Effects of olanzapine on ethanol withdrawal syndrome in rats

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gy 579 (2008) 208–214www.elsevier.com/locate/ejphar

European Journal of Pharmacolo

Effects of olanzapine on ethanol withdrawal syndrome in rats

Nasibe Unsalan a, Esra Saglam b, Hakan Kayir c, Tayfun Uzbay c,⁎

a Department of Psychiatry, Faculty of Medicine, Maltepe University, Istanbul, Turkeyb Department of Pharmacology, Faculty of Medicine, Maltepe University, Istanbul, Turkey

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

Received 5 June 2007; received in revised form 11 October 2007; accepted 16 October 2007Available online 25 October 2007

Abstract

The present study was designed to investigate the effects of olanzapine, a serotonin–dopamine antagonistic atypical antipsychotic agent, onethanol withdrawal syndrome in rats. Adult male Wistar rats were subjects. Ethanol (7.2%, v/v) was given to rats by a liquid diet for 21 days.Control rats were pair fed with an isocaloric liquid diet containing sucrose as a caloric substitute to ethanol. After 2nd, 4th and 6th h of ethanolwithdrawal, rats were observed for 5 min, afterwards withdrawal signs that included locomotor hyperactivity, agitation, stereotyped behavior,tremor, wet dog shakes, abnormal posture and abnormal gait were recorded or rated. Olanzapine (0.5, 1 and 2 mg/kg) and saline were injected tothe rats intraperitoneally 30 min before ethanol withdrawal assessment. A second series of injections was also given 30 min before the 6th-h-observation, and subjects were then tested for audiogenic seizures. Olanzapine (2 mg/kg) produced significant inhibitory effects on stereotypedbehaviors and wet dog shakes at the 6th h of ethanol withdrawal. Contrary, the same dose caused some increases in the intensity of posture andgait impairments at the 2nd h of ethanol withdrawal. In addition, that dose was found to be ineffective on agitation, tremor, tail stiffness andaudiogenic seizures. Our results suggest that acute olanzapine treatment has beneficial effects on stereotyped behavior and wet dog shakes, but italso has some adverse effects on posture and gait during ethanol withdrawal in rats. Overall, olanzapine does not seem to be an adequate andsuitable drug in controlling of ethanol withdrawal syndrome.© 2007 Elsevier B.V. All rights reserved.

Keywords: Olanzapine; Ethanol withdrawal syndrome; Ethanol dependence; (Rat)

1. Introduction

Ethanol, as a rewarding substance, is associated with therelease of dopamine in the pleasure center of the brain. Currentresearch related to the neurobiology of substance abuse supportsthe role of dopaminergic involvement in motivation, reward andreinforcement (Volkow et al., 2004). A strong body of evidenceindicates that ethanol activates dopamine release from thenucleus accumbens and extended amygdale (Di Chiara, 1995;Heimer et al., 1997). The action of ethanol on the mesolimbicpathway is considered to be strongly associated with suscep-tibility to alcoholism (Noble, 1996) and the development of

⁎ Corresponding author. Gulhane Military Medical Academy, Faculty ofMedicine, Department of Medical Pharmacology, Psychopharmacology Re-search Unit, Etlik 06018 Ankara, Turkey. Tel.: +90 312 304 4764; fax: +90 312304 2010.

E-mail addresses: [email protected], [email protected] (T. Uzbay).

0014-2999/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.ejphar.2007.10.024

craving and loss of control (Robinson and Berridge, 1993). Asmentioned before, the ethanol-related increase in dopaminerelease contributes to the rewarding and reinforcing effects ofethanol consumption (i.e. pleasurable feelings) (Kenna et al.,2004).

Serotonergic drugs are thought to have particular interactionin that point, especially because of the hypothesized linksbetween mood disorders and ethanol consumption. Neuro-chemical findings from clinical (Roy et al., 1987; Le Marquandet al., 1994) and experimental (Murphy et al., 1987) studiessuggested some significant chances in central serotonergicneurotransmission during ethanol consumption and/or with-drawal. It is also hypothesized that serotonin is also associatedwith the rewarding and reinforcing effects of ethanol consump-tion. During intoxication, there is an increase in serotoninfunctioning. Considerable experimental evidence suggests thatserotonin plays a crucial role in the impulsivity and cravingoften seen in alcoholics (Ciccocioppo, 1999) and is at least

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partly responsible for alcohol dependence (Myers and Martin,1973; Schuckit, 1996).

Olanzapine is an atypical antipsychotic agent and used intreatment of schizophrenia. Like other atypical antipsychotics, ithas a high binding ratio of serotonergic 5-HT2 to dopaminergicD2

receptors. Similar to clozapine, olanzapine is an antagonist ofdopamine receptors (D1–D4) and 5-HT2 receptor (Schatzberget al., 2003). Some clinical studies indicated that atypicalantipsychotics such as clozapine (Drake et al., 2000) andolanzapine (Hutchison et al., 2001, 2003) reduce ethanol cravingor consumption in social drinkers. Although, these observationsimply that olanzapine could be effective in treatment of ethanoldependence, there is no report investigating the effects ofolanzapine in alcohol dependent patients. On the other hand,ethanol withdrawal syndrome precipitated by discontinuingchronic ethanol intake is the most important evidence indicatingthe presence of physical ethanol dependence either in humans orin experimental animals (Jaffe, 1990; Uzbay et al., 1994; Brust,2004). However, effects of olanzapine on ethanol withdrawal orethanol dependence have been subjected neither to clinical norexperimental studies yet.

The main objective of the present study was to investigatethe effects of olanzapine, which is a dopamine and serotoninantagonist, on the signs of ethanol withdrawal syndrome in rats.Thus, we wanted to reveal whether olanzapine is effective onethanol dependence or not.

Doses were selected according to our preliminary tests andprevious studies. Since doses of olanzapine higher than 2 mg/kgcaused sedative effects in rats (Ortega-Alvaro et al., 2006),doses up to 2 mg/kg of olanzapine were used in the presentstudy.

2. Materials and methods

2.1. Animals and laboratory

All procedures in this study are in accordance with theGuide for the Care and Use of Laboratory Animals as adoptedby the National Institutes of Health (USA). Local ethicalcommittee approval was also taken. All efforts were made tominimize animal suffering to reduce the number of animalsused.

Adult maleWistar rats (216–318 g weight at the beginning ofthe experiments) were used (n=8 for each group). They werehoused in a quiet and temperature- and humidity-controlledroom (22±3 °C and 60±5%, respectively) in which a 12-h light/dark cycle was maintained (07:00–19:00 h light). Exposure toethanol and all behavioral experiments involved in ethanolwithdrawal syndrome were carried out in other separate andisolated laboratories, which have the same environmentalconditions with the colony room.

2.2. Chronic exposure to ethanol

For chronic ethanol exposure, the rats were housedindividually and ethanol was given in the modified liquid dietas previously described (Uzbay and Kayaalp, 1995). The rats

were given a modified liquid diet with or without ethanol adlibitum. No extra chow or water was supplied. The compositionof the modified liquid diet with ethanol is: cow milk 925 ml(Pinar, Turkey), 25–75 ml ethanol (96.5% ethyl alcohol; Tekel,Turkish State Monopoly), vitamin A 5000 LU (Akpa, Turkey)and sucrose 17 g (Uzbay and Kayaalp, 1995). This mixturesupplies 1000.7 kcal/L.

At the beginning of the study, rats were given the modifiedliquid diet without ethanol for 7 days. Then, liquid diet with2.4% ethanol was administered for 3 days. The ethanolconcentration was increased to 4.8% for the following 4 daysand finally to 7.2% for 21 days. Liquid diet was freshly prepareddaily and presented at the same time of the day (10:00 h). Theweight of the rats was recorded every day, and daily ethanolintake was measured and expressed as g per kg per day. Controlrats (n=8) were pair fed with an isocaloric liquid diet containingsucrose as a caloric substitute to ethanol.

2.3. Drug used in the study

Olanzapine was purchased from Lilly (USA). The drugswere dissolved in saline. Olanzapine or saline was injected torats intraperitoneally at a volume of 1 ml/200 g body weight.Drug solutions were prepared freshly in the morning just beforethe administration.

2.4. Evaluation of ethanol withdrawal syndrome

At the end of the exposure to 7.2% ethanol-containing liquiddiet, diet with ethanol was withdrawn and replaced withisocaloric ethanol free diet at 10:00 h. Ethanol-dependent ratswere then assigned into four groups randomly (n=8 for eachgroup). Olanzapine (0.5, 1 and 2 mg/kg) and saline wereinjected to the rats 30 min before ethanol withdrawal evaluation.The rats were then observed for 5 min at the 2nd, 4th and 6thhour of the withdrawal period. Injections were repeated 30 minbefore the 6th-h-observation. At each observation time, ratswere assessed simultaneously for the following behavioralconditions: agitation, tremor, stereotyped behavior, wet dogshakes, abnormal posture and gait. Locomotor activities of therats were also recorded (Opto Varimex Minor, Columbus, OH,USA) as a total of horizontal, vertical and ambulatory activitiesand expressed as mean±S.E.M. The subjects were returned totheir home cages between the observation periods.

Tremors and audiogenic seizures were assessed as incidence.Tremor was determined after lifting rats vertically by the tail;positive was assigned to rats showing clearly distinct forelimbtremor (Frye et al., 1983). Wet dog shakes behavior wasconsidered positive if they occurred at least three times duringthe observation period. Grooming, sniffing, head weaving,gnawing and chewing were observed as major stereotypedbehaviors during the ethanol withdrawal in the study. Totalnumber of stereotyped behaviors and wet dog shakes foreach observation period was calculated and expressed as mean±S.E.M. Agitation, abnormal posture, abnormal gait and tailstiffness were scored by using the rating scale as previouslydescribed (Uzbay et al., 1997) (Table 1).

Table 1Rating scale for agitation and stereotyped behavior signs induced by ethanolwithdrawal in rats

Signs Scoring

Agitation 0: no irritability or aggressive behavior1: rats showing mild or moderate irritability2: very irritable3: handling vocalization and moderately aggressive4: handling vocalization and very aggressive5: spontaneous vocalization and very aggressive

Tail stiffness 1: mild tail rigidity2: moderate tail rigidity3: tail rigid but mildly flexible during ambulation4: tail rigid and not flexible during ambulation5: tail very rigid and not flexible during ambulation

Abnormal posture 1: mild head-down, back-hunched2: moderate head-down, back-hunched3: prominent head-down, back-hunched4: in addition hind legs wide apart5: in addition fore limbs apart

Abnormal gait 1–2: mild difficulty in ambulating and rearing normal3–4: moderate difficulty in ambulating and rearing5: prominent difficulty in ambulating and no rearing

Uzbay et al. (1997).

Table 2Changes in weight gains of the rats fed by liquid diet with or without ethanol

Groups Body weight (g)

Beginning ofthe study

End of thestudy

Changes duringthe study

Control 228.42±4.93 236.58±4.90 +3.5%Ethanol-fed 266.34±4.51 271.91±4.98 +0.2%

Values are means±S.E.M.; g = gram; Control = liquid-diet fed without ethanol.

Fig. 1. Effects of olanzapine on the locomotor activities of the rats [n=8 for eachgroup; Olz., Olanzapine; h. hour; # Pb0.05 significantly different from Control(−), Student's t test; −: ethanol nondependent; +: ethanol dependent].

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Each group received a second injection of its original drug30 min before the 6th-h-observation. After 6 h of withdrawaltesting, rats were exposed to an audiogenic stimulus (100 dB)for 60 s in a separate and soundproof place in the laboratory.The incidence and latency of the audiogenic seizures wererecorded.

Control rats receiving no ethanol contained liquid diet werealso evaluated for ethanol withdrawal signs as parallel toethanol-dependent groups.

All experiments were carried out during the light period. Allratings were done by a naive observer, who was blind to whichtreatment the rats received.

2.5. Measurements of locomotor activity in naive control rats

Olanzapine (0.5, 1 and 2 mg/kg) and saline wereadministered in four groups of naive (not ethanol dependent)Wistar rats. Thirty minutes after the injections, rats were put intothe locomotor activity test apparatus and locomotor activities ofthe rats were measured for 30 min. The results of the locomotoractivity tests were expressed as mean±S.E.M.

2.6. Statistical analysis

Changes in locomotor activity and body weight of ethanol-dependent rats as compared to ethanol nondependent controlrats were analyzed by unpaired (between groups) Student's ttest. Analysis of variance (one-way ANOVA) followed by post-hoc Dunnett's test was used in the evaluation of the effects ofolanzapine on the locomotor hyperactivity, stereotyped beha-viors, wet dog shakes and latency of audiogenic seizures. Theeffects of olanzapine on the intensities of agitation, abnormalposture and abnormal gait in different groups were analyzed byKruskall–Wallis test followed by Mann–Whitney-U test.

Comparison of the incidence of tremor and audiogenic seizureswas done by Chi-square test. The level of significance was set atPb0.05 levels.

3. Results

3.1. Ethanol consumption of the rats

Daily ethanol consumption of the rats in control and olan-zapine treated groups ranged from 10.10±0.31 to 14.32±0.32 g/kg during the exposure to ethanol (7.2%). While there was adifference in consumption amounts between the days of the lasttwo weeks including 7.2% alcohol consumption, the differencebetween the two groups was not statistically significant.

Body weight changes of the ethanol-fed and control rats arepresented in Table 2. Body weights of the rats increasedprogressively during the study. An increase in body weight ofapproximately 0.2% (ethanol-fed) and 3.5% (control) comparedto initial weights was observed at the end of the study. Therewere no significant differences in body weight changes of therats compared to the beginning the study (Student's t test,PN0.05).

3.2. Behavioral changes during ethanol withdrawal

A significant locomotor hyperactivity was observed in theethanol-dependent groups at the 4th h and 6th h of thewithdrawal-testing period as compared to the ethanol nonde-pendent saline groups (Student's t test; Psb0.05) (Fig. 1). Otherbehavioral signs of ethanol withdrawal syndrome, such as

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agitation, stereotyped behaviors, tremor, tail stiffness, wet dogshakes, abnormal posture and gait, appeared during the wholeobservation period (Figs. 2 and 3).

Audiogenic seizures occurred at 6th h of ethanol withdrawalwith an incidence of 50% and latency of 14.75±6.54 s inethanol-dependent control group (Table 3). No ethanol with-drawal signs were observed in the ethanol nondependent rats.

3.3. Effects of olanzapine on ethanol withdrawal syndrome

Olanzapine did not produce any significant change onlocomotor hyperactivity at 4th and 6th h of ethanol withdrawal[F(3,28)=1.341 and F(3,28)=0.854; PsN0.05, respectively](Fig. 1).

However, olanzapine produced some significant inhibitoryeffects on stereotyped behaviors and wet dog shakes at 6th h ofethanol withdrawal [F(3,28)=3.259; P=0.036 and F(3,28)=3.010; P=0.047, respectively]. Post-hoc analysis of dataindicated that olanzapine (2 mg/kg) significantly reducedethanol withdrawal-induced stereotyped behaviors and wetdog shakes (Pb0.05, Dunnett's test) (Fig. 2A and B).

Contrary, it produced some significant increases in theintensity of abnormal posture and abnormal gait at 2nd h ofethanol withdrawal (KW=8.243; P=0.041 and KW=8.277;P=0.041, respectively). Post-hoc analysis of data indicated thatolanzapine (2 mg/kg) significantly increased abnormal posture

Fig. 2. Effects of olanzapine treatment on stereotyped behaviors (A) and wet dogshakes (B) during ethanol withdrawal syndrome (n=8 for each group; Olz. =Olanzapine; WDS = Wet dog shakes; h = hour; ⁎Pb0.05 significantly differentfrom Control).

Fig. 3. Effects of olanzapine treatment on abnormal posture (A) and abnormalgait (B) during ethanol withdrawal syndrome (n=8 for each group; Olz. =Olanzapine; h = hour; ⁎Pb0.05 significantly different from Control).

and abnormal gait during ethanol withdrawal (Pb0.05, Mann–Whitney-U test) (Fig. 3A and B).

Meanwhile, olanzapine did not produce any significanteffects on agitation, tail stiffness and tremor (data not shown). Itwas also ineffective both on the intensity and latency of theaudiogenic seizures (Table 3).

3.4. Effects of olanzapine on locomotor activity in ethanolnondependent (naive) rats

Olanzapine treatment (0.5, 1 and 2 mg/kg) did not cause anysignificant change on locomotor activity of the naive (no ethanoldependent) rats. These doses of olanzapine did not cause ab-normal posture and gait in these animals either (data not shown).

Table 3Effects of olanzapine on incidence of the audiogenic seizures in ethanol-dependent rats

Treatment Incidence (%) Latency (s)

Saline (ethanol dependent) 50 (4/8) 14.25±6.54Olanzapine (0.5 mg/kg) 50 (4/8) 14.50±3.77Olanzapine (1 mg/kg) 25 (2/8) – a

Olanzapine (2 mg/kg) 50 (4/8) 22.00±11.60

n=8 for each group; Figures in the parenthesis represent the number of animalsthat have seizure activity after audiogenic stimulus.a Statistical analyses were not performed in the groups having less than three

rats with seizure activity.

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4. Discussion

The main finding of the present study is, while olanzapine,an atypical antipsychotic agent, has inhibitory effects on somesigns of ethanol withdrawal such as stereotyped behavior andwet dog shakes, it increases the severity of abnormal postureand gait in ethanol-dependent rats. On the other hand, it wasfound to be ineffective on the other signs of ethanol withdrawal.Consistent with our previous findings (Uzbay et al., 1994, 1997,1998, 2000a,b), the present data demonstrated that daily ethanolconsumption higher than 10 g/kg for 21 consecutive daysproduced physical dependence in rats. Majchrowicz (1996) alsoshowed that dependence and signs of ethanol withdrawal couldbe produced in rats with 4-day intragastric administration of 9–15 g/kg of ethanol per day. Thus, we observed several signs ofethanol withdrawal such as locomotor hyperactivity, agitation,stereotyped behavior, tremor, tail stiffness, wet dog shakes andaudiogenic seizures. As we did not observe any significantchange on the locomotor activity in naive group, significanteffects of olanzapine on ethanol withdrawal syndrome mightnot be related to other nonspecific effects such as sedation,muscle relaxation or locomotor stimulation.

Alcoholism is one the most important major worldwidepublic health problem. Disulfiram (an aldehyde dehydrogenaseblocker), naltrexone (an opioid antagonist) and acomprosate (afunctional glutamate antagonist) were approved for thetreatment of alcohol dependence, but these medications areeffective on ethanol craving and drinking rather than treatmentof withdrawal syndrome (Heilig and Egli, 2006). Althoughattenuating the severity of ethanol withdrawal symptoms is alsoimportant, treatment choices are very limited except benzodia-zepines. New approaches and new drug choices are necessaryfor treatment of ethanol withdrawal syndrome.

As it is known, both dopaminergic and serotonergic systemsplay a crucial role in development of ethanol dependence(Kuriyama and Ohkuma, 1990). On the other hand, recentreports indicate that there is a relationship between schizophre-nia and ethanol dependence (Seeman et al., 2006; D'Souzaet al., 2006; Conroy et al., 2007). It could be expected that theremight be potential beneficial effects of new atypical antipsy-chotic drugs, such as clozapine, in the treatment of the signs ofethanol withdrawal as well as blocking craving effects ofethanol (Hutchison et al., 2003). Thus, in the present study, wetested the effects of atypical antipsychotic agent olanzapinewhich blocks serotonin 5-HT2 receptors as well as dopamineD2 receptors (Schatzberg et al., 2003). In our study, whileolanzapine inhibited stereotyped behaviors and wet dog shakes,it precipitated abnormal gait and posture during ethanolwithdrawal. Inhibitory effects of olanzapine on wet dogshakes and stereotyped behaviors may be explained by its sero-tonin 5-HT2 and dopamine D2 receptor antagonistic activity,respectively.

Several reports indicated the involvement of serotonin 5-HT2

receptors in the wet dog shake behaviors and blockade of 5-HT2

receptors by antagonists inhibited wet dog shakes in rats (Yapand Taylor, 1983; Fone et al., 1991; Takao et al., 1995). 5-HT2

antagonistic activity of olanzapine may be due to its inhibitory

effect on wet dog shakes. Inhibitory effects on stereotypedbehavior could also be explained by its dopamine D2 receptorantagonistic activity. As previously described, combinedstimulation of dopamine D1 and D2 receptors resulted indose-dependent behavioral activation associated with stereo-types in rats (Longoni et al., 1987; Dall'Olio et al., 1988).Evidence also supports the hypothesis that psychostimulantstereotypy is mediated through postsynaptic dopamine recep-tors (Feldman et al., 1997). Furthermore, it has been showedthat dopamine D2 receptor antagonists inhibit stereotypedbehaviors in rats (Magnusson et al., 1986). Thus, attenuationof ethanol withdrawal-induced stereotyped behavior by olanza-pine, which is a dopamine D2 receptor antagonist at the sametime, is not surprising. Our findings also suggest that theremight be a possible relationship between serotonin 5-HT2 anddopamine D2 receptors, and these signs of ethanol withdrawal.However, here, olanzapine decreased stereotypy and wet dogshakes at 6th h of ethanol withdrawal, but not at earlierintervals. Additional dose of olanzapine given before 6th-h-testing might be the explanation for the effect came into sight.

Precipitation of posture and gait abnormalities may also beexplained by dopamine D2 receptor antagonism. As known,dopamine D2 receptor antagonists cause some motor distur-bances and locomotor inhibition in rats (Hauber and Münkle,1997; Hauber et al., 1998; Hauber and Lutz, 1999). Althougholanzapine and other atypical antipsychotics do not cause extra-pyramidal side effects and motor disturbances like classicalantipsychotics (Schatzberg et al., 2003), ethanol withdrawalmay be augmented by their negative effects on posture and gait.If this explanation is true, olanzapine does not seem to be a safedrug during ethanol withdrawal.

Antipsychotic medications can lower the seizure threshold,increasing the chances of seizure induction (Alldredgge, 1999;Hedges et al., 2003). Some reports also mentioned seizures orlowered seizure threshold associated with olanzapine treatment(Lee et al., 1999; Woolley and Smith, 2001; Hedges et al.,2003; Camacho et al., 2005). Thus, we expected worsenedaudiogenic seizures by olanzapine treatment during ethanolwithdrawal. However, neither incidence nor latency ofaudiogenic seizures was affected by olanzapine treatment inethanol-dependent rats. Moreover olanzapine reduced theincidence of the audiogenic seizures at dose of 1 mg/kgwithout reaching to a statistically significant level. Discrepancymay be due to differences between the species. Rats may bemore resistant to effects of olanzapine on seizure threshold.Simply, our findings showing ineffectiveness of olanzapine onlocomotor hyperactivity, agitation, tail stiffness, tremors andaudiogenic seizures imply that these signs of ethanolwithdrawal may not be related to mechanisms modulated byolanzapine.

In conclusion, our results suggested that olanzapine inhibitsonly stereotyped behavior and wet dog shakes during ethanolwithdrawal in rats. While olanzapine treatment precipitatedabnormal posture and gait in early term of withdrawal, it wasalso ineffective on the other signs. Thus, it does not seem to bean adequate and suitable drug in controlling the ethanolwithdrawal syndrome.

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Acknowledgements

Authors would like to thank Dr. Murat Yildirim and Mr.Selami Alan for their valuable contribution to language of themanuscript and technical assistance during the study, respectively.

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