Biochemical and immunochemical studies of supraoptic and paraventricular cultures

A tissue culture model was established for the study of hypothalamic peptide synthesis and secretion. Microdissected explants of the paraventricular and supraoptic regions from Sprague-Dawley rats (neonates or young rats) were maintained in culture for up to 3 weeks. Studies were performed to evaluate vasopressin and oxytocin content (medium and tissue levels), immunocytochemical localization, and biosynthetic activity. Immunocytochemical staining for oxytocin, neurophysin, and neuron-specific enolase showed positive neurons in both the paraventricular and supraoptic cultures. In many cases, the neurons were large (30-40 microns) and bipolar, resembling the classic magnocellular neuron. Measurement of tissue and medium content showed the continued presence of vasopressin and oxytocin in the cultured explants. Even after 3 weeks, there were significant amounts of vasopressin present. Biosynthesis was evaluated by determining the incorporation of 35S-labeled cystine or cysteine into proteins and peptides. The medium and tissue extracts were separated by reverse-phase high-performance liquid chromatography. Results showed that most of the labeled peptides were released into the medium rather than stored. There were two labeled peaks in the medium, which chromatographically resembled native vasopressin and oxytocin. Treatment with a protein synthesis inhibitor, either puromycin or cycloheximide, resulted in a decrease in labeled peptides. A comparison of 35S-labeled cystine and cysteine showed that the latter was the label of choice, with significantly greater incorporation.