Alcoholism: Clinical and Experimental Research

Vol. 33, No. 6 June 2009

Effect of Acute Ethanol Administration on the Release of Opioid Peptides From the Midbrain Including the Ventral Tegmental Area
Samuel Jarjour, Li Bai, and Christina Gianoulakis

Background: Experimental evidence suggests that ethanol alters the activity of the endogenous opioid peptide systems in a dose and brain-region dependent manner. These alterations may inuence the processes of ethanol reward and reinforcement. Thus, it was the objective of this study to investigate the response of the 3 major opioid peptide systems (endorphins, enkephalins, and dynorphins) to acute ethanol administration, at the level of the midbrain including the ventral tegmental area (midbrain VTA), a region important for drug, including ethanol reinforcement. Methods: Using the in vivo microdialysis technique coupled with specic solid-phase radioimmunoassay for b-endorphin, met-enkephalin, and dynorphin A18, changes in the extracellular concentration of theses peptides at the level of midbrain VTA were determined at distinct time points following the administration of 0.0 (saline), 0.8, 1.2, 1.6, 2.0, and 2.4 g ethanol kg B.Wt. Results: A biphasic effect of ethanol on b-endorphin release was found, with low to medium (1.2, 1.6, and 2.0 g) but not high (2.4 g) doses of ethanol, inducing a signicant increase in the dialysate content of b-endorphin. A late increase in the dialysate content of dynorphin A18 was observed in response to the 1.2 g ethanol dose. However, none of the ethanol doses tested signicantly altered the content of met-enkephalin in the dialysate. Conclusions: The present ndings suggest that the ethanol-induced increase of b-endorphin release at the level of midbrain VTA may inuence alcohol reinforcement. Key Words: Alcohol, b-Endorphin, Met-Enkephalin, Dynorphin A18, In Vivo Microdialysis.

HE ENDOGENOUS OPIOID system is associated with a range of functions, including ethanol reward and reinforcement, and several lines of evidence suggest that enhanced activity of endogenous opioids in response to acute ethanol exposure mediate ethanol intake and preference (Gianoulakis, 2004; Olson et al., 1996; Oswald and Wand, 2004). Three endogenous opioid peptide families are distinguished, according to their high molecular weight precursors. They include the families of the pro-enkephalin, proopiomelanocortin (POMC), and pro-dynorphin-derived peptides. Several peptides are derived from pro-enkephalin, including met-enkephalin, met-enkephalin-Arg6-Phe7, metenkephalin-Arg6-Gly7-Leu8, and leu-enkephalin. Although widely distributed throughout the brain, enkephalinergic

From the Department of Psychiatry, McGill University (SJ, CG), Montreal, Quebec, Canada; Douglas Mental Health University Institute (SJ, LB, CG), Neuroscience Division, Montreal, Quebec, Canada; and Department of Physiology, McGill University (CG), Montreal, Quebec, Canada. Received for publication September 17, 2008; accepted January 23, 2008. Reprint requests: Christina Gianoulakis, PhD, Douglas Mental Health University Institute, Neuroscience Division, 6875 LaSalle Boulevard, Montreal, Quebec, H4H 1R3 Canada; Fax: 514-762-3034; E-mail: christina.gianoulakis@mcgill.ca Copyright 2009 by the Research Society on Alcoholism.
DOI: 10.1111/j.1530-0277.2009.00924.x
Alcohol Clin Exp Res, Vol 33, No 6, 2009: pp 10331043

perikarya are concentrated in basal ganglia and striatum (Finley et al., 1981; Sar et al., 1978). Enkephalins bind to both l and d opioid receptors, with some studies demonstrating 10 to 25 times higher afnity for d than l receptors (Akil et al., 1988; Charnes, 1989; Khachaturian et al., 1985) and other studies demonstrating similar afnities (Raynor et al., 1994). POMC-derived opioids include b-endorphin 131, b-endorphin 127, b-endorphin 126, a, b, and cmelanocyte stimulating hormones, adrenocorticotropic hormone, and corticotrophin-like intermediate lobe peptide. Cells synthesizing POMC-derived peptides are present in arcuate nucleus, nucleus tractus solitarius, and anterior and neurointrmediate lobes of the pituitary gland (Bronstein et al., 1992; Gee et al., 1983; Khachaturian et al., 1985; Watson et al., 1977). Projections from the POMC-producing neurons innervate many brain regions including nucleus accumbens (NAcb), amygdala, hypothalamus, and ventral tegmental area (VTA) (Khachaturian et al., 1985; Watson et al., 1977; Zakarian and Smyth, 1982). b-endorphin 131 binds with roughly equal afnity to l and d opioid receptors, presenting little afnity for j receptors. Pro-dynorphinderived peptides include dynorphin A(18; 117), dynorphin B(113; 1429; 129), and a b-neo-endorphins (Akil et al., 1984). Dynorphins bind preferentially to j opioid receptors (Akil et al., 1998; Quirion and Pert, 1981). Dynorphin synthesis is widely distributed in the brain, and j opioid receptors are found in cortex, amygdala, striatum, thalamus, hypothalamus, and VTA (Akil et al., 1984; Mansour et al., 1988).
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Several studies using opioid receptor agonists and antagonists have examined the role of the endogenous opioid system in mediating ethanol reinforcement by measuring the effects of disrupting opioid signaling on ethanol behaviors such as ethanol operant responding and place conditioning. In the absence of alcohol, activation of distinct opioid receptor types produces rewarding and aversive states (Bals-Kubik et al., 1989; Mucha and Iversen, 1984; van Ree et al., 1979; Van Wolfswinkel and van Ree, 1985). The nonspecic opioid receptor antagonists naloxone, naltrexone, and methylnaloxonium produce dose-dependent extinction patterns of operant responding for ethanol in trained monkeys, rats, and mice (Altshuler et al., 1980; Froehlich et al., 1990; Heyser et al., 1999; Hubbell et al., 1986; Hyytia and Sinclair, 1993; Middaugh et al., 1999; Samson and Doyle, 1985; Stromberg et al., 1998; Volpicelli et al., 1986). Comparably, microinjections of methylnaloxonium, in reward-relevant brain regions such as the VTA of the midbrain attenuated ethanol place preference conditioning (Bechtolt and Cunningham, 2005). Enkephalinase inhibitors, which enhance the actions of enkephalins, have been found to both decrease and increase voluntary drinking in rats and mice, depending on species and strain (Froehlich et al., 1991; George et al., 1991). Furthermore, d opioid receptor blockade, which may interfere with enkephalinergic neurotransmission, was shown to alter ethanol preference in some alcohol-preferring rats and mice (Froehlich et al., 1991; June et al., 1999; Le et al., 1993). How ever, the direction of the effects of d-opioid receptor blockade varies between studies and animal models used (Koenig and Olive, 2002). The j receptor agonist U50,488H decreased alcohol intake in rats, while pretreatment with a j opioid receptor antagonist reversed this effect (Lindholm et al., 2001). A low dose of a short-acting j receptor agonist increased operant responding for alcohol, while a higher dose attenuated it (Holter et al., 2000). Studies on the effects of ethanol on the activity of endogenous opioid peptides have supported a role of various components of the endogenous opioid system in mediating some of the acute responses to ethanol. Stimulation of b-endorphinergic activity has been observed following in vivo exposure to ethanol, in postmortem tissues from several brain regions including the VTA (Rasmussen et al., 1998), in dialysate samples from the shell region of the NAcb (Marinelli et al., 2003; Olive et al., 2001) and from the central amygdala (Lam et al., 2008), as well as following in vitro exposure to ethanol of hypothalamic and pituitary tissues (de Waele and Gianoulakis, 1993; Gianoulakis, 1990; Gianoulakis and Barcomb, 1987; Keith et al., 1986). Increased hypothalamic and pituitary POMC mRNA levels have been reported after acute (Rasmussen et al., 1998) and short chronic (Angelogianni and Gianoulakis, 1993; de Waele and Gianoulakis, 1994; Krishnan-Sarin et al., 1998) ethanol treatments, suggesting increased peptide biosynthesis. Alterations of enkephalinergic activity in response to ethanol have also been observed. Acute in vivo exposure to ethanol increased the met-enkephalin peptide content in postmortem tissues of the rat striatum

(Schulz et al., 1980; Seizinger et al., 1983) and in dialysate samples from the shell region of the NAcb (Marinelli et al., 2005), as well as the pro-enkephalin mRNA content in the rat hypothalamus and striatum (Li et al., 1998; Ng et al., 1996). However, the effects of acute ethanol administration on pro-enkephalin mRNA levels was not consistent, with both enhancement and attenuation of pro-enkephalin mRNA levels being observed, depending on the distinct brain region and time postethanol administration (Mendez and MoralesMulia, 2006). A transient reduction in the binding of the specic l opioid receptor agonist 3H-DAMGO was observed in the VTA following acute ethanol administration (Mendez et al., 2001), which could be attributed, at least in part, to an ethanol induced increase in the release of b-endorphin, met-enkephalin, or both. Reports on the effects of ethanol on dynorphin peptides are inconsistent. An increase in the extracellular concentration of dynorphin A18 was observed in the shell region of the NAcb following high ethanol doses (Marinelli et al., 2006), while other studies reported decrease (Seizinger et al., 1983), increase (Lindholm et al., 2000), or no effect (Nylander et al., 1994; Przewlocka et al., 1994) of ethanol on dynorphin peptides or prodynorphin mRNA content in distinct brain regions. The inconsistency among studies may be due to differences in the ethanol doses, duration of ethanol treatment, animal strains, and brain regions tested. Despite experimental evidence implicating the opioid system at the level of the VTA in ethanol reinforcement, few studies have examined the activity of endogenous opioid peptides in the VTA following acute ethanol administration. Thus, the main objective of the current investigation was to perform a dose response, time course study of the response of b-endorphin, met-enkephalin, and dynorphin A18 peptides at the level of midbrain including the VTA following the intraperitoneal (i.p.) injection of rats with various doses of ethanol, using the in vivo microdialysis technique coupled with solidphase radioimmunoassay (RIA).
MATERIALS AND METHODS Animals Male SpragueDawley rats (Charles River, St-Constant, QC, Canada), weighing 280 to 350 g, were used. Animals were housed individually to prevent gnawing of cannulae and had access to food and water ad libitum. A period of at least 1 week was allowed for acclimatization prior to initiation of experiments. Animals were kept in a temperature and humidity-controlled environment, on a 12-h light dark cycle (lights on at 8:00 am). Rats were treated in accordance with McGill Universitys Policy on the Handling and Treatment of Laboratory Animals and the Canadian Council on Animal Care guidelines. Every effort was taken to minimize the number of animals used and their suffering. Surgery Prior to surgical procedures, rats were anaesthetized with a cocktail of ketamine (50 mg kg), xylazine (5 mg kg), and acepromazine (1 mg kg). Guide cannulae (20 mm shaft length; 1 mm O.D.; S.P.E. Ltd., Concord, ON, Canada) were stereotaxically implanted

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according to the established coordinates from Solinas and colleagues (2004) ()6.0 mm anteroposterior from the bregma, 1 mm lateral from the midline, and 5.8 mm dorsoventral from the skull). Cannulae were anchored in place using 3 stainless steel screws and dental cement. The cannula passage was blocked with an obturator. A tether screw, to which microdialysis tethers would be connected, was set with dental cement. Animals were given 3 to 5 days to recover from surgery prior to habituation and in vivo microdialysis. Habituation and In Vivo Microdialysis Habituation and microdialysis were carried out in round, black, plastic testing cages measuring 31 (diameter) 30 (height) cm. On 3 occasions, given on alternate days, rats were housed in the testing cages overnight. On the following day, rats received single i.p. injections of sterile saline solution to acclimatize them to the manipulations during microdialysis testing. Rats remained un-tethered during habituation sessions. Following the third habituation session rats were weighed, obturators were removed, and microdialysis probes (20 mm shaft length, 0.6 mm O.D., 2 mm PES membrane, 15 kD cutoff; S.P.E.) were implanted under light isourane (Janssen, Toronto, ON, Canada) anaesthesia to a nal depth of 7.8 mm from the skull (Solinas et al., 2004). A syringe pump (Harvard Apparatus, South Natick, MA) was set to infuse articial cerebrospinal uid (aCSF: 124 mM sodium chloride, 3 mM potassium choride, 1 mM magnesium chloride-6H2O, 0.5 mM sodium phosphate monobasic-H2O, 5 mM sodium phosphate dibasic, 1.3 mM calcium chloride-2H2O, 0.2 mM l-ascorbic acid, 0.025% bovine serum albumin) overnight at a rate of 0.2 ll min. PTFE tubing (0.56 mm I.D.; Cole-Parmer, Vernon Hills, IL) connected the pump to a dual-channel swivel (Instech, Plymouth Meeting, PA). The swivel was counter-balanced on an arm that swung laterally and vertically with the animals movements. FEP tubing (0.12 mm I.D.; CSC, Montreal, QC, Canada) ran to and from the probe through a stainless steel spring tether that was connected to the tether screw. At 8:00 am of the following day, the pump rate was increased to 2.0 ll min. The ow rate of 2.0 ll min was selected on the basis of previous in vitro studies on the absolute and relative recovery of the 3 opioid peptides using the same type of probes as those used in the present study (Marinelli et al., 2003, 2004, 2005, 2006; Olive et al., 2001). Two hours were allowed for the animals to acclimatize to the higher ow rate and dialysate samples were then collected at 30-minute intervals for the duration of the study. Following 4 baseline dialysate collections, rats were injected with 0.0, 0.8, 1.2, 1.6, 2.0, and 2.4 g ethanol kg B.Wt., administered i.p. as 0, 5, 7.5, 10, 12.5, and 15% ethanol in 0.9% saline (v v), respectively, ensuring volumematched injections across dose groups. Collection of dialysate continued for 4 hours postinjection. All dialysate samples were collected in 500 ll polypropylene vials immersed in ice, and were promptly frozen in CO2 prior to storage at )70C. The present study used previously established implantation coordinates and microdialysis probes (Solinas et al., 2004) with main target site for probe implantation the region of VTA. Furthermore, dialysate samples from animals whose probes were positioned outside the VTA were not included in the analysis. However, considering the 2 mm length of the microdialysis probe used and the approximately 1 mm size of the VTA in the dorsal-ventral dimension (Paxinos and Watson, 1998) the origin of peptides in the dialysates may not be exclusively from the VTA but may include midbrain regions above or below the VTA as well. Thus, to more accurately portray the brain regions from which the microdialysis samples were taken, in the discussion we refer to midbrain region including the VTA (midbrain VTA) instead of exclusively VTA. Histology Following the microdialysis session the rats were euthanized with CO2 and brains were removed, snap-frozen in isopentane and stored

at )70C. Frozen brains were sectioned into 40 lm coronal slices and mounted on gelatin-coated slides. Slides were Nissl-stained and inspected for accuracy of probe placement. Animals whose VTA was not accurately targeted were excluded from peptide content assessment. Solid-phase RIA for the Opioid Peptides Dialysate peptide concentrations were determined using a solid phase RIA (after Maidment and Evans, 1991; Marinelli et al., 2003, 2005, 2006). Ninety-six removable-well microplates (Dynex Microlite 2, Chantilly, VA) were lled with 0.8 lg protein A (Sigma, St. Louis, MO) 100 ll 0.1 M sodium bicarbonate (pH approx. 8.4) and incubated for 24 h at 4C. The next day, wells were emptied and rinsed with 200 ll of RIA buffer (0.15 M potassium phosphate dibasic, 0.2 mM l-ascorbic acid, 0.1% Tween 20, 0.1% gelatin, pH adjusted to 7.4 with 10 N hydrochloric acid). A 50-ll aliquot of antiserum specic for b-endorphin (1:5000 dilution), met-enkephalin (1:5000 dilution), or dynorphin A18 (1:5000 dilution) was placed in each well and incubated for 24 hours at 4C. After 24-hour incubation at 4C wells were again rinsed with 200 ll of RIA buffer. Fifty microliters of appropriately diluted dialysate samples or standards (diluted in RIA buffer) were added to wells, and again incubated for 24 hours at 4C. Then, 50 ll of either iodinated b-endorphin [5000 to 6000 cpm 50 ll, specic activity (SA) = 74 TBq mmol; Amersham Pharmacia Biotech, Buckinghamshire, U.K.], iodinated met-enkephalin (5000 to 6000 cpm 50 ll, SA = 60 TBq mmol; Peninsula Laboratories, Inc., San Carlos, CA), or iodinated dynorphin A18 (5000 to 6000 cpm 50 ll, SA = 71 TBq mmol; Peninsula Laboratories Inc.) was added to each well, and incubated for 48 hours at 4C. Following this incubation, wells were emptied and rinsed with 200 ll of RIA buffer and placed into 5 ml polypropylene culture tubes and counted on a c-ray counter (Cobra II; Packard, Meriden, CT). The RIA detection limits were 0.5 pg well (0.14 fmol well) for b-endorphin, 0.5 pg well (0.87 fmol well) for met-enkephalin, and 0.05 pg well (0.05 fmol well) for dynorphin A18. The IC50s were 50 pg well (14.43 fmol well) for b-endorphin, 32 pg well (55.98 fmol well) for met-enkephalin, and 2 pg well (2.04 fmol well) for dynorphin A18. The antiserum used for b-endorphin was specic for the C-terminal of b-endorphin and recognized proopiomelanocortin, b-lipotropin, and both acetylated and nonacetylated forms of b-endorphin 131, 127, and 126. This antiserum did not recognize adrenocorticotropic hormone, a-melanotropin, or b-lipotropin fragments 165, 6267, and 8084 (Gianoulakis and Gupta, 1986). The met-enkephalin antiserum (Peninsula Laboratories, Inc.) specications, provided by the manufacturer, indicate that the antibody presents cross-reactivity with Met-enkephalin (100%), Leu-enkephalin (2.8%), Met-enkephalin-Arg-Phe (0.1%), and b-endorphin (0.1%), and no cross-reactivity with Met-enkephalin-Arg-Gly-Leu, dynorphin A117, adrenocorticotropic hormone, or endothelin-1. The specicity of the dynorphin A18 antiserum (Peninsula Laboratories Inc.), was indicated by the manufacturer to display cross-reactivity with dynorphin A18 (100%), large dynorphin, dynorphin A, dynorphin A113 and dynorphin A19 (less than 0.01%), and no cross-reactivity with dynorphin A17, dynorphin A16, dynorphin A617, dynorphin B, b-endorphin, Met-enkephalin, a-neoendorphin, and leuenkephalin-arg. Determination of Blood Alcohol Concentration Blood alcohol concentrations (BAC) were determined in a separate cohort of rats than those used for the microdialysis. Briey, 50 ll of tail blood samples obtained at 15, 30, 45, 60, 90, 120, 180, 300, and 360 minutes after the i.p. injection of the various doses of ethanol, were placed into 1.5 ml centrifuge tubes each containing 450 ll ice cold 6.25% trichloroacetic acid solution (Sigma-Aldrich, St. Louis, MO), mixed vigorously by vortexing, and centrifuged for 5 minutes

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at 6000 rpm. The supernatants were transferred into new 1.5 ml centrifuge tubes, frozen in dry ice, and stored at )70C for determination of the BAC. A commercially available ethanol assay kit (Diagnostic Chemicals Limited, Charlottetown, PEI, Canada) was used to determine the BAC according to the manufacturers instructions, based on a modied alcohol dehydrogenase spectrophotometric method (Lundquist, 1957). The BAC is expressed as mg ethanol per 100 ml blood. Data Analysis For the statistical evaluation of the differences in the BACs in response to various doses of ethanol, a 2-way mixed ANOVA was used with ethanol dose as the independent variable and time as the repeated variable. For each peptide (b-endorphin, met-enkephalin, or dynorphin A18) the stability of the basal concentration in the dialysates collected at the 4 time points prior to the i.p. injection was determined by 1-way repeated measures ANOVA with time as the repeated variable. The basal levels of each peptide in the dialysates were determined as the mean of the 4 basal collections prior to the i.p. injections. The basal dialysate concentrations of each peptide for the 6 ethanol dose groups (0.0, 0.8, 1.2, 1.6, 2.0, and 2.4 g ethanol kg B.Wt.) were analyzed using 1-way, independent samples ANOVAs. The effect of ethanol on the extracellular levels of each peptide was estimated as a percent change from the basal levels. The data of the percent change from baseline for each peptide were analyzed using a mixed 2-way ANOVA with ethanol dose as the independent variable and time as the repeated variable. Further analysis of main effects and interactions was done with simple ANOVAs and the Neuman Keuls multiple comparisons test. For all statistical evaluations, the DATASIM statistical software package for an IBM-compatible computer was used (DATASIM version 1.1, 1996). For all tests a p 0.05 was considered signicant.

RESULTS In the present investigation animals were injected i.p. with 5 doses of ethanol and a volume-matched saline control. Among the behavioral responses observed following ethanol administration was greater activity shortly after administration of the lower doses of ethanol (0.8 g ethanol kg B.Wt.), and behavioral depression and hypnosis following the higher doses of ethanol (2.0 and 2.4 g ethanol kg B.Wt.). BACs are

shown in Fig. 1. The mixed 2-way ANOVA indicated a significant main effect of dose [F(4, 162) = 21.82, p < 0.0001], time [F(9, 162) = 95.53, p < 0.0001], and a signicant interaction between dose and time factors [F(36, 162) = 7.75, p < 0.01]. Figure 2 shows the tracks left by the microdialysis probes, identied in 40 lm Nissl stained coronal sections, which were plotted on a stereotaxic map of the VTA )6.3, )6.04, and )5.8 mm from the bregma. The animals in which the VTA was considered to be accurately targeted were included in peptide content assessment and data analysis. Mean basal values of b-endorphin, met-enkephalin, and dynorphin A18 were assessed for each animal, using the initial four 30 minute dialysate collections taken prior to the i.p. injection of saline or ethanol solutions. One-way repeated measures ANOVA demonstrated no signicant difference between the basal concentrations at the 4 time points prior to the i.p. injection neither for b-endorphin [F(3,153) = 0.63; p > 0.05], nor for enkephalin [F(3,144) = 1.87; p > 0.05], nor for dynorphinA18 [F(3,129) = 1.40; p > 0.05], indicating a stable baseline for all 3 peptides. Thus, to determine the average basal concentration of each opioid peptide in the dialysates, the peptide concentration of all 4 basal dialysate samples of each experiment in the 6 dose groups were used. The average concentration for b-endorphin was 1722 182.3 pg 50 ll (496 .70 52.60 fmol 50 ll), for enkephalin was 91.61 15.13 pg 50 ll (160.3 26.47 fmol 50 ll), and for dynorphinA18 was 17.29 2.46 pg 50 ll (17.65 2.51 fmol 50 ll). For each peptide the basal dialysate concentration in each of the 6 dose groups is shown in Fig. 3AC. These basal values were statistically analyzed using a 1-way, independent variables ANOVA. The analysis revealed no signicant difference in the basal peptide values among the 6 dose groups for b-endorphin [F(5, 47) = 0.51, p > 0.05], met-enkephalin [F(5, 41) = 1.13, p > 0.05], and dynorphin A18 [F(5, 41) = 1.03, p > 0.05]. Figure 4 AE presents the changes in the dialysate b-endorphin content, following the i.p. injections of 0.0, 0.8, 1.2, 1.6, 2.0, and 2.4 g ethanol kg B.Wt., expressed as percent change from the basal values. The mixed, 2-way ANOVA, with ethanol doses the nonrepeated measure, and time the repeated measure demonstrated a signicant main effect of

Fig. 1. Blood alcohol concentrations from a separate cohort of animals than those used in the microdialysis experiments, following an i.p. injection with 0.8 (n = 4), 1.2 (n = 4), 1.6 (n = 4), 2.0 (n = 5), or 2.4 (n = 5) g ethanol kg B.Wt.

Fig. 2. A representative photomicrograph of a Nissl stained 40 lm coronal brain section. Arrow indicates probe location in the midbrain VTA region. Diagrams of coronal sections with solid lines indicating the neuroanatomic probe locations in the brains of the animals used for peptide content analysis. Numbers to the lower right of each diagram indicate the position anteroposterior to bregma, in mm.

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Fig. 3. Content of b-endorphin (A), met-enkephalin (B), and dynorphin A18 (C), in the dialysate samples for each ethanol dose group, collected from the midbrain VTA region under basal conditions. Bars represent the mean SEM. The number in parentheses indicates the number of animals in each dose group.

dose [F(5, 46) = 5.79, p = 0.0003], where rats administered 1.2, 1.6, or 2.0 g ethanol kg B.Wt. showed signicantly greater levels of b-endorphin than the saline group (p < 0.05). A signicant main effect of time [F(8, 368) = 17.32, p < 0.0001], and a signicant interaction between dose and time factors [F(40, 368) = 1.62, p < 0.02] were also found. NeumannKeuls post-hoc tests revealed that in the 1.2 g ethanol kg B.Wt. group, b-endorphin was signicantly higher than basal values at all time points postinjection, except for the 30-minute time point (p < 0.05). The changes in the dialysate b-endorphin content following the i.p. injection of 1.2 g ethanol were signicantly higher than

those observed in the saline-injected animals at 60, 150, 180, 210, and 240 minutes postinjection (p < 0.05). In response to the 1.6 g ethanol kg B.Wt. dose, the dialysate b-endorphin content was signicantly elevated from basal values at all time points (p < 0.05), and was signicantly higher than the saline-treated animals at all time points except the last (240 minutes postethanol) (p < 0.05). In the 2.0 g ethanol kg B.Wt. dose group, b-endorphin levels were signicantly higher than basal values at 120, 150, 180, 210, and 240 minutes (p < 0.05), and from those of the saline-treated animals, at 180, 210, and 240 minutes postinjection (p < 0.05). Changes in the dialysate b-endorphin content following the i.p. injection of either 0.8 or 2.4 g ethanol kg B.Wt. did not signicantly differ from those following the i.p. injection of saline (p > 0.05) or from each other. Figure 5 AE presents the changes in the dialysate met-enkephalin content following the i.p. injections of 0.0, 0.8, 1.2, 1.6, 2.0, and 2.4 g ethanol kg B.Wt, expressed as percentage of the basal values. The mixed, 2-way ANOVA demonstrated no signicant main effect of dose [F(5, 42) = 0.47 p > 0.05], a signicant main effect of time [F(8, 336) = 7.34, p < 0.0001], and no signicant interaction between dose and time factors [F(40, 336) = 1.02, p > 0.05]. The signicant main effect of time was associated with a gradual elevation above basal values with time, in the dialysate met-enkephalin content of all treatment groups. Figure 6 AE presents the changes in the dialysate dynorphin A18 content following the i.p. injection of 0.0, 0.8, 1.2, 1.6, 2.0, or 2.4 g ethanol kg B.Wt, expressed as percentage of basal values. The mixed, 2-way ANOVA, indicated no signicant main effect of the ethanol dose [F(5, 38) = 2.29, p > 0.5], a signicant main effect of time [F(8, 304) = 12.41, p < 0.0001], and a signicant interaction between dose and time factors [F(40, 304) = 1.88, (p < 0.5). The signicant main effect of time and signicant interaction of dose with the time factors were associated with an increase of the dynorphin A18 content in the dialysate with time for the 0.0, 0.8, and 1.2 g ethanol dose groups. Post-hoc analysis using the NewmanKeuls multiple comparison test indicated signicant elevations of dynorphin A18 above the basal values at 120, 150, 180, 210, and 240 minutes following the injection of saline (p < 0.05), at 210 and 240 minutes following the injection of 0.8 g ethanol kg (p < 0.05) and at 60, 90, 120, 150, 180, 210, and 240 minutes following the injection of 1.2 g ethanol kg B.Wt. (p < 0.05). In addition, the Newmann Keuls test indicated that following the 1.2 g ethanol dose the changes in the content of dynorphin A18 in the dialysates at 180, 210, and 240 minutes were signicantly higher than those observed following the injection of saline (p < 0.05). DISCUSSION The main ndings of the present investigation were (i) the biphasic effect of ethanol on the release of b-endorphin with moderate (1.2, 1.6, and 2.0 g ethanol kg B.Wt.) but not high (2.4 g ethanol kg B.Wt.) doses of ethanol inducing a

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Fig. 4. Direct comparison of the effect of 0.8 (n = 7), 1.2 (n = 8), 1.6 (n = 8), 2.0 (n = 8), and 2.4 (n = 10) g ethanol (ETOH) kg B.Wt. with the effect of saline (0.0 g ethanol kg B.Wt., n = 11) on the dialysate b-endorphin content at the level of midbrain VTA, expressed as percent change from basal levels. Error bars denote SEM. *Denotes signicant difference from the saline-treated group at the corresponding time points (p < 0.05), NeumanKeuls post hoc test. +Indicates signicant difference from basal values (p < 0.05), NeumanKeuls post hoc test.

signicant increase of b-endorphin release at the level of midbrain VTA (ii) the lack of a signicant effect of all doses of ethanol tested, compared to the saline control treatment, on the release of met-enkephalin, and (iii) a signicantly higher release of dynorphin A18 towards the end of the experimental session (180 to 240 minutes) in response to the 1.2 g ethanol dose than to the saline control treatment. The presence of the 3 opioid peptide systems within the VTA, (b-endorphin, met-enkephalin, and dynorphin A18,) as well as of the d, l, and j opioid receptors (Akil et al., 1984; Khachaturian et al., 1985) suggests a possible role of all 3 opioid peptides in modulating the effects of reinforcing agents. The increased release of b-endorphin in response to moderate doses of ethanol at the level of midbrain VTA observed in the present investigation is in agreement with previous reports demonstrating increased content of b-endorphin peptides in tissue extracts of the VTA and NAcb at 30 minutes following intragastric ethanol administration (Rasmussen et al., 1998). Since the presence of POMC-producing neurons has not been

demonstrated in either NAcb or the midbrain VTA, these regions must receive endorphinergic innervation from POMC-producing neurons located elsewhere in the brain. Evidence from a number of studies indicates that the major site of POMC producing cells in the brain is the arcuate nucleus of the hypothalamus while a smaller group of POMC-producing neurons is found in the nucleus tractus solitarious (Bronstein et al., 1992; Khachaturian et al., 1985). Short chronic treatments with ethanol have been shown to increase (Angelogianni and Gianoulakis, 1993; de Waele and Gianoulakis, 1994; Krishnan-Sarin et al., 1998), while prolonged chronic treatments with ethanol have been shown to decrease (Rasmussen and colleagues 2002) the hypothalamic content of POMC mRNA. Furthermore, studies by Rasmussen and colleagues (1998) demonstrated that acute intragastric administration of ethanol, sufcient to produce BACs higher than 100 mg dl, increased the hypothalamic content of POMC mRNA. However, intragastric administration of lower doses of ethanol, sufcient to produce

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Fig. 5. Direct comparison of the effect of 0.8 (n = 8), 1.2 (n = 7), 1.6 (n = 11), 2.0 (n = 6), and 2.4 (n = 8) g ethanol (ETOH) kg B.Wt. with that of saline (0.0 g ethanol kg B.Wt., n = 9) on dialysate met-enkephalin content at the level of midbrain VTA, expressed as percent change from basal levels. Error bars denote SEM.

BACs lower than 100 mg dl, did not signicantly alter the content of hypothalamic POMC mRNA. In the present studies i.p. administration of 1.2, 1.6, and 2.0 g ethanol kg B.Wt. produced BACs equal to or higher than 100 mg dl. Therefore, these doses of ethanol may be associated not only with increased release of b-endorphin in the midbrain VTA region but also with increased hypothalamic POMC mRNA content and increased synthesis of POMC peptides in the hypothalamus. In the present investigation, the ethanol-induced increase in the extracellular concentration of b-endorphin at the level of midbrain VTA was maintained for the entire experimental session. It is tempting then to speculate that the long-lasting increase in the release of b-endorphin observed at the level of midbrain VTA is associated with an ethanolinduced increase in the synthesis and post-translational processing of POMC by the POMC-producing neurons. On the other hand, the i.p. administration of 0.8 g ethanol kg B.Wt. that produces BACs lower than 100 mg dl is associated with neither increased content of hypothalamic POMC mRNA

(Rasmussen et al., 1998) nor increased b-endorphin release at the level of midbrain VTA. Among the ndings of the present studies was that the 2.4 g ethanol kg B.Wt. dose of alcohol failed to enhance the release of b-endorphin in the midbrain VTA, indicating a biphasic effect of ethanol with low and moderate but not high doses of ethanol inducing an increase in the extracellular concentration of b-endorphin in the midbrain VTA region. The exact mechanisms associated with this biphasic effect of ethanol are not known. However, there are previous studies reporting a biphasic effect of ethanol on the in vitro release of hypothalamic b-endorphin. Thus, studies using Sprague Dawley, AA and ANA rats, as well as C57BL 6 and DBA 2 mice demonstrated that in in vitro preparations of intact hypothalamus moderate concentrations of ethanol (20 mM) induced a more pronounced increase of b-endorphin release, than higher concentrations (30 to 40 mM), while when the concentration of ethanol in the incubation medium was increased to 60 mM it did not alter the b-endorphin release

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Fig. 6. Direct comparison of the effect of 0.8 (n = 7), 1.2 (n = 8), 1.6 (n = 8), 2.0 (n = 6), and 2.4 (n = 8) g ethanol (ETOH) kg B.Wt. with that of saline (0.0 g ethanol kg B.Wt., n = 7) on dialysate dynorphin A18 content at the level of midbrain VTA, expressed as percent change from basal levels. Error bars denote SEM. *Denotes signicant difference from the saline-treated group at the corresponding time points (p < 0.05), NeumanKeuls post hoc test. +Indicates signicant difference from basal values (p < 0.05), NeumanKeuls post hoc test.

from the basal levels (de Waele and Gianoulakis, 1993, 1994; de Waale et al., 1992). Using in vivo microdialaysis, a dosedependent biphasic effect of ethanol on the release of enkephalin peptides was also observed in the rat NAcb (Marinelli et al., 2005), providing further support for the presence of a similar biphasic effect of ethanol on b-endorphin release at the level of midbrain VTA. Furthermore, similar biphasic responses to ethanol have been observed for other neurotransmitter systems, including the midbrain dopaminergic system (Mocsary and Bradberry, 1996). Nevertheless, it must be noted that while biphasic effects of ethanol on dopaminergic activity are commonly reported, the particular doses that do, or do not elicit responses are inconsistent and vary among investigations. For instance, several reports indicate that dopaminergic activity only becomes augmented following doses both lower and higher than the 1.6 g ethanol kg B.Wt. dose (Blanchard et al., 1993; Di Chiara and Imperato, 1985; Gessa et al., 1985; Marinelli et al., 2003; Tizabi et al., 2002; Yan, 1999). While these differences may in some cases be

ascribed to variations of administration routes, and species strains used, they also suggest that the VTA b-endorphin is only one of many neurotransmitter systems that may mediate ethanols effects on the mesolimbic dopaminergic system. The experimental evidence indicating that the ethanolinduced A10 dopaminergic neuronal activity may be modulated by opioid receptors in the VTA (Chefer et al., 2005; Dalman and OMalley, 1999; Ford et al., 2006; Yoshida et al., 1993) is supported by the ethanol-induced increase of midbrain VTA b-endorphin release observed in the present studies. This nding is in agreement with reports on the stimulatory effect of ethanol on mesolimbic dopaminergic activity (Gessa et al., 1985; Imperato and Di Chiara, 1986; Marinelli et al., 2003). Furthermore, the rapid commencement of the increase in midbrain VTA b-endorphin release ts well with the ethanol-induced increase in the NAcb dopaminergic activity reported elsewhere (Gessa et al., 1985; Imperato and Di Chiara, 1986; Marinelli et al., 2003). In addition, the doses that increased the release of midbrain VTA b-endorphin have

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previously been shown to increase dopamine release in the NAcb (Imperato and Di Chiara, 1986; Marinelli et al., 2003; Yim et al., 2000). However, there are some apparent discrepancies, between b-endorphin release observed in the midbrain VTA and dopaminergic responses to alcohol reported previously including differences in the timing and duration of release (Imperato and Di Chiara, 1986; Kohl et al., 1998; Yim et al., 2000). Thus, in contrast to the protracted release of midbrain VTA b-endorphin, the increases of NAcb dopamine release by acute ethanol administration of doses comparable to those used in the present studies demonstrated a more rapid cessation, typically within approximately 1 to 2 hours postethanol (Imperato and Di Chiara, 1986; Kohl et al., 1998; Yim et al., 2000). However, both the BACs and the extracellular concentration of b-endorphin in the midbrain VTA remain high long after the NAcb dopamine levels return to basal, suggesting that the time prole of ethanolinduced dopaminergic activation is inuenced by a number of mechanisms. These mechanisms could include a modulatory role of VTA b-endorphin, an excitatory glutamatergic input, and an inhibitory input mediated by dopaminergic autoreceptors or GABAergic interneurons (Kohl et al., 1998; Rahman and McBride, 2001). The lack of a signicant effect of ethanol on midbrain VTA met-enkephalin and the observation that only the 1.2 g ethanol kg B.Wt. dose induced a small but signicant increase in the content of dynorphin A18 in the dialysate could indicate either that at the level of midbrain VTA these opioid peptide systems are not sensitive to ethanol or that due to methodological limitations the effects of ethanol could not be detected. Among the methodological limitations could be the low sensitivity of the microdialysis coupled with the RIA technique to detect small changes in the extracellular concentrations of these peptides. Techniques with higher sensitivity may be needed to detect the subtle effects of ethanol on the midbrain VTA met-enkephalin and dynorphin systems. In the dialysates from the midbrain VTA region the average basal concentration of b-endorphin (1722 182.3 pg 50 ll) is lower than the average basal concentration in dialysates from CeA (2394 237 pg 50 ll) (Lam et al., 2008) but higher than the basal concentration in dialysates from the NAcb (165 31 pg 50 ll) (Marinelli et al., 2003). The basal concentration of met-enkephalin in the dialysates from the midbrain VTA region (91.61 15.13 pg 50 ll) is similar to the average basal concentration in dialysates from the CeA (92.51 9.39 pg 50 ll) (Lam et al., 2008) and higher than the basal met-enkephalin concentration in dialysates from NAcb (39.82 10.29 pg 50 ll) (Marinelli et al., 2005). Previous studies at the level of NAcb (Marinelli et al., 2003, 2005) using the same technique (in vivo microdialysis coupled to solid phase RIA) a dose-dependent effect of ethanol on the release of met-enkephalin and b-endorphin peptides was demonstrated with low to medium, but not high doses of ethanol increasing the release of both peptides despite their low basal dialysate concentrations. The basal dynorphinA18 concentration in dialysates from the midbrain VTA region

(17.29 2.461 pg 50 ll) is lower than in dialysates from both CeA (55.04 10.49 pg 50 ll) (Lam et al., 2008) and NAcb (46.43 12.26 pg 50 ll) (Marinelli et al., 2006) and may be associated with lower sensitivity in detecting small concentration changes in response to ethanol exposure, accounting for the observation that only the 1.2 g ethanol dose induced detectable changes in the dialysate content of dynorphinA18. Furthermore, previous studies demonstrated that high but not low doses of ethanol increased the concentration of dynorphinA18. in dialysates from NAcb (Marinelli et al., 2006) and CeA (Lam et al., 2008). In the present studies, doses higher than 2.4 g ethanol kg B.Wt. were not tested because such high doses are known to induce depressedhypnotic, rather than reinforcing effects. Furthermore, due to low recovery rates in microdialysis studies of neuropeptides compared to monoamine and amino acid neurotransmitters, in the present study dialysate samples were collected at 30-minute intervals. The low recovery rates and the long interval of sample collection pose some limitations on (i) the rate of detection and temporal resolution and (ii) the sensitivity of the procedure. Thus, in interpreting the data it should be taken into consideration that transient changes in the dialysate concentrations of met-enkephalin and or dynorphin A18, in response to one or more ethanol doses may occur earlier than the 30-minute detection limit allowed by the design of the present study. Another important consideration in interpreting the data should be the relevance of the changes in the extracellular concentration of opioid peptides in response to i.p. bolus injections of various doses of ethanol observed in the present investigation, to the changes that may occur in response to voluntary ethanol consumption. In the present study, signicant effects of ethanol on the dialysate concentration of b-endorphin were observed in response to i.p. injections of 1.2 g kg or higher doses of ethanol. However, in ethanol self-administration studies most outbred rats do not consume more than 1 g kg and that in a more protracted period of time. Thus, it is not known whether b-endorphin release in the midbrain VTA region occurs in response to doses of ethanol achieved during voluntary consumption. In conclusion, the current investigation demonstrated that at the level of midbrain VTA systemic administration of ethanol mainly altered the release of b-endorphin in a dose-dependent manner with low to medium, but not high, doses of ethanol increasing b-endorphin release. This ethanol induced increase of b-endorphin release in the midbrain VTA region may play a signicant role in the ethanol-induced stimulation of the mesolimbic dopaminergic system and the initiation of the processes of ethanol reward and reinforcement. ACKNOWLEDGMENTS A portion of these data has been presented as a poster at the 2006 annual meeting of the Research Society on Alcoholism. The authors would like to thank Mr. Minh Lam for interesting discussions and suggestions.

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FUNDING SOURCE These experiments were funded through a grant from the Natural Sciences and Engineering Research Council of Canada. REFERENCES
Akil H, Bronstein D, Mansour A (1988) Overview of the endogenous opioid systems: anatomical, biochemical, and functional issues. in Endorphins, Opiates and Biochemical Processes (Rodgers RJ, Cooper SJ eds), pp 123. John Wiley & Sons Ltd., Chichester. Akil H, Owens C, Gutstein H, Taylor L, Curran E, Watson SJ (1998) Endogenous opioids: overview and current issues. Drug Alcohol Depend 51:127140. Akil H, Watson SJ, Young E, Lewis ME, Khachaturian H, Walker JM (1984) Endogenous opioids: biology and function. Annu Rev Neurosci 7:223255. Altshuler HL, Phillips PE, Feinhandler DA (1980) Iteration of ethanol selfadministration by naltrexone. Life Sci 26:679688. Angelogianni P, Gianoulakis C (1993) Chronic ethanol increases proopiomelanocortin gene expression in the hypothalamus. Neuroendocrinology 57:106114. Bals-Kubik R, Herz A, Shippenberg TS (1989) Evidence that the aversive effects of opioid antagonists and j-agonists are centrally mediated. Psychopharmacology (Berl) 98:203206. Bechtholt AJ, Cunningham CL (2005) Ethanol-induced conditioned place preference is expressed through a ventral tegmental area dependent mechanism. Behav Neurosc 119:213223. Blanchard BA, Steindorf S, Wang S, Glick SD (1993) Sex differences in ethanol-induced dopamine release in nucleus accumbens and in ethanol consumption in rats. Alcohol Clin Exp Res 17:968973. Bronstein DM, Schafer MK, Watson SJ, Akil H (1992) Evidence that betaendorphinis synthesized in cells in the nucleus tractus solitarious: detection of POMC mRNA. Brain Res 587:269275. Charnes ME (1989) Ethanol and opioid receptor signalling. Experientia 45:518528. Chefer VI, Czyzyk T, Bolan EA, Moron J, Pintar JE, Shippenberg TS (2005) Endogenous j-opioid receptor systems regulate mesoaccumbal dopamine dynamics and vulnerability to cocaine. J Neurosci 25:50295037. Dalman FC, OMalley KL (1999) j-opioid tolerance and dependence in cultures of dopaminergic midbrain neurons. J Neurosci 19:57505757. de Waele J-P, Gianoulakis C (1993) Effects of single and repeated exposure to ethanol on hypothalamic b-endorphin and CRH release by the C57BL 6 and DBA 2 strains of mice. Neuroendocrinology 57:700709. de Waele J-P, Gianoulakis C (1994) Enhanced activity of the brain b-endorphin system by free-choice ethanol drinking in C57BW6 but not DBA 2 mice. Eur J Pharmacol 258:119129. de Waele J-P, Papachristou D, Gianoulakis C (1992) The alcohol-preferring C57BL 6 mice present an enhanced sensitivity of the hypothalamic b-endorphin system to ethanol than the alcohol-avoiding DBA 2 mice. J Pharmacol Exp Ther 261:788794. Di Chiara G, Imperato A (1985) Ethanol preferentially stimulates dopamine release in the nucleus accumbens of freely-moving rats. Eur J Pharmacol 115:131132. Finley JCW, Maderdrut JL, Petrusz P (1981) The immunocytochemical localization of enkephalin in the central nervous system of the rat. J Comp Neurol 198:541565. Ford CP, Mark GP, Williams JT (2006) Properties and opioid inhibition of mesolimbic dopamine neurons vary according to target location. J Neurosci 26:27882797. Froehlich JC, Harts J, Lumeng L, Li T-K (1990) Naloxone attenuates voluntary ethanol intake in rats selectively bred for high ethanol preference. Pharmacol Biochem Behav 35:385390. Froehlich JC, Zweifel M, Harts J, Lumeng L, Li TK (1991) Importance of d opioid receptors in maintaining high alcohol drinking. Psychopharmacology (Berl) 103:467472.

Gee CE, Chen CL, Roberts JL, Thompson R, Watson SJ (1983) Identication of proopiomelanocortin neurons in rat hypothalamus by in situ cDNAmRNA hybridization. Nature 306:374376. George SR, Rolan L, Lui A, Naranjo CA (1991) Endogenous opioids are involved in the genetically determined high preference for ethanol consumption. Alcohol Clin Exp Res 15:668672. Gessa GL, Muntoni F, Collu M, Vargiu L, Mereu G (1985) Low doses of ethanol activate dopaminergic neurons in the ventral tegmental area. Brain Res 348:201203. Gianoulakis C (1990) Characterization of the effects of acute ethanol administration on the release of endorphin peptides by the rat hypothalamus. Eur J Pharmacol 180:2129. Gianoulakis C (2004) Endogenous opioids and addiction to alcohol and other drugs of abuse. Curr Top Med Chem 4:3950. Gianoulakis C, Barcomb A (1987) Effect of acute ethanol in vivo and in vitro on the b-endorphin system in the rat. Life Sci 40:1928. Gianoulakis C, Gupta A (1986) Inbred strains of mice with variable sensitivity to ethanol exhibit differences in the content and processing of b-endorphin. Life Sci 39:23152325. Heyser CJ, Roberts AJ, Schulteis G, Koob GF (1999) Central administration of an opiate antagonist decreases oral ethanol self-administration in rats. Alcohol Clin Exp Res 23:14681476. Holter SM, Henniger MS, Lipkowski AW, Spanagel R (2000) j-opioid receptors and relapse-like drinking in long-term ethanol-experienced rats. Psychopharmacology (Berl) 153:93102. Hubbell CL, Czirr SA, Hunter GA, Beaman CM, LeCann NC, Reid LD (1986) Consumption of ethanol solution is potentiated by morphine and attenuated by naloxone persistently across repeated daily administrations. Alcohol 3:3954. Hyytia P, Sinclair JD (1993) Responding for oral ethanol after naloxone treatment by alcohol-preferring AA rats. Alcohol Clin Exp Res 17:631 636. Imperato A, Di Chiara G (1986) Preferential stimulation of dopamine release in the nucleus accumbens of freely moving rats by ethanol. J Pharmacol Exp Ther 239:219228. June HL, McCane SM, Zink RW, Portoghese PS, Li TK, Froehlich JC (1999) The d2-opioid receptor antagonist naltriben reduces motivated responding for ethanol. Psychopharmacology (Berl) 147:8189. Keith LD, Crabbe JC, Robertson LM, Kendall JW (1986) Ethanol-stimulated endorphin and corticotropin secretion in vitro. Brain Res 367:222229. Khachaturian H, Lewis ME, Schafer MK-H, Watson SJ (1985) Anatomy of the CNS opioid systems. Trends Neurosci 8:111119. Koenig HN, Olive MF (2002) Ethanol consumption patterns and conditioned place preference in mice lacking preproenkephalin. Neurosci Lett 325:7578. Kohl RR, Katner JS, Chernet E, McBride WJ (1998) Ethanol and negative feedback regulation of mesolimbic dopamine release in rats. Psychopharmacology (Berl) 139:7985. Krishnan-Sarin S, Wand GS, Li XW, Portoghese PS, Froehlich JC (1998) Effect of l opioid receptor blockade on alcohol intake in rats bred for high alcohol drinking. Pharmacol Biochem Behav 59:627635. Lam MP, Marinelli PW, Bai L, Gianoulakis C (2008) Effects of acute ethanol on opioid peptide release in the central amygdale: an in vivo microdialysis study. Psychopharmacology 201:261271. Le AD, Poulous CX, Quan B, Chow S (1993) The effects of selective blockade of d and l opiate receptors on ethanol consumption by C57BL 6 mice in a restricted access paradigm. Brain Res 630:330332. Li X-W, Li T-K, Froehlich JC (1998) Enhanced sensitivity of the nucleus accumbens proenkephalin system to alcohol in rats selectively bred for alcohol preference. Brain Res 794:3547. Lindholm S, Ploj K, Franck J, Nylander I (2000) Repeated ethanol administration induces short- and long-term changes in enkephalin and dynorphin tissue concentrations in rat brain. Alcohol 22:165171. Lindholm S, Werme M, Brene S, Franck J (2001) The selective j-opioid receptor agonist U50,488H attenuates voluntary ethanol intake in the rat. Behav Brain Res 120:137146.

ACUTE ETHANOL ALTERS OPIOID PEPTIDE RELEASE IN THE VTA

1043

Lundquist F (1957) The determination of ethyl alcohol in blood and tissues, in Methods of Biochemical Analysis (Glick D ed.), pp 217251. Interscience Publishers, New York. Maidment NT, Evans CJ (1991) Measurement of extracellular neuropeptides in the brain: microdialysis linked to solid-phase radioimmunoassay with subfemtomole limits of detection. in Microdialysis in the Neurosciences (Robinson TE, Justice JB Jr eds), pp 275302. Elsevier Science Publishers, Amsterdam. Mansour A, Khachaturian H, Lewis ME, Akil H, Watson SJ (1988) Anatomy of CNS opioid receptors. Trends Neurosci 11:308314. Marinelli PW, Bai L, Quirion R, Gianoulakis C (2005) A microdialysis prole of met-enkephalin release in the rat nucleus accumbens following alcohol administration. Alcohol Clin Exp Res 29:18211828. Marinelli PW, Lam M, Bai L, Quirion R, Gianoulakis C (2006) A microdialysis prole of dynorphin A(1-8) release in the rat nucleus accumbens following alcohol administration. Alcohol Clin Exp Res 30:982990. Marinelli PW, Quirion R, Gianoulakis C (2003) A microdialysis prole of b-endorphin and catecholamines in the rat nucleus accumbens following alcohol administration. Psychopharmacology (Berl) 169:6067. Marinelli PW, Quirion R, Gianoulakis C (2004) An in vivo prole of b-endorphin release in the arcuate nucleus and nucleus accumbens following exposure to stress or alcohol. Neurosci 127:777784. Mendez M, Leriche M, Calva JC (2001) Acute ethanol administration differentially modulates l opioid receptors in the rat mesoaccumbens and mesocortical pathways. Mol Brain Res 94:148156. Mendez M, Morales-Mulia M (2006) Ethanol exposure differentially alters pro-enkephalin mRNA expression in regions of the mesocorticolimbic system. Psychopharmacology (Berl) 189:117124. Middaugh LD, Kelley BM, Cuison ER Jr, Groseclose CH (1999) Naltrexone effects on ethanol reward and discrimination in C57BL 6 mice. Alcohol Clin Exp Res 23:456464. Mocsary Z, Bradberry CW (1996) Effect of ethanol on extracellular dopamine in nucleus accumbens: comparison between Lewis and Fischer 344 rat strains. Brain Res 706:194198. Mucha RF, Iversen SD (1984) Reinforcing properties of morphine and naloxone revealed by conditioned place preferences: a procedural examination. Psychopharmacology (Berl) 82:241247. Ng GYK, ODowd BF, George SR (1996) Genotypic differences in mesolimbic enkephalin gene expression in DBA 2J and C57BL 6J inbred mice. Eur J Pharmacol 311:4552. Nylander I, Hyytia P, Forsander O, Terenius L (1994) Differences between alcohol-preferring (AA) and alcohol-avoiding (ANA) rats in the prodynorphin and proenkephalin systems. Alcohol Clin Exp Res 18:12721279. Olive MF, Koenig HN, Nannini MA, Hodge CW (2001) Stimulation of endorphin neurotransmission in the nucleus accumbens by ethanol, cocaine and amphetamine. J Neurosci 21:RC184. Olson GA, Olson RD, Kastin AJ (1996) Endogenous opiates: 1995. Peptides 17:14211466. Oswald LM, Wand GS (2004) Opioids and alcoholism. Physiol Behav 81:339 358. Paxinos G, Watson C (1998) The Rat Brain in Stereotaxic Coordinates. 4th ed. Academic Press, San Diego. Przewlocka B, Lason W, Przewlocki R (1994) Repeated ethanol differently affects opioid peptide biosynthesis in the rat pituitary. Neuroendocrinology 60:331336. Quirion R, Pert CB (1981) Dynorphins: similar relative potencies on l, d, and j-opiate receptors. Eur J Pharmacol 76:467468.

Rahman S, McBride WJ (2001) D1-D2 dopamine receptor interaction within the nucleus accumbens mediates long-loop negative feedback to the ventral tegmental area (VTA). J Neurochem 77:12481255. Rasmussen DD, Boldt BM, Wilkinson CW, Mitton DR (2002) Chronic daily ethanol and withdrawal: forebrain pro-opiomelanocortin gene expression and implications for dependence, relapse, and deprivation effect. Alcohol Clin Exp Res 26:535546. Rasmussen DD, Bryant CA, Boldt BM, Colasurdo EA, Levin N, Wilkinson CW (1998) Acute alcohol effects on opiomelanocortinergic regulation. Alcohol Clin Exp Res 22:789801. Raynor K, Kong H, Chen Y, Yasuda K, Yu L, Bell GI, Reisine T (1994) Pharmacological characterization of the cloned j-d-and l-opioid receptors. Mol Pharmacol 45:330334. van Ree JM, Smyth DG, Colpaert FC (1979) Dependence creating properties of lipotropin C-fragment (b-endorphin): evidence for its internal control of behavior. Life Sci 24:495502. Samson HH, Doyle TF (1985) Oral ethanol-self-administration in the rat: effect of naloxone. Pharmacol Biochem Behav 22:9299. Sar M, Stumpf WE, Miller RJ, Chang K-J, Cuatrecasas P (1978) Immunocytochemical localization of enkephalin in the rat brain and spinal cord. J Comp Neurol 182:1738. Schulz R, Wuster M, Duka T, Herz A (1980) Acute and chronic ethanol treatment changes endorphin levels in brain and pituitary. Psychopharmacology (Berl) 68:221227. Seizinger BR, Bovermann K, Maysinger D, Hollt V, Herz A (1983) Differential effects of acute and chronic ethanol treatment on particular opioid peptide systems in discrete regions of rat brain and pituitary. Pharmacol Biochem Behav 18:361369. Solinas M, Zangen A, Thiriet N, Goldberg SR (2004) b-Endorphin elevations in the ventral tegmental area regulate the discriminative effects of D-9-tetrahydrocannabinol. Eur J Neurosci 10:31833192. Stromberg MF, Casale M, Volpicelli JR, Obrien CP (1998) A comparison of the effects of the opioid antagonists naltrexone, naltrindole, and b-funaltrexamine on ethanol consumption in the rat. Alcohol 15:281289. Tizabi Y, Copeland RL Jr, Louis VA, Taylor RE (2002) Effects of combined systemic alcohol and central nicotine administration into ventral tegmental area on dopamine release in the nucleus accumbens. Alcohol Clin Exp Res 26:394399. Van Wolfswinkel L, van Ree JM (1985) Effects of morphine and naloxone on thresholds of ventral tegmental electrical self stimulation. Naunyn Schmiedebergs Arch Pharmacol 330:8492. Volpicelli JR, Davis MA, Olgin JE (1986) Naltrexone blocks the post-shock increase of ethanol consumption. Life Sci 38:841847. Watson SJ, Barchas JD, Li CH (1977) b-lipotropin: localization of cells and axons in rat brain by immunocytochemistry. Proc Natl Acad Sci U S A 74:51555158. Yan Q-S (1999) Extracellular dopamine and serotonin after ethanol monitored with 5-minute microdialysis. Alcohol 19:17. Yim HJ, Robinson DL, White M, Jaworski JN, Randall PK, Lancaster FE, Gonzales RA (2000) Dissociation between the time course of ethanol and extracellular dopamine concentrations in the nucleus accumbens after a single intraperitoneal injection. Alcohol Clin Exp Res 24:781788. Yoshida M, Yokoo H, Tanaka T, Mizoguchi K, Emoto H, Ishii H, Tanaka M (1993) Facilitatory modulation of mesolimbic dopamine neuronal activity by a mu-opioid agonist and nicotine as examined with in vivo microdialysis. Brain Res 624:277280. Zakarian S, Smyth DG (1982) Distribution of beta-endorphin-related peptides in rat pituitary and brain. Biochem J 202:561571.