Agmatine attenuates acquisition but not the expression of ethanol conditioned place preference in mice: a role for imidazoline receptors.

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.

D-Serine ameliorates neonatal PolyI:C treatment-induced emotional and cognitive impairments in adult mice.

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.

Involvement of neurosteroids in the anxiolytic-like effects of AC-5216 in mice.

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.

[Participation of GABA--benzodiazepine receptor complex in the anxiolytic effect of piracetam].

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.

alpha2-Adrenergic agonists antagonise the anxiolytic-like effect of antidepressants in the four-plate test in mice.

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.

Benzodiazepine antagonist Ro 15-1788 in self-poisoning. Diagnostic and therapeutic use.

Thirteen patients with benzodiazepine overdosage received the specific benzodiazepine antagonist Ro 15-1788. Intravenous administration of 1.5 to 10 mg reversed the central nervous system depression induced by different benzodiazepine compounds within one to two minutes of injection. These case reports indicate that Ro 15-1788 may be an effective tool in the primary management of self-poisoning.

Benzodiazepines and their antagonists: a pharmacoethological analysis with particular reference to effects on "aggression".

A review of the literature on chlordiazepoxide indicates that the traditional view of benzodiazepines as antiaggressive drugs represents an inaccurate generalization. In fact, highly variable findings have been reported with the most significant factor in the variability being the type of aggression studied. Furthermore, considerable controversy surrounds the nature of chlordiazepoxide's influence on social conflict in rodents, with opinion divided regarding the selectivity of its inhibitory effect on offensive responding. It is argued that inappropriate behavioural methodology may have substantially contributed to this controversy. A pharmacoethological approach to the analysis of drug effects on social behaviour is described and exemplified by new data on chlordiazepoxide and midazolam. Both agents inhibit offense at doses which do not result in general response inhibition, yet their overall behavioural profiles are somewhat different. Evidence is also presented indicating low dose behavioural activity of the benzodiazepine antagonist Ro15-1788 in two test situations and suggesting possible differences in the effects of Ro15-1788 and CGS8216 on novelty-related responding. Several avenues of research are discussed which may yield insights into the manner whereby benzodiazepines influence social patterns and the significance of benzodiazepine-GABA interactions in such processes.

What can be learned from the effects of benzodiazepines on exploratory behavior?

The purpose of this review is to assess the value of using tests of exploratory behavior to study the actions of benzodiazepines. The methods of measuring exploration and the factors influencing it are briefly described. The effects of benzodiazepines on exploratory behavior of rats and mice are reviewed; and the dangers of interpreting the results of such tests in terms of any of the clinical effects of the benzodiazepines is stressed. Finally, the interactions between benzodiazepines and other drugs acting at the GABA-benzodiazepine receptor complex are described. The results of these experiments caution against global classification of compounds as benzodiazepine "antagonists."

Behavioural pharmacology of food, water and salt intake in relation to drug actions at benzodiazepine receptors.

Drugs which are agonists at benzodiazepine receptors produce many interesting behavioural effects, and amongst these are the stimulation of food, water and salt intake. This review examines the evidence for benzodiazepine effects on these forms of ingestion, and makes tentative proposals about their modes of action. The recent advent of putative benzodiazepine antagonists and inverse agonists provides important new pharmacological tools for the analysis of factors which control ingestion. Preliminary data on examples of such drugs are considered. Anorectic effects of inverse agonists are described. It is clear, though, that the categorization of a drug in one test situation may not apply to another. For example, the compound Ro15-1788 appears as a specific antagonist in one test, a partial agonist in another, and apparently lacks effect in a third. We are not yet sufficiently forward in our understanding of drug actions at benzodiazepine receptors, and their interactions with particular test circumstances, to predict and account for divergent effects of this kind.

Attenuation of hormone responses to the 5-HT1A agonist ipsapirone by long-term treatment with fluoxetine, but not desipramine, in male rats.

The present study had two objectives: (1) to provide information on neuroendocrine challenge tests that can lead to diagnostic tests in humans; and (2) to confirm our previous observation that chronic fluoxetine selectively inhibits serotonin (5-HT1A) receptor function. We determined the effect of chronic fluoxetine and desipramine (DMI) on the hormone response to ipsapirone, a 5-HT1A agonist and a potential anxiolytic drug. Ipsapirone increased oxytocin, adrenocorticotropic hormone (ACTH), corticosterone, and prolactin, but not renin or vasopressin concentrations in plasma. Chronic fluoxetine, but not DMI, significantly inhibited the effect of ipsapirone on plasma oxytocin, ACTH and corticosterone concentrations. Chronic fluoxetine also reduced the Bmax for 3H-8-hydroxy-2-(dipropylamino) tetralin (3H-8-OH-DPAT) labelled 5-HT1A receptors in the midbrain. Neither antidepressant altered the density or affinity of 5-HT uptake sites. In conclusion, the present results confirm our previous results using 8-OH-DPAT as a challenge, and suggest that chronic 5-HT uptake inhibition results in adaptive changes leading to decreased function of the 5-HT1A receptor system. Finally, because ipsapirone may be administered to humans, it might be usable to evaluate 5-HT1A receptor function in depressed patients.

Flumazenil reduces midazolam-induced cognitive impairment without altering pharmacokinetics.

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

Diazepam attenuation of somatostatin binding and effect of somatostatin on accumulation of inositol 1,4,5-trisphosphate in the rat frontoparietal cortex.

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.

Absence of intrinsic antagonist actions of benzodiazepine antagonists on an exploratory model of anxiety in the mouse.

Recent reports suggest that antagonists of benzodiazepine receptors have intrinsic anxiogenic and convulsant properties. An exploratory model in the mouse, specific for anxiolytics, was employed to test further, the hypothesis that antagonists have intrinsic actions pharmacologically opposite to those of the benzodiazepines. The drugs Ro 15-1788 (ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H- imidiazo -[1,5-a] [1,4]benzodiazepine-3-carboxylate), CGS-8216 (2-phenylpyrazolo[4,3-c] quinolin -3[5H]one), beta-CCM (3-carbomethoxy-beta-carboline) and FG-7142 (N1-methyl-beta-carboline-3-carboxamide) all effectively blocked the anxiolytic actions of diazepam. None of these antagonists demonstrated intrinsic or anxiogenic or anxiolytic activity in this model system. These findings suggest that both the species and paradigm employed will determine the pharmacological profile of drugs which act at the GABA-benzodiazepine receptor complex.

Valproate potentiates and picrotoxin antagonizes the anxiolytic action of ethanol in a nonshock conflict task.

The purpose of this study was to examine the effects of the indirect GABA agonist valproate and the indirect GABA antagonist picrotoxin on the anxiolytic (anti-conflict) activity of ethanol in a behavioral conflict task that does not employ electroshock. This task (negative contrast) quantifies how animals respond to an abrupt, unexpected reduction in reward. Treatment with valproate alone did not elevated depressed behavior engendered by abrupt reduction in reward. However, when administered together with a sub-effective dose of ethanol (0.5 g/kg), valproate (50-200 mg/kg) dose-dependently potentiated the anxiolytic action of ethanol. Picrotoxin (2 mg/kg) antagonized the anxiolytic effects of a larger dose of ethanol (1.0 g/kg) given alone, as well as the ability of valproate to enhance the anxiolytic effects of smaller dose of ethanol (0.5 g/kg). As such, these data support a role for GABA in mediating the anxiolytic activity of ethanol.

Interoceptive stimuli produced by an anxiogenic drug are mimicked by benzodiazepine antagonists in rats pretreated with isoniazid.

Treatments which increase gamma-aminobutyric acid (GABA) neurotransmission or those which cause stimulation of benzodiazepine receptors, produce anxiolytic effects; but the converse (anxiogenic) effects have not been reported after suppression of either system alone. We report here that simultaneous inhibition of these two systems produces anxiogenic effects. After partial depletion of GABA by isoniazid, the benzodiazepine antagonists, RO 15-1788 and CGS8216, produced pentobarbital reversible anxiogenic effects in the pentylenetetrazol discrimination assay. These results support the hypothesis that GABA and endogenous anxiolytics mutually facilitate modulation of anxiety. They also indicate that there may exist endogenous anxiogenics which act at non-benzodiazepine recognition sites.

Involvement of 5-HT1A receptors in the antidepressant-like activity of gepirone in the forced swimming test in rats.

The antidepressant-like activity of gepirone, a drug with a high and selective affinity for 5-hydroxytryptamine1A (5-HT1A) receptors, was studied in the forced swimming test in rats. The drug, administered intraperitoneally in single doses of 2.5-20 mg/kg, potently and dose-dependently shortened the immobility time. The anti-immobility effect of gepirone (10 mg/kg) was dose-dependently antagonized by the 5-HT1A receptor and alpha 1-adrenoceptor antagonist, NAN-190 (0.25 and 0.5 mg/kg), the beta-adrenoceptor blocker with the affinity for 5-HT1A and 5-HT1B receptors, pindolol (2 and 4 mg/kg), the 5-HT1A, 5-HT2 and dopamine receptor blocker spiperone (0.01 and 0.03 mg/kg) and by the dopamine receptor antagonist, haloperidol (0.125 and 0.25 mg/kg). On the other hand, the non-selective 5-HT receptor antagonist, metergoline (2 and 4 mg/kg), the selective 5-HT2 receptor antagonist, ketanserin (1 and 2 mg/kg), the selective alpha 1-adrenoceptor blocker, prazosin (0.25 and 0.5 mg/kg) and the beta-blockers with no affinity for 5-HT receptors, betaxolol (4 and 8 mg/kg) and ICI 118,551 (4 and 8 mg/kg), did not affect the anti-immobility effect of gepirone. The effect of gepirone was not modified, either, in animals with a lesion of the 5-HT system, produced by p-chloroamphetamine (PCA, 2 x 10 mg/kg) or p-chlorophenylalanine (PCPA, 3 x 300 mg/kg). The results obtained suggest that the anti-immobility effect of gepirone is mediated by activation of 5-HT1A receptors, most probably located postsynaptically and that dopamine may be involved in this action.

Evidence for control of cardiac vagal tone by benzodiazepine receptors.

Previous work has suggested that vagal preganglionic neurons which project to the heart, are tonically inhibited by endogenous gamma-aminobutyric acid (GABA). This study tested the hypothesis that benzodiazepines, which are thought to act by enhancing GABAergic inhibition, would increase heart rate by suppressing cardiac vagal activity in anesthetized rats, and that pretreatment with Ro 15-1788, a specific benzodiazepine receptor antagonist, would prevent tachycardia induced by benzodiazepines. Midazolam (0.05-4 mg/kg i.v.), alprazolam (1 mg/kg i.v.) and chlordiazepoxide (10 and 20 mg/kg i.v.), all evoked significant increases in heart rate. Pretreatment with atropine methobromide (2 mg/kg i.v.) increased the basal heart rate and prevented tachycardia induced by benzodiazepines. Basal heart rate and blood pressure were unchanged after pretreatment with Ro 15-1788 (10 mg/kg), but subsequent administration of any of the benzodiazepines failed to elicit increases in heart rate in these animals. These findings suggest that benzodiazepines may be potent vagolytics and that this effect should be considered before these agents are administered to patients who have suffered a recent myocardial infarction, in whom vagal tone is thought to be protective against fatal ventricular arrhythmias.

Evidence for the involvement of 5-HT1A receptors in the anticonflict effect of ipsapirone in rats.

The anticonflict activity of ipsapirone, a non-benzodiazepine anxiolytic drug, with high affinity for 5-hydroxytryptamine1A (5-HT1A) receptors, was studied in the drinking conflict test in the rat. The drug, administered in doses 1.25-20 mg/kg increased the number of punished licks, with the maximum effect observed after doses of 5-20 mg/kg of ipsapirone. The anticonflict effect of ipsapirone (5 mg/kg) was dose-dependently antagonized by the 5-HT1A receptor, alpha 1-adrenoceptor and dopamine receptor antagonist, NAN-190 (0.25-1 mg/kg) and by the beta-adrenoceptor blocker, SDZ 21009, which also has a high affinity for 5-HT1A and 5-HT1B receptors (2-8 mg/kg). On the other hand, the non-selective 5-HT receptor antagonist, metergoline (2 and 4 mg/kg), the 5-HT2/5-HT1C receptor antagonist, ritanserin (0.25 and 0.5 mg/kg), the selective alpha 1-adrenoceptor blocker, prazosin (0.25-0.5 mg/kg) and the beta-blockers, betaxolol (8 mg/kg) and ICI 118 551 (8 mg/kg), which have no affinity for 5-HT receptors, did not affect the anticonflict action of ipsapirone. The effect of ipsapirone was also not modified in animals with lesions of 5-HT neurones, produced by p-chloroamphetamine (PCA--2 x 10 mg/kg). These results suggest that the anticonflict effect of ipsapirone in the Vogel test, results from its interaction with 5-HT1A receptors, which are probably located postsynaptically.

Mercury interaction with the GABA(A) receptor modulates the benzodiazepine binding site in primary cultures of mouse cerebellar granule cells.

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.