Elsevier

Neuroscience

Volume 272, 11 July 2014, Pages 29-33
Neuroscience

Stereological quantification of olfactory receptor neurons in mice

https://doi.org/10.1016/j.neuroscience.2014.04.050Get rights and content

Highlights

  • No data are available on the total number of olfactory receptor neurons (ORNs).

  • Stereological analyses were applied for the total quantification of ORNs in mice.

  • We concluded that the total number of ORNs was approximately 10,000,000.

Abstract

The total number of olfactory receptor neurons (ORNs) in the mouse main olfactory epithelium (MOE) was estimated using stereological sampling. Noses and skulls of male and female 8-week-old C57BL/6J mice were de-calcified, embedded in paraffin, cut into 10-μm-thick sections serially at 100-μm intervals, and processed for immunohistochemistry for the olfactory marker protein (OMP), a specific marker for ORNs. The number of OMP (+) receptor neurons was measured using an optical fractionator with the Stereo-Investigator software. The mean values of the total number of OMP (+) receptor neurons in the unilateral MOE were 5,140,000 ± 380,000 in males and 5,210,000 ± 380,000 in females, with no significant differences between the sexes. We concluded that the total number of ORNs in the unilateral MOE is approximately 5 × 106 in mice.

Introduction

In recent years, the olfactory system has attracted considerable interest because of important discoveries regarding the odorant (olfactory) receptors expressed by the olfactory receptor neurons (ORNs; Buck and Axel, 1991). Odorant detection is mediated by a huge number of receptor neurons which are located in the olfactory epithelium lining of the nasal cavity (Mori et al., 1999). Olfactory receptors have been extensively studied, and 1035, 1207, 811 and 387 receptors were reported in mice, rats, dogs and humans, respectively (Niimura and Nei, 2007). It was revealed that each receptor neuron expresses only one receptor type, and a group of receptor neurons that express the same receptor type send their axons to the same specific bulbar site, the olfactory glomerulus (Mombaerts, 2004).

Quantification of ORNs in the main olfactory epithelium (MOE) has been studied in several mammalian species, including rats, rabbits, dogs, pigs and humans (Allison and Warwick, 1949, Müller, 1955, Gasser, 1956, Moran et al., 1982, Meisami, 1989, Meisami et al., 1990, Youngentob et al., 1997). However, it is very surprising that no data are available at present on the total number of ORNs in mice (Mombaerts, 2004), for which there are abundant genetic data. The present study was undertaken to provide quantitative data on the total number of ORNs in C57BL/6J mice; therefore, we performed immunohistochemistry for the olfactory marker protein (OMP), which is the most commonly used marker for ORNs in many animals. Furthermore, we employed stereological analyses, the most reliable counting method for whole quantification, to estimate the total number of ORNs. Based on our data, we discuss the converging ratios of receptor neurons to projection neurons (mitral cells) of the olfactory bulb, which is the brain target of receptor neurons.

Section snippets

Animals

The experiments were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals, and the protocols used were approved by our Institutional Animal Care and Use Committee. All efforts were made to minimize the number of animals used and their suffering. Normal 8-week-old C57BL/6J mice (Japan SLC Inc., Hamamatsu, Japan) of both sexes were used in this experiment.

Tissue preparation

Mice (male n = 6; female n = 6) were euthanatized with sodium pentobarbital (100 mg/kg,

Results

The MOE, which contains numerous OMP (+) receptor neurons, was situated in the caudal two-thirds of the nasal cavity. Fig. 1A–G shows the whole MOE, which was sectioned at regular intervals and placed in a rostrocaudal sequence. The MOE appeared at the dorsal aspect of the nasal cavity with the vomeronasal organ situated ventrally near the nasal septum (Fig. 1A–C). The MOE extended laterally and ventrally in the nasal cavity (Fig. 1D, E), resulting in great expansion of the surface area of the

ORNs and OMP

The MOE consists of the following three major cell types: ORNs, supporting cells and basal cells. ORNs are primary neurons of the olfactory system and exhibit a unique character because they have a relatively short life-span of approximately 1 month and are continually replaced from the neural progenitor cells (basal cells) throughout life (Farbman, 2000). Therefore, it is difficult to distinguish between immature and mature receptor neurons of the MOE, where continued neurogenesis is taking

Acknowledgments

This work was supported by a Grant-in Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (23791886 and 25462661).

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