Strychnine-insensitive [H] glycine binding to synaptosomal membranes from the chick retina

The pharmacology and kinetics of strychnine-insensitive [[3]H] glycine binding to synaptic membranes from the outer (P₁) and the inner (P₂) plexiform layers of chick retina was studied. Inhibition curves for glycine, d-serine, 1-amincyclopropanecarboxylic acid (ACPC) and strychnine were analyzed by non-linear regression. Hill slopes for glycine and d-serine were not different from unity, whereas those for ACPC were < 1 in both fractions, revealing heterogeneity of binding sites in these membranes. Non-linear regression analysis of time course and saturation experiments strengthen the idea that [[3]H] glycine binds to more than one class of sites, with similar affinities at equilibrium. Antagonists of strychnine-insensitive glycine receptors in the CNS did not inhibit [[3]H] glycine binding to these membranes, which demonstrates that NMDA receptors in the retina have different structural requirements for ligand interaction at these sites. pH affected the specific binding, in agreement with the participation of specific amino acid residues at glycine binding sites on NMDA receptors, and also with functional studies in which the modulation of affinity at this site by protons has been observed. These results support previous studies regarding CPP and MK-801 binding, and provide evidence which indicates that the pharmacophore for glycine and other NMDA-related ligands is distinct for the retina, compared to the CNS, mainly regarding the effects of glycine-site antagonists.     

Taurine binding to membranes from rat brain regions

[³H]-taurine binding to membranes from different regions from rat brain was studied. Binding to membranes from cerebral cortex and its subcellular fractions, hypothalamus, olfactory bulb and cerebellum was measured. Binding to membranes from dorsal root ganglion was also determined. Na⁺-dependent taurine binding was consistently observed in all the membranes except those from dorsal root ganglion. A K_D = 4.06 μM was obtained for binding to membranes from cerebral cortex. Na⁺-dependent taurine binding was displaced by 20 μM strychnine or bicuculline. Na⁺-independent taurine binding with properties corresponding to a postsynaptic interaction could not be detected in any of the regions studied. The possibility of Na⁺-dependent taurine binding, representing binding to uptake sites or to postsynaptic receptors for GABA and glycine, is discussed.     

Strychnine binding associated with synaptic glycine receptors in rat spinal cord membranes: Ionic influences

Ammonium salts of some anions decrease the potency of glycine in inhibiting [³H]strychnine binding associated with synaptic glycine receptors. A correspondence exists between the ability of the ammonium salts of anions to increase the IC₅₀ of glycine in inhibiting the [³H]strychnine binding, their capacity to reduce the [³H] strychnine binding itself, and their capacity to reverse inhibitory postsynaptic potentials. The decrease of [³H]strychnine binding in the presence of chloride is abolished by sodium, while the decrease of the potency of glycine in inhibiting [³H]strychnine is not. Binding of [³H]strychnine is influenced by monovalent cations in a biphasic fashion. Concentrations of Li⁺, K⁺, and Na⁺ up to 150 mM decrease [³H]strychnine binding, while higher concentrations of the cations increase [³H]strychnine binding. Inhibition by glycine of [³H]strychnine binding is enhanced by low concentrations of these cations.     

Strychnine-insensitive [3H] glycine binding to synaptosomal membranes from the chick retina

The pharmacology and kinetics of strychnine-insensitive [[3]H] glycine binding to synaptic membranes from the outer (P₁) and the inner (P₂) plexiform layers of chick retina was studied. Inhibition curves for glycine, d-serine, 1-amincyclopropanecarboxylic acid (ACPC) and strychnine were analyzed by non-linear regression. Hill slopes for glycine and d-serine were not different from unity, whereas those for ACPC were < 1 in both fractions, revealing heterogeneity of binding sites in these membranes. Non-linear regression analysis of time course and saturation experiments strengthen the idea that [[3]H] glycine binds to more than one class of sites, with similar affinities at equilibrium. Antagonists of strychnine-insensitive glycine receptors in the CNS did not inhibit [[3]H] glycine binding to these membranes, which demonstrates that NMDA receptors in the retina have different structural requirements for ligand interaction at these sites. pH affected the specific binding, in agreement with the participation of specific amino acid residues at glycine binding sites on NMDA receptors, and also with functional studies in which the modulation of affinity at this site by protons has been observed. These results support previous studies regarding CPP and MK-801 binding, and provide evidence which indicates that the pharmacophore for glycine and other NMDA-related ligands is distinct for the retina, compared to the CNS, mainly regarding the effects of glycine-site antagonists.     

Reduction of [H]muscimol binding sites in rat dorsal spinal cord after neonatal capsaicin treatment

Two-day-old rats were pretreated with 50 mg/kg of capsaicin. After 3–4 months, specific binding of [³H]muscimol and [³H]strychnine was measured in membrane preparations from dorsal spinal cord. A 20–30% decrease of the number of [³H]muscimol binding sites was observed after capsaicin treatment. In contrast, [³H]strychnine binding was unchanged. The results provide indirect evidence for a presynaptic location of GABA receptors on capsaicin-sensitive primary afferent neurons.     

Glycine high affinity uptake and strychnine binding associated with glycine receptors in the frog central nervous system

Accumulation of [3H]glycine into synaptosomal fractions occurs by high affinity systems in cerebral cortex, optic tectum, brain stem and spinal cord of the frog. Specific [3H]strychnine binding which appears associated with postsynaptic glycine receptors is also demonstrable in these regions. By contrast, only very low levels of strychnine binding and high affinity glycine uptake occur in higher centers of the rat central nervous system. The relative potencies of small neutral amino acids in competing for [3H]strychnine binding are similar in frog brain and spinal cord. No evidence for a high affinity accumulation of [3H]taurine by synaptosomal fractions of frog spinal cord can be demonstrated. These observations favor glycine rather than taurine as an inhibitory transmitter in frog spinal cord. Moreover, these findings suggest that glycine may have a synaptic role in higher brain centers in the frog.     

The binding of [3H]-5-hydroxytryptamine to homogenates of human brain

Summary The binding of [³H]-5-hydroxytryptamine ([³H]-5-HT) to homogenates of human brain has been studied. The specific binding is saturable, with a K_d (frontal cortex) of 12±2nM, and is inhibited by non-radioactive 5-HT (IC₅₀=26 nM) and D-Lysergic acid diethylamide (IC₅₀=20 nM). Specific, but not non-specific binding of [³H]-5-HT was inhibited by incubation of the homogenates at 50 °C. The binding of [³H]-5-HT across the human brain was not uniform, the highest binding being found in the substantia nigra and hippocampus, and the lowest in the thalamus and pons. The K_d of the binding sites towards 5-HT did, however, appear to be similar for the different brain regions.     

Characterization of [3H]nemonapride binding to mouse brain dopamine D2 receptors assessedin vivo andex vivo for metabolic modeling in PET studies

Summary We characterized [³H]nemonapride ([³H]NEM, [³H]YM-09151-2) binding to dopamine D₂ receptors. In mice given [³H]NEM with and without sulpiride, thein vivo specific binding of the [³H]NEM to the D₂ receptors in the striatum was assessed: SB_{in vivo-1}, striatal uptake minus cerebellar uptake; and SB_{in vivo-2}, uptake in the control mice minus uptake in the sulpiride-treated mice. Tissue homogenates were divided into cytosol and the membrane binding fraction (MB). When the MB was incubatedin vitro with sulpiride, the dissociated and nondissociated fractions were defined as theex vivospecific binding (SB_{ex vivo}) and theex vivo nonspecific binding (NB_{ex vovo}), respectively. HPLC revealed that most of the radioactivity in the MB was [³H]NEM, whereas metabolites were found in the cytosol. In the striatum, the SB_{ex vivo} increased with time (50% of the total tissue uptake at 60 min), and was equivalent to the SB_{in vivo-2}. The SB_{in vivo-1} was comparable to the MB. In the cerebral cortex and cerebellum, the SB_{ex vivo} decreased with time and the SB_{ex vivo}/free [³H]NEM ratios were smaller than those in the striatum.     

Regional differences in binding of [H]LSD and [H]L-HT in calf hippocampal slices revealed by radioautography and rapid filtration studies

Previous radioautographic experiments demonstrated that binding sites labeled by [³H]5-HT and [³H]LSD in rat brain were seen in all layers of CA1, CA4 and the dentate gyrus but not in fields CA2 and CA3 of the hippocampus. In an attempt to confirm this observation we performed binding assays on homogenates from selected areas of calf hippocampus since the small size of the rat hippocampus precluded using preparations from this animal for this purpose. Studies on homogenates from calf hippocampal regions, were done after we determined that the binding to slices in vitro was similar in the calf and rat. Binding of both [³H]5-HT and [³H]LSD by homogenates of CA1 and dentate gyrus, but not of CA3, was saturable. These studies show that the qualitative differences in binding site distribution within the calf hippocampus seen by radioautography reflect quantitative differences in the densities of binding sites revealed by the homogenate studies.     

The glycine receptor in the mutant mouse spastic (spa): strychnine binding characteristics and pharmacology

There is a marked deficit in the binding of the glycine receptor antagonist strychnine to the CNS of the mutant mouse spastic. The characteristics and pharmacology of [3H]strychnine binding to washed homogenates of spastic and littermate control spinal cord and brainstem were investigated to determine the nature of this defect. The maximal binding of [3H]strychnine to spastic homogenates is approximately 20% the value obtained from littermate control homogenates; the affinity of [3H]strychnine binding is approximately 25% lower than littermate control values. The pharmacology of [3H]strychnine binding has the same rank order of potency in spastic and littermate control mice; however, there are small differences in the potency of several compounds in spastic vs littermate control animals. These results indicate that the glycine receptor alteration seen in the spastic mutant mouse is primarily due to a decrease in receptor number. They also suggest that the pharmacological characteristics of the glycine receptor in spastic are different from littermate control. The data do not permit a distinction between whether the strychnine binding sites found in spastic represent the same population seen in littermate control animals or are a subpopulation that is spared by the mutation.     

Evidence for loss of brain [H]spiroperidol and [H]ADTN binding sites in rabbit brain with aging

[³H]Spiroperidol and [³H]2-amino-6,7-dihydroxyl-1,2,3,4,-tetrahydronaphthalene hydrochloride (ADTN) binding were measured in various central nervous system regions of 5 month and 5.5 year old rabbits. In striatum, young animals had a 38% higher number of [³H]spiroperidol binding sites and a 140% higher number of [³H]ADTN binding sites than did the older animals. In frontal cortex and anterior limbic cortex there were respectively 42% and 26% more [³H]spiroperidol binding sites in the young animals. There was no change in the binding site number or affinity for [³H]spiroperidol in retina with aging. Pharmacological characterization demonstrated that [³H]spiroperidol binds to a dopamine receptor in striatum and to a serotonin receptor in cortex.     

Age-Related Changes in the N-Methyl- -aspartate Receptor Binding Sites within the Human Basal Ganglia

The present study examined the regional differences in dopamine transporter binding sites and NMDA receptor complex binding based on autoradiographic images obtained in postmortem sections of human normal brain tissues. In middle-aged control tissues, high and comparable levels of [³H]CFT binding were observed in the caudate nucleus, putamen, and accumbens nucleus without significant alteration along the rostrocaudal axis and ventral and dorsal parts of these nuclei. In aging normal brain tissues, dopamine binding sites for [³H]CFT were significantly reduced in the caudate nucleus, putamen, and accumbens nucleus. -[³H]Glutamate, [³H]MK-801, and [³H]glycine binding to the NMDA receptor complex was lower in aging brain tissues than in middle-aged controls. Significant correlation did occur between age and [³H]CFT binding and between age and -[³H]glutamate, [³H]MK-801, and [³H]glycine binding sites. These results demonstrate that the basal ganglia have age-associated reductions in dopamine transporter uptake and NMDA receptors. These data support hypoactive activity of the NMDA receptor complex system with advancing age. The dopamine transporter uptake and NMDA receptors appear to be vulnerable to the aging process in the basal ganglia.     

Quantitative autoradiography of [H]ouabain binding sites in rat brain

In vitro quantitative autoradiography was used to localize in rat brain binding sites for [³H]ouabain, an inhibitor of the Na⁺,K⁺-ATPase. High levels of [³H]ouabain binding sites were found in the superior and inferior colliculi, the mammillary nucleus, the interpeduncular nucleus, and in various divisions of the olfactory, auditory and somatomotor systems. The heterogeneous distribution of [³H]ouabain binding closely parallels the regional brain glucose consumption as determined by the [¹⁴C]deoxyglucose method. Lesion studies of the rat hippocampus using the excitotoxin, ibotenic acid, showed both a marked decrease of neuronal cell types on the injected side and a corresponding decrease in [³H]ouabain binding, indicating that some of the [³H]ouabain binding sites are localized to neurons. The close correlation between [³H]-ouabain binding and regional glucose utilization provides further evidence for a linkage between glucose utilization and the neuronal Na⁺,K⁺-ATPase.     

Localization of high affinity [H]glycine transport sites in the cerebellar cortex

A study was made of [³H]glycine uptake sites in a preparation greatly enriched in large pieces of the cerebellar glomeruli (glomerulus particles) and in morphologically well preserved slices of rat cerebellum. Electron microscopic autoradiography revealed that of the neurones in the cerebellar cortex only Golgi cells transported [³H]glycine at the low concentration used. Glial cells also took up [³H]glycine but to a lesser extent than the Golgi neurons. It was also confirmed that under comparable conditions Golgi cells transport [³H]GABA. Kinetic studies utilizing the Golgi axon terminal-containing glomerulus particles showed that glycine is a weak non-competitive inhibitor of [³H]GABA uptake (K_i over 600 μM vs theK_t of about 20 μM) and that GABA is an even weaker inhibitor of [³H]glycine uptake. These observations indicated that glycine and GABA do not share the same carrier. Quantitative electron microscopic autoradiography showed that the uptake of the two amino acids, in terms of the unit area of labelled Golgi axon terminals, was not additive. In contrast, their uptake in terms of unit protein was strictly additive. These observations, the first relating to unit volume and the latter to the total volume of Golgi terminals, are consistent with the view that there are two biochemically separate populations of Golgi neurons, one transporting glycine the other GABA. Saturable [³H]strychnine binding was detected in the preparations of glomerulus particles, but in comparison with those from the spinal cord the affinity was lower and [³H]strychnine was not displaced by glycine. Available information on glycine receptors, however, suggest that this should not exclude the possibility of strychnine resistant glycine receptors in the rat cerebellum.     

Effects of reboxetine and sertraline treatments alone and in combination on the binding properties of cortical NMDA and β1-adrenergic receptors in an animal model of depression

Summary. Changes to the binding properties of cortical N-methyl-D-aspartic acid (NMDA) and beta-adrenergic receptors have both been reported as potential indicators of antidepressant activity. In the present investigation we examined the effects of the noradrenaline reuptake inhibitor, reboxetine, the serotonin reuptake inhibitor, sertraline, alone and in combination on the binding properties of cortical NMDA receptors and cortical β₁-adrenoceptors following 14 days of treatment in the olfactory bulbectomized rat model of depression. A decrease in the potency of glycine to displace the strychnine insensitive glycine antagonist [³H] 5,7 dichlorokynurenic acid (5,7 DCKA) was observed in cortical homogenates of OB rats when compared to sham-operated controls. Similarly, treatment with the combination of reboxetine and sertraline for 14 days produced a decrease in the potency of glycine when compared to vehicle treated controls. By contrast neither olfactory bulbectomy or drug treatment significantly altered basal or glycine enhanced binding of the non-competitive NMDA antagonist [³H] MK-801 in cortical homogenates. Reboxetine alone, and in combination with sertraline, down-regulated [³H]-CGP 12177 (a selective β-adrenoceptor antagonist) binding in both OB and sham-operated animals. The lack of a bulbectomy effect in the [³H] CGP-12177 binding assay, and the fact that olfactory bulbectomy and antidepressant treatments produce a similar change to the potency of glycine at the NMDA receptor, suggests that these tests do not provide a neurochemical marker for either the behavioral hyperactivity deficit or antidepressant response in the model. Received February 6, 2000; accepted March 6, 2000     

Distribution of α-adrenergic, β-adrenergic and dopaminergic receptors in discrete hypothalamic areas of rat

Catecholamine receptor binding sites were measured in discrete hypothalamic nuclei or regions as well as in certain extrahypothalamic areas of the adult male rat. For each assay, discrete areas were microdissected from frozen tissue sections and pooled from several animals. Specific high affinity binding sites were assessed at fixed ligand concentrations for [³H]p-aminoclonidine (PAC) and [³H](2-C 2′,6′-(CH₃O)₂ phenoxyethylamino)-methylbenzodioxan (WB-4101) for α-adrenergic receptor sites, for [³H]dihydroalprenolol (DHA) for β-adrenergic receptor sites, and for [³H]2-amino-6, 7-dihydroxy-1,2,3,4-tetrahydronaphtalene (ADTN) and [³H]spiroperidol in the presence of cinanserin for dopaminergic receptor sites.Regional variations in [³H]WB-4101 binding were relatively small in magnitude, with most hypothalamic and extrahypothalamic areas possessing between 60 and 90% of the binding in frontal cortex. [³H]PAC binding showed a wider range of binding density across brain areas than did [³H]WB-4101, but, in general, variations in [³H]PAC binding paralleled those in [³H]WB-4101 binding. In hypothalamus, binding was characterized as being predominantly to α₁-receptors in the of [³H]WB-4101 and to α₂-receptors in the case of [³H]PAC. The medial hypothalamic areas exhibited a somewhat higher density of these α-adrenergic sites than did the lateral hypothalamus (perifornical hypothalamus and medial forebrain bundle). Also, the ratio of [³H]PAC to [³H]WB-4101 binding differed in different hypothalamic areas, ranging from 1.5:1 to 4:1. The median eminence was exceptional in that it contained appreciable [³H]PAC but no significant [³H]WB-4101 binding sites at the radioligand concentrations tested. Binding of [³H]DHA to β-adrenergic receptors varied over approximately a 3-fold range in the different hypothalamic areas, with binding highest in the medial forebrain bundle and the medial preoptic area, and lowest in the periventricular, dorsomedial and posterior hypothalamic nuclei, the median eminence and the zona incerta. The ratio of β-adrenergic to α-adrenergic binding sites was generally lower in the medial than in the lateral hypothalamic areas and higher in the extrahypothalamic areas examined than in the hypothalamus. With regard to [³H]spiroperidol and [³H]ADTN binding to dopaminergic sites, the striatum, nucleus accumbens and olfactory tubercle showed a greater density of [³H]spiroperidol than of [³H]ADTN sites, in contrast to the hypothalamus where [³H]ADTN binding was more predominant. Within the hypothalamus, [³H]ADTN binding was relatively uniform, while [³H]spiroperidol binding was quite high in four hypothalamic areas (lateral perifornical area, medial forebrain bundle, paraventricular and dorsomedial nuclei), intermediate in the median eminence and arcuate nucleus, and low or not detectable in all other hypothalamic areas.     

Autoradiographic localization of α5 subunit-containing GABAA receptors in rat brain

Multiple subtypes of GABA_A receptors are expressed in the rat central nervous system (CNS). To determine the distribution and proportion of α5 subunit containing receptors, quantitative autoradiographic analyses were performed with both [³H]L-655,708 and [³H]Ro15-1788, an α5 selective and a non selective benzodiazepine binding site ligand, respectively. High densities of [³H]L-655,708 binding sites were observed in hippocampus and olfactory bulb, where α5 receptors accounted for 20–35% of total [³H]Ro15-1788 binding sites. Low levels of [³H]L-655,708 sites were associated with the cortex as well as amygdala, thalamic, hypothalamic and midbrain nuclei. These observations indicate that although [³H]L-655,708 binding sites have an overall low expression in rat CNS, they may contribute significantly to GABAergic inhibition in specific brain regions.     

Autoradiographic localization and quantification of dopamine D2 receptors in normal human brain with [H]N-n-propylnorapomorphine

The precise distribution of dopamine receptors has been studied autoradiographically in the normal human brain using [³H]N-n-propylnorapomorphine ([³H]NPA) as a ligand. Preliminary experiments aimed at optimizing the binding assay conditions revealed that preincubation washing of caudate nucleus sections was a prerequisite to obtain a good ratio of specific to non-specific binding. The binding of [³H]NPA to caudate-putamen sections was saturable, stereospecific, reversible, of high affinity (K_d = 0.27–0.35 nM) and occurred at a single population of sites. Competition experiments with various drugs indicated that in the caudate-putamen the specific [³H]NPA binding sites possess the pharmacological features of the dopamine D₂ receptor. The highest levels of [³H]NPA binding sites were found in the caudate nucleus, putamen, globus pallidus and nucleus accumbens. There were also intermediate to low concentrations of the ³H-ligand in the hippocampus, the insular and cingular cortices and in the occipito-temporal gyrus, while almost undetectable levels of binding were found in the anteior frontal cortex. Thorough examination of the subregional distribution of [³H]NPA binding sites in the caudate-putamen-pallidum complex revealed heterogenous patterns of radioactivity. In these brain regions, the distribution of autoradiographic grains was punctate and islands of high and low densities were observed. Moreover, in the caudate nucleus, there was a subtle high lateral to low medial gradient in the topography of the [³H]NPA binding sites and a more pronounced gradient along the rostrocaudal axis; the highest levels of binding being located at the midbody of the nucleus. No gradients of [³H]NPA binding were observed in the putamen. The present data indicate that [³H]NPA is a suitable ligand for accurate autoradiographic labeling of dopamine D₂ receptors in human postmortem brain tissue and that dopamine receptors are heterogeneously distributed and topographically organized in patches and gradients in the basal ganglia regions.     

Binding of [H]glycine, [H]β-alanine and [H]strychnine in cultured rat spinal cord and brain stem

Cultures of rat brain stem and spinal cord were used to visualize binding sites for [³H]glycine, [³H]β-alanine and their antagonist [³H]strychnine by light microscopic autoradiography. In spinal cord cultures, all radio-ligands were bound mainly to large neurones, probably motoneurones whereas in brain stem cultures, both medium-sized and large neurones were labelled. In contrast, glial cells did not show binding sites for any of the compounds studied, suggesting that glial elements may not possess receptors for glycine and β-alanine.     

Atypical in vitro and in vivo binding of [3H]S-14506 to brain 5-HT1A receptors

Summary The tritiated derivative of the potent 5-HT_1A receptor agonist S-14506 {1[2-(4-fluorobenzoylamino)ethyl]-4-(7-methoxynaphtyl)piperazine} was tested for its capacity to selectively label the serotonin 5-HT_1A receptors both in vitro in the rat and the mouse brain, and in vivo in the mouse. In vitro studies showed that the pharmacological profile and the distribution of [³H]S-14506 specific binding sites (Kd=0.15 nM) in different brain regions matched perfectly those of the prototypical 5-HT_1A receptor ligand [³H]8-OH-DPAT. However, in the three regions examined (hippocampus, septum, cerebral cortex), the density of [³H]S-14506 specific binding sites was significantly higher (+ 66–90%) than that found with [³H]8-OH-DPAT. Whereas the specific binding of [³H]8-OH-DPAT was markedly reduced by GTP and Gpp(NH)p and increased by Mn²⁺, that of [³H]S-14506 was essentially unaffected by these compounds. In addition, the alkylating agent N-ethylmaleimide was much less potent to inhibit the specific binding of [³H]S-14506 than that of [³H]8-OH-DPAT. Measurement of in vivo accumulation of tritium one hour after i.v. injection of [³H]S-14506 to mice revealed marked regional differences, with about 2.5 times more radioactivity in the hippocampus than in the cerebellum. Pretreatment with 5-HT_1A receptor ligands prevented tritium accumulation in the hippocampus but not in the cerebellum. Autoradiograms from brain sections of injected mice confirmed the specific in vivo labeling of 5-HT_1A receptors by [³H]S-14506, therefore suggesting further developments with derivatives of this molecule for positron emission tomography in vivo in man.