Chem. Senses 29: 301-310,
2004
© Oxford University Press 2004
Morphological Evidence for Two Types of Mammalian Vomeronasal System
1 Department of Developmental Morphology, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashidai, Fuchu, Tokyo 183-8526, Japan, 2 Laboratory of Veterinary Ethology, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan, 3 CREST of Japan Science and Technology Corporation, Kawaguchi 332-0012, Japan, 4 Laboratory of Primates, Central Institute for Experimental Animals, 1430 Nogawa Miyamae-ku, Kawasaki 216-0001, Japan and 5 Department of Basic Technics and Facilities, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashidai, Fuchu, Tokyo 183-8526, Japan
Correspondence to be sent to: Dr Masumi Ichikawa, Department of Basic Technics and Facilities, Tokyo Metropolitan Institute for Neuroscience, 2–6 Musashidai, Fuchu, Tokyo 183-8526, Japan. e-mail: mich{at}tmin.ac.jp
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Abstract |
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The vomeronasal (VN) systems of rodents and opossums are of the segregated type, i.e alpha-subtype G protein Gi2- or Go-expressing VN neurons, which are sensory cells, project discretely to the rostral or caudal region of the accessory olfactory bulb (AOB). Although this zone-specific projection is believed to be a common feature for processing pheromones in mammals, we previously found a uniform-type VN system in goat in which only Gi2-expressing VN axons terminate at the AOB. In most mammals, it remains unclear whether their VN systems are of the segregated or uniform type. Therefore, we investigated morphologically the VN systems of different mammalian species (dog, horse, musk shrew and common marmoset). Consequently, all VN axons of the examined animals were positively stained with immunohistochemistry for Gi2 in the same way as that in the goat. On the other hand, we observed immunoreactivities against Go in the olfactory axons, but not in the VN axons. These results suggest that many mammals have uniform-type VN systems, and at least two types of VN systems exist in terrestrial mammals. This morphological evidence will help us determine the processing function of VN systems.
Key words: accessory olfactory bulb, G proteins, olfaction, pheromone, vomeronasal organ
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Introduction |
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It was first reported that a family of genes encoding candidate pheromone receptors is expressed in rat VN neurons by Dulac and Axel (1995
). These receptors (V1Rs) consist of seven transmembrane receptors (Dulac and Axel, 1995) and are expressed in microvilli and the knoblike structure exposed in the VN lumen (Takigami et al., 1999). Subsequently, another type of pheromone receptor (V2R) was reported (Herrada and Dulac, 1997; Matsunami and Buck, 1997; Ryba and Tirindelli, 1997). V1R-expressing VN neurons are distributed in the apical layer of the VN sensory epithelium (VSE) where Gi2-expressing VN neurons (Gi2-VN neurons) are located and V2Rs are distributed in the basal layer where the Go-VN neurons are located. Therefore, V1Rs were thought to be co-expressed in Gi2-VN neurons and V2Rs in Go-VN neurons (Dulac and Axel, 1995; Herrada and Dulac, 1997; Matsunami and Buck, 1997; Ryba and Tirindelli, 1997). Two populations of VN neurons are known to project their axons to segregated (rostral or caudal) regions of the AOB (Shinohara et al., 1992a; Jia and Halpern, 1996; Halpern et al., 1998). Furthermore, the two neural pathways, namely the rostral and caudal VN pathways, are independent of each other (Berghard et al., 1996; Sugai et al., 1997; Kumar et al., 1999; Halem et al., 2001). This segregated projection of VN neuron has been reported to play a critical role in pheromonal information processing (Brennan et al., 1999; Inamura et al., 1999; Krieger et al., 1999; Leinders-Zufall et al., 2000; Fieni et al., 2003). Although this segregated projection was until now believed to be a common feature in mammals, recently we have shown that the goat VN system has a pattern different from that of the rodent VN system (Takigami et al., 2000). In the goat VN system, only Gi2-VN neurons project throughout the glomeruli of the AOB (Takigami et al., 2000). Immunoreactivity (iR) for Go was not observed on the surface of VSE or in the VN axons. Thus, in that report, we suggested that the segregated projection is not a common feature of the mammalian VN system. The pheromonal influences on endocrine regulation via the VN system are expected to enable synthetic pheromones to control the mammalian endocrine system. However, there are few studies on the VN systems of mammals other than experimental animals such as the mouse, rat, rabbit and opossum. Therefore, it is necessary to determine whether most mammals have the same type of VN system as that of rodents. The aim of the present study was to examine AOBs in widely divergent mammalian species.
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Materials and methods |
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All experiments were conducted in accordance with the Guidelines for the Care and Use of Animals (Tokyo Metropolitan Institute for Neuroscience, 2000). Five mammalian species, namely guinea pig (Brown Takei, 650–700 g, six males; Seasco), beagle, (five males >1 year old; Veterinary Pharmacological Laboratory, Tokyo University of Agriculture and Technology), horse (thoroughbred, two 2-year-old females, one 13-year-old female; Japan Racing Association Equine Research Institute), musk shrew (five 8-week-old females, five 5-week-old males; Clea Japan Inc.) and common marmoset (3-year-old and 12-year-old females, one 16-year-old male, Central Institute for Experimental Animals) were used in the present study. Dogs are classified under the order Carnivora, and are known to have a keen sense of hearing and smell. Musk shrews are primitive animals that are classified under the order Insectivora. Horses are classified under the order Perissodactyla. In this study, we examined a strain of thoroughbred whose family has good running potential and is known to perform flehmen, which was originally proposed to facilitate pheromone testing (Ladewig and Hart, 1980
; Melese-d'Hospital and Hart, 1985). Common marmosets are classified under the order Primates. They are known to have specialized glands and exude characteristic odor. Western blot analysis
In all of the examined animals, the unfixed olfactory bulbs consisting of the AOB were removed immediately after killing and stored at –80°C until analysis. These olfactory bulbs were then cut into pieces in phosphate-buffered saline (PBS) containing several protease inhibitors and homogenized by sonication. All homogenates of the olfactory bulbs were denatured in sodium dodecyl sulfate under reducing conditions at 95°C and subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Proteins were blotted onto nitrocellulose membranes and incubated overnight with BlockAce for blocking at 4°C. The blots were incubated with the primary antibody—Gi2 diluted 1:1000 (Wako), or Go diluted to 0.25 µg/ml (MBL)—for 1 h at room temperature. After washing in TBS-T (20 mM Tris, 137 mM NaCl, 0.1% Tween 20), they were incubated with alkaline-phosphatase-conjugated goat anti-rabbit IgG (1:5000 dilution) for 1 h at room temperature. Bound antibodies were visualized by employing a Western-blue-stabilized substrate (Promega).
Preparation of AOB and VNO sections
The guinea pigs, musk shrews and common marmosets were deeply anesthetized with sodium pentobarbital. They were then killed by transcardial perfusion with 0.9% saline and 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer. The fixed olfactory bulbs, which contain the AOB, were removed and immersed in 4% PFA overnight. After the horses and dogs were killed, their brains and VN organs (VNO) were removed without perfusion and immersed in 4% PFA. Then they were placed in 30% sucrose/PBS solution for cryoprotection. AOBs were cut into 50 µm sagittal sections (horizontal sections in the guinea pigs and dogs) using a freezing microtome. These sections were suspended in 0.1 M PBS.
VNOs were removed from the fixed tissues of all the animals and immersed in decalcifying solution B (Wako). They were then immersed in 30% sucrose/PBS solution for cryoprotection. The sections were cut into 20 µm coronal cryosections using a Cryostat (Jung CM3000; Leica) and mounted on gelatin-coated slides. After drying overnight, they were stained with hematoxylin and eosin or Nissl’s stain and observed using an Axiophot (Zeiss).
Immunohistochemistry for Gi2 or Go
The AOB sections were incubated in a blocking solution containing 10% BlockAce (Dainippon Pharmaceutical), 3% goat serum and 0.05% Triton X-100 in PBS for 1 h. The sections were then incubated for 3 days with antibodies against Gi2 (diluted to 1:800; Wako) or against Go (diluted to 1 µg/ml; MBL) in PBS containing 10% BlockAce and 0.05% Triton X-100 at 4°C. After washing in PBS, the sections were incubated with biotinylated goat anti-rabbit IgG (Vector) in PBS with 10% BlockAce for 2 h at room temperature. After washing with PBS again, the sections were incubated with streptavidin–fluorescein (Amersham) in PBS for 1 h and then rinsed four times with PBS prior to observation under a fluorescence microscope (Axioplan 2; Zeiss).
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Results |
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Cross-reactivity of anti-Gi2 or anti-Go antibody among the examined animals
In the guinea pig, the AOB has a five-layer structure and a segregated-type VN system similar to that in rodents (Figure 1A). The distinct five-layer structure consists of a VN nerve (VNL), glomerulus (GL), mitral/tufted cell (MTL), myelinated nerve (MNL) and granule cell layer (GRL). By immunohistochemistry, Gi2-iR was found in a VN axon projecting to the rostral region of the AOB (Figure 1B) and Go-iR in a VN axon projecting to the caudal region (Figure 1C). Also, by immunohistochemistry of the main olfactory bulb (MOB) of all the examined animals, we detected iR for Go in their olfactory axons (Figure 2). It has previously been reported that Go is expressed in olfactory axons (Shinohara et al., 1992b).
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The Gi2 antiserum (Wako) was prepared from rabbit immunized with a synthetic peptide, LERIAQSDYI (160–169) of human. The Go-antiserum (MBL) was prepared from rabbit immunized with affinity-purified Go from a bovine brain. These antisera are not known to interact with the antigens of other species. Then, to confirm the interaction between the commercial antibodies used and Gi2 or Go in the examined animals, we performed Western blot analysis of anti-Gi2 and anti-Go antibodies using the olfactory bulbs of all the examined animals. In all cases, we detected single bands each corresponding to Gi2 (Mr = 40 000) and Go (Mr = 39 000; Figures 4B, 5B, 6B and 7B). The positions of these single bands were in agreement with the reported molecular masses of Gi2 (Graziano and Gilman, 1987
) and Go (Sternweis and Robishaw, 1984). Because these results show that the antibodies interact with Gi2 or Go in all the examined mammalian species, we used both polyclonal antibodies in the comparative observation by immunohistochemistry for Gi2 and Go.
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Comparative observation in VNO
In all the examined animals, VNOs were located in the basal regions of the nasal septum. In the coronal sections of the VNOs, VSE was located in the medial part (Figure 3A–E). On the opposite side of the VSE, a nonsensory epithelium was located. In the circumference of the VN lumen there were numerous VN glands. Blood vessels were localized at the lateral part of the lumen. The VNO consisted of the same components in all the examined animals, but the lumen form, the number of VN glands and the development of blood vessels varied.
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In the dog, the VN lumen was large and surrounded by many blood vessels (Figure 3B). In the horse, the VN lumen had a complicated form and numerous VN glands existed around it (Figure 3C); blood vessels were small in diameter, but numerous. In the musk shrew, we observed few VN glands and a blood vessel with a large diameter (Figure 3D). In the common marmoset, the VN lumen formed an ellipsoid (Figure 3E) and the VN glands were randomly distributed around the lumen with few blood vessels.
In the guinea pig VSE, many VN neurons were observed (Figure 3F). In the apical region, supporting cells were aligned. Basal cells, which were believed to be stem cells, were not observed in the guinea pig VSE. In the VSEs of the dog, horse, musk shrew and marmoset, VN neurons were located in two or three rows (Figure 3G–J,). Supporting cells were aligned in the same manner as that observed in rodents. Although no basal cells were found in the VSEs of the dog, musk shrew and marmoset, basal cells were observed in the VSE of the horse (Figure 3H).
Comparative immunohistochemistry of AOBs
In the guinea pig, the AOB is located at the dorsocaudal position of the olfactory bulb. The five-layer structure (VNL, GL, MTL, MNL and GRL) was distinctly observed (Figure 1A). Gi2-iR was observed in the rostral VNL and GL of the AOB (Figure 1B) and Go-iR was observed in the caudal VNL and GL (Figure 1C). The guinea pig was reconfirmed to have the segregated-type VN system (Sugai et al., 1997).
The dog AOB was located on the medial surface of the olfactory peduncle at the caudal margin of the MOB (Figure 4C). In these sections, three-layer structures—VNL, GL and MTL (or GRL)—were observed (Figure 4A). It was difficult to distinguish MT cells from GR cells. By immunohistochemistry, Gi2-iR was observed in the VNL and GL throughout the AOB (Figure 4D). Go-iR was not observed in the VNL in the AOB (Figure 4E). The dog was confirmed to have the uniform-type VN system.
The horse AOB was located in the dorsocaudal position of the olfactory bulb (Figure 5C). In the sagittal AOB sections of the horse, three-layer structures—VNL, GL, MTL (or GRL)—were observed (Figure 5A). VNL was thick in the horse AOB. Gi2-iR was observed in VNL and GL (Figure 5D), but not Go-iR (Figure 5E). The horse was found to have the uniform-type VN system.
The AOB of the musk shrew was located in the dorsocaudal region of the olfactory bulb (Figure 6C). In the sagittal AOB sections, four-layer structures—VNL, GL, MTL and GRL—were recognized (Figure 6A). Gi2-iR VN nerves were observed throughout VNL and GL (Figure 6D). In contrast, Go-iR VN nerves were not observed in VNL (Figure 6E). The musk shrew was confirmed to have the uniform-type VN system.
The common marmoset AOB was located on the olfactory peduncle (Figure 7C). In the sagittal AOB sections of the common marmoset, three-layer structures—VNL, GL and MTL (or GRL)—were identified (Figure 7A). The boundary between MTL and GRL could not be distinguished. All VN axons were stained with the anti-Gi2 antibody, but not with the anti-Go antibody (Figure 7D,E). Although iR for Go was found in the caudal part of VNL and GL (Figure 7E), the region was not stained with the anti-neural cell adhesion molecule antibody used as a marker of olfactory and vomeronasal nerves (data not shown). The common marmoset was confirmed to have the uniform-type VN system.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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In the previous study using rats and goats (Takigami et al., 2000
), the immunohistochemical results for Gi2 and Go in AOB were consistent with those in VSE. Although immunoreactivity for Gi2 or Go was detected in both VSE and AOB of rats, only nerve terminals with immunoreactivity for Gi2, but not Go, were observed in the goat VSE and AOB (Takigami et al., 2000). Therefore, immunohistochemistry results for Gi2 or Go in AOB were thought to reflect the existence of Gi2- or Go-VN neurons in VSE. In the present study, only Gi2-VN axons terminated at the AOBs of the dog, horse, musk shrew and marmoset (Figures 4–7). In MOBs of all examined animals, nerve terminals with immunoreactivity for Go were clearly observed (Figure 2). Because olfactory and VN system are anatomically separated (Winans and Scalia, 1970; Scalia and Winans, 1975), these results suggest that only Gi2-expressing VN neuron may exist in the VSE of uniform-type VN systems. Interestingly, although VNO had common structural components such as a sensory epithelium, a nonsensory epithelium, a VN lumen, VN glands and blood vessels, in all the examined animals (Figure 3A–E), thin sensory epitheliums were observed in the examined animals with the uniform-type VN system (Figure 3G–J). In contrast, in the segregated-type VN system, thick VSE was observed (Figure 3F) and many VN neurons were stratified (Figure 3F). The difference in the thickness of sensory epithelium may reflect the number of VN neurons.
In the olfactory sensory epithelium, olfactory neurons are discretely distributed among four zones and they project to four respective zones of MOB (zone-to-zone projections; Mori et al., 1999). The zone-to-zone projections depend on the expression of the olfactory receptor gene and play a critical role in axon guidance (Buck and Axel, 1991; Wang et al., 1998; Mombaerts et al., 1996; Mombaerts, 1999; Zou et al., 2001) and odor discrimination (Uchida et al., 2000). Thereby, mixed odors were expected to be divided into the respective components of the MOB. This anatomical filter is believed to be a critical mechanism of olfactory discrimination. In the VN systems of rodents and opossums, there was a similar segregated projection by the two populations of VN neurons to the olfactory system exists. In previous studies, different responses to various pheromones in the rostral or caudal region of AOB were reported (Brennan et al., 1999; Kumar et al., 1999; Halem et al., 2001). For example, when male mice are exposed to the urine of a diestrus female, increased neural activity is observed in the rostral side of AOB. The rostral side of the female AOB is associated with processing of the male pheromone signal and the strain recognition component (Brennan et al., 1999). Furthermore, the pheromones associated with aggressive behavior against strange males are processed in the caudal region of the male AOB (Brennan et al., 1999) and Gi2- and Go-expressing VN neurons respond to different urinary pheromones (Inamura et al., 1999). A recent study (Fieni et al., 2003) also reported that these VN neurons exhibit distinct electrophysiological properties in the amplitudes of their voltage-gated Na+ currents. In addition, it was also reported that the two types of VN neurons respond to different chemical stimuli (Leinders-Zufall et al., 2000) and possibly to different types of chemicals (Krieger et al., 1999). These data show that both VN pathways appear to be functional in rodents. In the present study, immunohistochemistry for Gi2 or Go revealed that no Go-VN axons project into AOBs of the dog, horse, musk shrew or marmoset (Figures 4–7). Our results suggest the possibility that the Go-VN pathway does not exist in uniform-type VN systems. Furthermore, genes encoding pheromone receptors have been reported in the goat (Wakabayashi et al., 2002). In goats, two types of V1R homologues and eight kinds of V2R homologues have been identified. The V2R homologues have many pseudogenes. On the other hand, one V1R homologue (gV1R1) has 40–50% homology to V1Rs and has an open reading frame encoding 309 amino acids. This evidence increases the possibility that only one pheromone receptor population is expressed in the uniform-type VN system. Consequently, the processing potentials in the uniform-type VN system are considered to be less than those in the segregated type.
The segregated-type VN system has been reported mainly in rodents and opossum. Rodents are the most common experimental animals and they have the highest number of strains in the Eutheria, thus it is generally believed that the results obtained using rodents are applicable to all mammals. In the present and previous studies (Takigami et al., 2000), mammals other than rodents were investigated. All the examined animals belonged to the Eutheria and their VN systems were found to be of the uniform type. Our results suggest that the VN systems of many terrestrial mammals are of the uniform type rather than the segregated type. In addition, because Gi2-VN neurons definitely exist in both types of VN systems, they have been conserved in most mammalian species during evolution. Gi2-VN neurons are expected to receive pheromones with a common effect in the mammalian VN system. To elucidate the function of the VN system in mammals, further comparative studies are necessary. In our next study, we will examine ontogenetically animals with the uniform-type VN system in an attempt to understand differences and similarities in features between the segregated- and uniform-type VN systems.
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This work was supported by CREST of Japan Science and Technology Corporation. We are grateful to Dr Toyohiko Yoshihara and his laboratory staff for collecting the horse tissue samples and to Drs Naotoshi Kanda and Hideshi Shibata for providing the experimental materials necessary for collecting the musk shrew and dog tissue samples. We would like to thank the staff of the Veterinary Pharmacological Laboratory at Tokyo University of Agriculture and Technology for supplying the dog tissue samples, and Drs Takefumi Kikusui and Kazuyuki Taniguchi for collecting the common marmoset tissue samples.
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References |
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Accepted February 13, 2004
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