Neuropharmacology Poster Session
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Introduction
Direct influences of the serotonin (5-HT) in drug dependence/withdrawal has started to be reported in the last two decades mainly in the conjunctions with the effects of alcohol, cocaine and opioids, but also barbiturates and benzodiazepines. The molecular biology of 5-HT receptor subtypes is exploding.
The selective agonists and antagonists for individual 5-HT receptor subtypes are developing and it is becoming increasingly more possible to manipulate the function of serotonin selectively at one set of receptor sites without disruption normal effects of this neurotransmitter at other sites. Therefore, the broader studies of the involvement of agonists/antagonists for different 5-HT receptor subtypes in drug dependence/withdrawal are important. The present paper describes results of our study in singly-housed male mice searching into the role of various serotonergics in the effects of benzodiazepine diazepam eliciting behavioural signs of withdrawal upon the ending of the chronic treatment with. Selected w ere: a) the indirect and thus, nonselective 5-HTergic drug fluoxetine inhibiting 5-HT neuronal uptake, b) 5-HT1A/1B receptor agonist eltoprazine, c) adrenergic-beta/5-HT1B antagonist isamoltane, d) 5-HT2A/2C antagonist ketanserine, e) D2 receptor antagonist with 5-HT influences (mainly at 5-HT3 receptors - Jones, 1990) tiapride. These drugs are reported as potential antianxiety or antiaggressive agents. Behavioural signs of diazepam withdrawal in singly-housed male mice on dyadic social interactions with the non-aggressive group-housed partners include the increase of either timidity (anxiety) or aggressivity.
Materials and Methods
Diazepam was administered to singly-housed mice repeatedly (5 mg/kg/day, orally by tube). A combined treatment of diazepam and either serotonergic agent (fluoxetine - 10 mg/kg or eltoprazine - 2.5 mg/kg or isamoltane - 2.5 mg/kg or ketanserine - 1 mg/kg or tiapride - 20 mg/kg) or water was performed 3 days prior to withdrawal in order to find out if it can prevent from signs of withdrawal and/or restore behaviour to a profile that does not differ from controls.
The behavioural model of "agonistic behaviour" During singly-housing adult male mice (strain ICR, TOP-VELAZ s.r.o., Prague, Czech Republic) were treated with diazepam (4 mg/kg/day, orally) for 11-14 days when the first paired social interactions in neutral cages (4 min) with non-aggressive group-housed partners started 30 min after the last dose given.
Further treatment was allocated randomly into two groups. In the experimental group, serotonergic agent was added to diazepam for additional 3 days. In the control group mice were treated at the same manner with combination of diazepam and water (0.1 ml/10 g/day).
The social interactions were observed 30 min after the last dose of the treatments and 2 - 7 days after the withdrawal. Behavioural interactions were video-taped and the analysis was processed using the computer-compatible system (OBSERVER 3.1, Noldus Information Technology b.v., Holland). Behavioural elements of four categories were recorded: sociable - social sniffing, following the partn er, climbing over the partner; timid - defensive posture (upright), escape, alert posture; aggressive - attack, aggressive unrest (threat), tail rattling; locomotor - walk, rear. The tested singly-housed males show differential spontaneous behaviour towards non-aggressive opponents. Some attack the opponents in control interactions (evaluated as "aggressive mice"), the others exhibit mainly defensive-escape behaviour but no attacks (evaluated as "timid mice"). The differences between the occurrence of behavioural acts in control and experimental interactions were evaluated by the non-parametric Mann-Whitney U test
Results
There were found no significant differences in behaviour of control and experimental groups of mice after the repeated diazepam treatment (non-shown results). The presented figures show differences between the occurrence of behavioural acts exhibited in control (the Zero line) and experimental interactions after the repeated treatment and withdrawal. As can be seen in Figures 1 and 2, the withdrawal (48 h) from the repeated diazepam (4 mg/kg) administration inhibiting aggressivity (Fig.1) and defensive-escape behaviour (timidity = anxiety; Fig. 2) in singly-housed male mice caused a "rebound phenomenon" in both these behavioural categories.
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Fig. 1: Diazepam withdrawal in aggressive singly-housed mice.
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Fig. 2: Diazepam withdrawal in timid singly-housed mice.
As can be seen in Figures 3 and 4, in both, aggressive (Fig. 3) and timid mice (Fig.4) fluoxetine prevents from behavioural signs of 48 h withdrawal which occur as increased either aggressivity or timidity (anxiety) when diazepam is given alone (see Fig. 1 and Fig. 2).
click to enlargeFig. 3: Withdrawal of combined treatment with diazepam+fluoxetine in aggressive singly-housed mice.
click to enlargeFig. 4: Withdrawal of combined treatment with diazepam+fluoxetine in timid singly-housed mice.
As can be seen in Figure 5, ketanserine (1 mg/kg) added in the last 3 days to diazepam repeated treatment (4 mg/kg daily for 2 weeks) prevented from the fast return to the control level of aggressivity upon the withdrawal (48 h). Ketanserine given alone (0.5-15.0 mg/kg) caused dose-dependent selective inhibition of mouse aggression (non-shown results).
click to enlargeFig. 5: Withdrawal of combined treatment with diazepam+ketanserine in aggressive singly-housed mice.
The acute combined treatment with diazepam and tiapride potentiated the antiaggressive effects (non-shown results). Tiapride (20mg/kg) when given concurrently with diazepam (4 mg/kg daily for 2 weeks) in the last 3 days of the repeated treatment prevented from the rebound phenomenon in mouse aggression upon the withdrawal (48 h), as can be seen in Figure 6.
click to enlargeFig. 6: Withdrawal of combined treatment with diazepam+tiapride in aggressive singly-housed mice.
The acute dose of isamoltane (2.5 mg/kg) significantly inhibited all aggressive acts and disinhibited locomotor activities in aggressive mice (non-shown results). As can be seen in Figure 7, the same dose co-administered the last 3 days before the withdrawal of repeated diazepam treatment was able to prevent from the increase of aggressive unrest and attacks but not the tail rattling (rosy columns). The increase of all these aggressive activities was the effect of diazepam withdrawal (blue columns).
click to enlargeFig. 7: Withdrawal of treatment with either diazepam or diazepam+isamoltane in aggressive singly-housed mice.
As can be seen in Figure 8, eltoprazine (E) at the antiaggressive dose of 2.5 mg/kg added in the last 3 days to diazepam (D) repeated treatment prevented the relatively fast return to baseline of aggression in singly-housed mice.
It was apparent after 3 days of withdrawal (columns of green shades), and even after 7 days of withdrawal (columns of purple shades). Also the disinhibiting eltoprazine effect on locomotion persisted.
click to enlargeFig. 8: Three and seven day withdrawal of treatment with either diazepam or diazepam+isamoltane in aggressive singly-housed mice.
Discussion and Conclusion
Accumulating evidence suggests that fluoxetine might be useful in the treatment of alcohol, cocaine, amphetamine and opioid dependence/withdrawal (Herbst et al., 1997; Schmitz et al., 1997). Despite that fluoxetine did not exhibit anxiolytic effect in singly-housed mice (inhibiton of timidity) it was able to prevent the increase of both timidity and aggressivity upon the diazepam withdrawal in the present study. Thus, our results bring the evidence that fluoxetine might also be considered as a drug of choice in the treatment of diazepam dependence/withdrawal.It has been suggested that 5HT2A/C receptors may be important targets for the development of atypical tranquillising drugs (Kleven and Koek, 1976) which could be implicated in a number of disease states, including drug dependence/withdrawal problems (Bryant et al., 1976). Our results received in the study with ketanserine support these suggestions. Barry et al. (1987) reported that the repeated administration of tiapride was not associated with any behavi oural withdrawal effects unlike diazepam. This as well as our results may contribute to the suggestion that tiapride could be considered not only an alternative to BZs for the treatment of alcohol withdrawal (Peters and Faulds, 1994) but also for the prevention of behavioural diazepam withdrawal signs. In spite of the evidence that isamoltane by inhibiting the terminal 5-HT autoreceptors increases the synaptic concentration of 5-HT (Renyi, 1991) its efficacy to suppress behavioural signs of diazepam withdrawal in aggressive mice was lower than of the other 5-HTergics used. This corresponds to the fact that 5-HT1B knock-out mice appeared more aggressive than its wildtype (Olivier, 1997), and that eltoprazine, a partial 5-HT1A/1B receptor agonist, elicited a long-lasting prevention of return to aggressivity during diazepam withdrawal. The 5-HT1B receptor is suggested (Olivier, 1997) to be important in the modulation of offensive aggression, as the blockade of the 5-HT1A receptor by the 5-HT1A receptor antagonis t WAY 1000635 did not diminish the antiaggressive effects of eltoprazine. In summary, fluoxetine, ketanserine and tiapride provided equivalent significant prevention of the occurrence of behavioural signs of 48 h diazepam withdrawal in singly-housed male mice on dyadic interactions with non-aggressive group-housed partners in the present study. Eltoprazine preventive influence lasted 1 week, and included also marked disinhibition of locomotor behaviour. Acknowledgement: The study was supported by the grant No. 3426-3 from the Internal Grant Agency of the Czech Ministry of Health (IGA MZ CR).
References
- Barry, JM et al. (1987): Pharmacol. Biochem. Behav., 27: 239-245
- Bryant, HU et al. (1996): Life Sci., 59(15): 1259-1268
- Herbst, MD et al. (1997): In: Abstracts of the 59th Annual Scientific Meeting College on problems of Drug Dependence, Nashville, USA, p. 62
- Jones, B. (1990): Drug News and Perspectives, 3: 106-111
- Kleven, MS and Koek, W (1996): J. Pharmacol. Exp. Therap., 276: 388-397
- Oliver, B (1997): Abstracts of "Ethological Pharmacology: Current State", St. Petersburg, Russia, p.51
- Peters, DH and Faulds, D. (1994): Drugs, 47(6): 1010-1032
- Schmitz, JM et al. (1997): In: Abstracts of the 59th Annual Scientific Meeting College on problems of Drug Dependence, Nashville, USA, p. 134
- Renyi, A (1991): Naunyn Schmiedebergs Arch. Pharmacol. 343(1): 1-6
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