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Early weaning effects on voluntary ethanol consumption and stress responsivity in rats
Physiology&Behavior,Vol. 40, pp. 673-676. Copyright©PergamonJournals Ltd., 1987. Printed in the U.S.A.
0031-9384/87 $3.00 + .00
Early Weaning Effects on Voluntary Ethanol Consumption and Stress Responsivity in Rats G. E. R O C K M A N , '
A. HALL, L. MARKERT
Department of Psychology, University of Winnipeg Winnipeg, MB Canada, R3B 2E9 AND G. B. G L A V I N 2
Department of Pharmacology and Therapeutics, University of Manitoba, Winnipeg, MB R e c e i v e d 9 M a r c h 1987 ROCKMAN, G. E., A. HALL, L. MARKERT AND G. B. GLAVIN. Early weaning effects on voluntary ethanol consumption and stress responsivity in rats. PHYSIOL BEHAV 40(5) 673-676, 1987.--The present study examined the effect of early maternal deprivation (early weaning) on voluntary ethanol consumption and responses to cold-immobilization stress in adult rats. Rats were weaned at 16 and 21 days of age and housed individually with food and water ad lib until they reached 190_+5 g at which time half of the animals from each group were exposed to increasing concentrations (3 to 9%) of ethanol in a free choice with water every alternate day. Following acquisition of ethanol drinking, animals were provided with water and ethanol (9%) daily for eight days. At the end of this period, animals were divided into stressed and non-stressed groups. Stressed animals were exposed to cold immobilization stress for 3 hr. Results showed that the early-weaned animals consumed significantly more ethanol as compared to normal-weaned animals. Stomach pathology data revealed little ulcer formation in water-only groups. However, normal-weaned/ethanol-exposed animals exhibited significantly more severe ulcers when compared to all other water- or ethanol-exposed groups. We suggest that early maternal deprivation may predispose animals to increased ethanol consumption as adults. Stress ulcer susceptibility in these animals was likely influenced by interactions among the effects of early weaning, ethanol intake and handling and needs further clarification. Stress
Ulcer
Ethanol
Early weaning
R E S E A R C H examining the effects of early maternal separation (early weaning) on subsequent biological and behavioral measures has yielded many interesting results. F o r example, it has been observed that rat pups separated early from their mothers demonstrated an unusually high incidence of gastric ulcers when exposed to restraint stress at a later time [3]. Subsequent studies have confn'med and expanded this initial finding. Specifically, early weaning has been shown to disturb thermoregulation [2], increase gastric acid secretion [1,2] and increase insomnia and quiet wakefulness [3]. In addition, the enhanced susceptibility to restraint stress-induced gastric ulcers of early weaned animals has recently been attributed to a decrease of fat stores in these animals [5]. Furthermore, when early weaned female rats grow to adulthood and are mated with normal weaned animals, their normally weaned progeny also exhibit an increased susceptibility to restraint induced gastric ulcers [12]. Consequently, it seems quite clear that early weaning in rats is a rather robust treatment resulting in many subsequent biological and behavioral alterations. While the vast majority of
research in this area has employed animal subjects, the question of whether such a phenomenon occurs in human subjects has been addressed. Specifically, 'it has been suggested that the incidence of peptic ulcers in adolescents may be preceded by an early life maternal separation or loss [4]. It therefore seems that early maternal separation in rats and possibly in humans may induce significant and similar biological and behavioral changes. Whether early weaning in rats alters other behaviors in addition to stress responsivity has, as yet, not been ascertained and is the focus of the present study. We investigated the effects of early weaning of male rats on subsequent voluntary ethanol consumption and response to cold-immobilization stress. METHOD
Subjects Twenty female and 10 male Wistar rats (Holtzman Co., Madison, WI) were used to breed 64 male offspring which served as subjects. During the gestation, birthing and pre-
~Supported by NSERC A2605. 2Supported by NSERC A1590.
673
674
ROCKMAN, H A L L , MARKERT AND G L A V I N
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3%
5%
7%
alternate day Eorly Weoned n = 1 7
9%
9%
9%
Ethanol Conc. ~
N o r m o l Weoned n = 1 5
FIG. 1. Ethanol consumption in terms of mean grams per kilogram for early-weaned (n= 17) and normal-weaned (n = 15) rats during both the alternate day and everyday ethanol exposure period. weaning periods, females were housed in standard birthing cages. Pups were designated, in a semi-random fashion, to be weaned at 16 (early) or 21 (normal) days after birth. In other words, care was taken to ensure that each weaning group (normal or early) contained a random sample of male pups from each litter. After weaning, animals were housed in standard individual cages. Those which were weaned at 16 days were provided with a fleece cage liner to prevent excessive loss of body heat. F o o d and water were available ad lib. Room temperature was regulated at 25°C with a 12 hr light/dark cycle (lights on at 0700 hr). When animals reached 190___5 g, rats from the early-weaned and normal-weaned groups were randomly assigned to either water-only or ethanol-exposed groups. Water-only groups received two tubes of water daily and were weighed every second day. Animals exposed to ethanol were treated in the following manner.
tube was changed upon each presentation to eliminate the possibility of formation of a position preference by the rats. The same alternate day presentation was continued for ethanol concentrations of 5%, 7% and finally 9% (v/v). Therefore, during this initial ethanol exposure phase rats were exposed to each concentration of ethanol (3 to 9% v/v) on four occasions totaling to 16 ethanol exposure days. At the end of the alternate day ethanol exposure procedure, all rats received the 9% (v/v) ethanol in an everyday free-choice with water for eight consecutive days. Therefore, the rats were exposed to ethanol for a grand total of 24 days prior to immobilization. Fluid consumption (both ethanol and water) and body weight were monitored for each day of ethanol presentation and ethanol intake in grams per kilogram was calculated for those days. Fluid leakage from the ethanol tubes was prevented by using double ballbearing valves at the end of the spouts.
Ethanol Exposure
Immobilization Stress
Two calibrated drinking tubes were attached to the left front of each cage. One contained tap water while the other initially contained a 3% (v/v) solution of ethanol. This concentration was presented every alternate day over an 8 day period; that is, every other day the rats received two tubes of water and on intervening days, they received one tube of water and one tube of ethanol. The position of the ethanol
Following the last day of ethanol exposure, all animals were deprived of food and ethanol but not water for 24 hours. Animals from the early-weaned/ethanol-exposed, early-weaned/water-only, normal-weaned/ethanol-exposed and normal-weaned/water-only groups were divided into stressed and non-stressed groups. Care was taken to ensure that a comparable number of high, medium and low ethanol
EARLY WEANING AND ETHANOL CONSUMPTION consuming animals were included in both the stress and non-stressed groups. This is important in light of data from this laboratory indicating a differential effect of stress depending upon pre-stress levels of ethanol consumption [9,10]. Following food deprivation, stressed animals were placed in standard Plexiglas immobilization tubes for 3 hr at 4--6°C. Non-stressed animals were handled at the beginning and at the end of the stress period but were not immobilized.
675 TABLE 1 SUMMARY OF STOMACH PATHOLOGY FOR EARLY-WEANED AND NORMAL-WEANED RATS (MEAN ± SEM)
Ulcer Incidence
Number of Ulcers
Cumulative Ulcer Length (nun)
Early-Weaned Stress
3/8
0.6 (0.4)
3.3 (2.3)
Normal-Weaned Stress
6/7
2.7 (1.3)
14.3 (5.4)
Early-Weaned No Stress
3/9
0.9 (0.6)
3.0 (2.2)
Normal-Weaned No Stress
1/8
0.8 (0.8)
3.6 (3.6)
Early-Weaned Stress
1/8
0.1 (0.1)
0.4 (0.4)
Normal-Weaned Stress
3/7
0.7 (0.4)
3.8 (1.9)
Early-Weaned No Stress
1/9
0.6 (0.2)
4.0 (4.5)
Normal-Weaned No Stress
2/8
0.5 (0.5)
4.2 (3.5)
Group Ethanol-Exposed
Stomach Pathology Following the immobilization period, all rats were sacrificed with CO2. Stomachs were excised, preserved in 10% formaldehyde (v/v) and examined for damage by an observer who was naive with respect to treatment conditions. The number (mean number of ulcers per rat per group), location (rumenal or glandular) and cumulative length (ulcer severity defined as mean ulcer length per rat per group) of the ulcers were ascertained by examination under a dissecting microscope with an ocular micrometer. Ulcer length (in ram) was determined by adding length plus width of each glandular ulcer.
Statistical Analysis Ethanol consumption for both early-weaned and normal-weaned animals was calculated in terms of mean grams per kilogram per day (g/kg). Mean grams per kilogram of ethanol consumed during exposure to each concentration of ethanol was calculated. In other words, the average group intake of ethanol was calculated for 3, 5, 7 and 9% ethanol during the alternate day ethanol exposure procedure. In addition, ethanol consumption during the subsequent eight everyday ethanol presentation was calculated as two periods of four days each. Hence, for purposes of data presentation and analysis, ethanol consumption is presented over 6 periods of 4 days each. All data were analyzed by repeated measures analysis of variance [group (early vs. normalweaned) X period (ethanol exposure period)], and appropriate post-hoc (Tukey) tests, and simple main effects analysis when interactions were significant. RESULTS
Figure 1 illustrates ethanol consumption for both earlyweaned and normal-weaned animals during the initial alternate day and subsequent everyday ethanol presentation schedule. Analysis of variance yielded a significant main effect of group, F(1,30)=7.44, p<0.01, and a significant main effect of period, F(5,150)=15.73, p<0.001. Furthermore, a significant group x period interaction was observed, F(5,150)=2.22, p<0.05. Post-hoc Tukey tests revealed that ethanol consumption for the early- and normal-weaned groups differed in that the early-weaned animals consumed significantly more ethanol (p<0.01) when exposed to 7 and 9% ethanol during the alternate day exposure schedule. The enhanced ethanol consumption observed among the earlyweaned animals was maintained during the subsequent everyday ethanol exposure periods (p<0.05). It should also be noted that among early-weaned rats, 16 out of 17 animals consumed ethanol to the extent that it comprised in excess of 60% of their total daily fluid intake. Finally, no significant differences were observed in body weight between the groups and no overt signs of ethanol induced intoxication were observed in any of the ethanol exposed animals at any time.
Water Only
Examination of stomach ulcer data as indicated in Table 1 revealed a very low degree of ulcer development in the water-only stressed animals. Alternatively, the normalweaned animals exposed to ethanol exhibited considerably greater ulcer severity as compared to earlyweaned/ethanol-exposed animals [Main effect of groups: F(1,28)=4.48, p<0.04. Main effect of stress: F(1,28)=3.90, p < 0 . 0 5 ] . No significant differences in ulcer frequency (mean number of ulcers per rat per group) or ulcer incidence (number of rats per group which developed ulcers) were observed. DISCUSSION
The results from the present study revealed several interesting findings. The most significant is that early-weaned animals, when exposed to ethanol in adulthood, consumed significantly more ethanol than normal-weaned controls. This finding clearly has profound implications for the materhal deprivation literature. While previous studies have demonstrated an enhanced ulcer proliferation in animals separated early from their mothers [1-3, 5], the present data indicate that an additional consequence of early maternal separation may be manifested in a greater propensity to consume ethanol. It is also important to note that not only did the early-weaned animals as a group consume more ethanol as compared to the normal-weaned animals, but the variability in amounts of ethanol consumed by the early weaned animals was relatively small. For example, in the last ethanol drinking period, 16 out of 17 early-weaned animals were drinking ethanol at a high preference ratio compared to water
676
ROCKMAN, HALL, MARKERT AND GLAVIN
(greater than 60%), whereas only 7 out of 15 normal-weaned animals consumed ethanol at this level. Clearly, the earlyweaned animals' drinking level and pattern of drinking was higher and more consistent than the normal-weaned animals. While the mechanism underlying the enhanced ethanol intake of the early-weaned rats is unknown at this time, studies in this laboratory are presently being conducted in an effort to determine whether neurochemical alterations induced by early weaning may account for the observed enhanced ethanol intake among these rats. The ulcer data from this study are interesting but somewhat contradictory. It was observed that the normal-weaned animals exposed to both ethanol and stress exhibited a significantly greater degree of ulcer severity as compared to the early-weaned/ethanol-exposed animals and all water-only control groups. This finding suggests that ethanol exacerbated ulcer development which is consistent with data from this laboratory indicating that ethanol, in some circumstances, can enhance stress ulcer formation [10,11]. In contrast, it appears that ethanol served to reduce ulcer development among early-weaned animals. This is especially important in view of the fact that the early-weaned animals consumed g r e a t e r amounts of ethanol prior to exposure to immobilization stress. The possibility that ethanol may have reduced ulcer severity in these animals is consistent with other studies demonstrating that different pre-stress levels of ethanol intake may, in some situations, have differential effects on ulcer severity [9,10]. This suggests the possibility that high ethanol consumption may mask the effects of stress, while low levels of ethanol intake may interact deleteriously with stress. This suggestion is supported by very recent data from this laboratory indicating that very high voluntary ethanol intake is associated with attenuated stress responses in environmentally enriched animals (manuscript in submission). The contradictory nature of the ulcer data from this study is illustrated by our failure to observe
enhanced ulcer proliferation in early-weaned/water/waterexposed animals. As mentioned previously, data from other studies demonstrated that early-weaned animals show enhanced ulcer development as a consequence of restraint stress [1-3, 5]. It is important to note that we employed a milder stress in the form of immobilization in this study, hence, this may account for lack of ulcer development in the early-weaned animals. In addition, at least two other factors may have contributed to the lack of ulcers in these animals. Rats in this study were stressed as adults at approximately 125 days of age. Previous studies demonstrating enhanced ulcer formation in early-weaned animals applied the stress treatment at 30 days of age [1-3]. Although 100-day-old early-weaned rats show enhanced ulcer formation in response to restraint stress [5], other researchers have suggested that stress responses in normal-weaned rats may diminish as animals age [7]. Consequently, in the present experiment, both the age of the animals when stressed and the time elapsed since the weaning manipulation may have contributed to the low ulcer formation. Finally, it is important to note that all animals in this study were handled initially every second day and subsequently everyday to correspond to the ethanol exposure schedule. This is important in light of data from this laboratory [8] as well as from others [6] indicating that handling alters neurochemical and behavioral responses to stress. It is therefore possible that handling p e r se may have contributed to ulcer development in normalweaned/non-stressed rats. Whether handling also altered the stress responses of the early-weaned animals in this study is unclear and we are presently investigating this issue.
ACKNOWLEDGEMENT The authors gratefully acknowledge the technical assistance provided by Michelle Del Rizzo.
REFERENCES 1. Ackerman, S. H. The developmental physiology of the stomach: possible contributions to the regulatory disturbances of duodenal ulcer disease. Proc Soc Exp Biol Med 175:119-126, 1984. 2. Ackerman, S. H. Early life events and peptic ulcer susceptibility: An experimental model. Brain Res Bull 5: 43--49, 1980. 3. Ackerman, S. H., M. A. Hofer and H. Weiner. Early maternal separation increases gastric ulcer risk in rats by producing a latent thermoregulatory disturbance. Science 201: 373-376, 1978. 4. Ackerman, S. H., S. Manaker and M. I. Cohen. Recent separation and the onset of peptic ulcer disease in older children and adolescents. Psychosom Med 43: 305-310, 1981. 5. Greenberg, D. and S. H. Ackerman. Reduced fat stores after early weaning: A correlate of vulnerability to stress ulcers. Physiol Behav 38: 375-379, 1986. 6. Irwin, J., P. Ahluwalia, R. M. Zacharko and H. Anisman. Central norepinephrine and plasma corticosterone following acute and chronic stressors: Influence of social isolation and handling. Pharmacol Biochem Behav 24:1151-1154, 1986.
7. Pare, W. P., G. P. Vincent and K. E. Isom. Age differences and stress ulcer in the rat. Exp Aging Res 5:31-42, 1979. 8. Rockman, G. E., A. M. Hall, L. Markert, G. B. Glavin and W. P. Pare. Ethanol-stress interaction: Immediate versus delayed effects of ethanol and handling on stress responses of ethanolconsuming rats. Alcohol 4: in press, 1987. 9. Rockman, G. E., A. Hall, J. Hong and G. B. Glavin. Unpredictable cold-immobilization stress effects on voluntary ethanol consumption in rats. Life Sci 40: 1245-1251, 1987. 10. Rockman, G. E., A. Hall and G. B. Glavin. Effects of restraint stress on voluntary ethanol intake and ulcer proliferation in rats. Pharmacol Biochem Behav 25: 1083-1087, 1986. 11. Rockman, G. E. and G. B. Glavin. Activity stress effects on voluntary ethanol consumption, mortality and ulcer development in rats. Pharmacol Biochem Behav 24: 869-873, 1986. 12. Skolnick, N. J., S. H. Ackerman, M. A. Hofer and H. Weiner. Vertical transmission of acquired ulcer susceptibility in the rat. Science 208: 1161-1163, 1980.
0031-9384/87 $3.00 + .00
Early Weaning Effects on Voluntary Ethanol Consumption and Stress Responsivity in Rats G. E. R O C K M A N , '
A. HALL, L. MARKERT
Department of Psychology, University of Winnipeg Winnipeg, MB Canada, R3B 2E9 AND G. B. G L A V I N 2
Department of Pharmacology and Therapeutics, University of Manitoba, Winnipeg, MB R e c e i v e d 9 M a r c h 1987 ROCKMAN, G. E., A. HALL, L. MARKERT AND G. B. GLAVIN. Early weaning effects on voluntary ethanol consumption and stress responsivity in rats. PHYSIOL BEHAV 40(5) 673-676, 1987.--The present study examined the effect of early maternal deprivation (early weaning) on voluntary ethanol consumption and responses to cold-immobilization stress in adult rats. Rats were weaned at 16 and 21 days of age and housed individually with food and water ad lib until they reached 190_+5 g at which time half of the animals from each group were exposed to increasing concentrations (3 to 9%) of ethanol in a free choice with water every alternate day. Following acquisition of ethanol drinking, animals were provided with water and ethanol (9%) daily for eight days. At the end of this period, animals were divided into stressed and non-stressed groups. Stressed animals were exposed to cold immobilization stress for 3 hr. Results showed that the early-weaned animals consumed significantly more ethanol as compared to normal-weaned animals. Stomach pathology data revealed little ulcer formation in water-only groups. However, normal-weaned/ethanol-exposed animals exhibited significantly more severe ulcers when compared to all other water- or ethanol-exposed groups. We suggest that early maternal deprivation may predispose animals to increased ethanol consumption as adults. Stress ulcer susceptibility in these animals was likely influenced by interactions among the effects of early weaning, ethanol intake and handling and needs further clarification. Stress
Ulcer
Ethanol
Early weaning
R E S E A R C H examining the effects of early maternal separation (early weaning) on subsequent biological and behavioral measures has yielded many interesting results. F o r example, it has been observed that rat pups separated early from their mothers demonstrated an unusually high incidence of gastric ulcers when exposed to restraint stress at a later time [3]. Subsequent studies have confn'med and expanded this initial finding. Specifically, early weaning has been shown to disturb thermoregulation [2], increase gastric acid secretion [1,2] and increase insomnia and quiet wakefulness [3]. In addition, the enhanced susceptibility to restraint stress-induced gastric ulcers of early weaned animals has recently been attributed to a decrease of fat stores in these animals [5]. Furthermore, when early weaned female rats grow to adulthood and are mated with normal weaned animals, their normally weaned progeny also exhibit an increased susceptibility to restraint induced gastric ulcers [12]. Consequently, it seems quite clear that early weaning in rats is a rather robust treatment resulting in many subsequent biological and behavioral alterations. While the vast majority of
research in this area has employed animal subjects, the question of whether such a phenomenon occurs in human subjects has been addressed. Specifically, 'it has been suggested that the incidence of peptic ulcers in adolescents may be preceded by an early life maternal separation or loss [4]. It therefore seems that early maternal separation in rats and possibly in humans may induce significant and similar biological and behavioral changes. Whether early weaning in rats alters other behaviors in addition to stress responsivity has, as yet, not been ascertained and is the focus of the present study. We investigated the effects of early weaning of male rats on subsequent voluntary ethanol consumption and response to cold-immobilization stress. METHOD
Subjects Twenty female and 10 male Wistar rats (Holtzman Co., Madison, WI) were used to breed 64 male offspring which served as subjects. During the gestation, birthing and pre-
~Supported by NSERC A2605. 2Supported by NSERC A1590.
673
674
ROCKMAN, H A L L , MARKERT AND G L A V I N
¥ \
r
v ® 0 E
2 -
, I i
E t3 r 4-, bJ
///
i// 1 -
/
/
XXX
y/
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/
/ I
3%
5%
7%
alternate day Eorly Weoned n = 1 7
9%
9%
9%
Ethanol Conc. ~
N o r m o l Weoned n = 1 5
FIG. 1. Ethanol consumption in terms of mean grams per kilogram for early-weaned (n= 17) and normal-weaned (n = 15) rats during both the alternate day and everyday ethanol exposure period. weaning periods, females were housed in standard birthing cages. Pups were designated, in a semi-random fashion, to be weaned at 16 (early) or 21 (normal) days after birth. In other words, care was taken to ensure that each weaning group (normal or early) contained a random sample of male pups from each litter. After weaning, animals were housed in standard individual cages. Those which were weaned at 16 days were provided with a fleece cage liner to prevent excessive loss of body heat. F o o d and water were available ad lib. Room temperature was regulated at 25°C with a 12 hr light/dark cycle (lights on at 0700 hr). When animals reached 190___5 g, rats from the early-weaned and normal-weaned groups were randomly assigned to either water-only or ethanol-exposed groups. Water-only groups received two tubes of water daily and were weighed every second day. Animals exposed to ethanol were treated in the following manner.
tube was changed upon each presentation to eliminate the possibility of formation of a position preference by the rats. The same alternate day presentation was continued for ethanol concentrations of 5%, 7% and finally 9% (v/v). Therefore, during this initial ethanol exposure phase rats were exposed to each concentration of ethanol (3 to 9% v/v) on four occasions totaling to 16 ethanol exposure days. At the end of the alternate day ethanol exposure procedure, all rats received the 9% (v/v) ethanol in an everyday free-choice with water for eight consecutive days. Therefore, the rats were exposed to ethanol for a grand total of 24 days prior to immobilization. Fluid consumption (both ethanol and water) and body weight were monitored for each day of ethanol presentation and ethanol intake in grams per kilogram was calculated for those days. Fluid leakage from the ethanol tubes was prevented by using double ballbearing valves at the end of the spouts.
Ethanol Exposure
Immobilization Stress
Two calibrated drinking tubes were attached to the left front of each cage. One contained tap water while the other initially contained a 3% (v/v) solution of ethanol. This concentration was presented every alternate day over an 8 day period; that is, every other day the rats received two tubes of water and on intervening days, they received one tube of water and one tube of ethanol. The position of the ethanol
Following the last day of ethanol exposure, all animals were deprived of food and ethanol but not water for 24 hours. Animals from the early-weaned/ethanol-exposed, early-weaned/water-only, normal-weaned/ethanol-exposed and normal-weaned/water-only groups were divided into stressed and non-stressed groups. Care was taken to ensure that a comparable number of high, medium and low ethanol
EARLY WEANING AND ETHANOL CONSUMPTION consuming animals were included in both the stress and non-stressed groups. This is important in light of data from this laboratory indicating a differential effect of stress depending upon pre-stress levels of ethanol consumption [9,10]. Following food deprivation, stressed animals were placed in standard Plexiglas immobilization tubes for 3 hr at 4--6°C. Non-stressed animals were handled at the beginning and at the end of the stress period but were not immobilized.
675 TABLE 1 SUMMARY OF STOMACH PATHOLOGY FOR EARLY-WEANED AND NORMAL-WEANED RATS (MEAN ± SEM)
Ulcer Incidence
Number of Ulcers
Cumulative Ulcer Length (nun)
Early-Weaned Stress
3/8
0.6 (0.4)
3.3 (2.3)
Normal-Weaned Stress
6/7
2.7 (1.3)
14.3 (5.4)
Early-Weaned No Stress
3/9
0.9 (0.6)
3.0 (2.2)
Normal-Weaned No Stress
1/8
0.8 (0.8)
3.6 (3.6)
Early-Weaned Stress
1/8
0.1 (0.1)
0.4 (0.4)
Normal-Weaned Stress
3/7
0.7 (0.4)
3.8 (1.9)
Early-Weaned No Stress
1/9
0.6 (0.2)
4.0 (4.5)
Normal-Weaned No Stress
2/8
0.5 (0.5)
4.2 (3.5)
Group Ethanol-Exposed
Stomach Pathology Following the immobilization period, all rats were sacrificed with CO2. Stomachs were excised, preserved in 10% formaldehyde (v/v) and examined for damage by an observer who was naive with respect to treatment conditions. The number (mean number of ulcers per rat per group), location (rumenal or glandular) and cumulative length (ulcer severity defined as mean ulcer length per rat per group) of the ulcers were ascertained by examination under a dissecting microscope with an ocular micrometer. Ulcer length (in ram) was determined by adding length plus width of each glandular ulcer.
Statistical Analysis Ethanol consumption for both early-weaned and normal-weaned animals was calculated in terms of mean grams per kilogram per day (g/kg). Mean grams per kilogram of ethanol consumed during exposure to each concentration of ethanol was calculated. In other words, the average group intake of ethanol was calculated for 3, 5, 7 and 9% ethanol during the alternate day ethanol exposure procedure. In addition, ethanol consumption during the subsequent eight everyday ethanol presentation was calculated as two periods of four days each. Hence, for purposes of data presentation and analysis, ethanol consumption is presented over 6 periods of 4 days each. All data were analyzed by repeated measures analysis of variance [group (early vs. normalweaned) X period (ethanol exposure period)], and appropriate post-hoc (Tukey) tests, and simple main effects analysis when interactions were significant. RESULTS
Figure 1 illustrates ethanol consumption for both earlyweaned and normal-weaned animals during the initial alternate day and subsequent everyday ethanol presentation schedule. Analysis of variance yielded a significant main effect of group, F(1,30)=7.44, p<0.01, and a significant main effect of period, F(5,150)=15.73, p<0.001. Furthermore, a significant group x period interaction was observed, F(5,150)=2.22, p<0.05. Post-hoc Tukey tests revealed that ethanol consumption for the early- and normal-weaned groups differed in that the early-weaned animals consumed significantly more ethanol (p<0.01) when exposed to 7 and 9% ethanol during the alternate day exposure schedule. The enhanced ethanol consumption observed among the earlyweaned animals was maintained during the subsequent everyday ethanol exposure periods (p<0.05). It should also be noted that among early-weaned rats, 16 out of 17 animals consumed ethanol to the extent that it comprised in excess of 60% of their total daily fluid intake. Finally, no significant differences were observed in body weight between the groups and no overt signs of ethanol induced intoxication were observed in any of the ethanol exposed animals at any time.
Water Only
Examination of stomach ulcer data as indicated in Table 1 revealed a very low degree of ulcer development in the water-only stressed animals. Alternatively, the normalweaned animals exposed to ethanol exhibited considerably greater ulcer severity as compared to earlyweaned/ethanol-exposed animals [Main effect of groups: F(1,28)=4.48, p<0.04. Main effect of stress: F(1,28)=3.90, p < 0 . 0 5 ] . No significant differences in ulcer frequency (mean number of ulcers per rat per group) or ulcer incidence (number of rats per group which developed ulcers) were observed. DISCUSSION
The results from the present study revealed several interesting findings. The most significant is that early-weaned animals, when exposed to ethanol in adulthood, consumed significantly more ethanol than normal-weaned controls. This finding clearly has profound implications for the materhal deprivation literature. While previous studies have demonstrated an enhanced ulcer proliferation in animals separated early from their mothers [1-3, 5], the present data indicate that an additional consequence of early maternal separation may be manifested in a greater propensity to consume ethanol. It is also important to note that not only did the early-weaned animals as a group consume more ethanol as compared to the normal-weaned animals, but the variability in amounts of ethanol consumed by the early weaned animals was relatively small. For example, in the last ethanol drinking period, 16 out of 17 early-weaned animals were drinking ethanol at a high preference ratio compared to water
676
ROCKMAN, HALL, MARKERT AND GLAVIN
(greater than 60%), whereas only 7 out of 15 normal-weaned animals consumed ethanol at this level. Clearly, the earlyweaned animals' drinking level and pattern of drinking was higher and more consistent than the normal-weaned animals. While the mechanism underlying the enhanced ethanol intake of the early-weaned rats is unknown at this time, studies in this laboratory are presently being conducted in an effort to determine whether neurochemical alterations induced by early weaning may account for the observed enhanced ethanol intake among these rats. The ulcer data from this study are interesting but somewhat contradictory. It was observed that the normal-weaned animals exposed to both ethanol and stress exhibited a significantly greater degree of ulcer severity as compared to the early-weaned/ethanol-exposed animals and all water-only control groups. This finding suggests that ethanol exacerbated ulcer development which is consistent with data from this laboratory indicating that ethanol, in some circumstances, can enhance stress ulcer formation [10,11]. In contrast, it appears that ethanol served to reduce ulcer development among early-weaned animals. This is especially important in view of the fact that the early-weaned animals consumed g r e a t e r amounts of ethanol prior to exposure to immobilization stress. The possibility that ethanol may have reduced ulcer severity in these animals is consistent with other studies demonstrating that different pre-stress levels of ethanol intake may, in some situations, have differential effects on ulcer severity [9,10]. This suggests the possibility that high ethanol consumption may mask the effects of stress, while low levels of ethanol intake may interact deleteriously with stress. This suggestion is supported by very recent data from this laboratory indicating that very high voluntary ethanol intake is associated with attenuated stress responses in environmentally enriched animals (manuscript in submission). The contradictory nature of the ulcer data from this study is illustrated by our failure to observe
enhanced ulcer proliferation in early-weaned/water/waterexposed animals. As mentioned previously, data from other studies demonstrated that early-weaned animals show enhanced ulcer development as a consequence of restraint stress [1-3, 5]. It is important to note that we employed a milder stress in the form of immobilization in this study, hence, this may account for lack of ulcer development in the early-weaned animals. In addition, at least two other factors may have contributed to the lack of ulcers in these animals. Rats in this study were stressed as adults at approximately 125 days of age. Previous studies demonstrating enhanced ulcer formation in early-weaned animals applied the stress treatment at 30 days of age [1-3]. Although 100-day-old early-weaned rats show enhanced ulcer formation in response to restraint stress [5], other researchers have suggested that stress responses in normal-weaned rats may diminish as animals age [7]. Consequently, in the present experiment, both the age of the animals when stressed and the time elapsed since the weaning manipulation may have contributed to the low ulcer formation. Finally, it is important to note that all animals in this study were handled initially every second day and subsequently everyday to correspond to the ethanol exposure schedule. This is important in light of data from this laboratory [8] as well as from others [6] indicating that handling alters neurochemical and behavioral responses to stress. It is therefore possible that handling p e r se may have contributed to ulcer development in normalweaned/non-stressed rats. Whether handling also altered the stress responses of the early-weaned animals in this study is unclear and we are presently investigating this issue.
ACKNOWLEDGEMENT The authors gratefully acknowledge the technical assistance provided by Michelle Del Rizzo.
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