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. 2022 Dec 1;119(49):e2209531119. doi: 10.1073/pnas.2209531119

Fig. 3.

Fig. 3.

Modulation of neural activity of Ubx+ TH neurons affects flight. (A) Optogenetic activation (red shade) of TH neurons expressing channelrhodopsin CsChrimson (TH>CsChrimson) induces spontaneous flight (Top: Cartoon representation of experimental results) (B and C). Optogenetic inhibition (blue shade) of TH neurons (TH>GtACR2) (b) and Ubx+ cells (Ubx>GtACR2) (c) by expressing GtACR2 reduces flight. In each figure, Left panels represent wing flapping frequency, and Right panels depict a histogram of average frequencies. Control flies are UAS-CsChrimson and UAS-GtACR (n = 6). (D and E) Confocal images showing TH (d) and Ubx cells (e) in VNC and brain. (F) Schematic illustration of the Split-Gal4 system based on the complementation between the two functional domains of Gal4, the DNA-binding (DBD) and transcription-activation (AD) domains. Each domain is fused to a heterodimerizing leucine zipper (Zip+ or Zip−) that promotes the fusion of the two domains when expressed in the same cell reconstituting transcriptional activity. This technique was used to generate Ubx∩TH-Gal4 lines. (G) Confocal images of the fly VNC (ventral side) and brain (anterior side) showing the UAS-Myr::GFP expression pattern driven by UbxGal4.DBD ∩ pleGal4.AD (Ubx∩TH-Gal4) defines a subset of TH neurons that express Ubx. (H) Flight patterns of flies before and after optogenetic activation of Ubx+ TH cells, (n = 8–9). (I and J) Flies with Ubx positive neurons inhibited by expression of the potassium channel encoded by the Homo sapiens KCNJ2 gene (Kir2.1) show reduced flight duration (I) and forced flight index (J); a reduction is also observed when Kir2.1 is expressed in the TH-Gal4 domain, and, notably, in the Ubx∩TH intersectional domain. See also Movie S4 (related to Fig. 3A), Movie S5 (related to Fig. 3B), Movie S6 (related to Fig. 3C), and Movie S7 (related to Fig. 3H). Error bars represent SEM. Significant values in all figures based on Mann–Whitney U test or one-way ANOVA with the post hoc Tukey–Kramer test: ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.