Ayahuasca

Alcohol & Alcoholism Vol. 43, No. 1, pp. 15 – 24, 2008 Advance Access publication 12 October 2007

doi:10.1093/alcalc/agm145

SEROTONERGIC ANTI-DEPRESSANTS AND ETHANOL WITHDRAWAL SYNDROME: A REVIEW

I. TAYFUN UZBAY*

Gulhane Military Medical Academy, Department of Medical Pharmacology, Psychopharmacology Research Unit, Etlik 06018 Ankara, Turkey

(Received 24 July 2007; in revised form 24 August 2007; accepted 3 September 2007; advance access publication 12 October 2007)

Abstract — Aim: To review laboratory findings on the effects of anti-depressant agents that interact with the serotonergic system on signs of ethanol withdrawal syndrome in rats. Method: Adult Wistar rats received a modified liquid diet to produce ethanol dependence. Signs of ethanol withdrawal, locomotor hyperactivity, stereotyped behaviour, tremor, wet dog shakes, agitation, and audiogenic seizures, were evaluated for the first 6 h of ethanol withdrawal. The effects of the anti-depressants fluoxetine, venlafaxine, escitalopram, tianeptine, and extract of Hypericum perforatum (St. John’s wort) (HPE) were examined. Results: Some beneficial effects of fluoxetine, tianeptine, HPE, escitalopram and venlafaxine on ethanol withdrawal signs were observed, ranked as follows: fluoxetine = tianeptine > HPE > escitalopram > venlafaxine. Conclusions: Tianeptine and fluoxetine seem to be potent pharmacologically active agents on ethanol withdrawal syndrome in rats. Thus, these anti-depressants may be useful in treatment of ethanol withdrawal syndrome in patients with alcoholism. In addition to serotonergic effects, interactions with nitrergic, glutamatergic, and adenosinergic systems may also provide a significant contribution to the beneficial effects of these drugs on ethanol withdrawal syndrome.

INTRODUCTION

Ethanol abuse and dependence remain among the most com- mon substance abuse problems worldwide. The discontinua- tion of chronic administration of ethanol is associated with excitatory withdrawal signs called ethanol withdrawal syn- drome. Ethanol withdrawal syndrome is the most impor- tant evidence, which indicates the development of a phys- ical dependence to ethanol (O’Brien, 1996). Although the signs of ethanol withdrawal syndrome in humans (Thompson, 1978) and rodents (Majchrowicz, 1975; Uzbay and Kayaalp, 1995; Uzbay et al ., 1997) have been well described, the mechanisms underlying physical dependence to ethanol and ethanol withdrawal syndrome are poorly understood. Among the numerous neurotransmitter systems implicated in the phar- macological effects of ethanol, the serotonergic system has received particular attention. Serotonergic system has been shown to play an important role in the regulation of ethanol intake, preference, and dependence via central mechanisms (Roy et al ., 1987; Rezvani et al ., 1990; Ferreria and Soares- DaSilva, 1991; McBride et al ., 1991; Sellers et al ., 1992; Wallis et al ., 1993; LeMarquand et al ., 1994; Uzbay et al ., 1998, 2000).

Depression is an important psychiatric disorder that affects individuals’ quality of life and social relations directly. Depression is characterized by emotional symptoms such as hopelessness, apathy, loss of self-confidence, sense of guilt, indecisiveness, and amotivation, as well as biological symp- toms like psychomotor retardation, loss of libido, sleep dis- turbances, and loss of appetite. When the symptoms are very severe, major depression is considered. The prevalence of major depression is approximately 9% both in the United

*Author to whom correspondence should be addressed at: Gulhane Military Medical Academy, Faculty of Medicine, Department of Medical Pharma- cology, Psychopharmacology Research Unit, Etlik 06018 Ankara, Turkey. Tel: +312-304 4764; Fax: +312-304 2010; E-mail: [email protected]; [email protected]

States and Europe (Fichter et al ., 1996; Lepine et al ., 1997). A decrease in serotonergic activity is associated with depres- sion. In experimental studies, a decrease in brain serotonergic activity due to social isolation is known for decades (Garat- tini et al ., 1967). Specifically, rodents show hyperactive and aggressive behaviour during long-term social isolation, which can be blocked with anti-depressant treatment (Garzon and del Rio, 1981). These social isolation forms based on sero- tonin deficiency are used as experimental depression model in rodents (Leonard, 1998). On the other hand, selective sero- tonin re-uptake inhibitors (SSRIs) and some post-synaptic receptor agonists, which increase serotonergic activity in the synaptic space, are used widely and effectively to treat depres- sion (Cowen, 1998; Vaswani et al ., 2003).

Alcoholism and depression are often associated in psy- chiatric patients. Many alcoholic patients have symptoms of depression (Weissman and Myers, 1980; Miguel-Hidalgo and Rajkowska, 2003). A positive association between high ethanol intake and a depression-like status has been suggested also in genetically selected ethanol-preferring AA rats (Kiian- maa et al ., 1991) and in fawn-hooded rats (Overstreet et al ., 1992). Previous studies from our laboratory also indicated a significant decrease in striatal serotonin levels of rats dur- ing early ethanol withdrawal (Uzbay et al ., 1998) and chronic ethanol consumption (Uzbay et al ., 2000). These observations imply that there might be a correlation between decreased serotonergic activity and ethanol dependence. Thus, drugs that increase serotonergic activity in synapses could be useful in treatment of ethanol withdrawal or dependence. Furthermore, some anti-depressant drugs are in general use for patients with ethanol dependence. They are mainly indicated in the treatment of ethanol withdrawal and combined psychiatric disorders (Miller, 1995; Favre et al ., 1997; Myrick et al ., 2001). Although effects of some serotonergic anti-depressants on ethanol intake and/or ethanol abuse have been investigated in several studies, data relating to the action of anti-depressant drugs during ethanol withdrawal period are very limited.

 The Author 2007. Published by Oxford University Press on behalf of the Medical Council on Alcohol. All rights reserved

16 I. T. UZBAY

An ethanol-dependent rat model was developed in our Psychopharmacology Research Unit by a modified liquid diet technique in 1995 (Uzbay and Kayaalp, 1995). Numerous experimental studies were, and are being performed in our laboratory to understand the mechanism and etiology of alcoholism. Some of these were involved in the effects of anti-depressant drugs on ethanol withdrawal syndrome in rats. In this review, it was aimed to analyse the results obtained from ethanol-dependent rat models treated with various anti- depressant drugs that interact with the serotonergic system via different mechanisms. A relationship between the drug effects and the signs of ethanol withdrawal has also been evaluated and discussed.

ETHANOL-DEPENDENT RAT MODEL BY THE MODIFIED LIQUID DIET

Subjects and laboratory Procedure in all studies was in accordance with the Guide for the Care and Use of Laboratory Animals as adopted by the National Institutes of Health (USA). Adult Wistar rats (182 – 339 g in weight at the beginning of the exper- iments) were subjects. They were housed in a quiet and temperature- and humidity-controlled room (22 ± 3◦C and 65 ± 5%, respectively), in which a 12-h light/dark cycle was maintained (07:00 – 19:00 light).

Chronic ethanol administration to rats For chronic ethanol administration, the rats were housed individually and ethanol was administered in the modified liquid diet as previously described (Uzbay and Kayaalp, 1995). The rats received a modified liquid diet with or without ethanol ad libitum. No extra chow or water was supplied. The composition of the modified liquid diet with ethanol was: cow milk 925 ml (Mis Süt, Turkey), 25 – 75 ml ethanol (96.5% ethyl alcohol; Tekel, Turkish State Monopoly), vitamin A 5000 IU (Akpa İlaç Sanayi, Turkey) and sucrose 17 g (Uzbay and Kayaalp, 1995). This mixture supplied 1000.7 kcal/l.

At the beginning of the study, rats were given the modified liquid diet without ethanol for 7 days. Then, liquid diet with 2.4% ethanol was administered for 3 days. The ethanol concentration was increased to 4.8% for the following 4 days and finally to 7.2% for another 21 days. Liquid diet was prepared on a daily basis and presented at the same time each day. The weight of the rats was recorded every day, and daily ethanol intake was measured and expressed as g/kg/day. Control rats were pair fed with an isocaloric liquid diet containing of sucrose as a caloric substitute to ethanol.

Drugs used and dose regimens in the studies Fluoxetine (2.5 – 10 mg/kg, Sigma Chemical — USA), an SSRI, venlafaxine (5 – 40 mg/kg, White Company, İstanbul — Turkey), a serotonin/noradrenalin re-uptake inhibitory agent, escitalopram (2.5 – 10 mg/kg, Lundbeck — Denmark), a bound- ing agent at the primary site of pre-synaptic serotonin trans- porter, tianeptine (5 – 20 mg/kg, Servier Laboratory — France), a serotonin uptake stimulatory agent, were injected in rats

intraperitoneally. Extract of Hypericum perforatum (HPE) was prepared by using aerial parts of St John’s wort at Anadolu University, Department of Pharmacognosy as pre- viously described (Ozturk et al ., 1996) and injected in rats intraperitoneally, as well.

Doses of the anti-depressants were selected from our preliminary experiments and previous studies. Since higher doses of anti-depressants used in our studies caused sedation and impairment of motor co-ordination, higher doses were not tested.

Evaluation of ethanol withdrawal syndrome At the end of 7.2% ethanol-containing liquid diet adminis- tration, ethanol was withdrawn from the daily diet. Ethanol- dependent rats were then assigned to several groups (N = 8 – 10 for each group). Anti-depressants and saline were injected in the rats 30 min before ethanol withdrawal test- ing. The rats were then observed for 5 min at the 2nd, 4th and 6th h of the withdrawal period. At each observa- tion time, the rats were assessed simultaneously for the fol- lowing behavioural conditions: agitation, tremor, stereotyped behaviour, wet dog shakes and audiogenic seizures as pre- viously described (Uzbay and Kayaalp, 1995; Uzbay et al ., 1997).

Ethanol consumption, weight changes and blood ethanol levels Daily ethanol consumption of the rats in the control and anti- depressant treated groups ranged from 10 to 17 g/kg during exposure to ethanol (7.2%).

No significant weight loss was observed in any of the studies, as body weights of the rats increased progressively during the study.

Blood ethanol levels were found higher than 150 mg/dl in ethanol feeding groups.

EFFECTS OF ANTI-DEPRESSANTS ON ETHANOL WITHDRAWAL SYNDROME

Fluoxetine Fluoxetine is an SSRI that exhibits anti-depressant activity in experimental models (Detke et al ., 1995; Contreras et al ., 2001) and clinical trials (Stokes and Holtz, 1997; Vaswani et al ., 2003). Fluoxetine increases serotonergic transmission in synaptic cleft (Stahl, 1996). Studies suggest that SSRIs, such as zimelidine, citalopram, and fluoxetine, may reduce ethanol consumption, and that is not thought to be an anti- depressant effect (Miller, 1995). Limited clinical studies indicated that fluoxetine reduces the extent of anxiety and depression during ethanol withdrawal (Romeo et al ., 2000) and at its anti-depressant doses; it is able to prevent relapses in patients with alcoholism (Janiri et al ., 1996).

A detailed study reported the effects of fluoxetine on sev- eral signs of ethanol withdrawal in rats (Uzbay et al ., 2004). In this study, fluoxetine inhibited withdrawal-induced loco- motor hyperactivity and attenuated the severity or incidence of the signs of ethanol withdrawal, such as agitation, increased

ANTI-DEPRESSANTS AND ETHANOL WITHDRAWAL 17

Table 1. Acute effects of ip administration of fluoxetine on the signs of ethanol withdrawal syndrome in rats

Doses and Ethanol withdrawal signs observation intervals LH Agitation Stereotype Tremors WDS AS

2nd h of EWS 2.5 mg/kg 0 + 0 + + − 5.0 mg/kg + + 0 + + −

10.0 mg/kg 0 + + + + − 4th h of EWS 2.5 mg/kg 0 0 0 + + − 5.0 mg/kg 0 0 0 + 0 −

10.0 mg/kg 0 0 0 0 + − 6th h of EWS a

2.5 mg/kg 0 0 0 + 0 + 5.0 mg/kg + + 0 + 0 +

10.0 mg/kg 0 + 0 + 0 +

(Uzbay et al ., 2004); ip, Intraperitoeal; EWS, Ethanol withdrawal syndrome; LH, Locomotor hyperactivity; WDS, Wet dog shake; AS, Incidence of audiogenic seizure; −, Not evaluated; 0, Ineffective; +, Statistically significant attenuation. a All doses were repeated 30 min before the 6th h of evaluation.

stereotyped behaviour, wet dog shakes, and tremors, dose- dependently. It also reduced markedly the incidence of audio- genic seizures (Table 1). Preventive effects of fluoxetine were seen particularly on agitation, wet dog shakes, tremors, and audiogenic seizures. Effective doses of fluoxetine did not cause any significant change in locomotor activities of the naı̈ve (not ethanol-dependent) rats. Moreover, the inhibitory effects of fluoxetine on the signs of ethanol withdrawal were specific and not related to other non-specific effects, such as sedation and muscle relaxation. These observations clearly showed that fluoxetine is a pharmacologically active agent on mechanisms involved in development of physical dependence on ethanol in rats, and it may have a potential therapeutic effect in the treatment of ethanol-type dependence.

Escitalopram Escitalopram is an active enantiomer of citalopram, which is an SSRI. It has a proven efficacy in the treatment of major depression, like other SSRIs. It has been shown in non- clinical and clinical experiments that it has greater efficacy than equivalent doses of citalopram (Auquier et al ., 2003; Lepola et al ., 2003; Sánchez et al ., 2004). Unlike classical SSRIs, it is bound at the primary site of pre-synaptic serotonin transporter (SERT) with a very high affinity, and it has higher serotonergic activity than the classical SSRIs (Sánchez et al ., 2004).

In the light of information above, it could be expected that escitalopram is more effective than the classical SSRIs, such as fluoxetine on ethanol withdrawal syndrome. Thus, the effects of escitalopram on ethanol dependence or ethanol withdrawal have been evaluated in our laboratory (Saglam et al ., 2006). In contrast to our expectations, in this study, escitalopram was found to be less effective when compared to fluoxetine. While fluoxetine had additional preventive effects on locomotor hyperactivity, agitation, and audiogenic seizures (Uzbay et al ., 2004), escitalopram was not effective

on these signs of ethanol withdrawal. Its beneficial effects on ethanol withdrawal syndrome were found to be limited. It only produced a significant attenuation on tremors (Table 2). Although it produced some significant decrease on stereo- typed behaviours and wet dog shakes, these effects were limited and not dose-dependent (Saglam et al ., 2006). Thus, results of this study suggest that escitalopram has some lim- ited beneficial effects on ethanol withdrawal syndrome in rats. However, it does not have superiority over fluoxetine for treat- ment of ethanol withdrawal syndrome in rats.

Venlafaxine Venlafaxine is a bicyclic phenylethylamine derivative which inhibits pre-synaptic re-uptake of serotonin, noradrenaline, and, to a lesser extent, dopamine (Holliday and Benfield, 1995). Thus, it increases serotonergic and noradrenergic transmission in synaptic cleft (Stahl, 1996). Venlafaxine exhibits an anti-depressant activity in experimental models and clinical trials (Mitchel and Fletcher, 1993; Holliday and Benfield, 1995; Dierick, 1997). Anxiety is also a sign of withdrawal of the drugs that were abused, and produced physical dependence, such as ethanol in humans (Schuckit, 2000; De Witte et al ., 2003) and rats (Gatch et al ., 2000). Additionally, several clinical reports have suggested that venlafaxine has beneficial effects in some kind of anxiety disorders (Gelenberg et al ., 2000; Ninan, 2000; Gorman, 2003).

Evidence also showed that venlafaxine strongly attenuated morphine withdrawal in rats (Lu et al ., 2001). However, stud- ies assessing the effect of venlafaxine on ethanol withdrawal syndrome or ethanol dependence were limited. Therefore, the first detailed study investigating the effects of venlafaxine effects on ethanol withdrawal syndrome were performed in our laboratory.

In this study, no prominent effect on locomotor hyperac- tivity, agitation, stereotyped behaviour and wet dog shake

18 I. T. UZBAY

Table 2. Acute effects of ip administration of escitalopram on the signs of ethanol withdrawal syndrome in rats

Doses and Ethanol withdrawal signs observation intervals LH Agitation Stereotype Tremors WDS AS

2nd h of EWS 2.5 mg/kg 0 0 0 0 + − 5.0 mg/kg 0 0 0 + + −

10.0 mg/kg 0 0 0 + 0 − 4th h of EWS

2.5 mg/kg 0 0 0 0 0 − 5.0 mg/kg 0 0 0 0 0 −

10.0 mg/kg 0 0 0 0 0 − 6th h of EWS a

2.5 mg/kg 0 0 0 0 + 0 5.0 mg/kg 0 0 + 0 0 0

10.0 mg/kg 0 0 0 0 0 0

(Saglam et al ., 2006); ip, Intraperitoneal; EWS, Ethanol withdrawal syndrome; LH, Locomotor hyperactivity; WDS, Wet dog shake; AS, Incidence of audiogenic seizure; −, Not evaluated; 0, Ineffective; +, Statistically significant attenuation. a All doses were repeated 30 min before the 6th h of evaluation.

Table 3. Acute effects of ip administration of venlafaxine on the signs of ethanol withdrawal syndrome in rats

Doses and Ethanol withdrawal signs observation intervals LH Agitation Stereotype Tremors WDS AS

2nd h of EWS 5.0 mg/kg 0 0 0 − 0 −

10.0 mg/kg 0 0 0 − 0 − 20.0 mg/kg 0 0 0 − 0 − 40.0 mg/kg 0 0 0 − 0 − 4th h of EWS

5.0 mg/kg 0 0 0 − 0 − 10.0 mg/kg 0 0 0 − 0 − 20.0 mg/kg 0 0 0 − 0 − 40.0 mg/kg 0 0 0 − 0 − 6th h of EWS a

5.0 mg/kg 0 0 0 − 0 0 10.0 mg/kg 0 0 + − 0 0 20.0 mg/kg 0 0 0 − 0 +$ 40.0 mg/kg 0 0 0 − 0 0

(Saglam et al ., 2004) ip, Intraperitoneal; EWS, Ethanol withdrawal syndrome; LH, Locomotor hyperactivity; WDS, Wet dog shake; AS, Incidence of audiogenic seizure; ($, significant prolonged latency); −, Not evaluated; 0, Ineffective; +, Statistically significant attenuation. a All doses were repeated 30 min before the 6th h of evaluation.

by acute venlafaxine treatment was observed. However, ven- lafaxine had some limited preventive effects on the audiogenic seizures. It significantly prolonged the latency of audiogenic seizures at the dose of 20 mg/kg and reduced the incidence of the seizures without reaching a statistically significant level at the dose of 40 mg/kg (Saglam et al ., 2004) (Table 3). As a result, venlafaxine did not seem to be an agent as effective as fluoxetine to control ethanol withdrawal syndrome.

Tianeptine

Tianeptine is a tricyclic drug that exhibits anti-depressant activity in experimental models (Curzon and Datla, 1993) and clinical trials (Guelfi, 1992; Saiz-Ruiz et al ., 1998). The neu- rochemical properties of tianeptine vary from those of other tricyclic and non-tricyclic anti-depressants. It is a unique type of anti-depressant that produces its effect by enhancing, rather than inhibiting, serotonin re-uptake (Mennini et al ., 1987).

ANTI-DEPRESSANTS AND ETHANOL WITHDRAWAL 19

Table 4. Acute effects of ip administration of tianeptine on the signs of ethanol withdrawal syndrome in rats

Doses and Ethanol withdrawal signs observation intervals LH Agitation Stereotype Tremors WDS AS

2nd h of EWS 5.0 mg/kg 0 0 0 0 + −

10.0 mg/kg 0 0 0 + + − 20.0 mg/kg + 0 + + + − 4th h of EWS 5.0 mg/kg 0 0 0 0 0 −

10.0 mg/kg 0 0 0 + 0 − 20.0 mg/kg 0 + + + + − 6th h of EWS a

5.0 mg/kg 0 0 0 + 0 0 10.0 mg/kg 0 + 0 + + + 20.0 mg/kg 0 + + + + +$

(Uzbay et al ., 2006) ip, Intraperitoneal; EWS, Ethanol withdrawal syndrome; LH, Locomotor hyperactivity; WDS, Wet dog shake; AS, Incidence of audiogenic seizure; ($, significant prolonged latency); −, Not evaluated; 0, Ineffective; +, Statistically significant attenuation. a All doses were repeated 30 min before the 6th h of evaluation.

Limited clinical studies indicated that tianeptine has bene- ficial effects for patients with alcoholism. Malka et al . (1992) showed that long-term tianeptine treatment results in marked and consistent improvement in depression and anxiety scores after alcohol withdrawal. In addition, Favre et al . (1997) sug- gested that tianeptine prevents alcoholic relapses in patients.

In experimental studies, Daoust et al . (1992) showed that tianeptine decreases ethanol intake of male Wistar rats without causing any significant change on either their food intake or body weight. File et al . (1993) also suggested that tianeptine is able to reverse the anxiogenic effects of ethanol with- drawal in the social interaction test in rats. However, ethanol withdrawal consists of more than anxiety. Other symptoms such as locomotor hyperactivity, agitation, increased stereo- typed behaviour and wet dog shakes, tremors, and audiogenic seizures also appear during ethanol withdrawal in rodents. In a recent study, Uzbay et al . (2006) reported results from a detailed study investigating the effects of both acute and chronic tianeptine treatment on ethanol withdrawal syndrome in rats. Both acute and chronic administration of tianep- tine attenuated severity of ethanol withdrawal syndrome dose-dependently. However, acute tianeptine treatment was found to be more effective than chronic treatment. While acute tianeptine treatment was effective on all the signs of ethanol withdrawal (locomotor hyperactivity, agitation, increased stereotyped behaviour, wet dog shakes, tremors, and audiogenic seizures) (Table 4), chronic treatment was inef- fective on locomotor hyperactivity and agitation. In addition, chronic tianeptine treatment did not produce any significant effect on ethanol intake of the rats. Results of this study indicated that tianeptine may be a potent and pharmacologi- cally active agent on ethanol withdrawal syndrome in rats. It may be useful in treatment of ethanol dependence as well as depression in patients with history of ethanol abuse.

Extract of Hypericum perforatum (HPE, St. John’s wort)

HPE has been usually called St John’s wort, and commonly used in folk medicine of several European countries. Several preclinical studies indicate that extract of the common plant HPE may be useful for treatment of disorders, especially depression, originating from the central nervous system. Thus, the anti-depressant-like effect of HPE has been reported in rodents (Butterweck et al ., 1997; Ozturk, 1997; De Vry et al ., 1999). Several meta-analyses and overviews of randomized clinical trials also consistently show that HPE displays a clear anti-depressant action and it has been used for the treatment of mild to moderate depression (Linde et al ., 1996; Melchart, 1996; Volz, 1997; Kasper and Dienel, 2002). HPE has some serotonergic properties reducing 5-HT re-uptake and inhibiting monoamine oxidase (MAO) activity (Neary and Bu, 1990; Perovic and Muller, 1995; Cott, 1997; Bennett et al ., 1998; Greeson et al ., 2001) like other anti-depressant drugs.

Some experimental studies have been reported involving the effects of HPE on ethanol abuse and dependence. It was suggested that HPE inhibits ethanol intake and preference in several strains of ethanol-preferring rats (De Vry et al ., 1999; Rezvani et al ., 1999; Perfumi et al ., 1999, 2001, 2002) and mice (Wright et al ., 2003). In a recent report, Perfumi et al . (2005) showed that HPE significantly reduced ethanol self-administration, while it did not modify saccharin self- administration. They also observed that HPE abolished the increased ethanol intake following ethanol deprivation. Thus, these results suggested that HPE might be a useful agent in the treatment of ethanol abuse and dependence.

Although the effects of HPE on ethanol preference and intake have been investigated in detailed studies, only one study investigating the effects of HPE on ethanol withdrawal syndrome was reported (Coskun et al ., 2006). In this study, HPE blocked both locomotor hyperactivity and stereotyped behaviours especially at 2nd and 6th h of ethanol with- drawal. In addition, it attenuated the incidence of tremor in

20 I. T. UZBAY

Table 5. Acute effects of ip administration of extract of Hypericum perforatum on the signs of ethanol withdrawal syndrome in rats

Doses and Ethanol withdrawal signs observation intervals LH Agitation Stereotype Tremors WDS AS

2nd h of EWS 25.0 mg/kg + − + 0 − − 50.0 mg/kg + − + 0 − −

100.0 mg/kg + − + 0 − − 200.00 mg/kg + − + 0 − − 4th h of EWS

25.0 mg/kg 0 − 0 0 − − 50.0 mg/kg 0 − 0 + − −

100.0 mg/kg + − + + − − 200.00 mg/kg 0 − 0 0 − − 6th h of EWS a

25.0 mg/kg + − + 0 − +$ 50.0 mg/kg + − + 0 − +$

100.0 mg/kg + − + 0 − + 200.00 mg/kg + − + 0 − 0

(Coskun et al ., 2006) ip, Intraperitoneal; EWS, Ethanol withdrawal syndrome; LH, Locomotor hyperac- tivity; WDS, Wet dog shake; AS, Incidence of audiogenic seizure; HPE, Extract of hypericum perforatum [Yield of HPE were 27.4% (w/v), doses were expressed as dried extract (mg/kg) body weight]; ($, significant prolonged latency); −, Not evaluated; 0, Ineffective; +, Statistically significant attenuation. a All doses were repeated 30 min before the 6th h of evaluation.

ethanol-dependent rats at 4th h of ethanol withdrawal. HPE (100 mg/kg) produced a significant attenuation in the inci- dence of the audiogenic seizures appearing in 6th h of ethanol withdrawal. Latency of the audiogenic seizures was also pro- longed significantly by HPE (25 and 50 mg/kg) treatment (Table 5). These results imply that HPE may be useful in the treatment of ethanol withdrawal syndrome.

DISCUSSION AND CONCLUSION

Effects of each anti-depressant on the signs of ethanol with- drawal during observation terms are shown in Tables 1 – 5. Their comparative effects were also summarized in Table 6. As shown in the Tables, treatment of tianeptine, fluoxetine, HPE, escitalopram, and venlafaxine have some beneficial effects on the signs of ethanol withdrawal in rats. Effec- tiveness of the anti-depressants was as follows: fluoxetine = tianeptine > HPE > escitalopram > venlafaxine. Since any significant changes on the open field locomotor activities in naı̈ve groups were not observed, the beneficial effects of the anti-depressants on ethanol withdrawal syndrome could not be due to other non-specific effects, such as sedation or mus- cle relaxation. As shown in Tables 1 – 5, actions of the drugs are either lost or weakened at the 4t h-h-withdrawal. This may be related to elimination of single dose of tested drugs. Thus, second injections were repeated before 6th-h-observations.

Neurochemical findings from clinical (Roy et al ., 1987; LeMarquand et al ., 1994) and experimental (Murphy et al ., 1987; Uzbay et al ., 1998, 2000) studies suggested significant changes in central serotonergic neurotransmission in ethanol dependence. On the other hand, we hypothesized that there

might be a significant association between decreased seroton- ergic activity and ethanol dependence (Uzbay et al ., 1998, 2000). Our findings indicated some beneficial effects on with- drawal signs treated by fluoxetine, escitalopram, and HPE to support this hypothesis.

As venlafaxine inhibits the re-uptake of serotonin more than noradrenaline, and even more than dopamine in synaptic cleft (Muth et al ., 1986; Holliday and Benfield, 1995), serotonergic property of this drug may also be responsible for its prolonging effects of latency of audiogenic seizures. Ineffectiveness of venlafaxine on other signs of withdrawal may be due to its stimulative effects on noradrenalin re- uptake. Thus, ethanol withdrawal syndrome is especially characterized by the signs of overactivity of the sympathetic nervous system (Linnoila et al ., 1987; De Witte et al ., 2003). Inhibition of noradrenaline re-uptake, besides serotonin, by venlafaxine and increased noradrenergic activity in synaptic cleft might mask or prevent its beneficial effects on locomotor hyperactivity, agitation, stereotyped behaviour, and wet dog shakes. Some increase in agitation scores by venlafaxine treatment (Saglam et al ., 2004) also supports this idea. Thus, signs such as agitation and hyperreflexia, during ethanol withdrawal are related to increased noradrenergic activity (Linnoila et al ., 1987).

The beneficial effects of HPE might also be related to serotonergic mechanisms. HPE has some serotonergic properties, reducing serotonin re-uptake and inhibiting MAO activity (Neary and Bu, 1990; Perovic and Muller, 1995; Calapai et al ., 1999) like other anti-depressant drugs.

Unlike the classical SSRI anti-depressants, escitalopram is bound at the primary site of SERT with a very high affinity. In the central nervous system, the concentration of active serotonin in the synaptic cleft is regulated by

ANTI-DEPRESSANTS AND ETHANOL WITHDRAWAL 21

Table 6. Comparative effects of anti-depressants on the signs of ethanol withdrawal syndrome in rats

Ethanol withdrawal signs

Drugs LH Agitation Stereotype Tremors WDS AS

Fluoxetine ↓↓ ↓↓ ↓ ↓↓↓ ↓↓↓ ↓↓↓ Tianeptine ↓ ↓↓ ↓↓ ↓↓↓ ↓↓ ↓↓↓ HPE ↓↓↓ − ↓↓↓ ↓ − ↓↓ Escitalopram 0 0 ↓ ↓↓ ↓↓ 0 Venlafaxine 0 0 0 0 0 ↓

LH, Locomotor hyperactivity; WDS, Wet dog shake; AS, Audiogenic seizure; HPE, Extract of Hypericum perforatum ; −, Not evaluated; 0, Ineffective; ↓, Mild inhibitory effect; ↓↓, Moderate inhibitory effect; ↓↓↓, High inhibitory effect.

SERT (Tatsumi et al ., 1997; Sanchez et al ., 2004). SERT is also responsible for termination or modulation of the action of serotonin released from the pre-synaptic neuron. Thus, escitalopram has higher serotonergic activity than the classical SSRIs (Lepola et al ., 2003; Sánchez et al ., 2004) and it could be expected that escitalopram is more effective than the classical SSRIs, such as fluoxetine on ethanol withdrawal syndrome. In contrast to our expectations, Saglam et al . (2006) found that escitalopram is less effective compared to fluoxetine. While fluoxetine had additional protective effects on locomotor hyperactivity, agitation, and audiogenic seizures (Uzbay et al ., 2004), escitalopram was not effective on these signs of ethanol withdrawal. These findings imply that more selective serotonergic activity does not mean more effectiveness on ethanol withdrawal syndrome. Further studies are needed to clarify the lower effectiveness of escitalopram on ethanol withdrawal syndrome.

On the other hand, additional effects of fluoxetine on nitric oxide (NO) may contribute to its stronger activity on ethanol withdrawal. Several studies have shown that NO synthase (NOS) inhibitors cause a prominent attenuation in the signs of ethanol withdrawal syndrome in rats (Uzbay and Oglesby, 2001). Wegener et al . (2003) suggested that local administration of serotonergic anti-depressants, such as fluoxetine, tianeptine, paroxetine, citalopram, and imipramine, significantly decrease hippocampal NOS activity in rat brain. In addition, previous studies indicated that fluoxetine has some NOS inhibitory effects in humans (Yaron et al ., 1999) and rats (Luo and Tan, 2001).

Different from SSRIs and other anti-depressants, tianeptine was shown to enhance serotonin uptake selectively in rat brain synaptosomes (Mennini et al ., 1987). Thus, this drug can be described as a serotonin re-uptake enhancer, an atypical anti- depressant. However, in a recent study from our laboratory, it was found that tianeptine and fluoxetine, but not venlafaxine, have similar discriminative stimulus properties in rats (Alici et al ., 2006). Clinical anti-depressant efficacy of tianeptine has also been found to be similar to that of SSRIs (Lŏo et al ., 1999; Waintraub et al ., 2000) and other tricyclic-depressants (Guelfi, 1992; Staner and Mendlewicz, 1993).

Recent studies indicate that anti-depressant effects of this drug may be attributable to non-serotonergic mechanisms, including its capacity to buffer excitatory amino acid recep- tors against stress (Kole et al ., 2002). Tianeptine reverses the adverse effects of stress on brain morphology and synaptic plasticity by reducing excessive accumulation of intracellular

calcium, which results from stress-induced excitatory amino acid activation (McEwen and Magarinos, 2001). It also pre- vents stress-induced increase in glutamate transporter mRNA levels in rat hippocampus (Reagan et al ., 2004). On the other hand, many studies have shown a clear role of exci- tatory amino acid stimulation in the development of ethanol dependence (Rossetti and Carboni, 1995; Tsai et al ., 1995; Hardy et al ., 1999). In addition, blockade of NMDA receptors markedly reduces ethanol withdrawal signs in rodents (Mor- riset et al ., 1990; Liljequist, 1991; Thomas et al ., 1997). Fur- thermore, adenosine A1 agonistic agents have also inhibitory effect on ethanol withdrawal syndrome in rats (Concas et al ., 1996; Kaplan et al ., 1999) and it has been shown that tianep- tine has anti-convulsant activity via adenosine A1 receptor stimulation (Uzbay et al ., 2007). In addition, similar to flu- oxetine, tianeptine also decreases hippocampal NOS activity in rats (Wegener et al ., 2003). Glutamatergic, nitrergic, and adenosinergic mechanisms may be responsible for the promi- nent beneficial effects of tianeptine on ethanol withdrawal syndrome.

In the light of the results gained by five anti-depressants agents, it can be concluded that fluoxetine and tianeptine are potent and pharmacologically active agents on ethanol with- drawal syndrome. They may be useful in treatment of ethanol dependence as well as depression in patients with history of ethanol abuse. HPE, escitalopram, and venlafaxine also have limited beneficial effects on some signs of withdrawal. In addition, anti-depressants did not cause any deteriorating effect on any of the signs of ethanol withdrawal syndrome. It implies that anti-depressants could also be used safely in patients suffering from alcoholism.

Acknowledgements — The author would like to thank Dr Gökhan Göktalay, Dr Murat Yildirim and Dr Hakan Kayir for their scientific contributions and valuable comments on the manuscript.

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