Figure 5. Calcium dynamics of ASER in response to repeated salt stimuli is altered in clh-1 mutants.

(a and c) Calcium responses of ASER stimulated by NaCl concentration changes between 50 mM and 25 mM after cultivation at 0 mM NaCl (a) or 100 mM NaCl (c) in the presence of food for 6 hr. A to I indicate the time points for calculation of R/R₀ changes. A, D, and G are the time points for pre-stimulus R/R₀, B, E, and H are the time points for peak R/R₀ during stimulation, C, F, and I are the time points for decayed R/R₀ during stimulation. The shaded region represents s.e.m., n ≧ 16 animals. (b and d) R/R₀ changes at each NaCl down-step stimulus (B - A, E - D, and H - G for the 1st, 2nd, and 3rd stimulus, respectively). 0 mM NaCl cultivated (b) or 100 mM cultivated (d). See Materials and methods for details. Bars and the error bars represent mean +/- s.e.m., dots represent individual trials. n ≧ 16 animals, Tukey’s test, **p<0.01, *p<0.05. +p<0.05, compared with clh-1(pe572) mutant.

Figure 5—figure supplement 1. clh-1 missense mutations affect calcium dynamics of ASER.

(a and b) Decay of ASER calcium response during 25 mM NaCl (a) and 50 mM NaCl (b) derived from Figure 5a. Differences of R/R₀ between the time points illustrated in Figure 5a (B to I) were calculated along with time as follows. Calculation of ‘decay1’ during 25 mM NaCl (a) was as follows; B - C for 1st, E - F for 2nd, H - I for 3rd. Calculation of ‘decay2’ during 50 mM NaCl (b) was as follows; C - D for 1st, F – G for 2nd. See Materials and methods for details. Bars and the error bars represent mean +/- s.e.m., dots represent individual trials. n ≧ 16 animals, Tukey’s test, ***p<0.001, **p<0.01, compared with wild type. +++p<0.001, +p<0.05, compared with clh-1(pe572) mutant. (c and d) Decays derived from Figure 5c as calculated above. Bars and the error bars represent mean +/- s.e.m., dots represent individual trials. n ≧ 16 animals, Dunnett’s test, **p<0.01, *p<0.05. (e) Expression level of the clh-1 promoter. Fluorescence intensity of the clh-1 promoter-driven mTFP relative to that of gcy-5 promoter-driven mCherry in ASER. Naive animals were directly measured after cultivation for 4 days at 50 mM NaCl in the presence of food. Otherwise, animals were further cultivated for 6 hr at either 0 mM or 100 mM NaCl in the presence of food. Bars and the error bars represent mean +/- s.e.m., dots represent individual trials. n ≧ 10 animals, Tukey’s test, *p<0.05.

Figure 5—figure supplement 2. Effect of clh-1(pe) mutations on calcium dynamics of ASER in starved animals.

(a and e) Calcium responses of ASER stimulated by NaCl concentration changes between 50 mM and 25 mM after cultivation at 0 mM NaCl (a) or 100 mM NaCl (e) for 6 hr in the absence of food. The shaded region represents s.e.m., n ≧ 8 animals. (b–d and f–h) R/R₀ changes at each NaCl down-step stimulus (b, f), decay of ASER calcium response during 25 mM NaCl (c, g) or 50 mM NaCl (d, h). 0 mM NaCl-cultivated (b–d) or 100 mM NaCl-cultivated (f–h). Calculation of R/R₀ changes and decays was performed as described in Figure 5a and Figure 5—figure supplement 1. See Materials and methods for details. Bars and the error bars represent mean +/- s.e.m., dots represent individual trials. n ≧ 8 animals, Dunnett’s test, ***p<0.001, **p<0.01, *p<0.05.