Where do pale and yellow signals go in the Drosophila brain? Michael B. Reiser, Aljoscha Nern, Arthur Zhao, Kit Longden, Gerry Rubin, Miriam Flynn, Connor Laughland, Henrique Ludwig, and Ruchi Parekh Janelia Research campus, HHMI aMe12 Rh6-R8 (yellow ommatidia) Rh3-R7 (pale ommatidia) aMe12 Recently characterized aMe12 cells extend along pale columns in the medulla aMe12 cells reconstructed in FAFB SS-TEM volume aMe12 pR7 pR8 yR7 yR8 aMe12 cells identify pale and yellow columns for targeted reconstruction pR7 yR7 pR8 yR8 total % of total % of R7 % of R8 % of pale % of yellow Dm9 (6) 70 61 98 170 399 14.90 12.19 18.97 42.11 57.89 Dm8 (14) 226 151 0 0 377 14.08 35.07 0.00 59.95 40.05 Tm_unc (14) 59 17 50 46 172 6.43 7.07 6.79 63.37 36.63 Tm_OTu (6) 75 90 0 0 165 6.16 15.35 0.00 45.45 54.55 Tm5c (6) 8 4 20 110 142 5.30 1.12 9.20 19.72 80.28 Tm20 (4) 5 2 65 62 134 5.01 0.65 8.99 52.24 47.76 Mi4 (4) 0 0 56 57 113 4.22 0.00 8.00 49.56 50.44 Mi15 (4) 6 6 27 73 112 4.18 1.12 7.08 29.46 70.54 Dm2 (4) 21 3 55 15 94 3.51 2.23 4.95 80.85 19.15 ML1 (4) 0 0 56 38 94 3.51 0.00 6.65 59.57 40.43 yR7 (3) 0 3 0 80 83 3.10 0.28 5.66 0.00 100.00 pR7 (2) 0 0 68 0 68 2.54 0.00 4.81 100.00 0.00 Mi1 (4) 0 0 30 29 59 2.20 0.00 4.18 50.85 49.15 L3 (4) 18 15 11 12 56 2.09 3.07 1.63 51.79 48.21 Dm11 (2) 23 26 3 2 54 2.02 4.56 0.35 48.15 51.85 Tm5a (2) 0 52 0 0 52 1.94 4.84 0.00 0.00 100.00 Mt_unc (7) 2 11 11 18 42 1.57 1.21 2.05 30.95 69.05 Tm5b (3) 20 5 2 11 38 1.42 2.33 0.92 57.89 42.11 L1 (4) 2 1 19 14 36 1.34 0.28 2.34 58.33 41.67 Mi9 (4) 3 8 24 0 35 1.31 1.02 1.70 77.14 22.86 aMe12 (2) 4 0 24 0 28 1.05 0.37 1.70 100.00 0.00 yR8 (2) 0 27 0 0 27 1.01 2.51 0.00 0.00 100.00 C2 (3) 7 13 6 0 26 0.97 1.86 0.42 50.00 50.00 pR8 (2) 25 0 1 0 26 0.97 2.33 0.07 100.00 0.00 ML2b (3) 0 0 25 0 25 0.93 0.00 1.77 100.00 0.00 Dm_unc (3) 5 1 9 5 20 0.75 0.56 0.99 70.00 30.00 Mi10 (1) 0 0 0 6 6 0.22 0.00 0.42 0.00 100.00 C3 (1) 0 0 0 3 3 0.11 0.00 0.21 0.00 100.00 ML2a (1) 0 0 0 2 2 0.07 0.00 0.14 0.00 100.00 unknown (143) 30 51 43 65 189 7.06 7.53 7.64 38.62 61.38 total 579 496 660 753 2677 100.00 100.00 100.00 46.28 46.66 pre-synaptic cell types post-synaptic cell types Pale Yellow DRA Eye Lamina Medulla Lobula Lobula Plate 300 350 400 450 500 500 600 650 0 0.5 1 R7 Rh3 Rh4 Rh5 Rh6 Rh1 R8 R1-6 ) c ( ) a ( (d) (b) Spectral sensitivity Wavelength (nm) Rh5 Rh6 Rh3 Rh1 Rh1 Rh1 Rh1 Rh5 Rh1 Rh1 Rh3 Rh3 Rh4 Rh6 Motion vision Phototaxis Color vision Polarized light vision Current Opinion in Neurobiology Drosophila eye background Behnia & Desplan (2015) 50 µm lamina medulla central brain R7 Example R7 manually traced through ~2200 sections Full adult fly brain (Zheng, et al. 2018; temca2data.org) CATMAID environment (Saalfeld, Cardona, et al. 2009) Dm8 Dm2 Dm9 Dm11 TmOTu Tm5c Tm20 Mi4 Mi15 ML1 Mi1 L3 Tm5a Tm5b L1 Mi9 C2 C3 ML2a/b Examples of major R7 and R8 target neurons reconstructed to identification R7 R8 100 500 50 20 inputs (post-) R7 R8 outputs (pre-) synapse count 7-column FIBSEM 2-column pale TEM 2-column yellow TEM Comparing the synapse counts from our 4-column reconstruction to those of Janelia FlyEM’s 7-column FIBSEM (Takemura, et al. 2015). Surprisingly we find a similar number of synapses. A partial explanation is that the TEM volume contains the lamina and connections. 15.4% of input and 7.4% of output synapses are found outside of the medulla. In the 7-column reconstruction, only 57% of R8’s pre-synapse partners could be identified; 48% for R7’s partners. Connectivity summary •’Greedy’ approach to targeted connectome of R7/R8 shown to be practical; propose to use as test case for new analysis methods. •Unique identity for many Tm cells proving difficult only using morphology -- more extensive reconstruction and connectivity required. •Several candidate pale or yellow ‘specialist’ target neurons identified. Ongoing work to verify these and search for evidence of potential opponency mechanism. • What would you do next?