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.     

Behavioral interactions between ethanol and imidazodiazepines with high affinities for benzodiazepine receptors

The intrinsic effects of two imidazodiazepines RO 15-3505 and RO 17-1812 on the behavior of mice in a holeboard test were investigated. The interactions of these two drugs with ethanol were also studied. RO 15-3505 (0.75-6.0 mg/kg) failed to significantly alter either exploratory head-dipping or locomotor activity when administered alone but doses of 0.75 and 1.5 mg/kg reversed the reduction in the number of head-dips caused by ethanol (2 g/kg) and partially reversed ethanol's locomotor stimulant action. In contrast, RO 17-1812 (0.75-6.0 mg/kg) increased locomotor activity when administered alone, and enhanced the reduction in exploration caused by ethanol. Neither RO 15-3505 nor RO 17-1812 altered blood alcohol concentrations suggesting a pharmacodynamic basis for these interactions. The results suggest that in the holeboard test the interactions of imidazodiazepines with ethanol are related to the nature of their interaction with benzodiazepine receptors, inverse agonists antagonising and agonists enhancing ethanol's effects on exploration.     

Effects of the imidazobenzodiazepine RO15-4513 on the stimulant and depressant actions of ethanol on spontaneous locomotor activity

The purpose of this study was to investigate the effects of the imidazobenzodiazepine RO15-4513, a partial inverse agonist at benzodiazepine (BDZ) receptors, on the stimulant and depressant actions of ethanol in mice. For comparative purposes, another BDZ inverse agonist, FG-7142, was examined as well. Neither RO15-4513 nor FG-7142 influenced the low-dose excitatory effects of ethanol on spontaneous locomotor activity. However, both RO15-4513 and FG-7142 significantly antagonized the depressant effects of ethanol, and this antagonism was completely reversed by pretreatment with the BDZ receptor antagonist, RO15-1788. These data suggest that RO15-4513 is capable of antagonizing only some of the behavioral effects of ethanol, and in particular, those responses to ethanol that are mediated by modulation of the GABA/BDZ-chloride channel receptor complex.     

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.     

The Anxiogenic β-Carboline FG 7142 Selectively Increases Dopamine Release in Rat Prefrontal Cortex as Measured by Microdialysis

The effect of the anxiogenic beta-carboline methyl-beta-carboline-3-carboxyamide (FG 7142) on dopamine release in prefrontal cortex and striatum in the awake freely moving rat was determined using the technique of microdialysis. FG 7142 (25 mg/kg, i.p.) caused a time-dependent increase in dopamine release in prefrontal cortex which was statistically significantly greater than the response to vehicle administration. Dopamine release in striatum was unaltered by FG 7142. Pretreatment of animals with the benzodiazepine antagonist Ro 15-1788 (30 mg/kg, i.p., 15 min prior to FG 7142 administration) completely abolished the increase in dopamine release caused by FG 7142 in prefrontal cortex. These data indicate that the anxiogenic benzodiazepine inverse agonist FG 7142 can selectively increase dopamine release in prefrontal cortex, and that this effect appears to be mediated via the gamma-aminobutyric acid/benzodiazepine receptor complex.     

GABAergic mechanisms in the electrophysiological actions of ethanol on cerebellar neurons

We have found that the partial inverse benzodiazepine agonists Ro 15-4513 and FG 7142 antagonize the depressant electrophysiologial effects of locally applied ethanol in the cerebellum. Although absolute tissue concentrations are not known, dose-response curves constructed using pressure-ejection doses as previously described (31, 25) we found that FG 7142 was more efficacious, but less potent than Ro 15-4513. Our observation that ethanol and inverse benzodiazepine agonists have interactions which are not competitive might suggest that these two drugs act through separate, but interactive mechanisms in order to produce the observed ethanol antagonism. If such independent interactions were mediated at different sites on a given macromolecular complex, such as the GABA_a/Cl^– channel, then one might expect to find allosteric interactions between those sites as well as with the functional response of the complex to GABA activation. Indeed, this hypothesis is consistent with the recent finding of Harris and collaborators that ethanol potentiates the inverse agonist actions of Ro 15-4513 and FG 7142. On the other hand, we were unable to find large ethanol-induced potentiations of GABA effects on all neurons which showed depressant responses to ethanol administration in rat cerebellum. However we did find that the GABA_a antagonist, bicuculline, blocks the depressant effects of ethanol on the same neurons. We conclude that the interaction between ethanol and GABA probably does not occur directly at the GABA_a receptor site, but that the GABA_a mechanism does play a permissive role in the ethanol-induced depressions of cerebellar Purkinje neurons. Thus, although a postsynaptic GABA_a mechanism may not be the primary locus of action at which ethanol causes depressant electrophysiological responses of neurons, activation of the GABA_a receptor may be required to make cerebellar Purkinje neurons responsive to the depressant actions of ethanol. Further investigations will be required to determine the pre vs postsynaptic nature of this interaction of ethanol with the GABA mechanism of action.Special issue dedicated to Dr. Erminio Costa     

The benzodiazepine receptor inverse agonists beta-CCM and RO 15-3505 both reverse the anxiolytic effects of ethanol in mice

The antagonistic effects of the benzodiazepine receptor inverse agonist beta-CCM (1 mg/kg) and of the partial inverse agonist RO 15-3505 (3 mg/kg) on the anxiolytic properties of ethanol (1 g/kg) in mice confronted with a light/dark choice procedure and with the staircase test were investigated. Both drugs reversed the effects of ethanol on some of the behavioral parameters, but beta-CCM alone elicited anxiogenic intrinsic effects. RO 15-3505 induced seizures in mice treated with a subconvulsant dose of pentylenetetrazole, the most efficient doses being 3 and 6 mg/kg. These data indicate that beta-CCM and RO 15-3505 can reverse some of the anxiolytic effects of ethanol, acting probably to oppose GABA function via the benzodiazepine receptor.     

Comparative study of the effect of 3-carboxy-beta-carboline methylamide and diazepam on rat behavior

The effects of benzodiazepine receptor agonist, diazepam, and inverse agonist, FG 7142, were examined. Strong antagonism between FG 7142 (10 mg/kg) and diazepam (1 mg/kg) activity was revealed in the open field test. On the other hand, both FG 7142 and diazepam inhibited isolation-induced intraspecies aggressive behaviour of rats. FG 7142 also reduced interspecies aggression of mouse-killing rats. The findings suggest that both diazepam and FG 7142 have antiaggressive properties in the isolation-induced aggression model, which are mediated by benzodiazepine receptors of the central nervous system.     

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.     

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.     

Reversal of the anti-conflict action of valproate by various GABA and benzodiazepine antagonists

The effects of RO 15-1788, RO 5-3663, picrotoxin and bicuculline on the anti-conflict properties of valproate were studied in rats using a modified Vogel 's conflict test procedure. A low dose of the benzodiazepine (BDZ) antagonist, RO 15-1788 (5 mg/kg), blocked the anti-punishment properties of valproate (400 mg/kg), whereas no antagonism was observed after a high dose (25 mg/kg) of the BDZ antagonist. High doses of RO 5-3663 or picrotoxin also reversed the anti-conflict action of valproate. Bicuculline did not change the effects of valproate in this test situation. The suppressive effect of valproate on locomotor activity was reversed by a low dose (5 mg/kg) of RO 15-1788, but not by the other antagonists. RO 5-3663 was the only antagonist which effectively reversed the muscle relaxant effects of valproate observed in a Rotarod performance test. These findings indicate that various pharmacological actions of valproate may be due to a complex interplay with several sites at the GABA-BDZ-receptor complex.     

Effect of the beta-carboline derivative FG 7142 on inhibition in hippocampal sections

The action of anxiogenetic derivative of beta-carboline FG7142 on evoked activity of neurons in hippocampal sections was investigated using extra- and intracellular recordings. The activity in CAI area was registered upon stimulation of Schaffer's collaterals (SC). Excitatory effect of FG7142 (5 microM) on population spike (PS) was blocked by simultaneous diazepam (5 microM) or RO15-1788 (5 microM) application. This suggests that FG7142 action is mediated by benzodiazepine receptors. To evaluate the action of FG7142 on hippocampal inhibitory processes paired-pulse stimulation of SC was used. It was shown that FG7142 (5 microM) increased 4-5 times the amplitude of the second PS, the amplitude of the first one being much less augmented (10-20%). Such an effect may probably be associated with specific action of GABA inhibition. The following application of diazepam (5 microM) partially reversed disinhibitory effect of FG7142. The main intracellular change observed was the appearance of the local depolarization following the single action potential (AP). With the application continued, this depolarization gave rise to the second AP. The data suggest that suppression of hippocampal inhibitory circuits may contribute to the development of the anxiety feeling.     

Selective Increase in Dopamine Utilization in the Shell Subdivision of the Nucleus Accumbens by the Benzodiazepine Inverse Agonist FG 7142

Abstract: We examined the effects of the benzodiazepine inverse agonist FG 7142 on dopamine metabolism in the core and shell subdivisions of the nucleus accumbens. FG 7142 (15 mg/kg i.p.) or vehicle was administered to adult male rats 30 min before they were killed. Selected brain regions, including samples from the whole nucleus accumbens as well as core and shell subdivisions, were collected and assayed for tissue concentrations of dopamine and its major metabolite, 3,4-dihydroxyphenylacetic acid. Consistent with previous reports, FG 7142 administration increased dopamine utilization in the medial prefrontal cortex but not the whole nucleus accumbens. Examination of subdivisions revealed that FG 7142 produced increased dopamine utilization in the shell subdivision of the nucleus accumbens. No effect of FG 7142 on dopamine utilization in the core region of the nucleus accumbens was observed. These data are discussed in terms of in vivo microdialysis studies reporting increased dopamine release in the nucleus accumbens after FG 7142 administration.     

Effect of an imidazobenzodiazepine, Ro15-4513, on the incoordination and hypothermia produced by ethanol and pentobarbital

The imidazobenzodiazepine, Ro15-4513, which is a partial inverse agonist at brain benzodiazepine receptors, reversed the incoordinating effect of ethanol in mice, as measured on an accelerating Rotarod. This effect was blocked by benzodiazepine receptor antagonists. In contrast, Ro15-4513 had no effect on ethanol-induced hypothermia in mice. However, Ro15-4513 reversed the hypothermic effect of pentobarbital, and, at a higher dose, also reversed the incoordinating effect of pentobarbital in mice. The data support the hypothesis that certain of the pharmacological effects of ethanol are mediated by actions at the GABA-benzodiazepine receptor-coupled chloride channel.     

Blockade of FG 7142-Induced Increased Dopamine Utilization by the Glycine/NMDA Receptor Antagonist (+)-HA 966

Abstract: Systemic administration of the anxiogenic benzodiazepine inverse agonist FG 7142 has been shown to increase selectively dopamine utilization in the medial prefrontal cortex and the shell, but not core, subregion of the nucleus accumbens. In the present study, we examined the functional interaction between benzodiazepine and N -methyl- d -aspartate receptor influences on dopamine utilization in these areas. Male Sprague-Dawley rats were pretreated with the glycine receptor antagonist (+)-HA 966 (15 mg/kg, i.p.) or saline 15 min before FG 7142 (20 mg/kg, i.p.) or vehicle administration. Subjects were killed 30 min later and assayed for tissue concentrations of dopamine and its major metabolite 3,4-dihydroxyphenylacetic acid in the core and shell subdivisions of the nucleus accumbens and the medial prefrontal cortex. (+)-HA 966 administration blocked FG 7142-induced increased dopamine utilization in both the medial prefrontal cortex and the shell subdivision of the nucleus accumbens. Results are discussed in terms of N -methyl- d -aspartate receptor influences on the response of mesoaccumbal dopamine neurons to stress.     

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.     

Comparison of anticonvulsant effect of ethanol against NMDA-, kainic acid- and picrotoxin-induced convulsions in rats

The anticonvulsant effect of ethanol against N-methyl-D-aspartic acid-(NMDA), kainic acid-, and picrotoxin-induced convulsions was studied in rats. Ethanol (2 g/kg, ip) offered protection against these agents, and it was most effective against picrotoxin and least effective against kainic acid. MK801, NMDA receptor antagonist, also provided protection against these agents. However, it was most effective against NMDA and least effective against kainic acid. Ineffective doses of MK801 (0.1 mg/kg, ip) and ethanol (0.5 g/kg, ip), when administered concurrently, had a facilitatory anticonvulsant effect, thereby providing protection against mortality following severe convulsions induced by NMDA or picrotoxin, but not against kainic acid. The protective effect of ethanol against NMDA- and kainic acid-induced neurotoxicity, in contrast to picrotoxin-induced toxicity, was not reversed by imidazodiazepine, Ro 15-4513, an ethanol antagonist. Furthermore, Ro 15-4513 did not produce any proconvulsant effect with NMDA or kainic acid. These findings suggested that the anticonvulsant actions of ethanol may be attributed to its ability to antagonize NMDA-mediated excitatory responses and facilitate the GABAergic transmission.     

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)     

Effects of GABA(A) compounds on mCPP drug discrimination in rats

Male Long-Evans rats were trained to discriminate mCPP (1.4 mg/kg, i.p.) from saline, using a two-lever, food-reinforced operant task. The GABA(A) antagonist, bicuculline (0.16-0.64 mg/kg), partially substituted for mCPP, whereas the benzodiazepine antagonist, flumazenil (1-10 mg/kg), and the benzodiazepine inverse agonist, Ro 15-4513 (0.25-2.5 mg/kg), failed to substitute for mCPP. Bicuculline produced no change in response rate, whereas Ro 15-4513 dose-dependently decreased responding. Flumazenil produced a small increase in response rates. Flumazenil (10 mg/kg), Ro 15-4513 (1.25 mg/kg), and the benzodiazepine agonists alprazolam (0.64 mg/kg) and diazepam (5 mg/kg) full agonist all failed to block the mCPP discriminative stimulus. When given in combination with mCPP, Ro15-4513 and alprazolam both produced lower response rates than did mCPP alone, whereas flumazenil and diazepam did not significantly alter response rates. These findings provide evidence that GABA(A) antagonists modulate the discriminative stimulus effects of mCPP, but that these effects are not mediated by activity at the benzodiazepine site.     

Effects of caffeine on social behavior, exploration and locomotor activity: interactions with ethanol

The effects of caffeine and its interaction with ethanol were examined in a test of social behavior and a holeboard test of exploration and locomotion. Male mice were injected i.p. with 15, 30 or 60 mg/kg caffeine alone or in combination with 2 g/kg ethanol. The animals were then put in pairs into a familiar arena, or examined individually in the holeboard. Only the highest dose of caffeine (60 mg/kg) had a significant effect on the time spent in social interaction and motor activity in the social behavior test: both measures were reduced. The duration and frequency of avoidance-irritability behavior was dose-dependently increased by caffeine. In the holeboard, caffeine caused a dose-dependent increase in locomotor activity. 30 mg/kg caffeine reversed the ethanol-induced reduction of time spent in social interaction, and 60 mg/kg caffeine antagonized the ethanol-induced increase in locomotor activity in both the social behavior and holeboard tests. Caffeine's effects on ethanol-induced behavioral changes are compared with those of other drugs.