Ultrastructural localization of oxytocin mRNA in the rat hypothalamus by in situ hybridization using a synthetic oligonucleotide

Abstract

In situ hybridization (ISH) was used to study at the electron microscope level, the subcellular localization of oxytocin (OT) mRNA in the rat hypothalamic magnocellular neurons. Rat brains were fixed with paraformaldehyde and glutaraldehyde and vibratome slices were incubated with a 25-base synthetic oligonucleotide complementary to OT mRNA and labelled at the 3′-end with [ 3H]dCTP. Hybridized slices were embedded in Epon after post-fixation with osmium tetroxide and cut into ultrathin sections that were processed for ultrastructural radioautography. OT mRNA was observed in magnocellular neurons of supra-optic and paraventricular nuclei in the vibratome sections. On ultrathin sections, the cytological preservation appeared to be satisfactory. Except for a few silver grains over the nucleus, sometimes close to its membrane, most grains were localized over the cytoplasm of some magnocellular neurons, where they frequently overlapped the endoplasmic reticulum. To decrease exposure time, ISH was also performed with OT probes labelled with a long tritiated tail. In this case, clusters of silver grains occurred over the cell nuclei not only in magnocellular neurons but also in non-secretory neurons and even in glial cells. However, an excess of poly C added to the hybridization buffer strongly decreased this non-cytoplasmic labelling. In conclusion, the results obtained with the short-tailed oligonucleotides demonstrate that these synthetic oligonucleotides have possible applications for the ultrastructural localization of mRNAs and constitute a powerful tool for the dynamic study of cellular mRNA processing in several physiological and experimental conditions.