Nonradioactive detection of vasopressin mRNA with in situ hybridization histochemistry

In situ hybridization histochemistry has been used successfully by many laboratories to detect mRNAs within single cells in the CNS. The detection of these hybrids in CNS tissue sections has been accomplished mainly with radioactive probes. However, radiolabeled probes have intrinsic limitations, including the long exposure time required for high resolution autoradiography and the inability to detect multiple RNA species within the same neuron. Here we report a new method to detect mRNA in situ using a synthetic DNA probe conjugated to alkaline phosphatase (AP). The probe was synthesized to be complementary to the glycoprotein coding region of vasopressin mRNA. Under normal hybridization conditions high resolution detection of vasopressin mRNA within individual neurons was routinely obtained within 8 h. The distribution of hybridization signal obtained with the AP-conjugated probe was identical to that observed with the same probe radiolabeled with [35S]dATP. Hybridization-positive neurons were found in all regions of the CNS that have been previously reported to synthesize vasopressin, including magnocellular neurons in the paraventricular nucleus (PVN) and the supraoptic nucleus. Small diameter neurons were also observed in the suprachiasmatic nucleus, the accessory PVN, the bed nucleus of the stria terminalis, and a cell group along the ventral surface of the optic tract. These results suggest that nonradioactive detection of neuropeptide mRNA in situ can be easily accomplished within 24 h. Furthermore, the improved resolution with AP-conjugated oligonucleotide probes should enhance efforts to study the regulation of gene expression in the nervous system.