Dual Anterograde and Retrograde Viral Tracing of Reciprocal Connectivity

  • Matthias G. Haberl
  • Melanie Ginger
  • Andreas FrickEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1538)


Current large-scale approaches in neuroscience aim to unravel the complete connectivity map of specific neuronal circuits, or even the entire brain. This emerging research discipline has been termed connectomics. Recombinant glycoprotein-deleted rabies virus (RABV ∆G) has become an important tool for the investigation of neuronal connectivity in the brains of a variety of species. Neuronal infection with even a single RABV ∆G particle results in high-level transgene expression, revealing the fine-detailed morphology of all neuronal features—including dendritic spines, axonal processes, and boutons—on a brain-wide scale. This labeling is eminently suitable for subsequent post-hoc morphological analysis, such as semiautomated reconstruction in 3D. Here we describe the use of a recently developed anterograde RABV ∆G variant together with a retrograde RABV ∆G for the investigation of projections both to, and from, a particular brain region. In addition to the automated reconstruction of a dendritic tree, we also give as an example the volume measurements of axonal boutons following RABV ∆G-mediated fluorescent marker expression. In conclusion RABV ∆G variants expressing a combination of markers and/or tools for stimulating/monitoring neuronal activity, used together with genetic or behavioral animal models, promise important insights in the structure–function relationship of neural circuits.

Key words

Connectome Neural circuits Sparse labeling Pseudotyping Axonal arbor Dendritic spines Projections Rabies virus Neuroanatomy 


  1. 1.
    Wickersham IR, Finke S, Conzelmann K-K, Callaway EM (2007) Retrograde neuronal tracing with a deletion-mutant rabies virus. Nat Methods 4:47–49CrossRefPubMedGoogle Scholar
  2. 2.
    Haberl MG, Viana da Silva S, Guest JM et al. (2015) An anterograde rabies virus vector for high-resolution large-scale reconstruction of 3D neuron morphology. Brain Struct Funct 220:1369. doi: 10.1007/s00429-014-0730-z.
  3. 3.
    Mebatsion T, Schnell MJ, Cox JH, Finke S, Conzelmann KK (1996) Highly stable expression of a foreign gene from rabies virus vectors. Proc Natl Acad Sci U S A 93:7310–7314CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Ginger M, Haberl M, Conzelmann K-K, Schwarz MK, Frick A (2013) Revealing the secrets of neuronal circuits with recombinant rabies virus technology. Front Neural Circuits 7:2PubMedPubMedCentralGoogle Scholar
  5. 5.
    Ginger M, Bony G, Haberl MG, Frick A (2015). Use of rhabdoviruses to study neural circuitry. In: Pattnaik AK, Witt MA (eds), Biology and pathogenesis of rhabdo- and filoviruses, 1st edn. Chapter 11, pages: 263–287Google Scholar
  6. 6.
    Paxinos G, Franklin KBJ (2008) The mouse brain in stereotaxic coordinates. Academic, San DiegoGoogle Scholar
  7. 7.
    Cetin A, Komai S, Eliava M, Seeburg PH, Osten P (2007) Stereotaxic gene delivery in the rodent brain. Nat Protoc 1:3166–3173CrossRefGoogle Scholar
  8. 8.
    Gerfen CR (2003) Basic neuroanatomical methods. Curr Protoc Neurosci Chapter 1:Unit 1.1PubMedGoogle Scholar
  9. 9.
    Osakada F, Mori T, Cetin AH, Marshel JH, Virgen B, Callaway EM (2011) New rabies virus variants for monitoring and manipulating activity and gene expression in defined neural circuits. Neuron 71:617–631CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Yonehara K, Farrow K, Ghanem A, Hillier D, Balint K, Teixeira M, Jüttner J, Noda M, Neve RL, Conzelmann K-K, Roska B (2013) The first stage of cardinal direction selectivity is localized to the dendrites of retinal ganglion cells. Neuron 79:1078–1085CrossRefPubMedGoogle Scholar
  11. 11.
    Wickersham IR, Sullivan HA, Seung HS (2013) Axonal and subcellular labelling using modified rabies viral vectors. Nat Commun 4:2332CrossRefPubMedGoogle Scholar
  12. 12.
    Wickersham IR, Sullivan HA, Seung HS (2010) Production of glycoprotein-deleted rabies viruses for monosynaptic tracing and high-level gene expression in neurons. Nat Protoc 5:595–606CrossRefPubMedGoogle Scholar
  13. 13.
    Osakada F, Callaway EM (2013) Design and generation of recombinant rabies virus vectors. Nat Protoc 8:1583–1601CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Ghanem A, Kern A, Conzelmann K-K (2012) Significantly improved rescue of rabies virus from cDNA plasmids. Eur J Cell Biol 91:10–16CrossRefPubMedGoogle Scholar
  15. 15.
    Kelly RM, Strick PL (2000) Rabies as a transneuronal tracer of circuits in the central nervous system. J Neurosci Methods 103:63–71CrossRefPubMedGoogle Scholar
  16. 16.
    Ragan T, Kadiri LR, Venkataraju KU, Bahlmann K, Sutin J, Taranda J, Arganda-Carreras I, Kim Y, Seung HS, Osten P (2012) Serial two-photon tomography for automated ex vivo mouse brain imaging. Nat Methods 9:255–258CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Dodt H-U, Leischner U, Schierloh A, Jährling N, Mauch CP, Deininger K, Deussing JM, Eder M, Zieglgänsberger W, Becker K (2007) Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain. Nat Methods 4:331–336CrossRefPubMedGoogle Scholar
  18. 18.
    Susaki EA, Tainaka K, Perrin D, Kishino F, Tawara T, Watanabe TM, Yokoyama C, Onoe H, Eguchi M, Yamaguchi S, Abe T, Kiyonari H, Shimizu Y, Miyawaki A, Yokota H, Ueda HR (2014) Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis. Cell 157:726–739CrossRefPubMedGoogle Scholar
  19. 19.
    Chung K, Wallace J, Kim S-Y, Kalyanasundaram S, Andalman AS, Davidson TJ, Mirzabekov JJ, Zalocusky KA, Mattis J, Denisin AK, Pak S, Bernstein H, Ramakrishnan C, Grosenick L, Gradinaru V, Deisseroth K (2013) Structural and molecular interrogation of intact biological systems. Nature 497:332–337CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Ginger M, Broser P, Frick A (2013) Three-dimensional tracking and analysis of ion channel signals across dendritic arbors. Front Neural Circuits 7:61PubMedPubMedCentralGoogle Scholar
  21. 21.
    Parekh R, Ascoli GA (2013) Neuronal morphology goes digital: a research hub for cellular and system neuroscience. Neuron 77:1017–1038CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Dercksen VJ, Hege H-C, Oberlaender M (2014) The filament editor: an interactive software environment for visualization, proof-editing and analysis of 3D neuron morphology. Neuroinformatics 12:325–339CrossRefPubMedGoogle Scholar
  23. 23.
    Xiao H, Peng H (2013) APP2: automatic tracing of 3D neuron morphology based on hierarchical pruning of a gray-weighted image distance-tree. Bioinformatics 29:1448–1454CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Matthias G. Haberl
    • 1
    • 2
  • Melanie Ginger
    • 1
    • 2
  • Andreas Frick
    • 1
    • 2
    Email author
  1. 1.Neurocentre Magendie, Physiopathologie de la plasticité neuronaleINSERMBordeauxFrance
  2. 2.Neurocentre Magendie, Physiopathologie de la plasticité neuronale, The Neuroscience Institute at BordeauxUniversity of BordeauxBordeauxFrance

Personalised recommendations