Department of Clinical Physiology, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakaecho Itabashiku, Tokyo 173-0015, Japan, 1 Third Division, Department of Medicine, Kobe University School of Medicine, Kobe 650-0017, Japan and 2 Division of Gastroenterology, National Kyushu Cancer Center, Fukuoka 811-1395, Japan
* Author to whom correspondence should be addressed: Kyoko Miyasaka, Department of Clinical Physiology, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakaecho Itabashiku, Tokyo 173-0015, Japan. Tel.: +81 3964 3241 (ext. 3088); Fax: +81 3579 4776; E-mail: miyasaka{at}tmig.or.jp
(Received 13 July 2004; first review notified 29 September 2004; accepted in revised form 17 February 2005; Advance Access publication 14 March 2005)
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ABSTRACT |
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INTRODUCTION |
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We had previously investigated the association between the CCK-AR gene polymorphism (Funakoshi et al., 2000) and alcohol dependence in humans, and found that the CCK-AR gene 81A/G polymorphism was associated with alcohol dependence in a Japanese population (Miyasaka et al., 2004
). Our recent investigation using the STC-1 murine neuroendocrine cell line showed that the 81A to G change decreased luciferase activities only slightly (not significantly) (Takata et al., 2002
). However, limitations in the experimental conditions suggest that those findings should be interpreted as inconclusive, because no human cell lines expressing CCK-AR are available.
Since we speculated that the 81A/G polymorphism might decrease the CCK-AR gene expression, in the present study, the role of CCK-AR in ethanol ingestion was examined using CCK-AR gene deficient (/) mice (Suzuki et al., 2001; Takiguchi et al., 2002
). The two-bottle choice protocol was conducted and the righting reflex was examined. Two types of CCK receptors (CCK-AR and -BR) have been cloned so far (Wank, 1995
). Although CCK-BR is widely distributed throughout the central nervous system, CCK-AR is found in specific regions, such as the amygdala, nucleus tractus solitarius, posterior nucleus accumbens, ventral tegmental area, substantia nigra and raphe nucleus. Furthermore, the amino acid sequence of rat CCK-BR is 48% identical to that of rat CCK-AR (Wank, 1995
), and the expression patterns of these receptors overlap in the brain (Hill et al., 1987
; Hughes et al., 1990
; Honda et al., 1993
). Although several pharmacological studies using CCK receptor antagonists have been reported (Hughes et al., 1990
; Wank, 1995
), cross-reactivity, in which substances that should react to CCK-AR also react to CCK-BR and vice versa, could not be excluded. Therefore, to determine the physiological role of CCK-AR more conclusively, we used CCK-AR(/) and BR(/) mice (Nagata et al., 1996
, Miyasaka et al., 2002
) as well as wild-type mice. These three types of mice are viable and are fertile into adulthood. Furthermore, the protein levels of dopamine 2 receptor (D2R) in the nucleus accumbens of these mice were determined by western blotting, because signaling through D2R has been known to govern physiological functions related to locomotion and drug abuse (Maldonado et al., 1997
; Usiello et al., 2000
), and because D2R was expressed highly in the striatal complex, where CCK-AR and BR were expressed.
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MATERIALS AND METHODS |
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The progenitor strain for CCK-AR(/) and BR(/) was C57BL/6J (Nagata et al., 1996; Takiguchi et al., 2002
). More than seven generations of backcrossing have been performed. Mice were fed commercial chow (CRF-1: Charles River Japan Inc., Yokohama). We used age-matched (810 months) male mice in the test. The animals were maintained in individual cages in an air-conditioned room at 21°C, with 55 ± 5% humidity, and with a 12-h light/dark photocycle (8 am8 pm) at the Tokyo Metropolitan Institute of Gerontology. The cage size was 18 x 30 x 11 cm.
Two-bottle choice protocol
The two-bottle choice protocol consisted of four cycles of 7 days each, with the animals having access to 3, 6, and 10% ethanol successively, always versus tap water. Subsequently, some of the wild-type mice were given access to 20% ethanol, and others were used for the examination of the righting reflex as described below. Fluid consumption and body weight were evaluated by measuring fluid volumes at 3 pm every Monday. The places of the two bottles were reversed relative to each other on each Wednesday and Friday. The mean body weight during each 7-day period was estimated, and fluid consumption/kg mean body wt was estimated.
For comparison, additional mice were offered saccharin (0.033%) and quinine (0.03 mM) each versus tap water.
Righting reflex
Mice (without overnight fasting) were given an i.p. injection of 4.5 g/kg ethanol solution (30%) between 10 and 11 am. A mouse was judged to have recovered the righting reflex if it righted itself successfully twice within 30 s after being turned upside down two times at each of the several scheduled times after the administration (Haseba et al., 1993). Scoring was as follows: no recovery = 0, recovery the first time but not the second = 1, successful both times but without walking = 2, and awake and walking = 3.
Determination of DR2 by western blotting
Three additional animals for each genotype were decapitated between 10 and 11 am without any treatment, and the whole brain was immediately removed. The nucleus accumbens was quickly dissected and then stored at 80°C for later experiments. Each tissue sample was separately homogenized in lysis buffer. Synaptic plasma membrane was isolated according to the method of Jones and Matus (1974).
The respective nucleus accumbens homogenates (100 Mg of protein) were separated on a sodium dodecyl sulfate7.5% polyacrylamide gel. The proteins were electrophoretically transferred on to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad Laboratories, Hercules, CA) and then blocked with 5% skim milk for 1 h at room temperature.
The membrane was incubated with the goat anti-human D2R(L/S) antibody (1:250; Alpha Diagnostics International, San Antonio, TX) overnight at 4°C. After three washes, the membrane was incubated with 1:15 000 horseradish peroxidase-linked secondary anti-goat IgG (H+L) antibody for 1 h at room temperature. After three further washes, immunoreactive bands were detected using the Enhanced Chemiluminescence assay system (ECL Plus, Amersham Biosciences, Buckinghamshire, UK) (Rogers et al., 1991).
Autoradiograms then underwent a semiquantitative densitometric analysis. The data were expressed as means ± SE. The optical density (OD) of the immunoreactive bands was calculated by using an NIH Image software package.
Statistical analysis
Results were analyzed by multiple analysis of variance (MANOVA) with repeated measures or by one-way ANOVA, followed in either case by Fisher's protected least significant difference (PLSD). P < 0.05 was considered to be significant.
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RESULTS |
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DISCUSSION |
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Experiments with rodents have provided considerable evidence that dopamine is involved in the self-administration of ethanol (Lanca et al., 1994; Phillips et al., 1998
). However, there has been a controversy concerning the role of dopamine in alcohol preference in rodents. In a previous report (George et al., 1995
), a low availability of synaptic dopamine was postulated to increase ethanol preference. In contrast, Phillips et al. (1998)
reported that alcohol preference and sensitivity are reduced in D2R(/) mice. In the present study, we observed that CCK-BR(/) mice showed a significant decrease in D2R protein expression in the nucleus accumbens, and they ingested less ethanol; in particular, their consumption of 10% ethanol was significantly lower than that of CCK-AR(/) mice. Thus, our present observation was compatible with that of Phillips et al. (1998)
, in which lower D2R function decreased the voluntary ingestion of ethanol.
On the other hand, CCK-AR(/) mice showed an increase in D2R expression and showed no aversion to the high concentrations of ethanol, although the preference ratio did not increase. There have been controversial reports on the phenotype of D2R(/) mice (Baik et al., 1995; Kelly et al., 1997
). Baik et al. (1995)
reported that D2R(/) mice showed reduced food and water intake as well as retarded growth, whereas Kelly et al. (1997)
did not observe any inhibition in the gain of body weight. In recent reports (Usiello et al., 2000
; Wang et al., 2000
), D2R has been shown to have two isoforms, a long form (D2R-L) and a short form (D2R-S). D2R-L was strongly expressed in the striatum and nucleus accumbens (Khan et al., 1998
). D2R-L (/) mice displayed reduced levels of locomotion and rearing behavior (Wang et al., 2000
). We have previously observed (Miyasaka et al., 2002
) that locomotor activity was increased in CCK-AR(/) mice and was decreased in CCK-BR(/) mice in a plus maze. Since an animal model with overexpressed D2R has not been available, it is unknown whether the increase in D2R level has an essential role in the increase of locomotor activity. Taken together, these findings suggest that although the mechanism underlying CCK-Rs modulation of D2R expression is unknown, the differences in ethanol ingestion between CCK-AR(/) and BR(/) mice might be related to the D2R expression level.
In summary, voluntary ingestion of ethanol differed between CCK-AR(/) and CCK-BR(/) mice. This difference might be attributable to the different levels of D2R expression in the nucleus accumbens between these genotypes.
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ACKNOWLEDGEMENTS |
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