David J. Sanger

Differences in anxiety-related behaviours and in sensitivity to diazepam in inbred and outbred strains of mice

Abstract Rationale: Natural strain differences exist in mice for behavioural traits such as emotional reactivity. Objective: The present experiments compared the behavioural profiles of nine strains of mice (BALB/c, C57BL/6, C3H, CBA, DBA/2, NMRI, NZB, SJL, Swiss) in two models of anxiety after the administration of the benzodiazepine diazepam. Methods: The tests used were the light/dark choice task and the elevated plus-maze, two well-validated anxiolytic screening tests. Results: In vehicle-treated animals, differences on variables designed to measure anxiety-related behaviours were observed in both tests. In the light/dark test, the strains could be divided into three distinct groups: two non-reactive strains (NZB and SJL), an intermediate-reactive group (C3H, CBA, DBA/2, NMRI, C57BL/6 and Swiss), and one highly reactive strain (BALB/c). In the elevated plus-maze, SJL, NMRI, CBA and, to a lesser extent, C3H strains of mice, consistently showed low levels of anxiety-related behaviours. Intermediate levels were seen in the Swiss and BALB/c strains, and high levels of emotional reactivity were seen in C57BL/6, DBA/2 and NZB. The strain distribution between the light/dark and the elevated plus-maze tests shows similarities and differences, suggesting that each of these experimental procedures represents a different set of behaviours. Marked differences between a number of strains of mice in their sensitivity to the anxiolytic-like action of diazepam were observed in both the light/dark and the elevated plus-maze tests. Mice of the BALB/c, Swiss and, to a lesser extent, CBA and C3H strains were responsive to diazepam in both tests, although in the case of CBA mice, effects may have been contaminated by behavioural suppression. SJL mice were largely unresponsive to the action of the benzodiazepine in both tests, whereas in C57, DBA/2, NMRI and NZB mice, diazepam produced positive effects only in the elevated plus-maze. Conclusion: The finding of differential strain distributions both with and without diazepam treatment in the light/dark and the elevated plus-maze tests, indicates that not all strains of mice are suitable for investigating the effects of GABA/BZ receptor ligands. This study may thus provide a useful guide for choosing the best strain of mice for studying the pharmacology of fear-related behaviours.

Risk Assessment Behaviour: Evaluation of Utility in the Study of 5-HT-Related Drugs in the Rat Elevated Plus-Maze Test

The present study compared the effects of a wide range of 5-hydroxytryptamine (5-HT)-modulating and potential anxiolytic agents in the rat elevated plus-maze using spatiotemporal (i.e., open arm time and entries) and ethologically derived measures (i.e., risk assessment activities and directed exploration). The drugs used were 5-HT1A receptor partial (buspirone and ipsapirone) and full (8-OH-DPAT and flesinoxan) agonists, mixed 5-HT2A/2C receptor antagonists (ritanserin, ketanserin, mianserin, and pirenperone), selective 5-HT3 receptor antagonists (ICS 205-930, MDL 72222, ondansetron, and (RS)-zacopride), and selective (fluoxetine, fluvoxamine, and zimelidine) and nonselective (imipramine) 5-HT reuptake inhibitors. Only buspirone and mianserin produced effects indicative of an anxiolytic-like action on the spatiotemporal measures. However, all 5-HT1A receptor ligands, as well as mianserin, ketanserin, ondansetron, and zacopride, decreased the number of aborted attempts at entry into open arms (risk assessment). In addition, buspirone, mianserin, and zacopride increased head-dipping (directed exploration). Among the 5-HT reuptake inhibitors, zimelidine reduced head-dipping and total entries. The present findings demonstrate that risk assessment responses are sensitive to the action of 5-HT1A receptor ligands, but their modulation by drugs targetting 5-HT2A, 5-HT2C, and 5-HT3 receptors was not convincingly established.

Recent developments in the behavioral pharmacology of benzodiazepine (ω) receptors: Evidence for the functional significance of receptor subtypes

Recent research in molecular biology has demonstrated the complexity of GABAA receptors and shown that benzodiazepine (BZ-omega) receptor subtypes have a structural reality. It is therefore appropriate to ask whether the different pharmacological effects produced by benzodiazepines (anticonvulsant activity, anxiety reduction, motor incoordination, learning deficits, characteristic discriminative stimulus effects, tolerance and dependence) are associated with activity at different receptor subtypes. The present paper reviews the literature dealing with the behavioral effects of novel BZ (omega) receptor ligands relevant to the question of the functional significance of the BZ1 (omega 1) and BZ2 (omega 2) receptor subtypes. The only drugs currently available with a considerable degree of selectivity are alpidem and zolpidem. These compounds have relatively high affinity for GABAA receptors containing the alpha 1 subunit (corresponding to the BZ1 (omega 1) subtype) and very low affinity for receptors with the alpha 5 subunit (corresponding to one type of BZ2 (omega 2) receptor). Pharmacological effects observed with these, and other, less selective compounds allow several tentative conclusions to be drawn: (a) Little is known of the role of subtype selectivity in anxiolytic or amnestic effects but compounds with low intrinsic activity may reduce anxiety without giving rise to sedation or motor incoordination and BZ1 (omega 1) selective drugs appear to disrupt memory only at sedative doses; (b) Selectivity for BZ1 (omega 1) receptors may be associated with sleep-inducing activity but not with motor incoordination, suggesting that BZ2 (omega 2) receptors may be of particular importance in mechanisms of muscle relaxation; (c) The discriminative stimulus effects of different BZ (omega) receptor ligands are not identical and differences may be related to receptor selectivity; (d) Compounds with BZ1 (omega 1) selectivity and compounds with low intrinsic activity produce little or no tolerance and dependence. A wider range of selective compounds will be necessary to investigate these factors in detail and many different pharmacological profiles can be expected from drugs with selectivity and different levels of intrinsic activity.

Characterisation of the effects of nicotine in the five-choice serial reaction time task in rats: antagonist studies.

Abstract Rationale: Nicotine has been shown to decrease reaction time and increase anticipatory responses in a five-choice serial reaction time task (5-CSRTT) in rats, but the receptor mechanisms mediating this effect remain unknown. Objectives: To evaluate further the effects of nicotine in this task and to characterise the receptors mediating these effects. Methods: Using a standard 5-CSRTT protocol, rats were trained to respond to a 0.5-s visual stimulus, which was reduced to 0.25 s for experimental sessions to induce a performance decrement. The effects of acute (0.03–0.3 mg/kg IP) and repeated (0.1 and 0.3 mg/kg IP for 5 days) nicotine were studied, as was the ability of mecamylamine (1 mg/kg IP), hexamethonium (5 mg/kg IP), dihydro-β-erythroidine (6 mg/kg IP) and methyllycaconitine (10 mg/kg IP) to antagonise the effects of acute nicotine. Results: Nicotine had no effect on accuracy, but decreased response latencies, improved performance in the less-well attended stimulus locations and increased inappropriate responding after both acute and repeated treatment. The data suggest that nicotine improves readiness to respond and improves target scanning, and decreases the ability to withhold premature responses (i.e. increased impulsivity). Except for the reduction in error latency, all of the effects of nicotine were antagonised by the non-selective, centrally acting antagonist mecamylamine, whereas the peripheral antagonist hexamethonium had no effect, demonstrating that nicotine’s actions are central in origin. Dihydro-β-erythroidine, a competitive nicotinic antagonist, antagonised all of the effects of nicotine. In contrast, the α7 antagonist methyllycaconitine had no significant effects against nicotine. Conclusions: These results demonstrate that the α7 receptor subtype is not involved in the effects of nicotine in the 5-CSRTT and that its effects are more likely to be mediated by a receptor(s) such as α4β2, α4β4 and/or α3β2 which is sensitive to antagonism by dihydro-β-erythroidine.

Dopamine D3 receptor agonists produce similar decreases in body temperature and locomotor activity in D3 knock-out and wild-type mice

The function of the dopamine (DA) D3 receptor, a member of the D2-like family, has not been firmly established. It has been reported that the potency of DA receptor agonists in producing hypothermia and hypolocomotion in rodents correlates more strongly with the in vitro affinity for, or potency (mitogenesis test) at the D3 than the D2 subtype. In order to investigate further the role of D3 receptors in hypothermia and hypolocomotion, we tested the effects of ip administration of three DA receptor agonists reported to be selective for the D3 receptor subtype (7-OH-DPAT, quinelorane and PD 128907) on core temperature and spontaneous locomotor activity in homozygous (D3-/-), heterozygous (D3+/-) mutant and wild-type (D3+/+) mice. Quinelorane (0.003-0.3 mg/kg), PD 128907 (1-10 mg/kg) and 7-OH-DPAT (0.1-3 mg/kg) induced hypothermia and decreased locomotion to a similar extent in the three genotypes. Additionally, the putatively selective DA D3 receptor antagonist PNU 99194A (3-20 mg/kg i.p.) increased locomotor activity in habituated mice and reversed the hypothermia induced by 30 microg/kg of quinelorane, with no apparent difference between D3-/-, D3+/- and D3+/+ genotypes. The spontaneous level of locomotor activity of mutants (D3-/- or D3+/-) was found to be either at, below, or above that of controls, with no consistent trend between different batches of mice. These results show that the presence of DA D3 receptors is not necessary for the expression of these effects induced by the three agonists or the antagonist supposedly selective for the D3 receptor subtype. This raises the question of the involvement of the D3 receptor in these behavioural effects and the issue of the in vivo selectivity of these four compounds for the D3 receptor subtype. Alternatively, possible adaptive mechanisms taking place in D3-/- mice might have compensated for the absence of DA D3 receptors.

Amisulpride from animal pharmacology to therapeutic action.

Amisulpride is a benzamide derivative with a unique neurochemical and psychopharmacological profile. This compound has selective affinity for human dopamine D3 and D2 receptor subtypes in vitro (binding constant, K. ~3 nmol/1) and blocks functional responses mediated by these receptors. In ex vivo binding studies, amisulpride is twice as selective for D3 as for D2 receptors. At low doses, it preferentially blocks presynaptic dopamine autoreceptors (increase in dopamine release in vivo in the rat olfactory tubercle, 50% effective dose, ED50 3.7 mg/kg), while postsynaptic dopamine receptor antagonism is apparent at higher doses (decrease in striatal acetylcholine levels, ED50 ~60 mg/kg). Amisulpride preferentially stimulates dopamine synthesis and displaces 3H-raclopride binding in vivo in the limbic system rather than the striatum. It antagonizes apomorphine-induced hypothermia in mice and amphetamine- induced hypermotility in rats at low doses (ED50 2-3 mg/kg), blocks apomorphine-induced climbing and spontaneous grooming in mice, blocks apomorphine-induced gnawing in rats at higher doses (ED5019-115 mg/kg) and does not induce catalepsy at 100 mg/kg. The preferential antagonism by amisulpride of presynaptic D2/D3 receptors is reflected behaviourally in the potent blockade of apomorphine-induced effects mediated by dopamine autoreceptors (yawning and hypomotility: ED50 0.2 and 0.3 mg/kg, respectively) compared with those mediated by postsynaptic D2 receptors (e.g. gnawing; EDSO115 mg/kg). Moreover, low doses of amisulpride induce prohedonic (potentiation of food-induced place preference) effects in rats. The atypical neurochemical and psychopharmacological profiles of amisulpride may explain its therapeutic efficacy on both positive and negative symptoms of schizophrenia

A Comparative Study of the Effects of Selective and Non-Selective 5-HT2 Receptor Subtype Antagonists in Rat and Mouse Models of Anxiety

Although there is some evidence that compounds acting at 5-HT2 receptors show anxiolytic activity, little is known about the specific involvement of the different 5-HT2 receptor subtypes in the modulation of anxiety-related responses. In the present study, the behavioural effects of mianserin, a non-selective 5-HT2 receptor antagonist, MDL 100,907, a selective 5-HT2A receptor antagonist, and SB 206553, a selective 5-HT2B/2C receptor antagonist, were investigated in two rat (the Vogel drinking conflict and the elevated plus-maze tests) and two mouse (i.e. the mouse defense test battery (MDTB) and the light/dark choice test) models of anxiety. Diazepam was used as a positive control. In the Vogel drinking test, mianserin (10 mg/kg) and SB 206553 (3-30 mg/kg), but not MDL 100,907, increased punished responding. Similarly, mianserin (1 mg/kg) and SB 206553 (3-10 mg/kg), but not MDL 100,907, increased entries into the open arms of the elevated plus-maze. These effects are consistent with anxiolytic-like actions of mianserin and SB 206553, although the magnitude of the effects of these two compounds was less than those of diazepam. In addition, in the MDTB, the 5-HT2 antagonists did not clearly affect the defensive reactions of mice exposed to a rat stimulus and they failed to reverse the avoidance of the illuminated box in the light/dark choice test. These results indicate a lack of anxiolytic-like action of the compounds in mice. These behavioural profiles suggest that blockade of the 5-HT2A receptor may not reduce anxiety and demonstrate that 5-HT2B and/or 5-HT2C receptor subtypes may be primarily involved in the anxiolytic-like effects of mianserin and SB 206553 in rats.

Orphanin FQ, a novel neuropeptide with anti-stress-like activity.

Potential anxiolytic-like properties of intracerebroventricular (i.c. v.) infusion of orphanin FQ (OFQ), a recently discovered neuropeptide, were investigated in the mouse defense test battery, a well-validated anxiolytic screening test. In this model, Swiss mice are directly confronted with a natural threat (a rat) as well as situations associated with this threat. Primary measures taken during and after rat confrontation were flight, risk assessment, defensive attack and escape attempts. Unlike the anxiolytic drug diazepam (3-10 microgram/5 microliter, i.c.v.), which affected all defensive responses, OFQ (0.3-3 nM/5 microliter) only clearly reduced defensive upright postures and biting reactions. Subjects displayed these latter defensive behaviors upon forced contact with the threat stimulus, a situation which is considered to be highly stressful. These results suggest that the OFQ system may not be primarily involved in anxiety-related responses including cognitive aspects (i. e., risk assessment), while it may play a role in the adaptative responses to unavoidable or extreme stress stimuli.

Comparison of the pharmacological profiles of the hypnotic drugs, zaleplon and zolpidem.

The BZ1 (omega 1)-selective compound, zolpidem, is a clinically effective hypnotic drug with a pharmacological profile which differs from those of benzodiazepine anxiolytics and hypnotics. Zaleplon (CL 284,846) has recently been described as a hypnotic agent which also has BZ1 (omega 1) receptor selectivity. The pharmacological effects of zolpidem and zaleplon were therefore compared in mice and rats. Both drugs blocked tonic convulsions induced in mice by pentylenetetrazole and electroconvulsive shock and clonic convulsions induced by isoniazid. Zaleplon was more potent than zolpidem but the maximal effect of zolpidem for increasing the latency to isoniazid-induced convulsions was greater than that of zaleplon. Little tolerance developed to the anticonvulsant effect of zaleplon against isoniazid-induced seizures following twice daily administration of 10 or 30 mg/kg for 10 days. Both compounds reduced locomotor activity and produced motor deficits in the rotarod and loaded grid tests in mice. However, while zaleplon produced all three effects at similar doses, zolpidem showed the greatest potency for reducing locomotion. Zaleplon and zolpidem also decreased locomotion and produced a rotarod deficit in rats. Again, the difference between the doses giving rise to these two effects was greater for zolpidem than for zaleplon. In a drug discrimination procedure using rats trained to discriminate a dose (5 mg/kg) of chlordiazepoxide, zaleplon produced partial substitution for chlordiazepoxide at doses which greatly reduced response rates. These results show that zaleplon and zolpidem have similar pharmacological profiles, presumably related to their BZ1 (omega 1) receptor selectivity. However, the difference between doses producing motor deficits (rotarod, loaded grid) and those giving rise to other effects (anticonvulsant, decreased locomotion) was greater for zolpidem than for zaleplon. This difference may be related to a greater in vivo intrinsic activity of zolpidem as indicated by the different efficacies of the two drugs to antagonise isoniazid-induced convulsions.

Differences in pharmacological profiles of a new generation of benzodiazepine and non-benzodiazepine hypnotics

The hypnotics, quazepam (a benzodiazepine), brotizolam (a thienotriazolodiazepine), zopiclone (a cyclopyrrolone) and zolpidem (an imidazopyridine) have a common ability to bind to the benzodiazepine recognition site (omega receptor) within the GABAA receptor. For this reason we compared their pharmacological profiles in mice. All compounds shared anticonvulsant and central depressant effects. However, the sedative activity of zolpidem appeared at much lower doses than did the anticonvulsant and myorelaxant effects but the opposite was observed with the other hypnotics. In contrast to brotizolam, quazepam and zopiclone, zolpidem did not increase food intake in mice placed in a novel environment, indicating that this drug lacks disinhibitory activity. Moreover the efficacy of zolpidem at the GABAA receptor, as indicated by its activity against convulsions induced by the GABA synthesis inhibitor, isoniazid, was much greater than that of other hypnotics. These results suggest that the hypnoselective properties observed with zolpidem might be related to a high selectivity for the omega 1 recognition site of the GABAA receptor coupled with a very high intrinsic activity.