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Construction of a taste-blind medaka fish and quantitative assay of its preference-aversion behavior.

Aihara Y, Yasuoka A, Iwamoto S, Yoshida Y, Misaka T, Abe K - Genes Brain Behav. (2008)

Bottom Line: We then generated a transgenic fish expressing dominant-negative Galpha(i2) both in T1R-expressing and in T2R-expressing cells.The feeding assay revealed that the transgenic fish was unable to show a preference for AN food and an aversion to DN food.The assay system was useful for evaluating taste-blind behaviors, and the results indicate that the two taste signaling pathways conveying preferable and aversive taste information are conserved in fish as well as in mammals.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.

ABSTRACT
In vertebrates, the taste system provides information used in the regulation of food ingestion. In mammals, each cell group within the taste buds expresses either the T1R or the T2R taste receptor for preference-aversion discrimination. However, no such information is available regarding fish. We developed a novel system for quantitatively assaying taste preference-aversion in medaka fish. In this study, we prepared fluorescently labeled foods with fine cavities designed to retain tastants until they were bitten by the fish. The subjects were fed food containing a mixture of amino acids and inosine monophosphate (AN food), denatonium benzoate (DN food) or no tastant (NT food), and the amounts of ingested food were measured by fluorescence microscopy. Statistical analysis of the fluorescence intensities yielded quantitative measurements of AN food preference and DN food aversion. We then generated a transgenic fish expressing dominant-negative Galpha(i2) both in T1R-expressing and in T2R-expressing cells. The feeding assay revealed that the transgenic fish was unable to show a preference for AN food and an aversion to DN food. The assay system was useful for evaluating taste-blind behaviors, and the results indicate that the two taste signaling pathways conveying preferable and aversive taste information are conserved in fish as well as in mammals.

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Designing fluorescent dye-labeled foods for medaka fish and detecting the food in the gastrointestinal tract(a) DiIC12(3)-labeled starch particles as labeled food: (i) schematic drawing, (ii) observation in the bright field and (iii) observation under fluorescence excitation. Each food particle forms a porous network in which starch, detergent, lipids, lipophilic dye and tastants are homogenously contained. Scale bar: 200 μm. (b) Images of a 20-dpf fish fed the fluorescent dye-labeled food (upper panels), and a fish that was not fed the food (lower panels). Bright-field images (panels in the left column) and fluorescence images (panels in the right column) are shown. The red fluorescence derived from DiIC12(3) was detected only in the abdominal region of the fish that were fed the food (upper panel). Scale bar: 500 μm. (c) Correlation between FI values and the amount of food ingested by the fish. The fish were fed foods containing no tastant (NT), amino acid–IMP mix (AN) and denatonium benzoate (DN). Each FI value is the sum of the pixel values in the fluorescence image of each fish. DiIC12(3) was extracted from the same fish and analyzed by HPLC. The amount of food ingested was estimated from the quantities of DiIC12(3) extracted. The FI values correlated with the amount of food ingested (r2 = 0.78).
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fig02: Designing fluorescent dye-labeled foods for medaka fish and detecting the food in the gastrointestinal tract(a) DiIC12(3)-labeled starch particles as labeled food: (i) schematic drawing, (ii) observation in the bright field and (iii) observation under fluorescence excitation. Each food particle forms a porous network in which starch, detergent, lipids, lipophilic dye and tastants are homogenously contained. Scale bar: 200 μm. (b) Images of a 20-dpf fish fed the fluorescent dye-labeled food (upper panels), and a fish that was not fed the food (lower panels). Bright-field images (panels in the left column) and fluorescence images (panels in the right column) are shown. The red fluorescence derived from DiIC12(3) was detected only in the abdominal region of the fish that were fed the food (upper panel). Scale bar: 500 μm. (c) Correlation between FI values and the amount of food ingested by the fish. The fish were fed foods containing no tastant (NT), amino acid–IMP mix (AN) and denatonium benzoate (DN). Each FI value is the sum of the pixel values in the fluorescence image of each fish. DiIC12(3) was extracted from the same fish and analyzed by HPLC. The amount of food ingested was estimated from the quantities of DiIC12(3) extracted. The FI values correlated with the amount of food ingested (r2 = 0.78).

Mentions: It is obvious that medaka fish utilize their visual and olfactory systems to search for food. They are usually more attracted to food floating on the surface of the water than that at the bottom, possibly because of the optical contrast between the food and the surface. This implied that it was necessary to design a food that possessed buoyancy in order to lure the fish. Fish also respond to chemical stimuli from a distance. For example, if a solution containing amino acids is dropped into the water, they respond by swimming vigorously and searching for food (Carr et al. 1977; Hara 2006; Lindsay & Vogt 2004). This results from the rapid diffusion and detected of the chemicals by the fish olfactory system. Because our aim was to evaluate the behavior triggered by taste stimuli, we required a food that retained its chemical compounds until it came into contact with the fish's taste buds. Our porous food matrix, consisting of starch, detergent and lipids (Fig. 2a, i), satisfied both these demands. The resulting food had a lower specific gravity than water, and its multiple cavities retained tastants and allowed them to be released in response to mechanical stimuli. In addition, the amphipathic matrix was labeled with a fluorescent dye, namely DiIC12(3), to allow quantification of the amount of food ingested (Fig. 2a, ii and iii).


Construction of a taste-blind medaka fish and quantitative assay of its preference-aversion behavior.

Aihara Y, Yasuoka A, Iwamoto S, Yoshida Y, Misaka T, Abe K - Genes Brain Behav. (2008)

Designing fluorescent dye-labeled foods for medaka fish and detecting the food in the gastrointestinal tract(a) DiIC12(3)-labeled starch particles as labeled food: (i) schematic drawing, (ii) observation in the bright field and (iii) observation under fluorescence excitation. Each food particle forms a porous network in which starch, detergent, lipids, lipophilic dye and tastants are homogenously contained. Scale bar: 200 μm. (b) Images of a 20-dpf fish fed the fluorescent dye-labeled food (upper panels), and a fish that was not fed the food (lower panels). Bright-field images (panels in the left column) and fluorescence images (panels in the right column) are shown. The red fluorescence derived from DiIC12(3) was detected only in the abdominal region of the fish that were fed the food (upper panel). Scale bar: 500 μm. (c) Correlation between FI values and the amount of food ingested by the fish. The fish were fed foods containing no tastant (NT), amino acid–IMP mix (AN) and denatonium benzoate (DN). Each FI value is the sum of the pixel values in the fluorescence image of each fish. DiIC12(3) was extracted from the same fish and analyzed by HPLC. The amount of food ingested was estimated from the quantities of DiIC12(3) extracted. The FI values correlated with the amount of food ingested (r2 = 0.78).
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Related In: Results  -  Collection

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fig02: Designing fluorescent dye-labeled foods for medaka fish and detecting the food in the gastrointestinal tract(a) DiIC12(3)-labeled starch particles as labeled food: (i) schematic drawing, (ii) observation in the bright field and (iii) observation under fluorescence excitation. Each food particle forms a porous network in which starch, detergent, lipids, lipophilic dye and tastants are homogenously contained. Scale bar: 200 μm. (b) Images of a 20-dpf fish fed the fluorescent dye-labeled food (upper panels), and a fish that was not fed the food (lower panels). Bright-field images (panels in the left column) and fluorescence images (panels in the right column) are shown. The red fluorescence derived from DiIC12(3) was detected only in the abdominal region of the fish that were fed the food (upper panel). Scale bar: 500 μm. (c) Correlation between FI values and the amount of food ingested by the fish. The fish were fed foods containing no tastant (NT), amino acid–IMP mix (AN) and denatonium benzoate (DN). Each FI value is the sum of the pixel values in the fluorescence image of each fish. DiIC12(3) was extracted from the same fish and analyzed by HPLC. The amount of food ingested was estimated from the quantities of DiIC12(3) extracted. The FI values correlated with the amount of food ingested (r2 = 0.78).
Mentions: It is obvious that medaka fish utilize their visual and olfactory systems to search for food. They are usually more attracted to food floating on the surface of the water than that at the bottom, possibly because of the optical contrast between the food and the surface. This implied that it was necessary to design a food that possessed buoyancy in order to lure the fish. Fish also respond to chemical stimuli from a distance. For example, if a solution containing amino acids is dropped into the water, they respond by swimming vigorously and searching for food (Carr et al. 1977; Hara 2006; Lindsay & Vogt 2004). This results from the rapid diffusion and detected of the chemicals by the fish olfactory system. Because our aim was to evaluate the behavior triggered by taste stimuli, we required a food that retained its chemical compounds until it came into contact with the fish's taste buds. Our porous food matrix, consisting of starch, detergent and lipids (Fig. 2a, i), satisfied both these demands. The resulting food had a lower specific gravity than water, and its multiple cavities retained tastants and allowed them to be released in response to mechanical stimuli. In addition, the amphipathic matrix was labeled with a fluorescent dye, namely DiIC12(3), to allow quantification of the amount of food ingested (Fig. 2a, ii and iii).

Bottom Line: We then generated a transgenic fish expressing dominant-negative Galpha(i2) both in T1R-expressing and in T2R-expressing cells.The feeding assay revealed that the transgenic fish was unable to show a preference for AN food and an aversion to DN food.The assay system was useful for evaluating taste-blind behaviors, and the results indicate that the two taste signaling pathways conveying preferable and aversive taste information are conserved in fish as well as in mammals.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.

ABSTRACT
In vertebrates, the taste system provides information used in the regulation of food ingestion. In mammals, each cell group within the taste buds expresses either the T1R or the T2R taste receptor for preference-aversion discrimination. However, no such information is available regarding fish. We developed a novel system for quantitatively assaying taste preference-aversion in medaka fish. In this study, we prepared fluorescently labeled foods with fine cavities designed to retain tastants until they were bitten by the fish. The subjects were fed food containing a mixture of amino acids and inosine monophosphate (AN food), denatonium benzoate (DN food) or no tastant (NT food), and the amounts of ingested food were measured by fluorescence microscopy. Statistical analysis of the fluorescence intensities yielded quantitative measurements of AN food preference and DN food aversion. We then generated a transgenic fish expressing dominant-negative Galpha(i2) both in T1R-expressing and in T2R-expressing cells. The feeding assay revealed that the transgenic fish was unable to show a preference for AN food and an aversion to DN food. The assay system was useful for evaluating taste-blind behaviors, and the results indicate that the two taste signaling pathways conveying preferable and aversive taste information are conserved in fish as well as in mammals.

Show MeSH
Related in: MedlinePlus