In situ hybridization of the vasopressin mRNA in the rat hypothalamus by use of a synthetic oligonucleotide probe

Abstract

A 22mer oligonucleotide complementary to the rat vasopressin (AVP) mRNA was synthesized, radioactively labeled with 32P, and was applied to in situ hybridization with the mRNA on paraffin sections of the rat hypothalamus. The AVP mRNA in magnocellular neurosecretory neurons of the paraventricular and the supraoptic nuclei was demonstrated by use of this synthetic probe. Specificity of the autoradiographic signals was confirmed by a competition test using an excess amount of unlabeled probe and an absorption test using a synthetic template of the probe. Further, the mRNA of vasotocin, the ancestral molecule of AVP, was hybridized weakly with the probe, showing that the present probe can discriminate a few base substitutions. Autoradiographic signals representing hybrids of the probe and the AVP mRNA were rarely found in the region rich in oxytocin neurons whose mRNA is homologous to the AVP mRNA. These results show that the in situ hybridization method using synthetic oligonucleotide probes can be a powerful and specific tool for the study of gene expression in the central nervous system.