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The ability of rats to form conditioned taste aversions to linoleic and oleic acids, focusing on the role of the chorda tympani nerve. The study uses the davis rig to measure taste aversion and examines the effects of licl and nacl injections on rats' preferences for these acids. The document also discusses the potential influence of texture on taste aversion and the relationship between corn oil and mineral oil.
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Leah Harris Lauren MurchisonSara Shields Jennifer Wallace
Submitted as partial fulfillment of the Senior Thesis requirement of thePsychology major at Wofford College
Both the World Health Organization and the International Obesity Task Force have classified the prevalence of obesity as a global epidemic. The primary causes of obesity are sedentary lifestyles and high- fat diets. Such diets are preferred due to their high palatability. Fats are broken down into free fatty acids, such as linoleic and oleic acids. In order to determine the neural pathway responsible for transducing these free fatty acids, the chorda tympani nerve was examined. In this study, a conditioned taste aversion to linoleic and oleic acid was measured following chorda tympani nerve avulsion as demonstrated in a taste-salient assay by means of the Davis Rig. Animals formed conditioned taste aversion to linoleic acid at concentrations exceeding 44 μM. After cutting the chorda tympani nerve, rats no longer avoided linoleic acid. No taste aversion to oleic acid was detected; therefore, a stimulus generalization between linoleic and oleic acids did not exist. A second experiment was conducted with intact chorda tympani nerve animals, which demonstrated an active avoidance to NaCl. This study shows that rats are able to detect fat. Furthermore, the chorda tympani nerve is responsible for relaying taste information to the brain. It is understood that this animal model is applicable to humans. Therefore, in the future, this taste research could be useful in formulating dietary foods that are more palatable without high fat content.
effect preference for free fatty acids (Kawai et al., 2003). Also, it was found that triglycerides are perceived by the lingual lipase and radioactive triolein activated the lingual lipase and allowed for the perception of triglycerides. The triolein was administered through filter paper on the circumvallate papilla of the rat tongues for varying times of 1, 5, or 10 seconds. This allowed fo r the amount administered to be regulated and for the triglyceride breakdown to be measured. The findings show that in a short amount of time, significant levels of free fatty acids were made by enzymes in our saliva. Kawai et al (2003) found additional evidence for chemoreception of fats in that rats prefer corn oil to mineral oil even though both oils have the same texture. Smith et al (2000) examined the contribution of orosensory factors in the ingestion of a sucrose and corn oil mixture. Seven conditioned taste aversion and stimulus generalization experiments determined that the corn oil stimulus was the salient orosensory element in sucrose/corn oil mixtures. Given that the texture of the solution is potentially responsible for conditioned taste aversions, the relationship between corn oil and mineral oil was also examined. Rats were able to discriminate between a sucrose/corn oil mixture and a sucrose/mineral oil mixture. Rats injected with LiCl, thus resulting in a conditioned taste aversion, cons umed much less of the sucrose/corn oil mixture than did the rats receiving control injections. Finally, Smith et al (2000) examined the role of linoleic acid, which is a free fatty acid cleaved from corn oil and is acquired from one’s diet. Upon producing a conditioned taste aversion to corn oil, rats were tested with linoleic acid and showed a stimulus generalization between the two solutions, avoiding both.
Recording from isolated rat taste receptor cells suggests that the extracellular application of linoleic acid inhibits the outward flow of potassium (K+), producing depolarization (Gilbertson et al, 1997). Oleic acid did not produce a response in taste receptor cells from fungiform papillae but both oleic and linoleic acid was found to inhibit outward K+ currents in taste receptor cells from the circumvallate papillae located on the posterior tongue (Hansen et al, 2003). In summary, it appears that lingual lipase can generate free fatty acids from dietary fat within seconds of contact allowing the chemoreception of free fatty acids, such as linoleic and oleic acid. It has been shown that chorda tympani nerve cuts prevent a conditioned taste aversion to linoleic acid in a two-bottle test. In this study, a conditioned taste aversion to linoleic and oleic acid was measured following chorda tympani nerve cuts as demonstrated in a taste-salient assay by means of the Davis Rig. METHODS Subjects Fifty-two male ( Sprague Dawley ) rats, at least sixty days old, were individually housed in a room lighted on a 12- hour cycle. Rats were fed (Harlan Teklab 8604 laboratory rodent diet) ad libitum throughout the thirty-two day experiment. All procedures were approved by the Animal Care and Use Committee at Wofford College. Chemical Stimuli Reagent grade chemicals were obtained from Sigma. All solutions were mixed twice daily with reagent grade chemicals and deionized water. In phase one, concentrations used during Davis Rig preference testing consisted of deionized water, 44,
and the head was rotated 60 degrees to expose the ear canal. Retractors were used to expose the tympanic membrane. Upon rupturing the tympanic membrane, the chorda tympani nerve was avulsed for rats in the chorda tympani transection group (CTX, n=32). The procedure was then repeated to transect the contralateral chorda tympani nerve. The SHAM group (n=20) underwent identical surgical procedures excluding the chorda tympani nerve avulsion. Following preference testing, the rats were euthanized by sodium pentobarbital overdose and tongues were extracted to allow histological verification of the bilateral nerve cuts. When the gustatory nerves were removed, the taste buds atrophy resulting in the loss of the presence of a taste pore for the fungiform papillae. Papillae were counted to determine if a difference existed between chorda tympani nerve cut (CTX) and SHAM rats. Data Analysis The results of our data were analyzed using SPSS. A univariant ANOVA test determined the significant main effects and interactions. Post hoc testing with the least significant difference (LSD) test determined the source of any statically significant effects. Re sults were deemed significant if p<0.05. RESULTS
As seen in table one, histological results confirm the chorda tympani nerve cuts were successful. The maximum percent of fungiform papillae with taste pores for the
Table 1. Results of histological verification of bilateral chorda tympani transections. CTX Left Pores3.2 Left FP55.8 Right Pores2.7 Right FP Total Pores Total FP % FP with Pores SE %:55.7 5.9 111.4 5.3% 0.8% Sham 66.8 74.5 69.8 77.4 136.6 151.9 89.5% 1.8%
Figure 1. CTA formation to LA (A) and OA (B) contingent upon injection condition in SHAM animals. (p<0.01) between LiCl and NaCl injections at each Stars represent significant differences concentration.
1B
44 OA (^) Concentrations88 OA 176 OA
Lick Ratio
SHAM/OA/LiClSHAM/OA/NaCl
1A
44 LA (^) Concentrations88 LA 176 LA
Lick Ratio
2A
44 LA (^) Concentrations88 LA 176 LA
Lick Ratio
CTX/LA/NaClCTX/LA/LiCl
2B
44 OA (^) Concentrations88 OA 176 OA
Lick Ratio
CTX/OA/LiClCTX/OA/NaCl
Figure 2.contingent CTA formation to LA (A) and OA (B) upon injection condition in CTX animals.(p<0.01) between LiCl and NaCl injections at Stars represent significant differences each concentration.
CTX group was 18%, whereas the minimum percent of fungiform papillae with taste pores for the SHAM group was 81%.
Linoleic or oleic acid taste aversions The ability to form a conditioned taste aversion to linoleic acid and oleic acid was tested within the SHAM and CTX operated animals for three different free fatty acid concentrations. Within the SHAM group, a main effect was found for the injection condition (F1,113=33.916, p<0.01) in the measurement of a taste aversion to linoleic acid. There was a significant interaction between injection group and concentration
4A
44 OA (^) Concentrations88 OA 176 OA
Lick Ratio
SHAM/LA/LiClSHAM/LA/NaCl 4B
44 LA (^) Concentrations88 LA 176 LA
Lick Ratio
SHAM/OA/LiClSHAM/OA/NaCl
Figure 4. Stimulus generalization from LA to OA (A) and OA to LA (B) with LiCl and NaCl injectionsat each concentration in SHAM animals.
surgery conditions. A main effect was found for the surgery condition ( F1,281=40.267, p<.001) in the measurement of a taste aversion to linoleic acid. There was a significant interaction between surgery condition, injection group, and concentration (F2,280=4.248, p<.05). Post hoc tests show significant avoidance of linoleic acid at 88 and 176 μM for SHAM animals receiving LiCl injections in comparison to CTX animals (Fig 3A). These data reveal the importance of the chorda tympani nerve in the formation of taste aversions. There was no significant main effect for the surgery condition when oleic acid was assessed. This is expected since SHAM animals did not form a taste aversion to oleic acid as shown in figure 1B (Fig 3B). Stimulus generalization Stimulus generalizations were assessed to determine if animals avoided solutions that had caused previous sickness. Animals receiving linoleic acid before injection were tested with oleic acid and vice versa. There was no main effect of injection on stimulus generalizations for linoleic to oleic acid (Fig 4A) or oleic to linoleic acid (Fig 4B). Since animals did not form a taste aversion to oleic acid, it was expected that animals would not
5B
0
1
Concentration88 uM OA
Lick Ratio
OA/LiClOA/NaCl
5A
0
1
Concentration88 uM LA
Lick Ratio
LA/LiClLA/NaCl
Figure 5.(B) contingent CTA formation to LA (A) and OA upon injection condition in SHAM animals during Phase 2. Stars representsignificant differences (p<0.01) between LiCl and NaCl injections at each concentration
6A
0
1
88 uM OA (^) Sucrose0.2 M 0.2 M NaCl 10 mM CitricAcid 0.2 mM Q-HCl Concentration
Lick Ratio
LA/LiClLA/NaCl
6B
0
1
88 uM LA (^) Sucrose0.2 M 0.2 M NaCl 10 mM CitricAcid 0.2 mM Q-HCl Concentrations
Lick Ratio
OA/LiClOA/NaCl
Figure 6. Stimulus generalization from linoleicacid (A) or oleic acid (B) to six tastants depending on injection condition. Starsrepresent significant differences (p<.01) between LiCl and NaCl injections for eachtastant.
form a stimulus generalization from oleic acid to linoleic acid. Linoleic or oleic acid taste aversions: Experiment 2 Experiment 2 utilized SHAM animals from Experiment 1 to determine if stimulus generalizations would be formed to six other tastants. Conditioned taste aversions to linoleic or oleic acid at 88 μM concentrations, dependent upon injection condition, were first assessed. A main effect was found for the injection condition (F1,350=10.172, p<.05) in the measurement of a taste aversion to linoleic acid (Fig 5A). As seen in figure 5B, a main effect was also found for oleic acid (F1,315=28.438, p<.001). Conditioned taste aversions to oleic acid were not found in experiment 1. Stimulus generalizations to 6 tastants Post hoc tests show significant reductions in lick ratios to 0.2M NaCl and 10μM citric acid tastants in linoleic groups receiving LiCl injections versus NaCl injections (Fig
It is suggested that the chorda tympani nerve is responsible for transducing free fatty acids such as linoleic and oleic acids (McCormack et al., 2003). Since rats with the chorda tympani nerve cut are unable to develop a conditioned taste aversion, it was hypothesized that these rats would not avoid linoleic acid. It was demonstrated that the chorda tympani nerve is necessary for developing a conditioned taste aversion to linoleic acid (Fig 3A). Interestingly, findings did not support that the chorda tympani nerve is necessary for developing a conditioned taste aversion to oleic acid (Fig 3B). This is a result of the aforementioned methodological drawback with the allotted time to demonstrate a conditioned taste aversion. The second experiment examined the stimulus generalization to oleic or linoleic acid and one of each of the prototypical tastants. In contrast to the first experiment, rats did form a conditioned taste aversion to oleic acid. This could be attributed to the rats being exposed to the pairing of oleic and linoleic acids for the second time, which could reinforce the original taste aversion. Also, in the first experiment only four trials of oleic acid were presented, while in the second experiment six trials were presented. Although there was a decrease in the amount of oleic acid consumed, the rats did not completely avoid it, therefore only a small taste aversion existed. In both LiCl and NaCl injection groups, rats avoided the NaCl tastant. This may be attributed to a stimulus generalization with the NaCl tasting similar to free fatty acids. Another possible reason could be that rats are unable to discriminate between LiCl and NaCl when drinking, thereby, associating the NaCl with the sickness following injection and not a taste component. Thus, avoidance either occurred as a result of a stimulus generalization to NaCl or NaCl being associated with the rat becoming sick.
In the future, this experiment could be modified to address the methodological issues regarding the limited exposure to oleic acid. Possible experiments would present the taste stimuli for either eight seconds or thirty seconds, followed by a two-bottle preference test. This change ensures that the data would accurately reflect whether or not a conditioned taste aversion developed. We plan to evaluate our methodology by examining the different intake behaviors during stimulus presentations of varied durations up to a one- hour two-bottle preference test. Future studies should also identify what information the chorda tympani nerve is transducing to the brain in response to the presence of free fatty acids. The role of the glossopharyngeal and greater superficial petrosal nerves should be thoroughly examined in a similar manner as the chorda tympani.
In summary, animals formed a conditioned taste aversion to linoleic acid at concentrations exceeding 44 μM. This demonstrated that the chorda tympani nerve is responsible for the neural signaling necessary for the development of conditioned taste aversions. An alternative theory is that the chorda tympani, glossopharyngeal, and the greater superficial petrosal nerves are all important for the neural signaling of free fatty acids. If any of these three nerves are compromised, the ability to form a conditioned taste aversion is lost. These research findings would support this hypothesis if it was found that all three nerves are involved in the taste pathway.
