HISTOCHEMICAL-LOCALIZATION OF NITRIC-OXIDE NEURONS IN THE HYPOTHALAMUS - ASSOCIATION WITH GONADOTROPIN-RELEASING-HORMONE NEURONS AND COLOCALIZATION WITH N-METHYL-D-ASPARTATE RECEPTORS

The neurotransmitter glutamate plays an important role in the control of gonadotropin-releasing hormone (GnRH) secretion. Recent evidence su ggests that the novel transmitter nitric oxide may also play a role in controlling GnRH release and may be an important mediator of glutamat e effects. To explore the role of nitric oxide in these events, the pr esent study determined the distribution of the enzyme which catalyzes nitric oxide production, nitric oxide synthase (NOS) in the hypothalam us, its association with GnRH neurons, and whether NOS neurons contain NMDA receptors. NOS was localized by staining hypothalamic sections f rom female rats for NADPH-diaphorase activity. Specific antibodies for GnRH and the NMDA R(1) receptor subunit were used for double-staining to determine NOS association with GnRH neurons and the presence of NM DA R(1) receptor subunits in hypothalamic NOS neurons. The studies sho wed intense NOS cell body and fiber staining in the organum vasculosum of the lamina terminalis (OVLT) where numerous GnRH cell bodies are l ocated. Other major GnRH cell body sites such as the median preoptic n ucleus (MPN) and medial preoptic area (MPOA) displayed moderate staini ng of NOS cell bodies and fibers. Intense NOS staining was also observ ed in the median eminence, ventromedial nucleus, paraventricular nucle us and supraoptic nucleus of the hypothalamus. While no GnRH neurons w ere found to double stain for NOS in the hypothalamus, GnRH neurons we re frequently surrounded by NOS neurons in the OVLT, MPN and MPOA with potential contacts between NOS and GnRH neurons in these areas. In ad dition, there was significant overlap of GnRH and NOS fibers in the la teral portion of the internal zone of the median eminence where GnRH f ibers and terminals converge. Double-staining studies for NADPH-diapho rase and NMDA R(1) receptor subunit showed that many NOS neurons in th e OVLT, MPOA, ventromedial nucleus, paraventricular nucleus and suprao ptic nucleus co-localize the NMDA R(1) receptor subunit. Localization of NMDA R(1) receptor subunit immunoreactivity in B-NOS neurons in the hypothalamus was further confirmed by using combined immunohistochemi stry-in situ hybridization. Finally, the functional importance of this co-localization was shown by the finding that central administration of a nitric oxide synthase inhibitor blocked the ability of NMDA to in duce LH secretion. Taken as a whole, these studies provide evidence wh ich support a role for nitric oxide as an important regulator of GnRH neurons in the female. They also suggest that hypothalamic NOS neurons are targets for glutamate regulation as evidenced by co-localization of the NMDA R(1) receptor subunit.