Alcohol and Alcoholism Advance Access published online on October 29, 2008
Alcohol and Alcoholism, doi:10.1093/alcalc/agn085
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The Novel µ-Opioid Receptor Antagonist, [N-Allyl-Dmt1]Endomorphin-2, Attenuates the Enhancement of GABAergic Neurotransmission by Ethanol
1 Department of Psychiatry, Duke University Medical Center, Durham, NC 27710, USA
2 Neurobiology Research Laboratory, VA Medical Center, NC 27705, USA
3 Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences and The Graduate School of Food and Medicinal Sciences, Kobe Gakuin University, Nishi-ku, Kobe 651-2180, Japan
4 Medicinal Chemistry Group, Laboratory of Pharmacology, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA
5 Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA
* Corresponding author: Department of Psychiatry, Duke University Medical Center, Room 24, Building 16, VA Medical Center, 508 Fulton Street, Durham, NC 27705, USA. Tel: +1-919-286-6810; Fax: +1-919-286-4662; E-mail: HSS{at}duke.edu
Received 15 January 2008; first review notified 24 June 2008; in revised form 19 August 2008; accepted 2 October 2008
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Aims: We investigated the effects of [N-allyl-Dmt1]endomorphin-2 (TL-319), a novel and highly potent µ-opioid receptor antagonist, on ethanol (EtOH)-induced enhancement of GABAA receptor-mediated synaptic activity in the hippocampus.Methods: Evoked and spontaneous inhibitory postsynaptic currents (eIPSCs and sIPSCs) were isolated from CA1 pyramidal cells from brain slices of male rats using whole-cell patch-clamp techniques.Results: TL-319 had no effect on the baseline amplitude of eIPSCs or the frequency of sIPSCs. However, it induced a dose-dependent suppression of an ethanol-induced increase of sIPSC frequency with full reversal at concentrations of 500 nM and higher. The non-specific competitive opioid receptor antagonist naltrexone also suppressed EtOH-induced increases in sIPSC frequency but only at a concentration of 60 µM.Conclusion: These data indicate that blockade of µ-opioid receptors by low concentrations of [N-allyl-Dmt1]endomorphin-2 can reverse ethanol-induced increases in GABAergic neurotransmission and possibly alter its anxiolytic or sedative effects. This suggests the possibility that high potency opioid antagonists may emerge as possible candidate compounds for the treatment of ethanol addiction.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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One of the most promising areas of recent research on addiction has been the development of safe and effective medications for addiction treatment. In the case of ethanol (EtOH) addiction, medication development is complicated because EtOH does not act through a specific receptor mechanism. Rather, it regulates many physiological processes that affect the synaptic actions of multiple neurotransmitters, including opioid peptides and gamma-aminobutyric acid (GABA). There is evidence that the endogenous opioid system participates in the development of ethanol abuse and addiction (Gianoulakis, 2004
), and that the µ-opioid receptor may mediate some aspects of ethanol dependence and its reinforcing effects (Froehlich, 1995; Ulm et al., 1995). For example, mice lacking the µ-opioid receptor consume less ethanol and exhibit less ethanol reward (Roberts et al., 2000) in the conditioned place preference paradigm (Hall et al., 2001). Thus, it seems that the µ-opioid receptor may play a role in the regulation of ethanol consumption. While ethanol consumption and relapse rates are altered by both µ- and 
-opioid receptor antagonists (Honkanen et al., 1996; Ciccocioppo et al., 2002), special attention has been given to the blockade of the µ-opioid receptor subtype because of its role in ethanol-drinking behaviors (Hyytia, 1993; Stromberg et al., 1998).
Most of the µ-opioid receptor antagonists, including naloxone and naltrexone, which are used clinically for the treatment of alcoholism, exhibit adverse side effects (see reviews by Herz (1997), and Heilig and Egli (2006)). Studies have shown that [Dmt1]endomorphin-1 (H-Dmt-Pro-Trp-Phe-NH2) and [Dmt1]endomorphin-2 (H-Dmt-Pro-Phe-Phe-NH2, TL-319) exhibit highly effective µ-opioid agonism exceeding that of the parent compound (Jinsmaa et al., 2004, 2006). Following direct chemical N-allylation, they were transformed into extraordinarily potent µ-opioid antagonists: [N-allyl-Dmt1]endomorphins-1 and -2 retained high µ-opioid receptor affinity (Kiµ = 0.26 nM and 0.45 nM, respectively) with potent µ-opioid antagonism (pA2 = 8.18 and 8.59, respectively) (Li et al., 2007). In exhibiting µ-opioid antagonism, [N-allyl-Dmt1]endomorphin-1 and -2 lacked inverse agonist properties and completely suppressed the profound withdrawal symptoms (jumping, tremors, wet dog shakes) that are elicited by both naloxone and naltrexone in acute morphine-treated mice, or greatly reduced the symptoms in morphine-dependent mice (Marczak et al., 2007). Furthermore, in in vitro studies using cell cultures, these endomorphin antagonists reversed the inhibition by naloxone and naltrexone on the binding of [35S]GTP
S, the biochemical assessment of G-protein interaction with opioid receptors, in isolated cell membranes from cells pretreated with morphine or ethanol (Marczak et al., 2007).
Ethanol promotes GABAA receptor-mediated synaptic inhibition, which likely underlies its anxiolytic and sedative effects. Despite the relevance of the µ-opioid receptor system to ethanol effects, the possible modulation of GABAergic inhibitory transmission by selective, high potency µ-opioid receptor antagonists in the presence of ethanol has not been assessed using synaptic currents in hippocampal neurons. We and others have previously shown that acute ethanol increases the amplitude of evoked inhibitory postsynaptic currents (eIPSCs) and the frequency of spontaneous inhibitory postsynaptic currents (IPSCs) and miniature IPSCs in pyramidal cells from hippocampal slices (Proctor et al., 1992; Weiner et al., 1997, Li et al., 2003, 2006b; Cha et al., 2006). The present results show that blockade of µ-opioid receptors by the specific µ-opioid receptor antagonist [N-allyl-Dmt1]endomorphin-2 (TL-319) at relatively low concentrations can reverse acute ethanol-induced increases in GABAergic neurotransmission in hippocampus, a critical brain area responsible for learning and memory.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Male, Sprague-Dawley rats (PD 25–35) were anesthetized with isoflurane and decapitated. Animals were handled and housed according to the guidelines of the National Institutes of Health Committee on Laboratory Animal Resources. All experimental procedures or protocols were approved by Animal Care and Use Committee of Duke University and Durham VA Medical Center. The brains were removed and placed in cold (4°C) artificial cerebrospinal fluid (aCSF) containing (in mM) 120 NaCl, 3.3 KCl, 1.23 NaH2PO4, 26 NaHCO3, 1.2 MgSO4, 1.8 CaCl2 and 10 D-glucose, pH 7.3, previously saturated with 95% O2/5% CO2. Hippocampal slices (300 µm) were cut on a vibratome (100PLUS, Ted Pella, Inc., Redding, CA, USA) and incubated in aCSF, continuously bubbled with 95% O2/5% CO2 at room temperature (20–24°C).
Neurons were visualized using an infrared differential interference contrast (IR-DIC) Zeiss Axioskop microscope and a 40x water-immersion objective (Zeiss, Oberkochen, Germany). The recording pipettes were filled with a solution containing (in mM) 130 CsCl, 10 HEPES, 4 Mg–ATP, 0.5 Tris–GTP and 4 QX-314, pH 7.25, 285 mOsm. Evoked and spontaneous synaptic currents were recorded using an Axopatch 200-B amplifier (Molecular Devices, Union City, CA, USA). Output current signals were DC-coupled to a digital oscilloscope (TDS 2014, Tektronix, Inc., Beaverton, OR, USA). Series resistance was monitored throughout the recording, and cells were discarded if the series resistance changed by >20%. The digitized data were also stored using Strathclyde Electrophysiology Software, Whole Cell Program (WINWCP) (Courtesy of Dr John Dempster) with an interface (BNC-2090, National Instruments, Austin, TX, USA) to a PC-based computer.
Evoked or spontaneous inhibitory postsynaptic currents (eIPSCs or sIPSCs) were recorded at –70 mV in the presence of D-(–)-2-amino-5-phosphonovaleric acid (APV, 50 µM) (ACROS, Geel, Belgium) and 6,7-dinitroquinoxaline-2,3-dione (DNQX, 20 µM) (Sigma Chemical Co., St Louis, MO, USA). Current pulses (100-µs duration) were delivered to the slice at a frequency of 0.05 Hz through a monopolar stimulation electrode placed 50–75 µm lateral to the recording pipette. At the holding potential of –70 mV, eIPSCs and sIPSCs were inward currents and were abolished by adding 20 µM bicuculline, confirming their mediation by GABAA receptors. Stable recordings of eIPSCs or sIPSCs were maintained for 60 min or more without noticeable changes in series resistance or input resistance. The chamber temperature was maintained at 34°C during recording.
TL-319 was synthesized using published methods (Li et al., 2006a), dissolved in dimethyl sulfoxide (DSMO) and diluted in the extracellular bath solution to the desired concentration prior to use. Ethanol was bathapplied.
IPSCs were analyzed off-line using Clampfit 9.2 (Molecular Devices, Union City, CA, USA) or Mini-analysis software 6.0.3 (Synaptosoft, Decatur, GA, USA). The data were expressed as mean ± SEM for graphics, and statistical inferences were made using Student's t-tests and one-way ANOVAs, followed by Bonferroni post hoc comparisons when appropriate. The Kolmogorov–Smirnov (K-S) test was employed to assess differences in the cumulative distributions of sIPSC frequency and amplitude across drug exposures, within cells. The threshold for statistical significance was set at P 
0.05 for all inferential statistical analyses.
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RESULTS |
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The effect of TL-319 was initially assessed on eIPSC amplitude and sIPSC frequency. Fig. 1A shows representative traces of eIPSCs evoked by single stimuli. Fig. 1B illustrates that 1 µM TL-319 did not alter the eIPSC amplitude: the average amplitude of eIPSCs was 196.2 ± 25.2 and 204.9 ± 39.8 pA before and after bath application of 1 µM TL-319, respectively; the paired t-test revealed no significant differences in the mean amplitude (P > 0.05, n = 7). Similarly, 1 µM TL-319 did not significantly alter the mean frequency of sIPSCs: control frequency, 4.55 ± 0.78 Hz, and during TL-319 application, 4.35 ± 0.69 Hz (paired t-test, P > 0.05, n = 7, data not shown).
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Since bath application of 60 mM EtOH reliably increases the frequency of sIPSCs in CA1 pyramidal cells (Li et al., 2006b
), we used this dose to assess the effects of TL-319. Fig. 2A shows representative examples of the effects of EtOH, and EtOH plus TL-319 on sIPSCs. EtOH significantly increased the frequency of sIPSCs and this was reversed by 1 µM TL-319. The cumulative probability analysis of sIPSC frequency indicated that cumulative inter-event intervals decreased after exposure to EtOH, shifting the distribution curve to the left (P < 0.01, K-S test, Fig. 2B). This EtOH-induced increase in sIPSC frequency was significantly reduced by 1 µM TL-319 (P < 0.01, K-S test, Fig. 2B). Neither EtOH nor TL-319 changed the distribution pattern of sIPSC amplitude (P > 0.05, K-S test, Fig. 2C).
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The effect of TL-319 on the EtOH-induced increase in sIPSC frequency was concentration dependent. While 10 nM TL-319 had no effect and 100 nM TL-319 attenuated EtOH-induced increases in sIPSC frequency in only two of seven pyramidal cells (a statistically non-significant effect), both 500 and 1000 nM TL-319 significantly attenuated the EtOH-induced increase in sIPSC frequency (one-way ANOVA F(3, 23) = 1.29, P = 9.42x10–5). Post hoc analyses revealed that TL-319 suppressed the EtOH-induced increase in the frequency of sIPSCs in a concentration-dependent manner (Fig. 2D).
The decay kinetics of sIPSCs were also unaffected by EtOH or TL-319. sIPSC decay kinetics under each condition were fitted as a biexponential equation. Representative examples are shown in Fig. 2E (top panel). There were no significant changes in the mean fast and slow decay times (tau) under either treatment condition, compared to control (Fig. 2E, bottom panel). This suggests a non-postsynaptic mechanism for the effect of TL-319 on EtOH-induced enhancement of sIPSCs.
Studies in both humans and animal models have shown that the non-selective µ-opioid receptor antagonist naltrexone reduces ethanol consumption (Croop et al., 1997; Krystal et al., 2001; Srisurapanont and Jarusuraisin, 2005). Therefore, we assessed the effects of naltrexone on EtOH-induced enhancement of IPSCs. Thirty micromolar naltrexone had no effect on EtOH-induced enhancement of sIPSC frequency; however, at 60 µM, naltrexone significantly reduced the increase in sIPSC frequency that was produced by 60 mM EtOH (Fig. 3A). The cumulative inter-event intervals shifted to the left after exposure to EtOH (P < 0.01, K-S test, Fig. 3B), and 60 µM naltrexone diminished this effect (P < 0.01, K-S test, Fig. 3B). While neither 30 µM nor 60 µM naltrexone altered the amplitude of sIPSCs (P > 0.05, K-S test, Fig. 3C), 60 µM naltrexone attenuated the EtOH-induced increase in sIPSC frequency (paired t-test, P < 0.05, n = 6) (Fig. 3D).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The µ-opioid receptor system represents a potential target for therapeutic treatment of ethanol dependence, particularly since its impact on the physiological effects of ethanol can be altered by high-potency antagonists. The present data show that TL-319, a selective and potent µ-opioid receptor antagonist (Li et al., 2006a
, 2007), attenuated EtOH-induced increases in inhibitory neurotransmission in the hippocampus, in the absence of any effect on baseline inhibitory neurotransmission. In addition, this novel compound appeared to be more potent than naltrexone in reversing the effects of EtOH on hippocampal inhibitory synaptic function.
The opioid neuromodulatory system is involved in both the mediation of acute ethanol effects and the development of addiction to ethanol. For example, in animal models, ethanol consumption is associated with the release of endogenous opioids (Schulz et al., 1980; De Waele et al., 1992; Li et al., 1998; Roberts et al., 2000), and the ethanol-induced opioid release is more pronounced in ethanol-preferring rats than in non-ethanol-preferring animals (De Waele et al., 1994). In addition, a significant correlation has been identified between the expression and availability of µ-opioid receptors during abstinence and ethanol craving in detoxified ethanol-dependent patients (Heinz et al., 2005).
Consistent with our previous reports (Li et al., 2003, 2006b), the present data indicate that acute application of ethanol results in a significant increase in GABAergic transmission in CA1 pyramidal cells in hippocampal slices. That the ethanol-induced increase in sIPSC frequency was attenuated by TL-319 suggests that the effect of ethanol on inhibitory function in the hippocampus could be driven, in part, by an activation of the µ-opioid receptor system. Although the precise mechanisms whereby these effects are mediated remain unclear, they are provocative because of the in vivo effects of specific µ-opioid receptor antagonists. For example, central or systemic administration of the specific µ-opioid receptor antagonists CTOP (Hyytia, 1993; Hyytia and Kiianmaa, 2001), β-funaltrexamine (Stromberg et al., 1998] or naloxonazine (Honkanen et al., 1996) reduces ethanol consumption in animal models. Thus, in light of the published literature (Marczak et al., 2007), the present data suggest a potential cellular mechanism whereby µ-opioid receptors may mediate some of the effects of ethanol. Although the hippocampus is not typically associated with motivational aspects of ethanol consumption, it is a model neural system for studying synaptic inhibition and the effects of addictive drugs, particularly ethanol. These findings further suggest that studies should be undertaken in other GABAergically mediated neural circuits that are associated with anxiolysis or reward stimuli.
The hippocampus is critically involved in learning and memory formation, a set of functions that are powerfully compromised by both acute and chronic ethanol consumption. Moreover, the hippocampus is heavily innervated by opioidergic neurons, including inhibitory interneurons in CA1 subregions (Crain et al., 1986; Drake and Milner, 2002). Electrophysiological findings indicate that activation of µ-opioid receptors by agonists suppresses the inhibitory activity mediated by GABAA and GABAB receptors in hippocampal CA1 pyramidal cells (Siggins and Zieglgansberger, 1981; Lupica, 1995; McQuiston, 2007). Indeed, two endogenous opioid peptides, endomorphin-1 and endomorphin-2, which exhibit high affinity and selectivity for µ-opioid receptors, have been identified in the brain (Zadina et al., 1997). These peptides are known to reduce GABAergic synaptic transmission in the nucleus of the solitary tract (Glatzer and Smith, 2005) and the nucleus ambiguus (Venkatesan et al., 2002], although Browning et al. (2002) reported that excitatory, but not inhibitory, synaptic transmission was blocked by the agonist [Met5]enkephalin, or selective opioid receptor antagonists, suggesting that opioid peptides exert an influence on selective pathways. Because we did not observe an effect of TL-319 on GABAA receptor-mediated IPSCs in the absence of EtOH, the present data appear to be inconsistent with a depressive effect of µ-opioid receptors on hippocampal GABAergic synaptic transmission. The functional role of the endogenous µ-opioid system in the presence of ethanol is unknown. Thus, the mechanisms that underlie the interaction between GABAergic neuromodulators, such as EtOH, and the endogenous µ-opioid system remain unclear and may involve other neuromodulatory systems.
Although naltrexone is one of three FDA-approved drugs for the treatment of alcoholism, it is a non-specific opioid antagonist and blocks three major types of endogenous opioid receptors (µ, and 
). Pharmacological studies indicate that naltrexone affects the activity of mesolimbic dopaminergic cells (Di Chiara, 1995) and blocks ethanol-induced increases in the extracellular concentration of dopamine in the nucleus accumbens (Benjamin et al., 1993; Gonzales and Weiss, 1998]. Furthermore, it suppresses ethanol consumption (Anton et al., 1999) and craving among alcoholics (O’Malley et al., 2002), effects which are associated with its action on opioid systems. Although a recent study reported that naltrexone enhanced ethanol consumption under certain circumstances (Juarez and Eliana, 2007), our electrophysiological data show that, similar to the action of TL-319, naltrexone also attenuates EtOH-induced increases in the frequency of sIPSCs. A similar result was reported in an in vivo study, in which pretreatment with 30 mg/kg naltrexone, but not 3 mg/kg, reduced ethanol-induced increases in the firing rate of dopamine neurons (Inoue, 2000). We cannot, however, exclude the possibility that naltrexone antagonizes the effect of EtOH on sIPSCs through multiple mechanisms (Gonzales and Weiss, 1998).
In conclusion, TL-319, a selective and potent µ-opioid receptor antagonist, significantly attenuates ethanol-induced increases in GABAergic synaptic activity and the effect was more than two orders of magnitude greater than the FDA-approved naltrexone. Although the neuronal mechanisms underlying µ-opioid receptor regulation of GABAergic function in the presence of ethanol are poorly understood, this potent new compound may represent a novel class of highly effective agents for investigating acute ethanol effects on synaptic transmission and may eventually find application in the clinical treatment of alcoholism.
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ACKNOWLEDGEMENTS |
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This work was supported by IMR grant to Q.L. and NIAAA grant AA-01489 to H.S.S., by VA senior Research Career Scientist Awards to H.S.S. and W.A.W., and in part by the Intramural Research Program of the NIH and NIEHS.
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