COEXPRESSION OF TYROSINE-HYDROXYLASE MESSENGER RNA-1 AND RNA-2 IN HUMAN VENTRAL MESENCEPHALON REVEALED BY DIGOXIGENIN-LABELED AND BIOTIN-LABELED OLIGODEOXYRIBONUCLEOTIDES

In situ hybridization experiments, using oligodeoxyribonucleotides specific for the two major expressed human tyrosine hydroxylase mRNAs, were performed on human brain sections at the level of the mesencephalon. The specificity of the probes was ascertained by Northern blot experiments carried out with independently in vitro synthesized human tyrosine hydroxylase mRNAs. For in situ hybridization experiments, oligodeoxyribonucleotides were labelled with nucleotides tagged with digoxigenin or biotin molecules. The hybridized oligonucleotides were detected by antibodies coupled with peroxidase and alkaline phosphatase enzymes, which yield, with appropriate substrates, brown and purple products, respectively. The simultaneous detection of the two mRNAs with digoxigeninated and biotinylated probes revealed that these two mRNAs are co-expressed in single cells. The purple product obtained with alkaline phosphatase exhibits a discrete distribution within the dopaminergic cells suggesting these mRNAs are associated with sub-cellular structures. Finally, a heterogeneity in the intensity of the labelling of reactive cells with both probes was visualized as well as the expression of the two mRNA species in neurites.