The administration of atomoxetine during alcohol
deprivation induces a time-limited increase in
alcohol consumption after relapse
Francisco Alén1,2*, Antonia Serrano2*, Miguel Ángel Gorriti1, Francisco Javier Pavón2, Laura Orio2, Raquel Gómez de Heras1, María Teresa Ramírez-López1, María Antón2, Miguel Ángel Pozo3and Fernando Rodríguez de Fonseca1,2
1Departamento de Psicobiología, Facultad de Psicología, Universidad Complutense de Madrid, 28224, Spain
2Instituto IBIMA, Unidad de Gestión Clínica de Salud Mental, Hospital Regional Universitario de Málaga, Universidad de Málaga,
29010, Spain
3Instituto Pluridisciplinar, Universidad Complutense de Madrid, 28224, Spain
Abstract
The administration of selective serotonin reuptake inhibitors (SSRIs) typically used as antidepressants increases alcohol consumption after an alcohol deprivation period in rats. However, the appearance of this effect after the treatment with selective noradrenaline reuptake inhibitors (SNRIs) has not been studied. In the present work we examined the effects of a 15-d treatment with the SNRI atomoxetine (1, 3 and 10 mg/kg, i.p.) in male rats trained to drink alcohol solutions in a 4-bottle choice test. The treatment with atomoxetine (10 mg/kg, i.p.) during an alcohol deprivation period increased alcohol consumption after relapse. This effect only lasted one week, disappearing thereafter. Treatment with atomoxetine did not cause a behavioral sensitized response to a challenge dose of amphetamine (1.5 mg/kg, i.p.), indicating the absence of a super-sensitive dopaminergic transmission. This effect is markedly different from that of SSRI antidepressants that produced both long-lasting increases in alcohol consumption and behavioral sensitization. Clinical implications are discussed.
Received 18 February 2014; Reviewed 26 March 2014; Revised 24 April 2014; Accepted 29 April 2014; First published online 15 July 2014
Key words:Alcoholism, antidepressants, atomoxetine, rat, relapse.
Introduction
Alcoholism is a chronic relapsing disorder associated with multiple medical consequences, including the affec-tive disorders (Kessler et al.,1996). Depressive symptoms can be observed in approximately 80% of alcoholics, of which a 30% meet criteria for major depression (Kessler et al.,1996; Torrens et al., 2005). Thus, a large number of alcoholic patients have been prescribed with anti-depressants, despite the lack of information on the poten-tial long-term effect of such therapy and the scarcity of conclusive studies on the efficacy of antidepressants for comorbid alcoholic/depressive patients (Pettinati, 2004; Torrens et al.,2005). Among the different antidepressants, selective serotonin reuptake inhibitors (SSRIs) are the most frequently prescribed. Recently, concerns about the use of SSRIs have been raised because of: (1) the potential
side effects (Coupland et al., 1996; Ashton and Young, 1999; Blier and Tremblay,2006); (2) the low efficacy on preventing relapse (Pettinati, 2004; Torrens et al., 2005); and (3) lack of effectiveness of antidepressants, especially SSRIs, for the treatment of clinical depression in alcoholic patients (Kranzler et al.,2006; Nunes et al.,2006). A recent clinical report suggests that SSRIs are less effective than selective noradrenaline reuptake inhibitors (SNRIs) for the treatment of alcoholism associated with psychi-atric comorbidity (Petrakis et al., 2012). Although these clinical observations remain to be fully confirmed, pre-clinical studies found a similar efficacy of either SSRIs or SNRIs in different models of alcoholism (De Vry et al.,1999).
The serotonin and noradrenaline transmission systems are key elements in the neuroadaptions associated with loss of control of drug use. Both serotonin and noradren-aline deficits have been linked to alcoholism and studies using animal models have demonstrated the efficacy of the administration of either SSRIs or SNRIs for alcoholism associated with stress and depression (Higley et al.,1998; Getachew et al.,2010). The normal serotonergic function needs to be balanced with respect to other modulatory systems (i.e. dopaminergic and noradrenergic) and it Address for correspondence: F. Alén and F. R. de Fonseca, Laboratorio de
Medicina Regenerativa, Instituto IBIMA, Hospital Regional Universitario de Málaga, Av. Carlos Haya 82, Sótano, Málaga 29010, Spain.
Tel.: +34669426548 Fax: +34952614012
appears that the most abused drugs, including alcohol, disrupt this equilibrium when consumed chronically (Tassin, 2008). Thus, it is feasible that the induction of an imbalanced serotonergic transmission, as a result of chronic administration of SSRIs, might have an impact on addictive behaviors. This assumption is supported by the induction of behavioral sensitization observed in animals treated with fluoxetine (Collu et al., 1997). If this is proven, such a side effect might limit the use of SSRI antidepressants as a therapy for alcoholism-associated affective disorders.
This hypothesis has been recently addressed in an ex-perimental approach using an animal model of alcohol-ism (Alén et al., 2013). In that study, the sub-chronic administration of SSRIs (fluoxetine or the noradrenaline/ serotonin reuptake inhibitor venlafaxine) during a period of alcohol deprivation enhanced the alcohol deprivation effect (ADE), leading to a sustained increase in alcohol consumption that lasted at least 5 wk. In addition,
fluoxetine-treated animals showed an enhanced response to amphetamine, indicating a sensitized state of the dopa-minergic reward system, as previously described (Collu et al., 1997). This sensitization was not observed in venlafaxine-treated animals. This study opens the debate on the adequacy of SSRI-based therapy for depression associated with alcoholism (see Nunes and Levin,2004), in the absence of longitudinal studies on the efficacy of SSRIs in the relapse rates of alcoholics affected of major depression (Pettinati,2004; Petrakis et al., 2012). This is especially relevant if we consider the need to address the treatment of depression without promoting relapse to alcohol consumption. As an alternative to SSRI, the use of SNRIs can be considered on the basis of the pre-clinical studies described above. However, there is no information on the effects of chronic SNRI treatment on alcohol consumption after relapse with the exception of the cited report of Petrakis et al. (2012) in veterans suffering comorbid alcoholism and affective disorders. In order to explore this hypothesis, the present study was designed to address the effects of the sub-chronic treatment with a SNRI during a period of alcohol depri-vation on the levels of alcohol consumption after relapse. We have selected atomoxetine, a SNRI widely used for attention-deficit/hyperactivity disorder (Newman et al., 2008; Robinson et al.,2008). Atomoxetine, an orally active propylamine derivative, was initially designed as an antidepressant that was proven to have clinical efficacy (Chouinard et al., 1984) with a clinical pharmacology profile in humans compatible with that of a selective nor-adrenergic uptake blocker (Zerbe et al.,1985). However, it is currently approved only for attention-defi cit/hyper-activity disorder (Newman et al.,2008). Its unique phar-macological profile allow us to test whether or not the enhancement of noradrenergic transmission will increase alcohol consumption when given along an alcohol-deprivation period, as observed after SSRI administration (Alén et al.,2013).
Methods
Animals
Thirty-two adult male Wistar rats (8 per group) (Harlan, Barcelona, Spain) weighing 375–425 g at the beginning of the experiments were housed individually under a 12 h light/dark cycle (lights off at 00:00 hours) in a room at constant temperature (23 ± 1 °C). Standard food and tap water were available ad-libitum at the home-cage. The animals were allowed to accustom to the housing facilities for 2 wk before the beginning of the alcohol self-administration protocol.
All experimental animal procedures were performed in compliance with the European Directive 2010/63/ EU on the protection of animals used for scientific pur-poses and with Spanish regulations (RD 53/2013 and 178/2004). All protocols were approved by the Ethics Com-mittee of the Complutense University of Madrid. Special care was taken to minimize the suffering and number of animals to achieve our research goals.
Drugs
Atomoxetine was purchased from Tocris-Bioscience (BiogenCientífica, Madrid, Spain). A fresh solution was prepared daily (before injection) by dissolving atomo-xetine hydrochloride in the vehicle (0.9% saline) solution. Atomoxetine was injected at different doses (1, 3 or 10 mg/kg, i.p.) in a volume of 2 ml/kg. The doses were chosen based on the literature reporting behavioral characterization of atomoxetine (Robinson et al., 2008; Economidou et al.,2011; Janak et al.,2012). Three alcohol solutions were prepared daily: 5, 10 and 20% ethanol in water (w/v). Amphetamine was obtained from Sigma-Aldrich (Spain), and administered i.p. to the animals prior to the locomotor sensitization test, in a dose of 1.5 mg/kg.
Non-operant alcohol self-administration and relapse model
We used the 4-bottle non-operant alcohol self-administration model (Hölter and Spanagel, 1999; Spanagel and Hölter, 1999) for training the animals to drink alcohol. After 1 wk of habituation to the animal room, all rats were given continuous access to tap water and to 5, 10 and 20% alcohol solutions in their home cages.
was monitored for three consecutive weeks, and changes in the patterns of alcohol consumption were monitored. To this end, alcohol consumption was measured on the 2nd, 4th and 7th day of each week.
Behavioral sensitization experiment
The same experimental conditions, previously described with atomoxetine and vehicle, were reproduced in another set of Wistar rats to establish their potential for inducing sensitization to the psychostimulant effects of the amphetamine.
Animals were trained to consume alcohol and sub-sequently injected with the doses of atomoxetine and ve-hicle for 15 d, similarly to the alcohol self-administration experiment. After treatment, locomotor activity was moni-tored for 30 min and then, the rats were returned to their home cages. The amphetamine challenge (1.5 mg/kg, i.p.) was administered on the 16th day for all treatment groups, and the differences in locomotion among the ex-perimental groups were monitored for the 30 min-post-injection interval. The testing and the sensitization phase were performed keeping the context constant for each animal (e.g. using the same chamber for each animal, procedure time, light intensity and so on).
Behavioral testing was performed within activity monitoring chambers (35 × 35 cm) equipped with 8 photo-cells evenly distributed in two rows of four photo-cells at 5 and 10 cm from thefloor. The number of times that each photo beam was broken was registered by a computer program devised for this purpose, and used as measure of loco-motion activity.
Data analysis
Data are expressed as means±S.E.M. The statistical analysis of the results was performed using GraphPad Prism ver-sion 5.04 (GraphPad Software Inc, USA). The non-operant alcohol self-administration results were evaluated by two-way analysis of variance (ANOVA) with repeated measures (factors: the treatment and time). Regarding locomotor activity, within- and between-groups analysis were performed by either one-way or two-way ANOVA (factors: the treatment and amphetamine challenge), respectively. In addition, multiple comparison post-hoc
tests were performed [Dunnett’s test (one-way ANOVA) and Bonferroni´s test (two-way ANOVA)]. A two-tailed
pvalue less than 0.05 was considered to be statistically significant.
Results
Effects of atomoxetine on weekly alcohol consumption (Fig. 1a–c)
Following the 15-d period of alcohol deprivation, the ethanol consumption was increased during the relapse in thefirst week in all groups of treatment (51% [vehicle
group], 97% [atomoxetine 3 mg/kg] and 416% [atomo-xetine 10 mg/kg] of increase, approximately), with the exception of animals receiving 1 mg/kg of atomoxetine, which showed a slight increase of 6%.
The weekly pattern of alcohol consumption was differ-ent in each week. During thefirst week (Fig. 1a), a two-way ANOVA indicated that only the treatment had a significant primary effect on ethanol intake [F3,84= 21.44,
p< 0.001], with no interaction between treatment and time [F6,84= 0.6726, n.s]. In fact, alcohol consumption was greater in the first 2 d for atomoxetine 3 and 10 mg/kg (**p <0.01) than vehicle. In the following days, only the group treated with atomoxetine 10 mg/kg
10
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Atomoxetine 3 mg/kg Atomoxetine 10 mg/kg
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(**p <0.01) remained high. During the second week (Fig. 1b), the time had a significant primary effect on ethanol intake [F2,84= 7.605,p= 0.0009], with no interaction between treatment and time [F6,84= 0.6259, n.s]. However, during the third week (Fig. 1c), the ethanol intake was found to be similar in all groups with no significant effects or interaction between the time and treatment. These results suggest that the effects observed for ato-moxetine were time-limited.
Effects of atomoxetine treatment on locomotor sensitization (Fig. 2)
One-way ANOVA indicated that sub-chronic treatment with atomoxetine had a significant primary effect on locomotion [F3,31= 3.945, p= 0.0182]. Thus, the post-hoc analysis showed that the atomoxetine induced a mild decrease in locomotion resulting significant at the doses of 3 and 10 mg/kg(#p< 0.05). However, the sub-chronic treatment with atomoxetine did not affect the psycho-stimulant response to amphetamine (1.5 mg/kg) [F3,31= 0.1379, n.s].
A two-way ANOVA indicated that only amphetamine had a significant main effect on locomotion [F1,56= 158.6,
p< 0.001], with no interaction between amphetamine and atomoxetine treatment [F3,56= 0.1418, n.s]. In fact, animals with amphetamine displayed an increased loco-motion compared with the control groups (***p< 0.001 and**p< 0.01). This result indicated that chronic treatment with atomoxetine does not result in sensitization of the reward system, an effect distinct to that described for the SSRI in a previous report (Alén et al.,2013).
Discussion
The mainfinding of this study is that atomoxetine, given during a period of abstinence from alcohol, produced time-limited effects on alcohol consumption after relapse. Moreover, atomoxetine did not result in behavioral sensi-tization in the context of alcohol consumption, suggesting a preservation of the mesolimbic dopaminergic reward system. This profile is very different when compared to that described for SSRIs such asfluoxetine (Alén et al., 2013). SSRI administration during alcohol deprivation produced increased alcohol consumption after relapse, an effect that lasted more than 5 wk, whereas atomoxetine effects disappeared within a week. This profile is much more favorable, considering the proposed role of noradre-naline in facilitating the extinction of contextual memories associated with the self-administration of abused drugs (Economidou et al.,2011; Janak et al.,2012).
The increase in alcohol consumption was observed at the highest atomoxetine dose administered (10 mg/kg), that it is known to block the dopamine transporter (Turner et al., 2013). However, the effect on increased alcohol consumption observed seems to be independent of the dopaminergic transmission, as it did not result
in behavioral sensitization. Supporting this hypothesis, methylphenidate, a drug used also for attention-deficit/ hyperactivity disorder, did not affect alcohol consump-tion (Soeters et al.,2008). Methylphenidate is a potent in-hibitor of the dopamine transporter, and a mild inin-hibitor of noradrenaline uptake, a pharmacological profile sym-metrical to that of atomoxetine. Whether the short-term effect of the high dose of atomoxetine is related to a loss of selectivity towards the noradrenaline transporter, or to the interaction of the drug with other targets such as the N-methyl-D-aspartate (NMDA) receptor (Udvardi et al., 2013), or the serotonin transporter (Ding et al., 2014) remains to be studied. This is an important limitat-ion on the clinical translatlimitat-ion of the present observatlimitat-ions, as Atomoxetine has been found to display differential selectivity in rats, non-human primates and clinical patients (Zerbe et al., 1985; Ding et al., 2014). In any case, and in rodents, the lack of effect of the intermediate dose on the ADE and the suppression of the ADE by the low dose are of a great interest. The low dose of atomoxetine used in this study, 1 mg/kg, is sufficient to block completely the noradrenaline transporter. Similar doses have been used in different tests of either impul-sivity (Robinson et al., 2008; Ansquer et al., 2013) or extinction of conditioned responses in the context of ad-diction (Economidou et al., 2011; Janak et al., 2012). Considering the study of the effects of atomoxetine in drug-seeking behaviors, the administration of 1 mg/kg of atomoxetine facilitated the extinction of cocaine-seeking behavior, in a similar way to that observed after multiple, but not discrete, stimuli associated with drug presentation (Janak et al., 2012). The authors of these
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Fig. 2. Average locomotor activity as indexed by the number of light beams broken during the 30 min after last drug injection on each drug treatment (vehicle, 1 mg/kg, 3 mg/kg or 10 mg/kg of atomoxetine), compared with the same measure after the amphetamine (1.5 mg/kg) challenge, 24 h after the last drug injection. Sub-chronic atomoxetine treatment slightly reduced the locomotion but did not affect the psychostimulant effect of amphetamine that was equal in all groups.
studies considered that the enhanced arousal derived from the increased noradrenergic transmission produced by atomoxetine strengthened the extinction of drug-paired stimuli. In our study we did not control for conditioned responses, since the animals resumed alcohol self-administration after the three bottles containing different percentages of ethanol were presented again. However, the absence of the ADE observed in the group treated with atomoxetine 1 mg/kg, suggests that similar effects to that described in the above-cited studies, might account for the reduction in alcohol consumption. Further studies, using operant models of alcohol self-administration, will help to confirm that the extinction of conditional cues associated with alcohol is sensitive to the effects of atomoxetine, the same as it has been described for cocaine and heroin (Economidou et al.,2011).
In summary, we have observed that the administration of a noradrenaline reuptake blocker during alcohol depri-vation produced time-limited effects on the consumption of alcohol after relapse. The effects were short lasting and not associated with behavioral sensitization, indicating a better pharmacological profile with respect to the com-monly prescribed SSRIs. Careful analysis of in-between species differences in the pharmacological profile of ato-moxetine have to be considered for a potential clinical translation of the present studies. Because atomoxetine is not used as an antidepressant, further studies using other SNRI antidepressants such as reboxetine or de-sipramine, should be addressed in order to confirm or not the existence of better clinical outcomes in alcoholics suffering depression when treated with these SNRIs.
Acknowledgments
The present work has been supported by grants from Plan Nacional Sobre Drogas (to FRF and LO [PR28/11-18295]) and the following grants from the Ministerio de Economía y Competitividad: Instituto de Salud ‘Carlos III’ (PI13/02261), Red de Trastornos Adictivos EU-ERDF (RD2012/0028/0001; grant EU-ERDF PAIDI CTS-433 and grant PI45403 from the Andalusian Ministry of Economy, Science and Innovation; and grant from the Fundación Eugenio Rodríguez Pascual (Spain).
Statements of Interest
There are no actual or potential conflicts of interest.
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