Fig. 6. Halothane increases [Ca²⁺]ⁱand hyperpolarizes motoneuron RP2. (A ) Halothane treatment increases [Ca²⁺]ⁱin larval motoneuron RP2. Changes in [Ca²⁺]ⁱin RP2 cell bodies are visualized using the genetically encoded Ca²⁺indicator UAS-GCaMP3  driven by ShakB-GAL4 . As shown in this false color image of four cells in two abdominal segments, [Ca²⁺]ⁱincreases with the addition of halothane (2.5 mM). Scale bar = 10 μm. (B ) Representative traces of calcium transients observed in the somata of RP2 neurons with halothane treatment. The entire soma was defined as the region of interest, and fluorescence intensity was converted into a percentage change (δF/F) as described earlier under Experimental Procedures. (B1 ) All cells produced an immediate response, with a sharp rise that occurred within a short period of halothane entering the chamber. [Ca²⁺]ⁱbegan to decay before halothane was removed. (B2 ) At higher concentrations, a pronounced undershoot, in which [Ca²⁺]ⁱdropped below baseline, followed halothane removal (arrow ). (B3 ) A minority of cells produced a delayed response at the time of halothane removal, consisting of a plateau with superimposed spiky transients (arrowhead ). (C ) The concentration–response relationship for the peak amplitude of the halothane-induced calcium transient response in RP2 neurons. RP2 responded to halothane in a concentration-dependent manner, with EC⁵⁰= 0.61 mM. Values are mean ± SEM (n = 3–8 preparations per data point). (D ) dRyr  mutations affect RP2’s response to halothane. RP2 neurons in larvae heterozygous for dRyr  ^E4340K , which causes hypersensitivity to halothane in adult flies, respond more strongly to 0.5 mM halothane than controls. In contrast, halothane appears to induce smaller responses in RP2 neurons heterozygous for the deletion mutant dRyr  ^δ25 and the nonsense mutation dRyr  ^Q3878X . Error bars  represent SEM, and asterisk  denotes statistical significance by one-way ANOVA and Bonferroni–Dunn post hoc  tests (P = 0.0484). (E ) dRyr  mutations reduce sensitivity to caffeine. RP2 motoneurons from larvae heterozygous for dRyr  ^Q3878X and dRyr  ^E4340K respond more weakly to 5 mM caffeine than controls (P = 0.0375 and P = 0.0453, one-way ANOVA and Bonferroni–Dunn post hoc  tests). (F ) Halothane hyperpolarizes wild-type RP2 neurons. In whole-cell current clamp recordings, halothane application produces a strong hyperpolarization that is proportional to halothane concentration. Recovery is delayed at 1 mM compared to 0.5 mM halothane. Membrane potentials are recorded using whole-cell current clamp recording. Values are mean ± SEM (n = 5–7). dRyr = Drosophila Ryr .