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. Author manuscript; available in PMC: 2021 Jul 2.

Published in final edited form as: Cell Rep. 2020 Sep 15;32(11):108138. doi: 10.1016/j.celrep.2020.108138

Figure 6. — (A) A schematic of the Drosophila brain shows a group of α/β_p Kenyon cell input neurons—_PLPPNs (pink)—projecting from the posterior lateral protocerebrum (PLP) to the dACA.

(B and C) A group of _PLPPNs (bright green) were identified in the screen using two different transgenic lines (B: R19H07-GAL4 and C: R20G07-GAL4).

(D) _PLPPNs were photo-labeled using the R19H07-GAL4 transgenic line; the sample was fixed, immuno-stained (nc82 antibody, blue), and imaged. The photo-labeled neurons show an overall similar morphology: their somata are located near the LH; they extend projections in the PLP, the superior clamp (SC), the SLP, and the dACA.

(E and F) The expression patterns observed for each of the GAL4 lines are broad and include many neurons; (E) the R19H07-GAL4 line drives expression strongly in many neurons, including the _PLPPNs, (F) whereas the R20G07-GAL4 line drives expression weakly in fewer neurons, including the _PLPPNs. These images were obtained from the FlyLight website.

(G) A split-GAL4 transgenic line driving expression strongly in the _PLPPNs was engineered using the R19H07 promoter region to drive the GAL4_AD domain and the R20G07 promoter region to drive the GAL4_DBD.

(H and I) The split-GAL4 line was used to drive the expression of the pre-synaptic marker synaptotagmin-fused GFP (H) or the post-synaptic maker DenMark (I).

(J and K) _PLPPN-like neurons were identified in the hemibrain connectome: (J) these neurons project from the PLP to the dACA, the SLP, and the SC; (K) their axonal terminals innervate the dACA, but not the CA or the vACA. The connectome recognizes four different types of _PLPPN, but the basis for this categorization is not clear (blue, red, green, and purple). The images in (J) and (K) were taken directly from the Neuprint platform. The neuropil domains are defined by Neuprint platform.

The following genotypes were used in this figure: (B and D) yw/yw; MB247-DsRed^unknown, UAS-C3PA-GFP^unknown/UAS-C3PA-GFP^attP40;UAS-C3PA-GFP^attP2, UAS-C3PA-GFP^VK00005,UAS-C3PA-GFP^VK00027/R19H07-GAL4^attP2; (C) yw/yw; UAS-C3PA-GFP^unknown/CyO;UAS-C3PA-GFP^attP2, R20G07-GAL4^attP2/MKRS; (G) yw/yw; MB247-DsRed^unknown, UAS-C3PA-GFP^unknown/CyO; UAS-C3PA-GFP^attP2, UAS-C3PA-GFP^VK00005,UAS-C3PA-GFP^VK00027/R20G07-GAL4_DBD^attP2,R19H07-GAL4_AD^VK00027; (H) yw/yw;Sp/CyO; R20G07-GAL4_DBD^attP2,R19H07-GAL4_AD^VK00027/UAS-synaptotagmin::GFP^unknown; and (I) yw/yw;UAS-DenMark²/CyO; R20G07-GAL4_DBD^attP2,R19H07-GAL4_AD^VK00027/Tm2. Scale bars are 20 μm (A–D), 100 μm (E–G), and 20 μm (H and I).