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Adrenalectomy prevents the development of alcohol preference in male rats
Alcohol, Vol. 13, No. 3,233-238, 1996 Published1996by ElsevierScienceInc. Printedin the USA.All rightsreserved 0741-8329/96$0.00 + .00 ELSEVIER 0741-8329(95)02043-8
Adrenalectomy Prevents the Development of Alcohol Preference in Male Rats F. L A M B L I N A N D PH. DE W I T T E l
Laboratory o f Psychobiology, 1, Place Croix du Sud, B-1348 Louvain-la-Neuve, Belgium Received 13 A p r i l 1995; Accepted 28 July 1995 LAMBLIN, F. AND PH. DE WlTTE. Adrenalectomyprevents the development of alcoholpreference in male rats. ALCOHOL 13(3) 233-238, 1996.-By forcing adrenalectomized (ADX) and sham-operated (SHAM) rats to chronically drink ethanol by mean of presentation of only one drinking bottle containing 10°70ethanol, no differences occurred between both groups. ADX and SHAM rats were then exposed to chronic alcoholization using an inhalation procedure. After sejourning 3 weeks into the alcoholization chamber, rats were submitted to a free-choice paradigm [water vs. a 10¢/0(v/v) ethanol solution]. The sham-operated rats presented an alcohol-induced behavioral preference towards alcohol whereas adrenalectomized animals never exhibited a preference to ethanol. In the adrenalectomized rats treatment with hydrocortisone (30/zg/ml) given orally during the pulmonary alcoholization failed to modify this preference whereas treatment with corticosterone (25 #g/ml) given orally abolished the difference with SHAM animals. These data showed that adrenalectomy prevented the development of ethanol preference and the clear involvement of the hypothalamo-pituitary-axis in alcohol preference. Adrenalectomy
Pulmonary alcoholization
Free choice
Corticosterone
submitted to a free-choice paradigm, which was only transient (3,4). Indeed, these studies showed a gradual increase in alcohol consumption of the ADX rats after 2 and 3 weeks of the free-choice paradigm. Corticosterone supplementation to these adrenalectomized rats (ADX) immediately restored the alcohol intake to the preoperative level; in contrast, aldosterone treatment did not modify ethanol consumption: The decreased ethanol intake by ADX rats is not due to enhancement of aversion to the alcoholized solution (3). Second, adrenalectomy of male rats may possibly be a protective factor for ethanol withdrawal seizures. In previous studies, we have clearly shown that there was no increase in tremor activity in adrenalectomized rats induced by a harmine tremorogenic agent after ethanol withdrawal (10). In adrenalectomized mice, Sze et al. also show a decrease of the sensitivity to audiogenic seizures upon ethanol withdrawal (16,17). The original susceptibility to ethanol withdrawal seizures can be restored by corticosterone treatment. In the present studies we have evaluated the ethanol consumption in a free-choice paradigm (10~/0 ethanol vs. water) in various groups of rats: in naive, in orally or chronically pulmonary alcoholized adrenalectomized, and shamoperated male Wistar rats. The effect of supplementation with either hydrocortisone or corticosterone was also investigated.
AN increasing number of studies have implicated the hypothalamo-pituitary-adrenal axis (HPA) interaction in many of the perturbations associated with either acute or chronic alcohol intoxication. During acute exposure to alcohol there is a potent activation of the HPA axis marked by an increased circulating plasma concentration of glucocorticoid level in rodents (2,6,7,14,15,18) and in man (5,19). However, the effects of chronic alcoholization on the circulating concentrations of glucocorticoids have not been clearly established. Some studies have reported a prolonged elevation of plasma glucocorticoid levels during the chronic alcoholization period (2,18,20) whereas other studies have shown a tolerance development by the corticosterone response to ethanol (8,9,15). Depression of the HPA axis activity has also been observed after chronic alcoholization in aged rats that showed enhanced corticosterone and ACTH secretions (13). During ethanol withdrawal, the peak values for glucocorticoids concentrations was at a maximum on the first day of the alcohol deprivation, which coincided with the behavioural and physiological symptoms of the withdrawal syndrome (1,18). These results suggest that there are modifications of the HPA function by alcohol, and, reciprocally, that the HPA axis secretions modify several of the behaviorial changes associated with alcohol. First, either surgical or chemical suppression of adrenal function decreased the ethanol intake in rats
To whom requests for reprints should be addressed. 233
234
LAMBLIN AND DE WITTE METHOD
Animals Male Wistar rats weighing 150-180 g at the start of the experiments, were divided in two groups: an A D X group and a SHAM group. After anaesthesia with chloral hydrate (400 mg/ 100 g b.wt.) in the A D X group, the two adrenal glands were removed by means of two dorsal incisions. Sham surgery consisted of bilateral incisions; the adrenal glands were localized but not removed. Following surgery, all animals were supplemented with 1% saline solution. Success of ADX surgery was verified at the end of the experiment by the assay of plama corticosterone levels by radioimmunoassay. ADX rats presenting levels of corticosterone higher than 3 ng/ml were disregarded; the levels in the sham-operated rats were higher than 70 ng/ml. Experimental Treatment ADX and SHAM rats were submitted to the same experimental procedures and were compared in the following experiments: 1. Naive ADX and naive SHAM rats (naive means without any prior contact with alcohol). 2. Orally chronically alcohol loaded rats. Both groups of rats were chronically loaded with alcohol by the presentation of one drinking bottle containing 10% alcohol (v/v) for 3 weeks. The alcohol consumption was recorded each day. 3. Chronically alcohol loaded by pulmonary alcoholization. 4. Chronically alcohol loaded by pulmonary alcoholization to the ADX rats treated with supplementation of hydrocortisone (30 #g/ml, Solu-cortef, Upjohn) The hydrocortisone was given per os in the drinking bottle throughout the whole alcoholization procedure. The hydrocortisone solution was prepared fresh every 2 days, the drinking bottle containing this solution was sheltered from light to prevent degradation of the hydrocortisone. The rat's intake was monitored throughout the alcoholization period to ensure a constant drug administration. The treatment was stopped when the rats were removed from the alcoholization chamber. 5. SHAM and SHAM treated with hydrocortisone (30 #g/ml) given per os were also chronically alcohol loaded by chronic pulmonary alcoholization as described for the ADX group. 6. ADX-treated and untreated alcoholized SHAM rats were chronically alcohol loaded by pulmonary alcoholization and supplemented with corticosterone (25 /xg/ml, Sigma) throughout the procedure. The corticosterone was dissolved in water and the rat's intake was monitored throughout the experiment to ensure a constant drug administration. The treatment was stopped when the rats were removed from the alcoholization chamber. Chronic Pulmonary Alcoholization The rats were individually housed and maintained in an isolated plastic chamber (160 × 60 x 60 cm). A mixture of alcohol and air was pulsed into the chamber via a mixing system allowing the quantity of alcohol to be increased (0 to 25 mg/l) every 2 days during the experimental procedure (11). Adult rats were kept for 20 days in total in the alcoholization chamber.
Blood Alcohol Level The blood alcohol level was regularly assayed during the alcoholization exposure. Blood samples (0.1 ml) were collected from the caudal portion of the tail and alcohol was assayed by the reduction of NADH to NAD in the alcohol dehydrogenase-based method (Boerhinger-Mannhein Kit). Free-Choice Paradigm A free-choice beverage procedure consisting of water vs. ethanol solution, 10% (v/v), was presented to the two groups of rats in each experimental condition. The free choice proposed to the rats following the chronic pulmonary alcoholization required two preliminary steps: a full beverage deprivation (the last 18 h of the alcohol intoxication period and the first 6 h of the withdrawal period), and the presentation of a 10% (v/v) ethanol solution as the only drinking fluid during the following 18 h. During the free-choice period (3 weeks at least), fluid consumption was recorded daily. The bottle's positions were randomly distributed to avoid position preference. Statistical Methods All data are presented as means + SD. According to the experiments the analysis of variance (ANOVA) or the ANOVA for repeated measures were applied. RESULTS
Free-Choice Paradigm Figure 1 shows the free-choice paradigm of naive ADX (n = 13) and SHAM rats (n = 17). The alcohol consumption of SHAM rats represented 50% of their total fluid intake whereas the alcohol drunk by the ADX rats reached only 20% of their fluid consumption. Despite the absence of difference in the total fluid intake between SHAM (mean daily intake 42 11.68 ml/kg/day) and ADX (mean daily intake 57 + 15.21 ml/kg/day), ethanol consumed by the SHAM was significantly greater than ethanol consumed by the ADX, F(1, 28) = 13.5,p < 0.001. Rats that were chronically administered alcohol orally, by means of the presentation of only one bottle containing ethanol (10% v/v) for a 3-week period, had a mean daily intake of 2 g/kg ethanol (i.e., 25 ml of ethanol 10% per day) (data not shown). In comparison to the free-choice experiment, the total fluid intake was significantly lower in both SHAM and ADX after presenting only one bottle containing ethanol. For this type of alcoholization, the difference in the alcohol intake between ADX rats (n = 6) and SHAM rats (n = 8) was not significant, F ( I , 12) = 0.46,p > 0.05. Chronic pulmonary alcoholization induced a higher mortality in the ADX group than the sham-operated rats. Commencing the experiment with 48 ADX rats (b.wt.: 220 9 g) and 22 SHAM rats (b.wt.: 280 4- 8 g), at the end of the alcoholisation period there were 25 ADX (b.wt.: 180 + 6 g) and 20 SHAM (b.wt.: 290 ± 12 g). Even though 50% of the ADX rats did not survive this period of chronic alcoholisation, there was no apparent explanation, because the cortisol levels were similar in the survivors and nonsurvivors. Just before their death the blood alcohol level of ADX rats exceeded 3 g/l. The mean blood alcohol level assayed in both groups of rats at the end of the period of alcoholisation did not differ significantly (data not shown). During the free choice procedure (Fig. 2) the SHAM group
ADRENALECTOMY PREVENTS ALCOHOL PREFERENCE
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FIG. 1. Free-choise paradigm: percent of alcohol intake by naive ADX (n = 13) and naive SHAM (n = 17) rats when submitted to an ethanol 10% (v/v/)/water choice. ***p < 0.001 (ANOVA for repeated measures).
(n = 20) drank significantly more ethanol than the ADX group (n = 25), F(I, 43) = 7 . 0 9 , p < 0.05. Groups of eight A D X and eight SHAM rats were used to start Experiments 4, 5, and 6. After supplementation with hydrocortisone during the pulmonary alcoholization procedure at a dose of 30 t~g/ml (equivalent to a dose of 4.8 m g / k g / day), the A D X alcoholized rats (n -- 6) showed a lower ethanol intake than the SHAM alcoholized rats (n = 6), F(1, 10)
= 19.14, p < 0.05 (Fig. 3).The ethanol ingested by these two groups is elevated to that of the preceeding groups of animals, which is due to the higher level of alcohol intoxication. Inside the alcoholization chamber the total fluid intake did not differ significantly between the ADX and SHAM groups of rats, F(1, 10) = 0.04,p > 0.05. Supplementation with hydrocortisone (30 /~g/ml) during the alcohol intoxication of SHAM rats (n = 8) did not modify
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FIG. 2. Free-choice paradigm: percent of alcohol intake by ADX (n = 25) and SHAM (n = 20) rats when submitted to an ethanol 10% (v/v)/water choice after a pulmonary alcoholization for 3 weeks. *p < 0.05 (ANOVA for repeated measures).
236
LAMBLIN A N D DE WITTE
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FIG. 3. Free-choice paradigm: percent alcohol intake by ADX (n = 6) and SHAM (n = 6) rats when submitted to an ethanol 10% (v/v)/water choice after a pulmonary alcoholization for 3 weeks. During this alcoholization, ADX were orally treated with hydrocortisone (4.8 mg/kg/day). *p < 0.05 (ANOVA for repeated measures). their c o n s u m e d e t h a n o l percentage in c o m p a r i s o n with untreated S H A M (n = 11). Figure 4 shows the high percentage (65%) o f alcohol intake in the two g r o u p s o f S H A M rats. T h e total fluid intake was n o t significantly different between these two groups, F(1, 17) = 0 . 0 9 , p > 0.05. C o r t i c o s t e r o n e (25 # g / m l ) given to A D X rats (n = 7) during the p u l m o n a r y a l c o h o l i z a t i o n p r o c e d u r e partially reversed the effect o f A D X o n alcochol preference in c o m p a r i s o n with Pulmonary
Alcoholized
, SHAM
the SHAM rats (n = 6) submitted to the same alcoholization procedure (Fig. 5), but there was no statistical difference between the two groups, F(I, 11) = 3 . 2 , p > 0.05. DISCUSSION
In vivo studies have revealed the existence of a close interaction between ethanol and the HPA axis [i.e., modulation of and
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FIG. 4. Free-choice paradigm: percent of alcohol intake by two groups of SHAM rats when submitted to an ethanol 10% (v/v)/water choice after a pulmonary alcoholization for 3 weeks. During this alcoholization a group of SHAM rats (n = 8) was orally treated with hydrocortisone and the other group of SHAM rats (n = 11) served as controls. There was no statistical difference between the two groups (ANOVA for repeated measures).
ADRENALECTOMY PREVENTS ALCOHOL PREFERENCE
Pulmonary Alcoholized,
SHAM
237
and CORTICOSTERONE-TREATED ADX
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FIG. 5. Free-choice paradigm: percent alcohol intake by ADX (n = 7) and SHAM (n = 6) rats when submitted to an ethanol 10% (v/v)/water choice after a pulmonary alcoholization for 3 weeks. During this alcoholization the ADX were orally treated with corticosterone. There was no statistical difference between the two groups (ANOVA for repeated measures).
the HPA function by ethanol and reciprocally modification of ethanol-induced behaviors (such as ethanol intake and ethanol withdrawal seizures) by the HPA axis secretions]. Those interactions may be mediated by each of the different structures that constitute this neurohormonal axis. In our experiments on male adrenalectomized rats, we have investigated this adaptation of the HPA axis by ethanol intake in naive, alcoholized, and alcoholized corticoid-treated rats. First, a free-choice paradigm that proposed an alternative between water and alcohol 10070 showed a decreased ethanol preference in ADX rats compared with SHAM rats. Fahlke et al. (3) showed a similar decrease in ethanol intake after adrenalectomy in male rats. Because ethanol consumption may act as a stress factor in rodents, the adrenalectomized rats showed an increased aversion to ethanol to prevent excessive stress-induced perturbations. Indeed, even if glucocorticoids were not directly implicated in the adaptive response to stress but were indirectly involved in blocking neuromediators released by stress, we can assume that glucocorticoids are augmented to prevent the organism to be overloaded (12). Suppression of glucocorticoid production by adrenalectomy may thus precipitate the complete development of stress manifestations. Second, by forcing the rats to drink ethanol by mean of presentation of only one drinking bottle containing 10°70 ethanol during a 3-week period, ADX and SHAM rats consumed similar amounts of liquid. This alcohol intake by ADX rats (that in the free-choice paradigm largely prefered water to alcohol) may be attributed to their requirement for saline (1 070) that was contained in the drinking bottle. During pulmonary alcoholization, rats inhaled alcohol in progressively increasing concentrations. Many of the ADX succumbed to this forced alcohol intoxication and it was necessary to stop the alcoholization procedure before reaching
an alcoholization level that would show a steady behavioral dependence when compared to the control group (i.e., the sham rats). The increased sensitivity to forced alcoholization and the resulting death in ADX group could be one of the manifestation of the acceleration to the effects of stress, because pulmonary alcoholization represented a powerful stress factor. Treatment of the A D X rats with hydrocortisone during the alcoholization procedure did not affect their alcohol consumption. Indeed, as was the case in untreated phlmonary alcoholized ADX and SHAM rats, the alcohol intake was significantly greater in the SHAM group than in the ADX group for an equivalent total fluid intake. This lack of effect of hydrocortisone on ethanol intake could by caused by either the incompleteness or inappropiate transformation of hydrocortisone to corticosterone or to the uselessness of this transformation in rats having normal circulating glucocorticoid levels. In addition, the integrity of the adrenals must be required for hydrocortisone metabolism. However, an alternative explanation may involve the possible weak hydrocortisone dosage for exerting a valuable physiological action. Nevertheless, chronic alcoholized corticosterone treated ADX and alcoholized untreated SHAM, do not present a significant difference in their alcohol intake. This result is consistent with the involvement of the HPA axis via corticosterone during ethanol intake as observed by Fahlke et al. (3). This study also showed the restoration of ethanol intake to the SHAM ethanol intake level following a corticosterone treatment in the ADX rats. ACKNOWLEDGEMENTS This study was supported by the FNRS (FRSM-LN 9.4552-93), the FDS 94, and Groupe Lipha.
238
L A M B L I N A N D DE W I T T E
REFERENCES 1. Adinoff, B.; Risher-Flowers, D.; De Jong, J.; Ravitz, B.; Bone G.; Nutt, D. J.; Roehirich, L.; Martin, P. R.; Linnoila, M. Disturbances of hypothalamic-pituitary-adrenal axis functioning during ethanol withdrawal in six men. Am. J. Psychiatry 148: 1023-1025; 1991. 2. Ellis, F. W. Effect of ethanol on plasma corticosterone levels. J. Pharmacol. Exp. Ther. 153:121-127; 1966. 3. Fahlke, C.; Engel, J. A.; Eriksson, P.; Hard, E.; Soderpalm, B. Involvement of corticosterone in the modulation of ethanol consumption in the rat. Alcohol 11:195-202; 1994. 4. Fahlke, C.; Hard, E.; Thomasson, R.; Engel, J. A.; Hansen, S. Metyrapone-induced suppression of corticosterone synthesis reduces ethanol consumption in high-preferring rats. Pharmacol. Biochem. Bahav. 48:977-981; 1994. 5. Jenkins, J. S.; Connolly, J. Adrenocortical response to ethanol in man. Br. Med. J. 2:804-805; 1968. 6. Kakihana, R.; Butte, J. C.; Hattaway, A.; Noble, E. P. Adrenalocortical response to ethanol in mice: modification by chronic ethanol consumption. Acta Endocrinol. (Copenh.) 67:653-664; 1971. 7. Kakihana, R.; Moore, J. A. Circadian rhythm of corticosterone in mice: The effect of chronic comsumption of alcohol. Psychopharmacology (Berlin) 46:301-305; 1976. 8. Knych, E. T.; Prohaska, J. R. Effect of chronic intoxication and naloxone on the ethanol induced increase in plasma corticosterone. Life Sci. 28:1987-1994; 1981. 9. Koranyi, L.; Endroczi, E.; Tal, E.; Levay, G. The effect of acute and chronic ethanol administration on serum corticosterone concentration in rats. Acta Physiol. Hung. 69:123-128; 1987. 10. Lamblin, F.; Meert, T. F.; De Witte, Ph. Adrenalectomy protects ethanol-withdrawn rats from harmine-induced tremor. Alcohol. In press, 1996.
11. Le Bourhis, B. Alcoolisation du rat par voie pulmonaire. C. R. Seances Soc. Biol. 169:898-904; 1975. 12. Munck, A.; Guyre, P. M.; Holdbrook, N. J. Physiological function of glucocorticoids in stress and their relation to pharmacological actions. Endocr. Rev. 5:25-44; 1984. 13. Nolan, C. J.; Bestervelt, L. L.; Mousigian, C. A.; Maimansomsuk, P.; Cai, Y.; Piper, W. N. Chronic ethanol consumption depresses hypothalamic-pituitary-adrenal function in aged rats. Life Sci. 49:1923-1928; 1991. 14. Pohorecky, L. A.; Rassi, E.; Weiss, J. M.; Michalak, V. Biochemical evidence for an interaction of ethanol and stress: Preliminary studies. Alcohol. Clin. Exp. Res. 4:423-426; 1980. 15. Spencer, R. L.; McEwen, B. Adaptation of the hypothalamicpituitary-adrenal axis to chronic ethanol stress. Neuroendocrinology 52:481-489; 1990. 16. Sze, P. Y.; Yanai, J.; Ginsburg, G. B. E. Adrenal glucocorticoids as a required factor in the development of ethanol withdrawal seizures in mice. Brain Res. 80:155-159; 1974. 17. Sze, P. Y. The permissive role of glucocorticoids in the development of ethanol dependence and tolerance. Drug Alcohol Depend. 2:381-396; 1977. 18. Tabakoff, B.; Jaffe, R. C.; Ritzmann, R. F. Corticosterone concentration in mice during drinking and ethanol withdrawal. J. Pharm. Pharmacol. 30:371-374; 1978. 19. Thiagarajan, A. B.; Mefford, I. N.; Eshay, R. C. Single dose ethanol administration activates the hypothalamic-pituitary-adrenal axis: Exploration of the mechanism of action. Neuroendocrinology 50:427-432; 1989. 20. Zola-Morgan, S.; Squire, L. R.; Amaral, D. G. Human amnesia and the medial temporal region: Enduring memory impairment following a bilateral lesion limited to field CA1 of the hippocampus. J. Neurosci. 6:2950-2967; 1986.
Adrenalectomy Prevents the Development of Alcohol Preference in Male Rats F. L A M B L I N A N D PH. DE W I T T E l
Laboratory o f Psychobiology, 1, Place Croix du Sud, B-1348 Louvain-la-Neuve, Belgium Received 13 A p r i l 1995; Accepted 28 July 1995 LAMBLIN, F. AND PH. DE WlTTE. Adrenalectomyprevents the development of alcoholpreference in male rats. ALCOHOL 13(3) 233-238, 1996.-By forcing adrenalectomized (ADX) and sham-operated (SHAM) rats to chronically drink ethanol by mean of presentation of only one drinking bottle containing 10°70ethanol, no differences occurred between both groups. ADX and SHAM rats were then exposed to chronic alcoholization using an inhalation procedure. After sejourning 3 weeks into the alcoholization chamber, rats were submitted to a free-choice paradigm [water vs. a 10¢/0(v/v) ethanol solution]. The sham-operated rats presented an alcohol-induced behavioral preference towards alcohol whereas adrenalectomized animals never exhibited a preference to ethanol. In the adrenalectomized rats treatment with hydrocortisone (30/zg/ml) given orally during the pulmonary alcoholization failed to modify this preference whereas treatment with corticosterone (25 #g/ml) given orally abolished the difference with SHAM animals. These data showed that adrenalectomy prevented the development of ethanol preference and the clear involvement of the hypothalamo-pituitary-axis in alcohol preference. Adrenalectomy
Pulmonary alcoholization
Free choice
Corticosterone
submitted to a free-choice paradigm, which was only transient (3,4). Indeed, these studies showed a gradual increase in alcohol consumption of the ADX rats after 2 and 3 weeks of the free-choice paradigm. Corticosterone supplementation to these adrenalectomized rats (ADX) immediately restored the alcohol intake to the preoperative level; in contrast, aldosterone treatment did not modify ethanol consumption: The decreased ethanol intake by ADX rats is not due to enhancement of aversion to the alcoholized solution (3). Second, adrenalectomy of male rats may possibly be a protective factor for ethanol withdrawal seizures. In previous studies, we have clearly shown that there was no increase in tremor activity in adrenalectomized rats induced by a harmine tremorogenic agent after ethanol withdrawal (10). In adrenalectomized mice, Sze et al. also show a decrease of the sensitivity to audiogenic seizures upon ethanol withdrawal (16,17). The original susceptibility to ethanol withdrawal seizures can be restored by corticosterone treatment. In the present studies we have evaluated the ethanol consumption in a free-choice paradigm (10~/0 ethanol vs. water) in various groups of rats: in naive, in orally or chronically pulmonary alcoholized adrenalectomized, and shamoperated male Wistar rats. The effect of supplementation with either hydrocortisone or corticosterone was also investigated.
AN increasing number of studies have implicated the hypothalamo-pituitary-adrenal axis (HPA) interaction in many of the perturbations associated with either acute or chronic alcohol intoxication. During acute exposure to alcohol there is a potent activation of the HPA axis marked by an increased circulating plasma concentration of glucocorticoid level in rodents (2,6,7,14,15,18) and in man (5,19). However, the effects of chronic alcoholization on the circulating concentrations of glucocorticoids have not been clearly established. Some studies have reported a prolonged elevation of plasma glucocorticoid levels during the chronic alcoholization period (2,18,20) whereas other studies have shown a tolerance development by the corticosterone response to ethanol (8,9,15). Depression of the HPA axis activity has also been observed after chronic alcoholization in aged rats that showed enhanced corticosterone and ACTH secretions (13). During ethanol withdrawal, the peak values for glucocorticoids concentrations was at a maximum on the first day of the alcohol deprivation, which coincided with the behavioural and physiological symptoms of the withdrawal syndrome (1,18). These results suggest that there are modifications of the HPA function by alcohol, and, reciprocally, that the HPA axis secretions modify several of the behaviorial changes associated with alcohol. First, either surgical or chemical suppression of adrenal function decreased the ethanol intake in rats
To whom requests for reprints should be addressed. 233
234
LAMBLIN AND DE WITTE METHOD
Animals Male Wistar rats weighing 150-180 g at the start of the experiments, were divided in two groups: an A D X group and a SHAM group. After anaesthesia with chloral hydrate (400 mg/ 100 g b.wt.) in the A D X group, the two adrenal glands were removed by means of two dorsal incisions. Sham surgery consisted of bilateral incisions; the adrenal glands were localized but not removed. Following surgery, all animals were supplemented with 1% saline solution. Success of ADX surgery was verified at the end of the experiment by the assay of plama corticosterone levels by radioimmunoassay. ADX rats presenting levels of corticosterone higher than 3 ng/ml were disregarded; the levels in the sham-operated rats were higher than 70 ng/ml. Experimental Treatment ADX and SHAM rats were submitted to the same experimental procedures and were compared in the following experiments: 1. Naive ADX and naive SHAM rats (naive means without any prior contact with alcohol). 2. Orally chronically alcohol loaded rats. Both groups of rats were chronically loaded with alcohol by the presentation of one drinking bottle containing 10% alcohol (v/v) for 3 weeks. The alcohol consumption was recorded each day. 3. Chronically alcohol loaded by pulmonary alcoholization. 4. Chronically alcohol loaded by pulmonary alcoholization to the ADX rats treated with supplementation of hydrocortisone (30 #g/ml, Solu-cortef, Upjohn) The hydrocortisone was given per os in the drinking bottle throughout the whole alcoholization procedure. The hydrocortisone solution was prepared fresh every 2 days, the drinking bottle containing this solution was sheltered from light to prevent degradation of the hydrocortisone. The rat's intake was monitored throughout the alcoholization period to ensure a constant drug administration. The treatment was stopped when the rats were removed from the alcoholization chamber. 5. SHAM and SHAM treated with hydrocortisone (30 #g/ml) given per os were also chronically alcohol loaded by chronic pulmonary alcoholization as described for the ADX group. 6. ADX-treated and untreated alcoholized SHAM rats were chronically alcohol loaded by pulmonary alcoholization and supplemented with corticosterone (25 /xg/ml, Sigma) throughout the procedure. The corticosterone was dissolved in water and the rat's intake was monitored throughout the experiment to ensure a constant drug administration. The treatment was stopped when the rats were removed from the alcoholization chamber. Chronic Pulmonary Alcoholization The rats were individually housed and maintained in an isolated plastic chamber (160 × 60 x 60 cm). A mixture of alcohol and air was pulsed into the chamber via a mixing system allowing the quantity of alcohol to be increased (0 to 25 mg/l) every 2 days during the experimental procedure (11). Adult rats were kept for 20 days in total in the alcoholization chamber.
Blood Alcohol Level The blood alcohol level was regularly assayed during the alcoholization exposure. Blood samples (0.1 ml) were collected from the caudal portion of the tail and alcohol was assayed by the reduction of NADH to NAD in the alcohol dehydrogenase-based method (Boerhinger-Mannhein Kit). Free-Choice Paradigm A free-choice beverage procedure consisting of water vs. ethanol solution, 10% (v/v), was presented to the two groups of rats in each experimental condition. The free choice proposed to the rats following the chronic pulmonary alcoholization required two preliminary steps: a full beverage deprivation (the last 18 h of the alcohol intoxication period and the first 6 h of the withdrawal period), and the presentation of a 10% (v/v) ethanol solution as the only drinking fluid during the following 18 h. During the free-choice period (3 weeks at least), fluid consumption was recorded daily. The bottle's positions were randomly distributed to avoid position preference. Statistical Methods All data are presented as means + SD. According to the experiments the analysis of variance (ANOVA) or the ANOVA for repeated measures were applied. RESULTS
Free-Choice Paradigm Figure 1 shows the free-choice paradigm of naive ADX (n = 13) and SHAM rats (n = 17). The alcohol consumption of SHAM rats represented 50% of their total fluid intake whereas the alcohol drunk by the ADX rats reached only 20% of their fluid consumption. Despite the absence of difference in the total fluid intake between SHAM (mean daily intake 42 11.68 ml/kg/day) and ADX (mean daily intake 57 + 15.21 ml/kg/day), ethanol consumed by the SHAM was significantly greater than ethanol consumed by the ADX, F(1, 28) = 13.5,p < 0.001. Rats that were chronically administered alcohol orally, by means of the presentation of only one bottle containing ethanol (10% v/v) for a 3-week period, had a mean daily intake of 2 g/kg ethanol (i.e., 25 ml of ethanol 10% per day) (data not shown). In comparison to the free-choice experiment, the total fluid intake was significantly lower in both SHAM and ADX after presenting only one bottle containing ethanol. For this type of alcoholization, the difference in the alcohol intake between ADX rats (n = 6) and SHAM rats (n = 8) was not significant, F ( I , 12) = 0.46,p > 0.05. Chronic pulmonary alcoholization induced a higher mortality in the ADX group than the sham-operated rats. Commencing the experiment with 48 ADX rats (b.wt.: 220 9 g) and 22 SHAM rats (b.wt.: 280 4- 8 g), at the end of the alcoholisation period there were 25 ADX (b.wt.: 180 + 6 g) and 20 SHAM (b.wt.: 290 ± 12 g). Even though 50% of the ADX rats did not survive this period of chronic alcoholisation, there was no apparent explanation, because the cortisol levels were similar in the survivors and nonsurvivors. Just before their death the blood alcohol level of ADX rats exceeded 3 g/l. The mean blood alcohol level assayed in both groups of rats at the end of the period of alcoholisation did not differ significantly (data not shown). During the free choice procedure (Fig. 2) the SHAM group
ADRENALECTOMY PREVENTS ALCOHOL PREFERENCE
Unalcoholized
235
, ADX
and
SHAM
I00
I
ADX i
90
I
m
SHAM
80 70 I
1;
60
o 508 40302010O
i
i
i
i
o
Days
FIG. 1. Free-choise paradigm: percent of alcohol intake by naive ADX (n = 13) and naive SHAM (n = 17) rats when submitted to an ethanol 10% (v/v/)/water choice. ***p < 0.001 (ANOVA for repeated measures).
(n = 20) drank significantly more ethanol than the ADX group (n = 25), F(I, 43) = 7 . 0 9 , p < 0.05. Groups of eight A D X and eight SHAM rats were used to start Experiments 4, 5, and 6. After supplementation with hydrocortisone during the pulmonary alcoholization procedure at a dose of 30 t~g/ml (equivalent to a dose of 4.8 m g / k g / day), the A D X alcoholized rats (n -- 6) showed a lower ethanol intake than the SHAM alcoholized rats (n = 6), F(1, 10)
= 19.14, p < 0.05 (Fig. 3).The ethanol ingested by these two groups is elevated to that of the preceeding groups of animals, which is due to the higher level of alcohol intoxication. Inside the alcoholization chamber the total fluid intake did not differ significantly between the ADX and SHAM groups of rats, F(1, 10) = 0.04,p > 0.05. Supplementation with hydrocortisone (30 /~g/ml) during the alcohol intoxication of SHAM rats (n = 8) did not modify
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FIG. 2. Free-choice paradigm: percent of alcohol intake by ADX (n = 25) and SHAM (n = 20) rats when submitted to an ethanol 10% (v/v)/water choice after a pulmonary alcoholization for 3 weeks. *p < 0.05 (ANOVA for repeated measures).
236
LAMBLIN A N D DE WITTE
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FIG. 3. Free-choice paradigm: percent alcohol intake by ADX (n = 6) and SHAM (n = 6) rats when submitted to an ethanol 10% (v/v)/water choice after a pulmonary alcoholization for 3 weeks. During this alcoholization, ADX were orally treated with hydrocortisone (4.8 mg/kg/day). *p < 0.05 (ANOVA for repeated measures). their c o n s u m e d e t h a n o l percentage in c o m p a r i s o n with untreated S H A M (n = 11). Figure 4 shows the high percentage (65%) o f alcohol intake in the two g r o u p s o f S H A M rats. T h e total fluid intake was n o t significantly different between these two groups, F(1, 17) = 0 . 0 9 , p > 0.05. C o r t i c o s t e r o n e (25 # g / m l ) given to A D X rats (n = 7) during the p u l m o n a r y a l c o h o l i z a t i o n p r o c e d u r e partially reversed the effect o f A D X o n alcochol preference in c o m p a r i s o n with Pulmonary
Alcoholized
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the SHAM rats (n = 6) submitted to the same alcoholization procedure (Fig. 5), but there was no statistical difference between the two groups, F(I, 11) = 3 . 2 , p > 0.05. DISCUSSION
In vivo studies have revealed the existence of a close interaction between ethanol and the HPA axis [i.e., modulation of and
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FIG. 4. Free-choice paradigm: percent of alcohol intake by two groups of SHAM rats when submitted to an ethanol 10% (v/v)/water choice after a pulmonary alcoholization for 3 weeks. During this alcoholization a group of SHAM rats (n = 8) was orally treated with hydrocortisone and the other group of SHAM rats (n = 11) served as controls. There was no statistical difference between the two groups (ANOVA for repeated measures).
ADRENALECTOMY PREVENTS ALCOHOL PREFERENCE
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237
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FIG. 5. Free-choice paradigm: percent alcohol intake by ADX (n = 7) and SHAM (n = 6) rats when submitted to an ethanol 10% (v/v)/water choice after a pulmonary alcoholization for 3 weeks. During this alcoholization the ADX were orally treated with corticosterone. There was no statistical difference between the two groups (ANOVA for repeated measures).
the HPA function by ethanol and reciprocally modification of ethanol-induced behaviors (such as ethanol intake and ethanol withdrawal seizures) by the HPA axis secretions]. Those interactions may be mediated by each of the different structures that constitute this neurohormonal axis. In our experiments on male adrenalectomized rats, we have investigated this adaptation of the HPA axis by ethanol intake in naive, alcoholized, and alcoholized corticoid-treated rats. First, a free-choice paradigm that proposed an alternative between water and alcohol 10070 showed a decreased ethanol preference in ADX rats compared with SHAM rats. Fahlke et al. (3) showed a similar decrease in ethanol intake after adrenalectomy in male rats. Because ethanol consumption may act as a stress factor in rodents, the adrenalectomized rats showed an increased aversion to ethanol to prevent excessive stress-induced perturbations. Indeed, even if glucocorticoids were not directly implicated in the adaptive response to stress but were indirectly involved in blocking neuromediators released by stress, we can assume that glucocorticoids are augmented to prevent the organism to be overloaded (12). Suppression of glucocorticoid production by adrenalectomy may thus precipitate the complete development of stress manifestations. Second, by forcing the rats to drink ethanol by mean of presentation of only one drinking bottle containing 10°70 ethanol during a 3-week period, ADX and SHAM rats consumed similar amounts of liquid. This alcohol intake by ADX rats (that in the free-choice paradigm largely prefered water to alcohol) may be attributed to their requirement for saline (1 070) that was contained in the drinking bottle. During pulmonary alcoholization, rats inhaled alcohol in progressively increasing concentrations. Many of the ADX succumbed to this forced alcohol intoxication and it was necessary to stop the alcoholization procedure before reaching
an alcoholization level that would show a steady behavioral dependence when compared to the control group (i.e., the sham rats). The increased sensitivity to forced alcoholization and the resulting death in ADX group could be one of the manifestation of the acceleration to the effects of stress, because pulmonary alcoholization represented a powerful stress factor. Treatment of the A D X rats with hydrocortisone during the alcoholization procedure did not affect their alcohol consumption. Indeed, as was the case in untreated phlmonary alcoholized ADX and SHAM rats, the alcohol intake was significantly greater in the SHAM group than in the ADX group for an equivalent total fluid intake. This lack of effect of hydrocortisone on ethanol intake could by caused by either the incompleteness or inappropiate transformation of hydrocortisone to corticosterone or to the uselessness of this transformation in rats having normal circulating glucocorticoid levels. In addition, the integrity of the adrenals must be required for hydrocortisone metabolism. However, an alternative explanation may involve the possible weak hydrocortisone dosage for exerting a valuable physiological action. Nevertheless, chronic alcoholized corticosterone treated ADX and alcoholized untreated SHAM, do not present a significant difference in their alcohol intake. This result is consistent with the involvement of the HPA axis via corticosterone during ethanol intake as observed by Fahlke et al. (3). This study also showed the restoration of ethanol intake to the SHAM ethanol intake level following a corticosterone treatment in the ADX rats. ACKNOWLEDGEMENTS This study was supported by the FNRS (FRSM-LN 9.4552-93), the FDS 94, and Groupe Lipha.
238
L A M B L I N A N D DE W I T T E
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