In situ hybridization histochemistry for the analysis of gene expression in the endocrine and central nervous system tissues: a 3-year experience

We report our experience in development of the in situ hybridization (ISH) procedure to detect messenger RNAs (mRNAs) coding for various molecules involved in endocrine glands and central nervous system activity, including mRNAs coding for endorphin precursors [preproenkephalin A (PPA), pro-opiocortin (POMC)], vasopressin, and transferrin. Various conditions of fixation and handling of the tissues were tested to establish optimal parameters for mRNA detection. Double-stranded DNA probes labeled by nick translation, synthetic oligonucleotides labeled at their 5' end, as well as single-stranded RNA probes were used, after incorporation of 32P- or 35S-labeled nucleotides. Specific requirements for efficient and reproducible ISH investigations are discussed. Cells expressing the PPA gene in the adrenal medulla and in the brain were detected by ISH. The results show that ISH is as sensitive as immunohistochemistry in detecting peptide-producing cells in the adrenal and that it allows detection of PPA cell bodies in brain in conditions in which they are inconstantly detected by immunohistochemistry. Unilateral destruction of substantia nigra provokes a dramatic decrease in the number of neurons expressing the PPA gene in the contralateral striatum. Cells expressing the POMC gene were detected in the pituitary of various species including man and in the rat arcuate nucleus. Neurons containing vasopressin mRNA were visualized in the supraoptic paraventricular and suprachiasmatic nucleus of the adult rat by using a synthetic oligonucleotide probe. Transferrin gene expression was shown in the central nervous system of the rat brain in two cell populations, the oligodendrocytes and the epithelial cells of the choroid plexus, by demonstration of simultaneous presence in them of transferrin immunoreactivity together with transferrin mRNA. These results show that the ISH procedure is a technique that can be routinely used to investigate gene transcription anatomically in complex heterocellular tissues such as the endocrine glands and the nervous system.