- Email: [email protected]
Learned preferences for the flavor of salted food
Physiology&Behavior.Vol. 54, pp. 999-1004, 1993
0031-9384/93 $6.00 + .00 Copyright© 1993PergamonPressLtd.
Printedin the USA.
Leamed Preferences for the Flavor of Salted Food S U S A N E. C O L D W E L L .1 A N D M I C H A E L G. T O R D O F F i "
*Department of Psychology, University of Pennsylvania, Philadelphia, PA 19104-6196 and fMonell Chemical Senses Center, 3500 Market Street, Philadelphia, PA 19104-3308 Received 15 F e b r u a r y 1993 COLDWELL, S. E. AND M. G. TORDOFF. Learnedpreferencesfor theflavor of saltedfood. PHYSIOL BEHAV 54(5) 9991004, 1993.--We examined whether rats can associate the flavor of their food with its salt content, and whether this association is influenced by sodium status during training and testing. During two pairs of 2-h training trials, rats ate flavored food containing 1.75% NaCI or an alternatively flavored unsalted food. The motivational state of the rats was manipulated prior to each trial by combined 48-h dietary sodium deprivation and furosemidetreatment (severe sodium depletion), 48-h dietary sodium deprivation (mild sodium deprivation), or continued maintenance on stock diet containing 1% NaC1 (sodium replete). When later given a choice between the two flavors, all rats preferred food containing the salt-paired flavor. The strength of this preference was unaffected by motivational state during training or by the salt content of the test foods, but was modulated by the motivational state of the rats during the preference test. Preference for the salt-paired flavor was strongest when rats were tested after severe sodium depletion,lessstrong after mild sodium deprivation,and absent when sodium replete. These resultsindicatethat deprivation state during training has little effect on learned preferences for the flavor of salted food but deprivation state during testing affects the expression of this learning. Salt intake
Latent learning
Deprivation state
Food preference
ANIMALS can readily learn about the availability of NaC1 in the absence of a need for sodium (3,12,18,22). The clearest evidence for this comes from studies demonstrating that sodiumreplete rats given flavored salty water show a preference for the flavor when later made sodium deficient (3,12,22). Such studies make a compelling case that salt-based flavor preferences can be acquired, and that the expression of these preferences is dependent on sodium status. However, to our knowledge, there has been no attempt to examine whether sodium need influences the acquisition of this learning. Given that sodium deficiency increases the motivation to search for (26) and ingest (5,9,15,24,28-31 ) salt, an obvious hypothesis is that preferences acquired by sodium-deficient rats would be stronger than those acquired by sodium-replete rats. On the other hand, findings that rats without an apparant need for salt develop preferences for sodium-paired flavors (3,12,22) argue that motivational state may be irrelevant for this form of learning. In the experiments described below, we investigated the importance of sodium need for the acquisition of preferences for the flavor of salty food. EXPERIMENT l: EFFECT OF SEVERE SODIUM DEPLETION OR MILD SODIUM DEPRIVATION ON THE ACQUISITION OF PREFERENCES FOR THE FLAVOR OF SALTED FOOD METHOD
the beginning of the experiment. They were maintained on a 12:12 h light:dark cycle with lights offat 1000 h. The rats were housed and trained individually in hanging wire cages measuring 19.5 X 17.5 X 24.5 cm. Maintenance diet was AIN-76A sodiumfree diet (1) fabricated by Dyets Inc. (Bethlehem, PA) to which was added 171 mmol/kg NaCl ( 1% NaCl wt./wt.). This diet was given in 8.0-cm diameter metal cups, which were attached to the inside of the cage by a metal spring. Rats always had deionized water to drink.
Manipulations of Sodium Balance Rats were trained and tested after one of two manipulations of sodium status (see Table l). Severely depleted rats were subjected to combined dietary sodium restriction and furosemide treatment. The rats' maintenance diet was replaced with a sodium-free AIN-76A diet (Na + content < 3 mmol/kg) for 48 h. After the first 24 h, each rat also received two injections of furosemide (5.0 rag, SC, in 0.5-ml 150 m M NaCl) spaced ~ 3 h apart. Rats treated in this manner lose 1.75-2.50 mmol Na ÷ and develop a vigorous appetite for NaCl (5,9,15,28-31). Mildly deprived rats were fed sodium-deficient diet for 48 h and received injections of 0.5-ml 150 m M NaCl at the time the experimental group received furosemide. Rats deprived of dietary sodium for 48 h lose minimal amounts of sodium, and restriction for much longer periods is required to produce a measurable appetite for NaCl solutions ( 1 l, 15,27).
Subjects and Maintenance
Training Procedure
Subjects were 28 male Sprague-Dawley CD rats (Charles River Laboratories, Wilmington, MA) weighing 300-400 g at
Each rat received four training trials according to the schedule described in Table 2. Training trials occurred during the first 2
Requests for reprints should be addressed to Susan E. Coldwell,Department of Psychology, 3815 Walnut Street, Philadelphia, PA 19104. 999
1000
COIDWELL
TABLE 1 SCHEMATIC OF PROCEDURES FOR MANIPULATIN(; SODIUM STATUS Day
Food During First 2 h of Dark
Food During Following 22 h
Na-ffee Na-free Training/Test Na-free Na-ffee Na-free
Na-free Na-free Na-free l% NaCI 1% NaCI 1% NaCI
Injection
Test Procedure
Furosemide
Mild Sodium Deprivation 1 2 3 4 5 6
Na-free Na-free Training/Test Na-ffee Na-ffee Na-free
Na-free Na-free Na-free 1% NaCI 1% NaC1 1% NaC1
Saline*
Na-free Na-free Training/Test Na-free Na-free Na-free
1% NaCI 1% NaCI 1% NaCI 1% NaCI 1% NaCI 1% NaCI
Following training, the rats were given four preference tests. Tests occurred in the first 2 h of the dark period. Each was a two-cup preference test between diets containing the flavors used in training. In the first two tests, both flavors were given in sodium-free diet. Half the rats in each group were tested in each sodium state on each test according to a counterbalanced design. T h e third a n d fourth tests were conducted in the same way as the first two except the choice was between flavors mixed in diet containing 1.75% NaCI. Intake of each diet was measured at 30, 60, a n d 120 rain. Because the pattern of results was the same for readings at all three time periods, only the results from the 120-min reading are presented here.
Statistical ,4nalyses
Sodium Replete 1 2 3 4 5 6
I()RDOFI
between subjects, a n d the order of flavor presentation was det e r m i n e d by a c o u n t e r b a l a n c e d ABBA design. During the first 2 h of the dark cycle on n o n t r a i n i n g days, rats were given 5 g of unflavored, sodium-free diet in the same m a n n e r (see Tables I and 23.
Severe Sodium Depletion I 2 3 4 5 6
ANI)
None
* No injections were administered in Experiment 2.
h of the dark period on the third day of each 6-day training cycle (see Table 1). Just before the lights went out, the rats' m a i n t e n a n c e diet was removed and each rat received a fixed ration of 5 g of flavored diet in a 5.5 X 5.5 X 3.0 cm square metal cup. T h e flavors were 0.1% (wt./wt.) imitation a l m o n d or imitation vanilla extract ( M c C o r m i c k brand). O n two of the cycles, the flavored diet contained 1.75% NaC1. O n the other two cycles, the flavored diet was sodium free. One flavor was always presented with the NaC1 diet a n d the other with the sodium-free diet. The flavor paired with NaC1 was counterbalanced
Preliminary analyses showed that there were no differential effects of the treatments on intake of a l m o n d or vanilla flavor and so results obtained with the two flavors were collapsed into salt-paired a n d unpaired categories. Subsequent analyses were performed using analyses of variance (ANOVAs). Training data in each experiment were analyzed by A N O V A s with a betweensubject factor o f training deprivation state (i.e., group) a n d within-subject factor of diet (unsalted vs. salted). W h e n appropriate, the significance of differences between individual pairs of means was d e t e r m i n e d using Tukey's H S D tests. Data collected during the tests were analyzed by A N O V A with a betweensubject factor of training deprivation state (mild vs. severe) a n d within-subject factors of test deprivation state (mild vs. severe) and flavor (unpaired vs. salt-paired). Separate analyses were performed for tests c o n d u c t e d with unsalted a n d salted vehicle. A probability value o f p < 0.05 was considered significant, Values reported in the text are m e a n s _+ standard errors. RESULTS Three rats were omitted from analyses: one rat in the severe depletion group failed to eat the flavored diet on any of the training trials, a n d two rats in the severe depletion group spilled food during a preference test.
TABLE 2 ORDER OF TRAINING AND TEST TRIALS IN EXPERIMENT l Deprivation State Before Trial Cycle
Severely Deplete Group
Train 1 Train 2 Train 3 Train 4 Test 1 Test 2 Test 3 Test 4
Severe Severe Severe Severe Mild or severe Severe or mild Mild or severe Severe or mild
Mildly Deprived Group Mild Mild Mild Mild Mild or severe Severe or mild Mild or severe Severe or mild
Food Flavor and NaC1 Content During Trial Either FI in Na-free diet or F2 in 1.75% NaCI diet Either FI in 1.75% NaC1 diet or F2 in Na-free diet Either FI in 1.75% NaCI diet or F2 in Na-free diet Either FI in Na-free diet or F2 in 1.75% NaC1 diet FI vs. F2 both in Na-free diet FI vs. F2 both in Na-free diet F1 vs. F2 both in 1.75% NaCI diet FI vs. F2 both in 1.75% NaCI diet
FI and F2 = almond or vanilla flavors. Their association with sodium-free or 1.75% NaCI diet was counterbalanced. F1 vs. F2: both flavors presented together. Treatments prior to the first pair and second pair of tests were counterbalanced.
FLAVOR PREFERENCE LEARNING
1001
Training
Deprivation State Before Each Training Trial
Table 3 shows average food intakes during the 2-h training trials. The lowest salted food intake by any rat was 1.70 g, and many rats ate all 5 g available to them. Intake of the two diets differed in training, F(1, 23) = 48.7, p < 0.01. There was also a significant interaction between group and diet, F(I, 23) = 54.0, p < 0.01. Post hoc analyses revealed that although the two groups ate similar amounts of salted food, the severely deplete group ate significantly less of the unsalted diet than did the mildly deprived group, sometimes only sampling the diet (i.e., eating 0.1-0.2 g). Intake of the salted and unsalted diets did not differ for the mildly deprived group; however, the severely deprived group ate significantly less unsalted than salted diet.
Preference Tests With Flavors Presented in Unsalted Food Regardless of whether the rats were tested under mild sodium deprivation or severe sodium depletion, they ate more of the previously salted flavor than the flavor previously given alone, F(1, 23) = 19.8, p < 0.01 (Fig. 1, top panels). Intake of the saltpaired flavor tended to be greater when rats were tested under mild deprivation than severe depletion, although this was not significant, F(I, 23) = 3.05, p = 0.09. This was partly because rats tested while severely sodium deplete ate less of both flavored diets than did rats tested while mildly deprived, F( 1, 23) = 11.8, p < 0.01. The only differential effect of sodium status during training was that rats trained while severely deplete ate less overall (i.e., less of both flavors combined) during the tests than did rats trained while mildly deprived, F(1, 23) = 7.01, p < 0.02. The magnitude of the preference was unaffected by deprivation state during training, F(1, 23) = 0.23, p > 0.64. One concern was that furosemide administered prior to tests might produce permanent changes in the animal's motivation for salt (25). Because of the counterbalanced design, this could influence preferences for those rats that were first tested after furosemide treatment and later tested following dietary restriction alone. We wanted to determine if this explanation might account for the preference for the salt-paired flavor seen in rats trained and tested under mild deprivation. We thus compared preferences for the salt-paired flavor in the mild deprivation test in rats that were tested first while mildly deprived to those that were tested first while severely depleted. Order of testing had no effect on this preference, rendering this explanation unlikely, F(I, 12)= 1.78, p = 0.21.
Preference Tests With Flavors Presented in Salted Food The results of tests with flavors presented in salted food were similar to the results with the unsalted food vehicle. Rats tested
TABLE 3 INTAKE OF FLAVORED FOOD DURING 2-h TRAINING TRIALS Condition During Training
Experiment 1 Mild deprivation (n = 14) Severe depletion (n = 11) Experiment 2 Replete (n = 11) Mild deprivation (n = 12)
Unsalted Food
Salted Food
4.01 + 0.17 1.30 + 0.42*
3.93 __+0.18 4.36 _ 0.231-
4.07 -4-0.28 3.46 _+0.28
3.29 _+0.28t 3.66 + 0.26
Values are mean _.+SE of the average of two trials with each food. * p < 0.05 relative to mild deprivation group. t P < 0.05 relative to intake of unsalted food.
Mild
Severe J Flavors in Unsalted Food
I
,
I~1
m
~
Unpaired Paired
~" 3 0
2° e-
"o oo
LL
Flavors in Salted Food 6
,ij dlj Mild
Severe
Mild
Severe
Deprivation State Before Test
FIG. 1. Intake of flavored diet during 2-h preference tests by two groups of rats (n = I 1 or 14). Light bars indicate intake of diet containing the flavor paired with NaC1 during training. Dark bars indicate intake of diet containing the flavorunpaired with NaCI during training. Rats were tested with both flavorsin food containing 0% NaC1(top) or 1.75%NaCl (bottom). Left panels show data for rats trained while deprived of NaC1 for 48 h (mild deprivation). Right panels show data for rats trained while deprived of NaCI for 48 h and also injected with furosemide (severe depletion). The left set of bars in each panel indicates intake when rats were tested after mild deprivation. The right set of bars indicates intake when rats were tested followingsevere depletion.
under either motivational state ate more of the salt-paired flavor than unpaired flavor, F(1, 23) = 24.0, p < 0.01 (Fig. 1, bottom panels). Intake of the salt-paired flavor was greater after severe depletion than mild deprivation, F(1, 23) = 6.94, p < 0.02. There were no significant differences in overall intake during tests following severe sodium depletion and tests following mild sodium deprivation, F(1, 23) = 3.80, p > 0.06. Sodium status during training had no influence on the results. Unlike the tests using unsalted diet, with salted diet there were no significant differences in overall food intake between rats that were trained under mild sodium deprivation and rats that were trained under severe sodium depletion, F(1, 23) = 1.32, p > 0.26. The magnitude of the preference was unaffected by deprivation state during training, F(I, 23) = 0.35, p = 0.56. EXPERIMENT 2: EFFECT OF MILD SODIUM DEPRIVATION COMPARED WITH REMAINING SODIUM REPLETE ON THE ACQUISITION OF PREFERENCES FOR THE FLAVOR OF SALTED FOOD The main finding of Experiment 1 was that rats demonstrated similar preferences for flavors paired with salt whether they were trained under mild deprivation or severe depletion. Although we anticipated that rats tested after severe sodium depletion would show this response, we did not anticipate finding strong preferences for the salt-paired flavor in rats trained and tested
('OI.DWEI.L A N D I O R D O F t ~
1002
under mild sodium deprivation. This procedure produces negligible sodium loss and has been shown not to affect intake of concentrated NaC1 solution or salted diet (5,11,15,27). To clarify whether the effect of 48-h sodium deprivation was replicable, in Experiment 2 we compared rats trained while mildly sodium deprived with a group maintained replete. The animals were tested while sodium replete, mildly deprived, and then severely depleted. We preceded training with two pretests: one was to confirm that the rats used here preferred unsalted to salted food [see (5)], and the other assessed pretraining flavor preferences in each subject. METHOD The design of Experiment 2 is shown in Table 4. Twentyfour naive rats of similar body weight to those used in Experiment 1 were adapted to the maintenance diet for 5 days and then received a 2-h preference test between unsalted and salted food. The next day, they received a 2-h choice test between almond and vanilla flavors presented in unsalted food. Both tests were conducted during the first 2 h of the dark cycle. Using the procedures outlined for Experiment 1, the rats then received two pairs of training trials, except that one group was subjected to mild sodium deprivation before each training trial, and the other group remained sodium replete through continued maintenance on the 1% NaCI maintenance diet. Following training, three 2-h preference tests were conducted, using unsalted food as the flavor vehicle. In the first test, rats were tested in the state in which they were trained (i.e., rats trained while mildly deprived were tested while mildly deprived). In the second test, the rats were tested in the opposite state (i.e., rats trained while mildly deprived were tested while replete). In the third test, all rats were tested after severe sodium depletion. The order of these tests was not counterbalanced to prevent possible long-term effects caused by sodium depletion (5,25).
Statistical Ana@ses Training data were analyzed in the same manner as in Experiment 1. Pretests in Experiment 2 were analyzed separately with ANOVAs. In the first pretest, a within-subject factor of diet type was used. In the second pretest, a between-subject factor of group and a within-subject factor of diet were used. The data collected during the three preference tests were analyzed by separate ANOVAs according to testing deprivation state. To control for potential differences in preexisting preferences for the two flavors, each A N O V A involved a comparison between pretraining vs. posttraining flavor intakes. The three-way interactions between test (pretest or test), group (mild deprivation or replete), and flavor (paired or unpaired) were assessed using planned comparisons. RESULTS
One rat in the replete training condition ate less than 1 G of flavored food on each trial and so was omitted from all analyses Pretesls The rats showed a strong preference for unsalted food over salted food [F(I, 23) = 37.8, p < 0.01; 2.66 ___0.32 g vs. 0.54 + 0.09 g, respectively]. There were no significant flavor preferences prior to training, F(I, 21) = 2.10, p > 0.16 (Fig. 2, top panel) and no significant differences in intake of each flavor between the two groups, F(1, 21) = 1.10, p > 0.30.
Training Training intakes are summarized in Table 3. There was no overall difference in intake of the salted and unsalted flavored
TABLE 4 ORDER OF PRETEST, FRAINING, AND TESt IN EXPERIMENT 2
I RIAtS
Deprivation State Before Thai
Cycle
Mildl~ Deprived Group
Replete Group
Food Flavor and NaCI Content During Training
Pretest 1 Pretest 2 Train 1
Replete Replete Mild
Replete Replete Replete
Train 2
Mild
Replete
Train 3
Mild
Replete
Train 4
Mild
Replete
Test 1 Test 2
Mild Replete Severe
Replete Mild Severe
Na-free vs. 1.75% NaC1 diet FI vs. F2 both in Na-free diet Either FI in Na-free diet or F2 in 1.75% NaCI diet Either FI in 1.75c7~NaC1 diet or F2 in Na-free diet Either FI in 1.75% NaC1 diet or F2 in Na-free diet Either FI in Na-free diet or F2 in 1.75% NaCI diet El vs. F2 both in Na-free diet FI vs. F2 both in Na-free diet FI vs. F2 both in Na-free diet
Test 3
F I and F2 = almond or vanilla flavors. Their association with sodiumfree or 1.75% NaCI diet was counterbalanced.
diets, F(1, 21) = 0.91, p > 0.35. There was a significant interaction between group and diet (F = 5.58, p < 0.03). Post hoc analyses revealed that the two groups did not differ in intake of either salted or unsalted flavored diet. However, rats trained replete ate significantly more unsalted food than salted food, while rats trained mildly deprived ate similar amounts of both diets.
Preference Tests Relative to intake during the pretest, both groups of rats increased intake of the salt-paired flavor during mild deprivation or severe depletion but not when replete [F( 1, 21 ) = 5.90, p < 0,03; F(1, 21) = 23.4, p < 0.01; F(I, 21) = 0.51, p = 0.48, respectively] (see Fig. 2). Intake of the unpaired flavor was unaffected by deprivation state. Intake of salt-paired food increased only slightly in the mild deprivation test, and for both this and the replete test intake of the salt-paired and unpaired flavors did not differ [mild deprivation test, F( 1, 21 ) = 1.80, p = 0.19; replete test F( 1, 21) = 0,00, p = 0.97]. However, rats showed significantly greater intake of the salt-paired than unpaired flavors during severe sodium depletion, F(1, 21) = 34.8, p < 0.01. On none of the tests was there a significant difference in the response of rats trained while mildly deprived or replete, There were nonsignificant tendencies in all three tests for rats trained while mildly deprived to eat more salt-paired food than those trained while replete [replete test, F(1, 21) = 2.37, p = 0.14: mild deprivation test, F(I, 21) = 1.97, p = 0.18; deplete test, F(I, 21) = 1.34, p = 0.26]. Because this trend appeared even in the replete test, it probably reflects a greater motivation for sodium in these rats due to repeated deprivation experiences (25), rather than superior salt-flavor learning. Attesting to the rats' larger body mass and greater familiarity with the test situation, total food intake during the three posttraining preference tests was higher than during the pretest [replete, F( 1, 21) = 49.4, p < 0.01; mild deprivation, F( 1, 21) = 36.2, p < 0.01, severe depletion, F(1, 21) = 23.4, p < 0.01].
FLAVOR PREFERENCE LEARNING
Pretest
1003
Unpaired Paired
Replete 1
~" ¢M
2
0 e"o o
o u.
~prlvatl 4
)eprivation Test
2
Replete
Mild
Deprivation State Before Each Training Trial
FIG. 2. Intake of flavored diet containing 0% NaCI during 2-h tests by
two groups of rats (n = I I or 12/group). Light bars indicate intake of diet containing the flavor paired with NaCl during training. Dark bars indicate intake of diet containing the flavor unpaired with NaCl during training. Top panel: test that preceded any experimental manipulations of sodium balance. Second panel: test that followed at least 3 days' maintenance on 1%NaCl diet (replete test). Third panel: test that followed 48 h of sodium deprivation (mild deprivation test). Bottom panel: test that followed 48 h of sodium deprivation and injection of furosemide (severe deprivation test).
DISCUSSION Rats exposed to a flavored, salted food acquired a preference for the flavor. The preference was exhibited by rats trained while severely sodium depleted, mildly sodium deprived or sodium replete, but only if they were tested while mildly sodium deprived or severely sodium depleted, not while sodium replete. These findings suggest that sodium status has little or no influence on the acquisition of salt-flavor preferences but has a critical influence on the expression of these preferences. The finding that deprivation state during training had little impact on learning about a flavor associated with salt was surprising given data suggesting that NaCl is treated differently by sodium-deprived and sodium-replete rats [e.g., (8,14)]. It implies that a mechanism independent of drive state is responsible for flavor-salt learning, and thus contradicts predictions made by drive-reduction theories of learning [e.g., (13,17,19)]. One ex-
planation for state-independent learning might involve the rat's innate recognition of saltiness (20). However, the literature concerning similar manipulations of other drive systems is complex and does not necessarily distinguish learned from innately recognized ingesta. Motivational state during training influences subsequent preferences for flavors associated with water by thirsty rats (23) and with sucrose or saccharin by hungry rats (6), but not with ethanol by hungry rats (10). Motivational state during testing influences the magnitude of preferences exhibited by rats trained to associate a flavor with water (23), sucrose (6), or ethanol (10), but not with saccharin (6,10). It seems unlikely that a unitary explanation can encompass these findings. The results found here are consistant with, and address several deficiencies in, previous studies of flavor-NaC1 learning (3,12,22). Two previous studies involved training sodium-replete rats to associate flavors with a low-concentration NaCl solution [(12) Exp. l; (22)]. This procedure complicates interpretation because the acquired preferences could be due to the association of the flavor with NaCl specifically or, more generally, with a preferred taste. By using food as the vehicle for the salt we avoided this problem, because sodium-replete rats never prefer salted to unsalted food [(2,4,5,7), pretest of Experiment 2]. Another difference from previous studies of flavor-NaC1 learning was that we tested for preferences of flavors embedded in both unsalted and salted media. The main reason for this was that tests conducted in unsalted media compare a somewhat novel diet (the salt-paired flavor now presented in a Na+-deficient diet) with a familiar diet (the unpaired flavor presented in the Na +deficient diet). Since the latter but not former was eaten during training, there is a possibility that intake of the salt-paired flavor might be inhibited by neophobia or a failure to generalize from salted to unsalted flavors. Similarly, Beauchamp and colleagues have stressed that context has a strong influence on salt preference (2), and such effects could potentially influence preferences for flavors paired with salt. Our finding that the general pattern of results was the same whether tests were conducted with flavors embedded in unsalted or salted vehicle suggests that such factors played little, if any, role in determining flavor preferences. One difference between tests conducted using salted and unsalted food was that rats with experience of severe sodium depletion tended to eat more salted food, irrespective of its flavor. This is consistent with findings that unsalted food intake is reduced during the 24 h after administration of large doses of furosemide, and that salt in food can enhance short-term food intake of furosemide-treated rats (5). The reduced unsalted food intake seen in the present experiment involved conditioned effects because rats trained while severely deplete had low unsalted food intake even during preference tests conducted in the mildly deprived state. One explanation for this is that malaise induced by furosemide or by severe sodium depletion produces a conditioned aversion to unsalted food that does not generalize to flavors presented in salted food [see (5) for discussion of malaise and salt preference]. One of the most intriguing results was that 48-h dietary sodium restriction was sufficient to expose preferences for flavors previously associated with salt (i.e., mild deprivation tests in Experiment I and 2). Such a benign procedure has little effect on plasma concentrations of aldosterone and angiotensin, and is insufficient to induce rats to increase intakes of concentrated NaC1 solutions (28). Moreover, in studies conducted under almost identical conditions to the present experiment, sodium deprivation for 2 days did not induce rats to consume salted food (0.12-8.0% NaC1) in preference to unsalted food (5). One explanation for our results is that while 48 h of deprivation is not enough to cause rats to overcome any inherent distaste they have for sodium, it is sufficient to induce a sodium drive. Since sodium content of the two choices is identical in a flavor pref-
1004
('OIDWELL
erence test, it may be sensitive enough to detect this drive, while tests between substances with and without sodium are not. ACKNOWLEDGEMENTS We would like to thank Rebecca Hughes and Michelle Ewell for their valuable technical assistance. Drs. G. Beauchamp and D. Reed provided
AND
I()RDOH:
valuable comments on earlier drafts of the manuscript. I his research was supported by the National Institute of Diabetes and Digestive and Kidney Diseases, grant DK-40099. It was first presented at the Joint Annual Meeting of the North American Association for tile Study of Obesity and the Society for the Study of Ingestive Behavior, Sacramento. CA, October 199 I.
REFERENCES 1. Ad hoc Committee on Standards for Nutritional Studies. Second report of the ad hoc committee on standards for nutritional studies. J. Nutr. 110:1726; 1980. 2. Beauchamp, G. K.; Bertino, M. Rats do not prefer salted solid food. J. Comp. Psychol. 99:240-247; 1985. 3. Berridge, K. C.; Schulkin, J. Palatability shift of a salt-associated incentive during sodium depletion. Q. J. Exp. Psychol. [B] 41:121138; 1989. 4. Bertino, M.; Beauchamp, G. K. Rats show varying salt preferences in liquid milk products. Appetite. 8:55-66; 1987. 5. Bertino, M.; Tordoff, M. G. Sodium depletion increases rats' preferences for salted food. Behav. Neurosci, 102:565-573; 1988. 6. Capaldi, E. D. Hunger and the learning of flavor preferences. In: Bolles, R. C., ed. The hedonics of taste. Hillsdale, N J: Lawrence Erlbaum Associates, lnc: 1991:127-142. 7. Collier, G.; Johnson, D. F. Consumption of salty food by rats: Regulation of sodium intake? Physiol. Behav. 52:541-546; 1992. 8. Contreras, R. J.; Frank, M. Sodium deprivation alters neural responses to gustatory stimuli. J. Gen. Physiol. 73:569-594; 1979. 9. Cruz, C. E.; Perelle,l. B.; Wolf, G. Methodologic aspects of sodium appetite: An addendum. Behav. Biol. 20:96-103; 1977. 10. Fedorchak, P. M.; Bolles, R. C. Hunger enhances the expression of calorie, but not taste mediated conditioned flavor preferences. J. Exp. Psychol. [Anim. Behav.] 13:73-79; 1987. 11. Fregley, M. J.; Harper, J. M.; Radford, E. P., Jr. Regulation of sodium chloride intake by rats. Am. J. Physiol. 209:287-292; 1965. 12. Fudim, O. K. Sensory preconditioning of flavors with a formalininduced sodium need. J. Exp. Psychol. [Anim. Behav.] 4:276-285: 1978. 13. Hull, C. L. Principles of behavior. New York: Appleton-CenturyCrofts; 1943. 14. Jacobs, K. M.; Mark, G. P.; Scott, T. R. Taste responses in the nucleus tractus solitarius of sodium-deprived rats. J. Physiol. 406: 393-410; 1988. 15. Jaloweic, J. E. Sodium appetite elicited by furosemide: Effects of differential dietary maintenance. Behav. Biol. 10:313-327: 1974. 16. Jalowiec, J. E.; Stricker, E. M. Restoration of body fluid balance following acute sodium deficiency in rats. J. Comp. Physiol. Psychol. 70:94-102: 1970.
17. Kendler, H. H. Drive interaction: 11. Experimental analysis of role of drive in learning theory. J. Exp. Psychol. 35:188-198; 1945. 18. Krieckhaus, E. E.; Wolf, G. Acquisition of sodium by rats: Interaction of innate mechanisms and latent learning. J. Comp. Physiol. Psychol. 65:197-20l: 1968. 19. Miller, N. E. Comments on multiple-process conceptions of learning. Psychol. Rev. 58:375-381; 1951. 20. Nachman, M. Taste preferences for sodium salts by adrenalectomized rats. J. Comp. Physiol. Psychol. 55:1124-1129; 1962. 21. Pfaffmann, C. Taste mechanisms in preference behavior. Am. J. Clin. Nutr. 5:142-147; 1957. 22. Rescorla, R. A.; Freberg, L. The extinction of within-compound flavor associations. Learn. Motiv. 9:411-427; 1978. 23. Revusky, S. H. Effects of thirst level during consumption of flavored water on subsequent preference. J. Comp. Physiol. Psychol. 66:777779; 1968. 24. Richter, C. P. Increased salt appetite in adrenalectomized rats. Am. J. Physiol. 115:155-161; 1936. 25. Sakai, R. R.; Fine, W. B.; Epstein, A. N.; Frankmann, S. P. Salt appetite is enhanced by one prior episode of sodium depletion in the rat. Behav. Neurosci. 101:724-731; 1987. 26. Schulkin, J. Innate, learned, and evolutionary factors in the hunger for salt. In: Friedman, M. I.; Tordoff, M. G.; Kare, M. R., eds. Chemical senses: Volume 4 appetite and nutrition. New York: Marcel Dekker, Inc.; 1991:211-237. 27. Striker, E. M.; Thiels, E.; Verbalis, J. G, Sodium appetite in rats alter prolonged dietary sodium deprivation: A sexually dimorphic phenomenon. Am. J. Physiol. 260:RI082-R1089; 1991. 28. Tordoff, M. G.; Fluharty, S. J.; Schulkin, J. Physiological consequences of NaCI ingestion by Na+-depleted rats. Am, J. Physiol. 261 :R289-R295; 1991. 29. Tordofl, M. G.; Schulkin, J,; Friedman, M. I. Hepatic contribution to satiation of salt appetite in rats. Am. J. Physiol. 25 I:R 1095-R 1102; 1986. 30. Tordoff, M. G.: Schulkin, J.; Friedman, M. 1. Further evidence for hepatic control of salt intake in the rat. Am. J. Physiol. 253:R444R449: 1987. 31. Woll; G. Refined salt appetite methodology for rats demonstrated by assessing sex differences. J. Comp. Physiol. Psychol. 96:10161021: 1982.
0031-9384/93 $6.00 + .00 Copyright© 1993PergamonPressLtd.
Printedin the USA.
Leamed Preferences for the Flavor of Salted Food S U S A N E. C O L D W E L L .1 A N D M I C H A E L G. T O R D O F F i "
*Department of Psychology, University of Pennsylvania, Philadelphia, PA 19104-6196 and fMonell Chemical Senses Center, 3500 Market Street, Philadelphia, PA 19104-3308 Received 15 F e b r u a r y 1993 COLDWELL, S. E. AND M. G. TORDOFF. Learnedpreferencesfor theflavor of saltedfood. PHYSIOL BEHAV 54(5) 9991004, 1993.--We examined whether rats can associate the flavor of their food with its salt content, and whether this association is influenced by sodium status during training and testing. During two pairs of 2-h training trials, rats ate flavored food containing 1.75% NaCI or an alternatively flavored unsalted food. The motivational state of the rats was manipulated prior to each trial by combined 48-h dietary sodium deprivation and furosemidetreatment (severe sodium depletion), 48-h dietary sodium deprivation (mild sodium deprivation), or continued maintenance on stock diet containing 1% NaC1 (sodium replete). When later given a choice between the two flavors, all rats preferred food containing the salt-paired flavor. The strength of this preference was unaffected by motivational state during training or by the salt content of the test foods, but was modulated by the motivational state of the rats during the preference test. Preference for the salt-paired flavor was strongest when rats were tested after severe sodium depletion,lessstrong after mild sodium deprivation,and absent when sodium replete. These resultsindicatethat deprivation state during training has little effect on learned preferences for the flavor of salted food but deprivation state during testing affects the expression of this learning. Salt intake
Latent learning
Deprivation state
Food preference
ANIMALS can readily learn about the availability of NaC1 in the absence of a need for sodium (3,12,18,22). The clearest evidence for this comes from studies demonstrating that sodiumreplete rats given flavored salty water show a preference for the flavor when later made sodium deficient (3,12,22). Such studies make a compelling case that salt-based flavor preferences can be acquired, and that the expression of these preferences is dependent on sodium status. However, to our knowledge, there has been no attempt to examine whether sodium need influences the acquisition of this learning. Given that sodium deficiency increases the motivation to search for (26) and ingest (5,9,15,24,28-31 ) salt, an obvious hypothesis is that preferences acquired by sodium-deficient rats would be stronger than those acquired by sodium-replete rats. On the other hand, findings that rats without an apparant need for salt develop preferences for sodium-paired flavors (3,12,22) argue that motivational state may be irrelevant for this form of learning. In the experiments described below, we investigated the importance of sodium need for the acquisition of preferences for the flavor of salty food. EXPERIMENT l: EFFECT OF SEVERE SODIUM DEPLETION OR MILD SODIUM DEPRIVATION ON THE ACQUISITION OF PREFERENCES FOR THE FLAVOR OF SALTED FOOD METHOD
the beginning of the experiment. They were maintained on a 12:12 h light:dark cycle with lights offat 1000 h. The rats were housed and trained individually in hanging wire cages measuring 19.5 X 17.5 X 24.5 cm. Maintenance diet was AIN-76A sodiumfree diet (1) fabricated by Dyets Inc. (Bethlehem, PA) to which was added 171 mmol/kg NaCl ( 1% NaCl wt./wt.). This diet was given in 8.0-cm diameter metal cups, which were attached to the inside of the cage by a metal spring. Rats always had deionized water to drink.
Manipulations of Sodium Balance Rats were trained and tested after one of two manipulations of sodium status (see Table l). Severely depleted rats were subjected to combined dietary sodium restriction and furosemide treatment. The rats' maintenance diet was replaced with a sodium-free AIN-76A diet (Na + content < 3 mmol/kg) for 48 h. After the first 24 h, each rat also received two injections of furosemide (5.0 rag, SC, in 0.5-ml 150 m M NaCl) spaced ~ 3 h apart. Rats treated in this manner lose 1.75-2.50 mmol Na ÷ and develop a vigorous appetite for NaCl (5,9,15,28-31). Mildly deprived rats were fed sodium-deficient diet for 48 h and received injections of 0.5-ml 150 m M NaCl at the time the experimental group received furosemide. Rats deprived of dietary sodium for 48 h lose minimal amounts of sodium, and restriction for much longer periods is required to produce a measurable appetite for NaCl solutions ( 1 l, 15,27).
Subjects and Maintenance
Training Procedure
Subjects were 28 male Sprague-Dawley CD rats (Charles River Laboratories, Wilmington, MA) weighing 300-400 g at
Each rat received four training trials according to the schedule described in Table 2. Training trials occurred during the first 2
Requests for reprints should be addressed to Susan E. Coldwell,Department of Psychology, 3815 Walnut Street, Philadelphia, PA 19104. 999
1000
COIDWELL
TABLE 1 SCHEMATIC OF PROCEDURES FOR MANIPULATIN(; SODIUM STATUS Day
Food During First 2 h of Dark
Food During Following 22 h
Na-ffee Na-free Training/Test Na-free Na-ffee Na-free
Na-free Na-free Na-free l% NaCI 1% NaCI 1% NaCI
Injection
Test Procedure
Furosemide
Mild Sodium Deprivation 1 2 3 4 5 6
Na-free Na-free Training/Test Na-ffee Na-ffee Na-free
Na-free Na-free Na-free 1% NaCI 1% NaC1 1% NaC1
Saline*
Na-free Na-free Training/Test Na-free Na-free Na-free
1% NaCI 1% NaCI 1% NaCI 1% NaCI 1% NaCI 1% NaCI
Following training, the rats were given four preference tests. Tests occurred in the first 2 h of the dark period. Each was a two-cup preference test between diets containing the flavors used in training. In the first two tests, both flavors were given in sodium-free diet. Half the rats in each group were tested in each sodium state on each test according to a counterbalanced design. T h e third a n d fourth tests were conducted in the same way as the first two except the choice was between flavors mixed in diet containing 1.75% NaCI. Intake of each diet was measured at 30, 60, a n d 120 rain. Because the pattern of results was the same for readings at all three time periods, only the results from the 120-min reading are presented here.
Statistical ,4nalyses
Sodium Replete 1 2 3 4 5 6
I()RDOFI
between subjects, a n d the order of flavor presentation was det e r m i n e d by a c o u n t e r b a l a n c e d ABBA design. During the first 2 h of the dark cycle on n o n t r a i n i n g days, rats were given 5 g of unflavored, sodium-free diet in the same m a n n e r (see Tables I and 23.
Severe Sodium Depletion I 2 3 4 5 6
ANI)
None
* No injections were administered in Experiment 2.
h of the dark period on the third day of each 6-day training cycle (see Table 1). Just before the lights went out, the rats' m a i n t e n a n c e diet was removed and each rat received a fixed ration of 5 g of flavored diet in a 5.5 X 5.5 X 3.0 cm square metal cup. T h e flavors were 0.1% (wt./wt.) imitation a l m o n d or imitation vanilla extract ( M c C o r m i c k brand). O n two of the cycles, the flavored diet contained 1.75% NaC1. O n the other two cycles, the flavored diet was sodium free. One flavor was always presented with the NaC1 diet a n d the other with the sodium-free diet. The flavor paired with NaC1 was counterbalanced
Preliminary analyses showed that there were no differential effects of the treatments on intake of a l m o n d or vanilla flavor and so results obtained with the two flavors were collapsed into salt-paired a n d unpaired categories. Subsequent analyses were performed using analyses of variance (ANOVAs). Training data in each experiment were analyzed by A N O V A s with a betweensubject factor o f training deprivation state (i.e., group) a n d within-subject factor of diet (unsalted vs. salted). W h e n appropriate, the significance of differences between individual pairs of means was d e t e r m i n e d using Tukey's H S D tests. Data collected during the tests were analyzed by A N O V A with a betweensubject factor of training deprivation state (mild vs. severe) a n d within-subject factors of test deprivation state (mild vs. severe) and flavor (unpaired vs. salt-paired). Separate analyses were performed for tests c o n d u c t e d with unsalted a n d salted vehicle. A probability value o f p < 0.05 was considered significant, Values reported in the text are m e a n s _+ standard errors. RESULTS Three rats were omitted from analyses: one rat in the severe depletion group failed to eat the flavored diet on any of the training trials, a n d two rats in the severe depletion group spilled food during a preference test.
TABLE 2 ORDER OF TRAINING AND TEST TRIALS IN EXPERIMENT l Deprivation State Before Trial Cycle
Severely Deplete Group
Train 1 Train 2 Train 3 Train 4 Test 1 Test 2 Test 3 Test 4
Severe Severe Severe Severe Mild or severe Severe or mild Mild or severe Severe or mild
Mildly Deprived Group Mild Mild Mild Mild Mild or severe Severe or mild Mild or severe Severe or mild
Food Flavor and NaC1 Content During Trial Either FI in Na-free diet or F2 in 1.75% NaCI diet Either FI in 1.75% NaC1 diet or F2 in Na-free diet Either FI in 1.75% NaCI diet or F2 in Na-free diet Either FI in Na-free diet or F2 in 1.75% NaC1 diet FI vs. F2 both in Na-free diet FI vs. F2 both in Na-free diet F1 vs. F2 both in 1.75% NaCI diet FI vs. F2 both in 1.75% NaCI diet
FI and F2 = almond or vanilla flavors. Their association with sodium-free or 1.75% NaCI diet was counterbalanced. F1 vs. F2: both flavors presented together. Treatments prior to the first pair and second pair of tests were counterbalanced.
FLAVOR PREFERENCE LEARNING
1001
Training
Deprivation State Before Each Training Trial
Table 3 shows average food intakes during the 2-h training trials. The lowest salted food intake by any rat was 1.70 g, and many rats ate all 5 g available to them. Intake of the two diets differed in training, F(1, 23) = 48.7, p < 0.01. There was also a significant interaction between group and diet, F(I, 23) = 54.0, p < 0.01. Post hoc analyses revealed that although the two groups ate similar amounts of salted food, the severely deplete group ate significantly less of the unsalted diet than did the mildly deprived group, sometimes only sampling the diet (i.e., eating 0.1-0.2 g). Intake of the salted and unsalted diets did not differ for the mildly deprived group; however, the severely deprived group ate significantly less unsalted than salted diet.
Preference Tests With Flavors Presented in Unsalted Food Regardless of whether the rats were tested under mild sodium deprivation or severe sodium depletion, they ate more of the previously salted flavor than the flavor previously given alone, F(1, 23) = 19.8, p < 0.01 (Fig. 1, top panels). Intake of the saltpaired flavor tended to be greater when rats were tested under mild deprivation than severe depletion, although this was not significant, F(I, 23) = 3.05, p = 0.09. This was partly because rats tested while severely sodium deplete ate less of both flavored diets than did rats tested while mildly deprived, F( 1, 23) = 11.8, p < 0.01. The only differential effect of sodium status during training was that rats trained while severely deplete ate less overall (i.e., less of both flavors combined) during the tests than did rats trained while mildly deprived, F(1, 23) = 7.01, p < 0.02. The magnitude of the preference was unaffected by deprivation state during training, F(1, 23) = 0.23, p > 0.64. One concern was that furosemide administered prior to tests might produce permanent changes in the animal's motivation for salt (25). Because of the counterbalanced design, this could influence preferences for those rats that were first tested after furosemide treatment and later tested following dietary restriction alone. We wanted to determine if this explanation might account for the preference for the salt-paired flavor seen in rats trained and tested under mild deprivation. We thus compared preferences for the salt-paired flavor in the mild deprivation test in rats that were tested first while mildly deprived to those that were tested first while severely depleted. Order of testing had no effect on this preference, rendering this explanation unlikely, F(I, 12)= 1.78, p = 0.21.
Preference Tests With Flavors Presented in Salted Food The results of tests with flavors presented in salted food were similar to the results with the unsalted food vehicle. Rats tested
TABLE 3 INTAKE OF FLAVORED FOOD DURING 2-h TRAINING TRIALS Condition During Training
Experiment 1 Mild deprivation (n = 14) Severe depletion (n = 11) Experiment 2 Replete (n = 11) Mild deprivation (n = 12)
Unsalted Food
Salted Food
4.01 + 0.17 1.30 + 0.42*
3.93 __+0.18 4.36 _ 0.231-
4.07 -4-0.28 3.46 _+0.28
3.29 _+0.28t 3.66 + 0.26
Values are mean _.+SE of the average of two trials with each food. * p < 0.05 relative to mild deprivation group. t P < 0.05 relative to intake of unsalted food.
Mild
Severe J Flavors in Unsalted Food
I
,
I~1
m
~
Unpaired Paired
~" 3 0
2° e-
"o oo
LL
Flavors in Salted Food 6
,ij dlj Mild
Severe
Mild
Severe
Deprivation State Before Test
FIG. 1. Intake of flavored diet during 2-h preference tests by two groups of rats (n = I 1 or 14). Light bars indicate intake of diet containing the flavor paired with NaC1 during training. Dark bars indicate intake of diet containing the flavorunpaired with NaCI during training. Rats were tested with both flavorsin food containing 0% NaC1(top) or 1.75%NaCl (bottom). Left panels show data for rats trained while deprived of NaC1 for 48 h (mild deprivation). Right panels show data for rats trained while deprived of NaCI for 48 h and also injected with furosemide (severe depletion). The left set of bars in each panel indicates intake when rats were tested after mild deprivation. The right set of bars indicates intake when rats were tested followingsevere depletion.
under either motivational state ate more of the salt-paired flavor than unpaired flavor, F(1, 23) = 24.0, p < 0.01 (Fig. 1, bottom panels). Intake of the salt-paired flavor was greater after severe depletion than mild deprivation, F(1, 23) = 6.94, p < 0.02. There were no significant differences in overall intake during tests following severe sodium depletion and tests following mild sodium deprivation, F(1, 23) = 3.80, p > 0.06. Sodium status during training had no influence on the results. Unlike the tests using unsalted diet, with salted diet there were no significant differences in overall food intake between rats that were trained under mild sodium deprivation and rats that were trained under severe sodium depletion, F(1, 23) = 1.32, p > 0.26. The magnitude of the preference was unaffected by deprivation state during training, F(I, 23) = 0.35, p = 0.56. EXPERIMENT 2: EFFECT OF MILD SODIUM DEPRIVATION COMPARED WITH REMAINING SODIUM REPLETE ON THE ACQUISITION OF PREFERENCES FOR THE FLAVOR OF SALTED FOOD The main finding of Experiment 1 was that rats demonstrated similar preferences for flavors paired with salt whether they were trained under mild deprivation or severe depletion. Although we anticipated that rats tested after severe sodium depletion would show this response, we did not anticipate finding strong preferences for the salt-paired flavor in rats trained and tested
('OI.DWEI.L A N D I O R D O F t ~
1002
under mild sodium deprivation. This procedure produces negligible sodium loss and has been shown not to affect intake of concentrated NaC1 solution or salted diet (5,11,15,27). To clarify whether the effect of 48-h sodium deprivation was replicable, in Experiment 2 we compared rats trained while mildly sodium deprived with a group maintained replete. The animals were tested while sodium replete, mildly deprived, and then severely depleted. We preceded training with two pretests: one was to confirm that the rats used here preferred unsalted to salted food [see (5)], and the other assessed pretraining flavor preferences in each subject. METHOD The design of Experiment 2 is shown in Table 4. Twentyfour naive rats of similar body weight to those used in Experiment 1 were adapted to the maintenance diet for 5 days and then received a 2-h preference test between unsalted and salted food. The next day, they received a 2-h choice test between almond and vanilla flavors presented in unsalted food. Both tests were conducted during the first 2 h of the dark cycle. Using the procedures outlined for Experiment 1, the rats then received two pairs of training trials, except that one group was subjected to mild sodium deprivation before each training trial, and the other group remained sodium replete through continued maintenance on the 1% NaCI maintenance diet. Following training, three 2-h preference tests were conducted, using unsalted food as the flavor vehicle. In the first test, rats were tested in the state in which they were trained (i.e., rats trained while mildly deprived were tested while mildly deprived). In the second test, the rats were tested in the opposite state (i.e., rats trained while mildly deprived were tested while replete). In the third test, all rats were tested after severe sodium depletion. The order of these tests was not counterbalanced to prevent possible long-term effects caused by sodium depletion (5,25).
Statistical Ana@ses Training data were analyzed in the same manner as in Experiment 1. Pretests in Experiment 2 were analyzed separately with ANOVAs. In the first pretest, a within-subject factor of diet type was used. In the second pretest, a between-subject factor of group and a within-subject factor of diet were used. The data collected during the three preference tests were analyzed by separate ANOVAs according to testing deprivation state. To control for potential differences in preexisting preferences for the two flavors, each A N O V A involved a comparison between pretraining vs. posttraining flavor intakes. The three-way interactions between test (pretest or test), group (mild deprivation or replete), and flavor (paired or unpaired) were assessed using planned comparisons. RESULTS
One rat in the replete training condition ate less than 1 G of flavored food on each trial and so was omitted from all analyses Pretesls The rats showed a strong preference for unsalted food over salted food [F(I, 23) = 37.8, p < 0.01; 2.66 ___0.32 g vs. 0.54 + 0.09 g, respectively]. There were no significant flavor preferences prior to training, F(I, 21) = 2.10, p > 0.16 (Fig. 2, top panel) and no significant differences in intake of each flavor between the two groups, F(1, 21) = 1.10, p > 0.30.
Training Training intakes are summarized in Table 3. There was no overall difference in intake of the salted and unsalted flavored
TABLE 4 ORDER OF PRETEST, FRAINING, AND TESt IN EXPERIMENT 2
I RIAtS
Deprivation State Before Thai
Cycle
Mildl~ Deprived Group
Replete Group
Food Flavor and NaCI Content During Training
Pretest 1 Pretest 2 Train 1
Replete Replete Mild
Replete Replete Replete
Train 2
Mild
Replete
Train 3
Mild
Replete
Train 4
Mild
Replete
Test 1 Test 2
Mild Replete Severe
Replete Mild Severe
Na-free vs. 1.75% NaC1 diet FI vs. F2 both in Na-free diet Either FI in Na-free diet or F2 in 1.75% NaCI diet Either FI in 1.75c7~NaC1 diet or F2 in Na-free diet Either FI in 1.75% NaC1 diet or F2 in Na-free diet Either FI in Na-free diet or F2 in 1.75% NaCI diet El vs. F2 both in Na-free diet FI vs. F2 both in Na-free diet FI vs. F2 both in Na-free diet
Test 3
F I and F2 = almond or vanilla flavors. Their association with sodiumfree or 1.75% NaCI diet was counterbalanced.
diets, F(1, 21) = 0.91, p > 0.35. There was a significant interaction between group and diet (F = 5.58, p < 0.03). Post hoc analyses revealed that the two groups did not differ in intake of either salted or unsalted flavored diet. However, rats trained replete ate significantly more unsalted food than salted food, while rats trained mildly deprived ate similar amounts of both diets.
Preference Tests Relative to intake during the pretest, both groups of rats increased intake of the salt-paired flavor during mild deprivation or severe depletion but not when replete [F( 1, 21 ) = 5.90, p < 0,03; F(1, 21) = 23.4, p < 0.01; F(I, 21) = 0.51, p = 0.48, respectively] (see Fig. 2). Intake of the unpaired flavor was unaffected by deprivation state. Intake of salt-paired food increased only slightly in the mild deprivation test, and for both this and the replete test intake of the salt-paired and unpaired flavors did not differ [mild deprivation test, F( 1, 21 ) = 1.80, p = 0.19; replete test F( 1, 21) = 0,00, p = 0.97]. However, rats showed significantly greater intake of the salt-paired than unpaired flavors during severe sodium depletion, F(1, 21) = 34.8, p < 0.01. On none of the tests was there a significant difference in the response of rats trained while mildly deprived or replete, There were nonsignificant tendencies in all three tests for rats trained while mildly deprived to eat more salt-paired food than those trained while replete [replete test, F(1, 21) = 2.37, p = 0.14: mild deprivation test, F(I, 21) = 1.97, p = 0.18; deplete test, F(I, 21) = 1.34, p = 0.26]. Because this trend appeared even in the replete test, it probably reflects a greater motivation for sodium in these rats due to repeated deprivation experiences (25), rather than superior salt-flavor learning. Attesting to the rats' larger body mass and greater familiarity with the test situation, total food intake during the three posttraining preference tests was higher than during the pretest [replete, F( 1, 21) = 49.4, p < 0.01; mild deprivation, F( 1, 21) = 36.2, p < 0.01, severe depletion, F(1, 21) = 23.4, p < 0.01].
FLAVOR PREFERENCE LEARNING
Pretest
1003
Unpaired Paired
Replete 1
~" ¢M
2
0 e"o o
o u.
~prlvatl 4
)eprivation Test
2
Replete
Mild
Deprivation State Before Each Training Trial
FIG. 2. Intake of flavored diet containing 0% NaCI during 2-h tests by
two groups of rats (n = I I or 12/group). Light bars indicate intake of diet containing the flavor paired with NaCl during training. Dark bars indicate intake of diet containing the flavor unpaired with NaCl during training. Top panel: test that preceded any experimental manipulations of sodium balance. Second panel: test that followed at least 3 days' maintenance on 1%NaCl diet (replete test). Third panel: test that followed 48 h of sodium deprivation (mild deprivation test). Bottom panel: test that followed 48 h of sodium deprivation and injection of furosemide (severe deprivation test).
DISCUSSION Rats exposed to a flavored, salted food acquired a preference for the flavor. The preference was exhibited by rats trained while severely sodium depleted, mildly sodium deprived or sodium replete, but only if they were tested while mildly sodium deprived or severely sodium depleted, not while sodium replete. These findings suggest that sodium status has little or no influence on the acquisition of salt-flavor preferences but has a critical influence on the expression of these preferences. The finding that deprivation state during training had little impact on learning about a flavor associated with salt was surprising given data suggesting that NaCl is treated differently by sodium-deprived and sodium-replete rats [e.g., (8,14)]. It implies that a mechanism independent of drive state is responsible for flavor-salt learning, and thus contradicts predictions made by drive-reduction theories of learning [e.g., (13,17,19)]. One ex-
planation for state-independent learning might involve the rat's innate recognition of saltiness (20). However, the literature concerning similar manipulations of other drive systems is complex and does not necessarily distinguish learned from innately recognized ingesta. Motivational state during training influences subsequent preferences for flavors associated with water by thirsty rats (23) and with sucrose or saccharin by hungry rats (6), but not with ethanol by hungry rats (10). Motivational state during testing influences the magnitude of preferences exhibited by rats trained to associate a flavor with water (23), sucrose (6), or ethanol (10), but not with saccharin (6,10). It seems unlikely that a unitary explanation can encompass these findings. The results found here are consistant with, and address several deficiencies in, previous studies of flavor-NaC1 learning (3,12,22). Two previous studies involved training sodium-replete rats to associate flavors with a low-concentration NaCl solution [(12) Exp. l; (22)]. This procedure complicates interpretation because the acquired preferences could be due to the association of the flavor with NaCl specifically or, more generally, with a preferred taste. By using food as the vehicle for the salt we avoided this problem, because sodium-replete rats never prefer salted to unsalted food [(2,4,5,7), pretest of Experiment 2]. Another difference from previous studies of flavor-NaC1 learning was that we tested for preferences of flavors embedded in both unsalted and salted media. The main reason for this was that tests conducted in unsalted media compare a somewhat novel diet (the salt-paired flavor now presented in a Na+-deficient diet) with a familiar diet (the unpaired flavor presented in the Na +deficient diet). Since the latter but not former was eaten during training, there is a possibility that intake of the salt-paired flavor might be inhibited by neophobia or a failure to generalize from salted to unsalted flavors. Similarly, Beauchamp and colleagues have stressed that context has a strong influence on salt preference (2), and such effects could potentially influence preferences for flavors paired with salt. Our finding that the general pattern of results was the same whether tests were conducted with flavors embedded in unsalted or salted vehicle suggests that such factors played little, if any, role in determining flavor preferences. One difference between tests conducted using salted and unsalted food was that rats with experience of severe sodium depletion tended to eat more salted food, irrespective of its flavor. This is consistent with findings that unsalted food intake is reduced during the 24 h after administration of large doses of furosemide, and that salt in food can enhance short-term food intake of furosemide-treated rats (5). The reduced unsalted food intake seen in the present experiment involved conditioned effects because rats trained while severely deplete had low unsalted food intake even during preference tests conducted in the mildly deprived state. One explanation for this is that malaise induced by furosemide or by severe sodium depletion produces a conditioned aversion to unsalted food that does not generalize to flavors presented in salted food [see (5) for discussion of malaise and salt preference]. One of the most intriguing results was that 48-h dietary sodium restriction was sufficient to expose preferences for flavors previously associated with salt (i.e., mild deprivation tests in Experiment I and 2). Such a benign procedure has little effect on plasma concentrations of aldosterone and angiotensin, and is insufficient to induce rats to increase intakes of concentrated NaC1 solutions (28). Moreover, in studies conducted under almost identical conditions to the present experiment, sodium deprivation for 2 days did not induce rats to consume salted food (0.12-8.0% NaC1) in preference to unsalted food (5). One explanation for our results is that while 48 h of deprivation is not enough to cause rats to overcome any inherent distaste they have for sodium, it is sufficient to induce a sodium drive. Since sodium content of the two choices is identical in a flavor pref-
1004
('OIDWELL
erence test, it may be sensitive enough to detect this drive, while tests between substances with and without sodium are not. ACKNOWLEDGEMENTS We would like to thank Rebecca Hughes and Michelle Ewell for their valuable technical assistance. Drs. G. Beauchamp and D. Reed provided
AND
I()RDOH:
valuable comments on earlier drafts of the manuscript. I his research was supported by the National Institute of Diabetes and Digestive and Kidney Diseases, grant DK-40099. It was first presented at the Joint Annual Meeting of the North American Association for tile Study of Obesity and the Society for the Study of Ingestive Behavior, Sacramento. CA, October 199 I.
REFERENCES 1. Ad hoc Committee on Standards for Nutritional Studies. Second report of the ad hoc committee on standards for nutritional studies. J. Nutr. 110:1726; 1980. 2. Beauchamp, G. K.; Bertino, M. Rats do not prefer salted solid food. J. Comp. Psychol. 99:240-247; 1985. 3. Berridge, K. C.; Schulkin, J. Palatability shift of a salt-associated incentive during sodium depletion. Q. J. Exp. Psychol. [B] 41:121138; 1989. 4. Bertino, M.; Beauchamp, G. K. Rats show varying salt preferences in liquid milk products. Appetite. 8:55-66; 1987. 5. Bertino, M.; Tordoff, M. G. Sodium depletion increases rats' preferences for salted food. Behav. Neurosci, 102:565-573; 1988. 6. Capaldi, E. D. Hunger and the learning of flavor preferences. In: Bolles, R. C., ed. The hedonics of taste. Hillsdale, N J: Lawrence Erlbaum Associates, lnc: 1991:127-142. 7. Collier, G.; Johnson, D. F. Consumption of salty food by rats: Regulation of sodium intake? Physiol. Behav. 52:541-546; 1992. 8. Contreras, R. J.; Frank, M. Sodium deprivation alters neural responses to gustatory stimuli. J. Gen. Physiol. 73:569-594; 1979. 9. Cruz, C. E.; Perelle,l. B.; Wolf, G. Methodologic aspects of sodium appetite: An addendum. Behav. Biol. 20:96-103; 1977. 10. Fedorchak, P. M.; Bolles, R. C. Hunger enhances the expression of calorie, but not taste mediated conditioned flavor preferences. J. Exp. Psychol. [Anim. Behav.] 13:73-79; 1987. 11. Fregley, M. J.; Harper, J. M.; Radford, E. P., Jr. Regulation of sodium chloride intake by rats. Am. J. Physiol. 209:287-292; 1965. 12. Fudim, O. K. Sensory preconditioning of flavors with a formalininduced sodium need. J. Exp. Psychol. [Anim. Behav.] 4:276-285: 1978. 13. Hull, C. L. Principles of behavior. New York: Appleton-CenturyCrofts; 1943. 14. Jacobs, K. M.; Mark, G. P.; Scott, T. R. Taste responses in the nucleus tractus solitarius of sodium-deprived rats. J. Physiol. 406: 393-410; 1988. 15. Jaloweic, J. E. Sodium appetite elicited by furosemide: Effects of differential dietary maintenance. Behav. Biol. 10:313-327: 1974. 16. Jalowiec, J. E.; Stricker, E. M. Restoration of body fluid balance following acute sodium deficiency in rats. J. Comp. Physiol. Psychol. 70:94-102: 1970.
17. Kendler, H. H. Drive interaction: 11. Experimental analysis of role of drive in learning theory. J. Exp. Psychol. 35:188-198; 1945. 18. Krieckhaus, E. E.; Wolf, G. Acquisition of sodium by rats: Interaction of innate mechanisms and latent learning. J. Comp. Physiol. Psychol. 65:197-20l: 1968. 19. Miller, N. E. Comments on multiple-process conceptions of learning. Psychol. Rev. 58:375-381; 1951. 20. Nachman, M. Taste preferences for sodium salts by adrenalectomized rats. J. Comp. Physiol. Psychol. 55:1124-1129; 1962. 21. Pfaffmann, C. Taste mechanisms in preference behavior. Am. J. Clin. Nutr. 5:142-147; 1957. 22. Rescorla, R. A.; Freberg, L. The extinction of within-compound flavor associations. Learn. Motiv. 9:411-427; 1978. 23. Revusky, S. H. Effects of thirst level during consumption of flavored water on subsequent preference. J. Comp. Physiol. Psychol. 66:777779; 1968. 24. Richter, C. P. Increased salt appetite in adrenalectomized rats. Am. J. Physiol. 115:155-161; 1936. 25. Sakai, R. R.; Fine, W. B.; Epstein, A. N.; Frankmann, S. P. Salt appetite is enhanced by one prior episode of sodium depletion in the rat. Behav. Neurosci. 101:724-731; 1987. 26. Schulkin, J. Innate, learned, and evolutionary factors in the hunger for salt. In: Friedman, M. I.; Tordoff, M. G.; Kare, M. R., eds. Chemical senses: Volume 4 appetite and nutrition. New York: Marcel Dekker, Inc.; 1991:211-237. 27. Striker, E. M.; Thiels, E.; Verbalis, J. G, Sodium appetite in rats alter prolonged dietary sodium deprivation: A sexually dimorphic phenomenon. Am. J. Physiol. 260:RI082-R1089; 1991. 28. Tordoff, M. G.; Fluharty, S. J.; Schulkin, J. Physiological consequences of NaCI ingestion by Na+-depleted rats. Am, J. Physiol. 261 :R289-R295; 1991. 29. Tordofl, M. G.; Schulkin, J,; Friedman, M. I. Hepatic contribution to satiation of salt appetite in rats. Am. J. Physiol. 25 I:R 1095-R 1102; 1986. 30. Tordoff, M. G.: Schulkin, J.; Friedman, M. 1. Further evidence for hepatic control of salt intake in the rat. Am. J. Physiol. 253:R444R449: 1987. 31. Woll; G. Refined salt appetite methodology for rats demonstrated by assessing sex differences. J. Comp. Physiol. Psychol. 96:10161021: 1982.