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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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Generation of transgenic medaka fish expressing a dominant-negative form of the Gαi2 subunit in taste receptor cells(a) The construct contains two medaka plc-β2 promoters that regulate rat gαi2S47C mutant and gfp genes. The two genes are in an inverted configuration with an I-SceI meganuclease site at each 3′ end. Scale bar: 1 kbp. (b) The transgenic fish express GFP in the regions where taste buds are distributed. GFP was detected in the lip (left panel) and pharyngeal regions (right panel) of the transgenic fish (10 dpf). Images were obtained by overlaying fluorescence images on bright-field images. Scale bar: 200 μm. (c) Coexpression of the transgenes and endogenous plc-β2 gene in the taste bud cells. In each experiment, fluorescence microscopy (panels in the left column) and in situ hybridization analyses (panels in the center column) were performed using the same section (upper or lower panels). The GFP signals were detected only in the cells expressing the endogenous plc-β2 gene (upper panels). The cells expressing GFP also expressed rat Gαi2 mRNA (lower panels). Scale bar: 10 μm.
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fig04: Generation of transgenic medaka fish expressing a dominant-negative form of the Gαi2 subunit in taste receptor cells(a) The construct contains two medaka plc-β2 promoters that regulate rat gαi2S47C mutant and gfp genes. The two genes are in an inverted configuration with an I-SceI meganuclease site at each 3′ end. Scale bar: 1 kbp. (b) The transgenic fish express GFP in the regions where taste buds are distributed. GFP was detected in the lip (left panel) and pharyngeal regions (right panel) of the transgenic fish (10 dpf). Images were obtained by overlaying fluorescence images on bright-field images. Scale bar: 200 μm. (c) Coexpression of the transgenes and endogenous plc-β2 gene in the taste bud cells. In each experiment, fluorescence microscopy (panels in the left column) and in situ hybridization analyses (panels in the center column) were performed using the same section (upper or lower panels). The GFP signals were detected only in the cells expressing the endogenous plc-β2 gene (upper panels). The cells expressing GFP also expressed rat Gαi2 mRNA (lower panels). Scale bar: 10 μm.

Mentions: Rat Gαi2 cDNA was obtained using the method described by Kusakabe et al. (2000). The cDNA fragment from the initiation codon to the Ser 47 codon was mutagenized using a TaKaRa LA Taq PCR kit (Takara Bio, Tokyo, Japan) and the primers 5′-gcgatatcatgggctgcaccgtgagcgccga-3′ and 5′-gcgatatcgTgcaccatcgtcaagcagatga-3′, producing an ApaLI recognition site. The cDNA fragment from the Ser 47 codon to the termination codon was also mutagenized using the same kit and the primers 5′-gcgatatctcagaagaggccacagtccttca-3′ and 5′-gcgatatcgtgcActtccctgattctccagc-3′, producing also an ApaLI recognition site. These fragments were digested by ApaLI and EcoRV and subcloned into the EcoRV site of pBluescript II SK(−) (Stratagene, La Jolla, Ca, USA) to obtain full-length rat Gαi2S47C cDNA. The cDNA was excised by EcoRV and inserted into the BamHI site of pmfplcb2-1.6 kb (Aihara et al. 2007) to produce a mfplcb2-1.6 kb-rGi2S47C fragment. pmfplcb2-1.6 kb-EGFP<Kan> (Aihara et al. 2007) was used as a backbone vector. The SV40 polyadenylation signal for the rat Galphai2S47C transcript was first subcloned into the SalI site of pmfplcb2-1.6 kb-EGFP<Kan>. The mfplcb2-1.6 kb-rGi2S47C fragment was then excised by NotI and SmaI and inserted into the XhoI site of mfplcb2-1.6 kb-EGFP<Kan>, linking the SV40 polyadenylation signal to produce the final construct (Fig. 4a). Injection of the transgene and establishment of transgenic lines were performed using the method described previously (Aihara et al. 2007). Briefly, three G0 fish exhibiting the green fluorescent protein (GFP) signal were crossed with wild-type fish to obtain the F1 generation. Two lines were found to inherit the transgene, and the one exhibiting the most intense GFP signal was crossed with wild-type fish to produce the F3 and F4 generations. Mainly F4 fish were used in the food preference–aversion assay. The siblings exhibiting no GFP signal were used as wild-type controls.


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)

Generation of transgenic medaka fish expressing a dominant-negative form of the Gαi2 subunit in taste receptor cells(a) The construct contains two medaka plc-β2 promoters that regulate rat gαi2S47C mutant and gfp genes. The two genes are in an inverted configuration with an I-SceI meganuclease site at each 3′ end. Scale bar: 1 kbp. (b) The transgenic fish express GFP in the regions where taste buds are distributed. GFP was detected in the lip (left panel) and pharyngeal regions (right panel) of the transgenic fish (10 dpf). Images were obtained by overlaying fluorescence images on bright-field images. Scale bar: 200 μm. (c) Coexpression of the transgenes and endogenous plc-β2 gene in the taste bud cells. In each experiment, fluorescence microscopy (panels in the left column) and in situ hybridization analyses (panels in the center column) were performed using the same section (upper or lower panels). The GFP signals were detected only in the cells expressing the endogenous plc-β2 gene (upper panels). The cells expressing GFP also expressed rat Gαi2 mRNA (lower panels). Scale bar: 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2667311&req=5

fig04: Generation of transgenic medaka fish expressing a dominant-negative form of the Gαi2 subunit in taste receptor cells(a) The construct contains two medaka plc-β2 promoters that regulate rat gαi2S47C mutant and gfp genes. The two genes are in an inverted configuration with an I-SceI meganuclease site at each 3′ end. Scale bar: 1 kbp. (b) The transgenic fish express GFP in the regions where taste buds are distributed. GFP was detected in the lip (left panel) and pharyngeal regions (right panel) of the transgenic fish (10 dpf). Images were obtained by overlaying fluorescence images on bright-field images. Scale bar: 200 μm. (c) Coexpression of the transgenes and endogenous plc-β2 gene in the taste bud cells. In each experiment, fluorescence microscopy (panels in the left column) and in situ hybridization analyses (panels in the center column) were performed using the same section (upper or lower panels). The GFP signals were detected only in the cells expressing the endogenous plc-β2 gene (upper panels). The cells expressing GFP also expressed rat Gαi2 mRNA (lower panels). Scale bar: 10 μm.
Mentions: Rat Gαi2 cDNA was obtained using the method described by Kusakabe et al. (2000). The cDNA fragment from the initiation codon to the Ser 47 codon was mutagenized using a TaKaRa LA Taq PCR kit (Takara Bio, Tokyo, Japan) and the primers 5′-gcgatatcatgggctgcaccgtgagcgccga-3′ and 5′-gcgatatcgTgcaccatcgtcaagcagatga-3′, producing an ApaLI recognition site. The cDNA fragment from the Ser 47 codon to the termination codon was also mutagenized using the same kit and the primers 5′-gcgatatctcagaagaggccacagtccttca-3′ and 5′-gcgatatcgtgcActtccctgattctccagc-3′, producing also an ApaLI recognition site. These fragments were digested by ApaLI and EcoRV and subcloned into the EcoRV site of pBluescript II SK(−) (Stratagene, La Jolla, Ca, USA) to obtain full-length rat Gαi2S47C cDNA. The cDNA was excised by EcoRV and inserted into the BamHI site of pmfplcb2-1.6 kb (Aihara et al. 2007) to produce a mfplcb2-1.6 kb-rGi2S47C fragment. pmfplcb2-1.6 kb-EGFP<Kan> (Aihara et al. 2007) was used as a backbone vector. The SV40 polyadenylation signal for the rat Galphai2S47C transcript was first subcloned into the SalI site of pmfplcb2-1.6 kb-EGFP<Kan>. The mfplcb2-1.6 kb-rGi2S47C fragment was then excised by NotI and SmaI and inserted into the XhoI site of mfplcb2-1.6 kb-EGFP<Kan>, linking the SV40 polyadenylation signal to produce the final construct (Fig. 4a). Injection of the transgene and establishment of transgenic lines were performed using the method described previously (Aihara et al. 2007). Briefly, three G0 fish exhibiting the green fluorescent protein (GFP) signal were crossed with wild-type fish to obtain the F1 generation. Two lines were found to inherit the transgene, and the one exhibiting the most intense GFP signal was crossed with wild-type fish to produce the F3 and F4 generations. Mainly F4 fish were used in the food preference–aversion assay. The siblings exhibiting no GFP signal were used as wild-type controls.

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