Aversive learning as a mechanism for lack of repeated anxiolytic-like effect in the elevated plus-maze

Rodents re-exposed to the elevated plus-maze no longer respond to anxiolytic-like drugs, such as benzodiazepines. This phenomenon is thought to be due to retrieval of aversive learning associated with the initial exploration of this potentially dangerous environment. Based on this assumption, one might expect the maintenance of the drug's anxiolytic-like effect in rodents already experienced in the elevated plus-maze if the acquisition and/or consolidation of this learning were impaired. Using male Wistar rats, we investigated whether the systemic administration of propranolol, at putative learning-impairing doses, prior to or immediately after the first (Trial 1) elevated plus-maze exposure would retain the midazolam anxiolytic-like effect on the second (Trial 2) exposure to this apparatus. There was an anxiolytic-like effect, characterized by an increase in the open-arms exploration, in response to 0.25 mg/kg of midazolam on Trial 2 only in rats administered with 20 mg/kg of propranolol before Trial 1. Although propranolol had a dose-dependent and behaviorally-selective anti-anxiety effect (significant at 20 mg/kg) on Trial 1, further minute-by-minute analysis confirmed the propranolol-induced learning acquisition deficit in this group on Trial 2. The knowledge of the environment actually contributes to the unresponsiveness to anxiolytic-like drugs observed in rats re-exposed to the elevated plus-maze.     

Scopolamine given pre-Trial 1 prevents the one-trial tolerance phenomenon in the elevated plus-maze Trial 2

A learned avoidance response has been one of the hypotheses proposed to explain the 'one-trial tolerance' (OTT) phenomenon, which represents a drug's loss of anxiolytic-like effect in the elevated plus-maze (EPM) test in experienced rodents. Based on these facts, if some kind of learning occurs throughout Trial 1, then an impairment of its acquisition would maintain the drug's anxiolytic-like effect on Trial 2. Using male Wistar rats, the present study examined whether scopolamine (SCO; 0.5-1.5 mg/kg), a drug that impairs learning acquisition, given 30 min prior to Trial 1, actually prevents the OTT phenomenon to either the midazolam (MDZ; 0.5 mg/kg) or the memantine (MEM; 8.0 mg/kg) anxiolytic-like effect on the EPM Trial 2 (48 h later). According to the results, both MDZ and MEM increased open-arm exploration (indicating anxiolysis) on Trial 2 only in rats that had been treated previously with 1.5 mg/kg SCO. These results were observed in the absence of change in general exploratory activity. The present findings suggest that SCO impaired the acquisition of the behavioral strategy to cope with the subsequent EPM exposure that supposedly underlies the OTT phenomenon, thereby revealing the anxiolytic-like effects of MDZ and MEM on Trial 2.     

One-trial tolerance to midazolam is due to enhancement of fear and reduction of anxiolytic-sensitive behaviors in the elevated plus-maze retest in the rat

The anxiolytic-like effects of benzodiazepines (BZDs) in rats is reduced after a single exposure to the elevated plus-maze test (EPM). Several hypotheses have been formulated but no conclusive explanation exists for this phenomenon called "one-trial tolerance." In this study, we examined this phenomenon further by carrying out an ethopharmacological analysis of the behavior of rats submitted to the EPM in two trials. Rats injected with saline before both trials (control), treated with 1.0 mg/kg of midazolam before both trials (MM), or only before Trial 2 (SM), were exposed to the EPM. The SM group did not differ from the controls in the Trial 1 and Trial 2 conditions. The MM group showed a clear anxioselective profile in Trial 1 and no anxiolytic-like effects in Trial 2. Whereas midazolam injected before the first trial caused no significant change in immobility, there was a pronounced increase in immobility during Trial 2 for all three conditions. These data suggest that the anxiolytic-like action of midazolam in the first trial gives way to the fear-related insensitive behaviors (phobic/avoidance responses) responsible for the one-trial tolerance to BZDs in Trial 2. Furthermore, an additional experiment showed that midazolam does not seem to affect the acquisition of the learned avoidance response since it is present upon retesting even after midazolam administration in Trial 1 (MS group). Rather, the present data suggest an emotional shift from Trial 1 to Trial 2, which leads to change in the responsiveness of the animals to BZDs.     

The gerbil elevated plus-maze II: anxiolytic-like effects of selective neurokinin NK1 receptor antagonists.

Neurokinin NK1 receptor antagonists may have therapeutic potential in the treatment of anxiety and depression. Species variants in the NK1 receptor result in reduced affinity of NK1 receptor antagonists at rat and mouse NK1 receptors, making it difficult to test NK1 antagonists in traditional preclinical models of anxiety and depression. Gerbil NK1 receptors are similar in homology to the human NK1 receptor. In a companion article, we described the anxiety-like behavioral profile of gerbils on an adapted elevated plus-maze, and the ability of anxiolytic drugs to produce anti-anxiety effects in the gerbil elevated plus-maze. The aim of the present study was to determine whether oral (p.o.) administration of the NK1 receptor antagonists MK-869, L-742,694, L-733,060, CP-99,994, and CP-122,721 produced anxiolytic-like effects in the gerbil elevated plus-maze. Upon testing, all five NK1 antagonists produced anxiolytic-like effects. MK-869 (0.01-3 mg/kg) was the most potent NK1 antagonist, producing anxiolytic-like effects on percentage of open arm time, percentage of open arm entries, stretch-attend postures, and head dips at 0.03-0.3 mg/kg doses. L-742,694 (1-30 mg/kg) and L-733,060 (1-10 mg/kg) produced anxiolytic-like effects on percentage of open arm time and stretch-attend postures at 3-10 mg/kg doses. CP-99,994 (3-30 mg/kg) only produced an anxiolytic-like effect on stretch-attend postures. CP-122,721 (3-30 mg/kg) produced an anxiolytic-like effect on percentage of open arm time at 30 mg/kg. The order of potency of the NK1 antagonists to increase percentage of open arm time was very similar to their potency to block NK1 agonist-induced foot-tapping. These studies demonstrate that neurokinin NK1 receptor antagonists produce anxiolytic-like effects in a novel gerbil elevated plus-maze, and suggest that this is an appropriate model to test NK1 antagonists for preclinical anxiolytic activity.     

Neuropharmacological analysis of caffeic acid in rats

The aim of the present study was to investigate the effect of intraperitoneal administration of caffeic acid (0.5, 1, 2, 4 or 8 mg/kg) on elevated plus-maze and open field tasks in rats and its possible neuroprotection/neurotoxicity using the comet assay. Caffeic acid at 1 mg/kg increased the number of entries and the time spent in the open arms on plus-maze, suggesting an anxiolytic-like effect when used in lower doses without affecting locomotion and exploration on the open field. Furthermore, a protective effect against hydrogen peroxide-induced oxidative damage on brain tissue was observed through the treatment with caffeic acid at 1 and 8 mg/kg. However, in the highest dose, caffeic acid induced DNA damage in brain tissue.     

Anxiolytic-Like Effect of baicalin and its additivity with other anxiolytics

Baicalin, a naturally occurring flavonoid, was previously reported to exert anxiolytic-like effects in the Vogel conflict test. In the present study, the anxiolytic effects of baicalin alone and in combination with other anxiolytics were tested in mice using the elevated plus-maze (EPM). Baicalin treatment (7.5 - 30 mg/kg) significantly increased entries into and time spent in open arms, indicative of an anxiolytic-like effect. Motor-depressive and myorelaxant side effects commonly associated with anxiolytics were not observed with baicalin at effective anxiolytic doses in the hole-board and horizontal wire tests, respectively. Co-administration of baicalin (3.75 mg/kg) with dl-tetrahydropalmatine ( dl-THP; 0.25 mg/kg), an anxiolytic-hypnotic alkaloid, both at sub-effective doses, induced an additive effect resulting in considerable anxiolysis. Similarly, an additive anxiolytic-like effect was observed with baicalin (3.75 mg/kg) and diazepam (DZ; 0.5 mg/kg). Results obtained from this study demonstrate the potential of baicalin as a candidate anxiolytic and its possible application in multidrug therapy. Abbreviations. BZS:benzodiazepine-binding site EPM:elevated plus-maze DZ:diazepam GABA (A):type A gamma-aminobutyric acid dl-THP: dl-tetrahydropalmatine.     

The gerbil elevated plus-maze I: behavioral characterization and pharmacological validation.

Several neurokinin NK1 receptor antagonists currently being developed for anxiety and depression have reduced affinity for the rat and mouse NK1 receptor compared with human. Consequently, it has proven difficult to test these agents in traditional rat and mouse models of anxiety and depression. This issue has been overcome, in part, by using non-traditional lab species such as the guinea pig and gerbil, which have NK1 receptors closer in homology to human NK1 receptors. However, there are very few reports describing the behavior of gerbils in traditional models of anxiety. The aim of the present study was to determine if the elevated plus-maze, a commonly used anxiety model, could be adapted for the gerbil. Using a specially-designed elevated plus-maze, gerbils exhibited an 'anxious' behavioral profile similar to that observed in rats and mice, i.e., reduced entries into, and time spent exploring, an open, aversive arm. The anxiolytic drugs diazepam (0.03-3 mg/kg i.p.), chlordiazepoxide (0.3-10 mg/kg i.p.), and buspirone (0.3-30 mg/kg s.c.) increased open arm exploration and produced anxiolytic-like effects on risk-assessment behaviors (reduced stretch-attend postures and increased head dips). Of particular interest, the antidepressant drugs imipramine (1-30 mg/kg p.o.), fluoxetine (1-30 mg/kg, p.o.) and paroxetine (0.3-10 mg/kg p.o.) each produced some acute anxiolytic-like activity, without affecting locomotor activity. The antipsychotic, haloperidol, and the psychostimulant, amphetamine, did not produce any anxiolytic-like effects (1-10 mg/kg s.c). The anxiogenic beta-carboline, FG-7142, reduced time spent in the open arm and head dips, and increased stretch-attend postures (1-30 mg/kg, i.p.). These studies have demonstrated that gerbils exhibit an anxiety-like profile on an elevated plus-maze, and that the gerbil elevated plus-maze may have predictive validity for anxiolytics, and antidepressants with potential anxiolytic-like effects.     

The anxiogenic-like and anxiolytic-like effects of MDMA on mice in the elevated plus-maze: a comparison with amphetamine

Many abused substances have been found to possess anxiogenic-like or/and anxiolytic-like properties. Discrepancies about the effects of MDMA, one of the most popular recreational drugs in recent years, on anxiety have been seen in the literature, and almost all of the data in this respect were derived from retrospective studies. The present study was thus designed to examine the drug's actions by using an animal model of anxiety, the elevated plus-maze test in male mice. Intraperitoneal MDMA at 1 mg/kg was ineffective, at 4 mg/kg decreased the percent of open arm entries (p < 0.01), and increased enclosed entries (p < 0.05), at 12 mg/kg had no significant effect, and at 20 mg/kg induced an increase of percent of open time (p < 0.01). As control drugs, amphetamine (0.5-4 mg/kg, i.p.) produced a dose-dependent, anxiogenic-like effect and diazepam (1 mg/kg, i.p.) induced an anxiolytic-like effect in the test. The results indicate that MDMA has anxiogenic-like properties at lower doses and anxiolytic-like at higher doses. The effects of MDMA and amphetamine on the mouse's responses to the plus-maze are compared. These findings provide a possible explanation for the controversies over MDMA's effects on anxiety in the literature.     

7-Nitroindazole, a nitric oxide synthase inhibitor, has anxiolytic-like properties in exploratory models of anxiety

The action of the novel nitric oxide synthase (NOS) inhibitor 7-nitroindazole (7-NI) was studied in different exploratory models of anxiety. In the rat plus-maze test, 7-NI potently increased time spent on open arms and percentage of open arm visits in a dose-dependent manner with the minimal effective dose of 40 mg/kg. 7-NI caused an anxiolytic-like effect in the rat social interaction test. The minimal dose increasing social interaction time was 20 mg/kg. However, the drug also produced a clear sedative effect occurring even at smaller doses (10 mg/kg) in the open field test. 7-NI also showed an anxiolytic-like profile in the mouse light-dark compartment test and in the elevated plus-maze test, but the doses required were higher (80–120 mg/kg) than in rat models. Also, the sedative effect occurred at these doses in open field. We failed to demonstrate any effect of L-arginine either in the rat elevated plus-maze test or in the open field test at doses up to 600 mg/kg IP. These results indicate that there are no major interspecies differences between rats and mice in respect of action of 7-NI. The clear anxiolytic-like action of the nitric oxide synthase inhibitor in four different models shows that nitric oxide is involved in the process of anxiety and that NOS could be a new target in developing anxiolytic drugs. Received: 1 November 1996/Final version: 27 January 1997     

Dexmedetomidine synergism with midazolam in the elevated plus-maze test in rats

The anxiolytic profile of dexmedetomidine, a novel, highly-selective ₂-adrenergic agonist, was examined in rats in the elevated plus-maze test when administered either alone or in combination with the benzodiazepine agonist midazolam. Dexmedetomidine, 0.1–10 µg/kg, was inactive in modifying the rats' behavioral response in this test. Midazolam, 0.1–10 mg/kg, dose-dependently produced an anxiolytic-like profile characterized by an increased time spent in the open arms of the elevated plus-maze. A combination of dexmedetomidine 0.5 µg/kg and midazolam 0.5 mg/kg produced a synergistic interaction. This heterergic interaction of dexmedetomidine on midazolam's anxiolytic-like profile was dose-dependently blocked by pretreatment with an ₂-adrenergic antagonist, atipamezole, 10–50 µg/kg, and a benzodiazepine antagonist flumazenil, 1.0 and 10 mg/kg, but not by the ₁-adrenergic antagonist, prazosin, 0.1–10 mg/kg. While the transmembrane signal transduction pathways for benzodiazepine- and ₂-agonist responses do not share any molecular component, there does appear to be crosstalk between these two systems. These may involve GABA or noradrenergic downstream effects of either dexmedetomidine or midazolam, respectively.     

Antianxiety effect of cannabidiol in the elevated plus-maze

In order to assess the presence of anxiolytic properties in cannabidiol (CBD) the drug was tested in an elevated plus-maze model of anxiety, in rats. Doses of 2.5, 5.0 and 10.0 mg/kg significantly increased the entry ratio (open/total number of entries), an anxiolytic-like effect. CBD at a dose of 20.0 mg/kg was no longer effective. None of the doses of CBD used changed total number of entries, a measure of total exploratory activity. Diazepam (2.0 mg/kg) also caused an anxiolytic-like effect in this model. These results indicate that CBD causes a selective anxiolytic effect in the elevated plusmaze, within a limited range of doses.     

Ethological comparison of the effects of a bovine alpha s1-casein tryptic hydrolysate and diazepam on the behaviour of rats in two models of anxiety

A bovine alpha s1-casein tryptic hydrolysate was previously demonstrated to display an anxiolytic-like activity in the conditioned defensive burying and in the elevated plus-maze models when i.p. injected. The present study assessed the anxiolytic-like effects of this tryptic hydrolysate after an oral administration in rats faced to the same behavioural situations using diazepam as a reference. In a first experiment, the behavioural effects of the hydrolysate in the conditioned defensive burying test were investigated at doses ranging 5-50 mg/kg. The results showed that the minimal dose required to elicit an anxiolytic-like activity is 15 mg/kg. In a second experiment, the alpha s1-casein tryptic hydrolysate (15 mg/kg, p.o.) was demonstrated to display an anxiolytic-like activity similar to diazepam (3 mg/kg, p.o.) in the conditioned defensive burying test and the elevated plus-maze. However, the ethological analysis of behaviour indicated that this hydrolysate has a different activity compared to diazepam. While diazepam induced a disinhibition state in rats, possibly related to the risk-taking behaviour observed after a benzodiazepine ingestion in humans, the tryptic hydrolysate did not display such a side effect. These results suggest that the mechanism of action of the bovine alpha s1-casein tryptic hydrolysate may differ from that of diazepam.     

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.     

Anxiolytic-like effects of 5-HT(1A) agonists in drug-naive and in benzodiazepine-experienced rats

The efficacy of two 5-HT(1A) receptor agonists in drug-naive and benzodiazepine-experienced rats was compared in two animal tests of anxiety, the social interaction and elevated plus-maze tests. Benzodiazepine-experienced rats were tested 48h after the last of 28 daily injections of diazepam (2mg/kg/day), a time at which there was no anxiogenic withdrawal response. S20499 (0.04, 0.2 and 1mg/kg) ((+)-8(4-[N-(5-methoxychroman-3yl)N-propylamine]butyl)-8-azaspirol[4,5]decane-7,9-dione) and buspirone (0.2mg/kg) significantly increased social investigation, indicating anxiolytic-like actions, and there was no significant modification in these effects as a result of the previous diazepam treatment. Both drugs also significantly reduced aggressive behaviours in both drug-naive and diazepam-experienced rats. An anxiolytic-like action in the elevated plus-maze was also indicated for S 20499 (0.04 and 0.2mg/kg) by an increase in the percentage of time spent on the open arms; this effect was not significantly changed by prior diazepam experience. Buspirone (0.2mg/kg) had no significant effects on the plus-maze. The results provide no evidence for reduced efficacy of 5-HT(1A) receptor agonists in animal tests of anxiety as a result of prior diazepam treatment.     

Behavioral characterization of acetaldehyde in C57BL/6J mice: locomotor, hypnotic, anxiolytic and amnesic effects

Rationale Acetaldehyde, the first metabolite of ethanol, was recently suggested to contribute to many behavioral effects of ethanol, although few studies have directly investigated the behavioral effects of acetaldehyde itself.Objectives The aim of the present study was to characterize the locomotor, hypnotic, anxiolytic-like and amnesic effects of acetaldehyde in C57BL/6J mice.Methods Increasing doses of acetaldehyde (0–300 mg/kg) were injected intraperitoneally and their effects on a series of representative behaviors were investigated. The locomotor effects of acetaldehyde were measured in activity boxes. The duration of the loss of righting reflex was used as an index of the hypnotic effects of acetaldehyde. The anxiolytic-like effects of acetaldehyde were tested with an elevated plus-maze and the amnesic effects with the one-trial passive avoidance test. Finally, brain and blood acetaldehyde concentrations were assessed.Results Acetaldehyde induced a significant hypolocomotor effect at 170 mg/kg and higher doses. In addition, the hypnotic effects of acetaldehyde were demonstrated by a loss of righting reflex after the administration of 170 and 300 mg/kg acetaldehyde. The elevated plus-maze showed that acetaldehyde does not possess anxiolytic-like properties. Finally, acetaldehyde (100–300 mg/kg) dose-dependently altered memory consolidation as shown by a reduced performance in the passive avoidance test.Conclusions The present results show that acetaldehyde induces sedative, hypnotic and amnesic effects, whereas it is devoid of stimulant and anxiolytic-like properties in C57BL/6J mice. However, the behavioral effects of acetaldehyde after intraperitoneal administration were apparent at very high brain concentrations. The present results also indicate that acetaldehyde is unlikely to be involved in the anxiolytic properties of ethanol in mice.     

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.     

Anxiolytic-like effects of olanzapine, risperidone and fluoxetine in the elevated plus-maze test in rats

In the present study was examined the effect of treatment with olanzapine or risperidone, given separately or in combination with fluoxetine, in the elevated plus-maze test (an animal model of anxiety) in male Wistar rats. The obtained results showed that treatment with olanzapine (1 mg/kg), risperidone (0.1 and 0.3 mg/kg) or fluoxetine (5 and 10 mg/kg) induced an anxiolytic-like effect in the elevated plus-maze test. Olanzapine, risperidone and fluoxetine, tested in doses effective in the model of anxiolytic-like actions, did not affect motor coordination, while olanzapine (3 mg/kg) and risperidone (0.3 mg/kg) produced a significant reduction of exploratory activity in the open field test. In a combination study, the anxiolytic-like effect of olanzapine or risperidone was significantly antagonized by co-treatment with fluoxetine. Additionally, co-treatment with olanzapine or risperidone and fluoxetine disturbed the motor coordination of rats in a rota-rod test. These findings indicate that olanzapine, risperidone and fluoxetine per se may be clinically effective in treating anxiety disorders, but their effects may be attenuated when they are used in combination with other medications.     

Biphasic effects of clonidine on conflict behavior: involvement of different alpha-adrenoceptors

The effect of the alpha-2-adrenoceptor agonist clonidine on anxiety-related behavior was investigated using two different rat anxiety models: a modified Vogel's drinking conflict model and Montgomery's elevated plus-maze. In both models biphasic dose-response curves were obtained; in a narrow low-dose range (6.25-10.0 micrograms/kg) the drug produced anxiolytic-like effects, while anxiogenic-like properties were found after higher doses (12.5-80.0 micrograms/kg). Attempts to block the effects obtained were made in Montgomery's elevated plus-maze. The specific alpha-2-adrenoceptor antagonist idazoxan blocked the anxiolytic-like effect but did not influence the anxiogenic-like activity. Conversely, the specific alpha-1-adrenoceptor antagonist prazosin blocked the anxiogenic-like effect but did not alter the anxiolytic-like activity. These findings may suggest that alpha-1- and alpha-2-adrenergic receptor mechanisms are reciprocally involved in anxiety-related behavior.     

Bidirectional effects of benzodiazepine binding site ligands in the elevated plus-maze: differential antagonism by flumazenil and beta-CCt

Recent research using genetically modified mice has pointed to the specific contribution of individual receptor subtypes to the various effects of benzodiazepines. The aim of this study was to examine the relative significance of alpha(1)-containing GABA(A) receptors in the effects of modulators at the benzodiazepine site in the elevated plus-maze (EPM) under dim red light in rats. We tested the effects of the non-selective antagonist flumazenil (0-20.0 mg/kg), the preferential alpha(1)-subunit selective antagonist beta-carboline-3-carboxylate-t-butyl ester (beta-CCt, 0-30.0 mg/kg), the non-selective agonist midazolam (0-2.0 mg/kg), the preferential alpha(1)-subunit selective agonist zolpidem (0-2.0 mg/kg) and the non-selective inverse agonist methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM, 0-2.0 mg/kg). The influence of flumazenil (10.0 mg/kg) and beta-CCt (30.0 mg/kg) on the effects of both kinds of agonists were also examined. The standard spatio-temporal parameters reflecting anxiety (percentage of open arm entries and time) and locomotion (closed and total arm entries) were analyzed. beta-CCt did not affect behavior, while flumazenil at the highest dose (20.0 mg/kg) decreased indices of open arm activity and total arm entries. Midazolam at the dose of 1.0 mg/kg significantly increased the percentage of open arm time, whereas at 2.0 mg/kg both anxiety-related parameters were increased. In contrast to the open arm entries, the open arm time was independent of the decreased closed arm entries, observed at 2.0 mg/kg. Flumazenil abolished these effects, whereas beta-CCt partially potentiated the anxiolytic actions of midazolam. Zolpidem significantly increased both open-arm indices at 1.0 mg/kg, but the effect was dependent on the decreased closed arm entries. The selectivity of the anxiolytic-like effects of zolpidem was further checked under brighter white illumination. In these settings, the influence on anxiety-related, but not activity-related parameters, was absent. All of the activity-related effects of midazolam and zolpidem were mainly counteracted by both antagonists. DMCM produced significant anxiogenic effects at 1.0 mg/kg (open arm time) and 2.0 mg/kg (both parameters). beta-CCt (30.0 mg/kg) and flumazenil at higher dose (20.0 mg/kg) antagonized the effects of DMCM. The results indicate the anxiolytic effects of a non-selective benzodiazepine site agonist involve a predominant role of subunits other than alpha(1), whereas the behavioral indices of the anxiolytic-like properties of an alpha(1)-selective ligand, if observed, depend on the experimental settings and the changes in locomotor activity, and hence were behaviorally non-specific. The present results generally correspond well to the behavioral findings with the genetically modified mice. On the other hand, the relative significance of the alpha(1)-subunit in the anxiogenic effects of DMCM could not be clearly deduced.     

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.