H]glutamate binding' (AiQsGB) had a heterogeneous distribution in rat brain with high levels observed in molecular layer of cerebellum, striatum, and lateral septum. AiQsGB was reduced in molecular layer of cerebellum in mice lacking Purkinje cells. AiQsGB appears to represent binding to the 'metabotropic' neuronal excitatory amino acid receptor linked to phosphoinositide metabolism.     

Loss of hippocampal [3H]TCP binding in Alzheimer's disease

We have previously demonstrated a marked loss in N-methyl-D-aspartate (NMDA) receptors in the hippocampus and cerebral cortex of patients dying with dementia of the Alzheimer type (DAT). In addition, we have found that the dissociative anesthetic N-(1-[2-thienyl]cyclohexyl)3,4-piperidine ([3H]TCP) binds to a site whose regional distribution is highly correlated with that of NMDA receptor sites. We studied the binding of [3H]TCP to sections of hippocampi from 8 controls, 12 patients with DAT and 7 patients with other dementias. [3H]TCP binding was significantly reduced in strata pyramidalia of CA1/CA2, CA3 and subiculum of DAT hippocampal formation compared to that of control. Labelled dissociative anesthetics could potentially be used with positron emission tomography in the diagnosis of DAT.     

The association of [3H]d-aspartate binding and high-affinity glutamate uptake in the human brain

The binding of [³H]d-aspartate ([³H]d-Asp) to human cerebellum homogenate was compared with the uptake of [³H]glutamate ([³H]Glu) by homogenates prepared from rapidly frozen human cerebral cortex. There was a close correlation between the potencies of a range of drugs for inhibiting the binding of [³H]d-Asp and the uptake of [³H]l-Glu. Compounds selective for postsynaptic Glu receptors were inactive. The findings are consistent with the labelling of high-affinity Glu uptake sites by [³H]d-Asp, which may be a valuable ligand for studying excitatory amino acid terminals in human brain.     

3H]TCP binding sites in Alzheimer's disease

Quantitative autoradiography was used to determine the density and distribution of [3H]1-[1-(2-thienyl)-cyclohexyl]piperidine ([3H]TCP) binding sites in human hippocampal tissue sections from control and Alzheimer's disease patients. Some Alzheimer's cases showed no changes in binding site density while other cases showed substantial declines in the CA1 region. [3H]TCP binding in the CA1 region from Alzheimer's patients was reduced an average of 40% while the other hippocampal regions were unaffected. It is proposed that the loss of [3H]TCP sites in the hippocampal CA1 region of certain Alzheimer's cases is associated with the greater cell loss observed in cases of severe Alzheimer's disease.     

Phencyclidine and related drugs bind to the activated N-methyl-D-aspartate receptor-channel complex in rat brain membranes

In functional studies, phenycyclidine (PCP) and similar drugs non-competitively antagonize neuronal responses to the excitatory amino acid, N-methyl-D-aspartate (NMDA). Here we show that, in crude postsynaptic densities from rat brain, the binding of [3H]TCP (a PCP analogue) was enhanced almost 4-fold by L-glutamate and NMDA, but not by quisqualate, kainate or gamma-aminobutyric acid. The potencies of excitatory amino acid agonists and antagonists in the [3H]TCP binding assay closely paralleled their affinities for NMDA-sensitive L-[3H]glutamate binding sites. In contrast, dissociative anaesthetics and sigma-opiates inhibited [3H]TCP binding (with a profile characteristic of PCP binding sites), but had no effect on L-[3H]glutamate binding. These data indicate that PCP binding sites are linked to NMDA receptors, and that PCP and related drugs bind preferentially to the activated configuration of the NMDA receptor channel complex.     

Autoradiographic distribution of phencyclidine receptors in the rat brain using [3H]1-(1-(2-thienyl)cyclohexyl)piperidine ([3H]TCP)

The distribution of phencyclidine (PCP) receptors in the rat brain was determined by autoradiography using 1-(1-(2-thienyl)cyclohexyl)piperidine ([3H]TCP). [3H]TCP appeared to bind to PCP receptors as only PCP-like drugs and sigma-opioids inhibited the binding of [3H]TCP. The areas of the rat brain with the highest density of radiolabeled binding sites were the superficial layers of cerebral cortex, hippocampus and dentate gyrus. Moderate densities of binding sites were found in the medial geniculate nuclei, caudate nucleus, nucleus accumbens, interpeduncular nucleus, superior colliculus, periaqueductal gray and cerebellum. Low densities of binding sites were observed in spinal cord, most of the brainstem, the substantia nigra and most of the hypothalamus.     

Characterization of binding sites for spider toxin, [3H]NSTX-3, in the rat brain

A group of spider toxins (JSTX, NSTX, argiopin, argiotoxin etc.) share a basic common structure and have been reported to block strongly quisqualate- and kainate-sensitive glutamate responses in vertebrate and invertebrate nervous systems. They are presumed to be potent antagonists of both quisqualate and kainate receptors and may serve as useful tools for characterizing these receptors. We report here the synthesis of tritium-labeled NSTX-3 and the characterization of its binding sites in the rat brain. We found that high- and low-affinity binding sites exist in the cerebellum (Kd = 7.75 and 202 nM, Bmax = 0.37 and 5.54 pmol/mg protein, respectively). Synthetic NSTX analogs strongly inhibited [3H]NSTX-3 binding in the cerebellum (IC50 = 10(-7)-10(-6) M), whereas competitive agonists of glutamate receptors (AMPA, quisqualate, NMDA, kainate, glutamate and aspartate) exhibited weak or no inhibitory effects.     

Autoradiographic characterization of [3H]L-glutamate binding sites in developing mouse cerebellar cortex.

Postnatal changes of [3H]L-glutamate binding sites in mouse cerebellum were studied by in vitro autoradiography. These sites were already present at birth, their density globally increased until postnatal day 25, and at all ages it was higher when Cl- and Ca2+ were present in the incubation buffer. At birth, these binding sites were diffused through the whole cerebellar mass, but became distinctly concentrated in the molecular and the internal granular layers by postnatal day 10. From this age on, binding site sensitivity to ions and glutamate analogues takes a different course in each layer. The external granular layer and the white matter never displayed significant amounts of binding. In the molecular layer the Cl-/Ca2+ effect increased during ontogeny until, in adults, the ion-dependent binding was threefold higher than the ion-independent binding. Quisqualate-sensitive sites accounted for 80% of the total binding sites already at postnatal day 15, while displacement by alpha-amino-3-hydroxy-methyl-4-isoxazolepropionic and ibotenic acids attained the maximum (68%) at postnatal day 60. N-Methyl-D-aspartate displaced glutamate binding (50%) only in the presence of Cl- and Ca2+. Starting from postnatal day 15, binding site density in the molecular layer of lobules VIb and VII of the vermis was lower than in other lobules. In the internal granular layer, the Cl-/Ca2+ effect observed in young animals decreased during development. These transient binding sites were sensitive to quisqualic and ibotenic acid. In adults, the majority of glutamate binding sites were ion-independent and mainly sensitive to D,L-amino-5-phospho-valeric acid and N-methyl-D-aspartate. Throughout development and in both layers, sites displaced by kainate were present at low density and sites displaced by D,L-2-amino-4-phosphonobutyric acid were not detected. The localized postnatal changes of the [3H]L-glutamate binding sites were correlated with the events occurring during growth and maturation of cerebellar structures. The increase of the Cl-/Ca(2+)-dependent binding in the molecular layer is simultaneous with the growth of Purkinje cell dendrites and of parallel fibres and with the formation of the synapses between them. This suggests that these binding sites are localized in these synapses. The changing pattern of sensitivity to different agonists during development might correspond to the maturation of these synapses. The low density of [3H]L-glutamate binding in the molecular layer of lobules VIb and VII probably indicates the presence of specific nerve projections to these areas.(ABSTRACT TRUNCATED AT 400 WORDS)     

Autoradiographic distribution of binding sites for the non-NMDA receptor antagonist CNQX in chick brain

The anatomical distribution of binding sites for the non-N-methyl-D-aspartate (non-NMDA) glutamate receptor antagonist [3H]6-cyano-7-nitroquinoxaline-2,3-dione ([3H]CNQX) in 1-day-old chick brain was investigated. Specific [3H]CNQX binding sites were widely distributed but were particularly densely localised in the molecular layer of the cerebellum. In the midbrain, the binding was comparatively low, except for relatively high levels in the nucleus isthmi of the optic lobe. The distribution of [3H]CNQX binding was markedly different to that of the NMDA receptor ligand [3H]MK-801. Overall, the localisation of [3H]CNQX binding was similar to the combined distributions of [3H]AMPA and [3H]kainate binding sites. Kainate inhibited [3H]CNQX binding throughout the brain. AMPA also inhibited [3H]CNQX binding in the fore- and midbrain, but the dense binding in the molecular layer of the cerebellum was notable in being AMPA-insensitive.     

Autoradiographic localization of dopamine D2 receptors in the rat brain using the new agonist [3H]N-0437

The selective dopamine D2 agonist [3H]N-0437 was used to label dopamine receptors in vitro in slide-mounted rat brain microtome sections. The characteristics of the binding of [3H]N-0437 to tissue section were similar to those observed previously in membrane preparations and indicated that this ligand labels sites with the properties of a dopamine D2 receptor. The regional distribution of these receptors was examined by autoradiography and quantified by computer-assisted microdensitometry. The highest densities of [3H]N-0437 sites were observed in the nucleus caudate-putamen, accumbens, olfactory tubercle, island of Calleja and the glomerular layer of the olfactory bulb. Lower densities of binding sites were seen in stratum griseum superficialis of the superior colliculus, substantia nigra pars compacta and area ventral tegmental, entorhinal cortex and in the molecular layer of the 9th and 10th lobules of the cerebellum. Very low densities were seen in the neocortex and hippocampal formation. The density of [3H]N-0437 binding sites in the rat striatum are higher than those observed with other dopamine D2 [3H]agonists and comparable to those seen with [3H]antagonists. [3H]N-0437 is a new useful tool for the anatomical localization of dopamine D2 receptors in brain.     

Septotemporal distribution of [3H]MK-801, [3H]AMPA and [3H]Kainate binding sites in the rat hippocampus

The distribution of glutamate receptors in transverse hippocampal sections has been well investigated. However, in spite of the known septotemporal gradients of hippocampal connectivity no systematic studies exist about the distribution of glutamate receptors along the septotemporal (longitudinal) hippocampal axis. Therefore, in the present study this issue was investigated using receptor autoradiography for the [³H]MK-801, [³H]AMPA and [³H]Kainate binding sites. Hippocampi from 30-day-old rats were sectioned perpendicularly to their longitudinal axis, yielding a total of 25–30 equidistantly spaced autoradiographs for each hippocampus. For each section layer-specific concentrations of binding sites were calculated by the aid of a computerized image analysing system. The dependency of concentrations of binding sites on the septotemporal position was evaluated by regression analysis. Gradients of binding were confined to distinct hippocampal layers. Significant septotemporal gradients of [³H]MK-801 binding were observed in selected layers of CA1 and the dentate gyrus, a septal to temporal decrease of binding in the oriens and radiatum layers of CA1 being most prominent. For [³H]AMPA, significant septotemporal gradients of binding were restricted to layers of CA3, CA4 and the dentate gyrus, with values generally increasing from septal to temporal levels. The observed septotemporal gradients possibly reflect functional segregations along the longitudinal hippocampal axis and could be important for the comparability of ligand binding studies using transverse hippocampal sections or hippocampal slice cultures.     

Evidence for heterogenous glycine domains but conserved multiple states of the excitatory amino acid recognition site of the NMDA receptor: regional binding studies with [3H]glycine and [3H]L-glutamate

Summary The possible heterogeneity of the agonist and glycine sites of the N-methyl-D-aspartate (NMDA) receptor-complex was examined using receptor binding techniques. Binding of [³H]L-glutamate ([³H]GLU) and [³H]glycine to synaptic membranes of cerebral and cerebellar cortices, and membranes of a granule cell preparation of rat cerebellum, was characterized. [³H]Glycine always labelled a single population of sites; densities of binding sites (B_max) in cortical, cerebellar and granule membranes were 3.1, 0.87 and 3.6 pmol/mg protein, respectively. Dissociation constants (K_d) in the same three preparations were 0.13, 0.31 and 1.9 M, respectively. In competition studies, D-cycloserine, but not D-serine and 7-chlorokynurenate, showed varying potency between the membrane preparations, and analysis of variance (ANOVA) revealed a significant interaction between ligands and membrane fractions. Binding of [³H]GLU was saturable and to a single population of sites: K_d 0.5–0.9 M and B_max 3.2–3.6 pmol/mg protein. In all three membrane preparations the rank order of potency of NMDA agonists as inhibitors of the binding of [³H]GLU was always L-aspartate>L-cysteate>L-cysteinesulphinate>L-serine-O-sulphate>ibotenate>L-homocysteate. NMDA, quinolinate and competitive NMDA antagonists were only weak inhibitors of the binding of [³H]GLU and never fully inhibited specific binding. Other subtype-selective excitatory amino acids were very weak or ineffective inhibitors of binding. Binding of NMDA agonists was better described by a two site model whereby the proportion of high affinity sites did not vary significantly across the three membrane preparations. Although the binding of [³H]GLU was relatively insensitive to NMDA itself and competitive NMDA antagonists, binding may be to a recognition site for NMDA-like agonists, since they fully inhibited specific binding. This excitatory amino acid recognition for NMDA agonists was conserved in the three membrane preparations. In cortical and granule membranes the B_max values for the binding of [³H]GLU and [³H]glycine had a stoichiometry of 1:1, whilst in cerebellar synaptic membranes this ratio was 4:1. Receptor autoradiography of NMDA-related [³H]GLU and [³H]glycine binding in tissue sections failed to reveal any differential labelling patterns in cerebral cortex and cerebellum. In the cerebellum, densities of silver grains found with both [³H]ligands were concentrated in the granule cell layer relative to the molecular layer, but the differences detected in membrane binding studies were not observed in cerebellum. Our findings suggest the existence of three types of heterogeneity for the glycine domain of the NMDA receptor: (1) differing affinities for glycine, (2) differing pharmacological profiles, and (3) differing stoichiometry in relation to the putative NMDA-like agonist site. Our evidence supports an hypothesis for the existence of multiple glycine domains which might differentially modulate NMDA-mediated neurotransmission.     

Profiles of [3H]N-[1-(2-thienyl)cyclohexyl]piperidine binding in brain synaptic membranes treated with Triton X-100

Binding of [3H]N-[1-(2-thienyl)cyclohexyl]piperidine (TCP) was examined using rat brain synaptic membranes treated with a low concentration of Triton X-100. This compound is assumed to be a non-competitive antagonist for the N-methyl-D-aspartate(NMDA)-sensitive subclass of central excitatory amino acid receptors. Binding was quite low but detectable in Triton-treated membranes irrespective of the incubation temperature, and the temperature-dependent portion of the binding was greatly reduced in these Triton-treated membranes. However, binding was drastically potentiated by the inclusion of L-glutamate and its analogous amino acids in a concentration-dependent manner at a concentration range of 10 nM to 0.1 mM. Agonists for the NMDA-sensitive subclass also potentiated binding, with agonists for the other subclasses being ineffective. Glycine at a concentration above 10 nM was not only effective as a stimulant of potentiated binding by glutamate, but was also active in enhancing binding in the absence of added glutamate. Glycine increased both the association and dissociation rates without significantly affecting the dissociation constant. Pharmacological profiles of binding in Triton-treated membranes were not significantly different from those in untreated membranes, except for that of haloperidol. Haloperidol is proposed to be highly selective for brain sigma-receptors on the basis of a potent inhibition of sigma-receptor binding. The inhibitory potency of this sigma-ligand was markedly attenuated in the presence of both glutamate and glycine in Triton-treated membranes, as compared with that in untreated membranes. These results suggest that [3H]TCP binding in Triton-treated membranes is a useful biochemical tool to evaluate predominantly the activated state of ion channels associated with the NMDA-sensitive receptors in terms of freedom from the confounding effects of endogenous amino acids.     

Distribution of [3H]MK-801, [3H]AMPA and [3H]Kainate binding sites in rat hippocampal long-term slice cultures isolated from external afferents

The distribution of ionotropic glutamate receptors in transverse hippocampal sections and along the septotemporal hippocampal axis can be correlated to hippocampal connectivity, in particular to area- and layer-specific termination zones of afferents. However, in isolated organotypic hippocampal slice cultures developing without extrinsic afferent input no systematic studies exist about the distribution of glutamate receptors. In the present study we used receptor autoradiography to examine [³H]MK-801, [³H]AMPA and [³H]Kainate binding sites in hippocampal slice cultures prepared from 6-day-old rats. After 24 days in vitro layer-specific concentrations of [³H]MK-801, [³H]AMPA and [³H]Kainate binding sites were compared to age-matched hippocampi in situ (P30 rats). An obvious difference between hippocampal slice cultures and hippocampi in situ was a changed distribution of binding sites among the hippocampal areas showing a relative increase of [³H]MK-801 and [³H]AMPA binding sites in CA3 as compared to CA1 and to the dentate gyrus in the cultures. In CA1, however, the relative layer-specific distributions of [³H]MK-801 and [³H]AMPA binding sites were identical in hippocampal slice cultures and in hippocampi in situ. Interestingly, layer-specific binding of [³H]Kainate in the cultures exceeded that in the hippocampi in situ 3–5 times. Moreover, in the cultures the binding of the three ligands varied systematically showing gradients along the ”superficiomembranal’’ axis. Cultures taken from different positions along the hippocampal axis differed with respect to concentrations of [³H]MK-801 and [³H]Kainate binding sites, but not of [³H]AMPA binding sites. The results suggest a massive sprouting and reorganisation of intrinsic projections in long-term hippocampal slice cultures.     

On the location of gamma-aminobutyrate and benzodiazepine receptors in the cerebellum of the normal C3H and Lurcher mutant mouse

Binding of gamma-aminobutyrate and benzodiazepine receptor ligands has been studied in the cerebellum of adult normal (C3H) and Lurcher mutant mice. The adult mutant has lost all Purkinje cells and more than 90% of the granule cells in the cerebellar cortex. When compared with their normal littermates Lurcher mice displayed large decreases in the number of high-affinity binding sites for [3H]muscimol, a synaptic gamma-aminobutyrate receptor ligand, in washed cerebellar homogenates. This observation was consistent with the extensive loss of gamma-aminobutyrate receptive Purkinje and granule cells from the Lurcher cerebellum. However, specific binding of the benzodiazepine-receptor ligand [3H]flunitrazepam to Lurcher cerebellum remained unchanged. Indeed quantitative autoradiography, employing [3H]flunitrazepam as a photoaffinity label, showed no significant differences in the density of labelling between Lurcher and normal littermate mice in any region of the cerebellum. These benzodiazepine binding sites in washed homogenates or tissue sections displayed a gamma-aminobutyrate-induced enhancement of [3H]flunitrazepam binding which occurred to the same extent in both Lurcher and normal cerebellum, a facilitatory effect which could be blocked by the addition of bicuculline methobromide. Our results suggest that a large proportion of the high-affinity, specific benzodiazepine binding sites in mouse cerebellum are not coupled to the synaptic gamma-aminobutyrate receptors thought to be labelled by high affinity [3H]muscimol binding. Further, that benzodiazepine binding sites do not appear to be enriched on either the soma or dendrites of Purkinje cells, as has been suggested from previous studies. Investigations at the electron microscope level are now required to elucidate the cellular location of benzodiazepine binding sites in the cerebellar cortex and to examine whether or not they are likely to be exposed to gamma-aminobutyrate in vivo.     

Distribution of nicotinic cholinergic receptors in the rat tel- and diencephalon: a quantitative receptor autoradiographical study using [3H]-acetylcholine, [alpha-125I]bungarotoxin and [3H]nicotine

The topographical distribution of [alpha-125I]bungarotoxin [125I]BTX, [3H]nicotine ([3H]Nic), [3H]acetylcholine ([3H]ACh) (in the presence of atropine) binding in rat tel- and diencephalon was investigated using a quantitative receptor autoradiographical technique. With the [3H]ACh and [3H]Nic radioligands, a strong labelling was observed in various thalamic nuclei, including the medial habenula, a moderate labelling in different areas of the cortex cerebri, the nucleus caudatus putamen, the nucleus accumbens and tuberculum olfactorium and a uniform weak labelling in the hypothalamus. When the binding data for [3H]Nic were plotted against binding data for [3H]ACh in various brain nuclei, a significant correlation was obtained. Considering [125I]BTX, the strongest labelling was observed in the lateral mammillary nucleus and the hilus gyrus dentatus of the hippocampal formation. A weak labelling occurred in areas such as the nucleus causatus putamen, the thalamus and the cerebral cortex. No significant correlation was therefore obtained between the degree of [125I]BTX binding in various brain nuclei and the degree of binding observed with [3H]Nic or [3H]ACh. The present results underline the view that the high-affinity [3H]Nic and [3H]ACh binding sites label the same cholinergic nicotinic receptor binding site, while [125I]BTX labels another subpopulation of nicotinic cholinergic receptors, predominantly found in discrete areas of the hypothalamus and the limbic cortex.     

Photoaffinity labeling and binding studies reveal the existence of two types of phencyclidine receptors in the NCB-20 cell line

The mouse neuroblastoma-Chinese hamster brain hybrid cell line NCB-20 is the only clonal cell line in which binding studies indicate the presence of phencyclidine (PCP) receptor-like sites. We report here that polypeptide components of NCB-20 cell PCP sites were identified with the photolabile PCP derivative [3H]N-[1-(3-azidophenyl)cyclohexyl]piperidine ([3H]AZ-PCP). The pharmacological selectivity of [3H]AZ-PCP binding (under reversible conditions) was similar to that observed for [3H]N-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP) binding to NCB-20 cell membranes. Inhibition of [3H]TCP binding by AZ-PCP, dexoxadrol or MK-801 was biphasic, suggesting the presence of two types of PCP sites on NCB-20 cells. Photolysis of NCB-20 cell membranes pre-equilibrated with [3H]AZ-PCP, followed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), revealed the presence of 5 major labeled bands (Mr 90,000, 68,000, 49,000, 40,000 and 33,000), a pattern similar to that observed for rat brain membranes. MK-801 and D-2-amino-5-phosphonovaleric acid (D-(-)-AP5) selectively inhibited the labeling of Mr 68,000 and 90,000 polypeptides. These results indicate that the labeled bands represent constituents of at least two different PCP binding proteins. The Mr 68,000 and 90,000 components appear to correspond to a high-affinity site, which comprises approximately 20% of total [3H]TCP sites in these cells, and exhibits the pharmacology expected for the PCP receptor of the N-methyl-D-aspartate (NMDA)-gated channel.