Taste preferences of female rats: Modification by neonatal hormones, food deprivation and prior experience

Physiology and Behavior. Vol. 4, pp. 935-943. Pergamon Press, 1969. Printed in Great Britain

Taste Preferences of Female Rats: Modification by Neonatal Hormones, Food Deprivation and Prior Experience G E O R G E N. W A D E A N D I R V I N G Z U C K E R

Department of Psychology, University of California, Berkeley, California, U.S.A. (Received 21 M a y 1969) WADE, G. N. AND I. ZUCKER. Taste preferences of female rats: modification by neonatal hormones, food deprivation and prior experience. PHYSlOL.BEHAV. 4 (6) 935-943, 1969.--Steroid treatments during the early neonatal period modify

adult taste preferences of female rats. A single injection of testosterone propionate on the fifth day of life greatly decreases saccharin preferences (S.P.) while treatment with estradiol benzoate results in increased saccharin consumption. Both early hormone treatments abolish sexual cyclicity and sexual receptivity. These results are related to hormonally-induced modifications in sexual differentiation of neuroendocrine systems. S.P. are increased by total food deprivation but decreased by 25 per cent food deprivation in 24 hr tests; some effects of food deprivation on taste preference persist and others first appear after ad libitum feeding schedules have been re-instated. Mechanisms by which food deprivation and taste preference may interact are considered. Prior experience with saccharin solutions is an important variable in the extent to which ovarian hormones regulate S.P. Thus, for the great majority of rats experienced in the selection of saccharin solutions, S.P. are unaltered by ovariectomy while among inexperienced rats, S.P. are much greater for intact than for ovariectomized females. Hormonal control of S.P. is therefore more important during the acquisition phase of the preference pattern than during the maintenance phase, when other, presumably learned, cues take precedence. Saccharin preference

Ovariectomy

Sex differentiation

Early experience

Food and fluid intake

SELECTION of non-nutritive sweet substances by rats is influenced by a number of physiological variables, including the animal's sex [22-24], condition of bodily need [1, 5] and endocrine status [24, 26]. The present report describes some of the ways in which sexual differentiation of the neuroendocrine system, food deprivation and prior taste experience interact to regulate saccharin and water intake in taste preference tests.

day to day. With the exception of Experiment 1, food intake was measured daily to the nearest 0.1 g. All animals were tested nightly for estrous behavior with the "fingering" method. This technique i~ highly reliable for plotting behavioral estrous cycles and requires only several seconds testing during which the anogenital region of the rat is manually stimulated [27].

GENERAL PROCEDURE

EXPERIMENT 1

Experimentally naive Sprague-Dawley rats were housed individually in a room illuminated from midnight to noon each day. Unless otherwise noted, each rat had continuous access to Simonson dry lab chow and to solutions available from two 100 ml Richter tubes mounted at the front of each cage. Every 24 hr fluid intake was measured and the tubes cleaned and refilled with fresh solutions. Except for an initial adaptation period, during which only water was available, rats were offered a choice between distilled water and a 0.75 per cent saccharin solution prepared by dissolving 7.5 g sodium-o-benzoic sulfimide per litre of distilled water. In all experiments tubes were maintained in a fixed position from

Treatment with estrogens or androgens during the early neonatal period profoundly alters sexual characteristics of female rats. In adulthood these animals display a syndrome of anovulatory sterility, decreased sexual receptivity and greatly diminished sensitivity to certain hormones [2, 25]. These effects are related to modifications in the differentiation of hypothalamic mechanisms for ovulation and sex behavior [2, 9, 11, 25], although the nature of the neural changes remains unspecified. The procedures which alter sexual differentiation of the brain also affect the expression of non-reproductive behaviors. Sex differences in hypothalamic hyperphagia [6], feeding

tThis research was supported by Grant HD-02982 from the National Institute of Child Health and Human Development and the Committee on Research, University of California. We are grateful to Terry Rechlitz, Larry Robidoux and George Paxinos for technical assistance. G.W. was supported by NDEA pre-doctoral fellowship 67-00858 and L Z. by Career Development Award K4-HD-42413 from the National Institute of Child Health and Human Development. 935

936

WADE AND ZUCKER

(Bell & Zucker, unpublished), aggressive and social behavior [3, 7, 16] are all influenced by early hormone treatments. Recently, taste preferences for sweet solutions were found to be much greater among female than male rats [22-24]. The existence of this sexually dimorphic behavior permits analyses, previously not possible, of the relation between sexual processes and taste patterns. The main purpose of the first experiment was to determine whether early hormone treatments which modify adult reproductive behaviors exert similar influences on preferences for sweet solutions.

Method Three groups of female rats were injected subcutaneously at 5 days of age with one of the following: 1 mg testosterone propionate* (TP); 50 ~tg estradiol benzoate (EB) or sesame oil (O). The hormones were diluted with sesame oil as vehicle such that all injections consisted of a final volume of 0.05 ml/rat. There were 20, 20 and 19 rats in the TP, EB and O groups, with body weights of 312, 324 and 267 g, respectively at 134 days of age (Day 1 of testing). Beginning on the ninth day of testing, and continuing until the experiment was terminated on Day 23, rats could drink either water or the saccharin solution, ad libitum. Since rats frequently display both position and tube preferences, a procedure was adopted for selecting the tube to receive saccharin (saccharin-destined tube). The saccharindestined tube was designated such that the ratio of water

A-WATER ONLY --

consumed from it and from the water-destined tube during the adaptation period was approximately equal for the three experimental groups. In this experiment the ratios were 0.75, 0.71 and 0.76 for the TP, EB and O groups, respectively. Thus, between groups, there was no overall bias for or against the saccharin tube.

Results Neonatal treatment with either EB or TP was very effective in abolishing sexual receptivity and cyclicity. None of the 40 hormone-treated females displayed a regular estrous cycle and only rarely were weak signs of heat detected. In contrast, 16 of 19 oil-injected rats displayed one or more regular estrous cycles of 4 or 5 days duration [27]. With water as the only available solution, mean fluid intake was greater for both EB and TP females than for O rats (p -< 0.002, p < 0.02, respectively, Mann-Whitney U test, with comparison across all days of testing, Fig. 1A); EB and TP groups did not differ significantly. Differences in water intake were not significant, however, when corrections were made for differences in body weight. Water consumption was 12.3, 11.9 and 11.4 ml/100 g body weight for the 3 groups, respectively. EB and TP females weighed substantially more than O rats. With both water and saccharin available (Days 9-23), EB females consumed more saccharin (Fig. 2B) than either the TP or O females (p < 0.002,p < 0.02, respectively). Saccharin

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FIG. 1. Mean fluid consumption of adult female rats treated with 50 f~g estradiol benzoate (EB), 1 mg testosterone propionate (TP) or oil vehicle on the fifth day after birth. Panel A shows results when only water was available and Panel B plots fluid intake when water and saccharin were available. *Estradiol benzoate (Progynon), progesterone (Proluton) and testosterone propionate (Oreton propionate) were generously supplied by the Schering Corporation, Bloomfield, N.J.

HORMONES AND TASTE PREFERENCES

937

intake was greater for O than for TP rats but the difference was not significant. During this same period TP-treated rats consumed more water (Fig. 2C) than EB females (p : 0.02) or O females (U = 122, 0.05 < p < 0.1, 2 tailed Mann-Whitney test). There were no differences on this measure between O and EB groups.

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FIG. 2. Saccharin preference (A), saccharin intake (B) and water intake (C) of adult female rats treated on the fifth day after birth with 50 ~g estradiol benzoate (EB), 1 mg testosterone propionate (TP) or oil vehicle. Saccharin preference is computed by obtaining the ratio of saccharin intake/total fluid intake × 100. An 0.75 per cent saccharin solution was used.

Total fluid intake (Fig. IB) was greater for EB females than for TP or oil rats (p < 0.002, p < 0.02, respectively); O and TP groups did not differ on this measure even when corrections were made for differences in body weight. The ratio of saccharin to total fluids (saccharin/saccharin + water) consumed was used as the index of saccharin preference (S.P.). Results, depicted in Fig. 2A clearly indicate that, evaluated relative to oil controls, rats injected with TP have lower S.P. (p < 0.05, across all days), whereas S.P. of females treated with EB are slightly elevated. The latter effect was not statistically significant, although S.P. of EB rats were, of course, significantly greater than those of TP rats (p < 0.02). EXPERIMENT 2 In the course of an earlier series of experiments we observed that pregnant rats partially deprived of food did not increase consumption of a 0.75 per cent saccharin solution in 24 hr tests [24]. Since total food deprivation greatly increases saccharin consumption in both short term [1] and long term [19]

tests, we have investigated more systematically the effects of different degrees of food deprivation on S.P. The results show that while total food deprivation greatly elevates preference for saccharin over water, absolute quantities of saccharin consumed are not increased, Partial (25 per cent) deprivation is accompanied by slightly decreased S.P. The effects of both degrees of food deprivation on food and fluid regulation persist and sometimes first appear after rats have been returned to ad libitum feeding schedules. Method Thirty-nine female rats previously used in an experiment that tested the effects of pseudopregnancy on S.P. [24] served as subjects. All rats were first allowed to resume estrous cycling and were then tested for an additional 5 days with ad libitum access to food, water and 0.75 per cent saccharin solution. The baseline data obtained during this 5-day period were used to divide the animals into three groups of equal size equated with respect to food intake, S.P. and as closely as possible, previous experimental treatment. The three groups were then subjected to different degrees of food deprivation. One group was not deprived (0 per cent deprivation); a second group was totally deprived of food for 4 consecutive days (100 per cent deprivation) and then returned to an ad libitum feeding schedule. A third group was restricted to 75 per cent of its daily baseline food intake for 10 days before ad libitum feeding was reinstated (25 per cent deprivation). Fresh food was provided each day between 9 and 11 a.m. Without exception, rats restricted to 75 per cent of their baseline intake, consumed all the food available to them. All groups had continuous free access to water and saccharin solutions. Results Both fluid consumption and taste preferences were influenced by 100 per cent food deprivation. S.P. were elevated during the four deprivation days (p < 0.01 across 4 days, Wilcoxon Test) as compared with the last pre-deprivation day. The effect was especially pronounced on the last two days of deprivation (Fig. 3B). The increase in S.P. was not due to increased consumption of saccharin, but rather to a proportionately large decrease in water intake (Fig. 4A). In fact, on the first and last deprivation days saccharin intake was less than during pre-deprivation testing (Fig. 4B). S.P. were markedly decreased upon reinstatement of free access to food (Fig. 3B). Decreased preference for the sweet solution persisted for at least 9 days after deprivation ceased and was attributable to the combination of a large increase in water intake (Fig. 4A) and a large decrease in saccharin consumption (Fig. 4B). Total fluid intake returned to pre-deprivation levels within three days after free access to food was restored (Fig. 3A). F o o d intake of totally deprived rats was markedly elevated over pre-deprivation levels for 10 days after ad libitum feeding was reinstated (Fig. 5). During this period water intake was increased and saccharin intake and S.P. were markedly depressed (Figs. 3 and 4). Fluid intake and taste preferences were only slightly affected during 10 days of 25 per cent food deprivation; S.P. were slightly lower than prior to deprivation (Fig. 3B). This is attributable to a small increase in water intake (Fig. 4A), saccharin consumption changed only minimally (Fig. 4B).

938

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FIG. 4. Mean water intake (A) and mean saccharin intake (B) of female rats subjected to various de~-ees of food deprivation.

Rats in the 100 per cent deprived group were completely without food for 4 days, beginning on Day 6; rats in the 25 per cent deprived group were restricted to 75 per cent of their daily preintervention food ration for 10 days, beginning on Day 6. An 0.75 per cent saccharin solution was used.

Rats offered continuous daily access to water and a saccharin solution show increasing preference for the sweet solution on successive test days; this has been termed the acquisition phase of the preference pattern [26]. After several weeks of daily exposure to both solutions, a stable preference pattern emerges and is maintained over extended periods (maintenance phase).

H O R M O N E S A N D TASTE P R E F E R E N C E S

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FIG. 6. Saccharin preferences of female rats experienced in the selection of saccharin solutions before and after ovariectomy or sham-ovariectomy. An 0.75 per cent saccharin solution was used. The rationale for subdividing the ovariectomized rats into two groups is discussed in the text.

940

WADE AND ZUCKER Results Changes in S.P. are shown in Fig. 6. Examination of individual records from each of 36 rats revealed that with the exception of 3 animals considered separately below, changes in taste preference were either very minimal or transient in nature. Thus, there were no permanent changes in S.P. for all of the sham-operated rats and for 15 of the 18 ovariectomized animals. The results for the 3 remaining ovariectomized rats were strikingly different. Pre-operatively each of these females had a high preference for saccharin, consuming approximately 90 per cent of fluids in the form of saccharin solution. Following gonadectomy, S.P. declined gradually for two of the animals and very rapidly (in one day) for the third. By the twelfth post-operative day these rats had completely reversed their original drinking patterns, and more than 90 per cent of their fluid intake was now in the form of water (Fig. 6). Sham operated and ovariectomized rats were well equated with respect to food intake during the pre-operative period (Fig. 7C). Beginning on the fourth post-operative day (p < 0.02) and continuing for the duration of the experiment, mean food consumption for the 15 spayed rats was greater than that of the sham-operates. This confirms a number of earlier studies reviewed by Kakolewski, Cox and Valenstein

Ovarian hormones exert a stimulatory effect on S.P. during the acquisition phase of testing [26]; among rats without prior experience with saccharin, intake of saccharin and preference for the sweet solution over water are much greater for intact than for ovariectomized females [26]. The present experiment evaluated the influence of ovariectomy on stable, well established S.P. F o r the great majority of rats experienced in the selection of saccharin solutions S.P. are unaltered by ovariectomy. Well established S.P. are thus independent of the ovarian hormones that facilitate original acquisition of the taste preference. Method Thirty-six female rats from Experiment 2 with continuous access to both 0.75 per cent saccharin solution and water during the preceding 85 days, were tested for 10 additional days (baseline recording) with ad libitum access to food, water and saccharin solution. Rats were divided into two equal groups, equated with respect to food intake, S.P. and fluid intake during the baseline period; they were also matched as closely as possible for previous experimental treatments. One group of rats was ovariectomized under ether anesthesia; females in the other group were sham-operated. Following surgery all animals were tested for 20 days under ad libitum conditions of food and fluid access.

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SUCCESSIVE 5-DAY PERIODS FIG. 7. Mean water intake (A), mean fluid intake (B) and mean food intake (C) of female rats experienced in the selection of saccharin solutions before and after ovariectomy or sham-ovariectomy. The rationale for subdividing the ovadectomized rats into two groups is discussed in the text.

HORMONES AND TASTE PREFERENCES rats showing the large post-operative decrease in S.P. was significantly lower than for the remaining ovariectomized subjects (p < 0.05, comparison of median values across Days 4-21) and not significantly different from that of sham-operates. Sham-operated and ovariectomized rats were originally well equated for fluid intake (water plus saccharin); however, with the separate consideration given the 3 ovariectomized rats that showed large post-operative decreases in S.P., the groups were less well matched and pre-operative fluid intake for 18 sham-operates was somewhat greater than for the main group of 15 spayed rats (67.2 vs. 62.5 ml/day, Fig. 7B). Fluid intake decreased for all groups during the first three post operative days. Beginning on the fourth day and continuing for the duration of the experiment, fluid intake of the 15 spayed rats was approximately equal to that of the preoperative period (62.9 vs. 62.5 ml) for a change of less than 1 per cent (Fig. 7B). This finding is interesting in light of the 18 per cent increase in food intake shown by these rats during the same post-operative period (from 22.7 to 26.8 g/day, Fig. 7C). Increased food intake following ovariectomy may result specifically in increased water intake, rather than in greater total fluid consumption p e r se. That this was not the case is illustrated in Fig. 7A; water intake of the 15 spayed rats was not significantly different from that of the sham-operates, although the increases in food intake for the two groups were 18 and 1.3 per cent, respectively, during Days 4-21. At least during the early stages following ovarieetomy, substantial increases in food intake may be effected without accompanying changes in fluid consumption. Unlike the main group of spayed rats, the three ovariectomized females showing the post-operative decrease in S.P. also decreased their fluid intake 21 per cent following surgery, from a pre-operative mean of 74.9 to 58.9 ml/day on Days4-21. During the same period, food intake increased 1.7 per cent, again demonstrating a degree of independence in changes of food and fluid regulation following withdrawal of ovarian hormones. Fluid intake of sham-operated rats decreased 8 per cent when values for post operative Days 4-21 are compared with those from the pre-operative period; food consumption increased 1.4 per cent during this time.

DISCUSSION

The results of the three experiments considered together reveal that a number of factors modulate saccharin preferences in female rats. Early hormone treatments modify taste preferences of female rats in adulthood. The results indicate a dissociation of neonatal hormone effects on reproductive and taste behaviors: Early androgen and estrogen injections each abolish sexual receptivity and cyclicity and the ability of female rats to respond to exogenous hormones in adulthood with a display of estrous behavior [17, 25]. These hormones do, however, exert opposite effects on adult S.P. ; neonatal androgen treatment suppresses S.P., while neonatal estrogens enhance consumption of and preference for the sweet solution. However, it is possible that neonatal treatment with a larger dose of estrogen might also suppress adult S.P. For all mammalian species studied to date [16, 17, 25] there exists a well defined period in development when

941 exogenously provided androgens induce behavioral masculinization of genetic females. These effects are thought to be accomplished largely through changes in the central nervous system [2, 9, 11, 25]. That neonatally androgenized female rats display S.P. in adulthood similar to those of genetic males and different from those of genetic females is consistent with the view that sexual differentiation determines the character of essentially non-reproductive behaviors as well as those directly concerned with propogation of the species. The alteration in sexual differentiation produced by early treatment with estrogen is different from that produced by early androgen treatment. The latter is thought to more closely mimic the ordinary course of differentiation in genetic males where secretions from the neonatal testis [18] act to suppress female behavior [10] and possibly also to organize male behavior patterns. Thus, although female reproductive behavior is absent in estrogenized females, there is no corresponding "masculinization" of taste preferences. The results for S.P. are similar to those reported by Gerall [8] for running activity. General bodily activity, as measured in running wheels is decreased by TP and increased by EB treatments early in life [8]. Activity, like S.P., is sexually dimorphic in adult rats; females are much more active than males [12]. Although early hormone treatments do affect adult S.P., one may not conclude that the effect is essentially central in nature. Early exposure to hormones may modify peripheral taste mechanisms, perhaps directly at the receptor level. In addition, the early hormone treatments which modify brain mechanisms concomitantly influence the pattern of steroid secretion in adulthood [2]. Differential effects on S.P. may be due to variations in adult circulating levels of hormones (activational effects) rather than to a central "organizational" action during the neonatal period [17]. Experiments designed to test these alternatives have yielded inconclusive results and no clear statement may be made at present. Donna Bell of this laboratory has demonstrated that early treatment with either EB or TP renders female rats insensitive to certain steroid hormones in adulthood (unpublished studies). The endpoints used in these studies were food intake and body weight. Thus, S.P. and general activity may be part of one hormone-sensitive neural system that is differentially affected by neonatal estrogen and androgen treatment, whereas sex behavior, gonadotrophin secretion, body weight and food intake are part of a second system similarly affected by both classes of steroid. The status of the animal's energy balance also affects saccharin preferences. In 24 hr (long term) tests, the rat's preference for saccharin over water is greatly increased by total food deprivation. These results agree with those for short term (1 hr) preference tests [1]. In Experiment 2 absolute quantities of saccharin consumed remained relatively constant during total food deprivation and the increase in S.P. was exclusively due to a great decrease in water intake. Since water intake decreases in hungry rats (whether or not offered saccharin solutions) increased saccharin preference by hungry rats may be part of an "eating" response rather than an attempt at obtaining hydration. It should be noted that in two studies which differed procedurally from Experiment 2 in important ways, absolute quantities of saccharin consumed were increased during deprivation [1, 19], although a third investigator [15 cited

942

WADE AND ZUCKER

by 13] reported changes similar to those described in Experiment 2. The stimulus for increased saccharin intake appears, within certain limits, to be quantitatively related to the degree of food deprivation. Restricting rats to 75 per cent of their daily food ration did not affect S.P. or intake while a 50 per cent reduction in caloric intake [5] or total elimination of food resulted in increased S.P. or saccharin intake. The nature of the adequate stimulus for changes in saccharin consumption remains unspecified. Metabolic consequences quantitatively related to food deprivation may induce changes in peripheral receptors or in the central mechanisms which modulate the affective state elicited by saccharin solutions [20, 21]. The increased food intake following termination of food restriction was accompanied by changed patterns of fluid regulation. With the reinstatement of ad libitum feeding, rats selectively decreased intake of saccharin solution and increased water intake. Although saccharin may have acquired aversive properties due to its association with extreme food deprivation [4, 13], it is more likely that the decrease in S.P. is directly related to increased food intake. We have noted that during pregnancy, pseudopregnancy and prepubertally, when food intake is elevated, S.P. are depressed [24]. None of these animals was ever subjected to food deprivation. Post-prandial drinking following food deprivation and weight loss may be relatively selective for water as compared with drinking during ad libitum feeding. Perhaps following deprivation rats are generally less responsive to certain taste characteristics of solutions. Finally, the degree to which ovarian hormones regulate S.P. of rats is influenced by the animals' prior experience with saccharin solutions (Experiment 3). Thus, acquisition of high S.P. is retarded among ovariectomized rats as compared with intact females, when none of the rats has experienced saccharin solutions prior to the first post-operative tests [26]. Once the preference for saccharin has been established, however, ovariectomy has no effect on S.P. of the great majority of rats. For these animals, S.P. have become

independent of the ovarian hormones that originally facilitated acquisition of the taste preference. Perhaps secondary cues, such as the position of the saccharin tube (which remained constant throughout the experiment) come to exert greater control over fluid selection than hormonal variables. These results are consistent with the demonstration that ovariectomized females whose S.P. have been elevated by exogenous gonadal hormones persist in their high S.P. long after hormone injections have been discontinued [26]. S.P. of 3 out of 18 rats declined precipitously following ovariectomy; these rats may have been exceptional in theft they never learned to depend on environmental cues such as tube position in selecting saccharin and their S.P., therefore, remained totally dependent on hormonal stimulation. One should not, however, on the basis of Experiment 3, conclude that ovarian hormones are generally unimportant in determining S.P. of experienced rats. For example, in a number of earlier experiments, each involving experienced saccharin selectors, S.P. were always greatly depressed during pregnancy and pseudopregnancy [24]. While these effects are possibly unrelated to hormonal changes, it seems much more likely that the taste changes are related to fluctuations in hormone secretion associated with these endocrine states. A number of experiments reviewed by [14] have demonstrated increased food intake and body weight following ovariectomy. In Experiment 3 food consumption increased 18 per cent but fluid intake increased less than 1 per cent after ovariectomy. We interpret this finding as follows: With the availability of a 0.75 per cent saccharin solution food intake remains unaffected but fluid intake rises significantly and rats over-hydrate themselves. Following ovariectomy there is a primary increase in food eaten but there need be no obligatory increase in fluid intake because the animals are already overdrinking. Similar changes in food intake but not in fluid intake occur during the first half of pregnancy and during pseudopregnancy in rats drinking water plus saccharin solutions [24]; animals not given access to saccharin solution increase both food and water intake in the early stages of pregnancy, during pseudopregnancy, and following ovariectomy (unpublished data).

REFERENCES

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