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Effects of early weaning and social isolation on subsequent alcohol intake in rats
A L C O H O L , Vol. 14, No. 2, pp. 175-180, 1997 Copyright © 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0741-8329/97 $17.00 + .00
PII S0741-8329(96)00141-3
ELSEVIER
Effects of Early Weaning and Social Isolation on Subsequent Alcohol Intake in Rats CLAUDIA
FAHLKE,*
ERNEST
HARD*
AND
C. J. P E T E R
ERIKSSON~
*Department of Psychology, GOteborgs University, GOteborg, Sweden tBiomedical Research Center, Alko Ltd, Helsinki, Finland R e c e i v e d 3 M a y 1996; A c c e p t e d 22 J u l y 1996 FAHLKE, C., E. H,~RD AND C. J. P. ERIKSSON. Effects of early weaningandsocialisolation on subsequent alcohol intake in rats. ALCOHOL 14(2) 175-180, 1997.--The present study investigated the influence of early weaning and separation from mother and littermates on voluntary ethanol intake and general activity during prepuberal age, and adult age corticosterone levels. On day 16 after birth the male offspring of a litter were divided in three groups, each subjected to a different rearing condition: 1) early weaned and isolated from its littermates; 2) early weaned but growing up together with two littermates; 3) staying with mother and two littermates. On day 25 the animals were tested for general activity including assessment of fearfulness. From day 30 all animals were given a free choice between water and ethanol solution. The ethanol concentration was increased by 2% during each of the following weeks until 10% was reached during the 5th week. Ten days later, after cessation of alcohol testing, blood samples were taken from the tail for assessment of plasma levels corticosterone. The isolated, early weaning pups displayed higher activity levels than both normally reared pups and group-living, early weaning pups. The quotient peripheral locomotion/total locomotion was lower for the isolated pups compared with the other groups, suggesting less fearfulness in the early weaned, isolated pups. For 2%, 4%, and 6% ethanol solutions the normalreared rats consumed more ethanol and displayed higher ethanol preference than either of the early weaned groups of animals. No group differences were observed either at 8% or 10% ethanol solutions. Levels of plasma corticosterone in adult age in the early weaned rats were slightly reduced, not reaching statistical significance, compared to the normally weaned animals. © 1997 Elsevier Science Inc. Early weaning
Social isolation
Alcohol intake
Locomotor activity
P R E M A T U R E weaning and separation of rat pups from their m o t h e r has long-term effects on behavior, h o r m o n a l responsiveness, and resistance to stress (15). A t adult age early w e a n e d rats are slower to acquire a conditioned reflex (16), and also show deficits in m e m o r y consolidation c o m p a r e d to normally weaned rats (17). As assessed by the open field test (2,9), early w e a n e d rats display increased timidity during prepuberal age (11). They also show increased risk to develop gastric ulcers when exposed to stressful situations, like restraint, in adult age (1). R o c k m a n et al. (18) investigated the effects of early weaning and separation from m o t h e r on ethanol intake in adult age. In the test of ethanol intake the male rats were exposed to successively increasing concentrations of ethanol from 3% to 9%. The early w e a n e d rats displayed an increase of ethanol intake at the higher concentrations of ethanol, 7% and 9%,
Corticosterone
c o m p a r e d to normally weaned rats. A t the lower concentrations ethanol intake did not differ b e t w e e n early and normally weaned animals. In that study (18), rat pups were either p r e w e a n e d at 16 days of age or normally w e a n e d at 21 days of age. I n d e p e n d e n t of age, weaning was imposed by removal of the pups from the m o t h e r and housing them in individual cages. Thus, besides preweaning, the 16-day-old pups were also exposed to isolation from their littermates. The effects of the experimental procedure on subsequent alcohol intake attributed to preweaning may thus be confounded by the influence of early social isolation. The present study addressed this problem. The experiment was therefore designed to separately assessed the influences of early weaning and of social isolation, respectively, on subsequent alcohol consumption. In the same way as in the study by R o c k m a n et al. (18), ethanol consumption was assessed by ex-
Requests for reprints should be addressed to Claudia Fahlke, Department of Psychology, GOteborgs University, Haraldsgatan 1, S-413 14 G6teborg, Sweden. 175
176
FAHLKE. HARI) AND ERIKSSON
posing the animals to successively increasing concentrations of ethanol. To avoid a long period of isolation preceding the test of the ethanol intake, we initiated testing at 30 days of age. In addition, tests of general activity and assessments of fearfulness were p e r f o r m e d to investigate if ethanol intake was related to some of these behavioral parameters. In the same vein, determinations of plasma eorticosterone levels were p e r f o r m e d at adult age. METHOD
Animals Twenty male and 20 virgin females of Wistar strain purchased by M611egaard Breeding Center, Denmark, were bred in the laboratory. The females were inspected daily for the occurrence of estrus. A female in estrus was placed together with a male in a breeding cage (Macrolon 4; 52 × 30 x 18 cm). The male was r e m o v e d from the cage when the female was found to be pregnant. The breeding cages were inspected each morning for the occurrence of birth of a litter. On the day after birth the litter was maximized to nine pups, in the first hand sparing male pups. Based on a criterion of at least six and at most nine male pups in a litter, 12 of the 20 litters born were selected for the experiment. On day 16 (day of birth = 0) the male offspring of a litter were divided in three groups, each subjected to a different rearing condition: A) early weaned and isolated from its littermates (n - 26): B) early weaned but growing up together with two littermates (n - 24): C) staying with m o t h e r and two littermates and normally weaned at 25 days of age (n = 26). If the litter contained nine males, three animals were allotted to each of the conditions by random sampling. If the litter contained eight males and one female, two males were allotted to condition A, two males and the female to condition B, and thrcc males to condition C. If the liUer contained only six males, two pups were allotted to condition A, two males and a female to condition B and C, respectively. From day 16 the pups reared under condition A were individually housed in cages (Macrolon 3 : 4 5 × 30 x 16 cm). The three pups from
300
each litter reared under condition B were placed in the same kind of cages as those under condition A. U n d c r both conditions the floor of the cages were covered by soft paper (Caydry) and a sheet of cotton. Two times per day the pups were afforded luke warm gruel (Baby Semp No. 2) in Petri dishes. The pups also had access to rodent pellet food (Ewes AB, S6dertfilje, Sweden) and a bottle with fiesh tap water. Body weights were estimated on each day from day 16 to day 25. The animal facilities had a room temperature of 22°C, a relative humidity of 55%, and a 12-h light:dark cycle starling at 0600 h. Subsequent observations on the various parameters were p e r l o r m e d on the males only.
General Activity Motor activity was assessed by test chambers made of Plexiglas boxes (70 × 70 x 35 cmi. Each test box contained two series of invisible infrared photocell beams (32 photocells, one high-level and one low-lcvcl series) to measure spontaneous motor behavior. The following variables were measured. Locomotion and peripheral locomotion were registered by the lower grid of infrared beams. Counts occurred when the rat moved horizontally, showing predominantly ambulatory behavior. The photocell beams were arranged to give a separate counting of locomotor activity located to the peripheral part of the testing box. Total activity was registered when any of the photocells were interrupted [for detailed description see (3)]. Motor activity was observed for 30 min. Between tests the chambers were cleaned with hot water.
Ethanol Consumption After the test of general activity on day 25 the animals were housed in individual cages (Macrolon 3 : 4 5 x 30 × 16 cm). From day 30 the cage was equipped with two bottles: one for an alcohol solution and one for tap water. Starting with an alcohol concentration of 2% (v/v) during the first week, the concentration was increased by 2% during each of the folk)wing weeks until 10% was reached during the 5th week.
-
e., 2 0 0 >., o ~1
100 '
_ /~- f
-o- early weaned (is°lated) ~ early weaned (groupliving)
j
0
10
I
I
I
20
30
40
I
I
50
60
r
70
A g e (days) FIG. 1. Dcvelopmcnt of body weight in normal-weaned, early weaned (groupliving), and early weaned (isolated) rats during the age period 16 60 days of age. The body weight of normally weaned rats are higher from day 17 up to day 30 (p < 0.001) compared to early weaned (group-living) and early weaned (isolated) rats. Data are expressed as group median.
I N F L U E N C E OF E A R L Y W E A N I N G A N D S E P A R A T I O N ON A L C O H O L I N T A K E TABLE 1
tubes. After centrifugation for 5 min at 1500 rpm, the plasma was collected and stored at - 7 0 ° C until analyzed for corticosterone content. Corticosterone was measured by the use of radioimmunoassay kits supplied by ICN Biomedicals (Carson, CA).
RESULTS OF TEST ON G E N E R A L ACTIVITY A T D A Y 30 IN N O R M A L L Y W E A N E D RATS, IN G R O U P LIVING, E A R L Y W E A N I N G A N D IN I S O L A T E D , E A R L Y W E A N I N G RATS Normally Weaned
Early Weaned Group-Living
Locomotion (number of counts) 838 _+ 129 Peripheral locomotion (number of counts) 395 + 57 Quotient (peripheral locomotion/ total locomotion) 0.47 Total activity (number of counts) 2138 + 305
Early Weaned Isolated
872 +_ 104
1027 ÷ 128"
390 _+ 45
393 _+ 70
0.44
0.39*
2215 _+248
2663 + 304*
177
Statistics
Median + MAD values are reported. *p < 0.01 compared to isolated, early weaned group and normally weaned group.
To minimize the influence of interlitter variability, the statistical analyses were performed in the following way. Within each original litter observations on the male pups reared under one condition and on those reared under another condition were ranked and treated according to the Mann-Whitney U-test procedure (4). The results from the various litters were pooled into one test variable by adding the U-test variables in main accordance with the procedure used in Mantel's test (14). In the present procedure, however, a more accurate approximation of the distribution of the test variable was achieved by help of the Edgeworth expansion and Sheppard's correction for continuity (5). Two-tailed levels of significance were applied in all comparisons. RESULTS
Compared to the normally weaned group of pups the development of body weight was retarded in both early weaning groups from day 17, the first day after the start of weaning (Fig. 1). The retardation was evident up to day 30 (p < 0.001), but no longer from day 40 and onwards. There were no differences in body weight between the two early weaning groups with the exception for days 17-19 when the isolated pups were slightly more retarded than the groupliving pups (p < 0.05). The results on the test for general activity are given in Table 1. The isolated, early weaning pups displayed higher activity levels than both normally reared pups and group-living, early weaning pups. The quotient peripheral locomotion/total locomotion was lower for the isolated pups compared with the two other groups. There were no statistically significant differences between the normally reared rats and the group-living, early weaning pups.
Measurements of fluid intake were performed twice a week when the tubes were cleaned and refilled with fresh ethanol solution and tap water. The consumption of ethanol solution was calculated as an index of ethanol preference (i.e., proportion of ethanol solution relative to total fluid intake in percent) and absolute ethanol intake (gram of ethanol/kg body weight/day). Body weight and food intake were recorded once a week during the whole test period. Corticosterone Levels During 10 days after the last test with the 10% alcohol solution the animals received only tap water. On the next day at 1300 h a blood sample was taken in a separate room, within 5 rain, from the tail for assessment of plasma level of corticosterone. Blood samples were collected in heparinized test
]
3 o
2HN I, IHI 0
4
6
8
10
Ethanol concentration (%) []
normal weaned [ ]
early weaned (isolated) •
early weaned (groupliving)
FIG. 2. Ethanol intake (g/kg/day) in normal-weaned, early weaned (group-living), and early weaned (isolated) rats for ethanol solutions of various concentrations. Median values are reported. *p < 0.05; **p < 0.01; ***p < 0.001 compared to normally weaned group vs. isolated, early weaning group or group-living, early weaning group.
178
F A H L K E , HA, R D A N D E R I K S S O N 7O ,-,
60 50 #
~
40
~
20
10 0 2
4
6
8
10
Ethanol concentration (%) []
normal weaned [ ]
early weaned (isolated)
•
early weaned (groupliving)
FIG. 3. Ethanol preference (%) in normal-weaned, early weaned (group-living), and early weaned (isolated) rats for ethanol solutions of various concentrations. Median values are reported. *p < 0.05: **p < 0.(/1 compared to normally weaned group vs. isolated, early weaning group or group-living, early weaning group.
Figure 2 illustrates ethanol intake and Figure 3 illustrates ethanol preference for the normal-weaned and both early weaned groups. For 2%, 4%, and 6% ethanol solutions the normal-reared rats consumed more ethanol and displayed higher preference ratios than either of the early weaned groups of animals. The isolated, early weaned and the groupliving, early weaned rats did not differ from each other. No group differences were observed either at 8% or 10% ethanol solutions. The median ethanol intake of the normal weaned rats raised from an intake of about 2.25 g/kg/day at 2% to a maximum of about 3.5 g/kg/day at 4% ethanol. A t higher con-
centrations the intake slowly dropped. By contrast, the early weaned rats consumed approximately the same amount of ethanol over all concentrations, 1.5 2.25 g/kg/day, lacking a clcar maximum. The total fluid intake was slightly higher in the normalweaned group than in the early weaned groups (Fig. 4). Food intake was slightly higher in the early weaned animals compared to the normal-weaned rats (Fig. 5). The estimations of plasma corticosterone gave the following result (median + M A D ) : normal weaned: 78.1 _+ 29.3; early weaned, group-living: 71.7 _+ 30.0, and early weaned,
250 -
~
200-
-~
150-
~
tO0500 2
4
6
8
10
Ethanol concentration (%) []
normal weaned [ ]
early weaned (isolated) •
early weaned (groupliving)
FIG. 4. Total fluid intake (ml/kg/day) during subsequent weeks of testing in normally weaned rats, in group-living, early weaning, and in isolated, early weaning rats. Median values are reported. *p < 0.05 compared to normally weaned group vs. isolated, early weaning group or group-living, early weaning group.
I N F L U E N C E OF E A R L Y W E A N I N G AND S E P A R A T I O N ON A L C O H O L I N T A K E 200-
179
*
15o \\ \\ \\ \\ \\ \\ \\ \\ \\ \\ \\ \\ \\ \\ \\ \\ \\
,.~ 100 .=~ o
50-
2
4
6
8
10
Ethanol concentration (%) []
normal weaned [ ]
early weaned (isolated) •
early weaned (groupliving)
FIG. 5. Food intake (g/kg/day) during subsequent weeks of testing in normally weaned rats, in group-living, early weaning, and in isolated, early weaning rats. Median values are reported. **p < 0.01; ***p < 0.001 compared to normally weaned group vs. isolated, early weaning group or group-living,early weaning group. isolated: 72.5 _+ 24.4. Statistical analysis indicated no significant group differences. DISCUSSION The present results indicate that early weaning and separation from mother causes a reduction in subsequent voluntary ethanol consumption during prepuberal age compared to normally weaned rats. The reduction of alcohol intake was restricted to the lower concentrations of ethanol solutions (2-6%), whereas no differences were observed at higher concentrations, (8-10%). The social rearing conditions during the early weaning period did not modify the main effect of early weaning on ethanol intake. The reduction of ethanol intake was of the same magnitude in the early weaned, isolated rats as in weaned, group-living rats. The results are at variance with those obtained by Rockman et al. (18) reporting an increase of ethanol intake in early weaned rats. The increase was evident only at the high concentrations of ethanol solutions, but not at the lower concentrations. The reason for these contradictory results is unknown, but some differences in experimental procedure may be of importance. The most obvious difference is that the ethanol intake of the early weaned rats was tested during prepuberal age in the present study, in contrast to in adult age in the study by Rockman et al. (18). If this procedural difference is decisive, it implies the interesting possibility that some of the early weaning effects in males do not make their appearance until after the surge of increasing testosterone secretion occurring at puberty. We have previously shown that puberty constitutes a dynamic phase in the development of alcohol intake (8), During this period male rats accelerate their ethanol intake and surpass the intake of female rats, which is higher during the prepuberal age period (8). Besides other procedural differences, such as manner of presentation of fluids to the animal, it should be mentioned that the present study applied a strict randomization when allotting animals to various groups. The statistical analysis of the results were performed in such a way
to avoid influences of statistical dependence between littermates on the analysis. The mechanisms behind the effects on ethanol intake, either increase or decrease, are unknown at the present time. However, some observations in the present study with possible relevance can be reported. Both isolated and group-living, early weaned rats share the lack of nutrition and care-taking provided by their mother. The data on body weight development suggest that substitution with artificial milk and food pellets was not sufficient to match the initial development of body weight, up to 30 days, in the normally weaned pups. It should be noted, however, that the group of normally weaned rats in each litter contained fewer animals than the normal number of pups, 8-12, in natural litters, thereby affording an opportunity to temporary overnutrition. The effects of lack of maternal care-taking cannot be assessed in the present experiment. However, previous studies have indicated that prolonged separation from the mother causes a reduction in the secretion of growth hormone (12). This effect is specifically dependent on absence of the tactile stimulation provided by the mother's licking of her pups (20). In the present study basal levels of plasma corticosterone in the early weaned rats at adult age were slightly reduced, not approaching statistical significance, compared to normally weaned rats. Previous studies showed that adult rats exposed to short, daily separations from mother (handling) during their lactation period display reduced corticosterone secretion in response to stressful events compared to nonhandled animals (13). They also display faster recovery to basal corticosterone levels after termination of stressful stimulation (10). If similar effects are obtained after early weaning and separation from day 16 of age seems to be unknown at the present time. Besides the present study, which however includes ethanol exposition during the time intervening between weaning and adulthood, no studies addressing this problem have been found. The acute effect of separation from the mother is a potentiation of the corticosterone response induced in the pup
180
FAHLKE, HARD
w h e n e x p o s e d to a novel e n v i r o n m e n t (21). The effect increases with increasing duration of separation up to at least 24 h. E x p o s u r e to high levels of c o r t i c o s t e r o n e during this period p e r m a n e n t l y affects the d e v e l o p m e n t of the nervous system (19). In the p r e s e n t c o n t e x t the p e r m a n e n t effects on corticost e r o n e secretion are of special i m p o r t a n c e , because low cortic o s t e r o n e levels in adult animals d e c r e a s e the intake of ethanol (6,7). In the p r e s e n t study early w e a n e d , isolated pups displayed higher levels of m o t o r activity than the early w e a n e d , groupliving pups and the normally w e a n e d rats. The isolated pups allocated p r o p o r t i o n a l l y m o r e of their activity to the central part of the testing a r e n a than the o t h e r two groups. Hesitation to e n t e r the central part of an a r e n a is c o n s i d e r e d as a sign of fear (2). T h e r e f o r e , this result may be i n t e r p r e t e d as indicating less fearfulness in the isolated pups c o m p a r e d to the o t h e r groups. This behavioral change is not a c o n s e q u e n c e of early weaning, but of early social isolation, because the b e h a v i o r of
AND ERIKSSON
the early w e a n e d , group-living pups did not deviate from that of the normally r e a r e d pups. O n e reason may be that early w e a n e d , isolated pups w e r e isolated during 1 wcek before the test of activity and possibly a d a p t e d to loneliness. For the group-living animals, either early or normally weaned, the activity test constituted the first occasion of separation from littermates, which may temporarily frighten them. It is a p p a r e n t that the degree of fearfulness displayed in the activity test bears no relationship to s u b s e q u e n t alcohol consumption. ACKNOWI.I~I)GEMI{NTS The present study was supported by grants from the Swedish Medical Research Council (K96-21P-11842-01A), the Bank of Sweden Tercentenary Foundation, the Swedish Council l'or Research in the Humanities and Social Sciences, the Swedish Alcohol Monopoly Foundation lk)r Alcohol Research (90/4i), Wilhelm and Martina Lundgren's Foundation, Clas Groschinsky's Memorial Foundation, Ernst Collianders Foundation. and Anna Ahrcnbcrg s Foundation.
REFERENCES
1. Ackcrman, S. H.: Hofer, M. A.; Weiner, H.: Early maternal separation increases gastric ulcer risk in rats by producing a latenl thermorcgulatory disturbance. Science 201:373-376; 1978. 2. Archer, J.: Tests for emotionality in rats and mice: A review. Anim. Behav. 21:205 235; 1973. 3. Archer, T.; Frcdriksson, A.: Functional changes implicating dopaminergic system following perinatal treatments. In: Symposium 5: Dcvelopmental aspects of the mono-aminergic system, Borghohn. Dev. Pharmacol. Ther. 18:201-222; 1992. 4. Bradley, J. W.: Distribution-free statistical tests. London: Prentic Hall; 1968. 5. Cramer, H.: Mathematical models of statistics. Princeton, NJ: Princeton University Press; 1946. 6. Fahlke, C.; Engel, J. A.; Eriksson, C. J. P.; H,~rd, E.; S(Sderpalm, B.: Involvement of corticosterone in the modulation of ethanol consumption in the rat. Alcohol 11:195 202: 1994. 7. Fahlke, C.; Engel, J. A.: Hf.rd, E.: Thomasson, R.: Hansen, S.: Metyrapone-induced suppression of corticosterone synthesis reduces ethanol consumption in high-preferring rats. Pharmacol. Biochem. Behav. 48:977-981 : 1994. 8. Fahlke, C.; Svensson, L.; Engel, J. A.; H~rd. E.: Sexual dimorphism in the ontogenetic development of preference for ethanol. Alcohol Alcohol. A73; 1988. 9. Hall, C. S.: Emotional behavior in the rat. Defecation and urination as measures of individual differences in emotionality. J. Comp. Psychol. 18:385~403: 1934. 10. Hess, J. L.: Denenberg, V. H.: Zarrow, M. X.; Pfeifer, W. D.: Modification of the corticosterone response curve as a function of handling in infancy. Physiol. Behav. 4:109-112; 1969. 11. Janus, K.: Effects of early separation of young rats from the mother on their open-field behavior. Physiol. Behav. 4:711-715: 1987.
12. Kuhn, C. M.; Butler. S. R.; Schanberg, S. M.: Sclectivc depression of serum growth hormone during maternal deprivation. Science 201 : 1034 1036:1978. 13. Levinc, S.: Infantile experience and resistance to physiological stress. Science 126:4(/5 4(16:1957. 14. Mantel, N.: Chi-square tests with one degree of freedom: extenskm of the Mantel-Maenszcl procedure. J. Am. Star. Assoc. 58:680-700: 1963. 15. Novakova, V.: Weaning of young rats: effect of time on behavior. Science 151:475-476; 1969. 16. Novakow~, V.: Koldovsky, O.: Faltin, J.: Hahn, P.: Flandera, V.: Conditioned reflex activity in male rats weaned normally or prematurely. Physiol. Bohemoslov. 12:325-329: 1963. 17. Novakova. V.: Koldovsky, O.: Faltin, J.; Hahn, P.: Flandera. V.: The effect of premature weaning and high fat diet on retention of memory trace in male rats. Physiol. Bohemoslov. 12:583-587; 1963. 18. Rockman, G. E.: Hail, A.; Markert, L.: Gavin. G. B.: Early weaning effects on voluntary ethanol consumption and stress responsitivity in rats. Physiol. Behav. 40:673-676: 1987. 19. Sapolsky, R. M.: Meany M. J.: Maturation of the adrenocortical stress response: Neuroendocrine control mechanisms and the stress hyporesponsive period. Brain Res. Rev. I 1:65 76: 1986. 20. Schanberg, S. M. Evoniuk, G.: Kuhn, C. M.: Tactile and nutritional aspects of maternal care: Specific regulators of neurocndocrine function and cellular development. Proc. Soc. Exp. Biol. Med. 175:135 146:1984. 21. Stanton, M. E.; Gutierrez, Y. R.: Levine, S.: Maternal deprivation potentiates pituitary-adrenal stress response in infant rats, Behav. Neurosci. 1{)2:692-70(1: 1988.
PII S0741-8329(96)00141-3
ELSEVIER
Effects of Early Weaning and Social Isolation on Subsequent Alcohol Intake in Rats CLAUDIA
FAHLKE,*
ERNEST
HARD*
AND
C. J. P E T E R
ERIKSSON~
*Department of Psychology, GOteborgs University, GOteborg, Sweden tBiomedical Research Center, Alko Ltd, Helsinki, Finland R e c e i v e d 3 M a y 1996; A c c e p t e d 22 J u l y 1996 FAHLKE, C., E. H,~RD AND C. J. P. ERIKSSON. Effects of early weaningandsocialisolation on subsequent alcohol intake in rats. ALCOHOL 14(2) 175-180, 1997.--The present study investigated the influence of early weaning and separation from mother and littermates on voluntary ethanol intake and general activity during prepuberal age, and adult age corticosterone levels. On day 16 after birth the male offspring of a litter were divided in three groups, each subjected to a different rearing condition: 1) early weaned and isolated from its littermates; 2) early weaned but growing up together with two littermates; 3) staying with mother and two littermates. On day 25 the animals were tested for general activity including assessment of fearfulness. From day 30 all animals were given a free choice between water and ethanol solution. The ethanol concentration was increased by 2% during each of the following weeks until 10% was reached during the 5th week. Ten days later, after cessation of alcohol testing, blood samples were taken from the tail for assessment of plasma levels corticosterone. The isolated, early weaning pups displayed higher activity levels than both normally reared pups and group-living, early weaning pups. The quotient peripheral locomotion/total locomotion was lower for the isolated pups compared with the other groups, suggesting less fearfulness in the early weaned, isolated pups. For 2%, 4%, and 6% ethanol solutions the normalreared rats consumed more ethanol and displayed higher ethanol preference than either of the early weaned groups of animals. No group differences were observed either at 8% or 10% ethanol solutions. Levels of plasma corticosterone in adult age in the early weaned rats were slightly reduced, not reaching statistical significance, compared to the normally weaned animals. © 1997 Elsevier Science Inc. Early weaning
Social isolation
Alcohol intake
Locomotor activity
P R E M A T U R E weaning and separation of rat pups from their m o t h e r has long-term effects on behavior, h o r m o n a l responsiveness, and resistance to stress (15). A t adult age early w e a n e d rats are slower to acquire a conditioned reflex (16), and also show deficits in m e m o r y consolidation c o m p a r e d to normally weaned rats (17). As assessed by the open field test (2,9), early w e a n e d rats display increased timidity during prepuberal age (11). They also show increased risk to develop gastric ulcers when exposed to stressful situations, like restraint, in adult age (1). R o c k m a n et al. (18) investigated the effects of early weaning and separation from m o t h e r on ethanol intake in adult age. In the test of ethanol intake the male rats were exposed to successively increasing concentrations of ethanol from 3% to 9%. The early w e a n e d rats displayed an increase of ethanol intake at the higher concentrations of ethanol, 7% and 9%,
Corticosterone
c o m p a r e d to normally weaned rats. A t the lower concentrations ethanol intake did not differ b e t w e e n early and normally weaned animals. In that study (18), rat pups were either p r e w e a n e d at 16 days of age or normally w e a n e d at 21 days of age. I n d e p e n d e n t of age, weaning was imposed by removal of the pups from the m o t h e r and housing them in individual cages. Thus, besides preweaning, the 16-day-old pups were also exposed to isolation from their littermates. The effects of the experimental procedure on subsequent alcohol intake attributed to preweaning may thus be confounded by the influence of early social isolation. The present study addressed this problem. The experiment was therefore designed to separately assessed the influences of early weaning and of social isolation, respectively, on subsequent alcohol consumption. In the same way as in the study by R o c k m a n et al. (18), ethanol consumption was assessed by ex-
Requests for reprints should be addressed to Claudia Fahlke, Department of Psychology, GOteborgs University, Haraldsgatan 1, S-413 14 G6teborg, Sweden. 175
176
FAHLKE. HARI) AND ERIKSSON
posing the animals to successively increasing concentrations of ethanol. To avoid a long period of isolation preceding the test of the ethanol intake, we initiated testing at 30 days of age. In addition, tests of general activity and assessments of fearfulness were p e r f o r m e d to investigate if ethanol intake was related to some of these behavioral parameters. In the same vein, determinations of plasma eorticosterone levels were p e r f o r m e d at adult age. METHOD
Animals Twenty male and 20 virgin females of Wistar strain purchased by M611egaard Breeding Center, Denmark, were bred in the laboratory. The females were inspected daily for the occurrence of estrus. A female in estrus was placed together with a male in a breeding cage (Macrolon 4; 52 × 30 x 18 cm). The male was r e m o v e d from the cage when the female was found to be pregnant. The breeding cages were inspected each morning for the occurrence of birth of a litter. On the day after birth the litter was maximized to nine pups, in the first hand sparing male pups. Based on a criterion of at least six and at most nine male pups in a litter, 12 of the 20 litters born were selected for the experiment. On day 16 (day of birth = 0) the male offspring of a litter were divided in three groups, each subjected to a different rearing condition: A) early weaned and isolated from its littermates (n - 26): B) early weaned but growing up together with two littermates (n - 24): C) staying with m o t h e r and two littermates and normally weaned at 25 days of age (n = 26). If the litter contained nine males, three animals were allotted to each of the conditions by random sampling. If the litter contained eight males and one female, two males were allotted to condition A, two males and the female to condition B, and thrcc males to condition C. If the liUer contained only six males, two pups were allotted to condition A, two males and a female to condition B and C, respectively. From day 16 the pups reared under condition A were individually housed in cages (Macrolon 3 : 4 5 × 30 x 16 cm). The three pups from
300
each litter reared under condition B were placed in the same kind of cages as those under condition A. U n d c r both conditions the floor of the cages were covered by soft paper (Caydry) and a sheet of cotton. Two times per day the pups were afforded luke warm gruel (Baby Semp No. 2) in Petri dishes. The pups also had access to rodent pellet food (Ewes AB, S6dertfilje, Sweden) and a bottle with fiesh tap water. Body weights were estimated on each day from day 16 to day 25. The animal facilities had a room temperature of 22°C, a relative humidity of 55%, and a 12-h light:dark cycle starling at 0600 h. Subsequent observations on the various parameters were p e r l o r m e d on the males only.
General Activity Motor activity was assessed by test chambers made of Plexiglas boxes (70 × 70 x 35 cmi. Each test box contained two series of invisible infrared photocell beams (32 photocells, one high-level and one low-lcvcl series) to measure spontaneous motor behavior. The following variables were measured. Locomotion and peripheral locomotion were registered by the lower grid of infrared beams. Counts occurred when the rat moved horizontally, showing predominantly ambulatory behavior. The photocell beams were arranged to give a separate counting of locomotor activity located to the peripheral part of the testing box. Total activity was registered when any of the photocells were interrupted [for detailed description see (3)]. Motor activity was observed for 30 min. Between tests the chambers were cleaned with hot water.
Ethanol Consumption After the test of general activity on day 25 the animals were housed in individual cages (Macrolon 3 : 4 5 x 30 × 16 cm). From day 30 the cage was equipped with two bottles: one for an alcohol solution and one for tap water. Starting with an alcohol concentration of 2% (v/v) during the first week, the concentration was increased by 2% during each of the folk)wing weeks until 10% was reached during the 5th week.
-
e., 2 0 0 >., o ~1
100 '
_ /~- f
-o- early weaned (is°lated) ~ early weaned (groupliving)
j
0
10
I
I
I
20
30
40
I
I
50
60
r
70
A g e (days) FIG. 1. Dcvelopmcnt of body weight in normal-weaned, early weaned (groupliving), and early weaned (isolated) rats during the age period 16 60 days of age. The body weight of normally weaned rats are higher from day 17 up to day 30 (p < 0.001) compared to early weaned (group-living) and early weaned (isolated) rats. Data are expressed as group median.
I N F L U E N C E OF E A R L Y W E A N I N G A N D S E P A R A T I O N ON A L C O H O L I N T A K E TABLE 1
tubes. After centrifugation for 5 min at 1500 rpm, the plasma was collected and stored at - 7 0 ° C until analyzed for corticosterone content. Corticosterone was measured by the use of radioimmunoassay kits supplied by ICN Biomedicals (Carson, CA).
RESULTS OF TEST ON G E N E R A L ACTIVITY A T D A Y 30 IN N O R M A L L Y W E A N E D RATS, IN G R O U P LIVING, E A R L Y W E A N I N G A N D IN I S O L A T E D , E A R L Y W E A N I N G RATS Normally Weaned
Early Weaned Group-Living
Locomotion (number of counts) 838 _+ 129 Peripheral locomotion (number of counts) 395 + 57 Quotient (peripheral locomotion/ total locomotion) 0.47 Total activity (number of counts) 2138 + 305
Early Weaned Isolated
872 +_ 104
1027 ÷ 128"
390 _+ 45
393 _+ 70
0.44
0.39*
2215 _+248
2663 + 304*
177
Statistics
Median + MAD values are reported. *p < 0.01 compared to isolated, early weaned group and normally weaned group.
To minimize the influence of interlitter variability, the statistical analyses were performed in the following way. Within each original litter observations on the male pups reared under one condition and on those reared under another condition were ranked and treated according to the Mann-Whitney U-test procedure (4). The results from the various litters were pooled into one test variable by adding the U-test variables in main accordance with the procedure used in Mantel's test (14). In the present procedure, however, a more accurate approximation of the distribution of the test variable was achieved by help of the Edgeworth expansion and Sheppard's correction for continuity (5). Two-tailed levels of significance were applied in all comparisons. RESULTS
Compared to the normally weaned group of pups the development of body weight was retarded in both early weaning groups from day 17, the first day after the start of weaning (Fig. 1). The retardation was evident up to day 30 (p < 0.001), but no longer from day 40 and onwards. There were no differences in body weight between the two early weaning groups with the exception for days 17-19 when the isolated pups were slightly more retarded than the groupliving pups (p < 0.05). The results on the test for general activity are given in Table 1. The isolated, early weaning pups displayed higher activity levels than both normally reared pups and group-living, early weaning pups. The quotient peripheral locomotion/total locomotion was lower for the isolated pups compared with the two other groups. There were no statistically significant differences between the normally reared rats and the group-living, early weaning pups.
Measurements of fluid intake were performed twice a week when the tubes were cleaned and refilled with fresh ethanol solution and tap water. The consumption of ethanol solution was calculated as an index of ethanol preference (i.e., proportion of ethanol solution relative to total fluid intake in percent) and absolute ethanol intake (gram of ethanol/kg body weight/day). Body weight and food intake were recorded once a week during the whole test period. Corticosterone Levels During 10 days after the last test with the 10% alcohol solution the animals received only tap water. On the next day at 1300 h a blood sample was taken in a separate room, within 5 rain, from the tail for assessment of plasma level of corticosterone. Blood samples were collected in heparinized test
]
3 o
2HN I, IHI 0
4
6
8
10
Ethanol concentration (%) []
normal weaned [ ]
early weaned (isolated) •
early weaned (groupliving)
FIG. 2. Ethanol intake (g/kg/day) in normal-weaned, early weaned (group-living), and early weaned (isolated) rats for ethanol solutions of various concentrations. Median values are reported. *p < 0.05; **p < 0.01; ***p < 0.001 compared to normally weaned group vs. isolated, early weaning group or group-living, early weaning group.
178
F A H L K E , HA, R D A N D E R I K S S O N 7O ,-,
60 50 #
~
40
~
20
10 0 2
4
6
8
10
Ethanol concentration (%) []
normal weaned [ ]
early weaned (isolated)
•
early weaned (groupliving)
FIG. 3. Ethanol preference (%) in normal-weaned, early weaned (group-living), and early weaned (isolated) rats for ethanol solutions of various concentrations. Median values are reported. *p < 0.05: **p < 0.(/1 compared to normally weaned group vs. isolated, early weaning group or group-living, early weaning group.
Figure 2 illustrates ethanol intake and Figure 3 illustrates ethanol preference for the normal-weaned and both early weaned groups. For 2%, 4%, and 6% ethanol solutions the normal-reared rats consumed more ethanol and displayed higher preference ratios than either of the early weaned groups of animals. The isolated, early weaned and the groupliving, early weaned rats did not differ from each other. No group differences were observed either at 8% or 10% ethanol solutions. The median ethanol intake of the normal weaned rats raised from an intake of about 2.25 g/kg/day at 2% to a maximum of about 3.5 g/kg/day at 4% ethanol. A t higher con-
centrations the intake slowly dropped. By contrast, the early weaned rats consumed approximately the same amount of ethanol over all concentrations, 1.5 2.25 g/kg/day, lacking a clcar maximum. The total fluid intake was slightly higher in the normalweaned group than in the early weaned groups (Fig. 4). Food intake was slightly higher in the early weaned animals compared to the normal-weaned rats (Fig. 5). The estimations of plasma corticosterone gave the following result (median + M A D ) : normal weaned: 78.1 _+ 29.3; early weaned, group-living: 71.7 _+ 30.0, and early weaned,
250 -
~
200-
-~
150-
~
tO0500 2
4
6
8
10
Ethanol concentration (%) []
normal weaned [ ]
early weaned (isolated) •
early weaned (groupliving)
FIG. 4. Total fluid intake (ml/kg/day) during subsequent weeks of testing in normally weaned rats, in group-living, early weaning, and in isolated, early weaning rats. Median values are reported. *p < 0.05 compared to normally weaned group vs. isolated, early weaning group or group-living, early weaning group.
I N F L U E N C E OF E A R L Y W E A N I N G AND S E P A R A T I O N ON A L C O H O L I N T A K E 200-
179
*
15o \\ \\ \\ \\ \\ \\ \\ \\ \\ \\ \\ \\ \\ \\ \\ \\ \\
,.~ 100 .=~ o
50-
2
4
6
8
10
Ethanol concentration (%) []
normal weaned [ ]
early weaned (isolated) •
early weaned (groupliving)
FIG. 5. Food intake (g/kg/day) during subsequent weeks of testing in normally weaned rats, in group-living, early weaning, and in isolated, early weaning rats. Median values are reported. **p < 0.01; ***p < 0.001 compared to normally weaned group vs. isolated, early weaning group or group-living,early weaning group. isolated: 72.5 _+ 24.4. Statistical analysis indicated no significant group differences. DISCUSSION The present results indicate that early weaning and separation from mother causes a reduction in subsequent voluntary ethanol consumption during prepuberal age compared to normally weaned rats. The reduction of alcohol intake was restricted to the lower concentrations of ethanol solutions (2-6%), whereas no differences were observed at higher concentrations, (8-10%). The social rearing conditions during the early weaning period did not modify the main effect of early weaning on ethanol intake. The reduction of ethanol intake was of the same magnitude in the early weaned, isolated rats as in weaned, group-living rats. The results are at variance with those obtained by Rockman et al. (18) reporting an increase of ethanol intake in early weaned rats. The increase was evident only at the high concentrations of ethanol solutions, but not at the lower concentrations. The reason for these contradictory results is unknown, but some differences in experimental procedure may be of importance. The most obvious difference is that the ethanol intake of the early weaned rats was tested during prepuberal age in the present study, in contrast to in adult age in the study by Rockman et al. (18). If this procedural difference is decisive, it implies the interesting possibility that some of the early weaning effects in males do not make their appearance until after the surge of increasing testosterone secretion occurring at puberty. We have previously shown that puberty constitutes a dynamic phase in the development of alcohol intake (8), During this period male rats accelerate their ethanol intake and surpass the intake of female rats, which is higher during the prepuberal age period (8). Besides other procedural differences, such as manner of presentation of fluids to the animal, it should be mentioned that the present study applied a strict randomization when allotting animals to various groups. The statistical analysis of the results were performed in such a way
to avoid influences of statistical dependence between littermates on the analysis. The mechanisms behind the effects on ethanol intake, either increase or decrease, are unknown at the present time. However, some observations in the present study with possible relevance can be reported. Both isolated and group-living, early weaned rats share the lack of nutrition and care-taking provided by their mother. The data on body weight development suggest that substitution with artificial milk and food pellets was not sufficient to match the initial development of body weight, up to 30 days, in the normally weaned pups. It should be noted, however, that the group of normally weaned rats in each litter contained fewer animals than the normal number of pups, 8-12, in natural litters, thereby affording an opportunity to temporary overnutrition. The effects of lack of maternal care-taking cannot be assessed in the present experiment. However, previous studies have indicated that prolonged separation from the mother causes a reduction in the secretion of growth hormone (12). This effect is specifically dependent on absence of the tactile stimulation provided by the mother's licking of her pups (20). In the present study basal levels of plasma corticosterone in the early weaned rats at adult age were slightly reduced, not approaching statistical significance, compared to normally weaned rats. Previous studies showed that adult rats exposed to short, daily separations from mother (handling) during their lactation period display reduced corticosterone secretion in response to stressful events compared to nonhandled animals (13). They also display faster recovery to basal corticosterone levels after termination of stressful stimulation (10). If similar effects are obtained after early weaning and separation from day 16 of age seems to be unknown at the present time. Besides the present study, which however includes ethanol exposition during the time intervening between weaning and adulthood, no studies addressing this problem have been found. The acute effect of separation from the mother is a potentiation of the corticosterone response induced in the pup
180
FAHLKE, HARD
w h e n e x p o s e d to a novel e n v i r o n m e n t (21). The effect increases with increasing duration of separation up to at least 24 h. E x p o s u r e to high levels of c o r t i c o s t e r o n e during this period p e r m a n e n t l y affects the d e v e l o p m e n t of the nervous system (19). In the p r e s e n t c o n t e x t the p e r m a n e n t effects on corticost e r o n e secretion are of special i m p o r t a n c e , because low cortic o s t e r o n e levels in adult animals d e c r e a s e the intake of ethanol (6,7). In the p r e s e n t study early w e a n e d , isolated pups displayed higher levels of m o t o r activity than the early w e a n e d , groupliving pups and the normally w e a n e d rats. The isolated pups allocated p r o p o r t i o n a l l y m o r e of their activity to the central part of the testing a r e n a than the o t h e r two groups. Hesitation to e n t e r the central part of an a r e n a is c o n s i d e r e d as a sign of fear (2). T h e r e f o r e , this result may be i n t e r p r e t e d as indicating less fearfulness in the isolated pups c o m p a r e d to the o t h e r groups. This behavioral change is not a c o n s e q u e n c e of early weaning, but of early social isolation, because the b e h a v i o r of
AND ERIKSSON
the early w e a n e d , group-living pups did not deviate from that of the normally r e a r e d pups. O n e reason may be that early w e a n e d , isolated pups w e r e isolated during 1 wcek before the test of activity and possibly a d a p t e d to loneliness. For the group-living animals, either early or normally weaned, the activity test constituted the first occasion of separation from littermates, which may temporarily frighten them. It is a p p a r e n t that the degree of fearfulness displayed in the activity test bears no relationship to s u b s e q u e n t alcohol consumption. ACKNOWI.I~I)GEMI{NTS The present study was supported by grants from the Swedish Medical Research Council (K96-21P-11842-01A), the Bank of Sweden Tercentenary Foundation, the Swedish Council l'or Research in the Humanities and Social Sciences, the Swedish Alcohol Monopoly Foundation lk)r Alcohol Research (90/4i), Wilhelm and Martina Lundgren's Foundation, Clas Groschinsky's Memorial Foundation, Ernst Collianders Foundation. and Anna Ahrcnbcrg s Foundation.
REFERENCES
1. Ackcrman, S. H.: Hofer, M. A.; Weiner, H.: Early maternal separation increases gastric ulcer risk in rats by producing a latenl thermorcgulatory disturbance. Science 201:373-376; 1978. 2. Archer, J.: Tests for emotionality in rats and mice: A review. Anim. Behav. 21:205 235; 1973. 3. Archer, T.; Frcdriksson, A.: Functional changes implicating dopaminergic system following perinatal treatments. In: Symposium 5: Dcvelopmental aspects of the mono-aminergic system, Borghohn. Dev. Pharmacol. Ther. 18:201-222; 1992. 4. Bradley, J. W.: Distribution-free statistical tests. London: Prentic Hall; 1968. 5. Cramer, H.: Mathematical models of statistics. Princeton, NJ: Princeton University Press; 1946. 6. Fahlke, C.; Engel, J. A.; Eriksson, C. J. P.; H,~rd, E.; S(Sderpalm, B.: Involvement of corticosterone in the modulation of ethanol consumption in the rat. Alcohol 11:195 202: 1994. 7. Fahlke, C.; Engel, J. A.: Hf.rd, E.: Thomasson, R.: Hansen, S.: Metyrapone-induced suppression of corticosterone synthesis reduces ethanol consumption in high-preferring rats. Pharmacol. Biochem. Behav. 48:977-981 : 1994. 8. Fahlke, C.; Svensson, L.; Engel, J. A.; H~rd. E.: Sexual dimorphism in the ontogenetic development of preference for ethanol. Alcohol Alcohol. A73; 1988. 9. Hall, C. S.: Emotional behavior in the rat. Defecation and urination as measures of individual differences in emotionality. J. Comp. Psychol. 18:385~403: 1934. 10. Hess, J. L.: Denenberg, V. H.: Zarrow, M. X.; Pfeifer, W. D.: Modification of the corticosterone response curve as a function of handling in infancy. Physiol. Behav. 4:109-112; 1969. 11. Janus, K.: Effects of early separation of young rats from the mother on their open-field behavior. Physiol. Behav. 4:711-715: 1987.
12. Kuhn, C. M.; Butler. S. R.; Schanberg, S. M.: Sclectivc depression of serum growth hormone during maternal deprivation. Science 201 : 1034 1036:1978. 13. Levinc, S.: Infantile experience and resistance to physiological stress. Science 126:4(/5 4(16:1957. 14. Mantel, N.: Chi-square tests with one degree of freedom: extenskm of the Mantel-Maenszcl procedure. J. Am. Star. Assoc. 58:680-700: 1963. 15. Novakova, V.: Weaning of young rats: effect of time on behavior. Science 151:475-476; 1969. 16. Novakow~, V.: Koldovsky, O.: Faltin, J.: Hahn, P.: Flandera, V.: Conditioned reflex activity in male rats weaned normally or prematurely. Physiol. Bohemoslov. 12:325-329: 1963. 17. Novakova. V.: Koldovsky, O.: Faltin, J.; Hahn, P.: Flandera. V.: The effect of premature weaning and high fat diet on retention of memory trace in male rats. Physiol. Bohemoslov. 12:583-587; 1963. 18. Rockman, G. E.: Hail, A.; Markert, L.: Gavin. G. B.: Early weaning effects on voluntary ethanol consumption and stress responsitivity in rats. Physiol. Behav. 40:673-676: 1987. 19. Sapolsky, R. M.: Meany M. J.: Maturation of the adrenocortical stress response: Neuroendocrine control mechanisms and the stress hyporesponsive period. Brain Res. Rev. I 1:65 76: 1986. 20. Schanberg, S. M. Evoniuk, G.: Kuhn, C. M.: Tactile and nutritional aspects of maternal care: Specific regulators of neurocndocrine function and cellular development. Proc. Soc. Exp. Biol. Med. 175:135 146:1984. 21. Stanton, M. E.; Gutierrez, Y. R.: Levine, S.: Maternal deprivation potentiates pituitary-adrenal stress response in infant rats, Behav. Neurosci. 1{)2:692-70(1: 1988.