Sexually Dimorphic Expression of Nitric Oxide Synthase in the Rat Accessory Olfactory Bulb.

Ital.J.Anat.Embryol., 101, Suppl.1, 164-165.

García-Ojeda E.*°, Rossi M.C.°, C. Crespo*, R.Arévalo,* J.R.Alonso*, Panzica G.C.°

*Dept.Cell Biology and Pathology, University of Salamanca (Spain), °Dept. Anatomy, Pharmacology and Forensic Med., University of Torino (Italy)

The accessory olfactory bulb (AOB) of the rat is part of the accessory olfactory pathway, a sexually dimorphic pathway driving olfactory stimuli (mainly pheromones) to the medial preoptic region, a crucial site for the regulation of masculine behaviour (Guillamon and Segovia, 1993). The AOB receives direct inputs from the vomeronasal organ and projects to the medial amygdala. Its volume and the size of neurons are larger in male than in female rat, and both are sensitive to perinatal treatment with steroid hormones. The AOB has a layered structure, and a specific distribution of NADPH-diaphorase activity (ND) along this structure has been recently demonstrated (Porteros et al., 1994). ND has been recognized as the neuronal nitric oxide sinthase (nNOS) and its distribution has been considered as a reliable marker of the distribution of NO producing elements. All layers of the AOB show high levels of ND positivity. In the present study we have serially cut at the transverse plane the olfactory bulbs of 5 male and 5 female rats from the same litter at the age of three months. Adjacent 25 µm-thick sections were collected in four different series that were stained with toluidine blue (Nissl stain), for ND histochemistry, or nNOS immunohistochemistry (K205 sheep anti-rat neuronal NOS antibody diluted 1:20,000, Herbison et al., 1996). The last 4th series was employed for control reactions. Only one AOB per animal was considered. The AOB volume was measured on coded material to prevent the a priori knowledge of the sex of the animal. To calculate the volume in Nissl stained sections, as well as the volume of nNOS positive structures in the same region, drawings of the sections were made with the aid of a camera lucida. In each drawing, the boundaries between layers were indicated. The layer areas were then measured using a graphics tablet connected to an Apple //e computer. The volumes were calculated considering the area of the structure and the distance between two consecutive sections (generally 100 µm). Visual inspection of the sections revealed that the ND staining is observed throughout the entire extension of the AOB, whereas the immunoreactivity for nNOS is lacking in the two more external layers: vomeronasal nerve layer (VL) and glomerular layer (GL), where only a few, scattered nNOS positive cells were observed. These layers were clearly distinguished after the immunohistochemical reaction. Measurements of the AOB volume either with the Nissl stain or with NOS immunoreactivity (including nNOS positive and nNOS negative regions) demonstrated a highly significant difference (Nissl stain, F=23, p<0.005; NOS, F=12, p<0,02), being the AOB larger in males than in females. Considering the differences in the measured volumes among Nissl- or nNOS-stained material, the double-tailed paired T-test gave a p=0.2 (not significant) confirming the former idea that nNOS immunostaining is a good marker of the region. This experiment confirmed the sexual dimorphism in the AOB volume in rat, being the structure larger in male than in female animals. Moreover, we have here confirmed that staining for nNOS is a valid alternative to the Nissl staining to identify the region from the adjacent olfactory structures. In fact, no statistically significant difference were detected among measuments of AOB performed on Nissl- or nNOS-stained material. Nevertheless, nNOS immunostaining is a specific marker only for the more internal layers: granule cell layer (GCL), internal plexiform layer (IPL) and external plexiform layer (EPL), whereas the immunostaining is lacking in the GL and VL. The mitral cells are dispersed in the EPL and they were included in this layer. A comparison of the volume of these three layers, identified in both male and female by means of nNOS immunoreactivity, demonstrated a sexually dimorphic distribution, having these layers separately or considered all together a larger volume in male rat. When considering the volume of these three layers in Nissl-stained sections, the similarity among Nissl-staining and nNOS immunohistochemistry become even closer (p=0.9) than considering the whole structure. The differences among male and female were respectively p<0.02 for Nissl stain, and p<0.03 for nNOS immunostaining. In conclusion, the population of cells and processes expressing nNOS in the rat AOB represents a sexually dimorphic population of this region and nNOS immunostaining could be employed as a reliable marker of the structure in future experimental works. Acknowledgements: Supported by grants of the "Junta de Castilla y León", DGICyT PB94-1388, EU (CT94-0472), MURST 40%.

References