Ontogeny of the rat hypothalamic nitric oxide synthase and colocalization with neuropeptides

The topographic ontogeny of nitric oxide synthase (NOS) within the paraventricular nucleus (PVN) of the rat hypothalamus was studied by nicotinamide adenine dinucleotide-diaphorase (NADPH-diaphorase) histochemistry. At Day 1 of postnatal life (P1), NOS-positive neurons were already present and achieved their maturity (in terms of perikarya number and dendritic arborization) about the time of weaning (P21). Across all ages studied (P1 to adulthood), intense NADPH-diaphorase staining was primarily confined within magnocellular cells of the PVN largely characterized by medium-sized (12-15 mum in diameter), ovoid bipolar neurons with prominent clear nuclei. To identify the neurosecretory cells of the adult PVN in which NOS was present, double-labeling studies were carried out via fluorescent immunocytochemistry. Magnocellular oxytocin (OT) and arginine vasopressin (AVP), as well as parvocellular corticotropin-releasing factor (CRF), were found to be colocalized with NOS. However, colocalization occurred significantly more frequently in OT-containing neurons, relative to AVP- or CRF-positive cells. Most of the colocalization occurring between NOS and OT was observed in the rostral constituent of the magnocellular subdivision of the PVN, as opposed to a more caudal defined PVN. To provide a distribution comparison of OT, AVP, and CRF to that of NOS in the adult PVN, in situ hybridization was carried out with (35)S-cRNA antisense probes for the aforementioned neuropeptides. The results obtained with this evaluation were correlated with NOS histochemistry in the same brain sections. As expected, specific labeling was observed for all three neuroactive substances over their topographically distinctive nuclei. Among these nuclei, labeling by the OT cRNA probe provided the closest topographical correlation of hybridized signal over NOS perikarya, thus reinforcing the tenet that a relatively small population of OT nerve cells are concurrently colocalized with the enzyme. Taken together, these results indicate that NOS is present in the PVN of the rat at all postnatal ages which we tested. They also indicate that among neurosecretory cells of the PVN, only OT prominently shared with NOS the same common nerve cell type. This suggests that NOS neurons may represent a distinct neuropil group among multiple neuroactive nuclei in the neuroendocrine hypothalamus. Finally, we demonstrate that NADPH-diaphorase histochemistry can be easily combined with immunocytochemical and in situ hybridization procedures to evaluate the colocalization and topographical distribution of NOS with other phenotypic neurons in the mammalian central nervous system.