Stimulant effect of the beta-carboline FG 7142 in the open-field test.

The anxiogenic activity of N-methyl-beta-carboline-3-carboxamide (FG 7142) is sometimes difficult to observe in rats. As the open field has recently been applied successfully to test the anxiogenic potential of n-butyl-beta-carboline-3-carboxylate (beta-CCB) in mice, a comparable experiment was performed with FG 7142 (1, 5, 10, 30 mg/kg i.p.) in rats. In contrast to the inhibitory effects measured with beta-CCB, FG 7142 significantly increased the ambulation and rearing scores and induced aggressivity in some animals. A differential sensitivity of mice and rats to beta-carbolines, predominant analeptic properties of FG 7142, and differences in the types of anxiety induced are proposed to account for this discrepancy.     

The effects of the beta-carboline FG 7142, on intracranial self-stimulation in the rat

The effects of FG 7142 were examined, alone and in combination with chlordiazepoxide, on self-stimulation of the mid-lateral hypothalamus. Rewarding stimuli were delivered according to a 10-sec variable-interval schedule of reinforcement. FG 7142 (1-20 mg/kg) produced a dose-related depression in responding, and chlordiazepoxide (5 mg/kg) enhanced it. When these two drugs were given together, response rates did not differ significantly from control rates.     

Effects of the beta-carboline FG 7142 on saccharin preference and quinine aversion in water-deprived rats

Three groups of water-deprived male rats were given the choice of 0.05% sodium saccharin solution and water; 0.0005% quinine solution and water; water in both containers. N'-Methyl-beta-carboline-3-carboxamide (FG 7142), a benzodiazepine receptor inverse agonist, dose-dependently (2.5-10 mg X kg-1 i.p.) reduced intake of fluid in each group. The saccharin-choice group were most sensitive, showing a significant reduction at 2.5 mg X kg-1. Preference for the saccharin solution was reduced by FG 7142 but it did not increase aversion to the quinine solution. Hence, FG 7142 did not generally increase the rejection of flavoured solutions in a two-bottle test. A previously proposed "proconflict" effect of FG 7142 is discussed, in the light of these results.     

Discriminative stimulus control by the anxiogenic beta-carboline FG 7142: generalization to a physiological stressor

Drug discrimination was employed to investigate the similarities between FG 7142-induced anxiogenesis and the stress produced by exposure to either a novel environment or to footshock. Eight rats were trained to discriminate between the stimulus properties of the beta-carboline FG 7142 (5.0 mg/kg) and its vehicle in a two-lever, food motivated operant task. Once trained, decreasing doses of FG 7142 produced fewer FG 7142-appropriate responses and the dose-response relationship yielded an ED50 of 1.45 mg/kg. Rats were subsequently subjected to two physiological/environmental stressors, footshock and novelty, and then tested in the discriminative paradigm. Exposure to novelty resulted in partial FG 7142-appropriate responding, whereas footshock sessions produced responding predominately on the FG 7142-appropriate lever. This is the first report of stimulus control by FG 7142 and it is likely that the interoceptive cue state produced by this compound is anxiogenic in nature, as reported to occur in man. The anxiogenic nature of the FG 7142 discriminative stimulus is supported by the generalization of FG 7142 to the state produced following stressful environmental manipulation.     

Anxiogenic beta-carboline FG 7142 produces activation of noradrenergic neurons in specific brain regions of rats.

By measuring the levels of two major metabolites of rat brain noradrenaline (NA), 3-methoxy-4-hydroxyphenylglycol (MHPG) and 3,4-dihydroxyphenylglycol (DHPG), we investigated the effects of anxiogenic beta-carboline FG 7142, an inverse agonist of benzodiazepine (BZD) receptors, on brain noradrenergic activity of rats. Thirty min after treatment with FG 7142 (15 mg/kg IP), levels of both MHPG and DHPG in the hypothalamus, amygdala and thalamus, but not in the hippocampus and cerebral cortex, significantly increased. These increases were significantly antagonized by pretreatment with BZD receptor antagonist Ro 15-1788 (15 mg/kg, IP). Sixty min after treatment with FG 7142 at the same dose, significant increases in both metabolite levels occurred in the hypothalamus, amygdala, thalamus and cerebral cortex, and increases in MHPG levels only were observed in the hippocampus. These increases were significantly blocked by pretreatment with alpha 2-adrenoreceptor agonist clonidine (100 microgram/kg, IP). The present findings suggest that FG 7142 can produce increases in brain noradrenergic activity in specific brain regions by interacting with BZD receptors, and may support the hypothesis that hyperactivity of brain noradrenergic systems may be one neural mechanism in provocation of aversive emotional changes (anxiety, fear or panic).     

Co-existence of kindling induced by the beta-carboline, FG 7142, and tolerance to diazepam following chronic treatment in mice

The effect of chronic treatment with the beta-carboline, FG 7142, followed by chronic treatment with diazepam (DZP) on the acute effects of DZP and FG 7142 were studied. Mice were treated for 16 days with FG 7142 (40 mg/kg i.p.) followed by treatment with DZP (5 or 20 mg/kg i.p.) for 9 days. At the end of this period, the anticonvulsant, antipunishment and locomotor sedative properties of a test dose of DZP were assessed, as were the convulsant properties of FG 7142. During the chronic treatment with FG 7142, 80% of the mice developed clonic convulsions in response to the beta-carboline, and this increased sensitivity to FG 7142 (kindling) remained following the chronic DZP treatment. Thus long-term treatment with DZP does not reverse the changes which occur during FG 7142-induced kindling. Chronic treatment with DZP for 9 days gave rise to tolerance to its pharmacological effects as assessed in the 4-plate test of antipunishment activity, in a test of locomotor sedation, and by its ability to increase the convulsant threshold of intravenously administered pentylenetetrazol. The development of tolerance to DZP was not affected by a prior chronic treatment with FG 7142. Nor were the acute effects induced by DZP altered by a prior chronic treatment with FG 7142. Apart from reducing its own convulsant threshold, chronic treatment with FG 7142 had no effect in any of the experiments. These results suggest that kindling induced by FG 7142 and tolerance to DZP depend on different mechanisms. In neither case were the pharmacological changes induced by chronic administration reflected by changes in the biochemical measures of the coupling between benzodiazepine binding sites and gamma-aminobutyric acid (GABA) receptors.     

Anxiogenic drugs beta-CCE and FG 7142 increase extracellular dopamine levels in nucleus accumbens

Two experiments were conducted to study the effects of anxiogenic drugs on dopamine release and metabolism in nucleus accumbens. Microdialysis probes were implanted into the nucleus accumbens, and rats were tested the day after implantation. In the first experiment, groups of rats received injections of saline, 1.25 or 2.5 mg/kg beta-CCE. In the second experiment, groups of rats received injections of saline, 10.0, 20.0 or 30.0 mg/kg FG-7142. Both drugs produced significant increases in dopamine release and metabolism in nucleus accumbens. Neither drug had significant effects on locomotor activity. These experimeriments indicate that exposure to anxiogenic drugs increases accumbens dopamine activity, an effect that is consistent with other studies showing that the mesolimbic dopamine system is responsive to stressful stimuli. In addition, these results demonstrate that drug-induced increases in accumbens dopamine release are not unique to drugs of abuse.     

Effects of the beta-carboline, FG 7142, in the social interaction test of anxiety and the holeboard: correlations between behaviour and plasma concentrations

The behavioural effects of the beta-carboline FG 7142 were investigated in the social interaction test of anxiety and the holeboard test of exploration and locomotor activity. FG 7142 (5-20 mg/kg) produced a significant decrease in the time spent in social interaction by pairs of rats, without an accompanying decrease in motor activity. This anxiogenic effect was highly correlated with the plasma concentrations of FG 7142 for the rats receiving 5 and 10 mg/kg doses, but not for those receiving the 20 mg/kg dose. In the holeboard, FG 7142 had no effect on exploratory head-dipping at the doses tested, but selectively reduced locomotor activity and the number of rears. The profile of FG 7142 in these tests is compared with those of the beta-carbolines, B-CCE and B-CCP.     

Inverse agonist properties of the FG 7142 discriminative stimulus.

A two-lever, food-motivated discrimination was established between the benzodiazepine receptor partial inverse agonist FG 7142 (5.0 mg/kg) and its vehicle. The FG 7142 discriminative stimulus was pharmacologically characterized by testing trained rats with a variety of benzodiazepine receptor ligands. Administration of the inverse agonist DMCM (0.15-0.30 mg/kg) dose-dependently mimicked the FG 7142 stimulus. In contrast, the benzodiazepine receptor agonist chlordiazepoxide, partial agonist ZK 91 296, mixed agonist/antagonist CGS 9896 and antagonist RO 15-1788 blocked the FG 7142 cue. These results indicate that the FG 7142 discriminative stimulus is based on its inverse agonist activity. The generalization of FG 7142 to the anxiogenic/convulsant compound pentylenetetrazole (PTZ), but not to the anorectic agent norfenfluramine, indicates that the anxiogenic properties of FG 7142, rather than its anorectic actions, may underlie the FG 7142 discriminative stimulus.     

The effects of drugs acting at the GABAA-receptor/ionophore after chemical kindling with the benzodiazepine receptor ligand FG 7142.

Repeated administration of the beta-carboline benzodiazepine receptor ligand FG 7142 produces sensitization to its effects so that full seizures develop (chemical kindling); initially it is only pro-convulsant. The present study investigated alterations in the function of drugs which act at the different sites at the gamma-aminobutyric acid (GABA) benzodiazepine receptor complex, after repeated administration of FG 7142. In FG 7142 kindled mice decreased anticonvulsant and hypothermic effects of the GABA agonist muscimol were observed. The hypothermic effects of the GABA agonist progabide were reduced. In contrast a small increase in the hypothermic effect of pentobarbitone was seen. The convulsant effects of bicuculline and picrotoxin were unaltered when they were given intravenously but marginally increased when they were given by the intraperitoneal route. No changes were seen in the hypothermic effects of these drugs. No significant changes were seen in the convulsant or hypothermic effects of pentylenetetrazol. These results suggest that kindling with FG 7142 may alter GABA receptor function.     

Evidence for noradrenergic involvement in mediating the FG 7142 discriminative stimulus.

Rats were trained to discriminate the stimulus properties of the benzodiazepine receptor partial inverse agonist beta-carboline-3-carboxylate acid methyl amide (FG 7142) (5.0 mg/kg) or the alpha 2-adrenergic receptor antagonist 17 alpha-hydroxyyohimban-16 alpha-carboxylic acid methyl ester (yohimbine) (3.0 mg/kg) from vehicle in a two-lever, food-motivated operant task. These compounds have in common a beta-carboline structure and anxiogenic behavioral profiles. The yohimbine discriminative stimulus was mimicked by the alpha 2-adrenergic receptor antagonist idazoxan and antagonized by the alpha 2-adrenergic receptor agonist clonidine, indicating that the yohimbine stimulus was mediated through the alpha 2-adrenergic receptor. The anxiogenic beta-carbolines FG 7142, 1,2,3,4-tetrahydro-beta-carboline (THBC), and norharmane, the anxiogenic/convulsant agent pentylenetetrazole (PTZ), and two physiological stressors failed to mimic the yohimbine discriminative stimulus. In contrast, both yohimbine and idazoxan dose responsively mimicked the anxiogenic FG 7142 stimulus. The present results demonstrate that an asymmetrical generalization exists between the discriminative stimuli produced by yohimbine and FG 7142. Furthermore, these data suggest that yohimbine can produce a multicomponent discriminative stimulus, part of which may be anxiogenic in nature. The ability of alpha 2-adrenergic receptor antagonists to mimic the FG 7142 cue suggests that activation of the noradrenergic system may underlie cues produced by benzodiazepine receptor inverse agonists.     

Chronic benzodiazepine treatment increases the effects of the inverse agonist FG7142

A schedule of treatment with the benzodiazepine, flurazepam, in mice for 7 days caused a significant enhancement of the convulsive effects of the partial inverse agonist FG7142. Full convulsions were seen with FG7142 after the chronic administration of flurazepam, although this compound does not cause convulsions in normal mice of the strain used. The change appeared to be maximal at 24 hr after the last dose of flurazepam and lasted for up to a week. The chronic treatment with flurazepam caused tolerance to the effects of flurazepam, but the tolerance was of shorter duration than the increase in the effects of FG7142. When the benzodiazepine antagonist, Ro 15-1788, was given with the flurazepam, the incidence of convulsions induced by FG7142 was no longer significant. Repeated administration of midazolam also slightly increased the effects of FG7142. Single doses of flurazepam or midazolam did not significantly alter the effects of FG7142, although some convulsions were seen.     

Beta-CCE and FG 7142 increase defensiveness during intraspecies encounters in mice

The effects were ascertained of two partial inverse agonists at benzodiazepine receptors (beta-CCE and FG 7142) on the incidence of timid (defensive-escape), aggressive, sociable and locomotor activities in both timid and aggressive singly-housed male mice, treated with drugs in paired interactions with untreated non-aggressive males. FG 7142 (5 mg/kg) and beta-CCE (8 mg/kg) increased defenses and escapes without producing other behavioral changes in timid mice. FG 7142 (20 mg/kg) and beta-CCE (1–8 mg/kg) moderately increased defenses and alert postures in aggressive mice and this effect was associated with marked reduction of aggressive behavior. FG 7142 (20 and 80 mg/kg) also decreased walking across cage in aggressive mice. It is concluded that beta-CCE and FG 7142 produced behavioral changes which could be interpreted as anxiogenic.     

Midazolam-induced hyperphagia and FG 7142-induced anorexia: behavioural characteristics in the rat

Non-deprived male rats, familiarized with a highly palatable diet, were treated with 0.3-10.0 mg/kg of the imidazobenzodiazepine midazolam. The increases in consumption of the food observed at larger doses of midazolam were due to increases in the duration of feeding, but not in the rate of eating. These, in turn, were due to increases in the duration of eating bouts, but not in their frequency. The beta-carboline FG 7142, a partial benzodiazepine receptor inverse agonist, reduced the consumption of the diet when it was injected at 10.0 and 15.0 mg/kg (IP). The overall duration of feeding was not affected at these doses in the 30 min test, but rate of eating was reduced. However, during the first 5 min interval of the test, when feeding behaviour was most motivated, FG 7142 did significantly reduce the duration of feeding. The effect depended upon a reduction in the duration of eating bouts, but not upon any change in their frequency. Hence, midazolam and FG 7142 had opposite effects on the duration of bouts of feeding. Both midazolam and FG 7142 reduced the frequencies of concurrent grooming, locomotor activity, and rearing in the test of palatable food consumption. Possible explanations for these effects are briefly considered.     

Inverse agonist properties of the THBC discriminative stimulus: asymmetrical generalization with FG 7142.

Male rats were trained to discriminate the stimulus properties of the beta-carbolines 1,2,3,4-tetrahydro-beta-carboline (THBC) (15.0 mg/kg) or FG 7142 (5.0 mg/kg) from vehicle in a two-lever, food-motivated operant task. Consistent with the serotonergic properties of THBC, administration of the 5HT1B agonists TFMPP and mCPP to THBC-trained rats resulted in THBC-appropriate responding. Norharmane, a beta-carboline metabolite of THBC, also mimicked the THBC discriminative stimulus. In contrast, the benzodiazepine receptor partial inverse agonist FG 7142, the anxiogenic/convulsant pentylenetetrazole (PTZ), two physiological stressors and the alpha 2 adrenergic antagonists yohimbine and idazoxan failed to produce THBC-appropriate responding. In the FG 7142-trained rats, THBC and norharmane dose-dependently mimicked the FG 7142 discriminative stimulus. This generalization was not based upon the serotonergic properties of THBC and norharmane since administration of the serotonin agonist mCPP to FG 7142-trained rats failed to produce FG 7142-appropriate responding. The ability of THBC to substitute for the FG 7142 discriminative stimulus was antagonized by the benzodiazepine receptor mixed agonist/antagonist CGS 9896 and the benzodiazepine receptor antagonist RO 15-1788, indicating that THBC produces an inverse agonist stimulus in FG 7142-trained rats. These results suggest that THBC produces a discriminative stimulus which consists of both serotonergic and inverse agonist components.     

Long-term treatment with antidepressant drugs reduces the sensitivity of cortical cholinergic neurons to the activating actions of stress and the anxiogenic drug FG 7142

Certain antidepressant drugs exert an anxiolytic action in both humans and rodents. The effects of long-term treatment with imipramine or mirtazapine, two antidepressant drugs with different mechanisms of action, on the response of cortical cholinergic neurons to foot-shock stress or to the anxiogenic drug FG 7142 were investigated in freely moving rats. Chronic treatment with imipramine or mirtazapine reduced the increase in cortical acetylcholine output induced by foot-shock stress by approximately 50%. The same treatment also reduced the sensitivity of cortical cholinergic neurons to the stimulatory effect of acute administration of FG 7142. In contrast, the administration of a single dose of either antidepressant 40 min before foot shock or FG 7142 injection failed to increase the threshold of excitability of cortical cholinergic neurons. These results demonstrate that long-term treatment with either imipramine or mirtazapine reduces the sensitivity of cortical cholinergic neurons to stress or to an anxiogenic drug with an efficacy similar to that of acute administration of benzodiazepines. The neurochemical mechanism responsible for regulation of cholinergic neuron sensitivity might contribute to the modulation of cognitive function associated with emotional and affective disorders.     

Selective changes in the in vivo effects of benzodiazepine receptor ligands after chemical kindling with FG 7142

It has recently been demonstrated that kindling occurs with repeated administration of the benzodiazepine "inverse agonist" FG 7142. The present study was an investigation of the effects of other ligands for the benzodiazepine receptor in mice kindled with FG 7142. It was shown that over a range of doses the lowering effects of FG 7142 on the seizure threshold were greater in kindled animals than in control. In contrast, the hypothermic effect of FG 7142 was unaltered. The effects of the partial inverse agonist CGS 8216 were unaltered. The effects of the full inverse agonist DMCM were unchanged except for an enhancement of its convulsant effect when infused at a concentration of 100 mu gm 1-1. Studies with the full agonist benzodiazepine, flurazepam and the full agonist beta-carboline, ZK 93423, showed small but significant reductions in their hypothermic effects. The sedative and anticonvulsant effects of flurazepam were unaltered, whereas the anticonvulsant effects of ZK 93423 were decreased in animals kindled with FG 7142. There was a pronounced reduction in the anticonvulsant and hypothermic effects of the partial agonist beta-carboline, ZK 91296. These data do not fit any simple explanation of kindling being due to a change in the function of benzodiazepine receptors, although they may offer some support for the idea that kindling with FG 7142 produces a change in the effects of all beta-carboline compounds which act at the benzodiazepine receptor.