Skip to main content
SpringerLink
Log in
SpringerLink
Log in

Combining Anterograde Tracing and Immunohistochemistry to Define Neuronal Synaptic Circuits

  • Protocol
  • First Online:
Transmission Electron Microscopy Methods for Understanding the Brain

Part of the book series: Neuromethods ((NM,volume 115))

  • 895 Accesses

Abstract

Connectivity among different brain regions has been studied since the original neuronal descriptions by Santiago Ramón y Cajal. Ultimately, only evidence of synapse proves actual connectivity between neurons originating from different brain regions. This report focuses on technical aspects of anterograde neuroanatomical tract tracing combined with immunohistochemical methods specific for ultrastructural analysis of neuronal contacts, synapses. Specifically, this technique combines peroxidase labeling of the anterograde tracer, biotinylated dextran amine (BDA 10 kDa) with immunoperoxidase-silver enhancement detection of a neuroactive substance present in the structures postsynaptic to tracer-labeled axon terminals. This technique is widely utilized to identify neuronal circuits as well as the neurochemical content of neurons that are implicated in incredibly complex and dynamic tasks. In this report we discuss technical steps in detail, as well as the technique’s advantages and limitations.

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 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
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

Notes

  1. 1.

    In our previously published work [6–8], we used mouse primary antisera directed against tyrosine hydroxylase (TH; Incstar Corp., Stillwater, MN) that was diluted 1:5000 with 0.1 M TBS and contained 0.1 % BSA.

  2. 2.

    The secondary antibody used in our experiments that visualized catecholamine neurons was goat anti-mouse secondary antibody conjugated to 1 nm gold particle (Auro Probe One GAM; RPN 471; Amersham Corp., Arlington Heights, IL) diluted 1:50 with the blocking solution.

Abbreviations

ABC:

Avidin–biotin complex

BDA:

Biotinylated dextran amine

BSA:

Bovine serum albumin

DAB:

Diaminobenzidine

HRP:

Horseradish peroxidase

OsO4 :

Osmium tetroxide

PB:

Phosphate buffer

PBS:

Phosphate buffer saline

TBS:

Tris-buffered saline

TH:

Tyrosine hydroxylase

References

  1. Elias LJ, Saucier DM (2005) Neuropsychology: clinical and experimental foundations. Pearson, London, p 560

    Google Scholar 

  2. Lanciego JL, Wouterlood FG (2011) A half century of experimental neuroanatomical tracing. J Chem Neuroanat 42(3):157–183

    Article  PubMed  Google Scholar 

  3. Veenman CL, Reiner A, Honig MG (1992) Biotinylated dextran amine as an anterograde tracer for single- and double-labeling studies. J Neurosci Methods 41(3):239–254

    Article  CAS  PubMed  Google Scholar 

  4. Reiner A et al (2000) Pathway tracing using biotinylated dextran amines. J Neurosci Methods 103(1):23–37

    Article  CAS  PubMed  Google Scholar 

  5. Reiner A, Honing MG (2006) Dextran amines: versatile tools for anterograde and retrograde studies of nervous system connectivity. In: Zaborszky L, Wouterlood FG, Lanciego JL (eds) Neuroanatomical tract-tracing 3: molecules, neurons, and systems. Springer, New York City, NY, pp 304–335

    Chapter  Google Scholar 

  6. Bajic D, Proudfit HK, Van Bockstaele EJ (2000) Periaqueductal gray neurons monosynaptically innervate extranuclear noradrenergic dendrites in the rat pericoerulear region. J Comp Neurol 427(4):649–662

    Article  CAS  PubMed  Google Scholar 

  7. Bajic D, Van Bockstaele EJ, Proudfit HK (2001) Ultrastructural analysis of ventrolateral periaqueductal gray projections to the A7 catecholamine cell group. Neuroscience 104(1):181–197

    Article  CAS  PubMed  Google Scholar 

  8. Bajic D, Van Bockstaele EJ, Proudfit HK (2012) Ultrastructural analysis of rat ventrolateral periaqueductal gray projections to the A5 cell group. Neuroscience 224:145–159

    Article  CAS  PubMed  Google Scholar 

  9. Bozzola JJ, Russell LD (1999) Specimen preparation for transmission electron microscopy. In: Bozzola JJ, Russell LD (eds) Electron microscopy : principles and techniques for biologists. Jones & Bartlett Publishers, Sudbury, MA, pp 16–47

    Google Scholar 

  10. Hayat MA (2000) Principles and techniques of electron microscopy: biological applications. Cambridge University Press, New York, NY

    Google Scholar 

  11. Gonzalo N et al (2001) A sequential protocol combining dual neuroanatomical tract-tracing with the visualization of local circuit neurons within the striatum. J Neurosci Methods 111(1):59–66

    Article  CAS  PubMed  Google Scholar 

  12. Wouterlood FG, Jorritsma-Byham B (1993) The anterograde neuroanatomical tracer biotinylated dextran-amine: comparison with the tracer Phaseolus vulgaris-leucoagglutinin in preparations for electron microscopy. J Neurosci Methods 48(1-2):75–87

    Article  CAS  PubMed  Google Scholar 

  13. Bajic D, Proudfit HK (2013) Projections from the rat cuneiform nucleus to the A7, A6 (locus coeruleus), and A5 pontine noradrenergic cell groups. J Chem Neuroanat 50–51:11–20

    Article  PubMed  Google Scholar 

  14. Bajic D, Proudfit HK (1999) Projections of neurons in the periaqueductal gray to pontine and medullary catecholamine cell groups involved in the modulation of nociception. J Comp Neurol 405(3):359–379

    Article  CAS  PubMed  Google Scholar 

  15. Scopsi L et al (1986) Silver-enhanced colloidal gold probes as markers for scanning electron microscopy. Histochemistry 86(1):35–41

    Article  CAS  PubMed  Google Scholar 

  16. Chan J, Aoki C, Pickel VM (1990) Optimization of differential immunogold-silver and peroxidase labeling with maintenance of ultrastructure in brain sections before plastic embedding. J Neurosci Methods 33(2-3):113–127

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Pickel VM, Chan J (1993) Electron microscopic immunocytochemical labeling of endogenous and/or transported antigens in rat brain using silver-intensified one-nanometer colloidal gold. In: Cuello AC (ed) Immunohistochemistry II. Willey, New York City, NY, pp 265–280

    Google Scholar 

  18. Peters A, Palay SL (1996) The morphology of synapses. J Neurocytol 25(12):687–700

    Article  CAS  PubMed  Google Scholar 

  19. Peters A, Palay SL, Webster HD (1992) Fine structure of the nervous system: neurons and their supporting cells, 3rd edn. Oxford University Press, New York, NY

    Google Scholar 

  20. Hunter EE, Silver M (1993) Practical electron microscopy: a beginner’s illustrated guide, 2nd edn. Cambridge University Press, New York, NY, p 188

    Book  Google Scholar 

  21. Bozzola JJ, Russell LD (1998) Electron microscopy, 2nd edn. Jones & Bartlett Publishers, Toronto, ON

    Google Scholar 

  22. Gray EG (1959) Axo-somatic and axo-dendritic synapses of the cerebral cortex: an electron microscope study. J Anat 93:420–433

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Brandt HM, Apkarian AV (1992) Biotin-dextran: a sensitive anterograde tracer for neuroanatomic studies in rat and monkey. J Neurosci Methods 45(1-2):35–40

    Article  CAS  PubMed  Google Scholar 

  24. Chamberlin NL et al (1998) Recombinant adeno-associated virus vector: use for transgene expression and anterograde tract tracing in the CNS. Brain Res 793(1-2):169–175

    Article  CAS  PubMed  Google Scholar 

  25. Gautron L et al (2010) Identifying the efferent projections of leptin-responsive neurons in the dorsomedial hypothalamus using a novel conditional tracing approach. J Comp Neurol 518(11):2090–2108

    Article  PubMed  PubMed Central  Google Scholar 

  26. Anderson KD, Karle EJ, Reiner A (1994) A pre-embedding triple-label electron microscopic immunohistochemical method as applied to the study of multiple inputs to defined tegmental neurons. J Histochem Cytochem 42(1):49–56

    Article  CAS  PubMed  Google Scholar 

  27. Freund TF, Antal M (1988) GABA-containing neurons in the septum control inhibitory interneurons in the hippocampus. Nature 336(6195):170–173

    Article  CAS  PubMed  Google Scholar 

  28. Omelchenko N, Bell R, Sesack SR (2009) Lateral habenula projections to dopamine and GABA neurons in the rat ventral tegmental area. Eur J Neurosci 30(7):1239–1250

    Article  PubMed  PubMed Central  Google Scholar 

  29. Omelchenko N, Sesack SR (2009) Ultrastructural analysis of local collaterals of rat ventral tegmental area neurons: GABA phenotype and synapses onto dopamine and GABA cells. Synapse 63(10):895–906

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This research was supported by (1) USPHS Grant DAO3980 from the National Institutes on Drug Abuse (NIDA) to Dr. Herbert K. Proudfit; (2) grants ANA#96002249 and NIDA 09082 to Dr. Elisabeth J. Van Bockstaele; as well as the (3) National Institutes of Health R03 DA030874 grant to Dr. Dusica Bajic.

Author information

Authors and Affiliations

  1. Department of Anesthesiology, Perioperative and Pain Medicine, Boston Children’s Hospital, 300 Longwood Ave, Bader 3, Boston, MA, 02115, USA

    Dusica Bajic M.D., Ph.D.

  2. Department of Anaesthesia, Harvard Medical School, Boston, MA, USA

    Dusica Bajic M.D., Ph.D.

Authors
  1. Dusica Bajic M.D., Ph.D.
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Dusica Bajic M.D., Ph.D. .

Editor information

Editors and Affiliations

  1. Department of Pharmacology and Physiolog, Drexel University, Philadelphia, Pennsylvania, USA

    Elisabeth J. Van Bockstaele

Rights and permissions

Reprints and permissions

Copyright information

© 2016 Springer Science+Business Media New York

About this protocol

Cite this protocol

Bajic, D. (2016). Combining Anterograde Tracing and Immunohistochemistry to Define Neuronal Synaptic Circuits. In: Van Bockstaele, E. (eds) Transmission Electron Microscopy Methods for Understanding the Brain. Neuromethods, vol 115. Humana Press, New York, NY. https://doi.org/10.1007/7657_2015_81

Download citation

  • DOI: https://doi.org/10.1007/7657_2015_81

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3638-0

  • Online ISBN: 978-1-4939-3640-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation