European Journal of Pharmacology 740 (2014) 53–57
Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Behavioural pharmacology
Assessment of pregnenolone effects on alcohol intake and preference in male alcohol preferring (P) rats Amir H. Rezvani n, Edward D. Levin Department of Psychiatry and Behavioral Sciences and Department of Psychology and Neuroscience, Duke University Medical Center, Durham, NC 27710, USA
art ic l e i nf o
a b s t r a c t
Article history: Received 8 May 2014 Received in revised form 30 June 2014 Accepted 1 July 2014 Available online 10 July 2014
Neuroactive steroids can modulate a variety of neurobehavioral functions via the GABAergic system. This study was conducted to determine the importance of the neurosteroid pregnenolone on the regulation of alcohol intake. The effects of acute and chronic administration of pregnenolone on alcohol intake were assessed in alcohol preferring (P) rats. The rats were injected i.p. with the vehicle or pregnenolone (25, 50 or 75 mg/kg) and their alcohol and water intake were recorded at 2, 4, 6 and 24 h. Also, the chronic effects of 50 mg/kg (i.p.) pregnenolone on alcohol intake were determined. Our results show that although the main effect of i.p. injection of pregnenolone in reducing alcohol intake was not quite significant compared with the vehicle, pregnenolone at 75 mg/kg significantly (Po 0.025) reduced alcohol intake. Regarding alcohol preference, acute administration of pregnenolone both at 50 mg/kg (P o0.05) and at 75 mg/kg (Po 0.025) significantly reduced alcohol preference. In chronic experiments pregnenolone given for 10 consecutive days did not show a significant effect on alcohol intake and alcohol preference. Overall, although pregnenolone given i.p. acutely and significantly reduced alcohol intake and preference, the fact that chronic treatment did not show an effect diminishes its promise to be considered for the treatment of alcoholism. However, its profile of effects might be different in human alcoholics. & 2014 Elsevier B.V. All rights reserved.
Keywords: Alcohol drinking Alcoholism Treatment Neuroactive steroids GABAergic system
1. Introduction Neuroactive steroids have been shown to exert a variety of behavioral effects including anxiolytic (Bitran et al., 1991; Wieland et al., 1991), sedative-hypnotic (Mendelson et al., 1987; Wiebe and Kavaliers 1988), anti-convulsant (Finn and Gee, 1994), cognitive impairing effects or cognitive improving effects (Flood et al., 1992; Marx et al., 2009; 2011; Ritsner et al., 2010; Savitz, 2010). Neurosteroids such as pregnenolone also contribute to the behavioral effects of alcohol both in rodents (O’Dell et al., 2004; Barbaccia et al., 1999) and humans, as it has been shown that alcohol ingestion increases the level of neurosteroids in both brain and plasma (Barbaccia et al., 1999; Pierucci-Lagha et al., 2006). The GABAergic systems play an important role in the regulation of alcohol intake in rodents and humans (June et al., 2003; Quintanilla et al., 2008; Agabio et al., 2012; Stopponi et al., 2012). The GABAergic neuroactive steroids have been shown to have alcohol-like discriminative stimulus properties in rodents (Shannon et al., 2005) and primates (Grant et al., 1997). These n Correspondence to: Department of Psychiatry and Behavioral Sciences, Duke University Medical Center, Box 104790 Durham, NC, 27710, USA. Tel.: þ 1 919 668 1880; fax: þ1 919 681 3416. E-mail address: [email protected] (A.H. Rezvani).
http://dx.doi.org/10.1016/j.ejphar.2014.07.003 0014-2999/& 2014 Elsevier B.V. All rights reserved.
steroids act as allosteric modulators of GABAA, NMDA glutamate and sigma receptors (Wu et al., 1991; Baulieu, 1997; Belelli and Lambert, 2005; Morrow et al., 2006). The neurosteroid pregnenolone has an opposing effect on NMDA glutamate and GABAA receptors; increasing NMDA glutamate receptor activity but decreasing GABAA receptor activity (Majewska et al., 1990,1992). Consistent with their specific actions on different receptors, neurosteroids have been implicated in several behavioral actions of alcohol including excessive drinking in rats (Besheer et al., 2010). In a clinical study, it has been shown that endogenous neurosteroids mediate some of the subjective effects of alcohol, as alcohol ingestion modifies neurosteroid hormones bidirectionally; i.e. increasing plasma pregnenolone and dehydroepiandrosterone (DHEA) concentrations and decreasing progesterone and alloprenanolone concentrations (Pierucci-Lagha et al., 2006). Although these findings suggest a role for these neurosteroids in some of the subjective effects of alcohol, they do not address the effects of these hormones on rewarding effects of alcohol. Recently, a group of researchers investigated the effect of pregnenolone on alcohol intake and found that pregnenolone when given systemically (i.p.), dose-dependently reduced 30-min operant alcohol self-administration in alcohol preferring P rats without having an impairing effect on their locomotion (Besheer et al., 2010). In this study the effect of pregnenolone on alcohol intake
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A.H. Rezvani, E.D. Levin / European Journal of Pharmacology 740 (2014) 53–57
was measured only for 30 min following the treatment using an operant behavior. Although this finding may be suggestive, it does not portray the effect of this compound post-30 min. Furthermore, the chronic effect of this compound on alcohol intake has not been studied and currently is unknown. Considering the important role of the GABAergic system in the brain in the regulation of alcohol intake, and the interaction of neurosteroids with the GABAergic system, we decided to study the effects of both acute and chronic administration of pregnenolone on alcohol intake in selectively-bred alcohol preferring (P) rats. It was hypothesized that pregnenolone, based on the existing literature, will reduce alcohol intake and preference in alcohol preferring rats.
2. Materials and methods 2.1. Animals Adult male selectively-bred alcohol preferring (P) rats were used. The rats were 5357 6.27 g at the initiation of the first treatment. This line of rats was originally derived from Wistar rats at Indiana University. We obtained these rats directly from a breeding colony maintained at Dr. Lawrence Lumeng's Laboratory at Indiana University, Indianapolis. Upon arrival at Duke University, they were kept in a standard laboratory with controlled temperature of 21 72 1C and humidity of 507 10 percent and reversed light cycle (lights off: 0700–2200). Rats were housed individually in specialized cages that were fitted with two 100-ml graduated Richter drinking tubes for the recording of water and alcohol consumption. The rats were initially given free access to a solution of 10% (v/v) alcohol for three consecutive days, and then had a free choice between water and alcohol solution (15%) in two different graduated Richter tubes for the remainder of the study. Three days of forced alcohol exposure made the rats accustomed to the taste and pharmacological effects of alcohol (Rezvani et al., 2009; 2010). Animals were fed 5001 Rodent Chow (Lab Diet, Brentwood, MO, USA) and water ad libitum. Rats were on alcohol and water for at least 6 weeks before the drug treatment began. All treatments and care of the animals were conducted according to a protocol approved by the Animal care and Use committee of Duke University in an AAALAC- approved facility. 2.2. Drug preparation Pregnenolone (5-Pregnen-3β-OL-20-ONE) was purchased from Steraloids, Inc., Newport RI, USA and was dissolved in a solution of 100% alcohol, then diluted with saline. The amount of alcohol in the drug solution and in the vehicle was equal to 0.1 g/kg. Solutions of alcohol were prepared from a solution of 100% ethanol mixed with tap water. Fresh alcohol and water bottles were presented to rats 15 min after each injection. 2.3. Experimental protocol The following experiments were carried out to study the effects of acute and chronic administration of pregnenolone on alcohol intake and preference in alcohol preferring rats.
drug or vehicle injected i.p. was 2 ml/kg. Treatments were given 15 min before alcohol exposure and alcohol intake and preference as well as water intake were measured at 2, 4, 6 and 24 h after the treatment. To avoid a possible carryover effect a 3-day interval was allowed between injections. Alcohol intake returned to the pretreatment baseline levels after this interval. 2.5. Chronic effects To determine the chronic effect of pregnenolone on alcohol intake, the same P rats were injected i.p. with either 50 mg of pregnenolone (n¼ 7) or the control vehicle (n ¼6) for 10 consecutive days. There was a 2-weeks interval between the last injection in the acute study and the initiation of the chronic study. The baseline for alcohol intake before the initiation of the chronic treatment was 4.72 70.4 g/kg/day. Alcohol intake and alcohol preference as well as water intake were measured at 6 and 24 h after each treatment. 2.6. Statistical analysis The results were evaluated for statistical significance by the analysis of variance with repeated measures of drug treatment dose and hour after administration. The drug doses were given in a counterbalanced order with the vehicle as the control. Planned comparisons between the vehicle administered control condition and each drug dose were made. As a follow-up to significant interactions, tests of the simple main effects were performed. Fisher Least Significant Difference (Fisher LSD) was used as posthoc test. The threshold of P o0.05 (two-tailed) was used as the threshold for significance. Alcohol preference (i.e. % alcohol intake) was calculated as the percent of alcohol consumed over total fluid intake (total alcohol intake/total fluid intake) 100 (Rezvani et al., 2010). Data values are expressed as means 7 S.E.M. 3. Results 3.1. Acute effects To study the acute effects of pregnenolone, rats (n¼ 13) were treated i.p. with 25, 50 or 75 mg/kg pregnenolone or the control vehicle and their alcohol and water intake were measured. In the dose-effect study, the main effects of i.p. administration of pregnenolone in reducing alcohol intake and alcohol preference were nearly significant (F(3,36) ¼2.76, P¼ 0.056 and F(3,36) ¼ 2.83, P¼ 0.052 for alcohol intake and alcohol preference, respectively). Compared with control vehicle, only the 75 mg/kg dose significantly (F(1,36)¼ 6.25, P o0.025) reduced alcohol intake (Fig. 1). Alcohol intake was significantly (P o0.005) reduced by acute administration of 75 mg/kg of pregnenolone at 4-h and 6-h time points but not at 2 or 24 h. The 25 and 50 mg/kg doses did not show a significant effect on alcohol intake (Fig. 1). Regarding alcohol preference, both 50 and 75 mg/kg doses significantly (F (1,36) ¼5.38, P o0.05 and F(1,36) ¼7.17, Po 0.025 for 50 and 75 mg/kg, respectively) reduced alcohol preference while the lower dose of 25 mg/kg dose did not show a significant effect (Fig. 2). Compared with control vehicle, no dose of pregnenolone had a significant effect on water or total fluid intake (Table 1).
2.4. Acute effects 3.2. Chronic effects After establishment of a stable level of alcohol drinking (4.88 70.19 g/kg/day), rats (n¼ 13) were injected i.p. with 25, 50 or 75 mg/kg pregnenolone or the same volume of the control vehicle following a counterbalanced order with random assignment. Thus, all animals received all treatments. The volume of
To study the chronic effects of pregnenolone on alcohol intake, rats were injected with 50 mg/kg of compound or vehicle for 10 consecutive days and their alcohol and water intake was recorded. No significant effects of pregnenolone were seen on alcohol
A.H. Rezvani, E.D. Levin / European Journal of Pharmacology 740 (2014) 53–57
55
Fig. 1. Acute effects of i.p. administration of pregnenolone on alcohol intake (g/kg) at different time points. Data represent means 7 S.E.M., n ¼13. Planned comparisons of pregnenolone doses vs. control for the main effect for all time points were made.
Fig. 2. Acute effects of i.p. administration of pregnenolone on alcohol preference (%) at different time points. Data represent means 7 S.E.M., n¼ 13. Planned comparisons of pregnenolone doses vs. control for the main effect for all time points were made.
Table 1 Effects of pregnenolone on total fluid intake (ml/kg) at different time points. Pregnenolone did not exert a significant effect on total fluid intake at any time points.
Dose-response, i.p. Vehicle 25 mg/kg 50 mg/kg 75 mg/kg
2 (H)
4 (H)
6 (H)
24 (H)
67 1 67 2 57 1 57 2
107 1 117 2 87 1 87 2
21 72 21 73 20 72 16 72
507 4 517 3 49 7 3 48 7 4
Data presented as means 7 S.E.M., n ¼13.
consumption or alcohol preference when 50 mg/kg of pregnenolone was administered i.p. for 10 consecutive days. At the 6 h time point there was a significant effect of days (F(9,99) ¼4.59, P o0.0005), but there was no significant linear or quadratic trends of increasing or decreasing alcohol consumption over days (Fig. 3). At the 24 h time point there was also a significant main effect of days (F(9,99) ¼4.59, P o0.0005) and there were significant linear and quadratic trends (Fig. 4). The pregnenolone X days interaction was not significant at either time point, therefore tests of the simple main effects of pregnenolone at each session was not performed.
Fig. 3. Chronic effects of i.p. administration of pregnenolone (50 mg/kg) on alcohol intake (g/kg) at the 6 h time point. Data represent means 7S.E.M., n ¼7 for pregnenolone group and n ¼6 for the control group.
4. Discussion The main goal of the present work was to investigate the potential role of the neuroactive neurosteroid pregnenolone on the regulation of alcohol intake. These studies examined the
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A.H. Rezvani, E.D. Levin / European Journal of Pharmacology 740 (2014) 53–57
Fig. 4. Chronic effects of i.p. administration of pregnenolone (50 mg/kg) on alcohol intake (g/kg) at the 24 h time point. Data represent means 7 S.E.M., n¼ 7 for pregnenolone group and n¼ 6 for the control group.
efficacy of acute and chronic pregnenolone treatment for reducing alcohol intake and preference in the selectively-bred male alcohol preferring P rats. The major finding was that an acute dose of 75 mg/kg pregnenolone (i.p.) reduced alcohol intake and preference when animal had continual free choice of water and alcohol (Figs. 1 and 2). Although this dose significantly reduced alcohol intake, the total fluid intake did not change suggesting a compensatory increase in water intake (Table 1). Our findings with i.p. administration are consistent with previous findings showing that the same dose of pregnenolone significantly reduces operant alcohol self-administration in the same line of rats (Besheer et al., 2010). Since medications for the treatment of alcoholism are taken chronically, we also assessed the effects of this compound over a 10-day period with one daily i.p. injection of 50 mg/kg of the drug. The 50 mg/kg dose was selected based on the fact that it was recently found that pregnenolone at this dose produced a significant increase in cerebral cortical allopregnanolone levels in the alcohol-experienced P rats but not in alcohol-naïve P rats (Besheer et al., 2010). These authors suggested that the decrease in alcohol intake after an acute administration of pregnenolone may be related in part to increased levels of the GABAergic neuroactive steroid allopregnanolone. However, contrary to the promising acute effect of pregnenolone on alcohol intake, the results from the chronic experiment were not encouraging. No significant pregnenolone effect was seen in the chronic study. If just first day was examined there was a significant (Po0.05) reduction in alcohol consumption at the 6 h time point but not at 24 h, similar to earlier acute results. Curiously, there was a significant effect of pregnenolone in reducing alcohol consumption on day 8 at the 6 but not at 24 h time point. It seems that this was an isolated finding. The lack of chronic effects of pregnenolone on alcohol intake can be explained by considering the observation that tolerance develops to the action of allopregnanolone even with only one acute treatment (Birzniece et al., 2006; Türkmen et al. 2011). However, the exact mechanism of allopregnanolone tolerance and its effects on the assembly of the GABAA and receptor composition are complex and not totally understood (Türkmen et al., 2011). Although the mechanism of this lack of effect after the first injection is not clear at this point, it might be due to a metabolic tolerance due to an enzymatic induction, or it might be a functional tolerance due to the effects of the compound on specific receptors in the brain. Alternatively, may be the 50 mg/kg dose was not sufficient enough to exert a prolonged significant effect on alcohol intake. Thus, further study is warranted with higher doses. However, the reduced efficacy of pregnenolone with chronic administration may have a kinetic nature that can be overcome by escalating dosing or imposing a drug-holiday schedule. Regardless of the mechanism of action, the profile of chronic effects
might be different in humans as the rate of metabolism in human is several folds lower than that in rats and the half-life of pregnenolone in rats is much shorter. In addition to dopaminergic systems, the GABAergic systems in the brain have been shown to play an important role in the regulation of alcohol intake (Besheer et al., 2010; Koob, 2003; 2004; Morrow et al., 2001; Ramaker et al., 2011). For instance, it has been suggested that GABAA receptors in the ventral pallidum may play an important role in alcohol drinking behavior (June et al., 2003) and similarly allopregnanolone, which acts as GABAA positive modulator, may have the same effect. Indeed, it has been shown that intrahippocampal administration of allopregnanolone significantly decreased voluntary alcohol consumption in nonselected rat line (Martin-García et al., 2007). Similar findings have been reported with systemic administration of allopregnanolone, a metabolite of pregnenolone, in the high alcohol drinking C57BL/ 6J inbred mouse strain, an effect that was blocked by the administration of finasteride, an inhibitor of allopregnanolone synthesis (Ford et al., 2005). Thus, it is possible that pregnenolone exerts its short-acting effect on alcohol consumption by activation of GABAergic neuroactive steroids such as allopregnanolone in the brain. Another possibility is the influence of pregnenolone on the dopamine transmission in the brain. The mesolimbic dopaminergic system has been repeatedly shown to play a critical role in the regulation of alcohol intake. Alcohol administration stimulates extracellular dopamine release in the striatum and the nucleus accumbens of rats (Brand et al., 2013; Imperato and Di Chiara, 1986;Yoshimoto et al., 1992). More specifically it was recently shown that dopamine D3 receptor is necessary for alcohol consumption in mice (Leggio et al., 2014). Furthermore, it was previously shown that, similar to alcohol, pregnenolone also dose-dependently increased dopamine release in the nucleus accumbens of rats (Barrot et al., 1999). It is possible that dopamine release within the reward system in the brain by pregnenolone administration may partially satisfy the need for dopamine in P rats, which have innate dopamine deficiency (McBride et al., 1993). Thus it can be speculated that pregnenolone action on the mesolimbic dopamine neurotransmission substitutes for the alcohol effect on dopamine, as both pregnenolone and alcohol stimulate extracellular dopamine release in the nucleus accumbens.
5. Conclusion The acute systemic administration of pregnenolone reduced alcohol intake and preference in alcohol preferring P rats; however the fact that the chronic systemic administration of pregnenolone lost its effect on the second day of the treatment is a concern for considering this compound for the treatment of alcoholism.
Acknowledgments We thank Drs. Lawrence Lumeng and Richard Bell from the Indiana University for providing alcohol preferring P rats. We also thank Andrew Lee of Duke University for editing the manuscript. This work was supported in part by the National Institute of Alcohol Abuse and Alcoholism at the National Institute of Health (Grant R24-AA015512-02).
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Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Behavioural pharmacology
Assessment of pregnenolone effects on alcohol intake and preference in male alcohol preferring (P) rats Amir H. Rezvani n, Edward D. Levin Department of Psychiatry and Behavioral Sciences and Department of Psychology and Neuroscience, Duke University Medical Center, Durham, NC 27710, USA
art ic l e i nf o
a b s t r a c t
Article history: Received 8 May 2014 Received in revised form 30 June 2014 Accepted 1 July 2014 Available online 10 July 2014
Neuroactive steroids can modulate a variety of neurobehavioral functions via the GABAergic system. This study was conducted to determine the importance of the neurosteroid pregnenolone on the regulation of alcohol intake. The effects of acute and chronic administration of pregnenolone on alcohol intake were assessed in alcohol preferring (P) rats. The rats were injected i.p. with the vehicle or pregnenolone (25, 50 or 75 mg/kg) and their alcohol and water intake were recorded at 2, 4, 6 and 24 h. Also, the chronic effects of 50 mg/kg (i.p.) pregnenolone on alcohol intake were determined. Our results show that although the main effect of i.p. injection of pregnenolone in reducing alcohol intake was not quite significant compared with the vehicle, pregnenolone at 75 mg/kg significantly (Po 0.025) reduced alcohol intake. Regarding alcohol preference, acute administration of pregnenolone both at 50 mg/kg (P o0.05) and at 75 mg/kg (Po 0.025) significantly reduced alcohol preference. In chronic experiments pregnenolone given for 10 consecutive days did not show a significant effect on alcohol intake and alcohol preference. Overall, although pregnenolone given i.p. acutely and significantly reduced alcohol intake and preference, the fact that chronic treatment did not show an effect diminishes its promise to be considered for the treatment of alcoholism. However, its profile of effects might be different in human alcoholics. & 2014 Elsevier B.V. All rights reserved.
Keywords: Alcohol drinking Alcoholism Treatment Neuroactive steroids GABAergic system
1. Introduction Neuroactive steroids have been shown to exert a variety of behavioral effects including anxiolytic (Bitran et al., 1991; Wieland et al., 1991), sedative-hypnotic (Mendelson et al., 1987; Wiebe and Kavaliers 1988), anti-convulsant (Finn and Gee, 1994), cognitive impairing effects or cognitive improving effects (Flood et al., 1992; Marx et al., 2009; 2011; Ritsner et al., 2010; Savitz, 2010). Neurosteroids such as pregnenolone also contribute to the behavioral effects of alcohol both in rodents (O’Dell et al., 2004; Barbaccia et al., 1999) and humans, as it has been shown that alcohol ingestion increases the level of neurosteroids in both brain and plasma (Barbaccia et al., 1999; Pierucci-Lagha et al., 2006). The GABAergic systems play an important role in the regulation of alcohol intake in rodents and humans (June et al., 2003; Quintanilla et al., 2008; Agabio et al., 2012; Stopponi et al., 2012). The GABAergic neuroactive steroids have been shown to have alcohol-like discriminative stimulus properties in rodents (Shannon et al., 2005) and primates (Grant et al., 1997). These n Correspondence to: Department of Psychiatry and Behavioral Sciences, Duke University Medical Center, Box 104790 Durham, NC, 27710, USA. Tel.: þ 1 919 668 1880; fax: þ1 919 681 3416. E-mail address: [email protected] (A.H. Rezvani).
http://dx.doi.org/10.1016/j.ejphar.2014.07.003 0014-2999/& 2014 Elsevier B.V. All rights reserved.
steroids act as allosteric modulators of GABAA, NMDA glutamate and sigma receptors (Wu et al., 1991; Baulieu, 1997; Belelli and Lambert, 2005; Morrow et al., 2006). The neurosteroid pregnenolone has an opposing effect on NMDA glutamate and GABAA receptors; increasing NMDA glutamate receptor activity but decreasing GABAA receptor activity (Majewska et al., 1990,1992). Consistent with their specific actions on different receptors, neurosteroids have been implicated in several behavioral actions of alcohol including excessive drinking in rats (Besheer et al., 2010). In a clinical study, it has been shown that endogenous neurosteroids mediate some of the subjective effects of alcohol, as alcohol ingestion modifies neurosteroid hormones bidirectionally; i.e. increasing plasma pregnenolone and dehydroepiandrosterone (DHEA) concentrations and decreasing progesterone and alloprenanolone concentrations (Pierucci-Lagha et al., 2006). Although these findings suggest a role for these neurosteroids in some of the subjective effects of alcohol, they do not address the effects of these hormones on rewarding effects of alcohol. Recently, a group of researchers investigated the effect of pregnenolone on alcohol intake and found that pregnenolone when given systemically (i.p.), dose-dependently reduced 30-min operant alcohol self-administration in alcohol preferring P rats without having an impairing effect on their locomotion (Besheer et al., 2010). In this study the effect of pregnenolone on alcohol intake
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was measured only for 30 min following the treatment using an operant behavior. Although this finding may be suggestive, it does not portray the effect of this compound post-30 min. Furthermore, the chronic effect of this compound on alcohol intake has not been studied and currently is unknown. Considering the important role of the GABAergic system in the brain in the regulation of alcohol intake, and the interaction of neurosteroids with the GABAergic system, we decided to study the effects of both acute and chronic administration of pregnenolone on alcohol intake in selectively-bred alcohol preferring (P) rats. It was hypothesized that pregnenolone, based on the existing literature, will reduce alcohol intake and preference in alcohol preferring rats.
2. Materials and methods 2.1. Animals Adult male selectively-bred alcohol preferring (P) rats were used. The rats were 5357 6.27 g at the initiation of the first treatment. This line of rats was originally derived from Wistar rats at Indiana University. We obtained these rats directly from a breeding colony maintained at Dr. Lawrence Lumeng's Laboratory at Indiana University, Indianapolis. Upon arrival at Duke University, they were kept in a standard laboratory with controlled temperature of 21 72 1C and humidity of 507 10 percent and reversed light cycle (lights off: 0700–2200). Rats were housed individually in specialized cages that were fitted with two 100-ml graduated Richter drinking tubes for the recording of water and alcohol consumption. The rats were initially given free access to a solution of 10% (v/v) alcohol for three consecutive days, and then had a free choice between water and alcohol solution (15%) in two different graduated Richter tubes for the remainder of the study. Three days of forced alcohol exposure made the rats accustomed to the taste and pharmacological effects of alcohol (Rezvani et al., 2009; 2010). Animals were fed 5001 Rodent Chow (Lab Diet, Brentwood, MO, USA) and water ad libitum. Rats were on alcohol and water for at least 6 weeks before the drug treatment began. All treatments and care of the animals were conducted according to a protocol approved by the Animal care and Use committee of Duke University in an AAALAC- approved facility. 2.2. Drug preparation Pregnenolone (5-Pregnen-3β-OL-20-ONE) was purchased from Steraloids, Inc., Newport RI, USA and was dissolved in a solution of 100% alcohol, then diluted with saline. The amount of alcohol in the drug solution and in the vehicle was equal to 0.1 g/kg. Solutions of alcohol were prepared from a solution of 100% ethanol mixed with tap water. Fresh alcohol and water bottles were presented to rats 15 min after each injection. 2.3. Experimental protocol The following experiments were carried out to study the effects of acute and chronic administration of pregnenolone on alcohol intake and preference in alcohol preferring rats.
drug or vehicle injected i.p. was 2 ml/kg. Treatments were given 15 min before alcohol exposure and alcohol intake and preference as well as water intake were measured at 2, 4, 6 and 24 h after the treatment. To avoid a possible carryover effect a 3-day interval was allowed between injections. Alcohol intake returned to the pretreatment baseline levels after this interval. 2.5. Chronic effects To determine the chronic effect of pregnenolone on alcohol intake, the same P rats were injected i.p. with either 50 mg of pregnenolone (n¼ 7) or the control vehicle (n ¼6) for 10 consecutive days. There was a 2-weeks interval between the last injection in the acute study and the initiation of the chronic study. The baseline for alcohol intake before the initiation of the chronic treatment was 4.72 70.4 g/kg/day. Alcohol intake and alcohol preference as well as water intake were measured at 6 and 24 h after each treatment. 2.6. Statistical analysis The results were evaluated for statistical significance by the analysis of variance with repeated measures of drug treatment dose and hour after administration. The drug doses were given in a counterbalanced order with the vehicle as the control. Planned comparisons between the vehicle administered control condition and each drug dose were made. As a follow-up to significant interactions, tests of the simple main effects were performed. Fisher Least Significant Difference (Fisher LSD) was used as posthoc test. The threshold of P o0.05 (two-tailed) was used as the threshold for significance. Alcohol preference (i.e. % alcohol intake) was calculated as the percent of alcohol consumed over total fluid intake (total alcohol intake/total fluid intake) 100 (Rezvani et al., 2010). Data values are expressed as means 7 S.E.M. 3. Results 3.1. Acute effects To study the acute effects of pregnenolone, rats (n¼ 13) were treated i.p. with 25, 50 or 75 mg/kg pregnenolone or the control vehicle and their alcohol and water intake were measured. In the dose-effect study, the main effects of i.p. administration of pregnenolone in reducing alcohol intake and alcohol preference were nearly significant (F(3,36) ¼2.76, P¼ 0.056 and F(3,36) ¼ 2.83, P¼ 0.052 for alcohol intake and alcohol preference, respectively). Compared with control vehicle, only the 75 mg/kg dose significantly (F(1,36)¼ 6.25, P o0.025) reduced alcohol intake (Fig. 1). Alcohol intake was significantly (P o0.005) reduced by acute administration of 75 mg/kg of pregnenolone at 4-h and 6-h time points but not at 2 or 24 h. The 25 and 50 mg/kg doses did not show a significant effect on alcohol intake (Fig. 1). Regarding alcohol preference, both 50 and 75 mg/kg doses significantly (F (1,36) ¼5.38, P o0.05 and F(1,36) ¼7.17, Po 0.025 for 50 and 75 mg/kg, respectively) reduced alcohol preference while the lower dose of 25 mg/kg dose did not show a significant effect (Fig. 2). Compared with control vehicle, no dose of pregnenolone had a significant effect on water or total fluid intake (Table 1).
2.4. Acute effects 3.2. Chronic effects After establishment of a stable level of alcohol drinking (4.88 70.19 g/kg/day), rats (n¼ 13) were injected i.p. with 25, 50 or 75 mg/kg pregnenolone or the same volume of the control vehicle following a counterbalanced order with random assignment. Thus, all animals received all treatments. The volume of
To study the chronic effects of pregnenolone on alcohol intake, rats were injected with 50 mg/kg of compound or vehicle for 10 consecutive days and their alcohol and water intake was recorded. No significant effects of pregnenolone were seen on alcohol
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Fig. 1. Acute effects of i.p. administration of pregnenolone on alcohol intake (g/kg) at different time points. Data represent means 7 S.E.M., n ¼13. Planned comparisons of pregnenolone doses vs. control for the main effect for all time points were made.
Fig. 2. Acute effects of i.p. administration of pregnenolone on alcohol preference (%) at different time points. Data represent means 7 S.E.M., n¼ 13. Planned comparisons of pregnenolone doses vs. control for the main effect for all time points were made.
Table 1 Effects of pregnenolone on total fluid intake (ml/kg) at different time points. Pregnenolone did not exert a significant effect on total fluid intake at any time points.
Dose-response, i.p. Vehicle 25 mg/kg 50 mg/kg 75 mg/kg
2 (H)
4 (H)
6 (H)
24 (H)
67 1 67 2 57 1 57 2
107 1 117 2 87 1 87 2
21 72 21 73 20 72 16 72
507 4 517 3 49 7 3 48 7 4
Data presented as means 7 S.E.M., n ¼13.
consumption or alcohol preference when 50 mg/kg of pregnenolone was administered i.p. for 10 consecutive days. At the 6 h time point there was a significant effect of days (F(9,99) ¼4.59, P o0.0005), but there was no significant linear or quadratic trends of increasing or decreasing alcohol consumption over days (Fig. 3). At the 24 h time point there was also a significant main effect of days (F(9,99) ¼4.59, P o0.0005) and there were significant linear and quadratic trends (Fig. 4). The pregnenolone X days interaction was not significant at either time point, therefore tests of the simple main effects of pregnenolone at each session was not performed.
Fig. 3. Chronic effects of i.p. administration of pregnenolone (50 mg/kg) on alcohol intake (g/kg) at the 6 h time point. Data represent means 7S.E.M., n ¼7 for pregnenolone group and n ¼6 for the control group.
4. Discussion The main goal of the present work was to investigate the potential role of the neuroactive neurosteroid pregnenolone on the regulation of alcohol intake. These studies examined the
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Fig. 4. Chronic effects of i.p. administration of pregnenolone (50 mg/kg) on alcohol intake (g/kg) at the 24 h time point. Data represent means 7 S.E.M., n¼ 7 for pregnenolone group and n¼ 6 for the control group.
efficacy of acute and chronic pregnenolone treatment for reducing alcohol intake and preference in the selectively-bred male alcohol preferring P rats. The major finding was that an acute dose of 75 mg/kg pregnenolone (i.p.) reduced alcohol intake and preference when animal had continual free choice of water and alcohol (Figs. 1 and 2). Although this dose significantly reduced alcohol intake, the total fluid intake did not change suggesting a compensatory increase in water intake (Table 1). Our findings with i.p. administration are consistent with previous findings showing that the same dose of pregnenolone significantly reduces operant alcohol self-administration in the same line of rats (Besheer et al., 2010). Since medications for the treatment of alcoholism are taken chronically, we also assessed the effects of this compound over a 10-day period with one daily i.p. injection of 50 mg/kg of the drug. The 50 mg/kg dose was selected based on the fact that it was recently found that pregnenolone at this dose produced a significant increase in cerebral cortical allopregnanolone levels in the alcohol-experienced P rats but not in alcohol-naïve P rats (Besheer et al., 2010). These authors suggested that the decrease in alcohol intake after an acute administration of pregnenolone may be related in part to increased levels of the GABAergic neuroactive steroid allopregnanolone. However, contrary to the promising acute effect of pregnenolone on alcohol intake, the results from the chronic experiment were not encouraging. No significant pregnenolone effect was seen in the chronic study. If just first day was examined there was a significant (Po0.05) reduction in alcohol consumption at the 6 h time point but not at 24 h, similar to earlier acute results. Curiously, there was a significant effect of pregnenolone in reducing alcohol consumption on day 8 at the 6 but not at 24 h time point. It seems that this was an isolated finding. The lack of chronic effects of pregnenolone on alcohol intake can be explained by considering the observation that tolerance develops to the action of allopregnanolone even with only one acute treatment (Birzniece et al., 2006; Türkmen et al. 2011). However, the exact mechanism of allopregnanolone tolerance and its effects on the assembly of the GABAA and receptor composition are complex and not totally understood (Türkmen et al., 2011). Although the mechanism of this lack of effect after the first injection is not clear at this point, it might be due to a metabolic tolerance due to an enzymatic induction, or it might be a functional tolerance due to the effects of the compound on specific receptors in the brain. Alternatively, may be the 50 mg/kg dose was not sufficient enough to exert a prolonged significant effect on alcohol intake. Thus, further study is warranted with higher doses. However, the reduced efficacy of pregnenolone with chronic administration may have a kinetic nature that can be overcome by escalating dosing or imposing a drug-holiday schedule. Regardless of the mechanism of action, the profile of chronic effects
might be different in humans as the rate of metabolism in human is several folds lower than that in rats and the half-life of pregnenolone in rats is much shorter. In addition to dopaminergic systems, the GABAergic systems in the brain have been shown to play an important role in the regulation of alcohol intake (Besheer et al., 2010; Koob, 2003; 2004; Morrow et al., 2001; Ramaker et al., 2011). For instance, it has been suggested that GABAA receptors in the ventral pallidum may play an important role in alcohol drinking behavior (June et al., 2003) and similarly allopregnanolone, which acts as GABAA positive modulator, may have the same effect. Indeed, it has been shown that intrahippocampal administration of allopregnanolone significantly decreased voluntary alcohol consumption in nonselected rat line (Martin-García et al., 2007). Similar findings have been reported with systemic administration of allopregnanolone, a metabolite of pregnenolone, in the high alcohol drinking C57BL/ 6J inbred mouse strain, an effect that was blocked by the administration of finasteride, an inhibitor of allopregnanolone synthesis (Ford et al., 2005). Thus, it is possible that pregnenolone exerts its short-acting effect on alcohol consumption by activation of GABAergic neuroactive steroids such as allopregnanolone in the brain. Another possibility is the influence of pregnenolone on the dopamine transmission in the brain. The mesolimbic dopaminergic system has been repeatedly shown to play a critical role in the regulation of alcohol intake. Alcohol administration stimulates extracellular dopamine release in the striatum and the nucleus accumbens of rats (Brand et al., 2013; Imperato and Di Chiara, 1986;Yoshimoto et al., 1992). More specifically it was recently shown that dopamine D3 receptor is necessary for alcohol consumption in mice (Leggio et al., 2014). Furthermore, it was previously shown that, similar to alcohol, pregnenolone also dose-dependently increased dopamine release in the nucleus accumbens of rats (Barrot et al., 1999). It is possible that dopamine release within the reward system in the brain by pregnenolone administration may partially satisfy the need for dopamine in P rats, which have innate dopamine deficiency (McBride et al., 1993). Thus it can be speculated that pregnenolone action on the mesolimbic dopamine neurotransmission substitutes for the alcohol effect on dopamine, as both pregnenolone and alcohol stimulate extracellular dopamine release in the nucleus accumbens.
5. Conclusion The acute systemic administration of pregnenolone reduced alcohol intake and preference in alcohol preferring P rats; however the fact that the chronic systemic administration of pregnenolone lost its effect on the second day of the treatment is a concern for considering this compound for the treatment of alcoholism.
Acknowledgments We thank Drs. Lawrence Lumeng and Richard Bell from the Indiana University for providing alcohol preferring P rats. We also thank Andrew Lee of Duke University for editing the manuscript. This work was supported in part by the National Institute of Alcohol Abuse and Alcoholism at the National Institute of Health (Grant R24-AA015512-02).
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