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Repeated ethanol administration induces short- and long-term changes in enkephalin and dynorphin tissue concentrations in rat brain
Alcohol 22 (2000) 165 ± 171
Repeated ethanol administration induces short- and long-term changes in enkephalin and dynorphin tissue concentrations in rat brain Sara Lindholma,*, Karolina Plojb, Johan Francka, Ingrid Nylanderb a
Clinical Alcohol and Drug Addiction Research, Department of Clinical Neuroscience, Magnus Huss, M4:02, Karolinska Hospital, Karolinska Institutet, SE-171 76 Stockholm, Sweden b Department of Pharmaceutical Biosciences, Division of Pharmacology, Uppsala University, Uppsala, Sweden Received 31 January 2000; received in revised form 5 July 2000; accepted 17 August 2000
Abstract Recently, we have shown that rats repeatedly treated with ethanol and/or cocaine have decreased k-opioid receptor mRNA levels in the mesolimbic system. The aim of the present study was to investigate the short- and long-term effects of repeated ethanol administration on opioid peptide concentrations in brain tissue of male Sprague ± Dawley rats. Dynorphin B (1 ± 13) (Dyn B) and Met-enkephalinArg6Phe7 (MEAP), endogenous ligands to k- and d-opioid receptors, respectively, were measured using radioimmunoassays. The rats were given either ethanol [intraperitoneal (ip), twice daily, 2 g/kg bw/dose] or saline for 13 consecutive days. Thirty min after the last ethanol dose on Day 13, the Dyn B tissue concentration was significantly decreased in the cingulate cortex. The MEAP tissue concentration was decreased in the hippocampus 5 days after the last ethanol injection as compared to saline-treated controls. Furthermore, the Dyn B and the MEAP concentrations were increased in the periaqueductal grey area (PAG) at this time point. Of particular interest were the significant increases in Dyn B tissue concentrations found in the nucleus accumbens (NAcc) at 30 min and at 21 days after the last ethanol dose. The results suggest that repeated ethanol administration induces both short- and long-term changes in the tissue concentrations of opioids in certain brain regions associated with motivation and reward. D 2001 Elsevier Science Inc. All rights reserved. Keywords: Opioids; Nucleus accumbens; Dynorphin B; Ethanol; Met-enkephalinArg6Phe7
1. Introduction Rodents with high or low preference for ethanol intake have differences in the mesolimbic reward system (Li et al., 1988). Such animal lines include the high- and low-preferring AA- and ANA-rats (Eriksson, 1968), and the P- and NP-rats (Li et al., 1979) as well as the C57BL/6 and DBA/2 mice strains (Gentry, 1985). Differences in basal as well as in ethanol-induced dopamine (DA) and/or opioid peptide turnover have been reported in these strains. Ethanol-preferring AA-rats have, for example, low basal tissue concentrations of both Met-enkephalinArg6Phe7 (MEAP) and dynorphin peptides in the nucleus accumbens (NAcc) as compared to ANA-rats, and the MEAP concentration increases after volitional ethanol intake in both
* Corresponding author. Tel.: +46-8-51774870; fax: +46-8-326369. E-mail address: [email protected] (S. Lindholm).
strains (Nylander et al., 1994). The ethanol-preferring C57BL/6(J) mouse has lower basal dynorphin peptides and prodynorphin mRNA levels in the NAcc (Jamensky & Gianoulakis, 1997; Ploj et al., 2000) as well as lower levels of b-endorphin- and enkephalin-like peptides in the NAcc as compared to the low-preferring DBA/2(J) mouse strain (de Waele & Gianoulakis, 1994; Ploj et al., 2000). Furthermore, differences in opioid-receptor density have been reported in high- and low-preferring rodents. The AA-rats have higher basal density of m-opioid receptor binding sites (de Waele et al., 1995), and the C57BL/6 mice have increased density of dopioid receptors (de Waele & Gianoulakis, 1997) as well as decreased density of k-opioid receptor binding sites (Jamensky & Gianoulakis, 1997) in the mesolimbic system compared to their low-preferring counterparts. Non-selective opioid receptor antagonists decrease volitional ethanol intake in several animal models; see Herz (1997). This effect is also seen with administration of selective d- (Froehlich et al., 1991; Franck et al., 1998)
0741-8329/00/$ ± see front matter D 2001 Elsevier Science Inc. All rights reserved. PII: S 0 7 4 1 - 8 3 2 9 ( 0 0 ) 0 0 11 8 - X
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and m-receptor antagonists (Hyytia, 1993; Krishnan-Sarin et al., 1998) although conflicting results have been reported (Honkanen et al., 1996; Stromberg et al., 1998). Agonists acting at m- and d-opioid receptors are self-administered in various animal models and produce place preference, see Koob et al. (1986), whereas k-opioid receptor agonists mainly have aversive properties (Bals-Kubik et al., 1989; Dykstra et al., 1997). Taken together, this indicates that the opioid peptide systems play a role in the propensity for ethanol intake in rodents. We have previously shown that a d-receptor antagonist decreases voluntary ethanol intake in rats, suggesting that the effects of ethanol may partly depend on activation of opioid receptors (Franck et al., 1998). In addition, we have also found that k-receptor mRNA levels decreased in the NAcc and in the ventral tegmental area (VTA) after repeated ethanol administration (Rosin et al., 1999). This kreceptor mRNA down-regulation may be interpreted as an adaptive response to increased mesolimbic dynorphin levels. In this study, it was hypothesized that long-term repeated administration of ethanol would alter the activity of the endogenous ligands to the d- and k-receptors, the enkephalins and dynorphins, respectively. Therefore, the effects of repeated ethanol administration on both enkephalin and dynorphin tissue concentrations were studied. The tissue concentrations of MEAP and dynorphin B (1 ± 13) (Dyn B) were determined in selected brain regions and in the pituitary gland of male Sprague ± Dawley rats 30 min after the last dose, i.e. during ethanol intoxication. In order to study the long-term effects of repeated ethanol administration, the tissue concentrations of MEAP and Dyn B were also measured at 5 and 21 days after the last dose. MEAP is exclusively cleaved from proenkephalin and was therefore measured as a marker for its precursor, whereas Dyn B was measured as a marker for the prodynorphin system. 2. Materials and methods 2.1. Animals Male Sprague ± Dawley rats (B&K Universal; Stockholm, Sweden) were used in all experiments. The rats (n = 72) were housed in groups of six in their normal cages in a temperature-controlled environment (20.5 2°C) on a 12-h light/dark cycle with access to food (B&K rat chow, B&K Universal) and tap water ad libitum. Rats weighed 228 ±347 g at the beginning of the experiments. All animal experiments were performed under an approved protocol in accordance with the Swedish Animal Protection Legislation. 2.2. Experimental design The rats were randomly divided into three experimental and three control groups. The animals in the experimental groups were administered ethanol intraperitoneally [ip; 2 g/
kg bw/dose, 18% (v/v), diluted in saline] twice daily at 9 a.m. and 3 p.m. for 13 consecutive days. Each experimental group had a corresponding control group. The controls were given saline in equal volumes at the same time points as the ethanol groups. Rats were killed by decapitation. In the first set of experiments, one group of ethanol-treated rats and one control group were decapitated 30 min after the last dose. In the second set of experiments, ethanol- and saline-treated rats were decapitated at 5 days after the last dose. A third set of rats was decapitated at 21 days after the last dose. During these two latter periods, the rats did not receive any treatment. 2.3. Dissection After decapitation, the pituitary gland and the brain were immediately removed and rapidly dissected on ice. The whole pituitary gland was extracted in all experiments. The hypothalamus was removed from the brain, which was then placed in a cooled brain blocker (AgnTho's AB; Stockholm, Sweden), and sliced manually with razor blades in coronal sections. The NAcc, hippocampus, cingulate cortex, VTA, dorsal striatum, and the periaqueductal grey area (PAG) were manually dissected out using a scalpel with guidance from the rat brain atlas of Paxinos and Watson (1986). The tissue samples were immediately frozen on dry ice and stored at ÿ 80°C until further analysis. 2.4. Extraction and separation of peptides Tissue extraction was performed with 1 M acetic acid. The samples were heated at 95°C for 5 min, and after cooling on ice, the tissues were homogenized by sonication using a Branson Sonifier. The samples were reheated at 95°C for 5 min, cooled on ice, and then centrifuged for 15 min at 12 000 g in a Beckman GS-15R Centrifuge. The samples were purified using a cation exchange procedure. The supernatants were applied onto small (1 ml) cation exchange columns containing SP Sephadex C-25 gel (Pharmacia Diagnostics; Uppsala, Sweden), and the opioid peptides were eluted with buffers containing mixtures of pyridine and formic acid. Peptides eluted in separate fractions depending on the buffer used (Bergstrom et al., 1983; Christensson-Nylander et al., 1985). MEAP and Dyn B were measured as markers for their respective precursor peptides. The fractions were dried in a vacuum centrifuge and stored at ÿ 20°C until peptide analysis. 2.5. Radioimmunoassay Dyn B and MEAP were measured with specific radioimmunoassays as previously described in detail (Nylander et al., 1997). Rabbit antisera for the peptides were used. The tracer peptides were labeled with 125I according to the chloramine T method and were purified with reversed phase HPLC using a gradient of 15± 40% acetonitrile in 0.04% trifluoroacetic acid.
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In the Dyn B radioimmunoassay, an aliquot of the sample (25 ml) was dissolved in methanol/0.1 M hydrochloric acid (1:1). The solution was incubated with 100 ml of antiserum (113+, final dilution, 1:562 500) and 100 ml of 125I-labelled peptide, both diluted in gelatin buffer. The gelatin buffer contained 0.15 M sodium chloride, 0.02% sodium azide, 0.1% gelatin, 0.1% Triton X-100, and 0.1% bovine serum albumin in a 0.05 M sodium phosphate buffer. Samples were incubated for 24 h at 4°C. To separate free and antibody-bound peptides, 100 ml of a sheep antirabbit antiserum (Pharmacia Decanting Suspension 3, Pharmacia Diagnostics) was added, and the samples were incubated for 1 h at 4°C. After centrifugation for 10 min at 12 000 g in a Beckman GS-15R Centrifuge, the radioactivity in the pellet was counted in a Wallac 1470 Wizard gamma counter. Dyn-32 and Dyn B-29 crossreact 100% and 1%, respectively with the Dyn B antiserum, whereas other dynorphin peptides, Met-enkephalin, Met-enkephalinArg6, Met-enkephalinArg6Gly7Leu8, Leuenkephalin, and Leu-enkephalinArg6, cross-react < 0.1%. Samples subjected to MEAP assay were oxidized prior the radioimmunoassay procedure, i.e. the samples were dissolved in acetic acid (1 M, 100 ml), and H2O2 (30%, 10 ml) was added. The samples were incubated at 37°C for 30 min and dried in a vacuum centrifuge. An aliquot of the sample (25 ml) was dissolved in methanol/0.1 M hydrochloric acid (1:1) and incubated with 100 ml of antiserum (90:3D, final dilution 1:180 000) and 100 ml of 125I-labelled peptide, both diluted in gelatin buffer. The gelatin buffer contained 0.15 M sodium chloride, 0.025 M EDTA, 0.1% gelatin, and 0.1% bovine serum albumin in 0.05 M sodium phosphate buffer. Samples were incubated for 24 h at 4°C. To separate free and antibody-bound peptides, a charcoal suspension (200 ml) consisting of 250 mg charcoal and 25mg dextran T-70 in 100 ml of sodium phosphate buffer (0.05 M) was added, and the samples were incubated for 10 min. The samples were centrifuged for 2 min at 12 000 g in a Beckman GS-
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15R Centrifuge, and the radioactivity in a 300 ml aliquot of the supernatant was counted in the Wallac 1470 Wizard gamma counter. Cross-reactivity of Met-enkephalin, Met-enkephalinArg6, Met-enkephalinArg6Gly7Leu8, Leu-enkephalin, Leu-enkephalinArg6, and dynorphin peptides was < 0.1% for the MEAP antiserum. 2.6. Statistical analysis The values obtained from the control rats decapitated 30 min after the last dose are expressed as fmol/mg tissue (mean S.E.M., wet weight; see Results). A statistical comparison of peptide immunoreactivity in brain regions from saline- and ethanol-treated rats were performed on three occasions using non-transformed data; 30 min after the morning dose on the last day of the treatment period, and 5 and 21 days after the last dose. In Figs. 1 and 2, the data from ethanol-treated rats are expressed as % of controls (mean S.E.M.), where the controls are shown as 100%. Comparisons across the different time points were not conducted since samples from each experimental time point were analyzed in separate radioimmunoassays. The samples from ethanol and control rats were at each separate time point handled together in the various sample-handling procedures, i.e. during the tissue homogenization and extraction, separation procedures, and statistical analysis. Differences between groups were analyzed with a one-way ANOVA combined with the Fisher post-hoc test using the Statview 4.5 software. A P-value < 0.05 was considered statistically significant. 3. Results Although no specific quantitative measures were used, mild physical signs of withdrawal including hyper-reactivity and irritability were observed at 5 days after the last administration of ethanol. At the other time points studied,
Fig. 1. Effects of repeated ethanol (2 g/kg bw ip, twice daily) or saline administration on the concentration of Dyn B in tissue from distinct rat brain regions and the pituitary gland (Pt); hypothalamus (HT), striatum (Str), NAcc, VTA, cingulate cortex (CC), hippocampus (HP), and the PAG. The Dyn B concentration was measured at 30 min, and 5 or 21 days after the last dose of ethanol or saline. The values represent % of control values (mean S.E.M.) where the controls are expressed as 100%. Comparisons between two separate groups at respective time points were made with one-way ANOVA combined with the Fisher post-hoc test using the Statview 4.5 software. * P < .05.
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Fig. 2. Effects of repeated ethanol (2 g/kg bw ip, twice daily) or saline administration on MEAP concentrations in tissue from distinct rat brain regions and the pituitary gland (Pt); hypothalamus (HT), striatum (Str), NAcc, VTA, cingulate cortex (CC), hippocampus (HP), and the PAG. The MEAP concentrations were measured at 30 min, and 5 or 21 days after the last dose of ethanol or saline. The values represent % of the control values (mean S.E.M.) where the controls are 100%. Comparisons between two separate groups at respective time points were made with one-way ANOVA combined with the Fisher post-hoc test using the Statview 4.5 software. * P < 0.05.
ethanol-treated rats were either intoxicated (30 min after the last dose) or seemingly unaffected by the treatment (21 days after the last dose). The mean body weights of the ethanol- and saline-treated groups were 247 3 and 244 2 g (mean S.E.M., n = 12), respectively at the start of the first set of experiments. After 13 days of treatment, rats repeatedly treated with ethanol had 18% less body weight as compared to the saline-treated animals (263 6 vs. 320 2 g, P < 0.01). This difference between the treatment groups was also found 5 days after the last dose but not after 21 days (data not shown). Although the relative levels of opioids in different brain regions were comparable between all time points, no statistical comparison could be conducted for samples from one experimental time point to another, since the samples from each time point were analyzed in separate radioimmunoassays. The basal values of the opioid tissue concentration in different brain regions and in the pituitary gland of saline-treated rats are shown for the 30-min control group. 3.1. Dyn B and MEAP tissue concentrations in salinetreated rats The highest Dyn B concentration in saline-treated rats decapitated 30 min after the last dose was found in the pituitary gland (86.3 8.3 fmol/mg tissue, mean S.E.M., n = 12). Moderate Dyn B concentrations were found in the hypothalamus (24.1 3.0 fmol/mg tissue, n = 12), PAG (19.3 3.6 fmol/mg tissue, n = 9), VTA (18.6 4.7 fmol/ mg tissue, n = 9), striatum (13.8 1.0 fmol/mg tissue, n = 12), hippocampus (9.8 1.3 fmol/mg tissue, n = 9), and the NAcc (6.7 0.6 fmol/mg tissue, n = 12), whereas a low Dyn B concentration was found in the cingulate cortex (1.9 0.2 fmol/mg tissue, n = 8). The highest MEAP tissue concentration of the brain regions studied in the control group decapitated 30 min after the last dose was found in the hypothalamus (54.9 3.4 fmol/mg tissue,
n = 12). Moderate MEAP tissue concentrations were found in the striatum (30.4 3.2 fmol/mg tissue, n = 10), PAG (25.1 5.5 fmol/mg tissue, n = 9), NAcc (25.1 3.6 fmol/ mg tissue, n = 10), and the VTA (22.5 4.1 fmol/mg tissue, n = 9), whereas lower MEAP concentrations were detected in the pituitary gland (3.5 0.7 fmol/mg tissue, n = 12), cingulate cortex (2.8 0.4 fmol/mg tissue, n = 10), and the hippocampus (2.2 0.3 fmol/mg tissue, n = 8) of the salinetreated rats. 3.2. Short-term effects of repeated ethanol administration on Dyn B concentration in brain tissue The effects of repeated ethanol administration followed by decapitation 30 min after the last dose on Dyn B tissue concentration in various brain regions and the pituitary gland as compared to the respective control group are shown in Fig. 1. The Dyn B tissue concentrations were significantly increased by 40 12% in the NAcc and decreased in the cingulate cortex by 35 15% in ethanol-treated rats as compared to their respective controls (Fig. 1, P < 0.05). It should be pointed out that although a marked tendency to a reduced Dyn B concentration was observed in the VTA, this change failed to reach statistical significance due to large variability within the control group ( P = 0.0649). No significant difference between the groups was found in any of the other brain regions studied. 3.3. Long-term effects of repeated ethanol administration on Dyn B concentration in brain tissue Five days after the last injection of ethanol, a significant increase ( + 48 14%) in Dyn B tissue concentration was found in the PAG (Fig. 1, P < 0.05) as compared to the control group. No significant difference between the groups was found in the other brain regions analyzed at this time point. Rats treated repeatedly with ethanol followed by 21
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days of recovery from treatment had a significantly increased Dyn B concentration in the NAcc by 39 12% (Fig. 1, P < 0.05) as compared to the control group. No significant difference between the groups was found in the other brain regions analyzed at this time point. 3.4. Short-term effects of repeated ethanol administration on MEAP concentration in brain tissue The effect of repeated administration with ethanol or saline on brain tissue MEAP concentration is shown in Fig. 2. No significant difference between the controls and ethanoltreated rats was found in any of the brain regions analyzed. 3.5. Long-term effects of repeated ethanol administration on MEAP concentration in brain tissue A significant decrease in MEAP concentration (ÿ30 8%) was found in the hippocampus at 5 days after the last ethanol dose as compared to their controls (Fig. 2, P < 0.05). In addition, the MEAP tissue concentration increased significantly ( + 45 11%) in the PAG in the same animals (Fig. 2, P < 0.05). No significant changes were observed in any other brain regions that were analyzed, neither at 5 or 21 days after the last dose (Fig. 2). 4. Discussion In this study, the tissue concentrations of MEAP and Dyn B were measured in selected brain regions and in the pituitary gland of rats after repeated ethanol administration. Peptide levels were determined both at 30 min, i.e. when ethanol was still present in brain and at 5 and 21 days after the last ethanol dose. A previous study showed that an acute intraperitoneal injection of 2 g/kg bw ethanol produced a significant blood alcohol concentration (ranging approximately between 175 and 190 mg%) that is stable during a post-injection period of 10 ± 120 min (Bloom et al., 1982). Ethanol in this dose is sedative, and may cause ataxia and decreased food intake (Bloom et al., 1982), which may explain the divergent body weight found between the groups. The dynorphins are mainly found in the pituitary ± hypothalamic axis, in the mesolimbic system, and in the striatonigral pathway, whereas the enkephalins are widely distributed in the brain (Christensson-Nylander et al., 1986; Watson et al., 1982). The dynorphins and the k-opioid receptors are in close proximity to the dopaminergic neurons of the mesolimbic and nigrostriatal systems (HerreraMarschitz et al., 1986), and interactions between DA and Dyn B have previously been demonstrated in the latter pathway (Herrera-Marschitz et al., 1986; You et al., 1994). Ethanol activates the dopaminergic mesolimbic system (Di Chiara & Imperato, 1985). The cell bodies of this system are located in the VTA (DahlstroÈm & Fuxe, 1964) with terminals in the NAcc and the prefrontal cortex (Anden et al.,
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1966; Ungerstedt, 1971), and the system is tonically controlled by opioid peptides (Spanagel et al., 1992). Stimulation of m-opioid receptors, located on GABA-containing interneurons in the VTA, leads to an increased extracellular DA concentration in the terminal regions (Spanagel et al., 1992). Local administration of d-opioid receptor agonists also increase (Spanagel et al., 1990), whereas k-opioid receptor agonists decrease the DA concentration in the NAcc (Di Chiara & Imperato, 1988; Spanagel et al., 1990). Chronic drug administration may cause adaptations in the central nervous system to counter the neurobiological effect of the drug. Between-system adaptations are interpreted as changes in other neurotransmitter systems that are not linked to the acute reinforcing effects of the drug but become present following chronic administration, see Koob and Le Moal (1997). A change in tissue concentration may be interpreted as altered transmitter synthesis, release, and/or degradation. Decreased peptide levels may, for instance, either result from long-term inhibition of neurons leading to decreased transmitter synthesis or a sudden increase in peptide release caused by depolarization resulting in depletion of the transmitter. The finding in the present study that the concentration of Dyn B is increased in the NAcc at 30 min after the last dose of ethanol may be interpreted as an example of a ``between-system'' adaptation, and is in agreement with previous studies on prodynorphin and kreceptor mRNA levels. A significant increase in Dyn B levels was also observed at 21 days after the last dose, suggesting that the effect of ethanol on this system may be long-lasting. Chronic administration of ethanol increases prodynorphin mRNA in the rat NAcc at 24 and 48 h after withdrawal (Przewlocka et al., 1997). We have previously shown that rats treated repeatedly with ethanol in similar doses as in the present study have decreased k-receptor mRNA levels in the NAcc and the VTA 4 h after the last dose (Rosin et al., 1999). The down-regulation of k-receptor mRNA levels in these brain regions may reflect an adaptive response of the receptor system to an increased activity in dynorphin nerve terminals. The dynorphins act at the kreceptors and may counteract the activation of DA nerve terminals induced by ethanol. The hippocampus is densely innervated by both enkephalinergic and dynorphinergic neurons (McGinty et al., 1982). This region is involved in learning and memory processing (DeJong, 1973; Simmons & Chavkin, 1996) but may also have a role in alcohol dependence (Anokhina, 1979; Seizinger et al., 1983). Differences in hippocampal Met-enkephalin tissue concentration have been found between ethanol preferring P-rats and non-preferring NP-rats (Froehlich et al., 1987). The hippocampal enkephalin system seems to be largely unaffected by chronic ethanol administration (Seizinger et al., 1983) at least when studied immediately after cessation of the ethanol treatment, a finding that was confirmed in this study. However, 5 days after the last dose, a decrease in MEAP tissue concentration was observed in the hippocampus,
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suggesting that during ethanol withdrawal the activity of the hippocampal proenkephalin system is decreased. Reduced hippocampal dynorphin concentration and mRNA levels have previously been reported after chronic ethanol administration (Seizinger et al., 1983; Przewlocka et al., 1992). With the experimental design and dosage used in the present study and the time points chosen herein, the Dyn B concentration was not affected. In the PAG of rats treated with ethanol, the Dyn B concentration was increased 5 days after the last dose as compared to the controls. In addition, the MEAP concentration was increased at the same time point. The PAG is an anatomically heterogeneous region (Bandler & Shipley, 1994), and the caudal ventrolateral PAG is connected to brain regions associated with reward and motivation such as the NAcc (Li et al., 1990). The PAG has been suggested to be involved in opioid withdrawal and to be sensitive to sensory stimuli (Brandao, 1993; Chieng & Christie, 1996). Increased levels of proenkephalin in the PAG have been reported during morphine withdrawal (Fukunaga et al., 1998), and enkephalinase inhibitors attenuate naloxoneprecipitated withdrawal symptoms in morphine-dependent rats (Haffmans & Dzoljic, 1987). The increase in opioid peptide concentration presented here suggests involvement of these peptide systems also after the termination of repeated ethanol administration. The Dyn B concentration was decreased in the cingulate cortex 30 min after the last ethanol dose compared to the control animals. The mesocortical DA pathway projects to the cingulate cortex where a high DA concentration is found (Reader & Grondin, 1987). This region, which is part of the limbic system, has a high density of opioid receptors (Mansour et al., 1987) and is associated with sensory stimuli as well as motivational behavior (Devinsky et al., 1995). The decreased Dyn B tissue concentration that was observed in the cingulate cortex suggests that adaptive changes in the dynorphin/k-receptor system are induced not only by cocaine (Unterwald et al., 1994) but also by repeated ethanol administration. In conclusion, repeated ethanol administration induced both immediate and long-term alterations in the tissue concentration of brain opioids. In particular, the Dyn B levels were significantly elevated in the NAcc not only 30 min after the last dose but also 21 days later, suggesting that repeated exposure to ethanol may lead to long-lasting alterations in the turnover of dynorphin in this brain region. Acknowledgments The authors thank Ms. Lisa Gustafsson and Ms. Helena Lundholm for skillful assistance in the radioimmunoassays. This study was supported by the Swedish Alcohol Research Fund (94/30:5, 95/2, 98/21), Swedish Medical Research Council (07164, K98-O4X-12588-01A), Fredrik och Ingrid Thurings Fund, and Karolinska Institutet Research Funds.
References Anden, N. E., Dahlstrom, A., Fuxe, K., Olson, L., & Ungerstedt, U. (1966). Ascending noradrenaline neurons from the pons and the medulla oblongata. Experientia 22, 44 ± 45. Anokhina, I. P. (1979). Neurochemical aspects in pathogenesis of alcohol and drug dependence. Drug Alcohol Depend 4, 265 ± 273. Bals-Kubik, R., Herz, A., & Shippenberg, T. S. (1989). Evidence that the aversive effects of opioid antagonists and kappa-agonists are centrally mediated. Psychopharmacology 98, 203 ± 206. Bandler, R., & Shipley, M. T. (1994). Columnar organization in the midbrain periaqueductal gray: modules for emotional expression? Trends Neurosci 17, 379 ± 389. Bergstrom, L., Christensson, I., Folkesson, R., Stenstrom, B., & Terenius, L. (1983). An ion exchange chromatography and radioimmunoassay procedure for measuring opioid peptides and substance P. Life Sci 33, 1613 ± 1619. Bloom, F., Lad, P., Pittman, Q., & Rogers, J. (1982). Blood alcohol levels in rats: non-uniform yields from intraperitoneal doses based on body weight. Br J Pharmacol 75, 251 ± 254. Brandao, M. L. (1993). Involvement of opioid mechanisms in the dorsal periaqueductal gray in drug abuse. Rev Neurosci 4, 397 ± 405. Chieng, B., & Christie, M. D. (1996). Local opioid withdrawal in rat single periaqueductal gray neurons in vitro. J Neurosci 16, 7128 ± 7136. Christensson-Nylander, I., Nyberg, F., Ragnarsson, U., & Terenius, L. (1985). A general procedure for analysis of proenkephalin B derived opioid peptides. Regul Pept 11, 65 ± 76. Christensson-Nylander, I., Herrera-Marschitz, M., Staines, W., Hokfelt, T., Terenius, L., Ungerstedt, U., Cuello, C., Oertel, W. H., & Goldstein, M. (1986). Striato-nigral dynorphin and substance P pathways in the rat: I. Biochemical and immunohistochemical studies. Exp Brain Res 64, 169 ± 192. DahlstroÈm, A., & Fuxe, K. (1964). Evidence for the existence of monoamine neurons in the central nervous system: I. Demonstration of monoamine-containing neurons in the cell bodies of brainstem neurons. Acta Physiol Scand 62(suppl. 232), 1. DeJong, R. N. (1973). The hippocampus and its role in memory. Clinical manifestations and theoretical considerations. J Neurol Sci 19, 73 ± 83. Devinsky, O., Morrell, M. J., & Vogt, B. A. (1995). Contributions of anterior cingulate cortex to behaviour. Brain 118, 279 ± 306. de Waele, J. P., & Gianoulakis, C. (1994). Enhanced activity of the brain beta-endorphin system by free-choice ethanol drinking in C57BL/6 but not DBA/2 mice. Eur J Pharmacol 258, 119 ± 129. de Waele, J. P., & Gianoulakis, C. (1997). Characterization of the mu and delta opioid receptors in the brain of the C57BL/6 and DBA/2 mice selected for their differences in voluntary ethanol consumption. Alcohol: Clin Exp Res 21, 754 ± 762. de Waele, J. P., Kiianmaa, K., & Gianoulakis, C. (1995). Distribution of the mu and delta opioid binding sites in the brain of the alcohol-preferring AA and alcohol-avoiding ANA lines of rats. J Pharmacol Exp Ther 275, 518 ± 527. Di Chiara, G., & Imperato, A. (1985). Ethanol preferentially stimulates dopamine release in the nucleus accumbens of freely moving rats. Eur J Pharmacol 115, 131 ± 132. Di Chiara, G., & Imperato, A. (1988). Opposite effects of mu and kappa opiate agonists on dopamine release in the nucleus accumbens and in the dorsal caudate of freely moving rats. J Pharmacol Exp Ther 244, 1067 ± 1080. Dykstra, L. A., Preston, K. L., & Bigelow, G. E. (1997). Discriminative stimulus and subjective effects of opioids with mu and kappa activity: data from laboratory animals and human subjects. Psychopharmacology (Berlin) 130, 14 ± 27. Eriksson, K. (1968). Genetic selection for voluntary alcohol consumption in the albino rat. Science 159, 739 ± 741. Franck, J., Lindholm, S., & Raaschou, P. (1998). Modulation of volitional ethanol intake in the rat by central delta-opioid receptors. Alcohol: Clin Exp Res 22, 1185 ± 1189.
S. Lindholm et al. / Alcohol 22 (2000) 165±171 Froehlich, J. C., Harts, J., Lumeng, L., & Li, T. K. (1987). Naloxone attenuation of voluntary alcohol consumption. Alcohol Alcohol, 333 ± 337 (suppl.). Froehlich, J. C., Zweifel, M., Harts, J., Lumeng, L., & Li, T. K. (1991). Importance of delta opioid receptors in maintaining high alcohol drinking. Psychopharmacology 103, 467 ± 472. Fukunaga, Y., Nishida, S., Inoue, N., Miyamoto, M., Kishioka, S., & Yamamoto, H. (1998). Time course of morphine withdrawal and preproenkephalin gene expression in the periaqueductal gray of rats. Brain Res Mol Brain Res 55, 221 ± 231. Gentry, R. T. (1985). An experimental model of self-intoxication in C57 mice. Alcohol 2, 671 ± 675. Haffmans, J., & Dzoljic, M. R. (1987). Inhibition of enkephalinase activity attenuates naloxone-precipitated withdrawal symptoms. Gen Pharmacol 18, 103 ± 105. Herrera-Marschitz, M., Christensson-Nylander, I., Sharp, T., Staines, W., Reid, M., Hokfelt, T., Terenius, L., Ungerstedt, U. (1986). Striato-nigral dynorphin and substance P pathways in the rat: II. Functional analysis. Exp Brain Res 64, 193 ± 207. Herz, A. (1997). Endogenous opioid systems and alcohol addiction. Psychopharmacology (Berlin) 129, 99 ± 111. Honkanen, A., Vilamo, L., Wegelius, K., Sarviharju, M., Hyytia, P., & Korpi, E. R. (1996). Alcohol drinking is reduced by a mu 1 ÿ but not by a delta-opioid receptor antagonist in alcohol-preferring rats. Eur J Pharmacol 304, 7 ± 13. Hyytia, P. (1993). Involvement of mu-opioid receptors in alcohol drinking by alcohol-preferring AA rats. Pharmacol, Biochem Behav 45, 697 ± 701. Jamensky, N. T., & Gianoulakis, C. (1997). Content of dynorphins and kappa-opioid receptors in distinct brain regions of C57BL/6 and DBA/2 mice. Alcohol: Clin Exp Res 21, 1455 ± 1464. Koob, G. F., & Le Moal, M. (1997). Drug abuse: hedonic homeostatic dysregulation. Science 278, 52 ± 58. Koob, G. F., Vaccarino, F. J., Amalric, M., & Bloom, F. E. (1986). Neurochemical substrates for opiate reinforcement. NIDA Res Monogr 71, 146 ± 164. Krishnan-Sarin, S., Wand, G. S., Li, X. W., Portoghese, P. S., & Froehlich, J. C. (1998). Effect of mu opioid receptor blockade on alcohol intake in rats bred for high alcohol drinking. Pharmacol, Biochem Behav 59, 627 ± 635. Li, T. K., Lumeng, L., McBride, W. J., & Waller, M. B. (1979). Progress toward a voluntary oral consumption model of alcoholism. Drug Alcohol Depend 4, 45 ± 60. Li, T. K., Lumeng, L., McBride, W. J., Murphy, J. M., Froehlich, J. C., & Morzorati, S. (1988). Pharmacology of alcohol preference in rodents. Adv Alcohol Subst Abuse 7, 73 ± 86. Li, Y. Q., Rao, Z. R., & Shi, J. W. (1990). Midbrain periaqueductal gray neurons with substance P- or enkephalin-like immunoreactivity send projection fibers to the nucleus accumbens in the rat. Neurosci Lett 119, 269 ± 271. Mansour, A., Khachaturian, H., Lewis, M. E., Akil, H., & Watson, S. J. (1987). Autoradiographic differentiation of mu, delta, and kappa opioid receptors in the rat forebrain and midbrain. J Neurosci 7, 2445 ± 2464. McGinty, J. F., Henriksen, S. J., Goldstein, A., Terenius, L., & Bloom, F. E. (1982). Opioid peptide identity and localization in hippocampus. Life Sci 31, 1797 ± 1800.
171
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, 1272 ± 1279. Nylander, I., Stenfors, C., Tan-No, K., Mathe, A. A., & Terenius, L. (1997). A comparison between microwave irradiation and decapitation: basal levels of dynorphin and enkephalin and the effect of chronic morphine treatment on dynorphin peptides. Neuropeptides 31, 357 ± 365. Paxinos, S., & Watson, C. (1986). The Rat Brain Atlas in Stereotaxic Coordinates. London: Academic Press. Ploj, K., Roman, E., Gustafsson, L., & Nylander, I. (2000). Basal levels and alcohol induced changes in nociceptin/orphanin FQ, dynorphin, and enkephalin levels in C57BL/6J mice. Brain Res Bull (in press). Przewlocka, B., Lason, W., & Przewlocki, R. (1992). Repeated ethanol administration decreases prodynorphin biosynthesis in the rat hippocampus. Neurosci Lett 134, 195 ± 198. Przewlocka, B., Turchan, J., Lason, W., & Przewlocki, R. (1997). Ethanol withdrawal enhances the prodynorphin system activity in the rat nucleus accumbens. Neurosci Lett 238, 13 ± 16. Reader, T. A., & Grondin, L. (1987). Distribution of catecholamines, serotonin, and their major metabolites in the rat cingulate, piriformentorhinal, somatosensory, and visual cortex: a biochemical survey using high-performance liquid chromatography. Neurochem Res 12, 1087 ± 1097. Ê ., Lindholm, S., Franck, J., & Georgieva, J. (1999). DownregulaRosin, A tion of kappa opioid receptor mRNA levels by chronic ethanol and repetitive cocaine in rat ventral tegmentum and nucleus accumbens. Neurosci Lett 275, 1 ± 4. Seizinger, B. R., 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, 361 ± 369. Simmons, M. L., & Chavkin, C. (1996). Endogenous opioid regulation of hippocampal function. Int Rev Neurobiol 39, 145 ± 196. Spanagel, R., Herz, A., & Shippenberg, T. S. (1990). The effects of opioid peptides on dopamine release in the nucleus accumbens: an in vivo microdialysis study. J Neurochem 55, 1734 ± 1740. Spanagel, R., Herz, A., & Shippenberg, T. S. (1992). Opposing tonically active endogenous opioid systems modulate the mesolimbic dopaminergic pathway. Proc Natl Acad Sci USA 89, 2046 ± 2050. Stromberg, M. F., Casale, M., Volpicelli, L., Volpicelli, J. R., & O'Brien, C. P. (1998). A comparison of the effects of the opioid antagonists naltrexone, naltrindole, and beta-funaltrexamine on ethanol consumption in the rat. Alcohol 15, 281 ± 289. Ungerstedt, U. (1971). Stereotaxic mapping of the monoamine pathways in the rat brain. Acta Physiol Scand, Suppl 367, 1 ± 48. Unterwald, E. M., Rubenfeld, J. M., & Kreek, M. J. (1994). Repeated cocaine administration upregulates kappa and mu, but not delta, opioid receptors. NeuroReport 5, 1613 ± 1616. Watson, S. J., Khachaturian, H., Akil, H., Coy, D. H., & Goldstein, A. (1982). Comparison of the distribution of dynorphin systems and enkephalin systems in brain. Science 218, 1134 ± 1136. You, Z. B., Herrera-Marschitz, M., Nylander, I., Goiny, M., O'Connor, W. T., Ungerstedt, U., Terenius, L. (1994). The striatonigral dynorphin pathway of the rat studied with in vivo microdialysis: II. Effects of dopamine D1 and D2 receptor agonists. Neuroscience 63, 427 ± 434.
Repeated ethanol administration induces short- and long-term changes in enkephalin and dynorphin tissue concentrations in rat brain Sara Lindholma,*, Karolina Plojb, Johan Francka, Ingrid Nylanderb a
Clinical Alcohol and Drug Addiction Research, Department of Clinical Neuroscience, Magnus Huss, M4:02, Karolinska Hospital, Karolinska Institutet, SE-171 76 Stockholm, Sweden b Department of Pharmaceutical Biosciences, Division of Pharmacology, Uppsala University, Uppsala, Sweden Received 31 January 2000; received in revised form 5 July 2000; accepted 17 August 2000
Abstract Recently, we have shown that rats repeatedly treated with ethanol and/or cocaine have decreased k-opioid receptor mRNA levels in the mesolimbic system. The aim of the present study was to investigate the short- and long-term effects of repeated ethanol administration on opioid peptide concentrations in brain tissue of male Sprague ± Dawley rats. Dynorphin B (1 ± 13) (Dyn B) and Met-enkephalinArg6Phe7 (MEAP), endogenous ligands to k- and d-opioid receptors, respectively, were measured using radioimmunoassays. The rats were given either ethanol [intraperitoneal (ip), twice daily, 2 g/kg bw/dose] or saline for 13 consecutive days. Thirty min after the last ethanol dose on Day 13, the Dyn B tissue concentration was significantly decreased in the cingulate cortex. The MEAP tissue concentration was decreased in the hippocampus 5 days after the last ethanol injection as compared to saline-treated controls. Furthermore, the Dyn B and the MEAP concentrations were increased in the periaqueductal grey area (PAG) at this time point. Of particular interest were the significant increases in Dyn B tissue concentrations found in the nucleus accumbens (NAcc) at 30 min and at 21 days after the last ethanol dose. The results suggest that repeated ethanol administration induces both short- and long-term changes in the tissue concentrations of opioids in certain brain regions associated with motivation and reward. D 2001 Elsevier Science Inc. All rights reserved. Keywords: Opioids; Nucleus accumbens; Dynorphin B; Ethanol; Met-enkephalinArg6Phe7
1. Introduction Rodents with high or low preference for ethanol intake have differences in the mesolimbic reward system (Li et al., 1988). Such animal lines include the high- and low-preferring AA- and ANA-rats (Eriksson, 1968), and the P- and NP-rats (Li et al., 1979) as well as the C57BL/6 and DBA/2 mice strains (Gentry, 1985). Differences in basal as well as in ethanol-induced dopamine (DA) and/or opioid peptide turnover have been reported in these strains. Ethanol-preferring AA-rats have, for example, low basal tissue concentrations of both Met-enkephalinArg6Phe7 (MEAP) and dynorphin peptides in the nucleus accumbens (NAcc) as compared to ANA-rats, and the MEAP concentration increases after volitional ethanol intake in both
* Corresponding author. Tel.: +46-8-51774870; fax: +46-8-326369. E-mail address: [email protected] (S. Lindholm).
strains (Nylander et al., 1994). The ethanol-preferring C57BL/6(J) mouse has lower basal dynorphin peptides and prodynorphin mRNA levels in the NAcc (Jamensky & Gianoulakis, 1997; Ploj et al., 2000) as well as lower levels of b-endorphin- and enkephalin-like peptides in the NAcc as compared to the low-preferring DBA/2(J) mouse strain (de Waele & Gianoulakis, 1994; Ploj et al., 2000). Furthermore, differences in opioid-receptor density have been reported in high- and low-preferring rodents. The AA-rats have higher basal density of m-opioid receptor binding sites (de Waele et al., 1995), and the C57BL/6 mice have increased density of dopioid receptors (de Waele & Gianoulakis, 1997) as well as decreased density of k-opioid receptor binding sites (Jamensky & Gianoulakis, 1997) in the mesolimbic system compared to their low-preferring counterparts. Non-selective opioid receptor antagonists decrease volitional ethanol intake in several animal models; see Herz (1997). This effect is also seen with administration of selective d- (Froehlich et al., 1991; Franck et al., 1998)
0741-8329/00/$ ± see front matter D 2001 Elsevier Science Inc. All rights reserved. PII: S 0 7 4 1 - 8 3 2 9 ( 0 0 ) 0 0 11 8 - X
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and m-receptor antagonists (Hyytia, 1993; Krishnan-Sarin et al., 1998) although conflicting results have been reported (Honkanen et al., 1996; Stromberg et al., 1998). Agonists acting at m- and d-opioid receptors are self-administered in various animal models and produce place preference, see Koob et al. (1986), whereas k-opioid receptor agonists mainly have aversive properties (Bals-Kubik et al., 1989; Dykstra et al., 1997). Taken together, this indicates that the opioid peptide systems play a role in the propensity for ethanol intake in rodents. We have previously shown that a d-receptor antagonist decreases voluntary ethanol intake in rats, suggesting that the effects of ethanol may partly depend on activation of opioid receptors (Franck et al., 1998). In addition, we have also found that k-receptor mRNA levels decreased in the NAcc and in the ventral tegmental area (VTA) after repeated ethanol administration (Rosin et al., 1999). This kreceptor mRNA down-regulation may be interpreted as an adaptive response to increased mesolimbic dynorphin levels. In this study, it was hypothesized that long-term repeated administration of ethanol would alter the activity of the endogenous ligands to the d- and k-receptors, the enkephalins and dynorphins, respectively. Therefore, the effects of repeated ethanol administration on both enkephalin and dynorphin tissue concentrations were studied. The tissue concentrations of MEAP and dynorphin B (1 ± 13) (Dyn B) were determined in selected brain regions and in the pituitary gland of male Sprague ± Dawley rats 30 min after the last dose, i.e. during ethanol intoxication. In order to study the long-term effects of repeated ethanol administration, the tissue concentrations of MEAP and Dyn B were also measured at 5 and 21 days after the last dose. MEAP is exclusively cleaved from proenkephalin and was therefore measured as a marker for its precursor, whereas Dyn B was measured as a marker for the prodynorphin system. 2. Materials and methods 2.1. Animals Male Sprague ± Dawley rats (B&K Universal; Stockholm, Sweden) were used in all experiments. The rats (n = 72) were housed in groups of six in their normal cages in a temperature-controlled environment (20.5 2°C) on a 12-h light/dark cycle with access to food (B&K rat chow, B&K Universal) and tap water ad libitum. Rats weighed 228 ±347 g at the beginning of the experiments. All animal experiments were performed under an approved protocol in accordance with the Swedish Animal Protection Legislation. 2.2. Experimental design The rats were randomly divided into three experimental and three control groups. The animals in the experimental groups were administered ethanol intraperitoneally [ip; 2 g/
kg bw/dose, 18% (v/v), diluted in saline] twice daily at 9 a.m. and 3 p.m. for 13 consecutive days. Each experimental group had a corresponding control group. The controls were given saline in equal volumes at the same time points as the ethanol groups. Rats were killed by decapitation. In the first set of experiments, one group of ethanol-treated rats and one control group were decapitated 30 min after the last dose. In the second set of experiments, ethanol- and saline-treated rats were decapitated at 5 days after the last dose. A third set of rats was decapitated at 21 days after the last dose. During these two latter periods, the rats did not receive any treatment. 2.3. Dissection After decapitation, the pituitary gland and the brain were immediately removed and rapidly dissected on ice. The whole pituitary gland was extracted in all experiments. The hypothalamus was removed from the brain, which was then placed in a cooled brain blocker (AgnTho's AB; Stockholm, Sweden), and sliced manually with razor blades in coronal sections. The NAcc, hippocampus, cingulate cortex, VTA, dorsal striatum, and the periaqueductal grey area (PAG) were manually dissected out using a scalpel with guidance from the rat brain atlas of Paxinos and Watson (1986). The tissue samples were immediately frozen on dry ice and stored at ÿ 80°C until further analysis. 2.4. Extraction and separation of peptides Tissue extraction was performed with 1 M acetic acid. The samples were heated at 95°C for 5 min, and after cooling on ice, the tissues were homogenized by sonication using a Branson Sonifier. The samples were reheated at 95°C for 5 min, cooled on ice, and then centrifuged for 15 min at 12 000 g in a Beckman GS-15R Centrifuge. The samples were purified using a cation exchange procedure. The supernatants were applied onto small (1 ml) cation exchange columns containing SP Sephadex C-25 gel (Pharmacia Diagnostics; Uppsala, Sweden), and the opioid peptides were eluted with buffers containing mixtures of pyridine and formic acid. Peptides eluted in separate fractions depending on the buffer used (Bergstrom et al., 1983; Christensson-Nylander et al., 1985). MEAP and Dyn B were measured as markers for their respective precursor peptides. The fractions were dried in a vacuum centrifuge and stored at ÿ 20°C until peptide analysis. 2.5. Radioimmunoassay Dyn B and MEAP were measured with specific radioimmunoassays as previously described in detail (Nylander et al., 1997). Rabbit antisera for the peptides were used. The tracer peptides were labeled with 125I according to the chloramine T method and were purified with reversed phase HPLC using a gradient of 15± 40% acetonitrile in 0.04% trifluoroacetic acid.
S. Lindholm et al. / Alcohol 22 (2000) 165±171
In the Dyn B radioimmunoassay, an aliquot of the sample (25 ml) was dissolved in methanol/0.1 M hydrochloric acid (1:1). The solution was incubated with 100 ml of antiserum (113+, final dilution, 1:562 500) and 100 ml of 125I-labelled peptide, both diluted in gelatin buffer. The gelatin buffer contained 0.15 M sodium chloride, 0.02% sodium azide, 0.1% gelatin, 0.1% Triton X-100, and 0.1% bovine serum albumin in a 0.05 M sodium phosphate buffer. Samples were incubated for 24 h at 4°C. To separate free and antibody-bound peptides, 100 ml of a sheep antirabbit antiserum (Pharmacia Decanting Suspension 3, Pharmacia Diagnostics) was added, and the samples were incubated for 1 h at 4°C. After centrifugation for 10 min at 12 000 g in a Beckman GS-15R Centrifuge, the radioactivity in the pellet was counted in a Wallac 1470 Wizard gamma counter. Dyn-32 and Dyn B-29 crossreact 100% and 1%, respectively with the Dyn B antiserum, whereas other dynorphin peptides, Met-enkephalin, Met-enkephalinArg6, Met-enkephalinArg6Gly7Leu8, Leuenkephalin, and Leu-enkephalinArg6, cross-react < 0.1%. Samples subjected to MEAP assay were oxidized prior the radioimmunoassay procedure, i.e. the samples were dissolved in acetic acid (1 M, 100 ml), and H2O2 (30%, 10 ml) was added. The samples were incubated at 37°C for 30 min and dried in a vacuum centrifuge. An aliquot of the sample (25 ml) was dissolved in methanol/0.1 M hydrochloric acid (1:1) and incubated with 100 ml of antiserum (90:3D, final dilution 1:180 000) and 100 ml of 125I-labelled peptide, both diluted in gelatin buffer. The gelatin buffer contained 0.15 M sodium chloride, 0.025 M EDTA, 0.1% gelatin, and 0.1% bovine serum albumin in 0.05 M sodium phosphate buffer. Samples were incubated for 24 h at 4°C. To separate free and antibody-bound peptides, a charcoal suspension (200 ml) consisting of 250 mg charcoal and 25mg dextran T-70 in 100 ml of sodium phosphate buffer (0.05 M) was added, and the samples were incubated for 10 min. The samples were centrifuged for 2 min at 12 000 g in a Beckman GS-
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15R Centrifuge, and the radioactivity in a 300 ml aliquot of the supernatant was counted in the Wallac 1470 Wizard gamma counter. Cross-reactivity of Met-enkephalin, Met-enkephalinArg6, Met-enkephalinArg6Gly7Leu8, Leu-enkephalin, Leu-enkephalinArg6, and dynorphin peptides was < 0.1% for the MEAP antiserum. 2.6. Statistical analysis The values obtained from the control rats decapitated 30 min after the last dose are expressed as fmol/mg tissue (mean S.E.M., wet weight; see Results). A statistical comparison of peptide immunoreactivity in brain regions from saline- and ethanol-treated rats were performed on three occasions using non-transformed data; 30 min after the morning dose on the last day of the treatment period, and 5 and 21 days after the last dose. In Figs. 1 and 2, the data from ethanol-treated rats are expressed as % of controls (mean S.E.M.), where the controls are shown as 100%. Comparisons across the different time points were not conducted since samples from each experimental time point were analyzed in separate radioimmunoassays. The samples from ethanol and control rats were at each separate time point handled together in the various sample-handling procedures, i.e. during the tissue homogenization and extraction, separation procedures, and statistical analysis. Differences between groups were analyzed with a one-way ANOVA combined with the Fisher post-hoc test using the Statview 4.5 software. A P-value < 0.05 was considered statistically significant. 3. Results Although no specific quantitative measures were used, mild physical signs of withdrawal including hyper-reactivity and irritability were observed at 5 days after the last administration of ethanol. At the other time points studied,
Fig. 1. Effects of repeated ethanol (2 g/kg bw ip, twice daily) or saline administration on the concentration of Dyn B in tissue from distinct rat brain regions and the pituitary gland (Pt); hypothalamus (HT), striatum (Str), NAcc, VTA, cingulate cortex (CC), hippocampus (HP), and the PAG. The Dyn B concentration was measured at 30 min, and 5 or 21 days after the last dose of ethanol or saline. The values represent % of control values (mean S.E.M.) where the controls are expressed as 100%. Comparisons between two separate groups at respective time points were made with one-way ANOVA combined with the Fisher post-hoc test using the Statview 4.5 software. * P < .05.
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Fig. 2. Effects of repeated ethanol (2 g/kg bw ip, twice daily) or saline administration on MEAP concentrations in tissue from distinct rat brain regions and the pituitary gland (Pt); hypothalamus (HT), striatum (Str), NAcc, VTA, cingulate cortex (CC), hippocampus (HP), and the PAG. The MEAP concentrations were measured at 30 min, and 5 or 21 days after the last dose of ethanol or saline. The values represent % of the control values (mean S.E.M.) where the controls are 100%. Comparisons between two separate groups at respective time points were made with one-way ANOVA combined with the Fisher post-hoc test using the Statview 4.5 software. * P < 0.05.
ethanol-treated rats were either intoxicated (30 min after the last dose) or seemingly unaffected by the treatment (21 days after the last dose). The mean body weights of the ethanol- and saline-treated groups were 247 3 and 244 2 g (mean S.E.M., n = 12), respectively at the start of the first set of experiments. After 13 days of treatment, rats repeatedly treated with ethanol had 18% less body weight as compared to the saline-treated animals (263 6 vs. 320 2 g, P < 0.01). This difference between the treatment groups was also found 5 days after the last dose but not after 21 days (data not shown). Although the relative levels of opioids in different brain regions were comparable between all time points, no statistical comparison could be conducted for samples from one experimental time point to another, since the samples from each time point were analyzed in separate radioimmunoassays. The basal values of the opioid tissue concentration in different brain regions and in the pituitary gland of saline-treated rats are shown for the 30-min control group. 3.1. Dyn B and MEAP tissue concentrations in salinetreated rats The highest Dyn B concentration in saline-treated rats decapitated 30 min after the last dose was found in the pituitary gland (86.3 8.3 fmol/mg tissue, mean S.E.M., n = 12). Moderate Dyn B concentrations were found in the hypothalamus (24.1 3.0 fmol/mg tissue, n = 12), PAG (19.3 3.6 fmol/mg tissue, n = 9), VTA (18.6 4.7 fmol/ mg tissue, n = 9), striatum (13.8 1.0 fmol/mg tissue, n = 12), hippocampus (9.8 1.3 fmol/mg tissue, n = 9), and the NAcc (6.7 0.6 fmol/mg tissue, n = 12), whereas a low Dyn B concentration was found in the cingulate cortex (1.9 0.2 fmol/mg tissue, n = 8). The highest MEAP tissue concentration of the brain regions studied in the control group decapitated 30 min after the last dose was found in the hypothalamus (54.9 3.4 fmol/mg tissue,
n = 12). Moderate MEAP tissue concentrations were found in the striatum (30.4 3.2 fmol/mg tissue, n = 10), PAG (25.1 5.5 fmol/mg tissue, n = 9), NAcc (25.1 3.6 fmol/ mg tissue, n = 10), and the VTA (22.5 4.1 fmol/mg tissue, n = 9), whereas lower MEAP concentrations were detected in the pituitary gland (3.5 0.7 fmol/mg tissue, n = 12), cingulate cortex (2.8 0.4 fmol/mg tissue, n = 10), and the hippocampus (2.2 0.3 fmol/mg tissue, n = 8) of the salinetreated rats. 3.2. Short-term effects of repeated ethanol administration on Dyn B concentration in brain tissue The effects of repeated ethanol administration followed by decapitation 30 min after the last dose on Dyn B tissue concentration in various brain regions and the pituitary gland as compared to the respective control group are shown in Fig. 1. The Dyn B tissue concentrations were significantly increased by 40 12% in the NAcc and decreased in the cingulate cortex by 35 15% in ethanol-treated rats as compared to their respective controls (Fig. 1, P < 0.05). It should be pointed out that although a marked tendency to a reduced Dyn B concentration was observed in the VTA, this change failed to reach statistical significance due to large variability within the control group ( P = 0.0649). No significant difference between the groups was found in any of the other brain regions studied. 3.3. Long-term effects of repeated ethanol administration on Dyn B concentration in brain tissue Five days after the last injection of ethanol, a significant increase ( + 48 14%) in Dyn B tissue concentration was found in the PAG (Fig. 1, P < 0.05) as compared to the control group. No significant difference between the groups was found in the other brain regions analyzed at this time point. Rats treated repeatedly with ethanol followed by 21
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days of recovery from treatment had a significantly increased Dyn B concentration in the NAcc by 39 12% (Fig. 1, P < 0.05) as compared to the control group. No significant difference between the groups was found in the other brain regions analyzed at this time point. 3.4. Short-term effects of repeated ethanol administration on MEAP concentration in brain tissue The effect of repeated administration with ethanol or saline on brain tissue MEAP concentration is shown in Fig. 2. No significant difference between the controls and ethanoltreated rats was found in any of the brain regions analyzed. 3.5. Long-term effects of repeated ethanol administration on MEAP concentration in brain tissue A significant decrease in MEAP concentration (ÿ30 8%) was found in the hippocampus at 5 days after the last ethanol dose as compared to their controls (Fig. 2, P < 0.05). In addition, the MEAP tissue concentration increased significantly ( + 45 11%) in the PAG in the same animals (Fig. 2, P < 0.05). No significant changes were observed in any other brain regions that were analyzed, neither at 5 or 21 days after the last dose (Fig. 2). 4. Discussion In this study, the tissue concentrations of MEAP and Dyn B were measured in selected brain regions and in the pituitary gland of rats after repeated ethanol administration. Peptide levels were determined both at 30 min, i.e. when ethanol was still present in brain and at 5 and 21 days after the last ethanol dose. A previous study showed that an acute intraperitoneal injection of 2 g/kg bw ethanol produced a significant blood alcohol concentration (ranging approximately between 175 and 190 mg%) that is stable during a post-injection period of 10 ± 120 min (Bloom et al., 1982). Ethanol in this dose is sedative, and may cause ataxia and decreased food intake (Bloom et al., 1982), which may explain the divergent body weight found between the groups. The dynorphins are mainly found in the pituitary ± hypothalamic axis, in the mesolimbic system, and in the striatonigral pathway, whereas the enkephalins are widely distributed in the brain (Christensson-Nylander et al., 1986; Watson et al., 1982). The dynorphins and the k-opioid receptors are in close proximity to the dopaminergic neurons of the mesolimbic and nigrostriatal systems (HerreraMarschitz et al., 1986), and interactions between DA and Dyn B have previously been demonstrated in the latter pathway (Herrera-Marschitz et al., 1986; You et al., 1994). Ethanol activates the dopaminergic mesolimbic system (Di Chiara & Imperato, 1985). The cell bodies of this system are located in the VTA (DahlstroÈm & Fuxe, 1964) with terminals in the NAcc and the prefrontal cortex (Anden et al.,
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1966; Ungerstedt, 1971), and the system is tonically controlled by opioid peptides (Spanagel et al., 1992). Stimulation of m-opioid receptors, located on GABA-containing interneurons in the VTA, leads to an increased extracellular DA concentration in the terminal regions (Spanagel et al., 1992). Local administration of d-opioid receptor agonists also increase (Spanagel et al., 1990), whereas k-opioid receptor agonists decrease the DA concentration in the NAcc (Di Chiara & Imperato, 1988; Spanagel et al., 1990). Chronic drug administration may cause adaptations in the central nervous system to counter the neurobiological effect of the drug. Between-system adaptations are interpreted as changes in other neurotransmitter systems that are not linked to the acute reinforcing effects of the drug but become present following chronic administration, see Koob and Le Moal (1997). A change in tissue concentration may be interpreted as altered transmitter synthesis, release, and/or degradation. Decreased peptide levels may, for instance, either result from long-term inhibition of neurons leading to decreased transmitter synthesis or a sudden increase in peptide release caused by depolarization resulting in depletion of the transmitter. The finding in the present study that the concentration of Dyn B is increased in the NAcc at 30 min after the last dose of ethanol may be interpreted as an example of a ``between-system'' adaptation, and is in agreement with previous studies on prodynorphin and kreceptor mRNA levels. A significant increase in Dyn B levels was also observed at 21 days after the last dose, suggesting that the effect of ethanol on this system may be long-lasting. Chronic administration of ethanol increases prodynorphin mRNA in the rat NAcc at 24 and 48 h after withdrawal (Przewlocka et al., 1997). We have previously shown that rats treated repeatedly with ethanol in similar doses as in the present study have decreased k-receptor mRNA levels in the NAcc and the VTA 4 h after the last dose (Rosin et al., 1999). The down-regulation of k-receptor mRNA levels in these brain regions may reflect an adaptive response of the receptor system to an increased activity in dynorphin nerve terminals. The dynorphins act at the kreceptors and may counteract the activation of DA nerve terminals induced by ethanol. The hippocampus is densely innervated by both enkephalinergic and dynorphinergic neurons (McGinty et al., 1982). This region is involved in learning and memory processing (DeJong, 1973; Simmons & Chavkin, 1996) but may also have a role in alcohol dependence (Anokhina, 1979; Seizinger et al., 1983). Differences in hippocampal Met-enkephalin tissue concentration have been found between ethanol preferring P-rats and non-preferring NP-rats (Froehlich et al., 1987). The hippocampal enkephalin system seems to be largely unaffected by chronic ethanol administration (Seizinger et al., 1983) at least when studied immediately after cessation of the ethanol treatment, a finding that was confirmed in this study. However, 5 days after the last dose, a decrease in MEAP tissue concentration was observed in the hippocampus,
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suggesting that during ethanol withdrawal the activity of the hippocampal proenkephalin system is decreased. Reduced hippocampal dynorphin concentration and mRNA levels have previously been reported after chronic ethanol administration (Seizinger et al., 1983; Przewlocka et al., 1992). With the experimental design and dosage used in the present study and the time points chosen herein, the Dyn B concentration was not affected. In the PAG of rats treated with ethanol, the Dyn B concentration was increased 5 days after the last dose as compared to the controls. In addition, the MEAP concentration was increased at the same time point. The PAG is an anatomically heterogeneous region (Bandler & Shipley, 1994), and the caudal ventrolateral PAG is connected to brain regions associated with reward and motivation such as the NAcc (Li et al., 1990). The PAG has been suggested to be involved in opioid withdrawal and to be sensitive to sensory stimuli (Brandao, 1993; Chieng & Christie, 1996). Increased levels of proenkephalin in the PAG have been reported during morphine withdrawal (Fukunaga et al., 1998), and enkephalinase inhibitors attenuate naloxoneprecipitated withdrawal symptoms in morphine-dependent rats (Haffmans & Dzoljic, 1987). The increase in opioid peptide concentration presented here suggests involvement of these peptide systems also after the termination of repeated ethanol administration. The Dyn B concentration was decreased in the cingulate cortex 30 min after the last ethanol dose compared to the control animals. The mesocortical DA pathway projects to the cingulate cortex where a high DA concentration is found (Reader & Grondin, 1987). This region, which is part of the limbic system, has a high density of opioid receptors (Mansour et al., 1987) and is associated with sensory stimuli as well as motivational behavior (Devinsky et al., 1995). The decreased Dyn B tissue concentration that was observed in the cingulate cortex suggests that adaptive changes in the dynorphin/k-receptor system are induced not only by cocaine (Unterwald et al., 1994) but also by repeated ethanol administration. In conclusion, repeated ethanol administration induced both immediate and long-term alterations in the tissue concentration of brain opioids. In particular, the Dyn B levels were significantly elevated in the NAcc not only 30 min after the last dose but also 21 days later, suggesting that repeated exposure to ethanol may lead to long-lasting alterations in the turnover of dynorphin in this brain region. Acknowledgments The authors thank Ms. Lisa Gustafsson and Ms. Helena Lundholm for skillful assistance in the radioimmunoassays. This study was supported by the Swedish Alcohol Research Fund (94/30:5, 95/2, 98/21), Swedish Medical Research Council (07164, K98-O4X-12588-01A), Fredrik och Ingrid Thurings Fund, and Karolinska Institutet Research Funds.
References Anden, N. E., Dahlstrom, A., Fuxe, K., Olson, L., & Ungerstedt, U. (1966). Ascending noradrenaline neurons from the pons and the medulla oblongata. Experientia 22, 44 ± 45. Anokhina, I. P. (1979). Neurochemical aspects in pathogenesis of alcohol and drug dependence. Drug Alcohol Depend 4, 265 ± 273. Bals-Kubik, R., Herz, A., & Shippenberg, T. S. (1989). Evidence that the aversive effects of opioid antagonists and kappa-agonists are centrally mediated. Psychopharmacology 98, 203 ± 206. Bandler, R., & Shipley, M. T. (1994). Columnar organization in the midbrain periaqueductal gray: modules for emotional expression? Trends Neurosci 17, 379 ± 389. Bergstrom, L., Christensson, I., Folkesson, R., Stenstrom, B., & Terenius, L. (1983). An ion exchange chromatography and radioimmunoassay procedure for measuring opioid peptides and substance P. Life Sci 33, 1613 ± 1619. Bloom, F., Lad, P., Pittman, Q., & Rogers, J. (1982). Blood alcohol levels in rats: non-uniform yields from intraperitoneal doses based on body weight. Br J Pharmacol 75, 251 ± 254. Brandao, M. L. (1993). Involvement of opioid mechanisms in the dorsal periaqueductal gray in drug abuse. Rev Neurosci 4, 397 ± 405. Chieng, B., & Christie, M. D. (1996). Local opioid withdrawal in rat single periaqueductal gray neurons in vitro. J Neurosci 16, 7128 ± 7136. Christensson-Nylander, I., Nyberg, F., Ragnarsson, U., & Terenius, L. (1985). A general procedure for analysis of proenkephalin B derived opioid peptides. Regul Pept 11, 65 ± 76. Christensson-Nylander, I., Herrera-Marschitz, M., Staines, W., Hokfelt, T., Terenius, L., Ungerstedt, U., Cuello, C., Oertel, W. H., & Goldstein, M. (1986). Striato-nigral dynorphin and substance P pathways in the rat: I. Biochemical and immunohistochemical studies. Exp Brain Res 64, 169 ± 192. DahlstroÈm, A., & Fuxe, K. (1964). Evidence for the existence of monoamine neurons in the central nervous system: I. Demonstration of monoamine-containing neurons in the cell bodies of brainstem neurons. Acta Physiol Scand 62(suppl. 232), 1. DeJong, R. N. (1973). The hippocampus and its role in memory. Clinical manifestations and theoretical considerations. J Neurol Sci 19, 73 ± 83. Devinsky, O., Morrell, M. J., & Vogt, B. A. (1995). Contributions of anterior cingulate cortex to behaviour. Brain 118, 279 ± 306. de Waele, J. P., & Gianoulakis, C. (1994). Enhanced activity of the brain beta-endorphin system by free-choice ethanol drinking in C57BL/6 but not DBA/2 mice. Eur J Pharmacol 258, 119 ± 129. de Waele, J. P., & Gianoulakis, C. (1997). Characterization of the mu and delta opioid receptors in the brain of the C57BL/6 and DBA/2 mice selected for their differences in voluntary ethanol consumption. Alcohol: Clin Exp Res 21, 754 ± 762. de Waele, J. P., Kiianmaa, K., & Gianoulakis, C. (1995). Distribution of the mu and delta opioid binding sites in the brain of the alcohol-preferring AA and alcohol-avoiding ANA lines of rats. J Pharmacol Exp Ther 275, 518 ± 527. Di Chiara, G., & Imperato, A. (1985). Ethanol preferentially stimulates dopamine release in the nucleus accumbens of freely moving rats. Eur J Pharmacol 115, 131 ± 132. Di Chiara, G., & Imperato, A. (1988). Opposite effects of mu and kappa opiate agonists on dopamine release in the nucleus accumbens and in the dorsal caudate of freely moving rats. J Pharmacol Exp Ther 244, 1067 ± 1080. Dykstra, L. A., Preston, K. L., & Bigelow, G. E. (1997). Discriminative stimulus and subjective effects of opioids with mu and kappa activity: data from laboratory animals and human subjects. Psychopharmacology (Berlin) 130, 14 ± 27. Eriksson, K. (1968). Genetic selection for voluntary alcohol consumption in the albino rat. Science 159, 739 ± 741. Franck, J., Lindholm, S., & Raaschou, P. (1998). Modulation of volitional ethanol intake in the rat by central delta-opioid receptors. Alcohol: Clin Exp Res 22, 1185 ± 1189.
S. Lindholm et al. / Alcohol 22 (2000) 165±171 Froehlich, J. C., Harts, J., Lumeng, L., & Li, T. K. (1987). Naloxone attenuation of voluntary alcohol consumption. Alcohol Alcohol, 333 ± 337 (suppl.). Froehlich, J. C., Zweifel, M., Harts, J., Lumeng, L., & Li, T. K. (1991). Importance of delta opioid receptors in maintaining high alcohol drinking. Psychopharmacology 103, 467 ± 472. Fukunaga, Y., Nishida, S., Inoue, N., Miyamoto, M., Kishioka, S., & Yamamoto, H. (1998). Time course of morphine withdrawal and preproenkephalin gene expression in the periaqueductal gray of rats. Brain Res Mol Brain Res 55, 221 ± 231. Gentry, R. T. (1985). An experimental model of self-intoxication in C57 mice. Alcohol 2, 671 ± 675. Haffmans, J., & Dzoljic, M. R. (1987). Inhibition of enkephalinase activity attenuates naloxone-precipitated withdrawal symptoms. Gen Pharmacol 18, 103 ± 105. Herrera-Marschitz, M., Christensson-Nylander, I., Sharp, T., Staines, W., Reid, M., Hokfelt, T., Terenius, L., Ungerstedt, U. (1986). Striato-nigral dynorphin and substance P pathways in the rat: II. Functional analysis. Exp Brain Res 64, 193 ± 207. Herz, A. (1997). Endogenous opioid systems and alcohol addiction. Psychopharmacology (Berlin) 129, 99 ± 111. Honkanen, A., Vilamo, L., Wegelius, K., Sarviharju, M., Hyytia, P., & Korpi, E. R. (1996). Alcohol drinking is reduced by a mu 1 ÿ but not by a delta-opioid receptor antagonist in alcohol-preferring rats. Eur J Pharmacol 304, 7 ± 13. Hyytia, P. (1993). Involvement of mu-opioid receptors in alcohol drinking by alcohol-preferring AA rats. Pharmacol, Biochem Behav 45, 697 ± 701. Jamensky, N. T., & Gianoulakis, C. (1997). Content of dynorphins and kappa-opioid receptors in distinct brain regions of C57BL/6 and DBA/2 mice. Alcohol: Clin Exp Res 21, 1455 ± 1464. Koob, G. F., & Le Moal, M. (1997). Drug abuse: hedonic homeostatic dysregulation. Science 278, 52 ± 58. Koob, G. F., Vaccarino, F. J., Amalric, M., & Bloom, F. E. (1986). Neurochemical substrates for opiate reinforcement. NIDA Res Monogr 71, 146 ± 164. Krishnan-Sarin, S., Wand, G. S., Li, X. W., Portoghese, P. S., & Froehlich, J. C. (1998). Effect of mu opioid receptor blockade on alcohol intake in rats bred for high alcohol drinking. Pharmacol, Biochem Behav 59, 627 ± 635. Li, T. K., Lumeng, L., McBride, W. J., & Waller, M. B. (1979). Progress toward a voluntary oral consumption model of alcoholism. Drug Alcohol Depend 4, 45 ± 60. Li, T. K., Lumeng, L., McBride, W. J., Murphy, J. M., Froehlich, J. C., & Morzorati, S. (1988). Pharmacology of alcohol preference in rodents. Adv Alcohol Subst Abuse 7, 73 ± 86. Li, Y. Q., Rao, Z. R., & Shi, J. W. (1990). Midbrain periaqueductal gray neurons with substance P- or enkephalin-like immunoreactivity send projection fibers to the nucleus accumbens in the rat. Neurosci Lett 119, 269 ± 271. Mansour, A., Khachaturian, H., Lewis, M. E., Akil, H., & Watson, S. J. (1987). Autoradiographic differentiation of mu, delta, and kappa opioid receptors in the rat forebrain and midbrain. J Neurosci 7, 2445 ± 2464. McGinty, J. F., Henriksen, S. J., Goldstein, A., Terenius, L., & Bloom, F. E. (1982). Opioid peptide identity and localization in hippocampus. Life Sci 31, 1797 ± 1800.
171
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, 1272 ± 1279. Nylander, I., Stenfors, C., Tan-No, K., Mathe, A. A., & Terenius, L. (1997). A comparison between microwave irradiation and decapitation: basal levels of dynorphin and enkephalin and the effect of chronic morphine treatment on dynorphin peptides. Neuropeptides 31, 357 ± 365. Paxinos, S., & Watson, C. (1986). The Rat Brain Atlas in Stereotaxic Coordinates. London: Academic Press. Ploj, K., Roman, E., Gustafsson, L., & Nylander, I. (2000). Basal levels and alcohol induced changes in nociceptin/orphanin FQ, dynorphin, and enkephalin levels in C57BL/6J mice. Brain Res Bull (in press). Przewlocka, B., Lason, W., & Przewlocki, R. (1992). Repeated ethanol administration decreases prodynorphin biosynthesis in the rat hippocampus. Neurosci Lett 134, 195 ± 198. Przewlocka, B., Turchan, J., Lason, W., & Przewlocki, R. (1997). Ethanol withdrawal enhances the prodynorphin system activity in the rat nucleus accumbens. Neurosci Lett 238, 13 ± 16. Reader, T. A., & Grondin, L. (1987). Distribution of catecholamines, serotonin, and their major metabolites in the rat cingulate, piriformentorhinal, somatosensory, and visual cortex: a biochemical survey using high-performance liquid chromatography. Neurochem Res 12, 1087 ± 1097. Ê ., Lindholm, S., Franck, J., & Georgieva, J. (1999). DownregulaRosin, A tion of kappa opioid receptor mRNA levels by chronic ethanol and repetitive cocaine in rat ventral tegmentum and nucleus accumbens. Neurosci Lett 275, 1 ± 4. Seizinger, B. R., 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, 361 ± 369. Simmons, M. L., & Chavkin, C. (1996). Endogenous opioid regulation of hippocampal function. Int Rev Neurobiol 39, 145 ± 196. Spanagel, R., Herz, A., & Shippenberg, T. S. (1990). The effects of opioid peptides on dopamine release in the nucleus accumbens: an in vivo microdialysis study. J Neurochem 55, 1734 ± 1740. Spanagel, R., Herz, A., & Shippenberg, T. S. (1992). Opposing tonically active endogenous opioid systems modulate the mesolimbic dopaminergic pathway. Proc Natl Acad Sci USA 89, 2046 ± 2050. Stromberg, M. F., Casale, M., Volpicelli, L., Volpicelli, J. R., & O'Brien, C. P. (1998). A comparison of the effects of the opioid antagonists naltrexone, naltrindole, and beta-funaltrexamine on ethanol consumption in the rat. Alcohol 15, 281 ± 289. Ungerstedt, U. (1971). Stereotaxic mapping of the monoamine pathways in the rat brain. Acta Physiol Scand, Suppl 367, 1 ± 48. Unterwald, E. M., Rubenfeld, J. M., & Kreek, M. J. (1994). Repeated cocaine administration upregulates kappa and mu, but not delta, opioid receptors. NeuroReport 5, 1613 ± 1616. Watson, S. J., Khachaturian, H., Akil, H., Coy, D. H., & Goldstein, A. (1982). Comparison of the distribution of dynorphin systems and enkephalin systems in brain. Science 218, 1134 ± 1136. You, Z. B., Herrera-Marschitz, M., Nylander, I., Goiny, M., O'Connor, W. T., Ungerstedt, U., Terenius, L. (1994). The striatonigral dynorphin pathway of the rat studied with in vivo microdialysis: II. Effects of dopamine D1 and D2 receptor agonists. Neuroscience 63, 427 ± 434.