Alcohol. Vol. 8. pp. 237-240. e Pergamon Press plc, 1991. Printed in lhe U.S.A.
0741-8329/91 $3.00 + .00
Morphine Increases Intake of Beer Among Rats M I C H A E L L. N I C H O L S , C H R I S T O P H E R L. H U B B E L L , M I C H A E L J. K A L S H E R A N D L A R R Y D. R E I D I
Deparonent of Psychology, Rensselaer Poh,technic htstitute, Troy, NY 12180-3590 R e c e i v e d 20 M a r c h 1990; A c c e p t e d 15 F e b r u a r y 1991 NICHOLS, M. L.. C. L. HUBBELL, M. J. KALSHER AND L. D. REID. Morphine increases intake of beer amon~,r.'qt~s! ALCOHOL g{4) 237-240, 1991.--Rats were maintained on a daily regimen of 22 h of water deprivation followed by a 2-h opportunity to take water and sweetened alcoholic beverage containing 12% ethanol. After 30 days, the alcoholic beverage was changed to beer containing either 3% or 6% ethanol. After 20 daily sessions with beer, they received, before the next session, an injection of saline. On the next day, they received a 1.0 mg/kg injection of morphine before the session. Morphine reliably increased rats' mean intake of both kinds of beer. Subsequently. the concentration of ethanol in each groups" beer was changed. The 3% group's beer was switched to 6%, and the 6% group's to 3%. Both groups altered their mean intake of beer in an apparent attempt to maintain intakes of nearly the same amount of ethanol, but presentation of 6% beer resulted in greater intakes of ethanol. Beer-intake
Morphine
Ethanol
Opioids
sessment of morphine's effects on intake of various kinds of alcoholic beverages addresses the issue of generality of the conclusion that the neural events initiated by a small dose of morphine increase propensity to take alcoholic beverages. An additional aim was to investigate the possibility that rats, like people, systematically adjust their consumption of alcoholic beverage (e.g., beer) to attain an optimal pharmacological effect, as indexed by g of pure ethanol consumed per kilogram of body weight (g/kg). Such assessments have important ramifications for current attempts to promote the use of low alcohol content beverages among people (4). Previous research on rats' beer drinking [e.g., (2, 7, 8)] has shown that, in general, level of consumption is inversely related to concentration of ethanol. Two interpretations have been applied to the data from these studies: (a) an orosensory interpretation in which decreased consumption of beer with increasing concentration of ethanol is presumed to be a function of palatability; and (b) an inebriation interpretation in which decreased consumption of beer with increasing levels of ethanol is presumed to be a function of rats' tendency to avoid high levels of inebriation which is assumed to be aversive. It is equally plausible to suggest that there exists in rats, as well as in people, optimal levels of alcohol-intake which are characterized by attempts to increase the positive affect achievable with ethanol, while minimizing potentially adverse effects. A methodological gap inherent in the prior studies of rats' drinking of beer that precludes an assessment of these various hypotheses is a failure to tabulate the amount of pure ethanol consumed by rats given the opportunity to drink beers of varying alcoholic content. Thus, a second aim was to assess how rats adjust their drinking
SMALL doses of morphine increase rats' intake of alcoholic beverages across a wide variety of circumstances [for a review, see (6)]. In general, the alcoholic beverages which investigators have used to assess morphine's effects have been either an ethanol solution which is ethanol diluted with only water, or a sweetened ethanol solution. The use of both of these kinds of beverage with rats can be criticized because of their potential extremes of palatability, one being too unpalatable and the other too palatable, to be relevant to people's use of alcoholic beverages. Rats' voluntary drinking of alcoholic beverages can be a model of a general propensity to take alcoholic beverages. Conclusions derived from such studies are apt to be applicable to the human conditions of alcohol abuse and alcoholism (6). Nevertheless, there are those who might criticize the use of palatable alcoholic beverages [e.g., (3)] because of a potential to confound the supposedly separable factors contributing to propensity to drink alcoholic beverages (palatability, calories, thirst quenching, and a pharmacological effect of ethanol). As long as the relevant behavior is ingestion of a beverage, however, palatability is an intrinsic factor that cannot be separated. If factors clearly related to ingestion (e.g., palatability) are always involved in drinking of alcoholic beverages, then all that can be done is to see if a manipulation (e.g., a drug's effect) holds across a wide range of these supposedly extraneous variables. Although it is surely not necessary for a model to have good face validity for that model to have relevance to its " r e a l " world conterpart, it is surely all right to have strong face validity. Consequently, intake of beer (of the kind people drink) was chosen to further assessthe small-dose-morphine effect. The astRequests for reprints should be addressed to L. D. Reid.
237
238
NICHOLS, HUBBELL. KALSHER AND REID
of beer depending on the concentration of ethanol. METHOD
Subjects The subjects were 24 male, Sprague-Dawley rats purchased from Taconic Farms (Germantown, NY). Upon arrival at the laboratory, they were individually housed in standard hanging cages in a windowless colony room maintained at 22°C with 12 h of artificial light per day, beginning at 0700 h. They were previously used as controls in a unrelated procedure (a test for conditioned place preferencesl in which they received extensive handling and injections of physiological saline.
Drugs and Solutions The dose of morphine sulfate was 1.0 mg/kg, This is a dose that previous research has shown to increase intake of typical alcoholic beverages used in laboratories (6). This dose of morphine produces no discernable sedative effects among rats. Placebos were of physiological saline, the vehicle of morphine. All injections were administered, subcutaneously 1.0 ml/kg, 15 min before presentation of fluids. The ethanol solution used initially in these procedures was a , solution containing 12% ethanol and 5% sucrose, i.e., 12 g of pure ethanol, 5 g of sucrose and 83 g of tap water. Solutions of beer contained either 3 or 6% ethanol. The 3% solution was 3 g pure ethanol and 97 g of a nonalcoholic malt beverage (Carling Black Label, G. Heileman Brewing Co., La Crosse, Wll. The 6% solution was 6 g of ethanol and 94 g of nonalcoholic beverage. Nonalcoholic malt beverages, however, can contain trace amounts of alcohol, with a legal maximum of 0.5% by volume, so o u r two types of beer may have contained somewhat more ethanol than 6 or 3%. All fluids were presented in glass bottles equipped with ballpoint sipping tubes. Despite the ballpoint tubes which usually limit spillage, we found that the carbonation of beer leads to excessive spillage (5). Therefore, spillage was collected in small plastic cups placed beneath the rats' cages, out of reach, directly under the sipping tubes of the beer bottles. Consequently. the spillage was taken into account. To reduce the amount of spillage, the level of carbonation was reduced by opening the container of the nonalcoholic beverage 24 h before preparing the beer solution. In a pilot study, we found that rats had no differential preference for either carbonated or partly decarbonated (flat) beer.
Procedure Throughout these procedures, the subjects were maintained on a daily regimen of 22 h of water deprivation followed by a 2-h period when fluids were available. Food was always available. Across the first 30 days, the rats were provided with a bottle of water and a bottle of sweetened ethanol solution during the daily. 2-h opportunity to take fluids. Toward the end of the 30 days, rats were taking a daily mean of between 1.5 and 2.4 g/kg daily. Then the ethanol solution was replaced with 3% beer for half the rats (i.e., 12 rats), while the other half received 6% beer. After 20 days of opportunity to take beer and water, the first injections were given. On the initial day of injections, half received placebo, and, on the next day, morphine. In order to control for potential effects associated with a particular day, the series of injections for the other rats began one day later. The rats continued the daily regimen; but, 6 days after the assessment of morphine's effects, rats being presented 6% beer
were presented 3% beer and those being presented 3% were presented 6%. The mean intake of the last 4 days before the change was taken as the index of a rat's prechange intake. The mean of two 4-day blocks of intake after the change was used to index a rat's response to the change in concentration of ethanol.
Measures. Data Reduction and Statistics Body weight, intake of water, and intake of ethanol solution or beer (corrected for spillage) were measured daily to the nearest 0.1 g. From these basic data. other scores were calculated, particularly (a) g of pure ethanol per kg of body weight (g/kg); and (b) preference ratios (g of ethanol divided by total intake of fluids). To assess morphine's effects, each measure was submitted to a 2 by 2 analysis of variance (ANOVA) for repeated measures having a factor associated with the two concentrations of ethanol (3 and 6%) and placebo versus drug. The results of the ANOVAs generally indicated that the factors were reliable sources of variance with each measure. Consequently, the appropriate Student's t-tests were performed to assess the effects of the small doses of morphine on each measure for each concentration of ethanol. To simplify the analyses of the data associated with changing the concentrations of ethanol, we collapsed the data into 4-day means for each rat. Given that there were two groups of rats (one switched from 3% to 6%, one from 6% to 3%) and 3 blocks of 4 days each (one prior to the switch and two subsequent to it), the data of each measure confon-n to a 2 by 3 ANOVA having repeated measures. RESULTS The data, summarized in Table I. show that morphine reliably increased intake of beer and reduced intake of water, thereby producing marked changes in preference ratios. Figure 1 depicts the results associated with changing the alcoholic content of the beer. The shift from 6 to 3% led to a decrease in g/kg of ethanol, but an increase in actual g of beer. The shift from 3 to 6% led to an increase in g/kg of ethanol and a reduction in g of beer. The ANOVAs for each measure associated with the change in concentration of ethanol indicate that neither the main effect associated with groups (group 6 to 3% versus group 3 to 6%) nor with blocks of days were reliable sources of variance. The interaction terms for each measure of fluid intake were all reliable sources of variance (all ps
0741-8329/91 $3.00 + .00
Morphine Increases Intake of Beer Among Rats M I C H A E L L. N I C H O L S , C H R I S T O P H E R L. H U B B E L L , M I C H A E L J. K A L S H E R A N D L A R R Y D. R E I D I
Deparonent of Psychology, Rensselaer Poh,technic htstitute, Troy, NY 12180-3590 R e c e i v e d 20 M a r c h 1990; A c c e p t e d 15 F e b r u a r y 1991 NICHOLS, M. L.. C. L. HUBBELL, M. J. KALSHER AND L. D. REID. Morphine increases intake of beer amon~,r.'qt~s! ALCOHOL g{4) 237-240, 1991.--Rats were maintained on a daily regimen of 22 h of water deprivation followed by a 2-h opportunity to take water and sweetened alcoholic beverage containing 12% ethanol. After 30 days, the alcoholic beverage was changed to beer containing either 3% or 6% ethanol. After 20 daily sessions with beer, they received, before the next session, an injection of saline. On the next day, they received a 1.0 mg/kg injection of morphine before the session. Morphine reliably increased rats' mean intake of both kinds of beer. Subsequently. the concentration of ethanol in each groups" beer was changed. The 3% group's beer was switched to 6%, and the 6% group's to 3%. Both groups altered their mean intake of beer in an apparent attempt to maintain intakes of nearly the same amount of ethanol, but presentation of 6% beer resulted in greater intakes of ethanol. Beer-intake
Morphine
Ethanol
Opioids
sessment of morphine's effects on intake of various kinds of alcoholic beverages addresses the issue of generality of the conclusion that the neural events initiated by a small dose of morphine increase propensity to take alcoholic beverages. An additional aim was to investigate the possibility that rats, like people, systematically adjust their consumption of alcoholic beverage (e.g., beer) to attain an optimal pharmacological effect, as indexed by g of pure ethanol consumed per kilogram of body weight (g/kg). Such assessments have important ramifications for current attempts to promote the use of low alcohol content beverages among people (4). Previous research on rats' beer drinking [e.g., (2, 7, 8)] has shown that, in general, level of consumption is inversely related to concentration of ethanol. Two interpretations have been applied to the data from these studies: (a) an orosensory interpretation in which decreased consumption of beer with increasing concentration of ethanol is presumed to be a function of palatability; and (b) an inebriation interpretation in which decreased consumption of beer with increasing levels of ethanol is presumed to be a function of rats' tendency to avoid high levels of inebriation which is assumed to be aversive. It is equally plausible to suggest that there exists in rats, as well as in people, optimal levels of alcohol-intake which are characterized by attempts to increase the positive affect achievable with ethanol, while minimizing potentially adverse effects. A methodological gap inherent in the prior studies of rats' drinking of beer that precludes an assessment of these various hypotheses is a failure to tabulate the amount of pure ethanol consumed by rats given the opportunity to drink beers of varying alcoholic content. Thus, a second aim was to assess how rats adjust their drinking
SMALL doses of morphine increase rats' intake of alcoholic beverages across a wide variety of circumstances [for a review, see (6)]. In general, the alcoholic beverages which investigators have used to assess morphine's effects have been either an ethanol solution which is ethanol diluted with only water, or a sweetened ethanol solution. The use of both of these kinds of beverage with rats can be criticized because of their potential extremes of palatability, one being too unpalatable and the other too palatable, to be relevant to people's use of alcoholic beverages. Rats' voluntary drinking of alcoholic beverages can be a model of a general propensity to take alcoholic beverages. Conclusions derived from such studies are apt to be applicable to the human conditions of alcohol abuse and alcoholism (6). Nevertheless, there are those who might criticize the use of palatable alcoholic beverages [e.g., (3)] because of a potential to confound the supposedly separable factors contributing to propensity to drink alcoholic beverages (palatability, calories, thirst quenching, and a pharmacological effect of ethanol). As long as the relevant behavior is ingestion of a beverage, however, palatability is an intrinsic factor that cannot be separated. If factors clearly related to ingestion (e.g., palatability) are always involved in drinking of alcoholic beverages, then all that can be done is to see if a manipulation (e.g., a drug's effect) holds across a wide range of these supposedly extraneous variables. Although it is surely not necessary for a model to have good face validity for that model to have relevance to its " r e a l " world conterpart, it is surely all right to have strong face validity. Consequently, intake of beer (of the kind people drink) was chosen to further assessthe small-dose-morphine effect. The astRequests for reprints should be addressed to L. D. Reid.
237
238
NICHOLS, HUBBELL. KALSHER AND REID
of beer depending on the concentration of ethanol. METHOD
Subjects The subjects were 24 male, Sprague-Dawley rats purchased from Taconic Farms (Germantown, NY). Upon arrival at the laboratory, they were individually housed in standard hanging cages in a windowless colony room maintained at 22°C with 12 h of artificial light per day, beginning at 0700 h. They were previously used as controls in a unrelated procedure (a test for conditioned place preferencesl in which they received extensive handling and injections of physiological saline.
Drugs and Solutions The dose of morphine sulfate was 1.0 mg/kg, This is a dose that previous research has shown to increase intake of typical alcoholic beverages used in laboratories (6). This dose of morphine produces no discernable sedative effects among rats. Placebos were of physiological saline, the vehicle of morphine. All injections were administered, subcutaneously 1.0 ml/kg, 15 min before presentation of fluids. The ethanol solution used initially in these procedures was a , solution containing 12% ethanol and 5% sucrose, i.e., 12 g of pure ethanol, 5 g of sucrose and 83 g of tap water. Solutions of beer contained either 3 or 6% ethanol. The 3% solution was 3 g pure ethanol and 97 g of a nonalcoholic malt beverage (Carling Black Label, G. Heileman Brewing Co., La Crosse, Wll. The 6% solution was 6 g of ethanol and 94 g of nonalcoholic beverage. Nonalcoholic malt beverages, however, can contain trace amounts of alcohol, with a legal maximum of 0.5% by volume, so o u r two types of beer may have contained somewhat more ethanol than 6 or 3%. All fluids were presented in glass bottles equipped with ballpoint sipping tubes. Despite the ballpoint tubes which usually limit spillage, we found that the carbonation of beer leads to excessive spillage (5). Therefore, spillage was collected in small plastic cups placed beneath the rats' cages, out of reach, directly under the sipping tubes of the beer bottles. Consequently. the spillage was taken into account. To reduce the amount of spillage, the level of carbonation was reduced by opening the container of the nonalcoholic beverage 24 h before preparing the beer solution. In a pilot study, we found that rats had no differential preference for either carbonated or partly decarbonated (flat) beer.
Procedure Throughout these procedures, the subjects were maintained on a daily regimen of 22 h of water deprivation followed by a 2-h period when fluids were available. Food was always available. Across the first 30 days, the rats were provided with a bottle of water and a bottle of sweetened ethanol solution during the daily. 2-h opportunity to take fluids. Toward the end of the 30 days, rats were taking a daily mean of between 1.5 and 2.4 g/kg daily. Then the ethanol solution was replaced with 3% beer for half the rats (i.e., 12 rats), while the other half received 6% beer. After 20 days of opportunity to take beer and water, the first injections were given. On the initial day of injections, half received placebo, and, on the next day, morphine. In order to control for potential effects associated with a particular day, the series of injections for the other rats began one day later. The rats continued the daily regimen; but, 6 days after the assessment of morphine's effects, rats being presented 6% beer
were presented 3% beer and those being presented 3% were presented 6%. The mean intake of the last 4 days before the change was taken as the index of a rat's prechange intake. The mean of two 4-day blocks of intake after the change was used to index a rat's response to the change in concentration of ethanol.
Measures. Data Reduction and Statistics Body weight, intake of water, and intake of ethanol solution or beer (corrected for spillage) were measured daily to the nearest 0.1 g. From these basic data. other scores were calculated, particularly (a) g of pure ethanol per kg of body weight (g/kg); and (b) preference ratios (g of ethanol divided by total intake of fluids). To assess morphine's effects, each measure was submitted to a 2 by 2 analysis of variance (ANOVA) for repeated measures having a factor associated with the two concentrations of ethanol (3 and 6%) and placebo versus drug. The results of the ANOVAs generally indicated that the factors were reliable sources of variance with each measure. Consequently, the appropriate Student's t-tests were performed to assess the effects of the small doses of morphine on each measure for each concentration of ethanol. To simplify the analyses of the data associated with changing the concentrations of ethanol, we collapsed the data into 4-day means for each rat. Given that there were two groups of rats (one switched from 3% to 6%, one from 6% to 3%) and 3 blocks of 4 days each (one prior to the switch and two subsequent to it), the data of each measure confon-n to a 2 by 3 ANOVA having repeated measures. RESULTS The data, summarized in Table I. show that morphine reliably increased intake of beer and reduced intake of water, thereby producing marked changes in preference ratios. Figure 1 depicts the results associated with changing the alcoholic content of the beer. The shift from 6 to 3% led to a decrease in g/kg of ethanol, but an increase in actual g of beer. The shift from 3 to 6% led to an increase in g/kg of ethanol and a reduction in g of beer. The ANOVAs for each measure associated with the change in concentration of ethanol indicate that neither the main effect associated with groups (group 6 to 3% versus group 3 to 6%) nor with blocks of days were reliable sources of variance. The interaction terms for each measure of fluid intake were all reliable sources of variance (all ps
