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APPETITE, OBESITY, DIGESTION, AND METABOLISM
Department of Psychological Sciences, Ingestive Behavior Research Center, and Integrative Graduate Program in Neuroscience, Purdue University, West Lafayette, Indiana 47907
Submitted 22 January 2004 ; accepted in final form 12 July 2004
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ABSTRACT |
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 TOP ABSTRACT EXPERIMENT 1: ELECTRONIC...EXPERIMENT 2: MACRONUTRIENT...EXPERIMENT 3: LEARNING ABOUT...EXPERIMENT 3aEXPERIMENT 3bEXPERIMENT 4: DUODENAL INFUSIONS...EXPERIMENT 4aEXPERIMENT 4bGENERAL DISCUSSIONGRANTSREFERENCES |
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food preference; duodenum; gustatory; macronutrients; Pavlovian conditioning
The experimental paradigm in which ingestion of arbitrary flavors is paired with intragastric (IG) infusions of particular macronutrients or diets (the "electronic esophagus" preparation) (6) has been used effectively to analyze what postoral effects different nutrients can have on the subsequent preferences for different flavors or tastes. The electronic esophagus strategy makes it practical to vary independently the stimuli in the mouth and stimuli in the GI tract, and the paradigm also makes it possible to specify the timing of the oral and postoral stimuli so that Pavlovian conditioning can be used to analyze underlying mechanisms. To take advantage of this preparation and to extend its capabilities, we have combined the electronic esophagus preparation with the classical conditioned taste aversion paradigm (11) into a conditioned intestinal taste aversion paradigm. With our combined protocol it is possible to ask whether the postoropharyngeal GI tract independently "tastes" nutrients. In the event that it does, the intestinal taste aversion paradigm also makes it possible to analyze what is learned about nutrients and how this information is used.
The present series of four experiments demonstrates that when a nutritive taste stimulus is delivered into either the stomach or the duodenum and is followed by lithium chloride-induced illness, oral preference for that same nutrient is subsequently reduced during the first period of contact by mouth. The experiments also illustrate that this GI taste may be specific to the nutrient composition of the stimuli infused, insofar as the intestinal taste aversion technique altered preference for two different macronutrients but not two pairs of nonnutrient flavor stimuli. The findings indicated that poisoning produced a preference shift in consumption of nutrients for which rats had no apparent prior oral experience. This outcome was replicated in several experiments that were designed to render implausible different potential accounts of how this preference shift might be attributable to direct contact between the IG infusates and oropharyngeal receptors at the time of poisoning.
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EXPERIMENT 1: ELECTRONIC ESOPHAGUS BASELINES |
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TOPABSTRACTEXPERIMENT 1: ELECTRONIC...EXPERIMENT 2: MACRONUTRIENT...EXPERIMENT 3: LEARNING ABOUT...EXPERIMENT 3aEXPERIMENT 3bEXPERIMENT 4: DUODENAL INFUSIONS...EXPERIMENT 4aEXPERIMENT 4bGENERAL DISCUSSIONGRANTSREFERENCES |
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Briefly, the flavors or conditioned stimuli (CSs) provided for oral consumption were the arbitrary nonnutritive tastants used by Lucas and Sclafani (19), and the IG-delivered infusates or unconditioned stimuli (UCSs) were either fat (corn oil) or carbohydrate (maltodextrin), also employed by Lucas and Sclafani. All animals were trained to associate one Kool-Aid flavor (CS+Fat) with an IG infusion of a corn oil emulsion and a second flavor (CS+Carb) with an equicaloric maltodextrin infusion. A third flavor (CS–) was paired with IG infusion of water. A subsequent test phase consisted of three separate two-bottle choice tests in which intakes of CS+Carb, CS+Fat, and CS– were compared.
Methods
Subjects. Subjects were twelve 90-day-old (275–300 g) naive male Sprague-Dawley albino rats (Harlan Sprague Dawley, Indianapolis, IN). They were housed individually in stainless steel cages in a colony with a 12:12-h light/dark cycle (lights on 0700). Water was available at all times. Food availability was as described below.
Surgery. Approximately 1 wk after arrival, animals were fasted overnight and surgically equipped with gastric catheters. Each animal was anesthetized (pentobarbital sodium, 60 mg/kg ip), then laparotomized, and a Silastic catheter (ID = 0.025 in.; OD = 0.047 in.) was introduced through a puncture wound in the greater curvature of the nonglandular stomach and advanced 1 cm into the organ. The catheter was secured in place by a stay suture attached to Marlex mesh on the serosal surface of the stomach. The entry point of the catheter on the gastric wall was closed with two concentric purse-string sutures, and a serosal tunnel on the fundus of the stomach was formed around the catheter with interrupted sutures. The catheter was then passed through the left abdominal wall and tunneled subcutaneously to a backmount (Instech Solomon, Plymouth Meeting, PA). Rats returned to presurgical body weight 7–10 days postsurgery while being maintained on ad libitum chow in their home cages. Catheters were flushed daily with 0.5–1.0 ml saline beginning 24 h after surgery.
Apparatus. Conditioning procedures were conducted in four identical cylindrical chambers (height, 16 in., diameter, 12 in.), constructed of clear Plexiglas with stainless steel grid floors. Two drinking bottles were mounted on the front of the cage, 1.5 in. from the floor and 2 in. apart, with openings 1 in. x 2 in. Spouts from the bottles protruded into the center of recessed areas 2 in. deep that the rats could access inside the cage. The animal's catheter was passed through a stainless steel spring, which was anchored to the backmount, and attached to an overhead swivel, allowing the animal to move freely about the test chamber. The tubing was attached to a 60-ml syringe in a syringe pump (Razel Scientific Instruments, Stamford, CT). The syringe pump was set at an infusion rate of 1.8 ml/min. It was activated by an animal licking at one of the drinking spouts in the cage, and the infusion stopped anytime the animal did not lick the spout for 1 s. Overall, this protocol generated a ratio of 0.58 ± 0.18 ml macronutrient infused per 1.0 ml nonnutrient flavor solution consumed. The monitoring equipment and computer software (GraphicState) were purchased from Coulbourn Instruments (Allentown, PA).
Oral and IG stimuli. Oral stimuli consisted of 0.2% sodium saccharin (Sigma, St. Louis, MO) solutions flavored with 0.05% cherry, grape, or orange unsweetened Kool-Aid (Kraft Foods, White Plains, NY). The IG carbohydrate stimulus was a 16% maltodextrin solution (Polycose, Ross Laboratories, Columbus, OH). The IG fat stimulus was an emulsion of 7.1% corn oil (Mazola, Bestfoods, Englewood Cliffs, NJ) and 0.6% emulsifier (Emplex, American Ingredients, Grandview, MO). The pH values for the two solutions were 7.31 for Polycose and 7.08 for corn oil. Both solutions had a viscosity of <100 cP (Brookfield Digital Viscometer Model DV-I+). The oral stimuli paired with fat, carbohydrate, and water infusion will be referred to as CS+Fat, CS+Carb, and CS–, respectively. Flavors paired with each IG stimulus were counterbalanced across subjects.
Pretraining procedure.
The rats were randomly assigned to one of two pretraining groups (n = 6 each) before being gradually reduced to 85% of their ad libitum postsurgical body weight. All rats were maintained at this weight throughout habituation, training, and testing. Based on the habituation procedure reported by Lucas and Sclafani (19), the long habituation group (group LH) was presented with two overnight exposures to 
30 ml of unflavored saccharin solution in a bottle on the front of their home cage. This exposure was followed by six daily 30-min habituation sessions in the conditioning chambers during which the animals were allowed unlimited access to the saccharin solution, but there was no IG infusion. Three daily 30-min sessions followed in which the animals were presented again with the unflavored saccharin solution and were infused IG with plain water on the schedule described above. Rats in the short habituation group (group SH) were given an abbreviated version of this procedure. Group SH received one overnight exposure to each of the three flavored CS solutions presented in a bottle on the home cage in counterbalanced order, followed by one 30-min habituation session in the conditioning chamber during which the animals were attached to the apparatus, but not presented with any oral or IG stimuli. These sessions for group SH were conducted coincident with the final four habituation days for group LH, so that training began on the same day for all animals. The rats were run in squads of four animals.
Training procedure.
The training procedure was the same for both groups. All animals were weighed and catheters were flushed with 0.5 ml saline before being attached to the infusion apparatus. Half the animals received the CS+Carb (with IG maltodextrin) on day 1 of training, and the other half received the CS+Fat (with IG corn oil). On day 2, all animals were presented with their CS– and water infusions. On day 3, animals received the opposite of what they were given on day 1 (i.e., CS+Carb and IG Polycose if they had CS+Fat and IG corn oil on day 1 and CS+Fat and IG corn oil if they had CS+Carb and IG maltodextrin on day 1). This 3-day sequence was repeated four times for a total of twelve 30-min training sessions. The placement of the drinking spout in the left or right slot was determined randomly each day. The amount of the CS consumed was determined by weighing bottles before and after each session. The amount of the UCS infused was determined by recording the volume on the syringe. To reduce the possibility of oral contact with the IG infusates, as each rat was detached from the apparatus, the catheter and area around the backmount was wiped with a paper towel and the catheter was again flushed with 0.5 ml saline. Three hours after each daily training session, the animals were fed their daily chow ration.
Test procedure. After training, all rats were given a 30-min two-bottle test session for each of six consecutive days. Half the animals were given a choice between the CS+Fat and the CS– for the first 2 days followed by the CS+Carb and the CS– for 2 days. The remaining half of the animals were given the CS+Carb and CS– test first followed by the CS+Fat and CS– test. All rats were then given the CS+Fat and the CS+Carb for 2 days. Left-right bottle position was determined randomly on the first day and alternated daily during testing. No IG infusions were given during test sessions.
Data analysis. Data in all experiments were analyzed using repeated-measures ANOVA. Individual comparisons were made using simple main effects or Newman-Keuls tests where appropriate. Significance level was set at P < .05 for all comparisons.
Results and Discussion
Three animals failed to complete the training protocol due to backmount failures. Their data were discarded. Final group sizes were n = 4 for LH and n = 5 for SH.
Training. Intake of the CS+Fat, CS+Carb, and CS– across 12 training sessions (4 per CS) is shown in Fig. 1. The animals developed a greater acceptance of the CS+Carb than either the CS+Fat or CS– in single-bottle training, although it appears that acceptance of all CSs increased over sessions. These observations were confirmed by an ANOVA, which indicated main effects of both session [F(3,15) = 6.94, P < 0.01] and CS type [F(2,10) = 14.42, P < 0.01]. Newman-Keuls analyses confirmed that intake of CS+Carb was significantly greater than intake of CS+Fat or CS–, whereas intake of CS+Fat and CS– did not differ. There was also a main effect of habituation treatment [F(1,5) = 9.67, P < 0.05] based on animals in group SH drinking more of all CSs than group LH. However, habituation treatment did not interact significantly with sessions or CS type.
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EXPERIMENT 2: MACRONUTRIENT INTAKE ALTERED BY INTESTINAL TASTE AVERSION |
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TOPABSTRACTEXPERIMENT 1: ELECTRONIC...EXPERIMENT 2: MACRONUTRIENT...EXPERIMENT 3: LEARNING ABOUT...EXPERIMENT 3aEXPERIMENT 3bEXPERIMENT 4: DUODENAL INFUSIONS...EXPERIMENT 4aEXPERIMENT 4bGENERAL DISCUSSIONGRANTSREFERENCES |
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Lucas and Sclafani (19) suggested that carbohydrates may be inherently more reinforcing than fats. However, it is also possible that, in addition to any difference in reinforcement, these macronutrients also affect preference by producing distinct interoceptive stimuli that are detected in the gut. To investigate this possibility more directly, in the present experiment we differentially associated the postoral stimuli produced by IG infusion of maltodextrin and corn oil, respectively, with illness (induced by injection of LiCl).
To accomplish this, naive rats were first given flavor-nutrient training, similar to that described in experiment 1. Then the rats received additional training in which licking produced two flavor CSs, one of which was paired with IG infusion of the maltodextrin, and the other paired with IG infusion of the corn oil. After each training session with one CS and IG infusate, the rats were injected with LiCl to induce mild malaise. After training sessions with the other CS and IG infusate, the rats were given an intraperitoneal saline injection. The effects of this differential training were evaluated with three types of two-bottle preference tests as well as with a single-bottle test. The different tests were designed to determine whether this type of intestinal taste aversion procedure altered oral preference for 1) the CSs that were consumed during intestinal taste aversion training of the IG infusates; 2) the originally trained CSs that were associated with IG infusions, but were not consumed during intestinal taste aversions training; and 3) the previously unconsumed macronutrient that had been infused IG and poisoned.
Methods
Subjects. Subjects were 32 naive animals of the same description and maintained under the same conditions as experiment 1. Two procedurally identical replications of 16 animals each were run.
Surgery and apparatus. Animals underwent the same surgical procedures and were trained and tested using the apparatus described for experiment 1.
Stimuli. Oral stimuli were 0.2% sodium saccharin solutions flavored with grape or cherry (first phase) and lemon-lime or orange (second phase) Kool-Aid. IG stimuli were the same nutrient solutions described in experiment 1. LiCl (0.15 M; Sigma, St. Louis, MO) was delivered intraperitoneally at a dose of 20 ml/kg. The pairings of flavored oral stimuli with IG infusates and drug stimuli were counterbalanced across animals.
Procedure. After recovery from surgery, animals were reduced to 85% of their ad libitum postsurgical body weights. They then received one overnight exposure each to 30 ml of the grape- and cherry-flavored saccharin solutions presented in counterbalanced order in the home cage. The animals then had one 30-min habituation session in which they were placed in the conditioning chamber and attached to the apparatus, but no oral or IG stimuli were presented. After this were eight daily 30-min training sessions in which the animals were provided with either the CS+Carb and IG maltodextrin or the CS+Fat and IG corn oil. The CS-nutrient pairs were presented on alternating days, for a total of four training sessions with each nutrient. The CS-nutrient pair used on the first day was counterbalanced across animals.
Animals were then given a single 30-min test session in which they were presented with both the CS+Fat and the CS+Carb, but there were no IG infusions. After this first test session, animals were given exposure to the novel lemon-lime- and orange-flavored solutions. First, they were given a 30 ml overnight exposure to each flavor in the home cage followed by one 30-min exposure session with each flavor in the conditioning chamber (counterbalanced order, no IG infusion). Animals were then given two aversion-training sessions. In these sessions, the animals received one of the new oral stimuli (counterbalanced) to drink paired with IG maltodextrin in one session and the other flavor paired with IG corn oil in the second session (counterbalanced order). For half of the animals, after the session in which they received IG maltodextrin, they were given an intraperitoneal injection of LiCl and given an intraperitoneal injection of saline after the session in which they received IG corn oil. The other half of the animals received the reverse nutrient-drug contingency (corn oil-LiCl, maltodextrin-saline). To assess the possibility that these nutrients are detected in the gut over different time courses, half of the animals in each of these groups received injections 15 min after the end of the session, while the other half received injections 4 h after the end of the session.
One 30-min test session was then conducted in which the animals were presented with the lemon-lime and orange solutions with no IG infusions. The 3-day aversion training and two-bottle test sequence was then repeated. Animals were then tested with the original CS+Carb and CS+Fat in a 30-min two-bottle test with no IG infusions. After this, two sets of single bottle tests were given: first, the CS+Carb and CS+Fat paired with the appropriate IG infusion (one session each, counterbalanced order), then the CS+Carb and CS+Fat alone with no IG infusions (one session each, counterbalanced order). Finally, all animals were presented with a two-bottle test in which they were allowed to consume orally the two infusates from bottles on the front of the home cage. Intake of the infusates was measured at 30 min and 1, 2, and 4 h.
Results and Discussion
Flavor-nutrient training. CS+Carb and CS+Fat intakes across the four flavor-nutrient training sessions are shown in Fig. 3. These data are similar to those in experiment 1, with greater intake of the CS+Carb than CS+Fat, but increasing intake of both CSs across training sessions. A repeated-measures ANOVA yielded main effects of session [F(3,90) = 53.47, P < 0.01] and CS type [F(1,30) = 61.56, P < 0.01], confirming the above observations. Poisoned nutrient (fat vs. carbohydrate) was included as a dummy variable. The lack of any effect of this variable indicates that both fat-poisoned and carbohydrate-poisoned groups demonstrated equivalent flavor-nutrient learning.
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Figure 4C depicts the results of the postaversion two-bottle CS+Carb vs. CS+Fat test, in which these two solutions were again presented to the animals in the absence of any IG infusion. The results of this test were very similar to those seen in the preaversion test, with poisoning of the paired nutrient having little effect on intake of these previously associated flavors. Confirming this, an ANOVA yielded no effect of poisoning [F(1,28) = 0.81, P > 0.05] but did yield a main effect of CS type [F(1,28) = 157.7, P < 0.05], indicating that the overall preference for CS+Carb compared with CS+Fat was maintained and not affected by having had one of these nutrients poisoned. In addition, single-bottle tests in which the animals were presented with either the CS+Carb or CS+Fat in separate sessions, one with no IG infusion and one with the appropriate IG infusion, produced no differences in intake based on whether the animals had been poisoned with IG corn oil or IG maltodextrin (data not shown, largest F = 0.68, P > 0.05).
Figure 5 shows the results from the first 30 min of the two-bottle test comparing consumption of the corn oil infusate with the maltodextrin infusate when animals were allowed to consume these solutions orally for the first time. Mean intake of each infusate for the poisoned and nonpoisoned treatment conditions, along with statistical analysis, is presented in the figure legend. Analysis of these data yielded a main effect of poisoning (poisoned vs. nonpoisoned solution) [F(1,27) = 10.25, P < 0.01], indicating that, overall, animals consumed more of the infusate paired with saline than that paired with LiCl. In addition, there was an interaction between poisoned intake and poisoned nutrient type (fat poisoned vs. carbohydrate poisoned) [F(1,27) = 93.65, P < 0.01], reflecting the fact that all animals consumed more of the maltodextrin solution, but that the difference between maltodextrin and corn oil intake was smaller for animals that had IG maltodextrin poisoned. Finally, there was a significant interaction between poisoning and injection delay [F(1,27) = 5.59, P < 0.05], indicating that animals given IG maltodextrin-LiCl training drank less during this test if they were injected at 15 min compared with 4 h, while animals given IG corn oil paired with LiCl drank less of the infusates if they had been injected at 4 h compared with 15 min. This may reflect differences in the temporal parameters of the stimulation produced by the two IG infusates. That is, the postoral stimulus effects of the maltodextrin solution may have been more readily detected, and thus associated with the illness, at 15 min postinfusion, while the corn oil was more readily detected after 4 h. However, this interpretation is not entirely supported by these results, as there did not seem to be greater learning about the stimuli at different times (i.e., greater suppression of the poisoned solution relative to the nonpoisoned solution), only greater overall suppression of both solutions.
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In summary, when animals had an opportunity to sample orally and ingest the two macronutrients (one of which had been poisoned) for the first time, they showed a significant reduction in preference for the poisoned nutrient. This indicates that 1) postoral stimulation by corn oil and maltodextrin is detectable and the two nutrients can be discriminated from one another and 2) learning about these postoral consequences can be translated into effects on oral consumption, even in the absence of prior oral experience with these solutions. In contrast, the prediction that the animals would learn an aversion to stimulation produced by gastric nutrient infusions and demonstrate that aversion by avoidance of arbitrary nonnutritive CSs associated with that same nutrient stimulus was not supported by the results of this experiment. In fact, in all tests of CS intake, preference for the CS+Carb was consistently demonstrated and seemed to be unaffected by which nutrient had been paired with LiCl.
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EXPERIMENT 3: LEARNING ABOUT INTESTINAL TASTES—TWO CONTROLS |
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TOPABSTRACTEXPERIMENT 1: ELECTRONIC...EXPERIMENT 2: MACRONUTRIENT...EXPERIMENT 3: LEARNING ABOUT...EXPERIMENT 3aEXPERIMENT 3bEXPERIMENT 4: DUODENAL INFUSIONS...EXPERIMENT 4aEXPERIMENT 4bGENERAL DISCUSSIONGRANTSREFERENCES |
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EXPERIMENT 3a |
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TOPABSTRACTEXPERIMENT 1: ELECTRONIC...EXPERIMENT 2: MACRONUTRIENT...EXPERIMENT 3: LEARNING ABOUT...EXPERIMENT 3aEXPERIMENT 3bEXPERIMENT 4: DUODENAL INFUSIONS...EXPERIMENT 4aEXPERIMENT 4bGENERAL DISCUSSIONGRANTSREFERENCES |
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To assess this learned safety hypothesis, in the present experiment we conducted intestinal taste aversion training first, pairing IG infusions of one of the two macronutrients with LiCl injections and the other with saline in the absence of any oral stimuli to maximize learning about the postoral nutrient stimulation. The animals were then trained in the "electronic esophagus" or flavor-nutrient learning paradigm. We predicted that the postoral stimulation of the nutrient poisoned during the prior intestinal taste aversion training would be a less effective UCS for flavor preference conditioning than the nonpoisoned nutrient.
Finally, we used a summation test as a further means of assessing learning about flavor CSs that have been associated with different IG UCSs. If poisoning the IG UCS has a decremental effect on the ability of its associated flavor CS to elicit intake, then adding this CS flavor to the poisoned infusate should reduce intake of that infusate more than adding the flavor CS that was not associated with the poisoned IG UCS.
Methods
Subjects. Subjects were 24 naive animals of the same description and maintained under the same conditions as described for experiment 1.
Surgery and apparatus. Animals underwent the same surgical procedures and were trained and tested using the same apparatus as described for experiment 1.
Stimuli. Oral and IG stimuli were the same as those used in experiment 1.
Procedure. After recovery from surgery, animals were reduced to 85% of their ad libitum postsurgical body weights. They then received two 30-min habituation sessions in which they were placed in the conditioning chamber and attached to the apparatus, but no oral or IG stimuli were presented. Animals were then given four aversion-training sessions. In these sessions, animals were placed in the infusion cages for 20 min and infusion of either 7.1% corn oil emulsion (2 sessions) or 16% maltodextrin (2 sessions) was administered for 10 s out of every minute at a rate of 1.8 ml/min for a total infusion volume of 6 ml. The session length and infusion volumes were based on data from aversion sessions in experiment 2, in which animals determined their own infusion volumes. Half of the rats were given an intraperitoneal injection of LiCl after the sessions in which they received IG maltodextrin and were given an intraperitoneal injection of saline after the session in which they received IG corn oil. The remaining rats received the reverse nutrient-drug contingency (corn oil-LiCl, maltodextrin-saline). After these pairings, all rats were given eight 30-min flavor-nutrient training sessions similar to those in experiment 2. The CS+Carb and CS+Fat (grape and cherry saccharin solutions, counterbalanced) were given alternately in separate sessions and paired with the appropriate IG infusion, using the parameters given in experiment 2.
After completion of this training phase, animals were given successively three types of tests. First, the animals were given one 30-min two-bottle preference test between the CS+Carb and CS+Fat with no IG infusions (an extinction test). Second, for an infusate oral intake test, a 4-h two-bottle preference test was then given in the home cage. Animals were presented with the corn oil emulsion and maltodextrin solution that had been previously administered exclusively IG, and they were allowed to orally consume these nutrients. Intake was measured at 30 min and 1, 2, and 4 h. Finally, the animals were given a set of two-bottle summation tests. All of these tests took place in the home cage and lasted 1 h, with intake measured at 30 min and 1 h. Flavors mixed into the nutrient solutions were 0.05% Kool-Aid. No saccharin was added to these solutions. On the first day, half the animals received one bottle containing the nutrient solution that had been paired with LiCl and one bottle containing that nutrient solution mixed with its associated CS flavor (i.e., CS+Carb for maltodextrin and CS+Fat for corn oil). The remaining half of the animals also received one bottle containing their poisoned nutrient solution and a second bottle containing that nutrient mixed with the opposite CS (i.e., CS+Carb for corn oil and CS+Fat for maltodextrin). On the second day, all animals again received one bottle of their poisoned nutrient solution and the second bottle contained that nutrient solution mixed with the opposite CS flavor from that given on the first day. On the third day, all animals received two bottles of their poisoned nutrient solution, one mixed with each of the two CS flavors.
Results and Discussion
Flavor-nutrient training. Figure 6A shows the results of four sessions of flavor-nutrient conditioning after intestinal taste aversion training with one of the two IG nutrients. Because we were interested in the effects of IG nutrient poisoning on learning these flavor associations, these data are presented as CS+poisoned nutrient and CS+nonpoisoned nutrient. Analysis of these data yielded no effect involving this poisoning variable [largest F = 1.51], indicating that flavor-nutrient learning was not affected by one of the IG nutrients being previously paired with LiCl. There was a main effect of nutrient [F(1,20) = 20.64, P < 0.01], due to an overall greater intake of the CS+Carb, and a main effect of session [F(3,60) = 121.69, P < 0.01], due to increasing intake as training progressed. These results indicate that this flavor-nutrient training was very similar to that seen in experiments 1 and 2, in which there was no prior experience with the IG nutrients.
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Infusate oral intake test. Intake during the first 30 min that the animals were offered the two infusates (previously only administered IG) for oral consumption is presented in Fig. 7. Mean intake of each infusate for the poisoned and nonpoisoned treatment conditions, along with statistical analysis, is presented in the figure legend. As in experiment 2, oral intake of the previously poisoned nutrient was suppressed relative to the nonpoisoned nutrient. ANOVA confirmed this observation, indicating a main effect of poisoning [F(1,22) = 16.86, P < 0.01]. There was also a main effect of nutrient [F(1,22) = 39.44, P < 0.01], indicating that animals consumed more of the carbohydrate solution than the fat solution regardless of which nutrient was poisoned. Furthermore, ANOVA yielded a significant nutrient x poisoned nutrient interaction [F(1,22) = 16.86, P < 0.01]. Post hoc analysis revealed that intake of maltodextrin was significantly greater than intake of corn oil only for rats that had been poisoned after IG corn oil infusions. This result indicated that IG maltodextrin poisoning was able to attenuate the apparent strong preference for the carbohydrate solution. This result also demonstrates that the learned association between LiCl and the orally detected components of these stimuli was maintained in the absence of the poison during the flavor-nutrient training.
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EXPERIMENT 3b |
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TOPABSTRACTEXPERIMENT 1: ELECTRONIC...EXPERIMENT 2: MACRONUTRIENT...EXPERIMENT 3: LEARNING ABOUT...EXPERIMENT 3aEXPERIMENT 3bEXPERIMENT 4: DUODENAL INFUSIONS...EXPERIMENT 4aEXPERIMENT 4bGENERAL DISCUSSIONGRANTSREFERENCES |
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The purpose of experiment 3b was to provide a more direct assessment of the reflux hypothesis. In this study, animals were trained using the same aversion procedure described for experiment 3a. However, in experiment 3b, the IG infusates were the same nonnutritive stimuli (i.e., lemon-lime- and orange-flavored saccharin solutions) that were used in experiment 2 to verify the efficacy of the LiCl. Thus it is clear that these stimuli are distinguishable when presented to rats orally. If LiCl injection produces reflux of these infusates, then the rats should be able to taste these flavors and associate them with illness. Accordingly, the rats should exhibit a preference for their nonpoisoned compared with their poisoned infusate when given the opportunity to orally ingest each substance. On the other hand, no preference between the poisoned and nonpoisoned infusates would make the hypothesis that the rats tasted the infusates during aversion training less plausible. Rather this outcome would bolster the idea that IG infusates differ in the extent to which they give rise to distinct nutritive postoral sensory consequences that can be associated with other events and that carbohydrates and fats may serve as more salient stimuli than the nonnutritive flavors.
Method
Subjects. Subjects were the same as those used in experiment 3a.
Stimuli. Stimuli were 0.2% saccharin solutions flavored with 0.05% lemon-lime- or orange-flavored Kool-Aid.
Procedure. As in experiment 3a, animals were given four 20-min training sessions. During two of these sessions, they received IG infusions of either the lemon-lime- or the orange-flavored saccharin solution using the same infusion parameters as for the maltodextrin and corn oil in experiment 3a. In the two alternating sessions, they received infusions of the opposite flavored solution. No oral stimuli were provided for the animals to consume during these sessions. Fifteen minutes after each session, all animals were given an intraperitoneal injection of either saline or LiCl. Animals were divided into two equal groups, counterbalanced with respect to the nutrient that had been poisoned in experiment 3a. For one group of animals, the lemon-lime solution was paired with LiCl and the orange solution with saline. The remaining animals were given the opposite pairings. The aversion training sessions were conducted under the same conditions as in experiment 3a. After this training, a two-bottle test session was conducted in the home cage in which the orange and lemon-lime solutions were presented simultaneously with no IG infusions, and intake of the solutions was measured at 30 min and 1, 2, and 4 h.
Results and Discussion
It can be seen in Fig. 9 that intake of the two flavored solutions did not differ during the first 30 min of the test session in which the two flavored saccharin solutions were presented orally. This observation was confirmed by an ANOVA that yielded no effect of poisoning and no significant interactions with poisoned flavor or previously poisoned nutrient (largest F = 0.22, P < 0.05). The low level of intake at 30 min leaves open the possibility that the failure to observe an aversion effect is due to a floor effect. However, this concern is reduced by the fact that there was no significant difference between intake of the poisoned and nonpoisoned flavors at any time point, although cumulative intake climbed to a mean of 37.4 g over 4 h (poisoned = 34.7 ± 5.0 g, nonpoisoned = 40.0 ± 4.4 g).
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EXPERIMENT 4: DUODENAL INFUSIONS IMPLICATE INTESTINAL CHEMORECEPTORS IN LEARNING ABOUT INTESTINAL TASTES |
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TOPABSTRACTEXPERIMENT 1: ELECTRONIC...EXPERIMENT 2: MACRONUTRIENT...EXPERIMENT 3: LEARNING ABOUT...EXPERIMENT 3aEXPERIMENT 3bEXPERIMENT 4: DUODENAL INFUSIONS...EXPERIMENT 4aEXPERIMENT 4bGENERAL DISCUSSIONGRANTSREFERENCES |
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EXPERIMENT 4a |
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TOPABSTRACTEXPERIMENT 1: ELECTRONIC...EXPERIMENT 2: MACRONUTRIENT...EXPERIMENT 3: LEARNING ABOUT...EXPERIMENT 3aEXPERIMENT 3bEXPERIMENT 4: DUODENAL INFUSIONS...EXPERIMENT 4aEXPERIMENT 4bGENERAL DISCUSSIONGRANTSREFERENCES |
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Methods
Subjects. Subjects were 32 naive rats of the same description and maintained under the same conditions as experiment 1.
Surgery and apparatus. Sixteen rats underwent the same surgical procedures for implantation of gastric cannulas as described in experiment 1. The remaining 16 rats underwent a similar procedure, but after being inserted into the stomach the cannula was advanced through the pyloric sphincter and anchored to the lumenal duodenal wall 4 cm distal to the pyloric sphincter. The rats were trained and tested using the same apparatus as described in experiment 1.
Stimuli. Oral and IG/intraduodenal stimuli were the same as those used in experiment 1.
Procedure. After recovery from surgery, the rats were reduced to 85% of their ad libitum postsurgical body weights. They then received two 30-min habituation sessions in which they were placed in the conditioning chamber and attached to the apparatus, but no oral or IG stimuli were presented. The rats were then given four aversion-training sessions, in which they were placed in the infusion cages for 20 min and either 7.1% corn oil emulsion or 16% maltodextrin (2 sessions each, alternating) was delivered IG or intraduodenally at a rate of 0.6 ml/min in a repeating cycle of 10 s on/10 s off for a total infusion volume of 6 ml. For half of the animals (gastric catheter n = 8; duodenal catheter n = 8), after the sessions in which they received IG/intraduodenal maltodextrin, they were given an intraperitoneal injection of LiCl and given an intraperitoneal injection of saline after the session in which they received IG/intraduodenal corn oil. The remaining rats received the reverse nutrient-drug contingency (corn oil-LiCl, maltodextrin-saline).
After completion of this training phase, the rats were given a 4-h two-bottle preference test between the corn oil and maltodextrin solutions in the home cage. Intake of the nutrient solutions was measured at 30 min and 1, 2, and 4 h.
Results and Discussion
The results from the first 30 min of the two-bottle infusate intake test for animals implanted with either gastric (left) or duodenal (right) catheters are shown in Fig. 10. Mean intake of each infusate for the poisoned and nonpoisoned treatment conditions, along with statistical analysis, is presented in the figure legend. There was no difference in intake of the nutrient solutions between the two catheter placements, and both groups of animals consumed greater amounts of the nonpoisoned solution than the poisoned. Both of these observations were confirmed statistically, as there was a main effect of poisoning [F(1,28) = 12.79, P < 0.01], but no significant effects involving catheter type [largest F(1,28) = 0.26]. Consistent with previous results, there was also an interaction between poisoned nutrient type and poisoning [F(1,28) = 9.33, P < 0.01], indicating that the intake of the poisoned solution was greater than the nonpoisoned for animals that had corn oil paired with LiCl, while intake of the two solutions did not differ for animals that had maltodextrin paired with LiCl. Again, there was an overall greater intake of maltodextrin than corn oil [F(1,28) = 7.99, P < 0.01].
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EXPERIMENT 4b |
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TOPABSTRACTEXPERIMENT 1: ELECTRONIC...EXPERIMENT 2: MACRONUTRIENT...EXPERIMENT 3: LEARNING ABOUT...EXPERIMENT 3aEXPERIMENT 3bEXPERIMENT 4: DUODENAL INFUSIONS...EXPERIMENT 4aEXPERIMENT 4bGENERAL DISCUSSIONGRANTSREFERENCES |
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To address these hypothetical concerns and to provide an additional control for reflux through the catheter, the present experiment 1) used groups of naive animals that had not previously been conditioned, thus eliminating any issue of preexposure; and 2) introduced several variations into the training paradigm. In particular, the last fractions of the nutritive CSs (corn oil and maltodextrin) were flushed into the GI tract and out of the catheters by a second infusion of a nonnutritive Kool-Aid solution administered without detaching the animals from the infusion line. Instead of the flavors employed in experiment 3b, nonnutritive grape and cherry flavorings that have been used extensively with the electronic esophagus preparation and repeatedly shown to be readily discriminated orally (19; also cf. experiment 1) were used to flush all nutrients into the intestines. In addition, instead of stomach catheters, duodenal catheters were employed for infusions, because arguably some backpressure in the catheter can occur with gastric infusions, and backpressure might increase the possibility of any leakage out of the catheter. Finally, the animals were not disconnected from the infusion lines until 15 min after the flavored saccharin flush of the catheter to provide additional certainty that any backpressure associated with the infusions would have dissipated.
Method
Subjects. Subjects were 32 naive animals of the same description and maintained under the same conditions as in experiment 1.
Surgery and apparatus. Animals were implanted with duodenal cannulas as described in experiment 4a; they otherwise underwent the same surgical procedures and were trained and tested using the same apparatus as described for previous experiments.
Stimuli. Stimuli were the same solutions described in experiment 1.
Procedure. After recovery from surgery, the rats were reduced to 85% of their ad libitum postsurgical body weights. They then received two 30-min habituation sessions on successive days in which they were placed in the conditioning chamber and attached to the apparatus, but no oral or intraduodenal stimuli were presented.
Animals were then given four 35-min training sessions (2 poisoned trials; 2 nonpoisoned) over four successive days. During the first 20 min of each session, animals were intraduodenally infused with either 7.1% corn oil (2 sessions) or 16% maltodextrin (2 sessions) at a rate of 0.6 ml/min in a repeating cycle of 10 s on/10 s off for a total infusion volume of 6 ml (same parameters as experiment 4a). After this nutrient infusion, and without detaching the infusion lines from the catheters, animals received an additional 1 ml infusion of either a grape-flavored (2 sessions) or cherry-flavored (2 sessions) saccharin solution using the same rate parameters. This saccharin infusion cleared the infusion line (dead space volume = 0.32 ml) and the lumen of the catheter (volume = 0.07 ml) of nutrients to ensure that any tastant that might leak when the animals were detached would be the flavored saccharin. The animals then remained in the conditioning chamber for an additional 15 min with the infusion line attached to the catheter. The particular flavor of the saccharin solution was consistently paired with one nutrient, such that animals always received corn oil followed by cherry and maltodextrin followed by grape or vice versa. Nutrient-flavor pairings and the order in which nutrient-flavor pairings were presented were counterbalanced. At the conclusion of each session, animals were removed from the conditioning chamber, the cannula and backmount areas were blotted with paper towels as further precaution against any inadvertent leakage, and cannulas were flushed with 0.5 ml saline. Animals then received an intraperitoneal injection of either LiCl or saline. For half the animals, the LiCl injections followed infusions with the corn oil emulsion, while saline injections followed the maltodextrin infusions. The remaining animals received the opposite nutrient-poisoning contingency. Similarly, half the animals received LiCl after infusion of the grape solution and saline after the cherry solution, while the other half received the opposite pairing.
On the 2 days after the completion of training, two-bottle test sessions were conducted in the home cage. On the first day, all animals received one bottle containing the corn oil emulsion and one bottle containing the maltodextrin solution. On the second day, all animals received one bottle containing the grape-flavored solution and one bottle containing the cherry-flavored solution. Intake of these solutions was recorded at 30 min, 90 min, and 4 h.
Results and Discussion
One animal became detached from the infusion apparatus during a training session and could conceivably have consumed infusate during training; therefore the data from this animal were discarded from the analysis. Figure 11 shows the intake of the poisoned and nonpoisoned nutrient solutions during the first 30 min of the two-bottle intake test. Figure 12 shows the intake of the poisoned and nonpoisoned nonnutritive flavor solutions during the same period. Mean intake of each infusate for the poisoned and nonpoisoned treatment conditions, along with statistical analysis, is presented in the figure legend.
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