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

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

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

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

The Role of Delta Opioid Receptors in Ethanol Consumption

different mechanisms in the central nervous system.

**4.1 Ethanol consumption in KOP-R knockout mice** 

**4.2 KOP-R activation and ethanol consumption** 

**consumption and seeking** 

**4. The role of the Kappa Opioid Peptide Receptor (KOP-R) in ethanol** 

2009; Ebner et al., 2010; Margolis et al., 2003; Shippenberg & Herz, 1986).

Mice with a genetic deletion of the KOP-R (mixed C57BL/6-129SvJ background) have reduced levels of ethanol intake in animals consuming 12% ethanol and saccharin using a two-bottle choice paradigm (Kovacs et al., 2005) (Table 1). In contrast to the MOP-R and DOP-R, stimulation of the KOP-R is associated with dysphoria, suppression of reward, induced states of aversion, reduced dopamine release in the NAc, inhibition of dopaminergic neurons in the VTA and promotion of negative reinforcement (Bruijnzeel,

Systemic administration of the KOP-R agonist, U50488, reduces intake of 10% ethanol using two-bottle choice paradigms (Lindholm et al., 2001; Nestby et al., 1999) (Table 1). Furthermore, administration of the KOP-R agonists, dynorphin1-17 or MR-2266-BS, prior to the first free-choice session following a period of forced ethanol exposure reduced preference for ethanol (Sandi et al., 1988; Sandi et al., 1990). The long-acting benzomorphan opioid compound, bremazocine, which acts as an antagonist at MOP-R and DOP-R and an agonist at KOP-R, was shown to more potently reduce 10% ethanol intake than the nonselective opioid antagonist, naltrexone (Nestby et al., 1999). However, as KOP-R agonists

and Seeking: Implications for New Treatments for Alcohol Use Disorders 213

Conversely, when ethanol (75-150 mg/kg) was combined with the MOP-R-preferring agonist, morphine (0.1 mg/kg), or the selective DOP-R agonist, TAN-67 (20 mg/kg, s.c.), at doses below the threshold to produce place preference on their own, there was an enhancement of place preference to ethanol (Matsuzawa et al., 1998; Matsuzawa et al., 1999a). Furthermore, naltrindole (3 mg/kg, s.c.) significantly attenuated the enhancement of the ethanol-induced (75 mg/kg, i.p) place preference produced by TAN67 (20 mg/kg, s.c.) suggesting DOP-R receptors may be involved in the rewarding mechanism of ethanol under psychological stress (Matsuzawa et al., 1999b). The attenuation of CPP to ethanol and increased conditioned taste aversion to ethanol by the DOP-R antagonist, naltrindole (Froehlich et al., 1998; Matsuzawa et al., 1998; Matsuzawa et al., 1999a; Matsuzawa et al., 1999b) may contribute, at least in part, to the reductions in ethanol consumption following administration of DOP-R antagonists (Hyytia & Kiianmaa, 2001; Krishnan-Sarin et al., 1995a; Krishnan-Sarin et al., 1995b; Nielsen et al., 2008). However, whether enhanced CPP to ethanol following treatment with TAN67 would explain the effects of DOP-R agonists on ethanol consumption and seeking is unclear. An increase in the rewarding actions of ethanol by TAN67 may subsequently lead to reduced consumption (van Rijn & Whistler, 2009) as less ethanol may be required to produce ethanol's effects. Conversely, activation of DOP-Rs and the subsequent increased rewarding effects of ethanol may have contributed to further increased levels of ethanol consumption following treatment with DOP-R agonists (Barson et al., 2009; Barson et al., 2010; van Rijn et al., 2010). Collectively, these studies suggest that activation and inhibition of DOP-R activity may modulate ethanol consumption via

hypothesized as DOP-R1 existing as a MOP-R/DOP-R heterodimer which opposes the actions of DOP-R2, which may exist as a DOP-R homomer (van Rijn & Whistler, 2009). Another recent study showed that DOP-R1 inhibition appears more important than DOP-R2 inhibition in yohimbine stress-induced reinstatement of ethanol-seeking in rats (Nielsen et al., 2011). Using the [35S]GTPS signaling assay in midbrain membranes prepared from yohimbine-treated rats previously responding for ethanol and subsequently extinguished, there was higher TAN67-mediated DOP-R1 activity (> 700-fold) than deltorphin II-mediated DOP-R2 activity (≥ 4-fold) compared to vehicle-treated rats (Nielsen et al., 2011). Pretreatment with the DOP-R antagonist, SoRI-9409, in yohimbine-treated rats reduced DOP-R1 activity by 37-fold, but did not change DOP-R2, MOP-R or KOP-R activity. The potent inhibition of TAN67-stimulated DOP-R1 activity in yohimbine-treated rat membranes by SoRI-9409, naltrindole and the DOP-R1 antagonist, 7-Benzylidenenaltrexone (BNTX) (Sofuoglu et al., 1993), further suggests that DOP-R1 inhibition is important for reducing yohimbine-induced ethanol-seeking (Nielsen et al., 2011). As SoRI-9409 effectively reduces ethanol consumption in rats and potently reduces DOP-R1 activity in brain membranes of high-ethanol consuming rats (Nielsen et al., 2008), DOP-R1 inhibition appears to play a role in both reducing reinstatement of ethanol-seeking and voluntary ethanol intake in rats. However, as SoRI-9409 inhibits both deltorphin II-mediated DOP-R2 analgesia and DPDPEmediated DOP-R1 analgesia in mice (Wells et al., 2001), it appears that the subtypes of the DOP-R have different roles in different behaviors and species.

#### **3.7 The role of the DOP-R in the rewarding effects of ethanol**

The DOP-R has been shown to play a role in the reinforcing effects of ethanol (Borg & Taylor, 1997; Froehlich et al., 1998; Froehlich et al., 1991; Shippenberg et al., 2008). Activation of DOP-Rs in the NAc and VTA leads to increased basal dopamine release (Borg & Taylor, 1997; Devine et al., 1993b; Herz, 1997; Vetulani, 2001). Dopamine release in the striatum is stimulated by ethanol, DOP-R agonists and enkephalinase inhibitors (Dourmap et al., 1990; Petit et al., 1986; Spanagel et al., 1990) and ethanol-induced release of dopamine in the striatum is blocked by DOP-R antagonists (Acquas et al., 1993; Widdowson & Holman, 1992). DOP-R agonists increase and DOP-R antagonists decrease, respectively, ethanolinduced place preference in rats (Bie et al., 2009; Matsuzawa et al., 1999a; Matsuzawa et al., 1999b) and DOP-R antagonists can make a nonaversive dose of alcohol aversive (Froehlich et al., 1998). A series of studies demonstrated that the DOP-R plays a role in conditioned place preference (CPP) to ethanol using a model of conditioned fear stress (Matsuzawa et al., 1998; Matsuzawa et al., 1999a; Matsuzawa et al., 1999b). In this model, ethanol produced a significant CPP only in rats that were exposed to an environment previously paired with an electric foot shock (Matsuzawa et al., 1998; Matsuzawa et al., 1999a; Matsuzawa et al., 1999b). Significant CPP to ethanol (300 mg/kg, intraperitoneal (i.p.) in rats was attenuated following systemic administration of the non-selective opioid receptor antagonist naloxone (1-3 mg/kg, subcutaneous (s.c.)), the MOP-R antagonist beta-funaltrexamine (3-10 mg/kg, i.p.), the DOP-R antagonist naltrindole (1-3 mg/kg, s.c.) and the KOP-R agonist, U50488 (0.3- 1 mg/kg, s.c.)(Matsuzawa et al., 1998; Matsuzawa et al., 1999a). This is in contrast to enhanced place preference to ethanol following systemic administration of the KOP-R antagonist, nor-binaltorphimine (nor-BNI; 3 mg/kg, i.p.) (Matsuzawa et al., 1999a).

hypothesized as DOP-R1 existing as a MOP-R/DOP-R heterodimer which opposes the actions of DOP-R2, which may exist as a DOP-R homomer (van Rijn & Whistler, 2009). Another recent study showed that DOP-R1 inhibition appears more important than DOP-R2 inhibition in yohimbine stress-induced reinstatement of ethanol-seeking in rats (Nielsen et al., 2011). Using the [35S]GTPS signaling assay in midbrain membranes prepared from yohimbine-treated rats previously responding for ethanol and subsequently extinguished, there was higher TAN67-mediated DOP-R1 activity (> 700-fold) than deltorphin II-mediated DOP-R2 activity (≥ 4-fold) compared to vehicle-treated rats (Nielsen et al., 2011). Pretreatment with the DOP-R antagonist, SoRI-9409, in yohimbine-treated rats reduced DOP-R1 activity by 37-fold, but did not change DOP-R2, MOP-R or KOP-R activity. The potent inhibition of TAN67-stimulated DOP-R1 activity in yohimbine-treated rat membranes by SoRI-9409, naltrindole and the DOP-R1 antagonist, 7-Benzylidenenaltrexone (BNTX) (Sofuoglu et al., 1993), further suggests that DOP-R1 inhibition is important for reducing yohimbine-induced ethanol-seeking (Nielsen et al., 2011). As SoRI-9409 effectively reduces ethanol consumption in rats and potently reduces DOP-R1 activity in brain membranes of high-ethanol consuming rats (Nielsen et al., 2008), DOP-R1 inhibition appears to play a role in both reducing reinstatement of ethanol-seeking and voluntary ethanol intake in rats. However, as SoRI-9409 inhibits both deltorphin II-mediated DOP-R2 analgesia and DPDPEmediated DOP-R1 analgesia in mice (Wells et al., 2001), it appears that the subtypes of the

The DOP-R has been shown to play a role in the reinforcing effects of ethanol (Borg & Taylor, 1997; Froehlich et al., 1998; Froehlich et al., 1991; Shippenberg et al., 2008). Activation of DOP-Rs in the NAc and VTA leads to increased basal dopamine release (Borg & Taylor, 1997; Devine et al., 1993b; Herz, 1997; Vetulani, 2001). Dopamine release in the striatum is stimulated by ethanol, DOP-R agonists and enkephalinase inhibitors (Dourmap et al., 1990; Petit et al., 1986; Spanagel et al., 1990) and ethanol-induced release of dopamine in the striatum is blocked by DOP-R antagonists (Acquas et al., 1993; Widdowson & Holman, 1992). DOP-R agonists increase and DOP-R antagonists decrease, respectively, ethanolinduced place preference in rats (Bie et al., 2009; Matsuzawa et al., 1999a; Matsuzawa et al., 1999b) and DOP-R antagonists can make a nonaversive dose of alcohol aversive (Froehlich et al., 1998). A series of studies demonstrated that the DOP-R plays a role in conditioned place preference (CPP) to ethanol using a model of conditioned fear stress (Matsuzawa et al., 1998; Matsuzawa et al., 1999a; Matsuzawa et al., 1999b). In this model, ethanol produced a significant CPP only in rats that were exposed to an environment previously paired with an electric foot shock (Matsuzawa et al., 1998; Matsuzawa et al., 1999a; Matsuzawa et al., 1999b). Significant CPP to ethanol (300 mg/kg, intraperitoneal (i.p.) in rats was attenuated following systemic administration of the non-selective opioid receptor antagonist naloxone (1-3 mg/kg, subcutaneous (s.c.)), the MOP-R antagonist beta-funaltrexamine (3-10 mg/kg, i.p.), the DOP-R antagonist naltrindole (1-3 mg/kg, s.c.) and the KOP-R agonist, U50488 (0.3- 1 mg/kg, s.c.)(Matsuzawa et al., 1998; Matsuzawa et al., 1999a). This is in contrast to enhanced place preference to ethanol following systemic administration of the KOP-R antagonist, nor-binaltorphimine (nor-BNI; 3 mg/kg, i.p.) (Matsuzawa et al., 1999a).

DOP-R have different roles in different behaviors and species.

**3.7 The role of the DOP-R in the rewarding effects of ethanol** 

Conversely, when ethanol (75-150 mg/kg) was combined with the MOP-R-preferring agonist, morphine (0.1 mg/kg), or the selective DOP-R agonist, TAN-67 (20 mg/kg, s.c.), at doses below the threshold to produce place preference on their own, there was an enhancement of place preference to ethanol (Matsuzawa et al., 1998; Matsuzawa et al., 1999a). Furthermore, naltrindole (3 mg/kg, s.c.) significantly attenuated the enhancement of the ethanol-induced (75 mg/kg, i.p) place preference produced by TAN67 (20 mg/kg, s.c.) suggesting DOP-R receptors may be involved in the rewarding mechanism of ethanol under psychological stress (Matsuzawa et al., 1999b). The attenuation of CPP to ethanol and increased conditioned taste aversion to ethanol by the DOP-R antagonist, naltrindole (Froehlich et al., 1998; Matsuzawa et al., 1998; Matsuzawa et al., 1999a; Matsuzawa et al., 1999b) may contribute, at least in part, to the reductions in ethanol consumption following administration of DOP-R antagonists (Hyytia & Kiianmaa, 2001; Krishnan-Sarin et al., 1995a; Krishnan-Sarin et al., 1995b; Nielsen et al., 2008). However, whether enhanced CPP to ethanol following treatment with TAN67 would explain the effects of DOP-R agonists on ethanol consumption and seeking is unclear. An increase in the rewarding actions of ethanol by TAN67 may subsequently lead to reduced consumption (van Rijn & Whistler, 2009) as less ethanol may be required to produce ethanol's effects. Conversely, activation of DOP-Rs and the subsequent increased rewarding effects of ethanol may have contributed to further increased levels of ethanol consumption following treatment with DOP-R agonists (Barson et al., 2009; Barson et al., 2010; van Rijn et al., 2010). Collectively, these studies suggest that activation and inhibition of DOP-R activity may modulate ethanol consumption via different mechanisms in the central nervous system.
