Relationship of Neuronal Nitric Oxide Synthase Immunoreactivity to GnRH Neurons in the Ovariectomized and Intact Female Rat

Abstract

The present study has used a rat neuronal nitric oxide synthase (nNOS) antibody to examine the relationship of nNOS immunoreactivity to GnRH neurons in the ovariectomized and intact diestrous and proestrous rat. A striking band of nNOS-immunoreactive cells was identified in the rostral preoptic area which began in the median preoptic nucleus and organum vasculosum of the lamina terminalis and formed an inverted Y-type distribution above the rostral third ventricle at the level of the anteroventral periventricular nucleus. Another band of nNOS-immunoreactivity was found extending through the internal zone of the median eminence into the arcuate nucleus. Although nNOS immunoreactivity was not detected within GnRH neuronal cell bodies in any of the experimental groups, GnRH perikarya located in the rostral preoptic area, but not elsewhere, were found to be surrounded by nNOS-containing cells. In the median eminence, nNOS and GnRH immunoreactivities were distributed separately in the internal and external zones, respectively. These results provide evidence that, regardless of their pattern of activity, GnRH neurons in the female rat do not express nNOS. Instead, a close anatomical relationship between nNOS-immunoreactive cells and GnRH perikarya and fibers has been identified within specific sub-regions of the rostral preoptic area and in the median eminence. Such findings are compatible with a role for NO at both sites in regulating the release of GnRH throughout the estrous cycle.

Synergistic action of advanced glycation end products and endogenous nitric oxide leads to neuronal apoptosis in vitro: A new insight into selective nitrergic neuropathy in diabetes

“…The sections were dried on gelatinecoated slides for 2 h at room temperature, then incubated with PBS containing 0.1% Triton X-100 and 5% of the serum of the species from which the secondary antibody was obtained. Then the slides were incubated with antibodies against nNOS (raised in sheep, K205 [22]; 1:2000) or tyrosine hydroxylase (TH; 1:200; Chemicon, Harrow, UK) at 4°C overnight and with advanced glycation endproducts (AGEs; clone 6D12; 2 µg/ml; Biologo) for 2 h at room temperature followed by detection with appropriate fluorescent conjugated secondary antibodies. The images were obtained using a laser-scanning confocal microscope (Leica TCS-DMRE, Bensheim, Germany).…”

Nitric oxide synthase and background adaptation in Xenopus laevis

“…Dilutions of the sera were made in filtered TBS supplemented with 0.3% Triton X-100. The specificity of these antisera in staining mammalian tissues has been largely demonstrated (de Vente et al, 1996a,b;Herbison et al, 1996;Tanaka et al, 1997;Yamada et al, 1997). In the single cGMP-immunostaining procedure, secondary antisera used were the CY3-labeled goat anti-rabbit IgG (Jackson; diluted 1:800) or the FITC-labeled rabbit anti-sheep (Calbiochem, La Jolla, CA; diluted 1:150), depending on the host species of the primary antiserum.…”

Coupling of Neuronal Nitric Oxide Synthase to NMDA Receptors via Postsynaptic Density-95 Depends on Estrogen and Contributes to the Central Control of Adult Female Reproduction

“…Sheep polyclonal antinNOS (1:3000) was a generous gift from Dr. P. C. Emson (Medical Research Council, Laboratory for Molecular Research, Cambridge, UK) (Herbison et al, 1996). Rabbit polyclonal anti-nNOS (61-700; 1:250) and anti-NR2B (71-8600; 1:500) were purchased from Zymed, described by Christopherson et al (1999), and previously characterized for immunocytochemistry by others (El-Husseini et al, 2000;Burette et al, 2002;Czaja et al, 2006).…”