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Taste responses to dilute sucrose solutions are modulated by stage of the estrous cycle and fenfluramine treatment in female rats
Physiology & Behavior 86 (2005) 265 – 271
Taste responses to dilute sucrose solutions are modulated by stage of the estrous cycle and fenfluramine treatment in female rats Deann P.D. Atchley, Karen L. Weaver, Lisa A. Eckel * Department of Psychology and Program in Neuroscience Florida State University Tallahassee, FL 32306-1270, United States
Abstract Meal size is decreased during the estrous stage of the rat’s ovarian reproductive cycle. This is mediated, in part, by estradiol’s ability to increase the strength by which negative-feedback signals function to inhibit meal size. For example, we recently reported that the anorectic effect of fenfluramine, a serotonin agonist, is enhanced during estrus. Here, we investigated whether a decrease in the strength of positivefeedback signals, like those related to the taste of food, contributes to the decrease in meal size observed either in estrous rats or following fenfluramine treatment. Rats were given brief access to six sucrose solutions (0.0, 0.025, 0.05, 0.1, 0.2, and 0.4 M) and the mean number of licks to these solutions was monitored in diestrous and estrous rats treated with 1 mg/kg fenfluramine or saline vehicle. Following saline treatment, estrous rats displayed fewer licks than diestrous rats to the 0.025 M sucrose solution. Following fenfluramine treatment, a decrease in the number of licks to 3 of the 5 sucrose solutions was observed in diestrous rats only. This decrease in sucrose palatability was limited to brief access tests, as overnight preference for the 0.025 M sucrose solution was not decreased by fenfluramine in either diestrous or estrous rats. Our findings suggest that estrous rats experience a decrease in the strength of positive-feedback signals elicited by a dilute sucrose solution and that the anorectic effect of fenfluramine is associated with a decline in positive-feedback signaling in the diestrous rat. D 2005 Elsevier Inc. All rights reserved. Keywords: Estrogen; Food intake; Taste preference; Serotonin
1. Introduction In the rat, and many other species, food intake is influenced by the ovarian reproductive cycle. For example, it is well established that rats display a 20 –40% decrease in food intake during estrus, relative to diestrous and proestrous stages [1– 5]. This estrous-related decrease in food intake is thought to be mediated by the preovulatory increase in estradiol secretion. Evidence in support of this hypothesis is derived primarily from studies of ovariectomized rats. Bilateral removal of the ovaries induces hyperphagia and weight gain [6,7], both of which may be prevented by a cyclic regimen of estradiol replacement alone [8]. Analyses of the spontaneous feeding patterns of gonadally intact and ovariectomized rats indicate that estradiol’s inhibitory effect on food intake is mediated by a decrease in meal size, not meal number [1,4,5]. In the rat, * Corresponding author. Tel.: +1 850 644 3480; fax: +1 850 644 7739. E-mail address: [email protected] (L.A. Eckel). 0031-9384/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2005.08.001
meal size appears to be directly controlled by the relative strength of positive- and negative-feedback signals generated by the stimulation of peripheral, preabsorptive receptors during bouts of ingestive behavior [9– 11]. Positivefeedback signals, generated by the taste, sight, and odor of food, function to sustain the meal. Negative-feedback signals, generated by food stimuli acting on preabsorptive receptors in the mouth to induce conditioned aversive reactions and in the stomach and small intestine to stimulate the release of satiety-inducing peptides like cholecystokinin (CCK), function to terminate the meal. Because estradiol does not interact directly with preabsorptive receptors, it is considered an indirect control of meal size that functions to modulate those factors involved in the direct control of meal size [5]. That is, the inhibitory effects of estradiol on meal size must be mediated by estradiol’s ability to increase negative-feedback signals, decrease positive-feedback signals, or both. Considerable evidence suggests that the estrous-related decrease in meal size is mediated, in part, by estradiol’s
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ability to increase the strength of negative-feedback, satiety signals. For example, the inhibitory effects of both endogenous and exogenous CCK are increased during estrus in cycling rats [12], and by estradiol treatment in ovariectomized rats [13 – 16]. In addition, we recently reported that the anorectic effect of fenfluramine, a serotonin (5-HT) agonist, is increased in estrous rats, relative to diestrous rats [17], and by estradiol treatment in ovariectomized rats [18]. Because fenfluramine has been found to decrease the palatability of a 2% sucrose solution during a brief-access, taste reactivity test in male rats [19], the anorectic action of fenfluramine may also involve a decrease in positive-feedback signaling during bouts of ingestive behavior. Thus, this action of fenfluramine may have contributed to our recent report of a differential anorectic response in estrous and diestrous rats following fenfluramine treatment [17]. Additional research, which dissociates fenfluramine’s ability to generate positive- and negative-feedback signals, is required to resolve this issue. Initial attempts to investigate estradiol’s ability to modulate the strength of positive-feedback signals, under test conditions in which negative-feedback signals were minimal or absent, produced mixed findings. For example, estradiol failed to decrease sham intake of a 0.4 M sucrose solution in ovariectomized rats fitted with open gastric fistulas which permitted the ingested food to drain out of the stomach [20]. Estradiol also failed to reduce the number of licks in ovariectomized rats consuming a 0.8 M sucrose solution during the first min of a test meal [21]. Together, these studies suggest that estradiol does not modulate the strength of positive-feedback signals generated during consumption of a highly palatable sucrose solution. There is, however, one recent report in which estradiol appeared to decrease the positive-feedback signal generated by consumption of a dilute sucrose solution. In this study, estradiol decreased the number of licks during brief (10 s) access to a 0.05 M sucrose solution in ovariectomized rats [22]. This suggests that additional research, involving a range of sucrose concentrations, should prove useful in determining whether estradiol’s ability to decrease meal size is mediated, in part, by decreased sensitivity to positive-feedback signals that function to sustain eating during a meal. The goals of the present study were to determine whether the strength of taste-related, positive-feedback signals is modulated by stage of the estrous cycle, and to determine whether our previous finding of an estrous-related increase in the anorectic response to fenfluramine involves decreased sensitivity to positive-feedback signals during bouts of ingestive behavior. To investigate these hypotheses, we used the Davis MS80 Rig to examine the licking responses of diestrous and estrous rats to a range of sucrose concentrations following fenfluramine and saline treatment. Access to sucrose solutions was brief (10 s) in order to isolate positive-feedback signals, which are typically evident within the first min of ingestion [23,24], from negativefeedback signals. A limitation of previous studies inves-
tigating the contribution of positive-feedback signals to the inhibitory effect of estradiol on meal size is the use of foodor water-restricted rats to ensure licking during exposure to novel tastants. Because the effect of such deprivation on taste responses remains unclear [25], our rats were tested in a food- and water-replete state. In a second experiment, we evaluated whether fenfluramine treatment or estrous cycle stage produces any long-term changes in sucrose preference.
2. Methods 2.1. Subjects Nineteen adult female Long – Evans rats (Charles River Laboratories), weighing 175 –225 g at the beginning of the experiment, were used as subjects. The animals were housed individually in a testing room maintained at 20 T 2 -C with a 12 : 12 h light:dark cycle (dark onset 1320 h). Rats had free access to tap water and laboratory chow (Purina 5001), except where noted otherwise. Animal usage and all procedures were in compliance with the Florida State University Institutional Animal Care and Use Committee. 2.2. Estrous cycles Vaginal mucosal samples were obtained daily between 1000– 1100 h. A cotton swab, moistened with physiological saline, was inserted into the vaginal canal and the resulting sample was transferred to a microscope slide, fixed with alcohol (Surgipath Cytology Spray, Richmond, IL), and examined under a light microscope at low magnification (10x). Stages of the estrous cycle were assigned using standard criteria [26,27]. Diestrus 1 (D1; also called metestrus) was characterized by leukocytes interspersed with occasional small clusters of non-nucleated cornified cells or leukocytes interspersed with nucleated epithelial cells. Diestrus 2 (D2) was characterized by leukocytes interspersed with nucleated epithelial cells. Proestrus (P) was characterized by large clumps of round, nucleated epithelial cells, the absence of leukocytes, and occasional small clusters of cornified cells. Estrus (E) was characterized by large clumps of non-nucleated, squamous cornified cells. Cycle stages encompassed the 24-h period ending at the time of sampling. Accordingly, E included the prior 12-h dark period when female rats ovulate and display changes in sexual receptivity, locomotor activity, and food intake (i.e., behavioral estrus). At study onset, all rats displayed regular, 4-day estrous cycles. 2.3. Davis MS80 rig Licking behavior during brief presentations of flavored solutions was monitored using the Davis MS80 Rig (Dilog Instruments and Systems, Tallahassee, FL). The Davis MS80 Rig consists of a Plexiglas chamber with an opening
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that allows access to one of eight drinking tubes positioned on a sliding platform. A shutter opens and closes to allow rats access to one of eight tubes for a given length of time. A computer controlled the movement of a platform that determined the order of tube presentations, and the opening and closing of a shutter that determined the length of access to a tube and the interval between tube presentations. When rats licked the spout of a tube, this completed a circuit which generated a current (< 60 nA) that was recorded by the computer as a lick. 2.4. Training Over a period of 20 daily sessions, 14 rats were individually trained to consume fluids in the Davis MS80 Rig. Although estrous cycles were monitored daily, training sessions were not cycle synchronized. For the first 3 training days, rats were transferred to the Davis MS80 Rig and a single drinking tube containing 0.2 M sucrose was presented for 15 min/day. Following each training session, rats were returned to their home cages. For the next 3 training days, rats were placed on a 23 h/day water-deprivation schedule. Following 23 h of water deprivation, rats were transferred to the Davis MS80 Rig and a single drinking tube containing 0.2 M sucrose was presented for 15 min/day. After each training session, rats were returned to their home cages where water was available for 1 h. On the seventh training day, rats were placed on a 12 h/day water-deprivation schedule. Following 12 h of water deprivation, rats were transferred to the Davis MS80 Rig and a single drinking tube containing 0.2 M sucrose was presented for 15 min and then rats were returned to their home cages where water was available for 12 h. The following day, a second drinking tube was added during the training session. During this phase of training, rats received a total of three presentations of 0.2 M sucrose from the 2 drinking tubes. Each presentation was 240 s long with a 10 s inter-trial interval. If rats failed to lick within the first 60 s of tube presentation, access to the tube ended and, 10 s later, the other tube was presented. These 2-tube trials, which continued for 4 days, allowed rats to adapt to the sound of the moving platform and the opening and closing of the shutter. The last phase of training was conducted in water-replete rats. On training days, rats were transferred to the Davis MS80 Rig where they received a total of 6 brief (10 s) presentations of 0.2 M sucrose from 2 drinking tubes. Once rats licked reliably during all six presentations (4 days), the next three training sessions included a total of 4 presentations of 0.2 M sucrose and 2 presentations of distilled water from the 2 drinking tubes. Finally, the last two training sessions were each lengthened to include a total of 3 presentations of 0.2 M sucrose, 1 presentation of distilled water, 3 presentations of 0.1 M sucrose, and 2 presentations of 0.05 M sucrose from 4 tubes. The addition of 0.05 and 0.1 M sucrose allowed rats to adapt to presentations of multiple concentrations of the same taste stimulus.
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2.5. Sucrose licking during short-term access Following training in the Davis MS80 Rig, a withinsubject design was used to examine the effects of fenfluramine on sucrose-elicited taste responses at 2 different stages of the estrous cycle. Intraperitoneal injections of 1.0 mg/kg fenfluramine (d-fenfluramine, Sigma Chemical, St. Louis, MO) or 1 ml/kg physiological saline vehicle were administered on days in which rats displayed vaginal cytology indicative of either D2 or E. Thus, each rat received a total of 4 test sessions (fenfluramine injection on D2 and E and saline injection on D2 and E). Injections were randomized such that some rats received their first injection of fenfluramine during D2, whereas other rats received their first injection of fenfluramine during E. The dose of fenfluramine was selected based on a previous report from our laboratory which demonstrated that 1.0 mg/kg fenfluramine potently reduced food intake in D2 and E rats for 6 h [17] . One h following drug injection, rats were transferred from their home cages to the Davis MS80 Rig and taste responses to varying concentrations of sucrose were assessed. During each of the 4 test sessions, rats received two exposures to six different sucrose solutions (0.0, 0.025, 0.05, 0.1, 0.2, and 0.4 M), presented in random order. Presentations lasted for 10 s with a 60 s limit to start licking. Failure to lick during the 60 s limit was occasionally observed during presentation of water and very dilute sucrose solutions. Inter-trial intervals were 10 s. The number of licks for each presentation was recorded and the mean number of licks for each solution was calculated for each test condition by averaging the number of licks during the two presentations of that solution. 2.6. Sucrose intake and preference test Analysis of the licking data during short-term access to varying concentrations of sucrose solutions revealed that the number of licks to 0.025 M sucrose was influenced by stage of the estrous cycle and by fenfluramine treatment. To determine whether such differences persist in the presence of postabsorptive cues, we used a within-subject design to examine the effects of fenfluramine on the consumption of, and preference for, 0.025 M sucrose during a longer-term (overnight) test. Five naı¨ve rats were housed in standard cages with free access to food and water. For three consecutive days, rats were presented with a bottle containing 0.025 M sucrose for 2 h during the light phase in order to adapt them to this novel taste. During D2 and E of the following two estrous cycles, rats received intraperitoneal injections of 1 ml/kg fenfluramine or 1 ml/kg saline vehicle. One h later, rats were given access to bottles containing a 0.025 M sucrose solution. Sucrose and water intake were measured the following morning at 0900 h. Sucrose preference ratios were calculated by dividing the amount of sucrose consumed by the total fluid (sucrose + water) consumed.
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Table 1 Rats displayed an increase in the mean number of licks with each successive increase in sucrose concentration Sucrose concentration
Number of licks
0.0 M
0.025 M
0.05 M
0.1 M
0.2 M
0.4 M
6.2 T 1.2a
15.9 T 2.1b
23.1 T 2.8c
38.5 T 3.2d
47.9 T 3.8e
54.8 T 3.0f
Values are means T SEM. Data are collapsed across 2 of the 3 factors of our analysis (stage of the estrous cycle and drug treatment) to illustrate the main effect of sucrose concentration [ F(5, 65) = 63.14, P < 0.0001]. Means with different letters are significantly different from each other, P < 0.05.
3. Results
successive increase in sucrose concentration, all P’s < 0.05. Our 3-factor ANOVA also revealed that the number of licks was influenced by an interactive effect of estrous cycle stage and drug treatment [ F(1, 13) = 7.43, P < 0.05], Fig. 1. Post hoc analyses revealed that estrous cycle-related changes in lick responses were limited to the 0.025 M sucrose solution. Following saline treatment, E rats displayed fewer licks to 0.025 M sucrose than D2 rats, P < 0.05, Fig. 1A. Following fenfluramine treatment, no estrous cycle-related changes in licking were detected. Interestingly, drug-related changes in licking were observed in D2 rats (Fig. 1B), but not in E rats (Fig. 1C). In D2 rats, fenfluramine treatment decreased the number of licks during presentation of the 0.025, 0.05, and 0.4 M sucrose solutions, P’s < 0.05. In E rats, licking was not influenced by fenfluramine treatment. The numbers of licks to water, 0.1, and 0.2 M sucrose were not influenced by stage of the estrous cycle or drug treatment.
3.1. Sucrose licking during short-term access
3.2. Sucrose intake and preference
The number of licks during short-term access to varying concentrations of sucrose solutions was influenced by a main effect of sucrose concentration [ F(5, 65) = 63.14, P < 0.0001], Table 1. To investigate this effect, data were collapsed across 2 of the 3 factors of our analysis (i.e., stage of the estrous cycle and drug treatment) and post hoc tests were used to investigate differences in the number of licks elicited by each sucrose concentration. These analyses revealed that the number of licks increased with each
Consumption of the 0.025 M sucrose solution was influenced by a main effect of drug treatment [ F(1, 4) = 9.84, P < 0.05], Table 2. Fenfluramine treatment decreased acceptance of the 0.025 M sucrose solution in D2 and E rats, relative to that observed following saline treatment, P’s < 0.05. Water consumption was not influenced by either stage of the estrous cycle or by drug treatment. All rats displayed a strong preference for the 0.025 M sucrose solution over water. Unlike the short-term access test,
2.7. Statistical analysis Data are presented as means T SEM. Sucrose licking during short-term access tests was analyzed using a 3-factor [estrous cycle stage (D2 and E) by drug treatment (fenfluramine and saline) by sucrose concentration (0.0, 0.025, 0.05, 0.1, 0.2, and 0.4 M)] repeated-measures analysis of variance (ANOVA) procedure. Overnight intakes of sucrose and water, and the sucrose preference ratios, were analyzed using 2-factor [estrous cycle stage (D2 and E) by drug treatment (fenfluramine and saline)] repeated-measures ANOVAs. Significant main or interaction effects ( P < 0.05) were examined using Tukey’s honestly significant difference test.
number of licks in 10 s
80 60
A
B D2/SAL E/SAL
C D2/SAL D2/FEN
E/SAL E/FEN
40
b
20 0
b
a 0.0 0.025 0.05
0.1
0.2
0.4
b
0.0 0.025 0.05
b
0.1
0.2
0.4
0.0 0.025 0.05
0.1
0.2
0.4
sucrose concentration (M) Fig. 1. Mean number of licks during brief access to water (0.0 M) and varying concentrations of sucrose solutions in D2 and E rats treated with saline and fenfluramine. Values are means T SEM. (A) An E-related decrease in the number of licks to 0.025 M sucrose was apparent following saline treatment. (B) During D2, fenfluramine decreased the number of licks to 0.025, 0.05, and 0.4 M sucrose. (C) Fenfluramine failed to decrease the number of licks to any concentration of sucrose during E. aE/SAL rats less than D2/SAL rats, P < 0.05. bD2/FEN rats less than D2/SAL rats, P < 0.05. Abbreviations: D2; diestrus 2, E; estrus, SAL; saline, FEN; fenfluramine.
D.P.D. Atchley et al. / Physiology & Behavior 86 (2005) 265 – 271 Table 2 Fenfluramine decreased overnight consumption of 0.025 M sucrose but failed to alter the preference ratio for 0.025 M sucrose
Sucrose intake (ml/20 h) Water intake (ml/20 h) Sucrose preference ratio
Saline treatment
Fenfluramine treatment
Diestrus
Estrus
Diestrus
Estrus
80.5 T 11.1
87.7 T 11.4
65.2 T 11.6*
70.4 T 18.9*
11.6 T 2.2
10.2 T 1.6
9.4 T 1.4
11.6 T 1.5
0.87 T 0.03
0.91 T 0.02
0.87 T 0.01
0.81 T 0.05
Values are means T SEM. Following saline and fenfluramine treatment, rats were given overnight (20 h) access to 0.025 M sucrose and water. Sucrose preference was determined by the ratio of sucrose consumed to total fluid consumed. *Less than saline-treated rats, P < 0.05.
preference for 0.025 M sucrose was not influenced either by stage of the estrous cycle or by drug treatment.
4. Discussion Consistent with previous studies [22,28], all rats displayed concentration-dependent, lick – response curves during brief access to a range of sucrose solutions that were presented in random order. When licking data were arranged as a function of ascending sucrose concentration, a reliable increase in the number of licks was apparent with each successive increment in sucrose concentration (Table 1). Changes in the number of licks, as a function of estrous cycle stage or drug treatment, were observed at 3 sucrose concentrations (Fig. 1). Stage of the estrous cycle influenced sucrose licking only at the lowest concentration of sucrose tested here; estrous rats displayed fewer licks to 0.025 M sucrose than diestrous rats. Interestingly, an inhibitory effect of fenfluramine on sucrose licking was apparent only in diestrous rats at some (i.e., 0.025, 0.05, and 0.4 M), but not all, sucrose concentrations. This decline in sucrose palatability was observed only during brief-access tests. Fenfluramine failed to reduce preference for the 0.025 M sucrose solution during an overnight, two-bottle choice test. The estrous-related decrease in licking responses to 0.025 M sucrose observed here is consistent with a previous study involving estradiol-treated, ovariectomized rats [22]. Using similar experimental paradigms, both studies revealed decreases in licking to dilute sucrose solutions during a time when estradiol’s behavioral effects on food intake are maximal. However, the present results are not consistent with two previous studies in which estradiol-treated, ovariectomized rats failed to decrease the number of licks during the first min of a 0.8 M sucrose test meal [21], and failed to decrease sham intake of a 0.4 M sucrose solution [20]. These discrepant findings may be related to differences in sucrose concentration that likely produced differences in the saliency of the gustatory stimuli across studies. In previous studies [20,21], the sucrose concentrations were the same or twice that of the highest concentration examined
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here. Indeed, at our highest sucrose concentration (0.4 M), the number of licks was not influenced by stage of the estrous cycle. Thus, estradiol’s ability to decrease positivefeedback signals from the mouth may depend upon the strength of the gustatory stimulus. An additional factor that may have contributed to the discrepant findings involves the animal’s internal state. In previous studies [20,21], rats were tested in a food-deprived state whereas our rats were tested in a food-replete state. This likely resulted in different internal drives to eat which may have influenced the strength of positive-feedback signals arising from the mouth. Finally, previous studies that have examined estradiol’s influence on taste in short-term access trials have been conducted in ovariectomized rats receiving estradiol replacement [20 –22]. Thus, our results extend previous studies by demonstrating, for the first time, that changes in endogenous hormone secretion, presumably the preovulatory rise in estradiol secretion, can decrease the positivefeedback signals arising from ingestion of a dilute sucrose solution. Although the anorectic effect of fenfluramine has been largely attributed to its ability to augment the satiating strength of negative-feedback signals from the gut (e.g., [29]), there is some evidence that it may also reduce the strength of positive-feedback signals from the mouth. For example, fenfluramine has been shown to decrease sham intake of a sucrose solution in rats fitted with open gastric fistulas [30], and increase aversive taste reactivity responses during intraoral infusions of a 2% sucrose solution [19]. Our finding, that fenfluramine decreased licking during brief access to 3 of the 5 sucrose concentrations (0.025, 0.05, and 0.4 M) in diestrous rats (Fig. 1B), is consistent with these previous studies in male rats. The significance of the lack of fenfluramine’s ability to produce a reliable decrease in the number of licks during presentations of 0.1 and 0.2 M sucrose is unclear; however, two points are worth mentioning. First, there was a trend for fenfluramine treatment to reduce the number of licks during presentation of each sucrose solution in diestrous rats. Clearly, the overall number of licks across all sucrose concentrations was reduced by fenfluramine treatment in the diestrous rat. Second, the variability in the number of licks produced by fenfluramine-treated, diestrous rats during presentation of 0.1 and 0.2 M sucrose was greater than that observed during presentation of the other sucrose concentrations. This may have limited our ability to detect a reliable inhibitory effect of fenfluramine on licking at these sucrose concentrations. Additional research, perhaps involving a longer training interval or a greater number of sucrose presentations during testing, would be helpful in resolving this issue. Interestingly, fenfluramine failed to modulate sucrose licking in estrous rats (Fig. 1C). At the lowest sucrose concentration (0.025 M), this may be related to a floor effect as the number of licks was already suppressed in estrous rats, relative to diestrous rats. However, a similar argument cannot be made for the highest sucrose concentration; both
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diestrous and estrous rats displayed a high number of licks, yet an inhibitory effect of fenfluramine was only apparent in diestrous rats. That fenfluramine failed to decrease the number of licks to a range of sucrose solutions in estrous rats suggests that an estrous-related decline in the strength of positive-feedback signals did not contribute to our earlier report that fenfluramine’s anorectic effects are increased in estrous rats, relative to diestrous rats [17]. Clearly, additional research is required to determine whether hormonal fluctuations across the rat’s estrous cycle differentially modulate positive- and negative-feedback signals generated during a meal. To investigate whether fenfluramine-induced changes in sucrose licking during brief-access tests are present only when postingestive signals are minimal and ingestion is driven largely by taste, we examined the effects of fenfluramine treatment on sucrose preference in rats given overnight access to water and a dilute sucrose solution. We choose 0.025 M sucrose based on the results of our briefaccess tests that revealed maximal estrous cycle- and fenfluramine-related differences at this sucrose concentration. In overnight tests, all rats displayed a clear preference for the dilute sucrose solution and no group differences in sucrose preference were detected. Thus, postingestive signals appear to override any effect of fenfluramine on positive-feedback signals generated during brief access to sweet solutions. Fenfluramine did, however, reduce overnight intake of the dilute sucrose solution in diestrous and estrous rats. This is consistent with fenfluramine’s ability to decrease intake of powdered chow during overnight intake tests in female rats [17]. However, unlike the overnight intake test involving a chow diet, an estrous-related increase in the inhibitory effects of fenfluramine on sucrose intake was not detected here. This may be related to differences in palatability between a dilute sucrose solution and laboratory chow. It is possible that the drive to consume the palatable sucrose solution overrides the hormonal influences that would normally function to decrease intake. Another possibility is that during estrus, rats were preferentially consuming the sucrose solution relative to their chow diet that was also freely available. Since overnight chow intake was not recorded, it is unknown whether differences in chow intake, and therefore total caloric intake, differed between estrous and diestrous rats. In summary, it is well established that alterations in circulating estradiol play an important role in the control of meal size in the female rat. Prevailing research suggests that the decline in meal size, observed following estradiol treatment in ovariectomized rats and during estrus in cycling rats, is mediated by increased sensitivity to negativefeedback controls of meal size (reviewed in [5]). Our present findings suggest that the estrous-related decrease in meal size may also involve a decrease in the positivefeedback controls of meal size. Here, we demonstrated a decline in licking during brief access to a 0.025 M sucrose solution in estrous rats, relative to diestrous rats. Because
others have failed to demonstrate similar results in rats consuming more palatable sucrose solutions [20,21], it appears that estradiol’s ability to modulate positive-feedback signals during ingestion is influenced by the strength of the gustatory stimulus. Thus, it will be important to determine the influence of diet palatability on estrousrelated changes in ingestive behavior in future studies. Our present findings also suggest that this estrous-related increase in the anorectic effects of fenfluramine does not involve a decrease in the positive-feedback controls of meal size. Here, a fenfluramine-induced decrease in sucrose licking during brief-access tests was observed only in diestrous rats and, in an overnight preference test, fenfluramine failed to decrease preference for a 0.025 M sucrose solution in either diestrous or estrous rats. Thus, the estrousrelated increase in the satiating effects of fenfluramine appear to rely upon postabsorptive properties of ingestion.
Acknowledgements We gratefully acknowledge the assistance and support of R. Henderson and P. Hendrick of the Technical Support Group at Florida State University Program in Neuroscience. We thank Dr. Kathleen Curtis for help with the design of this experiment. This research was supported by a National Institute of Mental Health Grant MH-63932 (LAE) and a National Institute of Health Training Grant in the Chemical Senses DC-000044 (DPDA).
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D.P.D. Atchley et al. / Physiology & Behavior 86 (2005) 265 – 271 [13] Butera PC, Bradway DM, Cataldo NJ. Modulation of the satiety effect of cholecystokinin by estradiol. Physiol Behav 1993;53:1235 – 8. [14] Butera PC, Xiong M, Davis RJ, Platania SP. Central implants of dilute estradiol enhance the satiety effect of CCK-8. Behav Neurosci 1996;110:823 – 30. [15] Geary N, Trace D, McEwen B, Smith GP. Cyclic estradiol replacement increases the satiety effect of CCK-8 in ovariectomized rats. Physiol Behav 1994;56:281 – 9. [16] Asarian L, Geary N. Cyclic estradiol treatment phasically potentiates endogenous cholecystokinin’s satiating action in ovariectomized rats. Peptides 1999;20:445 – 50. [17] Eckel LA, Rivera HM, Atchley DPD. The anorectic effect of fenfluramine is influenced by sex and stage of the estrous cycle in rats. Am J Physiol 2005;288:R1486 – 91. [18] Rivera HM, Eckel LA. The anorectic effect of fenfluramine is increased by estradiol treatment in ovariectomized rats. Physiol Behav 2005;86:331 – 7. [19] Barnfield A, Parker LA, Davies AM, Miles C. Fenfluramine-induced modification of palatability: analysis by the taste reactivity test. Pharmacol Biochem Behav 1994;48:875 – 9. [20] Geary N, Trace D, Smith GP. Estradiol interacts with gastric or pregastric food stimuli to decrease sucrose ingestion in ovariectomized rats. Physiol Behav 1995;57:155 – 8. [21] Hrupka BJ, Smith GP, Geary N. Ovariectomy and estradiol affect postingestive controls of sucrose licking. Physiol Behav 1997;61: 243 – 7.
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[22] Curtis KS, Davis LM, Johnson AL, Therrien KL, Contreras RJ. Sex differences in behavioral taste responses to and ingestion of sucrose and NaCl solutions by rats. Physiol Behav 2004;80:657 – 64. [23] Davis JD. Some new developments in the understanding of oropharyngeal and postingestional controls of meal size. Nutrition 1999; 15:32 – 9. [24] Davis JD, Smith GP. Analysis of lick rate measures the positive and negative feedback effects carbohydrates on eating. Appetite 1988; 11:229 – 38. [25] Scalera G. Taste preference and acceptance in thirsty and dehydrated rats. Physiol Behav 2000;71:457 – 68. [26] Freeman ME. The neuroendocrine control of the ovarian cycle of the rat. In: Knobil E, Neill JD, editors. The physiology of reproduction. Second edition. New York’ Raven Press, Ltd.; 1994. p. 613 – 58. [27] Long JA, Evans HM. The oestrous cycle in the rat and its associated phenomenon. In: Leuschner AO, editor. Memoirs of the University of California. Oxford’ University of California Press; 1922. p. 1 – 148. [28] Smith J. Microstructure of the rat’s intake of food, sucrose and saccharin in 24-hour tests. Neurosci Biobehav Rev 2000;24:199 – 212. [29] Simansky KJ. Serotonin and the structure of satiation. In: Smith GP, editor. Satiation: from gut to brain. Oxford’ Oxford University Press; 1998. p. 217 – 62. [30] Neill JC, Cooper SJ. Evidence for serotonergic modulation of sucrose sham-feeding in the gastric-fistulated rat. Physiol Behav 1988;44: 453 – 9.
Taste responses to dilute sucrose solutions are modulated by stage of the estrous cycle and fenfluramine treatment in female rats Deann P.D. Atchley, Karen L. Weaver, Lisa A. Eckel * Department of Psychology and Program in Neuroscience Florida State University Tallahassee, FL 32306-1270, United States
Abstract Meal size is decreased during the estrous stage of the rat’s ovarian reproductive cycle. This is mediated, in part, by estradiol’s ability to increase the strength by which negative-feedback signals function to inhibit meal size. For example, we recently reported that the anorectic effect of fenfluramine, a serotonin agonist, is enhanced during estrus. Here, we investigated whether a decrease in the strength of positivefeedback signals, like those related to the taste of food, contributes to the decrease in meal size observed either in estrous rats or following fenfluramine treatment. Rats were given brief access to six sucrose solutions (0.0, 0.025, 0.05, 0.1, 0.2, and 0.4 M) and the mean number of licks to these solutions was monitored in diestrous and estrous rats treated with 1 mg/kg fenfluramine or saline vehicle. Following saline treatment, estrous rats displayed fewer licks than diestrous rats to the 0.025 M sucrose solution. Following fenfluramine treatment, a decrease in the number of licks to 3 of the 5 sucrose solutions was observed in diestrous rats only. This decrease in sucrose palatability was limited to brief access tests, as overnight preference for the 0.025 M sucrose solution was not decreased by fenfluramine in either diestrous or estrous rats. Our findings suggest that estrous rats experience a decrease in the strength of positive-feedback signals elicited by a dilute sucrose solution and that the anorectic effect of fenfluramine is associated with a decline in positive-feedback signaling in the diestrous rat. D 2005 Elsevier Inc. All rights reserved. Keywords: Estrogen; Food intake; Taste preference; Serotonin
1. Introduction In the rat, and many other species, food intake is influenced by the ovarian reproductive cycle. For example, it is well established that rats display a 20 –40% decrease in food intake during estrus, relative to diestrous and proestrous stages [1– 5]. This estrous-related decrease in food intake is thought to be mediated by the preovulatory increase in estradiol secretion. Evidence in support of this hypothesis is derived primarily from studies of ovariectomized rats. Bilateral removal of the ovaries induces hyperphagia and weight gain [6,7], both of which may be prevented by a cyclic regimen of estradiol replacement alone [8]. Analyses of the spontaneous feeding patterns of gonadally intact and ovariectomized rats indicate that estradiol’s inhibitory effect on food intake is mediated by a decrease in meal size, not meal number [1,4,5]. In the rat, * Corresponding author. Tel.: +1 850 644 3480; fax: +1 850 644 7739. E-mail address: [email protected] (L.A. Eckel). 0031-9384/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2005.08.001
meal size appears to be directly controlled by the relative strength of positive- and negative-feedback signals generated by the stimulation of peripheral, preabsorptive receptors during bouts of ingestive behavior [9– 11]. Positivefeedback signals, generated by the taste, sight, and odor of food, function to sustain the meal. Negative-feedback signals, generated by food stimuli acting on preabsorptive receptors in the mouth to induce conditioned aversive reactions and in the stomach and small intestine to stimulate the release of satiety-inducing peptides like cholecystokinin (CCK), function to terminate the meal. Because estradiol does not interact directly with preabsorptive receptors, it is considered an indirect control of meal size that functions to modulate those factors involved in the direct control of meal size [5]. That is, the inhibitory effects of estradiol on meal size must be mediated by estradiol’s ability to increase negative-feedback signals, decrease positive-feedback signals, or both. Considerable evidence suggests that the estrous-related decrease in meal size is mediated, in part, by estradiol’s
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ability to increase the strength of negative-feedback, satiety signals. For example, the inhibitory effects of both endogenous and exogenous CCK are increased during estrus in cycling rats [12], and by estradiol treatment in ovariectomized rats [13 – 16]. In addition, we recently reported that the anorectic effect of fenfluramine, a serotonin (5-HT) agonist, is increased in estrous rats, relative to diestrous rats [17], and by estradiol treatment in ovariectomized rats [18]. Because fenfluramine has been found to decrease the palatability of a 2% sucrose solution during a brief-access, taste reactivity test in male rats [19], the anorectic action of fenfluramine may also involve a decrease in positive-feedback signaling during bouts of ingestive behavior. Thus, this action of fenfluramine may have contributed to our recent report of a differential anorectic response in estrous and diestrous rats following fenfluramine treatment [17]. Additional research, which dissociates fenfluramine’s ability to generate positive- and negative-feedback signals, is required to resolve this issue. Initial attempts to investigate estradiol’s ability to modulate the strength of positive-feedback signals, under test conditions in which negative-feedback signals were minimal or absent, produced mixed findings. For example, estradiol failed to decrease sham intake of a 0.4 M sucrose solution in ovariectomized rats fitted with open gastric fistulas which permitted the ingested food to drain out of the stomach [20]. Estradiol also failed to reduce the number of licks in ovariectomized rats consuming a 0.8 M sucrose solution during the first min of a test meal [21]. Together, these studies suggest that estradiol does not modulate the strength of positive-feedback signals generated during consumption of a highly palatable sucrose solution. There is, however, one recent report in which estradiol appeared to decrease the positive-feedback signal generated by consumption of a dilute sucrose solution. In this study, estradiol decreased the number of licks during brief (10 s) access to a 0.05 M sucrose solution in ovariectomized rats [22]. This suggests that additional research, involving a range of sucrose concentrations, should prove useful in determining whether estradiol’s ability to decrease meal size is mediated, in part, by decreased sensitivity to positive-feedback signals that function to sustain eating during a meal. The goals of the present study were to determine whether the strength of taste-related, positive-feedback signals is modulated by stage of the estrous cycle, and to determine whether our previous finding of an estrous-related increase in the anorectic response to fenfluramine involves decreased sensitivity to positive-feedback signals during bouts of ingestive behavior. To investigate these hypotheses, we used the Davis MS80 Rig to examine the licking responses of diestrous and estrous rats to a range of sucrose concentrations following fenfluramine and saline treatment. Access to sucrose solutions was brief (10 s) in order to isolate positive-feedback signals, which are typically evident within the first min of ingestion [23,24], from negativefeedback signals. A limitation of previous studies inves-
tigating the contribution of positive-feedback signals to the inhibitory effect of estradiol on meal size is the use of foodor water-restricted rats to ensure licking during exposure to novel tastants. Because the effect of such deprivation on taste responses remains unclear [25], our rats were tested in a food- and water-replete state. In a second experiment, we evaluated whether fenfluramine treatment or estrous cycle stage produces any long-term changes in sucrose preference.
2. Methods 2.1. Subjects Nineteen adult female Long – Evans rats (Charles River Laboratories), weighing 175 –225 g at the beginning of the experiment, were used as subjects. The animals were housed individually in a testing room maintained at 20 T 2 -C with a 12 : 12 h light:dark cycle (dark onset 1320 h). Rats had free access to tap water and laboratory chow (Purina 5001), except where noted otherwise. Animal usage and all procedures were in compliance with the Florida State University Institutional Animal Care and Use Committee. 2.2. Estrous cycles Vaginal mucosal samples were obtained daily between 1000– 1100 h. A cotton swab, moistened with physiological saline, was inserted into the vaginal canal and the resulting sample was transferred to a microscope slide, fixed with alcohol (Surgipath Cytology Spray, Richmond, IL), and examined under a light microscope at low magnification (10x). Stages of the estrous cycle were assigned using standard criteria [26,27]. Diestrus 1 (D1; also called metestrus) was characterized by leukocytes interspersed with occasional small clusters of non-nucleated cornified cells or leukocytes interspersed with nucleated epithelial cells. Diestrus 2 (D2) was characterized by leukocytes interspersed with nucleated epithelial cells. Proestrus (P) was characterized by large clumps of round, nucleated epithelial cells, the absence of leukocytes, and occasional small clusters of cornified cells. Estrus (E) was characterized by large clumps of non-nucleated, squamous cornified cells. Cycle stages encompassed the 24-h period ending at the time of sampling. Accordingly, E included the prior 12-h dark period when female rats ovulate and display changes in sexual receptivity, locomotor activity, and food intake (i.e., behavioral estrus). At study onset, all rats displayed regular, 4-day estrous cycles. 2.3. Davis MS80 rig Licking behavior during brief presentations of flavored solutions was monitored using the Davis MS80 Rig (Dilog Instruments and Systems, Tallahassee, FL). The Davis MS80 Rig consists of a Plexiglas chamber with an opening
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that allows access to one of eight drinking tubes positioned on a sliding platform. A shutter opens and closes to allow rats access to one of eight tubes for a given length of time. A computer controlled the movement of a platform that determined the order of tube presentations, and the opening and closing of a shutter that determined the length of access to a tube and the interval between tube presentations. When rats licked the spout of a tube, this completed a circuit which generated a current (< 60 nA) that was recorded by the computer as a lick. 2.4. Training Over a period of 20 daily sessions, 14 rats were individually trained to consume fluids in the Davis MS80 Rig. Although estrous cycles were monitored daily, training sessions were not cycle synchronized. For the first 3 training days, rats were transferred to the Davis MS80 Rig and a single drinking tube containing 0.2 M sucrose was presented for 15 min/day. Following each training session, rats were returned to their home cages. For the next 3 training days, rats were placed on a 23 h/day water-deprivation schedule. Following 23 h of water deprivation, rats were transferred to the Davis MS80 Rig and a single drinking tube containing 0.2 M sucrose was presented for 15 min/day. After each training session, rats were returned to their home cages where water was available for 1 h. On the seventh training day, rats were placed on a 12 h/day water-deprivation schedule. Following 12 h of water deprivation, rats were transferred to the Davis MS80 Rig and a single drinking tube containing 0.2 M sucrose was presented for 15 min and then rats were returned to their home cages where water was available for 12 h. The following day, a second drinking tube was added during the training session. During this phase of training, rats received a total of three presentations of 0.2 M sucrose from the 2 drinking tubes. Each presentation was 240 s long with a 10 s inter-trial interval. If rats failed to lick within the first 60 s of tube presentation, access to the tube ended and, 10 s later, the other tube was presented. These 2-tube trials, which continued for 4 days, allowed rats to adapt to the sound of the moving platform and the opening and closing of the shutter. The last phase of training was conducted in water-replete rats. On training days, rats were transferred to the Davis MS80 Rig where they received a total of 6 brief (10 s) presentations of 0.2 M sucrose from 2 drinking tubes. Once rats licked reliably during all six presentations (4 days), the next three training sessions included a total of 4 presentations of 0.2 M sucrose and 2 presentations of distilled water from the 2 drinking tubes. Finally, the last two training sessions were each lengthened to include a total of 3 presentations of 0.2 M sucrose, 1 presentation of distilled water, 3 presentations of 0.1 M sucrose, and 2 presentations of 0.05 M sucrose from 4 tubes. The addition of 0.05 and 0.1 M sucrose allowed rats to adapt to presentations of multiple concentrations of the same taste stimulus.
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2.5. Sucrose licking during short-term access Following training in the Davis MS80 Rig, a withinsubject design was used to examine the effects of fenfluramine on sucrose-elicited taste responses at 2 different stages of the estrous cycle. Intraperitoneal injections of 1.0 mg/kg fenfluramine (d-fenfluramine, Sigma Chemical, St. Louis, MO) or 1 ml/kg physiological saline vehicle were administered on days in which rats displayed vaginal cytology indicative of either D2 or E. Thus, each rat received a total of 4 test sessions (fenfluramine injection on D2 and E and saline injection on D2 and E). Injections were randomized such that some rats received their first injection of fenfluramine during D2, whereas other rats received their first injection of fenfluramine during E. The dose of fenfluramine was selected based on a previous report from our laboratory which demonstrated that 1.0 mg/kg fenfluramine potently reduced food intake in D2 and E rats for 6 h [17] . One h following drug injection, rats were transferred from their home cages to the Davis MS80 Rig and taste responses to varying concentrations of sucrose were assessed. During each of the 4 test sessions, rats received two exposures to six different sucrose solutions (0.0, 0.025, 0.05, 0.1, 0.2, and 0.4 M), presented in random order. Presentations lasted for 10 s with a 60 s limit to start licking. Failure to lick during the 60 s limit was occasionally observed during presentation of water and very dilute sucrose solutions. Inter-trial intervals were 10 s. The number of licks for each presentation was recorded and the mean number of licks for each solution was calculated for each test condition by averaging the number of licks during the two presentations of that solution. 2.6. Sucrose intake and preference test Analysis of the licking data during short-term access to varying concentrations of sucrose solutions revealed that the number of licks to 0.025 M sucrose was influenced by stage of the estrous cycle and by fenfluramine treatment. To determine whether such differences persist in the presence of postabsorptive cues, we used a within-subject design to examine the effects of fenfluramine on the consumption of, and preference for, 0.025 M sucrose during a longer-term (overnight) test. Five naı¨ve rats were housed in standard cages with free access to food and water. For three consecutive days, rats were presented with a bottle containing 0.025 M sucrose for 2 h during the light phase in order to adapt them to this novel taste. During D2 and E of the following two estrous cycles, rats received intraperitoneal injections of 1 ml/kg fenfluramine or 1 ml/kg saline vehicle. One h later, rats were given access to bottles containing a 0.025 M sucrose solution. Sucrose and water intake were measured the following morning at 0900 h. Sucrose preference ratios were calculated by dividing the amount of sucrose consumed by the total fluid (sucrose + water) consumed.
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Table 1 Rats displayed an increase in the mean number of licks with each successive increase in sucrose concentration Sucrose concentration
Number of licks
0.0 M
0.025 M
0.05 M
0.1 M
0.2 M
0.4 M
6.2 T 1.2a
15.9 T 2.1b
23.1 T 2.8c
38.5 T 3.2d
47.9 T 3.8e
54.8 T 3.0f
Values are means T SEM. Data are collapsed across 2 of the 3 factors of our analysis (stage of the estrous cycle and drug treatment) to illustrate the main effect of sucrose concentration [ F(5, 65) = 63.14, P < 0.0001]. Means with different letters are significantly different from each other, P < 0.05.
3. Results
successive increase in sucrose concentration, all P’s < 0.05. Our 3-factor ANOVA also revealed that the number of licks was influenced by an interactive effect of estrous cycle stage and drug treatment [ F(1, 13) = 7.43, P < 0.05], Fig. 1. Post hoc analyses revealed that estrous cycle-related changes in lick responses were limited to the 0.025 M sucrose solution. Following saline treatment, E rats displayed fewer licks to 0.025 M sucrose than D2 rats, P < 0.05, Fig. 1A. Following fenfluramine treatment, no estrous cycle-related changes in licking were detected. Interestingly, drug-related changes in licking were observed in D2 rats (Fig. 1B), but not in E rats (Fig. 1C). In D2 rats, fenfluramine treatment decreased the number of licks during presentation of the 0.025, 0.05, and 0.4 M sucrose solutions, P’s < 0.05. In E rats, licking was not influenced by fenfluramine treatment. The numbers of licks to water, 0.1, and 0.2 M sucrose were not influenced by stage of the estrous cycle or drug treatment.
3.1. Sucrose licking during short-term access
3.2. Sucrose intake and preference
The number of licks during short-term access to varying concentrations of sucrose solutions was influenced by a main effect of sucrose concentration [ F(5, 65) = 63.14, P < 0.0001], Table 1. To investigate this effect, data were collapsed across 2 of the 3 factors of our analysis (i.e., stage of the estrous cycle and drug treatment) and post hoc tests were used to investigate differences in the number of licks elicited by each sucrose concentration. These analyses revealed that the number of licks increased with each
Consumption of the 0.025 M sucrose solution was influenced by a main effect of drug treatment [ F(1, 4) = 9.84, P < 0.05], Table 2. Fenfluramine treatment decreased acceptance of the 0.025 M sucrose solution in D2 and E rats, relative to that observed following saline treatment, P’s < 0.05. Water consumption was not influenced by either stage of the estrous cycle or by drug treatment. All rats displayed a strong preference for the 0.025 M sucrose solution over water. Unlike the short-term access test,
2.7. Statistical analysis Data are presented as means T SEM. Sucrose licking during short-term access tests was analyzed using a 3-factor [estrous cycle stage (D2 and E) by drug treatment (fenfluramine and saline) by sucrose concentration (0.0, 0.025, 0.05, 0.1, 0.2, and 0.4 M)] repeated-measures analysis of variance (ANOVA) procedure. Overnight intakes of sucrose and water, and the sucrose preference ratios, were analyzed using 2-factor [estrous cycle stage (D2 and E) by drug treatment (fenfluramine and saline)] repeated-measures ANOVAs. Significant main or interaction effects ( P < 0.05) were examined using Tukey’s honestly significant difference test.
number of licks in 10 s
80 60
A
B D2/SAL E/SAL
C D2/SAL D2/FEN
E/SAL E/FEN
40
b
20 0
b
a 0.0 0.025 0.05
0.1
0.2
0.4
b
0.0 0.025 0.05
b
0.1
0.2
0.4
0.0 0.025 0.05
0.1
0.2
0.4
sucrose concentration (M) Fig. 1. Mean number of licks during brief access to water (0.0 M) and varying concentrations of sucrose solutions in D2 and E rats treated with saline and fenfluramine. Values are means T SEM. (A) An E-related decrease in the number of licks to 0.025 M sucrose was apparent following saline treatment. (B) During D2, fenfluramine decreased the number of licks to 0.025, 0.05, and 0.4 M sucrose. (C) Fenfluramine failed to decrease the number of licks to any concentration of sucrose during E. aE/SAL rats less than D2/SAL rats, P < 0.05. bD2/FEN rats less than D2/SAL rats, P < 0.05. Abbreviations: D2; diestrus 2, E; estrus, SAL; saline, FEN; fenfluramine.
D.P.D. Atchley et al. / Physiology & Behavior 86 (2005) 265 – 271 Table 2 Fenfluramine decreased overnight consumption of 0.025 M sucrose but failed to alter the preference ratio for 0.025 M sucrose
Sucrose intake (ml/20 h) Water intake (ml/20 h) Sucrose preference ratio
Saline treatment
Fenfluramine treatment
Diestrus
Estrus
Diestrus
Estrus
80.5 T 11.1
87.7 T 11.4
65.2 T 11.6*
70.4 T 18.9*
11.6 T 2.2
10.2 T 1.6
9.4 T 1.4
11.6 T 1.5
0.87 T 0.03
0.91 T 0.02
0.87 T 0.01
0.81 T 0.05
Values are means T SEM. Following saline and fenfluramine treatment, rats were given overnight (20 h) access to 0.025 M sucrose and water. Sucrose preference was determined by the ratio of sucrose consumed to total fluid consumed. *Less than saline-treated rats, P < 0.05.
preference for 0.025 M sucrose was not influenced either by stage of the estrous cycle or by drug treatment.
4. Discussion Consistent with previous studies [22,28], all rats displayed concentration-dependent, lick – response curves during brief access to a range of sucrose solutions that were presented in random order. When licking data were arranged as a function of ascending sucrose concentration, a reliable increase in the number of licks was apparent with each successive increment in sucrose concentration (Table 1). Changes in the number of licks, as a function of estrous cycle stage or drug treatment, were observed at 3 sucrose concentrations (Fig. 1). Stage of the estrous cycle influenced sucrose licking only at the lowest concentration of sucrose tested here; estrous rats displayed fewer licks to 0.025 M sucrose than diestrous rats. Interestingly, an inhibitory effect of fenfluramine on sucrose licking was apparent only in diestrous rats at some (i.e., 0.025, 0.05, and 0.4 M), but not all, sucrose concentrations. This decline in sucrose palatability was observed only during brief-access tests. Fenfluramine failed to reduce preference for the 0.025 M sucrose solution during an overnight, two-bottle choice test. The estrous-related decrease in licking responses to 0.025 M sucrose observed here is consistent with a previous study involving estradiol-treated, ovariectomized rats [22]. Using similar experimental paradigms, both studies revealed decreases in licking to dilute sucrose solutions during a time when estradiol’s behavioral effects on food intake are maximal. However, the present results are not consistent with two previous studies in which estradiol-treated, ovariectomized rats failed to decrease the number of licks during the first min of a 0.8 M sucrose test meal [21], and failed to decrease sham intake of a 0.4 M sucrose solution [20]. These discrepant findings may be related to differences in sucrose concentration that likely produced differences in the saliency of the gustatory stimuli across studies. In previous studies [20,21], the sucrose concentrations were the same or twice that of the highest concentration examined
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here. Indeed, at our highest sucrose concentration (0.4 M), the number of licks was not influenced by stage of the estrous cycle. Thus, estradiol’s ability to decrease positivefeedback signals from the mouth may depend upon the strength of the gustatory stimulus. An additional factor that may have contributed to the discrepant findings involves the animal’s internal state. In previous studies [20,21], rats were tested in a food-deprived state whereas our rats were tested in a food-replete state. This likely resulted in different internal drives to eat which may have influenced the strength of positive-feedback signals arising from the mouth. Finally, previous studies that have examined estradiol’s influence on taste in short-term access trials have been conducted in ovariectomized rats receiving estradiol replacement [20 –22]. Thus, our results extend previous studies by demonstrating, for the first time, that changes in endogenous hormone secretion, presumably the preovulatory rise in estradiol secretion, can decrease the positivefeedback signals arising from ingestion of a dilute sucrose solution. Although the anorectic effect of fenfluramine has been largely attributed to its ability to augment the satiating strength of negative-feedback signals from the gut (e.g., [29]), there is some evidence that it may also reduce the strength of positive-feedback signals from the mouth. For example, fenfluramine has been shown to decrease sham intake of a sucrose solution in rats fitted with open gastric fistulas [30], and increase aversive taste reactivity responses during intraoral infusions of a 2% sucrose solution [19]. Our finding, that fenfluramine decreased licking during brief access to 3 of the 5 sucrose concentrations (0.025, 0.05, and 0.4 M) in diestrous rats (Fig. 1B), is consistent with these previous studies in male rats. The significance of the lack of fenfluramine’s ability to produce a reliable decrease in the number of licks during presentations of 0.1 and 0.2 M sucrose is unclear; however, two points are worth mentioning. First, there was a trend for fenfluramine treatment to reduce the number of licks during presentation of each sucrose solution in diestrous rats. Clearly, the overall number of licks across all sucrose concentrations was reduced by fenfluramine treatment in the diestrous rat. Second, the variability in the number of licks produced by fenfluramine-treated, diestrous rats during presentation of 0.1 and 0.2 M sucrose was greater than that observed during presentation of the other sucrose concentrations. This may have limited our ability to detect a reliable inhibitory effect of fenfluramine on licking at these sucrose concentrations. Additional research, perhaps involving a longer training interval or a greater number of sucrose presentations during testing, would be helpful in resolving this issue. Interestingly, fenfluramine failed to modulate sucrose licking in estrous rats (Fig. 1C). At the lowest sucrose concentration (0.025 M), this may be related to a floor effect as the number of licks was already suppressed in estrous rats, relative to diestrous rats. However, a similar argument cannot be made for the highest sucrose concentration; both
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diestrous and estrous rats displayed a high number of licks, yet an inhibitory effect of fenfluramine was only apparent in diestrous rats. That fenfluramine failed to decrease the number of licks to a range of sucrose solutions in estrous rats suggests that an estrous-related decline in the strength of positive-feedback signals did not contribute to our earlier report that fenfluramine’s anorectic effects are increased in estrous rats, relative to diestrous rats [17]. Clearly, additional research is required to determine whether hormonal fluctuations across the rat’s estrous cycle differentially modulate positive- and negative-feedback signals generated during a meal. To investigate whether fenfluramine-induced changes in sucrose licking during brief-access tests are present only when postingestive signals are minimal and ingestion is driven largely by taste, we examined the effects of fenfluramine treatment on sucrose preference in rats given overnight access to water and a dilute sucrose solution. We choose 0.025 M sucrose based on the results of our briefaccess tests that revealed maximal estrous cycle- and fenfluramine-related differences at this sucrose concentration. In overnight tests, all rats displayed a clear preference for the dilute sucrose solution and no group differences in sucrose preference were detected. Thus, postingestive signals appear to override any effect of fenfluramine on positive-feedback signals generated during brief access to sweet solutions. Fenfluramine did, however, reduce overnight intake of the dilute sucrose solution in diestrous and estrous rats. This is consistent with fenfluramine’s ability to decrease intake of powdered chow during overnight intake tests in female rats [17]. However, unlike the overnight intake test involving a chow diet, an estrous-related increase in the inhibitory effects of fenfluramine on sucrose intake was not detected here. This may be related to differences in palatability between a dilute sucrose solution and laboratory chow. It is possible that the drive to consume the palatable sucrose solution overrides the hormonal influences that would normally function to decrease intake. Another possibility is that during estrus, rats were preferentially consuming the sucrose solution relative to their chow diet that was also freely available. Since overnight chow intake was not recorded, it is unknown whether differences in chow intake, and therefore total caloric intake, differed between estrous and diestrous rats. In summary, it is well established that alterations in circulating estradiol play an important role in the control of meal size in the female rat. Prevailing research suggests that the decline in meal size, observed following estradiol treatment in ovariectomized rats and during estrus in cycling rats, is mediated by increased sensitivity to negativefeedback controls of meal size (reviewed in [5]). Our present findings suggest that the estrous-related decrease in meal size may also involve a decrease in the positivefeedback controls of meal size. Here, we demonstrated a decline in licking during brief access to a 0.025 M sucrose solution in estrous rats, relative to diestrous rats. Because
others have failed to demonstrate similar results in rats consuming more palatable sucrose solutions [20,21], it appears that estradiol’s ability to modulate positive-feedback signals during ingestion is influenced by the strength of the gustatory stimulus. Thus, it will be important to determine the influence of diet palatability on estrousrelated changes in ingestive behavior in future studies. Our present findings also suggest that this estrous-related increase in the anorectic effects of fenfluramine does not involve a decrease in the positive-feedback controls of meal size. Here, a fenfluramine-induced decrease in sucrose licking during brief-access tests was observed only in diestrous rats and, in an overnight preference test, fenfluramine failed to decrease preference for a 0.025 M sucrose solution in either diestrous or estrous rats. Thus, the estrousrelated increase in the satiating effects of fenfluramine appear to rely upon postabsorptive properties of ingestion.
Acknowledgements We gratefully acknowledge the assistance and support of R. Henderson and P. Hendrick of the Technical Support Group at Florida State University Program in Neuroscience. We thank Dr. Kathleen Curtis for help with the design of this experiment. This research was supported by a National Institute of Mental Health Grant MH-63932 (LAE) and a National Institute of Health Training Grant in the Chemical Senses DC-000044 (DPDA).
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