Application of Virus Vectors for Anterograde Tract-Tracing and Single-Neuron Labeling Studies

  • Hiroyuki Hioki
  • Hisashi Nakamura
  • Takahiro Furuta
Part of the Neuromethods book series (NM, volume 110)


Elucidating neuronal circuits is fundamental issue for understanding how the brain works and implements higher-order functions. Various viral vectors have been developed and become valuable tools for the analysis of neuronal circuits in the central nervous system. Sindbis virus vector is very useful for anterograde labeling of neurons, since the vector expresses reporter protein rapidly and strongly. Furthermore, the vector makes it possible to visualize whole structures of a single neuron by injecting adequately diluted virus solution. After immunoperoxidase staining with a tyramine-based signal amplification technique, two-dimensional reconstruction of a single neuron is performed with a virtual slide system and graphic software. In this chapter, we describe a set of single-neuron tracing method in exact detail. On the other hand, Sindbis virus vector shows very high cytotoxicity by shutting off host cellular transcription and translation and is not suitable for the experiments requiring long-term expression of transgene. In the previous study, we developed novel lentivirus vector and succeeded in neuron-specific and high-level sustained gene expression. This novel vector is expected to be applied as a sensitive anterograde tracer in addition to Sindbis virus vector.


Sindbis virus Membrane-targeting signal Anterograde tracing Single-neuron tracing Reconstruction Lentivirus Tet-Off system 



This work was supported by Grants-in-Aid from The Ministry of Education, Culture, Sports, Science, and Technology (MEXT) and Japan Society for the Promotion of Science (JSPS); Grant numbers: for Scientific Research (24500408 and 15K14333 to H.H.; 24500408 and 15H04266 to T.F.); for Exploratory Research (15K14333 to H.H.); and for Scientific Research on Innovative Areas, “Neuronal Diversity and Neocortical Organization” (25123709 to H.H.), “Foundation of Synapse and Neurocircuit Pathology” (22110007 to H.H.), and “Adaptive Circuit Shift” (15H01430 to H.H.).


  1. 1.
    Dryga SA, Dryga OA, Schlesinger S (1997) Identification of mutations in a Sindbis virus variant able to establish persistent infection in BHK cells: the importance of a mutation in the nsP2 gene. Virology 228:74–83CrossRefPubMedGoogle Scholar
  2. 2.
    Furuta T, Tomioka R, Taki K, Nakamura K, Tamamaki N, Kaneko T (2001) In vivo transduction of central neurons using recombinant Sindbis virus: Golgi-like labeling of dendrites and axons with membrane-targeted fluorescent proteins. J Histochem Cytochem 49:1497–1508CrossRefPubMedGoogle Scholar
  3. 3.
    Furuta T, Timofeeva E, Nakamura K, Okamoto-Furuta K, Togo M, Kaneko T, Deschênes M (2008) Inhibitory gating of vibrissal inputs in the brainstem. J Neurosci 28:1789–1797CrossRefPubMedGoogle Scholar
  4. 4.
    Hioki H, Kameda H, Nakamura H, Okunomiya T, Ohira K, Nakamura K, Kuroda M, Furuta T, Kaneko T (2007) Efficient gene transduction of neurons by lentivirus with enhanced neuron-specific promoters. Gene Ther 14:872–882CrossRefPubMedGoogle Scholar
  5. 5.
    Hioki H, Kuramoto E, Konno M, Kameda H, Takahashi Y, Nakano T, Nakamura KC, Kaneko T (2009) High-level transgene expression in neurons by lentivirus with Tet-Off system. Neurosci Res 63:149–154CrossRefPubMedGoogle Scholar
  6. 6.
    Hioki H, Nakamura H, Ma YF, Konno M, Hayakawa T, Nakamura KC, Fujiyama F, Kaneko T (2010) Vesicular glutamate transporter 3-expressing nonserotonergic projection neurons constitute a subregion in the rat midbrain raphe nuclei. J Comp Neurol 518:668–686CrossRefPubMedGoogle Scholar
  7. 7.
    Hioki H, Okamoto S, Konno M, Kameda H, Sohn J, Kuramoto E, Fujiyama F, Kaneko T (2013) Cell type-specific inhibitory inputs to dendritic and somatic compartments of parvalbumin-expressing neocortical interneuron. J Neurosci 33:544–555CrossRefPubMedGoogle Scholar
  8. 8.
    Honda Y, Furuta T, Kaneko T, Shibata H, Sasaki H (2011) Patterns of axonal collateralization of single layer V cortical projection neurons in the rat presubiculum. J Comp Neurol 519:1395–1412CrossRefPubMedGoogle Scholar
  9. 9.
    Ito T, Hioki H, Nakamura K, Tanaka Y, Nakade H, Kaneko T, Iino S, Nojyo Y (2007) Gamma-aminobutyric acid-containing sympathetic preganglionic neurons in rat thoracic spinal cord send their axons to the superior cervical ganglion. J Comp Neurol 502:113–125CrossRefPubMedGoogle Scholar
  10. 10.
    Kuramoto E, Furuta T, Nakamura KC, Unzai T, Hioki H, Kaneko T (2009) Two types of thalamocortical projections from the motor thalamic nuclei of the rat: a single neuron-tracing study using viral vectors. Cereb Cortex 19:2065–2077CrossRefPubMedGoogle Scholar
  11. 11.
    Kuramoto E, Ohno S, Furuta T, Unzai T, Tanaka YR, Hioki H, Kaneko T (2015) Ventral medial nucleus neurons send thalamocortical afferents more widely and more preferentially to layer 1 than neurons of the ventral anterior-ventral lateral nuclear complex in the rat. Cereb Cortex 25:221–235CrossRefPubMedGoogle Scholar
  12. 12.
    Matsuda W, Furuta T, Nakamura KC, Hioki H, Fujiyama F, Arai R, Kaneko T (2009) Single nigrostriatal dopaminergic neurons form widely spread and highly dense axonal arborizations in the neostriatum. J Neurosci 29:444–453CrossRefPubMedGoogle Scholar
  13. 13.
    Nakamura K, Matsumura K, Kaneko T, Kobayashi S, Katoh H, Negishi M (2002) The rostral raphe pallidus nucleus mediates pyrogenic transmission from the preoptic area. J Neurosci 22:4600–4610PubMedGoogle Scholar
  14. 14.
    Nakamura K, Matsumura K, Hübschle T, Nakamura Y, Hioki H, Fujiyama F, Boldogköi Z, König M, Thiel HJ, Gerstberger R, Kobayashi S, Kaneko T (2004) Identification of sympathetic premotor neurons in medullary raphe regions mediating fever and other thermoregulatory functions. J Neurosci 24:5370–5380CrossRefPubMedGoogle Scholar
  15. 15.
    Nishino E, Yamada R, Kuba H, Hioki H, Furuta T, Kaneko T, Ohmori H (2008) Sound-intensity-dependent compensation for the small interaural time difference cue for sound source localization. J Neurosci 28:7153–7164CrossRefPubMedGoogle Scholar
  16. 16.
    Ohno S, Kuramoto E, Furuta T, Hioki H, Tanaka YR, Fujiyama F, Sonomura T, Uemura M, Sugiyama K, Kaneko T (2012) A morphological analysis of thalamocortical axon fibers of rat posterior thalamic nuclei: a single neuron tracing study with viral vectors. Cereb Cortex 22:2840–2857CrossRefPubMedGoogle Scholar
  17. 17.
    Paxinos G, Watson C (2007) The rat brain in stereotaxic coordinates. Academic, San DiegoGoogle Scholar
  18. 18.
    Suzuki Y, Kiyokage E, Sohn J, Hioki H, Toida K (2015) Structural basis for serotonergic regulation of neural circuits in the mouse olfactory bulb. J Comp Neurol 523:262–280CrossRefPubMedGoogle Scholar
  19. 19.
    Tamamaki N, Nakamura K, Furuta T, Asamoto K, Kaneko T (2000) Neurons in Golgi-stain-like images revealed by GFP-adenovirus infection in vivo. Neurosci Res 38:231–236CrossRefPubMedGoogle Scholar
  20. 20.
    Toribio R, Ventoso I (2010) Inhibition of host translation by virus infection in vivo. Proc Natl Acad Sci U S A 107:9837–9842PubMedCentralCrossRefPubMedGoogle Scholar
  21. 21.
    Nakamura H, Hioki H, Furuta T, Kaneko T (2015) Different cortical projections from three subdivisions of the rat lateral posterior thalamic nucleus: a single-neuron tracing study with viral vectors. Eur J Neurosci. 41:1294–1310.Google Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Hiroyuki Hioki
    • 1
  • Hisashi Nakamura
    • 1
  • Takahiro Furuta
    • 1
  1. 1.Department of Morphological Brain ScienceGraduate School of Medicine, Kyoto UniversityKyotoJapan

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