Histochemical Localization of Nitric Oxide Neurons in the Hypothalamus: Association with Gonadotropin-Releasing Hormone Neurons and Co-Localization with N-Methyl-D-Aspartate Receptors

Abstract

The neurotransmitter glutamate plays an important role in the control of gonadotropin releasing hormone (GnRH) secretion. Recent evidence suggests that the novel transmitter nitric oxide may also play a role in controlling GnRH release and may be an important mediator of glutamate effects. To explore the role of nitric oxide in these events, the present study determined the distribution of the enzyme which catalyzes nitric oxide production, nitric oxide synthase (NOS) in the hypothalamus, its association with GnRH neurons, and whether NOS neurons contain NMDA receptors. NOS was localized by staining hypothalamic sections from female rats for NADPH-diaphorase activity. Specific antibodies for GnRH and the NMDA R₁receptor subunit were used for double-staining to determine NOS association with GnRH neurons and the presence of NMDA R₁ receptor subunits in hypothalamic NOS neurons. The studies showed intense NOS cell body and fiber staining in the organum vasculosum of the lamina terminalis (OVLT) where numerous GnRH cell bodies are located. Other major GnRH cell body sites such as the median preoptic nucleus (MPN) and medial preoptic area (MPOA) displayed moderate staining of NOS cell bodies and fibers. Intense NOS staining was also observed in the median eminence, ventromedial nucleus, paraventricular nucleus and supraoptic nucleus of the hypothalamus. While no GnRH neurons were found to double stain for NOS in the hypothalamus, GnRH neurons were frequently surrounded by NOS neurons in the OVLT, MPN and MPOA with potential contacts between NOS and GnRH neurons in these areas. In addition, there was significant overlap of GnRH and NOS fibers in the lateral portion of the internal zone of the median eminence where GnRH fibers and terminals converge. Double-staining studies for NADPH-diaphorase and NMDA R₁ receptor subunit showed that many NOS neurons in the OVLT, MPOA, ventromedial nucleus, paraventricular nucleus and supraoptic nucleus co-localize the NMDA R₁ receptor subunit. Localization of NMDA R₁ receptor subunit immunoreactivity in B-NOS neurons in the hypothalamus was further confirmed by using combined immunohistochemistry-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 induce LH secretion. Taken as a whole, these studies provide evidence which 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 Ri receptor subunit.

Agouti-Related Peptide and MC3/4 Receptor Agonists Both Inhibit Excitatory Hypothalamic Ventromedial Nucleus Neurons

“…A number of different neuroactive substances have been reported in the VMH, including GABA, glutamate, nitric oxide, enkephalin, and dynorphin (Bhat et al, 1995;Commons et al, 1999;Ziegler et al, 2002;Slamberova et al, 2004;Lin et al, 2006). Glutamate appears to be the primary excitatory neurotransmitter in the medial hypothalamus (van den Pol et al, 1990), and glutamate agonists and antagonists have substantial effects on food intake (Hettes et al, 2003;Lee and Stanley, 2005).…”

Pituitary Adenylate Cyclase-activating Polypeptide Stimulates Nitric-oxide Synthase Type I Expression and Potentiates the cGMP Response to Gonadotropin-releasing Hormone of Rat Pituitary Gonadotrophs

“…These enzymes catalyze the formation of nitric oxide (NO), a highly reactive and diffusible gas that plays an important role as an inter-or intracellular messenger and exerts, at least in part, its effects via the activation of soluble guanylate cyclase (sGC) (19,20). NOS I has been shown to mediate the N-methyl-D-aspartate action on GnRH secretion in the hypothalamus (21,22), whereas an endothelial NOS III is involved in the estradiol-induced secretion of GnRH (23). The identification of NOS I in pituitary gonadotrophs and folliculostellate cells (24) has raised the possibility that NO may act as a regulator of pituitary activity.…”

Castration increases and androgens decrease nitric oxide synthase activity in the brain: Physiologic implications

“…NO functions as a neurotransmitter (2)(3)(4)(5)(6)(7)(8)(9)(10) and nNOS is present near gonadotropin-releasing hormone terminals (14) and in regions of the brain that appear to regulate emotional behaviors (15,16). Estradiol (E 2 ) up-regulates endothelial NOS in endothelial cells (17,18) and nNOS in the brain (18)(19)(20)(21), and many of the actions of E 2 on the brain have been suggested to be through a NO-mediated mechanism (2)(3)(4)(5)(6)(7)(8)(9)(10)(18)(19)(20)(21)(22). Administration of E 2 and progesterone to ovariectomized rats leads to an increase in the luteinizing hormone surge, the magnitude of which is significantly attenuated after administration of nNOS antisense oligonucleotides into the third ventricle (22), suggesting an intermediary role of NO in modulating some actions of E 2 by up-regulating nNOS activity.…”