ALCOHOLISM:CLINICAL AND EXPERIMENTAL RESEARCH Vol. 38, No. 1 January 2014 Intra-VTA Deltorphin, But Not DPDPE, Induces Place Preference in Ethanol-Drinking Rats: Distinct DOR-1 and DOR-2 Mechanisms Control Ethanol Consumption and Reward Jennifer M. Mitchell, Elyssa B. Margolis, Allison R. Coker, Daicia C. Allen, and Howard L. Fields Background: While there is a growing body of evidence that the delta opioid receptor (DOR) modu- lates ethanol (EtOH) consumption, development of DOR-based medications is limited in part because there are 2 pharmacologically distinct DOR subtypes (DOR-1 and DOR-2) that can have opposing actions on behavior. Methods: We studied the behavioral influence of the DOR-1-selective agonist [D-Pen2,D-Pen5]- Enkephalin (DPDPE) and the DOR-2-selective agonist deltorphin microinjected into the ventral tegmental area (VTA) on EtOH consumption and conditioned place preference (CPP) and the physio- logical effects of these 2 DOR agonists on GABAergic synaptic transmission in VTA-containing brain slices from Lewis rats. Results: Neither deltorphin nor DPDPE induced a significant place preference in EtOH-na€ıve Lewis rats. However, deltorphin (but not DPDPE) induced a significant CPP in EtOH-drinking rats. In con- trast to the previous finding that intra-VTA DOR-1 activity inhibits EtOH consumption and that this inhibition correlates with a DPDPE-induced inhibition of GABA release, here we found no effect of DOR-2 activity on EtOH consumption nor was there a correlation between level of drinking and deltorphin-induced change in GABAergic synaptic transmission. Conclusions: These data indicate that the therapeutic potential of DOR agonists for alcohol abuse is through a selective action at the DOR-1 form of the receptor. Key Words: Ethanol, Place Preference, Self-Administration, Delta Opioid Receptor, Reward. HERE ARE 4 distinct classes of opioid receptors: the tion, depending upon the behavioral state of the animal T mu opioid receptor (MOR), the delta opioid receptor (Mitchell et al., 2005; Walker and Koob, 2008). (DOR), the kappa opioid receptor (KOR), and the nocicep- The ventral tegmental area (VTA) is important for both tin receptor. There is evidence that endogenous opioids exert opioid reward and regulation of EtOH consumption in a strong regulatory action on ethanol (EtOH) consumption rodents. Alcohol-preferring rats self-administer EtOH in both humans and rodents and that selectively targeting (McBride et al., 1993) and its primary metabolite acetalde- each opioid receptor can either promote or inhibit EtOH hyde (Rodd-Henricks et al., 2002) directly into the VTA, and consumption. For example, MOR knockout mice drink less VTA cFOS expression increases after exposure to an EtOH- EtOH (Roberts et al., 2000), while DOR knockout mice associated context (Hill et al., 2007). Intra-VTA administra- drink more EtOH (Roberts et al., 2001). In addition, KOR tion of either the nonselective opioid-antagonist naltrexone antagonists can either promote or reduce EtOH consump- (Mitchell et al., 2009) or the MOR-selective antagonist CTOP reduces EtOH consumption in rats (Margolis et al., 2008), while intra-VTA administration of the DOR-antago- nist TIPP-Ψ increases EtOH consumption (Margolis et al., From the Ernest Gallo Clinic and Research Center (JMM, EBM, 2008). The nonselective opioid-antagonist methylnaloxoni- ARC, DCA, HLF), University of California, San Francisco, Emeryville, um also attenuates EtOH place preference in mice (Bechtholt California; and Department of Neurology (JMM, EBM, ARC, HLF), and Cunningham, 2005). University of California, San Francisco, California. Although there is only 1 gene encoding the DOR (Evans Received for publication January 30, 2013; accepted June 5, 2013. Reprint requests: Jennifer M. Mitchell, PhD, Ernest Gallo Clinic and et al., 1992; Simonin et al., 1994), there are 2 pharmacologi- Research Center, 5858 Horton Street, Suite #200, Emeryville, CA 94608; cally distinct classes of DOR ligands: DOR-1 and DOR-2, Tel.: 510-985-3921; Fax: 510-985-3101; E-mail: jennifer.mitchell@ucsf. both of which are blocked by the highly DOR-selective edu antagonist TIPP-Ψ (Schiller et al., 1993). The metabolically © Copyright 2013 by the Research Society on Alcoholism. stable enkephalin analog [D-Pen2,D-Pen5]-Enkephalin DOI: 10.1111/acer.12246 (DPDPE) is a highly selective DOR-1 agonist, while Alcohol Clin Exp Res, Vol 38, No 1, 2014: pp 195–203 195 196 MITCHELL ET AL. [D-Ala2]-Deltorphin II (deltorphin) is a highly selectively regulating reward and anxiety necessitates consideration of DOR-2 agonist (Fang et al., 1994; Horan et al., 1993; issues related to addiction and abuse liability. Therefore, it is Mattia et al., 1991; Vanderah et al., 1994). There is no cross- critical to determine whether these compounds are rewarding tolerance between these two DOR agonists (Sofuoglu et al., in ETOH-consuming animals. 1991a,b), and DOR-1 and DOR-2 ligands have different, Here, we used a conditioned place preference (CPP) para- sometimes opposing, effects on a variety of behaviors (Chur- digm to assess the rewarding effects of the DOR-1 agonist chill et al., 1995; Noble et al., 1996; van Rijn and Whistler, DPDPE and the DOR-2 agonist deltorphin in both EtOH- 2009; Yu and Bodnar, 1997). We previously reported that na€ıve and EtOH-drinking Lewis rats when administered into DOR-1 activation in the VTA of the rat decreases EtOH the VTA. We also examined the effects of the DOR-2 agonist consumption (Margolis et al., 2008). However, in mice, sys- deltorphin on VTA GABAA synaptic transmission to deter- temic administration of the DOR-2 antagonist, naltriben, mine whether the previously identified relationship between decreases EtOH consumption (van Rijn and Whistler, 2009), attenuated drinking and inhibition of GABA release in VTA suggesting opposing roles of DOR subtypes on drinking slices in response to a DOR-1 agonist would generalize to a behavior. To study the therapeutic potential of DOR ligands DOR-2 agonist. for alcohol abuse and related behavioral disorders, it is essen- tial to differentiate DOR-1 and DOR-2 effects on behavior MATERIALS AND METHODS and synaptic function in brain regions that contribute to reward. Animals We have previously shown that MOR and DOR-1 activity Sixty-one male Lewis rats (47 for behavioral experiments and 14 in the VTA has opposing effects on EtOH consumption. In for electrophysiology; Harlan Laboratories, Hayward, CA) weigh- contrast to the attenuation of EtOH consumption by intra- ing between 275 and 300 g on arrival were housed individually in a temperature-controlled colony room (21°C) on a 12-hour reversed VTA injection of the MOR-selective antagonist CTOP, light/dark cycle (lights off at 10:00 AM). For pharmacological stud- intra-VTA microinjection of the DOR-1 agonist DPDPE ies, all animals served as their own controls (within subjects design). decreases EtOH consumption through inhibition of GABA Additionally, each animal served in only a single pharmacology release in chronically drinking animals (Margolis et al., experiment (Fig. 1). Animals were all within the same age range at 2008). Furthermore, the DPDPE-induced presynaptic inhibi- the beginning of each experiment. All experiments were performed during the dark portion of the cycle. Rat chow and water were tion of GABA terminals is inversely correlated with EtOH available ad libitum throughout the experiment. During EtOH self- consumption (Margolis et al., 2008). However, the actions of administration periods (see below), 10% EtOH (v/v; Gold Shield, DOR-2 agonists on GABAergic synaptic transmission in Hayward, CA) was also available ad libitum. All experimental VTA neurons have not been studied. Further, although the protocols were approved by the Institutional Animal Care and Use evidence indicates that DOR ligands are potentially effective Committee and were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (NIH). therapeutics for alcohol abuse, the involvement of DOR in Fig. 1. Experimental timeline for place preference behavior (n = 37), ethanol (EtOH)-drinking experiments (n = 10), and electrophysiology (n = 14). Animals always had access to EtOH and H2O bottles when not in the conditioned place preference (CPP) apparatus. DOR-1 AND DOR-2 EFFECTS ON ETOH REWARD 197 EtOH Self-Administration Animals were counterbalanced such that an equal number of ani- mals received drug injections in the black versus white box. Animals EtOH was self-administered via a 2-bottle continuous access, were tested for expression of CPP 1 day after the final conditioning free-choice paradigm in which 1 bottle contained 10% EtOH (v/v) session. and the other bottle contained water. Sucrose was never added to the EtOH solution. Animals were weighed daily, and the amount of EtOH and water consumed was measured at the same time each VTA Microinjections day (10:00 AM). Bottles were identical, and their positions were Each injection was made using a 1-ll syringe (Hamilton, Reno, counterbalanced and rotated daily. Animals were maintained on NV) attached to 20 cm of PE 50 tubing connected to a 33-gauge the 2-bottle choice paradigm for at least 12 weeks until consump- injection cannula (Plastics One). Microinjections of 0.5 llvolumes tion had stabilized (defined as <15% change in drinking over 3 day were given at a rate of 0.25 ll/min using a syringe pump (KD Scien- bins) before surgery and experimentation commenced. Similarly, tific, Holliston, MA) into each side of the VTA. Injection cannulae following surgery, behavioral training did not begin until drinking extended 1.5 to 2 mm beyond guide cannula to reach a depth of had returned to presurgical levels (defined as no significant differ- 9.0 mm and were left in place for 1 minute following microinjec- ence between presurgical 3 day bin and final postsurgical 3 day tions to minimize the backflow of drug solution. For CPP experi- bin). For place preference experiments, animals always had access ments, microinjections were made directly prior to placement of to EtOH and H O bottles when not in the CPP chambers.