Effects of lifelong ethanol consumption on drinking behavior and motor impairment of alcohol-preferring AA and alcohol-avoiding ANA rats

Alcohol 23 (2001) 157 ± 166

Effects of lifelong ethanol consumption on drinking behavior and motor impairment of alcohol-preferring AA and alcohol-avoiding ANA rats Maija Sarviharjua,*, Pia Jaatinenb, Petri HyytiaÈa, Antti Hervonenc, Kalervo Kiianmaaa a

Department of Mental Health and Alcohol Research, National Public Health Institute, P.O. Box 719, FIN-00101, Helsinki, Finland b Department of Internal Medicine, Tampere University Hospital, FIN-33700, Tampere, Finland c School of Public Health, University of Tampere, FIN-33700, Tampere, Finland Received 6 July 2000; received in revised form 11 December 2000; accepted 18 December 2000

Abstract The effects of drinking ethanol throughout a lifetime on voluntary drinking behavior and ethanol-induced motor impairment were studied in alcohol-preferring AA (Alko, Alcohol) and alcohol-avoiding ANA (Alko, Non-Alcohol) rats of both sexes. At the age 3 months, the rats were tested for individual voluntary ethanol (10% [vol./vol.]) intake and ethanol-induced motor impairment (2 g/kg, i.p.). The rats were housed in group cages, half of them having 12% (vol./vol.) ethanol as the only source of fluid and the other half having free access to water. Food was always available for all animals. At the age of 23 months, their individual voluntary ethanol intake and ethanol-induced motor impairment were tested again. During forced drinking, the females of both strains consumed more ethanol than did the males. The ethanol consumption of the AA and ANA females and the ANA males increased significantly ( P < .001) with age, but a slight decrease was seen in the ethanol consumption of the AA males. Time  strain interaction showed a significant ( P < .05) difference in the ethanol consumption of male rats, with the AA males having a slight decrease in ethanol consumption with age, whereas the ANA males increased their ethanol consumption. After 19 months of forced ethanol exposure, AA males significantly decreased their individual voluntary ethanol consumption, and individual voluntary ethanol consumption by ethanol-exposed AA males was more pronounced ( P < .001) than that of the AA rats that had free access to water ( P < .05). For the female AA rats, those having free access to water significantly decreased their voluntary ethanol consumption ( P < .05), but those having ethanol only did not. No significant changes in voluntary ethanol consumption with age or with different exposures were seen in the ANA rats. Body weights were higher in the groups having access to water than in the ethanol-only groups, but the differences were not significant within the AA and ANA strains. The ANA rats were significantly heavier in all groups. These results indicate that the voluntarily nondrinking ANA rats can drink almost as much ethanol as the voluntarily drinking AA rats when they are forced to drink ethanol and that lifelong forced ethanol drinking does not change their inherent drinking habits. When sensitivity to ethanol was measured with the tilting-plane test, the old AA female rats were more sensitive to ethanol than were the young ones. The young ANA females were more sensitive than the AA females when tested at 4 months. In males, aging did not produce any differences in ethanol sensitivity. D 2001 Elsevier Science Inc. All rights reserved. Keywords: Long-term ethanol consumption; Voluntary ethanol drinking; Aging; Motor impairment

1. Introduction Intuitively, one would expect that exposing a rat or a person to ethanol for a long time would increase the preference for drinking ethanol and, conversely, depriving a rat or a person of ethanol would decrease the preference. * Corresponding author. Tel.: +358-9-133-2639; fax: +358-9-1333286. E-mail address: [email protected] (M. Sarviharju). Editor: S. Borg

Thus, the most common treatment for alcoholism has been a period of forced abstinence. The experimental evidence, however, is generally contrary to these expectations. Depriving a rat of ethanol increases the motivation and drinking when ethanol is returned (Sinclair & Senter, 1967). Similarly, forcing C57Bl mice to drink ethanol solution (by removing access to water) produced a temporary decrease in subsequent voluntary ethanol drinking (McClearn, 1972). The alcohol deprivation effect increases ethanol consumption not only after a few days without ethanol but also

0741-8329/01/$ ± see front matter D 2001 Elsevier Science Inc. All rights reserved. PII: S 0 7 4 1 - 8 3 2 9 ( 0 1 ) 0 0 1 3 2 - X

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after months of abstinence (Sinclair et al., 1973). The study with mice, however, included only 6 days of forced consumption, and it is possible that the expected increase may still be produced with very long periods of exposure. In the present study, we examined the extreme condition: the effect of essentially a lifetime of forced ethanol drinking on voluntary ethanol selection. Experimental studies elucidating the interactions of chronic ethanol exposure and the aging process are rather few in number. The interactions of aging and chronic ethanol ingestion have been studied in mice (Samorajski et al., 1982), where the mice were exposed to 10% ethanol as the only source of fluid from 12 to 30 months of age, and in rats (Pietrzak et al., 1989, 1990), where 15% ethanol also as the only fluid was used for as long as 25 months. Blokland et al. (1993) found that chronic ethanol consumption did not affect the performance of adult and old rats and does not necessarily lead to learning and memory impairments in the rat. Results of studies on cognitive functions (Becker et al., 1983; Riege et al., 1984), signal transduction mechanisms (Sun et al., 1987), and dendritic morphometrics of Purkinje neurons (Pentney & Quackenbush, 1990; Pentney & Quigley, 1987) seems to indicate independent or even opposite effects of aging and long-term ethanol consumption. We have previously used lifelong voluntary ethanol consumption (Hervonen et al., 1992; Jaatinen et al., 1992) of alcohol-preferring AA (Alko, Alcohol) rats (Eriksson, 1971) to study the effects of ethanol-aging interactions on ethanol-related behaviors as well as on the morphological and histochemical characteristics of the myocardium and peripheral sympathetic neurons (Jaatinen & Hervonen, 1994; Jaatinen et al., 1994). The alcohol-preferring AA (Alko, Alcohol) and alcoholavoiding ANA (Alko, Non-Alcohol) rat lines have been developed by selective outbreeding since 1963 in the Alcohol Research Center at the National Public Health Institute (formerly the Research Laboratories of Alko). The lines were originally produced by selective outbreeding for high and low levels of voluntary ethanol consumption (Eriksson, 1969, 1971; Eriksson & Rusi, 1981). These lines have been used extensively to study the factors controlling voluntary ethanol consumption (Sinclair et al., 1989). Several behavioral, metabolic, and central nervous system differences have been shown between the AA and the ANA lines (Eriksson, 1981). They have also been found to differ in their sensitivity to ethanol; the AA rats were previously found to be more resistant to ethanol-induced hypnosis (Rusi et al., 1977) and motor impairment (Malila, 1978) than were the ANA rats. Two large programs for the improvement of these lines in regard to health and genetics were conducted in the past few decades. First, a revitalization program was conducted in generation F37; the AA and ANA breeders were crossed with the F1 hybrids obtained from crossing Brown Norwegian and Lewis rats (Hilakivi et al., 1984; HyytiaÈ et al., 1987). Second, rederivation by hysterectomy to make the rats pathogen free was

carried out in generations F63 and F64 (Sarviharju & Jaakkola, 1994). After the revitalization, the lines did not differ significantly in ethanol-induced motor impairment, in the hypothermic effects of ethanol, or in the hypnotic effect (Hilakivi et al., 1984). However, the lines still showed a marked difference in their acetaldehyde metabolism, and the line difference in voluntary ethanol consumption remained the same (Koivisto et al., 1993). After the hysterectomy, the voluntary ethanol consumption of the AA rats was slightly decreased, but the line difference remained clear in the derived rats (Sarviharju & Jaakkola, 1994). The present study was designed to examine the effects of aging and lifelong forced ethanol exposure on voluntary ethanol consumption and ethanol sensitivity (motor impairment) in both sexes of AA and ANA rats. The forced ethanol feeding regimen was chosen because the ANA rats do not drink ethanol voluntarily (Eriksson, 1973). The present experimental setting was also used to compare the effects of aging and chronic ethanol exposure on the nervous systems of male versus female rats and of the AA versus ANA lines of rats, respectively (Lu et al., 1997; Riikonen et al., 1999; Rintala et al., 1997, 1998). For the administration of ethanol to animals, a liquid diet (Lieber & DeCarli, 1989; Lindros & JaÈrvelaÈinen, 1998), ethanol vapor (Goldstein & Pal, 1971), intragastric intubation (Tsukamoto et al., 1984), and ethanol in the drinking fluid (Keegan et al., 1995) have been used in other studies. Only the liquid diet and ethanol in the drinking fluid can be considered practical over a lifelong period. The present study used forced 12% ethanol; that is, ethanol solution as the only source of fluid for a lifelong ethanol ingestion. Preliminary results of the present study were reported at the Fourth IBRO World Congress of Neuroscience, Kyoto, 1995 (Sarviharju et al., 1995).

2. Materials and methods 2.1. Animals Altogether, 160 male and 159 female rats of the alcohol-preferring AA and the alcohol-avoiding ANA lines (generations F65, F67, and F69) were used for the study: 99 male AA rats, 98 female AA rats, 61 male ANA rats, and 61 female ANA rats. The rats were housed individually in stainless steel wire-mesh cages when voluntary ethanol consumption was measured and in groups of four or five in stainless steel cages during the forced ethanol exposure. The animals received standard food (RM1(E)SQC, SDS, Witham, Essex, England) ad libitum in powder form during the ethanol self-selection periods and in pellet form during the forced exposure. The rats were maintained under a 12± 12 h light ± dark cycle (lights on at 6:00 a.m.) at a temperature of 20 ‹ 1°C and a relative humidity of 50 ‹ 5%.

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All the animal experiments were approved by the Institutional Animal Care and Use Committee at Alko Group Ltd. 2.2. Ethanol consumption At the age of 3 ±4 months, the AA and ANA rats were randomly assigned to either the ethanol-fed or the water group. The experiment consisted of two kinds of ethanol exposure: (1) voluntary ethanol consumption for 1 month at the age of 3± 4 months and again at 23 months of age and (2) between these periods, forced ethanol consumption with 10% ±12% (vol./vol.) ethanol as the only source of fluid. The individual intake of water and ethanol was measured by giving the rats a free choice between water and 10% (vol./ vol.) ethanol for 3 weeks (Eriksson, 1968). The free-choice period was preceded by a 1-week habituation period, when only 10% (vol./vol.) ethanol solution was offered. The positions of the bottles during the self-selection period were switched weekly to counteract the effect of position preference. During the self-selection ethanol intake, food consumption and body weights were monitored weekly, and the values of the third week were used in the statistical analysis. When the forced ethanol exposure was started, 10% (vol./ vol.) ethanol was the only source of fluid for the first month and then 12% (vol./vol.) ethanol was the only source for the remaining time. The 12% ethanol solution was chosen because results of preliminary tests showed that the ANA rats refused to drink higher concentrations of ethanol, which resulted in dehydration. The control group had free access to water only. The body weights and the consumption of food and fluids were monitored monthly for a period of 1 week during the forced exposure. New groups were introduced to the feeding regimen every 12th month. The results of the three cohorts were pooled together. The blood ethanol levels of randomly selected rats from each group (n = 10) in generation F65 were measured by taking a 0.05-ml blood sample from the tip of the tail at 6:30 a.m. and determining the levels by gas chromatography (Eriksson, 1973). 2.3. Ethanol-induced motor impairment The AA rats of the F65 generation were tested twice for sensitivity to ethanol-induced motor impairment on the tilting-plane test (Arvola et al., 1958; Hellevuo et al., 1989): at the beginning (4 months old, ethanol naive) and at the end (24 months old) of the exposure. The ethanol-fed groups were not withdrawn from ethanol before the later sensitivity test. In this test, the animal was placed on a wire, cloth-covered plane, which was tilted at a constant speed from horizontal to vertical in 5 s. The tilting was automatically stopped by the rat's sliding backward to the lower edge of the plane, and the sliding angle was recorded. Each rat was given a three-trial pre-ethanol test and was then injected intraperitoneally with 2 g/kg of ethanol (12% [wt./vol.] ethanol in 0.9% saline) and tested again 30 min later. A 0.05-ml blood sample was taken from

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the tip of the tail after the tilting test to determine the blood ethanol concentration by gas chromatography (Eriksson, 1973). The difference in sliding angles before the injection of ethanol and 30 min after injection was then calculated and used as the measure of motor performance under the influence of ethanol. 2.4. Statistics Comparisons between the different treatment groups on free-choice ethanol drinking and on ethanol sensitivity at the ages of 4 and 24 months were performed with either unpaired or matched-pairs Student t tests. Data obtained from the forced ethanol drinking phases in caged groups from the three generations were pooled and expressed as the mean ethanol intake, mean total fluid intake, mean food intake, and mean body weight during five blocks of 3 or 4 months. The underlying assumption was that the differences between the mean values in each generation, at a particular age, were caused only by chance sampling differences; that is, that there were no real differences between the generations. Therefore, it was possible to obtain estimates of the variability between individual animals (e.g., in food intake), even though the group housing precluded taking individual measurements. These data were analyzed with the use of a two-way (treatment and line) analysis of variance (ANOVA) with repeated measures on one factor (time). Differences were considered significant when P < .05. 3. Results 3.1. Ethanol consumption During the self-selection periods, the AA rats consistently drank very large amounts of ethanol, whereas the ANA rats avoided it (Table 1). The difference in voluntary ethanol consumption between the strains was thus highly significant at both 4 months and 23 months of age (Table 1). The individual voluntary ethanol consumption by AA male rats decreased significantly after 20 months of forced ethanol exposure. The age-related decrease in voluntary ethanol consumption was more pronounced for the ethanol-exposed AA males than for the water-exposed rats. Voluntary ethanol consumption by AA female rats also decreased at 23 months of age compared with the initial consumption in the water-exposed group, but not in the ethanol-exposed group. No significant changes in voluntary ethanol consumption with age or with different exposures were seen in the ANA rats. The old AA female rats drank significantly more ethanol than did the old AA males. When the old ANA ethanol-exposed rats were moved from the 20month forced ethanol drinking to the water versus 10% ethanol self-selection situation, they still preferred water to ethanol. In the voluntary situation at the beginning and at the end, the AA rats consumed about 90% of their fluid

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Table 1 Daily ethanol consumption as absolute alcohol (g/kg/day) and body weights during the self-selection periods (means ‹ S.D.) Age 4 months

Age 23 months

Strain

Sex

Group

n

Weight (g)

Absolute alcohol (g/kg/day)

AA AA AA AA ANA ANA ANA ANA

M M F F M M F F

E W E W E W E W

52 47 50 48 34 29 33 28

329 ‹ 37 333 ‹ 46 203 ‹ 18 202 ‹ 19 397 ‹ 41e 388 ‹ 42 232 ‹ 21f 230 ‹ 24f

6.31 ‹ 1.88 6.03 ‹ 1.97 6.84 ‹ 1.82 6.58 ‹ 1.93 0.24 ‹ 0.35 0.13 ‹ 0.09 0.31 ‹ 0.73 0.32 ‹ 0.43

n

Weight (g)

Absolute alcohol (g/kg/day)

43 36 43 36 13 8 11 4

565 ‹ 83 548 ‹ 75 322 ‹ 54 304 ‹ 55 555 ‹ 77 598 ‹ 50 371 ‹ 54e 369 ‹ 72e

3.78 ‹ 1.27a,b 4.13 ‹ 1.27a,b 6.46 ‹ 2.07c 5.53 ‹ 1.82d 0.28 ‹ 0.16 0.32 ‹ 0.31 0.62 ‹ 0.45 0.37 ‹ 0.32

AA = Alcohol-preferring AA (Alko, Alcohol) rats; ANA = alcohol-avoiding ANA (Alko, Non-Alcohol) rats; E = ethanol-exposed group; W = waterexposed group; M = male; F = female. The mean consumption figures of the third week are used. a P < .001 compared initial versus final. b P < .001 compared with corresponding females. c P < .05 compared with water-exposed group. d P < .05 compared initial versus final. e P < .01 compared with corresponding AA group. f P < .001 compared with corresponding AA group.

intake as ethanol solution, and the ANA rats consumed only from 3% to 10%. There was no significant strain difference in ethanol consumption during the forced ethanol drinking, but the AA males showed a tendency ( P < .07) toward drinking more (Fig. 1). The females of both strains consumed significantly ( P < .001) more ethanol than did the males.

With age, the ethanol consumption of the AA and ANA females and the ANA males increased significantly ( P < .01; AA females: from 7.26 ‹ 0.24 to 7.76 ‹ 0.24; ANA females: from 6.16 ‹ 0.35 to 7.14 ‹ 0.28; ANA males: from 3.83 ‹ 0.26 to 4.99 ‹ 0.30 g/kg/day as absolute alcohol). The AA males, however, had a slight decrease (from 5.77 ‹ 0.16 to 5.15 ‹ 0.24 g/kg/day), thus causing the time  strain interaction to show a significant ( P < .05) difference for the male rats. The blood ethanol concentration values (mean ‹ S.D., expressed as millimoles per liter) during the forced exposure, measured early in the morning, were: 8.8 ‹ 6.2 for AA males; 6.9 ‹ 4.8 for AA females; 1.6 ‹ 2.8 for ANA males; and 3.9 ‹ 2.8 for ANA females. 3.2. Intake of food and fluid, body weights

Fig. 1. Intake of ethanol as absolute alcohol (g/kg/day) during lifelong forced ethanol consumption. The consumption was measured monthly during a 1-week period. Each point represents the mean consumption of 3 ± 4 months. Means ‹ S.E. are given. AA F = Female alcohol-preferring AA (Alko, Alcohol) rats; AA M = male AA rats; ANA F = female alcoholavoiding (Alko, Non-Alcohol) rats; ANA M = male ANA rats.

There were significant differences ( P < .01 for males and P < .001 for females) in the total fluid intake between the water-exposed and the ethanol-exposed groups throughout the experiment (Fig. 2). The waterexposed groups drank more in all other groups except for the AA males, for which the total fluid intake was higher in the ethanol-exposed group. The females drank more than the males. The intake of food in the ethanol-exposed groups was significantly lower ( P < .001 for the AA males and females and the ANA females and P < .01 for the ANA males) than in the water-exposed groups (Fig. 3). With age, food consumption decreased significantly ( P < .001) in all the groups. Ethanol constituted from about 25% to 30% of the total calories consumed in the AA ethanolexposed groups and slightly less (23% ± 26%) in the ANA ethanol-exposed groups. The total energy intake did not differ significantly between the water- and ethanol-exposed

M. Sarviharju et al. / Alcohol 23 (2001) 157±166

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Fig. 2. Intake of fluid (water or ethanol [EtOH]) during lifelong forced ethanol consumption. Each point represents the mean of 3 ± 4 months. Means ‹ S.E. are given. AA F = Female alcohol-preferring AA (Alko, Alcohol) rats; AA M = male AA rats; ANA F = female alcohol-avoiding (Alko, Non-Alcohol) rats; ANA M = male ANA rats.

groups or between the AA and the ANA strains. The total energy intake by the females of all treatment groups and of both strains was higher than that of the males. A signifi-

cant time  strain interaction on energy intake was found in AA and ANA male groups ( P < .05 for ethanol-exposed groups and P < .001 for water-exposed groups). A signifi-

Fig. 3. Intake of food during lifelong forced ethanol (EtOH) consumption. Each point represents the mean of 3 ± 4 months. Means ‹ S.E. are given. AA F = Female alcohol-preferring AA (Alko, Alcohol) rats; AA M = male AA rats; ANA F = female alcohol-avoiding (Alko, Non-Alcohol) rats; ANA M = male ANA rats.

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Fig. 4. Effect of lifelong forced ethanol (EtOH) consumption on body weight. Each point represents the mean of 3 ± 4 months. Means ‹ S.E. are given. AA F = Female alcohol-preferring AA (Alko, Alcohol) rats; AA M = male AA rats; ANA F = female alcohol-avoiding (Alko, Non-Alcohol) rats; ANA M = male ANA rats.

cant time  treatment interaction was found for the ANA males ( P < .01). There was no significant difference in body weight between the ethanol- and water-exposed groups within the AA or the ANA strain (Fig. 4). Body weights of all groups increased significantly ( P < .001) with age. The strain differences were significant ( P < .05 for the ethanol-exposed males, P < .001 for the ethanol-exposed females, and P < .001 for the water-exposed males and females) in all the groups, the ANA rats being heavier.

In the males, aging did not produce any differences in ethanol sensitivity. There was a significant difference in motor impairment ( P < .001) between the AA and the ANA females when tested at 4 months, the latter being more sensitive. At the age of 24 months, it was not possible to conduct the test with the ANA rats, because many of the old ANA rats had tumors in their abdomens.

3.3. Sensitivity to motor impairment

An intervening 19 months of forced ethanol drinking did not increase the voluntary consumption of ethanol at 23 months of age over that at 4 months of age in any of the groups. At most, for AA females it tended to weaken the

On the tilting-plane test, the old AA female rats were more sensitive to ethanol than the young ones were (Table 2).

4. Discussion

Table 2 Ethanol-induced motor impairment measured on a tilting plane Age 4 months

Age 24 months

Group

n

Performance

BEC (mM)

Group

n

Performance

BEC (mM)

AA, M ANA, M AA, F ANA, F

60 24 60 24

14.4 ‹ 0.8 16.2 ‹ 1.4 8.7 ‹ 0.8a 14.7 ‹ 1.6c

43.9 ‹ 0.8 43.4 ‹ 1.1 38.3 ‹ 0.8a 46.1 ‹ 2.1c

AA, AA, AA, AA,

22 20 17 21

14.9 ‹ 1.4 12.5 ‹ 1.4 16.8 ‹ 1.9b 17.8 ‹ 1.6b

43.4 ‹ 2.6 40.9 ‹ 2.5 41.1 ‹ 3.0 38.1 ‹ 2.5

E, M W, M E, F W, F

AA, F = Female alcohol-preferring AA (Alko, Alcohol) rats; AA, M = male AA rats; ANA, F = female alcohol-avoiding (Alko, Non-Alcohol) rats; ANA, M = male ANA rats; BEC = blood ethanol concentration; E = ethanol-exposed group; W = water-exposed group. Values show the difference as degrees between pre-ethanol value and test value (mean ‹ S.E.). Blood ethanol concentrations after testing at 30 min are given. a P < .001 compared with corresponding AA males. b P < .001 final at age 24 months versus initial at age 4 months. c P < .001 compared with corresponding AA females.

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decrease with age seen in other AA groups. All ANA groups continued to avoid almost all ethanol whenever water was available. This study was a continuation of previous work (Hervonen et al., 1992; Jaatinen et al., 1992), in which the interactions between aging and chronic ethanol exposure were studied in AA rats by using a voluntary ethanol feeding regimen. The effect of this feeding regimen on ethanol-related behaviors and longevity proved to be minimal. Therefore, in the present work, a heavier treatment with forced ethanol drinking and a higher ethanol concentration were used in an attempt to produce a model that parallels the lifelong drinking of human alcoholics. The original aim was to introduce the animals gradually to 15% (vol./vol.) ethanol, but this concentration proved to be too high for the ANA rats, which showed signs of dehydration on this concentration. Therefore, the 12% ethanol solution was used in the experiment. One problem associated with this design is the dropping out of subjects. Many rats died in the course of the experiment, which made the sample giving the final measurements very different from the original sample. This drop-out effect can be seen, for instance, in the total fluid intake of the ANA female water-exposed group, for which a considerable rise in water consumption was seen at the end of the experiment. This increase was probably due to the death of the weaker rats with lower fluid intakes. Changes over the time may have partly resulted from the dropping out of subjects and not from aging or ethanol exposure per se. However, all experimental groups lost subjects. It could be argued that the remaining animals were still a representative sample of each experimental group and that the observed differences between the groups were not affected. The health status of the experimental animals is essential in gerontological research. The health status of animals in lifelong experiments must be surveyed because of the high incidence of pathological changes associated with increasing age. In the present lifelong experiment, the environment was monitored carefully, and the health status of the animals was surveyed continuously. No pathological microbes were found in the serological analyses. Differences in circadian drinking rhythms between the AA and the ANA lines have been found elsewhere (Aalto, 1986). The AA rats have a tripodal pattern of ethanol intake during the dark period, but the ANA rats drink ethanol rather evenly throughout the dark period in the voluntary situation. The blood ethanol levels checked early in the morning showed that several AA rats had ethanol levels of about 10 mol/l, which are considered to cause inebriation in both human beings and experimental animals. The blood ethanol values of the ANA rats were naturally lower because of their lower intake of ethanol and of a different drinking rhythm. When ethanol was served as the only available fluid, a difference between the sexes was found in both strains,

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with the females consuming more ethanol throughout the exposure. This finding is consistent with results of earlier studies showing that females consume more ethanol (Eriksson & MalmstroÈm, 1967; Lancaster & Spiegel, 1992). The difference between sexes in voluntary ethanol consumption found in the old AA rats, but not in the young rats, may suggest different age-related alterations in sensitivity to ethanol or in ethanol metabolism between males and females. The difference between sexes itself in ethanol drinking may be due to a difference in ethanol metabolism. The ethanol metabolic rates of adult female rats and mice are faster than those of male animals (Eriksson & MalmstroÈm, 1967; Rachamin et al., 1980). Because immature and aged female animals (Collins et al., 1975; Rachamin et al., 1980) do not have faster ethanol elimination rates than males, the difference between the sexes of mature animals is believed to be of hormonal origin; that is, the presence of higher levels of progesterone or estrogen in mature females may enhance the rate of ethanol metabolism. The difference between sexes in ethanol intake may also be due to differences in brain dopamine systems (Blanchard & Glick, 1995), although dopamine does not seem to mediate ethanol reinforcement in AA rats (Kiianmaa et al., 1995; Nurmi et al., 1996). During the self-selection period, the young (4 month old) AA rats consumed amounts of ethanol similar to those drunk during the beginning of the forced exposure, but the old AA rats consumed less ethanol voluntarily than they did during the forced drinking. The reduction in voluntary intake of ethanol after long-term forced ethanol consumption in our study is parallel to the result obtained by McClearn (1972). In accordance with these observations, results of several studies showed old animals to consume less ethanol voluntarily than do young animals (Abel, 1978; Hervonen et al., 1992; Wood, 1976), although opposite results also were reported (Kahn, 1975; Wallgren & Forsander, 1963). Animals previously exposed to ethanol were shown to consume more ethanol solution than did nonexposed animals (Parisella & Pritham, 1964). In our study, only the AA females consumed more ethanol solution than did the nonexposed (water-exposed) controls. The rats were housed individually when their voluntary ethanol consumption was measured, whereas during the forced ethanol consumption, the rats were housed in groups of four or five animals. It is therefore possible that the housing conditions could have interfered with ethanol drinking, and slightly different results for voluntary ethanol consumption could have been obtained for group-housed animals. To what extent the rearing conditions influence drinking behavior has, to our knowledge, not been studied. It is also possible that changing the environment from that in which forced ethanol consumption had taken place for several months prevented an effect on voluntary consumption. The drinking behavior of the AA rats is in line with earlier observations (Hervonen et al., 1992), which seem to

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indicate that voluntary ethanol intake by AA male rats decreases with age. In all other groups of the present study, forced ethanol consumption increased with age. The ANA rats, given a free choice, drink virtually no ethanol. However, when no water is available, the ANA rats drink enough ethanol solution to maintain their body weights, and at the end of the exposure, the ANA females drank even more ethanol than did the AA males. This result is in line with that obtained by Sinclair and Suomela (1994). The ethanol consumption by the ANA rats increased gradually during the forced feeding, in line with the findings of Files et al. (1993), who showed that alcohol-nonpreferring (NP) rats consume ethanol under continuous access conditions and will slowly initiate ethanol self-administration, but to a lesser extent and at a slower rate than alcoholpreferring (P) rats. The ANA rats, however, when moved from the situation of forced ethanol exposure to that of voluntary ethanol consumption at the age of 23 months, dramatically returned to the ethanol-nonpreferring state and avoided ethanol as they usually do. This finding confirms the importance of genetic factors in controlling ethanol consumption by AA and ANA rats. Van Haaren and Anderson (1993) reported that male rats ingested ethanol solution with a higher ethanol concentration than did female rats to obtain maximum ethanol intake (g/kg) in a schedule-induction environment. Males also reached higher blood ethanol levels than did females. It is possible that the concentration of ethanol solution in the lifelong feeding regimen was too low for the AA male rats to reach their maximum consumption. The growth rate of all the experimental groups indicates that all the animals received an adequate quantity of food. In accordance with our previous report (Hervonen et al., 1992), the ethanol-exposed groups reduced their food intake so as to keep the sum of energy from food and ethanol constant (Eriksson, 1969; Forsander, 1988; Gill et al., 1996; Richter, 1941). In the present study, the ANA rats obtained from 19% to 26% of their daily energy intake from ethanol, and the AA rats obtained from 23% to 28%. This percentage is only a part of the maximum amount of ethanol that could have been metabolized (Lindros et al., 1972). It must, therefore, be assumed that ethanol consumption is seldom limited by the rate of ethanol oxidation and that other factors limit the intake of ethanol. For ANA rats, one such factor has been suggested to be the accumulation of acetaldehyde (Eriksson, 1980; Koivisto & Eriksson, 1997). ANA and AA rats have been shown to have different hypothalamic monoamine mechanisms controlling food intake, which could also partly account for their differential ethanol acceptance (Korpi et al., 1991). However, it is unlikely that animals with a preference for ethanol solutions consume ethanol as a food. Both the AA and the ANA rats have been reported to equally reduce their intake of calories from carbohydrates when given ethanol solution as their only fluid (Forsander, 1988), which is in line with the present

observations. Protein intake affects the ethanol consumption of AA rats through a factor correlated to ethanol elimination, and ethanol in turn reduces carbohydrate consumption, but the ethanol intake of ANA rats is not affected by the amount of protein ingested (Forsander & Sinclair, 1988). With age, the total fluid consumption decreased in the water-exposed groups, but it did not in the ethanol-exposed groups except for the AA males. Despite that the animals consumed unequal volumes of fluid, dehydration in the ethanol-exposed groups is unlikely because no growth retardation was seen in the ethanol-exposed rats, as would be expected if they suffered from chronic dehydration. Beilin et al. (1992) reported that plasma volumes remain unaltered with chronic administration of moderate amounts of ethanol. In male AA rats, aging did not produce any differences in sensitivity to ethanol-induced motor impairment, in line with our earlier results (Hervonen et al., 1992). In the female rats, on the contrary, an aging effect was seen: the old AA females showed an increased sensitivity compared with the findings for young AA females. In accordance with our findings, general motor activity was reported to be more impaired in old female C57BL/6J mice compared with findings for younger animals subsequent to an ethanol injection of 3 g/kg (Abel, 1978). Age differences in motor activity were not significant when the mice were injected with ethanol doses of 1 or 2 g/kg. Of the young rats, the AA females seemed to be less sensitive to ethanol-induced motor impairment than were the ANA females or the male rats of either line. No sex-based or line differences were observed, however, when the differences in blood ethanol concentration were taken into account. We also did not find any major sex-based or line (AA versus ANA) differences in the ethanol-induced degeneration of the central or peripheral nervous system, studied in the present lifelong feeding regimen (Lu et al., 1997; Riikonen et al., 1999; Rintala et al., 1997, 1998). In conclusion, voluntary ethanol consumption in AA rats decreased with age regardless of whether the rats were kept on only ethanol or only water for the intervening 19 months. The only significant effect of the treatment was that the decrease was significantly smaller for ethanol-exposed AA females than for water-exposed AA females. With age and under the forced ethanol exposure, ethanol consumption by the ANA female and male rats and by the AA female rats increased, but ethanol consumption by the AA males decreased. The old AA female rats were more sensitive to ethanol than were the young ones. For males, aging did not produce any differences in ethanol sensitivity. We have shown that the voluntarily nondrinking ANA rats drink ethanol almost as much as the voluntarily drinking AA rats when they are forced to drink ethanol and that lifelong forced ethanol drinking does not change their inherent drinking habits.

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Acknowledgments We thank Ms. Jaana Koukkarinen, Ms. Lea Karvonen, and Ms. Rina Leo-GranstroÈm, for taking good care of the animals; Ms. Hilkka Salohalla for excellent technical assistance; and Dr. David Sinclair for valuable comments and corrections. The participation of Dr. Jaatinen and Professor Hervonen in the study was supported by the Medical Research Fund of Tampere University Hospital.

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