Figure 2. OA1.0 can report the release of OA release in vivo.

(A) Schematic illustration depicting the experimental setup in which a transgenic fly expressing OA1.0 in the MB (MB247-LexA-driven) is fixed under a two-photon microscope (2PM) and a glass electrode is used to apply electrical stimuli near the MB.

(B) Example fluorescence image of OA1.0 expressed in the MB. The dotted circle represents the region of interest (ROI) used for subsequent analysis.

(C) Representative pseudocolor images (top) and corresponding traces (bottom) of the change in OA1.0 fluorescence in response to the indicated number of electrical stimuli in a control fly, a control fly treated with 100 μM epinastine (Ep), and an OAN (Tdc2-GAL4-driven) > TβH^RNAi fly. (D) Summary of peak ΔF/F₀ (left) and the signal-to-noise ratio (SNR, right) measured in response to electrical stimuli for the indicated conditions; n = 2–6 flies/group.

(E) Left: Time course of ΔF/F₀ measured in OA1.0-expressing flies in response to 50 electrical stimuli applied at 100 Hz; the rise and decay phases were fitted with a single-exponential function (red traces). Right: Summary of τ_on and τ_off; n = 3 flies/group.

(F) Schematic illustration depicting the experimental setup for optogenetic stimulation.

(G) Example dual-color fluorescence image of OA1.0 expressed in the MB (green, MB247-LexA-driven) and CsChrimson-mCherry expressed in OANs (red, Tdc2-GAL4-driven); the ΔF/F₀). The γ1-γ5 compartments of the MB are indicated using dashed lines.

(H) Representative pseudocolor images (top) and corresponding traces (bottom) of the change in OA1.0 fluorescence measured in response to the indicated number of optogenetic stimuli applied either in saline or 100 μM Ep.

(I) Summary of peak ΔF/F₀ measured in response to optogenetic stimuli; n = 8 flies/group. (J) Left: Time course of ΔF/F₀ measured in the γ3 compartment in response to a single laser pulse; the rise and decay phases were fitted with a single-exponential function (red traces). Right: Summary of τ_on and τ_off; n = 7 flies/group.

**p < 0.01, and n.s., not significant (for D, paired or unpaired Student’s t-test; for I, one-way ANOVA with Tukey’s post hoc test). Scale bar = 20 μm.