Comparison of the pharmacological properties of classical and novel BZ-omega receptor ligands

The experiments in this study compared the pharmacological properties of several BZ-omega receptor ligands, including the imidazobenzodiazepine imidazenil, the beta-carboline abecarnil, the pyridazinone Y-23684, the pyrido [1,2-a]benzimidazole RWJ 46771 and the 1,6-naphthyridin-2(1H)-one derivative SX-3228, with the prototypical BZs diazepam, clobazam and bretazenil. In in vitro experiments diazepam, bretazenil, imidazenil and Y-23684 displaced [3H]flumazenil binding non-selectively in membranes from rat cerebellum and spinal cord, two brain areas enriched in the BZ-omega 1 and BZ-omega 2 receptor subtypes, respectively. In contrast, abecarnil, RWJ 46771 and SX-3228 were more potent in displacing [3H]flumazenil binding to membranes from rat cerebellum than from spinal cord or hippocampus, indicating selectivity for the BZ-omega 1 receptor subtype. The in vivo experiments showed that all compounds increased the latency to clonic seizures produced by isoniazid. However, the maximal increase in latency induced by diazepam, clobazam, abecarnil, RWJ 46771 and SX-3228 was greater than that of bretazenil, imidazenil and Y-23684, thereby indicating that these latter compounds have low intrinsic efficacy. In the punished drinking, the punished lever pressing and the elevated plus-maze tests in rats, three models of anxiety, diazepam, clobazam and imidazenil elicited clear anxiolytic-like effects but at doses which were close to those producing hypolocomotion, ataxia and myorelaxation as measured in activity cages, the rotarod and the loaded grid tests, respectively. In contrast, bretazenil and Y-23684 induced anxiolytic-like activity at much lower doses than those which impaired motor performances. The magnitude of the positive effects of Y-23684 was similar to that of the reference BZs, suggesting that it may become a valuable alternative to currently used agents for the treatment of anxiety disorders. Abecarnil, RWJ 46771 and SX-3228 produced weaker or non-specific anxiolytic-like effects as they decreased anxiety-related behaviours at doses similar or close to those impairing motor performance. However, unlike the other compounds they induced myorelaxation at doses which were 3-10 times higher than those needed to produce decrease in exploratory activity. It is suggested that the behavioural profiles of abecarnil, RWJ 46771 and SX-3228 may be attributed to their selectivity for the BZ-omega 1 receptor subtype which may account for their sedative activity, thereby masking other effects including anxiolytic-like activity. This suggests that BZ receptor modulation of anxiety may involve BZ receptor subtypes other than BZ-omega 1.     

Interactions between LY354740, a group II metabotropic agonist and the GABA(A)-benzodiazepine receptor complex in the rat elevated plus-maze.

Flumazenil, a benzodiazepine (BZ) receptor antagonist, and naloxone, a non-selective mu-receptor antagonist, were used to investigate whether the anxiolytic action of LY354740 [1S,2S,5R,6S-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate monohydrate], a Group II metabotropic glutamate receptor agonist, was mediated through the benzodiazepine binding site on the GABA(A) receptor and opioid pathways. LY354740 (1.0-10.0 mg/kg i.p.) induced dose-dependent anxiolytic-like effects in the rat elevated plus-maze. The anxiolytic-like effects of LY354740 (10.0 mg/kg) and the benzodiazepine receptor agonist, chlordiazepoxide (CDP, 5.0 mg/kg i.p.) were blocked by flumazenil (15.0 mg/kg i.p.). By contrast, naloxone (10.0 mg/kg i.p.) failed to affect the anxiolytic-like effects of either LY354740 or CDP. The behaviour of animals treated with flumazenil or naloxone alone did not significantly differ from that of animals treated with vehicle alone. This study suggests that the anxiolytic-like effects of LY354740 on the elevated plus-maze may be directly or indirectly mediated by the benzodiazepine binding site on the GABA(A) receptor complex.     

The effects of compounds varying in selectivity as 5-HT(1A) receptor antagonists in three rat models of anxiety

Compounds varying in selectivity as 5-HT(1A) receptor antagonists have recently been reported to produce benzodiazepine-like antianxiety effects in mice. To assess the cross-species generality of these findings, the present experiments compared the effects of diazepam (0.625-5 mg/kg) with those of several non-selective (MM-77, 0.03-1 mg/kg and pindobind-5-HT(1A), 0.1-5 mg/kg) and selective (WAY100635, 0.01-10 mg/kg, p-MPPI, 0.01-3 mg/kg and SL88.0338, 0.3-10 mg/kg) 5-HT(1A) receptor antagonists in three well-validated anxiolytic screening tests in rats: punished lever-pressing, punished drinking, and the elevated plus-maze. In the punished lever-pressing conflict test, none of the 5-HT(1A) receptor antagonists modified rates of punished responding, whereas in the punished drinking test, WAY100635 (0.3-1 mg/kg), SL88.0338 (3-10 mg/kg), p-MPPI (1 mg/kg), MM-77 (0.03-0.3 mg/kg), but not pindobind-5-HT(1A), produced clear anticonflict activity. However, the increase in punished responding with the 5-HT(1A) compounds was smaller than that produced by diazepam, indicating weaker anxiolytic-like activity. In the elevated plus-maze test, WAY100635 (0.1-0.3 mg/kg), SL88.0338 (0.3-10 mg/kg), MM-77 (0.01-3 mg/kg), pindobind-5-HT(1A) (0.1-3 mg/kg), but not p-MPPI, showed anxiolytic-like activity on traditional behavioral indices, increasing the percentage of time spent in open arms and the percentage of open arm entries. As was the case in the punished drinking test, the magnitude of the positive effects of the 5-HT(1A) compounds was generally smaller than that of diazepam. Of the ethological measures recorded in the plus-maze, all compounds markedly decreased risk assessment (i.e. attempts) over the entire dose-range, but only diazepam clearly increased directed exploration (i.e. head-dipping). Although the present results demonstrate that 5-HT(1A) receptor antagonists elicit anxiolytic-like effects in rats, this action appears to be test-specific and, unlike previous findings in mice, smaller than that observed with benzodiazepines. The data are discussed in relation to the possible relevance of species differences in 5-HT(1A) receptor function and the nature of the anxiety response studied.     

Anticonflict effect of the glycineB receptor partial agonist, D-cycloserine, in rats. Pharmacological analysis

RATIONALE: Several studies have provided evidence that antagonists and partial agonists of glycine(B) receptors exhibit an anxiolytic-like activity in different animal models. OBJECTIVE: Using the conflict-drinking Vogel test in rats as a model, in the present study we examined the anxiolytic-like activity of D-cycloserine (DCS), a partial agonist of the glycine(B) site of the N-methyl-D-aspartate (NMDA) receptor complex. Diazepam was used as a reference drug. RESULTS: DCS (200 and 300 mg/kg) and diazepam (5 mg/kg) produced an anxiolytic-like effect in rats by increasing the number of shocks accepted. We also demonstrated that NMDA (15 mg/kg) reduced the anxiolytic-like activity of DCS (200 mg/kg), whereas glycine (800 mg/kg) and flumazenil (10 mg/kg) did not affect the anticonflict effect of DCS (200 mg/kg). The anticonflict effect of diazepam (5 mg/kg) was totally blocked by flumazenil (10 mg/kg). CONCLUSION: The obtained results have shown that DCS exhibits an anxiolytic-like activity which depends on NMDA receptors rather than on glycine(B) or benzodiazepine sites.     

Effects of melatonin and agomelatine in anxiety-related procedures in rats: interaction with diazepam.

The anxiolytic potential of melatonin and agomelatine, a potent MT(1/2) receptor agonist, and their combined effects with diazepam, were investigated in rats using the punished drinking test, the safety signal withdrawal operant paradigm, the elevated-plus-maze and hypophagia-induced novelty. In the punished drinking test, evening injections of melatonin (80 mg/kg, IP, but not 20 and 40 mg/kg) and agomelatine (40 mg/kg, IP) increased the number of foot shocks received. However, neither melatonin (40-80 mg/kg) nor agomelatine (20-40 mg/kg) released response suppression during the period associated with the safety signal withdrawal and affected rats' behaviour in the elevated-plus-maze. Furthermore, agomelatine (40 mg/kg) did not enhance food consumption in unfamiliar environment. However, the co-administration of melatonin (80 mg/kg) or agomelatine (20-40 mg/kg) with diazepam, at a dose (0.25 mg/kg) inactive on its own, induced an anxiolytic-like effect in the punished drinking test and the elevated plus-maze. These results indicate that, although mostly devoid of anxiolytic-like action per se, melatonin and agomelatine can potentiate the anxiolytic effects of diazepam.     

Selective activation of metabotropic G-protein-coupled glutamate 7 receptor elicits anxiolytic-like effects in mice by modulating GABAergic neurotransmission

We describe the anxiolytic-like effects of the first, selective metabotropic G-protein-coupled glutamate 7 (mGlu7) receptor agonist, N,N'-dibenzyhydryl-ethane-1,2-diamine dihydrochloride (AMN082), as measured in the modified stress-induced hyperthermia (SIH) and the four-plate tests. Administration of AMN082 (3-6 mg/kg intraperitoneally) to Swiss mice produced anxiolytic-like effects in the modified SIH and four-plate tests. Moreover, it was ineffective as an anxiolytic in the SIH test in mGlu7 receptor knockout mice as compared with wild-type C57BL/6J littermate controls. In contrast, diazepam (1.25-5 mg/kg) significantly reduced SIH in both the wild-type and knockout animals. The anxiolytic-like effect of AMN082 in the SIH paradigm was abolished by pretreatment with flumazenil (10 mg/kg intraperitoneally). This indicates an involvement of gamma-aminobutyric acid-ergic neurotransmission in AMN's anxiolytic actions. The results indicate that activation of the mGlu7 receptor produces anxiolytic-like effects via the modulation of the gamma-aminobutyric acid system.     

The influence of the benzodiazepine receptor antagonist flumazenil on the anxiolytic-like effects of CGP 37849 and ACPC in rats

In this paper we examined the effect of flumazenil (Ro 15-1788, 10 mg/kg), a benzodiazepine receptor antagonist, on the anticonflict activity of DL-(E)-2-amino-4-methyl-5-phosphono-3-pentenoic acid (CGP 37849), a competitive N-methyl-D-aspartate (NMDA) receptor antagonist, and 1-aminocyclopropanecarboxylic acid (ACPC), a partial agonist at glycine(B) receptors, in the Vogel conflict drinking test in rats. The effect of flumazenil on the anxiolytic-like (in the plus-maze test) and the anticonvulsant (in the maximal electroshock-induced seizures) activities of CGP 37849 in rats was also studied. Diazepam was used as a reference drug. CGP 37849 (2. 5-5 mg/kg), ACPC (50-200 mg/kg) and diazepam (2.5-5 mg/kg) significantly and dose-dependently increased the number of shocks accepted during experimental sessions in the conflict drinking test. Flumazenil partly but significantly reduced the anticonflict effect of CGP 37849, and it fully blocked the anticonflict effect of ACPC and diazepam. CGP 37849 (2.5-5 mg/kg) and diazepam (2.5-5 mg/kg) were also active in the plus-maze test, as they significantly increased the percentage of the time spent in and entries into the open arms of the plus-maze, both those effects having been antagonized by flumazenil. Flumazenil alone was inactive in both the conflict drinking and the plus-maze tests. In the maximal electroshock-induced seizures, both CGP 37849 (2.5-5 mg/kg) and diazepam (5-10 mg/kg) produced anticonvulsant effects, of which only that of diazepam was antagonized by flumazenil. The results of the present study showing antagonism of flumazenil towards the anxiolytic-like effects of CGP 37849 and ACPC suggest involvement of benzodiazepine receptors in such an activity of the NMDA and glycine(B) receptor ligands, respectively, which may be due to a possible interaction between NMDA and GABA/benzodiazepine systems. The lack of effect of the benzodiazepine antagonist on the anticonvulsant activity of CGP 37849 indicates that involvement of benzodiazepine receptors in the pharmacological action of the NMDA antagonist is not a general phenomenon.     

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/GABA(A) receptors are involved in magnesium-induced anxiolytic-like behavior in mice

Behavioral studies have suggested an involvement of the glutamate pathway in the mechanism of action of anxiolytic drugs, including the NMDA receptor complex. It was shown that magnesium, an NMDA receptor inhibitor, exhibited anxiolytic-like activity in the elevated plus-maze test in mice. The purpose of the present study was to examine interaction between magnesium and benzodiazepine/GABA(A) receptors in producing anxiolytic-like activity. We examined behavior of mice treated with magnesium and benzodiazepine/GABA(A) receptor ligands, in the elevated plus maze. The anxiolytic-like effect of magnesium (20 mg/kg) was antagonized by flumazenil (10 mg/kg) (benzodiazepine receptor antagonist) while combined treatment with the non-effective doses of magnesium (10 mg/kg) and benzodiazepines (diazepam (0.5 mg/kg) or chlordiazepoxide (2 mg/kg)) produced synergistic interaction (increased time in open arms and number of open arm entries) in this test. The obtained data indicate that benzodiazepine receptors are nvolved in the anxiolytic-like effects of magnesium.     

The anxiolytic-like effect of 5-HT1B receptor ligands in rats: a possible mechanism of action

We have examined the effect of lesions of 5-hydroxytryptamine (5-HT) neurons, produced by p-chloroamphetamine (p-CA; 2 x 10 mg kg(-1)), and the influence of flumazenil (Ro 15-1788, 10 mg kg(-1)), a benzodiazepine receptor antagonist, on the anxiolytic-like activity of CP 94253 (5-propoxy-3-(1,2,3,6-tetrahydro-4-pyridinyl)-1H-pyrrolo[3,2-b]pyridine), a 5-HT1B receptor agonist, SB 216641 (N-[3-[3-(dimethylamino)ethoxy]-4-methoxyphenyl]-2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)-[1,1'-biphenyl]-4-carboxamide), a 5-HT1B receptor antagonist, and GR 127935 (N-[4-methoxy-3-(4-methyl-l-piperazinyl)phenyl]-2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)-l, l'-biphenyl-4-carboxamide), a 5-HT1B/1D receptor antagonist, in the Vogel conflict drinking test in rats. Diazepam was used as a reference compound. CP 94253 (2.5 mg kg(-1)), SB 216641 (2.5 mg kg(-1)), GR 127935 (10 mg kg(-1)) and diazepam (5 mg kg(-1)) significantly increased the number of shocks accepted during experimental sessions in the conflict drinking test in vehicle- and p-CA-pretreated rats. Flumazenil did not change the anxiolytic-like effect of CP 94253 (2.5 mg kg(-1)), but wholly blocked the anxiolytic-like effects of SB 216641 (2.5 mg kg(-1)), GR 127935 (10 mg kg(-1)) and diazepam (5 mg kg(-1)). p-CA and flumazenil alone were inactive in the conflict drinking test. The results suggested that the anxiolytic-like effect of the 5-HT1B receptor ligands CP 94253, SB 216641 and GR 127935 was possibly linked to the postsynaptic 5-HT1B receptors or/and 5-HT1B heteroreceptors. The results suggested also that benzodiazepine receptors were indirectly involved in the effects of SB 216641 and GR 127935 (but not of CP 94253), which might have been due to a possible interaction between the 5-HT and the GABA/benzodiazepine systems.     

Interaction of the hypnotic lormetazepam with central benzodiazepine receptor subtypes omega 1, omega 2 and omega 3]

To clarify the action of lormetazepam, 3-hydroxybenzodiazepine, on the benzodiazepine (BZ) receptor subtypes, effects of lormetazepam on motor performance in the traction test and hexobarbital-induced loss of righting reflex in mice and the binding to BZ receptor subtypes were investigated in comparison with those of other BZ hypnotics. Lormetazepam prolonged the duration of hexobarbital-induced loss of righting reflex. The minimal effective dose (1 mg/kg, p.o.) was higher than that of flunitrazepam, lower than those of diazepam and zopiclone, and the same as those of triazolam and brotizolam. Lormetazepam showed the ataxic effect at 10 mg/kg, p.o., but the separation between its effective doses for the hypnotic and ataxic effects was the largest among the hypnotics tested. In the displacement study on [3H]flumazenil binding to cerebellar and spinal cord membranes, lormetazepam bound with a higher affinity to omega 1 receptor (Ki = 10 nM) than to omega 2 receptor (Ki = 29 nM). The GABA-ratios of lormetazepam to omega 1 and omega 2 receptors were 3.9 and 4.0, respectively; and they were higher and lower than those of flunitrazepam to omega 1 and omega 2 receptors, respectively. In the displacement study on [3H] Ro5-4864 binding to kidney membranes, lormetazepam bound with a lower affinity to the omega 3 receptor (Ki = 213 nM) than flunitrazepam. Thus, lormetazepam was suggested to be a potent hypnotic with weaker ataxic effects than other BZ hypnotics, which may be due to its selective and potent agonistic action on central omega 1 receptors.     

Anxiolytic-like effects of mGlu1 and mGlu5 receptor antagonists in rats

The purpose of the present study was to compare anxiolytic activity of the metabotropic glutamate receptor 1 (mGlu) antagonist, EMQMCM ((3-ethyl-2-methyl-quinolin-6-yl)-(4-methoxy-cyclohexyl)-methanone methanesulfonate) and the mGlu5 receptor antagonist MTEP ([(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine) and MPEP (2-methyl-6-(phenylethynyl)pyridine) in animal models of anxiety. In the elevated plus maze, diazepam (1 mg/kg), but not the mGlu1 or mGlu5 receptor antagonists induced anxiolytic-like effects. Meanwhile, MTEP (2.5 and 5 mg/kg), EMQMCM (5 mg/kg), and diazepam (2 mg/kg) all significantly inhibited fear potentiated startle. In the contextual fear conditioning test, MTEP (1.25 and 2.5 but not 5 mg/kg) and EMQMCM (0.6 to 5 mg/kg) attenuated freezing responding. In the Geller-Seifter conflict test, MPEP (1 and 3 mg/kg), MTEP (3 mg/kg), chlordiazepoxide (10 and 20 mg/kg) and midazolam (1 mg/kg) all facilitated punished responding, while ECMQCM failed to produce any significant effects up to 3 mg/kg dose. To summarise, the present data further support a significant anxiolytic potential of group I mGlu receptor antagonists, while suggesting the effects of mGlu1 receptor antagonists may depend on the experimental procedure and may be qualitatively different from those of mGlu5 receptor antagonists.     

Effects of antagonists at the NMDA receptor complex in two models of anxiety

The effects of an antagonist at the strychnine insensitive glycine site (5,7-dichlorokynurenic acid, i.c.v.), and of noncompetitive (MK-801, i.p.) and competitive (CGP 37849, i.p.; CGP 39551, i.p.; AP-7, i.c.v.) NMDA antagonists were compared with diazepam (i.p.) in two animal models of anxiety (the open field exploratory behavior of non-habituated rats, and the Vogel conflict test). All drugs when applied in appropriate doses increased punished drinking in the Vogel test, without producing any significant changes in free drinking and the stimulus threshold at their lowest anticonflict doses. The effective doses were as follows: diazepam 1.5 and 2.5 mg/kg; MK-801 0.005 and 0.01 mg/kg; CGP 39551 5.0 and 20.0 mg/kg; CGP 37849 1.0 and 2.5 mg/kg; 5,7-dichlorokynurenic acid 5.0 μg (i.c.v); AP-7 0.5 μg (i.c.v.). In the open field diazepam (0.05 mg/kg), MK-801 (0.1 mg/kg), CGP 37849 (0.01, 0.1, 1.0 mg/kg), and AP-7 2.5 μg (i.c.v.) significantly increased exploratory activity in the central sectors of the open field (anti-neophobic reaction), without changing motor activity of the rat. MK-801 at the highest tested dose of 0.2 mg/kg significantly stimulated animal locomotor activity. CGP 37849 in the largest dose examined (10 mg/kg) significantly depressed the motor behavior of rats. Overall, it appeared that different NMDA antagonists showed an anxiolytic-like profile, similar to that of the benzodiazepine diazepam. Among different NMDA receptor complex antagonists studied, CGP 37849 was characterized by the largest distinction between the doses showing an anxiolytic-like action in the open field test, and changing rat motor behavior.     

Withdrawal precipitation by benzodiazepine receptor antagonists in dogs chronically treated with diazepam or the novel anxiolytic and anticonvulsant β-carboline abecarnil

Summary The effects of the benzodiazepine (BZ) receptor antagonists flumazenil (Ro 15-1788) and the -carboline ZK 93426 were compared in dogs before and after chronic treatment with diazepam or the novel BZ receptor ligand abecarnil (ZK 112119). Abecarnil, -carboline, is thought to act as partial (low efficacy) and/or subtype selective agonist at central BZ receptors. Diazepam and abecarnil were administered at doses which, based on previous experiments on anticonvulsant activity, resulted in about equieffective drug concentrations during treatment. In dogs treated with diazepam, 6 mg/kg/day p.o., for 2 weeks, severe abstinence symptoms, including seizures, were precipitated in all animals by i. v. infusion of the BZ receptor antagonists, differences being found in the type of symptoms caused by flumazenil and ZK 93426. In dogs treated with abecarnil, 4 mg/kg/d s. c., for 6 weeks, only relatively mild abstinence symptoms were precipitated by infusion of flumazenil or ZK 93426, although pharmacologically active plasma concentrations of abecarnil had been maintained throughout the period of treatment. This suggests that BZ receptor ligands which act as partial and/or selective agonists might be more favourable than traditional agonists, such as diazepam, regarding the induction of physical dependence. Send offprint requests to W. Löscher at the above address     

Anticonflict-like effect of a prefrontal dopaminergic lesion in rats: permissive role of noradrenergic neurons

The effects of a perfrontal dopamine (DA) lesion were compared to those induced by a combined DA and noradrenaline (NA) lesion, to investigate the permissive role of prefrontal NA terminals in the anxiolytic-like effect of a prefrontal DA lesion. Lesions consisted of bilateral microinjections of 6-hydroxydopamine into the medial prefrontal cortex in rats either given desipramine (25mg/kg) before surgery (DA lesion) or not pretreated with desipramine (combined DA and NA lesions). Three weeks post-surgery, water-restricted rats, given saline or diazepam (2mg/kg), were subjected to a single session in the punished drinking test. Prefrontal DA lesions significantly increased punished drinking in saline-treated rats. This effect was not observed in rats with the dual prefrontal lesions. The ability of diazepam to increase punished drinking was not modified by either lesion. These data suggest that the integrity of NA afferents to the prefrontal cortex is necessary for the anxiolytic-like effect induced by the prefrontal DA lesion. The results also indicate that the effect of diazepam is not mediated by the prefrontal catecholamine afferents.     

Discriminative stimulus properties of the benzodiazepine receptor antagonist flumazenil

Rats were trained to discriminate the stimulus properties of the benzodiazepine (BZ) receptor antagonist flumazenil using a conditioned taste aversion procedure. On drug trials, fluid-restricted rats were injected with flumazenil (32 mg/kg), given access to a 0.25% saccharin solution for 30 min, and injected with LiCl (1.8 mEq/kg IP). On saline trials, injections of saline bracketed the period of saccharin consumption. Acquisition of the discriminated taste aversion, as measured by differential effects of drinking between saline and drug trials, developed after only five pairings of flumazenil with the LiCl injections. Flumazenil did not alter saccharin consumption in unconditioned controls (N=9) that never received LiCl. The discrimination was also measured by flumazenil's ability to reduce the preference for saccharin over tap water using two-bottle choice tests. Flumazenil demonstrated dose-dependent generalization upon decreasing the training dose as low as 1 mg/kg. Two other BZ receptor antagonists of different chemical structure, CGS 8216 and ZK 93426, substituted completely for the flumazenil stimulus. Partial generalization was exhibited to the partial inverse agonists FG 7142 and beta-CCE, while the full inverse agonists DMCM and PTZ failed to substitute for the flumazenil stimulus. The BZ receptor agonists diazepam and alprazolam failed to substitute for the flumazenil stimulus, although partial generalization was shown with CDP. The results suggest that the BZ receptor antagonist flumazenil may produce intrinsic discriminative stimulus effects that are independent from those of BZ receptor agonists or inverse agonists.     

Comparative assessment of the anxiolytic-like activities of honokiol and derivatives

Honokiol has previously been shown to be an effective anxiolytic-like agent in mice when administered for 7 days at 0.2 mg/kg/day prior to evaluation in an elevated plus-maze, while 20 mg/kg is required for efficacy as a single oral dose. The aim of this study was to find analogs of honokiol that are more effective for acute administration. Among the eight analogs evaluated, one partially reduced derivative of honokiol [3'-(2-propenyl)-5-propyl-(1,1'-biphenyl)-2,4'-diol] exhibited significant anxiolytic-like activity at 0.04 mg/kg. Following oral administration of 1 mg/kg of this analog, anxiolytic-like activity was clearly evident at 1 h, peaked at 3 h, and remained significant for longer than 4 h after treatment. Combined administration of the derivative with diazepam led to enhanced anxiolytic-like efficacy. Moreover, as with diazepam, the anxiolytic-like effect of the analog was reduced by flumazenil. In contrast, bicuculline, a GABA(A) antagonist, had no effect on the activity of the derivative. Taken together, these results suggest that this analog of honokiol acts at the benzodiazepine recognition site of the GABA(A)-benzodiazepine receptor complex.     

Effect of flumazenil and diazepam on transient actions in defensive burying elicited by the social interaction experience in rats

In the present work, we studied the effects of benzodiazepine (BZ) receptor antagonist, flumazenil, and of the agonist, diazepam, on social interaction-induced transient changes in defensive burying (DB). Enhanced defensive burying was observed after 1.5 min of social interaction experience, while a longer social interaction experience, 15 min, inhibited the expression of burying behavior. Defensive burying and social interaction paradigms have been used for the screening of compounds with anxiolytic potential and, more extensively, to study the neurobiology of anxiety. To elucidate the participation of the BZ receptor on transient changes induced by intervals of social interaction experience, its receptor antagonist, flumazenil (2.5, 5, and 10 mg/kg) was intraperitoneally injected (IP). Flumzenil enhanced in a dose-dependent manner, the blocking effect of the saline IP injection on facilitated DB in 1.5-min social interaction-experienced subjects. In addition, flumazenil enlarged in a dose-dependent manner the blocking effect of saline IP on defensive burying levels in animals exposed to social interaction experience for 15 min. To analyze the presumed participation of the BZ receptor mediating enhanced burying behavior levels in subjects exposed to 1.5 min of social interaction, a suboptimal dose of diazepam (0.25 mg/kg) was administered. Diazepam enhanced the saline IP elicited defensive burying reduction. Results are discussed in terms of the suggested BZ receptor mediation on transient changes in defensive burying elicited by social interaction experience.     

Characterization of the nociceptin receptor (ORL-1) agonist, Ro64-6198, in tests of anxiety across multiple species

Rationale Previous studies have demonstrated behaviors indicative of anxiolysis in rats pretreated with the nociceptin receptor (opioid receptor like-1, ORL-1) agonist, Ro64-6198. Objectives The aim of this study was to examine the effects of Ro64-6198 in anxiety models across three species: rat, guinea pig, and mouse. In addition, the receptor specificity of Ro64-6198 was studied, using the ORL-1 receptor antagonist, J-113397, and ORL-1 receptor knockout (KO) mice. Finally, neurological studies examined potential side effects of Ro64-6198 in the rat and mouse. Results Ro64-6198 (3–10 mg/kg) increased punished responding in a rat conditioned lick suppression test similarly to chlordiazepoxide (6 mg/kg). This effect of Ro64-6198 was attenuated by J-113397 (10 mg/kg), but not the mu opioid antagonist, naltrexone (3 mg/kg). In addition, Ro64-6198 (1–3 mg/kg) reduced isolation-induced vocalizations in rat and guinea pig pups. Ro64-6198 (3 mg/kg) increased the proportion of punished responding in a mouse Geller–Seifter test in wild-type (WT) but not ORL-1 KO mice, whereas diazepam (1–5.6 mg/kg) was effective in both genotypes. In rats, Ro64-6198 reduced locomotor activity (LMA) and body temperature and impaired rotarod, beam walking, and fixed-ratio (FR) performance at doses of 10–30 mg/kg, i.e., three to ten times higher than an anxiolytic dose. In WT mice, Ro64-6198 (3–10 mg/kg) reduced LMA and rotarod performance, body temperature, and FR responding, but these same measures were unaffected in ORL-1 KO mice. Haloperidol (0.3–3 mg/kg) reduced these measures to a similar extent in both genotypes. These studies confirm the potent, ORL-1 receptor-mediated, anxiolytic-like effects of Ro64-6198, extending the findings across three species. Ro64-6198 has target-based side effects, although the magnitude of these effects varies across species.     

Discriminative stimulus properties of the benzodiazepine receptor inverse agonist methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM)

The purpose of this study was to determine whether rats could be trained to discriminate the stimulus properties of the benzodiazepine (BZ) receptor inverse agonist DMCM from saline in a conditioned taste aversion paradigm. On a drug trial, water-deprived rats were injected with DMCM (0.55–0.6 mg/kg IP), allowed access to a 0.25% saccharin solution for 30 min, and then injected with LiCl. On non-drug trials, saline injections bracketed the drinking period. Conditioned controls were treated similarly with DMCM and saline on drug and non-drug trials, but received injections of saline instead of LiCl. At the completion of training, DMCM produced a 69% reduction of saccharin consumption on drug trials, compared with 23% for conditioned controls. The stimulus properties of DMCM were then measured by its ability to reduce the preference for saccharin over water in a two-bottle choice test. DMCM reduced saccharin preference in rats that received discrimination training from 68% to 19%, but did not alter saccharin preference in conditioned controls. Other compounds with varying activity at BZ receptors were evaluated for their ability to substitute for the discriminative stimulus effects of DMCM. Two BZ receptor inverse agonists, -CCE (10–18 mg/kg) and FG 7142 (3.2–18 mg/kg), substituted completely for DMCM. Partial substitution for DMCM was shown by the BZ receptor antagonist CGS 8216 (3.2–10 mg/kg) and the non-BZ convulsant pentylenetetrazol (10–20 mg/kg). The BZ receptor agonists chlordiazepoxide (0.32–5.0 mg/kg), diazepam (0.32–10 mg/kg), and alprazolam (0.1–3.2 mg/kg) and the BZ receptor antagonist flumazenil (1.0–32 mg/kg) failed to substitute for the DMCM stimulus. Pretreatment with flumazenil (1.0 mg/kg) blocked the stimulus effects of the training dose of DMCM and produced a shift to the right of the DMCM generalization curve. The pattern of compounds that substituted for the DMCM stimulus and the blockade of that stimulus by flumazenil indicate that the stimulus properties of DMCM are associated with its effects as a BZ receptor inverse agonist.