Umber of synapses on C4da and A08n neurons, and then calculated the percentage of synapses that contained A08n postsynapses andNATURE COMMUNICATIONS | (2019)ten:3506 | 41467-019-11408-1 | www.nature.comnaturecommunicationsNATURE COMMUNICATIONS | 41467-019-11408-ARTICLEaC2da C3da C4da A08nAnterior (A)Posterior (P) Lateral Medial LateralbC4da presynapse A08n postsynapsecFasrecGFPMerge27H06-LexA LexAop-syb-spGFP1-10, UAS-CD4-spGFP-11; 82E12-GaldA08n (Drep2-GFP) Abdominal segment five 6 C4da (Brpshort -mCherry)eC4da A A08nP Brpshort-mCherry Drep2-GFPshortMerge27H06-LexA LexAop-Brp-mCherry ; 82E12-Gal4 UAS-Drep2-GFPfC4da-A08n synapsesg500 Synaptic spots 400 300 200 100. P e. . oc st AS Pr Po ol R C a 8nhof total synapses one hundred 80 60 40 20C4da nsA08n ns100 50EMEM.ocolCbG4dFig. 1 Quantitative evaluation of C4da 08n neuron connectivity. a Schematic model of a larval brain with A08n neurons (green), C4da ( magenta), and C2daC3da axon projections (bluecyan) into the VNC and schematic expansion of abdominal segments 5 and six. b Schematic model of Syb-GRASPmediated GFP reconstitution. c C4da 08n Syb-GRASP signal in a single hemisegment showing anti-Fas3 staining (blue), reconstituted GFP (green), and merge. Scale bar = five . d C4da presynaptic (Brpshort-mCherry) and A08n postsynaptic (Drep2-GFP) marker expression and example confocal image of abdominal segments five and 6 displaying expressed markers in C4da (magenta) and A08n (green). e Hemisegment showing Brpshort-mCherry in C4da (magenta), Drep2-GFP in A08n (green), and merge. Scale bar = five . f Quantification of Syb-GRASP puncta when compared with synaptic marker colocalization. Syb-GRASP n = eight, colocalization n = 9 SD. g Quantification of C4da presynaptic and A08n postsynaptic marker puncta per hemisegment. n = 10 SD. h Comparison of EM and light microscopic marker colocalization analyses. Percentages of counted C4da presynapses PEG4 linker Autophagy connected to A08n or A08n postsynapses connected to C4da showed no important differences involving both techniques. EM: C4da n = 4, A08n n = 4, confocal: C4da n = ten, A08n n = 10. C4da: P = 0.06, A08n: P = 0.43 SD. Unpaired two-tailed t-testSyACColoc.NATURE COMMUNICATIONS | (2019)10:3506 | 41467-019-11408-1 | www.nature.comnaturecommunicationsARTICLEaC4da-A08n synapses 150 100 50 0 48 h 72 h 96 h 120 h Syb-GRASPNATURE COMMUNICATIONS | 41467-019-11408-bC4da-A08n synapsesColoc ns 100 50 0 48 h 72 h 96 h 120 h 27H06-LexA LexAop-Brpshort-mCherry; 82E12-Gal4 UAS-Drep2-GFP27H06-LexA LexAop-syb-spGFP1-10, UAS-CD4-spGFP-11; 82E12-GalcC4da presynapses500 400 300 200 one hundred 0 48 h 72 h 96 h 120 h 27H06-LexA LexAop-Brpshort-mCherry; 82E12-Gal4 UAS-Drep2-GFPdA08n postsynapses200 150 one hundred 50 0 48 hnsns72 h96 h120 h27H06-LexA LexAop-Brpshort-mCherry; 82E12-Gal4 UAS-Drep2-GFPeC4da-A08n synapses C4da presynapsesC4da-A08n synapses A08n postsynapses100 80 60 40 20 0 48 h 72 hfns100 80 60 40 20 0 48 h 72 hnsns96 h120 h96 h120 h27H06-LexA LexAop-Brpshort-mCherry; 82E12-Gal4 UAS-Drep2-GFP27H06-LexA LexAop-Brpshort-mCherry; 82E12-Gal4 UAS-Drep2-GFPFig. 2 Scalar improve of C4da 08n connectivity during larval growth. a Quantification of C4da 08n synapses in the course of improvement from 48 h AEL until 120 h AEL with Syb-GRASP. 48 h: n = 4, 72 h: n = ten, 96 h: n = ten, 120 h: n = 9. P 0.0001. ANOVA with various comparisons and Sidak’s post-hoc test (for exact P-values and statistics see Supplementary Information 1). b Quantification of C4da 08n synaptic connectivity through development from 48 h AEL until 120 h AEL applying co.
