Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Multiple neuroanatomical tract-tracing using fluorescent Alexa Fluor conjugates of cholera toxin subunit B in rats

Nature Protocols volume 4, pages 1157–1166 (2009)Cite this article

Abstract

Cholera toxin subunit B (CTB) is a highly sensitive retrograde neuroanatomical tracer. With the new availability of fluorescent Alexa Fluor (AF) conjugates of CTB, multiple neuroanatomical connections can be reliably studied and compared in the same animal. Here we provide a protocol that describes the use of AF-CTB for studying connections in the central nervous system of rats. The viscous properties of CTB allow small and discreet injection sites yet still show robust retrograde labeling. Furthermore, the AF conjugates are extremely bright and photostable, compared with other conventional fluorescent tracers. This protocol can also be adapted for use with other neuroanatomical tracers. Including a 7-d survival period, this protocol takes ∼11 to 12 d to complete in its entirety.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Photographs of key surgical steps (a–x) from the PROCEDURE (Steps 9–25).
Figure 2: Example injection sites and labeling patterns of AF 488 (green) and 594 (red) CTB.

Similar content being viewed by others

References

  1. Krout, K.E., Belzer, R.E. & Loewy, A.D. Brainstem projections to midline and intralaminar thalamic nuclei of the rat. J. Comp. Neurol. 448, 53–101 (2002).

    Article  PubMed  Google Scholar 

  2. Llewellyn-Smith, I.J., Martin, C.L., Arnolda, L.F. & Minson, J.B. Tracer-toxins: cholera toxin B-saporin as a model. J. Neurosc. Methods 103, 83–90 (2000).

    Article  CAS  Google Scholar 

  3. Luppi, P.-H., Fort, P. & Jouvet, M. Iontophoretic application of unconjugated cholera toxin B subunit (CTb) combined with immunohistochemistry of neurochemical substances: a method for transmitter identification of retrogradely labeled neurons. Brain Res. 534, 209–224 (1990).

    Article  CAS  PubMed  Google Scholar 

  4. Zin-Ka-Ieu, S., Roger, M. & Arnault, P. Direct contacts between fibers from the ventrolateral thalamic nucleus and frontal cortical neurons projecting to the striatum: a light-microscopy study in the rat. Anat. Embryol. 197, 77–87 (1997).

    Article  Google Scholar 

  5. Conte, W.L., Kamishina, H., Corwin, J.V. & Reep, R.L. Topography in the projections of lateral posterior thalamus with cingulate and medial agranular cortex in relation to circuitry for directed attention and neglect. Brain Res. 1240, 87–95 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Christianson, J.A. et al. Convergence of bladder and colon sensory innervation occurs at the primary afferent level. Pain 128, 235–243 (2007).

    Article  PubMed  Google Scholar 

  7. Kreier, F. et al. Tracing from fat tissue, liver, and pancreas: a neuroanatomical framework for the role of the brain in type 2 diabetes. Endocrinology 147, 1140–1147 (2006).

    Article  CAS  PubMed  Google Scholar 

  8. McDavid, S., Lund, J.P., Auclair, F. & Kolta, A. Morphological and immunohistochemical characterization of interneurons within the rat trigeminal motor nucleus. Neuroscience 139, 1049–1059 (2006).

    Article  CAS  PubMed  Google Scholar 

  9. Niedringhaus, M. et al. Brainstem sites controlling the lower esophageal sphincter and crural diaphragm in the ferret: a neuroanatomical study. Auton. Neurosci. 144, 50–60 (2008).

    Article  PubMed  Google Scholar 

  10. O'Malley, M.R. et al. Fluorescent retrograde axonal tracing of the facial nerve. Laryngoscope 116, 1792–1797 (2006).

    Article  PubMed  Google Scholar 

  11. Kamishina, H. et al. Cortical connections of the rat lateral posterior thalamic nucleus. Brain Res. 1264, 39–56 (2009).

    Article  CAS  PubMed  Google Scholar 

  12. Schofield, B.R., Schofield, R.M., Sorensen, K.A. & Motts, S.D. On the use of retrograde tracers for identification of axon collaterals with multiple fluorescent retrograde tracers. Neuroscience 146, 773–783 (2007).

    Article  CAS  PubMed  Google Scholar 

  13. Panchuk-Voloshina, N. et al. Alexa dyes, a series of new fluorescent dyes that yield exceptionally bright, photostable conjugates. J. Histochem. Cytochem. 47, 1179–1188 (1999).

    Article  CAS  PubMed  Google Scholar 

  14. Reiner, A. & Honig, M.G. Dextran amines: versatile tools for anterograde and retrograde studies of nervous system connectivity. In Neuroanatomical Tract-Tracing 3 (eds. Zaborszky, L., Wouterlood, F.G., & Lanciego, J.L.) 304–335 (Springer Science+Business Media Inc., New York, 2006).

    Chapter  Google Scholar 

  15. Angelucci, A., Clascá, F. & Sur, M. Anterograde axonal tracing with the subunit B of cholera toxin: a highly sensitive immunohistochemical protocol for revealing fine axonal morphology in adult and neonatal brains. J. Neurosc. Methods 65, 101–112 (1996).

    Article  CAS  Google Scholar 

  16. Ruigrok, T.J.H. & Apps, R. A light microscope-based double retrograde tracer strategy to chart central neuronal connections. Nat. Protoc. 2, 1869–1878 (2007).

    Article  CAS  PubMed  Google Scholar 

  17. Dederen, P., Gribnau, A. & Curfs, M. Retrograde neuronal tracing with cholera toxin B subunit: comparison of three different visualization methods. Histochem. J. 26, 856–862 (1994).

    Article  CAS  PubMed  Google Scholar 

  18. Chen, S. & Aston-Jones, G. Evidence that cholera toxin B subunit (CTb) can be avidly taken up and transported by fibers of passage. Brain Res. 674, 107–111 (1995).

    Article  CAS  PubMed  Google Scholar 

  19. Kobbert, C. et al. Current concepts in neuroanatomical tracing. Prog. Neurobiol. 62, 327–351 (2000).

    Article  CAS  PubMed  Google Scholar 

  20. Datiche, F. & Cattarelli, M. Reciprocal and topographic connections between the piriform and prefrontal cortices in the rat: a tracing study using the B subunit of the cholera toxin. Brain Res. Bull. 41, 391–398 (1996).

    Article  CAS  PubMed  Google Scholar 

  21. Tsumori, T., Yokota, S., Ono, K. & Yasui, Y. Organization of projections from the medial agranular cortex to the superior colliculus in the rat: a study using anterograde and retrograde tracing methods. Brain Res. 903, 168–176 (2001).

    Article  CAS  PubMed  Google Scholar 

  22. Paxinos, G. & Watson, C. The Rat Brain in Stereotaxic Coordinates (Elsevier Academic Press, San Diego, 2005).

    Google Scholar 

  23. Apps, R. & Ruigrok, T.J.H. A fluorescence-based double retrograde tracer strategy for charting central neuronal connections. Nat. Protoc. 2, 1862–1868 (2007).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We greatly thank Maggie Stoll for her continued technical help. We also thank the help of Dr. Stephen B. Lewis, Robin Barry and Bryan Thomasey for providing equipment for the video clips. We also appreciate further technical help from Elyse Morin and Rachel Tai. This study was supported in part by the NIMH Grant MH60399.

Author information

Authors and Affiliations

  1. Department of Physiological Sciences, University of Florida, Gainesville, Florida, USA

    William L Conte & Roger L Reep

  2. McKnight Brain Institute, University of Florida, Gainesville, Florida, USA

    William L Conte & Roger L Reep

  3. Department of Veterinary Medicine, Iwate University, Morioka, Japan

    Hiroaki Kamishina

Authors
  1. William L Conte
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hiroaki Kamishina
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Roger L Reep
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to William L Conte.

Supplementary information

Supplementary Video 1

Positioning of the animal in a stereotaxic device (Step 11). (MOV 13794 kb)

Supplementary Video 2a

Opening of the scalp and exposure of skull surface (Step 13). (MOV 14344 kb)

Supplementary Video 2b

Opening of the scalp and exposure of skull surface (Step 13). (MOV 14325 kb)

Supplementary Video 3a

Drilling of two separate burr holes (Steps 15-16). (MOV 13694 kb)

Supplementary Video 3b

Drilling of two separate burr holes (Steps 15-16). (MOV 14392 kb)

Supplementary Video 4a

Excision of the meninges (Step 17). (MOV 13682 kb)

Supplementary Video 4b

Excision of the meninges (Step 17). (MOV 13481 kb)

Supplementary Video 5a

Cleaning the surgical site, placement of gel foam, and closing of the wound (Step 25). (MOV 10188 kb)

Supplementary Video 5b

Cleaning the surgical site, placement of gel foam, and closing of the wound (Step 25). (MOV 10275 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Conte, W., Kamishina, H. & Reep, R. Multiple neuroanatomical tract-tracing using fluorescent Alexa Fluor conjugates of cholera toxin subunit B in rats. Nat Protoc 4, 1157–1166 (2009). https://doi.org/10.1038/nprot.2009.93

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2009.93

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing