Combining Anterograde Tracing and Immunohistochemistry to Define Neuronal Synaptic Circuits

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

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.

Keywords:

Anterograde tracer Biotinylated dextran amine BDA Electron microscopy Immunohistochemistry Synapse Tyrosine hydroxylase Ultrastructure 

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

Notes

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.

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of Anesthesiology, Perioperative and Pain MedicineBoston Children’s HospitalBostonUSA
  2. 2.Department of AnaesthesiaHarvard Medical SchoolBostonUSA

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