Cellular localization of nerve growth factor synthesis by in situ hybridization

Abstract

A very sensitive and specific method for in situ hybridization has been developed. This method detects low copy numbers of mRNA(NGF) transcripts in both tissue sections and cultured cells using 35S-labelled cRNA and oligonucleotide probes. In order to reduce the high nonspecific background occurring with 35S-labelled probes, prehybridization in the presence of non-labelled thio alpha UTP at pH 5.5 proved to be essential, together with a series of additional changes in the standard procedures for in situ hybridization. With this improved method it was possible to demonstrate that in tissues densely innervated by sensory (whisker pad) or both sympathetic and sensory (iris) fibers, NGF is synthesized not only by Schwann cells ensheathing these fibers, but also--and even to a much larger extent--by the target cells of the sensory and sympathetic neurons, i.e. epithelial cells, smooth muscle cells and fibroblasts. Moreover, in the sciatic nerve of newborn rats (where the mRNA(NGF) levels are 15 X higher than in adults) it was demonstrated that all Schwann cells have the capacity to express mRNA(NGF), not just those ensheathing the axons of NGF-responsive neurons.