The imidazobenzodiazepine Ro 15-4513 antagonizes methoxyflurane anesthesia

Parenteral administration of the imidazobenzodiazepine Ro 15-4513 (a high affinity ligand of the benzodiazepine receptor with partial inverse agonist qualities) produced a dose dependent reduction in sleep time of mice exposed to the inhalation anesthetic, methoxyflurane. The reductions in methoxyflurane sleep time ranged from approximately 20% at 4 mg/kg to approximately 38% at 32 mg/kg of Ro 15-4513. Co-administration of the benzodiazepine receptor antagonist Ro 15-1788 (16 mg/kg) or the inverse agonists DMCM (5-20 mg/kg) and FG 7142 (22.5 mg/kg) blocks this effect which suggests that the reductions in methoxyflurane sleep time produced by Ro 15-4513 are mediated via occupation of benzodiazepine receptors. Moreover, neither DMCM (5-20 mg/kg) nor FG 7142 (22.5 mg/kg) reduced methoxyflurane sleep time which suggests this effect of Ro 15-4513 cannot be attributed solely to its partial inverse agonist properties. These observations support recent findings that inhalation anesthetics may produce their depressant effects via perturbation of the benzodiazepine/GABA receptor chloride channel complex, and suggest that Ro 15-4513 may serve as a prototype of agents capable of antagonizing the depressant effects of inhalation anesthetics such as methoxyflurane.     

Anxiolytic effect of Kami-Shoyo-San (TJ-24) in mice: possible mediation of neurosteroid synthesis.

We assessed the anxiolytic effect of Kami-Shoyo-San (Jia-wei-xiao-yao-san; TJ-24), one of a traditional Chinese herbal medicine used for the treatment of menopausal anxiety, by the social interaction (SI) test in male mice. Acute administration of TJ-24 (25-100 mg/kg, p.o.), as well as the gamma-amino-butyric acidA/benzodiazepine (GABA(A)/BZP) receptor agonist diazepam (1-3 mg/kg, i.p.), dose dependently increased the SI time, respectively. The GABA(A) receptor antagonist picrotoxin blocked the effects of TJ-24 and diazepam. TJ-24-induced SI behavior was significantly blocked by the GABA(A)/BZP receptor inverse agonist Ro 15-4513 and the GABA(A)/BZP receptor antagonist flumazenil. In addition, 5alpha-reductase inhibitor finasteride potently blocked the effect of TJ-24 without attenuating the basal level by itself. These findings suggest that TJ-24 shows the anxiolytic effect through the neurosteroid synthesis followed by GABA(A)/BDZ receptor stimulations.     

Ro 15-1788 antagonizes the discriminative stimulus effects of diazepam in rats but not similar effects of pentobarbital

The benzodiazepine antagonist properties of Ro 15-1788 were evaluated in rats trained to discriminate between saline and either 1.0 mg/kg of diazepam or 10 mg/kg of pentobarbital in a two-choice discrete-trial shock avoidance procedure. When administered alone, 1.0 mg/kg of diazepam and 10 mg/kg of pentobarbital produced comparable amounts of drug-appropriate responding (> 84%), whether rats were trained to discriminate between diazepam or pentobarbital and saline. Ro 15-1788 (3–32 mg/kg, p.o.), administered 10 min before diazepam or pentobarbital, produced a dose-related blockade of the discriminative effects of diazepam in both groups of rats, but was completely ineffective in blocking the discriminative effects of pentobarbital. The dose-effect curve for the discriminative effects of diazepam was shifted to the right in a parallel fashion 3- and 13-fold by 10 and 32 mg/kg of Ro 15-1788, respectively, indicating that Ro 15-1788 acts as a surmountable, competitive antagonist of diazepam. When administered alone, Ro 15-1788 (32–100 mg/kg, p.o.) produced primarily saline-appropriate responding, although 100 mg/kg of Ro 15-1788 produced drug-appropriate responding in one out of eight rats. When administered orally 30 min after diazepam, Ro 15-1788 (32 mg/kg) completely reversed within 10 min the discriminative effects of diazepam. The blockade of diazepam's discriminative effects by 32 mg/kg of Ro 15-1788 appeared to last at least as long (approximately 2 hr) as the effects of diazepam alone.     

Ethanol-induced limb defects in mice: effect of strain and Ro15-4513

It is now thought that ethanol exerts many of its behavioral effects in the CNS by interaction with the gamma-aminobutyric acid (GABA) receptor, and it has been shown that the benzodiazepine reverse agonist Ro15-4513 reverses some of the CNS effects produced by ethanol. The hypothesis was tested that ethanol exerts its teratogenic effects through interaction with a putative embryonic GABA receptor by determining whether Ro15-4513 reverses ethanol-induced forelimb ectrodactyly in C57BL/6 mice. First, pregnant C57BL/6 dams were injected twice i.p. with ethanol (2.9 g/kg body weight, 4 hr apart) on day 10 of gestation: 49% of the fetuses were resorbed or dead and 46% of the survivors showed forelimb ectrodactyly. In contrast, when SWV mice were treated with ethanol, embryolethality was only 11.9% and no forelimb ectrodactyly was observed. In a second experiment, when ethanol (2.6 g/kg x 2) was administered to C57BL/6 mice, 34% resorptions and 31% forelimb ectrodactyly were observed. Ectrodactyly induced by ethanol was primarily of the forelimb and exclusively postaxial. Ethanol produced an unusual forelimb defect in a small number of instances where there was a postaxial autopod reduction defect coupled with a preaxial zeugopod reduction defect. Ro15-4513 administered alone (50 mg/kg x 2) was neither embryolethal nor teratogenic in C57BL/6 mice. To attempt to reverse the teratogenic effect of ethanol, dams that were injected 5 min before each ethanol administration with Ro15-4513 (0.5, 1, 2.5, 5, 10 mg/kg twice) showed no significant change in frequency of forelimb ectrodactyly compared to embryos treated with ethanol alone. However, resorptions increased significantly to 77% and 62% with the 5 and 10 mg/kg doses of Ro15-4513. Thus there appears to be an embryolethal interaction of Ro15-4513 with ethanol. Nevertheless, since Ro15-4513 did not reverse the teratogenic effect induced by ethanol, these results do not support the hypothesis that the teratogenic mechanism of ethanol is mediated through a putative embryonic GABA receptor.     

Characterization of Two Cerebellar Binding Sites of[3H]Ro 15-4513

Ro 15-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H- imidazo[1,5-a][1,4]benzodiazepine-3-carboxylate), a partial inverse agonist of central benzodiazepine receptors, binds to two distinct sites in the cerebellum. The binding to diazepam-sensitive (DZ-S) sites is displaced by different benzodiazepine receptor ligands, whereas the other site is insensitive to benzodiazepine agonists [diazepam-insensitive (DZ-IS)]. The binding of [3H]Ro 15-4513 was studied in pig cerebellar membranes and in receptors solubilized and purified from these. Micromolar concentrations of gamma-aminobutyric acid (GABA) decreased DZ-S binding at both 0 and 37 degrees C, whereas it had no effect on DZ-IS binding at 0 degrees C and was stimulatory at 37 degrees C. The pH profiles of [3H]Ro 15-4513 binding were quite similar in both binding sites in the pH range of 5.5-10.5 but differed at acidic pH values from those reported for flunitrazepam and Ro 15-1788 (flumazenil; ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H- imidazol[1,5-a][1,4]benzodiazepine-3-carboxylate) binding in DZ-S sites, suggesting that [3H]Ro 15-4513 does not interact with a histidine residue apparently present in the binding site. Zn2+, Cu2+, Co2+, and Ni2+ enhanced the binding to DZ-S sites, and the first three mentioned also enhanced the binding to DZ-IS sites. [3H]Ro 15-4513 binding activity was solubilized by various detergents. All detergents tested were more efficient in solubilizing DZ-S binding activity. High ionic strength improved especially the solubility of DZ-IS binding activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of Diethyl Pyrocarbonate Modification of Benzodiazepine Receptors on [3H]Ro 15-4513 Binding

The effects of treatment of brain membranes with diethyl pyrocarbonate (DEP), a histidine-modifying reagent, on the binding of 3H-labeled Ro 15-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a]- [1,4]benzodiazepine-3-carboxylate) and [3H]diazepam were compared. DEP pretreatment produced a dose-dependent decrease in [3H]diazepam binding, whereas low DEP concentrations enhanced the binding of [3H]Ro 15-4513. These effects were reversed by incubation with hydroxylamine after the treatment. The enhancement of [3H]Ro 15-4513 binding was due to an increase in the affinity of the binding sites (KD), without any effect on binding capacity (Bmax). The enhancement was perceived in cerebral cortical, cerebellar, and hippocampal membranes. DEP treatment decreased the displacement of [3H]Ro 15-4513 binding by diazepam and FG 7142 (N-methyl-beta-carboline-3-carboxamide) but not by Ro 15-4513 and Ro 19-4603 (tert-butyl-5,6-dihydro-5-methyl-6-oxo-4H-imidazol[1,5- a]thieno[2,3-f][1,4]diazepine-3-carboxylate). Although the stimulating effect of gamma-aminobutyric acid (GABA) on [3H]-diazepam binding was not affected by DEP treatment, such treatment reduced the inhibitory effect of GABA on [3H]Ro 15-4513 binding. The enhancement of [3H]Ro 15-4513 binding was observed in membranes pretreated with DEP in the presence of flunitrazepam, whereas such pretreatment reduced significantly the inhibitory effect of DEP on [3H]-diazepam binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of benzodiazepine receptors in the discriminative stimulus properties of delta-9-tetrahydrocannabinol

Twenty male Sprague-Dawley rats were trained to discriminate 3.0 mg/kg delta-9-tetrahydrocannabinol (THC) from its vehicle. Following acquisition of this discrimination animals were tested for generalization to 3.0 mg/kg diazepam. Thirteen animals showed a generalization from THC to diazepam, whereas the remaining seven animals did not. The generalization curve for diazepam was dose-dependent from 0.1 to 10.0 mg/kg in the first group; the latter group showed no generalization from THC at any dose of diazepam in this range. No differences were found between these groups in the generalization curve for THC. The benzodiazepine antagonist Ro 15-1788 (2.0 mg/kg) antagonized the generalization to diazepam in the group that discriminated diazepam as THC. In contrast, Ro 15-1788 increased THC lever responding of 10 mg/kg diazepam in the group which did not generalize from THC. Ro 15-1788 did not alter the discriminability of THC in either group. THC also showed partial generalization to pentobarbital (1 to 10 mg/kg). The generalization was again complete in one subgroup and absent in another, but there was only a 43 percent overlap between the subgroups found with testing for generalization to diazepam. The percent THC lever responding with 3.0 mg/kg pentobarbital was increased by Ro 15-1788 in the group which generalized to diazepam, but not the other group. These data suggest that the discriminative stimulus properties of THC may have some commonality with the effects of diazepam in a subpopulation of rats trained to discriminate THC. These THC-like effects of diazepam are probably mediated by benzodiazepine receptors since they are antagonized by a specific benzodiazepine receptor antagonist.     

Photoaffinity Labeling of Benzodiazepine Receptor Proteins with the Partial Inverse Agonist [3H]Ro 15–4513: A Biochemical and Autoradiographic Study

Photolabeling of the benzodiazepine receptor, which to date has been done with benzodiazepine agonists such as flunitrazepam, can also be achieved with Ro 15-4513, a partial inverse agonist of the benzodiazepine receptor. [3H]Ro 15-4513 specifically and irreversibly labeled a protein with an apparent molecular weight of 51,000 (P51) in cerebellum and at least two proteins with apparent molecular weights of 51,000 (P51) and 55,000 (P55) in hippocampus. Photolabeling was inhibited by 10 microM diazepam but not by 10 microM Ro 5-4864. The BZ1 receptor-selective ligands CL 218872 and beta-carboline-3-carboxylate ethyl ester preferentially inhibited irreversible binding of [3H]Ro 15-4513 to protein P51. Not only these biochemical results but also the distribution and density of [3H]Ro 15-4513 binding sites in rat brain sections were similar to the findings with [3H]flunitrazepam. Thus, the binding sites for agonists and inverse agonists appear to be located on the same proteins. In contrast, whereas [3H]flunitrazepam is known to label only 25% of the benzodiazepine binding sites in brain membranes, all binding sites are photolabeled by [3H]Ro 15-4513. Thus, all benzodiazepine receptor sites are associated with photolabeled proteins with apparent molecular weights of 51,000 and/or 55,000. In cerebellum, an additional protein (MW 57,000) unrelated to the benzodiazepine receptor was labeled by [3H]Ro 15-4513 but not by [3H]flunitrazepam. In brain sections, this component contributed to higher labeling by [3H]Ro 15-4513 in the granular than the molecular layer.     

Behavioral effects and benzodiazepine antagonist activity of Ro 15-1788 (flumazepil) in pigeons

The selective benzodiazepine receptor antagonist, Ro 15-1788, produced behavioral effects in pigeons at doses at least 100 times lower than those previously reported to possess intrinsic pharmacological activity in mammals. In contrast to its effects in mammalian species, in pigeons, Ro 15-1788 does not exhibit partial agonist activity. Key-peck responses of pigeons were studied under a multiple fixed-interval 3-min, fixed-interval 3-min schedule in which the first response after 3-min produced food in the presence of red or white keylights. In addition, every 30th response during the red keylight produced a brief electric shock (punishment). Under control conditions, punished responding was suppressed to 30% of unpunished response levels. Ro 15-1788 (0.01 mg/kg, i.m.) increased unpunished response rates by 33% without affecting rates of punished responding. Doses of 0.1 to 1.0 mg/kg Ro 15-1788 produced dose-related decreases in both punished and unpunished responding. As is characteristic of other benzodiazepines, midazolam (0.1 and 0.3 mg/kg, i.m.) markedly increased punished responding but had little effect on rates of unpunished responding. Ro 15-1788 antagonized the increases in punished responding and also reversed the rate-decreasing effects of higher doses of midazolam. However, the effectiveness of Ro 15-1788 as a benzodiazepine antagonist was limited by its intrinsic activity: rate-decreasing doses of Ro 15-1788 were unable to completely reverse behavioral effects of midazolam. Midazolam was an effective antagonist of the behavioral effects of Ro 15-1788 (up to 0.1 mg/kg) but midazolam did not influence the rate-decreasing effects of 1.0 mg/kg Ro 15-1788 across a 100-fold dose range. In the pigeon, the behavioral effects of relatively low doses of Ro 15-1788 (0.01-0.1 mg/kg) appear to be related to benzodiazepine receptor mechanisms, whereas other systems appear to be involved in the effects of higher doses.     

An interaction between benzodiazepines and neuroactive steroids at GABA A receptors in cultured hippocampal neurons

Neurosteroids are modulators of several receptors and ion channels and are implicated in the pathophysiology of several neuropsychiatric diseases including hepatic encephalopathy (HE). The neurosteroid, allopregnanolone, a positive allosteric modulator of GABA_A receptors, accumulates in the brains of HE patients where it can potentiate GABA_A receptor-mediated responses. Attenuation of the effects of neurosteroids on GABA-ergic neurotransmission is therefore of interest for the management of HE. In the present study, we determined the effect of the benzodiazepine partial inverse agonist, Ro15-4513, and the benzodiazepine antagonist, flumazenil on modulation of the GABA_A mediated chloride currents by allopregnanolone and on spontaneous synaptic activity in cultured hippocampal neurons using the patch-clamp technique. Allopregnanolone (0.03–0.3 μM), dose-dependently potentiated GABA-induced currents, an action significantly reduced by Ro15-4513 (10 μM). In contrast, flumazenil (10 μM) had no effect on the ability of allopregnanolone to potentiate GABA_A currents but it blocked the effects of Ro15-4513. The frequency of spontaneous synaptic activity was significantly reduced in the presence of allopregnanolone (0.1 μM) from 1.5 ± 0.7 to 0.1 ± 0.04 Hz. This action was partially reversed by Ro15-4513 (10 μM) but was not significantly influenced by flumazenil (10 μM). These findings suggest that the beneficial affects of Ro15-4513 in experimental HE result from attenuation of the effects of neurosteroids at GABA_A receptors. Our results may provide a rational basis for the use of benzodiazepine inverse agonists in the management and treatment of hepatic encephalopathy in patients with liver failure.     

Discriminative stimulus properties of oxazepam in the pigeon

Five pigeons were trained to discriminate IM injections of oxazepam (4.0 mg/kg) from vehicle with responding maintained under a fixed-ratio 30 schedule of food delivery. Under test conditions, responding increased in a dose-dependent manner in all pigeons after the administration of other benzodiazepines including diazepam (0.01-1.0 mg/kg), temazepam (0.01-3.0 mg/kg), halazepam (0.1-56.0 mg/kg), and midazolam (0.1-1.0 mg/kg) as well as the barbiturate pentobarbital (2.0-8.0 mg/kg) and the non-benzodiazepine anxiolytic CL 218,872 (1.0-8.0 mg/kg). At the higher doses of each of these compounds, over 80% of responding occurred on the oxazepam-appropriate key. Cocaine (0.5-4.0 mg/kg), bupropion (3.0-56.0 mg/kg) and nortriptyline (3.0-56.0 mg/kg) failed to substitute for oxazepam even at doses that decreased rates of responding. The discriminative stimulus (DS) effects of the lowest doses of oxazepam and CL 218,872 that produced 100% drug-appropriate responding were blocked by the benzodiazepine antagonist Ro 15-1788. This antagonism was reversed by increasing the dose of the agonists. The DS effects of diazepam were antagonized partially by Ro 15-1788 (3 of 5 pigeons), and the antagonism was reversed by higher doses of diazepam in two of these pigeons. The DS effects of pentobarbital were antagonized by Ro 15-1788 in 2 of 5 pigeons, but the blockade was not reversed by higher pentobarbital doses.     

Identification of the bovine gamma-aminobutyric acid type A receptor alpha subunit residues photolabeled by the imidazobenzodiazepine [3H]Ro15-4513

Ligands binding to the benzodiazepine-binding site in gamma-aminobutyric acid type A (GABA(A)) receptors may allosterically modulate function. Depending upon the ligand, the coupling can either be positive (flunitrazepam), negative (Ro15-4513), or neutral (flumazenil). Specific amino acid determinants of benzodiazepine binding affinity and/or allosteric coupling have been identified within GABA(A) receptor alpha and gamma subunits that localize the binding site at the subunit interface. Previous photolabeling studies with [(3)H]flunitrazepam identified a primary site of incorporation at alpha(1)His-102, whereas studies with [(3)H]Ro15-4513 suggested incorporation into the alpha(1) subunit at unidentified amino acids C-terminal to alpha(1)His-102. To determine the site(s) of photoincorporation by Ro15-4513, we affinity-purified ( approximately 200-fold) GABA(A) receptor from detergent extracts of bovine cortex, photolabeled it with [(3)H]Ro15-4513, and identified (3)H-labeled amino acids by N-terminal sequence analysis of subunit fragments generated by sequential digestions with a panel of proteases. The patterns of (3)H release seen after each digestion of the labeled fragments determined the number of amino acids between the cleavage site and labeled residue, and the use of sequential proteolytic fragmentation identified patterns of cleavage sites unique to the different alpha subunits. Based upon this radiochemical sequence analysis, [(3)H]Ro15-4513 was found to selectively label the homologous tyrosines alpha(1)Tyr-210, alpha(2)Tyr-209, and alpha(3)Tyr-234, in GABA(A) receptors containing those subunits. These results are discussed in terms of a homology model of the benzodiazepine-binding site based on the molluscan acetylcholine-binding protein structure.     

Ultrasonic vocalization in response to unavoidable aversive stimuli in rats: effects of benzodiazepines

The effects of two benzodiazepine derivatives (diazepam, 0.5-1 mg/kg; alprazolam, 1.25-2.5 mg/kg) on ultrasonic calling elicited in adult rats by unavoidable aversive stimuli (footshocks) were investigated. The results show that either diazepam or alprazolam affected the duration of ultrasonic calls. In particular, a significant decrease in the length of ultrasounds was found in the group of animals treated with these benzodiazepines. The effects of diazepam were counteracted by the benzodiazepine-antagonist Ro 15-1788. On the other hand, neither a neuroleptic agent, such as haloperidol (0.5-1 mg/kg), nor an antidepressant, such as desipramine (5-10 mg/kg) influenced the parameters of ultrasonic emission in this experimental situation. The present results suggest that ultrasonic vocalization in response to unavoidable aversive stimuli could be considered as a potential new tool for studying drugs with antianxiety properties.     

Convulsions induced by submaximal dose of pentylenetetrazol in mice are antagonized by the benzodiazepine antagonist Ro 15-1788

The effects of benzodiazepine antagonist Ro 15–1788, alone or with diazepam, were studied in mice on convulsions induced by pentylenetetrazol (PTZ). We found that Ro 15–1788 (1 mg/kg) was able to antagonize the anticonvulsive effects of diazepam (1 mg/kg), but also had, with submaximal doses of PTZ (65 mg/kg), its own anti-convulsive action. At very low doses (0.1 mg/kg), it even potentiated the anticonvulsive effects of diazepam (0.05 mg/kg). This dual action provides evidence for partial agonist properties of the antagonist Ro 15–1788.     

Benzodiazepine Prevention of Swim Stress-Induced Sensitization of Cortical Biogenic Amines: An in Vivo Microdialysis Study

In vivo microdialysis was used to determine the effect of diazepam, flumazenil and FG-7142 upon the biogenic amine response to acute and repeated swim stress in the medial prefrontal cortex of the rat. Acute swim stress increased norepinephrine levels, although dopamine and serotonin levels remained stable. Upon re-exposure to swim stress twenty-four hours later, sustained increases (200–300% of baseline) in all three biogenic amines were detected. This enhanced response to re-stress was not seen in rats pretreated with either a benzodiazepine agonist (diazepam, 2 mg/kg), an antagonist (flumazenil, 10 mg/kg), or an inverse agonist (FG-7142, 10 mg/kg) given prior to the first swim stress. Therefore, the sensitization of biogenic amine response to re-stress may be prevented by compounds which differ in their activity at the benzodiazepine receptor.     

Modulation of the Electrically Evoked Release of 5-[3H]Hydroxytryptamine from Rat Cerebral Cortex: Effects of Alpidem, CL 218872, and Diazepam

The effect of omega (benzodiazepine)-receptor agonists, antagonists, and inverse agonists on the electrically evoked release of 5-[3H]hydroxytryptamine ([3H]5-HT) was studied in superfused slices of the rat frontal cerebral cortex. The electrically evoked release of [3H]5-HT was enhanced by nanomolar concentrations of diazepam and the selective omega 1-receptor agonists alpidem and CL 218872. The omega 1/omega 2- and omega 1-receptor antagonists flumazenil and CGS 8216, respectively, did not modify the electrically evoked release of [3H]5-HT. The omega 3-receptor agonist Ro 5-4864 and the omega 1-receptor inverse agonist ethyl-beta-carboline-3-carboxylate on their own did not affect the electrically evoked release of [3H]5-HT. On the other hand, the inverse agonist 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylic acid methyl ester (DMCM), at micromolar concentrations, inhibited both the spontaneous and the evoked release of [3H]5-HT. The facilitation of the electrically evoked release of [3H]5-HT by diazepam, alpidem, or CL 218872 was potentiated by gamma-aminobutyric acid (GABA). Exposure to flumazenil and CGS 8216 antagonized the facilitation by diazepam, alpidem, or CL 218872 of [3H]5-HT release. The inhibition of the release of [3H]5-HT by DMCM was not modified by exposure to either flumazenil, CGS 8216, or GABA. The inhibitory effect of DMCM was not observed when monoamine oxidase activity was inhibited by pargyline.(ABSTRACT TRUNCATED AT 250 WORDS)     

The benzodiazepine recognition site inverse agonists Ro 15-4513 and FG 7142 both antagonize the EEG effects of ethanol in the rat

The aim of the present study was to compare the ability of Ro 15-4513 and FG 7142, two inverse agonists for benzodiazepine recognition sites, to antagonize the EEG effects of ethanol in freely moving rats. Ethanol (2.5 g/kg, p.o.) induced sedation and ataxia associated with a progressive suppression of the fast cortical activities and an enhancement of low frequencies in both cortical and hippocampal tracings. In contrast, Ro 15-4513 (2 mg/kg, i.p.) and FG 7142 (10 mg/kg, i.p.) both caused a state of alertness associated with desynchronized cortical activity and theta hippocampal rhythm as well as spiking activity which was predominantly observed in the cortical tracings. When rats were treated with FG 7142 or RO 15-4513 either before or after ethanol, a reciprocal antagonism of the behavioral and EEG effects of ethanol and of the partial inverse agonists was observed. These data support the view that the anti-ethanol effects of Ro 15-4513 may be related to its partial inverse agonist properties.     

Discriminative stimulus properties of methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM), an inverse agonist at benzodiazepine receptors

Rats (N = 8) were trained to discriminate the stimulus properties of the potent benzodiazepine (BZ) receptor inverse agonist methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) from saline in a two-lever operant task. The initial training dose of DMCM was 0.4 mg/kg at which the discrimination developed slowly; increasing the dose to 0.8 mg/kg resulted in rapid acquisition. However, since convulsions eventually developed during further training (sensitization), the training dose was finally individualized below the convulsive threshold (0.4-0.7 mg/kg). The DMCM cue was mimicked by FG 7142 (10 mg/kg), a non-convulsant anxiogenic beta-carboline, by pentylenetrazol (20-30 mg/kg), and by the GABA antagonist bicuculline (2 mg/kg). The DMCM cue was not, or marginally, blocked by diazepam (2.5 mg/kg) or pentobarbital (10-15 mg/kg). Furthermore, the BZ receptor antagonists CGS 8216 (2.5 mg/kg), ZK 93426 (20 mg/kg), and Ro 15-1788 (20-80 mg/kg) also did not, or only marginally, block the DMCM cue. However, the receptor antagonists (alone) substituted for DMCM although Ro 15-1788 was less effective. The partial BZ receptor agonist ZK 91296 (25 mg/kg), which is structurally similar to DMCM, blocked completely the DMCM stimulus effect. THIP (4 mg/kg) did not block the DMCM cue. To explain these results, we suggest that the repeated DMCM treatment, necessary for maintaining the discrimination, shifts the balancing point ("set-point") for positive (i.e., BZ-like) agonist efficacy versus inverse agonist efficacy, towards inverse action. This hypothesis was supported by the finding of an enhanced ability of GABA to reduce 3H-DMCM binding to cortical neuronal membranes of animals treated chronically with DMCM in a regimen similar to that used to maintain the DMCM discrimination. Furthermore, this treatment did not affect baseline 3H-DMCM binding, baseline or GABA stimulated 3H-diazepam binding, or 35S-TBPS binding (to chloride channels).     

Diazepam Sensitivity of the Binding of an Imidazobenzodiazepine, [3H]Ro 15-4513, in Cerebellar Membranes from Two Rat Lines Developed for High and Low Alcohol Sensitivity

Ro 15-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a] [1,4]benzodiazepine-3-carboxylate), a partial inverse agonist of brain benzodiazepine receptors, has been shown to antagonize some actions of ethanol. In addition to conventional benzodiazepine binding sites, Ro 15-4513 binds to a specific cerebellar protein, the binding of which has been shown to be insensitive to diazepam. The binding of [3H]Ro 15-4513 was studied in washed membranes of the cerebellum, hippocampus, and cerebral cortex of two rat lines developed for differences in their sensitivity to ethanol-induced motor impairment. Only minor differences were found in the estimated parameters (KD and Bmax) for the total specific binding between the rat lines. The main difference between the rat lines was, however, observed in the characteristics of the cerebellar binding, all of which was displaced by diazepam in most of the alcohol-sensitive [alcohol-nontolerant (ANT)] rats, in contrast to only approximately 75% displacement in most of the alcohol-insensitive [alcohol-tolerant (AT)] ones. The following cerebellar results were obtained with the major subgroups of both lines, i.e., with the AT rats chosen for the presence of the diazepam-insensitive binding and with the ANT rats chosen for its absence. The KD for the total specific [3H]Ro 15-4513 binding in the ANT animals was about half of that in the AT animals. No line difference was found in the Bmax of the binding in these rats. Photolabeling with [3H]Ro 15-4513 showed that the diazepam-insensitive binding was in a protein with a molecular weight of 55,000.(ABSTRACT TRUNCATED AT 250 WORDS)     

Benzodiazepine receptor mediated discriminative cues: Effects of GABA-ergic drugs and inverse agonists

Rats were exposed to a two-layer drug discrimination procedure using the benzodiazepine (BZ) receptor inverse agonists N′-methyl-β-carboline-3-carboxamide (FG 7142) or methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM). FG 7142 (30 mg/kg) failed to acquire discriminative stimulus control, although it did suppress responding. The same group of animals was trained successfully to discriminate diazepam (DZP, 2.5 mg/kg) from vehicle. The DZP cue was potentiated by the GABA agonist 4,5,6,7-tetrahydro-isoxazolo [5, 4-c] pyridin-3-ol (THIP, 1–3 mg/kg); THIP alone produced vehicle-appropriate responding. In addition, clonazepam (0.2 mg/kg) and chlordiazepoxide (5 mg/kg) substituted for DZP (with potencies of 7.5 and 0.25 times that of DZP, respectively). In antagonism tests, FG 7142 (5–17.5 mg/kg), methyl-β-carboline-3-carboxylate (β-CCM, 2.5 mg/kg), nicotine (0.3 mg/kg), harmaline (5 mg/kg) and naltrexone (10 mg/kg) did not effect, bicuculine (2 mg/kg) and DMCM (1 mg/kg) partially blocked, and the BZ receptor antagonist Ro 15–1788 (40 mg/kg) completely blocked the discriminative stimulus effects of DZP. In animals trained to discriminate DMCM (0.2 mg/kg) from vehicle, 95% substitution occured with bicuculline (2 mg/kg); DZP (1–5 mg/kg) completely antagonized DMCM. These results indicate that the DZP cue is mediated by GABA-coupled BZ receptors and that GABA may modulate the efficacy of a BZ at its receptor site. However, since inverse BZ receptor agonists (FG 7142, DMCM and β-CCM) were, at best, only marginally effective in antagonizing DZP, the DZP cue may be mediated by a distinct subclass of BZ receptors.