Cladistic analysis of olfactory and vomeronasal systems.

Most tetrapods possess two nasal organs for detecting chemicals in their environment, which are the sensory detectors of the olfactory and vomeronasal systems. The seventies' view that the olfactory system was only devoted to sense volatiles, whereas the vomeronasal system was exclusively specialized for pheromone detection was challenged by accumulating data showing deep anatomical and functional interrelationships between both systems. In addition, the assumption that the vomeronasal system appeared as an adaptation to terrestrial life is being questioned as well. The aim of the present work is to use a comparative strategy to gain insight in our understanding of the evolution of chemical "cortex." We have analyzed the organization of the olfactory and vomeronasal cortices of reptiles, marsupials, and placental mammals and we have compared our findings with data from other taxa in order to better understand the evolutionary history of the nasal sensory systems in vertebrates. The olfactory and vomeronsasal cortices have been re-investigated in garter snakes (Thamnophis sirtalis), short-tailed opossums (Monodelphis domestica), and rats (Rattus norvegicus) by tracing the efferents of the main and accessory olfactory bulbs using injections of neuroanatomical anterograde tracers (dextran-amines). In snakes, the medial olfactory tract is quite evident, whereas the main vomeronasal-recipient structure, the nucleus sphaericus is a folded cortical-like structure, located at the caudal edge of the amygdala. In marsupials, which are acallosal mammals, the rhinal fissure is relatively dorsal and the olfactory and vomeronasal cortices relatively expanded. Placental mammals, like marsupials, show partially overlapping olfactory and vomeronasal projections in the rostral basal telencephalon. These data raise the interesting question of how the telencephalon has been re-organized in different groups according to the biological relevance of chemical senses.

Introduction

Odor perception is initiated by interactions between odorants (sensu lato) and a diverse repertoire of receptors in sensory neurons (Ache and Young, 2005; Bargmann, 2006; Kaupp, 2010). Classical neuroanatomical studies have investigated the olfactory system in a number of species of different taxa including fishes (s.l.; Hara, 1975; Laberge and Hara, 2001; Hamdani el and Doving, 2007), amphibians (Duchamp-Viret and Duchamp, 1997), reptiles (Lohman and Smeets, 1993), birds (Rieke and Wenzel, 1978), and mammals (Shepherd, 1972). These reports have allowed the characterization of the olfactory “cortex” in most vertebrates. Also, neuroanatomical investigations lead to the identification of the vomeronasal “cortex” in mammals (Winans and Scalia, 1970; Raisman, 1972; Scalia and Winans, 1975). The main conclusions of all these reports were that chemosensory cortex lacks odotopy and that olfactory and vomeronasal projections reached adjacent, non-overlapping zones in the telencephalon.

During the last two decades, the cloning of different olfactory (Buck and Axel, 1991; Liberles and Buck, 2006) and vomeronasal (Dulac and Axel, 1995; Bargmann, 1997, 1999; Herrada and Dulac, 1997; Matsunami and Buck, 1997; Ryba and Tirindelli, 1997) receptors has allowed considerable progress in tracing olfactory and vomeronasal perception from receptors to the activity of sensory neurons to higher processing centers and, ultimately, to behavior (Buck, 1995, 1996, 2000, 2004; Mombaerts, 1996, 1999a,b, 2004; Mombaerts et al., 1996; Dulac, 1997, 2000; Dulac and Torello, 2003; Brennan and Keverne, 2004; Brennan and Zufall, 2006; Dulac and Wagner, 2006; Cleland, 2010). Interactions and synergic activation of both systems, however, occurs in early stages of neural processing (Xu et al., 2005; Slotnick et al., 2010).

The genetic and molecular approaches to the study of the organization of chemosensory systems have allowed a step forward to understand the organization of chemosensory systems. Genetic tracing of given receptors constitutes a valuable tool for this aim. The present review tries to provide a global panorama on the organization and evolution of olfactory and vomeronasal cortices with emphasis on recent advances including tract-tracing as well as genetic and molecular approaches. Own data of reptiles, marsupials and placental mammals are re-analyzed in the context of current knowledge of main taxa of vertebrates.

Taxonomic Overview of Vertebrates

Although a complete description of the taxonomic classification of vertebrates is out of the scope of the present manuscript, a brief outline of the main vertebrate taxa will be useful not only for the comparative strategy herein used but also to help non-familiarized readers to understand such organization. For the sake of clarity, the main taxonomic categories have been maintained but simplified. This hierarchical organization is explained below and has been adapted from Hickman and collaborators (Hickman et al., 2007).

The Phylum Chordata is defined by the presence of notochord. In its basic plan these animals are bilateral, coelomed, metameric, and cephalized. This Phylum includes the Groups Prochordata (with the Subphyla Urochordata –tunicates – and Cephalochordata –amphioxus) and Craniata (with the Subphylum Vertebrata). Within Subphylum Vertebrata are included the Superclasses Agnata (jawless, with the Classes Mixinoidea –mixins – and Cephalaspidomorpha –lampreys) and Gnathostomata (jawed, with the Classes Chondrichthyes – cartilaginous fishes – Osteichthyes –bony fishes – Amphibia, Reptilia, Aves, and Mammalia. The Class Mammalia includes the Subclasses Prototheria –monotremas – and Theria. The Subclasse Theria comprises the Infraclasses Metatheria – marsupials – and Eutheria with several Orders in which highlights the Order Primate. Finally, within the Order Primate consist of the Superfamilies Platyrrhini –new world monkeys – Catarrhini – old world monkeys – and Hominidae in which we are included.

Agnata, jawless

Among superclass Agnata most data in the literature regarding the organization of the olfactory system have been obtained in lampreys. Reports on the lamprey olfactory system has been devoted to the organization of the olfactory mucosa (Thornhill, 1967; Suzuki, 1984; VanDenbossche et al., 1995; Laframboise et al., 2007), olfactory bulbs (Iwahori et al., 1987a; Melendez-Ferro et al., 2001; Pombal et al., 2002), and telencephalon (Chiba, 1999; Pombal et al., 2002; Perez-Costas et al., 2004). The projections from the olfactory bulb identified medial and lateral olfactory tracts (LOT) reaching dorsal, lateral, and probably medial pallium as well as the posterior diencephalon as well as reciprocal, centrifugal projections to the bulb (Northcutt and Puzdrowski, 1988; Polenova and Vesselkin, 1993). On the other hand, the origin of the vomeronasal system is uncertain. It was classically considered that the vomeronasal system was developed by tetrapods and it was hypothesized to be an adaptation to terrestrial life (Eisthen, 1997). It has been demonstrated that lampreys detect pheromones (Fine and Sorensen, 2008) and display some genetic components of the vomeronasal system (Grus and Zhang, 2009). Also, an accessory olfactory organ has been shown to send separated projections from those of the main olfactory epithelium to the medial portion of the bulb (Ren et al., 2009). No further differential projections from this medial portion of the bulb to the pallium have been investigated. Therefore, data in the literature suggests that primordial elements of the vomeronasal system could be already present in jawless vertebrates.

Chondrichthyes, cartilaginous fishes

The olfactory system, including the olfactory epithelium (Ferrando et al., 2006, 2007, 2009; Zaccone et al., 2010) and the secondary connections from the olfactory bulbs have been investigated in sharks and rays (Smeets, 1983). Secondary olfactory centers have been neurochemically characterized as well (Yuen et al., 2005). Like lampreys, cartilaginous fishes display some genetic components of the vomeronasal system (Grus and Zhang, 2009). The distribution of G proteins suggests incipient segregated primary olfactory projections (Ferrando et al., 2009).

Osteichthyes, bony fishes

The teleosts olfactory system has been investigated including the olfactory organs (Wilson and Westerman, 1967; Westerman and Wilson, 1968; Goel, 1978; Jain and Sahai, 1991), olfactory bulbs (Kosaka and Hama, 1982; Alonso et al., 1989; Satou, 1990), and olfactory-recipient areas (Singru et al., 2003; Gaikwad et al., 2004). The olfactory projections from the olfactory bulbs have been investigated in teleosts, with studies including early anatomical reports (Scalia and Ebbesson, 1971; Finger, 1975; Murakami et al., 1983). These projections have been re-investigated using modern genetic approaches (Miyasaka et al., 2009). In teleost fishes, whose telencephalon is everted (Butler, 2000; Mueller and Wullimann, 2009), the secondary olfactory projections terminate in non-cortical areas of the telencephalon (Folgueira et al., 2004). Interestingly, three pathways from different receptors to different areas in the olfactory bulb and to different areas in the telencephalon have been characterized in teleost fishes devoted to detect social cues, sex pheromones, and food odors (Hamdani el and Doving, 2007). In fact, not only vomeronasal receptors have been described in several species (Pfister et al., 2007), but, in lungfishes, it has been recently reported a complete vomeronasal system (Gonzalez et al., 2010).

Amphibia

Studies on the amphibian olfactory system include reports focused in the olfactory epithelium (Getchell et al., 1989; Daston et al., 1990; Crowe and Pixley, 1992; Krishna et al., 1992), olfactory bulb (Scalia et al., 1991b), and olfactory-recipient areas (Gonzalez and Smeets, 1991; Petko and Santa, 1992; Marin et al., 1997; Brox et al., 2004). The differential projections from the olfactory and vomeronasal epithelia to the main and accessory olfactory bulbs (AOB) were reported in several species (Taniguchi et al., 2008). The amphibian AOB has been investigated as well (Saito et al., 2006). Secondary olfactory projections were early addressed in amphibians (Northcutt and Royce, 1975). The differential projections from the main and AOBs were reported later on reaching cortical and non-cortical areas of the telencephalon, respectively (Scalia, 1972; Scalia et al., 1991a). The vomeronasal system was fully described more recently including not only hodology, but neurochemical and gene expression characterization (Moreno et al., 2005; Moreno and Gonzalez, 2007). Interestingly, heterogeneous expression of G proteins have been reported in the amphibian olfactory and vomeronasal epithelia (Jungblut et al., 2009). Vomeronasal receptor genes have been reported to be also expressed in the main olfactory epithelium indicating that some pheromone-like triggered behaviors (Woodley, 2010) are mediated via the olfactory system in some amphibians species (Date-Ito et al., 2008).

Reptilia

Research on the reptilian olfactory system includes reports focused to olfactory receptors (Steiger et al., 2009), olfactory and vomeronasal epithelia (Wang and Halpern, 1982a,b, 1988; Iwahori et al., 1987b; Kondoh et al., 2010), olfactory bulbs (Iwahori et al., 1989a,b; Kosaka et al., 1991), and olfactory-recipient telencephalic areas (Smeets et al., 1986, 1987; Smeets, 1988; Smeets and Steinbusch, 1990). In reptiles, the main and AOBs projections were investigated in a number of species, mainly lizards and snakes (Gamble, 1952, 1956; Halpern, 1976; Lohman et al., 1988; Martinez-Garcia et al., 1991; Lanuza and Halpern, 1998). These projections have also reviewed (Lohman and Smeets, 1993). Both projections are reciprocated by backwards centrifugal projections (Martinez-Garcia et al., 1991; Lanuza and Halpern, 1998). The projections from the main olfactory bulb (MOB) are characterized by lateral, intermediate, and medial olfactory tracts (MOT; Figure 1). The lateral projection is the most robust projection and ends in the superficial layer of the reptilian lateral cortex (Figure 1). The projection from the AOB courses through the accessory olfactory tract (AOT) to reach the vomeronasal amygdala, within the reptilian amygdaloid complex (Martinez-Garcia et al., 1993; Martinez-Marcos et al., 1999). The vomeronasal amygdala is mainly composed by the nucleus sphericus, a folded, cortical-like structure. The vomeronasal projection ends in the inner layer of this nucleus (Figure 2). The projections from the main and AOBs in reptiles are summarized in Figure 3.

Figure 1

Bright field microscopic images from rostral to caudal of coronal sections of one brain hemisphere showing biotinylated dextran-amine labeling Nissl-counterstained in the snake (. (A) injection site in the MOB. (B–H) labeling in olfactory-recipient areas of the telencephalon. Scale bar A–D = 267; E–H = 400 μm.

Figure 2

Bright field microscopic images from rostral to caudal of coronal sections of one brain hemisphere showing biotinylated dextran-amine labeling Nissl-counterstained in the snake (. (A) injection site. (B–F) labeling in vomeronasal-recipient areas. Scale bar A = 160; B–C = 267; D–E = 400; F = 533 μm.

Figure 3

Schematic representation of main (red) and accessory (green) projections from rostral to caudal (A-D) areas in the snake (.

Aves

The avian olfactory system has been investigated from olfactory receptors (Nef et al., 1996) to olfactory epithelium (Slaby, 1987), olfactory bulb (Cobb, 1960; Ioale and Papi, 1989), and olfactory-recipient areas (Dietl and Palacios, 1988). The olfactory bulb projections in Aves were studied with Fink–Heimer technique demonstrating a bilateral non-symmetrical terminal field (Rieke and Wenzel, 1978). Using autoradiographic techniques, the avian olfactory bulb projections were compared to those of turtles. The main conclusions were that both projections were similar regarding “olfactory cortex,” but they do differ significantly regarding projections to the amgydaloid region, being this latter reduced to the nucleus taeniae in the case of birds (Reiner and Karten, 1985). These projections have been characterized using neural tracers as well (Ebinger et al., 1992). Avian olfaction have been largely related to navigation (Gagliardo et al., 2009). In fact, the olfactory forebrain is quite specialized in migratory birds like anseriforms (Ebinger et al., 1992). New techniques based on the combinatorial expression patterns of neural markers and developmental regulatory genes have allowed to identify zones in the olfatorecipient area of the ventral pallium comparable to piriform, entorhinal, amygdalopiriform, and amygdaloid cortices of mammals (Martinez-Garcia et al., 2002; Abellan et al., 2009). Finally, analysis of olfactory receptor gene repertoires show that in all species studied, amplified olfactory receptor sequences were predicted to be from potentially functional genes thus suggesting that olfaction in birds may be a more important sense than generally believed (Steiger et al., 2008; Balthazart and Taziaux, 2009). The issue of avian pheromones is still far from been resolved (Caro and Balthazart, 2010).

Metatheria, marsupials

Marsupials lack corpus callosum and consequently the telencephalon shows a different organization as compared to placental mammals. The rhinal fissure is located more dorsal in marsupials than in placental mammals and their anterior commissure is hypertrophied to communicate both hemispheres (Gloor, 1997). The olfactory epithelium (Kratzing, 1982) and olfactory bulbs are organized in a particular way in marsupials (Switzer and Johnson, 1977; Jia and Halpern, 2004). Olfactory projections were already investigated in the middle of twentieth century (Adey, 1953). The projections from the main and AOBs were described in opossums using the Fink–Heimer method (Scalia and Winans, 1975; Meyer, 1981; Shammah-Lagnado and Negrao, 1981). The olfactory-recipient and vomeronasal-recipient cortices occupied adjacent non-overlapping areas. Using neural tracers, the projections from the opossum main (Martinez-Marcos and Halpern, 2006) and accessory (Martinez-Marcos and Halpern, 1999b) olfactory bulbs were later re-investigated using modern neural tracers. The projections from the MOB course mainly via the LOT to reach the anterior olfactory nucleus, olfactory tubercle, nucleus of the LOT, anterior and posterolateral cortical amygdaloid nuclei and the piriform, and lateral entorhinal cortices (Figure 4). The projections from the AOB course through the AOT to reach the medial amgydaloid complex and the posteromedial cortical amygdaloid nucleus (Figure 5). Both projections converge at rostral levels such as the medial amgydala, whereas are non-overlapping at caudal levels (Figure 6). The differential expression of G proteins in the vomeronasal system (Halpern et al., 1995) and the cloning of the two families of vomeronasal receptors (Dulac and Axel, 1995; Herrada and Dulac, 1997; Matsunami and Buck, 1997; Ryba and Tirindelli, 1997) allowed to identify the differential projections to the anterior and posterior portions of the AOB (Jia and Halpern, 1996; Belluscio et al., 1999; Rodriguez et al., 1999). Both portions send convergent but also differential projections to the vomeronasal amygdala in opossums (Martinez-Marcos and Halpern, 1999b), which are also reciprocated by differential centrifugal projections to the anterior and posterior divisions of the AOB (Martinez-Marcos and Halpern, 1999a).

Figure 4

Bright field microscopic images from rostral to caudal of coronal sections of one brain hemisphere showing biotinylated dextran-amine labeling Nissl-counterstained in the opossum (. (A) injection site. (B–F) labeling in olfactory-recipient areas. Scale bar A, D, and F = 160; B, C, and E = 800 μm.

Figure 5

Bright field microscope images from rostral to caudal of coronal (A is sagittal) sections of one brain hemisphere showing biotinylated dextran-amine labeling Nissl-counterstained in the opossum (. (A) injection site. (B) labeling in vomeronasal-recipient areas. Scale bar A = 400; B–F = 800 μm.

Figure 6

Schematic representation of main (red) and accessory (green) projections from rostral to caudal (A-D) areas in the opossum (.

Eutheria, placental mammals

In placental mammals, the projections from the olfactory epithelium to the MOB (Clark, 1951) and from the bulb to the cortex (Clark and Meyer, 1947) were already addressed in the middle of the twentieth century. The characterization of projections from the main and AOBs were reported later on (Heimer, 1968; Winans and Scalia, 1970; Raisman, 1972; Price, 1973; Scalia and Winans, 1975; Devor, 1976; Skeen and Hall, 1977; Kosel et al., 1981; Schoenfeld and Macrides, 1984; Shipley and Adamek, 1984; De Carlos et al., 1989) including the centrifugal projections (Davis et al., 1978; de Olmos et al., 1978; Davis and Macrides, 1981; Shipley and Adamek, 1984; Coolen and Wood, 1998). As in marsupials, the projections from the MOB form a minor projection through the MOT and a major projection through the LOT to reach the anterior olfactory nucleus, taenia tecta, olfactory tubercle, the nucleus of the LOT, rostral levels of the medial amgydaloid complex and the anterior and posterolateral cortical amygdaloid nuclei as well as the piriform and lateral entorhinal cortices (Figure 7). The projections from the AOB (Larriva-Sahd, 2008) travel through the AOT to reach the cortex–amygdala-transition zone and the medial amgydaloid complex and posteromedial amygdaloid nucleus. Also, fibers course through the stria terminalis to reach the bed nucleus of the stria terminalis (Figure 8). Olfactory and vomeronasal projections converge at rostral levels of the basal telencephalon (Pro-Sistiaga et al., 2007) but not at caudal ones (Figure 9; Martinez-Marcos, 2009; Gutierrez-Castellanos et al., 2010). The differential expression of G proteins in the vomeronasal system (Halpern et al., 1995) and the cloning of two families of vomeronasal receptors (Dulac and Axel, 1995; Herrada and Dulac, 1997; Matsunami and Buck, 1997; Ryba and Tirindelli, 1997) led to the description of differential projections from the vomeronasal epithelium to the anterior and posterior divisions of the AOB (Jia and Halpern, 1996; Belluscio et al., 1999; Rodriguez et al., 1999). Both divisions of the AOB show convergent (Von Campenhause and Mori, 2000), but also partially divergent projections in the vomeronasal amygdala (Mohedano-Moriano et al., 2007) that are preserved in the hypothalamus (Mohedano-Moriano et al., 2008).

Figure 7

Bright field microscopic images from rostral to caudal of coronal (A is sagittal) sections of one brain hemisphere showing biotinylated dextran-amine labeling Nissl-counterstained in the rat (. (A) injection site. (B–H) labeling in olfactory-recipient areas. Scale bar A–B and D–H = 800; C = 400 μm.

Figure 8

Bright field microscopic images from rostral to caudal of coronal (A is sagittal) sections of one brain hemisphere showing biotinylated dextran-amine labeling Nissl-counterstained in the rat (. (A) injection site. (B–H) labeling in vomeronasal-recipient areas. Scale bar A = 400; B–C and E–H = 800; D = 160 μm.

Figure 9

Schematic representation of main (red) and accessory (green) projections from rostral to caudal (A-D) areas in the rat (.

Platyrrhini, new world monkeys

The MOB connections were recently characterized in the marmoset (Liebetanz et al., 2002). Injections of anterograde tracers revealed the medial and LOTs as well as the olfactory-recipient structures including the anterior olfactory nucleus, piriform cortex, tenia tecta, periamygdaloid cortex, and rostral entorhinal cortex. Centrifugal projections were also revealed originating in olfactory-recipient areas by using retrograde tracers. The vomeronasal system exists, but it is poorly developed in New World monkeys. In marmoset, that possess an intact vomeronasal organ, sequences of vomeronasal receptors (V1R) appear to correspond to pseudogenes (Giorgi and Rouquier, 2002). Neuron-specific markers have been reported to be expressed in the vomeronasal epithelium of different species of primates including marmoset (Dennis et al., 2004). Neurons in the AOB express markers typical of mitral/tufted cells (Nakajima et al., 2003). Marmoset, like other mammals and in contrast to rodents and opossums, display a homogeneous vomeronasal system where only V1R and Gi proteins are expressed (Takigami et al., 2004). To our knowledge, the secondary vomeronasal projections from the AOB have not been traced in marmosets.

Catarrhini, old world monkeys

Olfactory receptors are quite reduced in humans and great apes including a 40% of pseudogenes (Sharon et al., 1999). The projections from the MOB were traced in Old World monkeys using lesion–degeneration, autoradiographic (Turner et al., 1978), and neural tracing (Carmichael et al., 1994; Mohedano-Moriano et al., 2005) techniques. Among structures identified as olfactory-recipient were included the anterior olfactory nucleus, piriform cortex, ventral tenia tecta, olfactory tubercle, anterior cortical nucleus of the amygdala, periamygdaloid cortex, and olfactory division of the entorhinal cortex. Interestingly, olfactory fibers reach layer I of the medial amygdaloid complex. This structure has been traditionally described as a vomeronasal-recipient area, but recent data (Pro-Sistiaga et al., 2007; Kang et al., 2009) indicate that it is a mixed chemosensory structure receiving olfactory and vomeronasal inputs. Therefore, it could be hypothesized that this structure has changed from vomeronasal- to olfactory-recipient. Further investigations are needed to confirm this point (Figure 10).

Figure 10

Cladogram illustrating the main taxa analyzed in the present report and the main changes in the olfactory and vomeronasal systems occurred during evolution.

Hominidae, human beings

Olfactory receptor genes have been identified in humans (Ben-Arie et al., 1994; Glusman et al., 1996, 2000; Buettner et al., 1998; Rouquier et al., 1998), although up to 40% appear to correspond to pseudogenes (Sharon et al., 1999). In fact, evolutionary pressures have led to the loss of part of the olfactory receptor repertoire (Young et al., 2002; Gilad et al., 2003; Niimura and Nei, 2003). The characterizations of olfactory projections in human have been only addressed by indirect methods such as comparative approaches with other primates (Insausti, 1993; Insausti et al., 2002). Putative olfactory areas include the same structures identified in other primates such as anterior olfactory nucleus, piriform cortex, ventral tenia tecta, olfactory tubercle, anterior cortical nucleus of the amygdala, periamygdaloid cortex, and olfactory division of the entorhinal cortex. In humans, the vomeronasal system is vestigial. The vomeronasal organ is apparently only present during embryonic development and its presence is quite controversial in adult humans (Stensaas et al., 1991; Smith et al., 1998, 2001; Smith and Bhatnagar, 2000; Trotier et al., 2000; Abolmaali et al., 2001; Bhatnagar and Smith, 2001; Knecht et al., 2001, 2003; Meredith, 2001; Witt et al., 2002; Besli et al., 2004; Witt and Hummel, 2006; Witt and Wozniak, 2006). Putative pheromone receptors have been identified in human olfactory mucosa (Rodriguez et al., 2000; Rodriguez and Mombaerts, 2002). Further, to our knowledge, the human AOB has not been described. Therefore, the human “pheromonal-like” behaviors are probably mediated through the olfactory system (Mast and Samuelsen, 2009; Savic et al., 2009).

Conclusion

Olfaction plays a main role in most vertebrate taxa. Cladistic analysis of different vertebrates indicates that during evolution an early anatomical and functional subdivision of nasal chemical systems occurred – mainly olfactory and vomeronasal systems – including different receptors, and primary and secondary projection areas. The different chemical systems have suffered differential involution in given taxa due to evolutionary pressures.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.