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Long-lasting suppression of alcohol preference in rats following serotonin receptor blockade by ritanserin
Brurn Rcwirc,i~ Bullefm, Vol. 2X. pp. 493-496. Printed in the USA.
1992 Copyright
+
0361.9230/1)2 $5.00t .OO 1492 Pergamon Press Ltd.
RAPID COMMUNICATION
Long-Lasting Suppression of Alcohol Preference in Rats Following Serotoni~ Receptor Blockade by Ritanserin IZABELA
PANOCKA
AND MAURI210
MASSI”’
~epart~~ent of Behavioral P~~sju~ug~, institute of Genetics and A?~imai Breeding Polish Academy of Sciences, Jastszebiec, 05-551 Mrokow, Poland “Institute of Pharmacology, Universit’v of Camerino, 62032 Camerino. ftuly Received 7 June 1991 PANOCKA. I. AND M. MASSI. Long-lasting suppwssion ~~f‘nk~~ho~ prt$wntv in tim ./i)/kwing .seroionin rwplor hhckadt~ hj’ rmn.wrin. BRAIN RES BULL 28(3) 493-499, 199?.-Rats with developed preference for 3 % ethanol were injected subcutaneously (SC) with 10 mgikg of the 5HTZ antagonist ritanserin for 9 days. This resulted in a marked and significant suppression of alcohol preference, as compared to controls. The effect was very hong-las~ng, as shown by the fact that it was still evident up to 20 days after the end of the treatment. Since ritanserin shows some affinity also for D,-dopaminergic receptors (even though much lower than for 5HT, receptors). for comparison, other rats were injected SC for 9 days with 0.0625 mgkg of haloperidol or with its vehicle. The effect of haloperidol treatment was low and short-lasting. Depletion of endogenous serotonin by p-chlorophenylalanine (600 mg/kg x 3 days) completely abolished the suppression of alcohol preference by ritanserin. These results suggest that: I) the ritanserin-induced reduction of alcohol preference is not due to dopaminergic blockade, 2) that the effect of ritanserin is completely dependent on the endo~enous serotoninergic mechanisms. Ritanserm
Haloperidol
Alcohol preference -
p-Chlorophenylalanine
METHOD
A large body of evidence indicates that serotonin is involved in alcohol preference and in the maintainence of volitional alcohol intake (8. 17, 20). Serotonin uptake inhibitors, such as zimeldine and fluoxetine, which increase 5-HT concentration at ~stsyna~t~c level, as well as 5-W’ agonists are known to inhibit alcohol intake in rats and in humans (1, 5, 14, 19.
Animals Male Wistat rats (Charles River, Calco Co., Italy) weighing 300~0~ g were employed. They were kept in individu~ cages on a 12: 12 h light-dark cycle and fed ad lib (diet No. 4RF18, Mucedola, Settimo Milanese, Italy).
21. 23, 24).
However, it was recently shown that reduction of 3% alcohol intake in rats and decrease of alcohol intake in humans can also be achieved by the 5-HT receptor antagonist ritanserin (l&18). The present study was aimed to further evaluate the effect of ritanserin on alcohol intake in the rat, and to verify whether its effect is just dependent on its 5-HT blocking activity or on other aspects of the pharmacological profile of the molecule. Although ritanserin is highly selective for S-HT, and 5-HT,, receptors, it binds with lower affinity also to Hi-histamine and D,-dopamine receptors (10,12). Particular interest on the affinity of ritanserin for D2 receptors comes from the notion that the dopaminergic system is involved in the rewarding aspects of alcohol intake (3, 4, 9). ‘Requests for reprints Camerino (MC), Italy.
should be addressed
to Dr. Maurizio
Massi.
D?%@ I) Ritanserin was a generous gift of Janssen Pharmaceutics (Beerse, Belgium); 2) haloperidol (Haldol, Janssen Farmaceutici, Rome, Italy) and 3) p-chlorophenylalanine (PCPA; SIGMA, Milan, Italy) were purchased from commercial sources. Drug Administrati~?~ Ritanserin was dissolved at the concentration of 10 mg/ml in a vehicle containing 20% propylene glycol, 0.5% lactic acid and NaOH 2 N to adjust the pH to 5. Institute
493
of Pharmacology,
University
of Camerino,
Via Scalzino
5, 62032
494
PAhW’KA
w x z k
AND
MASS1
20-
o-,
1
I
,
1
I,
,
12345678
9 days
E
1
2
3
4
5
6
7
8
9
IO
days
FIG. 3. Alcohol preference in rats pretreated with FCPA (B and C) or with PCPA vehicle (A) and then treated with ritanserin (B) or its vehicle (A and C). Values are meanskS.E.M. of 5 rats (B) and 6 rats (A and C). Difference from controls as in Fig. I.
were given free choice between 3% alcohol and water (6,16).
011 I
Experimental Procedure I
I
I
I
3
5
I
1
I
7
I
I
9
1
,
1
11
13
I
1
15
1
1
I
17
I
I
19
days
FIG. 1. Alcohol preference in rats during (Panel A) and after (Panel B) treatment with ritanserin (BIT) or with its vehicle (CO). Values are means+ S.E.M. of 5 subjects. Statistical difference from controls: ‘p
The haloperidol solution purchased was diluted with distilled water to the concentration of 0.0625 mg/ml and the pH was adjusted to 5 with NaOH 2 N. Both drugs were administered subcutaneously (SC) in a volume of 1 ml/kg of body weight (b.wt.), once a day. PCPA was administered intraperitoneally (IP) as a fine suspension, obtained by dissolving the drug in NaOH 0.5 N and then neutralizing the solution with HCl 0.5 N. The same volume of NaOH 0.5 N was employed for the preparation of the control vehicle, at pH = 7. Induction of Ethanol Preference Animals were forced to drink 3% ethanol for a week. The following week they had access just to water and after that they
HAL
Experiment 1: Effect of subchronic ritanserin treatment on alcohol preference. Ten rats with developed preference for 3% alcohol were divided in 2 groups of 5 rats, receiving daily SC injection of ritanserin, or of its vehicle. Daily SC injections were given at 7:OO p.m. Both treatments started on the first day of free choice between alcohol and water and lasted for 9 days. Immediately after completion of ritanserin treatment, alcohol and water intake of the same rats were recorded during the 20day follow-up period, in which they received no treatment. Experiment 2: Effect of subchronic haioperidol treatment on alcohol preference. As in Experiment 1, animals were treated for 9 days either with 0.0625 mgkg of haloperidol (6 rats) or with vehicle (6 rats). This dose of haloperidol was chosen since it has been shown to markedly reduce bar pressing for alcohol (22), as well as the 24-h alcohol intake of geneticahy selected alcohol preferring rats (Panocka, unpublished data), while being low enough to avoid the problem of daily water intake reduction by the drug (13). Experiment 3: EfSect of ritanserin in PCPA-pretreated rats. In order to cause brain 5-HT depletion (2, 11, 25), PCPA. 300 mg/kg. was given IP twice a day at 1100 and 1800 h during the last three days of forced access to water. After PCPA pretreatment. rats were divided in 2 groups: one (5 rats) was treated with ritanserin 10 mg/kg and the other (6 rats) with its vehicle for 10 days. A third group of 6 rats was pretreated with the vehicle for PCPA and then injected with ritanserin vehicle for IO days. Statistical Analysis Data are reported as means~S.E.M. Statistical analysis was performed by means of split-plot analysis of variance with between-group comparisons for drug treatment and within-group comparisons for treatment day. Post hoc comparisons were carried out by means of the Dunnett’s test. Statistical significance was set at pCO.05.
0’ FIG. 2.
RESULTS I
I
I
12345678
I
1
1
t
1
I
9
days
Alcohol preference in rats treated with haloperidol (HAL) or with its vehicle (CO). Values are means kS.E.M. of 6 subjects. Difference from controls as in Fig. 1.
Experiment 1: Effect of Subchronic Ritanserin Treatment on Alcohol Preference As shown in Fig. lA, ritanserin treatment gradually reduced 3% alcohol preference. The overall analysis of variance revealed
RITANSERIN
495
AND ALCOHOL PREFERENCE
a potent treatment effect, F(l,8)=30.803. p
ofSubchronic Haloperidol Treatment on
As shown in Fig. 2, haloperidol treatment resulted in reduction of alcohol preference mainly in the first 5 days. The analysis of variance revealed significant treatment effect, F( 1.10) = 5.147, ~~0.05, as well as a marked time effect, F(8,80)= 2.806, p
Rats
As shown in Fig. 3. alcohol preference in PCPA-pretreated rats was not affected by ritanserin. The overall analysis of variance revealed the absence of significant treatment effect, F( 1.9) = 0.048. NS, and treatment-time interaction, F(9,81)=0.477, NS. Again, no significant difference was revealed between ritanserinand vehicle-treated rats in relation to total fluid and food intake (data not shown). On the other hand. PCPA pretreatment per se appeared to reduce alcohol preference, as shown in Fig. 3. The analysis of variance revealed a statistically significant PCPA effect. F(1,10)=5.459, pcO.05. Moreover, PCPA pretreatment resulted also in a significantIy larger total fluid and food intake in the first 5-6 days of treatment (data not shown).
DISCUSSION
The results of the present study clearly show that ritanserin potently suppresses 3% alcohol preference in the rat. thus confirming previous findings ( 16). Alcohol preference suppression by the drug was behaviorally selective. as shown by the fact that the dose of ritanserin used did not suppress either total fluid intake, or food intake [see also (IS)]. Moreover, our data indicate that the reduction of 3% alcohol preference by ritanserin is very long-lasting. as it was still evident even at 20 days after the end of the subchronic treatment. Even though ritanserin is known to produce a long-lasting occupation of cortical 5-HT, receptors (12). such a prolonged effect can hardly be explained on the basis of residual receptor blockade. Further studies are required to investigate whether such a long-lasting ritanserin effect might result from interference with the mechanisms involved in the maintenance of alcohol preference. As stated above, ritanserin shows also affinity for D,-dopamine receptors, even though this is about 80 times lower than that for the 5-HT, ones (12). However, the results obtained with haloperidol suggest that ritanserin reduction of alcohol preference is not due to dopaminer~ic blockade. Haloperidol. in fact, evoked a completely different effect with suppression of alcohol preference in the first 5 days of the treatment, and then gradual decrease of the effect over time apparently due to development of tolerance. Finally. endogenous 5-HT depletion by PCPA abolished the effect of ritanserin on 3% ethanol preference. thus providing clear-cut evidence in favor of the complete dependence of ritanserin effect on serotoninergic mechanisms. In other words, these findings suggest that the effect of ritanserin is not due to interference with receptors different from the serotoninergic ones. Recent studies have shown that ritanserin stimulates AI0 dopaminergic neurons in the ventral tegmental area, by blocking serotonin inhibition of their activity. which is mediated by S-HT, receptors in the prefrontal cortex (26.27). Activation of these neurons is strongly implicated in the rewarding properties of drugs of abuse including alcohol (3. 4. 7. 9. 28). thus, ritanserin effect on alcohol preference might be due to interference with the activity of dopaminergic neurons. Experiments are underway to evaluate this working hypothesis.
REFERENCES 1. Amit. Z.; Sutherland. E. A.; Gill, K.; Ogren, S. 0. Zimeldine: A review of its effects on ethanol consumption. Neurosci. Biobehav. Rev. 8:3S-54; 1984. 2. Carlton, S. M.; Steinman, J. L.; Hillman, G. R.; Willis, W. D. Differential effects of p-chlorophenylaianine on indoleamines in brainstem nuclei and spinal cord of rats. 11. Identi~cation of immu-
8. Geller, I. R.: Purdy, R.; Merritt. J. H. Alterations in ethanol pref9.
nohistochemically stained structures using computer-assisted image enhancement techniques. Brain Res. 426:310-322; 1987. 3. Di Chiara, G.; Imperato, A. Ethanol preferentially stimulates dopamine release in the nucleus accumbens of freely moving rats. Eur.
IO,
J. Pharmacol. 115:131-132: I985 G.: Imperato, A. Drugs abused by humans preferentially
12.
Il.
4. Di Chiara,
increase synaptic dopamine concentration in-the mesohmbic system of freelv moving rats. Proc. Natl. Acad. Sci. 85:5274-5277; 1988. 5. Engel, J. A.; F&Ike, C.; Hard, E.; Svensson, L. Effects of 5-HT,, receptor agonists on ethanol preference in the rat. Pharmacol. Toxicol. 67(Suppl. 1):21; 1990. 6.
Erikson. K. Factors affecting voluntary alcohol consumption in the albino rat. Ann. Zoll. Fennie. 6227-265; 1969.
7. Fadda,
F.; Mosca, E.: Colombo, G.; Gessa, G. L. Effect of spontaneous ingestion of ethanol on brain dopamine metabolism. Life Sci. 44:281-287; 1989.
13.
erence in the rat: The role of brain biogenic amines. Ann. NY Acad. Sci. 215:54-59; 1973. Gessa, G. L.; Muntoni, F.; Collu, M.; Vargiu. L.; Mereu. G. Low doses of ethanol activate dopaminergic neurons m the ventral tegmental area. Brain Res. 348:201-203; 1985. Hoyer, D. Molecular pharmacology and biology of 5-HT,, receptors. Trends Pharmacol. Sci. 9:89-94; 1988. Koe. B. K.: Weissman, A. p-Chlorophenylalanine: a specific depletor of brain serotonin. J. Pharmacol. Exp. Ther. 154:499-5 16; 1966. Leysen, J. E.; Gommeren, W.; Van Gompei, P.; Wynans. G.: Janssen. P. A. J.; Laduron, P. M. Receptor binding properties in vitro and in viva of ritanserin: A very potent and long-acting serotonin Sa antagonist. Mol. Pharmacol. 27600-61 1: 1985. Linseman, M. A. Effects of dopaminergic agents on alcohol consumption by rats in a limited access paradigm. Psychopharmacology (Berlin) loO:l95-200; 1990.
14. McBride. W. J.; Murphy, J. M.; Lumeng, L.; Li. T. K. Serotonin and ethanol preference. Recent Dev. Alcohol. 7P: 187-209; 1989. 15. Massi, M.; Marini, M. Effect of the 5-HT, antagonist ritanserin on food intake and on 5-HT-induced anorexia in the rat. Pharmacol. Biochem. Behav. 26:333-340; 1987.
I6
17
18. 19.
20.
21.
22.
Meert, T. F.; Awounters, F.; Melis, W. J. C.: Janssen P. A, J Ritanserin reduces alcohol preference and alcohol intake in rats given the choice between 3% alcohol and water. Pharmacol. (Life Sci. Adv.) 9:63-69; 1990. Melchior, C. L.; Myers, R. D. Genetic differences in ethanol drinking of the rat following injection of 6-OHDA. 5,6-DHT or 5.7-DHT into cerebral ventricles. Pharmacol. Biochem. Behav. S:6372; 1976. Monti, J. M.; Alterwain, P. Ritanserin decreases alcohol intake in chronic alcoholics. Lancet 337:16; 1991, Murphy, J. M.; Wailer, M. B.; Gatto, G. J.; McBride, W. J.; Lumeng, L.; Li, T. K. Monoamines uptake inhibitors attenuate ethanol intake in alcohol-preferring (P) rats. Alcohol 2:349-352; 1985. Myers, R. D.; Melchior, C. L. Alcohol and alcoholism: Role of serotonin. In: Essman, W. B., ed. Serotonin in health and disease. vol. 2. New York: Spectrum; 1977:373430. Naranjo, R. D.; Sellers, E. M.; Roach, C. A.; Woodley, D. V.; Sanchez-Craig, M.; Sykora, K. Zimelidine-induced variations in alcohol intake by nondepressed heavy drinkers. Clin. Phatmacol. Ther. 35:374-381; 1984. Pfeffer, A. 0.; Samson, H. H. Haloperidol and apomorphine effects on ethanol reinforcement in free-feeding rats. Pharmacol. Biothem. Behav. 29:343-350: 1988.
23
24
25.
26.
27.
28.
Rockman, 0. E.; Amit, Z.; Brown. Z. W.; Ggren, S. 0. An mvcstigation of the mechanisms of action of S-hydroxytryptamine in the suppression of alcohol intake. Neurophamracology 21:341-347; 1982. Rockman, G. E.; Amit, 2.; Carr. G.; Brown. Z W.; Ogren, S. 0 Attenuation of ethanol intake by S-hydroxytryptamine uptake blockade in laboratory rats: I. Involvement of brain 5-hydroxytryptamine in the mediation of the positive reinforcing properties of ethanol Arch. Int. Pharmacodyn. Ther. 241:245-259: 1979. Steinman. J. L.; Carlton, S. M.; Haber, B.: Willis, W. 1). Differential effects of p-chlorophenylalanine on indoleamines in brainstem nuclei and spinal cord of rats. I. Biochemical and behavioral analysis. Brain Res. 426:297-309: 1987. Svensson, T. H.; Tung, C. S.: Grenhoff, J. The 5-HT, antagonist ritanserin blocks the effect of prefrontal cortex inactivation on rat A10 dopamine neurons in vivn. Acta Physiol. Stand. 136:497498; 1989. Ugedo, L.; Grenhoff, J.: Svensson, T. H. Ritanserin. a 5.HTL receptor antagonist, activates midbrain dopamine neurons by blocking serotonergic inhibition. Psychopharmacology (Berlin) 98:45-50; 1989. Wise. A. R.; Bozarth, M. .4. Action of drugs of abuse on reward system: an update with specific attention to opiates. Pharmacol. Biochem. Behav. 17:239-243: 1982.
1992 Copyright
+
0361.9230/1)2 $5.00t .OO 1492 Pergamon Press Ltd.
RAPID COMMUNICATION
Long-Lasting Suppression of Alcohol Preference in Rats Following Serotoni~ Receptor Blockade by Ritanserin IZABELA
PANOCKA
AND MAURI210
MASSI”’
~epart~~ent of Behavioral P~~sju~ug~, institute of Genetics and A?~imai Breeding Polish Academy of Sciences, Jastszebiec, 05-551 Mrokow, Poland “Institute of Pharmacology, Universit’v of Camerino, 62032 Camerino. ftuly Received 7 June 1991 PANOCKA. I. AND M. MASSI. Long-lasting suppwssion ~~f‘nk~~ho~ prt$wntv in tim ./i)/kwing .seroionin rwplor hhckadt~ hj’ rmn.wrin. BRAIN RES BULL 28(3) 493-499, 199?.-Rats with developed preference for 3 % ethanol were injected subcutaneously (SC) with 10 mgikg of the 5HTZ antagonist ritanserin for 9 days. This resulted in a marked and significant suppression of alcohol preference, as compared to controls. The effect was very hong-las~ng, as shown by the fact that it was still evident up to 20 days after the end of the treatment. Since ritanserin shows some affinity also for D,-dopaminergic receptors (even though much lower than for 5HT, receptors). for comparison, other rats were injected SC for 9 days with 0.0625 mgkg of haloperidol or with its vehicle. The effect of haloperidol treatment was low and short-lasting. Depletion of endogenous serotonin by p-chlorophenylalanine (600 mg/kg x 3 days) completely abolished the suppression of alcohol preference by ritanserin. These results suggest that: I) the ritanserin-induced reduction of alcohol preference is not due to dopaminergic blockade, 2) that the effect of ritanserin is completely dependent on the endo~enous serotoninergic mechanisms. Ritanserm
Haloperidol
Alcohol preference -
p-Chlorophenylalanine
METHOD
A large body of evidence indicates that serotonin is involved in alcohol preference and in the maintainence of volitional alcohol intake (8. 17, 20). Serotonin uptake inhibitors, such as zimeldine and fluoxetine, which increase 5-HT concentration at ~stsyna~t~c level, as well as 5-W’ agonists are known to inhibit alcohol intake in rats and in humans (1, 5, 14, 19.
Animals Male Wistat rats (Charles River, Calco Co., Italy) weighing 300~0~ g were employed. They were kept in individu~ cages on a 12: 12 h light-dark cycle and fed ad lib (diet No. 4RF18, Mucedola, Settimo Milanese, Italy).
21. 23, 24).
However, it was recently shown that reduction of 3% alcohol intake in rats and decrease of alcohol intake in humans can also be achieved by the 5-HT receptor antagonist ritanserin (l&18). The present study was aimed to further evaluate the effect of ritanserin on alcohol intake in the rat, and to verify whether its effect is just dependent on its 5-HT blocking activity or on other aspects of the pharmacological profile of the molecule. Although ritanserin is highly selective for S-HT, and 5-HT,, receptors, it binds with lower affinity also to Hi-histamine and D,-dopamine receptors (10,12). Particular interest on the affinity of ritanserin for D2 receptors comes from the notion that the dopaminergic system is involved in the rewarding aspects of alcohol intake (3, 4, 9). ‘Requests for reprints Camerino (MC), Italy.
should be addressed
to Dr. Maurizio
Massi.
D?%@ I) Ritanserin was a generous gift of Janssen Pharmaceutics (Beerse, Belgium); 2) haloperidol (Haldol, Janssen Farmaceutici, Rome, Italy) and 3) p-chlorophenylalanine (PCPA; SIGMA, Milan, Italy) were purchased from commercial sources. Drug Administrati~?~ Ritanserin was dissolved at the concentration of 10 mg/ml in a vehicle containing 20% propylene glycol, 0.5% lactic acid and NaOH 2 N to adjust the pH to 5. Institute
493
of Pharmacology,
University
of Camerino,
Via Scalzino
5, 62032
494
PAhW’KA
w x z k
AND
MASS1
20-
o-,
1
I
,
1
I,
,
12345678
9 days
E
1
2
3
4
5
6
7
8
9
IO
days
FIG. 3. Alcohol preference in rats pretreated with FCPA (B and C) or with PCPA vehicle (A) and then treated with ritanserin (B) or its vehicle (A and C). Values are meanskS.E.M. of 5 rats (B) and 6 rats (A and C). Difference from controls as in Fig. I.
were given free choice between 3% alcohol and water (6,16).
011 I
Experimental Procedure I
I
I
I
3
5
I
1
I
7
I
I
9
1
,
1
11
13
I
1
15
1
1
I
17
I
I
19
days
FIG. 1. Alcohol preference in rats during (Panel A) and after (Panel B) treatment with ritanserin (BIT) or with its vehicle (CO). Values are means+ S.E.M. of 5 subjects. Statistical difference from controls: ‘p
The haloperidol solution purchased was diluted with distilled water to the concentration of 0.0625 mg/ml and the pH was adjusted to 5 with NaOH 2 N. Both drugs were administered subcutaneously (SC) in a volume of 1 ml/kg of body weight (b.wt.), once a day. PCPA was administered intraperitoneally (IP) as a fine suspension, obtained by dissolving the drug in NaOH 0.5 N and then neutralizing the solution with HCl 0.5 N. The same volume of NaOH 0.5 N was employed for the preparation of the control vehicle, at pH = 7. Induction of Ethanol Preference Animals were forced to drink 3% ethanol for a week. The following week they had access just to water and after that they
HAL
Experiment 1: Effect of subchronic ritanserin treatment on alcohol preference. Ten rats with developed preference for 3% alcohol were divided in 2 groups of 5 rats, receiving daily SC injection of ritanserin, or of its vehicle. Daily SC injections were given at 7:OO p.m. Both treatments started on the first day of free choice between alcohol and water and lasted for 9 days. Immediately after completion of ritanserin treatment, alcohol and water intake of the same rats were recorded during the 20day follow-up period, in which they received no treatment. Experiment 2: Effect of subchronic haioperidol treatment on alcohol preference. As in Experiment 1, animals were treated for 9 days either with 0.0625 mgkg of haloperidol (6 rats) or with vehicle (6 rats). This dose of haloperidol was chosen since it has been shown to markedly reduce bar pressing for alcohol (22), as well as the 24-h alcohol intake of geneticahy selected alcohol preferring rats (Panocka, unpublished data), while being low enough to avoid the problem of daily water intake reduction by the drug (13). Experiment 3: EfSect of ritanserin in PCPA-pretreated rats. In order to cause brain 5-HT depletion (2, 11, 25), PCPA. 300 mg/kg. was given IP twice a day at 1100 and 1800 h during the last three days of forced access to water. After PCPA pretreatment. rats were divided in 2 groups: one (5 rats) was treated with ritanserin 10 mg/kg and the other (6 rats) with its vehicle for 10 days. A third group of 6 rats was pretreated with the vehicle for PCPA and then injected with ritanserin vehicle for IO days. Statistical Analysis Data are reported as means~S.E.M. Statistical analysis was performed by means of split-plot analysis of variance with between-group comparisons for drug treatment and within-group comparisons for treatment day. Post hoc comparisons were carried out by means of the Dunnett’s test. Statistical significance was set at pCO.05.
0’ FIG. 2.
RESULTS I
I
I
12345678
I
1
1
t
1
I
9
days
Alcohol preference in rats treated with haloperidol (HAL) or with its vehicle (CO). Values are means kS.E.M. of 6 subjects. Difference from controls as in Fig. 1.
Experiment 1: Effect of Subchronic Ritanserin Treatment on Alcohol Preference As shown in Fig. lA, ritanserin treatment gradually reduced 3% alcohol preference. The overall analysis of variance revealed
RITANSERIN
495
AND ALCOHOL PREFERENCE
a potent treatment effect, F(l,8)=30.803. p
ofSubchronic Haloperidol Treatment on
As shown in Fig. 2, haloperidol treatment resulted in reduction of alcohol preference mainly in the first 5 days. The analysis of variance revealed significant treatment effect, F( 1.10) = 5.147, ~~0.05, as well as a marked time effect, F(8,80)= 2.806, p
Rats
As shown in Fig. 3. alcohol preference in PCPA-pretreated rats was not affected by ritanserin. The overall analysis of variance revealed the absence of significant treatment effect, F( 1.9) = 0.048. NS, and treatment-time interaction, F(9,81)=0.477, NS. Again, no significant difference was revealed between ritanserinand vehicle-treated rats in relation to total fluid and food intake (data not shown). On the other hand. PCPA pretreatment per se appeared to reduce alcohol preference, as shown in Fig. 3. The analysis of variance revealed a statistically significant PCPA effect. F(1,10)=5.459, pcO.05. Moreover, PCPA pretreatment resulted also in a significantIy larger total fluid and food intake in the first 5-6 days of treatment (data not shown).
DISCUSSION
The results of the present study clearly show that ritanserin potently suppresses 3% alcohol preference in the rat. thus confirming previous findings ( 16). Alcohol preference suppression by the drug was behaviorally selective. as shown by the fact that the dose of ritanserin used did not suppress either total fluid intake, or food intake [see also (IS)]. Moreover, our data indicate that the reduction of 3% alcohol preference by ritanserin is very long-lasting. as it was still evident even at 20 days after the end of the subchronic treatment. Even though ritanserin is known to produce a long-lasting occupation of cortical 5-HT, receptors (12). such a prolonged effect can hardly be explained on the basis of residual receptor blockade. Further studies are required to investigate whether such a long-lasting ritanserin effect might result from interference with the mechanisms involved in the maintenance of alcohol preference. As stated above, ritanserin shows also affinity for D,-dopamine receptors, even though this is about 80 times lower than that for the 5-HT, ones (12). However, the results obtained with haloperidol suggest that ritanserin reduction of alcohol preference is not due to dopaminer~ic blockade. Haloperidol. in fact, evoked a completely different effect with suppression of alcohol preference in the first 5 days of the treatment, and then gradual decrease of the effect over time apparently due to development of tolerance. Finally. endogenous 5-HT depletion by PCPA abolished the effect of ritanserin on 3% ethanol preference. thus providing clear-cut evidence in favor of the complete dependence of ritanserin effect on serotoninergic mechanisms. In other words, these findings suggest that the effect of ritanserin is not due to interference with receptors different from the serotoninergic ones. Recent studies have shown that ritanserin stimulates AI0 dopaminergic neurons in the ventral tegmental area, by blocking serotonin inhibition of their activity. which is mediated by S-HT, receptors in the prefrontal cortex (26.27). Activation of these neurons is strongly implicated in the rewarding properties of drugs of abuse including alcohol (3. 4. 7. 9. 28). thus, ritanserin effect on alcohol preference might be due to interference with the activity of dopaminergic neurons. Experiments are underway to evaluate this working hypothesis.
REFERENCES 1. Amit. Z.; Sutherland. E. A.; Gill, K.; Ogren, S. 0. Zimeldine: A review of its effects on ethanol consumption. Neurosci. Biobehav. Rev. 8:3S-54; 1984. 2. Carlton, S. M.; Steinman, J. L.; Hillman, G. R.; Willis, W. D. Differential effects of p-chlorophenylaianine on indoleamines in brainstem nuclei and spinal cord of rats. 11. Identi~cation of immu-
8. Geller, I. R.: Purdy, R.; Merritt. J. H. Alterations in ethanol pref9.
nohistochemically stained structures using computer-assisted image enhancement techniques. Brain Res. 426:310-322; 1987. 3. Di Chiara, G.; Imperato, A. Ethanol preferentially stimulates dopamine release in the nucleus accumbens of freely moving rats. Eur.
IO,
J. Pharmacol. 115:131-132: I985 G.: Imperato, A. Drugs abused by humans preferentially
12.
Il.
4. Di Chiara,
increase synaptic dopamine concentration in-the mesohmbic system of freelv moving rats. Proc. Natl. Acad. Sci. 85:5274-5277; 1988. 5. Engel, J. A.; F&Ike, C.; Hard, E.; Svensson, L. Effects of 5-HT,, receptor agonists on ethanol preference in the rat. Pharmacol. Toxicol. 67(Suppl. 1):21; 1990. 6.
Erikson. K. Factors affecting voluntary alcohol consumption in the albino rat. Ann. Zoll. Fennie. 6227-265; 1969.
7. Fadda,
F.; Mosca, E.: Colombo, G.; Gessa, G. L. Effect of spontaneous ingestion of ethanol on brain dopamine metabolism. Life Sci. 44:281-287; 1989.
13.
erence in the rat: The role of brain biogenic amines. Ann. NY Acad. Sci. 215:54-59; 1973. Gessa, G. L.; Muntoni, F.; Collu, M.; Vargiu. L.; Mereu. G. Low doses of ethanol activate dopaminergic neurons m the ventral tegmental area. Brain Res. 348:201-203; 1985. Hoyer, D. Molecular pharmacology and biology of 5-HT,, receptors. Trends Pharmacol. Sci. 9:89-94; 1988. Koe. B. K.: Weissman, A. p-Chlorophenylalanine: a specific depletor of brain serotonin. J. Pharmacol. Exp. Ther. 154:499-5 16; 1966. Leysen, J. E.; Gommeren, W.; Van Gompei, P.; Wynans. G.: Janssen. P. A. J.; Laduron, P. M. Receptor binding properties in vitro and in viva of ritanserin: A very potent and long-acting serotonin Sa antagonist. Mol. Pharmacol. 27600-61 1: 1985. Linseman, M. A. Effects of dopaminergic agents on alcohol consumption by rats in a limited access paradigm. Psychopharmacology (Berlin) loO:l95-200; 1990.
14. McBride. W. J.; Murphy, J. M.; Lumeng, L.; Li. T. K. Serotonin and ethanol preference. Recent Dev. Alcohol. 7P: 187-209; 1989. 15. Massi, M.; Marini, M. Effect of the 5-HT, antagonist ritanserin on food intake and on 5-HT-induced anorexia in the rat. Pharmacol. Biochem. Behav. 26:333-340; 1987.
I6
17
18. 19.
20.
21.
22.
Meert, T. F.; Awounters, F.; Melis, W. J. C.: Janssen P. A, J Ritanserin reduces alcohol preference and alcohol intake in rats given the choice between 3% alcohol and water. Pharmacol. (Life Sci. Adv.) 9:63-69; 1990. Melchior, C. L.; Myers, R. D. Genetic differences in ethanol drinking of the rat following injection of 6-OHDA. 5,6-DHT or 5.7-DHT into cerebral ventricles. Pharmacol. Biochem. Behav. S:6372; 1976. Monti, J. M.; Alterwain, P. Ritanserin decreases alcohol intake in chronic alcoholics. Lancet 337:16; 1991, Murphy, J. M.; Wailer, M. B.; Gatto, G. J.; McBride, W. J.; Lumeng, L.; Li, T. K. Monoamines uptake inhibitors attenuate ethanol intake in alcohol-preferring (P) rats. Alcohol 2:349-352; 1985. Myers, R. D.; Melchior, C. L. Alcohol and alcoholism: Role of serotonin. In: Essman, W. B., ed. Serotonin in health and disease. vol. 2. New York: Spectrum; 1977:373430. Naranjo, R. D.; Sellers, E. M.; Roach, C. A.; Woodley, D. V.; Sanchez-Craig, M.; Sykora, K. Zimelidine-induced variations in alcohol intake by nondepressed heavy drinkers. Clin. Phatmacol. Ther. 35:374-381; 1984. Pfeffer, A. 0.; Samson, H. H. Haloperidol and apomorphine effects on ethanol reinforcement in free-feeding rats. Pharmacol. Biothem. Behav. 29:343-350: 1988.
23
24
25.
26.
27.
28.
Rockman, 0. E.; Amit, Z.; Brown. Z. W.; Ggren, S. 0. An mvcstigation of the mechanisms of action of S-hydroxytryptamine in the suppression of alcohol intake. Neurophamracology 21:341-347; 1982. Rockman, G. E.; Amit, 2.; Carr. G.; Brown. Z W.; Ogren, S. 0 Attenuation of ethanol intake by S-hydroxytryptamine uptake blockade in laboratory rats: I. Involvement of brain 5-hydroxytryptamine in the mediation of the positive reinforcing properties of ethanol Arch. Int. Pharmacodyn. Ther. 241:245-259: 1979. Steinman. J. L.; Carlton, S. M.; Haber, B.: Willis, W. 1). Differential effects of p-chlorophenylalanine on indoleamines in brainstem nuclei and spinal cord of rats. I. Biochemical and behavioral analysis. Brain Res. 426:297-309: 1987. Svensson, T. H.; Tung, C. S.: Grenhoff, J. The 5-HT, antagonist ritanserin blocks the effect of prefrontal cortex inactivation on rat A10 dopamine neurons in vivn. Acta Physiol. Stand. 136:497498; 1989. Ugedo, L.; Grenhoff, J.: Svensson, T. H. Ritanserin. a 5.HTL receptor antagonist, activates midbrain dopamine neurons by blocking serotonergic inhibition. Psychopharmacology (Berlin) 98:45-50; 1989. Wise. A. R.; Bozarth, M. .4. Action of drugs of abuse on reward system: an update with specific attention to opiates. Pharmacol. Biochem. Behav. 17:239-243: 1982.