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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 6. Stromalin knockdown increases the number of dopaminergic synaptic vesicles.

Figure 6.

(A) Diagram of the heel region imaged in this experiment.

(B) Electron micrograph of the MB heel (left; scale bar 5µm). DAn axons expressing membrane-localized HRP were labeled with an electron dense stain (right; scale bar 250nm). Serial EM images were used to trace DAn processes (blue), synaptic vesicles (SV; red), larger sized dense core vesicle (DCV; red), and active zones as identified by T-bars (arrow).

(C) 3D reconstructions of serial EM images of a section of labeled DAn processes in the MB heel region from a control and a Stromalin KD brain. Scale bar 150nm.

(D) Sum of measures obtained from 3 control and 3 stromalinRNAi brains. Control: TH-GAL4>+, UAS-Dcr2, UAS-CD4:HRP. StromalinRNAi: TH-GAL4>UAS-SARNAi, UAS-Dcr2, UAS-CD4:HRP.

(E) Volume of DAn processes in the MB heel region sampled from control and stromalinRNAi brains. Control: TH-GAL4>+, UAS-Dcr2, UAS-CD4:HRP. StromalinRNAi: TH-GAL4>UAS-SARNAi, UAS-Dcr2, UAS-CD4:HRP. n=3. Mean±SEM.

(F) Despite sampling the same volume of DAn processes from both genotypes, Stromalin KD DAn processes contained 2.3-fold more vesicles than the controls (left). Frequency of DA vesicle sizes indicates the presence of 2 populations of vesicles: synaptic vesicles (≤50nm) and dense core vesicles (>50nm). Cumulative probability graph of vesicle sizes reveals Stromalin KD does not alter the proportion or size of vesicles (right). Control: TH-GAL4>+, UAS-Dcr2, UASCD4:HRP. StromalinRNAi: TH-GAL4>UAS-SARNAi, UAS-Dcr2, UAS-CD4:HRP.

(G) Stromalin KD DAn contain 2-fold more synaptic vesicles within 200nm of an active zone compared to control DAn. The numbers of DCVs within this proximity to an active zone was not different between genotypes. Control: TH-GAL4>+, UAS-Dcr2, UAS-CD4:HRP. StromalinRNAi: TH-GAL4>UAS-SARNAi, UAS-Dcr2, UAS-CD4:HRP. Mann-Whitney U, ****p<0.0001. n=52 control, n=74 StromalinRNAi. Mean±SEM.

(H) Graphical cartoon of representative control and Stromalin KD vesicle distributions within 200nm of an active zone. Scale bar 50nm.

See also Figure S6.