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Oral self-administration of γ-hydroxybutyric acid in the rat
eJp ELSEVIER
European Journal of Pharmacology285 (1995) 103-107
Short communication
Oral self-administration of y-hydroxybutyric acid in the rat Giancarlo Colombo a,,, Roberta Agabio b, Nadejda Balaklievskaia b, Giacomo Diaz c, Carla Lobina a, Roberta Reali b, Gian Luigi Gessa b a C.N.R. Center for Neuropharmacology, University of Cagliari, Cagliari, Italy b 'Bernard B. Brodie' Department of Neuroscience, University of Cagliari, Cagliari, Italy c Department of Cytomorphology, University of Cagliari, Cagliari, Italy
Received 27 July 1995; accepted 8 August 1995
Abstract
The present study describes the induction of y-hydroxybutyric acid (GHB) preference over water in rats. GHB solution (1% w / v in water) was initially offered as the sole fluid available for 14 consecutive days. Subsequently, rats were given a free choice of GHB solution and tap water for 20 consecutive weeks. Under the free-choice regimen, all rats showed periods of preference for GHB solution over water and periods of voluntary abstinence from GHB. On GHB-preference days, GHB was ingested at pharmacologically relevant doses. GHB intake occurred in 2-3 discrete episodes during the nocturnal phase. The development of an animal model of GHB self-administration may constitute a useful tool in the investigation of the neurobiological substrates of GHB-reinforcing properties. Keywords: GBH (y-hydroxybutyric acid); Voluntary y-hydroxybutyric acid intake; Alcoholism; (Rat)
1. Introduction
Administration of y-hydroxybutyric acid (GHB), a putative neurotransmitter or neuromodulator found in m a m m a l i a n brain, has been reported to produce a number of neuropharmacological effects both in laboratory animals and humans (see Cash, 1994). G H B has been clinically used as a general anaesthetic and hypnotic agent (Laborit et al., 1962) and in the treatment of narcolepsy (Lammers et al., 1993). More recently, G H B has been proposed as an effective agent in the p h a r m a c o t h e r a p y of alcoholism and opiate addiction. Indeed, G H B reduces alcohol craving and consumption (Gallimberti et al., 1992) and ameliorates symptoms of alcohol (Gallimberti et al., 1989) and heroin (Gallimberti et al., 1993) withdrawal syndrome. However, recent papers (Auerbach, 1990; Chin et al., 1992) and warning reports by U.S. agencies (Food and Drug Administration, 1991; Krawczeniuk, 1993)
* Corresponding author. C.N.R. Center for Neuropharmacology, c/o 'Bernard B. Brodie' Department of Neuroscience, University of Cagliari, Via Porcell 4, 1-09124 Cagliari, Italy. Tel.: +39 70 670961; fax: + 39 70 657237. 0014-2999/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0014-2999(95)00493-9
suggest a potential abuse liability of GHB. Administration of G H B allegedly produces subjective feelings of being 'high' and euphoria, rendering the drug compelling. More recently, Martellotta et al. (1995) found that G H B is able to induce conditioned place preference in rats, indicating that it possesses reinforcing properties. The present study describes the development of an animal model of G H B self-administration, which might constitute a useful experimental paradigm in the search of the biological basis of GHB-reinforcing effects.
2. Materials and methods 2.1. A n i m a l s
Twenty male Wistar rats (Charles River, Calco, CO, Italy), weighing 400-500 g and aged 4 months at the start of the study, were used. The rats were singly housed in standard plastic cages with wood chip bedding under a 12-h artificial light-dark cycle (lights on at 7:00 p.m.), at constant t e m p e r a t u r e of 22 + 2°C and relative humidity of 60%. Standard laboratory rat chow ( M I L Morini, San Polo d'Enza, RE, Italy) was pro-
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vided ad libitum to the rats throughout the experimental period. Rat body weights were monitored weekly. 2.2. Induction o f G H B preference
The rats were initially forced to drink 1% ( w / v ) G H B (sodium salt, from Sigma, St. Louis, MO, USA) in water, this being the sole drinking fluid available for 14 consecutive days. G H B intake was recorded daily at the beginning of the dark period (7:00 a.m.). On the fifteenth day, the rats were changed to a free-choice regimen. Two graduated bottles, containing tap water and 1% ( w / v ) G H B in water, respectively, were continuously offered for 20 consecutive weeks. G H B and water intakes were monitored daily at 7:00 a.m. Bottles were refilled every day with fresh solution and their position was reversed at random to avoid development of position habit. The criterion of preference for G H B was defined as a daily intake of G H B solution greater than 50% of the total fluid intake ( G H B - p r e f e r e n c e day).
3. R e s u l t s
During the 14 days of the no-choice period, the rats showed a stable intake of G H B , ranging 800-1200 mg/kg/day. Once G H B and water were offered under the freechoice procedure, preference for G H B was established in all rats and persisted for the entire experimental period. However, in order to simplify data layout, only the results of the first 20 consecutive days of the free-choice regimen are reported in detail. Fig. 1 shows the pattern of G H B and water intake on the first 20 consecutive days of the free-choice regimen by all rats used in the present study. All rats alternated periods of high daily intake of G H B with temporarily self-imposed cessation of G H B intake. A virtually equal n u m b e r of G H B - p r e f e r e n c e days (10.15 + 0.7 (mean + S.E.M.)) and water-preference days (9.85 + 0.7 (mean + S.E.M.)) was recorded. However, a large individual variability (with respect to other rats and by the same rat) in the duration of the G H B - and water-preference day sequences was observed. Indeed, G H B - and water-preference periods ranged from 1 to 12 (2.6 + 0.2 (mean + S.E.M.)) and 1 to 11 (2.7 + 0.2 (mean + S.E.M.)) consecutive days, respectively. On G H B - p r e f e r e n c e days, G H B consump-
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Fig. 2. Illustrative patterns of GHB (1% w/v) and total fluid intakes for 24 consecutive hours by rat No. 7 (top panel) and rat No. 13 (bottom panel) on day 5 and day 4, respectively, of the free-choice period. The hourly intake of GHB (e) is expressed on the left ordinate as milliliters of solution consumed by the rat and on the right ordinate as mg/kg. Water (©) intake is expressed on the left ordinate as milliliters consumed by the rat.
tion averaged 666.3 + 13.2 (mean + S.E.M.) m g / k g / day. On water-preference days, a compensatory increase in water consumption took place, so that the daily total fluid intake in each rat did not significantly change. No difference in the amount of food intake was observed between G H B - p r e f e r e n c e and waterpreference days. No signs of a withdrawal syndrome were observed on the water-preference days. Fig. 2 illustrates the one-day pattern of G H B and water intake by two rats on a G H B - p r e f e r e n c e day (rat No. 7 on day 5 (top panel) and rat No. 13 on day 4 (bottom panel), respectively). In agreement with the well-characterized drinking pattern of rats, G H B and
Fig. 1. Individual patterns of GHB (1% w/v) and total fluid intakes on 20 consecutive days during the free-choice period by all the rats (n = 20) tested in the present study. The daily intake of GHB (e) is expressed on the left ordinate as milliliters of solution consumed by the rat, and on the right ordinate as mg/kg. Total fluid intake (©) is expressed on the left ordinate as the sum of milliliters of GHB solution and tap water consumed by the rat.
106
G. Colombo et al. / European Journal of Pharmacology 285 (1995) 103-107
water intake took place predominantly during the nocturnal phase of the light-dark cycle. In both the above rats, about 75% of GHB consumption occurred in three separate binges evenly distributed over the 12-h dark period. Both the pattern of alternatively preferring GHB or water and the amount of GHB consumed did not vary during the remaining 17 weeks of monitoring. No rat showed any sign of withdrawal when GHB was finally removed at the end of the 20-week period.
4. Discussion In a previous experiment, we failed to establish GHB preference in GHB-naive rats offered 1% (w/v) GHB and water in a two-bottle free-choice paradigm. In contrast, the 14-day no-choice period described in the present study resulted in the induction of GHB preference over water in all the rats tested, as shown in Fig. 1 by the occurrence of daily intakes of GHB largely exceeding the amount of consumed water. The reason for this discrepancy between the two experiments might be the unpleasant taste of GHB (presented as sodium salt), which would prevent the rats from discovering its reinforcing properties. In the present study, these effects were revealed during the period of forced GHB intake. Voluntary intake of GHB occurred under a fluctuating pattern, showing a sequence of GHB-preference days followed by a sequence of water-preference days. Periods of G H B - a n d water-preference days alternated during the entire experimental period in each rat. Voluntary and temporary discontinuation of drug intake has been reported to occur in monkeys self-administering amphetamine, cocaine and ethanol (see Meisch and Stewart, 1994). Thus, although the alternation between GHB- and water-preference periods observed in the present study is not a unique and peculiar characteristic of voluntary GHB intake, at present the reason for this phenomenon escapes our understanding. It might be hypothesized that prolonged GHB consumption induces noxious and aversive effects that mask the reinforcing properties of the drug and lead the rats to temporarily avoid GHB. The accumulation of a product of GHB catabolism might be responsible for the toxic effects. Further studies are needed to verify this hypothesis. On the GHB-preference days, GHB consumption took place in distinct binges, interspaced by 3-5 h, during the dark phase of the light-dark cycle. In each binge, GHB intake occurred at pharmacologically relevant doses (100-300 mg/kg), well within the dose range reported to produce anxiolytic (Kr~iak et al., 1974) and discriminable (Colombo et al., 1995) effects, and establish place preference (Martellotta et al., 1995)
in the rat. Moreover, the 3-5 h interval between GHB binges is consistent with the pharmacokinetic characteristics of oral GHB in the rat (Lettieri and Fung, 1979), indicating a self-controlled adjustment of GHB dose by the rat over the 24-h cycle. In conclusion, these results (a) indicate that GHB can be voluntarily ingested by the rat after a period of forced GHB drinking, and (b) confirm that GHB possesses reinforcing properties. Indeed, it has recently been demonstrated by Martellotta et al. (1995) that GHB is capable of inducing place preference in rats. Future studies will address whether the reinforcing properties of GHB can give rise to potential abuse liability.
Acknowledgements The authors are grateful to Mrs. M. Dolores Sedda and Mrs. M. Elena Vincis for their technical assistance and Mr. Hugh Sugden for language editing of the manuscript.
References Auerbach, S., 1990, Multistate outbreak of poisoning associated with illicit use of gamma hydroxy butyrate, MMWR 39, 861. Cash, D.C., 1994, Gammahydroxybutyrate: an overview of the pros and cons for it being a neurotransmitter a n d / o r a useful therapeutic agent, Neurosci. Biobehav. Rev. 18, 291. Chin, M.Y., R.A. Kreutzer and J.E. Dyer, 1992, Acute poisoning from gamma-hydroxybutyrate in California, West. J. Med. 156, 380. Colombo, G., R. Agabio, C. Lobina, R. Reali, F. Fadda and G.L. Gessa, 1995, Symmetrical generalization between the discriminative stimulus effects of gamma-hydroxybutyric acid and ethanol: occurrence within narrow dose ranges, Physiol. Behav. 57, 105. Food and Drug Administration, 1991, Warning about GHB, J. Am. Med. Assoc. 285, 1802. Gallimberti, L., G. Canton, N. Gentile, M. Ferri, M. Cibin, S.D. Ferrara, F. Fadda and G.L. Gessa, 1989, Gamma-hydroxybutyric acid for the treatment of alcohol withdrawal syndrome, Lancet 2, 787. Gallimberti, L., M. Ferri, S.D. Ferrara, F. Fadda and G.L. Gessa, 1992, Gamma-hydroxybutyric acid in the treatment of alcohol dependence: a double-blind study, Alcohol. Clin. Exp. Res. 16, 673. Gallimberti, L., M. Cibin, P. Pagnin, R. Sabbion, P.P. Pani, R. Pirastu, S.D. Ferrara and G.L. Gessa, 1993, Gamma-hydroxybutyric acid for treatment of opiate withdrawal syndrome, Neuropsychopharmacology 9, 77. Krawczeniuk, A., 1993, The occurrence of gamma hydroxybutyric acid (GHB) in a steroid seizure, Microgram 26, 161. Kr§iak, M., D. Novakova, I. Paclt and R.U. Ostrovskaya, 1974, Effect of sodium hydroxybutyrate on behavior of mice after prolonged isolation, Bull. Exp. Biol. Med. 77, 288. Laborit, H., A. Larcan and A. Kind, 1962, Le gamma-hydroxybutyrate en anesthesie neuro-chirurgicale, Neuro-chirurgie 8, 10.
G. Colombo et al. / European Journal of Pharmacology 285 (1995) 103-107 Lammers, G.J., J. Arends, A.C. Declerck, M.D. Ferrari, G. Schouwink and J. Troost, 1993, Gammahydroxybutyrate and narcolepsy: a double-blind placebo-controlled study, Sleep 16, 216. Lettieri, J.T. and H.-O. Fung, 1979, Dose-dependent pharmacokinetics and hypnotic effects of sodium 3,-hydroxybutyrate in the rat, J. Pharmacol. Exp. Ther. 208, 7.
107
Martellotta, M.C., L. Fattore, G. Cossu and W. Fratta, 1995, Gamma-hydroxybutyric acid induces place-preference in rats, Behav. Pharmacol. 6 (Suppl. 1), 84. Meisch, R.A. and R.B. Stewart, 1994, Ethanol as a reinforcer: a review of laboratory studies of non-human primates, Behav. Pharmacol. 5, 425.
European Journal of Pharmacology285 (1995) 103-107
Short communication
Oral self-administration of y-hydroxybutyric acid in the rat Giancarlo Colombo a,,, Roberta Agabio b, Nadejda Balaklievskaia b, Giacomo Diaz c, Carla Lobina a, Roberta Reali b, Gian Luigi Gessa b a C.N.R. Center for Neuropharmacology, University of Cagliari, Cagliari, Italy b 'Bernard B. Brodie' Department of Neuroscience, University of Cagliari, Cagliari, Italy c Department of Cytomorphology, University of Cagliari, Cagliari, Italy
Received 27 July 1995; accepted 8 August 1995
Abstract
The present study describes the induction of y-hydroxybutyric acid (GHB) preference over water in rats. GHB solution (1% w / v in water) was initially offered as the sole fluid available for 14 consecutive days. Subsequently, rats were given a free choice of GHB solution and tap water for 20 consecutive weeks. Under the free-choice regimen, all rats showed periods of preference for GHB solution over water and periods of voluntary abstinence from GHB. On GHB-preference days, GHB was ingested at pharmacologically relevant doses. GHB intake occurred in 2-3 discrete episodes during the nocturnal phase. The development of an animal model of GHB self-administration may constitute a useful tool in the investigation of the neurobiological substrates of GHB-reinforcing properties. Keywords: GBH (y-hydroxybutyric acid); Voluntary y-hydroxybutyric acid intake; Alcoholism; (Rat)
1. Introduction
Administration of y-hydroxybutyric acid (GHB), a putative neurotransmitter or neuromodulator found in m a m m a l i a n brain, has been reported to produce a number of neuropharmacological effects both in laboratory animals and humans (see Cash, 1994). G H B has been clinically used as a general anaesthetic and hypnotic agent (Laborit et al., 1962) and in the treatment of narcolepsy (Lammers et al., 1993). More recently, G H B has been proposed as an effective agent in the p h a r m a c o t h e r a p y of alcoholism and opiate addiction. Indeed, G H B reduces alcohol craving and consumption (Gallimberti et al., 1992) and ameliorates symptoms of alcohol (Gallimberti et al., 1989) and heroin (Gallimberti et al., 1993) withdrawal syndrome. However, recent papers (Auerbach, 1990; Chin et al., 1992) and warning reports by U.S. agencies (Food and Drug Administration, 1991; Krawczeniuk, 1993)
* Corresponding author. C.N.R. Center for Neuropharmacology, c/o 'Bernard B. Brodie' Department of Neuroscience, University of Cagliari, Via Porcell 4, 1-09124 Cagliari, Italy. Tel.: +39 70 670961; fax: + 39 70 657237. 0014-2999/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0014-2999(95)00493-9
suggest a potential abuse liability of GHB. Administration of G H B allegedly produces subjective feelings of being 'high' and euphoria, rendering the drug compelling. More recently, Martellotta et al. (1995) found that G H B is able to induce conditioned place preference in rats, indicating that it possesses reinforcing properties. The present study describes the development of an animal model of G H B self-administration, which might constitute a useful experimental paradigm in the search of the biological basis of GHB-reinforcing effects.
2. Materials and methods 2.1. A n i m a l s
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vided ad libitum to the rats throughout the experimental period. Rat body weights were monitored weekly. 2.2. Induction o f G H B preference
The rats were initially forced to drink 1% ( w / v ) G H B (sodium salt, from Sigma, St. Louis, MO, USA) in water, this being the sole drinking fluid available for 14 consecutive days. G H B intake was recorded daily at the beginning of the dark period (7:00 a.m.). On the fifteenth day, the rats were changed to a free-choice regimen. Two graduated bottles, containing tap water and 1% ( w / v ) G H B in water, respectively, were continuously offered for 20 consecutive weeks. G H B and water intakes were monitored daily at 7:00 a.m. Bottles were refilled every day with fresh solution and their position was reversed at random to avoid development of position habit. The criterion of preference for G H B was defined as a daily intake of G H B solution greater than 50% of the total fluid intake ( G H B - p r e f e r e n c e day).
3. R e s u l t s
During the 14 days of the no-choice period, the rats showed a stable intake of G H B , ranging 800-1200 mg/kg/day. Once G H B and water were offered under the freechoice procedure, preference for G H B was established in all rats and persisted for the entire experimental period. However, in order to simplify data layout, only the results of the first 20 consecutive days of the free-choice regimen are reported in detail. Fig. 1 shows the pattern of G H B and water intake on the first 20 consecutive days of the free-choice regimen by all rats used in the present study. All rats alternated periods of high daily intake of G H B with temporarily self-imposed cessation of G H B intake. A virtually equal n u m b e r of G H B - p r e f e r e n c e days (10.15 + 0.7 (mean + S.E.M.)) and water-preference days (9.85 + 0.7 (mean + S.E.M.)) was recorded. However, a large individual variability (with respect to other rats and by the same rat) in the duration of the G H B - and water-preference day sequences was observed. Indeed, G H B - and water-preference periods ranged from 1 to 12 (2.6 + 0.2 (mean + S.E.M.)) and 1 to 11 (2.7 + 0.2 (mean + S.E.M.)) consecutive days, respectively. On G H B - p r e f e r e n c e days, G H B consump-
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Fig. 2. Illustrative patterns of GHB (1% w/v) and total fluid intakes for 24 consecutive hours by rat No. 7 (top panel) and rat No. 13 (bottom panel) on day 5 and day 4, respectively, of the free-choice period. The hourly intake of GHB (e) is expressed on the left ordinate as milliliters of solution consumed by the rat and on the right ordinate as mg/kg. Water (©) intake is expressed on the left ordinate as milliliters consumed by the rat.
tion averaged 666.3 + 13.2 (mean + S.E.M.) m g / k g / day. On water-preference days, a compensatory increase in water consumption took place, so that the daily total fluid intake in each rat did not significantly change. No difference in the amount of food intake was observed between G H B - p r e f e r e n c e and waterpreference days. No signs of a withdrawal syndrome were observed on the water-preference days. Fig. 2 illustrates the one-day pattern of G H B and water intake by two rats on a G H B - p r e f e r e n c e day (rat No. 7 on day 5 (top panel) and rat No. 13 on day 4 (bottom panel), respectively). In agreement with the well-characterized drinking pattern of rats, G H B and
Fig. 1. Individual patterns of GHB (1% w/v) and total fluid intakes on 20 consecutive days during the free-choice period by all the rats (n = 20) tested in the present study. The daily intake of GHB (e) is expressed on the left ordinate as milliliters of solution consumed by the rat, and on the right ordinate as mg/kg. Total fluid intake (©) is expressed on the left ordinate as the sum of milliliters of GHB solution and tap water consumed by the rat.
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water intake took place predominantly during the nocturnal phase of the light-dark cycle. In both the above rats, about 75% of GHB consumption occurred in three separate binges evenly distributed over the 12-h dark period. Both the pattern of alternatively preferring GHB or water and the amount of GHB consumed did not vary during the remaining 17 weeks of monitoring. No rat showed any sign of withdrawal when GHB was finally removed at the end of the 20-week period.
4. Discussion In a previous experiment, we failed to establish GHB preference in GHB-naive rats offered 1% (w/v) GHB and water in a two-bottle free-choice paradigm. In contrast, the 14-day no-choice period described in the present study resulted in the induction of GHB preference over water in all the rats tested, as shown in Fig. 1 by the occurrence of daily intakes of GHB largely exceeding the amount of consumed water. The reason for this discrepancy between the two experiments might be the unpleasant taste of GHB (presented as sodium salt), which would prevent the rats from discovering its reinforcing properties. In the present study, these effects were revealed during the period of forced GHB intake. Voluntary intake of GHB occurred under a fluctuating pattern, showing a sequence of GHB-preference days followed by a sequence of water-preference days. Periods of G H B - a n d water-preference days alternated during the entire experimental period in each rat. Voluntary and temporary discontinuation of drug intake has been reported to occur in monkeys self-administering amphetamine, cocaine and ethanol (see Meisch and Stewart, 1994). Thus, although the alternation between GHB- and water-preference periods observed in the present study is not a unique and peculiar characteristic of voluntary GHB intake, at present the reason for this phenomenon escapes our understanding. It might be hypothesized that prolonged GHB consumption induces noxious and aversive effects that mask the reinforcing properties of the drug and lead the rats to temporarily avoid GHB. The accumulation of a product of GHB catabolism might be responsible for the toxic effects. Further studies are needed to verify this hypothesis. On the GHB-preference days, GHB consumption took place in distinct binges, interspaced by 3-5 h, during the dark phase of the light-dark cycle. In each binge, GHB intake occurred at pharmacologically relevant doses (100-300 mg/kg), well within the dose range reported to produce anxiolytic (Kr~iak et al., 1974) and discriminable (Colombo et al., 1995) effects, and establish place preference (Martellotta et al., 1995)
in the rat. Moreover, the 3-5 h interval between GHB binges is consistent with the pharmacokinetic characteristics of oral GHB in the rat (Lettieri and Fung, 1979), indicating a self-controlled adjustment of GHB dose by the rat over the 24-h cycle. In conclusion, these results (a) indicate that GHB can be voluntarily ingested by the rat after a period of forced GHB drinking, and (b) confirm that GHB possesses reinforcing properties. Indeed, it has recently been demonstrated by Martellotta et al. (1995) that GHB is capable of inducing place preference in rats. Future studies will address whether the reinforcing properties of GHB can give rise to potential abuse liability.
Acknowledgements The authors are grateful to Mrs. M. Dolores Sedda and Mrs. M. Elena Vincis for their technical assistance and Mr. Hugh Sugden for language editing of the manuscript.
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