- Email: [email protected]
The orexin1 receptor antagonist SB-334867 dissociates the motivational properties of alcohol and sucrose in rats
BR A IN RE S EA RCH 1 3 91 ( 20 1 1 ) 5 4 –59
available at www.sciencedirect.com
www.elsevier.com/locate/brainres
Research Report
The orexin1 receptor antagonist SB-334867 dissociates the motivational properties of alcohol and sucrose in rats Bianca Jupp a,1 , Bedrija Krivdic a,b,1 , Elena Krstew a , Andrew J. Lawrence a,c,⁎ a
Florey Neuroscience Institutes, Parkville, Victoria 3010, Australia Department of Psychology, Royal Melbourne Institute of Technology University, Melbourne, Victoria 3000, Australia c Centre for Neuroscience, the University of Melbourne, Parkville, Victoria 3010, Australia b
A R T I C LE I N FO
AB S T R A C T
Article history:
A role for orexin A in mediating the primary and conditioned reinforcing effects of alcohol
Accepted 17 March 2011
has been established. It is unclear however whether the contribution of orexins to alcohol
Available online 23 March 2011
reward occurs independently of effects on appetite and feeding, and whether orexins regulate the motivation to consume alcohol compared to other rewards. To examine this
Keywords:
further here we investigate the effect of the orexin1 receptor antagonist, SB-334867, on self-
Orexin/Hypocretin
administration of alcohol (10% v/v) under both fixed (FR) and progressive ratio (PR)
SB-334867
schedules of reinforcement, and whether this differs from the motivation to administer a
Ethanol
natural food reward, sucrose (0.2–0.7% w/v) in alcohol preferring (iP) rats. SB-334867
Sucrose
treatment significantly reduced responding for both alcohol and sucrose under a FR3
Self-administration
schedule; however, at the same dose, reduced responding and break point for ethanol, but
Motivation
not sucrose, under a PR schedule. These findings for the first time implicate a role for orexins in the motivation to self-administer alcohol and suggest that this may occur independent of any generalized effect on appetitive drive. © 2011 Elsevier B.V. All rights reserved.
1.
Introduction
Orexinergic neurons originate in the hypothalamus and project widely throughout the neuraxis to areas associated with reward, learning and memory, emotion and attention (Peyron et al., 1998). While these neuropeptides are classically involved in a range of homeostatic mechanisms including arousal and feeding (de Lecea et al., 1998), their anatomic distribution suggests that they may contribute to reward processing. Indeed a large volume of literature has recently identified a role, particularly for orexin A, in aspects of motivation and reward of drugs of abuse and natural re-
inforcers (reviewed in Cason et al. (2010); Lawrence (2010); Thompson and Borgland (2010)). In particular, orexin A has been implicated in mediating both the primary and conditioned reinforcing effects of alcohol reward. Studies have demonstrated that central orexin A administration can selectively increase ethanol consumption (Schneider et al., 2007) while orexin1 receptor antagonists reduce ethanol intake in both operant self-administration (Lawrence et al., 2006; Richards et al., 2008) and two-bottle free choice (Moorman and Aston-Jones, 2009) paradigms in rats. Orexin1 receptor blockade has also been shown to essentially inhibit both stress and cue-induced reinstatement of previously
⁎ Corresponding author at: Florey Neuroscience Institutes, Royal Parade, Parkville, Victoria 3010, Australia. Fax: + 61 3 93481707. E-mail address: [email protected] (A.J. Lawrence). 1 Both authors contributed equally to this work. 0006-8993/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2011.03.045
BR A IN RE S E A RCH 1 3 91 ( 20 1 1 ) 5 4 –5 9
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extinguished alcohol-seeking (Jupp et al., 2011; Lawrence et al., 2006; Richards et al., 2008) and exposure to cues and contexts associated with alcohol availability activates orexinergic neurons within the lateral hypothalamus (Dayas et al., 2008; Hamlin et al., 2007). More recently, it has been shown that acute alcohol can increase the expression of orexin A mRNA and mature peptide in this region (Morganstern et al., 2010). Another study however found that blockade of orexin A signaling had mixed effects on the acquisition and expression of a conditioned place preference to ethanol in mice (Voorhees and Cunningham, 2011). In addition, a role for orexins in alcohol drinking, but not seeking, has been suggested (Dhaher et al., 2010). Given this, it is unclear whether the putative role of orexins in alcohol reward occurs independently from effects on feeding related systems. Further, whether orexins also play a role in the motivational aspects of alcohol consumption remains unknown. To further investigate these aspects, here we report a study investigating the effects of orexin1 receptor antagonism on the motivation to consume both alcohol and sucrose under a progressive ratio (PR) schedule of reinforcement. The results of this study for the first time demonstrate a role for orexinergic signaling in the motivation to self-administer alcohol but not sucrose suggesting that the role of orexins in the motivation to consume ethanol may occur independent from a generalized impact on appetitive drive.
2.
Results
2.1. Effect of SB-334867 on ethanol and sucrose self-administration The effect of different doses of SB-334867 on ethanol selfadministration was investigated on a FR3 schedule of reinforcement. In all cases, after the instrumental task was completed, 100 μl of the appropriate fluid was delivered into an adjacent receptacle for oral consumption. Prior to treatment with SB-334867, all rats acquired stable administration of 10% (v/v) ethanol responding on average of 119 ± 6 times per session, equating to a consumption of 0.79 g/kg of ethanol. This is comparable to previous studies in this line of rats (Lawrence et al., 2006). Two way repeated measures ANOVA revealed a main effect SB-334867 treatment (F(3,54) = 101.215, p < 0.0001) but no effect of dose (F(1,18) = 1.380, p > 0.05) for ethanol responding. Planned comparisons analysis revealed that SB-334867 had a significant effect to reduce responding for ethanol at both 5 mg/kg and 10 mg/kg doses (p < 0.00001 for both doses; Fig. 1A); however, no effect was observed for water responding (p > 0.05 for both doses, Fig. 1A). Given the robust effect of the lower dose (5 mg/kg) of SB334867 on alcohol responding, we tested this same dose on sucrose self-administration. Again, all animals acquired a stable level of responding for sucrose (0.2–0.7% w/v) prior to treatment with SB-334867, responding on average 106 ± 7 times per session, titrated to ensure similar response rates between sucrose and ethanol. A main effect of SB-334867 treatment was observed for sucrose self-administration (F(3,21) = 66.678, p < 0.0001), with planned comparisons analysis revealing that this effect was significant for sucrose (p > 0.01; Fig. 1B) but not for water responding (p> 0.05; Fig. 1B).
Fig. 1 – Effect of SB-334867 treatment on FR3 responding for ethanol and sucrose. (A) SB-334867 treatment significantly reduced responding for ethanol at both 5 mg/kg and 10 mg/kg doses on a FR3 schedule of reinforcement. Neither dose had an effect on water responding. *p < 0.05 mixed factorial ANOVA with planned comparisons. (B) SB-334867 treatment also significantly reduced responding for sucrose at 5 mg/kg but had no effect on water responding. *p< 0.05, one way repeated measures ANOVA with planned comparisons.
The effect of SB-334867 treatment on responding for either ethanol or sucrose was also investigated. A main effect of treatment (F(1,16) = 57.508, p < 0.001 and reinforcer (F(1,16) = 7.603, p < 0.05) was observed, with planned comparisons analysis revealing a significantly greater effect of SB-334867 (5 mg/kg) treatment to reduce responding for ethanol when compared to sucrose (p < 0.001). No effect was observed for responding between the two reinforcers following vehicle treatment.
2.2. Effect of SB-334867 on progressive ratio responding for ethanol and sucrose Rats again acquired stable self-administration of 10% ethanol on a FR3 schedule of reinforcement (98 ± 9 responses per session, 0.67 g/kg of ethanol) prior to investigating the effect of SB-334867 treatment on self-administration on a progressive ratio schedule of reinforcement. A main effect of treatment was found on a PR schedule (F(3,51) = 43.365, p < 0.00001; Fig. 2A) and planned comparisons analysis revealed a significant reduction in ethanol responding following SB-334867 treatment (5 mg/kg) when compared to vehicle (p < 0.05, Figs. 2A and 3A); however, no effect was observed for water responding (p > 0.05; Fig. 2A). A main effect of treatment was observed for
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Fig. 2 – Effect of SB-334867 treatment on PR responding and break point for ethanol and sucrose. SB-334867 treatment significantly reduced responding (A) and break point (C) for ethanol but not water on a PR schedule of reinforcement. No effect of SB-334867 treatment was found on PR responding (B) or break point (D) for sucrose or water. *p < 0.05, one way repeated measures ANOVA with planned comparisons.
ethanol break point (F(3,51) = 88.738, p < 0.00001; Fig. 2C), with planned comparisons revealing an effect of SB-334867 treatment to significantly reduce break point for ethanol (p < 0.001) but not water (p > 0.05; Fig. 2C). Planned comparison analysis to investigate the effect of SB-334867 on self-administration of sucrose revealed no effect of treatment (5 mg/kg) on sucrose or water (F(1,7) = 1.431, p > 0.05, Figs. 2B and 3B) responding under the PR schedule, or sucrose or water break point (F(1,7) = 1.143, p > 0.05; Fig. 2D). A comparison between the effect of SB-334867 treatment on active lever responding for either ethanol or sucrose under a PR schedule was also investigated. A main effect of reinforcer was observed (F(1,24) = 5.631, p < 0.05), with planned comparisons analysis revealing a significant reduction in responding for ethanol compared to sucrose following SB-334867 (5 mg/kg) treatment (p < 0.01). No difference was observed between ethanol and sucrose responding following vehicle treatment.
3.
Discussion
We, and others, have previously demonstrated a role for orexin A signaling in the primary and conditioned rewarding effects of alcohol (Lawrence et al., 2006; Moorman and Aston-Jones, 2009; Richards et al., 2008). Despite evidence for orexins in the motivational aspects of other drugs of abuse (Borgland et al., 2009; Espana et al., 2010; Hollander et al., 2008), the role of
orexins in the motivation to consume alcohol is less well established. Further, it is unclear whether the role that orexins play in alcohol reward occurs independent from any more generalized effects on feeding related systems, given the calorific value of alcohol. Here we demonstrate that antagonism of orexin1 receptors reduces both responding and breakpoint for alcohol on a progressive ratio schedule, for the first time implicating orexin A in the motivation to self-administer alcohol. Further, the contribution of orexin A to motivation for alcohol reward appears to occur independently from any nonspecific effect on appetitive drive given that the same dose of SB-334867 had no effect on the breakpoint for sucrose selfadministration.
3.1. Antagonism of orexin 1 receptors reduces self-administration of alcohol and sucrose SB-334867 treatment significantly reduced volitional alcohol self-administration on a FR3 ratio of responding at the two doses investigated (5 mg/kg and 10 mg/kg respectively). This finding is in-keeping with previous reports from our lab and others (Lawrence et al., 2006; Richards et al., 2008) albeit at a lower dose than has been previously reported in iP rats. A notable observation is that previous studies from our laboratory (for example Lawrence et al. (2006)) used SB-334867 obtained as a gift from the manufacturer, while the present study utilized SB-334867 purchased from Tocris. Nevertheless,
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Fig. 3 – Effect of SB-334867 treatment on cumulative PR responding for ethanol and sucrose. SB-334867 had a significant effect to reduce PR responding for ethanol (A) but not sucrose (B). *p < 0.05 repeated measures ANOVA with planned comparisons.
the effects on operant alcohol self-administration observed with both samples are similar, the only difference being the reduced dose required with the current supply of SB-334867. For example, in our original study we found that 20 mg/kg of SB-334867 reduced alcohol responding by approximately 50% (Lawrence et al., 2006), whereas in the current study the batch of SB-334867 used gave a reduction in ethanol responding of approximately 75% at a dose of only 5 mg/kg. Importantly, all of these doses fall within the range that has previously been demonstrated to be effective in vivo (Boutrel et al., 2005; Harris et al., 2005; Richards et al., 2008). The dose of SB-334867 used in the present study is an important point, given the recent observation that systemic treatment with an orexin2 receptor antagonist can reduce alcohol, but not saccharin, responding under a similar FR3 operant schedule (Shoblock et al., 2011). Indeed, our studies on the effects of SB-334867 on selfadministration were all performed in the same colony of inbred rats. Consequently, it is most likely that the effects observed are mediated via antagonism of orexin1 receptors, rather than representing off-target effects of SB-334867. Nevertheless, given the findings by Shoblock and colleagues, combined with the high density of orexin2 receptors in the nucleus accumbens (Trivedi et al., 1998), a putative role for orexin2 receptors in ethanol reward cannot be ruled out. Now that selective orexin2 receptor antagonists are more widely available, future studies will be able to address this issue in greater depth.
57
Antagonism of orexin1 receptors using a 5 mg/kg dose was also found to significantly reduce sucrose self-administration on the same schedule of responding as for alcohol. Antagonism of orexin1 receptors has previously been shown to reduce sucrose consumption at a higher dose than that utilized in the current study (Cason et al., 2010), however other studies have shown SB334867 to be ineffective at reducing sucrose self-administration at doses that regulate alcohol intake in an outbred strain of rat (Richards et al., 2008). It is possible that this discrepancy reflects differences in the reinforcement schedule used (FR1 vs FR3), or the type of administration (sucrose drinking vs sweet pellets) or the use of inbred vs an outbred strain. Similar differences have been observed for self-administration of other natural reinforcers, where doses of 10 and 20 mg/kg SB-334867 are sufficient to reduce responding for high fat chow on a FR1 schedule (Nair et al., 2008) but unsuccessful for normal chow on a PR schedule (Borgland et al., 2009). Interestingly however, our data clearly demonstrate a differential ability of the same dose of SB-334867 (5 mg/kg) to reduce ethanol responding over sucrose responding. Thus, planned comparisons of SB-334867 on FR3 responding for ethanol compared to sucrose indicated a significantly greater effect of treatment on ethanol responding compared to sucrose. These findings provide a possible link between these data and previous studies (e.g. Richards et al. (2008)) suggesting the potential for a window in doses between impacting upon ethanol self-administration over a more naturalistic reward. While no effect was observed for SB-334867 treatment on water responding, given the low level of responding following vehicle treatment it is difficult to ascertain whether this was due to a floor effect. Previous studies however have found that at doses within the range used in the current study, SB-334867 had no effect on EEG measures of arousal and sleep (Ishii et al., 2005). Therefore it is more likely that the reduction in responding for both ethanol and sucrose is due to effects on reward rather than as a result of sedation following treatment.
3.2. SB-334867 treatment reduces progressive ratio responding for alcohol but not sucrose The ability of FR schedules to quantitatively assess the reinforcing potential of a reward and level of disruption of this by a pharmacological challenge is limited (Arnold and Roberts, 1997; Richardson and Roberts, 1996). Progressive ratio schedules, beyond overcoming this limitation, also provide the opportunity to assess the motivational component of selfadministration. Antagonism of orexin1 receptors by SB-334867 resulted in a significant reduction in responding and break point for alcohol when assessed on a PR schedule of reinforcement, in-keeping with our findings and others, that orexins are seemingly involved in mediating the rewarding aspects of alcohol. This finding however also implicates a role for orexins in the motivational drive for alcohol consumption. Previous data have demonstrated an orexinergic component in the motivational properties of a number of drugs of abuse including cocaine and nicotine (Borgland et al., 2009; Hollander et al., 2008); however, this has to date not been assessed for alcohol. As mentioned previously, it is unclear whether the role of orexins in the rewarding effects of alcohol occurs independently from any more general effects on appetite and feeding behavior. To investigate this we also examined the effect of SB-334867
58
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treatment on PR for sucrose; however, we found no effect on responding or break point, at the same dose that was capable of dramatically reducing the breakpoint for alcohol responding. This finding is contrary to previous studies investigating PR responding for sucrose pellets and other natural rewards such as high fat chow following SB-334867 administration (Borgland et al., 2009; Choi et al., 2010; Espana et al., 2010). It should be noted however that these studies were conducted with higher doses of SB-334867 than used in the current study, suggesting that the lack of effect in our hands may be a consequence of antagonist dose. There is some suggestion that at high doses SB334867 may also have effects to block orexin2 receptors (Scammell and Winrow, 2011). Indeed there is evidence that orexin2 receptor antagonism is able to reduce ethanol selfadministration independent on any effects on saccharin selfadministration (Shoblock et al., 2011). It has previously been demonstrated however that low doses of SB-334867, similar to those used in the current study, have a selective effect on motivated responding for highly-salient reinforcers independent of effects on more general reward processes relative to hunger and satiety given that no effect of orexin1 receptor antagonism has been observed for PR responding for normal chow or consumption of freely available food in rats (Borgland et al., 2009; Hollander et al., 2008). Indeed our finding that SB-334867 reduced responding significantly more for ethanol when compared with sucrose on both FR3 and PR schedules of reinforcement further supports this. Given this, the current study suggests that the contribution of orexins to the motivation to consume alcohol likely occurs independent from a nonspecific effect on appetitive drive in general. Moreover, these data also provide evidence for the potential existence of a “window” between a role for orexins in motivational properties of alcohol compared to a natural reinforcer.
4.
Experimental procedures
All experiments were performed in accordance with the Prevention of Cruelty to Animals Act, 1986 under the guidelines of the National Health and Medical Research Council Code of Practice for the Care and Use of Animals for Experimental Purposes in Australia. Inbred alcohol-preferring (iP) rats were obtained from the breeding colony at the Florey Neuroscience Institutes, University of Melbourne. Parental stock had previously been obtained from Professor T.K. Li (while at Indiana University, Indianapolis). Rats were maintained on a 12 h light–dark cycle with ad libitum access to food and water. Adult age-matched male iP rats were trained to either ethanol (10% v/v; n = 38) or sucrose (0.2–0.7% w/v; n =16) in daily 20 min operant sessions in sound-attenuated operant chambers (Med Associates, VT, USA) using a fixed ratio 3 (FR3) administration schedule as previously described (Cowen et al., 2005) conducted 5 days per week. The concentration of sucrose was titrated for each animal to result in a similar level of responding for sucrose when compared to ethanol. Reward availability (0.1 ml of either ethanol (10% v/v) or sucrose (0.2–0.7% w/v), the latter titrated so that sucrose responding was similar in terms of lever presses to that for ethanol) was conditioned with the presence of an olfactory cue (S+; two drops of vanilla essence
placed in a cap directly below the active lever) and a one-second light stimulus (CS+), located over the active lever, illuminated when the FR3 requirement was met. Both active and inactive lever responses were recorded during each session. Rats that achieved levels of active lever responding greater than 90 with less than 10% variability were included in the study. Progressive ratio (PR) responding was assessed during a 90 min session, using a reinforcement schedule previously described (Besheer et al., 2008), where the response requirement for reward delivery increased by 1 following each reward. Break point was defined as the highest response ratio completed (which was also equal to the total number of rewards received).
4.1. Effect of SB-33867 on ethanol and sucrose self-administration The effect of SB-334867 on responding on a FR3 schedule of reward was assessed in a subset of animals over two consecutive days following acquisition of stable responding, with rats receiving vehicle treatment (1.5% DMSO i.p.) the day prior to SB-334867 treatment (Tocris, Bristol UK) (Ethanol: 5 mg/ kg i.p.; n = 10, or 10 mg/kg i.p.; n = 10. Sucrose: 5 mg/kg i.p. only; n = 8. N.B. Sucrose was only tested at 5 mg/kg as no significant difference between doses was found for ethanol responding). Rats were injected 30 min prior to each session. Both active and inactive lever responses were recorded during each session.
4.2. Effect of SB-334867 on Ethanol and sucrose progressive ratio responding The effect of SB-334867 on progressive ratio responding was assessed in the remainder of the animals. After acquisition of stable ethanol responding, using a counter-balanced design, animals were subjected to a PR session once per week over a three week period, consisting of treatment with either vehicle or SB-334867 (5 mg/kg i.p.; n = 18). Sucrose trained animals were also subjected to vehicle and SB-334867 treatment (5 mg/ kg i.p.; n = 8). Both active and inactive lever responses and associated break points were recorded during each session.
4.3.
Statistical analysis
Differences in FR3 responding for ethanol following SB-334867 administration were assessed using a mixed factorial ANOVA with planned comparison analysis for dose and treatment. The effect of SB-334867 treatment on self-administration of sucrose, progressive ratio responding and break point for sucrose and ethanol was assessed using one way repeated measures ANOVA with planned comparisons for water or ethanol responding.
Acknowledgments These studies were funded by the National Health and Medical Research Council of Australia (project grant 508976) of which AJL is a Senior Fellow (454303); the Pratt and Besen Foundations and the Victorian Government's Operational Infrastructure Support Program.
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available at www.sciencedirect.com
www.elsevier.com/locate/brainres
Research Report
The orexin1 receptor antagonist SB-334867 dissociates the motivational properties of alcohol and sucrose in rats Bianca Jupp a,1 , Bedrija Krivdic a,b,1 , Elena Krstew a , Andrew J. Lawrence a,c,⁎ a
Florey Neuroscience Institutes, Parkville, Victoria 3010, Australia Department of Psychology, Royal Melbourne Institute of Technology University, Melbourne, Victoria 3000, Australia c Centre for Neuroscience, the University of Melbourne, Parkville, Victoria 3010, Australia b
A R T I C LE I N FO
AB S T R A C T
Article history:
A role for orexin A in mediating the primary and conditioned reinforcing effects of alcohol
Accepted 17 March 2011
has been established. It is unclear however whether the contribution of orexins to alcohol
Available online 23 March 2011
reward occurs independently of effects on appetite and feeding, and whether orexins regulate the motivation to consume alcohol compared to other rewards. To examine this
Keywords:
further here we investigate the effect of the orexin1 receptor antagonist, SB-334867, on self-
Orexin/Hypocretin
administration of alcohol (10% v/v) under both fixed (FR) and progressive ratio (PR)
SB-334867
schedules of reinforcement, and whether this differs from the motivation to administer a
Ethanol
natural food reward, sucrose (0.2–0.7% w/v) in alcohol preferring (iP) rats. SB-334867
Sucrose
treatment significantly reduced responding for both alcohol and sucrose under a FR3
Self-administration
schedule; however, at the same dose, reduced responding and break point for ethanol, but
Motivation
not sucrose, under a PR schedule. These findings for the first time implicate a role for orexins in the motivation to self-administer alcohol and suggest that this may occur independent of any generalized effect on appetitive drive. © 2011 Elsevier B.V. All rights reserved.
1.
Introduction
Orexinergic neurons originate in the hypothalamus and project widely throughout the neuraxis to areas associated with reward, learning and memory, emotion and attention (Peyron et al., 1998). While these neuropeptides are classically involved in a range of homeostatic mechanisms including arousal and feeding (de Lecea et al., 1998), their anatomic distribution suggests that they may contribute to reward processing. Indeed a large volume of literature has recently identified a role, particularly for orexin A, in aspects of motivation and reward of drugs of abuse and natural re-
inforcers (reviewed in Cason et al. (2010); Lawrence (2010); Thompson and Borgland (2010)). In particular, orexin A has been implicated in mediating both the primary and conditioned reinforcing effects of alcohol reward. Studies have demonstrated that central orexin A administration can selectively increase ethanol consumption (Schneider et al., 2007) while orexin1 receptor antagonists reduce ethanol intake in both operant self-administration (Lawrence et al., 2006; Richards et al., 2008) and two-bottle free choice (Moorman and Aston-Jones, 2009) paradigms in rats. Orexin1 receptor blockade has also been shown to essentially inhibit both stress and cue-induced reinstatement of previously
⁎ Corresponding author at: Florey Neuroscience Institutes, Royal Parade, Parkville, Victoria 3010, Australia. Fax: + 61 3 93481707. E-mail address: [email protected] (A.J. Lawrence). 1 Both authors contributed equally to this work. 0006-8993/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2011.03.045
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extinguished alcohol-seeking (Jupp et al., 2011; Lawrence et al., 2006; Richards et al., 2008) and exposure to cues and contexts associated with alcohol availability activates orexinergic neurons within the lateral hypothalamus (Dayas et al., 2008; Hamlin et al., 2007). More recently, it has been shown that acute alcohol can increase the expression of orexin A mRNA and mature peptide in this region (Morganstern et al., 2010). Another study however found that blockade of orexin A signaling had mixed effects on the acquisition and expression of a conditioned place preference to ethanol in mice (Voorhees and Cunningham, 2011). In addition, a role for orexins in alcohol drinking, but not seeking, has been suggested (Dhaher et al., 2010). Given this, it is unclear whether the putative role of orexins in alcohol reward occurs independently from effects on feeding related systems. Further, whether orexins also play a role in the motivational aspects of alcohol consumption remains unknown. To further investigate these aspects, here we report a study investigating the effects of orexin1 receptor antagonism on the motivation to consume both alcohol and sucrose under a progressive ratio (PR) schedule of reinforcement. The results of this study for the first time demonstrate a role for orexinergic signaling in the motivation to self-administer alcohol but not sucrose suggesting that the role of orexins in the motivation to consume ethanol may occur independent from a generalized impact on appetitive drive.
2.
Results
2.1. Effect of SB-334867 on ethanol and sucrose self-administration The effect of different doses of SB-334867 on ethanol selfadministration was investigated on a FR3 schedule of reinforcement. In all cases, after the instrumental task was completed, 100 μl of the appropriate fluid was delivered into an adjacent receptacle for oral consumption. Prior to treatment with SB-334867, all rats acquired stable administration of 10% (v/v) ethanol responding on average of 119 ± 6 times per session, equating to a consumption of 0.79 g/kg of ethanol. This is comparable to previous studies in this line of rats (Lawrence et al., 2006). Two way repeated measures ANOVA revealed a main effect SB-334867 treatment (F(3,54) = 101.215, p < 0.0001) but no effect of dose (F(1,18) = 1.380, p > 0.05) for ethanol responding. Planned comparisons analysis revealed that SB-334867 had a significant effect to reduce responding for ethanol at both 5 mg/kg and 10 mg/kg doses (p < 0.00001 for both doses; Fig. 1A); however, no effect was observed for water responding (p > 0.05 for both doses, Fig. 1A). Given the robust effect of the lower dose (5 mg/kg) of SB334867 on alcohol responding, we tested this same dose on sucrose self-administration. Again, all animals acquired a stable level of responding for sucrose (0.2–0.7% w/v) prior to treatment with SB-334867, responding on average 106 ± 7 times per session, titrated to ensure similar response rates between sucrose and ethanol. A main effect of SB-334867 treatment was observed for sucrose self-administration (F(3,21) = 66.678, p < 0.0001), with planned comparisons analysis revealing that this effect was significant for sucrose (p > 0.01; Fig. 1B) but not for water responding (p> 0.05; Fig. 1B).
Fig. 1 – Effect of SB-334867 treatment on FR3 responding for ethanol and sucrose. (A) SB-334867 treatment significantly reduced responding for ethanol at both 5 mg/kg and 10 mg/kg doses on a FR3 schedule of reinforcement. Neither dose had an effect on water responding. *p < 0.05 mixed factorial ANOVA with planned comparisons. (B) SB-334867 treatment also significantly reduced responding for sucrose at 5 mg/kg but had no effect on water responding. *p< 0.05, one way repeated measures ANOVA with planned comparisons.
The effect of SB-334867 treatment on responding for either ethanol or sucrose was also investigated. A main effect of treatment (F(1,16) = 57.508, p < 0.001 and reinforcer (F(1,16) = 7.603, p < 0.05) was observed, with planned comparisons analysis revealing a significantly greater effect of SB-334867 (5 mg/kg) treatment to reduce responding for ethanol when compared to sucrose (p < 0.001). No effect was observed for responding between the two reinforcers following vehicle treatment.
2.2. Effect of SB-334867 on progressive ratio responding for ethanol and sucrose Rats again acquired stable self-administration of 10% ethanol on a FR3 schedule of reinforcement (98 ± 9 responses per session, 0.67 g/kg of ethanol) prior to investigating the effect of SB-334867 treatment on self-administration on a progressive ratio schedule of reinforcement. A main effect of treatment was found on a PR schedule (F(3,51) = 43.365, p < 0.00001; Fig. 2A) and planned comparisons analysis revealed a significant reduction in ethanol responding following SB-334867 treatment (5 mg/kg) when compared to vehicle (p < 0.05, Figs. 2A and 3A); however, no effect was observed for water responding (p > 0.05; Fig. 2A). A main effect of treatment was observed for
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Fig. 2 – Effect of SB-334867 treatment on PR responding and break point for ethanol and sucrose. SB-334867 treatment significantly reduced responding (A) and break point (C) for ethanol but not water on a PR schedule of reinforcement. No effect of SB-334867 treatment was found on PR responding (B) or break point (D) for sucrose or water. *p < 0.05, one way repeated measures ANOVA with planned comparisons.
ethanol break point (F(3,51) = 88.738, p < 0.00001; Fig. 2C), with planned comparisons revealing an effect of SB-334867 treatment to significantly reduce break point for ethanol (p < 0.001) but not water (p > 0.05; Fig. 2C). Planned comparison analysis to investigate the effect of SB-334867 on self-administration of sucrose revealed no effect of treatment (5 mg/kg) on sucrose or water (F(1,7) = 1.431, p > 0.05, Figs. 2B and 3B) responding under the PR schedule, or sucrose or water break point (F(1,7) = 1.143, p > 0.05; Fig. 2D). A comparison between the effect of SB-334867 treatment on active lever responding for either ethanol or sucrose under a PR schedule was also investigated. A main effect of reinforcer was observed (F(1,24) = 5.631, p < 0.05), with planned comparisons analysis revealing a significant reduction in responding for ethanol compared to sucrose following SB-334867 (5 mg/kg) treatment (p < 0.01). No difference was observed between ethanol and sucrose responding following vehicle treatment.
3.
Discussion
We, and others, have previously demonstrated a role for orexin A signaling in the primary and conditioned rewarding effects of alcohol (Lawrence et al., 2006; Moorman and Aston-Jones, 2009; Richards et al., 2008). Despite evidence for orexins in the motivational aspects of other drugs of abuse (Borgland et al., 2009; Espana et al., 2010; Hollander et al., 2008), the role of
orexins in the motivation to consume alcohol is less well established. Further, it is unclear whether the role that orexins play in alcohol reward occurs independent from any more generalized effects on feeding related systems, given the calorific value of alcohol. Here we demonstrate that antagonism of orexin1 receptors reduces both responding and breakpoint for alcohol on a progressive ratio schedule, for the first time implicating orexin A in the motivation to self-administer alcohol. Further, the contribution of orexin A to motivation for alcohol reward appears to occur independently from any nonspecific effect on appetitive drive given that the same dose of SB-334867 had no effect on the breakpoint for sucrose selfadministration.
3.1. Antagonism of orexin 1 receptors reduces self-administration of alcohol and sucrose SB-334867 treatment significantly reduced volitional alcohol self-administration on a FR3 ratio of responding at the two doses investigated (5 mg/kg and 10 mg/kg respectively). This finding is in-keeping with previous reports from our lab and others (Lawrence et al., 2006; Richards et al., 2008) albeit at a lower dose than has been previously reported in iP rats. A notable observation is that previous studies from our laboratory (for example Lawrence et al. (2006)) used SB-334867 obtained as a gift from the manufacturer, while the present study utilized SB-334867 purchased from Tocris. Nevertheless,
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Fig. 3 – Effect of SB-334867 treatment on cumulative PR responding for ethanol and sucrose. SB-334867 had a significant effect to reduce PR responding for ethanol (A) but not sucrose (B). *p < 0.05 repeated measures ANOVA with planned comparisons.
the effects on operant alcohol self-administration observed with both samples are similar, the only difference being the reduced dose required with the current supply of SB-334867. For example, in our original study we found that 20 mg/kg of SB-334867 reduced alcohol responding by approximately 50% (Lawrence et al., 2006), whereas in the current study the batch of SB-334867 used gave a reduction in ethanol responding of approximately 75% at a dose of only 5 mg/kg. Importantly, all of these doses fall within the range that has previously been demonstrated to be effective in vivo (Boutrel et al., 2005; Harris et al., 2005; Richards et al., 2008). The dose of SB-334867 used in the present study is an important point, given the recent observation that systemic treatment with an orexin2 receptor antagonist can reduce alcohol, but not saccharin, responding under a similar FR3 operant schedule (Shoblock et al., 2011). Indeed, our studies on the effects of SB-334867 on selfadministration were all performed in the same colony of inbred rats. Consequently, it is most likely that the effects observed are mediated via antagonism of orexin1 receptors, rather than representing off-target effects of SB-334867. Nevertheless, given the findings by Shoblock and colleagues, combined with the high density of orexin2 receptors in the nucleus accumbens (Trivedi et al., 1998), a putative role for orexin2 receptors in ethanol reward cannot be ruled out. Now that selective orexin2 receptor antagonists are more widely available, future studies will be able to address this issue in greater depth.
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Antagonism of orexin1 receptors using a 5 mg/kg dose was also found to significantly reduce sucrose self-administration on the same schedule of responding as for alcohol. Antagonism of orexin1 receptors has previously been shown to reduce sucrose consumption at a higher dose than that utilized in the current study (Cason et al., 2010), however other studies have shown SB334867 to be ineffective at reducing sucrose self-administration at doses that regulate alcohol intake in an outbred strain of rat (Richards et al., 2008). It is possible that this discrepancy reflects differences in the reinforcement schedule used (FR1 vs FR3), or the type of administration (sucrose drinking vs sweet pellets) or the use of inbred vs an outbred strain. Similar differences have been observed for self-administration of other natural reinforcers, where doses of 10 and 20 mg/kg SB-334867 are sufficient to reduce responding for high fat chow on a FR1 schedule (Nair et al., 2008) but unsuccessful for normal chow on a PR schedule (Borgland et al., 2009). Interestingly however, our data clearly demonstrate a differential ability of the same dose of SB-334867 (5 mg/kg) to reduce ethanol responding over sucrose responding. Thus, planned comparisons of SB-334867 on FR3 responding for ethanol compared to sucrose indicated a significantly greater effect of treatment on ethanol responding compared to sucrose. These findings provide a possible link between these data and previous studies (e.g. Richards et al. (2008)) suggesting the potential for a window in doses between impacting upon ethanol self-administration over a more naturalistic reward. While no effect was observed for SB-334867 treatment on water responding, given the low level of responding following vehicle treatment it is difficult to ascertain whether this was due to a floor effect. Previous studies however have found that at doses within the range used in the current study, SB-334867 had no effect on EEG measures of arousal and sleep (Ishii et al., 2005). Therefore it is more likely that the reduction in responding for both ethanol and sucrose is due to effects on reward rather than as a result of sedation following treatment.
3.2. SB-334867 treatment reduces progressive ratio responding for alcohol but not sucrose The ability of FR schedules to quantitatively assess the reinforcing potential of a reward and level of disruption of this by a pharmacological challenge is limited (Arnold and Roberts, 1997; Richardson and Roberts, 1996). Progressive ratio schedules, beyond overcoming this limitation, also provide the opportunity to assess the motivational component of selfadministration. Antagonism of orexin1 receptors by SB-334867 resulted in a significant reduction in responding and break point for alcohol when assessed on a PR schedule of reinforcement, in-keeping with our findings and others, that orexins are seemingly involved in mediating the rewarding aspects of alcohol. This finding however also implicates a role for orexins in the motivational drive for alcohol consumption. Previous data have demonstrated an orexinergic component in the motivational properties of a number of drugs of abuse including cocaine and nicotine (Borgland et al., 2009; Hollander et al., 2008); however, this has to date not been assessed for alcohol. As mentioned previously, it is unclear whether the role of orexins in the rewarding effects of alcohol occurs independently from any more general effects on appetite and feeding behavior. To investigate this we also examined the effect of SB-334867
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treatment on PR for sucrose; however, we found no effect on responding or break point, at the same dose that was capable of dramatically reducing the breakpoint for alcohol responding. This finding is contrary to previous studies investigating PR responding for sucrose pellets and other natural rewards such as high fat chow following SB-334867 administration (Borgland et al., 2009; Choi et al., 2010; Espana et al., 2010). It should be noted however that these studies were conducted with higher doses of SB-334867 than used in the current study, suggesting that the lack of effect in our hands may be a consequence of antagonist dose. There is some suggestion that at high doses SB334867 may also have effects to block orexin2 receptors (Scammell and Winrow, 2011). Indeed there is evidence that orexin2 receptor antagonism is able to reduce ethanol selfadministration independent on any effects on saccharin selfadministration (Shoblock et al., 2011). It has previously been demonstrated however that low doses of SB-334867, similar to those used in the current study, have a selective effect on motivated responding for highly-salient reinforcers independent of effects on more general reward processes relative to hunger and satiety given that no effect of orexin1 receptor antagonism has been observed for PR responding for normal chow or consumption of freely available food in rats (Borgland et al., 2009; Hollander et al., 2008). Indeed our finding that SB-334867 reduced responding significantly more for ethanol when compared with sucrose on both FR3 and PR schedules of reinforcement further supports this. Given this, the current study suggests that the contribution of orexins to the motivation to consume alcohol likely occurs independent from a nonspecific effect on appetitive drive in general. Moreover, these data also provide evidence for the potential existence of a “window” between a role for orexins in motivational properties of alcohol compared to a natural reinforcer.
4.
Experimental procedures
All experiments were performed in accordance with the Prevention of Cruelty to Animals Act, 1986 under the guidelines of the National Health and Medical Research Council Code of Practice for the Care and Use of Animals for Experimental Purposes in Australia. Inbred alcohol-preferring (iP) rats were obtained from the breeding colony at the Florey Neuroscience Institutes, University of Melbourne. Parental stock had previously been obtained from Professor T.K. Li (while at Indiana University, Indianapolis). Rats were maintained on a 12 h light–dark cycle with ad libitum access to food and water. Adult age-matched male iP rats were trained to either ethanol (10% v/v; n = 38) or sucrose (0.2–0.7% w/v; n =16) in daily 20 min operant sessions in sound-attenuated operant chambers (Med Associates, VT, USA) using a fixed ratio 3 (FR3) administration schedule as previously described (Cowen et al., 2005) conducted 5 days per week. The concentration of sucrose was titrated for each animal to result in a similar level of responding for sucrose when compared to ethanol. Reward availability (0.1 ml of either ethanol (10% v/v) or sucrose (0.2–0.7% w/v), the latter titrated so that sucrose responding was similar in terms of lever presses to that for ethanol) was conditioned with the presence of an olfactory cue (S+; two drops of vanilla essence
placed in a cap directly below the active lever) and a one-second light stimulus (CS+), located over the active lever, illuminated when the FR3 requirement was met. Both active and inactive lever responses were recorded during each session. Rats that achieved levels of active lever responding greater than 90 with less than 10% variability were included in the study. Progressive ratio (PR) responding was assessed during a 90 min session, using a reinforcement schedule previously described (Besheer et al., 2008), where the response requirement for reward delivery increased by 1 following each reward. Break point was defined as the highest response ratio completed (which was also equal to the total number of rewards received).
4.1. Effect of SB-33867 on ethanol and sucrose self-administration The effect of SB-334867 on responding on a FR3 schedule of reward was assessed in a subset of animals over two consecutive days following acquisition of stable responding, with rats receiving vehicle treatment (1.5% DMSO i.p.) the day prior to SB-334867 treatment (Tocris, Bristol UK) (Ethanol: 5 mg/ kg i.p.; n = 10, or 10 mg/kg i.p.; n = 10. Sucrose: 5 mg/kg i.p. only; n = 8. N.B. Sucrose was only tested at 5 mg/kg as no significant difference between doses was found for ethanol responding). Rats were injected 30 min prior to each session. Both active and inactive lever responses were recorded during each session.
4.2. Effect of SB-334867 on Ethanol and sucrose progressive ratio responding The effect of SB-334867 on progressive ratio responding was assessed in the remainder of the animals. After acquisition of stable ethanol responding, using a counter-balanced design, animals were subjected to a PR session once per week over a three week period, consisting of treatment with either vehicle or SB-334867 (5 mg/kg i.p.; n = 18). Sucrose trained animals were also subjected to vehicle and SB-334867 treatment (5 mg/ kg i.p.; n = 8). Both active and inactive lever responses and associated break points were recorded during each session.
4.3.
Statistical analysis
Differences in FR3 responding for ethanol following SB-334867 administration were assessed using a mixed factorial ANOVA with planned comparison analysis for dose and treatment. The effect of SB-334867 treatment on self-administration of sucrose, progressive ratio responding and break point for sucrose and ethanol was assessed using one way repeated measures ANOVA with planned comparisons for water or ethanol responding.
Acknowledgments These studies were funded by the National Health and Medical Research Council of Australia (project grant 508976) of which AJL is a Senior Fellow (454303); the Pratt and Besen Foundations and the Victorian Government's Operational Infrastructure Support Program.
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