Effects of risperidone, quetiapine and ziprasidone on ethanol withdrawal
syndrome in rats
Ipek Komsuoglu Celikyurt, Hakan Kayir, Guner Ulak, Faruk B. Erden,
Gokhan K. Ulusoy, Tayfun I. Uzbay
PII:
DOI:
Reference:

S0278-5846(10)00480-X
doi: 10.1016/j.pnpbp.2010.12.009
PNP 7843

To appear in:

Progress in Neuropsychopharmacology & Biological Psychiatry

Received date:
Revised date:
Accepted date:

4 November 2010
9 December 2010
9 December 2010

Please cite this article as: Celikyurt Ipek Komsuoglu, Kayir Hakan, Ulak Guner, Erden
Faruk B., Ulusoy Gokhan K., Uzbay Tayfun I., Eects of risperidone, quetiapine and
ziprasidone on ethanol withdrawal syndrome in rats, Progress in Neuropsychopharmacology
& Biological Psychiatry (2010), doi: 10.1016/j.pnpbp.2010.12.009

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Effects of risperidone, quetiapine and ziprasidone on ethanol

withdrawal syndrome in rats

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Ipek Komsuoglu Celikyurta, Hakan Kayirb, Guner Ulaka, Faruk B. Erdena,

Gokhan K. Ulusoyb, Tayfun I. Uzbayb*

Kocaeli University, Faculty of Medicine, Department of Pharmacology, Izmit - Turkey

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Gulhane Military Medical Academy, Faculty of Medicine Department of Medical

Pharmacology, Psychopharmacology Research Unit, Ankara - Turkey

*Address for correspondence

Prof. I. Tayfun Uzbay
Gulhane Military Medical Academy,
Faculty of Medicine Department of Medical Pharmacology,
Psychopharmacology Research Unit,
Etlik 06018 Ankara TURKEY
Tel: + 90 312 304 4764
Fax: + 90 312 304 2010
E-Mail: tuzbay@gata.edu.tr
tuzbay@gmail.com

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ABBREVIATION LIST
5-hydroxytryptamine

ANOVA:

Analysis of variance

dB:

Decibel

EWS:

Ethanol withdrawal syndrome

Qtp:

Quetiapine

Risp:

Risperidone

S.E.M.:

The standard error of the mean

TUBITAK:

Scientific and Technological Research Council of Turkey

Zip:

Ziprasidone

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5-HT:

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ABSTRACT
Comorbid substance use in schizophrenic patients is common, and substance dependence is a

predictive factor for psychosis. The present study was designed to investigate the effects of

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risperidone, quetiapine and ziprasidone, atypical antipsychotic drugs, on ethanol withdrawal

syndrome (EWS) in rats. Adult male Wistar rats were used in the study. Ethanol (7.2%, v/v)

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was given to rats via a liquid diet for 21 days. An isocaloric liquid diet without ethanol was

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given to control rats. Risperidone (1 and 2 mg/kg), quetiapine (8 and 16 mg/kg), ziprasidone
(0.5 and 1 mg/kg) and vehicle were injected into rats intraperitoneally at 1.5 and 5.5 hrs of

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ethanol withdrawal. At the 2nd, 4th and 6th hrs of ethanol withdrawal, rats were observed for 5
min, and withdrawal signs that included locomotor hyperactivity, stereotyped behavior,

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abnormal gait and posture, tail stiffness and agitation were recorded or rated. Following the
observations at the 6th h, the rats were tested for audiogenic seizures. All three drugs had some

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significant inhibitory effects on EWS-induced behavioral signs beginning at the 2nd h of

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withdrawal. The drugs also significantly reduced the incidence of audiogenic seizures.
Overall, risperidone and quetiapine seemed to be more effective than ziprasidone in
ameliorating the withdrawal signs. Doses of the drugs used in the present study did not
produce any significant changes in locomotor activities of nave rats. Our results suggest that
risperidone, quetiapine and ziprasidone had beneficial effects on EWS in rats. Thus, these
drugs may be helpful for controlling withdrawal signs in ethanol-dependent patients.
Keywords: Ethanol withdrawal; Risperidone; Quetiapine; Ziprasidone; Rat(s)

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1. Introduction
Epidemiologic studies and clinical assessment of schizophrenic populations indicate

that there is a marked relationship between schizophrenia and addictive behaviors.

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Observations of the general population have recognized a high risk of alcohol dependence or
substance abuse in subjects presenting with criteria for schizophrenia (Regier et al., 1990;

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Batel, 2000). It has also been shown that schizophrenia is four times more frequent among

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alcoholic than non-alcoholic subjects (Helzer and Pryzbeck, 1988). Thus, comorbid substance
use in schizophrenic patients is common, and substance abuse may be acceptable as a

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predictive factor for psychosis (Hambrecht and Hafner, 1996).

Ethanol abuse and dependence is one of the most important worldwide public health

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problems. The discontinuation of chronic administration of ethanol is associated with

excitatory withdrawal signs called ethanol withdrawal syndrome (EWS). EWS is the most

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important evidence indicating the development of physical dependence on ethanol (Jaffe,

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1990). Although attenuating the severity of EWS is very important, current treatment choices
are very limited except for the use of benzodiazepines. Acamprosate (a glutamate antagonist),
naltrexone (an opioid antagonist) and disulfiram (an aldehyde dehydrogenase blocker) are
approved for the treatment of ethanol dependence, but these medications are effective in
attenuating ethanol cravings and consumption rather than treatment of EWS (Heilig and Egli,
2006). New approaches and new drug choices are necessary for treatment of EWS.
A strong body of evidence indicates that ethanol activates dopamine release from the
nucleus accumbens and extended amygdala (DiChiara, 1995; Heimer et al., 1997). The action
of ethanol on the mesolimbic pathway is considered to be strongly associated with
susceptibility to alcoholism (Noble, 1996) and the development of craving and loss of control
(Robinson and Berridge, 1993). Neurochemical findings from clinical (Roy et al., 1987; Le
Marquand et al., 1994) and experimental (Murphy et al., 1987; Uzbay et al., 1998; Uzbay et

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al., 2000; Uzbay, 2008) studies have also suggested that significant changes in central
serotonergic neurotransmission occur during ethanol consumption and/or withdrawal.

Substantial experimental evidence indicates that serotonin has a critical role in impulsivity

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and craving, which are frequently seen in alcoholics (Ciccocioppo, 1999), and serotonin is at
least partly responsible for alcohol dependence (Myers and Martin, 1973; Schuckit, 1996). As

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a result, dopaminergic and serotonergic systems play an important role in the development of
ethanol dependence. Thus, drugs that affect the central dopaminergic and serotonergic

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systems may be helpful for controlling ethanol abuse and dependence.

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Atypical antipsychotic drugs are widely used in patients with schizophrenia. In

contrast to classical (or typical) antipsychotics, these drugs have substantially lower risks of

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extrapyramidal symptoms, including tardive dyskinesia. They also modulate serotonergic and
dopaminergic receptors (Schatzberg et al., 2003). Although several reports investigating the

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effects of new atypical antipsychotics, such as clozapine, risperidone, quetiapine and

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aripiprazole, on ethanol abuse and dependence have been published, many of them examined
ethanol consumption, preference or craving (Drake et al., 2000; Ingman et al., 2003; Potvin et
al., 2003; Martinotti et al., 2008; Vergne and Anton, 2010). These observations imply that
new atypical antipsychotic drugs could be effective in the treatment of ethanol dependence,
but there are limited reports investigating the effects of these drugs on EWS. In our previous
studies, we tested the effects of clozapine and olanzapine, atypical antipsychotic drugs, on
EWS in rats. We observed some beneficial effects of clozapine on EWS (Kayir and Uzbay,
2008). On the other hand, while olanzapine inhibited the intensity of some symptoms such as
stereotyped behavior and wet dog shakes, it had some adverse effects on other signs such as
abnormal gait and posture (Unsalan et al., 2008). Effects of risperidone, quetiapine and
ziprasidone, relatively new and effective atypical drugs, on EWS have not been subjected to
clinical or experimental studies yet.

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The main objective of the present study was to investigate the effects of risperidone,
quetiapine and ziprasidone on the signs of EWS in rats. Thus, the current study was focused

on revealing whether these drugs are effective in attenuating ethanol withdrawal or not.

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2. Materials and methods

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2.1. Animals and laboratory

All procedures in this study were in accordance with the Guide for the Care and Use of

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Laboratory Animals as adopted by the National Institutes of Health (USA). Local ethical
committee approval was also attained, numbered 08/36 K-R on April 4, 2008. All efforts were

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made to minimize animal suffering and to reduce the number of animals used.
Adult male Wistar rats (220-320 g at the beginning of the experiments) were used (n=

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8 for each group). They were housed in a quiet and temperature- and humidity-controlled

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room (223 C and 605%, respectively) in which a 12-h light/dark cycle was maintained
(07:00-19:00 h light). Exposure to ethanol and all behavioral experiments involved in

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examining EWS were carried out in other separate and isolated laboratories, which had the
same environmental conditions as the colony room.
2.2. Chronic exposure to ethanol
For chronic ethanol exposure, the rats were housed individually and ethanol was given
in the modified liquid diet as previously described (Uzbay and Kayaalp, 1995). The rats were
given 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 as follows: 925-975
mL cow milk (Danone, Turkey), 25-75 mL ethanol (96.5% ethyl alcohol; Tekel, Turkish State
Monopoly), 5000 IU vitamin A (Aksu Farma, Turkey) and 17 g sucrose (Uzbay and Kayaalp,
1995). This mixture supplies 1000.7 kcal/L.
At the beginning of the study, all of the rats were given the modified liquid diet
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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 21 days. Liquid diet was freshly prepared daily and presented at the same time of

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the day (09:00 h). The weight of the rats was recorded every day, and daily ethanol intake
was measured and expressed as g per kg per day. Control rats (n=8) were pair fed with an

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isocaloric liquid diet containing sucrose as a caloric substitute for ethanol.

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2.3 Drug used in the study

Risperidone (Teva Ltd. API Division, Israel), quetiapine (Sanovel, Turkey) and

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ziprasidone (Pfizer, Turkey) were dissolved in vehicle (0.1% acetic acid). All drugs and
vehicle were injected into rats intraperitoneally at a volume of 1 mL/200 g body weight.

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Drug solutions were prepared freshly in the morning just before administration.

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2.4. Evaluation of EWS

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At the end of the exposure to the 7.2% ethanol-containing liquid diet, diet with
ethanol was withdrawn and replaced with isocaloric ethanol free diet at 09:30 h. Ethanoldependent rats were then assigned into seven groups randomly (n=8 for each group).
Risperidone (1 and 2 mg/kg), quetiapine (8 and 16 mg/kg), ziprasidone (0.5 and 1 mg/kg) and
vehicle were injected into the rats 30 min before ethanol withdrawal evaluation. At the 2nd, 4th
and 6th hrs of ethanol withdrawal, rats were observed for 5 min, and withdrawal signs
including locomotor hyperactivity, stereotyped behavior, abnormal posture and gait, tail
stiffness and agitation were recorded or rated as previously described (Uzbay and Kayaalp,
1995; Uzbay et al., 1997; Kayir and Uzbay, 2008).
Locomotor activities of the rats were recorded using an open-field locomotor activity
test apparatus (Opto Varimex Minor, Columbus, OH, USA) as a total of horizontal, vertical
and ambulatory activities and expressed as mean S.E.M.
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Grooming, sniffing, head weaving, gnawing and chewing were observed as major
stereotyped behaviors during the ethanol withdrawal in the study. The total number of

stereotyped behaviors for each observation period was calculated and expressed as mean

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S.E.M. Abnormal gait and posture, agitation and tail stiffness were scored using the rating
scale as previously described (Uzbay et al., 1997).

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Each group received a second injection of its original drug 30 min before the 6th h of
observation. After 6 h of withdrawal testing, rats were exposed to an audiogenic stimulus

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(100 dB) for 60 s in a separate and soundproof place in the laboratory. The incidence and

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latency of the audiogenic seizures were recorded.

Control rats receiving liquid diet without ethanol were also evaluated for ethanol
withdrawal signs parallel to ethanol-dependent groups.

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All experiments were carried out during the light period. All ratings were scored by a

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naive observer who was blind to the treatments that the rats received.

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2.5. Measurements of locomotor activity in naive control rats

The experimental drugs and vehicle were administered in seven groups of naive (not
ethanol-dependent) Wistar rats. Thirty minutes after the injections, rats were put into the
locomotor activity test apparatus and locomotor activities of the rats were measured for 30
min. The results of the locomotor activity tests were expressed as mean S.E.M.
2.6. Statistics
Changes in locomotor activity and body weight of ethanol-dependent rats as compared
to ethanol non-dependent control rats were analyzed by two-way analysis of variance
(ANOVA) tests (treatment x time). A two-way ANOVA (treatment x time) followed by
Bonferroni or Students t-test was used in the evaluation of the effects of the drugs on the
locomotor hyperactivity, stereotyped behaviors, latency of audiogenic seizures, abnormal gait

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and posture, agitation and tail stiffness in different treatment groups. Comparison of the
incidence of audiogenic seizures was completed using a chi-square test. A two-way ANOVA

(group x time) was used to compare ethanol consumptions of the groups given the ethanol-

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containing liquid diet. The effects of risperidone, quetiapine and ziprasidone on locomotor
activities of the nave rats were also analyzed by a two-way ANOVA (group x time) followed

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by Bonferroni test. The level of significance was set at p<0.05.

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3. Results
3.1. Rat ethanol consumption and body weight gain

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Daily ethanol consumption of the rats in control and drug-treated groups ranged from
9.230.85 to 15.720.88 g/kg during the exposure to 7.2% ethanol. While there was a

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difference in consumption amounts between the days of the last two weeks including the
7.2% alcohol consumption, the difference between the groups was not statistically significant.

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The results of the two-way ANOVA on ethanol consumption data revealed significant effects

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for time [F(13,637)=9.694, p<0.0001] and interaction [F(78,637)=3.911, p<0.0001] effects

but not a group effect [F(6,49)=2.018, p=0.081]. These data indicate that there was no
significant difference in ethanol consumption between the groups. However, ethanol
consumption of the rats significantly increased during exposure to the liquid diet.
Body weight changes of the ethanol-fed and control rats are presented in Table 1.
Body weights of the rats increased slightly during the study in ethanol feeding groups. There
was a 4.5% increase in body weight during the study in the ethanol fed groups. In control rats,
an increase in body weight of approximately 17.6% was observed. This increase in body
weight of the rats was statistically significant compared to the beginning of the study
(Students t-test, p<0.05).

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3.2. Behavioral changes during ethanol withdrawal
A two-way ANOVA revealed that there was a significant difference between the

locomotor activities of the ethanol-dependent and non-dependent rats during the withdrawal

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testing [treatment effect: F(1,12)= 8.625; p=0.012]. The locomotor activity values changed
during the time course of withdrawal [time effect: F(2,24)=47.627; p<0.0001], but the

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difference between groups was not dependent on the time course [interaction: F(2,24)=2.334;
p>0.05]. Post-hoc analysis indicated significant locomotor hyperactivity in the ethanol-

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dependent groups at the 2nd and 4th hrs of the withdrawal testing period compared to the

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ethanol non-dependent vehicle groups (p<0.05) (Fig. 1). Other behavioral signs of EWS, such
as stereotyped behaviors, abnormal posture and gait, tail stiffness and agitation, were apparent

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during the whole observation period (Fig. 2-7 A-D, dark bars).
Audiogenic seizures occurred at the 6th h of ethanol withdrawal with an incidence of

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50% and latency of 12.25 6.55 s in the ethanol-dependent control group (Table 2). No

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ethanol withdrawal signs were observed in the ethanol non-dependent rats.

3.3. Effects of risperidone on EWS
Risperidone produced significantly affected EWS-induced locomotor hyperactivity
[treatment effect: F(2,20)=14.354; p<0.0001] with a significant interaction [F(4,40)=15.615;
p<0.0001] with testing time point. Post-hoc analysis of the data indicated that 1 and 2 mg/kg
risperidone significantly reduced ethanol withdrawal-induced locomotor hyperactivity at the
2nd h but not the 4th and 6th hrs of ethanol withdrawal (p<0.05, Bonferroni test) (Fig. 2A).
Similarly, risperidone significantly affected stereotyped behaviors observed during
ethanol withdrawal [treatment effect: F(2,20)=6.138; p=0.008]. The effect of testing time and
interaction between treatment and time were not significant for stereotyped behaviors [time
effect: F(2,40)= 2.873; p>0.05 and interaction: F(4,40)= 1.417; p>0.05]. Post-hoc analysis of

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the data indicated that 1 and 2 mg/kg risperidone significantly reduced EWS-induced
stereotyped behaviors at the 2nd h of withdrawal (p< 0.05, Bonferroni test), and this inhibitory

effect on stereotyped behaviors was not observed at the 4th and 6th hrs of ethanol withdrawal

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(ps>0.05, Bonferroni test, Fig. 2B).

Risperidone significantly affected abnormal posture [F(2,20)=74.108; p<0.0001, Fig.

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3A], abnormal gait [F(2,20)=154.417; p<0.0001, Fig. 3B], tail stiffness [F(2,20)=43.786;

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p<0.0001, Fig. 3C] and agitation [F(2,20)=43.786; p<0.0001, Fig. 3D] at the 2nd, 4th and 6th
hrs of ethanol withdrawal. Post-hoc analysis of data obtained from the whole observation

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period indicated that all doses of risperidone were significantly effective on all these signs,
except the 2 mg/kg dose did not affect abnormal posture (ps<0.05, Bonferroni test).

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Risperidone (1 and 2 mg/kg) also significantly reduced the incidence of audiogenic

seizures appearing at the 6th h of withdrawal (p<0.05, chi-square test) (Table 2).

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3.4. Effects of quetiapine on EWS

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Quetiapine significantly affected EWS-induced locomotor hyperactivity [treatment

effect: F(2,21)=171.139; p<0.0001] with a significant interaction [F(4,42)=4.882; p=0.003]
with testing time point. Post-hoc analysis of the data indicated that 8 and 16 mg/kg quetiapine
significantly reduced EWS-induced locomotor hyperactivity at the 2nd h of ethanol
withdrawal (p<0.05, Bonferroni test, Fig. 4A).
Quetiapine significantly affected stereotyped behaviors observed during EWS
[treatment effect: F(2,21)=33.140; p<0.0001, Fig. 4B]. The effect of testing time and
interaction between treatment and time were not significant for stereotyped behaviors [time
effect: F(2,42)=2.303; p>0.05 and interaction: F(4,42)=0.903; p>0.05]. Post-hoc analysis of
the data indicated that 16 mg/kg quetiapine significantly inhibited (ps<0.05, Bonferroni test)

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the stereotyped behaviors at the 2nd and 4th hrs of EWS, and this activity was not observed at
the 6th h of testing (Fig. 4B).

Quetiapine also significantly affected abnormal posture [F(2,21)=68.665; p<0.0001,

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Fig. 5A], abnormal gait [F(2,21)=91.263; p<0.0001, Fig. 5B], tail stiffness [F(2,21)=21.754;
p<0.0001, Fig. 5C] and agitation [F(2,21)=14.646; p<0.0001, Fig. 5D] at the 2nd, 4th and 6th

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hrs of ethanol withdrawal. Post-hoc analysis of the data obtained from the whole observation

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period indicated that both doses of quetiapine had significant effects on abnormal gait and
posture and tail stiffness (ps<0.05, Bonferroni test). However, the inhibitory effect of

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quetiapine on agitation was significant only at the 8 mg/kg dose (ps<0.05, Bonferroni test).
Quetiapine (8 and 16 mg/kg) also significantly reduced the incidence of audiogenic

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seizures appearing at the 6th h of withdrawal (p<0.05, chi-square test) (Table 2).

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3.5. Effects of ziprasidone on EWS

Ziprasidone significantly affected EWS-induced locomotor hyperactivity [treatment

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effect: F(2,21)=226.464; p<0.0001] with a significant interaction [F(4,42)=2.737; p=0.041]

with testing time point. Post-hoc analysis of the data indicated that 1 mg/kg ziprasidone
significantly reduced EWS-induced locomotor hyperactivity at the 2nd and 6th hrs of EWS
(p<0.05, Bonferroni test, Fig. 6A).
Ziprasidone did not significantly affect stereotyped behaviors observed during EWS
[treatment effect: F(2,21)=0.800; p>0.05, Fig. 6B]. The effect of testing time and interaction
between treatment and time were also not significant for stereotyped behaviors [time effect:
F(2,42)=0.155; p>0.05 and interaction: F(4,42)=1.288; p>0.05].
Ziprasidone also significantly affected abnormal posture [F(2,21)=11.972; p<0.0001,
Fig. 7A], abnormal gait [F(2,21)=18.702; p<0.0001, Fig. 7B], tail stiffness [F(2,21)=9.647;
p<0.0001, Fig. 7C] and agitation [F(2,21)=7.177; p=0.004, Fig. 7D] during EWS. Post-hoc

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analysis of the data obtained from the whole observation period indicated that ziprasidone had
inhibitory effects on abnormal posture and abnormal gait only at the 6th h of EWS.

Ziprasidone inhibited abnormal posture at a dose of 0.5 mg/kg but not at 1 mg/kg. However, 1

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mg/kg ziprasidone affected abnormal gait (ps<0.05, Bonferroni test, Fig. 7B). Tail stiffness
was reversed by ziprasidone at all time points and drug doses, except at the 6th h at the 1

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mg/kg dose (Fig. 7C). The inhibitory effect of ziprasidone on agitation was significant at the

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2nd and 6th hrs of observation at both doses. (ps<0.05, Bonferroni test).
At the dose of 1 mg/kg, ziprasidone significantly reduced the incidence of audiogenic

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seizures (p<0.05, chi-square test) (Table 2).

3.6. Effects of risperidone, quetiapine and ziprasidone on locomotor activity in ethanol non-

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dependent (naive) rats

Risperidone (1 and 2 mg/kg) significantly affected locomotor activity of nave

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(ethanol non-dependent) rats during a parallel time course with EWS experiments [treatment

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effect: F(2,21)=18.203; p<0.0001, Fig. 8A]. Measurement time had no effect on the results
[time effect: F(2,42)=1.248; p>0.05], but the effect of risperidone on locomotor activity
interacted with the measurement time [interaction: F(4,42)=3.951; p=0.008]. Post-hoc
analysis indicated that 1 and 2 mg/kg risperidone significantly decreased locomotor activity of
the nave rats only at the 6th h time point (ps<0.05, Bonferroni test, Fig. 8A).
Quetiapine (8 and 16 mg/kg) also significantly affected locomotor activity of nave
rats [treatment effect: F(2,21)=11.205; p<0.0001, Fig. 8B]. Measurement time independently
changed the locomotor activity counts [time effect: F(2,42)=13.948; p<0.0001, interaction:
F(4,42)=0.699;p> 0.05]. Post-hoc analysis indicated that the 16 mg/kg dose but not the 8
mg/kg dose of quetiapine significantly decreased locomotor activity of the nave rats at all
three time points (ps<0.05, Bonferroni test, Fig. 8B).

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Ziprasidone (0.5 and 1 mg/kg) significantly affected locomotor activity of nave rats
[treatment effect: F(2,21)=10.935; p<0.0001, Fig. 8C]. Measurement time also had a

significant effect on the locomotor activity counts [time effect: F(2,42)=4.164; p=0.022]. A

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significant interaction between treatment and time was also obtained [interaction:
F(4,42)=4.269; p=0.005]. Post-hoc analysis indicated that both doses (0.5 and 1 mg/kg) of

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ziprasidone significantly decreased locomotor activity of the nave rats at the 2nd h time point,
and 1 mg/kg ziprasidone had a significant effect at the 6th h time point (ps<0.05, Bonferroni

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test, Fig. 8C).

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4. Discussion

In this study, risperidone, quetiapine and ziprasidone, relatively new atypical

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antipsychotic drugs, had inhibitory effects on the behavioral signs of EWS such as locomotor
hyperactivity, stereotyped behavior, abnormal posture and gait, tail stiffness and agitation.

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Moreover, they were found to be effective on audiogenic seizures during EWS. Consistent

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with our previous findings (Uzbay et al., 1994; Uzbay et al., 1997; Uzbay et al., 1998; Uzbay
et al., 2000a,b; Uzbay et al., 2004a; Unsalan et al., 2008), the present data demonstrated that
daily ethanol consumption over 9 g/kg for 15 consecutive days produced physical dependence
in rats. Majchrowicz (1975) also showed that dependence and signs of ethanol withdrawal
could be produced in rats with intragastric administration of 9-15 g/kg of ethanol per day for
four days. We did not measure blood ethanol levels in the present study. However, we
detected high blood ethanol levels in our previous studies with the same method (Uzbay et al.,
1994; Uzbay and Kayaalp, 1995; Uzbay et al., 1997; Uzbay et al., 1998; Uzbay and Erden,
2003; Uzbay et al., 2004b; Kayir and Uzbay, 2008). In addition, we observed several signs of
ethanol withdrawal such as locomotor hyperactivity, stereotyped behaviors, abnormal posture
and gait, tail stiffness, agitation and audiogenic seizures. Audiogenic seizure response is
considered as the most reliable and easily quantifiable behavioral sign of EWS in rats

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(Majchrowicz, 1975; Trzaskowska et al., 1989; Uzbay and Kayaalp, 1995). The incidence of
audiogenic seizures observed in the present study was very similar to that of our previous

studies (Uzbay et al., 1994; Uzbay and Kayaalp, 1995; Uzbay et al., 1997; Uzbay et al.,

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2000a).

In the present study, we did not observe any body weight losses in the rats fed by

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liquid diet with ethanol. Even a slight body weight loss in the rats fed with an ethanolcontaining liquid diet can be accepted as normal (Parale and Kulkarni, 1986; Larue-

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Achagiotis et al., 1990; Uzbay and Kayaalp, 1995). In fact, an increase in body weight in

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control rats compared to initial weights was observed at the end of the study.
Drug doses were selected according to our preliminary tests and previous studies.
Because doses of the drugs higher than used in the study caused some sedative effects or

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slight extrapyramidal symptoms such as tremor in rats, doses up to 2, 16 and 1 mg/kg

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risperidone, quetiapine and ziprasidone, respectively, were used in the present study. In nave
rats, both doses of risperidone and ziprasidone and a higher dose of quetiapine (16 mg/kg)

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reduced locomotor activity significantly at the 2nd h of ethanol withdrawal. However, the
effects of risperidone on locomotor activity disappeared at later observation periods. Even
after a second injection before the 6th h of observation period, risperidone did not significantly
affect locomotor activity of the nave rats. Quetiapine (8 mg/kg) also did not significantly
affect locomotor activities at all observation periods. Thus, the observed effects of these drugs
on EWS seem to be specific and not dependent on other non-specific effects such as sedation
and muscle relaxation. Ziprasidone exhibited some significant inhibitory effects on locomotor
activities of nave rats except at the 4th h of the observation period. Thus, some significant
inhibitory effects induced by ziprasidone may be related to its sedative effects. However,
because some limited beneficial effects of ziprasidone, for example inhibition of audiogenic
seizures, appeared as a result of only the higher dose, we did not use lower doses of this drug

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in the present study. While risperidone and ziprasidone caused significant reductions in
locomotor activities of nave rats during the first injections and observation period, these

inhibitory effects did not occur during the following observation periods, even after the

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second injections. It is difficult to explain this occurrence with the present data, but it may be
related to acute unlike effects of the drugs in the rats followed by a tolerance development.

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Both dopaminergic and serotonergic systems play a crucial role in the development of
ethanol dependence (Kuriyama and Ohkuma, 1990; Uzbay, 2008), and recent reports indicate

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that there is a relationship between schizophrenia and ethanol dependence (DSouza et al.,

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2006; Seeman et al., 2006; Conroy et al., 2007). There may be potential beneficial effects of
new atypical antipsychotic drugs in the treatment of the signs of ethanol withdrawal as well as
blocking the craving effects of ethanol (Drake et al., 2000; Hutchison et al., 2003). In recent

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studies from our laboratory, we investigated and reported the effects of olanzapine (Unsalan

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et al., 2008) and clozapine (Kayir and Uzbay, 2008), another atypical antipsychotic drug, on
EWS in ethanol-dependent rats. In the present study, we tested the effects of three additional,

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relatively new atypical antipsychotic agents: risperidone, quetiapine and ziprasidone, which
blocks serotonin 5-HT2 receptors as well as dopamine D2 receptors (Stefan et al., 2002;
Schatzberg et al., 2003). In our previous study, clozapine inhibited locomotor hyperactivity,
stereotyped behaviors, wet dog shakes, tremors and tail stiffness during ethanol withdrawal.
Thrasher et al. (1999) suggested that ethanol-stimulated locomotor activity was reduced by
clozapine treatment in mice. Unsalan et al. (2008) also observed inhibitory effects on
stereotyped behavior and wet dog shakes in ethanol-dependent rats by olanzapine treatment.
Our results regarding the beneficial effects of risperidone and quetiapine on ethanol
withdrawal-induced locomotor hyperactivity and stereotyped behaviors are in line with the
results of these studies. Marked inhibitory effects of risperidone and quetiapine on locomotor

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hyperactivity and stereotyped behaviors may be explained by their dopamine D2 receptor
antagonistic activity.

As previously described, combined stimulation of dopamine D1 and D2 receptors

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resulted in dose-dependent behavioral activation associated with stereotypes in rats (Longoni

et al., 1987; DallOlio et al., 1988). Evidence also supports the hypothesis that

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pyschostimulant-induced stereotypy is mediated through postsynaptic dopamine receptors

(Feldman et al., 1997). Furthermore, it has been shown that dopamine D2 receptor antagonists

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inhibit stereotyped behaviors in rats (Magnusson et al., 1986). Thus, attenuation of ethanol

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withdrawal-induced stereotyped behaviors by risperidone and quetiapine, which are both

dopamine D2 receptor antagonists, is not surprising.
In contrast to results obtained with olanzapine (Unsalan et al., 2008), in the present

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study, risperidone, quetiapine and ziprasidone did not cause any precipitation of posture and

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gait abnormalities in ethanol-withdrawn rats. Conversely, they significantly inhibited the

intensity of abnormal posture and gait that occurred during ethanol withdrawal. Atypical

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antipsychotic drugs cause fewer extrapyramidal symptoms at effective doses. This may be
related to their potent 5-HT2A and relatively weak D2 receptor blocking properties (Meltzer,
1995). Our findings regarding abnormal posture and gait are in line with this general
information. Precipitation of abnormal posture and gait in ethanol-dependent rats by
olanzapine treatment may be due to another property of this drug.
In the present study, we observed some biphasic profiles with risperidone and
ziprasidone treatments on abnormal posture. These drugs were found to be effective on
abnormal posture at their low doses, and this effect disappeared at higher doses. This situation
was not observed for other behavioral signs of ethanol withdrawal. Furthermore, these drugs
did not exhibit biphasic effects on locomotor activity in non-dependent rats. The biphasic

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effects may be related to insufficient selectivity in the scoring method used to evaluate
abnormal posture during ethanol withdrawal in the present study.

In our study, all three atypical antipsychotics were found to be effective on other

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behavioral signs of EWS such as tail stiffness and agitation. Agitation, together with
locomotor hyperactivity, may be a reflection of anxiety occurring during ethanol withdrawal.

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Previous studies showed that antagonism of 5-HT2 receptors, especially the 5-HT2C subtype,
reduced anxiogenic behaviors during ethanol withdrawal (Lal et al., 1993; Overstreet et al.,

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2003; Overstreet et al., 2006). Risperidone, quetiapine and ziprasidone have antagonistic

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activity at the 5-HT2A and 5-HT2C receptors. Affinity of quetiapine for 5-HT2A receptors is
lower than risperidone and ziprasidone (Stefan et al., 2002; Schatzberg et al., 2003). Although
literature on the role of 5-HT2A receptors in ethanol abuse and dependence is very limited, an

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antagonism at 5-HT2A receptors could be responsible for the beneficial effects of the drugs on

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EWS. These three drugs also bind to a range of non-dopaminergic and non-serotonergic
targets, including glutamate, histamine, alpha-adrenergic and muscarinic receptors (Nasrallah,

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2008). Interaction with other neurotransmitter systems may contribute to their beneficial
effects on all of the signs of ethanol withdrawal. For insitance, all three agents used in the
present study have antagonistic activity at noradrenergic 1 receptors, and it was shown that
1-noradrenergic receptor antagonism blocked acute withdrawal signs in ethanol-dependent
rats (Walker et al., 2008).
Antipsychotic medications can lower the seizure threshold and increase the chance of
seizure induction (Alldregge, 1999; Hedges et al., 2003). Thus, antipsychotic treatments
during ethanol withdrawal may worsen audiogenic seizures. However, in our study, all three
agents significantly reduced the incidence of audiogenic seizures. Previously, we showed that
neither clozapine (Kayir and Uzbay, 2008) nor olanzapine (Unsalan et al., 2008) treatments
affected the incidence and the latency of audiogenic seizures in ethanol-dependent rats. Our

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previous findings showing the ineffectiveness of clozapine and olanzapine on audiogenic
seizures imply that this sign of ethanol withdrawal may not be related to mechanisms

modulated by clozapine and olanzapine. Our results also indicate that atypical antipsychotics

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are safer than classical agents during ethanol withdrawal in alcoholic patients with epilepsy.
Many reports have indicated marked reductions in the brain 5-HT levels of ethanol-

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withdrawn rats after seizures induced by audiogenic stimulus (Mirowski et al., 1995; Uzbay et
al., 1998; Yu et al., 2000). In addition, it has been suggested that bromocriptine, a

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dopaminergic agonist, prevents (Trzaskowska et al., 1989) or prolongs (Uzbay et al., 1994)

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the latency of audiogenic seizures in ethanol-dependent rats. Thus, all of these data indicate
that increased serotonergic and dopaminergic activity may be a protective response to the
audiogenic seizures that occur during ethanol withdrawal in rats. Because of their

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dopaminergic and serotonergic antagonistic properties, the beneficial effects of risperidone,

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quetiapine and ziprasidone could not be explained via their interactions with serotonergic
and/or dopaminergic receptor systems. Also, because H1 receptor antagonists increased

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seizure susceptibility in rodents (Gerald and Richter, 1976; Sturman et al., 2001), the H1
receptor antagonistic property of the atypical antipsychotics could not be responsible for the
inhibitory effects on audiogenic seizures. However, Semenova and Ticku (1997) showed that
treatment with the 2 adrenoceptor blockers reduced the sensitivity of mice to audiogenic
convulsions. The blocking effect at 2 receptors by risperidone and quetiapine may be
responsible for their beneficial effects on audiogenic seizures. The effect of ziprasidone on 2
receptors is controversial, and it is not as clear as risperidone and quetiapine. In the present
study, risperidone and quetiapine were found to be more effective than ziprasidone on
audiogenic seizures. This finding may also be due to antagonistic efficacies of these drugs on
2 receptors. Overall, in contrast to clozapine and olanzapine, marked inhibition of audiogenic
seizures by risperidone, quetiapine and ziprasidone treatments is a very interesting finding.

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However, we cannot currently give a definitive explanation of how these drugs inhibited the
audiogenic seizures. Further studies are necessary to elucidate the mechanisms behind this

phenomenon.

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5. Conclusion

Our results suggest that risperidone, quetiapine and ziprasidone had significant

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beneficial effects on EWS in rats. Thus, these drugs may be helpful for controlling some
withdrawal symptoms in ethanol-dependent patients. Moreover, we observed that risperidone

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and quetiapine were more effective than ziprasidone. Further research is needed to explain the

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differences between the activities of these three drugs.

Acknowledgements

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This study was supported by the Scientific and Technological Research Council of
Turkey (TUBITAK, Project Number: 105S387, SBAG-3194). The authors would like to

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thank Mr. Selami Alan for his technical assistance during the study. Language edition of the

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manuscript has been made by American Journal Experts (USA).

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Figure legends
Figure 1. Changes in locomotor activity during the first 6 h of ethanol withdrawal (n=8 for

each group; h=hour; * p<0.05 significantly different from control, Students t-test).

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Figure 2. Effects of risperidone treatments on the locomotor activities (A) and stereotyped
behaviors (B) of rats during ethanol withdrawal [n=8 for each group; Risp=risperidone;

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h=hour; Control (+), ethanol-withdrawn group treated with vehicle; * p<0.05 significantly

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different from Control (+), Bonferroni test].

Figure 3. Effects of risperidone treatment on abnormal posture (A), gait (B), tail stiffness (C)

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and agitation (D) during ethanol withdrawal [n=8 for each group; Risp=risperidone; h=hour;
Control (+), ethanol-withdrawn group treated with vehicle; * p<0.05 significantly different

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from Control (+), Bonferroni test].

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Figure 4. Effects of quetiapine treatments on the locomotor activities (A) and stereotyped
behaviors (B) of rats during ethanol withdrawal [n=8 for each group; Qtp=quetiapine; h=hour;

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Control (+), ethanol-withdrawn group treated with vehicle; * p<0.05 significantly different
from Control (+), Bonferroni test].
Figure 5. Effects of quetiapine treatment on abnormal posture (A), gait (B), tail stiffness (C)
and agitation (D) during ethanol withdrawal [n=8 for each group; Qtp=quetiapine; h=hour;
Control (+), ethanol-withdrawn group treated with vehicle; * p<0.05 significantly different
from Control (+), Bonferroni test].
Figure 6. Effects of ziprasidone treatments on the locomotor activities (A) and stereotyped
behaviors (B) of rats during ethanol withdrawal [n=8 for each group; Zip=ziprasidone; h=
hour; Control (+), ethanol-withdrawn group treated with vehicle; * p<0.05 significantly
different from Control (+), Bonferroni test].

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Figure 7. Effects of ziprasidone treatment on abnormal posture (A), gait (B), tail stiffness (C)
and agitation (D) during ethanol withdrawal [n=8 for each group; Zip=ziprasidone; h=hour;

Control (+), ethanol-withdrawn group treated with vehicle; * p<0.05 significantly different

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from Control (+), Bonferroni test].

Figure 8. Effects of risperidone (A), quetiapine (B) and ziprasidone (C) on locomotor activity

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in nave (ethanol non-dependent) rats [n=8 for each group; Risp=risperidone; Qtp=quetiapine;

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Zip=ziprasidone; h= hour; * p<0.05 significantly different from control, Bonferroni test].

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Table 1

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Body weight (g)

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

Beginning of the study

End of the Study

Control (-)
(n=8)

240.17 6.39

282.50 5.93*

Control (+)
and drugs
(n=56)

233.75 9.83

244.37 7.82

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Groups

Changes during the study

+ 17.6%

+ 4.5%

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* p< 0.05, Students t test; Control (-), groups fed without ethanol; Control (+), groups fed
with ethanol containing liquid diet.

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Table 2
Effects of risperidone, quetiapine and ziprasidone on the audiogenic seizures in ethanol-

Treatment

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dependent rats.
Incidence (%)

Latency (sec)
12.25 6.55

50 (4/8)*

Risperidone (1 mg/kg)

0 (0/8)*

Risperidone (2 mg/kg)

0 (0/8)*

Quetiapine (8 mg/kg)

12.5 (1/8)*

Quetiapine (16 mg/kg)

0 (0/8)*

25 (2/8)

12.5 (1/8)*

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Control (ethanol-dependent)

Ziprasidone (0.5 mg/kg)

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Ziprasidone (1 mg/kg)

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n= 8 for each group; * p<0.05 significantly different from control (chi-square test); figures in
the parenthesis represent the number of animals that have seizure activity after audiogenic
stimulus; -: statistical analyses were not performed and data were not given in the groups
having less than three rats with seizure activity.

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Research Highlights
Ethanol consumption for 21 days caused physical dependence in rats.

Risperidone, quetiapine and ziprasidone attenuated ethanol withdrawal syndrome.

Effective doses of drugs did not affect locomotory activity of nave animals.

Risperidone and quetiapine seemed to be more effective than ziprasidone.

These drugs may be helpful for controlling of ethanol withdrawal syndrome.

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