Alcohol & Alcoholism Vol. 40, No. 4, pp. 291–296, 2005 doi:10.1093/alcalc/agh133 Advance Access publication 3 May 2005

EFFECT OF HYPERICUM PERFORATUM CO2 EXTRACT ON THE MOTIVATIONAL PROPERTIES OF ETHANOL IN ALCOHOL-PREFERRING RATS MARINA PERFUMI*, LAURA MATTIOLI, LAURA FORTI, MAURIZIO MASSI and ROBERTO CICCOCIOPPO

Department of Pharmacological Sciences and Experimental Medicine, University of Camerino, via Scalino 3, 62032 Camerino (MC), Italy

(Received 29 September 2004; first review notified 22 October 2004; in final revised form 26 November 2004; accepted 26 November 2004)

Abstract — Aims: Extracts of Hypericum perforatum (HPE) attenuate voluntary ethanol intake in different lines of alcohol-preferring rats. The present study evaluated the effect of the intragastric (IG) administration of a CO2 Hypericum perforatum extract (HPCO2) on

operant ethanol self-administration, as well as on voluntary ethanol intake, after a period of ethanol deprivation in genetically selected Downloaded from https://academic.oup.com/alcalc/article/40/4/291/127971 by guest on 29 September 2021 Marchigian Sardinian alcohol-preferring rats. Methods: HPCO2 was administered by means of an indwelling IG catheter, 1 h before the tests. For the self-administration experiments, the rats were trained to self-administer 10% (v/v) ethanol in 30-min daily sessions under a fixed ratio 1 schedule of reinforcement. HPCO2 was also tested on 0.2% w/v saccharin self-administration. For the ethanol deprivation experiments, rats that had a previous experience with voluntary ethanol drinking were deprived of ethanol for 9 days, whereas water and food were freely available; HPCO2 was given by IG injection 1 h before the ethanol re-presentation. Results: HPCO2 in doses of 31 or 125 mg/kg but not 7 mg/kg, significantly reduced ethanol self-administration, while it did not modify saccharin self-administration. The same doses of the extract abolished the increased ethanol intake following ethanol deprivation. Conclusions: These findings provide evidence that HPCO2 markedly reduces the reinforcing properties of ethanol in the self- administration paradigm, as well as the increase of ethanol intake following ethanol deprivation. These findings further support the view that the use of HPE may represent an interesting pharmacological approach in the treatment of alcohol abuse and alcoholism.

INTRODUCTION 1998, 2001; Singer et al., 1999; Vormfelde and Poser, 2000; Nathan, 2001; Cervo et al., 2002; Keller et al., 2003). Extracts of Hypericum perforatum (HPE), the common plant In the present work, hyperforin enriched plant extract, usually referred to as St John’s wort, are known to exert namely a CO2 Hypericum perforatum extract (HPCO2), was antidepressant effects in humans (Volz, 1997; Laakmann used to further investigate the mechanisms accounting for the et al., 1998; Linde and Mulrow, 2000; Vormfelde and Poser, control of ethanol intake by HPE, under operant conditions. 2000; Barnes et al., 2001; Kasper, 2001; Van Grup, 2002; For this purpose, using Marchigian Sardinian alcohol-

Muller, 2003; Rodriguez-Landa and Contreras, 2003) and an preferring (msP) rats, the HPCO2 extract was tested on the oral antidepressant-like action in laboratory animals, in different self-administration of 10% ethanol under a fixed ratio 1 (FR1) experimental models (Butterweck et al., 1997; Bennett et al., schedule of reinforcement. It is well known that the data 1998; Gambarana et al., 1999, 2001; Nathan, 1999; Perfumi obtained under operant paradigms provide a closer measure on et al., 1999; Greeson et al., 2001). Recent reports have also the reinforcing value of the self-administered drug, thus described the anxiolytic, analgesic and memory enhancing offering important advantages over the home-cage two-bottle properties of HPE in rats (Kumar et al., 2000, 2001; choice paradigm (Samson et al., 1988, 1990, 1998; Weiss Vandenbogaerde et al., 2000; Khalifa, 2001; Klusa et al., et al., 1993; Vacca et al., 2002; Samson and Czachowski, 2001; Flausino et al., 2002; Skalisz et al., 2004). Catania et al. 2003). Moreover, in order to evaluate the selectivity of the

(2003) have also demonstrated that HPE attenuates nicotine effect of HPCO2, its ability to affect 0.2% saccharin self- withdrawal signs in mice. administration was studied. In the last few years, several studies have shown that HPE Finally, to extend our study, we investigated the effect of reduces voluntary ethanol intake in the home-cage two-bottle HPCO2 on the alcohol deprivation effect (ADE) in the msP choice paradigm in alcohol-preferring rats (De Vry et al., 1999; rats under the home-cage two-bottle choice condition. ADE is Perfumi et al., 1999, 2001, 2002, 2003; Rezvani et al., 1999; a temporary increase in the voluntary ethanol intake found in Panocka et al., 2000) and C57BL/6J mice (Wright et al., 2003). different animal species after a period of alcohol abstinence The mechanisms accounting for the inhibition of ethanol intake and has been proposed as an experimental model of alcohol in alcohol-preferring rats and mice by HPE remain unknown. relapses in human alcoholics (Spanagel, 2000; Boening et al., Hyperforin has been suggested to be the primary bioactive 2001; McBride et al., 2002). ingredient responsible for the effect of HPE on ethanol intake (Perfumi et al., 2001; Wright et al., 2003), as well as for its antidepressant-like action (Bhattacharya et al., 1998; MATERIALS AND METHODS Chatterjee et al., 1998a,b; Laakmann et al., 1998; Muller et al., Animals Male genetically selected alcohol-preferring rats, weight between 250–300 g (at the beginning of the experiment), were *Author to whom correspondence should be addressed at: Marina Perfumi, Department of Pharmacological Sciences and Experimental Medicine, used. Animals were bred at the Department of Pharmacological University of Camerino, via Scalino 3, 62032 Camerino (MC), Italy. Tel.: +39 Sciences and Experimental Medicine of the University of 0737 403310; Fax: +39 0737 630618; E-mail: [email protected] Camerino (Marche, Italy) for 41 generations, starting from the

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© The Author 2005. Published by Oxford University Press on behalf of the Medical Council on Alcohol. All rights reserved 292 M. PERFUMI et al. Sardinian alcohol-preferring (sP) rats of the 13th generation (0.2% w/v), a stimulus light (‘house light’) located on the front provided by the Department of Neurosciences of the University panel was turned on for 1.0 s (that corresponds to the duration of Cagliari (Gessa et al., 1991). They are referred to as of the syringe pump activation). Lever presses during this period Marchigian Sardinian (msP) rats. The animals were kept in a of time were counted but did not lead to further infusions. A PC room with a reverse 12 h:12 h light/dark cycle (lights off at station controlled the delivery of fluids, presentation of visual 10:00 a.m.), at a temperature of 20–22C and a humidity of stimuli and recording of the behavioural data. 45–55%. The rats were allowed free access to tap water and food pellets (4RF18, Mucedola, Settimo Milanese, Italy). Alcohol self-administration training procedures At the age of 2 months, the msP rats were selected for their The animals were trained to self-administer 10% (v/v) ethanol preference for 10% (v/v) ethanol solution, offering them a free in 30-min daily sessions under an FR1 schedule of choice between water and 10% ethanol 24 h a day for 15 days. reinforcement, wherein each response resulted in the delivery Water and 10% ethanol were provided in graduated drinking of 0.1 ml of fluid (Weiss et al., 1993). To facilitate the tubes equipped with metallic drinking spouts. The rats acquisition of level pressing during the first 3 days of training, selected for the following experiments had a 24-h ethanol the rats were placed on a restriction schedule, limiting the intake of ~6 g/kg with an ethanol preference [ml of ethanol water availability to 2 h/day. During this time, responses at the Downloaded from https://academic.oup.com/alcalc/article/40/4/291/127971 by guest on 29 September 2021 solution/ml of total fluids (water + 10% ethanol) ingested in lever were reinforced by the delivery of a 0.2% (w/v) 24 h 100] >80%. All the msP rats (n = 57) that were saccharin solution into the drinking receptacle under an FR1 subjected to this screening procedure reached the selection schedule, throughout the daily 30-min sessions. During all criteria and were therefore used in the experiments. Different subsequent training and testing, water was freely available in groups of msP rats were used in each experimental procedure. the home cages. After the successful acquisition of saccharin- Animal testing was carried out according to the European reinforced responding, the rats were trained to self-administer Communities Council Directive of 24 November 1986 10% ethanol. From the first day, the rats began to press for (86/609/EEC). 10% ethanol and a stimulus light located on the front panel (‘house light’) was turned on for 1.0 s. Drugs Saccharin self-administration training procedures HPCO2, containing 24.33% hyperforin and 0.08% hypericin, was provided by Indena, Milan, Italy. It was emulsified in For saccharin self-administration, the training procedure was 0.1% Tween 80 and tap water, just before administration, and identical to that described for alcohol self-administration, given by intragastric (IG) injection at doses of 7, 31 and except that lever pressing during the first 3 days of water 125 mg/kg. deprivation was reinforced by water delivery, and after the successful acquisition of operant responding, the animals IG administration immediately received 0.2 % (w/v) saccharin. From the first day, The rats were implanted with an indwelling IG catheter before the rats began to press for saccharin and a stimulus light located the experiments. IG administration by means of an indwelling on the front panel (‘house light’) was turned on for 1.0 s. catheter was adopted to avoid any disturbance to the animal during or just before the experiments. Effect of HPCO2 on ethanol self-administration The rats were anaesthetized by an intramuscular injection of During the last 4 days of self-administration training (pre- Zoletil 100 (Virbac, Milan, Italy), which contains Tiletamine treatment), the animals were given an IG administration of the and Zolazepam. A polyethylene (PE) catheter (PE-50, Clay drug vehicle in order to familiarize the animals with the Adams) was permanently implanted into the stomach, injection procedure. After the acquisition of a stable 10% according to the method of Lukas and Moreton (1979). The ethanol self-administration baseline (9 days), eight msP rats

PE tubing ran subcutaneously to reach the skin between the received HPCO2 (7, 31 or 125 mg/kg/10 ml) or vehicle in four scapulae, where it was exteriorized. The rats were then different experimental sessions (at intervals of 3 days), allowed a week to recover from the IG surgery. Before the according to a within-subject design. The IG injection took experiments, they were familiarized with the administration place 1 h before the 10% ethanol self-administration. procedure. The number of responses at both active and inactive levers was recorded for the entire period of the experiment. Between The self-administration apparatus the drug testing session, ethanol self-administration baseline The self-administration stations consisted of operant was always re-established. conditioning chambers (Med Associate, Inc.) enclosed in sound-attenuating, ventilated environmental cubicles. Each Effect of HPCO2 on saccharin self-administration chamber was equipped with a drinking reservoir (volume To control for the behavioural selectivity of the effect of capacity: 0.2 ml), positioned 4 cm above the grid floor in the HPCO2 on ethanol-related responding, this drug was tested on centre of the front panel of the chamber, and two retractable the self-administration of a different reward. For this purpose levers located 3 cm, one to the right and the other to the left of saccharin was adopted because the sweet taste represents, for the drinking receptacle. An infusion pump was activated by msP rats, a reinforcer comparable to ethanol. responses on the right or ‘active’, lever, while responses on Thus, the msP rats (n = 8) were trained to respond for 0.2% the left, or ‘inactive’ lever were recorded but did not result in saccharin until a stable self-administration baseline (8 days) activation of the pump. Activation of the pump resulted in the was reached. As in the previous experiment during the last delivery of 0.1 ml of fluid (either ethanol, saccharin or water). 4 days of training (pre-treatment), before the self-administration During the infusion of ethanol (10% v/v) or saccharin sessions, the animals were given an IG administration of the HYPERICUM PERFORATUM AND MOTIVATION FOR ALCOHOL 293 vehicle to familiarize them with the injection procedure. A Subsequently, the rats received HPCO2 (7, 31 or 80 VEH 125 mg/kg/10 ml) or vehicle in four different experimental HPCO2 7 mg/kg sessions (at intervals of 3 days), according to a within-subject 70 HPCO2 31 mg/kg design. The IG injection took place 1 h before the saccharin HPCO2 125 mg/kg self-administration. Between the drug-testing session, 60 saccharin self-administration baseline was always re- min) 50

established. The number of responses at both the active and Number of

inactive lever was recorded for the entire period of the ** experiment. es (30 40

± SEM) 30 Effect of HPCO2 on voluntary ethanol intake after a period of ethanol deprivation Respons 20

Mean ( For 35 consecutive days (pre-deprivation period), the rats were ** Downloaded from https://academic.oup.com/alcalc/article/40/4/291/127971 by guest on 29 September 2021 allowed free home-cage access to 10% ethanol and tap water. 10 Ethanol and water intakes were recorded daily at 10:00 a.m., just before the beginning of the dark phase of the inverse 0 light/dark cycle. The position (to the right or left) of the ethanol Treatment and the water drinking tubes was changed daily to avoid the B development of preference for a particular side (right or left). 140 After 35 days, the rats were divided into two groups of similar ethanol intake. A group of 33 animals was deprived of 120 ethanol, but not of water, for nine consecutive days. The other group of eight msP rats had free access to both water and 100 ethanol. After 9 days of ethanol deprivation, the 33 deprived min)

Number of rats were divided into four subgroups that received HPCO2 (7, 80 31 or 125 mg/ kg 10 ml) or vehicle, according to a between- es (30 subject design. The group of non-deprived msP rats received

± SEM) 60 only the vehicle under the same experimental condition.

Respons The IG administration took place at 9:00 a.m., that is, 1 h 40 before the beginning of the dark phase. Ethanol, water and Mean ( food intakes were measured 1, 2 and 24 h after ethanol 20 representation. Statistical analysis 0 Treatment Data are reported as mean ± SEM. To evaluate the effect of HPCO on ethanol and saccharin self-administration, the 2 Fig. 1. Effect of IG HPCO2 (7, 31 and 125 mg/kg) or its vehicle (VEH) results were analysed by a one-way analysis of variance on: (A) 10% alcohol self-administration in msP rats (n = 8); (B) 0.2% (ANOVA) with repeated measures (Latin Square design). To saccharin self-administration in msP rats (n = 8). Values represent the evaluate the effect of HPCO2 on ethanol intake after the mean ± SEM of the number of lever presses for ethanol. Difference deprivation, a split-plot ANOVA, with between-group from vehicles: **P < 0.01; where not indicated, the difference was not comparisons for drug treatment and within-group statistically significant. comparisons for time was used. Post hoc comparisons were made by means of the Dunnett’s test. Statistical significance influenced by treatment with HPCO2 [F(3,7) = 0.368; NS]. On was set at P < 0.05. the first day after the end of treatment, lever pressing for ethanol returned to baseline levels (data not shown). A closer analysis of the pattern of lever pressing showed RESULTS that, in the control group, ethanol reinforced responses mostly occurred (~80%) during the first 10-min session bin. During Effect of HPCO2 on ethanol self-administration the remaining 20 min, only a few scattered responses were During the training period, the rats reached a baseline number detected. Latency of response to the lever was very short and of lever presses for ethanol between 40 and 75 in the 30-min in all vehicle-treated animals, with the first lever pressing sessions. As shown in Fig. 1A, HPCO2 reduced ethanol self- occurring within 10 s from the beginning of the session. In the administration in a dose-dependent manner. The ANOVA animals treated with HPCO2, a delay was observed in the revealed a statistically significant treatment effect first lever pressing, which, in the rats administered with

[F(3,7) = 58.843; P < 0.01]. The dose of 7 mg/kg of HPCO2 the highest dose of the drug, occurred within a few minutes did not significantly modify ethanol self-administration, while from the beginning of the session. In addition, in the animals the doses of 31 and 125 mg/kg reduced the number of lever treated with 31 and 125 mg/kg of HPCO2, lever pressing was presses for ethanol by 37.2 (P < 0.01) and 81.8% (P < 0.01), not clustered during the first 10-min bin but, rather, it respectively. The responses at the inactive lever were almost was homogeneously scattered throughout the entire 30-min absent during the training and treatment period, and were not session. 294 M. PERFUMI, et al. Effect of HPCO on saccharin self-administration 2 A As shown in Fig. 1B, IG administration of HPCO2 (7, 31 or PRE-DEPRIVATION POST-DEPRIVATION 8

125 mg/kg) did not significantly modify the number of + * responses for saccharin self-administration [F(3,7) = 0.572; 7 NON-DEPRIVED NS]. Responses at the inactive lever were almost absent during DEPRIVED ) the whole experiment and were never influenced by HPCO2 6 treatment [F(3,7) = 0.572; NS]. 5 Effect of HPCO2 on voluntary ethanol intake after a period of ethanol deprivation 4 As shown in Fig. 2A, ethanol deprivation for 9 days induced a 3

statistically significant increase in rats with voluntary ethanol (g/kg Intake Ethanol intakes at 1, 2 or 24 h after ethanol representation compared 2 *+ with rats that were not ethanol deprived [F(1,14) = 10.909; Downloaded from https://academic.oup.com/alcalc/article/40/4/291/127971 by guest on 29 September 2021

** P < 0.01]. Moreover, the ANOVA also revealed a statistically 1 ++ significant increase when the post-deprivation intake was 0 compared with that of the same animals in the pre-deprivation 1 2 24 1224 h period [F(1,14) = 10.648; P < 0.01]. On the other hand, no significant differences in food intake were observed between B the pre- and post-deprivation period [F(1,14) = 0.681; NS] POST-DEPRIVATION (data not shown). 8

As shown in Fig. 2B, IG treatment with HPCO2 significantly reduced the increased ethanol intake following a period of 7 VEH HPCO 7 mg/kg ethanol deprivation [F(3,29) = 13.628; P < 0.01]. Post hoc 2 6 HPCO 31 mg/kg comparisons revealed that the administration of 31 and 2 HPCO2 125 mg/kg ** 125 mg/kg of HPCO2 significantly reduced the increased 5 ethanol intake induced by deprivation at 1, 2 and 24 h after 4 ** access to ethanol, while 7 mg/kg of HPCO2 did not show any significant modification of ethanol intake. The reduction of 3 ethanol drinking elicited by HPCO2 was no longer evident after Ethanol Intake (g/kg) Intake Ethanol 48 h. Nevertheless, no rebound of ethanol drinking was observed 2 in the animals. The concomitant water intake was not modified * by HPCO treatment [F(3,29) = 2.87; NS]. The treated rats 1 * 2 ** ingested more water than controls, but this difference never ** 0 reached statistical significance (data not shown). 1224 h Moreover, 24-h food intake of ethanol-deprived rats was not significantly affected by HPCO2 treatment [F(3,29) = 1.703; Fig. 2. (A) Voluntary 10% alcohol intake before and after the deprivation NS] (data not shown). phase, in the control group of alcohol-deprived (n = 8) and non-deprived (n = 8) msP rats after re-access to alcohol. Both groups had free access to alcohol for 35 consecutive days before the beginning of the deprivation DISCUSSION phase. Alcohol non-deprived (control) rats had continuous access to alcohol during the deprivation phase (9 days) as well. Values are mean ± Several studies have shown that HPE markedly reduces SEM of ethanol drinking. Difference between deprived and non-deprived voluntary ethanol intake in several lines of alcohol-preferring rats: *P < 0.05; **P < 0.01; difference in alcohol deprived rats before and rats (for a review see Rezvani et al., 2003) and alcohol- after deprivation: +P < 0.05; ++P < 0.01. (B) Effect on voluntary 10% preferring C57BL/6J mice (Wright et al., 2003). The effect is alcohol intake, of different doses of IG HPCO2 (7, 31 and 125 mg/kg) or behaviourally selective, since doses that reduce ethanol intake its vehicle (VEH), in the 1st, 2nd and 24th h of re-access to alcohol in do not modify the simultaneous intake of food, water or alcohol-deprived msP rats. Values are mean ± SEM of ethanol drinking. Difference from controls: *P < 0.05; **P < 0.01; where not indicated, saccharin solution (Perfumi et al., 2001, 2003). Until now, the the difference was not statistically significant. effect of the extracts has been investigated only under conditions of voluntary access to home-cage ethanol, which does not allow one to fully evaluate the motivation of the (Spanagel and Holter, 2000; McBride et al., 2002). Thus, the animal for ethanol. On the other hand, the operant self- present study evaluated whether the doses of HPCO2 that administration represents a paradigm in which the animal is reduce voluntary ethanol intake are also able to reduce ethanol required to express a deeply motivated behaviour to obtain self-administration in operant cages, as well as the increased ethanol reinforcement (Samson and Czachowski, 2003). ethanol intake following a period of ethanol deprivation. Another interesting paradigm to evaluate the motivation for The results clearly showed that, under operant conditions, ethanol is the voluntary ethanol intake following a period HPCO2 reduces ethanol intake over the same range of doses of ethanol deprivation; animals show a pronounced increase in that in previous studies have been shown to reduce home-cage ethanol intake immediately after ethanol representation, which ethanol drinking. The effect observed is behaviourally is considered to be an expression of craving for ethanol selective because, under identical experimental conditions, HYPERICUM PERFORATUM AND MOTIVATION FOR ALCOHOL 295 saccharin self-administration was not modified. Consistent Barnes, J., Anderson, L. A. and Phillipson, J. D. (2001) St John’s wort responding at the inactive lever was not affected by HPCO at (Hypericum perforatum L.): a review of its chemistry, pharmacology 2 and clinical properties. Journal of Pharmacy and Pharmacology 53, the doses used in the present study. Overall, these results are 583–600. consistent with the previous reports showing the selectivity of Bennett, D. A., Phun, L., Polk, J. F. et al. (1998) Neuropharmacology the effect of HPE on the home-cage two-bottle choice ethanol of St John’s wort (Hypericum). The Annals of Pharmacotherapy 32, drinking paradigm (Perfumi et al., 2003). 1201–1208. Results of the ADE experiment showed that the msP rats Bhattacharya, S. K., Chakrabarti, A. and Chatterjee, S. S. (1998) Active profile of two hyperforin-containing hypericum extracts in display a pronounced increase in ethanol drinking following a behavioral models. Pharmacopsychiatry 31 (Suppl. 1), 22–29. period of deprivation. This finding is consistent with the Boening, J. A. L., Lesch, O. M., Spanagel, R. et al. (2001) results obtained in other lines of alcohol-preferring animals Pharmacological relapse prevention in alcohol dependence: from (Sinclair and Li, 1989; McKinzie et al., 1998; Agabio et al., animal models to clinical trials. Alcoholism: Clinical and 2000; Rodd-Henricks et al., 2000; Serra et al., 2002, 2003; Experimental Research 25, 127S–131S. Butterweck, V., Wall, A., Lieflander-Wulf, U. et al. (1997) Effects of total Colombo et al., 2003; Vengeliene et al., 2003) and confirms extract and fractions of Hypericum perforatum in animal assays for that the msP rats represent a suitable animal model to study antidepressant activity. Pharmacopsychiatry 30 (Suppl. 2), 117–124. Catania, M. A., Firenzuoli, F., Crupi, A. et al. (2003) Hypericum Downloaded from https://academic.oup.com/alcalc/article/40/4/291/127971 by guest on 29 September 2021 alcohol craving. HPCO2 also evoked a pronounced and long lasting effect on the increased motivation for ethanol observed perforatum attenuates nicotine withdrawal signs in mice. Psychopharmacology 169, 186–189. after a period of ethanol deprivation. The doses of 31 and Chatterjee, S. S., Bhattacharya, S. K., Wonnemann, M. et al. (1998a) 125 mg/kg of HPCO2 were able to completely abolish the Hyperforin as a possible antidepressant component of Hypericum alcohol deprivation effect. Again, the effect was behaviourally extracts. Life Sciences 63, 499–510. selective since food and water intake were not affected. Chatterjee, S. S., Noldner, M., Koch, E. et al. (1998b) Antidepressant Overall, the present results, obtained under high activity of Hypericum perforatum and hyperforin: the neglected possibility. Pharmacopsychiatry 31, 7–15. motivational conditions (i.e. operant self-administration and Cervo, L., Rozio, M., Ekalle-Soppo, C. B. et al. 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