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Roles of 5-HT3 and opioid receptors in the ethanol-induced place preference in rats exposed to conditioned fear stress
Life Sciences, Vol. 64, No. 21. pp. PL 241~249,1999 CopyTight 0 1999 Ekvier science Inc. Printed in the USA. All rights reserwd
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ROLES OF S-HT3 AND OPIOID RECEPTORS IN THE ETHANOL-INDUCED PLACE PREFERENCE IN RATS EXPOSED TO CONDITIONED FEAR STRESS
Shigeki Matsuzawa”‘, Tsutomu Suzuki’, Miwa Misawa’ and Hiroshi Nagase3 ‘Department of Pharmacology, School of Pharmacy, Hoshi University, Shinagawa-ku, Tokyo 142, Japan; ‘Research Center, Kyorin Pharmaceutical Co. Ltd., Tochigi 329-01, Japan; 3Basic Research Laboratories, Toray Industries, Inc., Kamakura 248, Japan (Submitted November 10, 1998; accepted January 9, 1999; received in final form February 9, 1999)
Abstract: ‘Ihe effect of the selective 5-HT3 receptor antagonist ondansetron on the ethanol-induced place preference in rats exposed to conditioned fear stress, which stimulates the release of endogenous opioid peptides (B-endorphin and enkephalins), was investigated using the conditioned place preference paradigm. In addition, we also examined the effect of ondansetron on the ethanolinduced place preference enhanced by the administration of p- and Sopioid receptor agonists (exogenous opioids). The administration of ethanol (300 mg/kg, i.p.) induced a significant place preference in rats exposed to conditioned fear stress. pretreatment with ondansetron (0.01 and 0.1 mgkg, s.c.) effectively attenuated this ethanol-induced place preference. When the r-opioid receptor agonist morphine (0.1 mg/kg, s.c.) or the selective S-opioid rece tor agonist 2-methyl-4aa-(3hydroxyphenyl)-l,2,3,4,4a,5,12,12aa+ctahydroquinolino [2,3,3-g ‘; isoquinoline (TAN-63 20 mgkg, s.c.) was administered in combination with 75 mg/kg ethanol (which tended to produce a place preference), the ethanol-induced place preference was significantly enhanced. The selective Wopioid receptor antagonist Mimaltrexamine at a dose of 10 mg/kg significantly attenuated the enhancement of the ethanol-induced place preference produced by morphine. Ondansetron (0.1 mg/kg, s.c.) also significantly a&muted the enhancement of the ethanol-induced place preference produced by morphine. Furthermore, the selective b-opioid receptor antagonist naltrindole at a dose of 3 mglkg significantly attenuated the enhancement of the ethanol-induced place preference produced by TAN-67. Ondansetron (0.1 mg/kg, s.c.) slightly, but significantly, attenuated the enhancement of the ethanol-induced place preference produced by TAN-67. These results suggest that 5-HT3 receptors may be involved in the rewarding mechanism of ethanol under psychological stress, and may play an important role in the rewarding effect of ethanol through the activation of CLand fi-opioid receptors. 0 1999 Ekevier Sciencehc.
Key Words: ethanol, conditioned place preference paradigm, conditioned fear stress, rewarding effect, 5-HT, receptors, opioid receptors
Introduction ‘l?eF is considerable evidence that a subtype of serotonin (5-m receptors, 5-HT3 receptor, is mvolved in modulating the activity of the mesolimbic dopamine system, which plays a critical role in the rewarding mechanisms of many abused drugs including ethanol (1). Receptor binding studies have demonstrated that there arc high to moderate densities of 5X-B receptors in mesolimbic dopamine-innervated areas such as the nucleus accumbens (2-4), which nzceives serotonergic innervation from the dorsal raphe nucleus (5). These findings suggest that 5-HT3 Corresponding Author: Tsutomu Suzuki, Ph.D., Department of Pharmacology, Hoshi University, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan. Tel & Fax: 81-3-5498-5787 E-mail: [email protected]
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receptors may play an important role in the rewarding effect of ethanol. In fact, ethanol stimulates the release of %-IT in the nucleus accumbens (6) and increases dopamine levels in the nucleus accumbens through the activation of 5-HT3 receptors (7). Moreover, it has been reported that the increase in dopamine release in the nucleus accumbens induced by ethanol is attenuated by treatment with 5-HT3-receptor antagonists (6,8). Interestingly, behavioral studies have shown that 5-HT3 receptor antagonists can attenuate ethanol intake in rats (9,lO). Thus, 5-HT3 receptors may play a role in the rewarding effect of ethanol. The endogenous opioid system, which is known to modulate dopamine release in the mesolimbic dopamine system, also plays a key role in the rewarding mechanism of ethanol (11,12). It has been demonstrated that ethanol stimulates the release of Bendorphin and enkephalins (13), which activate the mesolimbic dopamine system. These observations support the hypothesis that ethanol may produce a rewarding effect through the activation of P- and/or b-opioid receptors. It is important to note that B-funaltrexamine (a selective k-opioid receptor antagonist) (14) and naltrindole (a selective B-opioid receptor antagonist) (15) decrease ethanol intake in rats. Gn the other hand, Williams and Woods (16) demonstrated that selective antagonism at the P- or h-opioid receptor did not reduce ethanol intake in rhesus monkeys. The reason for this discrepancy is not clear. However, we previously demonstrated that ethanol produces a place preference (as an index of its rewarding effect) in rats exposed to foot shock stress (17) and conditioned fear stress (18) using the conditioned place preference paradigm, and that Bfunaltrexamine and naltrindole attenuate this ethanol-induced place preference. These findings support the notion that the effect of ethanol on central dopaminergic systems may be mediated by endogenous opioid systems, and that the activation of c- and/or 8-opioid receptors may be important for the ethanol-induced increase in dopamine release. Widdowson and Holman (19) demonstrated that the ethanol-induced increase in basal dopamine release can be reversed by opioid receptor antagonists such as naloxone (a nonselective opioid receptor antagonist) and ICI 174864 (a selective h-opioid receptor antagonist), and suggested that ethanol may indirectly regulate dopamine release through the release of endogenous opioid receptor ligands such as enkephalins. Therefore, P- and Lopioid systems may play important roles in the rewarding effect of ethanol. The above findings lead to the possibility that 5-HT3 receptors may be involved in the rewarding effect of ethanol through the activation of endogenous opioid systems. To date, however, no studies have been conducted to examine this hypothesis. The first aim of the present study was to investigate the effect of the selective 5-HT3 receptor antagonist ondansetron on the ethanol-induced place preference in rats exposed to conditioned fear stress, which stimulates the release of endogenous opioid peptides (P-endorphin and enkephalins), using the conditioned place preference paradigm. The effect of ondansetron on the ethanol-induced place preference enhanced by the administration of P- and B-opioid receptor agonists (exogenous opioids) was also examined. Metbods The present study was conducted in accordance with the Guiding Principles for the Care and Use of Laboratory Animals, Hoshi University, as adopted by the Committee on Animal Research of Hoshi University, which is accredited by the Ministry of Education, Science, Sports and Culture of Japan.
Animals: Male Sprague-Dawley
rats (Tokyo Experimental Animal, Inc., Tokyo, Japan), weighing 170-220 g, were housed m groups of 4 in a temperature-controlled room (22 + 1 “C) with a 12-h light-dark cycle (light on 8:00 a.m. to 8:00 p.m.). Food and water were available aa’ libirum.
Apparatus: The test box consisted of a shuttlebox (30 x 60 x 30 cm) which was divided into two compartments of equal size. One compartment was white with a textured floor and the other was black with a smooth floor. The test box was placed under conditions of dim illumination (40 lux) and masking white noise.
Habituation to the test box: On days 1 and 2, the partition separating the two compartments was raised 12 cm above the floor, and a neutral platform was inserted along the seam separating the compartments. Non-treated rats were placed on the platform of the test box and allowed to move freely in the test box for 15 min.
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Pre-conditioning test (measurement of pre-conditioning scores): On day 3, as the habituation session, non-treated rats were placed on the platform of the test box and allowed to move freely in the test box for 15 min. The time spent in each ~rn~ent during the 15min session was measured automatically in a blind fashion by an infrared beam sensor (m-80; Natsume Seisakusho, Tokyo, Japan). Place conditioning: On days 4,6,8 and 10, the rats were individually subjected to intermittent electric foot shocks (10 min, 0.6 mA, 1 s on, 4 s off) through stainless stee1 fIoor grids by a shock generator (IT-3 O’Hara, Tokyo, Japan) in a gray shock chamber (27 x I8 x 27 cm). Twenty-four h after the foot shocks (on days 5, 7, 9 and ll), the rats were again individually placed in the same shock chamber without foot shocks for 10 min. All of the rats were then immediately injected with ethanol or saline and confined for 30 min to the non-preferred side in the preconditioning test following drug injection and to the preferred side in the pi-~nditio~ng test following saline injection on alternate days (2 for ethanol: 2 for saline). Post-conditioning test (measurement of post-conditioning scores): On day 12, as the p~~nditio~~ test session, the rats were placed on the platform of the test box and allowed to move freely in the test box for 15 min. The time spent in each compartment during a 15-min session was measured. Injection procedure: Ci~ntrol rats were injected with saline instead of ethanol in each conditioning session. Morphine (0.1 mglkg, s.c.) and TAN-67 (20 mgkg, S.C.> we= @a@j concomitantly with ethanol. Ondansetron (0.01 and 0.1 mgkg, s.c.) was injected 30 min before treatment with ethanol alone or ethanol in combination with morphine or TAN-67. BFNA (IO mg/kg, i.p.) and NTI (3 mg/kg, s.c.) were injected 24 h and 45 ruin before treatment with ethanol in ~mb~ation with morphine or TAN-67, respectively. Drugs: ?he drugs used in this study were ethanol (Wake Pure Chemical, Osaka, Japan), morphine hydrochloride (Sankyo Co., Tokyo, Japan), TAN-67 (2-methyl-4aa-(3-hydroxyphenyl)1,~,3,4,4a,S,lZ,l2aa~~yd~u~o~no [2,3,3-g] isoquinoline), ondansetron hy~~o~de (Nrsshin Flour Milling Co., Saitama, Japan), B-FNA (~-~~x~~ hy~~~o~de) and NTI (naltrmdole methanesulfonate). B-FNA, NTI and TAN-67 were synthesized by us. All of the drugs were dissolved in saline. Ethanol was used at doses of 75 and 300 mg/kg. It was diluted to form a 20 (v/v) % solution and injected intraperitoneally in different volumes. lAata analysis: Conditioning scores represent the difference in the time spent on the ethanolpaired side in the post-conditioning test minus the time spent on the non-preferred side in the preconditioning test, and are expressed as means * S.E.M. The dose-response was analyzed using a one-way analysis of variance (ANOVA). The Wilcoxon test was used to determine whether ~dividu~ doses produced significant ~nditio~ng. Results Effect of ondansetron on the ethanol-induced place preference under conditioned few stress: As shown in Fig. 1, ethanol at a dose of 300 mg/kg produced a significant place preference (P < 0.01) in rats exposed to conditioned fear stress. Ondansetron (0.01 and 0.1 mgkg; P
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Effects of NTIand ondansetron on the ethanol+TAN474nduced place preference under conditioned fear stress: The effects of pretreatment with NTI and ondansetron on the ethanol+TAN-67-induced place preference ate shown in Fig. 3. TAN-67 at a dose of 20 mg/kg (PcO.01) significantly enhanced the ethanol-induced place preference. NTI at a dose of 3 mg/kg significantly attenuated the place preference (P~0.01). Ondansetron (0.1 mg/kg) also significantly attenuated the place preference (P~0.05). lhe doses of TAN-67 and NTI used in this study induced neither significant place preference nor place aversion.
Discussion The mesolimbic dopamine system, which contains key components of the reward pathway [ventral tegmental ama (cell body) to the nucleus accumbens (nerve terminal)], plays a critical role in the rewarding effect of abused drugs (1). It has been reported that ethanol stimulates the firing rate of dopamine neurons in the ventral tegmental area and increases extracellular dopamine
fj 250 _m n x 200 .‘ (0 n 6, 150 2 u L 0 100 8 5ii
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Effect of ondansetron on the ethanol-induced place preference under conditioned fear stress. The ordinate represents preference for the drug-paired place. Each column represents the mean with S.E.M. of 8 animals. ## P c 0.01 vs. saline-treated control group. * P c 0.05 and ** P < 0.01 vs. ethanol alone-treated group. concentrations in the rat nucleus accumbens (20). In addition, it is conceivable that activation of the mesolimbic dopamine system mediated by stimulation of the endogenous opioid system may be an important mechanism underlying the rewarding effect of ethanol (11,12). In fact, ethanol is well known to stimulate the release of endogenous opioid peptides (&endorphin and enkephalins) in the mesolimbic dopamine system (13). Moreover, morphine increases ethanol intake (21-23), while naloxone (a non-selective opioid receptor antagonist) decreases ethanol intake (14,21,22,24,25). In addition, both selective p- and S-opioid receptor antagonists decrease ethanol intake (14,15). Recently, we found that naloxone, IHtmaltrexamine (a selective eopioid receptor antagonist) and naltrindole (a selective S-opioid receptor antagonist) attenuate the ethanol-induced place preference under foot shock stress (17) and conditioned fear stress (18), which am well known to stimulate the release of endogenous opioid peptides (13,26-30) and dopamine (31-36) in the mesolimbic
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Morphine Ethanol 75 mg/kg, i.p. + 0.1 Morphine 0.1 mg/kg, s.c.(mg/kg, __) B_FNA 10
B-FNA 10
ondansetron 0.1
Fig. 2 Effects of B-FNA and ondansetron on the enhancement of the ethanol-induced place preference induced by morphine under conditioned fear stress. The ordinate represents preference for the drug-paired place. Each column represents the mean with S.E.M. of 8 animals. ## P < 0.01 vs. saline-treated control group. ** P < 0.01 vs. ethanoltmorphine-treated group.
dopamine system. These findings suggest that activation of the endogenous opioid system, and especially CL-and h-opioid receptors, may be critically linked to the rewarding effect of ethanol under stress. On the other hand, it has also been demonstrated that mesolimbic dopamine neurons receive serotonergic innervation from the dorsal raphe nucleus (5). 5-HT3 receptors are located in moderate to high densities in the mesolimbic dopamine system (2-4), and the 5-HT? receptor agonist 2methyld-HT stimulates dopamine release in the nucleus accumbens (37). These findings support the possibility that 5-HT may facilitate the mesolimbic dopamine system through the activation of 5HT3 receptors. Furthermore, the firing rate of dorsal raphe nucleus neurons is enhanced by ethanol (38), and ethanol increases the concentration of 5-HT in the nucleus accumbens (6). Campbell and McBride (39) indicated that 5-HT3 receptors are involved in the ethanol-induced increase in dopamine release in the nucleus accumbens. Moreover, conditioned fear stress stimulates the release of 5-HT in the mesolimbic dopamine system including the nucleus accumbens (36). Based on these findings, the present experiment was designed to study the role of 5-HI? receptors in the rewarding effect of ethanol using the conditioned place preference paradigm combined with conditioned fear stress. The present study demonstrated that the 5-HD receptor antagonist ondansetron attenuates the ethanol-induced place preference in rats exposed to conditioned fear stress. Several behavioral studies have shown that 5-HT3 receptor antagonists suppress ethanol intake (9,10), and block the discriminative stimulus effect of ethanol (40). Jankowski and Kostowski (41) found that the 5-HT3 receptor antagonist ICS 205-930, when injected bilaterally into the nucleus accumbens, significantly reduces ethanol intake. Further, in vivo microdialysis studies have shown that 5-HT3 receptor antagonists such as ondansetron and ICS 205-930 inhibit
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Ethanol 75 mg/kg, i.p. TAN+-67 20 mg/kg, S.C. NTI 3
TAN-67 (:g,kg,
NTI s.c;
Ondansetron 0.1
Fig. 3 Effects of NTI and ondansetron on the enhancement of the ethanol-induced place preference induced by TAN-67 under conditioned fear stress. The ordinate represents preference for the drug-paired place. Each column represents the mean with S.E.M. of 8 animals. ##P c 0.01 vs. saline-treated control group. * P < 0.05 and ** P < 0.01 vs. ethanol+TAN-67-treated group. the increase in dopamine and 5-HT levels in the nucleus accumbens produced by the systemic or local administration of ethanol (8,42-44). Thus, it appears that 5-I-IT3 receptors, perhaps through an increase in dopamine release in the nucleus accumbens, may play a role in the development of the rewarding effect of ethanol. In the present study, we found that the k-opioid receptor agonist morphine and the selective S-opioid receptor agonist TAN-67 each significantly enhanced the ethanol-induced place preference. These results may be supported the previous findings: 1) under conditioned fear stress (but not non-stressed condition), ethanol produced a significant place preference at 150 and 300 mg/kg, but not 75, 600 and 1200 mg/kg (indicating a bell-shaped effect of ethanol over the dose range used), and 300 mg/kg was more efficacious than 150 mg/kg (45), 2) the development of morphmeinduced place preference was enhanced under conditioned fear stress (data not shown), suggesting that the rewarding effect of ethanol may be potentiated by conditioned fear stress through the stimulation of release of the endogenous opioid peptides such as Bendorphin and enkephalins. Furthermore, in the present study, we found that the morphine- and TAN-67-enhanced place preferences were blocked by the selective r-opioid receptor antagonist B-FNA and the selective bopioid receptor antagonist NTI, respectively. These results suggest that the place preference produced by ethanol in combination with morphine may be mediated through the activation of CIopioid receptors, while that in combination with TAN-67 may be mediated through the activation of s-opioid receptors. Based on these results, we investigated the roles of 5-HT3 receptors and endogenous opioid systems (W and S-opioid receptors) in the development of the ethanol-induced place preference. It has been demonstrated that the activation of 5-I-IT3 receptors (6,7) and
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endogenous opioid systems (IL- and s-opioid receptors) (12) contributes to ethanol-induced dopamine release in the rat nucleus accumbens. The present study found that ondansetron attenuates the enhancement of the ethanol-induced place preference produced by morphine (a p-opioid receptor agonist) or TAN-67 (a selective S-opioid receptor agonist). Behavioral studies have demonstrated that the place preference induced by morphine can be blocked by 5-I-IT3 receptor antagonists such as ICS205-930 (46) and MDL 72222 (47). Moreover, ICS 205-930 (43,48) and another 5-HT3 receptor antagonist granisetron (49) also attenuate the morphine-induced increase in extracellular dopamine concentrations in the nucleus accumbens. These findings indicate that 5-HT3 receptors may contribute to the rewarding effect of morphine and to the morphine-induced dopamine release in the nucleus accumbens. Therefore, our findings suggest that the functional interaction between 5I-IT3receptors and or-or 8-opioid receptors in the mesolimbic dopamine system may participate in the rewarding effxt of ethanol. This suggestion seems to be supported by the following findings: TAN-67 enhances the morphine-induced place preference through the activation of s-opioid receptors (50), and a s-opioid receptor agonist produces a conditioned place preference which can be attenuated by a dopamine receptor antagonist (5 1). In the present study, ondansetron had a greater attenuating effect on the morphine-enhanced ethanol-induced place preference than on the TAN-67-enhanced ethanol-induced place preference; ondansetron almost completely attenuated the ethanoltmorphine-induced place preference but only partially attenuated the ethanoltTAN-67-induced place preference. Suzuki et al. (51) reported that the place preference induced by the selective 8r-opioid receptor agonist DPDPE was abolished by the selective dopamine Dl receptor antagonist SCH23390, but not by the selective dopamine D2 receptor antagonist sulpiride, and that the place preference induced by the selective 8%opioid receptor agonist DELT ([D-Ala2]deltorphin II) was not abolished by either SCH23390 or sulpiride. We also found that the ethanol-induced place preference enhanced by morphine can be attenuated by either SCH23390 or sulpiride, and that the ethanol-induced place preference enhanced by TAN-67 can be attenuated by SCH23390, but not by sulpiride (unpublished data). These findings suggest the possibility that the rewarding effect provoked by the activation of s-opioid receptors may, at least in part, involve some dopamine-independent mechanisms. Therefore, it is likely that ondansetron attenuated the place preference produced by ethanol in combination with morphine (ILopioid receptor-mediated ethanol reward) more than that in combination with TAN-67 (b-opioid receptor-mediated ethanol reward). In summary, the 5-I-D’?receptor antagonist ondansetron attenuated the ethanol-induced place preference in rats exposed to conditioned fear stress. Furthermore, ondansetron attenuated the enhancement of the ethanol-induced place preference produced by the r-opioid receptor agonist morphine and by the selective S-opioid receptor agonist TAN-67. These results suggest that 5-HT3 receptors may be involved in the rewarding effect of ethanol under psychological stress, and may participate in the rewarding effect of ethanol through the activation of CLor s-opioid receptors. Acknowledgments
This work was supported by the Ministry of Education, Science, Sports, and Culture of Japan and by the Ministry of Health and Welfare of Japan to T. Suzuki. We wish to thank Ms. Yuko Shimada for her expert technical assistance. References
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ROLES OF S-HT3 AND OPIOID RECEPTORS IN THE ETHANOL-INDUCED PLACE PREFERENCE IN RATS EXPOSED TO CONDITIONED FEAR STRESS
Shigeki Matsuzawa”‘, Tsutomu Suzuki’, Miwa Misawa’ and Hiroshi Nagase3 ‘Department of Pharmacology, School of Pharmacy, Hoshi University, Shinagawa-ku, Tokyo 142, Japan; ‘Research Center, Kyorin Pharmaceutical Co. Ltd., Tochigi 329-01, Japan; 3Basic Research Laboratories, Toray Industries, Inc., Kamakura 248, Japan (Submitted November 10, 1998; accepted January 9, 1999; received in final form February 9, 1999)
Abstract: ‘Ihe effect of the selective 5-HT3 receptor antagonist ondansetron on the ethanol-induced place preference in rats exposed to conditioned fear stress, which stimulates the release of endogenous opioid peptides (B-endorphin and enkephalins), was investigated using the conditioned place preference paradigm. In addition, we also examined the effect of ondansetron on the ethanolinduced place preference enhanced by the administration of p- and Sopioid receptor agonists (exogenous opioids). The administration of ethanol (300 mg/kg, i.p.) induced a significant place preference in rats exposed to conditioned fear stress. pretreatment with ondansetron (0.01 and 0.1 mgkg, s.c.) effectively attenuated this ethanol-induced place preference. When the r-opioid receptor agonist morphine (0.1 mg/kg, s.c.) or the selective S-opioid rece tor agonist 2-methyl-4aa-(3hydroxyphenyl)-l,2,3,4,4a,5,12,12aa+ctahydroquinolino [2,3,3-g ‘; isoquinoline (TAN-63 20 mgkg, s.c.) was administered in combination with 75 mg/kg ethanol (which tended to produce a place preference), the ethanol-induced place preference was significantly enhanced. The selective Wopioid receptor antagonist Mimaltrexamine at a dose of 10 mg/kg significantly attenuated the enhancement of the ethanol-induced place preference produced by morphine. Ondansetron (0.1 mg/kg, s.c.) also significantly a&muted the enhancement of the ethanol-induced place preference produced by morphine. Furthermore, the selective b-opioid receptor antagonist naltrindole at a dose of 3 mglkg significantly attenuated the enhancement of the ethanol-induced place preference produced by TAN-67. Ondansetron (0.1 mg/kg, s.c.) slightly, but significantly, attenuated the enhancement of the ethanol-induced place preference produced by TAN-67. These results suggest that 5-HT3 receptors may be involved in the rewarding mechanism of ethanol under psychological stress, and may play an important role in the rewarding effect of ethanol through the activation of CLand fi-opioid receptors. 0 1999 Ekevier Sciencehc.
Key Words: ethanol, conditioned place preference paradigm, conditioned fear stress, rewarding effect, 5-HT, receptors, opioid receptors
Introduction ‘l?eF is considerable evidence that a subtype of serotonin (5-m receptors, 5-HT3 receptor, is mvolved in modulating the activity of the mesolimbic dopamine system, which plays a critical role in the rewarding mechanisms of many abused drugs including ethanol (1). Receptor binding studies have demonstrated that there arc high to moderate densities of 5X-B receptors in mesolimbic dopamine-innervated areas such as the nucleus accumbens (2-4), which nzceives serotonergic innervation from the dorsal raphe nucleus (5). These findings suggest that 5-HT3 Corresponding Author: Tsutomu Suzuki, Ph.D., Department of Pharmacology, Hoshi University, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan. Tel & Fax: 81-3-5498-5787 E-mail: [email protected]
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receptors may play an important role in the rewarding effect of ethanol. In fact, ethanol stimulates the release of %-IT in the nucleus accumbens (6) and increases dopamine levels in the nucleus accumbens through the activation of 5-HT3 receptors (7). Moreover, it has been reported that the increase in dopamine release in the nucleus accumbens induced by ethanol is attenuated by treatment with 5-HT3-receptor antagonists (6,8). Interestingly, behavioral studies have shown that 5-HT3 receptor antagonists can attenuate ethanol intake in rats (9,lO). Thus, 5-HT3 receptors may play a role in the rewarding effect of ethanol. The endogenous opioid system, which is known to modulate dopamine release in the mesolimbic dopamine system, also plays a key role in the rewarding mechanism of ethanol (11,12). It has been demonstrated that ethanol stimulates the release of Bendorphin and enkephalins (13), which activate the mesolimbic dopamine system. These observations support the hypothesis that ethanol may produce a rewarding effect through the activation of P- and/or b-opioid receptors. It is important to note that B-funaltrexamine (a selective k-opioid receptor antagonist) (14) and naltrindole (a selective B-opioid receptor antagonist) (15) decrease ethanol intake in rats. Gn the other hand, Williams and Woods (16) demonstrated that selective antagonism at the P- or h-opioid receptor did not reduce ethanol intake in rhesus monkeys. The reason for this discrepancy is not clear. However, we previously demonstrated that ethanol produces a place preference (as an index of its rewarding effect) in rats exposed to foot shock stress (17) and conditioned fear stress (18) using the conditioned place preference paradigm, and that Bfunaltrexamine and naltrindole attenuate this ethanol-induced place preference. These findings support the notion that the effect of ethanol on central dopaminergic systems may be mediated by endogenous opioid systems, and that the activation of c- and/or 8-opioid receptors may be important for the ethanol-induced increase in dopamine release. Widdowson and Holman (19) demonstrated that the ethanol-induced increase in basal dopamine release can be reversed by opioid receptor antagonists such as naloxone (a nonselective opioid receptor antagonist) and ICI 174864 (a selective h-opioid receptor antagonist), and suggested that ethanol may indirectly regulate dopamine release through the release of endogenous opioid receptor ligands such as enkephalins. Therefore, P- and Lopioid systems may play important roles in the rewarding effect of ethanol. The above findings lead to the possibility that 5-HT3 receptors may be involved in the rewarding effect of ethanol through the activation of endogenous opioid systems. To date, however, no studies have been conducted to examine this hypothesis. The first aim of the present study was to investigate the effect of the selective 5-HT3 receptor antagonist ondansetron on the ethanol-induced place preference in rats exposed to conditioned fear stress, which stimulates the release of endogenous opioid peptides (P-endorphin and enkephalins), using the conditioned place preference paradigm. The effect of ondansetron on the ethanol-induced place preference enhanced by the administration of P- and B-opioid receptor agonists (exogenous opioids) was also examined. Metbods The present study was conducted in accordance with the Guiding Principles for the Care and Use of Laboratory Animals, Hoshi University, as adopted by the Committee on Animal Research of Hoshi University, which is accredited by the Ministry of Education, Science, Sports and Culture of Japan.
Animals: Male Sprague-Dawley
rats (Tokyo Experimental Animal, Inc., Tokyo, Japan), weighing 170-220 g, were housed m groups of 4 in a temperature-controlled room (22 + 1 “C) with a 12-h light-dark cycle (light on 8:00 a.m. to 8:00 p.m.). Food and water were available aa’ libirum.
Apparatus: The test box consisted of a shuttlebox (30 x 60 x 30 cm) which was divided into two compartments of equal size. One compartment was white with a textured floor and the other was black with a smooth floor. The test box was placed under conditions of dim illumination (40 lux) and masking white noise.
Habituation to the test box: On days 1 and 2, the partition separating the two compartments was raised 12 cm above the floor, and a neutral platform was inserted along the seam separating the compartments. Non-treated rats were placed on the platform of the test box and allowed to move freely in the test box for 15 min.
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Pre-conditioning test (measurement of pre-conditioning scores): On day 3, as the habituation session, non-treated rats were placed on the platform of the test box and allowed to move freely in the test box for 15 min. The time spent in each ~rn~ent during the 15min session was measured automatically in a blind fashion by an infrared beam sensor (m-80; Natsume Seisakusho, Tokyo, Japan). Place conditioning: On days 4,6,8 and 10, the rats were individually subjected to intermittent electric foot shocks (10 min, 0.6 mA, 1 s on, 4 s off) through stainless stee1 fIoor grids by a shock generator (IT-3 O’Hara, Tokyo, Japan) in a gray shock chamber (27 x I8 x 27 cm). Twenty-four h after the foot shocks (on days 5, 7, 9 and ll), the rats were again individually placed in the same shock chamber without foot shocks for 10 min. All of the rats were then immediately injected with ethanol or saline and confined for 30 min to the non-preferred side in the preconditioning test following drug injection and to the preferred side in the pi-~nditio~ng test following saline injection on alternate days (2 for ethanol: 2 for saline). Post-conditioning test (measurement of post-conditioning scores): On day 12, as the p~~nditio~~ test session, the rats were placed on the platform of the test box and allowed to move freely in the test box for 15 min. The time spent in each compartment during a 15-min session was measured. Injection procedure: Ci~ntrol rats were injected with saline instead of ethanol in each conditioning session. Morphine (0.1 mglkg, s.c.) and TAN-67 (20 mgkg, S.C.> we= @a@j concomitantly with ethanol. Ondansetron (0.01 and 0.1 mgkg, s.c.) was injected 30 min before treatment with ethanol alone or ethanol in combination with morphine or TAN-67. BFNA (IO mg/kg, i.p.) and NTI (3 mg/kg, s.c.) were injected 24 h and 45 ruin before treatment with ethanol in ~mb~ation with morphine or TAN-67, respectively. Drugs: ?he drugs used in this study were ethanol (Wake Pure Chemical, Osaka, Japan), morphine hydrochloride (Sankyo Co., Tokyo, Japan), TAN-67 (2-methyl-4aa-(3-hydroxyphenyl)1,~,3,4,4a,S,lZ,l2aa~~yd~u~o~no [2,3,3-g] isoquinoline), ondansetron hy~~o~de (Nrsshin Flour Milling Co., Saitama, Japan), B-FNA (~-~~x~~ hy~~~o~de) and NTI (naltrmdole methanesulfonate). B-FNA, NTI and TAN-67 were synthesized by us. All of the drugs were dissolved in saline. Ethanol was used at doses of 75 and 300 mg/kg. It was diluted to form a 20 (v/v) % solution and injected intraperitoneally in different volumes. lAata analysis: Conditioning scores represent the difference in the time spent on the ethanolpaired side in the post-conditioning test minus the time spent on the non-preferred side in the preconditioning test, and are expressed as means * S.E.M. The dose-response was analyzed using a one-way analysis of variance (ANOVA). The Wilcoxon test was used to determine whether ~dividu~ doses produced significant ~nditio~ng. Results Effect of ondansetron on the ethanol-induced place preference under conditioned few stress: As shown in Fig. 1, ethanol at a dose of 300 mg/kg produced a significant place preference (P < 0.01) in rats exposed to conditioned fear stress. Ondansetron (0.01 and 0.1 mgkg; P
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Effects of NTIand ondansetron on the ethanol+TAN474nduced place preference under conditioned fear stress: The effects of pretreatment with NTI and ondansetron on the ethanol+TAN-67-induced place preference ate shown in Fig. 3. TAN-67 at a dose of 20 mg/kg (PcO.01) significantly enhanced the ethanol-induced place preference. NTI at a dose of 3 mg/kg significantly attenuated the place preference (P~0.01). Ondansetron (0.1 mg/kg) also significantly attenuated the place preference (P~0.05). lhe doses of TAN-67 and NTI used in this study induced neither significant place preference nor place aversion.
Discussion The mesolimbic dopamine system, which contains key components of the reward pathway [ventral tegmental ama (cell body) to the nucleus accumbens (nerve terminal)], plays a critical role in the rewarding effect of abused drugs (1). It has been reported that ethanol stimulates the firing rate of dopamine neurons in the ventral tegmental area and increases extracellular dopamine
fj 250 _m n x 200 .‘ (0 n 6, 150 2 u L 0 100 8 5ii
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0 Saline
Ethanol 300 mg/kg, i.p. 0.1
0.01
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Ondansetron (mg/kg,
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0.1 s.c.)
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Effect of ondansetron on the ethanol-induced place preference under conditioned fear stress. The ordinate represents preference for the drug-paired place. Each column represents the mean with S.E.M. of 8 animals. ## P c 0.01 vs. saline-treated control group. * P c 0.05 and ** P < 0.01 vs. ethanol alone-treated group. concentrations in the rat nucleus accumbens (20). In addition, it is conceivable that activation of the mesolimbic dopamine system mediated by stimulation of the endogenous opioid system may be an important mechanism underlying the rewarding effect of ethanol (11,12). In fact, ethanol is well known to stimulate the release of endogenous opioid peptides (&endorphin and enkephalins) in the mesolimbic dopamine system (13). Moreover, morphine increases ethanol intake (21-23), while naloxone (a non-selective opioid receptor antagonist) decreases ethanol intake (14,21,22,24,25). In addition, both selective p- and S-opioid receptor antagonists decrease ethanol intake (14,15). Recently, we found that naloxone, IHtmaltrexamine (a selective eopioid receptor antagonist) and naltrindole (a selective S-opioid receptor antagonist) attenuate the ethanol-induced place preference under foot shock stress (17) and conditioned fear stress (18), which am well known to stimulate the release of endogenous opioid peptides (13,26-30) and dopamine (31-36) in the mesolimbic
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Morphine Ethanol 75 mg/kg, i.p. + 0.1 Morphine 0.1 mg/kg, s.c.(mg/kg, __) B_FNA 10
B-FNA 10
ondansetron 0.1
Fig. 2 Effects of B-FNA and ondansetron on the enhancement of the ethanol-induced place preference induced by morphine under conditioned fear stress. The ordinate represents preference for the drug-paired place. Each column represents the mean with S.E.M. of 8 animals. ## P < 0.01 vs. saline-treated control group. ** P < 0.01 vs. ethanoltmorphine-treated group.
dopamine system. These findings suggest that activation of the endogenous opioid system, and especially CL-and h-opioid receptors, may be critically linked to the rewarding effect of ethanol under stress. On the other hand, it has also been demonstrated that mesolimbic dopamine neurons receive serotonergic innervation from the dorsal raphe nucleus (5). 5-HT3 receptors are located in moderate to high densities in the mesolimbic dopamine system (2-4), and the 5-HT? receptor agonist 2methyld-HT stimulates dopamine release in the nucleus accumbens (37). These findings support the possibility that 5-HT may facilitate the mesolimbic dopamine system through the activation of 5HT3 receptors. Furthermore, the firing rate of dorsal raphe nucleus neurons is enhanced by ethanol (38), and ethanol increases the concentration of 5-HT in the nucleus accumbens (6). Campbell and McBride (39) indicated that 5-HT3 receptors are involved in the ethanol-induced increase in dopamine release in the nucleus accumbens. Moreover, conditioned fear stress stimulates the release of 5-HT in the mesolimbic dopamine system including the nucleus accumbens (36). Based on these findings, the present experiment was designed to study the role of 5-HI? receptors in the rewarding effect of ethanol using the conditioned place preference paradigm combined with conditioned fear stress. The present study demonstrated that the 5-HD receptor antagonist ondansetron attenuates the ethanol-induced place preference in rats exposed to conditioned fear stress. Several behavioral studies have shown that 5-HT3 receptor antagonists suppress ethanol intake (9,10), and block the discriminative stimulus effect of ethanol (40). Jankowski and Kostowski (41) found that the 5-HT3 receptor antagonist ICS 205-930, when injected bilaterally into the nucleus accumbens, significantly reduces ethanol intake. Further, in vivo microdialysis studies have shown that 5-HT3 receptor antagonists such as ondansetron and ICS 205-930 inhibit
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Ethanol 75 mg/kg, i.p. TAN+-67 20 mg/kg, S.C. NTI 3
TAN-67 (:g,kg,
NTI s.c;
Ondansetron 0.1
Fig. 3 Effects of NTI and ondansetron on the enhancement of the ethanol-induced place preference induced by TAN-67 under conditioned fear stress. The ordinate represents preference for the drug-paired place. Each column represents the mean with S.E.M. of 8 animals. ##P c 0.01 vs. saline-treated control group. * P < 0.05 and ** P < 0.01 vs. ethanol+TAN-67-treated group. the increase in dopamine and 5-HT levels in the nucleus accumbens produced by the systemic or local administration of ethanol (8,42-44). Thus, it appears that 5-I-IT3 receptors, perhaps through an increase in dopamine release in the nucleus accumbens, may play a role in the development of the rewarding effect of ethanol. In the present study, we found that the k-opioid receptor agonist morphine and the selective S-opioid receptor agonist TAN-67 each significantly enhanced the ethanol-induced place preference. These results may be supported the previous findings: 1) under conditioned fear stress (but not non-stressed condition), ethanol produced a significant place preference at 150 and 300 mg/kg, but not 75, 600 and 1200 mg/kg (indicating a bell-shaped effect of ethanol over the dose range used), and 300 mg/kg was more efficacious than 150 mg/kg (45), 2) the development of morphmeinduced place preference was enhanced under conditioned fear stress (data not shown), suggesting that the rewarding effect of ethanol may be potentiated by conditioned fear stress through the stimulation of release of the endogenous opioid peptides such as Bendorphin and enkephalins. Furthermore, in the present study, we found that the morphine- and TAN-67-enhanced place preferences were blocked by the selective r-opioid receptor antagonist B-FNA and the selective bopioid receptor antagonist NTI, respectively. These results suggest that the place preference produced by ethanol in combination with morphine may be mediated through the activation of CIopioid receptors, while that in combination with TAN-67 may be mediated through the activation of s-opioid receptors. Based on these results, we investigated the roles of 5-HT3 receptors and endogenous opioid systems (W and S-opioid receptors) in the development of the ethanol-induced place preference. It has been demonstrated that the activation of 5-I-IT3 receptors (6,7) and
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endogenous opioid systems (IL- and s-opioid receptors) (12) contributes to ethanol-induced dopamine release in the rat nucleus accumbens. The present study found that ondansetron attenuates the enhancement of the ethanol-induced place preference produced by morphine (a p-opioid receptor agonist) or TAN-67 (a selective S-opioid receptor agonist). Behavioral studies have demonstrated that the place preference induced by morphine can be blocked by 5-I-IT3 receptor antagonists such as ICS205-930 (46) and MDL 72222 (47). Moreover, ICS 205-930 (43,48) and another 5-HT3 receptor antagonist granisetron (49) also attenuate the morphine-induced increase in extracellular dopamine concentrations in the nucleus accumbens. These findings indicate that 5-HT3 receptors may contribute to the rewarding effect of morphine and to the morphine-induced dopamine release in the nucleus accumbens. Therefore, our findings suggest that the functional interaction between 5I-IT3receptors and or-or 8-opioid receptors in the mesolimbic dopamine system may participate in the rewarding effxt of ethanol. This suggestion seems to be supported by the following findings: TAN-67 enhances the morphine-induced place preference through the activation of s-opioid receptors (50), and a s-opioid receptor agonist produces a conditioned place preference which can be attenuated by a dopamine receptor antagonist (5 1). In the present study, ondansetron had a greater attenuating effect on the morphine-enhanced ethanol-induced place preference than on the TAN-67-enhanced ethanol-induced place preference; ondansetron almost completely attenuated the ethanoltmorphine-induced place preference but only partially attenuated the ethanoltTAN-67-induced place preference. Suzuki et al. (51) reported that the place preference induced by the selective 8r-opioid receptor agonist DPDPE was abolished by the selective dopamine Dl receptor antagonist SCH23390, but not by the selective dopamine D2 receptor antagonist sulpiride, and that the place preference induced by the selective 8%opioid receptor agonist DELT ([D-Ala2]deltorphin II) was not abolished by either SCH23390 or sulpiride. We also found that the ethanol-induced place preference enhanced by morphine can be attenuated by either SCH23390 or sulpiride, and that the ethanol-induced place preference enhanced by TAN-67 can be attenuated by SCH23390, but not by sulpiride (unpublished data). These findings suggest the possibility that the rewarding effect provoked by the activation of s-opioid receptors may, at least in part, involve some dopamine-independent mechanisms. Therefore, it is likely that ondansetron attenuated the place preference produced by ethanol in combination with morphine (ILopioid receptor-mediated ethanol reward) more than that in combination with TAN-67 (b-opioid receptor-mediated ethanol reward). In summary, the 5-I-D’?receptor antagonist ondansetron attenuated the ethanol-induced place preference in rats exposed to conditioned fear stress. Furthermore, ondansetron attenuated the enhancement of the ethanol-induced place preference produced by the r-opioid receptor agonist morphine and by the selective S-opioid receptor agonist TAN-67. These results suggest that 5-HT3 receptors may be involved in the rewarding effect of ethanol under psychological stress, and may participate in the rewarding effect of ethanol through the activation of CLor s-opioid receptors. Acknowledgments
This work was supported by the Ministry of Education, Science, Sports, and Culture of Japan and by the Ministry of Health and Welfare of Japan to T. Suzuki. We wish to thank Ms. Yuko Shimada for her expert technical assistance. References
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