**6. Sigma Receptors and ethanol consumption**

Sigma receptors (SIG-R) were originally categorized as members of the opioid receptor family (Quirion et al., 1992) although more recent studies suggested SIG-Rs are unique binding sites including phencyclidine binding sites (Gundlach et al., 1985; Gundlach et al., 1986; Martin et al., 1976; Walker et al., 1990). Furthermore, the opioid antagonist, naltrexone, is not reported to have activity at the SIG-R (Holtzman, 1989; Vaupel, 1983).

The SIG-Rs antagonist, BD1047, reduces ethanol-induced locomotion, ethanol-induced place preference and taste conditioning in Swiss mice (Maurice et al., 2003). Conversely, the SIG-R agonist, PRE-084, increased ethanol CPP without effects on ethanol-induced locomotion (Maurice et al., 2003). A series of studies have shown the SIG-R to play a role in ethanol consumption and seeking in Sardinian alcohol-preferring (sP) rats (Sabino et al., 2011; Sabino et al., 2009a; Sabino et al., 2009b) (Table 1). Administration of the SIG-R antagonists, BD1063 NE-100, selectively reduces ethanol consumption, responding for ethanol and also prevents the increase in ethanol intake after an ethanol-deprivation period in rats (Sabino et al., 2009b). Chronic administration of the SIG-R agonist, DTG, increased responding for ethanol in rats, an effect that was blocked with BD-1063 pretreatment (Sabino et al., 2011). However, DTG treatment also increased responding for saccharin and sucrose (Sabino et al., 2011). Chronic administration of DTG to naive rats resulted in increased mRNA expression of MOP-R and DOP-R, but not KOP-R, in the VTA (Sabino et al., 2011). This increased opioid mRNA expression was suggested to be responsible for the excessive alcohol intake following DTG treatment, in agreement with previous studies reporting the importance of DOP-R activity in maintaining high ethanol intake in alcohol-preferring rats (Froehlich et al., 1991) and the roles of MOP-R and DOP-R activation on VTA-mediated dopamine release (Devine et al., 1993b).

### **7. Brain-region specific roles of opioid receptors in ethanol consumption and seeking**

The mesolimbic dopamine system plays a key role in mediating the reinforcing properties of ethanol and other drugs of abuse (Herz, 1997). Moreover, ethanol reinforcement and high

The Role of Delta Opioid Receptors in Ethanol Consumption

[3H]DPDPE autoradiography

Pro-enkephalin mRNA expression

hybridization and densitometry

Microdialaysis/ radioimmunoassa

enkephalin release and content

by in situ

y of Met-

al., 2009; Nielsen et al., 2008).

Acute ethanol 2.5 g/kg , i.p.

Acute ethanol 2.5 g/kg, i.p.

Acute ethanol treatment 0.5, 1, 2.5 g/kg i.p.

Wistar rats

Wistar rats

Wistar rats

**AUDs** 

and Seeking: Implications for New Treatments for Alcohol Use Disorders 217

DOP-Rs in specific brain regions have different roles in ethanol consumption and seeking. This is further supported by the increased ethanol consumption observed following administration of the DOP-R antagonist, TIPP-Ψ, into the VTA (Margolis et al., 2008) compared to reduced responding for ethanol following administration of naltrindole, given either systemically (Krishnan-Sarin et al., 1995a) or microinjected into the cerebroventricles, the NAc and basolateral amygdala (BLA), but not into the VTA (Hyytia & Kiianmaa, 2001). Furthermore, the reductions in ethanol consumption following administration of DOP-R antagonists into the cerebroventricles, the NAc and BLA are consistent with the reductions in ethanol consumption following systemic administration of DOP-R antagonists (Franck et al., 1998; Hyytia & Kiianmaa, 2001; June et al., 1999; Krishnan-Sarin et al., 1995b; Marinelli et

> Substantia nigra, Frontal cortex Prefrontal cortex

medial)

posterior)

VTA

caudate putamen (posterior)

Prefrontal cortex

Nucleus accumbens

Caudate Putamen

Prefrontal cortex

Table 2. Table of DOP-R activity in brain regions following acute ethanol treatment in rats.

In view of the potential development of opioid-receptor selectively acting compounds for the treatment of AUDs, it is important to consider adverse effects associated with opioid receptor activity. A number of adverse effects have been reported with the use of the non-

**8. Preclinical considerations of targeting the DOP-R for the treatment of** 

Nucleus accumbens shell Nucleus accumbens core caudate putamen (anterior-

caudate putamen (medial-

Nucleus accumbens shell Nucleus accumbens core

**DOP-R** 

Increased Increased Increased Increased Increased Increased

Increased

Reduced

Reduced Increased Increased Increased

Increased release Reduced content; Reduced content No change **Reference** 

Mendez et al., 2004

Mendez & Morales-Mulia, 2006

Mendez et al., 2010

**Strain Model Assay Brain tissue Effect on** 

alcohol drinking behavior have been suggested to involve the ethanol-induced activation of endogenous opioid systems (Froehlich et al., 1991). Ethanol may alter opioidergic transmission at different levels, including opioid peptide biosynthesis and release, as well as binding to opioid receptors. Ethanol stimulates the activity of the endogenous opioids for MOP-R (-endorphins) and DOP-R (enkephalins) leading to an enhanced release of dopamine in the mesolimbic pathway (Herz, 1997). Activation of MOP-R in the VTA leads to dopamine release in the NAc (Leone et al., 1991; Spanagel et al., 1992), however modulation of dopamine terminal activity is also reported to involve DOP-R (Borg & Taylor, 1997; Widdowson & Holman, 1992).

### **7.1 Brain-region specific activity of DOP-R with ethanol consumption**

Studies have shown that ethanol differentially alters opioid receptor binding in brain tissue and neuroblastoma cell lines depending on the conditions of study (Charness et al., 1993; Charness et al., 1986). Rat brain membranes treated with ethanol show inhibited enkephalin and DOP-R binding (Hiller et al., 1981). However, treatment of DOP-R-expressing neuroblastoma x glioma NG108-15 hybrid cells with ethanol (200 mM for 4 days or 25 mM for 2 weeks) results in increased DOP-R gene expression and increases in DOP-R binding sites (Charness et al., 1993; Charness et al., 1986), which was hypothesized to be due to a neuronal adaption to ethanol involving changes in receptor density. The discrepancies in ethanol responses in these studies may be explained by differences in ethanol doses and route of administration, time of exposure to the drug, time elapsed after ethanol administration at the moment the experiment was carried out, and receptor ligand used. A series of studies have demonstrated that an acute injection of ethanol given to rats results in altered DOP-R binding affinity, mRNA expression and release of enkephalins (Mendez et al., 2010; Mendez & Morales-Mulia, 2006; Mendez et al., 2004)(Table 2). The most consistent changes in DOP-R activity following acute ethanol treatment have been increased binding and met-enkephalin release, but not content, in mesocorticolimbic and nigrostriatal pathways (Mendez et al., 2010; Mendez et al., 2004). A number of studies have shown that rats given chronic or long-term access to ethanol have altered DOP-R activity in mesocorticolimbic and nigrostriatal pathways (Table 3). Long-term ethanol exposure in rats has been shown to increase DOP-R binding and supersensitivity of striatal DOP-Rs which has been suggested to be due to decreased endogenous peptide release (Lucchi et al., 1985; Lucchi et al., 1984). In comparison, the effects of long-term ethanol treatment in mice have been less consistent. CF-1 mice given ethanol for 5 days had an intermediate DOP-R binding site compared to a high and a low affinity DOP-R binding site in control mice (Hynes et al., 1983). Higher enkepahlin degrading enzyme activity was detected in the striatum of alcohol preferring C57/BL6 mice compared to the alcohol-avoiding DBA/2 mice (Winkler et al., 1998). However, enkephalin degrading enzyme activity was reduced in the hypothalamus, but not the striatum, following long-term ethanol treatment and ethanol withdrawal (Winkler et al., 1998). Furthermore, a higher density of DOP-Rs was found in the VTA and NAc of C57/BL6 mice compared to DBA/2 mice (Moller et al., 2002).

#### **7.2 Brain region-specific effects on ethanol consumption by DOP-R ligands**

The different effects on ethanol consumption by cerebral injections of the DOP-R agonists, DPDPE and DALA, (Barson et al., 2009; Barson et al., 2010; Margolis et al., 2008) suggest that

alcohol drinking behavior have been suggested to involve the ethanol-induced activation of endogenous opioid systems (Froehlich et al., 1991). Ethanol may alter opioidergic transmission at different levels, including opioid peptide biosynthesis and release, as well as binding to opioid receptors. Ethanol stimulates the activity of the endogenous opioids for MOP-R (-endorphins) and DOP-R (enkephalins) leading to an enhanced release of dopamine in the mesolimbic pathway (Herz, 1997). Activation of MOP-R in the VTA leads to dopamine release in the NAc (Leone et al., 1991; Spanagel et al., 1992), however modulation of dopamine terminal activity is also reported to involve DOP-R (Borg & Taylor,

Studies have shown that ethanol differentially alters opioid receptor binding in brain tissue and neuroblastoma cell lines depending on the conditions of study (Charness et al., 1993; Charness et al., 1986). Rat brain membranes treated with ethanol show inhibited enkephalin and DOP-R binding (Hiller et al., 1981). However, treatment of DOP-R-expressing neuroblastoma x glioma NG108-15 hybrid cells with ethanol (200 mM for 4 days or 25 mM for 2 weeks) results in increased DOP-R gene expression and increases in DOP-R binding sites (Charness et al., 1993; Charness et al., 1986), which was hypothesized to be due to a neuronal adaption to ethanol involving changes in receptor density. The discrepancies in ethanol responses in these studies may be explained by differences in ethanol doses and route of administration, time of exposure to the drug, time elapsed after ethanol administration at the moment the experiment was carried out, and receptor ligand used. A series of studies have demonstrated that an acute injection of ethanol given to rats results in altered DOP-R binding affinity, mRNA expression and release of enkephalins (Mendez et al., 2010; Mendez & Morales-Mulia, 2006; Mendez et al., 2004)(Table 2). The most consistent changes in DOP-R activity following acute ethanol treatment have been increased binding and met-enkephalin release, but not content, in mesocorticolimbic and nigrostriatal pathways (Mendez et al., 2010; Mendez et al., 2004). A number of studies have shown that rats given chronic or long-term access to ethanol have altered DOP-R activity in mesocorticolimbic and nigrostriatal pathways (Table 3). Long-term ethanol exposure in rats has been shown to increase DOP-R binding and supersensitivity of striatal DOP-Rs which has been suggested to be due to decreased endogenous peptide release (Lucchi et al., 1985; Lucchi et al., 1984). In comparison, the effects of long-term ethanol treatment in mice have been less consistent. CF-1 mice given ethanol for 5 days had an intermediate DOP-R binding site compared to a high and a low affinity DOP-R binding site in control mice (Hynes et al., 1983). Higher enkepahlin degrading enzyme activity was detected in the striatum of alcohol preferring C57/BL6 mice compared to the alcohol-avoiding DBA/2 mice (Winkler et al., 1998). However, enkephalin degrading enzyme activity was reduced in the hypothalamus, but not the striatum, following long-term ethanol treatment and ethanol withdrawal (Winkler et al., 1998). Furthermore, a higher density of DOP-Rs was found in the VTA and

**7.1 Brain-region specific activity of DOP-R with ethanol consumption** 

NAc of C57/BL6 mice compared to DBA/2 mice (Moller et al., 2002).

**7.2 Brain region-specific effects on ethanol consumption by DOP-R ligands** 

The different effects on ethanol consumption by cerebral injections of the DOP-R agonists, DPDPE and DALA, (Barson et al., 2009; Barson et al., 2010; Margolis et al., 2008) suggest that

1997; Widdowson & Holman, 1992).

DOP-Rs in specific brain regions have different roles in ethanol consumption and seeking. This is further supported by the increased ethanol consumption observed following administration of the DOP-R antagonist, TIPP-Ψ, into the VTA (Margolis et al., 2008) compared to reduced responding for ethanol following administration of naltrindole, given either systemically (Krishnan-Sarin et al., 1995a) or microinjected into the cerebroventricles, the NAc and basolateral amygdala (BLA), but not into the VTA (Hyytia & Kiianmaa, 2001). Furthermore, the reductions in ethanol consumption following administration of DOP-R antagonists into the cerebroventricles, the NAc and BLA are consistent with the reductions in ethanol consumption following systemic administration of DOP-R antagonists (Franck et al., 1998; Hyytia & Kiianmaa, 2001; June et al., 1999; Krishnan-Sarin et al., 1995b; Marinelli et al., 2009; Nielsen et al., 2008).


Table 2. Table of DOP-R activity in brain regions following acute ethanol treatment in rats.
