Visualization by positron emission tomography of the apparent regional heterogeneity of central type benzodiazepine receptors in the brain of living baboons

The feasibility of visualizing the heterogeneity of benzodiazepine (BDZ) receptors in the brain of living baboons was investigated using Positron Emission Tomography. Ethyl 8-fluoro-5,6-dihydro-5-methyl 6-oxo-4H-imidazo (1,5-a) (1, 4) benzodiazepine-3-carboxylate (RO 15 1788) labelled by carbon 11 (11C-RO 15 1788) was I.V. injected for the "in vivo" labelling of the central type BDZ receptors. Displacement experiments were performed 20 minutes after the administration of the radioligand by two different cold drugs: RO 15 1788 which has an equal affinity for central type BDZ receptors, and propyl B-Carboline-3-carboxylate (B-CCP) which favours the sites located in the cerebellum. Different sensitivities to these two drugs displacement of 11C-RO 15 1788 binding "in vivo" were observed: on the one hand in the regional localization of the displacement, and on the other hand, in the amount of the radioactivity displaced. The apparent interregional heterogeneity of the displacement seen in the cerebellum and in the temporal cortex are discussed in terms of discrepancies observed "in vitro" at physiological temperature, between cerebellar and non-cerebellar BDZ central type binding sites.     

Subcellular Localization of "Peripheral-Type" Binding Sites for Benzodiazepines in Rat Brain

The binding of [3H]Ro 5-4864, a specific ligand for "peripheral-type" benzodiazepine binding sites and [3H]Ro 15-1788, a specific ligand for the central benzodiazepine receptors, was determined in subcellular fractions of rat brain. As previously reported, the highest levels of "peripheral-type" benzodiazepine binding sites and benzodiazepine receptors were found in the crude P1 and P2 fractions, respectively. Purification of these crude fractions revealed that high levels of both [3H]Ro 5-4864 and [3H]Ro 15-1788 binding were present in the mitochondrial and synaptosomal fractions. In contrast, the purified nuclei and myelin contained low levels of both [3H]Ro 5-4864 and [3H]Ro 15-1788 binding.     

Changes in in vivo binding of 3H-Ro 15-1788 in mouse brain by reserpine

The effects of reserpine on the in vivo binding of ³H-Ro 15-1788, (Ro 15-1788: ethyl 8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5a] [1,4]benzodiazepine-3-carboxylate) a selective benzodiazepine antagonist, in the mouse brain were investigated. The biodistributions of tracer amounts of ³H-Ro 15-1788 in mice were significantly altered by pretreatment with reserpine (2.5 or 5.0 mg/kg, 24 h before the tracer administration). The time courses of radioactivity in the brain and the blood following i.v. injection of ³H-Ro 15-1788 with carrier Ro 15-1788 were not changed by pretreatment with reserpine, which suggested that the specific binding process might be altered by reserpine. The degree of alteration in the in vivo binding of ³H-Ro 15-1788 seemed to be dependent upon the dose of reserpine and the duration after the treatment of reserpine. The maximum changes in the biodistribution of ³H-Ro 15-1788 were observed at 1 day after injection of reserpine. The body temperature and the brain monoamine contents (dopamine, norepinephrine and 5-hydroxytryptamine) in mice were measured as indicators of pharmacological effects of reserpine, and good relationships to the degree of changes in the biodistribution of ³H-Ro 15-1788 and either the body temperature or brain monoamine contents, were observed. Furthermore, the changes in the biodistribution of ³H-Ro 15-1788 in the reserpinized mice were significantly suppressed by antidepressant imipramine treatment. These results suggest that it would be possible to detect the in vivo drug interaction with brain benzodiazepine receptors in the living human brain using ¹¹C-Ro 15-1788 and positron emission tomography (PET).     

In vivo binding of (3H)Ro 15-1788 in mice: comparison with the in vivo binding of (3H)flunitrazepam

Benzodiazepine binding sites have generally been labelled with benzodiazepine agonists: (3H)flunitrazepam and (3H)diazepam in vivo. We studied the in vivo binding of the antagonist (3H)Ro 15-1788 in mice and compared it to the in vivo binding of (3H)flunitrazepam. For this in vivo labelling, mice were injected with labelled and unlabelled ligands. Animals were then sacrificed and bound radioactivity was measured after homogenization of the excised brain and filtration of the homogenate. (3H)Ro 15-1788 is a better tool than (3H)flunitrazepam for in vivo labelling of benzodiazepine receptors since 1) it labels specifically the central type binding sites, 2) injection of 4 times less (3H)Ro 15-1788 (50 microCi/kg) than (3H)flunitrazepam (200 microCi/kg) produced the same amount of bound radioactivity, 3) 70-90% of the total (3H)Ro 15-1788 present in the brain is membrane bound instead of 45-55% with (3H)flunitrazepam, 4) maximal binding of (3H)Ro 15-1788 is reached within 3 min, 5) only 5% of the membrane bound (3H)Ro 15-1788 is nonspecific instead of 15% for (3H)flunitrazepam.     

Temperature Dependence of [3H]Ro 15–1788 Binding to Benzodiazepine Receptors in Human Postmortem Brain Homogenates

The temperature dependence of in vitro binding of [3H]Ro 15-1788 to benzodiazepine receptors in human postmortem neocortex and neocerebellum homogenates was studied. An increase of the equilibrium dissociation constants (KD) from 1.40 nmol/L and 1.04 nmol/L at 4 degrees C to 6.10 nmol/L and 8.91 nmol/L at 37 degrees C was found for neocortex and neocerebellum, respectively. In contrast, maximal binding (Bmax) remained in the range of 30-35 fmol/mg for neocortex and 24-27 fmol/mg of tissue (wet weight) for neocerebellum at all the temperatures. The KD of 6.10 nmol/L for neocortex at 37 degrees C in vitro is of the same order as the KD of 10 nmol/L obtained by positron emission tomography for [11C]Ro 15-1788 binding to benzodiazepine receptors in the human neocortex in vivo. The differences in KD between in vitro and in vivo benzodiazepine receptor binding to human neocortex and cerebellum seem to be due at least partially to temperature differences of in vitro and in vivo studies.     

3H]Ro 15-1788 binding sites to brain membrane of the saltwater Mugil cephalus

The equilibrium binding parameters of the benzodiazepine antagonist [3H]Ro 15-1788 (8-fluoro-3-carboethoxy-5,6-dihydro-5-methyl-6-oxo-4H-imidazol-[1,5-a]-1,4 benzodiazepine) were evaluated in brain membranes of the saltwater teleost fish, Mugil cephalus. To test receptor subtype specificity, displacement studies were carried out by competitive binding of [3H]Ro 15-1788 against six benzodiazepine receptor ligands, flunitrazepam [5-(2-fluoro-phenyl)-1,3-dihydro-1-methyl-7-nitro-2H-1,4-benzodiazepin-2-one], alpidem [N,N-dipropyl-6-chloro-2-(4-chlorophenyl)imidazo[1,2-a]pyridine-3-acetamide], zolpidem [N,N-6 trimethyl-2-(4-methyl-phenyl)imidazo[1,2-a]pyridine-3-acetamide hemitartrate], and beta-CCM (methyl beta-carboline-3-carboxylate). Saturation studies showed that [3H]Ro 15-1788 bound saturatably, reversibly and with a high affinity to a single class of binding sites (Kd value of 1.18-1.5 nM and Bmax values of 124-1671 fmol/mg of protein, depending on brain regions). The highest concentration of benzodiazepine recognition sites labeled with [3H]Ro 15-1788 was present in the optic lobe and the olfactory bulb and the lowest concentration was found in the medulla oblongata, cerebellum and spinal cord. The rank order of displacement efficacy of unlabelled ligands observed suggested that central-type benzodiazepine receptors are present in one class of binding sites (Type I-like) in brain membranes of Mugil cephalus. Moreover, the uptake of 36Cl- into M. cephalus brain membrane vesicles was only marginally stimulated by concentrations of GABA that significantly enhanced the 36Cl- uptake into mammalian brain membrane vesicles. The results may indicate a different functional activity of the GABA-coupled chloride ionophore in the fish brain as compared with the mammalian brain.     

Binding of the benzodiazepine ligand [H]-Ro 15–1788 to brain membrane of the saltwater fish Mullus surmuletus

The distribution and the pharmacological properties of the binding of the benzodiazepine receptor antagonist [³H]-Ro 15–1788 (8-fluoro-3-carboethoxy-5,6-dihydro-5-methyl-6-oxo-4H imidazol [1,5-a] 1,4 benzodiazepine) were compared in some brain membranes of the saltwater teleost fish, Mullus surmuletus: only a single population of [³H]-Ro 15–1788 binding sites was detected. The binding was saturable and reversible with a high affinity, revealing a significant population of binding sites (K_d value of 2.1 ± 0.2 nM and B_max value of 1400-900 fmol mg⁻¹ of protein, depending on fish length). The highest concentration of benzodiazepine recognition sites labelled with [³H]-Ro 15–1788 was present in the optic lobe and the olfactory bulb and the lowest concentration was found in the medulla oblongata, cerebellum and spinal cord. In order to explore behavioural selectivity as a consequence of multiple receptor subtypes, six benzodiazepine receptor ligands, flunitrazepam (5-(2-fluoro-phenyl)-1,3,dihydro-1-methyl-7-nitro-2H-1,4-benzodiazepine-2-one), alpidem, (N,N-dipropyl-6-chloro-2-(4-chlorophenyl) imidazo [1,2-a] pyridine-3-acetamide) zolpidem {N,N,6, trimethyl-2-(4-methyl-phenyl) imidazo [1,2-a] pyridine-3-acetamide hemitartrate}, methyl β carboline-3-carboxylate (βCCM), Ro 15–1788 and Ro 5–4864 (4′-chlorodiazepam), were tested in vitro by binding of [³H]-Ro 15–1788 to membrane preparations from various brain areas of Mullus surmuletus. Displacement studies showed a similar rank order of efficacy of various unlabelled ligands. In all regions of the brain and in the spinal cord, GABA potentiate [³H]-flunitrazepam binding in a similar order, suggesting that the BDZ recognition sites are part of the GABA_A receptor structure. These results suggest that central-type benzodiazepine receptors are present in one class of benzodiazepine binding sites in the saltwater teleost fish brain of Mullus surmuletus (type I-like). Here we report initial evidence of homogeneity of subtypes of central benzodiazepine receptors in the spinal cord of the saltwater teleost fish, Mullus surmuletus.     

In vitro interaction of premazepam with benzodiazepine receptors in rat brain regions

Premazepam (PRZ) in vitro competitively displaced 3H-diazepam (DIA), 3H-flunitrazepam (FLU) and 3H-RO 15-1788 from their binding sites on rat brain synaptosomes, with a potency intermediate to other benzodiazepines (BDZs), and Hill coefficients near 1 in different brain regions. Incubation at 37 degrees C reduced premazepam's affinity for BDZ receptors to a lower extent than other benzodiazepines and had no effect on the Hill coefficient. The IC50 of PRZ on 3H-RO 15-1788 and 3H-FLU binding was markedly reduced by GABA in rat cortex, like those of reference classical BDZs, but was GABA-independent in the cerebellum. The IC50 of the BDZ antagonist, RO 15-1788 was unaffected by GABA in both brain areas. The possibility that PRZ behaves as a partial agonist in the cortex and as an antagonist in the cerebellum is discussed.     

Attenuating effect of diazepam on stress-induced increases in noradrenaline turnover in specific brain regions of rats: antagonism by Ro 15-1788

One-hour immobilization stress increased levels of the major metabolite of brain noradrenaline (NA), 3-methoxy-4-hydroxyphenyl-ethyleneglycol sulfate (MHPG-SO4), in nine brain regions of rats. Diazepam at 5 mg/kg attenuated the stress-induced increases in MHPG-SO4 levels in the hypothalamus, amygdala, hippocampus, cerebral cortex and locus coeruleus (LC) region, but not in the thalamus, pons plus medulla oblongata excluding the LC region and basal ganglia. The attenuating effects of the drug on stress-induced increases in metabolite levels in the above regions were completely antagonized by pretreatment with Ro 15-1788 at 5 or 10 mg/kg, a potent and specific benzodiazepine (BDZ) receptor antagonist. When given alone, Ro 15-1788 did not affect the increases in MHPG-SO4 levels. Behavioral changes observed during immobilization stress such as vocalization and defecation, were also attenuated by diazepam at 5 mg/kg and this action of diazepam was antagonized by Ro 15-1788 at 10 mg/kg, which by itself had no effects on these behavioral measurements. These findings suggest: (1) that diazepam acts via BDZ receptors to attenuate stress-induced increases in NA turnover selectively in the hypothalamus, amygdala, hippocampus, cerebral cortex and LC region and (2) that this decreased noradrenergic activity might be closely related to relief of distress-evoked hyperemotionality, i.e., fear and/or anxiety in animals.     

Specific 3H-DMCM binding to a non-benzodiazepine binding site after silver ion treatment of rat brain membranes

Specific binding of the BZ-receptor ligand 3H-DMCM to rat cortical membranes was dramatically enhanced by preincubation of the homogenate with 0.1 mM silver (Ag+) nitrate. The binding was completely inhibited by midazolam. Nevertheless, the pharmacological specificity of the Ag+-enhanced 3H-DMCM binding was different from that of BZ-receptors. Furthermore, the Bmax value, the regional distribution and the molecular target size determined by radiation inactivation analysis of the Ag+-enhanced binding site were different from those of BZ-receptors. The results indicate that Ag+-enhanced 3H-DMCM binding represent a high affinity metal complex formation between 3H-DMCM and an unknown brain specific protein of approximately 100,000 daltons molecular weight.     

Effects of benzodiazepines on single unit activity in the substantia nigra pars reticulata

Intravenous administration of two benzodiazepines, flurazepam and diazepam, had an inhibitory effect on the firing rates of neurons of the substantia nigra pars reticulata, a brain region with an identified GABAergic innervation. Diazepam was more potent than flurazepam. Bicuculline and picrotoxin, two drugs which block GABAergic transmission, and caffeine and theophylline, two methylxanthines which inhibit benzodiazepine binding, all reversed the inhibition produced by diazepam. The action of theophylline was less consistent than that of caffeine. Similarly, Ro 15–1788, an imidazodiazepine which putatively functions as a specific benzodiazepine antagonist, reversed the diazepam-induced inhibition. These findings are consistent with previous reports which suggest that the benzodiazepines may act through a GABAergic mechanism. In a separate group of experiments, caffeine or Ro 15–1788 was administered alone. While caffeine excited all reticulata cells tested. Ro 15–1788, the more specific benzodiazepine antagonist, generally had little excitatory effect. These results suggest: 1) that cells of the substantia nigra pars reticulata may not receive a substantial, tonic inhibition mediated by an endogenous benzodiazepine-like substance; and 2) that the methylxanthines may increase reticulata cell firing, at least in part, through mechanisms unrelated to the blockade of benzodiazepine receptors.     

Benzodiazepine receptors on human blood platelets

Binding studies conducted on membrane preparation from human platelets using (3H) Ro5-4864 and (3H) diazepam showed specific and saturable binding. Scatchard analysis revealed a single class of binding sites with KD = 10.8 +/- 0.9 nM and Bmax = 775 +/- 105 fmol/mg protein for (3H) Ro5-4864 and KD = 10.5 +/- 1.1 nM and Bmax = 133 +/- 19 fmol/mg for (3H) diazepam. We were unable to detect any GABA binding site on crude membrane preparation, nor did GABA enhance the binding of (3H) Ro5-4864 or (3H) diazepam. This suggests that benzodiazepine receptors are uncoupled to GABA system on human platelets. Ro15-1788, a specific antagonist for "central type" benzodiazepine (BDZ) binding sites was inactive in displacing (3H) Ro5-4864 from membrane receptors, while PK 11195 (a specific ligand for the "peripheral type" receptor) was the most potent of the drugs tested in inhibiting (3H) Ro5-4864 binding. These results indicate that human blood platelets bear "peripheral-type" BDZ receptor. Moreover, we could not detect any (3H) propyl beta carboline specific binding on platelet membranes. Results on benzodiazepine receptors on human circulating lymphocytes are also reported and similarity in pharmacological properties with platelet benzodiazepine receptors is suggested.     

Benzodiazepine antagonist Ro 15-1788: binding characteristics and interaction with drug-induced changes in dopamine turnover and cerebellar cGMP levels

The recently discovered benzodiazepine antagonist Ro 15-1788 was characterized in binding studies, and its potency and selectivity were determined in vivo by interaction with drug-induced changes in dopamine turnover and cerebellar cGMP level. Ro 15-1788 reduced [3H]flunitrazepam binding in the brain in vivo with a potency similar to that of diazepam and effectively inhibited [3H]diazepam binding in vitro (IC50 = 2.3 +/- 0.6 nmol/liter). [3H]Ro 15-1788 bound to tissue fractions of rat cerebral cortex with an apparent dissociation (KD) of 1.0 +/- 0.1 nmol/liter. The in vitro potency of various benzodiazepines in displacing [3H]Ro 15-1788 from its binding site was of the same rank order as found previously in [3H]diazepam binding. Autoradiograms of [3H]Ro 15-1788 binding in sections of rat cerebellum showed the same distribution of radioactivity as with [3H]flunitrazepam. The attenuating effect of diazepam on the chlorpromazine- or stress-induced elevation of homovanillic acid in rat brain was antagonized by Ro 15-1788. Among a series of compounds which either decreased or increased the rat cerebellar cGMP level, only the effect of benzodiazepine receptor ligands (diazepam, zopiclone, CL 218 872) was antagonized by Ro 15-1788. Thus, Ro 15-1788 is a selective benzodiazepine antagonist acting at the level of the benzodiazepine receptor in the central nervous system. Peripheral benzodiazepine binding sites in kidney and schistosomes were not affected by Ro 15-1788.     

Interaction of piracetam with 3H-imipramine binding sites and the GAMA-benzodiazepine receptor complex of brain membranes

Piracetam at a concentration of 10(-6) M was shown to behave as a noncompetitive inhibitor of 3H-imipramine specific binding to rat brain membranes. At the same time piracetam failed to influence specific binding of 3H-mianserin to membranes of guinea-pig cerebellum, which is indicative of its inability to suppress histamine H1 receptors, a component of 3H-imipramine specific binding sites. At a concentration of 10(-4) M piracetam does not change specific binding of 3H-flunitrazepam to rat hippocampal membranes in the absence of GABA, but in the presence of 5 X 10(-5) M GABA, like atypical tranquilizer mebicar, acts as a competitor of 3H-flunitrasepam binding. Though Ro-15 1788 did not suppress anxyolytic piracetam (and mebicar) effect, our results give evidence of a possible involvement of GABA-benzodiazepine supramolecular complex in the anxiolytic activity of piracetam.     

On the benzodiazepine binding pocket in GABAA receptors

Benzodiazepines are used for their sedative/hypnotic, anxiolytic, muscle relaxant, and anticonvulsive effects. They exert their actions through a specific high affinity binding site on the major inhibitory neurotransmitter receptor, the gamma-aminobutyric acid, type A (GABA(A)) receptor channel, where they act as positive allosteric modulators. To start to elucidate the relative positioning of benzodiazepine binding site ligands in their binding pocket, GABA(A) receptor residues thought to reside in the site were individually mutated to cysteine and combined with benzodiazepine analogs carrying substituents reactive to cysteine. Direct apposition of such reactive partners is expected to lead to an irreversible site-directed reaction. We describe here the covalent interaction of alpha(1)H101C with a reactive group attached to the C-7 position of diazepam. This interaction was studied at the level of radioactive ligand binding and at the functional level using electrophysiological methods. Covalent reaction occurs concomitantly with occupancy of the binding pocket. It stabilizes the receptor in its allosterically stimulated conformation. Covalent modification is not observed in wild type receptors or when using mutated alpha(1)H101C-containing receptors in combination with the reactive ligand pre-reacted with a sulfhydryl group, and the modification rate is reduced by the binding site ligand Ro15-1788. We present in addition evidence that gamma(2)Ala-79 is probably located in the access pathway of the ligand to its binding pocket.     

Benzodiazepine Antagonist Ro 15–1788: Binding Characteristics and Interaction with Drug-Induced Changes in Dopamine Turnover and Cerebellar cGMP Levels

Abstract: The recently discovered benzodiazepine antagonist Ro 15-1788 was characterized in binding studies, and its potency and selectivity were determined in vivo by interaction with drug-induced changes in dopamine turnover and cerebellar cGMP level. Ro 15-1788 reduced [³H]flunitrazepam binding in the brain in vivo with a potency similar to that of diazepam and effectively inhibited [³H]diazepam binding in vitro (IC₅₀= 2.3 ± 0.6 nmol/liter). [³H]Ro 15-1788 bound to tissue fractions of rat cerebral cortex with an apparent dissociation constant ( K _D) of 1.0 ± 0.1 nmol/liter. The in vitro potency of various benzodiazepines in displacing [³H]Ro 15-1788 from its binding site was of the same rank order as found previously in [³H]diazepam binding. Autoradiograms of [³H]Ro 15-1788 binding in sections of rat cerebellum showed the same distribution of radioactivity as with [³H]flunitrazepam. The attenuating effect of diazepam on the chlorpromazine- or stress-induced elevation of homovanillic acid in rat brain was antagonized by Ro 15-1788. Among a series of compounds which either decreased or increased the rat cerebellar cGMP level, only the effect of benzodiazepine receptor ligands (diazepam, zopiclone, CL 218 872) was antagonized by Ro 15-1788. Thus, Ro 15-1788 is a selective benzodiazepine antagonist acting at the level of the benzodiazepine receptor in the central nervous system. Peripheral benzodiazepine binding sites in kidney and schistosomes were not affected by Ro 15-1788.     

Differences in the mechanism of the antihypoxic action of benzodiazepine receptor agonists and muscimol

Neuropharmacological analysis of previously revealed antihypoxic activity of benzodiazepines (BDZ) has been performed in experiments on mice exposed to hypoxia. Antihypoxic effect of diazepam is shown to be antagonized by the central BDZ receptor blocker, Ro 15-1788. A certain degree of antihypoxic activity also abolished by Ro 15-1788 is exhibited by hypothetical ligands of BDZ receptors: inosin, nicotinamide, ethyl-beta-carboline-3-carboxylate. The effect of dipyridamole, a drug with high affinity for BDZ receptors of the peripheral type is not antagonized by Ro 15-1788, another evidence of Ro 15-1788 affinity precisely to the central BDZ receptors. GABA-mimetics (muscimol and GABA cetyl ester) were also found to have marked antihypoxic activity. Unlike BDZ receptor agonists, this effect is reduced by bicuculline and not by Ro 15-1788. The data obtained suggest that antihypoxic activity of BDZ is caused by their direct interaction with the central BDZ receptors, probably with the type which is not modulated by GABAA receptors.     

Benzodiazepines and synaptic processing in the spatial domain within the cat's primary somatosensory cortex

In the primary somatosensory cortex of cats, the size of the receptive fields (RFs) of cutaneously responsive neurones is under the control of gamma-aminobutyric acid (GABA) mediated inhibition when the cells are situated in rapidly adapting (RA) background regions. Cells located in slowly adapting (SA) or low-velocity rapidly adapting (LVRA) background regions do not appear to be affected by GABA significantly in the spatial domain, although other response properties such as threshold and firing pattern are under the influence of bicuculline methiodide (BMI) sensitive processes. The GABA receptor is one component of the oligomeric complex that includes the benzodiazepine (Bzd) binding site, the barbiturate recognition site, and the Cl- ionophore. Owing to current debates about the possible existence of endogenous ligands of Bzd receptors, we have examined whether Bzd agonists, in addition to GABA and BMI, have RF-modulating actions on RA S1 neurones and have assessed the effectiveness of the Bzd antagonist, Ro 15-1788, in this experimental paradigm. Ro 15-1788 is an imidazobenzodiazepine that acts as a specific competitive antagonist of Bzds by exerting high-affinity interactions with that Bzd receptor through which anticonvulsant effects of flurazepam (flu) and diazepam are expressed. This has been shown previously in neurochemical, behavioral, neurological, and pharmacological studies. Ro 15-1788 has little or no affinity for nonneuronal binding sites in the CNS. Ro 15-1788 binding does not displace GABA from its own binding site but does compete for all major Bzd ligands that act as pharmacological agonists and inverse agonists of the Bzd receptor through which anticonvulsant and convulsant effects are expressed. Bzd agonists elevated the threshold for somatic activation, depressed spontaneous activity, and decreased RF size. One exception in this regard was midazolam, which sometimes decreased somatic thresholds and increased spontaneous discharges. These latter effects were reversed at higher doses of the agonist. BMI returned RFs to control sizes when the drug was administered concurrently with Bzd agonists, or it caused RFs to assume greater than normal sizes, depending on the strength of current ejecting the antagonist. Ro 15-1788 given alone decreased response thresholds, increased spontaneous firing, and sometimes enlarged RFs. This antagonist also reversed the RF size-decreasing action of flu, diazepam, and midazolam. Quantitative analyses of air-puffer responses evoked from low-threshold, S1 cells revealed that Bzds do not selectively attenuate spatial summation, but that they act preferentially in the surround, or in the peripheral, regions of cutaneous excitatory RFs.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of benzodiazepine receptors with fluorescent ligands

Fluorescein conjugates of the high-affinity benzodiazepine receptor ligands Ro 15-1788 and Ro 7-1986 were synthesized. The binding of these fluorescent ligands (BD 621 and BD 607) to benzodiazepine receptors was characterized by direct fluorescence measurement. Both the equilibrium dissociation constants (KD) of BD 621 and BD 607 and the maximum number of binding sites (Bmax) estimated by fluorescence monitoring were consistent with values obtained by using radioligand binding techniques. The binding of BD 621 and BD 607 assessed by fluorescence measurement was reversible, abolished by photoaffinity labeling with Ro 15-4513, and unaffected by a variety of substances that do not bind to benzodiazepine receptors. The potencies of chemically diverse benzodiazepine receptor compounds to inhibit fluorescent ligand binding were highly correlated (r = 0.94, P less than 0.001), with potencies obtained from radioligand binding techniques. These findings demonstrate the feasibility of using direct fluorescence measurement techniques to quantitate ligand-receptor interactions.     

Benzodiazepine receptor binding in vivo with [3H]-Ro 15-1788

In vivo benzodiazepine receptor binding has generally been studied by "ex vivo" techniques. In this investigation, we identify the conditions where [3H]-Ro 15-1788 labels benzodiazepine receptors by true "in vivo" binding, i.e. where workable specific to nonspecific ratios are obtained in intact tissues without homogenization or washing. [3H]-Flunitrazepam and [3H]-clonazepam did not exhibit useful in vivo receptor binding.