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Aims: Danshensu [3-(3,4-dihydroxyphenyl)-lactic acid], a phenylpropanoid compound isolated from Prunella vulgaris var. lilacina, is a well-known antioxidant. Although its antioxidant activity and cardioprotective effect have been reported, the pharmacological properties of danshensu in the central nervous system remain unclear. We investigated whether danshensu exerts anxiolytic-like activity in mice., Main Methods: We conducted monoamine oxidase A (MAO-A) inhibition assay on danshensu in vitro, and behavioral tests including the elevated plus-maze test (EPM), the hole-board test, the rotarod test and the open field test were employed., Key Findings: We found that danshensu significantly inhibited the activity of MAO-A in vitro. The administration of danshensu (3 or 10mg/kg) produced a significant anxiolytic-like effect in the EPM and hole-board test. In addition, no changes in the spontaneous locomotor activity and no myorelaxant effects were observed compared to the control group; these effects were confirmed with the open field test and the rotarod test. Moreover, the anxiolytic-like properties of danshensu were antagonized by a dopamine D1 receptor antagonist (SCH 23390) but not by a 5-HT1A receptor antagonist (WAY 100635) or an α1-adrenergic receptor antagonist (prazosin)., Significance: These results indicate that danshensu exerts its anxiolytic-like properties, in part, through dopaminergic neurotransmitter signaling., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The histamine H4 receptor (H4R) is expressed primarily on cells involved in inflammation and immune responses. Recently, it has been reported the functional expression of H4R within neurons of the central nervous system, but their role has been poorly understood. The present study aimed to elucidate the physiopathological role of cerebral H4R in animal models by the intracerebroventricular administration of the H4R agonist VUF 8430 (20-40 μg per mouse). Selectivity of results was confirmed by the prevention of the effects produced by the H4R antagonist JNJ 10191584 (3-9 mg/kg p.o.). Neuronal H4R activation induced acute thermal antinociception, indicating that neuronal histamine H4R might be involved in the production of antinociception in the absence of an inflammatory process. An anxiolytic-like effect of intensity comparable to that exerted by diazepam, used as reference drug, was produced in the light-dark box test. VUF 8430 reversed the scopolamine-induced amnesia in the passive avoidance test and showed anorexant activity in food deprived mice. Conversely, the H4R activation did not modify the immobility time in the tail suspension test. Rotarod performance test was employed to demonstrate that the effects observed following the administration of VUF 8430 and JNJ 10191584 were not due to impaired motor function of animals. Furthermore, both compounds did not alter spontaneous mobility and exploratory activity in the hole board test. These results show the antinociceptive, antiamnesic, anxiolytic and anorexant effects induced by neuronal H4R agonism, suggesting that H4 modulators may have broader utility further the control of inflammatory and immune processes., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The present study investigated the effect of agmatine on acquisition and expression of ethanol conditioned place preference (CPP) and its modulation by imidazoline agents. Swiss albino mice were treated intraperitoneally with saline or agmatine (20-40 mg/kg) before injection of ethanol (1.25 mg/kg) during conditioning days or on a test day (20-120 mg/kg), to observe the effect on acquisition or expression of CPP, respectively. Agmatine inhibited the acquisition but not the expression of ethanol CPP. Furthermore, both the I₁ receptor antagonist, efaroxan (9 mg/kg) and the I₂ receptor antagonist, BU224 (5 mg/kg) attenuated the agmatine-induced inhibition of the ethanol CPP acquisition. In contrast, the I₂ receptor agonist, 2-BFI (5 mg/kg) and I₁ receptor agonist, moxonidine (0.4 mg/kg) alone, or a combination of their subeffective doses, significantly attenuated the effect of agmatine (20 mg/kg) on acquisition of ethanol CPP. Agmatine or imidazoline agents alone produced neither place preference nor aversion, and at the doses used in the present study did not affect locomotor activity. Thus, agmatine attenuates the acquisition of ethanol CPP at least in part by imidazoline (I₁ or I₂) receptors. In future studies, agmatine or agents acting at the imidazoline receptors could be explored for their therapeutic potential in ethanol dependence.

Melatonin (MLT) is a neurohormone known to be involved in the regulation of anxiety. Most of the physiological actions of MLT in the brain are mediated by two high-affinity G-protein-coupled receptors, denoted MT(1) and MT(2). However, the particular role of these receptors in anxiety remains to be defined. Here we used a novel MT(2)-selective partial agonist, UCM765 to evaluate the involvement of MT(2) receptors in anxiety. Adult male rats were acutely injected with UCM765 (5-10-20mg/kg), MLT (20mg/kg) or diazepam (DZ, 1mg/kg). Anxiety-related behaviors were assessed in the elevated plus maze test (EPMT), novelty suppressed feeding test (NSFT) and open field test (OFT). UCM765 at the dose of 10mg/kg showed anxiolytic-like properties by increasing the time spent in the open arm of the EPMT, and by reducing the latency to eat in a novel environment in the NSFT. In the EPMT, animals treated with UCM765 (10mg/kg) or MLT (20mg/kg) spent more time in the open arms compared to vehicle-treated animals, but to a lesser extent compared to DZ (1mg/kg). In the NSFT, all treatments similarly decreased the latency to eat in a novel environment compared to vehicle. UCM765 and MLT did not affect the total time and the number of entries into the central area of the OFT, but unlike DZ, did not impair locomotion. The anxiolytic effects of UCM765 and MLT in the EPMT and the NSFT were blocked using a pre-treatment with the MT(1)/MT(2) antagonist luzindole (10mg/kg) or the MT(2) antagonist 4P-PDOT (10mg/kg). These results demonstrated, for the first time, the anxiolytic properties of UCM765 and suggest that MT(2)-receptors may be considered a novel target for the development of anxiolytic drugs., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Polyriboinosinic-polyribocytidilic acid (polyI:C) is a synthetic analog that elicits viral-like immune responses in mammals. We have recently found that polyI:C treatment in neonatal mice induced abnormalities of emotional, cognitive, and sensorimotor gating and dysfunction of glutamatergic neurotransmission in adulthood. In this study, we investigated the effect of the NMDA-receptor co-agonist D-serine on polyI:C-induced behavioral abnormalities in mice. Neonatal ICR mice were repeatedly injected with polyI:C for 5 days from postnatal day 2 to 6. At 10 weeks, sensorimotor gating function was analyzed in the prepulse inhibition (PPI) test. Emotional function was analyzed in open field and social interaction tests. Cognitive function was analyzed by novel object recognition tests. D-Serine dose-dependently improved polyI:C-induced impairment of emotional and cognitive behaviors whereas it had no effect on PPI deficit in adults. The ameliorating effects of D-serine were antagonized by pretreatment with an NMDA-receptor antagonist, MK-801. Although the mRNA level of D-amino acid oxidase (DAAO) was increased in the prefrontal cortex and hippocampus of neonatal polyI:C-treated mice in adulthood, no changes were observed in D-serine content and DAAO enzymatic activity. These results suggest that D-serine ameliorates emotional and cognitive impairments of the polyI:C-treated mice through potentiating NMDA receptor activity.

Several lines of evidence support the involvement of serotonergic (5-HT) neurons of the median raphe nucleus (MRN) in anxiety-like behaviour. In this context, it is known that blockade of 5-HT(1A) somatodendritic autoreceptors in the midbrain raphe nuclei increases the firing rate of these neurons, disinhibiting 5-HT release in postsynaptic target areas such as amygdala, hippocampus and periaqueductal grey matter (PAG). However, while activation of 5-HT(1A) or 5-HT(2) receptors in forebrain targets such as the amygdala or hippocampus enhances anxiety-like behaviours in rodents, stimulation of both receptor subtypes in the midbrain PAG markedly reduces anxiety-like behaviour. In view of these findings, the present study investigated whether the anti-anxiety effects induced by pharmacological disinhibition of 5-HT neurons in the MRN are attenuated by the blockade of 5-HT(2) receptors within the PAG. Mice received combined intra-PAG injection with ketanserin (10 nmol/0.1 μl), a 5-HT(2) receptor antagonist, followed by intra-MRN injection of WAY-100635 (5.6 nmol/0.1 μl), a highly selective 5-HT(1A) receptor antagonist. They were then individually exposed to the elevated plus-maze (EPM), with the videotaped behavioural sessions subsequently scored for both conventional and ethological measures. The results confirmed that intra-MRN infusion of WAY100635 reduces behavioural indices of anxiety without significantly altering general activity measures, and further showed that this effect was completely blocked by intra-PAG pretreatment with an intrinsically-inactive dose of ketanserin. Together, these results suggest that 5HT(2) receptor populations located within the midbrain PAG play a significant role in the reduction of anxiety observed following disinhibition of 5-HT neurons in the MRN., (Copyright © 2011. Published by Elsevier B.V.)

The neuropeptide galanin has been recognized as a possible neurotransmitter/neuromodulator, and in addition has been implicated in anxiety- and depression-related behaviors. The present study demonstrates increased locomotion and rearing after galanin (0.3mg/kg) that was given intraperitoneally (i.p.) to intact Wistar rats which were tested 1h later in the open field (OF). These effects, which suggest an anxiolytic-like action, were blocked by i.p. administered peptidic galanin antagonist M40. Further, the locomotion increase caused by galanin and the inhibitory effect of M40 persisted for 48h without additional treatment. Rats exposed to restraint stress (lasting 60min) for three consecutive days and tested 1h after stress termination exhibited reduced locomotion and exploration in the OF. Galanin (0.3 and 1.0mg/kg) given immediately after each stress exposure prevented the decrease of locomotion and exploration induced by stress in all trials. When the test was repeated 6 days later without stress and galanin treatment the reduction of locomotion produced by stress persisted; the anti-stress behavioral effects of both galanin doses were also present. Testing performed on the 12th day after the last stress and galanin treatment with 0.3mg/kg revealed an increased locomotion compared with unstressed and stress-exposed rats. Our results demonstrate that behavioral effects of the peptide galanin are evident even after i.p. administration. These results also suggest that galanin elicits stress-modulatory action, and support the notion that the galaninergic system may serve as a drug target in stress-related conditions., (Copyright © 2011. Published by Elsevier Ireland Ltd.)

Previous studies in our laboratory showed that cannabinoid CB1 receptor knockout mice (CB1-/-) presented increased anxiety-like behaviours that did not respond to the anxiolytic actions of benzodiazepines. These results suggest that the pharmacological effects of benzodiazepines may involve the participation of cannabinoid CB1 receptors. Therefore, the purpose of this study was to examine the effects of alprazolam and the cannabinoid CB1 receptor antagonist AM251 on behavioural assays (light-dark box test, neurological severity score and step-down inhibitory avoidance test) and on the functional activity of the CB1 receptor (WIN-55,212-stimulated [(35)S] guanosine triphosphate (GTP) gamma binding autoradiography).The administration of alprazolam (40 microg/kg, intraperitoneal (i.p.)) decreased anxiety-like behaviours in the light-dark box test and significantly reduced WIN-55,212-stimulated [(35)S]GTPgamma binding autoradiography in the amygdala and in the CA1 field of the hippocampus, but was without effects on CA2, CA3 and the dentate gyrus (DG) of the hippocampus. The administration of AM251 (3 mg/kg, i.p.) blocked the anxiolytic action of alprazolam (40 microg/kg, i.p.), significantly reduced the sedative (ataxia, neurological severity score in the 0.5 cm bar) and the amnesic actions (short time term memory (1 h after electric shock)) of alprazolam (0.5 mg/kg, i.p.).Taken together, these findings revealed that cannabinoid CB1 receptor plays a pivotal role in the pharmacological actions of benzodiazepines. Furthermore, these results suggest that blockade of cannabinoid CB1 receptors may be useful in the treatment of patients with problems related to the consumption of benzodiazepines. Further clinical trials are needed to test this hypothesis.

AC-5216, a ligand for the translocator protein (18 kDa) (TSPO), previously called the peripheral benzodiazepine receptor (PBR), produces anxiolytic-like effects mediated by TSPO in animal models of anxiety. Since stimulation of TSPO is considered to promote the synthesis of neurosteroids, we investigated the possible role of endogenous neurosteroids that positively act on the GABA(A) receptor in the anxiolytic-like effects of AC-5216. In our experiments, the effects of trilostane and finasteride, two inhibitors of steroidogenic enzymes, and picrotoxin, a GABA(A) receptor-gated Cl(-) channel blocker, on the anxiolytic-like effects of AC-5216 were examined in the social interaction test in mice. Also, the anxiolytic-like effects of allopregnanolone and progesterone were examined. The anxiolytic-like effects of AC-5216 (0.1 mg/kg, p.o.) were inhibited by trilostane (10-30 mg/kg, s.c.), finasteride (10-30 mg/kg, s.c.), and picrotoxin (0.03-0.3 mg/kg, s.c.), while those of diazepam (0.1 mg/kg, p.o.) were inhibited by picrotoxin only. The anxiolytic-like effects of progesterone (1-3 mg/kg, s.c.) were inhibited by finasteride (3-30 mg/kg) and picrotoxin (0.1-0.3 mg/kg), although those of allopregnanolone (10 mg/kg, s.c.) were inhibited by picrotoxin only. These results demonstrate that the anxiolytic-like effects of AC-5216 are due to newly synthesized neurosteroids that enhance GABA(A) receptor function.

Food-deprived Wistar rats were exposed to a fixed-time 60-s food delivery schedule until they developed schedule-induced polydipsia. Every fifth lick was then followed by an electric shock during two, signalled, 5-min periods, which ran concurrently with the food delivery schedule. Shock intensities were adjusted to reduce licking to 60-70% of the unpunished licking rates. The benzodiazepine full agonists, diazepam (0.3-3.0 mg/kg), chlordiazepoxide (0.3-10.0 mg/kg), oxazepam (0.3-3.0 mg/kg) and the benzodiazepine partial agonist, RU-32698 (3.0-17.0 mg/kg), led to increases in punished responding at intermediate doses and decreases at the highest doses tested. All benzodiazepine agonists brought about dose-dependent decreases in unpunished schedule-induced polydipsia, with doses required to reduce drinking proving higher than doses required to increase punished schedule-induced polydipsia. The antipunishment effect of 0.3 mg/kg of diazepam was dose-dependently antagonized by flumazenil and the benzodiazepine inverse agonist, RU-34000. Flumazenil effects, however, could reflect actions of flumazenil as a partial inverse agonist at GABAA receptors. RU-32698 at 10.0 mg/kg further facilitated the rate-increasing effect of 0.3 mg/kg of diazepam, but at 17.0 mg/kg partially blocked such antipunishment effect. Overall, the present results extend the similarities of the effects of benzodiazepine compounds on adjunctive and operant patterns of behaviour by showing similar interactions within the benzodiazepine receptor complex.

It is established that bicuculline, picrotoxin, and flumazenil (agents blocking different sites of GABA receptor) decrease the anxiolytic effect of piracetam as manifested in the conflict situation test. The most pronounced interaction was observed between piracetam and flumazenyl. On the background of antagonist action, piracetam inhibited the effects of flumazenil (but not those of bicuculline and picrotoxin). Based on these data, it is assumed that the anxiolytic effect of piracetam is mediated to some extent by benzodiazepine site of the GABA-benzodiazepine receptor complex.

The activity of the novel antidepressant agomelatine was evaluated in three models of anxiety and compared with that of melatonin and two anxiolytics, diazepam and buspirone. All drugs were tested 2 h before and 2 h after the dark phase of the diurnal cycle. Morning and evening agomelatine (10-75 mg/kg) administration increased animals' responses in the elevated plus maze and Vogel tests. Melatonin (10-75 mg/kg) enhanced open arms exploration in the evening experiment and was inactive in the Vogel test. In the conditioned ultrasonic vocalization test, agomelatine, but not melatonin, was active in the morning and evening experiment. Melatonin antagonist, S22153 (20 mg/kg), enhanced the action of morning and evening agomelatine administration in the Vogel and conditioned ultrasonic vocalization tests, while in the elevated plus maze test, S22153 inhibited effects of evening but not morning melatonin and agomelatine administration. These results indicate the involvement of both the melatonin and the 5-HT2C receptors in the mechanism of anxiolytic-like action of agomelatine.

Selective serotonin reuptake inhibitors (SSRIs) and serotonin/noradrenaline reuptake inhibitors (SNRIs) has been reported to be efficient in anxiety disorders. Some animal models have demonstrated an anxiolytic-like effect following acute administration, however, it is not yet known how noradrenergic receptors are implicated in the therapeutic effects of antidepressants (ADs) in anxiety. The effects of two alpha(2)-adrenoceptor agonists (clonidine, guanabenz) on anxiolytic-like effect of two SSRIs (paroxetine and citalopram) and two SNRIs (venlafaxine and milnacipran) were evaluated in the four-plate test (FPT) in mice. Paroxetine (4 mg/kg), citalopram (8 mg/kg), venlafaxine (8 mg/kg), and milnacipran (8 mg/kg) administered intraperitoneally (i.p.) increased the number of punishments accepted by mice in the FPT. Clonidine (0.0039-0.5 mg/kg) and guanabenz (0.03-0.5mg/kg) had no effect on the number of punishments accepted by mice. Clonidine (0.03 and 0.06 mg/kg) and guanabenz (0.125 and 0.5 mg/kg) (i.p. -45 min) reversed the anti-punishment effect of paroxetine, citalopram, venlafaxine and milnacipran (i.p. -30 min). But if the antidepressants are administered 45 min before the test and alpha(2)-adrenoceptor agonists 30 min before the test, alpha(2)-adrenoceptor agonists failed to alter the anti-punishment effect of antidepressants. The results of this present study indicate that alpha(2)-adrenoceptor agonists antagonise the anxiolytic-like effect of antidepressants in mice when they are administered 15 min before the administration of antidepressant suggesting a close inter-regulation between noradrenergic and serotoninergic system in the mechanism of SSRIs and SNRIs in anxiety-like behaviour.

The selective serotonin reuptake inhibitor antidepressant fluoxetine inhibits alprazolam metabolism in vivo by inhibition of the cytochrome P450 3A4 enzyme. Citalopram is a selective serotonin reuptake inhibitor antidepressant that has not yet been fully evaluated with respect to its potential for cytochrome P450 3A4-mediated drug interactions in vivo. Building on the existing in vitro and in vivo evidence that suggest a minimal effect of citalopram on cytochrome P450 3A4, we hypothesized that therapeutic doses of citalopram (20 mg/d), as compared with fluoxetine (20 mg/d), would cause less impairment in the metabolism of the probe drug alprazolam (1 mg) through inhibition of the cytochrome P450 3A4 isozyme as measured by pharmacokinetic and pharmacodynamic parameters in vivo. We found that fluoxetine prolonged the half-life of alprazolam by 16% and increased the area under the curve 0-infinity of alprazolam by 32%, while citalopram did not affect these parameters, although the time of maximum concentration of alprazolam was prolonged by 30 minutes after citalopram administration. Neither selective serotonin reuptake inhibitor significantly affected the pharmacodynamic profile of alprazolam. This experiment suggests differential effects by citalopram and fluoxetine on alprazolam kinetics.

This study was performed to investigate whether nitric oxide (NO) precursor (L-arginine), NO donor (S-nitroso-N-acetylpenicillamine, SNAP) and NO synthase inhibitors [N(G)-nitro-L-arginine-methylester (L-NAME) and N(G)-nitro-L-arginine (L-NOARG)] modulate morphine-induced anxiolytic effects in the plus-maze. L-Arginine (100, 200 and 300 mg kg(-1), i.p.) and SNAP (4, 8 and 10 mg kg(-1), i.p.) reduced the anxiolytic effect of morphine (20 mg kg(-1), s.c.). L-NAME (10, 20 and 40 mg/kg, i.p.) and L-NOARG (10, 15 and 20 mg kg(-1), i.p.) enhanced the anxiolytic effects of morphine (20 mg kg(-1), s.c.). On the other hand, L-arginine and SNAP increased the morphine-induced locomotor activity. L-NAME decreased the morphine-induced locomotor activity, but L-NOARG did not modify the morphine-induced locomotor activity. Therefore, these results suggest that the anxiolytic effects of morphine can be modulated by NO systems., (Copyright 2003 S. Karger AG, Basel)

As part of an effort to identify naturally occurring GABA(A) receptor benzodiazepine binding site (BDS) ligands from traditional medicinal herbs, we previously reported that flavonoid derivatives isolated from Scutellaria baicalensis (S. baicalensis) Georgi exhibited significant affinities for the BDS. The present study describes the characterization of 5,7-dihydroxy-6-methoxyflavone (oroxylin A), one of the major components of the herbal extract. Oroxylin A inhibited [3H]flunitrazepam binding to rat cerebral cortical membrane with a IC(50) value of 1.09+/-0.07 microM. A GABA ratio of 1.09+/-0.04 suggests that oroxylin A interacts as an antagonist at the recognition site. In neuropharmacological studies, oral administration of oroxylin A (3.75-60 mg kg(-1)) did not result in significant changes in animal models routinely employed for benzodiazepine (BD) evaluation. However, oroxylin A selectively abolished the anxiolytic, myorelaxant and motor incoordination, but not the sedative and anticonvulsant effects elicited by diazepam, a BDS agonist. These results add oroxylin A to the list of CNS active flavonoids, and as the first naturally occurring member endowed with selective antagonistic actions via the BDS.

Objectives: Intravenous midazolam is used as an in vivo biomarker of hepatic cytochrome P450 (CYP) 3A activity. Midazolam is a central nervous system depressant and can produce cognitive impairment. The purpose of this study was 2-fold: (1) to determine whether administration of intravenous flumazenil given before intravenous midazolam minimizes cognitive impairment and (2) to determine whether flumazenil pretreatment has an effect on midazolam pharmacokinetics during hepatic CYP3A phenotyping., Methods: Eleven healthy subjects (8 men) received intravenous flumazenil (0.005 mg/kg) or placebo followed 7 minutes later by intravenous midazolam (0.025 mg/kg) in a randomized, double-blind crossover study. Plasma midazolam concentrations were obtained before dosing and at 5, 30, 60, 120, 240, 300, and 360 minutes after dosing and were assayed by liquid chromatography-tandem mass spectrometry. Midazolam pharmacokinetics were determined by noncompartmental methods. The two 1-sided tests procedure was used to compare area under the curve (AUC) between study phases. Data were log-transformed before analysis, and bioequivalence criteria were applied. Digit symbol substitution tests, performed before dosing and at 5, 30, 60, 120, 240, 300, and 360 minutes after dosing, were used to measure cognition. General linear modeling was used to compare scores between study phases., Results: Midazolam AUC extrapolated to infinity [AUC(0-infinity)] between phases was bioequivalent. The AUC ratio (flumazenil plus midazolam/midazolam) was 0.99, with a 90% confidence interval of 0.98 to 1.00. Statistically significant differences(P

This study evaluated partial antagonism of tiletamine-zolazepam (TZ) anesthesia in cheetahs (Acinonyx jubatus) and differences between two benzodiazepine antagonists, flumazenil and sarmazenil, in this species. Four cheetahs were anesthetized three times at an interval of 14 days with an average intramuscular dose of 4.2 mg/kg TZ. In trials 2 and 3 flumazenil at 0.031 mg/kg and sarmazenil at 0.1 mg/kg, respectively, were applied intramuscularly 30 min after initial TZ injection. There was a highly significant difference between the duration of TZ anesthesia with and without antagonist. Use of the antagonists significantly shortened duration and recovery and eliminated excitatory behavior during the recovery phase. No significant differences could be determined between the two antagonists. We recommend the use of sarmazenil and flumazenil to antagonize TZ anesthesia in cheetahs.

The present study examined the influence of dexefaroxan, a potent and selective alpha(2)-adrenoceptor antagonist, on cognitive performance in rodents. In young adult rats, dexefaroxan reversed the deficits induced by UK 14304 [5-bromo-N-(4,5-dihydro-1-H-imidazol-2-yl)-6-quinoxalinamine], scopolamine, and diazepam in a passive avoidance task. In this test, dexefaroxan also attenuated the spontaneous forgetting induced by a 15-week training-testing interval. Moreover, dexefaroxan, given immediately after training, increased the memory performance of rats trained with a weak electric footshock in the passive avoidance test, facilitated spatial memory processes in the Morris water maze task in rats, and increased the performance of mice in an object recognition test. Thus, dexefaroxan appears to have a promnesic effect in these tests by facilitating the processes of memory retention, rather than acquisition or other noncognitive influences. The facilitatory effects of dexefaroxan in young adult rats persisted even after a 21- to 25-day constant subcutaneous infusion by using osmotic minipumps, indicating that tolerance to the promnesic effect of the drug did not occur during this prolonged treatment interval. Furthermore, in the passive avoidance and Morris water maze tests, dexefaroxan ameliorated the age-related memory deficits of 24-month-old rats to a level that was comparable to that of young adult animals, and reversed the memory deficits induced by excitotoxin lesions of the nucleus basalis magnocellularis region. Together, these findings support a potential utility of dexefaroxan in the treatment of cognitive deficits occurring in Alzheimer's disease.

Neurochemical investigations of the anti-anxiety action of ethanol demonstrate that increased dopaminergic, noradrenergic, and serotonergic activity mediates the anxiolytic actions of ethanol. Results of studies with animals and human beings also confirm an involvement of the sympathetic nervous system in the behavioral actions of ethanol. Because enhanced sympathetic activity increases the release of norepinephrine from sympathetic terminals, the interruption of normal sympathetic activity might disrupt the anxiolytic action of ethanol. The present study examined the effect of chemical sympathectomy by reserpine pretreatment on the subsequent anxiolytic action of ethanol. Seventy-one, female, Sprague-Dawley rats were administered reserpine (0 or 5 mg/kg), followed 17-19 h later by ethanol (0, 0.5, or 1.0 g/kg). Animals were then tested in the elevated plus maze. Compared with saline pretreatment, which did not attenuate the anxiolytic actions of a 1.0-g/kg dose of ethanol, reserpine pretreatment completely blocked the anxiolytic action of a 1.0-g/kg dose of ethanol. Reserpine, by itself, did not possess anxiolytic or anxiogenic actions. Because the anxiolytic action of ethanol involves increased catecholamine activity and the depletion of norepinephrine and dopamine from sympathetic nerve terminals by reserpine blocked the anxiolytic action of ethanol, it is concluded that the anxiolytic action of ethanol requires the presence of normal catecholamine activity. We suggest that prostaglandin activity normally evoked by enhanced sympathetic nervous system activity might be involved in the anxiolytic action of ethanol.

The outflow of [(3)H]5-hydroxytryptamine ([(3)H]5-HT) from electrically stimulated rat cortical slices was measured to ascertain the modulatory role of endogenous cholecystokinin (CCK) on the amine outflow and to test the hypothesis that different anxiolytic compounds inhibit 5-HT secretion. The [(3)H]5-HT outflow evoked at 10 Hz was increased up to +30% by CCK(4) 300-1000 nM, the effect being prevented by the CCK(B) receptor antagonist GV 150013, 3 nM. The limited sensitivity to CCK(4) seemed to depend on 5-HT auto-receptor feedback because pre-treatment with 100 nM methiothepin enhanced the [(3)H]5-HT outflow and lowered the CCK(4) threshold concentration from 300 to 30 nM. In addition, pre-treatment with 1 microM bacitracin to inhibit CCK metabolism increased [(3)H]5-HT efflux. This effect was concentration-dependently counteracted by GV150013 suggesting the presence of an endogenous CCK positive modulation. GV150013 30 nM, the 5-HT(1A) partial agonist buspirone 300 nM and the GABA(A) receptor modulator diazepam 10 nM, known to have anxiolytic properties, all significantly reduced the [(3)H] amine outflow from cortical slices by about 20%. This inhibition depended on their interaction with their respective receptors, which seemed to restrain the activity of functionally interconnected glutamatergic interneurones. In fact, APV (50 microM) and NBQX (10 microM) prevented the effect of the anxiolytic compounds. Thus, anxiolytic drugs with different receptor targets can reduce 5-HT outflow by dampening the glutamatergic signal, and in turn, the secretory process of the serotonergic nerve ending.

Background and Objective: Midazolam may occasionally precipitate hostility and violence instead of tranquility. We characterized these episodes, their rate of occurrence, the potential paradoxical responders and possible predisposing circumstances among patients undergoing lower body surgery under spinal or epidural anaesthesia and midazolam sedation., Patients and Methods: Fifty-eight patients who fulfilled the study entry criteria and who underwent surgery within a 3-month period in a large metropolitan, university-affiliated hospital were enrolled. Sedation and restlessness in all patients were controlled by midazolam administered intravenously by the attending anaesthesiologist; these parameters were later objectively confirmed by recorded actigrams. If "paradoxical" events occurred, flumazenil 0.1 mg 10 s-1 was injected until the aberrant behaviour ceased. Patients with paradoxical reactions were later compared with matched control patients selected from the study group to identify epidemiological characteristics., Results: The incidence of paradoxical events was 10.2% (six out of 58 patients, confidence limits 2.3-18.3%) and they occurred 45-210 min after sedation started; the only independent predictor was an age older than that of the entire study group. The mean cumulative and per weight doses of midazolam were similar for both the experimental and the study groups of patients: 7.3 +/- 2.8 to 10.1 +/- 3.6 mg, and 0.1 +/- 0.04 to 0.12 +/- 0.05 mg kg-1. Flumazenil 0.2-0.3 mg (range 0.1-0.5 mg) effectively stopped the midazolam-induced paradoxical activity within 30 s and surgery continued uneventfully., Conclusions: Flumazenil completely reverses midazolam-induced paradoxical reactions and they are more frequent in older patients.

Nicotine has been reported to reduce anxiety in humans and in a number of animal tests. In the social interaction test of anxiety, administration of low doses of nicotine into the dorsal raphé nucleus (DRN) increases the time spent in social interaction without producing accompanying changes in locomotor activity, suggesting that nicotine acts specifically to reduce anxiety in this brain region. The present study examined the ability of the high-affinity competitive nicotinic receptor antagonist dihydro-beta-erythroidine hydrobromide (DH beta E) to antagonise the anxiolytic effect of nicotine following intra-DRN infusion using the social interaction test. The increase in social interaction observed after administration of nicotine (5 ng) into the DRN was completely reversed by coadministration of 100 ng DH beta E. DH beta E (100 ng), when administered alone into the DRN, did not modify the time spent in social interaction. However, it did significantly increase locomotor activity, and this effect was not antagonised by coadministration of nicotine (5 ng) into the DRN. Because of the pharmacological profile of DH beta E, our results suggest that the anxiolytic effect of nicotine in the DRN is mediated by the alpha 4 beta 2 nicotinic receptor subtype.

Mercury compounds are neurotoxic compounds with a great specificity for cerebellar granule cells. The interaction of mercury compounds with proteins in the central nervous system may underlie some of their effects on neurotransmission. In this work we study the interaction of mercuric chloride (HgCl2) and methylmercury (MeHg) with the GABA(A) receptor in primary cultures of cerebellar granule cells. Both compounds increased, dose dependently, the binding of [3H]flunitrazepam to the benzodiazepine recognition site. EC50 values for this effect were 3.56 and 15.24 microM for HgCl2 and MeHg, respectively, after 30 min exposure of intact cultured cerebellar granule cells. The increase of [3H]flunitrazepam binding by mercury compounds was completely inhibited by the GABA(A) receptor antagonists bicuculline and picrotoxinin, and by the organochlorine pesticide alpha-endosulfan. It was also partially inhibited by the anion transporter blocker DIDS, however this effect could be due to a possible chelation of mercury by DIDS. Intracellular events, like intracellular calcium, kinase activation/inactivation or antioxidant conditions did not affect [3H]flunitrazepam binding or its increase induced by mercury compounds. The sulfhydryl alkylating agent N-ethylmaleimide mimicked the effect of mercury compounds on [3H]flunitrazepam binding suggesting a common mechanism. We conclude that mercury compounds interact with the GABA(A) receptor by the way of alkylation of SH groups of cysteinyl residues found in GABA(A) receptor subunit sequences.

From June 1998 to August 1999, 39 California sea lions (Zalophus californianus) were immobilized at a rehabilitation center in northern California (USA) using medetomidine plus zolazepam and tiletamine (MZT), alone and in combination with isoflurane, with atipamezole reversal. Animals were given 70 microg/kg medetomidine with 1 mg/kg of a 1:1 solution of tiletamine and zolazepam intramuscularly. Mean (+/-SD) time to maximal effect was 5+/-3 min. At the end of the procedure, animals were given 200 microg/kg atipamezole intramuscularly. Immobilization and recovery times were, respectively, 28+/-18 and 9+/-7 min for 15 animals maintained with MZT alone and 56+/-47 and 9+/-6 min for 18 animals intubated and maintained with isoflurane. One mortality occurred during anesthesia. Other disadvantages of the MZT combination included some prolonged ataxia, weakness and disorientation during recovery. However, the use of MZT resulted in faster induction and a more reliable plane of anesthesia that was reversible with atipamezole and safer than other previously used intramuscular agents. Physiological parameters including heart rate, respiratory rate, temperature, pulse oximeter saturation, and end-tidal carbon dioxide were monitored.

Intracerebroventricular injection of neuropeptide Y (NPY) produces potent 'anxiolytic' effects in animal models of anxiety. Administration of opioid receptor antagonists suppresses NPY-induced food intake and thermogenesis. The present study examined whether the opiate antagonist naloxone would also suppress the 'anxiolytic' effects of neuropeptide Y. Following training and stabilization of responding in an operant conflict model of anxiety, rats were injected with either NPY or diazepam. Both NPY (veh., 2, 4, 6 microg, i.c.v.) and chlordiazepoxide (veh., 2, 4, 6 mg/kg, i.p.) produced a dose-dependent increase in punished responding in the conflict test. The 'anxiolytic' effects of NPY were not blocked by the administration of flumazenil (3, 6, 12 mg/kg, i.p.). The administration of naloxone (0.25-2.0 mg/kg, s.c.) antagonized the effects of NPY. Central administration of the selective mu opiate antagonist CTAP (1 microg, i.c.v.) partially blocked NPY-induced conflict responding. These results support the hypothesis that NPY may play an important role in experimental anxiety independent of the benzodiazepine receptor and further implicate the opioid system in the behavioral expression of anxiety.

Objective: To determine the effect of a constant-rate infusion of fentanyl on minimum alveolar concentration (MAC) of isoflurane and to determine the interaction between fentanyl and a benzodiazepine agonist (diazepam) and antagonist (flumazenil) in isoflurane-anesthetized dogs., Animals: 8 mixed-breed adult dogs., Procedure: Dogs were anesthetized with isoflurane 3 times during a 6-week period. After a 30-minute equilibration period, each MAC determination was performed in triplicate, using standard techniques. Fentanyl was administered as a bolus (10 microg/kg of body weight, IV) that was followed by a constant infusion (0.3 microg/kg per min, IV) throughout the remainder of the experiment. After determining isoflurane-fentanyl MAC in triplicate, each dog received saline (0.9% NaCl) solution, diazepam, or flumazenil. After 30 minutes, MAC was determined again., Results: Fentanyl significantly decreased isoflurane MAC (corrected to a barometric pressure of 760 mm Hg) from 1.80+/-0.21 to 0.85+/-0.14%, a reduction of 53%. Isoflurane-fentanyl-diazepam MAC (0.48+/-0.29%) was significantly less than isoflurane-fentanyl-saline MAC (0.79+/-0.21%). Percentage reduction in isoflurane MAC was significantly greater for fentanyl-diazepam (74%), compared with fentanyl-saline (54%) or fentanyl-flumazenil (61%). Mean fentanyl concentrations for the entire experiment were increased over time and were higher in the diazepam group than the saline or flumazenil groups., Conclusions and Clinical Relevance: Fentanyl markedly decreased isoflurane MAC in dogs. Diazepam, but not flumazenil, further decreased isoflurane-fentanyl MAC. Our results indicate that diazepam enhances, whereas flumazenil does not affect, opioid-induced CNS depression and, possibly, analgesia in dogs.

Although it is widely believed that the anxiolytic effects of benzodiazepines are mediated through facilitation of GABA(A) receptor function, behavioural studies have to date provided rather weak support for this hypothesis. In particular, considerable inconsistency has been noted both for the effects of GABAergic manipulations in animal models of anxiety and the ability of GABA(A) receptor antagonists to block the anxiolytic effects of diazepam (DZ) and chlordiazepoxide. In view of the sensitivity of the murine plus-maze to the anxiety-modulating effects of GABAergic agents as well as classical benzodiazepines, the current study examined the extent to which the anxiolytic actions of valproic acid (VPA) and DZ in this test involve picrotoxin (PX)-sensitive receptor mechanisms. Subjects were male DBA/2 mice, test duration was 5 min, and ethological scoring methods were employed. Our results show that, while devoid of intrinsic behavioural effects under present test conditions, PX (0.25-0.5 mg/kg) selectively antagonised the anxiolytic-like (but not other) effects of VPA (400 mg/kg). In contrast, the same doses of PX failed to block any of the behavioural changes induced by DZ (1.5 mg/kg), including disinhibition of open arm exploration. These data suggest that the plus-maze anxiolytic effects of DZ in DBA/2 mice are not mediated through PX-sensitive GABA(A) receptors. Further studies will be required to assess the generality of present findings to other mouse strains, species and behavioural paradigms.

Numerous reports in both humans and animals have confirmed that benzodiazepines produce amnesia; however, mechanisms mediating this effect are not clear. In view of the important role of brain somatostatin (SRIF) in the cognitive function of rats, this study sought to determine if the benzodiazepine, diazepam, alters somatostatinergic system in the rat frontoparietal cortex. Intraperitoneal (i.p.) administration of diazepam (5 mg/kg/day) to male Wistar rats (200-250 g) for 3 or 7 days decreased the number of SRIF receptors (26 and 37%, respectively) in synaptosomes from the frontoparietal cortex, without influencing their apparent affinity. This decrease in the tracer binding was not attributable to a direct effect of diazepam on SRIF receptors, because no decrease of SRIF binding was induced by a large concentration of diazepam (10(-4) M) when the drug was added to a preparation of synaptosomes from frontoparietal cortex of untreated rats. To determine if the effect of diazepam on SRIF binding is related to the binding of diazepam to its recognition site on the GABA(A) receptor, a benzodiazepine antagonist, 2-phenylpyrazolo[3,4-c]quinolin-3(5H)-one (CGS 8216) was administered before the diazepam injection. Pretreatment with CGS 8216 (20 mg/kg/day, i.p.) blocked completely the diazepam-induced decrease in the number of SRIF receptors. CGS 8216 alone had no observable effect. The decrease in the number of 125I-Tyr11-SRIF receptor induced by diazepam was accompanied by a decrease in the effect of SRIF, after 15 seconds of stimulation, on inositol 1,4, 5-trisphosphate (IP3) mass accumulation in the rat frontoparietal cortex at 3 (64%) or 7 days (59%) after its administration. Diazepam alone had no observable effect on mass accumulation of IP3. After 14 days of daily diazepam injections, the levels of binding of 125I-Tyr11-SRIF in the frontoparietal cortex returned to control values, coinciding with the tolerance that develops to this benzodiazepine agonists when administered chronically. The decrease in IP3 levels was still observed after 14 days (57%) diazepam administration. Diazepam and CGS 8216 did not affect SRIF-like immunoreactivity levels in the frontoparietal cortex at the three time intervals studied (3, 7 or 14 days). The alteration of frontoparietal cortex SRIF receptor-effector system after 3 or 7 days of diazepam treatment suggests that somatostatinergic neurotransmission plays a role in the mechanism of diazepam action on memory.

Clinical trials have extensively reported the ability of Hypericum perforatum extracts to exert a significant antidepressant activity. Hypericin, the main constituent of H. perforatum extract, is no more regarded as the active principle of the antidepressant activity of the drug. Hence, the question of which constituents are involved in the basic activity of the total extract, is still waiting for an answer. In the present study we focused our attention on the potential anxiolytic activity of H. perforatum total extract, and of some pure components such as protohypericin and a fraction containing hypericin and pseudohypericin. Herein we report that the total extract of H. perforatum increases the locomotor activity in the open field and exerts anxiolytic activity in the light-dark test, whereas the single components did not show any effect. Interestingly, the anxiolytic activity of the total extract was blocked by pretreatment of rats with the benzodiazepine antagonist Flumazenil, hence suggesting an implication of benzodiazepine receptor activation in the anxiolytic effect of H. perforatum extract. Electrophysiological studies, performed to gain more information on the mechanism of action, showed that hypericin reduced the GABA-activated chloride currents, while pseudohypericin did an opposite effect. Furthermore, both hypericin and pseudohypericin inhibited the activation of NMDA receptors.

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.

Rationale: The triazolobenzodiazepine alprazolam has a unique clinical profile compared to most other benzodiazepines (e.g. diazepam, chlordiazepoxide), in that it is used to treat panic disorder and is effective in depression, two disorders that are usually treated with anti-depressants. Previous drug discrimination studies suggested that alprazolam has stimulus properties in common with antidepressants., Objective: In the present study, the discriminative stimulus properties of alprazolam were investigated to test more conclusively the role of benzodiazepine receptors and whether alprazolam has stimulus properties in common with antidepressants., Methods: Male Wistar rats (n=12) were trained to discriminate between alprazolam (2.0 mg/kg, PO) and vehicle in an operant two-lever drug discrimination procedure under a tandem VI40"-FR10 schedule of reinforcement. Generalization and antagonism tests were carried out under 2 min extinction., Results: In generalization tests, a number of benzodiazepines (alprazolam, chlordiazepoxide, midazolam, lorazepam) and the barbiturate pentobarbital substituted completely, while zolpidem and abecarnil substituted partially for alprazolam. In contrast, no significant degree of generalization to the antidepressants imipramine and fluvoxamine and the putative antidepressants buspirone and flesinoxan was found. In antagonism studies alprazolam could be antagonized (almost) completely by flumazenil, partially by pentylenetetrazole, but not by methyl 6, 7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM), N-methyl-beta-carboline-3-carboxamide (FG-7142) and picrotoxin., Conclusions: These results show that the discriminative stimulus properties of alprazolam are mediated by benzodiazepine receptors and that the finding that antidepressants share discriminative stimulus effects with alprazolam may have limited generality.

Twelve babirusa (Babyrousa babyrussa) (four females/eight males) were immobilized 30 times during a 4-yr interval. Significantly higher premedication and immobilizing doses were needed for females than for males (P < 0.05). An i.m. preanesthetic xylazine dose of 1.88 +/- 0.37 mg/kg (range = 1.20-2.12 mg/kg) was used for females and 1.22 +/- 0.16 mg/kg (range = 0.82-1.43 mg/kg) for males. After xylazine, the animals were induced with i.m. tiletamine/zolazepam; females received 2.20 +/- 0.47 mg/kg (range = 1.78-3.33 mg/kg) and males received 1.71 +/- 0.34 mg/kg (range = 1.08-2.05 mg/kg). Anesthesia was reversed with yohimbine (0.14 +/- 0.03 mg/kg; range = 0.07-0.20 mg/kg) and flumazenil (1 mg flumazenil/20 mg zolazepam) either i.m. or i.v. This anesthetic combination produced smooth induction, good relaxation, and sufficient immobilization to perform routine diagnostic and therapeutic procedures (venipuncture, hoof and tusk trims, transportation, radiographs, ultrasound examination, weight determinations, and skin biopsies). Supplemental ketamine HCl or isoflurane was administered to two animals to effectively deepen or prolong the anesthetic plane, with no resultant adverse effects.

Background: Benzodiazepines used for transesophageal echocardiography (TEE) sedation may be associated with postprocedural psychomotor effects that are undesirable., Hypothesis: We hypothesize that flumazenil can reverse cognitive and motor effects from benzodiazepine, promoting earlier return to baseline function., Methods: We prospectively evaluated the cognitive and motor function of patients who did or did not receive flumazenil following TEE. Patients' gait, level of drowsiness, and recall of items learned before and after benzodiazepine administration were evaluated before TEE, as well as immediately and 30 min after the procedure., Results: Of 207 patients (123 men and 84 women), 93 (45%) were given flumazenil 0.2 or 0.4 mg intravenously, and 113 (55%) were not. The baseline characteristics of the patients who received flumazenil were not significantly different from those who did not receive flumazenil, with the exception of a higher mean dosage of midazolam administered to the flumazenil group. In addition, patients in the flumazenil group appeared more drowsy immediately following TEE. When adjusted for age and midazolam dosage, there were no differences, at any time, between the two groups in gait or recall of items learned prior to sedation. however, at 30 min following TEE, the flumazenil group was able to recall a larger number of new items learned immediately after the procedure (1.92/3 vs. 1.61/3, p = 0.02) than did patients in the group not receiving flumazenil. No adverse effects were encountered in any patient., Conclusion: Flumazenil appears safe and effective in reversing anterograde amnesic effects of benzodiazepine following TEE, but has no effects on retrograde amnesia and does not promote earlier return of motor function to baseline. It is useful in clinical situations where high dosages of benzodiazepine have been used and/or excessive drowsiness is evident following TEE. Routine use of the drug, however, is not necessary.

We reported previously that the neurokinin substance P has anxiolytic-like effects when administered into the nucleus basalis of the rat ventral pallidum. The present study aimed to determine the possible involvement of the neurokinin-1 receptor in the anxiolytic action of intrabasalis substance P injection. Behavioral testing was performed in the rat elevated plus-maze model of anxiety. Microinjection of substance P (1 ng) into the nucleus basalis increased sojourn times on the open arms, excursions into the end of the open arms and scanning over the edge of an open arm, indicative of an anxiolytic-like profile. The non-peptide neurokinin-1 receptor antagonist WIN51,708, administered i.p. 20 min prior to intrabasalis substance P injection, antagonized the anxiolytic effects of the neurokinin in a dose-dependent manner. WIN51,708 at 10 mg/kg diminished, while at the higher dose of 20 mg/kg the antagonist completely blocked, the effect of substance P on anxiety-related behaviors. These findings suggest that the anxiolytic-like effects of substance P in the nucleus basalis are mediated through neurokinin-1 receptive sites.

We measured nasal resistance (Rn) while awake, during midazolam-induced sedation and after antagonism with flumazenil (n = 9). Nasal and oral airflow were measured. Rn was calculated by dividing the difference between maximal nasal mask and oropharyngeal pressures by inspiratory airflow at minimum pharyngeal pressure. During sedation, two subjects developed obstructive apnoeic events and four subjects had snoring events. Each apnoea was ended by mechanisms other than a change in breathing route. After antagonism with flumazenil, apnoeic and snoring events were abolished. Rn during midazolam sedation (median 1.46 (25th percentile 1.00, 75th 2.61) kPa litre-1 s) was significantly greater than before midazolam (0.29 (0.25, 0.48) kPa litre-1 s) and after flumazenil (0.41 (0.25, 0.58) kPa litre-1 s) (P < 0.01 in each subject). We conclude that midazolam increased Rn, sometimes leading to obstruction, and flumazenil abolished this increase in Rn.

The effect of benzodiazepines on the nociceptive threshold was studied in rats using the tail-flick and the formalin tests. Systemic injection of midazolam (10 mg/kg, i.p.) induced a significant decrease of the tail-flick latency and produced a long-lasting nociceptive effect in the formalin test, thus characterising a hyperalgesic state. The hyperalgesia induced by midazolam in the tail-flick test was blocked by flumazenil, a specific antagonist for benzodiazepine sites associated with GABA(A) receptors. Picrotoxin, a Cl- channel blocker, inhibited midazolam-induced hyperalgesia in both tests. Midazolam caused hyperalgesia when administered intracerebroventricularly (i.c.v.; 25 microg) but not intrathecally (i.t.; 75 microg). I.c.v. but not i.t. (5 microg) injection of flumazenil suppressed the hyperalgesia induced by midazolam (10 mg/kg, i.p.). Combination of non-hyperalgesic doses of diazepam (10 mg/kg, i.p.) or ethanol (0.48 g/kg, oral) with midazolam (5 mg/kg, i.p.) also induced hyperalgesia. Our results demonstrate that midazolam and diazepam alone or in combination with ethanol can produce hyperalgesia by interacting with GABA(A) receptors at the supraspinal level in rats. The risk of hyperalgesia should be taken in account when these drugs are used in combination in humans.

Although it is widely believed that benzodiazepines reduce anxiety through positive allosteric modulation of the GABA(A)-chloride channel complex, this is not the only mechanism through which agents of this class can modify CNS function. Furthermore, a significant number of reports of apparent flumazenil blockade of diazepam anxiolysis in animal models have paid limited attention to possible intrinsic behavioral actions of the antagonist per se. In the present study, ethological methods were employed to assess in detail the effects of diazepam, flumazenil, and their combination on the behavior of male DBA/2 mice in the elevated plus-maze paradigm. In two experiments, diazepam (1.5 mg/kg) alone reduced open-arm avoidance and increased head dipping, whereas flumazenil (10-40 mg/kg) alone was without significant behavioral effect. However, with the sole exception of head dipping, prior administration of flumazenil (10 and 40 mg/kg) failed to block the behavioral effects of diazepam under present test conditions. These findings imply that the anxiolytic effects of diazepam in the mouse plus-maze are not mediated through flumazenil-sensitive benzodiazepine receptors and that alternate mechanisms must be considered.

This study investigated a role of cholecystokinin (CCK) in the anxiolytic-like action of morphine, an agonist of mu-opioid receptors, in the rat plus-maze model of anxiety. The acute administration of morphine (1 mg/kg) induced a significant increase of exploratory activity in the plus-maze, but did not affect the locomotor activity in the motility test. The higher dose of morphine (2.5 mg/kg) tended to decrease the locomotor activity and, therefore, did not cause the anxiolytic-like action in the plus-maze. The other drugs (naloxone, BOC-CCK-4, L-365,260) and their combinations with morphine (0.5-1 mg/kg) did not affect the locomotor activity of rats. The opioid antagonist naloxone itself (0.5 mg/kg) did not change the exploratory activity in the plus-maze, but potently antagonized the anxiolytic-like action of morphine (1 mg/kg). An agonist of CCK(B)receptors BOC-CCK-4 (1-50 microgram/kg) induced a dose-dependent anxiogenic-like action in the plus-maze. Nevertheless, only one dose of BOC-CCK-4 (10 microgram/kg) completely reversed the action of morphine. Also, one dose of CCK(B)receptor antagonist L-365,260 (10 microgram/kg) was effective to modify the behaviour of rats in the elevated plus-maze. Namely, this dose of L-365,260 increased the ratio between open and total arm entries, a behavioural measure believed to reflect the anxiolytic-like action in the elevated plus-maze. The combination of L-365,260 (100 microgram/kg) with the sub-effective dose of morphine (0.5 mg/kg) caused the anxiolytic-like action in the plus-maze not seen if the drugs were given alone. In conclusion, morphine induces a potent anxiolytic-like action in the elevated plus-maze and CCK is acting as an endogenous antagonist of this effect of morphine., (Copyright 1999 Harcourt Brace & Co. Ltd.)

To evaluate methodology for in vivo interaction studies of benzodiazepines (BZs) and ketoconazole (KCZ) in animal models, this study assessed the pharmacokinetics and electroencephalographic (EEG) effect of KCZ, and suitable dosage regimens of KCZ to maintain sufficiently high KCZ concentrations to inhibit metabolism of BZs in rats. Rats were injected intraperitoneally (i.p.) with KCZ 10 mg kg(-2). No significant EEG change was detected regardless of serum KCZ concentration, indicating that the EEG changes after both BZ and KCZ administration can be attributed entirely to BZ. Serum KCZ concentrations showed an apparent nonlinear pattern of decline with a short half-life (1.38 h). An additional dose of 5 mg kg(-1) i.p. given 180 min after the initial dose sustained KCZ concentrations above 2 pg mL(-1) until at least 500 min after the initial dose. These results provide the basis for design of animal models for in vivo assessment of interactions of BZs and KCZ.

A multiple-trial passive avoidance paradigm was used to examine and compare the ability for estrogen replacement to attenuate learning and memory deficits produced by the muscarinic antagonist scopolamine and the benzodiazepine lorazepam. The multiple-trial paradigm was used in order to distinguish effects on acquisition from effects on retention. Estrogen replacement significantly attenuated a scopolamine-induced deficit on passive avoidance acquisition, but not retention. The ability for estrogen to attenuate the effect of scopolamine on acquisition was observed only when the analysis was limited to animals with serum levels of estradiol <200 pg/ml, suggesting that higher levels of estradiol were ineffective. This observation is consistent with at least one recent study showing dose-related effects of estrogen on ChAT-like immunoreactivity in the basal forebrain and supports the hypothesis that effects of estrogen on basal forebrain cholinergic neurons can help to reduce cognitive deficits associated with cholinergic impairment. Estrogen replacement was also observed to protect against a lorazepam-induced impairment on passive avoidance retention. This effect was observed specifically in animals that received estrogen prior to and during training and was not due to any effect of estrogen on serum levels of lorazepam following acute lorazepam administration. Collectively, these data demonstrate the ability for estrogen replacement to attenuate specific pharmacologically induced impairments in learning and retention and provide additional clues as to potential mechanisms by which estrogen replacement may help to reduce cognitive deficits associated with aging and Alzheimer's disease in postmenopausal women., (Copyright 1998 Academic Press.)

Midazolam is a commonly used benzodiazepine that is ideal for conscious sedation during a variety of procedures. Laryngospasm is listed by the manufacturer as a rare side effect of midazolam. Flumazenil is a competitive inhibitor of the benzodiazepines that is available for reversal at the end of such a procedure or when too much sedation has been achieved. We present a case of a 61-year-old man who was to undergo conscious sedation before cardioversion. Shortly after receiving midazolam, he developed laryngospasm and resultant respiratory distress; the laryngospasm was reversed by a small dose of flumazenil. The symptoms recurred after approximately 25 minutes and were again reversed with flumazenil. Neither midazolam-induced laryngospasm nor its reversal with flumazenil have previously been reported. A brief discussion regarding laryngospasm and the use of flumazenil are included.

Several factors suggest that endogenous benzodiazepines and gamma-amino-butyric acid may be involved in pathophysiology of hepatic encephalopathy (HE). Contrasting opinions exist on the therapeutic efficacy of flumazenil in the treatment of HE. This study was planned to assess the efficacy of flumazenil by a double-blind, placebo-controlled, crossover design in a large and selected population of cirrhotic patients in stage 4a HE admitted to intensive care units over a 4-year period. Out of 236 patients selected for the study, 132 received flumazenil, whereas 131 patients received placebo. Improvement of the neurological score was documented in 31 patients (23%) of flumazenil group and in two patients (1.5%) of placebo group (p < 0.001) during the first study period, whereas during the crossover period, improvement of the neurological score was documented in seven patients (5.3%) of the flumazenil group and in none of the placebo group (p = 0.022). Improvements in EEG tracings were observed in 44 patients (33.3%) of flumazenil group and in five patients (3.8%) of placebo group (p < 0.001) during the first study period; during the crossover period, improvements in EEG tracings were observed in 10 patients (7.5%) of the flumazenil group and in two patients (1.5%) of the placebo group (p = 0.040). The presence of benzodiazepines was detected in the serum of three responders and in two non-responders. The presence of diazepam and NN-desmethyl diazepam was documented in two responders; prior intake of synthetic diazepam was later confirmed in these patients. The results of our study suggest that flumazenil is beneficial only in a selected subset of cirrhotic patients with severe HE; the applicability of this treatment to unselected patients with hepatic coma or to cirrhotic patients with less severe HE still remains to be determined.

A prospective, randomly controlled study was conducted to test the effect of continuous flumazenil infusion in preventing complications arising from severe benzodiazepine (BZ) intoxication. Patients who were believed to be suffering benzodiazepine intoxication and whose Glasgow Coma Scale (GCS) score was below 10 were enrolled after showing a clear-cut response to flumazenil 0.5 or 1 mg (an improvement by 4 or more on the GCS). The patients were consecutively enrolled and randomized into two groups: a continuous infusion group (CI, n = 50) who were immediately given flumazenil 0.5 mg/h for 5 hours, and a control group (CIN, n = 50). Age, sex, incidence of underlying disease, GCS score at several time points, and complication rate were compared in the two groups. Although the CI group had a higher GCS score at most time points, the complication rate did not significantly differ between the two groups (14 of 36 in the CI group v 12 of 38 in the CIN group, P = .684). A greater incidence of underlying disease and an older age seemed to contribute to the higher complication rates in both groups. Several patients (in both groups) resedated into deeper coma after showing an initial response to flumazenil or after the cessation of flumazenil infusion. For severe BZ intoxication, treatment with flumazenil infusion should still be considered skeptically and should not be recommended as routine management BZ-intoxicated patients with an underlying disease, an older age, and resedation into a deep comatose state after showing an initial response to flumazenil should be treated in an intensive care unit.

Aims: The effects of rifampicin on the pharmacokinetics and pharmacodynamics of buspirone, a non-benzodiazepine anxiolytic agent, were investigated., Methods: In a randomized, placebo-controlled cross-over study with two phases, 10 young healthy volunteers took either 600 mg rifampicin or matched placebo once daily for 5 days. On day 6, 30 mg buspirone was administered orally. Plasma buspirone concentrations and effects of buspirone were measured up to 10 h., Results: The total area under the plasma buspirone concentration-time curve after rifampicin was 10.4% (95% CI, 6.3-14.5%) of that after placebo (1.64+/-0.35 ng ml(-1) h vs 22.0+/-15.1 ng ml(-1) h (mean+/-s.d.); P< 0.01). Rifampicin decreased the peak plasma concentration of buspirone from 6.6+/-3.7 ng ml(-1) to 0.84+/-0.23 ng ml(-1) (P< 0.01) and the half-life from 2.8+/-0.7 h to 1.3+/-0.5 h (P< 0.01). A significant (P<0.05) reduction in the effects of buspirone was observed in three of the six psychomotor tests employed (postural sway test with eyes closed, subjective drowsiness and overall drug effect) after rifampicin pretreatment., Conclusions: The strong interaction between rifampicin and buspirone is probably mostly due to enhanced CYP3A4-mediated first-pass metabolism of buspirone. Buspirone will most likely show a greatly reduced anxiolytic effect when used together with rifampicin or other potent inducers of CYP3A4 such as phenytoin and carbamazepine.

Changes in body weight (BW) in female rats treated for 5 weeks (wk) with weekly subcutaneous implantation of silastic capsules containing different benzodiazepines (BZs): diazepam (DZ) 90, 180, 360 and 540 mg wk-1; nordiazepam (ND) 600 mg wk-1; oxazepam (OX) 600 mg wk-1 and flunitrazepam (FN) 540 mg wk-1 and in male rats exposed to DZ (540 mg wk-1) were evaluated herein. Rats (female and male) implanted with empty capsules served as controls. The BW gain was significantly higher in male than in female rats (both DZ-treated and controls). The BW gain increased with increasing doses of DZ but slowed with time of exposure. In comparison to control rats, the BW gain was significantly higher in DZ-(540 mg wk-1) and OX- but not in ND- and FN-treated female rats. However, the differences between BZs were not of statistical significance. In rats exposed to empty capsules (male, female); DZ (male); ND and OX (female) the BW gain increased with time (1-4 wk) while in rats exposed to DZ and FN (female) the BW stabilised within 2 wk. Acute injection of the central BZ receptor antagonist, flumazenil (40 mg kg-1, i.v., 5th wk of chronic exposure), tended to inhibit the time-related BW gain in rats exposed to empty capsules (male, female), DZ (male), ND and OX (female) but did not affect the BW in DZ- (540 mg wk-1) and FN-exposed rats (female) where BW stabilised prior to FLU injection. Repeated administration of flumazenil (30 mg kg-1 wk-1, i.p.) did not affect the BW gain in DZ- and ND-treated female rats. The present data indicate that different BZs have different effects on BW gain in the rat suggesting that different subtypes of BZ receptors are involved.

The purpose of this study was to determine if flumazenil is absorbed from broncho-pulmonary tissue after intratracheal administration and whether therapeutically significant plasma concentrations can be obtained. Six elective surgical patients received a dose of 1.0 mg flumazenil in 10 mL saline intratracheally during general anesthesia. Blood samples were drawn for 6 hours after administration and plasma concentrations were determined by gas chromatography-mass spectrometry (GC-MS). An average peak plasma flumazenil concentration of 65.9 +/- 43.1 ng/mL was attained within 1 minute after administration. No patients reported chest discomfort or dyspnea upon awakening and there were no other side effects noted. Administration of flumazenil via an endotracheal tube results in rapid attainment of therapeutic blood levels.

The effects of gamma-hydroxybutyrate (GHB), a product of gamma-aminobutyric acid (GABA) metabolism which possesses neuromodulatory properties in brain, were investigated in the elevated plus maze in rats. The number of entries and the time spent in the open arms of the maze were increased by GHB (50, 150, 250 mg/kg i.p.). This is classically considered as indicative of an anxiolytic effect of the drug. There was no sedative effect at these doses as measured by the spontaneous locomotor activity in the actimeter or the total number of arm entries. The anxiolytic properties of GHB were reversed by neither the GHB receptor antagonist, NCS-382 (6,7,8,9-tetrahydro-5(H)-5-olylidene acetic acid) (300 mg/kg i.p.), nor the opioid receptor antagonist, naloxone (10 mg/kg i.p.). However the anti-anxiety effect of GHB was antagonized by the benzodiazepine receptor antagonist, flumazenil (10 mg/kg i.p.), suggesting an interaction of GHB with the GABA(A) receptor complex which mediates the anti-anxiety effect of benzodiazepines.