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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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.
Luo, L., Callaway, E. M. & Svoboda, K. Genetic dissection of neural circuits: a decade of progress. Neuron 98, 256–281 (2018).
Ugur, B., Chen, K. & Bellen, H. J. Drosophila tools and assays for the study of human diseases. Dis. Model. Mech. 9, 235–244 (2016).
Venken, K. J. T., Simpson, J. H. & Bellen., H. J. Genetic manipulation of genes and cells in the nervous system of the fruit fly. Neuron 72, 202–230 (2011).
Ohyama, T. et al. A multilevel multimodal circuit enhances action selection in Drosophila. Nature 520, 633–639 (2015).
Zheng, Z. et al. A complete electron microscopy volume of the brain of adult Drosophila melanogaster. Cell 174, 730–743 (2018).
Takemura, S.-Y, Lu, Z. & Meinertzhagen, I. A. Synaptic circuits of the Drosophila optic lobe: the input terminals to the medulla. J. Comp. Neurol. 509, 493–513 (2008).
Talay, M. et al. Transsynaptic mapping of second-order taste neurons in flies by trans-Tango. Neuron 96, 783–795 (2017).
Huang, T.-H. et al. Tracing neuronal circuits in transgenic animals by transneuronal control of transcription (TRACT). eLife 6, e32027 (2017).
Dong, M. et al. Sv2 is the protein receptor for botulinum neurotoxin A. Science 312, 592–596 (2006).
Montal, M. Botulinum neurotoxin: a marvel of protein design. Annu. Rev. Biochem. 79, 591–617 (2010).
Pirazzini, M., Rossetto, O., Eleopra, R. & Montecucco, C. Botulinum neurotoxins: biology, pharmacology, and toxicology. Pharmacol. Rev. 69, 200–235 (2017).
Pauli, A. et al. Cell-type-specific TEV protease cleavage reveals cohesin functions in Drosophila neurons. Dev. Cell 14, 239–251 (2008).
Kubala, M. H., Kovtun, O., Alexandrov, K. & Collins, B. M. Structural and thermodynamic analysis of the GFP:GFP-nanobody complex. Protein Sci. 19, 2389–2401 (2010).
Riabinina, O. et al. Improved and expanded Q-system reagents for genetic manipulations. Nat. Methods 12, 219–222 (2015).
Washbourne, P., Pellizzari, R., Baldini, G., Wilson, M. C. & Montecucco, C. Botulinum neurotoxin types A and E require the SNARE motif in SNAP-25 for proteolysis. FEBS Lett. 418, 1–5 (1997).
Gupta, G. D. et al. Analysis of endocytic pathways in Drosophila cells reveals a conserved role for GBF1 in internalization via GEECs. PLoS ONE 4, e6768 (2009).
Tobin, W. F., Wilson, R. I. & Lee, W.-C. A. Wiring variations that enable and constrain neural computation in a sensory microcircuit. eLife 6, e24838 (2017).
Masse, N. Y., Turner, G. C. & Jefferis, G. S. X. E. Olfactory information processing in Drosophila. Curr. Biol. 19, R700–R713 (2009).
Turner, G. C., Bazhenov, M. & Laurent, G. Olfactory representations by Drosophila mushroom body neurons. J. Neurophysiol. 99, 734–746 (2008).
Murthy, M., Fiete, I. & Laurent, G. Testing odor response stereotypy in the Drosophila mushroom body. Neuron 59, 1009–1023 (2008).
Caron, S. J. C., Ruta, V., Abbott, L. F. & Axel., R. Random convergence of olfactory inputs in the Drosophila mushroom body. Nature 497, 113–117 (2013).
Gruntman, E. & Turner, G. C. Integration of the olfactory code across dendritic claws of single mushroom body neurons. Nat. Neurosci. 16, 1821–1829 (2013).
Nern, A., Pfeiffer, B. D., Svoboda, K. & Rubin., G. M. Multiple new site-specific recombinases for use in manipulating animal genomes. Proc. Natl Acad. Sci. USA 108, 14198–14203 (2011).
Aso, Y. et al. The neuronal architecture of the mushroom body provides a logic for associative learning. eLife 3, e04577 (2014).
Couto, A., Alenius, M. & Dickson., B. J. Molecular, anatomical, and functional organization of the Drosophila olfactory system. Curr. Biol. 15, 1535–1547 (2005).
Fishilevich, E. & Vosshall, L. B. Genetic and functional subdivision of the Drosophila antennal lobe. Curr. Biol. 15, 1548–1553 (2005).
Rybak, J. et al. Synaptic circuitry of identified neurons in the antennal lobe of Drosophila melanogaster. J. Comp. Neurol. 524, 1920–1956 (2016).
Klapoetke, N. C. et al. Independent optical excitation of distinct neural populations. Nat. Methods 11, 338–346 (2014).
Dolan, M.-J. et al. Communication from learned to innate olfactory processing centers is required for memory retrieval in Drosophila. Neuron 100, 651–668 (2018).
Huoviala, P. et al. Neural circuit basis of aversive odour processing in Drosophila from sensory input to descending output. Preprint at bioRxiv https://doi.org/10.1101/394403 (2018).
Dolan, M.-J. et al. Neurogenetic dissection of the Drosophila lateral horn reveals major outputs, diverse behavioural functions, and interactions with the mushroom body. eLife 8, e43079 (2019).
Butcher, N. J., Friedrich, A. B., Lu, Z., Tanimoto, H. & Meinertzhagen., I. A. Different classes of input and output neurons reveal new features in microglomeruli of the adult Drosophila mushroom body calyx. J. Comp. Neurol. 520, 2185–2201 (2012).
Frechter, S. et al. Functional and anatomical specificity in a higher olfactory centre. eLife 8, e44590 (2019).
Felsenberg, J. et al. Integration of parallel opposing memories underlies memory extinction. Cell 175, 709–722 (2018).
Sayin, S. et al. A neural circuit arbitrates between persistence and withdrawal in hungry Drosophila. Neuron 104, 544–558 (2019).
Kazama, H. & Wilson, R. I. Origins of correlated activity in an olfactory circuit. Nat. Neurosci. 12, 1136–1144 (2009).
Kornfeld, J. & Denk, W. Progress and remaining challenges in high-throughput volume electron microscopy. Curr. Opin. Neurobiol. 50, 261–267 (2018).
Schlegel, P., Costa, M. & Jefferis, G. S. X. E. Learning from connectomics on the fly. Curr. Opin. Insect Sci. 24, 96–105 (2017).
Bates, A. S., Janssens, J., Jefferis, G. S. X. E. & Aerts, S. Neuronal cell types in the fly: single-cell anatomy meets single-cell genomics. Curr. Opin. Neurobiol. 56, 125–134 (2019).
Lacy, D. B., Tepp, W., Cohen, A. C., DasGupta, B. R. & Stevens, R. C. Crystal structure of botulinum neurotoxin type A and implications for toxicity. Nat. Struct. Biol. 5, 898–902 (1998).
Cachero, S. & Jefferis., G. S. X. E. Drosophila olfaction: the end of stereotypy? Neuron 59, 843–845 (2008).
Pfeiffer, B. D. et al. Refinement of tools for targeted gene expression in Drosophila. Genetics 186, 735–755 (2010).
Pfeiffer, B. D., Truman, J. W. & Rubin, G. M. Using translational enhancers to increase transgene expression in Drosophila. Proc. Natl Acad. Sci. USA 109, 6626–6631 (2012).
Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009).
R Core Team. R: A Language and Environment for Statistical Computing (R Foundation for Statistical Computing, 2016).
Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).
Cachero, S. et al. BAcTrace a new tool for retrograde tracing of neuronal circuits. Zenodo https://doi.org/10.5281/zenodo.3797211 (2020).
Chiang, A.-S. et al. Three-dimensional reconstruction of brain-wide wiring networks in Drosophila at single-cell resolution. Curr. Biol. 21, 1–11 (2011).
Costa, M., Manton, J. D., Ostrovsky, A. D., Prohaska, S. & Jefferis, G. S. X. E. NBLAST: rapid, sensitive comparison of neuronal structure and construction of neuron family databases. Neuron 91, 293–311 (2016).
Bates, A. S. et al. Complete connectomic reconstruction of olfactory projection neurons in the fly brain. Curr. Biol. 30, 3183–3199.e6 (2020).
Kohl, J., Ostrovsky, A. D., Frechter, S. & Jefferis., G. S. X. E. A bidirectional circuit switch reroutes pheromone signals in male and female brains. Cell 155, 1610–1623 (2013).
Davletov, B., Bajohrs, M. & Binz, T. Beyond Botox: advantages and limitations of individual botulinum neurotoxins. Trends Neurosci. 28, 446–452 (2005).
Phan, J. et al. Structural basis for the substrate specificity of tobacco etch virus protease. J. Biol. Chem. 277, 50564–50572 (2002).
Jeanne, J. M., Fişek, M. & Wilson, R. I. The organization of projections from olfactory glomeruli onto higher-order neurons. Neuron 98, 1198–1213.e6 (2018).
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.
The authors declare no competing interests.
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.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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.
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.
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.
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.
(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.
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.
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.
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.
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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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