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Hypothalamic hyperphagic rats overeat bitter sucrose octa acetate diets but not quinine diets
Physiology & Behavior, Vol. 22, pp. 759-766. PergamonPress and BrainResearch Publ., 1979. Printedin the U.S.A.
Hypothalamic Hyperphagic Rats Overeat Bitter Sucrose Octa Acetate Diets but Not Quinine Diets I A N T H O N Y S C L A F A N I , P A U L F. A R A V I C H A N D J E F F R E Y S C H W A R T Z
Brooklyn College o f the City University o f N e w York Brooklyn, N Y 11210 ( R e c e i v e d 28 S e p t e m b e r 1978) SCLAFANI, A., P. F. ARAVICH AND J. SCHWARTZ. Hypothalamic hyperphagic rats overeat bitter sucrose octa acetate diets but not quinine diets. PHYSIOL. BEHAV. 22(4) 759-766, 1979.--Female rats were made hyperphagic with knife cuts or lesions in the ventromedial hypothalamus (VMH) and their aversion to food made bitter by adulteration with quinine or sucrose octa acetate (SOA) was examined. In short-term choice tests VMH obese rats, as well as controls, strongly preferred a 0.1% quinine diet to a 1.0% SOA diet. Yet, in 24 hr intake tests VMH obese rats overate, relative to controls, the 1% SOA diet, but underate the 0.1% quinine diet. VMH rats in both dynamic and static stages also overate SOA diets in concentrations up to 16%. However, VMH obese rats underate a 1% SOA diet when previously fed a 0.1% quinine diet. The results indicate that the VMH rat's finickiness to quinine diets may not be due to bitter taste alone, but may result from toxic postingestive effects of quinine and the development of a conditioned taste aversion. Hypothalamic hyperphagia and obesity Conditioned taste aversion
Finickiness
AN IMPORTANT characteristic of the hyperphagia-obesity syndrome produced by ventromedial hypothalamic (VMH) damage is its dependence upon diet palatability. VMHdamaged rats (lesions or knife cuts) readily overeat and gain weight on palatable foods, but fail to do so, and may even undereat when offered unpalatable diets [7, 13, 18, 22, 34, 36]. The finickiness of VMH-damaged rats to unpalatable foods has typically been demonstrated by adulterating standard laboratory chow with various substances (cellulose, kaolin, sodium chloride), but the most widely used adulterant has been quinine, a bitter-tasting alkaloid [13, 18, 20, 22, 28, 34, 36]. The enhanced aversion displayed by VMH-damaged animals to quinine-adulterated foods has often been interpreted as the result of a lesion-produced increase in the affective response to taste and other stimuli (e.g., [2, 15, 16]). Recent studies, however, demonstrate that the VMH-damaged rat's finickiness to quinine diets is largely, if not completely, due to obesity rather than to VMH damage per se [6, 7, 34], which confirms the early findings of Teitelbaum [36]. Furthermore, quinine finickiness is not an exclusive property of VMH obese animals since dietary obese rats display an enhanced aversion to quinine diets [33]. VMH obese rats, as well as dietary obese animals, also display a reduced willingness to work for food [30,33], and fail to increase their activ-
Quinine
Sucrose octa acetate
ity when food deprived [31,32] so that their rejection of quinine-adulterated diets has been attributed to a decrease in food motivation which makes them less willing to tolerate bitter-tasting foods. The present study further examined hypothalamic finickiness by comparing the effects of two bitter adulterants, quinine hydrochloride and sucrose octa acetate (SOA), on the food intake of VMH damaged and intact rats. The results revealed that while VMH obese rats undereat quinine adulterated diets, they overeat more bitter SOA adulterated diets. The implications of these findings to current concepts of hypothalamic finickiness and diet palatability are discussed. EXPERIMENT 1 In unpublished experiments we have observed that VMH obese rats (lesions or knife cuts) who undereat a 0.1% quinine-adulterated diet subsequently undereat a diet adulterated with 1.0% sucrose octa acetate (SOA). SOA is a bitter tasting compound which has only occasionally been used in studies of ingestive behavior [4, 8, 19, 39]. In order to compare the relative bitterness or acceptability of the 0.1% quinine and 1.0% SOA adulterated diets brief (20 min/day) two-choice preference tests were conducted with VMH obese and normal rats.
1This research was supported by Grants 11380 and 11802 from the Faculty Research Award Program of the City University of New York. Portions of this report were presented at the Eastern Psychological Association Meeting in April, 1977. The authors are grateful to Dr. Carlos Grijalva and Dr. Steven Kieffer for their critical reading of an earlier draft of this paper. Reprint requests should be sent to Anthony Sclafani, Department of Psychology, Brooklyn College, Brooklyn, NY 11210.
C o p y r i g h t © 1979 B r a i n R e s e a r c h P u b l i c a t i o n s Inc.--0031-9384/79/040759-08502.00/0
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SCLAFANI, ARAVICH AND SCHWARTZ METHOD
Animals
Six control and six VMH obese rats were used. The animals were females of the CFE strain (Charles River Lab, MA), and had been used previously in a study of food motivated bar pressing [29]. They were individually housed in wire mesh cages in a colony room maintained at 21°C and under a 12:12 light-dark cycle. The V M H obesity was produced by placing bilateral parasagittal knife cuts between the medial and lateral hypothalamus according to the procedure of Sclafani [28]. The present experiment began 21 to 29 weeks after surgery at which time the VMH knife cut rats weighed considerably more than did the sham operated controis (501.7 vs 362 g, p<0.01), and were eating significantly more Purina chow diet (37.0 vs 25.7 g/day, p<0.01). Procedure
At the start of the experiment the animals were adapted to a Purina meal diet (dry powder form of Purina chow) for 10 days and double-size cages (16x 10x 17 in.) for two days. The Purina meal was presented in a 4 oz glass j a r located within a 16 oz glass jar which was used to collect spillage. The rats were then food deprived and allowed 20 rain/day access to two food jars containing the Purina meal diet. Water was available ad lib through a drinking spout located on the front wall of the cage midway between the two food jars. After 8 days of adaptation to this feeding schedule the rats were given a choice between 0.1% quinine adulterated diet and a 1.0% SOA adulterated diet for 10 days. The right-left position of the diets was alternated daily. Throughout the 10 day test 20 rain intake was measured to the nearest 0.1 g, and the rats were given chow supplements to maintain their body weight at apprxoimately 85% of their ad lib level. The diets were prepared using quinine hydrochloride (Sigma Chemical Co.), sucrose octa acetate (Nutritional Biochemicals Co.), and dry Purina meal. The appropriate amount of quinine (1 g in 999 g of meal) and SOA (10 g in 990 g of meal) was mixed thoroughly into the meal using a stirring tool attached to an electric drill. The efficacy of the mixing procedure was established by mixing 1 g of black powder (Sudan Black) in 999 g of meal and observing an even distribution of black particles throughout the diet. RESULTS AND DISCUSSION
When switched from the pellet to the powder form of the Purina diet the VMH obese rats were no longer hyperphagic and ate only slightly more than did the controls when food was available ad lib (19.1 vs 18.4 g/day), and significantly less than controls during the restricted feeding periods (3.9 vs 6.1 g/20 min, p<0.05). The results of the SOA vs quinine preference test are presented in Fig. 1. Analysis of variance indicated that during the 10 day test the VMH and control groups ate significantly more quinine diet than SOA diet (p<0.01), and that the VMH rats ate less food than did the controls (p<0.01). Examination of the individual intake scores revealed that five of the six VMH rats had a strong preference for the quinine diet, with quinine diet representing 78 to 91% of their total food intake, while one VMH rat had a weaker preference for the quinine diet (56%). In the control group, five of the six rats showed a very strong preference for the quinine diet (91 to 98%), but one rat preferred the SOA diet (quinine
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FIG. 1. Mean ( _+ SEM) 20 min food intake of VMH (n=6) and
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diet intake 31% of total intake). The mean quinine diet preferences of the VMH and control groups, therefore, were similar (81.5 vs 84.9%). These results demonstrate that both VMH obese and control animals find a 0.1% quinine diet more acceptable, and presumably less bitter, than a 1.0% SOA diet during shortterm preference tests. The fact that the VMH obese rats ate less food during the 20 rain/day tests than did the controls is consistent with previous reports of decreased food motivation in hypothalamic obese animals (e.g., [30,34]).
EXPERIMENT 2 Based on the preference data obtained in Experiment 1 VMH obese rats would be expected to be more finicky to a 1.0% SOA diet during 24 hr intake (no choice) tests than they are to a 0.1% quinine diet. Our initial findings indicated that this is not the case, however, in rats tested first with the quinine diet and then with the SOA diet (Sclafani and Aravich, unpublished data). The present experiment attempted to replicate these results and controlled for the order of diet presentation. METHOD
Animals
Ten control and 12 VMH obese rats singly housed in standard-size mesh cages were used. The animals were
H Y P O T H A L A M I C F I N I C K I N E S S TO BITTER DIETS females of the C F E strain previously used in a study of food motivated bar pressing [29] and had no prior experience with adulterated diets. The VMH obesity was produced by placing bilateral parasagittal knife cuts between the medial and lateral hypothalamus according to the procedure of Sclafani [28]. The present experiment started 23 weeks after surgery at which time the V M H knife cut rats weighed considerably more than did the sham operated controls (443 vs 298 g, p <0.01), and were eating significantly more Purina chow diet (29.8 vs 18.4 g/day, p<0.01). Procedure
At the start of the experiment the rats were switched from Purina chow (pellets) to Purina meal (powder) which was presented in the feeding jars described in Experiment 1. After 12 days adaptation to the powdered diet the VMH and control groups were each divided into two subgroups equated for body weight and food intake. The VMH-1 and CON-1 subgroups were given a 0.1% quinine diet as their only food for three days, followed by five days on unadulterated powder diet (henceforth called plain diet), and then three days of a 1.0% SOA diet. The VMH-2 and CON-2 subgroups were similarly treated except that they received the SOA diet first and the quinine diet second. Following the second diet adulteration test the subgroups were given plain powdered food for six days and were then returned to the pellet diet. RESULTS The results of the diet adulteration tests are summarized in Fig. 2 and were evaluated using analysis of variance and inidividual t-tests. Data from one CON-1 rat was excluded because the animal inadvertently received the wrong test diet. The VMH-1 rats ate more plain diet than did the controls (0<0.01), but when offered the quinine diet they reduced their food intake more (0<0.01), and underate compared to the CON-1 rats (0<0.01). When subsequently offered the SOA diet the VMH-1 rats again reduced their food intake more (0<0.01) and underate (0<0.01) relative to controls. Examination of the daily intake scores (Fig. 2) revealed that the VMH-1 rats significantly (0<0.05) increased their SOA intake from Day 1 to Day 3 of testing, and by Day 3 their SOA intake was no longer significantly below that of the CON-1 rats. The VMH-1 rats ate more SOA diet than quinine diet, with this difference being significant for their intakes on the third day of each test (11.0 vs 5.8 g, p<0.05). The VMH-2 rats consumed more of the plain diet than did the CON-2 rats (0<0.01), and when offered the SOA diet they displayed no change in their intake and continued to overeat relative to controls (0<0.01), even on the first day of the diet (Fig. 2). When subsequently offered the quinine diet the VMH-2 rats reduced their food intake more than did the controls (0<0.05), but they ate the same amount of quinine diet as did the controls. Both subgroups decreased (0 <0.05) their quinine intake over the three day test (Fig. 2). The quinine diet intake of the VMH-2 rats was reliably less than their SOA diet intake (0<0.01). Compared to the VMH-1 subgroup the VMH-2 rats consumed significantly more SOA diet (22.2 vs 8.0 g/day, p<0.01) and quinine diet (12.0 vs 6.1 g/day, p<0.02). The plain diet intakes of the VMH-1 and VMH-2 subgroups (21.1 vs 21.9 g/day) were similar, as were their body weights (436.8 vs 438.8 g).
761
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FIG. 2. Mean ( _+ SEM) 24 hr food intake of VMH and control subgroups during three day tests with 0.1% quinine hydrochloride (QHCL) diet and 1.{)% sucrose octa acetate (SOA) diet. VMH-1 (n=6) and CON-1 (n=4) subgroups were tested with the quinine diet first and the SOA diet second, while the VMH-2 (n=6) and CONo2 (n=5) subgroups were tested in the reverse order. Individual data points represent, from left to right, the mean intake on Days 1, 2 and 3 of the diet adulteration tests. The black bars represent the mean three day plain powder intakes prior to each diet test. At the start of the experiment when switched from the pellet to the powder form of the Purina diet the VMH and control rats decreased their food intake (0<0.01) but the V M H subgroups displayed significantly greater (p<0.01) reductions (VMH-I: 29.4 to 21.1 g/day; VMH-2:30.1 to 20.9 g/day, three day means) than did the control subgroups (CON-l: 17.7 to 17.6 g/day; C O N - 2 : 1 9 . 1 to 16.5 g/day). Conversely, when returned to the pellet diet at the end of the experiment the VMH subgroups increased (0<0.01) their food intake (VMH-I: 20.2 to 36.1 g/day; VMH-2:22.7 to 39.4 g/day) more than did the control subgroups (CON-l: 16.4 to 21.5 g/day; CON-2:17.2 to 22.6 g/day). DISCUSSION
The aversion displayed by the VMH-1 rats to both the quinine diet and the SOA diet confirms unpublished data obtained in our laboratory. However, the finding that the VMH-1 rats displayed a greater rejection of the quinine diet than of the SOA diet is inconsistent with the quinine diet preference observed in Experiment 1. Even more surprising are the results obtained with the VMH-2 rats. When first offered the SOA diet the VMH-2 rats displayed no aversion to the bitter food, and when subsequently given the quinine
762 diet they did not undereat it relative to the controls. Furthermore, the VMH-2 rats were less finicky to the quinine diet than were the VMH-1 rats. Thus, order of diet presentation is an important factor determining the reaction of VMH rats to bitter diets. Prior exposure to the quinine diet produced finickiness to the SOA diet, while prior exposure to the SOA diet reduced the finickiness to the quinine diet. The results of this experiment along with those of Experiment 1 seriously question the traditional interpretation of quinine diet finickiness. If VMH rats, and to a lesser extent control animals, reject a 0.1% quinine adulterated diet because of its bitter taste, then they should display a greater rejection of the less preferred 1.0% SOA diet, irrespective of its order of presentation. An alternative explanation of quinine diet finickiness is that it results, at least in part, from the postingestive effects of quinine. Although ignored in most taste experiments, quinine is known to have widespread systemic and central actions, and is fatal at high doses [27]. In particular quinine is a local irritant and when taken orally can produce gastric pain, nausea, and vomiting in humans [27]. Sucrose octa acetate, on the other hand, appears to be free of toxic effects [14,21], and can be ingested in large amounts by rats (see Experiment 3). Thus, the VMH-1 rats may have rejected the quinine diet because of its toxic postingestive effects, while the VMH-2 rats may have accepted the more bitter SOA diet because it did not produce such effects. The toxicity interpretation of quinine finickiness can account for the significant order effect observed in the present experiment. That is, if the VMH-1 rats rejected the quinine diet because of its aversive aftereffects, they should have developed a conditioned aversion to its bitter taste [11]. This would explain why they subsequently rejected the bitter, but nontoxic SOA diet, and extinction of the conditioned taste aversion can explain why their SOA diet intake increased over the three day test. The VMH-2 rats, on the other hand, when first offered the SOA diet should have learned that its bitter taste was safe since it was not associated with toxic aftereffects [17], and therefore the rats would have been less likely to form a conditioned taste aversion to the subsequently offered quinine diet. This would explain why the VMH-2 rats were less finicky to the quinine diet than were the VMH-1 rats, and extinction of the learned safety would explain why their quinine diet intake decreased over the three day test. Additional findings consistent with the toxicity interpretation of quinine finickiness can be cited. De Ruiter et al. [4] have noted that mice made obese with goldthioglucose (GTG) injections lose considerable body weight when maintained on a 0.4% quinine diet, but do not lose weight when given an equally bitter 1% SOA diet. Similarly, Kratz et al. [19] have reported recently that normal rats eat less, spill more food, and lose more weight when maintained on a 0.75% quinine sulfate diet than when given an equally bitter 4% SOA diet, and the authors implicated the pharmacological effects of quinine as being responsible for these results. Finally, we have found that normal rats (a) initially prefer a 0.1% quinine diet to a 1.0% SOA diet during 24 hr two-choice tests, but with continued experience with the diets they all switch their preference to the SOA diet over the quinine diet; (b) lose more weight when maintained on a 0.1% quinine diet (no choice) than when maintained on a 1.0% SOA diet; and (c) develop a conditioned aversion to a saccharin solution which is paired with the intragastric intubation of quinine (Aravich and Sclafani, unpublished data).
S C L A F A N I , ARAVICH A N D S C H W A R T Z In apparent conflict with the toxicity interpretation of quinine finickiness are the findings of two published studies. Epstein and Teitelbaum [5] reported that rats feeding themselves intragastrically did not decrease their food intake when the diet was adulterated with 0.05% quinine for three days, while rats feeding orally decreased their intake when given the same diet. However, since the intragastrically fed rats ate less food and were lower in body weight (see also [35]) than were the orally fed rats they may have been more resistant to the feeding suppressive effect of the quinine. More recently, Peck [23] has reported that quinine diet aversion is not due to postingestive effects because rats orally fed a 1.2% quinine diet spilled more food and lost more weight than did rats fed an unadulterated diet but intubated with equivalent amounts of quinine. However, the quinine intubated group appeared to be as hypophagic as the quinine fed group, and at least initially lost body weight. Also, the precise effects of the quinine intubation on food intake and body weight cannot be evaluated since there was no control intubated group. Furthermore, in neither of these two studies was quinine intubation paired with a novel taste stimulus so that the rats may not have formed a conditioned taste aversion, which may be the primary mechanism by which quinine adulteration reduces food intake.
EXPERIMENT 3 The failure of the VMH-2 rats in Experiment 2 to decrease their food intake when presented with the 1.0% SOA diet conflicts with the well known finickiness of VMH obese animals. It may be that the 1.0% diet is not bitter enough to reveal VMH finickiness, even though the diet is less preferred to the 0.1% quinine diet (Experiment 1), and is unpleasant to human taste. Experiment 3 examined this possibility by offering VMH and control rats more concentrated SOA diets. Since the previous results demonstrated the importance of the order of diet presentation, the rats were given the SOA diets in either increasing or decreasing concentrations. METHOD
Animals
The ten control and 12 VMH obese rats of Experiment 2 were used.
Procedure
Six days after the end of Experiment 2 the animals were returned to the powder diet for an adaptation period of 9 days. The VMH and control groups were then divided into new subgroups equated for food intake, body weight, and subgroup membership in Experiment 2. The VMH-A (body weight=450.0 g) and CON-A (294.0 g) subgroups were given SOA diets in an ascending order of 1%, 2%, 4%, 8% and 16% concentrations. Each SOA diet was presented for three days, and was followed by five days of plain powder diet to allow the rats to recover any weight lost on the SOA diet. The VMH-D (455.0 g) and CON-D (298.4 g) subgroups were tested in a similar manner except that the SOA diets were presented in a descending order of concentration. Following these SOA tests and five days on plain powder diet all subgroups were given the 16% SOA diet for three days, plain diet for five days, and a 24% SOA diet for three days.
H Y P O T H A L A M I C F I N I C K I N E S S TO BITTER DIETS
763
RESULTS The results of the first part of this experiment are summarized in Fig. 3. An overall analysis of variance indicated that the group (VMH vs CON) vs order (ascending vs descending) vs SOA concentration interaction was highly significant (p<0.001) and therefore the different subgroups are discussed separately. As illustrated in Fig. 3, the VMH-A rats ate more (p<0.01) food during the SOA tests than did the CON-A rats, and both subgroups showed significant (p<0.01), although relatively small decreases in intake as the SOA concentration increased (subgroup by diet interaction not significant). Thus, even on the 16% SOA diet the VMH-A rats consumed more (.0<0.05) than did the CON-A rats. Analysis of the VMH-D and CON-D results, on the other hand, revealed that the subgroup effect was not significant, but the diet effect and subgroup by diet interaction were (p<0.001). That is, while both groups increased their food intake as SOA concentration decreased, the VMH-D rats displayed greater changes than did the controls (p<0.01). Furthermore, whereas the VMH-D animals ate significantly (p <0.01) more than did the controls on the 0 to 2% SOA diets, they ate less than controls on the 10% diet, although this difference failed to be significant. A comparison between the VMH-A and VMH-D subgroups indicated that their overall food intake during the SOA tests did not reliably differ, but the intake of the VMH-D subgroup was more affected by concentration than was the intake of the VMH-A subgroup (p<0.001). In particular, the VMH-D rats ate significantly less (.o<0.05) of the 16% diet, but more (p<0.01) of the 1% and 2% diets than did the V M H - A subgroup. On the other hand, there was no significant difference between the CON-A and CON-D subgroups, and both decreased their food intake as SOA concentration increased. In the second part of the experiment when tested with the 16% and 24% SOA diets the intakes of the two V M H subgroups did not differ and therefore the rats were combined into one group. As illustrated in Fig. 4, when presented with the 16% diet both the VMH and control groups reduced their intake (p<0.001) and although the VMH rats displayed a greater reduction (p<0.01), their intake of the 16% SOA diet was slightly above that of the controls. When subsequently given the 24% SOA diet the VMH rats again reduced their food intake more 09<0.01) than did the controls, and now also ate significantly less (p<0.05) than did the controls.
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DISCUSSION
The results of this experiment indicate that hypothalamic finickiness can be demonstrated with SOA adulterated diets but only at very high concentrations (16 or 24%). Furthermore, V M H obese rats, unlike controls, are sensitive to the order of diet presentation. When offered the SOA diets in increasing concentrations the V M H - A rats apparently habituated to the bitter taste of SOA and overate even the 16% diet, which is extremely bitter to human taste. The VMH-D rats, on the other hand, when presented with the diets in a descending order underate the 16% SOA diet relative to the controls and VMH-A animals. The enhanced aversion displayed by the VMH obese rats to the 16% and 24% SOA diets cannot be conclusively attributed to taste alone, because such high concentrations of SOA alter the nutritional composition of the diet. However, SOA is thought to be hydrolyzed into dextrose, fructose, and
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FIG. 4. Mean ( _+ SEM) 24 hr food intake of VMH (n=12) and control (n=10) groups during three day tests with 16% and 24% sucrose octa acetate (SOA) diets. acetic acid, all of which are physiological and non-toxic substances [14], and there is little reason to suspect the postingestive effects of SOA as being responsible for the finickiness displayed by the VMH rats. EXPERIMENT 4 Recent studies have reported that maintaining VMH lesioned or knife cut rats on a quinine adulterated diet im-
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S C L A F A N I , ARAVICH A N D S C H W A R T Z
mediately after surgery completely blocks the hyperphagia and obesity that would otherwise appear [6, 9, 34]. This effect has been attributed to the bitter taste of the quinine diet, but the results of the two preceding experiments question this interpretation. Experiment 4 tested the prediction that VMH damaged rats would overeat and gain weight when offered bitter SOA diets immediately following surgery. VMH electrolytic lesions were used in order to demonstrate that the effects obtained in Experiments 2 and 3 are not dependent upon the use of VMH knife cuts. METHOD A I1im a Is
Twenty-two female rats of the C F E strain weighing 240280 g were used. The rats were singly housed in wire mesh cages as in the previous experiments.
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S u r g e l 3'
Bilateral VMH electrolytic lesions were performed under Equi-Thesin anesthesia (2.5 ml/kg bw) in 12 rats, while the remaining 10 rats received sham lesions. The VMH lesions were made by passing 1.5 mA anodal current for 30 sec through the exposed tip of a platinum-iridium electrode positioned at the DeGroot [3] atlas coordinates AP 5.8, H -3.0, L 1.0. At the end of the experiment the brains of the VMH lesioned rats were removed and histological sections through the lesion sites were prepared. Procedure
For eight days prior to surgery the rats were adapted to a 1.0% SOA diet presented in the feeding jars described in Experiment 1. Following surgery the rats were maintained on the 1.0% SOA diet for six days, and were then given, for three days each, SOA diets in concentrations of 2%, 4%, 8% and 16%. Next the rats were offered unadulterated diet for 6 days before being given a final three day test with the 16% SOA diet. RESULTS Data are presented from seven VMH lesioned rats and eight sham operated controls. The remaining seven rats were excluded from the study because of postoperative death or poorly placed lesions. The lesions of the seven rats included in the experiment were localized in the lateral aspect of the VMH and bilaterally destroyed the lateral edge of the ventromedial nucleus and the tisssue medial to the fornix. Preoperatively the VMH and control groups were well matched in food intake (19.8 vs 20.0 g/day) and body weight (259.4 vs 265.0 g). As illustrated in Fig. 5, following surgery the VMH rats increased their food intake by 71% and as a result rapidly gained weight (5.5 g/day) during the six days on the 1.0% SOA diet. The VMH rats then decreased their food intake and rate of weight gain as the SOA concentration of the diet increased. Compared to controls, however, the VMH rats overate (p<0.01) all of the SOA diets, even the 16% diet, although a significant group by diet interaction (p<0.01) indicated the VMH rats were more affected by the changes in SOA concentration than were the controls. By the last day of the 16% SOA diet the VMH rats weighed 308.4 g compared to the controls who weighed 278.6 g (p<0.05). When switched from the 16% SOA diet to the plain diet
FIG. 5. Mean ( _+ SEM) 24 hr food intake ofVMH (n=7) and control (n=8) groups during three day periods on diets adulterated with sucrose octa acetate (SOA) in concentrations ranging from 0% to 16%. Data from the first test (period 6) on the 16% SOA diet are presented twice in the figure. VMH electrolytic lesions and sham lesions were performed at time indicated by arrow. both groups increased their food intake (p<0.01), but the increase was significantly greater (p<0.01) for the VMH rats than for the controls (Fig. 5). When subsequently returned to the 16% SOA diet after six days on the plain food both groups decreased 09<0.01) their food intake, with the VMH rats again displaying the greatest change (p<0.01), and they now ate less (p<0.05) of the 16% diet than did the controls. The VMH rats also ate less (p<0.01) than they did when first offered the 16% SOA diet. DISCUSSION The results confirm the prediction that VMH damaged rats would overeat and gain weight when maintained on bitter tasting SOA diets following surgery. Therefore, the failure of the VMH damaged rats to overeat quinine diets postoperatively [6, 9, 34] does not appear to be due to the bitter taste of the diets, but rather may result from their postingestive effects. The VMH rats in the present experiment were finicky, however, in that their food intake was more influenced by SOA adulteration than was the intake of the control group. Furthermore, when returned to the 16% SOA diet after being on the plain diet for six days the VMH rats not only decreased their food intake more, but actually underate compared to the controls. The fact that they overate the 16% SOA diet the first time it was offered, but underate it the second time suggests that the VMH rats habituated to the bitter taste of SOA when presented with SOA diets of increasing concentration, which is consistent with the results obtained in Experiment 3. The VMH rats were heavier at the time of the second 16% SOA test than they were at the time of the first test (342.3 vs 299.3 g), and increased body weight may therefore have contributed to their rejection of the 16% SOA diet during the second test. G E N E R A L DISCUSSION The exaggerated quinine diet aversion of VMH-damaged
H Y P O T H A L A M I C F I N I C K I N E S S TO BITTER DIETS rats was first demonstrated in 1950 [22] and has been confirmed in numerous experiments since then. The implicit assumption of these experiments is that the small amounts of quinine used to adulterate food do not have postingestive consequences, but this assumption is questionable in light of recent findings [19]. The present results provide strong, albeit circumstantial, evidence that VMH finickiness to quinine adulterated food is due to the toxic rather than the taste effects of quinine. Further research is needed, however, to document the postingestive effects of quinine adulterated diets. The V M H rat's aversion to quinine solutions [2] should also be reexamined in view of these results. If, as the present findings suggest, VMH rats reject quinine diets because of their toxic aftereffects this does not contradict the motivational interpretation of quinine-diet finickiness. That is, VMH obese rats may undereat quinine diets because their reduced food motivation makes them less tolerant of the toxic effects produced by the quinine. Presumably the toxicosis produced by the quinine diets (0.1 to 0.2%) typically used in VMH studies is relatively mild since control rats lose little or no weight on these diets, and both control and VMH animals can be maintained on these diets for several weeks without obvious ill-effects [6,34]. Furthermore, VMH rats will overeat and gain weight on quinine adulterated foods if the rats are ovariectomized [9], or if the palatability of the diet is improved by increasing its fat content [24]. Nevertheless, the postingestive effects of quinine appear sufficiently aversive to produce anorexia in motivationally weakened animals, such as VMH obese, dietary obese, and recovered L H lesioned rats [7, 33, 34, 37]. However, the possibility that VMH damage or obesity exaggerate the toxic effects of quinine, for example by altering gastrointestinal motility [26], or the conditionability of the animal to aversive visceral stimuli [38,40] (but see [12,25]) cannot be discounted. In addition to challenging the traditional interpretation of quinine diet aversion the present results raise questions concerning the concepts of hypothalamic finickiness and diet palatability. The fact that VMH damaged rats, both static and dynamic, overeat diets adulterated with as much as 16% SOA indicate either that VMH rats are much less finicky to unpalatable diets than previously thought, or that the bitter taste of SOA has only minimal effects on diet palatability. The second possibility is suggested by the findings that the VMH rats in Experiment 2 significantly reduced their food intake (by 28%) when diet texture was changed from pellet to powder, but yet did not reduce their intake when the food was made bitter with 1% SOA adulteration (VMH-2 subgroup).
765 Rats and other animals appear to have an innate aversion to bitter taste [10], but they also appear to adapt or habituate to it. Guinea pigs, for example, display a strong aversion to SOA solutions in two bottle preference tests with water, but if they have been maintained on a S O A solution for some time, this aversion disappears [39]. Preliminary findings in our laboratory indicate that a similar effect can be obtained with rats using SOA adulterated food. The VMH-2 rats in Experiment 2, therefore, may not have appeared finicky to the 1% SOA diet because they rapidly habituated to its bitter taste. It is possible that a finer temporal analysis, i. e. an examination of individual meals, would have revealed a finickiness to the SOA diet. In support of this suggestion, De Ruiter et al. [4] have reported that, compared to controls, GTG obese mice display a significantly greater reduction in the size of their first meal (following a 24 hr fast) when switched to a 0.5% SOA diet although their long term food intake and body weight are not affected by SOA adulteration. With SOA diets more concentrated than 1% habituation to the bitter taste may be slower and less complete, and this would explain the significant diet concentration and order effects obtained in Experiments 3 and 4. Thus, when SOA concentration was gradually increased the VMH rats overate even the 16% SOA diet, but when the 16% SOA diet was the first presented (Experiment 3), or was returned after a period on plain food (Experiment 4), the V M H rats underate it relative to controls. In contrast to the situation with SOA diets animals would not be expected to habituate to the bitter taste of quinine adulterated diets if the quinine produces toxic postingestive effects. In fact, their aversion to the taste should increase rather than decrease as they eat the diet, experience its toxic effects, and develop a conditioned taste aversion. Consistent with this interpretation are our findings that normal rats eventually reverse their initial preference for the 0.1% quinine diet over the 1.0% SOA diet (Aravich and Sclafani, unpublished data). Thus, in the long run, the palatability of food, at least in the case of bitter diets, may be more determined by its postingestive rather than preingestive (orosensory) effects. Garcia [11], Cabanac [11 and others have also emphasized the importance of postingestive events in determining the hedonic response to food. The results of this study, therefore, do not require a revision of the concept of hypothalamic finickiness: V M H hyperphagia and obesity remains highly dependent upon diet palatability. However, the concept of diet palatability, in particular the determinants of palatability, should be reevaluated in view of the present findings.
REFERENCES
1. Cabanac, M. Physiological role of pleasure. Science 173:11031107, 1971. 2. Corbit, J. D. Hyperphagic hyperreactivity to adulteration of drinking water with quinine HCL. J. comp. physiol. Psychol. 60: 123-124, 1965. 3. De Groot, J. The Rat Forebrain in Stereotaxic Coordinates. Amsterdam: N. V. Noord-Hollandsche Uitgevers, 1967. 4. De Ruiter, L., P. R. Wiepkema and J,,Reddingius. Ethological and neurological aspects of the regulation of food intake. Ann. N.Y. Acad. Sci. 157: 1204-1216, 1969. 5. Epstein, A. N. and P. Teitelbaum. Regulation of food intake in the absence of taste, smell, and other oropharyngeal sensations. J. comp. physiol. Psychol. 55: 753-759, 1962.
6. Ferguson, N. B. L. and R. E. Keesey. Effect of a quinineadulterated diet upon the level of body weight maintained by male rats with ventromedial hypothalamic lesions. J. comp. physiol. Psychol. 89: 478--488, 1975. 7. Franklin, K. B. J. and L. J. Herberg. Ventromedial syndrome: The rats "finickiness" results from the obesity, not from the lesions. J. comp. physiol. Psychol. 87: 410-414, 1974. 8. Fuller, J. L. Genetic aspects of regulation of food intake. Adv. psychosom. Med. 7: 2-24, 1972. 9. Gale, S. K. and A. Sclafani. Comparison of ovarian and hypothalamic obesity syndromes in the female rat: Effects of diet palatability on food intake and body weight. J. comp. physiol. Psychol. 91: 381-392, 1977.
766 10. Garcia, J. and W. G. Hankins. The evolution of bitter and the acquisition of toxiphobia. In: Olfaction and Taste, edited by D. A. Denton and J. P. Coghlan. New York: Academic Press, 1975, pp. 39--47. 11. Garcia, J., W. G. Hankins and K. W. Rusiniak. Behavioral regulation of the milieu interne in man and rat. Science 185: 824-831, 1974. 12. Gold, R. M. and D. M. Proulx. Bait-shyness acquisition is impaired by VMH lesions that produce obesity. J. comp. physiol. Psychol. 79: 201-209, 1972. 13. Graft, H. and E. Stellar. Hyperphagia, obesity, and finickiness. J. comp. physiol. Psychol. 55: 418-424, 1962. 14. Green, M. W. Sucrose octa acetate as a possible bitter stomachic. Bull. hath. Formul. Comm. Am. pharm. Ass. 10: 131-133, 1942. 15. Grossman, S. P. The VMH: A center for affective reactions, satiety, or both? Physiol. Behuv. 1: 1-10, 1966. 16. Grossman, S. P. Aggression, avoidance, and reaction to novel environments in female rats with ventromedial hypothalamic lesions. J. comp. physiol. Ps'ychol. 78: 274-283, 1972. 17. Kalat, J. W. and P. Rozin. "Learned safety" as a mechanism in long-delay taste-aversion learning in rats. J. comp. physiol. Psycho/. 83: 198-207, 1973. 18. Kennedy, G. C. The hypothalamic control of food intake in rats. Proc. R. Soc. Ser. B. 137: 535-549, 1950. 19. Kratz, C. M., D. A. Levitsky and S. Lustick. Differential effects of quinine and sucrose octa acetate on food intake in the rat. Physiol. Behav. 20: 665-667, 1978. 20. Levison, M. J., G. P. Frommer and W. B. Vance. Palatability and caloric density as determinants of food intake in hyperphagic and normal rats. Physiol. Behav. 10: 455-462, 1973. 21. Linegar, C. R. Acute and chronic studies on sucrose octa acetate by the oral method. Bull. hath. Forrnul. Comm. Am. pharm. Ass. 11: 5%63, 1943. 22. Miller, N. E., C. J. Bailey and J. A. F. Stevenson. Decreased "hunger" but increased food intake resulting from hypothalamic lesions. Science 112: 256-259, 1950. 23. Peck, J. W. Rats defend different body weights depending on palatability and accessibility of their food. J. comp. physiol. Psychol. 92: 555-570, 1978. 24. Peters, R. H., L. L. Luttmers, M. W. Gunion and P. J. Wellman. Ventromedial hypothalamic syndrome: Finickiness? Physiol. Behav. 20: 27%285, 1978. 25. Peters, R. H. and M. J. Reich. Effects of ventromedial hypothalamic lesions on conditioned sucrose aversions in rats. J. comp. physiol. Psychol. 84: 502-506, 1973.
SCLAFANI, ARAVICH AND SCHWARTZ 26. Ralph, T. L. and P. E. Sawchenko. Differential effects of lateral and ventromedial hypothalamic lesions on gastrointestinal transit in the rat. Brain Res. Bull. 3: 11-14, 1978. 27. Rollo, I. M. Drugs used in the chemotherapy of malaria. In: The Pharmacological Basis o f Therapeutics, edited by L. S. Goodman and A. Gilman. New York: MacMillan, 1975, pp. 10451068. 28. Sclafani, A. Neural pathways involved in the ventromedial hypothalamic lesion syndrome in the rat. J. comp. physiol. Psychol. 77: 70-96, 1971. 29. Sclafani, A. Food motivation in hypothalamic hyperphagic rats reexamined. Neurosci. Biobehav. Rev. 2: 33%355, 1978. 30. Sclafani, A. and L. Kluge. Food motivation and body weight levels in hypothalamic hyperphagic rats: A dual lipostat model of hunger and appetite. J. comp. physiol. Psychol. 86: 28-46, 1974. 31. Sclafani, A. and A. Rendel. Food deprivation-induced activity in normal and hypothalamic obese rats. Behav. Biol. 22: 244-255, 1978. 32. Sclafani, A. and A. Rendel. Food deprivation-induced activity in dietary obese, dietary lean, and normal-weight rats. Behav. Biol. 24: 220-228, 1978. 33. Sclafani, A. and D. Springer. Dietary obesity in adult rats: Similarities to hypothalamic and human obesity syndromes. Physiol. Behav. 17: 461-471, 1976. 34. Sclafani, A., D. Springer and L. Kluge. Effects of quinine adulterated diets on the food intake and body weight of obese and non-obese hypothalamic hyperphagic rats. Physiol. Behav. 16: 631-640, 1976. 35. Snowdon, C. T. Motivation, regulation, and the control of meal parameters with oral and intragastric feeding. J. comp. physiol. Psychol. 69: 91-100, 1969. 36. Teitelbaum, P. Sensory control ofhypothalamic hyperphagia. J. comp. physiol. Psychol. 48: 156-163, 1955. 37. Teitelbaum, P. and A. N. Epstein. The lateral hypothalamic syndrome: recovery of feeding and drinking after lateral hypothalamic lesions. Psychol. Rev. 69: 74-90, 1962. 38. Thomas, J. B. and D. A. Smith. VMH lesions facilitate baitshyness in the rat. Physiol. Behav. 15: 7-11, 1975. 39. Warren, R. P. and C. Pfaftmann. Early experience and taste aversion. J. comp. physiol. Psychol. 52: 263-266, 1959. 40. Weisman, R. N., L. W. Hamilton and P. L. Carlton. Increased conditioned gustatory aversion following VMH lesions in rats. Physiol. Behav. 9: 801-804, 1972.
Hypothalamic Hyperphagic Rats Overeat Bitter Sucrose Octa Acetate Diets but Not Quinine Diets I A N T H O N Y S C L A F A N I , P A U L F. A R A V I C H A N D J E F F R E Y S C H W A R T Z
Brooklyn College o f the City University o f N e w York Brooklyn, N Y 11210 ( R e c e i v e d 28 S e p t e m b e r 1978) SCLAFANI, A., P. F. ARAVICH AND J. SCHWARTZ. Hypothalamic hyperphagic rats overeat bitter sucrose octa acetate diets but not quinine diets. PHYSIOL. BEHAV. 22(4) 759-766, 1979.--Female rats were made hyperphagic with knife cuts or lesions in the ventromedial hypothalamus (VMH) and their aversion to food made bitter by adulteration with quinine or sucrose octa acetate (SOA) was examined. In short-term choice tests VMH obese rats, as well as controls, strongly preferred a 0.1% quinine diet to a 1.0% SOA diet. Yet, in 24 hr intake tests VMH obese rats overate, relative to controls, the 1% SOA diet, but underate the 0.1% quinine diet. VMH rats in both dynamic and static stages also overate SOA diets in concentrations up to 16%. However, VMH obese rats underate a 1% SOA diet when previously fed a 0.1% quinine diet. The results indicate that the VMH rat's finickiness to quinine diets may not be due to bitter taste alone, but may result from toxic postingestive effects of quinine and the development of a conditioned taste aversion. Hypothalamic hyperphagia and obesity Conditioned taste aversion
Finickiness
AN IMPORTANT characteristic of the hyperphagia-obesity syndrome produced by ventromedial hypothalamic (VMH) damage is its dependence upon diet palatability. VMHdamaged rats (lesions or knife cuts) readily overeat and gain weight on palatable foods, but fail to do so, and may even undereat when offered unpalatable diets [7, 13, 18, 22, 34, 36]. The finickiness of VMH-damaged rats to unpalatable foods has typically been demonstrated by adulterating standard laboratory chow with various substances (cellulose, kaolin, sodium chloride), but the most widely used adulterant has been quinine, a bitter-tasting alkaloid [13, 18, 20, 22, 28, 34, 36]. The enhanced aversion displayed by VMH-damaged animals to quinine-adulterated foods has often been interpreted as the result of a lesion-produced increase in the affective response to taste and other stimuli (e.g., [2, 15, 16]). Recent studies, however, demonstrate that the VMH-damaged rat's finickiness to quinine diets is largely, if not completely, due to obesity rather than to VMH damage per se [6, 7, 34], which confirms the early findings of Teitelbaum [36]. Furthermore, quinine finickiness is not an exclusive property of VMH obese animals since dietary obese rats display an enhanced aversion to quinine diets [33]. VMH obese rats, as well as dietary obese animals, also display a reduced willingness to work for food [30,33], and fail to increase their activ-
Quinine
Sucrose octa acetate
ity when food deprived [31,32] so that their rejection of quinine-adulterated diets has been attributed to a decrease in food motivation which makes them less willing to tolerate bitter-tasting foods. The present study further examined hypothalamic finickiness by comparing the effects of two bitter adulterants, quinine hydrochloride and sucrose octa acetate (SOA), on the food intake of VMH damaged and intact rats. The results revealed that while VMH obese rats undereat quinine adulterated diets, they overeat more bitter SOA adulterated diets. The implications of these findings to current concepts of hypothalamic finickiness and diet palatability are discussed. EXPERIMENT 1 In unpublished experiments we have observed that VMH obese rats (lesions or knife cuts) who undereat a 0.1% quinine-adulterated diet subsequently undereat a diet adulterated with 1.0% sucrose octa acetate (SOA). SOA is a bitter tasting compound which has only occasionally been used in studies of ingestive behavior [4, 8, 19, 39]. In order to compare the relative bitterness or acceptability of the 0.1% quinine and 1.0% SOA adulterated diets brief (20 min/day) two-choice preference tests were conducted with VMH obese and normal rats.
1This research was supported by Grants 11380 and 11802 from the Faculty Research Award Program of the City University of New York. Portions of this report were presented at the Eastern Psychological Association Meeting in April, 1977. The authors are grateful to Dr. Carlos Grijalva and Dr. Steven Kieffer for their critical reading of an earlier draft of this paper. Reprint requests should be sent to Anthony Sclafani, Department of Psychology, Brooklyn College, Brooklyn, NY 11210.
C o p y r i g h t © 1979 B r a i n R e s e a r c h P u b l i c a t i o n s Inc.--0031-9384/79/040759-08502.00/0
760
SCLAFANI, ARAVICH AND SCHWARTZ METHOD
Animals
Six control and six VMH obese rats were used. The animals were females of the CFE strain (Charles River Lab, MA), and had been used previously in a study of food motivated bar pressing [29]. They were individually housed in wire mesh cages in a colony room maintained at 21°C and under a 12:12 light-dark cycle. The V M H obesity was produced by placing bilateral parasagittal knife cuts between the medial and lateral hypothalamus according to the procedure of Sclafani [28]. The present experiment began 21 to 29 weeks after surgery at which time the VMH knife cut rats weighed considerably more than did the sham operated controis (501.7 vs 362 g, p<0.01), and were eating significantly more Purina chow diet (37.0 vs 25.7 g/day, p<0.01). Procedure
At the start of the experiment the animals were adapted to a Purina meal diet (dry powder form of Purina chow) for 10 days and double-size cages (16x 10x 17 in.) for two days. The Purina meal was presented in a 4 oz glass j a r located within a 16 oz glass jar which was used to collect spillage. The rats were then food deprived and allowed 20 rain/day access to two food jars containing the Purina meal diet. Water was available ad lib through a drinking spout located on the front wall of the cage midway between the two food jars. After 8 days of adaptation to this feeding schedule the rats were given a choice between 0.1% quinine adulterated diet and a 1.0% SOA adulterated diet for 10 days. The right-left position of the diets was alternated daily. Throughout the 10 day test 20 rain intake was measured to the nearest 0.1 g, and the rats were given chow supplements to maintain their body weight at apprxoimately 85% of their ad lib level. The diets were prepared using quinine hydrochloride (Sigma Chemical Co.), sucrose octa acetate (Nutritional Biochemicals Co.), and dry Purina meal. The appropriate amount of quinine (1 g in 999 g of meal) and SOA (10 g in 990 g of meal) was mixed thoroughly into the meal using a stirring tool attached to an electric drill. The efficacy of the mixing procedure was established by mixing 1 g of black powder (Sudan Black) in 999 g of meal and observing an even distribution of black particles throughout the diet. RESULTS AND DISCUSSION
When switched from the pellet to the powder form of the Purina diet the VMH obese rats were no longer hyperphagic and ate only slightly more than did the controls when food was available ad lib (19.1 vs 18.4 g/day), and significantly less than controls during the restricted feeding periods (3.9 vs 6.1 g/20 min, p<0.05). The results of the SOA vs quinine preference test are presented in Fig. 1. Analysis of variance indicated that during the 10 day test the VMH and control groups ate significantly more quinine diet than SOA diet (p<0.01), and that the VMH rats ate less food than did the controls (p<0.01). Examination of the individual intake scores revealed that five of the six VMH rats had a strong preference for the quinine diet, with quinine diet representing 78 to 91% of their total food intake, while one VMH rat had a weaker preference for the quinine diet (56%). In the control group, five of the six rats showed a very strong preference for the quinine diet (91 to 98%), but one rat preferred the SOA diet (quinine
,-, 5.0 v
i,i ,~
~--~ 0.1%QHCL ~1.0% m SOA
1
4.0
Z c~ 0 0 Ii
3.0-
z
2.0
o N
+
1.0
0 VMH
CON
FIG. 1. Mean ( _+ SEM) 20 min food intake of VMH (n=6) and
control (n=6) rats over 10 day preference test with 0.1% quinine hydrochloride (QHCL) diet and 1.0% sucrose octa acetate (SOA) diet.
diet intake 31% of total intake). The mean quinine diet preferences of the VMH and control groups, therefore, were similar (81.5 vs 84.9%). These results demonstrate that both VMH obese and control animals find a 0.1% quinine diet more acceptable, and presumably less bitter, than a 1.0% SOA diet during shortterm preference tests. The fact that the VMH obese rats ate less food during the 20 rain/day tests than did the controls is consistent with previous reports of decreased food motivation in hypothalamic obese animals (e.g., [30,34]).
EXPERIMENT 2 Based on the preference data obtained in Experiment 1 VMH obese rats would be expected to be more finicky to a 1.0% SOA diet during 24 hr intake (no choice) tests than they are to a 0.1% quinine diet. Our initial findings indicated that this is not the case, however, in rats tested first with the quinine diet and then with the SOA diet (Sclafani and Aravich, unpublished data). The present experiment attempted to replicate these results and controlled for the order of diet presentation. METHOD
Animals
Ten control and 12 VMH obese rats singly housed in standard-size mesh cages were used. The animals were
H Y P O T H A L A M I C F I N I C K I N E S S TO BITTER DIETS females of the C F E strain previously used in a study of food motivated bar pressing [29] and had no prior experience with adulterated diets. The VMH obesity was produced by placing bilateral parasagittal knife cuts between the medial and lateral hypothalamus according to the procedure of Sclafani [28]. The present experiment started 23 weeks after surgery at which time the V M H knife cut rats weighed considerably more than did the sham operated controls (443 vs 298 g, p <0.01), and were eating significantly more Purina chow diet (29.8 vs 18.4 g/day, p<0.01). Procedure
At the start of the experiment the rats were switched from Purina chow (pellets) to Purina meal (powder) which was presented in the feeding jars described in Experiment 1. After 12 days adaptation to the powdered diet the VMH and control groups were each divided into two subgroups equated for body weight and food intake. The VMH-1 and CON-1 subgroups were given a 0.1% quinine diet as their only food for three days, followed by five days on unadulterated powder diet (henceforth called plain diet), and then three days of a 1.0% SOA diet. The VMH-2 and CON-2 subgroups were similarly treated except that they received the SOA diet first and the quinine diet second. Following the second diet adulteration test the subgroups were given plain powdered food for six days and were then returned to the pellet diet. RESULTS The results of the diet adulteration tests are summarized in Fig. 2 and were evaluated using analysis of variance and inidividual t-tests. Data from one CON-1 rat was excluded because the animal inadvertently received the wrong test diet. The VMH-1 rats ate more plain diet than did the controls (0<0.01), but when offered the quinine diet they reduced their food intake more (0<0.01), and underate compared to the CON-1 rats (0<0.01). When subsequently offered the SOA diet the VMH-1 rats again reduced their food intake more (0<0.01) and underate (0<0.01) relative to controls. Examination of the daily intake scores (Fig. 2) revealed that the VMH-1 rats significantly (0<0.05) increased their SOA intake from Day 1 to Day 3 of testing, and by Day 3 their SOA intake was no longer significantly below that of the CON-1 rats. The VMH-1 rats ate more SOA diet than quinine diet, with this difference being significant for their intakes on the third day of each test (11.0 vs 5.8 g, p<0.05). The VMH-2 rats consumed more of the plain diet than did the CON-2 rats (0<0.01), and when offered the SOA diet they displayed no change in their intake and continued to overeat relative to controls (0<0.01), even on the first day of the diet (Fig. 2). When subsequently offered the quinine diet the VMH-2 rats reduced their food intake more than did the controls (0<0.05), but they ate the same amount of quinine diet as did the controls. Both subgroups decreased (0 <0.05) their quinine intake over the three day test (Fig. 2). The quinine diet intake of the VMH-2 rats was reliably less than their SOA diet intake (0<0.01). Compared to the VMH-1 subgroup the VMH-2 rats consumed significantly more SOA diet (22.2 vs 8.0 g/day, p<0.01) and quinine diet (12.0 vs 6.1 g/day, p<0.02). The plain diet intakes of the VMH-1 and VMH-2 subgroups (21.1 vs 21.9 g/day) were similar, as were their body weights (436.8 vs 438.8 g).
761
FIRST TEST
8
SECOND TEST
=JO
l_l.I 20
z (2) I0 0 0 I_i_
VMH-2 CON-2
VMH-2 CON-2
VMH-I CON-I
VMH-I CON-I
~0 I,I z0 z
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PLAIN DIET
~
TEST DIET
FIG. 2. Mean ( _+ SEM) 24 hr food intake of VMH and control subgroups during three day tests with 0.1% quinine hydrochloride (QHCL) diet and 1.{)% sucrose octa acetate (SOA) diet. VMH-1 (n=6) and CON-1 (n=4) subgroups were tested with the quinine diet first and the SOA diet second, while the VMH-2 (n=6) and CONo2 (n=5) subgroups were tested in the reverse order. Individual data points represent, from left to right, the mean intake on Days 1, 2 and 3 of the diet adulteration tests. The black bars represent the mean three day plain powder intakes prior to each diet test. At the start of the experiment when switched from the pellet to the powder form of the Purina diet the VMH and control rats decreased their food intake (0<0.01) but the V M H subgroups displayed significantly greater (p<0.01) reductions (VMH-I: 29.4 to 21.1 g/day; VMH-2:30.1 to 20.9 g/day, three day means) than did the control subgroups (CON-l: 17.7 to 17.6 g/day; C O N - 2 : 1 9 . 1 to 16.5 g/day). Conversely, when returned to the pellet diet at the end of the experiment the VMH subgroups increased (0<0.01) their food intake (VMH-I: 20.2 to 36.1 g/day; VMH-2:22.7 to 39.4 g/day) more than did the control subgroups (CON-l: 16.4 to 21.5 g/day; CON-2:17.2 to 22.6 g/day). DISCUSSION
The aversion displayed by the VMH-1 rats to both the quinine diet and the SOA diet confirms unpublished data obtained in our laboratory. However, the finding that the VMH-1 rats displayed a greater rejection of the quinine diet than of the SOA diet is inconsistent with the quinine diet preference observed in Experiment 1. Even more surprising are the results obtained with the VMH-2 rats. When first offered the SOA diet the VMH-2 rats displayed no aversion to the bitter food, and when subsequently given the quinine
762 diet they did not undereat it relative to the controls. Furthermore, the VMH-2 rats were less finicky to the quinine diet than were the VMH-1 rats. Thus, order of diet presentation is an important factor determining the reaction of VMH rats to bitter diets. Prior exposure to the quinine diet produced finickiness to the SOA diet, while prior exposure to the SOA diet reduced the finickiness to the quinine diet. The results of this experiment along with those of Experiment 1 seriously question the traditional interpretation of quinine diet finickiness. If VMH rats, and to a lesser extent control animals, reject a 0.1% quinine adulterated diet because of its bitter taste, then they should display a greater rejection of the less preferred 1.0% SOA diet, irrespective of its order of presentation. An alternative explanation of quinine diet finickiness is that it results, at least in part, from the postingestive effects of quinine. Although ignored in most taste experiments, quinine is known to have widespread systemic and central actions, and is fatal at high doses [27]. In particular quinine is a local irritant and when taken orally can produce gastric pain, nausea, and vomiting in humans [27]. Sucrose octa acetate, on the other hand, appears to be free of toxic effects [14,21], and can be ingested in large amounts by rats (see Experiment 3). Thus, the VMH-1 rats may have rejected the quinine diet because of its toxic postingestive effects, while the VMH-2 rats may have accepted the more bitter SOA diet because it did not produce such effects. The toxicity interpretation of quinine finickiness can account for the significant order effect observed in the present experiment. That is, if the VMH-1 rats rejected the quinine diet because of its aversive aftereffects, they should have developed a conditioned aversion to its bitter taste [11]. This would explain why they subsequently rejected the bitter, but nontoxic SOA diet, and extinction of the conditioned taste aversion can explain why their SOA diet intake increased over the three day test. The VMH-2 rats, on the other hand, when first offered the SOA diet should have learned that its bitter taste was safe since it was not associated with toxic aftereffects [17], and therefore the rats would have been less likely to form a conditioned taste aversion to the subsequently offered quinine diet. This would explain why the VMH-2 rats were less finicky to the quinine diet than were the VMH-1 rats, and extinction of the learned safety would explain why their quinine diet intake decreased over the three day test. Additional findings consistent with the toxicity interpretation of quinine finickiness can be cited. De Ruiter et al. [4] have noted that mice made obese with goldthioglucose (GTG) injections lose considerable body weight when maintained on a 0.4% quinine diet, but do not lose weight when given an equally bitter 1% SOA diet. Similarly, Kratz et al. [19] have reported recently that normal rats eat less, spill more food, and lose more weight when maintained on a 0.75% quinine sulfate diet than when given an equally bitter 4% SOA diet, and the authors implicated the pharmacological effects of quinine as being responsible for these results. Finally, we have found that normal rats (a) initially prefer a 0.1% quinine diet to a 1.0% SOA diet during 24 hr two-choice tests, but with continued experience with the diets they all switch their preference to the SOA diet over the quinine diet; (b) lose more weight when maintained on a 0.1% quinine diet (no choice) than when maintained on a 1.0% SOA diet; and (c) develop a conditioned aversion to a saccharin solution which is paired with the intragastric intubation of quinine (Aravich and Sclafani, unpublished data).
S C L A F A N I , ARAVICH A N D S C H W A R T Z In apparent conflict with the toxicity interpretation of quinine finickiness are the findings of two published studies. Epstein and Teitelbaum [5] reported that rats feeding themselves intragastrically did not decrease their food intake when the diet was adulterated with 0.05% quinine for three days, while rats feeding orally decreased their intake when given the same diet. However, since the intragastrically fed rats ate less food and were lower in body weight (see also [35]) than were the orally fed rats they may have been more resistant to the feeding suppressive effect of the quinine. More recently, Peck [23] has reported that quinine diet aversion is not due to postingestive effects because rats orally fed a 1.2% quinine diet spilled more food and lost more weight than did rats fed an unadulterated diet but intubated with equivalent amounts of quinine. However, the quinine intubated group appeared to be as hypophagic as the quinine fed group, and at least initially lost body weight. Also, the precise effects of the quinine intubation on food intake and body weight cannot be evaluated since there was no control intubated group. Furthermore, in neither of these two studies was quinine intubation paired with a novel taste stimulus so that the rats may not have formed a conditioned taste aversion, which may be the primary mechanism by which quinine adulteration reduces food intake.
EXPERIMENT 3 The failure of the VMH-2 rats in Experiment 2 to decrease their food intake when presented with the 1.0% SOA diet conflicts with the well known finickiness of VMH obese animals. It may be that the 1.0% diet is not bitter enough to reveal VMH finickiness, even though the diet is less preferred to the 0.1% quinine diet (Experiment 1), and is unpleasant to human taste. Experiment 3 examined this possibility by offering VMH and control rats more concentrated SOA diets. Since the previous results demonstrated the importance of the order of diet presentation, the rats were given the SOA diets in either increasing or decreasing concentrations. METHOD
Animals
The ten control and 12 VMH obese rats of Experiment 2 were used.
Procedure
Six days after the end of Experiment 2 the animals were returned to the powder diet for an adaptation period of 9 days. The VMH and control groups were then divided into new subgroups equated for food intake, body weight, and subgroup membership in Experiment 2. The VMH-A (body weight=450.0 g) and CON-A (294.0 g) subgroups were given SOA diets in an ascending order of 1%, 2%, 4%, 8% and 16% concentrations. Each SOA diet was presented for three days, and was followed by five days of plain powder diet to allow the rats to recover any weight lost on the SOA diet. The VMH-D (455.0 g) and CON-D (298.4 g) subgroups were tested in a similar manner except that the SOA diets were presented in a descending order of concentration. Following these SOA tests and five days on plain powder diet all subgroups were given the 16% SOA diet for three days, plain diet for five days, and a 24% SOA diet for three days.
H Y P O T H A L A M I C F I N I C K I N E S S TO BITTER DIETS
763
RESULTS The results of the first part of this experiment are summarized in Fig. 3. An overall analysis of variance indicated that the group (VMH vs CON) vs order (ascending vs descending) vs SOA concentration interaction was highly significant (p<0.001) and therefore the different subgroups are discussed separately. As illustrated in Fig. 3, the VMH-A rats ate more (p<0.01) food during the SOA tests than did the CON-A rats, and both subgroups showed significant (p<0.01), although relatively small decreases in intake as the SOA concentration increased (subgroup by diet interaction not significant). Thus, even on the 16% SOA diet the VMH-A rats consumed more (.0<0.05) than did the CON-A rats. Analysis of the VMH-D and CON-D results, on the other hand, revealed that the subgroup effect was not significant, but the diet effect and subgroup by diet interaction were (p<0.001). That is, while both groups increased their food intake as SOA concentration decreased, the VMH-D rats displayed greater changes than did the controls (p<0.01). Furthermore, whereas the VMH-D animals ate significantly (p <0.01) more than did the controls on the 0 to 2% SOA diets, they ate less than controls on the 10% diet, although this difference failed to be significant. A comparison between the VMH-A and VMH-D subgroups indicated that their overall food intake during the SOA tests did not reliably differ, but the intake of the VMH-D subgroup was more affected by concentration than was the intake of the VMH-A subgroup (p<0.001). In particular, the VMH-D rats ate significantly less (.o<0.05) of the 16% diet, but more (p<0.01) of the 1% and 2% diets than did the V M H - A subgroup. On the other hand, there was no significant difference between the CON-A and CON-D subgroups, and both decreased their food intake as SOA concentration increased. In the second part of the experiment when tested with the 16% and 24% SOA diets the intakes of the two V M H subgroups did not differ and therefore the rats were combined into one group. As illustrated in Fig. 4, when presented with the 16% diet both the VMH and control groups reduced their intake (p<0.001) and although the VMH rats displayed a greater reduction (p<0.01), their intake of the 16% SOA diet was slightly above that of the controls. When subsequently given the 24% SOA diet the VMH rats again reduced their food intake more 09<0.01) than did the controls, and now also ate significantly less (p<0.05) than did the controls.
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5 DAY PERIODS FIG. 3. Mean ( -+ SEM) 24 hr food intake of VMH and control subgroups during three day tests on diets adulterated with sucrose octa acetate (SOA) in concentrations ranging from 0 to 16%. VMH-A (n=6) and CON-A (n=5) subgroups received the diets in an ascending order of concentrations, while VMH-D (n = 6) and CON-D (n=5) subgroups received the diets in a descending order. -~ u 50 ,,,, < I--- 20
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DISCUSSION
The results of this experiment indicate that hypothalamic finickiness can be demonstrated with SOA adulterated diets but only at very high concentrations (16 or 24%). Furthermore, V M H obese rats, unlike controls, are sensitive to the order of diet presentation. When offered the SOA diets in increasing concentrations the V M H - A rats apparently habituated to the bitter taste of SOA and overate even the 16% diet, which is extremely bitter to human taste. The VMH-D rats, on the other hand, when presented with the diets in a descending order underate the 16% SOA diet relative to the controls and VMH-A animals. The enhanced aversion displayed by the VMH obese rats to the 16% and 24% SOA diets cannot be conclusively attributed to taste alone, because such high concentrations of SOA alter the nutritional composition of the diet. However, SOA is thought to be hydrolyzed into dextrose, fructose, and
I
PLAINDIET
["--I TEST DIET
FIG. 4. Mean ( _+ SEM) 24 hr food intake of VMH (n=12) and control (n=10) groups during three day tests with 16% and 24% sucrose octa acetate (SOA) diets. acetic acid, all of which are physiological and non-toxic substances [14], and there is little reason to suspect the postingestive effects of SOA as being responsible for the finickiness displayed by the VMH rats. EXPERIMENT 4 Recent studies have reported that maintaining VMH lesioned or knife cut rats on a quinine adulterated diet im-
764
S C L A F A N I , ARAVICH A N D S C H W A R T Z
mediately after surgery completely blocks the hyperphagia and obesity that would otherwise appear [6, 9, 34]. This effect has been attributed to the bitter taste of the quinine diet, but the results of the two preceding experiments question this interpretation. Experiment 4 tested the prediction that VMH damaged rats would overeat and gain weight when offered bitter SOA diets immediately following surgery. VMH electrolytic lesions were used in order to demonstrate that the effects obtained in Experiments 2 and 3 are not dependent upon the use of VMH knife cuts. METHOD A I1im a Is
Twenty-two female rats of the C F E strain weighing 240280 g were used. The rats were singly housed in wire mesh cages as in the previous experiments.
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16%
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= VMH CON
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5 DAY PERIODS
S u r g e l 3'
Bilateral VMH electrolytic lesions were performed under Equi-Thesin anesthesia (2.5 ml/kg bw) in 12 rats, while the remaining 10 rats received sham lesions. The VMH lesions were made by passing 1.5 mA anodal current for 30 sec through the exposed tip of a platinum-iridium electrode positioned at the DeGroot [3] atlas coordinates AP 5.8, H -3.0, L 1.0. At the end of the experiment the brains of the VMH lesioned rats were removed and histological sections through the lesion sites were prepared. Procedure
For eight days prior to surgery the rats were adapted to a 1.0% SOA diet presented in the feeding jars described in Experiment 1. Following surgery the rats were maintained on the 1.0% SOA diet for six days, and were then given, for three days each, SOA diets in concentrations of 2%, 4%, 8% and 16%. Next the rats were offered unadulterated diet for 6 days before being given a final three day test with the 16% SOA diet. RESULTS Data are presented from seven VMH lesioned rats and eight sham operated controls. The remaining seven rats were excluded from the study because of postoperative death or poorly placed lesions. The lesions of the seven rats included in the experiment were localized in the lateral aspect of the VMH and bilaterally destroyed the lateral edge of the ventromedial nucleus and the tisssue medial to the fornix. Preoperatively the VMH and control groups were well matched in food intake (19.8 vs 20.0 g/day) and body weight (259.4 vs 265.0 g). As illustrated in Fig. 5, following surgery the VMH rats increased their food intake by 71% and as a result rapidly gained weight (5.5 g/day) during the six days on the 1.0% SOA diet. The VMH rats then decreased their food intake and rate of weight gain as the SOA concentration of the diet increased. Compared to controls, however, the VMH rats overate (p<0.01) all of the SOA diets, even the 16% diet, although a significant group by diet interaction (p<0.01) indicated the VMH rats were more affected by the changes in SOA concentration than were the controls. By the last day of the 16% SOA diet the VMH rats weighed 308.4 g compared to the controls who weighed 278.6 g (p<0.05). When switched from the 16% SOA diet to the plain diet
FIG. 5. Mean ( _+ SEM) 24 hr food intake ofVMH (n=7) and control (n=8) groups during three day periods on diets adulterated with sucrose octa acetate (SOA) in concentrations ranging from 0% to 16%. Data from the first test (period 6) on the 16% SOA diet are presented twice in the figure. VMH electrolytic lesions and sham lesions were performed at time indicated by arrow. both groups increased their food intake (p<0.01), but the increase was significantly greater (p<0.01) for the VMH rats than for the controls (Fig. 5). When subsequently returned to the 16% SOA diet after six days on the plain food both groups decreased 09<0.01) their food intake, with the VMH rats again displaying the greatest change (p<0.01), and they now ate less (p<0.05) of the 16% diet than did the controls. The VMH rats also ate less (p<0.01) than they did when first offered the 16% SOA diet. DISCUSSION The results confirm the prediction that VMH damaged rats would overeat and gain weight when maintained on bitter tasting SOA diets following surgery. Therefore, the failure of the VMH damaged rats to overeat quinine diets postoperatively [6, 9, 34] does not appear to be due to the bitter taste of the diets, but rather may result from their postingestive effects. The VMH rats in the present experiment were finicky, however, in that their food intake was more influenced by SOA adulteration than was the intake of the control group. Furthermore, when returned to the 16% SOA diet after being on the plain diet for six days the VMH rats not only decreased their food intake more, but actually underate compared to the controls. The fact that they overate the 16% SOA diet the first time it was offered, but underate it the second time suggests that the VMH rats habituated to the bitter taste of SOA when presented with SOA diets of increasing concentration, which is consistent with the results obtained in Experiment 3. The VMH rats were heavier at the time of the second 16% SOA test than they were at the time of the first test (342.3 vs 299.3 g), and increased body weight may therefore have contributed to their rejection of the 16% SOA diet during the second test. G E N E R A L DISCUSSION The exaggerated quinine diet aversion of VMH-damaged
H Y P O T H A L A M I C F I N I C K I N E S S TO BITTER DIETS rats was first demonstrated in 1950 [22] and has been confirmed in numerous experiments since then. The implicit assumption of these experiments is that the small amounts of quinine used to adulterate food do not have postingestive consequences, but this assumption is questionable in light of recent findings [19]. The present results provide strong, albeit circumstantial, evidence that VMH finickiness to quinine adulterated food is due to the toxic rather than the taste effects of quinine. Further research is needed, however, to document the postingestive effects of quinine adulterated diets. The V M H rat's aversion to quinine solutions [2] should also be reexamined in view of these results. If, as the present findings suggest, VMH rats reject quinine diets because of their toxic aftereffects this does not contradict the motivational interpretation of quinine-diet finickiness. That is, VMH obese rats may undereat quinine diets because their reduced food motivation makes them less tolerant of the toxic effects produced by the quinine. Presumably the toxicosis produced by the quinine diets (0.1 to 0.2%) typically used in VMH studies is relatively mild since control rats lose little or no weight on these diets, and both control and VMH animals can be maintained on these diets for several weeks without obvious ill-effects [6,34]. Furthermore, VMH rats will overeat and gain weight on quinine adulterated foods if the rats are ovariectomized [9], or if the palatability of the diet is improved by increasing its fat content [24]. Nevertheless, the postingestive effects of quinine appear sufficiently aversive to produce anorexia in motivationally weakened animals, such as VMH obese, dietary obese, and recovered L H lesioned rats [7, 33, 34, 37]. However, the possibility that VMH damage or obesity exaggerate the toxic effects of quinine, for example by altering gastrointestinal motility [26], or the conditionability of the animal to aversive visceral stimuli [38,40] (but see [12,25]) cannot be discounted. In addition to challenging the traditional interpretation of quinine diet aversion the present results raise questions concerning the concepts of hypothalamic finickiness and diet palatability. The fact that VMH damaged rats, both static and dynamic, overeat diets adulterated with as much as 16% SOA indicate either that VMH rats are much less finicky to unpalatable diets than previously thought, or that the bitter taste of SOA has only minimal effects on diet palatability. The second possibility is suggested by the findings that the VMH rats in Experiment 2 significantly reduced their food intake (by 28%) when diet texture was changed from pellet to powder, but yet did not reduce their intake when the food was made bitter with 1% SOA adulteration (VMH-2 subgroup).
765 Rats and other animals appear to have an innate aversion to bitter taste [10], but they also appear to adapt or habituate to it. Guinea pigs, for example, display a strong aversion to SOA solutions in two bottle preference tests with water, but if they have been maintained on a S O A solution for some time, this aversion disappears [39]. Preliminary findings in our laboratory indicate that a similar effect can be obtained with rats using SOA adulterated food. The VMH-2 rats in Experiment 2, therefore, may not have appeared finicky to the 1% SOA diet because they rapidly habituated to its bitter taste. It is possible that a finer temporal analysis, i. e. an examination of individual meals, would have revealed a finickiness to the SOA diet. In support of this suggestion, De Ruiter et al. [4] have reported that, compared to controls, GTG obese mice display a significantly greater reduction in the size of their first meal (following a 24 hr fast) when switched to a 0.5% SOA diet although their long term food intake and body weight are not affected by SOA adulteration. With SOA diets more concentrated than 1% habituation to the bitter taste may be slower and less complete, and this would explain the significant diet concentration and order effects obtained in Experiments 3 and 4. Thus, when SOA concentration was gradually increased the VMH rats overate even the 16% SOA diet, but when the 16% SOA diet was the first presented (Experiment 3), or was returned after a period on plain food (Experiment 4), the V M H rats underate it relative to controls. In contrast to the situation with SOA diets animals would not be expected to habituate to the bitter taste of quinine adulterated diets if the quinine produces toxic postingestive effects. In fact, their aversion to the taste should increase rather than decrease as they eat the diet, experience its toxic effects, and develop a conditioned taste aversion. Consistent with this interpretation are our findings that normal rats eventually reverse their initial preference for the 0.1% quinine diet over the 1.0% SOA diet (Aravich and Sclafani, unpublished data). Thus, in the long run, the palatability of food, at least in the case of bitter diets, may be more determined by its postingestive rather than preingestive (orosensory) effects. Garcia [11], Cabanac [11 and others have also emphasized the importance of postingestive events in determining the hedonic response to food. The results of this study, therefore, do not require a revision of the concept of hypothalamic finickiness: V M H hyperphagia and obesity remains highly dependent upon diet palatability. However, the concept of diet palatability, in particular the determinants of palatability, should be reevaluated in view of the present findings.
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