Anatomy of the olfactory mucosa

Handbook of Clinical Neurology, Vol. 164 (3rd series) Smell and Taste R.L. Doty, Editor https://doi.org/10.1016/B978-0-444-63855-7.00004-6 Copyright © 2019 Elsevier B.V. All rights reserved

Chapter 4

Anatomy of the olfactory mucosa IGNACIO SALAZAR1*, PABLO SANCHEZ-QUINTEIRO1, ARTHUR W. BARRIOS2, MANUEL LÓPEZ AMADO3, AND
A. VEGA4 JOSE 1

Department of Anatomy, Animal Production and Veterinary Clinical Sciences, Unit of Anatomy and Embryology, Faculty of Veterinary, University of Santiago de Compostela, Lugo, Spain

2

Laboratory of Histology, Embryology and Animal Pathology, Faculty of Veterinary Medicine, University Nacional Mayor of San Marcos, Lima, Peru 3 4

Department of Otorhinolaryngology, University Hospital La Coruña, La Coruña, Spain

Unit of Anatomy, Department of Morphology and Cell Biology, Faculty of Medicine, University of Oviedo, Oviedo, Spain

Abstract The classic notion that humans are microsmatic animals was born from comparative anatomy studies showing the reduction in the size of both the olfactory bulbs and the limbic brain relative to the whole brain. However, the human olfactory system contains a number of neurons comparable to that of most other mammals, and humans have exquisite olfactory abilities. Major advances in molecular and genetic research have resulted in the identification of extremely large gene families that express receptors for sensing odors. Such advances have led to a renaissance of studies focused on both human and nonhuman aspects of olfactory physiology and function. Evidence that olfactory dysfunction is among the earliest signs of a number of neurodegenerative and neuropsychiatric disorders has led to considerable interest in the use of olfactory epithelial biopsies for potentially identifying such disorders. Moreover, the unique features of the olfactory ensheathing cells have made the olfactory mucosa a promising and unexpected source of cells for treating spinal cord injuries and other neural injuries in which cell guidance is critical. The olfactory system of humans and other primates differs in many ways from that of other species. In this chapter we provide an overview of the anatomy of not only the human olfactory mucosa but of mucosae from a range of mammals from which more detailed information is available. Basic information regarding the general organization of the olfactory mucosa, including its receptor cells and the large number of other cell types critical for their maintenance and function, is provided. Cross-species comparisons are made when appropriate. The polemic issue of the human vomeronasal organ in both the adult and fetus is discussed, along with recent findings regarding olfactory subsystems within the nose of a number of mammals (e.g., the septal organ and Gr€ uneberg ganglion).

INTRODUCTION The olfactory mucosa is the mucus-secreting membrane in the upper recesses of the nose that contains cells responsible for initiating olfactory sensations. In humans, this mucosa retains many features of those of mammals with more complex olfactory systems, despite being housed in a nose with a less convoluted set of turbinates, the thin

shelves of bone covered with a mucus membrane and erectile tissue that extend into each side of the nasal cavity from each lateral wall. Based on classic comparative anatomy, such as the size of the olfactory bulbs relative to the brain and a reduction in the size of limbic brain regions associated with olfactory function, humans—along with Old World primates—have been historically considered “microsmatic” mammals. However,

*Correspondence to: Ignacio Salazar, Unit of Anatomy and Embryology, Faculty of Veterinary, Av. Carballo Calero s/n, 27002 Lugo, Spain. Tel: +34-639454437, E-mail: [email protected]

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some modern workers have argued against this concept for several reasons. For example, if one focuses solely on structural volumes, the olfactory bulb of the human is larger than the entire brain of some rodents. A recent review points out that humans rival many other mammals in terms of their olfactory abilities and concludes that poor human olfaction is a myth generated in the 19th century (McGann, 2017). It is clear that the major characteristics of the olfactory mucosa in so-called macrosmatic mammals, as well as its neurogenesis and neural regeneration, remain intact in humans (Girard et al., 2011; Feron et al., 2013). For example, the olfactory epithelium of nearly all land mammals, including humans, is a pseudostratified columnar epithelium. There are, however, considerable interspecies differences in the types and frequencies of occurrence of olfactory receptors. In humans, olfactory receptor genes have about 900 potential coding regions, of which only around 35% are functional (Glusman et al., 2001; Olender et al., 2008). This contrasts with many other mammals. In the African elephant, for example, the functional receptor gene number is close to 2000 (Niimura et al., 2014). In murine rodents, the functional olfactory receptor genes number around 1000. Surprisingly, the number of function olfactory receptors in humans (396) excels the number found in other primates, including chimpanzees (380), orangutans (296), rhesus macaques (309), and the common marmoset (356) (Laska and Salazar, 2015). Unlike humans, a number of forms, including most lagomorphs, rodents, and ungulates, are “obligatory nose breathers.” Whether such nonhuman mammals employ retronasal olfaction, i.e., stimulation of olfactory receptors via the nasal pharynx from mouth-borne substances, is unknown but questionable on anatomical grounds. This concept is of particular interest in light of the important role this process plays in contributing to the flavor of foods in humans (Rozin, 1982; Burdach and Doty, 1987; Shepherd, 2010; Bojanowski and Hummel, 2012). In this chapter, we provide a basic overview of the anatomy of not only the human olfactory mucosa, but of mucosae from a range of mammals from which more detailed information is available, most notably the laboratory mouse, Mus musculus. Its focus is on classic anatomy rather than on function, in light of the large literature that largely addresses function. The reader is referred to recent reviews by Escada et al. (2009), Borgmann-Winter et al. (2015), Chen et al. (2014), Dennis et al. (2015), Ding and Xie (2015), and Mackay-Sim et al. (2015) that largely focus on function. In these reviews, 1286 references are listed, of which only 159 were common to two or more of the reviews. After omitting these papers, 87.9% of the remaining studies were on original research. Of these, 28.3% focused on humans and 46.3% on mice or rats.

Other lab animals, domestic animals, and nonmammals comprised the remainder.

THE HUMAN OLFACTORY MUCOSA General organization The human olfactory mucosa not only harbors the olfactory receptor cells critical for initiating smell perception, but a range of cell types essential for their maintenance and function (DeMaria and Ngai, 2010). Therefore, knowledge of the general organization of the olfactory mucosa is a basic requirement for understanding the olfactory system in general. This mucosa is located on sectors of the lateral and medial walls and roof of the nasal cavity, as well as part of the turbinates, especially the region of the superior turbinate (Fig. 4.1) (Gilroy et al., 2012). Like most other land mammals, the human mucosa is characterized by a well-defined epithelium, a basal lamina, and a lamina propria (Polyzonis et al., 1979; Moran et al., 1982a; Jafek, 1983), although species differences exist (Borgmann-Winter et al., 2015). In humans this epithelium lines 3% and 5% of the nasal cavity, an area whose extension can vary from 200 to 1000 mm2. This contrasts with murine rodents, where 45%–50% of the nasal cavity is lined by olfactory mucosa (Sorokin, 1988; Lane et al., 2002; Harkema et al., 2006; Chamanza and Wright, 2015). Unfortunately, it is difficult to define the exact area of the human olfactory mucosa given its heterogeneity and diversity and the fact that it is challenging to differentiate it from surrounding tissue either in vivo (endoscopy) or postmortem, regardless of whether the samples are fresh or fixed (Fig. 4.2). There is lack of uniformity in the thickness of the olfactory epithelium and its underlying lamina propria, and regions of the epithelium become replaced over time with intermingled islands of respiratory epithelium (Naessen, 1970; Nakashima et al., 1984; Morrison and Costanzo, 1992; Paik et al., 1992). Such replacement is cumulative throughout life, reflecting age-related processes, medical conditions, and exposures to xenobiotics (Paik et al., 1992; Getchell et al., 1993; Kern, 2000; Mackay-Sim et al., 2006; Doty and Kamath, 2014). Given the heterogeneity and diversity in gross anatomy and the cytoarchitecture of the human olfactory mucosa, it is not surprising that its description varies in the literature. Nevertheless, its general organization is the same as that of the murine species, although it typically has a more diffuse laminar organization (Holbrook et al., 2011). In humans, the basal cells have more rounded bodies and it is more difficult to differentiate between globose basal cells and horizontal basal cells than in rodent forms. Such cells are the progenitors of other cell types within the epithelium (Hahn et al., 2005). Additional

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Fig. 4.1. Schematic representation of the medial wall (A), and lateral wall (B) of the nasal cavity of man. The dashed black lines outline the zones where the plates of olfactory mucosa are more frequent. (C and D) represent a frontal section of the human head, focused on the nasal cavity at the middle (C) and posterior (D) thirds of the nasal cavity. The selected areas correspond with the sections in (A) and (B) of Fig. 4.2. Adapted from Sch€unke, M., Schulte, E., Schumacher, U. 2015. Prometheus. Texto y Atlas de Anatomı´a 3ª edicio´n, vol. 3. Panamericana, Madrid.

types of cells have been described in both human and murine olfactory epithelia, including microvillar cells— cells similar to brush cells found throughout the lower respiratory organs (Moran et al., 1982a,b; Morrison and Costanzo, 1990, 1992; Pfister et al., 2012). As described later in the chapter, humans lack a functional vomeronasal organ (VNO), and some species, notably murine rodents, have specialized chemoreceptors on the anterior septum (septal organ) and the anterior nasal chamber (Gr€ uneberg ganglion). Prior to entering the brain via the foramina of the cribriform of the ethmoid bone, the unmyelinated receptor cell axons, which collectively make up cranial nerve I, form into bundles of about 200 axons apiece. Each bundle is encapsulated in olfactory ensheathing cells which have properties similar to astrocytes and Schwann cell mesaxons. These bundles are further encased by olfactory nerve fibroblasts. A distinct population of microglia and macrophages is found between the olfactory ensheathing cells, which serve to block the invasion

of viruses and xenobiotics into the brain via the olfactory nerves (Smithson and Kawaja, 2010). The olfactory pathways can be a direct passage that bypasses the blood brain barrier for such invasion (Jackson et al., 1979; Doty, 2008).

Olfactory biopsies and olfactory ensheathing cell explants The human olfactory mucosa takes on special interest from a medical perspective, since it can be biopsied in living humans (Lovell et al., 1982; Lanza et al., 1994; Feron et al., 1998, 2005; Jafek et al., 2002; Benítez-King et al., 2011; Ono et al., 2012), potentially yielding tissue of value in the early diagnosis of some neurodegenerative and neuropsychiatric disorders (Arnold et al., 1998; Doty, 2012; Buron and Bulbena, 2013; Casjens et al., 2013; Seligman et al., 2013; Auster et al., 2014; Doty et al., 2015; Field, 2015). Different instruments, some of them commercially available, have been designed to obtain

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Fig. 4.2. Frontal sections of the middle (A) and posterior (B and C) thirds of the human nasal cavity. The dashed red lines outline the zones that were sampled for the histology of the olfactory epithelium (Hematoxylin-eosin and Masson’s trichromic stained sections). Serial sections of the nasal mucosa lining of the concha nasalis superior, which consists of respiratory (RE) and olfactory (OE) epithelium (D: Hematoxylin-eosin-stained sections; E: immunohistochemistry for S100 protein). The structural differences between RE and OE can be clearly observed in (F–G) pictures, especially because the occurrence in the OE of abundant supporting cells (sc) that reach the lumen of the nasal cavity. Scale bars: D and E, 200 mm; F, 50 mm; G and H, 20 mm.

biopsies of the olfactory mucosa (Lovell et al., 1982; Lanza et al., 1994; Feron et al., 1998, 2005; Jafek et al., 2002; Benítez-King et al., 2011; Ono et al., 2012). However, such biopsies are somewhat impractical and, from other perspectives, have yielded little knowledge beyond that already gained from autopsies. Moreover, the success of obtaining olfactory epithelium from biopsied samples, even after

repeated sampling, has been reported to range from 25% to 76% (Feron et al., 1998; Lane et al., 2002; Pinna F de et al., 2013). Despite claims of no adverse effects of biopsy on olfactory function (Lanza et al., 1994; Andrews et al., 2016; Holbrook et al., 2016; Godoy et al., 2019), sample sizes have been small and age groups limited to those whose epithelium is unlikely to be compromised.

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Fig 4.2—Cont’d

That being said, biopsy of olfactory material has other potential uses. Thus, harvested cells can be explanted into damaged cell regions, most notably the spinal cord, to enhance neural regeneration and axonal guidance (Barnett and Riddell, 2004; Franssen et al., 2007; Mackay-Sim and St John, 2011; Chou et al., 2014; Li et al., 2015). Based on the remarkable ability of olfactory ensheathing cells to guide axonal outgrowth, transplanting them has opened up the possibility for aiding neural regeneration in different kinds of human nerve injuries (for review, see Chou et al., 2014). In rodents, such transplants have facilitated peripheral nerve regeneration (Radtke et al., 2009), spinal cord regeneration (Ramer et al., 2004), and, in a murine model of Parkinson’s disease, regeneration of severed nigrostriatal dopaminergic axons (Teng et al., 2008). Differences in morphology and gene expression patterns have been observed between olfactory ensheathing cells from the olfactory mucosa and from the olfactory bulb (Gu
erout et al., 2010; Kueh et al., 2011; Verbeurgt et al., 2014), impacting the regulation of the inflammatory response and axonal guidance (Honor
e et al., 2012). In general, explants from the olfactory bulb have proved more effective in mediating regeneration and functional connections than those cultured from the olfactory mucosa (Tabakow et al., 2014). Although the transplantation of olfactory ensheathing cells into spinal injuries of humans is reasonably safe (Li et al., 2015; Mackay-Sim and St John, 2011; Rao et al., 2014), the results of clinical trials have shown

limited success. Nonetheless, one study has reported remarkable success in the functional regeneration of supraspinal connections in a patient with a transected spinal cord following transplantation of olfactory ensheathing cells obtained from one of his olfactory bulbs excised via a craniotomy (Tabakow et al., 2014). This work, along with animal studies showing greater efficacy with bulbar explants, has led to explorations using fresh cadavers to develop minimally invasive means of obtaining olfactory bulb tissues, such as the keyhole supraorbital craniotomy with an eyebrow incision (Czyz et al., 2015).

The human VNO Despite claims that humans possess a functioning VNO, a structure described in detail later in this review for the mouse, there is no credible evidence for this claim. Humans lack the requisite vomeronasal nerves and accessory olfactory bulbs found in species with a functioning organ. Nonetheless, a rudiment of this structure is found in many human noses, with variable accounts as to its prevalence. Some have argued the so-called VNO pouch is found as a bilateral structure in all normal adult humans (revised by Monti-Bloch et al., 1998), whereas others estimate its presence is only in 39% of patients (Johnson et al., 1985). More recently, Stoyanov et al. (2016) noted its bilateral presence in only 2.3%, and unilateral presence of 24.7% of the patients

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they investigated (Stoyanov et al., 2016). The putative human VNO has been variously named, being called the olfactory pit (Feng et al., 1997), vomeronasal cavity (Trotier et al., 2000), vomeronasal duct (Abolmaali et al., 2001), putative VNO (Knecht et al., 2001), or simply vomeronasal epithelium (Witt et al., 2002). The reader is referred to more comprehensive reviews on this topic (e.g., Meredith, 2001; Evans, 2003; Stowers and Spehr, 2015). Descriptions about a presumptive VNO in human fetuses are quite interesting and relevant to the controversy surrounding the concept of an adult human VNO. Following different strategies and different methods, some of the most cited publications indicate that a vomeronasal sensory epithelium is present and well defined during the early human fetal period (Kreutzer and Jafek, 1980; Nakashima et al., 1985; Boehm and Gasser, 1993; Kjaer and Fisher-Hansen, 1996; Elmas et al., 2003). Some have suggested that this epithelium contains functional chemosensory neurons (Takami et al., 2001) and produces cells that migrate towards the brain (Takami et al., 2016). However, the presence of an accessory olfactory bulb in fetuses is controversial, being noted by some anatomists (Humphrey, 1940; Bossy, 1980; Chuah and Zheng, 1987) but not others (Macchi, 1951; Bhatnagar et al., 1987; Witt and Hummel, 2006). Connections between the vomeronasal sensory epithelium and the olfactory bulb have only been demonstrated in early development (M€ uller and O’Rahilly, 2004). Evidence that the presumptive VNO regresses in the prenatal period is quite solid and was provided by such early anatomists as Mihalkovics (1898) and Parker (1922) and by more recent workers such as D
enes et al. (2015). The presumptive VNO and accessory olfactory bulb in human fetuses appears to represent a clear example of a process of involution or regression (Salazar and Sánchez-Quinteiro, 2009).

mammals, primarily mice, although the general patterns are evident in most mammalian forms.

Olfactory epithelium In the human, the olfactory placode, the primordium of the olfactory epithelium, has thickened and has become invaginated by embryonic day 11.5 (E11.5; equivalent to Theiler Stage 19, TS19), at which time it can be clearly identified as epithelium (Treloar et al., 2010). From that stage of development, the cellular differentiation becomes apparent and the disposition of cells in rows are increasingly evident (Cuschieri and Bannister, 1975a,b). Before birth, the olfactory epithelium exhibits its characteristic structure, although during development most of the mitotic figures are found apically; postnatally cell proliferation is characteristic of the basal epithelium (Smart, 1971). In humans and most other mammals, the cells types of this pseudostratified columnar neuroepithelium are arranged in three layers: apical, intermediate, and basal. The apical layer is populated by the sustentacular (supporting) and microvillar cells; the intermediate layer consists of mature and immature olfactory sensory neurons arranged in rows; the basal layer is comprised of round global basal cells and elongated horizontal basal cells, also arranged in rows. A matrix of connective tissue is the main constituent of the lamina propria, which also contains blood and lymphatic vessels, axon bundles, and Table 4.1 Summary of the components that make up the olfactory mucosa Sustentacular cells Neurons Epithelium

DETAILS OF THE OLFACTORY MUCOSA OF MAMMALS The olfactory mucosa of mammals, as in most other vertebrates, has a well-organized morphology with two different compartments, the epithelium and the lamina propria, separated by a basal lamina (Table 4.1; Fig. 4.3). Because of the diverse nature of the many cell types, the number of biochemical markers available to define the specific characteristics of such cells is large and becomes even more diverse and complex when the markers include gene expression (Nickell et al., 2012; Heron et al., 2013; Mackay-Sim et al., 2015; Saraiva et al., 2015). Described in the following text are details of the major components of the mammalian olfactory mucosa. Most of the cited work comes from nonhuman

Basal cells

Mature Immature Globose Horizontal

Bowman’s gland

Excretory canals

Connective tissue

Different cells

Bowman’s gland

Excretory canals

Basal lamina Olfactory mucosa

Olfactory ensheathing cells and mesenchymal stem cells Lamina propria

Collagen and extracellular matrix Vessels

smooth muscle cells endothelial cells olfactory

Nerve bundles

vomeronasal trigeminal

A basal lamina establishes the limit between the epithelium and the lamina propria.

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Fig. 4.3. Diagrammatic reconstruction (top) and hematoxylineosin-stained section (bottom) of the olfactory mucosa, showing the olfactory epithelium (OE) and the lamina propria (LP). Basal lamina, horizontal bar. Bg, Bowman’s gland; supporting cells (1); mature and immature neurons (2); basal cells (3). Scale bar: 50 mm.

different cellular elements, including olfactory ensheathing cells and mesenchymal stem cells. Bowman’s glands are found in both the olfactory epithelium and the lamina propria. Some authors also distinguish between different subtypes of the microvillar cells depending upon their morphologic characteristics (Jourdan, 1975; Moran et al., 1982a; Rowley et al., 1989; Carr et al., 1991; Morrison and Costanzo, 1992; Miller et al., 1995). A third type of global basal cell has been described. Such cells are in touch with the basal lamina at an adherenstype junction (Holbrook et al., 1995; Mackay-Sim et al., 2015). Data regarding morphology, topography, and antigen expression profiles are usually enough to identify the cell types and their anatomical peculiarities in the olfactory epithelium (Murdoch and Roskams, 2007). Mature olfactory sensory neurons, located apically close to and under the sustentacular cells, express olfactory marker protein; immature olfactory sensory neurons, located basally close to and above the global basal cells, express the growth-associated protein GAP43; and both mature and immature olfactory sensory neurons express the neural cell adhesion molecule (NCAM) (Calof et al.,

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2002; Illing et al., 2002). Interestingly, olfactory marker protein, a brain protein unique to mature olfactory receptor neurons (Margolis, 1972), is now known to be expressed in nonolfactory tissue (e.g., in certain cells of the bladder and thyroid; Kang et al., 2015). Moreover, it has been demonstrated that olfactory marker protein is involved in certain olfactory kinetics, and that it plays an interesting role as a controller of several facets of the odorant response (Dibattista and Reisert, 2016). One of the major difficulties in identifying the stem cell population of the olfactory epithelium lies in defining the constituent cell characteristics exclusively on the basis of topography, morphology, or mitotic activity (Beites et al., 2005). Therefore, it is challenging to find corresponding molecular markers for each type of cell (Calof and Chikaraishi, 1989; Calof et al., 2002; Kawauchi et al., 2004). It should be borne in mind that the stem cell types usually reflect a heterogeneous cell population (G€oritz and Fris
en, 2012) and that speciesspecificity marker expression can complicate the identification of specific cell types (Grompe, 2012). The recent realization that the adult olfactory epithelium contains active global basal and quiescent horizontal basal stem cells (Schnittke et al., 2015) appears plausible as this occurs in other tissues (Li and Clevers, 2010). The global basal cells have a somewhat round morphology and sit close to and under the immature olfactory sensory neurons. GBC-1, p75NGFR, and cytokeratin are usually the selected markers to label them. Horizontal basal cells are flattened cells, lie deepest in the olfactory epithelium underneath the global basal cells, and express ICAM-1, cytokeratins 5 and 14, and a surface glycoprotein that binds to the lectin BS-I (Holbrook et al., 1995; Goldstein and Schwob, 1996). The regenerative capacity of the olfactory epithelium implies that certain cells have the specific tasks of ensuring the renewal of its neural and nonneural cells. Basal cells are believed to be the stem cells of the epithelium, among other reasons because the mitotic cell populations are located in the basal epithelium throughout the postnatal period. However, there is some controversy regarding the contribution of each type of basal cell in the process of regeneration. It appears evident that global basal cells give rise to neurons (Graziadei, 1973; Caggiano et al., 1994), but there is some doubt about their capacity for regenerating all the different cell types of the olfactory epithelium (Murdoch and Roskams, 2007). The opinion of a number of authors is that such cells give rise to neurons and nonneural cells (Huard et al., 1998), but others suggest that it is possible to distinguish different types of these cells and only a subpopulation of them are the neural stem cells, at least in the rodent (Jang et al., 2003). Despite the fact that the mitotic activity of the horizontal basal cells is limited and that they do not express

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neuronal markers (Carter et al., 2004), these cells are capable of regenerating neuronal and nonneuronal cells after severe damage of the tissue (Leung et al., 2007). However, it was necessary to clarify whether this ability was exclusively a response to the injury or was actually a physiologic process (Duggan and Ngai, 2007). The answer comes from Iwai et al., who demonstrated that regenerative capacity is a characteristic of these cells both in normal neuronal turnover and after acute lesions. These investigators presented evidence that the first action of horizontal basal cells was to give rise to global basal cells and olfactory sensory neurons, and then, at lower frequency, to form sustentacular and/or Bowman’s gland or duct cells (Iwai et al., 2008).

The lamina propria The morphology of the lamina propria is considerably more complex than that of the olfactory epithelium, and consequently the cells that comprise this region are mainly identified using specific markers. As a result, mucous and serous cells, pigment cells, acinar and duct cells of Bowman glands, endothelial cells, cells associated with inflammation and immunity (neutrophils, plasma cells, monocytes, macrophages), Schwann cells, olfactory epithelial cells, and microvillar cells, have been identified (Lindsay et al., 2010; Chen et al., 2014; Borgmann-Winter et al., 2015; Dennis et al., 2015; Ding and Xie, 2015; Mackay-Sim et al., 2015). As mentioned earlier, the primary interest of many researchers in the lamina propria is the fact that the olfactory ensheathing cells are promising candidates for promoting central nervous system repair, most notably that of injured spinal cords (Ramón-Cueto and Avila, 1998; Su and He, 2010; Raisman et al., 2012; Roet and Verhaagen, 2014; Yang et al., 2014). Contrary to what is currently the case in animal experimentation, mice are not the preferred animal models for laboratory studies of these cells and their applications, with most studies having been performed on rats (Devon and Doucette, 1992; Li et al., 1997; Imaizumi et al., 1998; RamónCueto et al., 1998). However, some interesting data using the mouse have been published on this topic (Au and Roskams, 2003; Shyu et al., 2008; Chehrehasa et al., 2012; Nazareth et al., 2015). Furthermore, mice are the only mammalian species in which the common neural crest cell origin of the olfactory epithelial and Schwann cells has been demonstrated (Forni et al., 2011). Previous data on this point were from chicken embryos (Barraud et al., 2010).

The olfactory subsystems within the nose The topographic epithelial distribution of olfactory receptor cells in mice and some other nonhuman species

is heterogeneous, albeit concentrated into four different regions: (1) the mucosa that lines the walls of the nasal cavity and ethmoturbinates, which gives rise to the main olfactory epithelium; (2) a small part of the inferior nasal septum, which gives rise to the septal organ; (3) a cluster of neurons in the nasal vestibule that gives rise to the ganglion of Gr€uneberg; and (4) the medial wall of the duct inside the VNO, which gives rise to its sensory epithelium (Figs. 4.4 and 4.5; Barrios et al., 2014a). The organization of the chemoreceptors into those four olfactory sensory areas has led to the introduction of new terminology to describe them, namely the olfactory subsystems (Breer et al., 2006; Storan and Key, 2006). The projection of each structure to the olfactory bulb can become a criterion to extend the number of such subsystems to eight. Whereas the septal organ and Gr€uneberg ganglion receptors have a single projection to the main olfactory bulb, the four subregions have been distinguished in the main olfactory epithelium projecting to the four main areas of the olfactory bulb (Ressler et al., 1993; Vassar et al., 1993). In the VNO neural epithelium, a distinction has been made between the apical and basal regions which, in this case, project to the anterior and posterior parts of the accessory olfactory bulb (Jia and Halpern, 1996; Salazar and Sánchez-Quinteiro, 2003). However,

Fig. 4.4. Schematic drawings of the rodent nasal septum (A) and the medial aspect of the nasal cavity (B), showing the locations of the main olfactory epithelium (1), septal organ (2), vomeronasal organ (3) and Gr€ uneberg ganglion (4) with indication of epithelial thickness (yellow, thin; green, medium; blue, thick). From Barrios, W.A., Nun˜ez, G., Sa´nchez-Quinteiro, P., et al. 2014a. Anatomy, histochemistry and immunohistochemistry of the olfactory subsystems in mice. Front Neuroanat 8, 63. https://doi.org/10.3389/ fnana.2014.00063.

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Fig. 4.5. The nasal cavity of the mouse divided into 35 20-section segments and a final 10-section transverse segment (Fig. 4.5A, left). Representation of a segment (Fig. 4.5A, inset at right) and section (Fig. 4.5B) of the MOE, of the SO (Fig. 4.5C), of the VNO (Fig. 4.5D) and of the GgG (Fig. 4.5E). Scale bars: 1 mm. The on-line atlas of the murine nasal cavity is available at http://www.usc. es/anatembriol/ (see Barrios et al., 2014a,b).

the axonal projection from the main olfactory epithelium to the bulb shows a certain degree of overlap (Munger et al., 2009). Three of the morphologic features of the mouse olfactory epithelium deserve mention. First, it is relatively thicker in its dorsal, medial, and posterior sectors, reflecting a greater number of cells in these areas. Second, a small patch of this epithelium is located in an isolated area on the inferior nasal septum, namely, the septal organ. Third, it is similar or slightly greater in thickness than its corresponding lamina propria. This is in contrast to other macrosmatic species in which the lamina propria is thicker in many areas than in the olfactory epithelium (Barrios et al., 2014b). The other two olfactory subsystem structures differ markedly from the main olfactory epithelium. The Gr€ uneberg ganglion lacks an epithelial organization. Rather, it is a small clustered and/or isolated

neuronal population that expresses olfactory marker protein located at the anterior end of the nasal cavity (Roppolo et al., 2006; Storan and Key, 2006). The vomeronasal epithelium belongs to a morphologic and independent olfactory system comprised of the VNO, the anterior olfactory bulb, and the vomeronasal amygdala with the corresponding nerves and tracts as connections between them. The VNO is a bilateral and complex structure located on the floor of the nasal cavity, adjacent to the nasal septum and close to the vomer. It is comprised of a cylindrical tube and duct surrounded by glands, vessels, nerves, and connective tissue. A thin lamina of bone envelops the major elements of the organ. In the mouse, the vomeronasal duct is blind at its caudal end but communicates anteriorly with the nasal cavity via its duct. Its sensory epithelium is limited to the central segment of the organ, exclusively to its medial

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Fig. 4.5—Cont’d

wall (Fig. 4.5D). Similar to the main olfactory epithelium, this epithelium is histologically defined by the presence of basal cells, neurons, and supporting cells. However, the vomeronasal receptors are hidden inside a blind pouch, in opposition to the general rule that the olfactory receptors are directly exposed to the external environment—one of the reasons for suspecting that the olfactory epithelium has developed the ability to continually replace neurons (Graziadei and MontiGraziadei, 1979). Indeed, this difference in the access of the receptors to the external environment is perhaps the most striking morphologic difference between the

two systems, although, unlike the main olfactory receptor cells whose receptor proteins are located on cilia, the receptor proteins of the vomeronasal receptor cells are located on microvillae. For stimuli to reach the receptors of the VNO, they must, at least in the case of the hamster, be pumped in via vasomotor movements (Meredith et al., 1980). While it is presumably also the case in other rodents and other vertebrates (Matsuda et al., 1996; Canto and Suburo, 1998), this has not been empirically demonstrated, and only rarely has the manner in which stimuli enter into the VNO been studied in nonrodent mammals (Salazar et al., 2008).

ANATOMY OF THE OLFACTORY MUCOSA

HOW DOES OLFACTION DIFFER FROM THE OTHER MAJOR SENSES? Living organisms are in permanent relationship with their external environment via their sense organs. At the same time, thanks to the senses, organisms receive information about what is happening within their own body as well. However, the sense organs are usually treated according to the broad Aristotelian division, that is to say, the five classic sense organs: sight, hearing, touch, smell, and taste. In this classification the communication is exclusively confined to the external environment. The mechanisms of reception, transduction, transport, processing, and storing of information are common to the five olfactory subsystems, but their traits differ from one to the other. In the case of reception, the phenomenon most directly related to the topic, the olfactory sensory receptors show three main features: (i) the signal must be of a chemical nature—as for the sense of taste—and can either be volatile or nonvolatile; (ii) they are found in neuronal endings—as in the case of the sense of touch—therefore they must be considered as primary sensory receptors; and (iii) the receptors are placed inside the nasal cavity with a specific distribution that demarcates an olfactory area, the olfactory mucosa, which is well differentiated from the surrounding, mostly respiratory, tissue. A remarkable trait of the sense of smell is the ability of its receptor proteins to detect and process hundreds of thousands of different odors (Axel, 2005; Buck, 2005). After the seminal contribution of Axel and Buck (Buck and Axel, 1991), receptor identification and function have become a burgeoning field of research, especially within the field of molecular biology (Dalton and Lomvardas, 2015; Hanchate et al., 2015). This topic is covered in depth in another chapter of this volume. A striking difference between olfaction and other sensory systems is the regenerative capacity of its neurons (Cajal, 1928; Graziadei and Monti-Graziadei, 1978, 1979, 1983; Doucette, 1990; Schwob, 2002)— neurons whose precursors are located both within the subventricular zone and the neuroepithelium of the nasal cavity (Graziadei, 1973; Moulton, 1974; Calof and Chikaraishi, 1989; Caggiano et al., 1994). In mammals, the neurogenesis persists during the adult lifespan, and the loss of smell sensitivity with age is to a large degree related to the loss of the regenerative capacity of its neurons. However, this age-related phenomenon, well known for decades, is far from being understood (Wilson and Raisman, 1980; Doty et al., 1984; Loo et al., 1996; Weiler and Farbman, 1997; Robinson et al., 2002; Kondo et al., 2010; Suzukawa et al., 2011; Brann and Firestein, 2014). Multiple factors contribute to age-related loss, including altered nasal engorgement,

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increased propensity to nasal disease, cumulative damage of the olfactory epithelium from viral and other environmental insults, decreases in mucosal metabolizing enzymes, ossification of cribriform plate foramina, loss of selectivity of receptor cells to odorants, changes in the olfactory neurotransmitter and neuromodulator systems, and neuronal expression of aberrant proteins associated with neurodegenerative disease (Doty and Kamath, 2014). As for most of the stem cell populations, it is assumed that the function, potency, or even the remaining replicative cycles of the olfactory cells, typically declines with increasing age (Signer and Morrison, 2013). To what degree genetics are involved in creating heterogeneity of this process is unknown.

DIVERSITY OF OLFACTORY SYSTEMS AMONG THE MAMMALIA Following classic anatomical and macroscopic criteria, one of the most remarkable differences of the overall olfactory system of mammals lies in its extreme diversity. In most mammals, sheer examination of the external surface of the brain allows one to draw a clear distinction between the endbrain and other brain divisions, a part of which is the rhinencephalon or the olfactory brain. Likewise, there are evident and significant differences in the form and size of the rhinencephalon between species—differences that are obvious without the need of specific measurement (Fig. 4.6). Based on these observations, Paul Broca, a prominent 19th century anatomist, classified mammals into two groups: osmatic species endowed with a well-developed rhinencephalon and anosmatic species with a feeble or underdeveloped rhinencephalon (Broca, 1878, 1879). Such classification was soon modified by Turner, another illustrious anatomist, who further divided this classification into three sectors: (i) macrosmatic, where the rhinencephalon is largely developed, (ii) microsmatic, where the rhinencephalon is relatively feeble, and (iii) anosmatic, where the rhinencephalon is apparently entirely absent (Turner, 1890). Turner’s classification remains popular today, although it is limited by the difficulty in establishing precise boundaries between the first two of these groups.

Species differences in the complexity of the nasal cavity The topography of the olfactory mucosa is critically dependent upon the anatomy of the nasal cavity. Following a comparative approach, and considering that the diversity of size, form, and other characteristics of the mammalian brain might have an impact on the anatomy and physiology other organic tissues (Stephan et al., 1988), it is of interest to consider the possibility of a relationship between (a) microsmatic (macrosmatic) and/or

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Fig. 4.6. Schematic representation of lateral views of the brains of selected mammals. Dark green, rhinencephalon. Examples of microsmatic mammal, rat (1); microsmatic mammal, man (2); and anosmatic mammal, whale (3). Adapted from Nieuwenhuys, R., ten Donkelaar, H.J., Nicholson, C. 1998. The central nervous system of vertebrates. Springer, Berlin.

gyrencephalic (lysencephalic) mammals and (b) the general organization and constitution of the nasal cavity. The nasal cavity is a structurally complex segment of the respiratory system, and in most mammals it is divided into two chambers by a nasal septum. Its function is obviously related to the sense of smell, but it also includes other functions such as filtering, humidifying, and warming the inhaled air. There are tremendous variations between species not only in the form and size (length, height, and width) of the nasal cavity but also in its content. For example, some animals, such as the dog, have very complex patterns of turbinates (conchae) relative to those of the human that are shown in Fig. 4.1. In all species, the turbinates are thin shelves of bone covered with a mucus membrane and erectile tissue, including the olfactory mucosa. They project into each side of the nasal cavity from its lateral walls. Three general types have been defined, although more complex classifications can be envisioned. The first type, which is seen in humans and most primates, is the simplest and comprises an unbranched lamellar projection, as depicted in Fig. 4.1. The second type, or double scroll, branches once and shows coiled free ends, as seen in many rodents (Fig. 4.5D). The third, or branched type, is defined by the extended branching of turbinates resulting in a labyrinthine mass (Parsons, 1971). The dog is a prototypical example of this type which is common among cats, bears, and many other canids. While differences in turbinal sizes occur among different breeds of dogs, this complex turbinate system is relatively uniform, being comprised of ethmoturbinates, a series of typically six ectoturbinates (lateral), and four endoturbinates (medial) as well as the ventral concha (formerly called the maxilloturbinates) (Fig. 4.7).

Fig. 4.7. Transverse frozen sections of the posterior third of the nasal cavity of the adult dog. D, dorsal nasal concha. Ectoturbinates are identified by Arabic numerals (1–5) and endoturbinates by roman numerals (II–IV). Adapted from Barrios, W.A., Sa´nchez-Quinteiro ,P., Salazar, I. 2014b. Dog and mouse: towards a balanced view of the mammalian olfactory system. Front Neuroanat 8, 106. https://doi.org/10.3389/ fnana.2014.00106.

A rostral projection of endoturbinates defines the dorsal concha (formerly called the nasoturbinates), while a part of endoturbinates (II) constitutes the middle concha.

ANATOMY OF THE OLFACTORY MUCOSA From a topographical point of view, dorsal, middle, ventral conchae, and ethmoturbinates can be described (Barrios et al., 2014b). All of them are highly branched, meaning that they almost completely fill the nasal cavity and consequently the meatuses are quite reduced (Fig. 4.7). The nasal volumes can fluctuate considerably based upon the engorgement of the covering erectile tissue. The relationship between olfactory acuity and the complexity of the nasal turbinates is not entirely clear. In many forms, the surface area of the olfactory mucosa appears to be largely dictated by the surface area of the ethmoidal turbinates. The olfactory epithelium lines sectors of the lateral and medial (nasal septum) walls, part of the roof, and a large extension of the ethmoidal turbinates, which project to the frontal sinus laterally and anteriorly. It is quite clear that the more branched or convoluted the ethmoturbinates are, the larger the surface area of the olfactory neuroepithelium and, hence, presumably greater olfactory acuity. If one takes into account the surface area of the cilia, the number of cilia preceptor cells, and the estimated number of the olfactory receptor cells, the surface area upon which odorants can be absorbed onto the olfactory receptors of the German shepherd dog has been calculated to cover 7.85 m2, or several times the area of the dog’s body surface; i.e., about 12,168 square inches (Moulton, 1977). Using similar calculations for humans, the estimated ciliary surface area is only around 0.0056 m2 or 9 square inches. Thus, the ciliary surface of the dog is over a 1000 times greater than that of a human!

Species differences in brain regions associated with olfaction To our knowledge, there are no empirical calculations across a range of species between the olfactory epithelial area and the areas of the cortex involved in olfactory processing, although one would expect such an association. In most mammals, the neopallium, the youngest part of pallium (the layers of gray and white matter that cover the upper surface of the cerebrum), is proportionally greater than the rhinencephalon, although notable exceptions can be found in insectivores, and the form and the external appearance of both parts is quite different. With regard to the external appearance, while the rhinencephalon always shows a smooth surface in mammals, the neopallium is not uniform and varies according to the species involved. In some cases, it may also have a smooth surface, which means that it maintains the typical appearance of the cerebral hemispheres at the early stage of development throughout life; in other cases, the neopallium is more sophisticated due to the presence of grooves that mark the characteristics folds of the cerebral

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surface boundaries. Mammals belonging to the first group are named lissencephalic (smooth brain), and those belonging to the second group are called gyrencephalic (convoluted-braind or folded-brain). Recently, a compelling and innovative approach to explain the folding process (De Juan Romero et al., 2015) has been added to the several existing theories (Kelava et al., 2013), theories that arise from or are fostered by evolutionary studies of the brain (Krubitzer, 2007; Ziles et al., 2013; Hoffman, 2014). One conclusion that can be drawn from these evolutionary studies is that there is a close relationship between brain size and body size, as well as between brain size and convolutions. It is widely accepted that big brains are folded and small brains are smooth. In the same way, another relationship could be established between the size of the rhinencephalon and the external appearance of the neopallium. Comparative gross anatomy studies have shown that a greater development of the rhinencephalon corresponds to a less convoluted brain or, in other words, that there is an inversely proportional relationship between microsmatic animals and gyrencephaly (Nieuwenhuys et al., 1998).

CONCLUDING REMARKS In man, the olfactory mucosa is confined to the area of the main olfactory epithelium, unlike the situation in a number of other mammals, most notably rodents. However, there is general agreement that the human olfactory mucosa is structurally similar to the general vertebrate pattern (reviewed by Graziadei, 1971) and that its surface area is equal to or even greater than that of many rodents. Moreover, it is now widely accepted that the human olfactory system is not degenerate, but reflects an evolutionary adaptation, along with that of other primates, critical for its own survival. Indeed, olfactory function seems to be a window into the overall health of the brain, reflecting early stages of neurodegeneration so evident in modern humans whose evolution was seemingly not designed to result in up to a hundred years of longevity. That being said, the olfactory mucosa and other elements of the human olfactory system do differ considerably from those of many other mammals, which, in turn, also exhibit diversity (Ache and Young, 2005). For example, human olfactory epithelia and olfactory bulbs are structurally less homogenous and uniform than their rodent counterparts, at least in the adult. Whether this reflects more direct exposure of humans to viruses and xenobiotics than incurred by other species, particularly ones confined to a uniform laboratory environment, or other factors is unclear. Humans and Old World primates lack the nasal diversity of mucosal chemoreceptors observed in rodents, i.e., a functional intranasal ganglion of Gr€uneberg, a septal organ, and

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a VNO (Salazar and Sánchez-Quinteiro, 2009), and their peripheral olfactory apparatus is dwarfed by the sheer size of that of dogs and other canids who depend more upon olfaction for their everyday survival. Nocturnal and crepuscular rodents are also more dependent than humans on their sense of smell for detecting predators, aggressive conspecifics, mates, and detecting foods such as seeds that often are not very odiferous. Thus, to properly understand the relative involvement of the sense of smell in general and the olfactory mucosa in particular, it is necessary to understand their function within species-specific umwelts and the related evolutionary constraints (Dobzhansky, 1973).

ACKNOWLEDGMENTS We thank D. Salazar and N. Vandenberghe for intellectual support and helpful comments and J. Feito for his technical assistance. Our thanks to the editor Prof. R. Doty for his enlightening and constructive comments and suggestions throughout the review process. W.A.B. thanks the Spanish Ministry of Foreign Affairs and Cooperation for an AECID grant. Private financial support is gratefully acknowledged (I.S.).

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