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. Author manuscript; available in PMC: 2020 Jan 2.
Published in final edited form as: Neuron. 2018 Nov 28;101(1):103–118.e5. doi: 10.1016/j.neuron.2018.11.003

Figure 4. Stromalin knockdown in dopamine neurons increases levels of presynaptic synaptotagmin:GFP without altering other neuroanatomical markers.

Figure 4.

(A) Schematic diagram of the DAn (PPL1) innervation pattern of the MB lobe neuropil. The PPL1 DAn send axonal projections to the vertical but not the horizontal lobes of the MB neuropil.

(B) The number of PPL1 DAn soma, visualized in the dorsal posterior brain using membrane-localized or nuclear-localized GFP, was not altered by Stromalin KD. Control: TH-GAL4>+, UAS-Dcr2, UAS-mCD8:GFP or TH-GAL4>+, UAS-Dcr2, UAS-GFPnls. StromalinRNAi: TH-GAL4>UAS-SARNAi, UAS-Dcr2, UAS-mCD8:GFP or TH-GAL4>UAS-SARNAi, UAS-Dcr2, UAS-GFPnls.

(C) Stromalin KD in DAn did not affect dopaminergic innervation of the of the MB neuropil, visualized using membrane-localized GFP. Quantitation of fluorescence intensity across MB lobe regions is shown in the right bar plot. MB lobes are outlined with a dotted line. Control: TH-GAL4>+, UAS-Dcr2, UAS-mCD8:GFP. StromalinRNAi: TH-GAL4>UAS-SARNAi, UAS-Dcr2, UAS-mCD8:GFP. n=19–20. Mean±SEM. Scale bar 20µm.

(D) Stromalin KD in DAn resulted in elevated levels of the presynaptic marker, syt:GFP across all sections of the vertical lobes and heel. Quantitation of fluorescence intensity across regions of interest are shown in the right bar plot. MB lobes are outlined with a dotted line. Control: TH-GAL4>+, UAS-Dcr2, UASsyt:GFP. StromalinRNAi: TH-GAL4>UAS-SARNAi, UAS-Dcr2, UAS-syt:GFP. MannWhitney U, ***p<0.001. n=20. Mean±SEM. Scale bar 20µm.

(E) Stromalin KD increased dopaminergic syt:GFP levels during the middle to late 3rd instar larval stage in the MB heel (arrowhead). MB vertical lobe, junction and heel are outlined with a white dotted line. Quantitation of fluorescence intensity in the heel region is shown in the right bar plot. Quantitation of fluorescence intensity in the vertical lobes is presented in Figure S4. Control: THGAL4>+, UAS-Dcr2, UAS-syt:GFP, UAS-myr-tdTom. StromalinRNAi: THGAL4>UAS-SARNAi, UAS-Dcr2, UAS-syt:GFP, UAS-myr-tdTom. Mann-Whitney U, *p<0.05. n=11–15. Mean±SEM. Scale bar 20µm.

(F) Pan-neuronal stromalinRNAi expression increased syt:GFP across the whole adult Drosophila brain. Control: nSyb-GAL4>+, UAS-Dcr2, and UAS-syt:GFP. StromalinRNAi: nSyb-GAL4>UAS-SARNAi, UAS-Dcr2, and UAS-syt:GFP. MannWhitney U, ***p<0.001. n=13–14. Mean±SEM. Scale bar 100µm.

(G) Reducing Stromalin in MBn increased syt:GFP levels in the adult Drosophila MBn. Control: nSyb-GAL4>+, UAS-Dcr2, and UAS-syt:GFP. StromalinRNAi: nSyb-GAL4>UAS-SARNAi, UAS-Dcr2, and UAS-syt:GFP. Mann-Whitney U, ****p<0.0001. n=13. Mean±SEM. Scale bar 50µm.

See also Figure S4.