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Effects of Preweanling Ethanol Odor Exposure on Ethanol Preference
Alcohol, Vol. 15, No. 3, pp. 213–217, 1998 © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0741-8329/98 $19.00 1 .00
PII S0741-8329(97)00122-5
Effects of Preweanling Ethanol Odor Exposure on Ethanol Preference MICHELLE D. BANNOURA, KIMBERLY S. KRAEBEL, LINDA P. SPEAR AND NORMAN E. SPEAR Center for Developmental Psychobiology, Binghamton University, P.O. Box 6000, Binghamton, NY Received 25 June 1996; Accepted 27 June 1997 BANNOURA, M. D., K. S. KRAEBEL, L. P. SPEAR AND N. E. SPEAR. Effects of preweanling ethanol odor exposure on ethanol preference. ALCOHOL 15(3) 213–217, 1998.—Preweaning exposure to a foreign odor has been shown to alter later responding to that odor. Early experience with ethanol odor may alter not only responding to ethanol odor, but also intake of ethanol solutions. The present study tested the effects of exposure to ethanol odor prior to weaning on ethanol odor preference and ethanol consumption. Sprague–Dawley-derived rats were exposed to the odor of 100% ethanol from postnatal day 1 to 22 in the home cage. An odor preference test was conducted on postnatal day 14 and a two-bottle ethanol intake test was conducted after weaning. In both odor preference and intake tests, animals previously exposed to ethanol odor exhibited a greater preference for ethanol than controls. The results demonstrate that early experience with the odor of ethanol can increase ingestion of ethanol later in life. © 1998 Elsevier Science Inc. Ethanol preference
Ethanol odor
Preweanling experience
results in a preference for those odors later in life (2,3,6,7,15). When rat pups were reared in a home cage containing a foreign odor, such as peppermint oil, a preference for the foreign odor was demonstrated up to 100 days later (3,7,11,13). The present experiment combines the above lines of research by investigating the effects of ethanol odor exposure in the home cage throughout the preweaning period. Pups were exposed to ethanol odor from postnatal day 1 through day 22. Ethanol odor preference was tested on postnatal day 14 and ethanol intake was tested from postnatal days 23 to 25.
EXPOSURE to ethanol early in development has been shown to alter later responding to ethanol, with the nature of the alteration depending on the circumstances of the exposure (16,18–21,23). Changes in ethanol ingestion were recorded following the pairing of ethanol odor and internal malaise in weanling rats (19,23). When internal malaise was paired with ethanol odor on a single occasion, a significant decrease in ethanol intake was observed in weanlings, but not adults, 24 h after the pairing (21). When multiple pairings of ethanol odor and internal malaise were given during the preweaning period, ethanol intake decreased in adulthood, 38 days after the final exposure (20). Weanlings given a pairing of ethanol odor and internal malaise decreased their ethanol intake 7 days later, whereas control animals exposed to ethanol odor unpaired with malaise significantly increased their ethanol intake compared to ethanol naive controls (19). The results demonstrate that reinforcement contingencies associated with ethanol odor exposure alters subsequent ethanol consumption, whereas mere exposure to ethanol odor also may alter responding to ethanol. Concomitant with studies showing an alteration in responding to ethanol following prior ethanol experience, others have shown that early experience with other foreign odors
METHOD
Subjects The offspring of 18 Sprague–Dawley-derived dams born and bred in our colony at Binghamton University served as the subjects in this experiment. Births were checked daily between 1500 and 1800 h, and the first day pups were found in the cage was considered postnatal day 0. Litters were culled to 10 pups (five male and five female when possible) within 24 h of birth. Weaning on postnatal day 21 was accomplished by removing the dam from the cage. Pups remained in the home cage with littermates until the time of ethanol intake testing.
Requests for reprints should be addressed to Norman E. Spear, Center for Developmental Psychobiology, Binghamton University, P.O. Box 6000, Binghamton, NY 13902-6000. Tel: (607) 777-2663; Fax: (607) 777-2677.
213
214 Twenty-four hours prior to the beginning of the ethanol intake test weanlings were removed from the home cage and individually housed. All animals were maintained on a 16:8 light:dark schedule at 23–25˚C under relatively constant humidity. Ethanol Odor Exposure Ten litters were exposed to ethanol odor in their home cage (standard maternity cage 47 3 25.4 3 20.3 cm) from postnatal day 1 through 22. Pure ethanol, 200 proof, was placed on cotton in a Plexiglas holder at each end of the home cage. The Plexiglas holders fit snugly into the ends of the maternity cage and had approximately 50 small holes drilled in the face. The holders were constructed so the odor could move into the cage while the animals could not physically contact the cotton. At 0900 every morning 30 ml of 200 proof ethanol was applied to the cotton in each end of the cage. Thus, animals were exposed to the odor of 60 ml of pure ethanol each day. The cotton in each end of the home cage was replaced each day prior to the application of ethanol. The ethanol odor-exposed litters were maintained in a room separate from the main colony on the same light:dark schedule at a similar temperature and humidity. Other than daily odor exposure and weekly cage cleaning, the litters were left undisturbed until postnatal day 14. Eight control litters were exposed to water odor following the same procedures used for ethanol odor exposure. Distilled water, 30 ml, was placed on cotton in Plexiglas holders in each end of the home cage. The control litters were housed in the main colony room. The function of the water odor control group was to equate the disturbance of the litter during the preweanling period. Odor Preference Test On postnatal day 14, an odor preference test was conducted to assess the relative preference for ethanol odor in comparison to a novel odor. The odor preference test was conducted at 1300 h, 4 h following the daily application of odor in the home cage. The timing of the odor preference test was chosen based on the blood alcohol levels of pups on postnatal day 16. Pups never displayed any overt signs of motor impairment or intoxication. Two pups from each litter, one male and one female, were removed from the home cage, taken to a separate experimental room, weighed, and kept in a holding cage for no more than 15 min prior to testing. The odor preference test was conducted in a clear Plexiglas chamber (28 3 10.5 3 12.5 cm) with cotton placed in each end. Pure ethanol, 3 ml, was placed on the cotton at one end of the apparatus and clove oil, 0.3 ml, was placed on the cotton at the other end. These odors and their concentrations were chosen on the basis of pilot data showing approximately equal preference for each odor in this combination by naive rats on postnatal day 14. Each preference test was 2 min long. Each pup was picked up and replaced on the midline at the start of the second minute. The relative left–right position of the ethanol odor was altered by changing the orientation of the pup’s head at the beginning of the second test minute. This was done to control for potential side preferences independent of odor preference. The time over each odor was recorded for each minute. The pup was considered over an odor when the whole head and both front paws were across the midline. Fresh cotton and odors were used for each pup. Pups used in the odor preference test were humanely sacrificed following odor preference testing to avoid exposing the rest of the litter to the testing odors.
BANNOURA ET AL. Blood Ethanol Concentration Analysis The concentration of ethanol in the blood of the ethanol odor-exposed litters was determined on postnatal day 16. Eight pups were decapitated at each of five time points following the daily application of ethanol odor in the home cage. Blood was collected at 15, 30, 60, 120, and 300 min following the application of ethanol to the home cage. Blood was collected in a 1.5-ml vial containing 0.10 ml of heparin sodium salt solution. The ethanol content was determined by head space gas chromatographic analysis. The gas chromatograph contained a 6-foot stainless steel column packed with Poropak Q and utilized nitrogen as the carrier gas. The blood sample, 25 ml, placed in an airtight vial, was kept in a 55˚C water bath for 8 min. An airtight syringe was used to extract 1 ml of the head space gas from the sample vial and inject the gas onto the column of the gas chromatograph. The retention time for the ethanol peak was 2.6 min. Concentration of ethanol in the blood was measured against a standard concentration curve. Ethanol Intake Test Animals were tested for 3 days beginning on postnatal day 23. For 23 h each day animals were given free access to one bottle containing a 6% (v/v) ethanol solution and one bottle containing tap water. Each morning the bottles were removed, weighed, and refilled with fresh solutions. While the bottles were off the cage, animals were weighed. The left– right position of the bottles was changed each day to control for possible side preferences. Food was freely available throughout the test period. Experimental Design and Statistical Analysis All data were analyzed by analysis of variance (ANOVA) with Preweaning Experience (ethanol odor or control) and Sex (female or male) serving as between-group factors in all analyses. Odor preference data were analyzed as a 2 3 2 3 2 mixed design with Test Minute (1, 2) serving as a withingroups factor. Ethanol intake, both percent total fluid intake and g/kg intake, was analyzed as a 2 3 2 3 3 mixed design with Test Day (1, 2, 3) serving as a within-groups measure. When significant main effects or interactions were found, a Newman–Keuls post hoc test was used to determine significant differences between means. Statistical significance was determined with alpha less than or equal to 0.05 in all tests. RESULTS
Odor Preference Test Pups exposed to ethanol odor throughout the preweaning period spent significantly more time over the ethanol odor than pups that had never been exposed to ethanol odor (Fig. 1). This effect was confirmed by a significant main effect of preweaning experience, F(1, 32) 5 5.36. A significant main effect of test minute, F(1, 32) 5 10.53, was also found. Post hoc analysis of the data collapsed across preweanling experience showed that animals spent significantly less time over the ethanol odor during the second test minute (17.6 6 2.07 s) than the first minute (23.9 6 1.59 s). Blood Ethanol Concentration The concentration of ethanol in the trunk blood remained relatively constant across the 300-min period of analysis. The highest level reached was 26.34 mg/dl at 120 min after the appli-
PREWEANLING ETHANOL ODOR EXPOSURE
215 firmed the influence of ethanol odor exposure on the intake of a 6% ethanol solution after weaning. Ethanol intake (g/kg) was also influenced by an interaction of Sex 3 Test Day, F(2, 28) 5 3.80. Females decreased their g/kg ethanol intake (day 1 5 3.5 6 0.6 g/kg, day 3 5 2.5 6 0.5 g/kg) whereas males showed no significant change in intake across the 3 test days (day 1 5 3.3 6 0.5, day 3 5 4.1 6 0.8 g/kg). DISCUSSION
FIG. 1. Ethanol odor preference on postnatal day 14. Total time over ethanol during a 2-min test. Data are collapsed over test minute.
cation of ethanol odor to the home cage (Table 1). Blood ethanol concentration was not significantly influenced by either time after application, F(4, 26) 5 2.00, or sex, F(1, 26) , 1.0. Ethanol Intake Test The percentage of total fluid intake accounted for by ethanol solution was significantly greater in pups exposed to ethanol odor than in control animals (Fig. 2A). This effect was confirmed by a significant main effect of preweaning experience, F(1, 14) 5 5.49. A significant interaction of Sex 3 Test Day, F(2, 28) 5 4.31, reflected a difference in the pattern of intake between males and females. Post hoc analysis of the data collapsed across preweanling experience revealed that females significantly decreased their ethanol intake from day 1 (17.8 6 2.8 %) to day 3 (12.1 6 1.9 %), whereas males significantly increased their intake from test day 1 (17.8 6 2.7 %) to 3 (21.7 6 3.4 %). These effects were not sufficiently robust, however, to result in a significant gender difference in percentage ethanol intake on any of the test days. Gram per kilogram (g/kg) ethanol intake displayed a pattern of results similar to the results obtained with the percentage total fluid intake measure. Preweaning experience with ethanol odor resulted in significantly greater ethanol intake (g/kg) compared to water controls (Fig. 2B). A significant main effect of preweaning experience, F(1, 14) 5 7.0, con-
TABLE 1 BLOOD ETHANOL CONCENTRATION ON POSTNATAL DAY 16 Time After Odor Application (Minutes)
15 30 60 120 300
Blood Ethanol Concentration [mg/dl (SE)]
8.99 (1.6) 15.86 (3.8) 16.11 (4.7) 26.34 (6.2) 13.16 (8.4)
The present results demonstrate that exposure to ethanol odor in the home cage throughout the preweaning period alters preweaning and postweaning responding to ethanol. Pups exposed to ethanol odor in the home cage showed a greater preference for ethanol odor on postnatal day 14. Weanlings previously exposed to ethanol odor ingested significantly more of a 6% ethanol solution than control animals. The increased preference for ethanol odor suggests that the animals associated the appetitive and familiar qualities of the home cage with the odor of ethanol. Studies have shown that preweanling pups will demonstrate a preference for home cage odor over clean bedding or soiled bedding from other conspecifics (2,6,15). Similarly, there is an increased preference for a foreign odor that was paired with the dam and/or siblings during the preweaning period (1,3,7,11,13). Enhanced preference for foreign odors experienced in association with the dam were found when the odor exposure was brief as 2 days (11) or with quantities small as 0.2 ml applied directly to the dam (3). Indirect exposure to ethanol odor also appears to result in an increased preference for ethanol. In a preliminary study from our lab we observed that control litters housed in the same room as litters being explicitly exposed to ethanol odor exhibited levels of ethanol odor preference and ethanol intake similar to the animals explicitly exposed to more proximal, and hence more intense, ethanol odor. These findings suggest that even relatively weak exposure to the odor of ethanol in the home cage can increase preference for ethanol. Together the present data with that from other studies suggest that exposure to a foreign odor during the preweanling period, even indirectly, can alter later responding to the exposed odor. As with odor preference, simple exposure to a foreign odor produces an increase in the intake of a diet tainted with that odor. Preweanling rats exposed to peppermint odor from postnatal day 1 to 19 showed a preference for a diet containing peppermint extract at weaning when given a choice between peppermint- and lemon-tainted diet (17). Similarly, weanlings exposed to a foreign odor for 30 min ingested significantly more diet containing that odor during the 24-h period following odor exposure (14). The effect of odor exposure on diet choice was observed only at weaning; when the same procedure was used to expose older animals to a foreign odor, no preference for the odor-tainted diet was observed. The effect of early odor exposure on subsequent food choice appears to be relatively long lasting. For instance, exposure to ethanol odor for 35 min on postnatal day 21 resulted in greater intake of an ethanol solution 1 week later (19). Thus, as with odor preference, simple exposure to an odor can result in an increased intake of diets or solutions containing that odor. Overall the data indicate that young animals may be “programmed” to attend to odors and flavors encountered in the home nest as an adaptive mechanism, perhaps to avoid potentially poisonous foods. Support for this hypothesis comes
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BANNOURA ET AL.
FIG. 2. Total ethanol intake in a two bottle choice test. Ethanol intake is expressed as the total percentage of the total fluid intake during the 3-day test (A) and as the total gram of ethanol per kilogram of body weight intake during the 3 test days (B). All intake data are summed over the 3-day intake test.
from studies in which pups artificially reared away from conspecifics do not preferentially choose a feeding site near a conspecific or a site marked by the feces of a conspecific, although pups reared with their dam and littermates preferentially eat from feeding sites near a conspecific or from a site marked by the feces of a conspecific (10). Rat pups will avoid a food to which the adults of the colony have developed a conditioned taste aversion prior to the birth of the pup and which the pups themselves never sampled (12). When the pups were weaned and removed from the colony they continued to avoid the food to which their parents had an aversion. Thus, social factors, especially those associated with the home cage and adult conspecifics, exert a very strong influence over the ingestive behavior of animals after weaning (9). It may not be the case, however, that it is necessary for the foreign odor to be associated with the home cage for the early experience to have an impact on later preferences. Simple exposure to a foreign odor may be sufficient to produce an enhanced preference for the odor at test. When rat pups were exposed to peppermint odor away from the home cage during the preweaning period they displayed a preference for peppermint odor 24 h following the last odor exposure (17). When pups were exposed to lemon odor for 3 min on postnatal day 15 they expressed a preference for lemon over a novel odor when tested immediately following the odor exposure (5). Thus, simple exposure to an odor can produce a subsequent preference for that odor. It is possible that the exposure effect displayed by pups in the present study may be the result of pairing ethanol odor with the appetitive cues of the home cage or simple familiarity associated with repeat exposures. The pharmacological effects of ethanol present a unique dimension to the use of ethanol as an odorant. Although foreign odors, such as peppermint, function only as an odor, ethanol functions as both an odor and pharmacological agent. The
blood ethanol concentrations attained by the pups in the current study were low albeit notable. The influence of the pharmacological effects of ethanol is difficult to separate from that of exposure to the odor of ethanol, given the evidence that preweanling rats detect their own expired ethanol after administration of ethanol [e.g., (18)]. The confounding of chemosensory and pharmacological consequences of ethanol exposure make it difficult to make definite conclusions as to the critical element of ethanol odor exposure. Based on the findings of studies involving other odorants and the low BACs registered in the present experiment, it is reasonable to suggest that the odor of ethanol and not the pharmacological effects was the critical factor in the present effects of early odor exposure, but further study is required for a more definitive conclusions. The present results indicate that exposure to ethanol odor during the preweaning period can be a simple and effective process to enhance ethanol ingestion in older animals. Numerous paradigms have been developed to induce ethanol ingestion in rats, a species that typically avoids ethanol solutions. Most of these methods involve periods of fluid deprivation, palatable liquid diets containing ethanol, or lengthy initiation periods to produce significant ethanol ingestion. Our data demonstrate that simple exposure to ethanol odor during the preweaning period will enhance ingestion of a 6% ethanol solution by 50% in a two-bottle choice test at weaning. Although the longevity of the effect of preweaning ethanol odor exposure has yet to be tested, other studies suggest that the influence of early odor experience may extend into adulthood. For instance, when lemon odor was placed in the home cage throughout the preweaning period, a preference for lemon odor was recorded on postnatal day 141, the last day tested (7). Offspring of dams fed a garlic-containing solution throughout the preweanling period ingested more garlic solution in a two-bottle (water vs. garlic solution) test at 73–78
PREWEANLING ETHANOL ODOR EXPOSURE
217
days of particularly when the garlic solution was available for the first 5 days after weaning (4). Thus, preweanling odor exposure may exert a long-term influence on behavior. One potential implication of the data presented in this study involves the influence of the early home environment on alcohol consumption in humans. Could the presence of alcohol in the home influence the drinking habits in adolescence and adulthood of individuals reared in such a home? One study showed that Scottish and English children were capable of correctly identifying alcohol by the smell over 80% of the time (8). A second study from Michigan suggests that children whose parents are heavy drinkers were more likely to correctly identify the odor of alcohol than children whose parents drank moderately or not at all (22). Other studies have
shown that the children of alcoholics tend to drink more than children of nonalcoholics (24). Although genetics undoubtedly plays an important role in alcoholism, the home environment may also exert a significant influence on responding to alcohol. ACKNOWLEDGEMENTS
The research presented in this papers was supported by grants 5R01AA10223 to Norman E. Spear, R01AA10228 to Linda P. Spear, and 5F31AA05386 a Predoctoral Fellowship to Michelle D. Bannoura. The grants and Fellowship were awarded by the National Institute of Alcohol Abuse and Alcoholism. The authors would like to express their gratitude to Teri Tanenhaus, Tammy Allen, and Norm Richter for their assistance.
REFERENCES 1. Brake, S. C.: Suckling infant rats learn a preference for a novel olfactory stimulus paired with milk delivery. Science 211:506–508; 1981. 2. Brown, R. E.: Preferences of pre- and postweanling Long–Evans rats for nest odors. Physiol. Behav. 29:865–874; 1982. 3. Brunjes, P. C.; Alberts, J. R.: Olfactory stimulation induces filial preferences for huddling in rat pups. J. Comp. Physiol. Psychol. 93:548–555; 1979. 4. Capretta, P. J.; Rawls, L. H., III.: Establishment of a flavor preference in rats: Importance of nursing and weaning experience. J. Comp. Physiol. Psychol. 86:670–673; 1974. 5. Caza, P. A.; Spear, N. E.: Short-term exposure to an odor increases its subsequent preference in preweanling rats: A descriptive profile of the phenomenon. Dev. Psychobiol. 17:407–422; 1984. 6. Cornwell-Jones, C.; Sobrian, S. K.: Development of odor-guided behavior in Wistar and Sprague–Dawley rat pups. Physiol. Behav. 19:685–688; 1977. 7. Echandia, E. L. R.; Foscolo, M.; Broitman. S. T.: Preferential nesting in lemon-scented environment in rats reared on lemonscented bedding from birth. Physiol. Behav. 29:47–49; 1982. 8. Fossey, E.: Identification of alcohol by smell among young children: An objective measure of early learning in the home. Drug Alcohol Depend. 34:29–35; 1993. 9.Galef, B. G., Jr.: Development of flavor preferences in man and animals: The role of social and nonsocial factors. In: Aslin, R. N.; Alberts, J. R.; Petersen, M. R., eds. Development of perception vol. 1. New York: Academic Press; 1981. 10. Galef, B. G., Jr.: Development of olfactory control of feeding-site selection in rat pups. J. Comp. Physiol. Psychol. 95:615–622; 1981. 11. Galef, B. G., Jr.: Acquisition and waning of exposure-induced attraction to a nonnatural odor in rat pups. Dev. Psychobiol. 15:479–490; 1982. 12. Galef, B. G., Jr.; Clark, M. M.: Social factors in the poison avoidance and feeding behavior of wild and domesticated rat pups. J. Comp. Physiol. Psychol. 75:341–357; 1971.
13. Galef, B. G., Jr.; Kaner, H. C.: Establishment and maintenance of preference for natural and artificial olfactory stimuli in juvenile rats. J. Comp. Physiol. Psychol. 94:588–595; 1980. 14. Galef, B. G., Jr.; Kennett, D. J.: Different mechanisms for social transmission of diet preference in rat pups of different ages. Dev. Psychobiol. 20:209–215; 1987. 15. Gregory, E. H.; Pfaff, D. W.: Development of olfactory-guided behavior in infant rats. Physiol. Behav. 6:573–576; 1971. 16. Hunt, P. S.; Kraebel, K. S.; Rabine, H.; Spear, L. P.; Spear, N. E.: Enhanced ethanol intake in preweanling rats following exposure to ethanol in a nursing context. Dev. Psychobiol. 26:133–153; 1993. 17. Leon, M.; Galef, B. G., Jr.; Behse, J. H.: Establishment of pheromonal bonds and diet choice in young rats by odor preexposure. Physiol. Behav. 18:387–391; 1977. 18. Molina, J. C.; Chotro, M. G.; Spear, N. E.: Early (preweanling) recognition of alcohol’s orosensory cues resulting from acute ethanol intoxication. Behav. Neural Biol. 51:307–325; 1989. 19. Molina, J. C.; Serwatka, J.; Spear, N. E.: Changes in alcohol intake resulting from prior experience with alcohol odor in young rats. Pharmacol. Biochem. Behav. 21:387–391; 1984. 20. Molina, J. C.; Serwatka, J.; Spear, N. E.: Alcohol drinking patterns of young adult rats as a function of infantile aversion experiences with alcohol odor. Behav. Neural Biol. 46:257–271; 1986. 21. Molina, J. C.; Serwatka, J.; Spear, L. P.; Spear, N. E.: Differential ethanol olfactory experiences affect ethanol ingestion in preweanlings but not in older rats. Behav. Neural Biol. 44:90–100; 1985. 22. Noll, R. B.; Zucker, R. A.; Greenberg, G. S.: Identification of alcohol by smell among preschoolers: Evidence for early socialization about drugs occurring in the home. Child Dev. 61:1520– 1527; 1990. 23. Serwatka, J.; Molina, J. C.; Spear, N. E.: Weanlings’ transfer of conditioned ethanol aversion from olfaction to ingestion depends on the unconditioned stimulus. Behav. Neural Biol. 45:57–70; 1986. 24. U.S. Department of Health and Human Services Alcohol and Health.: Eighth Special Report to the U.S. Congress; 1993.
PII S0741-8329(97)00122-5
Effects of Preweanling Ethanol Odor Exposure on Ethanol Preference MICHELLE D. BANNOURA, KIMBERLY S. KRAEBEL, LINDA P. SPEAR AND NORMAN E. SPEAR Center for Developmental Psychobiology, Binghamton University, P.O. Box 6000, Binghamton, NY Received 25 June 1996; Accepted 27 June 1997 BANNOURA, M. D., K. S. KRAEBEL, L. P. SPEAR AND N. E. SPEAR. Effects of preweanling ethanol odor exposure on ethanol preference. ALCOHOL 15(3) 213–217, 1998.—Preweaning exposure to a foreign odor has been shown to alter later responding to that odor. Early experience with ethanol odor may alter not only responding to ethanol odor, but also intake of ethanol solutions. The present study tested the effects of exposure to ethanol odor prior to weaning on ethanol odor preference and ethanol consumption. Sprague–Dawley-derived rats were exposed to the odor of 100% ethanol from postnatal day 1 to 22 in the home cage. An odor preference test was conducted on postnatal day 14 and a two-bottle ethanol intake test was conducted after weaning. In both odor preference and intake tests, animals previously exposed to ethanol odor exhibited a greater preference for ethanol than controls. The results demonstrate that early experience with the odor of ethanol can increase ingestion of ethanol later in life. © 1998 Elsevier Science Inc. Ethanol preference
Ethanol odor
Preweanling experience
results in a preference for those odors later in life (2,3,6,7,15). When rat pups were reared in a home cage containing a foreign odor, such as peppermint oil, a preference for the foreign odor was demonstrated up to 100 days later (3,7,11,13). The present experiment combines the above lines of research by investigating the effects of ethanol odor exposure in the home cage throughout the preweaning period. Pups were exposed to ethanol odor from postnatal day 1 through day 22. Ethanol odor preference was tested on postnatal day 14 and ethanol intake was tested from postnatal days 23 to 25.
EXPOSURE to ethanol early in development has been shown to alter later responding to ethanol, with the nature of the alteration depending on the circumstances of the exposure (16,18–21,23). Changes in ethanol ingestion were recorded following the pairing of ethanol odor and internal malaise in weanling rats (19,23). When internal malaise was paired with ethanol odor on a single occasion, a significant decrease in ethanol intake was observed in weanlings, but not adults, 24 h after the pairing (21). When multiple pairings of ethanol odor and internal malaise were given during the preweaning period, ethanol intake decreased in adulthood, 38 days after the final exposure (20). Weanlings given a pairing of ethanol odor and internal malaise decreased their ethanol intake 7 days later, whereas control animals exposed to ethanol odor unpaired with malaise significantly increased their ethanol intake compared to ethanol naive controls (19). The results demonstrate that reinforcement contingencies associated with ethanol odor exposure alters subsequent ethanol consumption, whereas mere exposure to ethanol odor also may alter responding to ethanol. Concomitant with studies showing an alteration in responding to ethanol following prior ethanol experience, others have shown that early experience with other foreign odors
METHOD
Subjects The offspring of 18 Sprague–Dawley-derived dams born and bred in our colony at Binghamton University served as the subjects in this experiment. Births were checked daily between 1500 and 1800 h, and the first day pups were found in the cage was considered postnatal day 0. Litters were culled to 10 pups (five male and five female when possible) within 24 h of birth. Weaning on postnatal day 21 was accomplished by removing the dam from the cage. Pups remained in the home cage with littermates until the time of ethanol intake testing.
Requests for reprints should be addressed to Norman E. Spear, Center for Developmental Psychobiology, Binghamton University, P.O. Box 6000, Binghamton, NY 13902-6000. Tel: (607) 777-2663; Fax: (607) 777-2677.
213
214 Twenty-four hours prior to the beginning of the ethanol intake test weanlings were removed from the home cage and individually housed. All animals were maintained on a 16:8 light:dark schedule at 23–25˚C under relatively constant humidity. Ethanol Odor Exposure Ten litters were exposed to ethanol odor in their home cage (standard maternity cage 47 3 25.4 3 20.3 cm) from postnatal day 1 through 22. Pure ethanol, 200 proof, was placed on cotton in a Plexiglas holder at each end of the home cage. The Plexiglas holders fit snugly into the ends of the maternity cage and had approximately 50 small holes drilled in the face. The holders were constructed so the odor could move into the cage while the animals could not physically contact the cotton. At 0900 every morning 30 ml of 200 proof ethanol was applied to the cotton in each end of the cage. Thus, animals were exposed to the odor of 60 ml of pure ethanol each day. The cotton in each end of the home cage was replaced each day prior to the application of ethanol. The ethanol odor-exposed litters were maintained in a room separate from the main colony on the same light:dark schedule at a similar temperature and humidity. Other than daily odor exposure and weekly cage cleaning, the litters were left undisturbed until postnatal day 14. Eight control litters were exposed to water odor following the same procedures used for ethanol odor exposure. Distilled water, 30 ml, was placed on cotton in Plexiglas holders in each end of the home cage. The control litters were housed in the main colony room. The function of the water odor control group was to equate the disturbance of the litter during the preweanling period. Odor Preference Test On postnatal day 14, an odor preference test was conducted to assess the relative preference for ethanol odor in comparison to a novel odor. The odor preference test was conducted at 1300 h, 4 h following the daily application of odor in the home cage. The timing of the odor preference test was chosen based on the blood alcohol levels of pups on postnatal day 16. Pups never displayed any overt signs of motor impairment or intoxication. Two pups from each litter, one male and one female, were removed from the home cage, taken to a separate experimental room, weighed, and kept in a holding cage for no more than 15 min prior to testing. The odor preference test was conducted in a clear Plexiglas chamber (28 3 10.5 3 12.5 cm) with cotton placed in each end. Pure ethanol, 3 ml, was placed on the cotton at one end of the apparatus and clove oil, 0.3 ml, was placed on the cotton at the other end. These odors and their concentrations were chosen on the basis of pilot data showing approximately equal preference for each odor in this combination by naive rats on postnatal day 14. Each preference test was 2 min long. Each pup was picked up and replaced on the midline at the start of the second minute. The relative left–right position of the ethanol odor was altered by changing the orientation of the pup’s head at the beginning of the second test minute. This was done to control for potential side preferences independent of odor preference. The time over each odor was recorded for each minute. The pup was considered over an odor when the whole head and both front paws were across the midline. Fresh cotton and odors were used for each pup. Pups used in the odor preference test were humanely sacrificed following odor preference testing to avoid exposing the rest of the litter to the testing odors.
BANNOURA ET AL. Blood Ethanol Concentration Analysis The concentration of ethanol in the blood of the ethanol odor-exposed litters was determined on postnatal day 16. Eight pups were decapitated at each of five time points following the daily application of ethanol odor in the home cage. Blood was collected at 15, 30, 60, 120, and 300 min following the application of ethanol to the home cage. Blood was collected in a 1.5-ml vial containing 0.10 ml of heparin sodium salt solution. The ethanol content was determined by head space gas chromatographic analysis. The gas chromatograph contained a 6-foot stainless steel column packed with Poropak Q and utilized nitrogen as the carrier gas. The blood sample, 25 ml, placed in an airtight vial, was kept in a 55˚C water bath for 8 min. An airtight syringe was used to extract 1 ml of the head space gas from the sample vial and inject the gas onto the column of the gas chromatograph. The retention time for the ethanol peak was 2.6 min. Concentration of ethanol in the blood was measured against a standard concentration curve. Ethanol Intake Test Animals were tested for 3 days beginning on postnatal day 23. For 23 h each day animals were given free access to one bottle containing a 6% (v/v) ethanol solution and one bottle containing tap water. Each morning the bottles were removed, weighed, and refilled with fresh solutions. While the bottles were off the cage, animals were weighed. The left– right position of the bottles was changed each day to control for possible side preferences. Food was freely available throughout the test period. Experimental Design and Statistical Analysis All data were analyzed by analysis of variance (ANOVA) with Preweaning Experience (ethanol odor or control) and Sex (female or male) serving as between-group factors in all analyses. Odor preference data were analyzed as a 2 3 2 3 2 mixed design with Test Minute (1, 2) serving as a withingroups factor. Ethanol intake, both percent total fluid intake and g/kg intake, was analyzed as a 2 3 2 3 3 mixed design with Test Day (1, 2, 3) serving as a within-groups measure. When significant main effects or interactions were found, a Newman–Keuls post hoc test was used to determine significant differences between means. Statistical significance was determined with alpha less than or equal to 0.05 in all tests. RESULTS
Odor Preference Test Pups exposed to ethanol odor throughout the preweaning period spent significantly more time over the ethanol odor than pups that had never been exposed to ethanol odor (Fig. 1). This effect was confirmed by a significant main effect of preweaning experience, F(1, 32) 5 5.36. A significant main effect of test minute, F(1, 32) 5 10.53, was also found. Post hoc analysis of the data collapsed across preweanling experience showed that animals spent significantly less time over the ethanol odor during the second test minute (17.6 6 2.07 s) than the first minute (23.9 6 1.59 s). Blood Ethanol Concentration The concentration of ethanol in the trunk blood remained relatively constant across the 300-min period of analysis. The highest level reached was 26.34 mg/dl at 120 min after the appli-
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215 firmed the influence of ethanol odor exposure on the intake of a 6% ethanol solution after weaning. Ethanol intake (g/kg) was also influenced by an interaction of Sex 3 Test Day, F(2, 28) 5 3.80. Females decreased their g/kg ethanol intake (day 1 5 3.5 6 0.6 g/kg, day 3 5 2.5 6 0.5 g/kg) whereas males showed no significant change in intake across the 3 test days (day 1 5 3.3 6 0.5, day 3 5 4.1 6 0.8 g/kg). DISCUSSION
FIG. 1. Ethanol odor preference on postnatal day 14. Total time over ethanol during a 2-min test. Data are collapsed over test minute.
cation of ethanol odor to the home cage (Table 1). Blood ethanol concentration was not significantly influenced by either time after application, F(4, 26) 5 2.00, or sex, F(1, 26) , 1.0. Ethanol Intake Test The percentage of total fluid intake accounted for by ethanol solution was significantly greater in pups exposed to ethanol odor than in control animals (Fig. 2A). This effect was confirmed by a significant main effect of preweaning experience, F(1, 14) 5 5.49. A significant interaction of Sex 3 Test Day, F(2, 28) 5 4.31, reflected a difference in the pattern of intake between males and females. Post hoc analysis of the data collapsed across preweanling experience revealed that females significantly decreased their ethanol intake from day 1 (17.8 6 2.8 %) to day 3 (12.1 6 1.9 %), whereas males significantly increased their intake from test day 1 (17.8 6 2.7 %) to 3 (21.7 6 3.4 %). These effects were not sufficiently robust, however, to result in a significant gender difference in percentage ethanol intake on any of the test days. Gram per kilogram (g/kg) ethanol intake displayed a pattern of results similar to the results obtained with the percentage total fluid intake measure. Preweaning experience with ethanol odor resulted in significantly greater ethanol intake (g/kg) compared to water controls (Fig. 2B). A significant main effect of preweaning experience, F(1, 14) 5 7.0, con-
TABLE 1 BLOOD ETHANOL CONCENTRATION ON POSTNATAL DAY 16 Time After Odor Application (Minutes)
15 30 60 120 300
Blood Ethanol Concentration [mg/dl (SE)]
8.99 (1.6) 15.86 (3.8) 16.11 (4.7) 26.34 (6.2) 13.16 (8.4)
The present results demonstrate that exposure to ethanol odor in the home cage throughout the preweaning period alters preweaning and postweaning responding to ethanol. Pups exposed to ethanol odor in the home cage showed a greater preference for ethanol odor on postnatal day 14. Weanlings previously exposed to ethanol odor ingested significantly more of a 6% ethanol solution than control animals. The increased preference for ethanol odor suggests that the animals associated the appetitive and familiar qualities of the home cage with the odor of ethanol. Studies have shown that preweanling pups will demonstrate a preference for home cage odor over clean bedding or soiled bedding from other conspecifics (2,6,15). Similarly, there is an increased preference for a foreign odor that was paired with the dam and/or siblings during the preweaning period (1,3,7,11,13). Enhanced preference for foreign odors experienced in association with the dam were found when the odor exposure was brief as 2 days (11) or with quantities small as 0.2 ml applied directly to the dam (3). Indirect exposure to ethanol odor also appears to result in an increased preference for ethanol. In a preliminary study from our lab we observed that control litters housed in the same room as litters being explicitly exposed to ethanol odor exhibited levels of ethanol odor preference and ethanol intake similar to the animals explicitly exposed to more proximal, and hence more intense, ethanol odor. These findings suggest that even relatively weak exposure to the odor of ethanol in the home cage can increase preference for ethanol. Together the present data with that from other studies suggest that exposure to a foreign odor during the preweanling period, even indirectly, can alter later responding to the exposed odor. As with odor preference, simple exposure to a foreign odor produces an increase in the intake of a diet tainted with that odor. Preweanling rats exposed to peppermint odor from postnatal day 1 to 19 showed a preference for a diet containing peppermint extract at weaning when given a choice between peppermint- and lemon-tainted diet (17). Similarly, weanlings exposed to a foreign odor for 30 min ingested significantly more diet containing that odor during the 24-h period following odor exposure (14). The effect of odor exposure on diet choice was observed only at weaning; when the same procedure was used to expose older animals to a foreign odor, no preference for the odor-tainted diet was observed. The effect of early odor exposure on subsequent food choice appears to be relatively long lasting. For instance, exposure to ethanol odor for 35 min on postnatal day 21 resulted in greater intake of an ethanol solution 1 week later (19). Thus, as with odor preference, simple exposure to an odor can result in an increased intake of diets or solutions containing that odor. Overall the data indicate that young animals may be “programmed” to attend to odors and flavors encountered in the home nest as an adaptive mechanism, perhaps to avoid potentially poisonous foods. Support for this hypothesis comes
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FIG. 2. Total ethanol intake in a two bottle choice test. Ethanol intake is expressed as the total percentage of the total fluid intake during the 3-day test (A) and as the total gram of ethanol per kilogram of body weight intake during the 3 test days (B). All intake data are summed over the 3-day intake test.
from studies in which pups artificially reared away from conspecifics do not preferentially choose a feeding site near a conspecific or a site marked by the feces of a conspecific, although pups reared with their dam and littermates preferentially eat from feeding sites near a conspecific or from a site marked by the feces of a conspecific (10). Rat pups will avoid a food to which the adults of the colony have developed a conditioned taste aversion prior to the birth of the pup and which the pups themselves never sampled (12). When the pups were weaned and removed from the colony they continued to avoid the food to which their parents had an aversion. Thus, social factors, especially those associated with the home cage and adult conspecifics, exert a very strong influence over the ingestive behavior of animals after weaning (9). It may not be the case, however, that it is necessary for the foreign odor to be associated with the home cage for the early experience to have an impact on later preferences. Simple exposure to a foreign odor may be sufficient to produce an enhanced preference for the odor at test. When rat pups were exposed to peppermint odor away from the home cage during the preweaning period they displayed a preference for peppermint odor 24 h following the last odor exposure (17). When pups were exposed to lemon odor for 3 min on postnatal day 15 they expressed a preference for lemon over a novel odor when tested immediately following the odor exposure (5). Thus, simple exposure to an odor can produce a subsequent preference for that odor. It is possible that the exposure effect displayed by pups in the present study may be the result of pairing ethanol odor with the appetitive cues of the home cage or simple familiarity associated with repeat exposures. The pharmacological effects of ethanol present a unique dimension to the use of ethanol as an odorant. Although foreign odors, such as peppermint, function only as an odor, ethanol functions as both an odor and pharmacological agent. The
blood ethanol concentrations attained by the pups in the current study were low albeit notable. The influence of the pharmacological effects of ethanol is difficult to separate from that of exposure to the odor of ethanol, given the evidence that preweanling rats detect their own expired ethanol after administration of ethanol [e.g., (18)]. The confounding of chemosensory and pharmacological consequences of ethanol exposure make it difficult to make definite conclusions as to the critical element of ethanol odor exposure. Based on the findings of studies involving other odorants and the low BACs registered in the present experiment, it is reasonable to suggest that the odor of ethanol and not the pharmacological effects was the critical factor in the present effects of early odor exposure, but further study is required for a more definitive conclusions. The present results indicate that exposure to ethanol odor during the preweaning period can be a simple and effective process to enhance ethanol ingestion in older animals. Numerous paradigms have been developed to induce ethanol ingestion in rats, a species that typically avoids ethanol solutions. Most of these methods involve periods of fluid deprivation, palatable liquid diets containing ethanol, or lengthy initiation periods to produce significant ethanol ingestion. Our data demonstrate that simple exposure to ethanol odor during the preweaning period will enhance ingestion of a 6% ethanol solution by 50% in a two-bottle choice test at weaning. Although the longevity of the effect of preweaning ethanol odor exposure has yet to be tested, other studies suggest that the influence of early odor experience may extend into adulthood. For instance, when lemon odor was placed in the home cage throughout the preweaning period, a preference for lemon odor was recorded on postnatal day 141, the last day tested (7). Offspring of dams fed a garlic-containing solution throughout the preweanling period ingested more garlic solution in a two-bottle (water vs. garlic solution) test at 73–78
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days of particularly when the garlic solution was available for the first 5 days after weaning (4). Thus, preweanling odor exposure may exert a long-term influence on behavior. One potential implication of the data presented in this study involves the influence of the early home environment on alcohol consumption in humans. Could the presence of alcohol in the home influence the drinking habits in adolescence and adulthood of individuals reared in such a home? One study showed that Scottish and English children were capable of correctly identifying alcohol by the smell over 80% of the time (8). A second study from Michigan suggests that children whose parents are heavy drinkers were more likely to correctly identify the odor of alcohol than children whose parents drank moderately or not at all (22). Other studies have
shown that the children of alcoholics tend to drink more than children of nonalcoholics (24). Although genetics undoubtedly plays an important role in alcoholism, the home environment may also exert a significant influence on responding to alcohol. ACKNOWLEDGEMENTS
The research presented in this papers was supported by grants 5R01AA10223 to Norman E. Spear, R01AA10228 to Linda P. Spear, and 5F31AA05386 a Predoctoral Fellowship to Michelle D. Bannoura. The grants and Fellowship were awarded by the National Institute of Alcohol Abuse and Alcoholism. The authors would like to express their gratitude to Teri Tanenhaus, Tammy Allen, and Norm Richter for their assistance.
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