HIGH ALCOHOL PREFERRING (HAP) AND LOW ALCOHOL PREFERRING (LAP) RATS SHOW ALTERED PROOPIOMELANOCORTIN (POMC) MESSENGER RNA EXPRESSION IN THE ARCUATE NUCLEUS

Hiroshi Kinoshita1,*, Michael S. Harbuz2, Minori Nishiguchi1, Harumi Ouchi1, Takako Minami1, Takao Utsumi3, Hiroyuki Motomura4 and Shigeru Hishida1

1 Department of Legal Medicine and 3 Department of Ophthalmology, Hyogo College of Medicine, 1-1, Mukogawa-cho, Nishinomiya, Hyogo, 663-8501, Japan, 2 LINE, Dorothy Hodgkin Building, Whitson Street, Bristol, BS1 3NY, UK and 4 Forensic Science Laboratory, Hyogo Prefectural Police Headquarters, 4-1, Shimoyamate-dori 5-chome, Chuo-ku, Kobe, 650-8510, Japan

* Author to whom correspondence should be addressed at: Department of Legal Medicine, Hyogo College of Medicine, 1-1, Mukogawa-cho, Nishinomiya, Hyogo, 663-8501, Japan. Tel.: +81 798 45 6578; Fax: +81 798 49 3279; E-mail: kinochin{at}hyo-med.ac.jp

(Received 23 October 2003; first review notified 28 February 2004; in revised form accepted 10 May 2004; accepted 12 May 2004)


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Aims and Methods: We have investigated proopiomelanocortin (POMC) and neuropeptide Y (NPY) gene expression in the arcuate nucleus of the hypothalamus comparing high alcohol preference (HAP) rats and low alcohol preference (LAP) rats under basal conditions using in situ hybridization histochemistry.

Results: A significantly higher expression of POMC mRNA was observed in HAP rats compared with LAP rats. In contrast, no difference in NPY mRNA expression was observed between the two rat lines.

Conclusions: These data suggest that an endogenous opioid system may contribute to alcohol preference in HAP/LAP rat lines.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Alcoholism is a serious problem in many developed countries, but the mechanisms underlying this condition are not well understood. Alcohol preference is an important factor in the development of alcoholism. However, its regulatory mechanism remains to be established. Different alcohol preference rodent lines, developed by selective breeding, are widely used as animal models in the study of alcoholism (Li et al., 1994Go; McBride and Li, 1998Go). Recently, high alcohol preferring rat lines (HAP) and low alcohol preferring rat lines (LAP) were developed from a Wistar rat colony by Hishida (Hishida, 1996Go). Several different characteristics have been observed between these two lines, such as altered aldehyde dehydrogenase (ALDH) activity, cytosolic ALDH (ALDH1) polymorphism, dopamine and serotonin release in the nucleus accumbens following methamphetamine administration and metabolic capacity of acetaldehyde (Hishida, 1996Go; Negoro et al., 1997Go; Yamauchi et al., 2000Go; Nishiguchi et al., 2002Go). However, little is known concerning the factors regulating ethanol preference between these two rat lines.

A number of neuropeptides have been implicated in changes in sensitivity/preference to alcohol (McBride and Li, 1998Go). A role for the endogenous opioid system, including ß-endorphin, has been suggested to be involved in controlling ethanol consumption (Maderia and Paula-Barbosa, 1999Go). ß-endorphin, one of the endogenous opioid peptides, originates from the post-translational processing of proopiomelanocortin (POMC). POMC is the precursor to ß-endorphin, {alpha}-melanocyte-stimulating hormone and other peptides which have a variety of biological functions in the brain (Raffin-Sanson et al., 2003Go). A difference in hypothalamic POMC mRNA expression has been noted between Alko Alcohol (AA) and Alko non-Alcohol (ANA) rats, rat lines selected on the basis of their voluntary ethanol consumption (Gianoulakis et al., 1992Go), suggesting a role for POMC-derived peptides in alcohol preference. Neuropeptide Y (NPY) is a 36 amino acid peptide, with a widespread distribution in the CNS (Tatemoto et al., 1982Go). Recent studies have shown a role for NPY in the regulation of alcohol consumption. Ethanol consumption and sensitivity are inversely related to brain NPY levels (Thele et al., 1998Go). The difference in mRNA expression and tissue content of NPY may contribute to alcohol preference (Hwang et al., 1999Go; Caberlotto et al., 2001Go). The arcuate nucleus (ARC) in the hypothalamus is one of the major sites of both POMC and NPY biosynthesis in the brain (Chronwall et al., 1985Go; Maderia and Paula-Barbosa, 1999Go). POMC and NPY gene expression and alcohol preference in HAP/LAP rat lines have not previously been assessed.

The present study was designed to investigate the expression of POMC and NPY mRNA in ARC of both HAP/LAP rat lines using quantitative in situ hybridization histochemistry. POMC mRNA expression in brain is influenced by circulating glucocorticoid (Beaulieu et al., 1988Go), therefore, we also measured plasma corticosterone concentrations.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
HAP and LAP rat lines were developed in our laboratory by selective inbreeding from a Wistar colony of rats (Hishida, 1996Go; Nishiguchi et al., 2002Go). Alcohol-naive male rats used in this experiment were obtained from the 28th generation of the HAP rat line and the 32nd generation of the LAP rat line, and these were compared with commercially available Wistar rats (Charles River Japan Inc, Shiga, Japan) with a weight range of 290–340 g. All animals were housed in a 12 h light/12 h darkness cycle in a temperature and humidity controlled environment with free access to food and water. Animals were sacrificed by decapitation and trunk blood was collected. Brains were rapidly removed, frozen on dry ice and stored at –80°C until sectioning. Twelve-micrometre thick sections containing the ARC were cut and thaw-mounted on APS-coated slides, and stored at –80°C before hybridization. Trunk blood was collected for the measurement of plasma corticosterone. This study was approved by the Animal Investigation Committee, Hyogo College of Medicine.

In situ hybridization histochemistry (ISHH)
ISHH was carried out as described previously (Harbuz et al., 1994Go; Kinoshita et al., 2001Go). In brief, the brain sections were fixed in 4% formaldehyde in phosphate-buffered saline (PBS) for 5 min, rinsed twice in PBS, and treated in 0.25% acetic anhydride in 0.1 M triethanolamine/0.9% NaCl for 10 min. The sections were passed through 70 (1 min), 80 (1 min), 95 (2 min) and 100% (1 min) ethanol, 100% chloroform (5 min), and 100 (1 min) and 95% (1 min) ethanol for dehydration, delipidation and partial rehydration.

The probes used for POMC and NPY were 48-mer oligonucleotides complementary to part of the exonic mRNA sequence, respectively (Perkin-Elmer, Warrington, UK). The probes were 3'-end labeled with [35S]deoxy ATP (1000 µCi/mmol, Amersham Biosciences, Piscataway, NJ) by terminal deoxyribonucleotidyl transferase (Roche Diagnostics GmbH, Mannheim, Germany) and column purified by Nuc trap nucleotide purification kit (Stratagene, La Jolla, CA). The specific activities of the probes for POMC and NPY were 2.14 x 1018 and 4.12 x 1018 dpm/mol, respectively. Approximately 100 000 c.p.m. probe (per 45 µl) were applied to each slide. Hybridization was performed overnight at 37°C. All the sections were processed at the same time. The sections were washed in four 15 min rinses of 1 x SSC (1 x SSC = 0.15 M NaCl/0.015 M sodium citrate, pH 7.0) at 55°C, followed by two 30 min washes in 1 x SSC at room temperature to remove non-specific binding before two short water rinses and air drying. The sections, together with 14C-labelled autoradiographic microscale (RPA504, Amersham Biosciences, Piscataway, NJ) were exposed to Hyperfilm MP autoradiography film (Amersham Pharmacia Biotech, Buckinghamshire, UK). The autoradiographic image was measured as previously described (Harbuz et al., 1994Go) using a computer-assisted image analysis system (Image 1.6.1, developed by W. Rasband, NIH, Bethesda, MD) run on Apple Macintosh. The results are presented as the percentage change from Wistar group.

Total plasma corticosterone was measured by enzyme immunoassay kit (Assay Designs Inc., Ann Arbor, MI), and the protocol was in accordance with the manufacturer's specifications. The optical density was measured by Fluostar galaxy (BMG labtechnologies, Offenburg, Germany). The data are expressed as mean ± SEM. All groups within each data set were compared by one-way ANOVA followed by Fisher PLSD test for multiple comparisons. A value of P < 0.05 was considered significant.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Figure 1(a) shows representative images of POMC mRNA expression in ARC in each group. Figure 1(b) shows the results of quantitative image analysis of this data. POMC mRNA expression in HAP rats was significantly higher than that of LAP rats (one-way ANOVA: F2,13 = 4.835, P = 0.0269). The representative images of NPY mRNA expression in each group are shown in Fig. 2(a). There was no significant difference in NPY mRNA expression among the three groups (Fig. 2b). Plasma corticosterone concentrations were 88.1 ± 15.3 ng/ml in HAP rat, 83.8 ± 10.5 ng/ml in LAP rat and 66.2 ± 23.7 ng/ml in Wistar rat, respectively. No significant difference was observed between the three groups.



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Fig. 1. Representative images of POMC mRNA in the arcuate nucleus. (A) High alcohol preference (HAP), (B) low alcohol preference (LAP) and (C) Wistar (Wistar) rats. 3V: Third ventricle. (b) POMC mRNA levels in the hypothalamic arcuate nucleus of each rat line. Values are presented as mean ± SEM for n = 4–6 rats/group. *P < 0.05 compared with LAP (one-way ANOVA followed by Fisher PLSD test).

 


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Fig. 2. (a) Representitive images of NPY mRNA in the arcuate nucleus. (A) High alcohol preference (HAP), (B) low alcohol preference (LAP) and (C) Wistar (Wistar) rats. 3V: Third ventricle. (b) NPY mRNA levels in the hypothalamic arcuate nucleus of each rat line. Values are presented as mean ± SEM for n = 4–6 rats/group.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The present study demonstrated that HAP rats showed significantly higher expressions of POMC mRNA in ARC in comparison to LAP rats, under basal conditions. A difference in POMC mRNA expression has also been reported between AA/ANA rat lines, using northern blot hybridization (Gianoulakis et al., 1992Go) and ISHH (Marinelli et al., 2001Go). These results confirm the strong expression of basal POMC mRNA in ARC observed in HAP rats, the independent high alcohol preference rat lines selected by the method of Iso and Sakaki (Iso and Sakaki, 1982Go) and developed by selective breeding (Hishida, 1996Go) used in the present study. A similar difference in POMC mRNA content in the hypothalamus was also reported comparing the C57BL/6 and DBA/2 mice, which show different voluntary ethanol consumption (De Waele et al., 1992Go). This evidence supports the involvement of POMC in alcohol preference.

Addictive drugs such as cocaine, amphetamine and ethanol activate dopaminergic neurotransmission, and the development of drug dependence is associated with alterations in dopaminergic activity in the limbic system (Tiihonen et al., 1995Go). An interaction between the opioid system and the dopaminergic system has been proposed, such as ß-endorphinergic neuron, projecting from the ARC (De Waele et al., 1995Go; Herz, 1997Go). In a previous study, no difference in basal extracellular levels of dopamine was observed in the nucleus accumbens between HAP and LAP rat (Yamauchi et al., 2000Go). This suggests that different POMC mRNA expression in ARC under basal conditions may be involved in altered alcohol preference in this HAP/LAP rat line, as has also been reported in the AA/ANA rat lines. High POMC mRNA expression may be associated with increased release of POMC products, such as ß-endorphin, {alpha}-melanocyte-stimulating hormone and other peptides. The details of the functional role of these peptides in the regulation of alcohol preference are not known. It also remains to be determined whether this high POMC mRNA expression acts directly to influence the high sensitivity of dopaminergic neurons or not. Further studies are required to clarify these mechanisms. POMC mRNA expression in the ARC is regulated by circulating glucocorticoids (Beaulieu et al., 1988Go). In our study, no difference in plasma corticosterone concentrations were observed among the three rat lines, suggesting that the differences in POMC mRNA observed were not due to differences in basal corticosterone.

In contrast to POMC mRNA, no difference in NPY mRNA expression in ARC between these three related rat lines was observed in this study. Similar results suggesting a lack of involvement of hypothalamic NPY mRNA expression has been previously reported comparing AA and ANA rats (Caberlotto et al., 2001Go). It has been suggested that the NPY in the hypothalamic nuclei may be more involved in feeding behavior (Brady et al., 1990Go; Hwang et al., 1999Go), rather than in ethanol preference. However, since NPY is related to various physiological effects, not only regulation of food intake but also regulation of anxiety (Heilig et al., 1992Go), and ethanol has an anxiolytic effect (Eckardt et al., 1998Go), these effects may partially contribute to alcohol drinking. Decreased NPY concentrations are implicated in anxiety and alcohol drinking behaviours (Pandey, 2003Go).

The present results indicate that the newly developed rat lines, HAP and LAP showed altered POMC mRNA expression, but no difference in NPY mRNA expression associated with the different alcohol preference in these lines. These results suggest that differences in endogenous opioid systems may be involved in alcohol preference. The role of endogenous opioid systems could represent an interesting target for the study of ethanol preference. The detailed mechanisms underlying the differences between these lines are not clear and additional studies will be required to clarify the involvement of ß-endorphin or other POMC products on the mechanisms of ethanol preference.


    ACKNOWLEDGEMENTS
 
We are grateful to Mr Kunihisa Hamada (Common Research Laboratory, Hyogo College of Medicine) for his technical advice and support. This work was partially supported by a grant-in-aid for Encouragement of Young Scientists, No 15689012 from the Japanese Society for the Promotion of Science.


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