Immunofluorescence of Vasopressin and Oxytocin in the Rat Hypothalamo-neurohypophypopseal System

The present paper deals with the development of an immunofluorescence procedure that allows specific localization of vasopressin and oxytocin in the hypothalamo-neurohypophyseal system(hnx) of the rat. Antibodies against arginine vasopressin (AVP), lysine-vasopressin (LVP) and oxytocin were raised by injecting these hormones that were covalently bound to thyroglobulin into rabbits. The vasopressin-immunized rabbits showed periods of diabetes insipidus, while histoloty of the "hns revealed an intact neurosecretory system with signs of increased endogenous hormone synthesis in the supraoptic nucleus and increased release in the neuro-hypophysis of some rabbits. The daily water intake of the oxytocin-immunized rabbits was similar to that of control rabbits. The development of antibodies against vasopressin as measured in the immunofluorescence procedure showed a course that was quite different from the curve of the titer as determined by radioimmunoassay (RIA). Also the specificity of the antibodies used in the immunofluorescence procedure was found to be quite different from their specificity in a RIA system. Potency and specificity of the antibodies have to be studied therefore within the immunofluorescence procedure itself. Using freshly frozen acetone-postfixed hypotalami or pituitaries, no sharp localization of immunofluorescence could be obtained in the HNS. Therefore prefixation was performed. Both, the type and the duration of prefixation revealed quite different results regarding the immunofluorescence in the neurosecretory cell boides in the hypotalamus and of their endings in the neurohypophysis. The best immunofluorescence results were obtained using 6 hours glyoxal-prefixation for the hypothalamus and 24 hours formalin-prefixation for the pituitary. The cross-reaction of the antibodies for oxytocin or vasopressin was tested on synthetic hormones that were bound to CNBR-activated agarose beads and mounted on glass sides. All anti-plasmas showed cross-reaction on beads containing the heterologou- antigen. The plasmas were purified by incubation with beads containing the heterologous hormone until the cross-reacting component had been removed. Using purified antibodies, the distribution of oxytocin and vasopressin cells within the HNS was investigated. More oxytocin containing cells were localized in the rostral part and more vasopressin in the caudal part of both, the supraoptic (SON) and paraventricular nucleus (PVN). Comparable percentages of oxytocin and vasopressin containing cells were found in the SON and PVN. The absolute amount of oxytocin containing cells was 2.5 times more in the SON than in the PVN, which seems to contradict the "classical" view that the PVN predominantly or entirely synthetizes oxytocin. In addition, fluorescence was found using antobodies against vasopressin in the suprachiasmatic nucleus in Wistar rats and heterozygous Brattleboro rats, but not in this nucleus of homozygous Brattleboros.