Fig. 5: Mirana downregulation reduces synaptic vesicle release and connectivity to postsynaptic cells. | Nature Communications

Fig. 5: Mirana downregulation reduces synaptic vesicle release and connectivity to postsynaptic cells.

From: Temporal transcriptional regulation of mitochondrial morphology primes activity-dependent circuit connectivity

Fig. 5

A–B” Live imaging of steady state synaptic vesicle release using UAS-Syt1-mCherry::pHluorin pH sensitive fluorescence probe in control (A–A”) and Mirana-RNAi100127 (B–B”). C Ratio of green (pHluorin, cyan arrowheads) to red (mCherry) fluorescence and number of puncta in control (n = 6) and Mirana-RNAi condition (n = 7). D–F’, I TransTango anterograde postsynaptic labeling of Mirana downregulation, with an expression of UAS-Mirana-RNAi100127 (n = 20 optic lobes) and Mirana_gRNA; UAS-UM-Cas9 (Supplementary Fig. S5, n = 25 optic lobes) compared to control (n = 17 optic lobes). Visible M-DCN axons in yellow arrowheads. Developmental Mirana depletion (from L1 till eclosion, F–F’, I, n = 11 optic lobes). G, G’ Silencing of DCNs by overexpression of conductive form of potassium channel dORK (UAS-dORK1Δ-C(1), n = 18 optic lobes). H, H’ Nonconductive form of dORK (UAS-dORK1Δ-NC(1), n = 10 optic lobes) used as an additional control, showed no effect on TransTango labeling. DCNs expressing myrGFP on the cellular membrane are shown in green (D, D’, E, E’, F, G, H) and postsynaptic cells in magenta (merged picture) and grayscale (D, D”, E, E”, F, F’, G, G’, H, H’). Postsynaptic connectivity was analyzed by manually counting cell bodies in Medulla (white arrowheads) labeled with dsRed fluorescence (magenta, merged picture – D–H and grayscale – D”, E”, F’, G’, H’), including all cells with weak or strong labeling to reveal all potential connections. Scale bar 20 µm. Object orientation behavior of flies with Mirana downregulation in DCNs in Buridan assay (J). K, L Individual fly trajectory during recording. K’, L’ Heat map of arena occupancy at the population level, n = 125 control and 135 RNAi100127 flies. M Absolute stripe deviation measurement, showing that Control flies (W+; UAS-KK40D30B;ato-Gal414A,UAS-CD4::GFP) normally walk more straight (represented in lover ASD value) than flies with Mirana downregulation (W+;UAS-Mirana-RNAi;ato-Gal414A,UAS-CD4::GFP). Me medulla, Lo lobula, OC optic chiasm. Ordinary one-way ANOVA with the Kruskal–Wallis test was used for the behavioral experiment and unpaired t-test with Welch’s correction for synaptic marker counting and trans-TANGO labeling analysis. Exact p values. *p < 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, ns not significant.

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