Stereological quantification of olfactory receptor neurons 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).
References (24)
- et al.
A novel multigene family may encode odorant receptors: a molecular basis for odor recognition
Cell
(1991) - et al.
Determination of functionally essential neuronal population of the olfactory epithelium for nipple search and subsequent suckling behavior in newborn rats
Brain Res
(2009) A proposed relationship between increases in the number of olfactory receptor neurons, convergence ratio and sensitivity in the developing rat
Dev Brain Res
(1989)Odorant receptor gene choice in olfactory sensory neurons: the one receptor-one neuron hypothesis revisited
Curr Opin Neurobiol
(2004)- et al.
A re-estimation of the number of glomeruli and mitral cells in the olfactory bulb of rabbit
Brain Res
(1998) - et al.
Odorant threshold following methyl bromide- induced lesions of the olfactory epithelium
Physiol Behav
(1997) - et al.
Effects of postnatal thyroid hormone deficiency on neurogenesis in the juvenile and adult rat
Neurobiol Dis
(2009) Estimation of nuclear population from microtome sections
Anat Rec
(1946)- et al.
Quantitative observations on the olfactory system of the rabbit
Brain
(1949) Cell biology of olfactory epithelium
The minimum number of neurons in the central olfactory pathway in relation to its function: a retrograde fiber tracing study
Chem Senses
Olfactory nerve fibers
J Gen Physiol
Cited by (29)
Bio-inspired encoding for a real-time and stable single component odor detection with a highly-redundant optical artificial olfactory system
2022, Sensors and Actuators B: ChemicalCitation Excerpt :A striking characteristic of olfaction is the redundancy of ORNs with the same nominal functionality. For instance, in mice, little above 1000 different receptors are replicated in up to 5 million olfactory receptor neurons (ORNs) in each nasal cavity [12]. Processing such a large number requires a precise organization of the olfactory pathway.
3.24 - Compartmentalization of the Mammalian Olfactory System
2020, The Senses: A Comprehensive Reference: Volume 1-7, Second EditionScaling Principles of Distributed Circuits
2019, Current BiologyCitation Excerpt :Two studies support this assumption. At least in mice, glomerular size is proportional to OSN number [18] (flies being a notable exception), and OSN size does not significantly vary across mice, rats, and rabbits [19–21]. We found that glomerular numbers and sizes increased with glomerular layer volumes (Figures 1F, 1G, and S1D).
Effects of various lengths of hypoglossal nerve resection on motoneuron survival
2019, Journal of Clinical NeuroscienceCitation Excerpt :XII neurons have advantages for quantitative analyses of motor neurons because the XII nucleus has a clear nuclear border and easily identified large neurons. Further, the design-based stereology approach with systemic random sampling, which was employed in this study using Stereo-Investigator software, is recommended for the estimation of total neuron number and has been widely used in recent studies [1,6–10]. It seems plausible that neuron survival rates differ depending on the nerve insult.
Comparison of unbiased estimation of neuronal number in the rat hippocampus with different staining methods
2015, Journal of Neuroscience MethodsCitation Excerpt :The stereology was established by G. Buffon in 1733 (Miyamoto, 1994), which is based on statistical sampling principle and Cavalieri's principle (fundamental principles of geometry). Stereological design-based method is widely used in neuroscience and which can offer unbiased, accurate and precise estimates of neuronal number in a defined brain region (Kawagishi et al., 2014; West et al., 1991). The optical fractionator is a three dimensional probe used in stereology for number counting, it is very efficient and the estimate requires relatively thick sections (≥20 μm after processing and cover slipping) (Wirenfeldt et al., 2003).
A genetic platform for functionally profiling odorant receptors in olfactory cilia ex vivo
2022, Science Signaling