Skip to main content
SpringerLink
Log in

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

  • Protocol
  • First Online:
Receptor and Ion Channel Detection in the Brain

Part of the book series: Neuromethods ((NM,volume 169))

  • 565 Accesses

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 189.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 249.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 329.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. 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 49:1497–1508

    Article  CAS  Google Scholar 

  2. 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 28:7153–7164

    Article  CAS  Google Scholar 

  3. 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 22:4600–4610

    Article  CAS  Google Scholar 

  4. 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 24:5370–5380

    Article  CAS  Google Scholar 

  5. 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 502:113–125

    Article  CAS  Google Scholar 

  6. 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 28:1789–1797

    Article  CAS  Google Scholar 

  7. 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 518:668–686

    Article  CAS  Google Scholar 

  8. 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 523:262–280

    Article  CAS  Google Scholar 

  9. 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 29:444–453

    Article  CAS  Google Scholar 

  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 19:2065–2077

    Article  Google Scholar 

  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 25:221–235

    Article  Google Scholar 

  12. Kuramoto E, Pan S, Furuta T, Tanaka YR, Iwai H, Yamanaka A, Ohno S, Kaneko T, Goto T, Hioki H (2017) Individual mediodorsal thalamic neurons project to multiple areas of the rat prefrontal cortex: a single neuron-tracing study using virus vectors. J Comp Neurol 525:166–185

    Article  Google Scholar 

  13. Kuramoto E, Iwai H, Yamanaka A, Ohno S, Seki H, Tanaka YR, Furuta T, Hioki H, Goto T (2017) Dorsal and ventral parts of thalamic nucleus submedius project to different areas of rat orbitofrontal cortex: a single neuron-tracing study using virus vectors. J Comp Neurol 525:3821–3839

    Article  CAS  Google Scholar 

  14. 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 22:2840–2857

    Article  Google Scholar 

  15. 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

    Article  Google Scholar 

  16. 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 519:1395–1412

    Article  Google Scholar 

  17. 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 228:74–83

    Article  CAS  Google Scholar 

  18. Toribio R, Ventoso I (2010) Inhibition of host translation by virus infection in vivo. Proc Natl Acad Sci U S A 107:9837–9842

    Article  CAS  Google Scholar 

  19. 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 63:149–154

    Article  CAS  Google Scholar 

  20. Paxinos G, Watson C (2007) The rat brain in stereotaxic coordinates. Academic Press, San Diego

    Google Scholar 

  21. 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 38:231–236

    Article  CAS  Google Scholar 

  22. 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 33:544–555

    Article  CAS  Google Scholar 

  23. 23. Okamoto S, Sohn J, Tanaka T, Takahashi M, Ishida Y, Yamauchi K, Koike M, Fujiyama F, Hioki H (2020) Overlapping Projections of Neighboring Direct and Indirect Pathway Neostriatal Neurons to Globus Pallidus External Segment. iScience 23(9):101409

    Google Scholar 

  24. Okamoto S, Yamauchi K, Sohn J, Takahashi M, Ishida Y, Furuta T, Koike M, Fujiyama F, Hioki H (2020) Exclusive labeling of direct and indirect pathway neurons in the mouse neostriatum by an adeno-associated virus vector with Cre/lox system. STAR Protoc 2(1):100230

    Google Scholar 

Download references

Acknowledgments

This study was supported by Grants-in-Aid for Scientific Research (21H02592 to H.H.; 21H03529 to T.F.), for Early-Career Scientists (JP20K15918 to H.N.), and for Scientific Research on Innovative Area “Resonance Bio” (JP18H04743 to H.H.) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT). It was also supported by the program for Brain Mapping by Integrated Neurotechnologies for Disease Studies (Brain/MINDS) from Japan Agency for Medical Research and development (AMED) (JP21dm0207112 to H.H. and T.F.), Moonshot Research and Development Program from the Japan Science and Technology Agency (JST) (JPMJMS2024-7 to H.H.), Fusion Oriented Research for disruptive Science and Technology (FOREST) from JST (JP20350060 to H.H.), a Grant-in-Aid from the Research Institute for Diseases of Old Age at the Juntendo University School of Medicine (X2001 to H.H.), and MEXT Private University Research Branding Project (Juntendo University).

Author information

Authors and Affiliations

  1. Department of Cell Biology and Neuroscience, Juntendo University Graduate School of Medicine, Tokyo, Japan

    Hiroyuki Hioki

  2. Division of Microscopic and Developmental Anatomy, Department of Anatomy, Kurume University School of Medicine, Kurume, Japan

    Hisashi Nakamura

  3. Department of Oral Anatomy and Neurobiology, Osaka University Graduate School of Dentistry, Suita, Japan

    Takahiro Furuta

Authors
  1. Hiroyuki Hioki
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hisashi Nakamura
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Takahiro Furuta
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Hiroyuki Hioki .

Editor information

Editors and Affiliations

  1. Instituto de Investigación en Discapacidades Neurológicas (IDINE), Departamento de Ciencias Médicas, Facultad de Medicina, Universidad Castilla-La Mancha, Albacete, Spain

    Rafael Lujan

  2. Pharmacology Unit, Department of Pathology and Experimental Therapeutics, School of Medicine and Health Sciences, IDIBELL, Universitat de Barcelona, L’Hospitalet de Llobregat, Barcelona, Spain

    Francisco Ciruela

Rights and permissions

Reprints and permissions

Copyright information

© 2021 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Hioki, H., Nakamura, H., Furuta, T. (2021). Application of Virus Vectors for Anterograde Tract-Tracing and Single-Neuron Labeling Studies. In: Lujan, R., Ciruela, F. (eds) Receptor and Ion Channel Detection in the Brain. Neuromethods, vol 169. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1522-5_21

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-1522-5_21

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1521-8

  • Online ISBN: 978-1-0716-1522-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation