BAcTrace, a tool for retrograde tracing of neuronal circuits in Drosophila

Abstract

Animal behavior is encoded in neuronal circuits in the brain. To elucidate the function of these circuits, it is necessary to identify, record from and manipulate networks of connected neurons. Here we present BAcTrace (Botulinum-Activated Tracer), a genetically encoded, retrograde, transsynaptic labeling system. BAcTrace is based on Clostridium botulinum neurotoxin A, Botox, which we engineered to travel retrogradely between neurons to activate an otherwise silent transcription factor. We validated BAcTrace at three neuronal connections in the Drosophila olfactory system. We show that BAcTrace-mediated labeling allows electrophysiological recording of connected neurons. Finally, in a challenging circuit with highly divergent connections, BAcTrace correctly identified 12 of 16 connections that were previously observed by electron microscopy.

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Fig. 1: System design.
Fig. 2: BAcTrace in tissue culture.
Fig. 3: BAcTrace in vivo.
Fig. 4: BAcTrace expression in ORNs labels connected PNs.
Fig. 5: BAcTrace reveals connections between PNs and LHNs.

Data availability

All data necessary for confirming the conclusions presented in the article are represented fully within the article. In addition, all raw and processed brain images and blots are freely available from the authors. All fly strains used are listed in Supplementary Tables 12–15 and are available either from the Bloomington Stock Center (Supplementary Table 17) or from the authors. All plasmids are available upon request from the authors and their sequences are deposited in GenBank as described in Supplementary Tables 9 and 12–14. Brain confocal stacks used for making ORN and LHN plots are available from the Zenodo repository47. Source data are provided with this paper.

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Acknowledgements

This work was supported by a Dorothy Hodgkin Fellowship from the Royal Society to S.C. (DH120072), ERC Starting (211089) and Consolidator (649111) grants and core support from the MRC (MC-U105188491) to G.S.X.E.J. We are grateful to B. Davletov (University of Sheffield), E. Ferrari (University of Lincoln) and J. Arsenault (University of Toronto) for sharing reagents and advice in early stages of the project. We acknowledge the Bloomington Drosophila Stock Center for fly stocks (NIH P40OD018537), the Developmental Studies Hybridoma Bank for antibodies and the Drosophila Genomics Resource Center (NIH grant 2P40OD010949) for plasmids. Finally, we thank R. Benton and all members of the Jefferis group for many insightful comments on the manuscript.

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Authors

Contributions

S.C. and G.S.X.E.J. conceived the project, obtained funding and supervised the research. S.C., L.B., A.M. and A.S. designed, cloned, purified proteins, conducted tissue culture experiments and analyzed the results. S.C., M.G. and B.S. designed and conducted BAcTrace experiments in flies, stained, imaged and analyzed the results. S.F. designed, conducted and analyzed the results of electrophysiology experiments. Y.A. made VT033006-LexA–P65 and obtained MFCO data. A.S.B. summarized electron microscopy connectivity data for LHNs. S.C. and G.S.X.E.J wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to Sebastian Cachero or Gregory S. X. E. Jefferis.

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The authors declare no competing interests.

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Peer review information Nina Vogt was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 BoNT/A1 mechanism of action in vertebrates.

Adapted from.52.

Extended Data Fig. 2 Testing BAcTrace in tissue culture.

a, Western blot analysis of S2 cell extracts transfected and incubated with decreasing amounts of toxin. b, Constructs used in (C) and (F). c, Immunochemistry of S2 cells transfected with sensors and TEV variants. d, Prediction of N-glycosylation sites on TEV (http://www.cbs.dtu.dk/services/NetNGlyc/). e, TEV structure (PDB id 1lvb)53 with potential glycosylation sites from (D) highlighted in blue. Bound pseudo-substrate shown in yellow. f, Western blot analysis of wild type and TEVT173V ability to cleave their target sequence. * non-specific band. g, Western blot analysis of cleavage efficiency under varying concentrations of hTfR::GFP receptor. Epitopes detected by antibodies are indicated in bold in (A), (C), (F) and (G). Experiments in (A), (F) and (G) were done once. Empty arrowheads in (A), (C), (F) and (G) indicate uncleaved while solid arrowheads indicate cleaved products. Source data

Extended Data Fig. 3 Expression strength of VT033006-LexA::P65.

a, V5 tag immunostaining of QF2::V5::hSNAP25::Syx in VK000018. b, GFP immunostaining of Syb::GFP in VK000037. Images in (A) and (B) are representative of stainings from 3 animals. c, Boxplot showing the fluorescence quantification per glomerulus for (A) and (B); each glomerulus was measured in 6 ALs from 3 brains. Leftmost panel in (A) and (B) are maximum intensity projections while all other panels are single slices from confocal microscopy stacks. Epitopes detected by antibodies are indicated in bold in (A) and (B). Scale bars 30 µm. Boxplot shows the median of the measurements. Lower and upper hinges correspond to the first and third quartiles; the upper whisker extends from the hinge to the largest value no further than 1.5. * inter-quartile range from the hinge. The lower whisker extends from the hinge to the smallest value at most 1.5 * inter-quartile range of the hinge.

Extended Data Fig. 4 TEVT173V modestly increases BAcTrace efficiency.

a) BAcTrace labelling of PNs from sparse KC Donors driven by MB005C using TEV variants. The panels for each condition are representative of stainings from 3 animals. (b) Constructs used in (C). (c) Targeting TEV to the synapse using the toxin receptor. The panels for each condition are representative of stainings from 4 animals. Maximum intensity projections of registered confocal stacks from age matched animals; images taken using the same microscope settings. Epitopes detected by antibodies are indicated in bold in (A) and (C). Genotypes for each panel are described in Supplementary Table 16. Scale bars 30 µm.

Extended Data Fig. 5 BAcTrace expression in subsets of KCs induces labelling in PNs.

a, Negative control missing a split Gal4 hemidriver. Donor KCs: b, all, c, α’/β’, d, γ and e, α/β. f, Higher magnification view of the MB calyx and LH of a brain with Donor MB607B neurons (same as in D). The brain was mounted dorsal side up to provide better image quality of the MB calyx and LH. Within each lobe subtypes are: γ lobe: main (m) and dorsal (d), α’/β’ lobe: anterior-posterior (ap) and middle (m) and α/β: posterior (p), core (c) and surface (s). Images in A-F are representative of stainings from at least 3 animals. Maximum intensity projections of registered confocal stacks from age matched animals; (A), (B), (C) and (D) taken using the same microscope settings. Epitopes detected by antibodies are indicated in bold in each panel. Number of KCs per subtype from24. Genotypes for each panel are described in Supplementary Table 16. Scale bars 30 µm.

Extended Data Fig. 6 BAcTrace expression in ORNs labels connected PNs.

(a) BAcTrace expression in specific ORNs induces labelling in connected PNs (dotted lines). Top panels shows maximum intensity projections of affine registered confocal stacks. Bottom 5 panels show single slices from the same specimen. Epitopes detected by antibodies are indicated in bold in each panel. (b) BAcTrace labelling quantification in older animals (10-12 days old) for the same crosses shown in Fig. 4c. Sample size: Or83c n = 10ALs, Or88a n = 10ALs and Or92a n = 12ALs. Genotypes for each panel are described in Supplementary Table 16. Scale bars 30 µm.

Extended Data Fig. 7 BAcTrace expression in LHNs induces labelling in PNs.

a, Expression of LHN driver lines tested. Anti-GFP immunostaining against UAS-csChrimson::mVenus in attP18. The name of each line is indicated on the top left and the cell type in each line on the top right. Images adapted from31 b, For each line animals of two ages were dissected. Several LHs were imaged (sample size: n number on top left corner of each image), registered to a template and averaged to produce the different panels. Bottom two panels on the right are a negative control made from line LH196 with one hemidriver missing. Epitopes detected by antibodies are indicated in bold in each panel. Genotypes for each panel are described in Supplementary Table 16. Scale bars (A) 30 µm and (B) 10 µm.

Extended Data Fig. 8 BAcTrace variability for PD2a1/b1 and AV1a1 LHNs.

BAcTrace labelled PNs by toxin expression in: a, PD2a1/b1 neurons (LH989) and b, AV1a1 neurons (LH1983). Images in (A) and (B) are representative of stainings from 5 and 7 animals respectively. Images are maximum intensity projections of confocal stacks, affine registered to a template for the AL region. The surface outside the neuropil region was masked out to avoid obscuring the glomeruli. Epitopes detected by antibodies are indicated in bold. Genotypes for each panel are described in Supplementary Table 16. Scale bars 10 µm.

Extended Data Fig. 9 BAcTrace reveals connections between PNs and LHNs.

a, Split Gal4 lines LH173 and LH1139 drive GFP expression in cell types PV5c1 and AV6a1, respectively. Images for a representative brain of 5 animals for each line. AL: Antennal Lobe. b, Single slices from a representative AL and corresponding LH showing PNs labelled by expression of toxin in PV5c1 and AV6a1. Images are representative of PV5c1 (9 animals) and AV6a1 (7 animals). c, 3D renderings summarizing BAcTrace results for PV5c1 and AV6a1. d, Forward and reciprocal synapses between PNs for the 15 glomeruli analyzed in Fig. 5 and PD2a1/b1 (top) and AV1a1 (bottom) as assessed by EM. The number next to the glomerular identity of the PN is its EM identification number. Epitopes detected by antibodies are indicated in bold in (A) and (B). Genotypes for each panel are described in Supplementary Table 16. Scale bars (A) 50 µm and (B) 10 µm.

Extended Data Fig. 10 LHN-PN connectivity.

Summary of published data for the 4 LHN cell types analyzed. R/S: average expression level for Receptor and Sensor (see Extended Data Fig. 3). BT: BAcTrace results. EM: Electron Microscopy connectivity data. EPhys: Electrophysiological recordings from LHNs during opto-stimulation of PNs54. Cell types PD2a1/b1: ML9+ML8 and AV1a1: V3 from ref. 53. LM: Light Microscopy overlap between single cell PNs labelled with a membrane marker and imaged by confocal microscopy and EM LHNs48,50. The number in column 1 (Glom.) indicates how many individual PNs contribute to the total number of synapses counted by EM.

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Supplementary Figs. 1–8, Notes 1–10, Tables 1–17, Protocols 1 and 2, and References.

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Source Data Fig. 2

Western blot films used to make Fig. 2a,d.

Source Data Extended Data Fig. 2

Western blot films used to make Extended Data Fig. 2a,f,g.

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Cachero, S., Gkantia, M., Bates, A.S. et al. BAcTrace, a tool for retrograde tracing of neuronal circuits in Drosophila. Nat Methods 17, 1254–1261 (2020). https://doi.org/10.1038/s41592-020-00989-1

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