HCN is necessary for the normal activity of bitter-sensing GRNs (bGRNs), although expressed in sGRNs.

Representative 5 sec-long traces of sensillum recording with either caffeine or sucrose at the indicated concentrations, shown along with box plots of spiking frequencies. (A) Caffeine-evoked bitter spiking responses of WT, the Ih-deficient mutants, Ihf03355 and Ih-TG4.0/+, and the genomic rescue, Ihf03355;{Ih}/+. (B) Sucrose responses were similar among the genotypes tested in (A). (C) Ih RNAi knockdown in sGRNs, but not bGRNs, reduced the bGRN responses to 2-mM caffeine. (D) Ih RNAi knockdown in sGRNs increased the sGRN responses to 50-mM sucrose. (E) Introduction of the Ih-RF cDNA in sGRNs, but not bGRNs, of Ihf03355 restored the bGRN response to 2-mM caffeine. (F) For sucrose responses, introduction of Ih-RF to bGRNs increased the spiking frequency. Letters indicate statistically distinct groups (a and b): Tukey’s test, p<0.05 (A), Dunn’s, p<0.05 (F). §: Welch’s ANOVA, Games-Howell test, p<0.05. #: Dunn’s test, p < 0.05. Numbers in gray indicate the number of tested naïve bristles, which are from at least 3 individuals.

Sensillum potential (SP) is reduced in HCN-deficient animals.

(A) Schematic diagram illustrating the sensillum potential in the taste bristle sensilla (Left). Black upward arrow indicates ion transport by pumps and transporters in support cells from the hemolymph to the sensillum lymph, which are physiologically separated by tight junctions between support cells. The resulting transcellular disparity of ions leads to a positive sensillum potential. Representative traces of potentials measured to evaluate SP (Right). Raw: the potential reading upon the contact of the recording electrode with the sensillum bristle tip (black). DC bias: the potential reading upon impalement of the head by the recording electrode (gray). Red line indicates the difference between raw and DC bias, which represents the sensillum potential. The values resulting from the subtraction of the data between 20 to 60 sec after the initial contact (time indicated by the purple double headed arrow) were averaged to determine SP. (B) Photographs of impaled flies for DC bias determination at indicated sites. (C) DC bias values obtained from indicated body parts. There is no statistical significance between the body sites (ANOVA Repeated Measures). (D) Photos before (top) and after (bottom) deflection of an i-type bristle. (E) Sensillum potential traces as a function of time from WT and nompCf00642. Bristle bending started at 10 sec, and the duration is marked by an orange double headed arrow. (F) The peak SP changes of WT and nompCf00642were compared. (G and H) SP was reduced in i- (G) and s-type (H) bristles of the indicated Ih-deficient mutants, relative to WT. (I and J) Ih RNAi in sGRNs reduced SPs of the i- and s-type bristles. (K and L) The SP of Ihf03355 was restored by Ih-RF expression in GRNs (red for sGRNs, blue for bGRNs). ###: Dunn’s, p<0.001. * and ***: Tukey’s, p<0.05 and p<0.001, respectively. §: Welch’s ANOVA, Games-Howell test, p<0.05. Letters indicate statistically distinct groups: Tukey’s test, p < 0.05. Numbers in gray indicate the number of naive bristles tested in at least three animals.

Inactivation of sGRNs raises bGRN activity and SP, both of which are reversed by Ih deficiency.

(A) The bGRN spiking was increased in response to the indicated bitters in Gr64af mutants impaired in sucrose and glucose sensing. Ber: 0.5, Lob: 0.5, NMM: 2, Caf: 2 (i-type) and 0.09 (s-type), Umb: 0.1, TPH: 1 mM. * and ***: Student’s t-test, p<0.01 and p<0.001, respectively. (B and C) Silencing by Kir2.1 (B), but not blocking chemical synaptic transmission (C), in sGRNs increased the spiking of bGRNs stimulated by 2-mM caffeine, which was reversed in Ihf03355 (B). #: Dunn’s, p<0.05. (D) Silencing sGRNs by Kir2.1 increased SP. #: Dunn’s, p<0.05. (E) The increased bGRN spiking in Gr64af was restored to WT levels by Ih deficiencies. Letters indicate significantly different groups (Tukey’s, p<0.05). Caffeine 2 mM was used (B, C and E). (F) Regardless of bristle type, SP was increased upon sGRN inactivation, which was reduced by Ih deficiencies. p-r: Dunn’s test, p<0.05. a-c: Welch’s ANOVA, Games-Howell test, p<0.05. Numbers in gray indicate the number of naïve bristles in at least 3 animals.

HCN suppresses HCN-expressing GRNs and increases SP.

(A) HCN misexpressed in bGRNs flattened the dose dependence to caffeine. (B) HCN ectopically expressed in bGRNs elevates sGRN responses to sucrose. (C) Overexpression of HCN in sGRNs reduced the sGRN responses to sucrose 5 sec after the initial contact. (D) Ih misexpression in bGRNs increased SP in i- and s-type bristles, which correlates with laterally increased sGRN activity (B). (E) Ih RNAi knockdown in sGRNs (Gr64f-Gal4 cells) dramatically elevates spiking frequencies in response to 1-, 5-, 10-, and 50-mM sucrose. *, **, and ***: Tukey’s, p<0.05, p<0.01, and p<0.001, respectively (A,D,E). # and ##: Dunn’s, p<0.05 and p < 0.01 between genotypes, respectively (B,C,E). ‡: Dunn’s, p < 0.05 between responses to different sucrose concentrations (B,C). §: Welch’s ANOVA, Games-Howell test, p<0.05 (E). The numbers in grey indicate the number of tested naïve bristles in at least three animals.

Sweetness in the diet decreases SP, bGRN activity, and bitter avoidance.

(A) Sweetness in the media reduced the 2-mM caffeine-evoked bGRN spiking, which was fully recovered in four-hour incubation with sorbitol only food. Ihf03355 was affected by the type of the media more severely than WT. O/N: overnight incubation with sorbitol only (grey) or sucrose food (red). (B) The SP of Ihf03355 bristle sensilla showed dysregulated reduction after four-hour and overnight incubation on sweet media. These reductions started to be recovered in one-hour feeding and were nearly fully recovered in four-hour feeding on the indicated sorbitol only food. (C) Caffeine (Caf) avoidance was assessed with capillary feeder assay (CAFE). Ih is required for robust caffeine avoidance for flies maintained on sweet cornmeal food (sweet exposure +: filled boxes). Ihf03355 flies avoided 4 mM caffeine like WT flies when separated from sweet food for 20 hrs (blank boxes). (D) Ih RNAi knockdown in sGRNs (Gr64f-Gal4) but not bGRNs (Gr66a-Gal4) led to relatively poor avoidance to caffeine after feeding on the sweet diet with sucrose. Suc: sucrose, and Sor: sorbitol Letters indicate statistically distinct groups: a-f, Dunn’s, p < 0.05 (A,B). * and ***: Tukey’s, p<0.05 and <0.001, respectively. (E) Illustration depicting the flies’ sweet feeding niche in overripe fruit (Left), leading to prolonged exposure of sGRNs to the sweetness (Right). (F) A schematic model of gustatory homeostasis in Drosophila bristle sensilla. Despite the prolonged sweetness in the environment robustly and frequently stimulating sGRNs, the sGRN activity is moderated by HCN to preserve the sensillum potential, which is required for normal bGRN responsiveness (Left). When HCN in sGRNs is incapacitated, sGRNs can become overly excited by sweetness of overripe fruit and deplete the sensillum potential, resulting in decreased bGRN activity and bitter avoidance (Right).