Multiple high-affinity [H]serotonin binding sites in human frontal cortex

High-affinity [³H]serotonin (5-hydroxytryptamine, 5-HT) binding sites from human frontal cortex can be divided into at least 3 pharmacological subtypes (5-HT_1A, 5-HT_1B and 5-HT₃) based on affinity for [³H]serotonin and spiperone. All 3 sites are solubilized by 3% Triton X-100, 1% Tween-80 and can be enriched by serotonin-linked-Sepharose 4B affinity chromatography. However, 5-HT₃ sites are more sensitive to heat inactivation, long-term storage, and sulfhydrylalkylation. The pharmacological profiles are distinct for the spiperone-insensitive 5-HT_1B and 5-HT₃ sites in both human and bovine cortex. In addition, evidence is presented for the existence of a novel, low concentration [³H]serotonin binding site in human cortex.     

The heterogeneity of central benzodiazepine receptor subtypes in the human hippocampal formation,frontal cortex and cerebellum using [3H]flumazenil and zolpidem

The ability of clonazepam and zolpidem to displace [3H]flumazenil binding was measured in the human hippocampal formation, frontal cortex (BA9) and the cerebellum using in situ radioligand binding and autoradiography. The use of high resolution phosphorimaging in all regions indicated the displacement of [3H]flumazenil by clonazepam was monophasic with K(i) values ranging from 2.73+/-0.17 to 6.49+/-0.21 nM. [3H]flumazenil binding that was not displaced by clonazepam ranged from 3.39+/-0.86 to 7.15+/-1.11%. The ability of zolpidem to displace [3H]flumazenil was also monophasic in the frontal cortex and cerebellum with K(i) values of 37.53+/-1.79 and 31.80+/-1.68 nM, respectively. In contrast, within all hippocampal regions, zolpidem displacement of [3H]flumazenil was biphasic, with K(i) values for the high affinity site ranging from 0.13+/-0.04 to 0.54+/-0.03 nM, whereas the low affinity site was between 84.98+/-1.58 and 98.84+/-1.89 nM. In addition, zolpidem insensitive [3H]flumazenil binding was observed to vary markedly between brain regions, ranging between 37.85+/-1.60 and 6.13+/-0.83%. In conclusion, the present results indicate that in situ radioligand binding and high-resolution phosphorimaging techniques can be utilized to measure the differential displacement of [3H]flumazenil by zolpidem and clonazepam. Moreover, our data suggests that the differential distribution of the zolpidem insensitive component of [3H]flumazenil binding is an indicator of GABA/BZ receptors assembled by different subunits within the human brain.     

Characterization of [3H]pentazocine binding sites in post-mortem human frontal cortex

Summary We investigated binding characteristics of [³H](+)-pentazocine in homogenates of post-mortem human frontal cortex. At equilibrium, specific binding was linear with protein concentration, was saturable, reversible, stereoselective, heat-labile and was nearly absent in the white matter. Saturation experiments revealed a K _d of 3.68 ± 0.46nM and a B_max of 0.636 ± 0.107 pmol/mg protein. The rank order of K_i values of competing substances was: haloperidol < N,N-di(o-tolyl)guanidine (DTG) < (+)-SKF 10,047 < (—)-SKF 10,047. We also examined the influence of age, gender, hemisphere, post-mortem time and storage time of brain tissue at –80 °C on [³H](+)-pentazocine binding sites. Of these variables, only age was significantly related to [³]H](+)-pentazocine binding (diminished binding with increasing age). Together, our results demonstrate the presence of specific [³H](+)-pentazocine binding sites in post-mortem human brain tissue. Furthermore, the binding sites decrease with increasing age and are apparently independent of gender, hemisphere, post-mortem time and storage time of brain tissue.     

[H]WIN 35,428 binding in the human brain

The binding of [³H]WIN 35,428 was studied in post-mortem human brain, including extrastriatal regions. In the putamen, dopamine almost completely inhibited the [³H]WIN 35,428 binding. Paroxetine inhibited the binding with similar affinity as cocaine, in the range 200–300 nM. In the frontal cortex, [³H]WIN 35,428 labelled cocaine- and alaproclate sensitive binding sites, of which a major fraction was of protein nature. The elucidation of the cocaine sensitive sites in the frontal cortex should be the subject of further research.     

Laminar distribution of NMDA receptors in cat and monkey visual cortex visualized by [3H]-MK-801 binding

Glutamate is the major excitatory neurotransmitter of the mammalian central nervous system. Two major classes of glutamate receptors have been reported. The actions of glutamate on its N-methyl-D-aspartate (NMDA)-type receptor may underlie developmental and adult plasticity as well as neurotoxicity. The NMDA-type of glutamate receptor in cat and monkey visual cortex was visualized by means of in vitro receptor autoradiography with the noncompetitive NMDA-receptor antagonist [³H]-MK-801. The kinetics, performed on tissue sections, revealed an apparently single, saturable site with an approximate dissociation constant (KD) of 18.5 nM in cat and 15.9 nM in monkey visual cortex. Autoradiography, performed on frontal sections of cat and monkey visual cortex, revealed a heterogeneous laminar distribution of NMDA receptors. Cat areas 17,18,19, and the lateral suprasylvian areas exhibited a similar NMDA-receptor distribution. In these areas, NMDA receptors were most prominent in layer II and the upper part of layer III. In monkey striate cortex, NMDA receptors were primarily concentrated in layers II, upper III, IVc, V, and VI. In monkey secondary visual cortex, [³H]-MK-801 labeling was most prominent in layers II, V, and VI; whereas in the temporal visual areas included in this study layer II displayed the heaviest receptor labeling. In neither cat nor monkey could we observe significant differences in NMDA-receptor distribution between different retinotopic subdivisions within a single visual area. Neither did we detect any periodic changes in NMDA-receptor distribution that would correspond to the compartments defined by cytochrome-oxidase in monkey V1 and V2. © 1993 Wiley-Liss, Inc.     

Transient increase in the in vivo binding of the benzodiazepine antagonist [3H]flumazenil in deafferented visual areas of the adult mouse brain

Flumazer it is an imidazobenzodiazepine, an antagonist of central benzodiazepine (BDZ) receptors. BDZ binding sites are a modulatory component located on the gamma-aminobutyric acid (GABA) receptor macromolecule. We studied the effect of monocular enucleation on [³H]flumazenil binding in deprived and intact visual areas and nonvisual areas of the adult mouse brain under in vivo conditions. [³H]flumazenil binding was examined at seven time points up to 56 days postenucleation. In some monocularly deprived mice, changes in local blood flow accompanied with the BDZ receptor response were evaluated by coinjection of [3H]flumazenil and ^9mTc-HMPAO. Monocular enucleation produced a transient increase in [³H]flumazenil binding in the deprived visual cortex and superior colliculus. At 17 days postenucleation, [³H]flumazenil binding in the anterior and posterior portions of the visual cortex and the superior colliculus increased by 28%, 15% and 23%, respectively, and declined to control levels at 45 days postenucleation. The increase in [³H]flumazenil was accompanied with a decrease in blood flow. Alterations in BDZ receptors and blood flow were selective to deprived visual structures. The regional correlation between the metabolic deficit and the BDZ response provides further support that the increase in BDZ receptor binding is confined to regions of reduced neuronal activity. [¹¹C]flumazenil is an excellent radiotracer for in vivo imaging of benzodiazepine receptors in human brain using positron emission tomography (PET). This study suggests the suitability of [¹¹C]flumazenil for in vivo PET study of BDZ receptor response to deafferentation of visual structures in human brain. © 1994 Wiley-Liss, Inc.     

[H]MK-801 binding to the NMDA receptor complex, and its modulation in human frontal cortex during development and aging

[³H]MK-801 binding was found to decline with age in well washed membranes from human frontal cortex taken from an age series from 24 weeks gestation to 100 years old. The decline was significant under basal conditions (no added modulators) (P < 0.01), and highly significant under stimulation with glutamate, glycine and spermidine alone and in combination (P < 0.001). Scatchard analysis in the presence of glutamate and glycine showed this decline was due to a loss in the number [³H]MK-801 binding sites rather than a change in the affinity of the binding site. There was a highly significant age related reduction in the attenuation of [³H]MK-801 binding by zinc (P < 0.001). In foetal and neonatal cases up to 7 weeks of age spermidine behaved in an antagonistic manner, inhibiting rather than stimulating [³H]MK-801 binding, when alone or in the presence of glutamate and glycine. The changes in influence of glutamate, glycine, spermidine and zinc on [³H]MK-801 binding during development and aging were not due to other pre- or postmortem factors. The reverse effect of spermidine in the foetal and neonatal cases has therapeutic implications in the treatment of neonates with antiischaemic agents whose action involves the polyamine site.     

Subcortical projections to the hippocampal formation in squirrel monkey (Saimiri sciureus)

Subcortical afferents to the hippocampal formation in squirrel monkey were investigated using horseradish peroxidase as a retrograde marker. Labeled cells were found in the medial septal area, the diagonal band of Broca, anterior and laterodorsal thalamic nuclei, reuniens and periventricular thalamic nuclei, lateral hypothalamus, supramamillary nucleus, and the dorsal and superior central midbrain raphe nuclei. These results in a primate confirm previous findings in rats and cats with the exception of the noradrenergic cell groups, where the interpretation of retrograde label was hampered by high levels of endogenous pigment.     

Enhancement of immunoreactivity for NF-κB in the hippocampal formation and cerebral cortex of Alzheimer's disease

The distribution of nuclear factor-kappa B (NF-κB) was investigated immunohistochemically in the hippocampal formation, entorhinal cortex, middle temporal gyrus and visual cortex of Alzheimer's disease (AD) and control postmortem cases using a polyclonal antibody against the (NF-κB) p65 subunit. In AD cases, prominent staining for (NF-κB) was seen in neurons and their processes, neurofibrillary tangles and dystrophic neurites. In control cases, only weak staining of some neurons was obtained. The neuronal staining observed in AD was strongest in the hippocampal formation and entorhinal cortex, less in the middle temporal gyrus and least in the visual cortex. There was no difference between AD and control cases in the staining of glial cells and vascular walls. These results suggest that enhanced expression of neuronal (NF-κB) occurs in areas affected by AD pathology.     

Guanine based purines inhibit [(3)H]glutamate and [(3)H]AMPA binding at postsynaptic densities from cerebral cortex of rats

Extracellular guanine-based purines (GBPs) have been implicated in neuroprotective effects against glutamate toxicity by modulating the glutamatergic system through mechanisms without the involvement of G proteins. Accordingly, GBPs have been shown to inhibit the binding of glutamate and its analogs in different brain membrane preparations. However, brain membrane preparations used for these studies are comprised of both post- and pre-neuronal and glial synaptic components. In this study we investigated the ability of GBPs to displaced glutamate and AMPA binding at postsynaptic densities (PSDs). PSDs are markedly prominent in glutamatergic synapses and retains the native apposition of membrane components and post synaptic receptors. The PSD fraction was prepared from cerebral cortex of Wistar rats and it was characterized as PSDs by electron microscopy and by an enrichment of PSD-95, a protein marker of PSDs (90% of immunodetection). Moreover, we detected an enrichment of glutamate receptors subunits that including NR1 subunit of NMDA receptors and GluR1 subunit of AMPA receptors. GppNp (poor hydrolyzable GTP analog) and GMP displaced 40 and 36% of glutamate binding, respectively, and guanosine only 23%. AMPA binding was not affected by guanosine and was inhibited 21 and 25% by GppNp and GMP, respectively. Hence, this study demonstrates that guanine based purines inhibited glutamate and AMPA binding at postsynaptic membrane preparations, contributing for a better understanding of the mechanisms by which GBPs antagonize glutamatergic neurotoxicicity, e.g. the possible involvement of glutamatergic postsynaptic receptors in their neuroprotective roles.     

Autoradiographic distribution of binding sites for the non-NMDA receptor antagonist [H]CNQX in human motor cortex, brainstem and spinal cord

The distribution of non-NMDA receptors in the normal human motor cortex, brainstem and spinal cord has been investigated using [³H]CNQX. In the motor and premotor cortex, specific [³H]CNQX binding was present in all cortical laminae with the highest density of binding sites in laminae I, II and the upper part of III. In the normal brainstem, non-NMDA receptors labelled by [³H]CNQX had a heterogenous distribution. Brainstem motor nuclei subserving eye movements, which tend to be spared in motor neuron disease (MND), had a higher density of [³H]CNQX binding sites compared to other cranial nerve motor nuclei (VII, X, XII) which tend to be affected. Specific [³H]CNQX binding was present throughout the spinal grey matter, the greatest density of binding being found in the substantia gelatinosa. Excitotoxicity at non-NMDA receptors has been implicated in chronic neurodegenerative diseases such as motor neuron disease. This study suggests that the density of non-NMDA receptors, labelled by [³H]CNQX, does not account for selective vulnerability of motor neurons in this disorder.     

Regional distribution of high-affinity [H]somatostatin binding sites in the human brain

The distribution of high-affinity binding sites for [³H]somatostatin has been studied in membrane preparations from a number of regions of normal human brain. The highest densities of binding sites (> 48 fmol/mg protein) were found in the cerebral and cerebellar cortices and the hippocampus, with intermediate binding densities (30–46 fmol/mg protein) being present in the basal ganglia, amygdala, septum and claustrum. The lowest densities of binding sites (<14 fmol/mg protein) were observed in the hypothalamus, thalamus and substantia nigra. The binding of [³H]somatostatin in both the frontal cortex and cerebellar cortex demonstrated pharmacological specificity, since somatostatin-28, but not somatostatin-28_1–12 or Des AA^{1,2,4,5,12,13}, -Trp⁸-somatostatin, competed for the binding sites. Scatchard analysis of the binding in both frontal cortex and cerebellar cortex revealed the presence of two classes of high-affinity binding sites.     

Antipsychotic drug interactions with specific [3H]ketanserin binding sites in membrane fragments derived from human prefrontal cortex and human amygdala

Two regions of the brain potentially significant for psychopathology in schizophrenia are the prefrontal cortex and the amygdala. Antipsychotic compounds bind at serotonin receptors in human prefrontal cortex. We hypothesized that the serotoninergic antagonist [3H]ketanserin would label similar sets of binding sites in these two brain regions. Further, we hypothesized that all antipsychotic compounds would show appreciable affinity for binding sites labeled by [3H]ketanserin in the prefrontal cortex. Our findings indicate some differences in [3H]ketanserin binding between prefrontal cortex and amygdala. We also observed that several antipsychotic compounds had very high affinity for the [3H]ketanserin binding sites in prefrontal cortex.     

[3H]5-HT binding in post-mortem human cerebral cortex: methodological considerations

Summary The effects of different membrane preparations and assay conditions on [³H]5-HT binding to post-mortem human cortical tissue was studied. Optimal binding necessitated thorough removal of endogenous 5-HT and this was achieved either by hypotonic lysis or by preincubation of the membranes at 37C. Calcium chloride (4 mM) increased specific [³H]5-HT binding. The further addition of ascorbic acid (5.7 mM) or ascorbic acid and clorgyline (10 M) reduced specific [³H]5-HT binding.     

3H]MK-801 binding to the NMDA receptor complex, and its modulation in human frontal cortex during development and aging.

[3H]MK-801 binding was found to decline with age in well washed membranes from human frontal cortex taken from an age series from 24 weeks gestation to 100 years old. The decline was significant under basal conditions (no added modulators) (P less than 0.01), and highly significant under stimulation with glutamate, glycine and spermidine alone and in combination (P less than 0.001). Scatchard analysis in the presence of glutamate and glycine showed this decline was due to a loss in the number of [3H]MK-801 binding sites rather than a change in the affinity of the binding site. There was a highly significant age related reduction in the attenuation of [3H]MK-801 binding by zinc (P less than 0.001). In foetal and neonatal cases up to 7 weeks of age spermidine behaved in an antagonistic manner, inhibiting rather than stimulating [3H]MK-801 binding, when alone or in the presence of glutamate and glycine. The changes in influence of glutamate, glycine, spermidine and zinc on [3H]MK-801 binding during development and aging were not due to other pre- or postmortem factors. The reverse effect of spermidine in the foetal and neonatal cases has therapeutic implications in the treatment of neonates with antiischaemic agents whose action involves the polyamine site.     

Alterations in [3H]spiperone binding in human caudate nucleus, substantia nigra and frontal cortex in the Shy-Drager syndrome and Parkinson's disease

[3H]Spiperone binding was investigated in the caudate nucleus, substantia nigra (s. nigra) and frontal cortex of control subjects and of patients with Parkinson's disease and the Shy-Drager syndrome. Binding sites for [3H]spiperone were interpreted as dopamine receptors in caudate and s. nigra, and as 5-hydroxytryptamine (5-HT) receptors in frontal cortex. Scatchard analysis showed that the Bmax (maximal number of binding sites) in caudate was similar in the 3 groups, whereas in s. nigra the Bmax was reduced by approximately 60% in both Parkinsons disease and Shy-Drager syndrome. The dissociation constant (Kd) for [3H]spiperone binding in s. nigra was similar in the 3 groups. In caudate nucleus, the Kd was similar in control and Parkinson groups; however, there was a significant increase in the dissociation constant in the caudate nucleus from cases of Shy-Drager syndrome. No differences in binding characteristics were observed in the frontal cortex. These results are taken to reflect a loss of dopamine receptor sites in the s. nigra in both Parkinson's disease and Shy-Drager syndrome, and a reduced affinity of dopamine receptor sites in the caudate nucleus in Shy-Drager syndrome.     

[3H]cocaine binding in brain is inhibited by tris (hydroxymethyl) aminomethane

High-affinity binding of [³H]cocaine to membranes of mouse cerebral cortex is inhibited by Tris (hydroxymethyl) aminomethane, the buffer commonly used in receptor binding assays. This inhibition is not due to an effect of ionic strength in general. Comparison of binding in Tris buffer with that in sodium phosphate buffer indicates a more than 4-fold higher K_d in the former buffer, with no differences in the B_max values.     

3H]1-[2-(2-thienyl)cyclohexyl]piperidine labels two high-affinity binding sites in human cortex: further evidence for phencyclidine binding sites associated with the biogenic amine reuptake complex

Previous work demonstrated two high-affinity PCP binding sites in guinea pig brain labeled by [3H]TCP (1-(1-[2-thienyl]cyclohexyl)piperidine): site 1 (N-methyl-D-aspartate [NMDA]-associated) and site 2 (dopamine-reuptake complex associated). The present study examined brain membranes prepared from various species, including human, for the presence of site 2, defined as binding in the presence of (+)-5-methyl-10,11-dihydro-5H-dibenzo [a, d]cyclohepten-5,10-imine maleate ((+)-MK801) minus binding in the presence of 10 microM TCP (nonspecific binding). Studies were conducted in absence of sodium which was found to be inhibitory to [3H]TCP binding. The results demonstrated detectable levels of site 2 in brain membranes of guinea pig, rabbit, pig, mouse, sheep, and human but not in the rat or chicken. Using human cortical membranes, site 2 was the predominant binding site. Detailed studies conducted with human cortical tissue showed that high-affinity dopamine (1-[2- [bis(4-fluorophenyl)-methoxy]ethyl]-4-(3-phenylpropyl)piperazine (GBR12909)], [1,2]benzo(b)thiophenylcyclo-hexylpiperidine (BTCP), and serotonin (fluoxetine) uptake inhibitors produced a wash-resistant inhibition of [3H]TCP binding to site 2, but not site 1. Preincubation of guinea pig brain membranes with BTCP was shown to produce an increase in the dissociation rate of [3H]TCP from PCP site 2. Structure activity studies with various uptake inhibitors showed that GBR12909, benztropine, fluoxetine, and BTCP have higher affinity for site 2 than for site 1. (+)-MK801, ketamine, and tiletamine were very selective for site 1, whereas dexoxadrol and TCP were moderately selective for site 1. These results suggest that human cortex possesses high-affinity PCP binding sites associated with biogenic reuptake binding sites, and that guinea pig brain, but not rat brain, may be an appropriate animal model for studying PCP site 2 in human brain.     

Alpha-1 and alpha-2 adrenoceptor binding in cerebral cortex: Competition studies with [3H]prazosin and [3H]idazoxan

Summary The tritiated adrenergic antagonists prazosin ([³H]PRZ) and idazoxan ([³H]IDA, or RX-781094) bind specifically and with high affinity in membrane preparations from cerebral cortex to alpha-1- and alpha-2-adrenoceptors respectively. Saturation experiments, performed to determine the density of receptors (B_max; maximum binding capacity) and the dissociation constant (K_d 25 °C), were analyzed by the methods of Eadie and Hofstee, iterative modelling, and the procedure of Hill. The pharmacologic properties and specificity of the labelling was verified by displacement experiments using alpha-adrenergic antagonists and agonists. The antagonist drugs showed the following order of potency to displace [³H]prazosin: prazosin phentolamine corynanthine > pyrextramine yohimbine piperoxan > benextramine > idazoxan; for the agonists: clonidine (–)-noradrenaline (–)-adrenaline phenylephrine, while other drugs, such as (–)-propranolol, dopamine, (–)-isoproterenol and serotonin only competed with the alpha-1-ligand at concentrations above 20 M. The alpha₂-sites labelled by [³H]idazoxan were characterized by the antagonist displacement sequence idazoxan phentolamine > yohimbine = > piperoxan pyrextramine benextramine prazosin corynanthine. The agonists order of potency to compete with [³H]idazoxan was clonidine phenylephrine = > (–)-adrenaline > (–)-noradrenaline, and for other related drugs it was (–)-propranolol dopamine serotonin > (–)-isoproterenol. These competition experiments clearly showed two pharmacologically distinct sites, but question the relative specificity of some of the adrenergic drugs.Abreviations [³H]PRZ [³H]prazosin - [³H]IDA [³H]idazoxan - B_max maximum binding capacity - K_d dissociation constant - IC 50 inhibitory concentration that reduces binding by 50% - K_i inhibition-dissociation constant - nH Hill coefficient - CMC coefficient of multiple correlation - fmol/mg p fentomoles per mg of protein - nM nanomolar Recipient of a F.R.S.Q. Studentship.     

Specific in vitro binding of (S,S)-[3H]MeNER to norepinephrine transporters

The aim of this study was to determine the selectivity of (S,S)-2-(alpha-(2-methoxyphenoxy)benzyl)morpholine (MeNER) binding to norepinephrine transporters (NET). Quantitative autoradiography studies of NET binding were performed in brains of wildtype mice and those of mutant mice lacking one or two alleles of the NET gene. [3H]MeNER binding in the wildtype mouse brains was consistent with previously reported distributions of NET. Highest levels were found in the locus coeruleus, thalamus, hypothalamus, and bed nucleus of stria terminalis. Specific binding in these regions was approximately 50% in the heterozygous NET mice and negligible in the NET knockout mice. Binding in the wildtype mouse brains was displaced by the NET ligand, nisoxetine, but not by the serotonin or dopamine transporter blockers, citalopram or GBR 12935. [3H]MeNER displayed much higher affinity for NET than for SERT or DAT in homogenate binding studies. Each of these features supports the binding specificity of this candidate in vivo NET ligand.