Presynaptic Opioid Receptors Regulate Ethanol Actions in Central Amygdala

Endogenous opioid systems are implicated in the reinforcing effects of ethanol consumption. For example, opioid receptor (DOR) knockout (KO) mice show greater ethanol consumption than wild-type (WT) mice (Roberts et al., 2001). To explore the neurobiological correlates underlying these behaviors, we examined effects of acute ethanol application in brain slices from DOR KO mice using whole-cell patch recording techniques. We examined the central nucleus of amygdala (CeA) because the CeA is implicated in alcohol reinforcement (Koob et al., 1998). We found that the acute ethanol effects on GABAA receptormediated inhibitory postsynaptic currents (IPSCs) were greater in DOR KO mice than in WT mice. Ethanol increased the frequency of miniature IPSCs (mIPSCs) significantly more in DOR KO mice than in WT mice. In CeA of WT mice, application of ICI 174864 [[allyl]2-Tyr-amino-isobutyric acid (Aib)-Aib-Phe-LeuOH], a DOR inverse agonist, augmented ethanol actions on mIPSC frequency comparable with ethanol effects seen in DOR KO mice. Superfusion of the selective DOR agonist D-Pen,DPen-enkephalin decreased the mean frequency of mIPSCs; this effect was reversed by the DOR antagonist naltrindole. These findings suggest that endogenous opioids may reduce ethanol actions on IPSCs of CeA neurons in WT mice through DOR-mediated inhibition of GABA release and that the increased ethanol effect on IPSCs in CeA of DOR KO mice could be, at least in part, due to absence of DOR-mediated inhibition of GABA release. This result supports the hypothesis that endogenous opioid peptides modulate the ethanol-induced augmentation of GABAA receptor-dependent circuitry in CeA (Roberto et al., 2003). Endogenous opioid systems regulate alcohol-seeking behavior because opiate receptor antagonists reduce ethanol consumption in human alcoholics and in a rat model of relapse (Froehlich, 1996; Herz, 1997). The opiate antagonist naltrexone is currently available as an adjunct medication for treatment of alcoholism. However, not all alcoholic patients respond to this medicine. In this context, the involvement of different opioid receptor subtypes on the ethanol reinforcement is under investigation. Although pharmacological studies using subtype-specific antagonists show the consistent involvement of opioid receptors (MORs) in ethanol reinforcement, the involvement of opioid receptors (DORs) is controversial (Matsuzawa et al., 1999; Mhatre et al., 2000; Ciccocioppo et al., 2002; Ingman et al., 2003). Recent studies using knockout (KO) mice suggest that MOR and DOR may act in an opposing manner to regulate alcohol consumption. MOR KO mice do not self-administer alcohol, whereas DOR KO mice consume more alcohol than wild-type (WT) controls (Roberts et al., 2000, 2001). In addition, DOR and MOR KO mice show differences in baseline emotional responses: DOR KO mice exhibit increased anxiety-like behavior relative to WT mice, whereas MOR KO mice exhibit less anxiety (Filliol et al., 2000; Roberts et al., 2001; Sasaki et al., 2002). FurThis work was supported by the National Institute on Alcohol Abuse and Alcoholism Integrative Neuroscience Initiative on Alcoholism (Grant UO1 AA013498 to G.R.S. and S.D.M.), by the National Institute on Drug Abuse (Grant R01 DA03665 to G.R.S.), and by a Veterans Administration Merit Review (to S.D.M.). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.106.112722. ABBREVIATIONS: MOR, opioid receptor; DOR, opioid receptor; KO, knockout; WT, wild type; CeA, central nucleus of the amygdala; IPSC, inhibitory postsynaptic current; ACSF, artificial cerebrospinal fluid; RMP, resting membrane potential; eIPSC, evoked IPC; AP-5, D-2-amino-5phosphonovalerate; DNQX, 6,7-dinitroquinoxaline-2,3-dione; CGP 55845, 3-N[1-(S)-(3,4-dichlorophenyl)ethyl]amino-2-(S)-hydroxypropyl-p-benzyl-phosphinic acid; I/O, input/output; TTX, tetrodotoxin; mIPSC, miniature IPSC; ICI 174864, [allyl]2-Tyr-amino-isobutyric acid (Aib)-Aib-PheLeu-OH; DPDPE, D-Pen,D-Pen-enkephalin; CRF, corticotropin-releasing factor. 0022-3565/07/3202-917–925 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 320, No. 2 U.S. Government work not protected by U.S. copyright 112722/3174349 JPET 320:917–925, 2007 Printed in U.S.A. 917 at A PE T Jornals on A uust 0, 2017 jpet.asjournals.org D ow nladed from thermore, the anxiolytic effect of ethanol is greater in DOR KO mice than WT mice (Roberts et al., 2001). These behaviors, possibly reflecting innate responses to stress, may play a role in regulating ethanol consumption in DOR and MOR mice. The amygdala formation plays a critical role in emotion and the response to stress (for review, see LeDoux, 2003). Because stress has long been considered to contribute to ethanol-seeking behavior in humans (Brown et al., 1995), the amygdala may have a significant role in regulating alcohol consumption. In addition, the central nucleus of the amygdala (CeA), as part of the extended amygdala, has been implicated in the positive reinforcing effects of ethanol in animal models (Koob et al., 1998). Microinjection of opioid antagonists into CeA produced significant reductions in ethanol self-administration, suggesting that the CeA is a critical site for opioid modulation of ethanol drinking (Heyser et al., 1999; Foster et al., 2004). In CeA, GABAergic neurons are the most abundant cell type (Sun and Cassell, 1993). Behavioral studies indicate that the CeA GABAergic system mediates the rewarding effect of ethanol (Hyytiä and Koob, 1995). A recent in vitro electrophysiological study demonstrated that ethanol enhances GABAergic inhibitory postsynaptic currents (IPSCs) in CeA at both preand postsynaptic sites (Roberto et al., 2003). Endogenous opioids are localized within GABAergic neurons in CeA (Veinante et al., 1997; Cassell et al., 1998), and acute ethanol increases c-Fos expression in enkephalin-containing GABAergic neurons in the CeA (Morales et al., 1998; Criado and Morales, 2000), suggesting an interaction between GABAergic and endogenous opioid systems on ethanol drinking. Therefore, we examined the ethanol modulation of GABAA receptor function in DOR KO mice using whole-cell patch recordings of CeA slices in vitro. Portions of this study have been reported in abstract form (Kang-Park et al., 2004; Park et al., 2004). Materials and Methods Generation of Knockout Mice. The methods for generation of the DOR KO mice have been described in detail in reports by Roberts et al. (2000, 2001). A total of 32 male homozygous -opioid receptor KO and 28 WT litter mate mice shipped from The Scripps Research Institute (La Jolla, CA) were used in this experiment. The genetic background of these mice was a hybrid C57BL/6Orl 129/SV strain. We housed the mice one to three per cage in a temperature-controlled room in which the lights were on a 12-h light/dark cycle with lights off at 10:00 AM. All experimental procedures were approved by the Duke University Medical Center and the Durham Veterans Affairs Medical Center Institutional Animal Care and Use Committees, in accordance with the Guide for the Care and Use of Laboratory Animals. Whole-Cell Recordings. Brains were rapidly removed from 5to 6-month old DOR KO or WT mice under halothane anesthesia. We immersed intact brains in ice-cold oxygenated (95% O2-5% CO2) artificial cerebrospinal fluid (ACSF). We cut coronal slices (300 m, between bregma 1.0 to approximately 1.9 mm; Paxinos and Franklin, 2004) using a Vibraslice (model 752; Campden Instruments Ltd., Leicester, UK) and incubated them in ACSF containing 120 mM NaCl, 3.3 mM KCl, 1.23 mM NaH2PO4, 25 mM NaHCO3, 2 mM CaCl2, 0.9 mM MgSO4, and 10 mM glucose that was continuously bubbled with 95% O2-5% CO2 at room temperature (for brain dissection and slicing only, ACSF CaCl2 was reduced to 0.5 mM). After a minimum of a 1-h incubation, we transferred a single slice to the recording chamber (volume, 0.5 ml) in which oxygenated ACSF was superfused over submerged slices at a rate of approximately 3 to 4 ml/min. We viewed individual cells with an upright fixed-stage microscope (Zeiss Axioskop; Carl Zeiss Inc., Thornwood, NY) equipped with a water immersion objective (40 , 0.75 numerical aperture, NA), IR filtered light, differential interference contrast optics, and a Hitachi (Tokyo, Japan) charge-coupled device camera. For recording, patch pipettes were pulled from borosilicate glass capillary tubing (1.5-mm o.d., 1.05-mm i.d.; World Precision Instruments, Sarasota, FL) using a Flaming-Brown horizontal microelectrode puller (model P-97; Sutter Instrument, Novato, CA). We filled the pipettes (input resistance, 2–5 M ) with the following recording solution: 75 mM potassium gluconate, 70 mM KCl, 10 mM HEPES, 2 mM MgCl2, 4 mM Mg-ATP, and 0.3 mM Tris-GTP, pH 7.25 (285 mOsM). Liquid junction potentials were not measured or compen-