Modification of γ-Aminobutyric AcidA Receptor Binding and Function by N-Ethoxycarbonyl-2-Ethoxy-1,2-Dihydroquinoline In Vitro and In Vivo: Effects of Aging

The irreversible protein-modifying reagent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) was used to investigate binding site characteristics on the gamma-aminobutyric acidA (GABAA) receptor complex. In vitro, preincubation with EEDQ led to a concentration-dependent decrease in receptor number for benzodiazepine, t-butylbicyclophosphorothionate (TBPS), and GABA binding sites in cerebral cortex. The effect was maximal at the highest concentration of EEDQ used (10(-4) M) and was greatest for the benzodiazepine site. Pretreatment of membranes with the benzodiazepine antagonist Ro 15-1788, 1 or 10 microM, or the agonist lorazepam, 10 microM, largely prevented the effects of EEDQ. Scatchard analysis indicated no effect of EEDQ, 10(-4) M, on apparent affinity, but a decrease in receptor density for each site. Administration of EEDQ to mice, 12.5 mg/kg i.p., led to a substantial (55-65%) decrease in number of benzodiazepine binding sites in cortex after 4 h. Slightly smaller changes were observed for TBPS and GABA binding. No changes were observed in apparent affinity at any site. Prior administration of Ro 15-1788, 5 mg/kg, prevented the effect of EEDQ on benzodiazepine binding. Density of benzodiazepine binding sites gradually recovered over time, and receptor density returned to control values by 96 h after EEDQ injection. Number of binding sites in cortex for TBPS and GABA also increased over time after EEDQ. Benzodiazepine sites in cerebellum were decreased proportionally to cortex after EEDQ, and increased over a similar time course. Function of the GABAA receptor in chloride uptake in cortex was markedly reduced (65%) by EEDQ.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for diabetes-induced alterations in the sulfation of heparin sulfate intestinal epithelial cells

Two compounds with high affinity for the "peripheral type" benzodiazepine binding sites, PK 11195 (an isoquinoline derivative) and RO5-4864 (a benzodiazepine derivative) can modify the sensitivity of DBA/2J mice to audiogenic seizures. RO5-4864 (1-15 mg/kg) facilitates in a dose-dependent manner the audiogenic seizures and PK 11195 (2-5 mg/kg) antagonizes the RO5-4864 effects. At these doses PK 11195 alone does not modify the sensitivity to audiogenic seizures, but at doses between 20-80 mg/kg it protects DBA/2J mice against audiogenic seizures. By contrast PK 11195 is inactive against the facilitation of audiogenic seizures by ethyl-beta-carboline-3-carboxylate (a brain benzodiazepine receptor inverse agonist) and against the seizure elicited in absence of noise stimuli by RO5-4864 at doses between 20-40 mg/kg. These results suggest that facilitation by RO5-4864 of the audiogenic seizures and its antagonism by PK 11195 are mediated by the peripheral type benzodiazepine binding sites and agree with the thermodynamic analysis of the binding data which suggested that RO5-4864 might be an agonist and PK 11195 an antagonist. The good correlation between pharmacological effects and the occupancy degree of the binding sites as measured by the displacement of the "in vivo" [3H]-PK 11195 binding give an additional support to binding sites mediated effects.     

Effects of Chronic Chlorpromazine Treatment on Peripheral Benzodiazepine Binding Sites in Heart, Kidney, and Cerebral Cortex of Rats

Daily intraperitoneal administration to rats of 5 mg/kg of chlorpromazine (CPZ) for 21 days induced a significant up-regulation (51%) of peripheral benzodiazepine binding sites (PBSs) in cerebral cortex and a down-regulation of PBSs in the heart (25%) and kidney (14%), whereas no alteration in [3H]flunitrazepam binding in cerebral cortex was observed. [3H]PK 11195 binding to cerebral cortex returned to normal following 5 days of CPZ withdrawal, whereas the density of PBSs in the heart and kidney remained reduced. The affinity of PBSs for the ligand [3H]PK 11195 in the cerebral cortex and heart was not affected by the drug treatment or withdrawal. The CPZ-induced alterations in PBSs may be relevant to the effects of the drug on CNS and/or peripheral organs.     

Melatonin reverses pinealectomy-induced decrease of benzodiazepine binding in rat cerebral cortex

Pinealectomy of rats resulted in significant depression of benzodiazepine receptors (assessed by [³H]flunitrazepam binding) in cerebral cortex 3–14 days after surgery without affecting their affinity significantly. A single s.c. injection of melatonin (800 μg/kg body wt) restored the depressed brain benzodiazepine receptor sites. Single melatonin injections (up to 1600 μg/kg) to intact rats did not affect brain benzodiazepine binding when injected at either morning or evening hours. Daily melatonin treatment to intact rats for 5 days augmented benzodiazepine receptor density in brain (morning injections) or its dissociation constant (evening injections). Melatonin added in vitro to rat cerebral cortex membranes only slightly depressed [³H]flunitrazepam binding at 100 μM concentrations. These results point out a link between pineal activity and benzodiazepine receptor function in rats. They also indicate that pharmacological doses of melatonin affect benzodiazepine binding sites in rat cerebral cortex.     

The role of benzodiazepine receptors in the induction of differentiation of HL-60 leukemia cells by benzodiazepines and purines

A series of benzodiazepines was evaluated for their capacity to induce the differentiation of HL-60 acute promyelocytic leukemia cells. Benzodiazepines were effective initiators of maturation in the concentration range of 50 to 150 microM. The possible involvement of benzodiazepine receptors in mediating the differentiation induced by these agents was investigated. The presence of high affinity, peripheral type benzodiazepine binding sites (KD = 7.3 nM, TB = 14.5 pmol/mg protein with Ro5-4864) was demonstrated in HL-60 membranes. The occupancy of peripheral type high affinity benzodiazepine receptors by various benzodiazepines showed some correlation (r = 0.76) with their differentiation-inducing capabilities, but binding potencies were 1,000-fold higher than the concentrations required to produce differentiation. A class of benzodiazepine receptors with lower binding affinity was also detected in HL-60 membranes (KD = 28.6 microM; TB = 199 pmol/mg protein with diazepam). A higher level of correlation (r = 0.88) was demonstrated between benzodiazepine occupancy of these lower affinity receptors and the capacity to induce maturation. Significantly, benzodiazepine concentrations needed for low affinity binding and induction of differentiation were the same (25-200 microM), suggesting that low affinity benzodiazepine receptors may be involved in the induction process. We have shown that the molecular form responsible for the induction of the differentiation of HL-60 cells to mature forms by 6-thioguanine (TGua) is the free base, TGua, itself [Ishiguro, Schwartz, and Sartorelli (1984) J. Cell. Physiol., 121:383-390]. Since hypoxanthine (Hyp) and inosine (Ino) have been identified as putative endogenous ligands for high affinity benzodiazepine receptors in brain tissue, the potential involvement of benzodiazepine receptors in the differentiation of HL-60 cells by the purines was investigated. Physiological purines such as Hyp and Ino were inactive in displacing the benzodiazepines from their high and low affinity binding sites in HL-60 membranes. In contrast, TGua caused inhibition of benzodiazepine binding to high and low affinity sites. The inhibition of Ro5-4864 binding to high affinity binding sites by TGua appeared to be due to the binding of TGua to membranes through the formation of a mixed disulfide between the 6-thiopurine and protein thiols, since the inhibition was reversed by the presence of 2-mercaptoethanol. The findings suggest a possible relationship between the occupancy of benzodiazepine receptors by TGua and the induction of leukemic cell differentiation.     

Opposite effects of two ligands for peripheral type benzodiazepine binding sites, PK 11195 and RO5-4864, in a conflict situation in the rat

The effects of two drugs acting at the peripheral type benzodiazepine binding sites, PK 11195 and RO5-4864, were examined in shock-induced suppression of drinking in rats. These two compounds have opposite effects : RO5-4864 (3.1-1205 mg/kg i.p.) enhanced whereas PK 11195 (25-50 mg/kg i.p.) decreased the punished responding, and PK 11195 (6.25 mg/kg, a dose which did not alter the punished responding) blocked the proconflict action of RO5-4864 (6.25 and 12.5 mg/kg). The effects of RO5-4864 and PK 11195 were not antagonized by RO15-1788, a selective antagonist of the central benzodiazepine site. In addition, PK 11195 (6.25 mg/kg) did not reverse the proconflict effect of two beta-carbolines : beta-CEE and FG 7142. AS picrotoxin did not change the punished responding, these data imply that the effects of RO5-4864 and PK 11195 on the one hand and those of chlordiazepoxide and beta-carbolines on the other hand are differentially mediated and suggest that the peripheral type benzodiazepine binding sites are involved in this conflict model.     

Chronic nicotine administration does not increase nicotinic receptors labeled by [125I]epibatidine in adrenal gland,superior cervical ganglia,pineal or retina

Neuronal nicotinic acetylcholine receptors (nAChRs) were measured in CNS and peripheral tissues following continuous exposure to saline or nicotine hydrogen tartrate (3.3 or 10 mg/kg/day) for 14 days via osmotic pumps. Initially, binding of [3H](-)nicotine, [3H]cytisine and [3H]epibatidine to nAChRs was compared to determine the suitability of each for these kinds of studies. The predominant nAChR labeled by agonists in the cerebral cortex is an alpha 4 beta 2 subtype, whereas the predominant nicotinic receptors in the adrenal gland, superior cervical ganglia and pineal gland contain an alpha 3 subunit, and they do not bind either [3H](-)nicotine or [3H]cytisine with high affinity. In retina some nAChRs bind all three ligands with high affinity, and others appear to bind only [3H]epibatidine. Thus, only [3H]epibatidine had high enough affinity to be useful for measuring the nAChRs in all of the tissues. The receptors from nicotine-treated rats were then measured using [125I]epibatidine, which has binding characteristics very similar to [3H]epibatidine. Treatment with the two doses of nicotine hydrogen tartrate increased binding sites in the cerebral cortex by 40% and 70%, respectively. In contrast, no significant changes in the density of receptor binding sites were found in the adrenal gland, superior cervical ganglia, pineal gland or retina. These data indicate that chronic administration of nicotine even at high doses does not increase all nicotinic receptor subtypes, and that receptors containing alpha 3 subunits may be particularly resistant to this nicotine-induced change.     

CL 218,872 Binding to Benzodiazepine Receptors in Rat Spinal Cord: Modulation by γ-Aminobutyric Acid and Evidence for Receptor Heterogeneity

The binding of the triazolopyridazine CL 218,872 to central benzodiazepine receptors identified with [3H]Ro 15-1788 was studied in extensively washed homogenates of rat spinal cord and cerebral cortex. CL 218,872 displacement curves were shallow in both spinal cord (nH = 0.67) and cortex (nH = 0.54), suggesting the presence of type 1 and type 2 benzodiazepine receptors in both tissues. CL 218,872 had lower affinity in spinal cord (IC50 = 825 nM) than cortex (IC50 = 152 nM), possibly reflecting the presence of fewer type 1 sites in the cord. Activating gamma-aminobutyric acid (GABA) receptors with 10 microM muscimol resulted in a two- to threefold increase in CL 218,872 affinity in both tissues without changes in the displacement curve slope. This indicates that GABA enhances CL 218,872 affinity for both type 1 and type 2 sites in both spinal cord and cerebral cortex.     

Brain benzodiazepine binding sites in ethanol dependent and withdrawal states

The brain benzodiazepine system has been implicated to be important in both the mechanism, and treatment of ethanol related syndromes. In this report evidence is presented which indicates that "peripheral type" benzodiazepine binding sites are probably more relevant than "central type" receptors for the neurochemical consequences of ethanol dependence and withdrawal states. Utilizing radioreceptor binding techniques 20-50% increases in the binding of [3H]RO-5-4864 (a "peripheral type" ligand) to brain membranes derived from rat cerebral cortex, cerebellum and hippocampus are observed in ethanol dependent rats. These increases persist for 3 days after cessation of ethanol. The number of [3H]RO-5-4864 binding sites in cerebellum returns to normal during 4-7 days after ethanol withdrawal. In all brain areas examined no changes were observed in the "central type" benzodiazepine receptor as judged by [3H]-ethyl-Beta-carboline-3-carboxylate, BCCE binding. Scatchard analysis revealed that the number of [3H]RO-5-4864 binding sites is increased in each brain area while the affinity was unchanged.     

Pharmacological evaluation of an [(123)I] labelled imidazopyridine-3-acetamide for the study of benzodiazepine receptors

In vitro binding of the iodinated imidazopyridine, N',N'-dimethyl-6-methyl-(4'-[(123)I]iodophenyl)imidazo[1,2-a]pyridine-3-acetamide [(123)I]IZOL to benzodiazepine binding sites on brain cortex, adrenal and kidney membranes is reported. Saturation experiments showed that [(123)I]IZOL, bound to a single class of binding site (n(H)=0.99) on adrenal and kidney mitochondrial membranes with a moderate affinity (K(d)=30 nM). The density of binding sites was 22+/-6 and 1.2+/-0.4 pmol/mg protein on adrenal and kidney membranes, respectively. No specific binding was observed in mitochondrial-synaptosomal membranes of brain cortex. In biodistribution studies in rats, the highest uptake of [(123)I]IZOL was found 30 min post injection in adrenals (7.5% ID/g), followed by heart, kidney, lung (1% ID/g) and brain (0.12% ID/g), consistent with the distribution of peripheral benzodiazepine binding sites. Pre-administration of unlabelled IZOL and the specific PBBS drugs, PK 11195 and Ro 5-4864 significantly reduced the uptake of [(123)I]IZOL by 30% (p<0.05) in olfactory bulbs and by 51-86% (p<0.01) in kidney, lungs, heart and adrenals, while it increased by 30% to 50% (p<0.01) in the rest of the brain and the blood. Diazepam, a mixed CBR-PBBS drug, inhibited the uptake in kidney, lungs, heart, adrenals and olfactory bulbs by 32% to 44% (p<0.01) but with no effect on brain uptake and in blood concentration. Flumazenil, a central benzodiazepine drug and haloperidol (dopamine antagonist/sigma receptor drug) displayed no effect in [(123)I]IZOL in peripheral organs and in the brain. [(123)I]IZOL may deserve further development for imaging selectively peripheral benzodiazepine binding sites.     

Independent benzodiazepine and beta-carboline binding sites in the brain of aggressive and anxious-defensive mice

Distribution of specific 3H-flunitrazepam and 3H-beta-carboline-3-carboxylate binding sites in the brain regions of aggressive and timid-defensive mice was investigated before and after subchronic injection of diazepam (5 mg/kg). The absence of differences between the affinity and concentration of 3H-flunitrazepam binding sites in diencephalon and brain cortex in aggressive and defensive mice may be explained by general benzodiazepine receptor reaction on isolation and agonistic interaction stress. Significant predominance of 3H-beta-carboline-3-carboxylate binding sites in the brain cortex, as compared to the concentration of 3H-flunitrazepam binding sites suggests the presence of specific binding sites for beta-carbolines, which have specific distribution in the brain.     

CHARACTERIZATION OF BENZODIAZEPINE BINDING SITES IN CULTURED CELLS OF NEURAL ORIGIN

Abstract— The binding of [³H]diazepam to benzodiazepine receptors was investigated in cultured cell lines of neural origin. Two cell lines, the rat C6 glioma and mouse NB41A3 neuroblastoma possess large numbers of benzodiazepine binding sites, while the other neural cell lines examined had significantly fewer benzodiazepine binding sites. [³H]diazepam binding to membranes prepared from C6 or NB41A3 cells was saturable and of a relatively high affinity ( K _D± 12 and 20 n m , respectively) when compared with rat cerebral cortex ( K _D± 4.6 n m ). A single class of binding sites in both cell lines was demonstrated by Scatchard analysis. The maximum binding capacities ( B _max) in the C6 and NB41A3 cell lines were found to be 10 and 3.5 fold higher than in rat cerebral cortex, respectively. In contrast to the rat cerebral cortex, binding of [³H]diazepam in cultured cells was not displaced by the clinically active benzodiazepines clonazepam and oxazepam while the clinically inactive benzodiazepine Ro 5-4864 potently inhibited the binding of [³H]diazepam in both neural cell lines. In toto , this data suggests a change in the benzodiazepine binding sites in cultured cells of neural origin to that found in peripheral (kidney) tissue. The observation that cell lines derived from both neuronal and glial elements contain large numbers of benzodiazepine binding sites also suggests benzodiazepine receptors in the central nervous system may not be confined to a single cell type.     

Changes in the characteristics of low affinity GABA binding sites elicited by Ro15-1788

3H-GABA binding was studied in cortical membranes from cerebral cortex of handling-habituated and naive rats after the in vitro addition of Ro15-1788. At low concentrations (10(-8), 10(-9) M) Ro15-1788 increased the total number of low affinity 3H-GABA binding sites in brain tissue from naive rats but failed to modify 3H-GABA binding in tissue from handling-habituated ones. On the contrary, Ro15-1788 at higher concentrations (10(-5), 10(-6)M) decreased the total number of low affinity 3H-GABA binding sites in tissue from handling-habituated rats but failed to modify 3H-GABA binding in tissue from naive animals. Ro15-1788 (10(-7)M) failed to modify significantly low affinity 3H-GABA binding in membranes from both naive and handling-habituated rats. However, this concentration abolished the effect of beta-carbolines and diazepam on 3H-GABA binding in membranes from naive and handling-habituated rats, respectively. The changes in the affinity of 3H-GABA binding were inversely related to the changes in the number. The results suggest that: a) the action "in vitro" of Ro15-1788 on low affinity 3H-GABA binding depends from its concentration at the benzodiazepine recognition sites; b) the benzodiazepine recognition site has a modulatory role in the control of the function of GABA-ergic receptor. Our data might explain the conflicting results obtained with this compound "in vivo".     

Presence of a peripheral type benzodiazepine binding site on the macrophage; its possible role in immunomodulation

Saturable binding site for 3H-flunitrazepam (KD = 43 +/- 7 nM, Bmax = 391 +/- 58 fmoles/cell, i.e. 250,000 sites/cell) is characterized on Mouse peritoneal inflammatory macrophages. The affinity for different ligands (PK 11195 greater than Ro 5-4864 greater than diazepam greater than flunitrazepam greater than clonazepam greater than Ro 15-1788) shows that this site is of peripheral type. In vivo the humoral response in Mice to Sheep red blood cells was stimulated by administration of 1 mg/kg of PK 11195 (+85%), Ro 5-4864 (+80%) and diazepam (+58%). Clonazepam and Ro 15-1788 are devoid of activity. This suggests that molecules which show affinity for the "peripheral type" benzodiazepine binding site might modulate the immune response.     

Peripheral-type benzodiazepine receptors in human cerebral cortex, kidney, and colon.

The specific binding of [3H]PK 11195 and [3H]Ro 5-4864 to human cerebral cortex, kidney, and colon membranes was studied in order to determine whether peripheral type benzodiazepine receptors (PBR) characteristics located in human tissues are similar to those located in calf or rat tissues. While [3H]PK 11195 (0.05-10 nM, final concentration) bound with high affinity (KD about 2 nM) to human cerebral cortex, kidney, and colon membranes, yielding maximal numbers of binding sites of 255 +/- 23, 1908 +/- 28, and 1633 +/- 98 fmol/mg protein, respectively, the specific binding of [3H]Ro 5-4864 (1.25-40 nM, final concentration), was barely detectable (nonspecific binding about 90% of the total binding). Furthermore, unlabeled PK 11195 was two orders of magnitude more potent than unlabeled Ro 5-4864 in displacing [3H]PK 11195 specific binding from human cerebral cortex and kidney membranes. These results indicate that PBR binding characteristics located in human tissues are similar (but not identical) to those located in calf tissues, but not to those located in rat tissues.     

The postnatal development of benzodiazepine receptor sites in the chick optic lobe is modulated by environmental lighting.

The present paper describes the ability of benzodiazepine receptor sites to undergo light mediated-plastic changes during the early postnatal development of the chick optic lobe. The postnatal development pattern of these receptors was studied under different levels of light stimulation, i.e. normal-, light-and dark-rearing. At hatching the specific binding of [3H]Flunitrazepam was 0.23 +/- 0.01 pmol/mg protein. The developmental profile shows a sharp and transient peak of receptor overexpression between the 1st and the 2nd postnatal day in three experimental groups. Between the 2nd and the 6th day significant differences were found between the three groups, being this difference maximal during the peak of overexpression. In fact, on the 2nd day the specific [3H]Flunitrazepam binding showed an increase of 17% (P < 0.0005) and a decrease of 34% (P < 0.0005) for light- and dark-reared animals as compared with normally-reared ones. The changes in receptor density were transient since from the 6th day onward they gradually disappeared, being almost identical in the three groups by the day 15. At this moment the number of benzodiazepine receptor sites stabilized at the adult level. Scatchard analysis at the 2nd postnatal day revealed that the differences observed in the high affinity benzodiazepine binding sites between the three groups were due to modifications in the total number of binding sites while the affinity remained unchanged. The maximal number of binding sites were: 2.76 +/- 0.03, 3.40 +/- 0.01 and 1.46 +/- 0.11 pmol/mg protein in normally-, light- and dark-reared chicks, respectively; while the apparent dissociation constants were unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective inhibition of the binding of 3H-(3-MeHis2) thyrotropin releasing hormone to rat amygdala membranes by some naturally occurring cannabinoids

The effect of naturally occurring cannabinoids, delta 9-tetrahydrocannabinol (THC), cannabinol (CBN) and cannabidiol (CBD), on the brain receptors for thyrotropin releasing hormone (TRH) was investigated. TRH receptors were labeled with 3H-(3-MeHis2)TRH (3H-MeTRH). 3H-MeTRH bound specifically to rat brain membranes at a single high affinity site with a Bmax value of 49.2 +/- 0.96 fmol per mg protein and a Kd value of 3.83 +/- 0.12 nM. The binding of 3H-MeTRH to whole brain membranes was inhibited when rats were injected intraperitoneally with 3 to 30 mg/kg of THC. The extent of inhibition in the binding at 10 and 30 mg/kg was similar. THC (10 mg/kg) significantly inhibited the binding of 3H-MeTRH to amygdala membranes but did not affect the binding to membranes prepared from hippocampus, septum, cortex, striatum and the rest of the brain. THC, CBN and CBD in doses of 3 to 30 mg/kg did not affect the binding of 3H-MeTRH to hypothalamic membranes. All the three cannabinoids at 30 mg/kg inhibited the binding of 3H-MeTRH to amygdala membranes. The inhibition in the binding of 3H-MeTRH by the cannabinoids was due to changes in the Kd values but the Bmax values remained unchanged. It is concluded that both psychotomimetic and nonpsychotomimetic cannabinoids inhibit the binding of 3H-MeTR to amygdala membranes selectively, which is accomplished by decreases in the affinity of the ligand to receptors, and the amygdala may be an important brain area in some of the actions of cannabinoids.     

Decreased alpha 1-adrenoceptor responsiveness and density in liver cells of thyroidectomized rats

The effects of hypothyroidism on the hepatic alpha 1-receptor system were studied in isolated rat liver cells. Phenylephrine and vasopressin caused concentration-dependent activation of glycogen phosphorylase and release of 45Ca from 45Ca-loaded cells in either normal or thyroidectomized rats. However, the magnitude of both responses to phenylephrine was markedly suppressed after thyroidectomy and could be restored to near normal levels by in vivo treatment with 1-triiodothyronine (0.25 mg/kg/day) for 4 days. The potency of vasopressin to induce phosphorylase activation and 45Ca release was only slightly reduced by thyroidectomy. Binding of [3H]prazosin to putative alpha 1-receptors in purified liver plasma membranes revealed that the above changes were accompanied by a decrease in the density of binding sites from 567 +/- 51 fmol/mg of protein in controls to 326 +/- 51 fmol/mg in thyroidectomized rats and a return to 498 +/- 23 fmol/mg in thyroidectomized rats treated with 1-triiodothyronine. The affinity of binding sites for [3H]prazosin or for alpha-receptor agonists was the same in the three groups of rats and affinity for epinephrine was unaffected by the presence of guanyl-5'-yl imidodiphosphate (30-100 microM). From these findings, it appears that a reduction in the number of hepatic alpha 1-receptors is responsible for the selective decrease in alpha-adrenergic responses in the hypothyroid rat liver. These changes are opposite to those previously reported for hepatic beta-receptors.     

Regulation of the mouse aggressive behavior (pharmacologic analysis of the GABAergic mechanism)

Ethological procedures were used to study the effects of GABA-positive drugs on aggression in male albino mice kept in isolation (opponent test). The results revealed several variants of antiaggressive effects of the tested GAB Aergic drugs: 1) antiaggressive, re-socializing of GABAA agonists muscimol (0.125 and 0.5 mg/kg) and THIP (2.0 mg/kg), and GABAB agonist baclofen (2.5-10 mg/kg); 2) antiaggressive, sedative of GABAB agonists baclofen (12.5 mg/kg), phenibut (50-100 mg/kg), and inhibitor of GABA transamininase sodium valproate (100 mg/kg); 3) antiaggressive, anxiogenic for muscimol (1 mg/kg), THIP (5 mg/kg), and sodium valproate (25-50 mg/kg).     

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.