From Cell to Beak: In-Vitro and In-Vivo Characterization of Chicken Bitter Taste Thresholds

Abstract

Bitter taste elicits an aversive reaction, and is believed to protect against consuming poisons. Bitter molecules are detected by the Tas2r family of G-protein-coupled receptors, with a species-dependent number of subtypes. Chickens demonstrate bitter taste sensitivity despite having only three bitter taste receptors-ggTas2r1, ggTas2r2 and ggTas2r7. This minimalistic bitter taste system in chickens was used to determine relationships between in-vitro (measured in heterologous systems) and in-vivo (behavioral) detection thresholds. ggTas2r-selective ligands, nicotine (ggTas2r1), caffeine (ggTas2r2), erythromycin and (+)-catechin (ggTas2r7), and the Tas2r-promiscuous ligand quinine (all three ggTas2rs) were studied. Ligands of the same receptor had different in-vivo:in-vitro ratios, and the ggTas2r-promiscuous ligand did not exhibit lower in-vivo:in-vitro ratios than ggTas2r-selective ligands. In-vivo thresholds were similar or up to two orders of magnitude higher than the in-vitro ones.

Keywords: T2R; Tas2r; avian; bitter taste; calcium imaging; chicken; ggTas2r; in-vitro; in-vivo; threshold.

Figure 1

Functional analyses of chicken Tas2rs. The cDNAs of the three chicken Tas2rs were transiently transfected in HEK 293T-Gα16gust44 cells and challenged with different concentrations of (+)-catechin (upper panel) and quinine hydrochloride (lower panel). The changes in fluorescence (ΔF/F) were monitored and plotted against the log compound concentration (x-axis). Raw calcium traces obtained with 1000 µM (+)-catechin (upper right panel) and 30 µM quinine hydrochloride (lower right panel), respectively, are diplayed next to the corresponding dose-response curves (scale: y-axis, 500 relative fluorescence units (RFU); x-axis, time in minutes (max 2 min)) r1 = ggTas2r1; r2 = ggTas2r2; r7 = ggTas2r7; Mock = empty plasmid which represents negative control).

Figure 2

Effects of different concentrations of bitter tastants on consumption parameters. Tastant side consumption (filled bars: (A) Nicotine; (B) Caffeine; (C) Erythromycin; (D) (+)-Catechin) and water side consumption per chick (open bars) as percentage of control, during 24 h. Consumption parameters were normalized to the distilled water control group (=100%, indicated by black line at 100). Bars represent consumption (represented as percentage of control group) ±SEM. * Significantly different (p ≤ 0.05) from control group using Dunnett’s Method.

Figure 3

Relative mRNA abundance of ggTas2r-encoding genes (ggTas2r1, ggTas2r2, ggTas2r7) on E19 (A) and at 21 days (B) with the ggTas2r2 serving as the set-point gene (relative expression set to 1, n = 6), obtained from data published in Cheled-Shoval et al. [18]. Values are presented as mean fold change ±SEM. Differences among the ggTas2r1 and ggTas2r7 genes within the palate: means without a common letter differ significantly (p < 0.05); differences between the tested genes (ggTas2r1 and ggTas2r7) and the control gene (ggTas2r2) within the palate: means with an asterisk (*) differ significantly from ggTas2r2.

Figure 4

Comparison of in-vivo and in-vitro thresholds. Blue, orange and green colors represent the in-vivo:in-vitro ratios for ggTas2r1, ggTas2r2 and ggTas2r7, respectively.