1 Department of Food and Biotechnology, Dongseo University, Busan, South Korea, 2 Department of Neuropsychiatry, School of Medicine, Paik Institute for Clinical Research, Inje University, Busan, South Korea, 3 Department of Psychiatry, College of Medicine, The Catholic University of Korea, Seoul, South Korea, 4 Department of BioSystems, Korea Advanced Institute of Science and Technology, Daejeon 305-701, South Korea and 5 Department of Psychiatry, Medical University of South Carolina, SC, USA
* Author to whom correspondence should be addressed at: Young-Hoon Kim, Paik Institute for Clinical Research, Inje University, 633-165, Gaegu-dong, Jin-gu, Busan, South Korea. Tel.: +82-51-890-6189; Fax: +82-51-894-6709; E-mail: npkyh{at}chol.com
(Received 3 February 2004; first review notified 20 March 2004; revised and accepted 16 November 2004)
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ABSTRACT |
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INTRODUCTION |
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Although the neurochemical mechanisms underlying the attenuation of ethanol intake by opioid receptor antagonists have yet to be clearly elucidated, some interaction with the dopaminergic neurotransmission system seems to be involved. The mesolimbic dopamine pathway that projects from the ventral tegmental area (VTA) to the nucleus accumbens (NA) has been implicated as a major site for the reinforcing actions of many addictive drugs including ethanol (Wise, 1978; Wise and Bozarth, 1981
; Wise and Rompre, 1989
; Koob et al., 1992
, 1998
; Di Chiara, 1995
; Everitt and Wolf, 2002
; Kelley and Berridge, 2002
; Weiss and Porrino, 2002
). Systemic ethanol administration increases the dopamine concentration in the NA (Yoshimoto et al., 1991
; Blomqvist et al., 1993
; Mocsary and Bradberry, 1996
; Yim et al., 1998
). Weiss et al. (1990
, 1993
) found that the oral administration of ethanol increases extracellular dopamine in the NA, which provides more direct evidence for the role of dopamine in the ethanol reward (Weiss et al., 1990
, 1993
). In addition, both the NA and VTA are rich in opioid peptides and receptors (Wamsley et al., 1980
; Lewis et al., 1983
; Dilts and Kalivas, 1989
, 1990
). The afferent projections to the NA from the VTA provide a potential substrate by which endogenous opioids may modulate the dopaminergic and rewarding effects of ethanol (Khachaturian et al., 1993
; De waele et al., 1995
). In fact, the non-selective opioid antagonist naltrexone has been reported to inhibit the rise in extracellular dopamine concentrations elicited by the reverse microdialysis of ethanol (Benjamin et al., 1993
; Gonzales and Weiss, 1998
).
Tyrosine hydroxylase (TH) is a major regulatory, rate-limiting enzyme in the synthesis of dopamine. Since the enzyme activity and protein levels are determined in dissected tissue homogenates of relatively small volume areas such as the catecholaminergic cell bodies and their projections, minor changes in the enzyme activity and the number of active enzyme molecules cannot be detected clearly. For this reason, an in situ hybridization technique has been applied to gain a semi-quantitative measure of enzyme mRNA in individual cells or cell clusters. Thus, assessing the levels of TH mRNA by in situ hybridization following chronic ethanol consumption with and without concomitant naltrexone administration might shed light on the mechanisms underlying the effect of naltrexone on the ethanol reinforcement system.
The aim of this study is to determine whether the effect of chronic ethanol to increase TH expression depends upon an opioid-mediated reward mechanism that is attenuated by naltrexone. We sought to examine changes in TH mRNA level by in situ hybridization in the VTA and substantia nigra (SN) and changes in the levels of dopamine and its metabolites by high performance liquid chromatography (HPLC), following regular ethanol consumption for 4 weeks with and without concomitant naltrexone administration.
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METHODS |
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In situ hybridization
Labelling of RNA probe with digoxigenin-11-UTP (DIG-11-UTP) was performed according to the manufacturer's recommendation. TH cDN A cloned into pBluescriptSK+ (Stratagene, La Jolla, CA) was given from Dr Kyong-Tai Kim (Pohang University of Science and Technology, Chae and Kim, 1995) After being linearized with PstI, the template cDNA was gel-purified and 1 µg of purified cDNA was used for in vitro transcription (corresponding to the antisense strand of 1.0 kb) with T7 polymerase and digoxigenin-labelled UTP according to the manufacturer's instructions (Dig RNA labeling kit; Boehringer Mannheim, Germany). The sense-stranded cRNA probe was synthesized from the same template cDNA but linearized with KpnI and driven by T3 polymerase. The transcription reactions were set up by mixing the following items at room temperature (RT): 8 µl H2O-DEPC, 5 µl linearized DNA (1 g), 2 µl 10x transcription buffer, 2 µl NTP labelling mixture (10 mM ATP, CTP, GTP, 6.5 mM UTP, 3.5 mM DIG-11-UTP), 1 µl RNase inhibitor (20 U/µl), 2 µl RNA polymerase (T7 or SP6, 20 U/µl). The reaction was performed at 37°C for 2 h and subsequently stopped by degrading the plasmid with 2 µl DNase (RNase free, 10 U/µl) for 15 min at 37°C. cRNA fragments were precipitated with 1/10 vol. of 4 M LiCl and 2.5 vol. of ethanol at 80°C for at least 1 h. The precipitated cRNA was recovered in 100 µl H2O-DEPC.
Rats were anaesthetized with pentobarbital (75 mg/kg, i.p.) and transcardially perfused with ice-cold PBS (pH 7.4) followed by ice-cold 4% paraformaldehyde in PBS. The brain was postfixed in the same fixative for 2 h and then cryoprotected in 30% sucrose-PBS overnight. They were then frozen by immersion in isopentane cooled at 80°C and stored at 80°C until use. Serial tissue sections were cut on a cryostat (1020 µm) and thaw-mounted on clean RNase free slides (DAKO BioTek Solution, USA). Before acetylation, sections were fixed with 4% paraformaldehyde in PBS for 10 min. The acetylation was carried out in 0.25% (v/v) acetic anhydride in 0.1 M triethanolamine-HCl (pH 8.0) at RT for 10 min. After ethanol dehydration, sections were hybridized in a buffer containing 50% deionized formamide, 300 mM NaCl, 1x Denhardt's solution, 50 mM Tris-Cl pH 8.0, 2 mM EDTA, 10% dextran sulphate, 0.25 mg/ml yeast tRNA and with 200 ng/ml of either antisense or sense cRNA probe. The hybridization was performed overnight at 58°C in a chamber humidified with 50% deionzed formamide/4x SSC. After RNase A (20 µg/ml, AMRESCO, USA) treatment at RT, non-specifically hybridized probe was washed away through several post-hybridization steps in a shaking water bath starting in 2x SSC and ending with a high-stringency washing in 0.1xSSC at 60°C. A final wash in 0.5x SSC was done at RT. DIG-labelled hybrids were immunologically detected using DIG Nucleic Acid Detection Kit (Boehringer Mannheim, Germany) as recommended by the manufacturer. Briefly, the slides were incubated with anti-digoxigenin Fab-fragments (1:500) and then stained with a freshly prepared solution of 4-nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolylphosphate (NBT-BCIP). After 24 h, the reaction was stopped in 10 mM Tris-Cl pH 8.0, 1 mM EDTA and coverslipped in Kaiser's glycerol gelatin. Slides were examined on microscope and the amount of TH mRNA was analysed by a Image-Pro Plus program (Media Cybernetics) using optical density.
HPLC analysis of dopamine and its metabolites
Each isolated brain region (cerebral cortex, striatum, hypothalamus, hippocampus, midbrain, and pons and medulla oblongata) was exposed to microwaves for 1 s to stop the enzymatic reaction of the dopaminergic enzyme system. The dopamine concentrations were determined by micropore reverse-phase HPLC. We used a 20 µl sample, and a mobile phase [2.5 mM 1-octane sulfate, 17:83 of methanol:0.1 M KH2PO4 (pH 3.2)] that included 10 µM EDTA was pumped through the C18 column (4.6 x 150 mm, Nakalai) at 0.9 ml/min. We used the pump of model 307 (Gilson, France) and electrochemical detector (Toa company, Japan) for this study. The total detection time was 25 min. The applied potential was 0.75 V and the sensitivity was maintained at 16 nA.
Statistical analyses
The significance of the differences in the expression levels of the TH mRNA and the levels of dopamine and its metabolites between the different treatment groups was determined by ANOVA and the Scheffe test was used for the comparisons among the means.
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RESULTS |
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DISCUSSION |
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Neural projections from the SN are directed towards the striatum, and pairs of these from the VTA area towards the NA. Since chronic ethanol consumption did not cause any significant change in the TH mRNA level in the SN, the levels of dopamine and its metabolites in the striatum were expected to be unchanged. On the other hand, because chronic ethanol consumption caused an increase in the TH mRNA level in the VTA, the levels of dopamine and its metabolites in the NA were expected to be increased. However, no significant difference in the levels of dopamine and its metabolites was found between the control and ethanol-treated group in most of the brain regions including the striatum.
Since the NA is a very small part attached to the striatum, we did not separate the NA from the striatum. In other words, the effects of ethanol on the dopamine metabolism in the NA could not be specifically examined. The lack of such separation is a limitation in the current data; hence, thus we will employ some methods to separate the NA from the striatum in future.
However, in the striatum only, the administration of naltrexone concomitant with ethanol consumption significantly increased the dopamine level whereas it did not cause any changes in the levels of its metabolites. The increase in the dopamine level may, thus, be due to increased dopamine turnover. Since the chronic ethanol consumption without naltrexone treatment did not induce any increase in the dopamine level in the striatum, we can interpret that chronic naltrexone might contribute to the increase in the dopamine level, regardless of chronic ethanol treatment. This suggests that the increased dopamine level induced by chronic naltrexone treatment in our striatal sample might arise from an increase in the dopamine synthesis in the VTANA system. As a matter of fact, we should admit that changes in TH mRNA and dopamine levels were not evaluated in a naltrexone-alone-treated group in this study. Thus, it might be the spurious result, without comparison with a naltrexone-alone-treated group, that the increase in the dopamine level in the striatum during chronic ethanol consumption is totally due to chronic naltrexone treatment. However, Middaugh et al. (2003) have recently found that extracellular dopamine levels of naltrexone- alone-treated rats are not significantly different from those of the control group. Thus, it seems that the increased dopamine level induced by chronic naltrexone treatment might arise from an increase in the dopamine synthesis in the VTANA system, which will be further studied in the near future.
Previous reports have suggested that the ethanol-induced increase in dialysate dopamine level in the NA is attenuated by naltrexone treatment (Benjamin et al., 1993; Gonzales and Weiss, 1998
). These reports only investigated the effects of a single pretreatment with naltrexone on acute ethanol treatment in the NA. In these reports, dopamine release was antagonized by acute naltrexone administration via the mu receptor in the striatum. However, in our report, the increased level of dopamine observed in the striatum following chronic ethanol administration with naltrexone treatment is considered to be a direct effect of chronic naltrexone on the dopaminergic system.
In conclusion, the current study investigated the action of naltrexone on decreasing alcohol consumption. The antagonistic effect of naltrexone against the change in the TH mRNA level after chronic ethanol consumption supports the previous hypothesis that naltrexone attenuates the rewarding properties of ethanol by interfering with the ethanol-induced stimulation of the mesolimbic dopaminergic pathway. Another preliminary finding that chronic naltrexone administration causes an increase in the striatal dopamine level requires further investigation with an appropriately designed trial.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Blomqvist, O., Engel, J. A., Nissbrandt, H. et al. (1993) The mesolimbic dopamine-activating properties of ethanol are antagonized by mecamylamine. European Journal of Pharmacology 249, 207213.[CrossRef][ISI][Medline]
Chae, H. D. and Kim, K. T. (1995) Cytosolic calcium is essential in the basal expression of tyrosine hydroxylase gene. Biochemical and Biophysical Research Communications 606, 659666.
De Waele, J. P., Kiianmaa, K. and 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. Journal of Pharmacology and Experimental Therapeutics 275, 518527.[Abstract]
Di Chiara, G. (1995) The role of dopamine in drug abuse viewed from the perspective of its role in motivation. Drug and Alcohol Dependence 38, 95137.[CrossRef][ISI][Medline]
Dilts, R. P. and Kalivas, P. W. (1989) Autoradiographic localization of mu-opioid and neurotension receptors within the mesolimbic dopamine system. Brain Research 488, 311327.[CrossRef][ISI][Medline]
Dilts, R. P. and Kalivas, P. W. (1990) Autoradiographic localization of delta opioid receptors within the mesocorticolimbic dopamine system using radioiodinated [2-D-penicillamine, 5-D-penicillamine]enkephalin (125I-DPDPE). Synapse 6, 121132.[CrossRef][ISI][Medline]
Everitt, B. J. and Wolf, M. E. (2002) Psychomotor stimulant addiction: a neural systems perspective. Journal of Neuroscience 22, 33133320.
Froehlich, J., Zweifel, M., Harts, J. et al. (1991) Importance of delta opioid receptors in maintaining high alcohol drinking. Psychopharmacology 103, 467472.[ISI][Medline]
Gianoculakis, C. and de Waele, J. P. (1994) Genetics of alcoholism: role of endogenous opioid system. Metabolic Brain Disease 9, 105131.[ISI][Medline]
Gianoulakis, C. (1996) Implications of endogenous opioids and dopamine in alcoholism: human and basic science studies. Alcohol and Alcoholism 31, 15.[ISI][Medline]
Gonzales, R. A. and Weiss, F. (1998) Suppression of ethanol-reinforced behavior by naltrexone is associated with attenuation of the ethanol-induced increase in dialysate dopamine levels in the nucleus accumbens. Journal of Neuroscience 18, 1066310671.
Herz, A. (1997) Endogenous opioid system and alcohol addiction. Psychopharmacology 129, 99111.[CrossRef][ISI][Medline]
Kelley, A. E. and Berridge, K. C. (2002) The neuroscience of natural rewards: relevance to addictive drugs. Journal of Neuroscience 22, 33063311.
Khachaturian, H., Schaefer, M. and Lewis, M. E. (1993) Anatomy and function of the endogenous opiod systems. In Opioids, Vol. 1, Akil, H. and Simon, E. J. eds, pp. 471497. Springer, New York.
Koob, G. F. (1992) Drugs of abuse: anatomy, pharmacology and function of reward pathway. Trends in Pharmacological Sciences 13, 177184.[CrossRef][ISI][Medline]
Koob, G. F., Roberts, A. J., Schulteis, G. et al. (1998) Neurocircuitry targets in ethanol reward and dependence. Alcoholism: Clinical and Experimental Research 22, 39.[ISI][Medline]
Kranzler, H. R., Tennen, H., Penta, C. et al. (1997) Targeted naltrexone treatment of early problem drinkers. Addictive Behaviors 22, 431436.[CrossRef][ISI][Medline]
Lewis, M. E., Khachaturian, H. and Watson, S. J. (1983) Comparative distribution of opiate receptors and three opioid peptide neuronal systems in rhesus monkey central nervous system. Life Sciences 33(Suppl. 1), 239242.[CrossRef][ISI][Medline]
Middaugh, L. D. and Bandy, A. L. (2000) Naltrexone effects on ethanol consumption and response to ethanol conditioned cues in C57BL/6 mice. Psychopharmacology (Berl) 151, 321327.[CrossRef][ISI][Medline]
Middaugh, L. D., Kelley, B. M., Cuison, J. R. et al. (1999) Naltrexone effects on ethanol reward and discrimination in C57BL/6 mice. Alcoholism: Clinical Experimental Research 23, 456464.[ISI][Medline]
Middaugh, L. D., Szumlinski, K. K., Van Patten, Y. et al. (2003) Chronic ethanol consumption by C57BL/6 mice promotes tolerance to its interoceptive cues and increases extracellular dopamine, an effect blocked by naltrexone. Alcoholism: Clinical and Experimental Research 27, 18921900.[CrossRef][ISI][Medline]
Mocsary, Z. and Bradberry, C. (1996) Effect of ethanol on extracellular dopamine in nucleus accumbens: comparison between Lewis and Fisher 344 rat strains. Brain Research 706, 194198.[CrossRef][ISI][Medline]
O'Brien, C. P., Volpicelli, L. A. and Volpicelli, J. R. (1996) Naltrexone in the treatment of alcoholism: a clinical review. Alcohol 3, 3539.[CrossRef]
O'Malley, S. S. (1995) Intergration of opioid antagonists and psychosocial therapy in the treatment of narcotic and alcohol dependence. Journal of Clinical Psychiatry 56(Suppl. 7), 3038.[Medline]
O'Malley, S. S., Jaffe, A. J., Chang, G. et al. (1992) Naltrexone and coping skills therapy for alcohol dependence. Archives of General Psychiatry 49, 881887.[Abstract]
Self, D. W. and Nestler, E. J. (1995) Molecular mechanism of drug reinforcement and addiction. Annual Review of Neuroscience 18, 463495.[CrossRef][ISI][Medline]
Swift, R. M. (1995) Effect of naltrexone on human alcohol consumption. Journal of Clinical Psychiatry 56(Suppl. 7), 2429.[ISI][Medline]
Swift, R. M., Whelihan, W., Kuznestsov, O. et al. (1994) Naltrexone induced alterations in human ethanol intoxication. The American Journal of Psychiatry 151, 14631467.[Abstract]
Volpicelli, J. R., Alterman, A. I., Hayashida, M. et al. (1992) Naltrexone in the treatment of alcohol dependence. Archives of General Psychiatry 49, 886880.
Volpicelli, J. R., Clay, K. L., Watson, N. T. et al. (1995) Naltrexon in the treatment of alcoholism: predicting response to naltrexone. Journal of Clinical Psychiatry 56(Suppl.), 3944.[ISI][Medline]
Wamsley, J. K., Young, S. D. and Kuhar, M. J. (1980) Anatomical localization of enkephalin immunoreactive sites in rat forebrain. Advances in Biochemical Psychopharmacology 22, 257270.[Medline]
Wise, R. A. (1978) Catecholamine theories of reward: a critical review. Brain Research 152, 215247.[CrossRef][ISI][Medline]
Wise, R. A. and Bozarth, M. A. (1981) Brain substrates for reinforcement and drug self-administration. Progress in Neuro-psychopharmacology 5, 467474.[CrossRef]
Wise, R. A. and Rompre, P. P. (1989) Brain dopamine and reward. Annual Review of Psychology 40, 191225.[CrossRef][ISI][Medline]
Weiss, F., Mitchiner, M., Bloom, F. E. et al. (1990) Free-choice responding for ethanol versus water in alcohol preferring (P) and unselected wistar rats is differentially modified by naloxone, bromocriptine, and methysergide. Psychopharmacology (Berl) 101, 178186.[ISI][Medline]
Weiss, F., Lorang, M. T., Bloom, F. E. et al. (1993) Oral alcohol self-administration stimulates dopamine release in the rat nucleus accumbens: genetic and motivational determinants. Journal of Pharmacology and Experimental Therapeutics 267, 250258.[Abstract]
Weiss, F. and Porrino, L. J. (2002) Behavioral neurobiology of alcohol abuse: recent advances and challenges. Journal of Neuroscience 22, 33323337.
Williams, K. L. and Woods, J. H. (1999) Conditioned effects produced by naltrexone doses that reduce ethanol-reinforced responding in rhesus monkeys. Alcoholism: Clinical Experimental Research 23, 708715.[CrossRef][ISI][Medline]
Yim, H. J., Schallert, T., Randall, P. K. et al. (1998) Comparison of local and systemic ethanol effects on extracellullar dopamine concentration in rat nucleus accumbes by microdialysis. Alcoholism: Clinical and Experimental Research 22, 367374.[ISI][Medline]
Yoshimoto, K., Mcbride, W. and Lumeng, L. (1991) Alcohol stimulates the release of dopamine and serotonin in the nucleus accumbens. Alcohol 9, 1722.[CrossRef][ISI]
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