**3. The role of the Delta Opioid Peptide Receptor (DOP-R) in ethanol consumption and seeking**

#### **3.1 Ethanol consumption in DOP-R knockout mice**

Studies in mice with a genetic deletion of the DOP-R (C57BL/6 or mixed C57/129sv background) have increased levels of ethanol intake when compared to wild-type mice (Roberts et al., 2001; van Rijn & Whistler, 2009) (Table 1). This suggests that decreased DOP-R activity is associated with high ethanol consumption. This may be related to the increased anxiety levels in DOP-R knockout mice as anxiety-like responses were reversed by the consumption of ethanol in the DOP-R knockout mice (Roberts et al., 2001).

#### **3.2 DOP-R activation and ethanol consumption**

Ethanol has been shown to stimulate the activity of the endogenous opioids, -endorphins and enkephalins, which target the MOP-R and DOP-R, respectively, to increase basal dopamine release in the mesolimbic pathway (Herz, 1997). Activation of the enkephalinergic system and occupation of DOP-Rs has been proposed to be important for the maintenance of high voluntary ethanol intake (Froehlich et al., 1991). The enkephalinase inhibitor, thiorphan, which potentiates the actions of the endogenous opioids for the DOP-R by protecting the enkephalins from degradation, increased ethanol, but not water, intake in alcohol-preferring rats (Froehlich et al., 1991).

Recently, further studies have examined the effects of activation of the DOP-R on ethanol consumption using various DOP-R agonists (Barson et al., 2009; Barson et al., 2010; van Rijn et al., 2010) (Table 1). In support of the earlier studies, systemic administration of the DOP-R agonist, SNC80, in mice and microinjections of the DOP-R agonist, DALA (7-14 nM), into the NAc and hypothalamic paraventricular nucleus (HPN) of rats lead to increased ethanol consumption (Barson et al., 2009; Barson et al., 2010; van Rijn et al., 2010). In mice trained using the 10% ethanol 4 hr limited access paradigm, SNC80 increased ethanol consumption (van Rijn et al., 2010). In rats trained to consume up to 7% ethanol for 12 h each day with incremental increases in the concentration of ethanol every four days, the administration of DALA into the NAc selectively increased ethanol consumption over food and water (Barson et al., 2009). Similarly, the effects of DALA administered into the HPN were selective for ethanol over food and water consumption (Barson et al., 2010). DOP-R agonists have also been shown to reduce ethanol consumption in rats (Margolis et al., 2008) and mice (van Rijn & Whistler, 2009). Systemic administration of the selective DOP-R agonist, TAN67, reduced voluntary ethanol intake in mice using a 4 hr limited ethanol access paradigm (van Rijn & Whistler, 2009). Following administration of the DOP-R agonist, DPDPE (10 mM), into the ventral tegmental area (VTA) of rats using continuous access to 10% ethanol paradigm, ethanol intake was reduced in low but not high ethanol consuming rats (Margolis et al., 2008). It is hypothesized that DOP-Rs in the VTA play a protective role against elevated ethanol consumption (Margolis et al., 2008).

ethanol in rhesus monkeys, except when it was co-administered with naltrexone (Williams & Woods, 1998). These studies suggest that naltrexone's ability to reduce ethanol

Studies in mice with a genetic deletion of the DOP-R (C57BL/6 or mixed C57/129sv background) have increased levels of ethanol intake when compared to wild-type mice (Roberts et al., 2001; van Rijn & Whistler, 2009) (Table 1). This suggests that decreased DOP-R activity is associated with high ethanol consumption. This may be related to the increased anxiety levels in DOP-R knockout mice as anxiety-like responses were reversed by the

Ethanol has been shown to stimulate the activity of the endogenous opioids, -endorphins and enkephalins, which target the MOP-R and DOP-R, respectively, to increase basal dopamine release in the mesolimbic pathway (Herz, 1997). Activation of the enkephalinergic system and occupation of DOP-Rs has been proposed to be important for the maintenance of high voluntary ethanol intake (Froehlich et al., 1991). The enkephalinase inhibitor, thiorphan, which potentiates the actions of the endogenous opioids for the DOP-R by protecting the enkephalins from degradation, increased ethanol, but not water, intake in

Recently, further studies have examined the effects of activation of the DOP-R on ethanol consumption using various DOP-R agonists (Barson et al., 2009; Barson et al., 2010; van Rijn et al., 2010) (Table 1). In support of the earlier studies, systemic administration of the DOP-R agonist, SNC80, in mice and microinjections of the DOP-R agonist, DALA (7-14 nM), into the NAc and hypothalamic paraventricular nucleus (HPN) of rats lead to increased ethanol consumption (Barson et al., 2009; Barson et al., 2010; van Rijn et al., 2010). In mice trained using the 10% ethanol 4 hr limited access paradigm, SNC80 increased ethanol consumption (van Rijn et al., 2010). In rats trained to consume up to 7% ethanol for 12 h each day with incremental increases in the concentration of ethanol every four days, the administration of DALA into the NAc selectively increased ethanol consumption over food and water (Barson et al., 2009). Similarly, the effects of DALA administered into the HPN were selective for ethanol over food and water consumption (Barson et al., 2010). DOP-R agonists have also been shown to reduce ethanol consumption in rats (Margolis et al., 2008) and mice (van Rijn & Whistler, 2009). Systemic administration of the selective DOP-R agonist, TAN67, reduced voluntary ethanol intake in mice using a 4 hr limited ethanol access paradigm (van Rijn & Whistler, 2009). Following administration of the DOP-R agonist, DPDPE (10 mM), into the ventral tegmental area (VTA) of rats using continuous access to 10% ethanol paradigm, ethanol intake was reduced in low but not high ethanol consuming rats (Margolis et al., 2008). It is hypothesized that DOP-Rs in the VTA play a protective role against elevated

**3. The role of the Delta Opioid Peptide Receptor (DOP-R) in ethanol** 

consumption of ethanol in the DOP-R knockout mice (Roberts et al., 2001).

consumption and seeking may be mediated via a non-MOP-R.

**3.1 Ethanol consumption in DOP-R knockout mice** 

**3.2 DOP-R activation and ethanol consumption** 

alcohol-preferring rats (Froehlich et al., 1991).

ethanol consumption (Margolis et al., 2008).

**consumption and seeking** 

#### **3.3 Ethanol consumption and seeking with DOP-R antagonists**

The administration of DOP-R antagonists decreases ethanol consumption and seeking in rats (Franck et al., 1998; Hyytia & Kiianmaa, 2001; June et al., 1999; Krishnan-Sarin et al., 1995b; Marinelli et al., 2009; Nielsen et al., 2011; Nielsen et al., 2008). However, DOP-R antagonists also have been shown to not affect (Ingman et al., 2003; Stromberg et al., 1998a) or increase ethanol intake (Margolis et al., 2008) (Table 1). Factors likely contributing to these disparities include the length of ethanol exposure, the route of drug administration, different types of drinking models that induce low, moderate and high ethanol consumption, the rodent strains used (such as alcohol-preferring rats) and potential different roles of subtypes of the DOP-R on ethanol-mediated behaviors.


Table 1. The roles of opioid receptors in ethanol consumption and seeking in rats and mice. 1. Roberts et al., 2000; 2. Hall et al., 2001; 3. Becker et al., 2002; 4. Roberts et al., 2001; 5. Kovacs et al., 2005; 6. Critcher et al., 1983; 7. Stromberg et al., 1998a; 8. Gardell et al., 1996; 9. Hyytia & Kiianmaa, 2001; 10. Franck et al., 1998; 11. Ciccocioppo et al., 2002a; 12. June et al., 1999; 13. Ingman et al., 2003; 14. Krishnan-Sarin et al., 1995a; 15. Krishnan-Sarin et al., 1995b; 16. Mitchell et al., 2005; 17. Holter et al., 2000; 18. Reid & Hunter, 1984; 19. Lindholm et al., 2001. 20. Nielsen et al., 2008; 21. Margolis et al., 2008; 22. Barson et al., 2009; 23. Barson et al., 2010; 24. van Rijn & Whistler, 2009; 25. van Rijn et al., 2010; 26. Walker et al., 2011 ; 27. Walker & Koob, 2008; 28. Marinelli et al., 2009; 29. Nielsen et al., 2011; 30. Le et al., 1999; 31. Sabino et al., 2011; 32. Kuzmin et al., 2007; 33. Ciccocioppo et al., 2007; 34. Ciccocioppo et al., 2003; 35. Ciccocioppo et al., 1999; 36. Simms et al., 2008; 37. Economidou et al., 2008; 38. Ciccocioppo et al., 2002b. 39. Martin-Fardon et al., 2000; 40. Cifani et al., 2006 41. Ciccocioppo et al., 2004; 42. Sakoori & Murphy, 2008; 43. Sabino et al., 2009a; 44. Sabino et al., 2009b; 45. Economidou et al., 2006; 46. Sandi et al., 1990.; 47. Stromberg et al., 2002; 48. Stromberg et al., 1998b.

The Role of Delta Opioid Receptors in Ethanol Consumption

and Seeking: Implications for New Treatments for Alcohol Use Disorders 211

for up to 28 days compared with vehicle-treated rats (Nielsen et al., 2008). When the daily administrations of SoRI-9409 were ceased after 28 days, ethanol consumption was maintained at approximately one-half of the baseline drinking levels of post-vehicle-treated rats, for a further 28 days. Cessation of SoRI-9409 treatment did not result in any significant escalation in ethanol consumption suggesting that multiple administrations of SoRI-9409 produce long-lasting and permanent effects on the modulation of ethanol consumption. This further suggests that inhibition of DOP-Rs directly reduces ethanol intake during the

A major problem in treating AUDs is the high rate of relapse, which can be triggered by reexposure to cues or an environment previously associated with alcohol use and also by stress. An effective pharmacological treatment for AUDs would ideally prevent relapse of alcohol-seeking in addition to reducing consumption of ethanol. Naltrexone reduces alcohol- and cue-induced reinstatement, but not foot-shock stress-induced reinstatement of ethanol-seeking in rodents (Ciccocioppo et al., 2002a; Le et al., 1999; Liu & Weiss, 2002). It has been shown that DOP-Rs, rather than MOP-Rs, are important for cue-induced reinstatement of ethanol-seeking, as the DOP-R antagonist, naltrindole, reduces cue-induced reinstatement of ethanol-seeking behavior in rodents more effectively than the MOP-R selective antagonists, naloxonazine or CTOP (Ciccocioppo et al., 2002a; Marinelli et al., 2009). Furthermore, recent studies show that the DOP-R antagonist, SoRI-9409, effectively and dose-dependently reduces yohimbine stress-induced reinstatement of ethanol-seeking in rats (Nielsen et al., 2011). This study further demonstrated that the DOP-R plays a greater role than MOP-R or KOP-R in yohimbine stress-induced reinstatement of ethanol-seeking in rats as TAN67- and Deltorphin II-mediated DOP-R activity, using the [35S]GTPS coupling assay in midbrain membranes, were increased in membranes of yohimbine-treated ethanolextinguished rats compared to vehicle-treated rats. Moreover, the increase in DOP-Rmediated [35S] GTPγS stimulation observed in yohimbine-treated ethanol-trained rats was absent in naive (non ethanol-trained) rats, suggesting that a history of ethanol selfadministration plays an important role in the regulation of DOP-R signaling. In contrast with DOP-R activity, there were no changes in DAMGO-mediated MOP-R or (–)U50488 mediated KOP-R activity in ethanol-trained rats, further supporting studies showing DOP-Rs rather than MOP-Rs are important for reinstatement of ethanol-seeking (Ciccocioppo et

Although one DOP-R gene has been cloned (Evans et al., 1992; Kieffer et al., 1992), two DOP-R subtypes, DOP-R1 and DOP-R2, have been pharmacologically identified *in vivo* and in binding studies in rodent brain membranes (Buzas et al., 1994; Mattia et al., 1991; Negri et al., 1991; Sofuoglu et al., 1991; Zaki et al., 1996). However, the roles of DOP-R subtypes in ethanol-mediated behaviors are not well defined. Studies in mice suggest DOP-R1 and DOP-R2 may have opposing effects on voluntary ethanol intake (van Rijn & Whistler, 2009) such that the DOP-R1 agonist, TAN67, and the DOP-R2 antagonist, naltriben, both reduce ethanol consumption. These different roles of DOP-R1 and DOP-R2 on ethanol intake are

escalation and maintenance of drinking in high-ethanol–consuming rats.

**3.5 DOP-R antagonists in models of relapse to ethanol-seeking** 

al., 2002a; Marinelli et al., 2009).

**3.6 DOP-R subtypes in ethanol consumption and seeking** 

#### **3.4 DOP-R antagonists in high drinking rats**

DOP-R antagonists reduce ethanol consumption in high-ethanol consuming or alcoholpreferring strains of rats (Hyytia & Kiianmaa, 2001; Krishnan-Sarin et al., 1995a; Krishnan-Sarin et al., 1995b; Nielsen et al., 2008). In studies using alcohol-preferring (P) rats, the DOP-R antagonists naltrindole, ICI 174864 and naltriben all effectively reduced ethanol consumption using the two-bottle choice 10% ethanol paradigm (Krishnan-Sarin et al., 1995a; Krishnan-Sarin et al., 1995b). Naltrindole given systemically in the maximally effective dose (15 mg/kg) produced a long-lasting reduction in ethanol intake for at least 8 h following a single dose and for at least 28 h following a second dose given 4 h later (Krishnan-Sarin et al., 1995a). Although the effects of naltrindole were selective for ethanol over water intake, naltrindole also suppressed the intake of saccharin solutions either with or without ethanol (Krishnan-Sarin et al., 1995a). In comparison, another study by the same group showed that systemic administration of the DOP-R2-selective antagonist, naltriben, (6 mg/kg in two doses) reduced ethanol consumption but not water consumption (Krishnan-Sarin et al., 1995b). Furthermore, it was shown that naltriben reduced the intake of solutions containing ethanol with saccharin and ethanol with quinine but did not affect the intake of either saccharin or quinine solutions alone. Taken together, this suggests that the suppressive effects of naltriben are selective for ethanol. In high-drinking AA (Alko, Alcohol) rats trained to operantly respond for 10% ethanol, intracerebroventricular (i.c.v.) administration of naltrindole, but not the MOP-R antagonist, CTOP, produced a significant and dose-dependent suppression of responding for ethanol (Hyytia & Kiianmaa, 2001). Furthermore, both naltrindole and CTOP suppressed responding in non-ethanol preferring Wistar rats suggesting that the DOP-R may play a greater role than the MOP-R in suppressing responding in high drinking rats, regardless of strain. This is further supported by other studies using models of low to moderate ethanol or limited access paradigms in which DOP-R antagonists do not reduce ethanol consumption (Ingman et al., 2003; Margolis et al., 2008; Stromberg et al., 1998a).

The recent recharacterization of a rat model of voluntary high ethanol intake using intermittent access to 20% ethanol without the use of any initiation procedures (Simms et al., 2008; Wise, 1973) has led to the investigation of the roles of opioid receptors and effects of opioid ligands in high-ethanol consuming, albeit non-ethanol preferring, rats. Using this model of high ethanol intake, the naltrexone-derived DOP-R antagonist, SoRI-9409, was shown to be threefold more effective and selective in reducing ethanol intake than naltrexone in Long-Evans rats (Nielsen et al., 2008). SoRI-9409 more potently reduced high ethanol intake using the model of intermittent access to 20% ethanol compared to less potent reductions in moderate ethanol intake using a model of continuous access to 10% ethanol. The effects of single doses of SoRI-9409 were long-lasting such that reductions in high ethanol intake were observed after 24 h of access, in comparison to naltrexone which was only effective after short access periods (30 min). The effects of SoRI-9409, unlike naltrexone, were also selective for ethanol such that water intake was not reduced. Furthermore, SoRI-9409 did not reduce sucrose intake at doses that effectively reduced ethanol intake. Using the high drinking model of intermittent access to 20% ethanol, daily systemic administrations of SoRI-9409 to Long Evans rats for 28 days selectively reduced the onset and escalation of ethanol intake and continued to selectively reduce ethanol intake by > 50%

DOP-R antagonists reduce ethanol consumption in high-ethanol consuming or alcoholpreferring strains of rats (Hyytia & Kiianmaa, 2001; Krishnan-Sarin et al., 1995a; Krishnan-Sarin et al., 1995b; Nielsen et al., 2008). In studies using alcohol-preferring (P) rats, the DOP-R antagonists naltrindole, ICI 174864 and naltriben all effectively reduced ethanol consumption using the two-bottle choice 10% ethanol paradigm (Krishnan-Sarin et al., 1995a; Krishnan-Sarin et al., 1995b). Naltrindole given systemically in the maximally effective dose (15 mg/kg) produced a long-lasting reduction in ethanol intake for at least 8 h following a single dose and for at least 28 h following a second dose given 4 h later (Krishnan-Sarin et al., 1995a). Although the effects of naltrindole were selective for ethanol over water intake, naltrindole also suppressed the intake of saccharin solutions either with or without ethanol (Krishnan-Sarin et al., 1995a). In comparison, another study by the same group showed that systemic administration of the DOP-R2-selective antagonist, naltriben, (6 mg/kg in two doses) reduced ethanol consumption but not water consumption (Krishnan-Sarin et al., 1995b). Furthermore, it was shown that naltriben reduced the intake of solutions containing ethanol with saccharin and ethanol with quinine but did not affect the intake of either saccharin or quinine solutions alone. Taken together, this suggests that the suppressive effects of naltriben are selective for ethanol. In high-drinking AA (Alko, Alcohol) rats trained to operantly respond for 10% ethanol, intracerebroventricular (i.c.v.) administration of naltrindole, but not the MOP-R antagonist, CTOP, produced a significant and dose-dependent suppression of responding for ethanol (Hyytia & Kiianmaa, 2001). Furthermore, both naltrindole and CTOP suppressed responding in non-ethanol preferring Wistar rats suggesting that the DOP-R may play a greater role than the MOP-R in suppressing responding in high drinking rats, regardless of strain. This is further supported by other studies using models of low to moderate ethanol or limited access paradigms in which DOP-R antagonists do not reduce ethanol consumption (Ingman et al., 2003; Margolis

The recent recharacterization of a rat model of voluntary high ethanol intake using intermittent access to 20% ethanol without the use of any initiation procedures (Simms et al., 2008; Wise, 1973) has led to the investigation of the roles of opioid receptors and effects of opioid ligands in high-ethanol consuming, albeit non-ethanol preferring, rats. Using this model of high ethanol intake, the naltrexone-derived DOP-R antagonist, SoRI-9409, was shown to be threefold more effective and selective in reducing ethanol intake than naltrexone in Long-Evans rats (Nielsen et al., 2008). SoRI-9409 more potently reduced high ethanol intake using the model of intermittent access to 20% ethanol compared to less potent reductions in moderate ethanol intake using a model of continuous access to 10% ethanol. The effects of single doses of SoRI-9409 were long-lasting such that reductions in high ethanol intake were observed after 24 h of access, in comparison to naltrexone which was only effective after short access periods (30 min). The effects of SoRI-9409, unlike naltrexone, were also selective for ethanol such that water intake was not reduced. Furthermore, SoRI-9409 did not reduce sucrose intake at doses that effectively reduced ethanol intake. Using the high drinking model of intermittent access to 20% ethanol, daily systemic administrations of SoRI-9409 to Long Evans rats for 28 days selectively reduced the onset and escalation of ethanol intake and continued to selectively reduce ethanol intake by > 50%

**3.4 DOP-R antagonists in high drinking rats** 

et al., 2008; Stromberg et al., 1998a).

for up to 28 days compared with vehicle-treated rats (Nielsen et al., 2008). When the daily administrations of SoRI-9409 were ceased after 28 days, ethanol consumption was maintained at approximately one-half of the baseline drinking levels of post-vehicle-treated rats, for a further 28 days. Cessation of SoRI-9409 treatment did not result in any significant escalation in ethanol consumption suggesting that multiple administrations of SoRI-9409 produce long-lasting and permanent effects on the modulation of ethanol consumption. This further suggests that inhibition of DOP-Rs directly reduces ethanol intake during the escalation and maintenance of drinking in high-ethanol–consuming rats.
