Beta-CCT, a selective BZ-omega1 receptor antagonist, blocks the anti-anxiety but not the amnesic action of chlordiazepoxide in mice.

The aim of this study was to test further the hypothesis that different benzodiazepine (BZ-omega) receptor subtypes may mediate anxiolytic and amnesic effects of BZ agonists, using the selective BZ-omega1 receptor antagonist beta-CCT (beta-carboline-3-carboxylate t-butyl-ester). Experiments were performed in Swiss mice using the elevated plus-maze anxiety test and two learning tasks - passive avoidance and the radial arm maze. In the elevated plus-maze test, beta-CCT (30 mg/kg, i.p.) completely abolished the increase in open-arm entries induced by the BZ chlordiazepoxide (5mg/kg, i.p.). Chlordiazepoxide decreased retention latency in the passive avoidance step-through procedure, and increased the number of errors in the radial arm maze. These effects were not modified by beta-CCT. Except for a slight, albeit significant, amnesic effect in the passive avoidance test, beta-CCT was devoid of intrinsic activity when administered alone. These results are in agreement with previous studies using selective BZ-omega1 agonists, and thus provide further evidence that BZ-omega1 receptors may be involved in the anxiolytic but not in the amnesic effects of BZ agonists.

Benzodiazepine receptor function in the chick social separation-stress procedure.

The role of benzodiazepine (BZ) receptors in modulating social separation-induced distress vocalizations (DVocs) and stress-induced analgesia (SIA) were examined in 8-day-old cockerels (Gallus gallus). In Experiment 1, the BZ agonist chlordiazepoxide (CDP; 5.0 mg/kg) reversed both DVocs and SIA in isolated chicks. Coadministration of the BZ antagonist flumazenil (0.01, 0.03, or 0.10 mg/kg) reversed CDP anxiolytic effects. In Experiment 2, the BZ agonists alprazolam (ALP; 0.065, 0.125, 0.25, or 1.50 mg/kg) and lorazepam (LOR; 0.125, 0.25, 0.50, or 1.0 mg/kg) dose dependently reversed social separation-induced DVocs and SIA. The ED50s for ALP and LOR in attenuating DVocs were 0.19 and 0.34 mg/kg, respectively. These data strongly support the theory that CDP anxiolytic effects are mediated by BZ receptor activity in the chick social separation procedure (Experiment 1) and that this model is sensitive to BZ agonists of different potencies (Experiment 2).

Naloxone blocks anxiolytic-like effects of benzodiazepines in Swiss but not in Balb/c mice.

The ability of naloxone to block the effects of the benzodiazepines chlordiazepoxide and diazepam was evaluated in Swiss and Balb/c mice subjected to the light/dark choice test of anxiety or to a choice paradigm for measuring spontaneous exploratory behaviour. In Swiss mice, naloxone (5 or 10 mg/kg) completely or partially suppressed the anxiolytic-like effects of chlordiazepoxide (5 mg/kg) and diazepam (1 mg/kg) in the light/dark test. Naloxone alone was ineffective. None of these compounds affected locomotion in the free exploratory test. In Balb/c mice, naloxone did not reduce the anxiolytic-like action of benzodiazepines in the light/dark test. Moreover, naloxone did not antagonize the decrease in neophobia observed after anxiolytic treatment in Balb/c mice in the free exploratory paradigm. In this strain, benzodiazepines produced an increase of locomotor activity, whereas naloxone decreased it. The stimulant effects of benzodiazepines on locomotor activity were abolished by naloxone. As naloxone (2 mg/kg) reversed the morphine-induced hyperthermia both in Swiss and in Balb/c mice, differences in possible pharmacokinetic factors between the two strains can be ruled out as an explanation for the failure of naloxone to antagonize anxiolytic-like effects in Balb/c mice. Therefore, the ability of naloxone to reverse anxiolytic effects does not hold for all strains of mice.

Anxiolytic effects of chlordiazepoxide blocked by injection of GABAA and benzodiazepine receptor antagonists in the region of the anterior basolateral amygdala of rats.

The amygdala is a structure that is often implicated in the regulation of anxiety responses. Many studies have shown that injection of benzodiazepines into the amygdala produces an anxiolytic effect. The present study was undertaken to determine whether the anxiolytic effect of administration of systemic benzodiazepine (chlordiazepoxide) might be blocked by local injection of a benzodiazepine receptor antagonist (flumazenil) or GABAA receptor antagonist (bicuculline methiodide; BMI) into the region of the anterior basolateral amygdala (BLA) of rats using an ethologically based test of anxiety, the social interaction test. Injection of flumazenil or BMI into the BLA of rats was found to reverse the anxiolytic effects of peripherally administered chlordiazepoxide. These results suggest a major role for the BLA in mediating the anxiolytic effects of benzodiazepines.

Nitrous oxide anxiolytic effect in mice in the elevated plus maze: mediation by benzodiazepine receptors.

In earlier research, we have hypothesized that exposure to nitrous oxide (N2O) produces an anxiolytic effect that is mediated by benzodiazepine (BZ) receptors. The present research was conducted to characterize pharmacologically the behavioral effects of N2O in comparison with a BZ standard, chlordiazepoxide (CP), in the mouse elevated plus maze. Exposure to increasing levels of N2O produced a concentration-related increase in the percent of total entries into and the percent of total time spent on the open arms, a pattern of response similar to that induced by CP. These effects on N2O and CP were both antagonized by pretreatment with the BZ receptor blocker flumazenil (FLU). In another experiment, mice made tolerant to CP also exhibited a cross-tolerance to N2O. These results support the hypothesis that the anxiolytic effect of N2O is mediated by BZ receptors.

Effects of nefiracetam on drug-induced impairment of latent learning in mice in a water finding task.

We investigated the effects of nefiracetam (DM-9384), a pyrrolidone derivative, on chlordiazepoxide-, apomorphine-, and methamphetamine-induced impairment of latent learning in a water finding test in mice. Pretreatment with nefiracetam reversed the inhibitory effects of chlordiazepoxide and apomorphine, but not those of methamphetamine, on latent learning. The ameliorative effects of nefiracetam on apomorphine-induced, but not chlordiazepoxide-induced impairment of latent learning were antagonized by scopolamine. These results provide further evidence that nefiracetam has anti-amnesic effects. Further, it is suggested that the cholinergic neuronal system may be involved in the ameliorative effects exerted by nefiracetam on apomorphine-induced impairment of latent learning.

Effects of intracranial infusions of chlordiazepoxide on spatial learning in the Morris water maze. II. Neuropharmacological specificity.

In the preceding paper it was found that infusions of chlordiazepoxide (CDP) into the medial septal region, but not several other regions possessing a high density of benzodiazepine receptors, impaired spatial learning, but not cue learning or swim speed, in the Morris water maze. The present investigation sought to further characterize the neuropharmacological profile of this effect. Initially, it was reconfirmed that systemically administered CDP impaired spatial learning, but not cue learning or swim speed, in the water maze. Additionally, it was found that systemically administered scopolamine, a muscarinic antagonist, impaired both spatial and cue learning, but not swim speed, confirming the detrimental effects of cholinergic hypofunction on maze learning. In new rats, a dose-response assessment revealed that 60 and 30 nmol, but not 10 nmol, CDP infused into the medial septum impaired spatial learning, but not cue learning or swim speed. On the following day, rats from each dose group, now undrugged, acquired a reversed platform location at control levels, suggesting that the previously observed impairment was not due to a neurotoxic effect. Additionally, it was found that systemically administered flumazenil (10 mg/kg) blocked the spatial learning deficit produced by the 60 nmol dose of CDP infused into the medial septum. However, intraseptal infusions of flumazenil (10, 20, or 30 nmol) failed to attenuate the spatial learning deficit produced by systemically administered CDP. Finally, systemically administered tetrahydroaminoacridine (1 or 3 mg/kg), an acetylcholinesterase inhibitor, failed to attenuate the spatial learning deficit produced by intraseptal CDP (60 nmol). Together these results implicate benzodiazepine receptors in the medial septum in the amnesic actions of CDP but suggest that additional sites also mediate this action. The present results fail to support the idea that the spatial learning deficit produced by intraseptal infusions of CDP is due to a suppression of septo-hippocampal cholinergic activity and it is proposed that CDP impairs spatial learning by exacerbating hippocampal inhibition by inhibiting septo-hippocampal GABAergic projection neurons.

Effects of post-training administration of (-)-baclofen and chlordiazepoxide on memory retention in ICRC Swiss mice: interactions with GABAA and GABAB receptor antagonists.

The effects of post-training administration of chlordiazepoxide and (-)-baclofen on memory retention was studied in ICRC Swiss mice by measuring the retest stepdown latency 24 hr after foot-shock in a passive avoidance task. Chlordiazepoxide 20 mg/kg impaired memory retention and a similar effect was produced by 10 mg/kg of diazepam. The effect of chlordiazepoxide was antagonised when combined with picrotoxin but not by the addition of a specific GABAB antagonist CGP 35348. The effect of chlordiazepoxide on memory retention seems to be mediated by action at the GABAA-benzodiazepine receptor complex. (-) Baclofen, the active isomer of the GABAB agonist enhanced memory in ICRC mice and this effect was antagonised by CGP 35348 at a dose of 10 mg/kg. The inactive isomer of baclofen, (+)-baclofen did not produce any effect. This indicates that GABAB receptors contribute to the effects of (-)-baclofen on memory.

Chlordiazepoxide-induced working memory impairments: site specificity and reversal by flumazenil (RO15-1788).

The following studies examined the dose and time dependence, site specificity, and reversibility of chlordiazepoxide (CDP)-induced working memory impairments in adult male Sprague-Dawley rats. The rats were tested in a delayed non-match-to-sample radial-arm maze task in which a 1-h delay was imposed between the first four (predelay) and all subsequent (postdelay) arm choices. Intraperitoneal (ip) injection of 2.5 or 5.0 but not 1.25 mg/kg CDP immediately following the predelay session impaired performance in the task. CDP increased the number of errors and decreased the number of correct choices during the postdelay session. The observed working memory impairments also appeared to be site specific since injection of CDP into the medial septum, but not into the anterior amygdala nuclei, immediately following the predelay session also impaired working memory in a dose-related manner. Furthermore, there was a time window for CDP-induced working memory impairments since intraseptal injection of the drug immediately but not 15 min following the predelay session disrupted memory. This observation suggests that the performance deficits reflect disrupted working memory and not proactive effects on performance or the induction of state-dependent learning. In the final experiment, rats were injected ip with either saline or an amnestic dose of CDP (5.0 mg/kg) following the predelay session and then were immediately infused with 10 nmol flumazenil (RO15-1788), a benzodiazepine receptor antagonist or vehicle, into either the medial septum or anterior nuclei of the amygdala. Intraseptal injection of flumazenil prevented the working memory impairments produced by ip injection of CDP. In contrast, intra-amygdala injection of flumazenil did not attenuate, enhance, or modify the CDP-induced working memory impairment. These observations suggest that CDP disrupts working memory by interacting with benzodiazepine receptors in the medial septum.

Possible involvement of nitric oxide in chlordiazepoxide-induced anxiolysis in mice.

Mice challenged with the anxiolytic benzodiazepine chlordiazepoxide exhibited significant increases in the percent of total entries into and percent of total time spent on open arms of an elevated plus maze. Systemic pretreatment with the nitric oxide synthase-inhibitor L-NG-nitro arginine (L-NOARG) antagonized these effects of chlordiazepoxide. This inhibitory effect of L-NOARG was stereospecifically and completely reversed by intracerebroventricular administration of L-arginine but not D-arginine. These findings suggest a possible role of nitric oxide in the anxiolytic effect of chlordiazepoxide in the elevated plus maze.

Dissociation between the effects of benzodiazepine receptor agonists on behavioral vigilance and responsitivity.

The effects of benzodiazepine receptor (BZR) full agonists chlordiazepoxide and midazolam, and the partial agonist beta-carboline ZK 91,296 on the rat's performance in a simple reaction time paradigm were examined. This task required the animals to respond to a rarely and unpredictably occurring brief (50 ms) visual stimulus. Non-parametric measures of signal sensitivity and response bias derived from signal-detection theory were used as a basis for the dissociation between the effects of these drugs on attentional abilities and general responsivity. The dose-dependent effects of midazolam (0.1-3.13 mg/kg) on signal sensitivity and general responsivity occurred in parallel. In contrast, the effects of chlordiazepoxide (1.56-12.5 mg/kg) on signal sensitivity were largely independent from effects on response bias. The partial agonist ZK 91,296 (0.39-25 mg/kg) in general had little effect on performance. The effects of the highest doses of chlordiazepoxide and midazolam were reversed by the co-administration of the BZR antagonist Ro15-1788 (15 mg/kg). Additionally, extension of the stimulus presentation time to 500 ms decreased the magnitude of the effect of chlordiazepoxide on signal sensitivity. These results support the hypothesis that BZR agonist-induced disruption of attentional abilities is not necessarily confounded by effects on general responsivity or sedation, and thus may represent a discrete pharmacological property of BZR-agonists.

Pindolol antagonizes the effects on hippocampal rhythmical slow activity of clonidine, baclofen and 8-OH-DPAT, but not chlordiazepoxide and sodium amylobarbitone.

Buspirone, benzodiazepines, barbiturates and ethanol all reliably reduce the frequency of reticular-elicited hippocampal rhythmical slow activity. In the present experiments we tested a number of drugs which are not usually used for treating generalized anxiety disorders but which have been reported to have some anxiolytic properties. Clonidine (0.3 mg/kg, i.p.), baclofen (6 mg/kg, i.p.) and 8-hydroxy-di-n-propylamino tetralin (8-OH-DPAT) (2.5 mg/kg, i.p.) all reduced the frequency of rhythmical slow activity. The effect of all three drugs was reduced by the 5-hydroxytryptamine 1a antagonist pindolol (2 mg/kg, i.p.). Pindolol had no effect on the reduction in rhythmical slow activity produced by sodium amylobarbitone, as has been previously reported for the benzodiazepine chlordiazepoxide. Flumazenil (10 mg/kg, i.p.), a benzodiazepine receptor antagonist, reduced the effects of chlordiazepoxide (5 mg/kg, i.p.), but not buspirone (10 mg/kg, i.p.). A combination of the selective beta 1 adrenergic receptor antagonist metoprolol (20 mg/kg, i.p.) and the beta 2 adrenergic receptor antagonist ICI 118,551 (4 mg/kg, i.p.) did not reduce the effects of either buspirone (10 mg/kg, i.p.) or diazepam (1 mg/kg, i.p.). These data show that there are at least two separate routes through which anxiolytic agents reduce the frequency of hippocampal rhythmical slow activity. Buspirone, clonidine, baclofen and 8-OH-DPAT act via a system dependent on 5-hydroxytryptamine 1a receptor activation. Benzodiazepines act via activation of the benzodiazepine receptor and probably share with barbiturates action at the GABA-benzodiazepine-chloride ionophore complex but do not produce their effects, directly or indirectly, by 5-hydroxytryptamine 1a receptor activation.

Spinally mediated antinociception following intrathecal chlordiazepoxide--further evidence for a benzodiazepine spinal analgesic effect.

This is a report of the results of 25 experiments in five rats investigating the dose-response relationship for the antinociceptive effects of chlordiazepoxide given intrathecally in the dose range 0.03-0.9 mumol and a further 40 experiments in eight rats investigating the actions of flumazenil and naloxone on this effect. Electrical-current thresholds for pain were measured in the skin of the tail and neck of rats with previously implanted lumbar subarachnoid catheters. Intrathecal chlordiazepoxide produced spinally mediated antinociception, i.e. rises in the current threshold for pain in the tail without a significant change in the neck. This antinociceptive effect was dose dependent. Flumazenil 16.5 mumol kg-1 i.p. reduced the response caused by chlordiazepoxide 0.6 mumol by 78 +/- 6% (mean +/- SEM). By contrast, the same dose of flumazenil did not significantly affect the antinociceptive effect of an equipotent dose of intrathecal fentanyl 0.74 nmol. Naloxone 0.38 mumol kg-1 i.p. abolished the spinally mediated antinociception caused by fentanyl (96 +/- 7% suppression) but did not significantly reduce the effect of chlordiazepoxide (27 +/- 13% suppression). However, a higher dose of naloxone (6.1 mumol kg-1 i.p.) caused significant partial suppression (79 +/- 10.7%) of chlordiazepoxide spinal antinociception. We conclude that chlordiazepoxide produces an antinociceptive effect by combination with benzodiazepine receptors in the spinal cord.

Effects of chlordiazepoxide and putative anxiogenics on conditioned suppression in rats.

This paper reports two experiments. In Experiment 1, the effects of chlordiazepoxide alone and in combination with a series of putative antagonists at various sites on the GABA/benzodiazepine receptor complex on conditioned suppression of operant behavior in rats were assessed. Response rates during presentation of a stimulus associated with shock (CS responding) and when only positive reinforcement is effective (pre-CS responding) were analysed. Chlordiazepoxide (10 mg/kg) significantly increased CS responding. This effect was significantly antagonised by Ro15-1788 (10 mg/kg) and by picrotoxin (1.5 mg/kg), but not by bicuculline (1.5 mg/kg) or by delta-amino-n-valeric acid (10 or 20 mg/kg). Chlordiazepoxide also significantly, albeit more slightly, increased pre-CS responding and none of the other drugs tested significantly antagonised this action, though Ro15-1788 plus chlordiazepoxide resulted in pre-CS response rates not significantly different from either chlordiazepoxide alone or control. These interactions are discussed in the context of the proposed GABA/benzodiazepine receptor complex with the conclusion that drug effects at the benzodiazepine- and picrotoxin-sensitive channel sites have an important role in mediating anxiolytic action. However, behavioral evidence of an important role for GABAa or GABAb receptors remains very limited. The second experiment studied the intrinsic actions of bicuculline, picrotoxin, and Ro15-1788 on conditioned suppression. Responding during a conditioned stimulus associated with a mild (0.125 to 0.15 mA) electric shock (CS responding) and a control rate of responding (pre-CS responding) were recorded. Bicuculline (1.5 mg/kg) and Ro15-1788 (10 mg/kg) did not significantly affect either response rate.(ABSTRACT TRUNCATED AT 250 WORDS)

Sabeluzole improves social recognition and antagonizes chlordiazepoxide's effect on habituation in the rat.

The memory enhancing properties of sabeluzole were evaluated in two experimental paradigms in rats. First, we determined the protective action of sabeluzole against a chlordiazepoxide-induced impairment of habituation. Sabeluzole (5 or 25 mg/kg, SC) was administered 1 h before and chlordiazepoxide (20 mg/kg, SC) immediately after the acquisition session. In the retention session 72 h later, chlordiazepoxide-treated animals displayed higher locomotor and rearing activities and this effect was blocked by pretreatment with sabeluzole. The results suggest that sabeluzole prevented the amnesic effect of chlordiazepoxide. The second paradigm was a social recognition test in which the behaviour toward a familiar or a novel conspecific was investigated. Time spent in social investigation and time spent sniffing of scent traces left on the floor was estimated during exposure of an adult to a juvenile male rat. Sabeluzole (25 mg/kg, SC) was injected into the adults immediately after the first exposure. Reexposure to the same or a novel juvenile was performed 120 min later. In contrast to control, sabeluzole-treated animals showed a significant reduction in social investigation during the second exposure to the same juvenile. Time spent sniffing the floor was significantly decreased in sabeluzole-treated males. Since there was no effect on investigation of a novel juvenile, results suggest that sabeluzole-treated rats are able to remember longer the individual characteristics of juvenile rat obtained through olfactory cues.

Chronic anxiolytic treatment effects on conflict behavior in the rat.

The present studies examined the effects of chronic posttest treatment with the antipanic agent alprazolam (ALP) or the traditional anxiolytic agents chlordiazepoxide (CDP) and phenobarbital (PhB) on conflict behavior. In daily ten-minute sessions, water-deprived rats were trained to drink from a tube which was occasionally electrified (0.25 or 0.5 mA). Electrification was signalled by a tone. Chronic ALP (10 mg/kg/day), CDP (40 mg/kg/day), PhB (80 mg/kg/day) or vehicle were injected IP after conflict testing (in some experiments again 12-16 hours later) for a minimum of 6 weeks. Chronic ALP (but not CDP or PhB) resulted in a time-dependent increase in punished responding, with a latency to onset of 3-4 weeks; this effect was not antagonized by the benzodiazepine antagonist Ro15-1788. These data support the hypothesis that conflict paradigms may serve as animal models for the study of antipanic agents. Moreover, these data suggest that not all anxiolytics will exhibit antipanic efficacy.

Inhibition by antimanic drugs of hyperactivity induced by methamphetamine-chlordiazepoxide mixture in mice.

The effects of lithium chloride and other antimanic drugs on locomotor hyperactivity induced by a mixture of methamphetamine (MAMP) and chlordiazepoxide (CDZP) were examined in mice, using an Animex activity meter. CDZP (12.5 mg/kg) given SC in combination with MAMP (1 mg/kg) caused a marked increase in locomotor activity, as compared with that in mice treated with MAMP alone. However, when CDZP (12.5 mg/kg) was administered together with 0.5 or 2.0 mg/kg of MAMP, no significant enhancement was observed. Lithium (2 and 3 mEq/kg, IP) and carbamazepine (4 and 8 mg/kg, IP) inhibited the hyperactivity induced by the MAMP (1 mg/kg)-CDZP (12.5 mg/kg) mixture to the level of activity in animals treated with MAMP (1 mg/kg) alone. Lithium and carbamazepine alone at these doses caused no significant inhibition of locomotor activity in saline- or MAMP-treated mice. Haloperidol (0.1 mg/kg, IP) and chlorpromazine (0.5 mg/kg, IP) decreased the MAMP-CDZP mixture-induced hyperactivity without significantly inhibiting locomotor activity in the saline- or MAMP-treated group. However, haloperidol (0.2 mg/kg) and chlorpromazine (1 mg/kg) alone significantly inhibited locomotor activity in all of the saline-, MAMP- and MAMP-CDZP mixture-treated groups. These results indicate that antimanic drugs selectively inhibit the hyperactivity induced by the MAMP-CDZP mixture, but that neuroleptics are less selective in inhibiting the hyperactivity.

Picrotoxinin receptor ligand blocks anti-punishment effects of alcohol.

Ethanol at low doses produces a release of punished responding in an operant rat conflict test similar to that observed for benzodiazepines and phenobarbital. It has been hypothesized that these anti-punishment effects are mediated via the GABA-benzodiazepine receptor-ionophore complex but not at the benzodiazepine binding site. In the present study isopropylbicyclophosphate (IPPO), which binds at the picrotoxinin site, reversed the release of punished responding produced by ethanol, pentobarbital and chlordiazepoxide; at low doses IPPO (less than 10 micrograms/kg) appeared to be most effective against ethanol but at higher doses (greater than 15 micrograms/kg) was also effective against pentobarbital and chlordiazepoxide. At still higher doses IPPO produced a decrease in punished and unpunished responding. These results suggest that the "anxiolytic" actions of ethanol may involve a direct action on the GABA-benzodiazepine receptor-ionophore complex and this action may underlie some of the intoxicating effects of ethanol.

Differential effects of Ro15-1788 in actions of chlordiazepoxide and ethanol.

Three behavioral tests, namely, runway activity, horizontal dowel test and hypothermia, were used to compare the effects of Ro15-1788, a specific benzodiazepine antagonist, on the common neuropharmacological actions of chlordiazepoxide (CDP) and ethanol in C57BL/6J mice. Ro15-1788 completely reversed the CDP-induced inhibition of runway activity and incoordination on a horizontal dowel, but only partially antagonized the hypothermic effects of CDP. The latter phenomenon was likely to be due to the rapid elimination of Ro15-1788, but could also be due to the fact that hypothermia might not be a specific action of CDP. The sedative actions of ethanol were not antagonized at all by Ro15-1788. In fact, Ro15-1788 potentiated the incoordinating effect of ethanol as determined by the horizontal dowel test such that mice injected with Ro15-1788/ethanol had lower brain ethanol levels than mice injected with vehicle/ethanol when they fell off the dowel. In contrast, mice injected with Ro15-1788/CDP took longer to fall off and had significantly higher CDP levels at fall-off than mice injected with vehicle/CDP. The stimulatory effect of a low dose of ethanol on runway activity was reversed by Ro15-1788. These data are discussed in terms of the possible mechanisms of actions for CDP and ethanol.