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Carbohydrate- and protein-conditioned flavor preferences: Effects of nutrient preloads
Physiology & Behavior, Vol. 59, No. 3,467-474, 1996 Copyright © 1996 Elsevier Science Inc. Printed in the USA. All rights reserved 0031-9384/96 $15.00 + .00
ELSEVIER
0031-9384(95)02085-A
Carbohydrate- and Protein-Conditioned Flavor Preferences: Effects of Nutrient Preloads C A T A L I N A PI~REZ, K A R E N A C K R O F F AND ANTHONY SCLAFANI 1
Department of Psychology, Brooklyn College and the Graduate School, The City University of New York, Brooklyn, NY 11210 USA Received 10 May 1995 PI~REZ, C., K. ACKROFF AND A. SCLAFANI. Carbohydrate- and protein-conditionedflavor preferences: Effects of nutrient pre,!oads. PHYSIOL BEHAV 59(3) 467-474, 1996.--Food-deprived rats were trained to associate one flavor (CS P~°t) with intragastric (IG) infusions of protein (PROT; 10% calcium caseinate), a second flavor (CS cH°) with IG infusic,ns of carbohydrate (CHO; 10% protein), and a third flavor ( C S - ) with IG water infusions during 30 min/day training sessions. (The CS flavors were cherry, grape, and orange saccharin solutions.) In subsequent two-bottle tests the rats reliably preferred both the CS Prot and CS CliO to the C S - and equally preferred the CS r~°t and CS cH°. The preference for the two nutrient-paired flavors was not altered by IG preloads of PROT or CHO delivered as three loads 120, 40, and 5 min prior to testing. However, single oral + gastric preloads of CS cH° + IG CHO and CS Pr°t + IG PROT 45 min prior to test selectively increased the preference for the CS r~°t and CS cH°, respectively. In subsequent gasn'ic-only and oral-only tests single IG preloads of PROT and CHO, but not CS Pr°t and CS cH° preloads, selectively altered the rats' preference for CS crt° vs. CS r~°t. In a second experiment with new rats, oral + gastric preloads again selectively altered the preference for the CS cH° vs. CS ~°t, but gastric-only preloads failed to have this effect. These results demonstrate that rats can learn to associate different flavors with the postingestive effects of different nutrients, and modify their flavor preferences after nutrient preloads. Oral + gastric preloads were most effective in altering flavor preferences, whereas gastric-only preloads had inconsistent effects and oral-only preloads were ineffective. Intragastric conditioning
Polycose
Calcium caseinate
RATS readily learn to associate flavors with the postingestive actions of nutrients. These nutrient-conditionedflavor preferences have been demonstrated using a variety of training procedures, and with specific macronutrients (carbohydrate, protein, fat) as well as complete diets ,(5,12). In the typical procedure, one cue flavor is paired with a nutritive solution and a second cue flavor is paired with a nonnuu'itive solution (e.g., saccharin solution). Flavor preferences are then assessed in two choice tests with the two cue flavors. We recently observed that rats can also acquire multiple flavor-nutrient associations (11). An oral delay training procedure was used in which different cue flavors were available for I0 min followed, after a 10-min delay, by presentation of one of two isocaloric nutrient solutions or a nonnutritive solution. The animals displayed similar preferences for each pair of two nutrient-paired flavors (carbohydrate and protein, carbohydrate and fat, or protein and fat) and preferred both to a third flavor that had no nutritive consequences. Other investigators have also reported that isocaloric solutions
Gastric preloads
Oral preloads
of different nutrients (sucrose, Polycose, corn oil, ethanol) condition comparable flavor preferences (9,10,16,18). The "equipotentiality" of different nutrients to support flavor preference conditioning has suggested that the preferences are reinforced by postingestive signals related to the energy value of the nutrients; such learning is often referred to as calorie based (6,9,10,17). This interpretation is questioned, however, by findings from our laboratory that isocaloric solutions of glucose and fructose differ substantially in their postingestive reinforcing effects (1,13,14). Furthermore, some data suggest that animals can discriminate between the postingestive effects of different nutrients at isocaloric concentrations. Baker et al. (3) reported that the preference acquired for a protein-paired flavor was suppressed by an intragastric (IG) protein preload but not by a carbohydrate preload. These findings suggest that animals leam to associate flavors with the specific nutrient value of food rather than, or in addition to, the food's energy yield. The finding (3) cited above that a protein preload suppressed
To whom requests for reprints should be addressed.
467
468
PI~REZ, ACKROFF AND SCLAFANI
the preference for a protein-paired flavor does not conclusively demonstrate that the rats learned that the flavor represented protein. Conceivably, protein preloads could reduce the preference for any nutrient-paired flavor. In fact, Bartness and Rowland (4) reported that IG protein preloads suppressed the intake of both protein and carbohydrate in rats self-selecting their diet from separate macronutrient sources. Note also that Mehiel and Bolles (9) reported that an IG carbohydrate preload did not reduce the rats' preference for a carbohydrate-paired flavor. In view of these results, the present study further investigated the nutrient specificity of conditioned flavor preferences. Rats were trained to associate different cue flavors with isocaloric IG infusions of protein and carbohydrate. The influence of carbohydrate and protein preloads on the rats' preference for the two nutrient-paired flavors was then determined. The rats were given three types of preloads to assess the relative importance of oral and postoral cues in nutrient selection. With the gastric-only preloads they were infused IG with a fixed amount of carbohydrate or protein prior to the flavor preference test. With the oral + gastric preloads the rats were infused IG with carbohydrate or protein as they drank the flavored solution associated with each nutrient. Finally, with the oral-only preloads, the rat drank a fixed amount of the flavored solutions but received no IG infusions. EXPERIMENT 1A: INITIAL CONDITIONING The ability of rats to learn preferences for flavors associated with the postingestive actions of protein and carbohydrate has been demonstrated in prior experiments using separate groups of animals (2,3,14,15). In this experiment we investigated whether the same group of animals would acquire preferences for different flavors paired with IG protein and carbohydrate infusions. Preferences were assessed by giving the rats the choice between each nutrient-paired flavor and a third flavor that had been paired with noncaloric infusions. In addition, the rats' preference for the protein-paired and carbohydrate-paired flavors was assessed directly in two-choice tests. METHOD
Subjects Fourteen Sprague-Dawley-derived adult female rats obtained from Charles River Breeding Laboratory (Wilmington, MA) were used. The rats weighed 270-314 g at the start of the experiment. They were individually housed in stainless steel cages in a vivarium maintained at 21°C under a 12:12 h light:dark cycle. The rats were fed powdered Purina Chow (No. 5001) and tap water as noted below.
Surgery The rats were surgically implanted with stainless steel gastric cannulas according to the method described by Elizalde and Sclafani (7).
Apparatus lntragastric infusions were accomplished using an "electronic esophagus" apparatus according to the procedure described by Elizalde and Sclafani (7). In brief, stainless steel rodent cages were modified to have a slot in the floor that permitted two catheters attached to the rat's gastric cannula to be connected to a dual-channel infusion swivel located below the cage; the catheters were protected by a flexible stainless steel spring. Tygon tubing connected the swivel to two peristaltic infusion pumps. The
pumps were operated automatically by drinkometer circuits and a microcomputer whenever the rat drank from sipper tubes located at the front of the cage. The flow rate of the pumps was 1.3 m l / m i n and they were controlled by computer software to infuse ~ 1 ml of fluid for each 1 ml of fluid orally consumed.
Test Solutions The cue solutions or conditioned stimuli (CS) consisted of 0.2% saccharin (Sigma Chemical Co., St. Louis, MO) and 0.05% unsweetened cherry, grape, and orange Kool-Aid flavors (General Foods, White Plains, NY) dissolved in tap water. An unflavored 0.2% saccharin solution was also used for pretraining purposes. The nutritive infusates consisted of 10% Polycose solution (Ross Laboratories, Columbus, OH) and a 10% calcium caseinate solution (Bio-Serv, Frenchtown, NJ). The nutrient solutions were prepared on a weight/weight basis and were isocaloric at 0.4 kcal/g. The nonnutritive infusate was water. The specific flavors paired with the Polycose, calcium caseinate, and water infusions were counterbalanced across subjects. The nutrientpaired flavors are referred to as the CS+ or, more specifically, as the CS c n ° and the c s P r ° t ; the water-paired flavor is referred to as the C S - .
Procedure The rats were allowed to recover from surgery for 1 week and were then familiarized with a 0.2% saccharin solution by giving them unlimited access to the solution and tap water for 2 days. The animals were then transferred to the test cages, where they lived until the end of the study. The next day they were placed on a food restriction schedule; food was restricted to 12 g / d a y for 20 days and then 14 g / d a y for the remainder of the experiment. The rats were accustomed to drink the saccharin solution first without concurrent infusions (four 30 m i n / d a y sessions) then with concurrent IG water infusions (four sessions). At the start of formal training, the rats were divided into two subgroups (n = 7 each). One subgroup was trained with one flavored solution (the CS ~°t) paired with IG infusions of 10% calcium caseinate and a second flavor ( C S - ) paired with IG water infusions. Training and testing were conducted 30 m i n / d a y over two 8-day cycles. On days 1-6 of each cycle the rats were given one-bottle access to the CS P~°t and C S - on alternating days. On days 7 and 8 of each cycle the rats were given the choice between the CS r'~°t and C S - , each paired with its appropriate infusion. The left-right positions of the CS solutions were alternated during training and testing to reduce side preference effects. Following the second cycle, the rats were given two additional 8-day training-test cycles with a new flavor (the CS cn°) paired with IG infusions of 10% Polycose and the same C S - flavor paired with IG water. The second subgroup was treated in an identical manner except that it was trained with the CS c " ° first and the CS re°t second. After the two training-test cycles with the second nutrient, all rats were given the choice between the CS cH° vs. the CS r'r°t, each paired with its appropriate nutrient infusion, for six 30 m i n / d a y sessions. Throughout the experiment water bottles were removed 1 h before the daily test sessions. Food rations and water were given 1.5-2 h after the test.
Statistical Analyses CS+ and C S - intakes were averaged over 3-day periods during training, and 2-day periods on preference test days. Analysis of the CS ClIO vs. CS Prot preference test was based on the last two test sessions. The preference data were also expressed as
CARBOHYDRATE AND PROTEIN PREFERENCES
r///~
CS F'~
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flavor paired with noncaloric infusions. They reported that the rats' preference for the CS Fv°t flavor over the C S - flavor was blocked by an IG protein preload but not by an isocaloric carbohydrate preload. The present experiment extended this design by comparing the effects of isocaloric protein and carbohydrate preloads on the rats' preference for the CS P~°t vs. CS cH° flavors. METHOD
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Two-Bottle Tests
FIG. 1. Experiment 1A. Mean+ SEM intake of CS Pr°t, CSCHO, and CSsolutions during two-bottle preference tests of the second training-test cycle with each nutrient; each bar represents the mean of two 30 min/day sessions. Percentages atop bars indicate the percent intake of that solution.The CSPr°t, CScH°, and CS- were paired with IG infusions of calcium caseinate,Polycose, and water, respectively. percent CS+ intake [(CS+ intake/total intake) × 100]. The data were analyzed using repeated-measures analysis of variance (ANOVA) followed by tests of simple main effects when appropriate (Crunch Statistical Package, Crunch Software, Inc.). To simplify presentation, only data from the second test-training cycle with each nutrient are discussed. RESULTS In the one-bottle training sessions of the second cycle, the rats consumed comparable amounts of the CS P~°t, CS cH°, and C S solutions (10.7, 10.0, and 10.3 g / 3 0 min, respectively) and consequently were infused with equivalent amounts of calcium caseinate, Polycose, and water. As indicated in Fig. 1, in the two-bottle choice tests l:he animals consumed more CS P~°t than CS and more CS cH° than CS, F(1, 13) = 129.4, p < 0.001. The absolute intakes of the CS P~°t and CS cH° were similar as were the percent intakes: 91% and 89%, respectively. In the choice test with the two nutrient-paired flavors, the rats did not reliably differ in their intakes of the two CS+ solutions; they consumed 45% of their intake as ('S P~°t and 55% as CS cH° (Fig. 1). EXPERIMENT 1B: EFFECT OF GASTRIC PRELOADS The rats in Experiment 1A acquired comparable preferences for the flavors paired with the IG protein and IG carbohydrate infusions. It is not known from these results whether the animals distinguished between the two nutrient infusions or treated them as identical caloric sources. Experiment 1B addressed this question by determining if altering the rats' protein and carbohydrate repletion states would alter their flavor preferences. In a prior experiment related to this issue, Baker et al. (3) trained rats to prefer a flavor paired with IG protein infusions over a C S -
After the completion of Experiment 1A, 13 rats had functional gastric cannulas. They were divided into two groups matched for CS r~°t and CS cH° preference and previous training experience (order in which they received the nutrients). One group (n = 6) was tested for its preference following gastric preloads of protein (10% casein) whereas the other group (n = 7) was tested following carbohydrate gastric preloads (10% Polycose). Using a testing procedure adapted from Baker et al. (3), the rats were given 7.5 g nutrient preloads in 2.5 g doses at 120, 40, and 5 min before the 30-min preference tests with the CS P~°t and csCH°; the CSs were each paired with their appropriate nutrient infusions during the preference tests. The rats were tested twice under these conditions with the left-right position of the CSs alternating. They were then given two additional preference tests without preloads followed by two more preload tests. In these later tests, 15 g IG preloads were delivered in 5 g doses at 120, 40, and 5 min prior to the preference tests. RESULTS Figure 2A presents the data from the 7.5 g preload tests. When not preloaded, both groups consumed similar amounts of CS cH° and CS Pr°t (these data are from Experiment 1A). Following the nutrient preloads, both groups tended to consume more CS ~°t than CS cH°, but the intake differences were not significant. Within-group comparisons revealed that, compared to the no-preload test, following the carbohydrate preload the rats tended to consume less CS cH° solution but only the overall preload effect was significant, F(I, 5)=7.86, p < 0 . 0 5 . The protein preload did not have a significant effect on CS+ intakes relative to the no-preload condition. The two groups did not reliably differ in their CS+ intakes following the 15 g carbohydrate and protein preloads (Fig. 2B). Within-group comparisons indicated that the carbohydrate preload produced a general suppression in CS+ intakes [preload main effect, F(1, 5 ) = 28.50, p < 0.01]. The protein preload did not alter CS+ intakes relative to the no-preload condition. EXPERIMENT 1C: ORAL AND GASTRIC PRELOADS The IG nutrient preloads used in Experiment 1B failed to differentially alter the preference for the CS P~°t and CS cH° flavors. The preload parameters were based on those described by Baker et al. (3) that were effective in suppressing a CS r~°t preference. Possible reasons for the discrepant results are considered in the General Discussion. In the present experiment a different preload protocol was used in an attempt to alter the rats' preference for the CS Pr°t and CS cH° flavors. Li and Anderson (8) previously reported that rats, given the choice of high- and low-protein diets, consumed more high-protein diet after a lowprotein premeal than after a high-protein premeal. The isocaloric premeals were orally consumed 45 min prior to the diet choice test. Following this procedure, the rats in Experiment 1C were given a protein or carbohydrate "premeal" 45 min prior to the CS Pr°t vs. CS cH° choice test. The "premeals" consisted of
470
PI~REZ, ACKROFF AND SCLAFANI
Two-Bottle Tests r///~
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rats were given 7.5 g of the CS ~°t to drink paired with a 7.5 g IG infusion of 10% casein on test days 3 and 4, and 7.5 g of the CS cH° to drink paired with 7.5 g infusion of 10% Polycose on test days 5 and 6. In the gastric-only preload test, the rats were given gastric infusions of 10% casein (7.5 g) for 2 days followed by gastric infusions of 10% Polycose (7.5 g) for 2 days. In the oral-only preload tests, the rats were given 7.5 g of the CS P~°t to drink for 2 days followed by 7.5 g of the CS cH° to drink for 2 days. The remaining rats were given the carbohydrate preload first and protein preload second in each of the three preload test cycles. The left-right position of the CS cH° and CS r%t solutions during the two-bottle tests alternated daily. Data analysis was performed using the mean intakes during the two tests conducted under each preload condition.
4
t-
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PROT
i
NONE
Preloads
ClIO
FIG. 2. Experiment lB. Mean + SEM intake of CS Pr°t and CS cH° during two-bottle preference tests; each bar represents the mean of two 30 min/day sessions. Percentages atop bars indicate the percent intake of that solution. The CS Pr°t and CS cH° were paired with IG infusions of calcium caseinate and Polycose, respectively. In the preload tests the rats received IG infusions of 7.5 g (top) or 15 g (bottom) of protein (PROT; six rats) or carbohydrate (CHO; seven rats) divided into three doses at 120, 40, and 5 rain before the two-bottle preference tests.
access to a fixed amount of the CS Pr°t drink paired with an equivalent amount drate, respectively, infused IG. These ("premeals") proved very effective in preferences, and we next compared the only and oral-only preloads.
and CS c n ° solutions to of protein and carbohyoral + gastric preloads altering the rats' C S + effectiveness of gastric-
METHOD
The 13 rats from Experiment 1B were studied. Their preference for the two nutrient-paired CSs was measured over three test cycles that involved oral + gastric, gastric-only, and oral-only preloads, in that order. Each test cycle included six 30 m i n / d a y preference tests with the CS Pr°t and CS cH°, each paired with their respective nutrient infusions. No preloads were given on the first 2 test days, whereas different nutrient preloads were given on the next 2 and last 2 test days. All preloads were given 45 min before the CS P~°t vs. CS cn° preference test. The rats had 15 min to complete the oral preloads, although most animals finished the preload within 10 min. In the oral + gastric preload tests, half the
There was no effect of the order in which the protein and carbohydrate preloads were given and the data from the 13 rats were combined. As illustrated in Fig. 3A, C S + preferences changed as a function of the oral + gastric nutrient preload [nutrient preload × CS interaction, F(1, 12) = 26.74, p < 0.001]. Following the protein preload the rats drank more CS c n ° than CS ~°t ( p < 0.001) whereas following the carbohydrate preload they drank more CS vr°t than CS cn°, although this latter difference was not reliable ( p = 0.08). The percent intakes as CS r'~°t were 26% and 61%, respectively, following the protein and carbohydrate preloads. In addition, the rats consumed more CS cH° after the protein preload than after the carbohydrate preload ( p < 0.001) and more CS P~°t after the carbohydrate preload than after the protein preload ( p < 0.01). Compared to the no-preload baseline, the protein preload suppressed ( p < 0.05) CS P~°t intake and tended to increase CS cH° intake [preload x CS interaction, F ( I , 12) = 8.31, p < 0.05]. The carbohydrate preload did not affect CS pr°t intake, relative to baseline, and slightly suppressed CS cH° intake. Figure 3B shows the effect of the gastric-only preloads on C S + preferences. Following the protein preload the rats consumed more ( p < 0.01) CS cH° than CS r~°t whereas following the carbohydrate preload they consumed more ( p < 0.01) CS r~°t than CS cn° [nutrient preload x CS interaction, F(I, 12) = 23.64, p < 0.001]. The percent intakes as CS Pr°t were 27% and 76%, respectively, following the protein and carbohydrate preloads. Also, the rats consumed more ( p < 0.01) CS cH° after the protein preload than after the carbohydrate preload, and more ( p < 0.01) CS P~°t after the carbohydrate preload than after the protein preload. Compared to the no-preload baseline, the carbohydrate preload reliably altered C S + preference [preload × CS interaction, F ( I , 12) = 5.02, p < 0.05]. The protein preload also tended to shift the C S + preference relative to the baseline condition but the preload × CS interaction was not significant. As shown in Fig. 4, the oral-only preloads did not differentially alter the preference for the C S + solutions. After both the CS r~°t and CS cH° preloads the rats consumed more CS cH° than CS pint, F(I, 12)= 11.84, p < 0.01. The CS cH° preference was slightly but not reliably greater following the oral preloads than in the no-preload condition. EXPERIMENT 2 Experiment 1 showed that, under some test conditions, rats can discriminate between the postingestive effects of protein and carbohydrate and following a gastric preload of one nutrient they shift their preference to a flavor associated with IG infusions of the other nutrient. Why this IG preload effect was obtained with
CARBOHYDRATE AND PROTEIN PREFERENCES
1A. Data are reported from 19 rats that completed the initial training phase and 17 rats that completed the preload phase of the experiment.
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The test solutions used were the same as in Experiment 1 except that, because of experimenter error, two rather than one C S - flavors were used. That is, the rats were first trained with cherry- and grape-flavored saccharin solutions as the CS P~t (or CS cH°) and the C S - . They were next trained with orange- and strawberry-flavored saccharin solutions as the CS c n ° (or CS r~°t) and C S - .
Procedure
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PROT
Gastric-Only
CHO
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Experiment 2A. The experimental procedure was similar to that of Experiment 1A except for the following differences. The rats were food restricted to 14 g of powdered chow throughout the experiment. They were divided into two subgroups ( n = 12 each) that were trained with the CS Pr°t first and CS cH° second or in the reverse order. Each training-test cycle consisted of four one-bottle training sessions followed by two two-bottle choice tests. Different C S - flavors were used in the CS r~°t and CS cH° training cycles. At the end of the last training-test cycle the rats' preference for the CS Pr°t vs. CS cH° was assessed over four 30 m i n / d a y sessions. Experiment 2B. The rats were divided into two groups matched for their CS P'°t vs. CS cH° preference and previous experience
e-
2 0
T w o - B o t t l e Tests 0 NONE
PROT
r////~ CS r'=
CHO
r/HA
C S c"°
Preloads Oral-Only
FIG. 3. Experiment lC. Mean + SEM intake of CS Pr°t and CS cH° during two-bottle preference tests; each bar represents the mean of two 30 rain/day sessions. Percentages atop bars indicate the percent intake of that solution. The CS Pr°t and CS cH° were paired with 1G infusions of calcium caseinate and Polycose, respectively. In the oral + gastric preload tests (top) the rats (n = 13) drank 7.5 g of the CS Pr°t or CS ca° paired with IG infusions of 7.5 g protein (PROT) and carbohydrate (CHO), respectively, 45 min before the two-bottle preference tests. In the gastriconly preload test (bottom) lhe rats were infused IG with 7.5 g protein or carbohydrate 45 min before the two-bottle preference tests.
10
42% t-
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the single IG preloads in Experiment 1C but not with divided preloads in Experimen'L 1B is not clear. In addition to the difference in how the preloads were delivered, in Experiment IC the rats were tested wit~a oral + gastric preloads before the gastric-only test. Conceivably, the oral + gastric preloads may have sensitized the rats to re,;pond to postingestive cues produced by IG nutrient infusions. This possibility was examined in the present experiment using new rats. The animals were tested with oral + gastric and gastric-only preloads as in Experiment IC but with the order of the preloads counterbalanced. METHOD
Subjects The subjects were 24 rats of the same type and source as those used in Experiment 1. The rats were implanted with gastric cannulas and tested in infusion cages as described in Experiment
O
NONE
CS ~*'
CSc"°
Preloads
FIG. 4. Experiment 1C. Mean + SEM intake of CS ~°t and CS cH° during two-bottle preference tests; each bar represents the mean of two 30 min/day sessions. Pe~entages at~,obars indicate the percent intake of that solution. The CS and CS were paired with IG infusions of calcium caseinate and Polycose, respectively. In the oral-only preload tests the rats (n = 13) drank 7.5 g of the C S Pr°t or CS cn° without concurrent infusions 45 rain before the two-bottle preference tests.
472
Pt~REZ, ACKROFF AND SCLAFANI
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0.01) CS cH° than CS p~°t after the protein preload, and tended to drink more CS P~°t than CS cH° after the carbohydrate preload [nutrient × CS interaction, F(1, 16) = 58.8, p < 0.001]. Also, the rats consumed more ( p < 0.01) CS cH° after the protein preload than after the carbohydrate preload, and more ( p < 0.01) CS P'°t after the carbohydrate preload than after the protein preload. Compared to the no-preload condition, the oral + gastric protein preload altered the rats' C S + preference [nutrient preload X CS interaction, F(1, 16)= 7.96, p < 0.05]. The rats consumed less CS Pr°t ( p < 0.05) and slightly more CS cH° after the protein preload compared to the no-preload condition. The carbohydrate oral + gastric preload produced only a marginal change ( p < 0.07) in C S + preference compared to the no-preload condition. In the gastric-only preload condition the rats did not respond differentially to the two nutrient preloads. They consumed more CS cH° than CS Pr°t after both the protein and carbohydrate preloads [CS main effect, F(1, 16)= 7.23, p < 0.02], although the difference tended to be greater after the protein preload.
Two-Bottle Tests v///.4
T w o - B o t t l e Tests
FIG. 5. Experiment 2A. Mean + SEM intake of CS ~°t, CS cH°, and CSsolutions during two-bottle preference tests of the second training-test cycle with each nutrient; each bar represents the mean of two 30 rain/day sessions. Percentages atop bars indicate the percent intake of that solution. The CS ~°t, CSc"°, and CS- were paired with IG infusions of calcium caseinate, Polycose, and water, respectively.
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(order of nutrient conditioning). One group (n = 8) was first tested with the oral + gastric preloads and then gastric-only preloads; the other group (n = 9) was tested in the reverse order. Within each subgroup, half of the rats received the protein preload first and the carbohydrate preload second; the other half received the nutrient preloads in the reverse order. The preloads were administered as in Experiment 1C.
CS P'"
t-
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RESULTS t'-
Experiment 2A The rats consumed comparable amounts of the CS ~°t, CS cH°, and C S - during the second one-bottle training cycle with each nutrient (10.4, 10.7, 10.9 g / 3 0 min, respectively). As illustrated in Fig. 5, in the choice tests they preferred both the CS P~°t and CS cry° to the C S - , F(1, 18)= 98.5, p < 0.001. The CS cH° preference (85%) was stronger than the CS r~°t preference (70%) [nutrient × CS interaction, F(1, 17) = 5.02, p < 0.04]. In the subsequent choice tests with the two nutrient-paired flavors, the rats did not differ in their intakes of the C S + solutions; they consumed 47% of their intake as CS pr°t and 53% as CS cH° (Fig.
5). Experiment 2B Preliminary analysis revealed that the order of preload testing was not a significant factor and therefore the data from the two groups were combined. The type of preload (oral + gastric or gastric-only) did, however, affect the rats' responses to the nutrient preloads [preload type X preioad nutrient X CS interaction, F(1, 16) = 5.69, p < 0.05]. As illustrated in Fig. 6, in the oral + gastric preload condition the rats consumed more ( p <
F=
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8
v
6
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PROT
CHO
Preloads
FIG. 6. Experiment 2B. Mean + SEM intake of CS ~°t and CS cH° during two-bottle preference tests; each bar represents the mean of two 30 min/day sessions. Percentages atop bars indicate the percent intake of that solution. The CS Prot and CS CHO were paired with IG infusions of calcium caseinate and Polycose, respectively. In the oral + gastric preload tests (top) the rats ( n = 19) drank 7.5 g of the CS ~°t or CS cH° paired with IG infusions of 7.5 g protein (PROT) and carbohydrate (CHO), respectively, 45 min before the two-bottle preference tests. In the gastriconly preload test (bottom) the rats were infused intragastrically with 7.5 g protein or carbohydrate 45 rain before the two-bottle preference tests.
CARBOHYDRATE AND PROTEIN PREFERENCES
Compared to the no-preload condition, the gastric protein preload tended to reduce CS r'~°' intake and increase CS cH° intake but the nutrient preload × CS interaction was not reliable ( p < 0.07). The carbohydrate gastric preload did not alter CS+ intakes compared to the no-preload baseline. GENERAL DISCUSSION In confirmation of prior studies (2,3,14,15), the rats in this study acquired strong preferences for flavors paired with IG infusions of protein and carbohydrate. The new finding here is that the protein- and carbohydrate-conditioned preferences were obtained in the same a~imals. Furthermore, the preferences for the two nutrient-paired flavors were equivalent as measured by CS r'~°t vs. csCH°choice tests. The rats also displayed comparable preferences for the CS r'~°t and CS cH° flavors relative to the C S - flavor in Experiment 1, although in the second experiment their intake of the CS r~,t was somewhat less than their CS cH° intake relative to the C S - . This may have resulted because the rats in the second experiment received less training and had two different CS flavors to learn; perhaps protein-conditioned preferences are acquired more slowly than carbohydrate-conditioned preferences. Nevertheles:~, when tested for their CS P~°t vs. CS cH° preference, the rats in Experiment 2, like those in the first experiment, did not prefer one nutrient-paired flavor over the other. We recently reported that rats acquire comparable preferences for flavors paired with the consumption of iso~:aloric solutions of casein and Polycose solutions using an oral-delay conditioning paradigm (11). The present findings confirm these results and demonstrate that the nutrients have equivalent postingestive reinforcing effects. In both studies the casein and Polycose provided the same caloric benefit ~:o the animals (i.e., the nutrient solutions were isocaloric and the amounts consumed/infused were the same). The similar flavcr preferences conditioned by casein and Polycose may have resulted because, as proposed by some investigators (9,17,18), the unconditioned stimulus (US) mediating reinforcement is related to the nutrients' energy value. In particular, Tordoff (17) proposed that the US may be a signal generated by hepatic fuel oxidatiorL. However, if animals attend only to the energy value of foods, this would not facilitate their selection of a nutritionally balanced diet. The present study investigated, therefore, whether rats could distinguish between the postingestive actions of carbohydrate and protein by measuring their flavor preferences after nutrient: preloads. In Experiment IB, giving the rats divided protein and carbohydrate preloads over a 2-h period failed to alter their selection of the CS r'r°t and CS cH° in the subsequent choice test. This protocol was based on the repot: of Baker et al. (3) that an IG protein preload, but not a carbohydrate preload, suppressed the rats' preference for a protein-paired flavor. The current study and the Baker et al. (3) experinaent differed in several respects, which may account for the discrepant results. In particular, Baker et al. (3) used minimally (4 h) deprived rats, trained and tested them with CS r'~°t and C S - flavors only, and did not infuse them during the CS er°t vs. CS choice test. Given the negative results of Experiment 1B, we next investigated the effects of oral + gastric preloads on the preference for the nutrient-paired flavcrs (Experiment IC). This manipulation provided the animals with both orosensory and viscerosensory information about the preload and was analogous to the orally consumed "premeals" used in earlier food selection studies (8,20). When prefed one flavor with its paired IG nutrient
473
infusion, the rats showed a shift in preference to the alternate nutrient-paired flavor in the CS P~°t vs. CS cn° choice test. The rats showed a similar preference shift when next tested with IG-only preloads in Experiment 1C. However, when these manipulations were repeated with new rats in Experiment 2B, the oral + IG preloads again produced nutrient-specific preference shifts, but now the IG-only preloads did not. Taken together, the results of Experiments 1B, 1C, and 2B indicate that IG-only preloads do not consistently alter the rats' acquired preferences for flavors paired with protein and carbohydrate. The reason for this inconsistency is not certain. Mixed results have also been reported in studies that assessed the effects of IG preloads on macronutrient self-selection. Bartness and Rowland (4) observed that a carbohydrate preload reduced subsequent carbohydrate but not protein intake, whereas a protein preload suppressed both protein and carbohydrate intake. Also, both preloads suppressed fat intake. Zeggeren and Li (19) reported that IG carbohydrate preloads selectively reduced the intake of a high-carbohydrate food more than a high-protein diet, but they did not measure the effects of a protein preload. Mehiel and Bolles (9), on the other hand, reported that an IG carbohydrate preload did not selectively reduced the intake of two flavors that were previously paired with carbohydrate and ethanol consumption. In contrast to the IG-only preloads, the oral + gastric preloads produced very similar preference shifts in Experiments 1C and 2B. This was not due solely to the oral cues provided by the preload. The oral-only preload in Experiment 1C did not cause the animals to shift their preference from the preload flavor to the alternate flavor. (The failure of oral-only preloads to produce a preference shift was confirmed in additional tests conducted with the rats from Experiment 2.) These data indicate that the combined actions of orosensory and viscerosensory cues are more effective than either type of cue alone in modifying the rat's subsequent food choices. This is perhaps not surprising because the animal has more information to guide its selection, and flavor and postingestive consequences of foods are normally closely linked under natural feeding conditions. An important aspect of the present results is that both the protein and carbohydrate preloads (oral + gastric) shifted the rats' preference to the opposite nutrient. The protein preload, however, was more effective than the carbohydrate preload in altering subsequent preference. That is, following the CS Pr°t + IG protein the rats displayed a 74-79% preference for the CS cH°, whereas following the CS cH° preload the rats showed only a 61-63% CS r~°t preference in Experiments 1 and 2. This could reflect a general asymmetry between the effects of the two types of nutrient preloads, which may be related to a tighter regulation of protein than carbohydrate intake. Alternatively, the apparent enhanced effectiveness of the protein preloads may be due to the particular test conditions of the present study. Note that the rats were fed restricted amounts of a relatively high-protein food (lab chow), and thus their need for nonprotein energy may have been greater than their need for protein. The influence of protein and carbohydrate preloads on nutrient selection should be studied under varied conditions. Little is known about the viscerosensory stimuli generated by nutrients. The present results do not refute the hypothesis that different nutrients produce a common energy-related signal that reinforces flavor preferences. They do indicate, however, that nutrient-specific signals are generated by the pre- and/or postabsorptive actions of nutrients. In flavor-nutrient conditioning, therefore, animals acquire knowledge about the specific nutritive value of foods.
474
PI~REZ, A C K R O F F A N D S C L A F A N I
ACKNOWLEDGEMENTS This research was supported by a grant from the National Institute of Diabetes and Digestive and Kidney Diseases (DK-31135) and a NIMH
Research Scientist Award (MH-00983) to Anthony Sclafani. Polycose and Kool-Aid were generously donated by Ross Laboratories and General Foods, respectively.
REFERENCES 1. Ackroff, K.; Sclafani, A. Flavor preferences conditioned by sugars: Rats learn to prefer glucose over fructose. Physiol. Behav. 50:815824; 1991. 2. Baker, B. J.; Booth, D. A. Preference conditioning by concurrent diets with delayed proportional reinforcement. Physiol. Behav. 46:585-590; 1989. 3. Baker, B. J.; Booth, D. A.; Duggan, J. P.; Gibson, E. L. Protein appetite demonstrated: Learned specificity of protein-cue preference to protein need in adult rats. Nutr. Res. 7:481-487; 1987. 4. Barmess, T. J.; Rowland, N. Dietary self-selection in normal and diabetic rats after gastric loads of pure macronutrients. Physiol. Behav. 31:546-554; 1983. 5. Booth, D. A. Food-conditioned eating preferences and aversions with interoceptive elements: Conditioned appetites and satieties. Ann. NY Acad. Sci. 443:22-41; 1985. 6. Capaldi, E. D.; Campbell, D. H.; Sheffer, J. D.; Bradford, J. P. Conditioned flavor preferences based on delayed caloric consequences. J. Exp. Psychol. [Anim. Behav. Proc.] 13:150-155; 1987. 7. Elizalde, G.; Sclafani, A. Flavor preferences conditioned by intragastric Polycose: A detailed analysis using an electronic esophagus preparation. Physiol. Behav. 47:63-77; 1990. 8. Li, E. T. S.; Anderson, G. H. Meal composition influences subsequent food selection in the young rat. Physiol. Behav. 29:779-783; 1982. 9. Mehiel, R.; Bolles, R. C. Learned flavor preferences based on caloric outcome. Anita. Learn. Behav. 12:421-427; 1984. 10. Mehiel, R.; Bolles, R. C. Learned flavor preferences based on calories are independent of initial hedonic value. Anim. Learn. Behav. 16:383-387; 1988. 11. P6rez, C.; Lucas, F.; Sclafani, A. Carbohydrate, fat and protein condition similar flavor preferences in rats using an oral-delay conditioning procedure. Physiol. Behav. 57:549-554; 1995.
12. Sclafani, A. Nutritionally based learned flavor preferences in rats. In: Capaldi, E. D.; Powley, T. L., eds. Taste, experience, and feeding. Washington, DC: American Psychological Association; 1990:139156. 13. Sclafani, A.; Ackroff, K. Glucose- and fructose-conditioned flavor preferences in rats: Taste vs. postingestive conditioning. Physiol. Behav. 56:399-405; 1994. 14. Sclafani, A.; Cardieri, C.; Tucker, K.; Blusk, D.; Ackroff, K. Intragastric glucose but not fructose conditions robust flavor preferences in rats. Am. J. Physiol. 265:R320-R325; t993. 15. Sclafani, A.; Nissenbaum, J. W. Robust conditioned flavor preference produced by intragastric starch infusions in rats. Am. J. Physiol. 255:R672-R675; 1988. 16. Sherman, J. E.; Hickis, C. F.; Rice, A. G.; Rusiniak, K. W.; Garcia, J. Preferences and aversions for stimuli paired with ethanol in hungry rats. Anim. Learn. Bebav. 1 l:101-106; 1983. 17. Tordoff, M. G. Metabolic basis of learned food preferences. In: Friedman, M. 1.; Tordoff, M. G.; Kare, M. R., eds. Appetite and nutrition. New York: Marcel Dekker; 1991:239-260. 18. Tordoff, M. G.; Tepper, B. J.; Friedman, M. I. Food flavor preferences produced by drinking glucose and oil in normal and diabetic rats: Evidence for conditioning based on fuel oxidation. Physiol. Behav. 41:481-487; 1987. 19. Van Zeggeren, A.; Li, E. T. S. Food intake and choice in lean and obese Zucker rats after intragastric carbohydrate preloads. J. Nutr. 120:309-316; 1990. 20. Wurtman, J. J.; Moses, P. L.; Wurtman, R. J. Prior carbohydrate consumption affects the amount of carbohydrate that rats choose to eat. J. Nutr. 113:70-78; 1983.
ELSEVIER
0031-9384(95)02085-A
Carbohydrate- and Protein-Conditioned Flavor Preferences: Effects of Nutrient Preloads C A T A L I N A PI~REZ, K A R E N A C K R O F F AND ANTHONY SCLAFANI 1
Department of Psychology, Brooklyn College and the Graduate School, The City University of New York, Brooklyn, NY 11210 USA Received 10 May 1995 PI~REZ, C., K. ACKROFF AND A. SCLAFANI. Carbohydrate- and protein-conditionedflavor preferences: Effects of nutrient pre,!oads. PHYSIOL BEHAV 59(3) 467-474, 1996.--Food-deprived rats were trained to associate one flavor (CS P~°t) with intragastric (IG) infusions of protein (PROT; 10% calcium caseinate), a second flavor (CS cH°) with IG infusic,ns of carbohydrate (CHO; 10% protein), and a third flavor ( C S - ) with IG water infusions during 30 min/day training sessions. (The CS flavors were cherry, grape, and orange saccharin solutions.) In subsequent two-bottle tests the rats reliably preferred both the CS Prot and CS CliO to the C S - and equally preferred the CS r~°t and CS cH°. The preference for the two nutrient-paired flavors was not altered by IG preloads of PROT or CHO delivered as three loads 120, 40, and 5 min prior to testing. However, single oral + gastric preloads of CS cH° + IG CHO and CS Pr°t + IG PROT 45 min prior to test selectively increased the preference for the CS r~°t and CS cH°, respectively. In subsequent gasn'ic-only and oral-only tests single IG preloads of PROT and CHO, but not CS Pr°t and CS cH° preloads, selectively altered the rats' preference for CS crt° vs. CS r~°t. In a second experiment with new rats, oral + gastric preloads again selectively altered the preference for the CS cH° vs. CS ~°t, but gastric-only preloads failed to have this effect. These results demonstrate that rats can learn to associate different flavors with the postingestive effects of different nutrients, and modify their flavor preferences after nutrient preloads. Oral + gastric preloads were most effective in altering flavor preferences, whereas gastric-only preloads had inconsistent effects and oral-only preloads were ineffective. Intragastric conditioning
Polycose
Calcium caseinate
RATS readily learn to associate flavors with the postingestive actions of nutrients. These nutrient-conditionedflavor preferences have been demonstrated using a variety of training procedures, and with specific macronutrients (carbohydrate, protein, fat) as well as complete diets ,(5,12). In the typical procedure, one cue flavor is paired with a nutritive solution and a second cue flavor is paired with a nonnuu'itive solution (e.g., saccharin solution). Flavor preferences are then assessed in two choice tests with the two cue flavors. We recently observed that rats can also acquire multiple flavor-nutrient associations (11). An oral delay training procedure was used in which different cue flavors were available for I0 min followed, after a 10-min delay, by presentation of one of two isocaloric nutrient solutions or a nonnutritive solution. The animals displayed similar preferences for each pair of two nutrient-paired flavors (carbohydrate and protein, carbohydrate and fat, or protein and fat) and preferred both to a third flavor that had no nutritive consequences. Other investigators have also reported that isocaloric solutions
Gastric preloads
Oral preloads
of different nutrients (sucrose, Polycose, corn oil, ethanol) condition comparable flavor preferences (9,10,16,18). The "equipotentiality" of different nutrients to support flavor preference conditioning has suggested that the preferences are reinforced by postingestive signals related to the energy value of the nutrients; such learning is often referred to as calorie based (6,9,10,17). This interpretation is questioned, however, by findings from our laboratory that isocaloric solutions of glucose and fructose differ substantially in their postingestive reinforcing effects (1,13,14). Furthermore, some data suggest that animals can discriminate between the postingestive effects of different nutrients at isocaloric concentrations. Baker et al. (3) reported that the preference acquired for a protein-paired flavor was suppressed by an intragastric (IG) protein preload but not by a carbohydrate preload. These findings suggest that animals leam to associate flavors with the specific nutrient value of food rather than, or in addition to, the food's energy yield. The finding (3) cited above that a protein preload suppressed
To whom requests for reprints should be addressed.
467
468
PI~REZ, ACKROFF AND SCLAFANI
the preference for a protein-paired flavor does not conclusively demonstrate that the rats learned that the flavor represented protein. Conceivably, protein preloads could reduce the preference for any nutrient-paired flavor. In fact, Bartness and Rowland (4) reported that IG protein preloads suppressed the intake of both protein and carbohydrate in rats self-selecting their diet from separate macronutrient sources. Note also that Mehiel and Bolles (9) reported that an IG carbohydrate preload did not reduce the rats' preference for a carbohydrate-paired flavor. In view of these results, the present study further investigated the nutrient specificity of conditioned flavor preferences. Rats were trained to associate different cue flavors with isocaloric IG infusions of protein and carbohydrate. The influence of carbohydrate and protein preloads on the rats' preference for the two nutrient-paired flavors was then determined. The rats were given three types of preloads to assess the relative importance of oral and postoral cues in nutrient selection. With the gastric-only preloads they were infused IG with a fixed amount of carbohydrate or protein prior to the flavor preference test. With the oral + gastric preloads the rats were infused IG with carbohydrate or protein as they drank the flavored solution associated with each nutrient. Finally, with the oral-only preloads, the rat drank a fixed amount of the flavored solutions but received no IG infusions. EXPERIMENT 1A: INITIAL CONDITIONING The ability of rats to learn preferences for flavors associated with the postingestive actions of protein and carbohydrate has been demonstrated in prior experiments using separate groups of animals (2,3,14,15). In this experiment we investigated whether the same group of animals would acquire preferences for different flavors paired with IG protein and carbohydrate infusions. Preferences were assessed by giving the rats the choice between each nutrient-paired flavor and a third flavor that had been paired with noncaloric infusions. In addition, the rats' preference for the protein-paired and carbohydrate-paired flavors was assessed directly in two-choice tests. METHOD
Subjects Fourteen Sprague-Dawley-derived adult female rats obtained from Charles River Breeding Laboratory (Wilmington, MA) were used. The rats weighed 270-314 g at the start of the experiment. They were individually housed in stainless steel cages in a vivarium maintained at 21°C under a 12:12 h light:dark cycle. The rats were fed powdered Purina Chow (No. 5001) and tap water as noted below.
Surgery The rats were surgically implanted with stainless steel gastric cannulas according to the method described by Elizalde and Sclafani (7).
Apparatus lntragastric infusions were accomplished using an "electronic esophagus" apparatus according to the procedure described by Elizalde and Sclafani (7). In brief, stainless steel rodent cages were modified to have a slot in the floor that permitted two catheters attached to the rat's gastric cannula to be connected to a dual-channel infusion swivel located below the cage; the catheters were protected by a flexible stainless steel spring. Tygon tubing connected the swivel to two peristaltic infusion pumps. The
pumps were operated automatically by drinkometer circuits and a microcomputer whenever the rat drank from sipper tubes located at the front of the cage. The flow rate of the pumps was 1.3 m l / m i n and they were controlled by computer software to infuse ~ 1 ml of fluid for each 1 ml of fluid orally consumed.
Test Solutions The cue solutions or conditioned stimuli (CS) consisted of 0.2% saccharin (Sigma Chemical Co., St. Louis, MO) and 0.05% unsweetened cherry, grape, and orange Kool-Aid flavors (General Foods, White Plains, NY) dissolved in tap water. An unflavored 0.2% saccharin solution was also used for pretraining purposes. The nutritive infusates consisted of 10% Polycose solution (Ross Laboratories, Columbus, OH) and a 10% calcium caseinate solution (Bio-Serv, Frenchtown, NJ). The nutrient solutions were prepared on a weight/weight basis and were isocaloric at 0.4 kcal/g. The nonnutritive infusate was water. The specific flavors paired with the Polycose, calcium caseinate, and water infusions were counterbalanced across subjects. The nutrientpaired flavors are referred to as the CS+ or, more specifically, as the CS c n ° and the c s P r ° t ; the water-paired flavor is referred to as the C S - .
Procedure The rats were allowed to recover from surgery for 1 week and were then familiarized with a 0.2% saccharin solution by giving them unlimited access to the solution and tap water for 2 days. The animals were then transferred to the test cages, where they lived until the end of the study. The next day they were placed on a food restriction schedule; food was restricted to 12 g / d a y for 20 days and then 14 g / d a y for the remainder of the experiment. The rats were accustomed to drink the saccharin solution first without concurrent infusions (four 30 m i n / d a y sessions) then with concurrent IG water infusions (four sessions). At the start of formal training, the rats were divided into two subgroups (n = 7 each). One subgroup was trained with one flavored solution (the CS ~°t) paired with IG infusions of 10% calcium caseinate and a second flavor ( C S - ) paired with IG water infusions. Training and testing were conducted 30 m i n / d a y over two 8-day cycles. On days 1-6 of each cycle the rats were given one-bottle access to the CS P~°t and C S - on alternating days. On days 7 and 8 of each cycle the rats were given the choice between the CS r'~°t and C S - , each paired with its appropriate infusion. The left-right positions of the CS solutions were alternated during training and testing to reduce side preference effects. Following the second cycle, the rats were given two additional 8-day training-test cycles with a new flavor (the CS cn°) paired with IG infusions of 10% Polycose and the same C S - flavor paired with IG water. The second subgroup was treated in an identical manner except that it was trained with the CS c " ° first and the CS re°t second. After the two training-test cycles with the second nutrient, all rats were given the choice between the CS cH° vs. the CS r'r°t, each paired with its appropriate nutrient infusion, for six 30 m i n / d a y sessions. Throughout the experiment water bottles were removed 1 h before the daily test sessions. Food rations and water were given 1.5-2 h after the test.
Statistical Analyses CS+ and C S - intakes were averaged over 3-day periods during training, and 2-day periods on preference test days. Analysis of the CS ClIO vs. CS Prot preference test was based on the last two test sessions. The preference data were also expressed as
CARBOHYDRATE AND PROTEIN PREFERENCES
r///~
CS F'~
I
I CS-
~oo
91% 14 89% 12 .=_ 10 E
o~
flavor paired with noncaloric infusions. They reported that the rats' preference for the CS Fv°t flavor over the C S - flavor was blocked by an IG protein preload but not by an isocaloric carbohydrate preload. The present experiment extended this design by comparing the effects of isocaloric protein and carbohydrate preloads on the rats' preference for the CS P~°t vs. CS cH° flavors. METHOD
~4
45%
8
Z 6
0
469
i
4 2 0
Two-Bottle Tests
FIG. 1. Experiment 1A. Mean+ SEM intake of CS Pr°t, CSCHO, and CSsolutions during two-bottle preference tests of the second training-test cycle with each nutrient; each bar represents the mean of two 30 min/day sessions. Percentages atop bars indicate the percent intake of that solution.The CSPr°t, CScH°, and CS- were paired with IG infusions of calcium caseinate,Polycose, and water, respectively. percent CS+ intake [(CS+ intake/total intake) × 100]. The data were analyzed using repeated-measures analysis of variance (ANOVA) followed by tests of simple main effects when appropriate (Crunch Statistical Package, Crunch Software, Inc.). To simplify presentation, only data from the second test-training cycle with each nutrient are discussed. RESULTS In the one-bottle training sessions of the second cycle, the rats consumed comparable amounts of the CS P~°t, CS cH°, and C S solutions (10.7, 10.0, and 10.3 g / 3 0 min, respectively) and consequently were infused with equivalent amounts of calcium caseinate, Polycose, and water. As indicated in Fig. 1, in the two-bottle choice tests l:he animals consumed more CS P~°t than CS and more CS cH° than CS, F(1, 13) = 129.4, p < 0.001. The absolute intakes of the CS P~°t and CS cH° were similar as were the percent intakes: 91% and 89%, respectively. In the choice test with the two nutrient-paired flavors, the rats did not reliably differ in their intakes of the two CS+ solutions; they consumed 45% of their intake as ('S P~°t and 55% as CS cH° (Fig. 1). EXPERIMENT 1B: EFFECT OF GASTRIC PRELOADS The rats in Experiment 1A acquired comparable preferences for the flavors paired with the IG protein and IG carbohydrate infusions. It is not known from these results whether the animals distinguished between the two nutrient infusions or treated them as identical caloric sources. Experiment 1B addressed this question by determining if altering the rats' protein and carbohydrate repletion states would alter their flavor preferences. In a prior experiment related to this issue, Baker et al. (3) trained rats to prefer a flavor paired with IG protein infusions over a C S -
After the completion of Experiment 1A, 13 rats had functional gastric cannulas. They were divided into two groups matched for CS r~°t and CS cH° preference and previous training experience (order in which they received the nutrients). One group (n = 6) was tested for its preference following gastric preloads of protein (10% casein) whereas the other group (n = 7) was tested following carbohydrate gastric preloads (10% Polycose). Using a testing procedure adapted from Baker et al. (3), the rats were given 7.5 g nutrient preloads in 2.5 g doses at 120, 40, and 5 min before the 30-min preference tests with the CS P~°t and csCH°; the CSs were each paired with their appropriate nutrient infusions during the preference tests. The rats were tested twice under these conditions with the left-right position of the CSs alternating. They were then given two additional preference tests without preloads followed by two more preload tests. In these later tests, 15 g IG preloads were delivered in 5 g doses at 120, 40, and 5 min prior to the preference tests. RESULTS Figure 2A presents the data from the 7.5 g preload tests. When not preloaded, both groups consumed similar amounts of CS cH° and CS Pr°t (these data are from Experiment 1A). Following the nutrient preloads, both groups tended to consume more CS ~°t than CS cH°, but the intake differences were not significant. Within-group comparisons revealed that, compared to the no-preload test, following the carbohydrate preload the rats tended to consume less CS cH° solution but only the overall preload effect was significant, F(I, 5)=7.86, p < 0 . 0 5 . The protein preload did not have a significant effect on CS+ intakes relative to the no-preload condition. The two groups did not reliably differ in their CS+ intakes following the 15 g carbohydrate and protein preloads (Fig. 2B). Within-group comparisons indicated that the carbohydrate preload produced a general suppression in CS+ intakes [preload main effect, F(1, 5 ) = 28.50, p < 0.01]. The protein preload did not alter CS+ intakes relative to the no-preload condition. EXPERIMENT 1C: ORAL AND GASTRIC PRELOADS The IG nutrient preloads used in Experiment 1B failed to differentially alter the preference for the CS P~°t and CS cH° flavors. The preload parameters were based on those described by Baker et al. (3) that were effective in suppressing a CS r~°t preference. Possible reasons for the discrepant results are considered in the General Discussion. In the present experiment a different preload protocol was used in an attempt to alter the rats' preference for the CS Pr°t and CS cH° flavors. Li and Anderson (8) previously reported that rats, given the choice of high- and low-protein diets, consumed more high-protein diet after a lowprotein premeal than after a high-protein premeal. The isocaloric premeals were orally consumed 45 min prior to the diet choice test. Following this procedure, the rats in Experiment 1C were given a protein or carbohydrate "premeal" 45 min prior to the CS Pr°t vs. CS cH° choice test. The "premeals" consisted of
470
PI~REZ, ACKROFF AND SCLAFANI
Two-Bottle Tests r///~
CS ~=
~
CS c"°
7.5 g nutrient preload: 3 x 2.5 g 12 t-. _ 10 O o3 v
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RESULTS 0 NONE
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PROT
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rats were given 7.5 g of the CS ~°t to drink paired with a 7.5 g IG infusion of 10% casein on test days 3 and 4, and 7.5 g of the CS cH° to drink paired with 7.5 g infusion of 10% Polycose on test days 5 and 6. In the gastric-only preload test, the rats were given gastric infusions of 10% casein (7.5 g) for 2 days followed by gastric infusions of 10% Polycose (7.5 g) for 2 days. In the oral-only preload tests, the rats were given 7.5 g of the CS P~°t to drink for 2 days followed by 7.5 g of the CS cH° to drink for 2 days. The remaining rats were given the carbohydrate preload first and protein preload second in each of the three preload test cycles. The left-right position of the CS cH° and CS r%t solutions during the two-bottle tests alternated daily. Data analysis was performed using the mean intakes during the two tests conducted under each preload condition.
4
t-
2 O 0 NONE
PROT
i
NONE
Preloads
ClIO
FIG. 2. Experiment lB. Mean + SEM intake of CS Pr°t and CS cH° during two-bottle preference tests; each bar represents the mean of two 30 min/day sessions. Percentages atop bars indicate the percent intake of that solution. The CS Pr°t and CS cH° were paired with IG infusions of calcium caseinate and Polycose, respectively. In the preload tests the rats received IG infusions of 7.5 g (top) or 15 g (bottom) of protein (PROT; six rats) or carbohydrate (CHO; seven rats) divided into three doses at 120, 40, and 5 rain before the two-bottle preference tests.
access to a fixed amount of the CS Pr°t drink paired with an equivalent amount drate, respectively, infused IG. These ("premeals") proved very effective in preferences, and we next compared the only and oral-only preloads.
and CS c n ° solutions to of protein and carbohyoral + gastric preloads altering the rats' C S + effectiveness of gastric-
METHOD
The 13 rats from Experiment 1B were studied. Their preference for the two nutrient-paired CSs was measured over three test cycles that involved oral + gastric, gastric-only, and oral-only preloads, in that order. Each test cycle included six 30 m i n / d a y preference tests with the CS Pr°t and CS cH°, each paired with their respective nutrient infusions. No preloads were given on the first 2 test days, whereas different nutrient preloads were given on the next 2 and last 2 test days. All preloads were given 45 min before the CS P~°t vs. CS cn° preference test. The rats had 15 min to complete the oral preloads, although most animals finished the preload within 10 min. In the oral + gastric preload tests, half the
There was no effect of the order in which the protein and carbohydrate preloads were given and the data from the 13 rats were combined. As illustrated in Fig. 3A, C S + preferences changed as a function of the oral + gastric nutrient preload [nutrient preload × CS interaction, F(1, 12) = 26.74, p < 0.001]. Following the protein preload the rats drank more CS c n ° than CS ~°t ( p < 0.001) whereas following the carbohydrate preload they drank more CS vr°t than CS cn°, although this latter difference was not reliable ( p = 0.08). The percent intakes as CS r'~°t were 26% and 61%, respectively, following the protein and carbohydrate preloads. In addition, the rats consumed more CS cH° after the protein preload than after the carbohydrate preload ( p < 0.001) and more CS P~°t after the carbohydrate preload than after the protein preload ( p < 0.01). Compared to the no-preload baseline, the protein preload suppressed ( p < 0.05) CS P~°t intake and tended to increase CS cH° intake [preload x CS interaction, F ( I , 12) = 8.31, p < 0.05]. The carbohydrate preload did not affect CS pr°t intake, relative to baseline, and slightly suppressed CS cH° intake. Figure 3B shows the effect of the gastric-only preloads on C S + preferences. Following the protein preload the rats consumed more ( p < 0.01) CS cH° than CS r~°t whereas following the carbohydrate preload they consumed more ( p < 0.01) CS r~°t than CS cn° [nutrient preload x CS interaction, F(I, 12) = 23.64, p < 0.001]. The percent intakes as CS Pr°t were 27% and 76%, respectively, following the protein and carbohydrate preloads. Also, the rats consumed more ( p < 0.01) CS cH° after the protein preload than after the carbohydrate preload, and more ( p < 0.01) CS P~°t after the carbohydrate preload than after the protein preload. Compared to the no-preload baseline, the carbohydrate preload reliably altered C S + preference [preload × CS interaction, F ( I , 12) = 5.02, p < 0.05]. The protein preload also tended to shift the C S + preference relative to the baseline condition but the preload × CS interaction was not significant. As shown in Fig. 4, the oral-only preloads did not differentially alter the preference for the C S + solutions. After both the CS r~°t and CS cH° preloads the rats consumed more CS cH° than CS pint, F(I, 12)= 11.84, p < 0.01. The CS cH° preference was slightly but not reliably greater following the oral preloads than in the no-preload condition. EXPERIMENT 2 Experiment 1 showed that, under some test conditions, rats can discriminate between the postingestive effects of protein and carbohydrate and following a gastric preload of one nutrient they shift their preference to a flavor associated with IG infusions of the other nutrient. Why this IG preload effect was obtained with
CARBOHYDRATE AND PROTEIN PREFERENCES
1A. Data are reported from 19 rats that completed the initial training phase and 17 rats that completed the preload phase of the experiment.
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The test solutions used were the same as in Experiment 1 except that, because of experimenter error, two rather than one C S - flavors were used. That is, the rats were first trained with cherry- and grape-flavored saccharin solutions as the CS P~t (or CS cH°) and the C S - . They were next trained with orange- and strawberry-flavored saccharin solutions as the CS c n ° (or CS r~°t) and C S - .
Procedure
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PROT
Gastric-Only
CHO
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Experiment 2A. The experimental procedure was similar to that of Experiment 1A except for the following differences. The rats were food restricted to 14 g of powdered chow throughout the experiment. They were divided into two subgroups ( n = 12 each) that were trained with the CS Pr°t first and CS cH° second or in the reverse order. Each training-test cycle consisted of four one-bottle training sessions followed by two two-bottle choice tests. Different C S - flavors were used in the CS r~°t and CS cH° training cycles. At the end of the last training-test cycle the rats' preference for the CS Pr°t vs. CS cH° was assessed over four 30 m i n / d a y sessions. Experiment 2B. The rats were divided into two groups matched for their CS P'°t vs. CS cH° preference and previous experience
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FIG. 3. Experiment lC. Mean + SEM intake of CS Pr°t and CS cH° during two-bottle preference tests; each bar represents the mean of two 30 rain/day sessions. Percentages atop bars indicate the percent intake of that solution. The CS Pr°t and CS cH° were paired with 1G infusions of calcium caseinate and Polycose, respectively. In the oral + gastric preload tests (top) the rats (n = 13) drank 7.5 g of the CS Pr°t or CS ca° paired with IG infusions of 7.5 g protein (PROT) and carbohydrate (CHO), respectively, 45 min before the two-bottle preference tests. In the gastriconly preload test (bottom) lhe rats were infused IG with 7.5 g protein or carbohydrate 45 min before the two-bottle preference tests.
10
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the single IG preloads in Experiment 1C but not with divided preloads in Experimen'L 1B is not clear. In addition to the difference in how the preloads were delivered, in Experiment IC the rats were tested wit~a oral + gastric preloads before the gastric-only test. Conceivably, the oral + gastric preloads may have sensitized the rats to re,;pond to postingestive cues produced by IG nutrient infusions. This possibility was examined in the present experiment using new rats. The animals were tested with oral + gastric and gastric-only preloads as in Experiment IC but with the order of the preloads counterbalanced. METHOD
Subjects The subjects were 24 rats of the same type and source as those used in Experiment 1. The rats were implanted with gastric cannulas and tested in infusion cages as described in Experiment
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Preloads
FIG. 4. Experiment 1C. Mean + SEM intake of CS ~°t and CS cH° during two-bottle preference tests; each bar represents the mean of two 30 min/day sessions. Pe~entages at~,obars indicate the percent intake of that solution. The CS and CS were paired with IG infusions of calcium caseinate and Polycose, respectively. In the oral-only preload tests the rats (n = 13) drank 7.5 g of the C S Pr°t or CS cn° without concurrent infusions 45 rain before the two-bottle preference tests.
472
Pt~REZ, ACKROFF AND SCLAFANI
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0.01) CS cH° than CS p~°t after the protein preload, and tended to drink more CS P~°t than CS cH° after the carbohydrate preload [nutrient × CS interaction, F(1, 16) = 58.8, p < 0.001]. Also, the rats consumed more ( p < 0.01) CS cH° after the protein preload than after the carbohydrate preload, and more ( p < 0.01) CS P'°t after the carbohydrate preload than after the protein preload. Compared to the no-preload condition, the oral + gastric protein preload altered the rats' C S + preference [nutrient preload X CS interaction, F(1, 16)= 7.96, p < 0.05]. The rats consumed less CS Pr°t ( p < 0.05) and slightly more CS cH° after the protein preload compared to the no-preload condition. The carbohydrate oral + gastric preload produced only a marginal change ( p < 0.07) in C S + preference compared to the no-preload condition. In the gastric-only preload condition the rats did not respond differentially to the two nutrient preloads. They consumed more CS cH° than CS Pr°t after both the protein and carbohydrate preloads [CS main effect, F(1, 16)= 7.23, p < 0.02], although the difference tended to be greater after the protein preload.
Two-Bottle Tests v///.4
T w o - B o t t l e Tests
FIG. 5. Experiment 2A. Mean + SEM intake of CS ~°t, CS cH°, and CSsolutions during two-bottle preference tests of the second training-test cycle with each nutrient; each bar represents the mean of two 30 rain/day sessions. Percentages atop bars indicate the percent intake of that solution. The CS ~°t, CSc"°, and CS- were paired with IG infusions of calcium caseinate, Polycose, and water, respectively.
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(order of nutrient conditioning). One group (n = 8) was first tested with the oral + gastric preloads and then gastric-only preloads; the other group (n = 9) was tested in the reverse order. Within each subgroup, half of the rats received the protein preload first and the carbohydrate preload second; the other half received the nutrient preloads in the reverse order. The preloads were administered as in Experiment 1C.
CS P'"
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6 4 2 0
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RESULTS t'-
Experiment 2A The rats consumed comparable amounts of the CS ~°t, CS cH°, and C S - during the second one-bottle training cycle with each nutrient (10.4, 10.7, 10.9 g / 3 0 min, respectively). As illustrated in Fig. 5, in the choice tests they preferred both the CS P~°t and CS cry° to the C S - , F(1, 18)= 98.5, p < 0.001. The CS cH° preference (85%) was stronger than the CS r~°t preference (70%) [nutrient × CS interaction, F(1, 17) = 5.02, p < 0.04]. In the subsequent choice tests with the two nutrient-paired flavors, the rats did not differ in their intakes of the C S + solutions; they consumed 47% of their intake as CS pr°t and 53% as CS cH° (Fig.
5). Experiment 2B Preliminary analysis revealed that the order of preload testing was not a significant factor and therefore the data from the two groups were combined. The type of preload (oral + gastric or gastric-only) did, however, affect the rats' responses to the nutrient preloads [preload type X preioad nutrient X CS interaction, F(1, 16) = 5.69, p < 0.05]. As illustrated in Fig. 6, in the oral + gastric preload condition the rats consumed more ( p <
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6
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FIG. 6. Experiment 2B. Mean + SEM intake of CS ~°t and CS cH° during two-bottle preference tests; each bar represents the mean of two 30 min/day sessions. Percentages atop bars indicate the percent intake of that solution. The CS Prot and CS CHO were paired with IG infusions of calcium caseinate and Polycose, respectively. In the oral + gastric preload tests (top) the rats ( n = 19) drank 7.5 g of the CS ~°t or CS cH° paired with IG infusions of 7.5 g protein (PROT) and carbohydrate (CHO), respectively, 45 min before the two-bottle preference tests. In the gastriconly preload test (bottom) the rats were infused intragastrically with 7.5 g protein or carbohydrate 45 rain before the two-bottle preference tests.
CARBOHYDRATE AND PROTEIN PREFERENCES
Compared to the no-preload condition, the gastric protein preload tended to reduce CS r'~°' intake and increase CS cH° intake but the nutrient preload × CS interaction was not reliable ( p < 0.07). The carbohydrate gastric preload did not alter CS+ intakes compared to the no-preload baseline. GENERAL DISCUSSION In confirmation of prior studies (2,3,14,15), the rats in this study acquired strong preferences for flavors paired with IG infusions of protein and carbohydrate. The new finding here is that the protein- and carbohydrate-conditioned preferences were obtained in the same a~imals. Furthermore, the preferences for the two nutrient-paired flavors were equivalent as measured by CS r'~°t vs. csCH°choice tests. The rats also displayed comparable preferences for the CS r'~°t and CS cH° flavors relative to the C S - flavor in Experiment 1, although in the second experiment their intake of the CS r~,t was somewhat less than their CS cH° intake relative to the C S - . This may have resulted because the rats in the second experiment received less training and had two different CS flavors to learn; perhaps protein-conditioned preferences are acquired more slowly than carbohydrate-conditioned preferences. Nevertheles:~, when tested for their CS P~°t vs. CS cH° preference, the rats in Experiment 2, like those in the first experiment, did not prefer one nutrient-paired flavor over the other. We recently reported that rats acquire comparable preferences for flavors paired with the consumption of iso~:aloric solutions of casein and Polycose solutions using an oral-delay conditioning paradigm (11). The present findings confirm these results and demonstrate that the nutrients have equivalent postingestive reinforcing effects. In both studies the casein and Polycose provided the same caloric benefit ~:o the animals (i.e., the nutrient solutions were isocaloric and the amounts consumed/infused were the same). The similar flavcr preferences conditioned by casein and Polycose may have resulted because, as proposed by some investigators (9,17,18), the unconditioned stimulus (US) mediating reinforcement is related to the nutrients' energy value. In particular, Tordoff (17) proposed that the US may be a signal generated by hepatic fuel oxidatiorL. However, if animals attend only to the energy value of foods, this would not facilitate their selection of a nutritionally balanced diet. The present study investigated, therefore, whether rats could distinguish between the postingestive actions of carbohydrate and protein by measuring their flavor preferences after nutrient: preloads. In Experiment IB, giving the rats divided protein and carbohydrate preloads over a 2-h period failed to alter their selection of the CS r'r°t and CS cH° in the subsequent choice test. This protocol was based on the repot: of Baker et al. (3) that an IG protein preload, but not a carbohydrate preload, suppressed the rats' preference for a protein-paired flavor. The current study and the Baker et al. (3) experinaent differed in several respects, which may account for the discrepant results. In particular, Baker et al. (3) used minimally (4 h) deprived rats, trained and tested them with CS r'~°t and C S - flavors only, and did not infuse them during the CS er°t vs. CS choice test. Given the negative results of Experiment 1B, we next investigated the effects of oral + gastric preloads on the preference for the nutrient-paired flavcrs (Experiment IC). This manipulation provided the animals with both orosensory and viscerosensory information about the preload and was analogous to the orally consumed "premeals" used in earlier food selection studies (8,20). When prefed one flavor with its paired IG nutrient
473
infusion, the rats showed a shift in preference to the alternate nutrient-paired flavor in the CS P~°t vs. CS cn° choice test. The rats showed a similar preference shift when next tested with IG-only preloads in Experiment 1C. However, when these manipulations were repeated with new rats in Experiment 2B, the oral + IG preloads again produced nutrient-specific preference shifts, but now the IG-only preloads did not. Taken together, the results of Experiments 1B, 1C, and 2B indicate that IG-only preloads do not consistently alter the rats' acquired preferences for flavors paired with protein and carbohydrate. The reason for this inconsistency is not certain. Mixed results have also been reported in studies that assessed the effects of IG preloads on macronutrient self-selection. Bartness and Rowland (4) observed that a carbohydrate preload reduced subsequent carbohydrate but not protein intake, whereas a protein preload suppressed both protein and carbohydrate intake. Also, both preloads suppressed fat intake. Zeggeren and Li (19) reported that IG carbohydrate preloads selectively reduced the intake of a high-carbohydrate food more than a high-protein diet, but they did not measure the effects of a protein preload. Mehiel and Bolles (9), on the other hand, reported that an IG carbohydrate preload did not selectively reduced the intake of two flavors that were previously paired with carbohydrate and ethanol consumption. In contrast to the IG-only preloads, the oral + gastric preloads produced very similar preference shifts in Experiments 1C and 2B. This was not due solely to the oral cues provided by the preload. The oral-only preload in Experiment 1C did not cause the animals to shift their preference from the preload flavor to the alternate flavor. (The failure of oral-only preloads to produce a preference shift was confirmed in additional tests conducted with the rats from Experiment 2.) These data indicate that the combined actions of orosensory and viscerosensory cues are more effective than either type of cue alone in modifying the rat's subsequent food choices. This is perhaps not surprising because the animal has more information to guide its selection, and flavor and postingestive consequences of foods are normally closely linked under natural feeding conditions. An important aspect of the present results is that both the protein and carbohydrate preloads (oral + gastric) shifted the rats' preference to the opposite nutrient. The protein preload, however, was more effective than the carbohydrate preload in altering subsequent preference. That is, following the CS Pr°t + IG protein the rats displayed a 74-79% preference for the CS cH°, whereas following the CS cH° preload the rats showed only a 61-63% CS r~°t preference in Experiments 1 and 2. This could reflect a general asymmetry between the effects of the two types of nutrient preloads, which may be related to a tighter regulation of protein than carbohydrate intake. Alternatively, the apparent enhanced effectiveness of the protein preloads may be due to the particular test conditions of the present study. Note that the rats were fed restricted amounts of a relatively high-protein food (lab chow), and thus their need for nonprotein energy may have been greater than their need for protein. The influence of protein and carbohydrate preloads on nutrient selection should be studied under varied conditions. Little is known about the viscerosensory stimuli generated by nutrients. The present results do not refute the hypothesis that different nutrients produce a common energy-related signal that reinforces flavor preferences. They do indicate, however, that nutrient-specific signals are generated by the pre- and/or postabsorptive actions of nutrients. In flavor-nutrient conditioning, therefore, animals acquire knowledge about the specific nutritive value of foods.
474
PI~REZ, A C K R O F F A N D S C L A F A N I
ACKNOWLEDGEMENTS This research was supported by a grant from the National Institute of Diabetes and Digestive and Kidney Diseases (DK-31135) and a NIMH
Research Scientist Award (MH-00983) to Anthony Sclafani. Polycose and Kool-Aid were generously donated by Ross Laboratories and General Foods, respectively.
REFERENCES 1. Ackroff, K.; Sclafani, A. Flavor preferences conditioned by sugars: Rats learn to prefer glucose over fructose. Physiol. Behav. 50:815824; 1991. 2. Baker, B. J.; Booth, D. A. Preference conditioning by concurrent diets with delayed proportional reinforcement. Physiol. Behav. 46:585-590; 1989. 3. Baker, B. J.; Booth, D. A.; Duggan, J. P.; Gibson, E. L. Protein appetite demonstrated: Learned specificity of protein-cue preference to protein need in adult rats. Nutr. Res. 7:481-487; 1987. 4. Barmess, T. J.; Rowland, N. Dietary self-selection in normal and diabetic rats after gastric loads of pure macronutrients. Physiol. Behav. 31:546-554; 1983. 5. Booth, D. A. Food-conditioned eating preferences and aversions with interoceptive elements: Conditioned appetites and satieties. Ann. NY Acad. Sci. 443:22-41; 1985. 6. Capaldi, E. D.; Campbell, D. H.; Sheffer, J. D.; Bradford, J. P. Conditioned flavor preferences based on delayed caloric consequences. J. Exp. Psychol. [Anim. Behav. Proc.] 13:150-155; 1987. 7. Elizalde, G.; Sclafani, A. Flavor preferences conditioned by intragastric Polycose: A detailed analysis using an electronic esophagus preparation. Physiol. Behav. 47:63-77; 1990. 8. Li, E. T. S.; Anderson, G. H. Meal composition influences subsequent food selection in the young rat. Physiol. Behav. 29:779-783; 1982. 9. Mehiel, R.; Bolles, R. C. Learned flavor preferences based on caloric outcome. Anita. Learn. Behav. 12:421-427; 1984. 10. Mehiel, R.; Bolles, R. C. Learned flavor preferences based on calories are independent of initial hedonic value. Anim. Learn. Behav. 16:383-387; 1988. 11. P6rez, C.; Lucas, F.; Sclafani, A. Carbohydrate, fat and protein condition similar flavor preferences in rats using an oral-delay conditioning procedure. Physiol. Behav. 57:549-554; 1995.
12. Sclafani, A. Nutritionally based learned flavor preferences in rats. In: Capaldi, E. D.; Powley, T. L., eds. Taste, experience, and feeding. Washington, DC: American Psychological Association; 1990:139156. 13. Sclafani, A.; Ackroff, K. Glucose- and fructose-conditioned flavor preferences in rats: Taste vs. postingestive conditioning. Physiol. Behav. 56:399-405; 1994. 14. Sclafani, A.; Cardieri, C.; Tucker, K.; Blusk, D.; Ackroff, K. Intragastric glucose but not fructose conditions robust flavor preferences in rats. Am. J. Physiol. 265:R320-R325; t993. 15. Sclafani, A.; Nissenbaum, J. W. Robust conditioned flavor preference produced by intragastric starch infusions in rats. Am. J. Physiol. 255:R672-R675; 1988. 16. Sherman, J. E.; Hickis, C. F.; Rice, A. G.; Rusiniak, K. W.; Garcia, J. Preferences and aversions for stimuli paired with ethanol in hungry rats. Anim. Learn. Bebav. 1 l:101-106; 1983. 17. Tordoff, M. G. Metabolic basis of learned food preferences. In: Friedman, M. 1.; Tordoff, M. G.; Kare, M. R., eds. Appetite and nutrition. New York: Marcel Dekker; 1991:239-260. 18. Tordoff, M. G.; Tepper, B. J.; Friedman, M. I. Food flavor preferences produced by drinking glucose and oil in normal and diabetic rats: Evidence for conditioning based on fuel oxidation. Physiol. Behav. 41:481-487; 1987. 19. Van Zeggeren, A.; Li, E. T. S. Food intake and choice in lean and obese Zucker rats after intragastric carbohydrate preloads. J. Nutr. 120:309-316; 1990. 20. Wurtman, J. J.; Moses, P. L.; Wurtman, R. J. Prior carbohydrate consumption affects the amount of carbohydrate that rats choose to eat. J. Nutr. 113:70-78; 1983.