INSERM U288, Neuropsychopharmacologie Moléculaire, Cellulaire et Fonctionnelle, Faculté de Médecine Pitié-Salpêtrière, 91 Boulevard de lHôpital, 75634 Paris Cedex 13,
1 CNRS UMR-7593, Personnalité et conduites adaptatives, IFR (70) des Neurosciences, CHU Pitié-Salpêtrière, 75013 Paris, France and
2 Department of Psychiatry, University of Würzburg, Füchsleinstrasse 15, 97080 Würzburg, Germany
Received 28 February 2003; in revised form 3 April 2003; accepted 3 April 2003
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ABSTRACT |
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INTRODUCTION |
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In addition, the gene coding for the 5-HT transporter (5-HTT) has been considered as a candidate gene in numerous studies of alcohol dependence (Gorwood et al., 2000; Heinz et al., 2000
; Preuss et al., 2000
, 2001
; Thompson et al., 2000
), and treatment with SSRIs (i.e. zimeldine, citalopram, fluoxetine and fluvoxamine) was reported to decrease the desire to drink alcohol in alcohol-dependent subjects (Lejoyeux, 1996
). However, not all studies have confirmed that this effect persists throughout treatment (Gorelick and Paredes, 1992
), and the usefulness of SSRIs to help alcoholic subjects to reduce their alcohol intake is still a matter of debate (Naranjo and Knoke, 2001
). Because knock-out mice with targeted disruption of the 5-HTT gene can be considered as a model of whole-life treatment with SSRI (Bengel et al., 1998
), they offer a relevant opportunity to assess whether or not long-term inactivation of 5-HT re-uptake yields a sustained decrease in alcohol intake. Using a free-choice procedure, we thus compared the spontaneous ethanol consumption in these mutants (5-HTT/) and paired wild-type (5-HTT+/+) mice, and investigated their respective response to chronic treatment with fluoxetine.
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MATERIALS AND METHODS |
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Procedures involving animals and their care were conducted in conformity with the institutional guidelines that are in compliance with national and international laws and policies (Council directive no. 87-848, 19 October 1987, Ministère de lAgriculture et de la Forêt, Service Vétérinaire de la Santé et de la Protection Animale; permissions No. 75-116 to M.H. and No. 6269 to L.L.).
Alcohol self-administration procedure
Male mice were used at 3 months of age when body weight in each genotype equally ranged between 28 and 30 g. Mice were then housed singly in standard laboratory cages (Macrolon type 2 cages, 22 x 16 x 14 cm). Two tubes (20 ml) were placed on each cage, one containing tap water and the other filled with water supplemented with varying concentrations of ethanol. Mice had continuous free access to the drinking tips of both tubes. The position of the tubes was changed every 2 days in order to avoid possible bias due to place preference. Tubes were filled with freshly prepared liquids every 2 days. Food was provided ad libitum.
Homozygous mutants and wild-type mice were exposed to a progressively increasing concentration of ethanol (0 to 20% ethanol in water within 24 days) under the free-choice procedure described by Crabbe et al.(1996). During the first 4 days, both tubes were filled with tap water. Then, mice were offered 3% ethanol (v/v) versus water for 4 days, and 6% ethanol versus water for the following 4 days. For the next step, the ethanol concentration was raised to 10% for 6 days, then to 15% for another 6 days, and finally to 20% for 1624 days (stabilization period).
Fluoxetine treatment
Fluoxetine [10 mg/kg/day intraperitoneally (i.p.)] or saline (5 ml/kg/day i.p.) was injected daily at 18:00 h for 10 days under the 20% ethanol/water conditions.
Ethanol and total fluid intake
Fluid intake and body weight were determined every 2 days between 17:00 and 18:00 h. The number of grams of ethanol consumed and volumes of total liquid intake/kg body weight/24 h were determined for each mouse.
Statistical analyses
Data were analysed by a two-way analysis of variance (ANOVA) and unpaired t-test, with Welchs corrections when necessary. Statistical significance was set at P ≤ 0.05.
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RESULTS |
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Body weight
As compared with values just before treatment (28.52 ± 0.11 and 28.63 ± 0.18 g in 5-HTT+/+ and 5-HTT/ mice, respectively, means ± SEM, n = 16 in each group), a slight decrease in body weight was noted in both wild-type (1.4 ± 0.2%) and knock-out (1.3 ± 0.9%) mice at the end of the 10-day treatment with fluoxetine (10 mg/kg/day). In contrast, body weight increased slightly in both wild-type (+1.8 ± 0.8%) and knock-out (+1.6 ± 0.7%) mice that had received saline for 10 days.
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DISCUSSION |
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Several 5-HT re-uptake inhibitors, including the long-acting fluoxetine, have been found to decrease alcohol intake in dependent alcoholic patients (Naranjo et al., 1990, 1994
; Lejoyeux, 1996
), as well as in animal models of ethanol intake (Haraguchi et al., 1990
; Higgins et al., 1992
; Maurel et al., 1999
). However, although in rat models the SSRI-induced reduction in ethanol intake has been established by several studies (see Maurel et al., 1999
), the long-term efficiency and usefulness of SSRIs in the treatment of alcohol dependence in human subjects continues to be a matter of debate (Gorelick and Paredes, 1992
; Naranjo and Knoke, 2001
). It has notably been emphasized that although all SSRIs reduce alcohol consumption, the degree of this effect varies markedly from one SSRI to another, and is not directly related to the potency of these drugs to inhibit 5-HT re-uptake. This led to the assumption that SSRI-induced reduction in alcohol intake may not be caused solely by 5-HT re-uptake inhibition, but may also involve additional effects that are highly variable among these drugs. This could be especially true for fluoxetine, because this compound, which has been shown to exert higher anti-alcohol effects than other SSRIs (Maurel et al., 1999
), is the least selective of these drugs with regard to 5-HT re-uptake inhibition, and is also a rather potent 5-HT2C receptor antagonist (Sanchez and Hyttel, 1999
).
In order to explore further the effect of SSRIs on ethanol intake in C57BL/6J mice, which are well characterized as alcohol-appetant (preferring) mice (Yoshimoto and Komura, 1989), we used genetically paired mutants with targeted disruption of the gene encoding the 5-HTT, responsible for 5-HT re-uptake (Bengel et al., 1998
). Indeed, homozygous 5-HTT/ mice, which can be considered as a model of whole-life treatment with SSRI, have been shown to exhibit adaptive changes in 5-HT neurotransmission similar to those observed after chronic treatment with these drugs (Fabre et al., 2000
; Mannoury la Cour et al., 2001
). Using a free-choice procedure of progressive alcohol self-administration, we observed that the spontaneous alcohol intake was markedly lower in 5-HTT/ mutants than in wild-type mice. In agreement with previous observations in rats (Haraguchi et al., 1990
; Maurel et al., 1999
), chronic treatment with fluoxetine was found to decrease significantly alcohol consumption in wild-type mice. However, this treatment did not affect alcohol consumption in 5-HTT/ mutants, as expected from an action of fluoxetine through the blockade of 5-HT re-uptake. Accordingly, it can be assumed that the inhibitory effect of fluoxetine on alcohol intake is really caused by 5-HTT blockade and does not involve secondary effects of the drug at other molecular targets.
Interestingly, total fluid intake (water plus ethanol solution) was not significantly different in 5-HTT/ mutants and paired wild-type mice, and was also unaffected by fluoxetine treatment, indicating that the reduction in alcohol consumption caused by 5-HTT inactivation was not the consequence of a general decrease in drinking behaviour. However, a slight decrease in body weight was noted in fluoxetine-treated mice, as expected from a limited reduction in food intake by this drug (Heisler et al., 1999). Interestingly, this effect was observed not only in wild-type mice, but also in 5-HTT/ mutants, suggesting that it might involve actions of fluoxetine at a target(s) other than the 5-HTT. Accordingly, 5-HT re-uptake appears to be a relevant target for treatments aimed at selectively reducing alcohol intake in dependent alcoholic subjects.
In conclusion, the present findings showed that inactivation of 5-HT re-uptake leads to a sustained decrease in alcohol intake in 5-HTT/ mutant mice, and that the inhibitory effect of fluoxetine on alcohol intake is the consequence of its direct interaction with the transporter responsible for 5-HT re-uptake in wild-type mice.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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