A Simple and Reliable Technique to Combine Oligonucleotide Probe in Situ Hybridization with Neuronal Tract Tracing in Vertebrate Embryos

Abstract

We describe a simple and reliable combination of in situ hybridization with neuronal tracing. The technique uses recent advances in the field of neuronal tract tracing including fast diffusing, low molecular weight dextran amines and fade resistant fluorescent dyes, and combines them with in situ hybridization using a sensitive Oligonucleotide probe. Using this technique we have investigated the mRNA encoding the trkB receptor for brain-derived neurotrophic factor in identified facial and vestibular afferent and efferent neurons. We found very low levels of trkB mRNA in facial efferent neurons, whereas in the vestibular afferent neurons, clear labeling for the trkB mRNA could be seen. This technique can be applied to the developing embryo to study topology of a variety of cellular markers with reference to neuronal population or fibers identified by their origin or target.

Key Words:

• amines
• fluorescent dextran
• in situ hybridization