Department of Psychology, Göteborg University, Box 500 SE - 405 30 Göteborg, Sweden and
1 Department of Mental Health and Alcohol Research, National Public Health Institute, POB 719, FIN-00101, Helsinki, Finland
Received 15 May 1999; in revised form 20 September 1999; accepted 5 October 1999
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ABSTRACT |
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INTRODUCTION |
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By selective breeding for high or low voluntary alcohol intake, two rat lines, alcohol-preferring (AA) and alcohol-avoiding (ANA), have been developed at the Biomedical Research Center of Alko Ltd, Helsinki, Finland (Eriksson, 1968, 1971
; Hilakivi et al., 1984
). A series of investigations has been devoted to search for physiological differences between the two lines potentially contributing to the differential intake of ethanol. These studies included investigations of neurochemical, metabolic, and behavioural parameters (Sinclair et al., 1989
; Kiianmaa et al., 1991
).
In view of the suggested relationship between corticosterone and alcohol intake, the present study examined if genetic lines of rats with high and low ethanol intake respond to manipulations of the hypothalamicpituitaryadrenal (HPA) axis. Thus, using the AA and ANA rats, we investigated the effects of adrenalectomy and subsequent treatment with corticosterone on voluntary alcohol intake.
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MATERIALS AND METHODS |
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At the time of the experiment, the animals were about 3 months of age and were placed individually in the stainless-steel wire cages. Initially, for a habituation period of 10 days, animals had 10% (v/v) ethanol solution as the sole drinking fluid (Richter tubes). After this habituation, they were given a choice between water and 10% ethanol solution for a further 2-week period. Fluid intake was measured daily and the bottles were cleaned and refilled with fresh beverages twice a week. On the basis of their ethanol intake (expressed as g/kg/day of absolute ethanol) and ethanol preference (i.e. proportion of ethanol solution intake relative to total fluid consumption as a percentage) during this period, the animals were assigned to matching groups and taken for surgery. Body weight and food intake were recorded once a week for all animals throughout the experiment.
Adrenalectomy and corticosterone treatment
The animals were subjected to adrenalectomy (AA, n = 10; ANA, n = 10) or sham surgery (controls; AA, n = 9, ANA, n = 9). Rats were anaesthetized with sodium metohexital (Brietal, Lilly; 100 mg/kg, i.p.) and the adrenal glands were removed through bilateral flank incisions. Control animals underwent the same treatment, except that the adrenal glands were left in situ. The rats regained consciousness in a warm (32°C) incubator, and were then replaced in their home cages, which were equipped with a NaCl pellet fastened to a wire hanging down from the cage top. Intake of fluids was recorded daily for 1 week following surgery.
Corticosterone (purchased from Sigma, Aldrich, Sweden) was administered to the adrenalectomized AA and ANA rats by dissolving corticosterone in equal amounts (25 mg/1000 ml fluid) in each of the drinking fluids presented to the animals (a dose of approximately 2.6 mg/kg/day; for method see Akana et al., 1985 and Fahlke, 1994). The remaining two groups (AA and ANA controls) served as controls for the corticosterone treatment. Intake of fluids was recorded daily for a further 2-week period.
Hormonal assay
To verify the removal of the adrenal glands, assessment of plasma levels of corticosterone was performed. Blood samples were collected from each rat at the end of the three different phases (baseline, adrenalectomy, and corticosterone treatment) of the experiment. Blood samples were taken 3 to 6 h after lights were switched on by tail nicking while the rats were briefly restrained in a wooden box. The blood samples (100200 µl) were collected in heparinized test tubes and the blood was separated by centrifugation for 5 min at 1500 rpm. The plasma was collected and stored at 70°C until the levels of corticosterone were estimated with radioimmunoassay, using kits supplied by ICN Biomedicals (Carson, CA, USA).
Statistics
Because the distribution of ethanol intake is too skewed to permit statistical testing with parametric tests, non-parametric methods were used in the statistical treatment of the data (StatView, Abacus software) and results are presented as median ± median absolute deviation (MAD). For each individual, the average intakes of fluids (ethanol or water) and food during the different periods (baseline, adrenalectomy, first and second weeks of corticosterone treatment) of the experiment were computed. Differences in average fluids or food consumption between two phases (baseline vs adrenalectomy) and adrenalectomy vs week 1 or week 2 of corticosterone replacement) were calculated for each rat. Groups were compared on the basis of these difference scores by the MannWhitney U-test. Within-group comparisons were analysed with the Wilcoxon matched-pairs signed-ranks test. Two-tailed levels of significance were used.
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RESULTS |
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As seen in Table 1, there were no statistically significant differences in total fluid intake or water intake between the adrenalectomized and control group during the 2 weeks of corticosterone treatment, probably due to the large distribution seen in the adrenalectomized animals. However, within-group comparisons showed that total fluid intake and water intake by the first and second week of corticosterone treatment was significantly higher than during the baseline period in the adrenalectomized group (total fluid intake: week 1, Z = 2.80, P = 0.01, and week 2, Z = 2.70, P = 0.01; water intake: week 1, Z = 2.80, P = 0.01 and week 2, Z = 2.39, P = 0.02). Total fluid intake and water intake did not change significantly during the corresponding period in the control group.
Adrenalectomy caused a decrease in body weight and food intake, compared to control rats (body weight: U = 21, P < 0.05; food intake: U = 21, P < 0.05; Table 1). Treatment of adrenalectomized animals with corticosterone caused an increase in both variables; thus there were no significant differences between adrenalectomized and control animals during the corticosterone treatment period.
ANA animals
In contrast to the AA rats, there were no alterations in ethanol drinking after removing the adrenal glands and following corticosterone replacement in the ANA rats. Thus, adrenalectomized and control rats did not differ significantly in ethanol intake and ethanol preference during any of the three different periods (baseline, adrenalectomy and subsequent treatment with corticosterone) of the experiment (Fig. 2).
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Plasma corticosterone
Table 3 shows plasma levels of corticosterone in blood samples obtained from all adrenalectomized and control animals at the end of the three periods: baseline (adrenally intact), adrenalectomy, and corticosterone replacement. All adrenalectomized rats had lower plasma levels of corticosterone, compared to their respective controls (AA, U = 0, P < 0.001; ANA, U = 0, P < 0.001).
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DISCUSSION |
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One possible explanation for the lack of effect of adrenalectomy on ethanol intake in the ANA group is that these animals cannot lower their ethanol intake much more. This is in agreement with previous studies showing that the effect of adrenalectomy as well as short-term treatment with corticosterone on ethanol intake is dependent on the pre-operative levels of ethanol drinking. Thus, it was found that ethanol intake remained entirely unaffected by adrenalectomy and subsequent corticosterone treatment in an unselected group of animals showing spontaneously low preference for ethanol with an average intake of 0.5 g ethanol/kg/day (Fahlke et al., 1994a). Furthermore, Fahlke et al. (1994b) found that there were no alterations in voluntary ethanol intake following treatment with the 11ß-hydroxylase inhibitor metyrapone, which blocks the formation of corticosterone (Ganong, 1987
), in low-preferring animals. Neither did metyrapone and corticosterone, administered in combination, affect ethanol intake in these animals (Fahlke et al., 1994b
). In contrast to the short-term treatment studies, long-term treatment of adrenalectomized animals with daily substitution of a constant corticosterone signal over several weeks enhances ethanol intake to levels above those normally observed in animals of an unselected strain of rats (Hansen et al., 1994
; Fahlke et al., 1995
). In addition, it was also found recently that adrenally intact low-preferring animals triple their daily ethanol intake by intracerebroventricular infusions of corticosterone (Fahlke et al., 1996
). Furthermore, a recent study employing the taste reactivity test (Berridge, 1996
) suggested that prolonged exposure to exogenous corticosterone might increase alcohol consumption by enhancing the sensory reward value of ethanol (Söderpalm and Hansen, 1999
). In the present study, the animals received short-term treatment with the steroid by dissolving corticosterone in equal amounts in each of the drinking fluids. All ANA animals, regardless of whether they were subjected to adrenalectomy or sham surgery, showed a lower total fluid intake than the AA animals (P < 0.001). It is possible that the amount of corticosterone ingested was not sufficient to facilitate ethanol drinking in ANA rats. Further studies are needed to explore whether long-term treatment with corticosterone, e.g. by subcutaneous implantation of corticosterone, or by exposing the brain alone to corticosterone, stimulates ethanol drinking, not only in ANA animals, but also in AA animals.
In line with earlier studies (Fahlke et al., 1994a, 1995
; Hansen et al., 1995
), the present study found that removal of the adrenal glands caused a decrease in body weight and food intake which was restored with corticosterone administration. In a related area of research, it has been found that corticosterone appears to act in the paraventricular nucleus of the hypothalamus to modulate food intake. For example, Tempel et al. (1992) have shown that corticosterone implants in the hypothalamic nucleus reproduce the effect of peripheral corticosterone injections. Thus, corticosterone can influence many behaviours (McEwen et al., 1986
) and the brain (McEwen, 1991
) by affecting the nerve cell surface to alter, for instance, ion permeability or neurotransmitter release (McEwen, 1991
; Moore and Orchinik, 1991
; Orchinik et al., 1994
). In a recent study by Fahlke and Hansen (1999), it was found that corticosterone partly acts within the ventral striatopallidal system to facilitate alcohol intake in rats. Thus, we found that animals bearing unilateral corticosterone implants in the ventral striatum showed a selective increase in voluntary alcohol consumption, whereas ethanol intake was not affected when corticosterone was implanted into the septum, hippocampus or thalamus. Furthermore, manipulation of corticosterone causes an array of changes in several components of the HPA system (McEwen et al., 1986
; Dallman et al., 1992
; Joels and De Kloet, 1992
) such as changed secretion of vasopressin, ß-endorphin, corticotropin-releasing factor (CRF) and adrenocorticotropic hormone (ACTH). Some of these components, e.g. CRF (George et al., 1990
; Ehlers et al., 1992
), ß-endorphin (Sandi et al., 1989
; Froehlich et al., 1990
) and ACTH (Krishnan et al., 1991
), may be involved in the modulation of ethanol intake. In fact, Gianoulakis et al. (1992) found that there are genetically determined differences in the pituitary and brain ß-endorphin system between ANA and AA rats, with the latter group having higher content of ß-endorphin and pro-opiomelanocortin (POMC; a protein which is subsequently cleaved into ACTH, ß-endorphin and other biologically active peptides; Herbert et al., 1980), which may be important in controlling the differences in voluntary alcohol consumption exhibited by these animals. Further studies are needed to specify the involvement of the HPA system in ethanol drinking in the genetic lines of AAA and ANA rats.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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