Targeted invalidation of CCK<Subscript>2</Subscript> receptor gene induces anxiolytic-like action in light–dark exploration,but not in fear conditioning test

Rationale Evidence suggests that γ-aminobutyric acid (GABA) and cholecystokinin (CCK) have opposite roles in the regulation of anxiety. Objectives The aim of our work was to study the behaviour of CCK₂ receptor deficient mice in light–dark exploration and fear conditioning tests. Moreover, the action of diazepam and methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM), having the opposite effect on GABA_A receptors, was evaluated on the exploratory behaviour in these mice. Expression levels of GABA_A receptor subunit genes were also measured. Methods Light–dark exploration and fear conditioning tests were used to determine changes in anxiety of mice. The action of diazepam (0.5–2 mg/kg i.p.) and DMCM (0.25–1 mg/kg i.p.) was studied in the light–dark box. The effect of DMCM was also evaluated in the motor activity test to demonstrate that its anti-exploratory action was not related to motor suppression. Expression levels of GABA_A receptor subunit genes were determined by means of real-time polymerase chain reaction (qRT-PCR). Results Female mice lacking CCK₂ receptors displayed increased exploratory activity in the light–dark box compared to their wild-type (+/+) littermates. Locomotor activity in the motility boxes and the intensity of freezing did not differ in wild-type (+/+) and homozygous (−/−) mice. Treatment with diazepam (0.5 mg/kg) increased the number of transitions in wild-type (+/+) animals, whereas in homozygous (−/−) mice diazepam (0.5–2 mg/kg) reduced exploratory activity. Administration of DMCM (0.25–1 mg/kg) induced an anxiogenic-like effect in homozygous (−/−) mice, but did not change their locomotor activity. Gene expression analysis established a 1.6-fold increase in the expression of the α2 subunit of GABA_A receptors in the frontal cortex of homozygous (−/−) mice. Conclusion Genetic invalidation of CCK₂ receptors induced an anxiolytic-like action in exploratory, but not in conditioned models of anxiety. The observed reduction in anxiety in homozygous (−/−) mice is probably related to an increased function of GABAergic system in the brain.     

Altered pain sensitivity and morphine-induced anti-nociception in mice lacking CCK<Subscript>2</Subscript> receptors

Rationale. Cholecystokinin (CCK) interacts with the endopioid system in the regulation of various physiological functions, including the control of pain sensitivity, motor activity and emotional behaviour. Objective. The aim of the present work was to study the pain sensitivity, morphine-induced antinociception and density of opioid receptors in mice lacking CCK₂ receptors. Methods. Plantar analgesia and hotplate tests were used to evaluate pain sensitivity and morphine-induced antinociception. The parameters of opioid receptors were analysed by using [³H]-diprenorphine binding. Results. In the plantar analgesia test the latency of hind paw withdrawal was significantly increased in CCK₂ receptor deficient mice compared to wild-type (+/+) littermates. The treatment with saline reversed the reduced pain sensitivity in heterozygous (+/−) and homozygous (−/−) mice. The administration of morphine (1 mg/kg) induced a significantly stronger antinociceptive effect in homozygous (−/−) mice compared with wild-type (+/+) animals. In the hotplate test, only homozygous (−/−) mutant mice displayed the delayed latency of hind paw licking/shaking in comparison with wild-type (+/+) mice. The injection of saline and isolation of mice for 30 min reversed the delayed response in homozygous (−/−) mice. However, in this test, the anti-nociceptive action of morphine (5–10 mg/kg) in mutant mice did not differ from that in wild-type (+/+) littermates. By contrast, the jump latency was decreased in both homozygous (−/−) and heterozygous (+/−) mice in the hotplate test. The increased density of opioid receptors was established in the striatum of homozygous (−/−) mice. Conclusion. It is apparent that the targeted mutagenesis of the CCK₂ receptor gene has different effects on the sensitivity of opioid receptors in various brain structures. This is a probable reason for the altered pain sensitivity and morphine-induced antinociception in mutant mice compared to wild-type (+/+) littermates. Electronic Publication     

Effect of noradrenergic system on the anxiolytic-like effect of DOI (5-HT2A/2C agonists) in the four-plate test

Rationale Selective serotonin reuptake inhibitors and serotonin and noradrenaline reuptake inhibitors demonstrated an anxiolytic-like effect in the four-plate test (FPT). (+/−)-1-(2,5-Dimethoxy-4-iodophenyl)-2-aminopropane (DOI; a 5-HT_2A receptor agonist) also possessed strong anxiolytic-like effect in the same test. A 5-HT_2A mechanism seems to be implicated in the mechanism of action of both antidepressants and DOI in this test. On the other hand, the α-adrenergic ligands have also demonstrated an activity in other models of anxiety. A previous study demonstrated that the α₂-adrenoceptor agonists abolished the anxiolytic-like effect of antidepressants. Objectives The aim of the present study was to evaluate the role of noradrenergic system on the regulation of 5-HT₂ receptors implicated in the DOI anxiolytic-like activity in the FPT. Methods First, the effect of noradrenergic and serotonergic lesions on DOI anxiolytic-like activity was studied in the FPT. Second, the effect of co-administration of α-adrenoceptor ligands and DOI was evaluated in the same test. Results The noradrenergic and serotonergic lesions had no effect on DOI (1 mg/kg) anti-punishment activity in the FPT. Adrafinil 0.25 and 4 mg/kg (an α₁-adrenoceptor agonist), prazosin 0.5 and 2 mg/kg (an α₁-adrenoceptor antagonist) and idazoxan 1 and 4 mg/kg (an α₂-adrenoceptor antagonist) did not modify the activity of DOI. Clonidine 0.06 mg/kg, guanabenz 0.125 and 0.5 mg/kg (two α₂-adrenoceptor agonists) and guanfacine 0.06 and 0.125 mg/kg (a specific α_2A-adrenoceptor agonist) completely abolished DOI-induced increase in punished passages. Conclusion These results indicate that the DOI seems to act on the 5-HT₂ receptors post-synaptically located. The effect of DOI is regulated by the α₂-adrenoceptors.     

Involvement of diazepam-insensitive benzodiazepine receptors in the suppression of DOI-induced head-twitch responses in diabetic mice

Rationale We previously reported that the head-twitch responses induced by the 5-HT₂ receptor agonist (±)-2,5-dimethoxy-4-iodoamphetamine (DOI) (DOI-HTRs) were decreased in streptozotocin-induced diabetic mice.Objectives We examined the involvement of γ-aminobutyric acid (GABA)/benzodiazepine system on the suppression of DOI-HTRs in diabetic mice.Results The benzodiazepine receptor antagonist flumazenil (0.1–1 mg/kg, i.v.) dose-dependently and significantly increased DOI-HTRs in diabetic mice to the same levels as in nondiabetic mice. However, flumazenil (0.1–1 mg/kg, i.v.) did not affect DOI-HTRs in nondiabetic mice. The benzodiazepine receptor agonist diazepam (0.1–1 mg/kg, i.p.) had no effect on DOI-HTRs in either nondiabetic or diabetic mice. The GABA_A receptor antagonist bicuculline (0.1–1 mg/kg, i.p.) and the benzodiazepine receptor partial inverse agonist Ro 15-4513 (0.1–1 mg/kg, i.v.) dose-dependently and significantly suppressed DOI-HTRs in nondiabetic mice to the same levels as in diabetic mice. Ro 15-4513-induced reduction of DOI-HTRs in nondiabetic mice was completely antagonized by flumazenil (1 mg/kg, i.v.), but not diazepam (0.3 mg/kg, i.p.).Conclusions We suggest that the abnormal diazepam-insensitive benzodiazepine receptor function partly underlies the suppression of DOI-HTRs in diabetic mice.     

Decreased prepulse inhibition and increased sensitivity to muscarinic,but not dopaminergic drugs in M<Subscript>5</Subscript> muscarinic acetylcholine receptor knockout mice

Rationale Schizophrenic patients show decreased measures of sensorimotor gating, such as prepulse inhibition of startle (PPI). In preclinical models, these measures may be used to predict antipsychotic activity. While current antipsychotic drugs act largely at dopamine receptors, the muscarinic acetylcholine receptors offer promising novel pharmacotherapy targets. Of these, the M₅ receptor gene was recently implicated in susceptibility to schizophrenia. Due to the lack of selective ligands, muscarinic receptor knockout mice have been generated to elucidate the roles of the five receptor subtypes (M₁–M₅). Objectives Here, we used M₅ receptor knockout (M₅−/−) mice to investigate the involvement of M₅ receptors in behavioral measures pertinent to schizophrenia. We tested the hypothesis that disruption of M₅ receptors affected PPI or the effects of muscarinic or dopaminergic agents in PPI or psychomotor stimulation. Materials and methods We measured PPI in M₅−/−, heterozygous and wild-type mice without drugs, and with clozapine (0.56–3.2 mg/kg) or haloperidol (0.32–3.2 mg/kg) alone, and as pretreatment to d-amphetamine. In addition, we evaluated locomotor stimulation by the muscarinic antagonist trihexyphenidyl (0.56–56 mg/kg) and by cocaine (3.2–56 mg/kg). Results The M₅−/− mice showed decreased PPI relative to wild-type mice, and clozapine appeared to reduce this difference, while haloperidol increased PPI regardless of genotype. The M₅−/− mice also showed more locomotor stimulation by trihexyphenidyl than wild-type mice, while cocaine had similar effects between genotypes. Conclusions These data suggest that disruption of the M₅ receptor gene affected sensorimotor gating mechanisms, increased sensitivity to clozapine and to the psychostimulant effects of muscarinic antagonists without modifying the effect of dopaminergic drugs.     

Lack of generalisation between the GABA<Subscript>A</Subscript> receptor agonist,gaboxadol, and allosteric modulators of the benzodiazepine binding site in the rat drug discrimination procedure

Introduction The binding sites for γ-aminobutyric acid (GABA) and GABA_A receptor agonists are located differently from the binding sites for benzodiazepine receptor agonists. Furthermore, the major pharmacological effects of benzodiazepine receptor agonists and the GABA_A receptor agonist gaboxadol (4,5,6,7-tetrahydrroisoxazolo(5,4-c)pyridin-3-ol, THIP) are mediated by different GABA_A receptor subunit compositions; that is, gaboxadol may interact primarily with extra-synaptically located α₄β_2/3δ-containing receptors and benzodiazepines with the synaptically located α₁β_2/3γ₂-containing receptors. Objectives The aim of the present study was to address if this different receptor subtype selectivity was reflected in vivo. Materials and methods A two-lever liquid reinforced operant discrimination procedure was conducted. Three groups of rats were trained to discriminate gaboxadol, diazepam and zolpidem 5.5, 1.5 and 0.7 mg/kg i.p., respectively, from vehicle. Results Substitution tests showed that gaboxadol-trained animals failed to recognize diazepam (0.75–1.5 mg/kg), zolpidem (0.4–0.7 mg/kg), zopiclone (2.5 mg/kg), zaleplon (1.0–1.5 mg/kg) or indiplon (0.31 mg/kg). In contrast, all benzodiazepine receptor agonists, but not gaboxadol (4.5–5.5 mg/kg), generalised to the discriminative stimulus in diazepam- and zolpidem-trained animals. Discussion In agreement with these data, the competitive benzodiazepine receptor antagonist flumazenil (10 mg/kg s.c.) antagonised the discriminative stimulus of zolpidem but not of gaboxadol. Interaction tests showed no synergistic interaction of concomitant administration of gaboxadol and zolpidem or diazepam. Conclusion Previous studies have shown that gaboxadol and benzodiazepines interact with different receptor populations, and the present study confirms that in vivo functional consequences of this receptor selectivity exist in the form of differential behavioural responses in rats.     

Pharmacological evaluation of IQM-95,333, a highly selective CCKA receptor antagonist with anxiolytic-like activity in animal models

1. The pyridopyrimidine derivative IQM-95,333 ((4aS,5R)-2-benzyl-5-[N^α-tert-butoxicarbonyl)L-tryptophyl]amino-1,3dioxoperhydropyrido[1,2-c]pyrimidine), a new non-peptide antagonist of cholecystokinin type A (CCK_A) receptors, has been evaluated in vitro and in vivo in comparison with typical CCK_A and CCK_B receptor antagonists, such as devazepide, lorglumide, L-365,260 and PD-135,158.
2. IQM-95,333 displaced [³H]-CCK-8S binding to CCK_A receptors from rat pancreas with a high potency in the nanomolar range. Conversely, the affinity of this new compound at brain CCK_B receptors was negligible (IC₅₀>10 μM). IQM-95,333 was a more selective CCK_A receptor ligand than devazepide and other CCK_A receptor antagonists.
3. Like devazepide, IQM-95,333 was a more potent antagonist of CCK-8S- than of CCK-4-induced contraction of the longitudinal muscle from guinea-pig ileum, suggesting selective antagonism at CCK_A receptors.
4. IQM-95,333 and devazepide were also potent inhibitors of CCK-8S-stimulated amylase release from isolated pancreatic acini, a CCK_A receptor-mediated effect. The drug concentrations required (IC₅₀s around 20 nM) were higher than in binding studies to pancreas homogenates.
5. Low doses (50–100 μg kg⁻¹, i.p.) of IQM-95,333 and devazepide, without any intrinsic effect on food intake or locomotion, blocked the hypophagia and the hypolocomotion induced by systemic administration of CCK-8S, two effects associated with stimulation of peripheral CCK_A receptors.
6. IQM-95,333 showed an anxiolytic-like profile in the light/dark exploration test in mice over a wide dose range (10–5,000 μg kg⁻¹). Typical CCK_A and CCK_B antagonists, devazepide and L-365,260 respectively, were only effective within a more limited dose range.
7. In a classical conflict paradigm for the study of anxiolytic drugs, the punished-drinking test, IQM-95,333, devazepide and L-365,260 were effective within a narrow dose range. The dose-response curve for the three drugs was biphasic, suggesting that other mechanisms are operative at higher doses.
8. In conclusion, IQM-95,333 is a potent and selective CCK_A receptor antagonist both in vitro and in vivo with an anxiolytic-like activity in two different animal models, which can only be attributed to blockade of this CCK receptor subtype.

     

Changes in relative potency among positive GABA<Subscript>A</Subscript> receptor modulators upon discontinuation of chronic benzodiazepine treatment in rhesus monkeys

Rationale Benzodiazepine treatment can result in dependence as evidenced by signs of withdrawal upon discontinuation of use. Objective Positive GABA_A receptor modulators were examined for their capacity to attenuate flumazenil-like discriminative stimulus effects (i.e., withdrawal) that emerge upon discontinuation of chronic benzodiazepine treatment. Methods Rhesus monkeys receiving chronic diazepam (5.6 mg⁻¹ kg⁻¹ 24 h⁻¹ p.o.) discriminated flumazenil (0.1 mg/kg s.c.) from vehicle. Results Upon discontinuation of diazepam treatment, responding switched from the vehicle to the flumazenil lever, although at different times among monkeys. The shorter-acting benzodiazepine lorazepam (3.2 mg⁻¹ kg⁻¹ 8 h⁻¹) was substituted for diazepam and, 11 h after lorazepam, monkeys consistently responded on the flumazenil lever. Flumazenil-lever responding after acute lorazepam deprivation was attenuated not only by benzodiazepines (lorazepam and midazolam) but also by positive GABA_A receptor modulators acting at neuroactive steroid (pregnanolone and alfaxalone) and barbiturate sites (pentobarbital). Deprivation-induced responding on the flumazenil lever was not attenuated by low efficacy positive GABA_A modulators (bretazenil and L-838,417) or non-GABA_A receptor ligands (ketamine and cocaine). Neuroactive steroids were relatively more potent than other positive GABA_A receptor modulators in attenuating deprivation-induced flumazenil-lever responding, as compared to their relative potency in monkeys discriminating midazolam and otherwise not receiving benzodiazepine treatment. Conclusions These results suggest that positive GABA_A receptor modulators acting at different sites attenuate withdrawal induced by discontinuation of benzodiazepine treatment, consistent with previous studies suggesting that the same compounds attenuate flumazenil-precipitated withdrawal. Differences in the relative potency of positive modulators as a function of acute versus chronic benzodiazepine treatment suggest that neuroactive steroids, in particular, are especially potent in benzodiazepine-dependent animals.     

Dopamine D3 receptor knock-out mice exhibit increased behavioral sensitivity to the anxiolytic drug diazepam

Dopamine D₃ receptors (DRsD3) seem to have a pivotal role in mood disorders. Using the elevated plus maze (EPM) and the novelty-induced grooming test (NGT), we assessed the responses of DRD3-deficient (D₃^−/−) mice to the acute treatment (different testing time) with the anxiolytic drug, diazepam. D₃^−/− mice treated with diazepam (0.1 or 0.5 mg/kg) exhibited a better behavioral response in the EPM than their wild type (WT). Furthermore, in D₃^−/− mice, but not in WT, 1 mg/kg diazepam induced anxiolytic effects at all testing times. The contribution of DRsD3 in the anxiolytic effects of diazepam was confirmed by similar results obtained in EPM by using the selective DRD3 antagonist U99194A (10 mg/kg) in combination with diazepam, in WT animals. D₃^−/− mice treated with diazepam (all doses), also showed a decrease in grooming behavior. However, the [³H]flunitrazepam autoradiographic analysis revealed no significant changes in D₃^−/− mice compared to WT, suggesting that if γ-aminobutyric acid receptor GABA_A changes are involved, they do not occur at the level of binding to benzodiazepine site. These data suggest that D₃^−/− mice exhibit low baseline anxiety levels and provide the evidence that the DRD3 is involved in the modulation of benzodiazepine anxiolytic effects.     

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

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

Clozapine binds preferentially to cortical D1-like dopamine receptors in the primate brain: a PET study

Rationale The D₁-like dopamine receptors have been suggested to play a role in the pathophysiology and treatment of schizophrenia. Previous positron emission tomography studies have demonstrated that the atypical antipsychotic clozapine occupies D₁-like dopamine receptors in the striatum in clozapine-treated patients. Objectives The aim of the present study was to compare striatal and cortical D₁-like dopamine receptor occupancy by clozapine in the primate brain. Methods Three monkeys were each examined three times at the same day with the radioligand (+)−[¹¹C]NNC 112. The first measurement was at baseline conditions, the second after 1.5 mg/kg and the third after 6 mg/kg clozapine IV. To compare regional levels of nonspecific binding in brain regions, an additional monkey was examined using the inactive enantiomer (−)−[¹¹C]NNC 112. Receptor occupancy was calculated using both the equilibrium–ratio analysis and the simplified reference tissue model. Results After 1.5 mg/kg the D₁-like dopamine receptor occupancy ranged from 30 to 38% in the striatum, whereas the range was 51 to 57% in the frontal cortex. After 6.0 mg/kg the occupancy was 53 to 64% in the striatum and 63 to 83% in the frontal cortex. The differences between striatal and cortical D₁-like receptors occupancy were between 12 and 25%. The study with (−)−[¹¹C]NNC 112 did not show regional differences in nonspecific binding that might explain the regional differences in occupancy. Conclusions The higher D₁-like dopamine receptor occupancy in the frontal cortex may reflect a different distribution of the D₁ and D₅ dopamine receptor subtypes among brain regions and different affinity of clozapine for the two subtypes. The finding supports the suggestion that binding to D₁-like dopamine receptors may explain clozapine’s atypical drug actions.     

Additive effects of cannabinoid CB1 receptors blockade and cholecystokinin on feeding inhibition

Cannabinoid CB1 receptor and cholecystokinin-1 (CCK₁) receptors are located in peripheral nerve terminals of the gut, where they mediate satiety signals. Here we describe a detailed analysis of the interaction of both receptors in the control of feeding of food-deprived rats. Male Wistar rats were deprived for food 24 h before testing. Rats were pre-treated with SR141716A (Rimonabant) or WIN 55,212-2 before CCK-8 sulphated administration and tested for food intake 60, 120 and 240 min after last drug injection. In parallel, the effect of Lorglumide - a CCK₁ receptor antagonist - pre-treatment was evaluated on feeding behaviour after SR141716A administration. Results show that SR141716A activates c-Fos expression in brainstem areas receiving vagal inputs. Blockade of CB1 receptors with SR141716A (1 mg/kg) reduces feeding and display additive satiety induction with the CCK₁ receptor agonist CCK-8 sulphated (5, 10, 25 μg/kg). The effect of SR141716A is not blocked by Lorglumide (10 mg/kg), indicating independent sites of action. Conversely, the administration of the CB1 agonist WIN 55,212-2 (2 mg/kg) reduced satiety induced by CCK-8. In conclusion, these results report additive anorectic actions for CCK1 activation and peripheral CB1 receptor blockade providing a framework for combined therapies in the treatment of eating disorders.     

Absence of amino acid-induced glomerular hyperfiltration in dopamine D3 receptor knockout mice

Pharmacological inhibition of receptors of the dopamine D₂-like family has been shown to abolish the glomerular hyperfiltration in response to amino acids. To further discriminate between the receptor subtypes within the D₂-like family, we investigated the effects of amino acid infusion on renal function in dopamine D₃ receptor knockout (–/–) mice. In clearance experiments pentobarbital-anesthetized D₃ receptor (–/–) and wild-type (+/+) mice were infused with Ringer solution at baseline, followed by a continuous infusion of mixed amino acids (10%). Baseline glomerular filtration rate (GFR), assessed by renal clearance of [³H]-inulin, was the same in D₃ receptor (–/–) mice (0.56±0.08 ml/min per g kidney weight) and wild-type animals (0.56±0.04 ml/min per g kw). During infusion of amino acids, GFR was significantly elevated by 50% in D₃ receptor (+/+) mice. In contrast, this amino acid-induced response of GFR was abolished in D₃ receptor (–/–) mice. Baseline urinary water and sodium excretion was not significantly different in both groups of mice. As observed in GFR, these renal excretory parameters were significantly elevated during amino acid infusion in D₃ receptor (+/+) but not in D₃ receptor (–/–) mice. Time controls, constantly infused with Ringer solution, did not show significant changes in GFR, renal water or sodium excretion during the entire experiment, indicating stable experimental conditions. Taken together, the data underline the involvement of dopamine D₂-like receptors in the renal response to amino acid infusion and, in addition, attribute this effect to the dopamine D₃ receptor subtype.     

Antinociceptive effects of haloperidol and its metabolites in the formalin test in mice

Rationale Formalin-induced pain is reduced in sigma-1 (σ₁) receptor knockout mice; therefore, we hypothesized that haloperidol and its metabolites I and II, which have affinity for σ₁ receptors, may modulate formalin-induced pain. Results Intraplantar administration of formalin (2.5%) to CD-1 mice produced a biphasic period of pain. Haloperidol (0.03–1 mg/kg, s.c.) and reduced haloperidol (metabolite II, 0.25–8 mg/kg, s.c.) dose-dependently inhibited both phases of formalin-induced pain. Haloperidol metabolite I (4–128 mg/kg, s.c.) also produced dose-dependent antinociception in the second phase of the formalin test, but was less potent and effective against first-phase pain. Haloperidol metabolite III (16 and 128 mg/kg) and (−)sulpiride (200 mg/kg), which have no affinity for σ₁ receptors, did not produce significant antinociception in either phase of the formalin test. The order of potency of the drugs to produce their antinociceptive effect [haloperidol > metabolite II > metabolite I ≫ metabolite III= (−)sulpiride=inactive] correlated with their affinity for σ₁ receptors, but not with their affinity for σ₂ or dopamine D₂ receptors. Naloxone (1 mg/kg, s.c.) did not antagonize the antinociception induced by haloperidol and its metabolites. None of the antinociceptive drugs in the formalin test produced any antinociception in the tail flick test. Conclusion These results suggest that the antinociceptive effect of haloperidol and its metabolites in the formalin test is not due to unspecific/generalised inhibition of nociception or modulation of opioid receptors, and that it may be related, at least partially, to the ability of these drugs to interact with σ₁ receptors.     

Stereoselective discriminative stimulus effects of zopiclone in rhesus monkeys

Abstract Rationale. The behavioral effects of racemic zopiclone are similar to those of benzodiazepines that positively modulate GABA at the GABA_A receptor complex; however, it is not clear how enantiomers or metabolites of zopiclone contribute to the benzodiazepine-like behavioral effects of racemic zopiclone. Objectives. Racemic zopiclone, its (R)- and (S)- enantiomers, and the (S)-N-desmethyl metabolite, were evaluated for discriminative stimulus effects in untreated and diazepam treated rhesus monkeys. Methods. One group of monkeys discriminated the benzodiazepine midazolam and another group, treated daily with the benzodiazepine diazepam (5.6 mg/kg, PO), discriminated the benzodiazepine antagonist flumazenil. Results. (RS)-Zopiclone (0.32–17.8 mg/kg) and (S)-zopiclone (0.1–10 mg/kg) substituted with similar potencies for midazolam (≥80% midazolam-appropriate responding). The midazolam-like discriminative stimulus effects of (RS)-zopiclone were antagonized by flumazenil (pK _B=7.52). (R)-Zopiclone occasioned a maximum 45% midazolam-appropriate responding at a dose of 100 mg/kg; (S)-desmethylzopiclone produced saline-appropriate responding up to a dose of 100 mg/kg. All four test compounds occasioned predominantly vehicle-appropriate responding in diazepam treated monkeys discriminating flumazenil. (RS)-Zopiclone (10 mg/kg) attenuated the discriminative stimulus effects of flumazenil in diazepam treated monkeys. Conclusions. These results clearly demonstrate that in rhesus monkeys the discriminative stimulus effects of zopiclone are stereoselective and qualitatively similar to those of midazolam. These results fail to show any benzodiazepine-like or benzodiazepine antagonist-like discriminative stimulus effects for (S)-N-desmethylzopiclone, suggesting that any behavioral (e.g. anxiolytic) effects of this compound are not the result of actions at benzodiazepine receptors. Electronic Publication     

Negative GABA<Subscript>A</Subscript> modulators attenuate the discriminative stimulus effects of benzodiazepines and the neuroactive steroid pregnanolone in rhesus monkeys

Rationale Negative GABA_A modulators (i.e., inverse agonists) might be useful for identifying mechanisms at the GABA_A receptor complex that mediate the effects of positive GABA_A modulators, especially those for which there are no available competitive antagonists. Objective Drug discrimination was used to examine antagonism of a 5-beta neuroactive steroid (pregnanolone) and a benzodiazepine (midazolam) by several negative GABA_A modulators in rhesus monkeys. Methods One group of monkeys (n=5) received 5.6 mg kg⁻¹ day⁻¹ of diazepam (p.o.) and discriminated the benzodiazepine antagonist flumazenil (0.1 or 0.32 mg/kg s.c.); another group of monkeys (n=5) discriminated the benzodiazepine midazolam (0.32 mg/kg s.c.). Results In diazepam-treated monkeys, negative GABA_A modulators with increasing efficacy, including Ro 15-4513, ethyl β-carboline-3-carboxylate (β-CCE), methyl β-carboline-3-carboxylate (β-CCM) and methyl-6,7-dimethoxyl-4-ethyl-β-carboline-3-carboxylate (DMCM), substituted for flumazenil. In monkeys discriminating midazolam, pregnanolone occasioned high levels of midazolam-lever responding, and these effects were attenuated by β-CCE and β-CCM, but not by flumazenil, Ro 15-4513, or DMCM. The midazolam discriminative stimulus also was attenuated by β-CCM and DMCM; Schild analysis was consistent with a simple competitive interaction between midazolam and β-CCM but not between midazolam and DMCM. Conclusions Negative modulators are qualitatively similar to neutral modulators in diazepam-treated animals; however, interactions between negative modulators and midazolam suggest that different receptors mediate the effects of some (DMCM) and not other (β-CCM) negative modulators. Negative modulators at benzodiazepine sites exert efficacy-dependent antagonism of positive modulators at neuroactive steroid sites. Without competitive antagonists at neuroactive steroid or barbiturate sites, negative modulators could prove useful for examining the mechanism of action of different classes of positive GABA_A modulator.     

The role of 5-HT receptor subtypes in the anxiolytic effects of selective serotonin reuptake inhibitors in the rat ultrasonic vocalization test

 We evaluated whether the anxiolytic effects of selective serotonin reuptake inhibitors (SSRIs) in the rat ultrasonic vocalization (USV) test are preferentially mediated by (indirect) activation of 5-HT_1A, 5-HT_1B/1D, 5-HT_2A, 5-HT₃ or 5-HT₄ receptors. The SSRIs, paroxetine (ED₅₀ in mg/kg, IP: 6.9), citalopram (6.5), fluvoxamine (11.7) and fluoxetine (>30), dose dependently reduced shock-induced USV. The effects of paroxetine (3.0 mg/kg, IP) were not blocked by the selective 5-HT_1A receptor antagonist, WAY-100635 (3.0 mg/kg, IP), the 5-HT_1B/1D receptor antagonist, GR 127935 (30 mg/kg, IP), the nonselective 5-HT_2A receptor antagonists, ritanserin (3.0 mg/kg, IP) and ketanserin (1.0 mg/kg, IP), the 5-HT₃ receptor antagonist, ondansetron (0.1 mg/kg, IP), or the 5-HT₄ receptor antagonist, GR 125487D (3.0 mg/kg, SC). In contrast, the selective 5-HT_2A receptor antagonist, MDL 100,907 (0.1 mg/kg, IP), completely prevented the paroxetine-induced reduction of USV. Under similar conditions, WAY-100635 blocked the anxiolytic-like effects of the selective 5-HT_1A receptor agonist, 8-OH-DPAT [(±)-8-hydroxy-2-(di-n-propylamino)tetralin, 1.0 mg/kg, IP], and ritanserin, ketanserin, and MDL 100,907 blocked the anxiolytic-like effects of the mixed 5-HT_2A/2C receptor agonist, DOI [1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane, 3.0 mg/kg, IP]. WAY-100635 (1.0 mg/kg, IP) in combination with ritanserin (3.0 mg/kg, IP), but not ondansetron (0.1 mg/kg, IP), GR 125487D (3.0 mg/ kg, SC), or GR 127935 (30 mg/kg, IP), attenuated the USV reducing effects of paroxetine. Although the results suggest that selective stimulation of 5-HT_1A and 5-HT_2A receptors produces a decrease of USV, we postulate that only 5-HT_2A receptors play a pivotal role in the effects of SSRIs in this model of anxiety. Received: 19 May 1997 / Final version: 21 July 1997     

Effects of 5-HT1A receptor agonists,partial agonists and a silent antagonist on the performance of the conditioned emotional response test in the rat

In the present study, the effects of 5-HT_1A receptor ligands with varying degrees of intrinsic activity at the 5-HT_1A receptor were examined in the conditioned emotional response (CER) test and their effects compared to those of the benzodiazepine receptor agonists, diazepam and chlordiazepoxide. Diazepam (3.0 mg/kg) and chlordiazepoxide (3.0 mg/kg), and the 5-HT_1A receptor partial agonists, ipsapirone (10.0 mg/kg) and gepirone (3.0 mg/kg), alleviated conditioned suppression of lever pressing. The 5-HT_1A receptor partial agonist, buspirone (0.1–1.0 mg/kg), the 5-HT_1A receptor agonist, 8-OH-DPAT (0.01– 0.10 mg/kg), and the 5-HT_1A receptor antagonist, WAY-100635 (0.03–3.0 mg/kg), had no effects on conditioned fear. Neither enhancing the level of food deprivation nor pretreatment with the amnesic agent scopolamine induced anxiolytic-like effects in the present CER test. The anxiolytic-like effects of ipsapirone in this test were completely reversed by WAY-100635. These results indicate that 5-HT_1A agonist, but not antagonist actions, induce an anxiolytic effect in the CER test in rats. Received: 13 March 1996/Final version: 8 July 1996     

Adenosinergic system is involved in development of diazepam tolerance in mice

In the present study the effect of adenosinergic system on the development of diazepam tolerance to motor disturbances in mice was investigated. Diazepam tolerance was obtained by administration of diazepam at a dose of 5.0 mg/kg, s.c. for ten consecutive days. On the 1st and the 10th day of the experiment motor impairments were measured in two behavioural tests: rota-rod and chimney test. We showed that acute diazepam injection produced significant motor impairments in mice and that effect was decreased by repeated diazepam treatment, confirming the development of tolerance to the motor impairing effect of diazepam. We demonstrated that adenosine A₁ and/or A_2A receptor agonists: CPA (0.025 and 0.05 mg/kg, i.p.), CGS 21680 (0.1 and 0.2 mg/kg, i.p.), NECA (0.005 and 0.01 mg/kg, i.p.) pretreatment with diazepam were able to attenuate the development of diazepam tolerance and adenosine receptor antagonists: DPCPX (1.0 and 3.0 mg/kg, i.p.), DMPX (3.0 and 6.0 mg/kg, i.p.) and caffeine (10.0 and 20.0 mg/kg, i.p.) induced the opposite effect. The most apparent effects were obtained by non-selective agonist (NECA) and antagonist (caffeine) of adenosine receptors. We conclude that adenosinergic system plays an important role in mechanisms underlying the development of benzodiazepine tolerance.