SIMULTANEOUS DETECTION OF NEUROPEPTIDES AND MESSENGER-RNA IN THE MAGNOCELLULAR HYPOTHALAMONEUROHYPOPHYSEAL SYSTEM BY A COMBINATION OF NONRADIOACTIVE IN-SITU HYBRIDIZATION HISTOCHEMISTRY AND IMMUNOHISTOCHEMISTRY

A protocol was developed combining non-radioactive in situ hybridizati on histochemistry with enzyme based immunohistochemistry, detect the e xpression of mRNA in phenotypically defined neurons. Free-floating bra in sections were hybridized with the oligonucleotide probes which have been 3'-end labelled with biotin-11-dUTP. The hybridized probe was vi sualized by a combined avidin-biotin bridge method, anti-avidin immuno histochemistry, and horseradish peroxidase detection using diaminobenz idine as a substrate. The in situ hybridization step yielded a very st able reaction product enabling subsequent immunohistochemical reaction s using horseradish peroxidase and benzidine dihydrochloride as a chro mogen. Magnocellular neurons of the hypothalamo-neurophypophysial syst em synthesize either vasopressin or oxytocin; water deprivation and ch ronic saline ingestion are potent stimuli for the expression of both o f the genes encoding these neuropeptides. A number of other neuropepti des with putative transmitter action are synthesized in magnocellular neurons during such stimulation. Experiments were performed to explore whether neuropeptide Y immunoreactivity is present within magnocellul ar vasopressin mRNA-expressing neurons of the hypothalamo-neurophypoph ysial system. The results clearly demonstrated that neuropeptide Y-imm unoreactive elements were present within a number of magnocellular vas opressin mRNA-containing cells. In addition, immunohistochemical detec tion of the neuropeptides ocytocin and cholecystokinin was carried out on sections hybridized non-radioactively for vasopressin; as expected vasopressin mRNA did not co-exist with cholecystokinin, whereas a few oxytocin immunoreactive neurons in osmotically stimulated animals als o contained vasopressin mRNA. The developed method makes possible the immunohistochemical detection of intracellular antigens with concomita nt detection of intracellular mRNA.