Distinct axonal projections from two types of olfactory receptor neurons in the middle chamber epithelium of Xenopus laevis Shoko Nakamuta, Nobuaki Nakamuta & Kazuyuki Taniguchi
Cell and Tissue Research ISSN 0302-766X Volume 346 Number 1 Cell Tissue Res (2011) 346:27-33 DOI 10.1007/s00441-011-1238-y
1 23
Your article is protected by copyright and all rights are held exclusively by SpringerVerlag. This e-offprint is for personal use only and shall not be self-archived in electronic repositories. If you wish to self-archive your work, please use the accepted author’s version for posting to your own website or your institution’s repository. You may further deposit the accepted author’s version on a funder’s repository at a funder’s request, provided it is not made publicly available until 12 months after publication.
1 23
Author's personal copy Cell Tissue Res (2011) 346:27–33 DOI 10.1007/s00441-011-1238-y
SHORT COMMUNICATION
Distinct axonal projections from two types of olfactory receptor neurons in the middle chamber epithelium of Xenopus laevis Shoko Nakamuta & Nobuaki Nakamuta & Kazuyuki Taniguchi
Received: 20 May 2011 / Accepted: 1 September 2011 / Published online: 22 September 2011 # Springer-Verlag 2011
Abstract Most vertebrates have two olfactory organs, the olfactory epithelium (OE) and the vomeronasal organ. African clawed frog, Xenopus laevis, which spends their entire life in water, have three types of olfactory sensory epithelia: the OE, the middle chamber epithelium (MCE) and the vomeronasal epithelium (VNE). The axons from these epithelia project to the dorsal part of the main olfactory bulb (d-MOB), the ventral part of the MOB (v-MOB) and the accessory olfactory bulb, respectively. In the MCE, which is thought to function in water, two types of receptor neurons (RNs) are intermingled and express one of two types of G-proteins, Golf and Go, respectively. However, axonal projections from these RNs to the v-MOB are not fully understood. In this study, we examined the expression of G-proteins by immunohistochemistry to reveal the projection pattern of olfactory RNs of Xenopus laevis, especially those in the MCE. The somata of Golf- and Go-positive RNs were separately situated in the upper and lower layers of the MCE. The former were equipped with cilia and the latter with microvilli on their apical surface. These RNs are S. Nakamuta : N. Nakamuta : K. Taniguchi (*) Laboratory of Veterinary Anatomy, Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka, Iwate 020-8550, Japan e-mail:
[email protected] S. Nakamuta e-mail:
[email protected] N. Nakamuta e-mail:
[email protected] S. Nakamuta : N. Nakamuta : K. Taniguchi United Graduate School of Veterinary Sciences, Gifu University, 1-1 Yanagido, Gifu, Gifu 501-1193, Japan
suggested to project to the rostromedial and the caudolateral regions of the v-MOB, respectively. Such segregation patterns observed in the MCE and v-MOB are also present in the OE and olfactory bulbs of most bony fish. Thus, Xenopus laevis is a very interesting model to understand the evolution of vertebrate olfactory systems because they have a primitive, fish-type olfactory system in addition to the mammalian-type olfactory system. Keywords Amphibian . Olfactory system . G-protein . Immunohistochemistry . Projection
Introduction Most vertebrates have two distinct olfactory organs, the olfactory epithelium (OE) and the vomeronasal organ (VNO). The axons from these organs project to the main olfactory bulb (MOB) and the accessory olfactory bulb (AOB), respectively (Mori et al. 1999; Keverne 1999). The VNO is supposed to appear during the evolution of vertebrates in accordance with the change in the life styles from aquatic to terrestrial (Bertmar 1981; Taniguchi et al. 2008). In fact, fish lack the VNO, whereas most terrestrial vertebrates possess the VNO, except for birds and humans (Døving and Trotier 1998). Amphibians are located at a turning point in the evolution of the olfactory organs. Most amphibians spend their life in water at their larval stage and adapt to the terrestrial environment through metamorphosis (Reiss and Eisthen 2008). The morphology of their olfactory organs is different between larva and adult. Unlike other amphibians, African clawed frog, Xenopus laevis, spends their entire life in water even at the adult stage. Therefore, the olfactory system of this frog is unique and possibly gives us important keys to understand the evolution of vertebrate olfactory systems.
Author's personal copy 28
In the olfactory organs of rodents, the OE contains ciliated olfactory receptor neurons (RNs), which express odorant receptors (OR), whereas the vomeronasal epithelium (VNE) contains microvillous vomeronasal RNs, which express type I or type II vomeronasal receptors (V1R or V2R). The OR, V1R and V2R are olfactory receptors that belong to the G-protein coupled receptor families and they are coupled with different α subunits of G-proteins: Golf, Gi2 and Go, respectively (Buck and Axel 1991; Dulac and Axel 1995; Herrada and Dulac 1997). In the olfactory organs of frogs, the nasal cavity is divided into three compartments: principal, middle and inferior chambers. The principal chamber is lined with the OE and the inferior chamber with the VNE. The middle chamber, lined with the non-sensory epithelium in most frogs, is lined with the sensory epithelium called middle chamber epithelium (MCE) in Xenopus laevis (Reiss and Eisthen 2008). The OE contains ciliated RNs, the VNE microvillous RNs and the MCE both ciliated and microvillous RNs (Oikawa et al. 1998; Hansen et al. 1998). These ultrastructural properties suggest that the MCE is a remainder of the primitive OE of fish and that the OE and VNE have phylogenetically originated from it. The axonal projections from these epithelia have been revealed by Di-I labeling and lectin histochemistry: the axons from the OE, MCE and VNE project to the dorsal part of the MOB (d-MOB), the ventral part of the MOB (v-MOB) and the AOB, respectively (Hofmann and Meyer 1991; Franceschini et al. 1992; Reiss and Burd 1997; Saito and Taniguchi 2000). In addition, the expression of OR and V1R in the OE, OR, V1R and V2R in the MCE, and V2R in the VNE, as well as the co-expression of OR with Golf, V1R with Gi2 and V2R with Go have been demonstrated by in situ hybridization (Date-Ito et al. 2008). According to them, the somata of the Golf-expressing RNs and those of the Go-expressing RNs are separately situated in the upper and lower layers of the MCE. However, it remains unknown whether these RNs project their axons separately to the v-MOB. In this study, we examined the expression patterns of G-proteins by immunohistochemistry in the olfactory organs and olfactory bulbs of Xenopus laevis and suggested the axonal projections of RNs intermingled in the MCE.
Materials and methods Animals Fourteen female adult Xenopus laevis, 89–96 g body weight, were purchased from Hamamatsu Biological Materials (Shizuoka, Japan). All procedures were in accordance with the Guideline for Care and Use of Animal Experiments and approved by the animal care
Cell Tissue Res (2011) 346:27–33
committee at Iwate University. After anesthesia by cooling on ice and an intraperitoneal injection of 0.65 mg/g body weight of sodium pentobarbital, animals were sacrificed by cardiac perfusion with Ringer’s solution followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The head was cut off and immersed in the same fixative overnight at 4°C. The brain was removed and the upper jaw was decalcified in 10% ethylenediamine tetra-acetic acid in 0.1 M phosphate buffer (pH 7.4) at 4°C for 3 days. All specimens were cryoprotected in a sucrose gradient, embedded in an O.C.T. compound (Sakura Finetek, Tokyo, Japan) and cut at 15–20 μm thickness with a cryostat. Sections were airdried and processed for immunohistochemistry or lectin histochemistry. Some of the sections were stained with hematoxylin-eosin for general histological examination. Immunohistochemistry Immunohistochemistry was performed with a Gαs/olf antibody (Santa Cruz, CA, USA: rabbit pAb, sc-383, 1:100–1:1,000), a Gαo antiboby (Millipore, Billerica, USA: mouse mAb, MAB 3073, 1:500–1:1,000) and three Gαi2 antibodies (Wako, Osaka, Japan: rabbit pAb, #012-15571; Santa Cruz: mouse mAb, sc-80007 and rabbit pAb, sc-7276). We also examined the olfactory organs and brains of Xenopus laevis by the use of other antibodies: anti-X. laevis Gαolf (gift from Dr. HaginoYamagishi, rabbit pAb; cited in Hagino-Yamagishi and Nakazawa, 2011) and anti-Gαo (MBL, Nagoya, Japan: #551, rabbit pAb) and confirmed that they gave consistent results with those of the above-mentioned antibodies. An immunofluorescence method was used for olfactory organs and an avidin–biotin peroxidase complex (ABC) method for the brain. After washing in phosphate-buffered saline (PBS) followed by 0.1% Triton X-100 in PBS (PBT), sections of the olfactory organs were treated with 2% normal goat serum in PBS for 30 min at room temperature (RT) to block non-specific binding. Then, they were incubated with one of the primary antibodies at 4°C overnight. After washing, the sections were incubated for 2 h at RT with fluorescent-labeled secondary antibodies: Alexa Fluor 594-goat anti-rabbit IgG (Molecular Probes, CA, USA: A-11037, 1:2,000) or Alexa Fluor 594-goat anti-mouse IgG (Molecular Probes: A-11032, 1:2,000). The sections were examined with a confocal laser scanning microscope C2 (Nikon, Tokyo, Japan). The sections of the brain were washed in PBS and PBT, incubated in 0.3% H2O2 in methanol for 30 min at RT and blocked with 2% normal donkey serum in PBS for 30 min at RT. Then, the sections were incubated with one of the primary antibodies at 4°C overnight. The sections were washed and incubated with
Author's personal copy Cell Tissue Res (2011) 346:27–33
29
biotinylated-donkey anti-rabbit or anti-mouse secondary antibodies (Jackson ImmunoResearch, PA, USA: 711-066152 or 715-066-151, 1:2,000) for 30 min at 37°C. After washing, the sections were incubated with Vectastain ABC reagent (Vector Laboratories, CA, USA) for 1 h at RT. After washing, the sections were colorized with 0.05 M Tris-HCl (pH 7.6) containing 0.01% 3-3’ diaminobenzidine tetrahydrochloride (DAB) and 0.003% H2O2 at RT for 5 min. All antibodies were diluted in 1% bovine serum albumin in PBS. Negative control staining was performed with PBS in place of the primary antibodies. No specific staining was observed in the control slides. We
performed a pre-absorption test with corresponding blocking peptides if available and confirmed the elimination of immunoreactivity (data not shown).
Fig. 1 Hematoxylin-eosin staining (a–e) and lectin histochemistry for SBA (f–j) in the olfactory organs (a–c, f–h) and olfactory bulbs (d, e, i, j) of Xenopus laevis. a–c In the olfactory epithelium (OE, a), the middle chamber epithelium (MCE, b) and the vomeronasal epithelium (VNE, c), nuclei of supporting cells are located in the upper layer and the nuclei of receptor neurons are in the middle and lower layers. d, e The horizontal sections in the dorsal (d) and ventral (e) parts of the forebrain. Rostral (R) is right, lateral (L) is top and medial (M) is bottom. The main olfactory bulb (MOB) is located in the rostral aspect of the forebrain and the accessory olfactory bulb (AOB) is ventral and caudolateral to the MOB. The glomerular layers are encircled by
dotted lines in (d) and (e). f–h Lectin histochemistry for SBA in the OE (f), the MCE (g) and the VNE (h). The supranuclear cytoplasm of the supporting cells of the OE and the apical surface of the OE and the MCE are labeled with SBA. The somata in any of these epithelia are not labeled with SBA. i, j Lectin histochemistry for SBA in the dorsal (i) and ventral (j) parts of the OB. The dorsal part of the MOB (dMOB) is not labeled with SBA, whereas the ventral part of the MOB (v-MOB) and the AOB are labeled with SBA. Sp supporting cells, ORN olfactory receptor neurons, RN receptor neurons, VRN vomeronasal receptor neurons, ON olfactory nerve, VN vomeronasal nerve. Bars 50 μm (a–c, f–h), 500 μm (d, e, i, j)
Lectin histochemistry Lectin histochemistry with a biotinylated lectin Soybean agglutinin (SBA) (Vector Laboratories) was performed by the ABC method. After washing, the sections were incubated in 0.3% H2O2 in methanol and blocked with 1% bovine serum albumin in PBS for 30 min at RT. After washing, the sections were incubated with SBA (0.001 mg/ml) at 4°C
Author's personal copy 30
Cell Tissue Res (2011) 346:27–33
overnight, followed by the Vectastain ABC reagent for 30 min at 37°C. After washing, the sections were colorized at RT for 5 min. Negative control staining was performed with PBS in place of SBA. No specific staining was observed in the control slides (data not shown). Double-labeling immunohistochemistry Double-labeling immunohistochemistry was performed to examine the co-localization of two antigens in the same section. Primary antibodies were used in a cocktail: Gαs/olf (Santa Cruz: rabbit pAb, sc-383, 1:100) and Gαo (Millipore: mouse mAb, MAB3073, 1:1,000), Gαs/olf and acetylated tubulin (Sigma-Aldrich: mouse mAb, #7451, 1:1,000) and Gαo (MBL: rabbit pAb, #551, 1:1,000) and acetylated tubulin. Secondary antibodies were used in a cocktail: Alexa Fluor 488-goat anti-rabbit IgG (Molecular Probes: A-11034, 1:2,000) and Alexa Fluor 594-goat anti-mouse IgG (1:2,000), or Alexa Fluor 594-goat anti-rabbit IgG (1:2,000) and Alexa Fluor 488-goat anti-mouse IgG (Molecular Probes: A-11029, 1:2,000). The sections were examined with a confocal laser scanning microscope C2 (Nikon).
Results In the olfactory organs of Xenopus laevis, the nuclei of supporting cells were located in the upper layer and the nuclei of receptor neurons were located in the middle and basal layers of the OE (Fig. 1a), the MCE (Fig. 1b) and the VNE (Fig. 1c). The olfactory nerve (ON) terminated in the glomerular layer (GL) of the MOB in the rostral aspect of the forebrain (Fig. 1d, e). The vomeronasal nerve (VN) ran along the ventrolateral aspect of the ON and terminated in the GL of the AOB in the ventral and caudolateral aspect of the forebrain (Fig. 1e). Although the supranuclear cytoplasm of the supporting cells of the OE and the apical surface of both OE and MCE were labeled with lectin SBA, the somata were not labeled with SBA in any of these epithelia (Fig. 1f–h). The d-MOB was not labeled with SBA, whereas the GL of the v-MOB and the AOB were labeled with SBA (Fig. 1i, j). In the OE, the cilia on the apical surface and the somata situated in the lower two-thirds were Golf-positive (Fig. 2a). However, no immunopositive reaction for Go was observed in the OE (Fig. 2b). In the VNE, no immunopositive reaction for Golf was observed (Fig. 2c). However, the apical surface and the somata were Gopositive in the VNE (Fig. 2d). In the MCE, the somata in the middle one-third were positive for Golf (Fig. 3a), whereas those in the lower one-third were positive for Go (Fig. 3a’). The immunoreactivities for Golf and Go did not
Fig. 2 Immunohistochemistry for G-proteins in the OE and the VNE. a The cilia on the apical surface (arrows) and the somata in the lower two-thirds of the OE are positive for Gαs/olf. b The OE is negative for Gαo. c The VNE is negative for Gαs/olf. d The apical surface (arrows) and the somata in the lower two-thirds of the VNE are positive for Gαo. Bars 50 μm
overlap with each other (Fig. 3a”). On the apical surface of the MCE, cilia and microvilli-like structures were Golf- and Go-positive, respectively (Fig. 3b–b”). The Golf-positive cilia, hair-like protrusions from the apical surface (Fig. 3b, c, arrows), were also positive for acetylated tubulin (Fig. 3c–c”). Longer, acetylated tubulin-positive and Golf-negative cilia were of the supporting cells (Fig. 3c”, red). The microvilli-like structures, short protrusions (Fig. 3d, arrowheads), were positive for Go and negative for acetylated tubulin (Fig. 3d–d”). In addition, no immunoreactivity was observed for any of the three Gαi2 antibodies in the olfactory organs of Xenopus laevis (data not shown). In the olfactory bulbs of Xenopus laevis, the GL’s of the d-MOB were positive for Golf and negative for Go (Fig. 4a, b). The GL of the v-MOB was further subdivided into two regions, i.e., the rostromedial (RM) and the caudolateral (CL) regions, by the expression pattern of G-proteins (Fig. 4c, d). The RM regions were positive for Golf and negative for Go, whereas the CL regions were negative for Golf and positive for Go (Fig. 4c, d). The GL’s of the AOB were negative for Golf and positive for Go (Fig. 4c, d).
Author's personal copy Cell Tissue Res (2011) 346:27–33
31
Fig. 3 Double-labeling immunohistochemistry for Gαs/olf and Gαo (a–a”, b–b”), Gαs/olf and acetylated tubulin (c–c”) and Gαo and acetylated tubulin (d–d”) in the MCE. The higher magnification views of the apical surface are shown in b–b”, c–c” and d–d”. The right panels are the merged images of the left and middle panels. a–a” Gαs/ olf-positive receptor neurons (a, red) are situated in the middle one-third and Gαo-positive receptor neurons (a’, green) are in the lower one-third. Open arrowheads and double open arrowheads indicate the apical dendrites and the somata of the Gαs/olf-positive (a) and Gαopositive receptor neurons (a’), respectively. On the apical surface, the cilia (b, arrows) and the microvilli-like structures (b’, arrowheads) are positive for Gαs/olf and Gαo, respectively. The cilia are positive for acetylated tubulin (AcT) (c’ and d’). Gαs/olf-positive cilia (c, arrows) are also positive for AcT (c”, yellow). Gαo-positive microvilli-like structures (d, arrowheads) are negative for AcT (d”). Bars 50 μm (a–a”), 20 μm (b–b”, c–c”, d–d”)
Discussion To date, the relationship between ultrastructural properties of the RNs (Oikawa et al. 1998; Hansen et al. 1998) and Gproteins expressed in the olfactory organs of Xenopus laevis (Date-Ito et al. 2008) was unknown. In this study, immunohistochemistry for G-proteins indicated that the OE contains Golf-positive RNs, the VNE contains Gopositive RNs and the MCE contains both Golf-positive and Go-positive RNs. Such immunostaining patterns for Golf and Go are consistent with the expression patterns for corresponding mRNAs revealed by Date-Ito et al. (2008). Although both the OE and MCE of Xenopus laevis contain a small number of Gi mRNA-expressing cells (Date-Ito et al. 2008), there was no immnoreactivity for any of the three Gi2 antibodies used in this study. Probably, all these antibodies, raised against mammalian Gi2 protein, might have no cross-reactivity to that of Xenopus laevis. In addition, the immunoreactivity on the apical surface indicated that the Golf-positive cells were ciliated and the Go-positive cells were microvillous in any of the three olfactory sensory epithelia. Such relationships between the
ultrastructure and G-protein expression in the RNs have been so far described in the olfactory organs of both fish and rodents and speculated to be the conserved feature during vertebrate evolution (Hansen et al. 2004). This notion is supported by our observation that found such relationships in the amphibian. Lectin histochemistry for SBA made it possible to distinguish the d-MOB from v-MOB and AOB (Hofmann and Meyer 1991; Franceschini et al. 1992; Reiss and Burd 1997; Saito and Taniguchi 2000). In this study, it has been revealed by the combination of SBA-labeling and immunohistochemistry for G-proteins that the ON and GL of the d-MOB were positive for Golf and the VN and GL of the AOB were positive for Go, suggesting that the Golf-positive RNs send axons from the OE to the d-MOB and the Go-positive RNs from the VNE to the AOB. These data are consistent with the axonal projections revealed by DiI-labeling (Reiss and Burd 1997; Saito and Taniguchi 2000). Thus, the validity of the G-protein-immunohistochemistry to speculate about the axonal projections in the olfactory system has been confirmed. The GL of the v-MOB was subdivided into
Author's personal copy 32
Fig. 4 Immunohistochemistry for Gαs/olf (a, c) and for Gαo (b, d) in the olfactory bulbs of Xenopus laevis. The horizontal sections in the dorsal parts (a, b) and the ventral parts (c, d) of the forebrain are shown. Rostral is right, lateral is top and medial is bottom in each figure. a, b The dorsal part of the MOB (d-MOB) is positive for Gαs/ olf and negative for Gαo. c, d The ventral part of the MOB is subdivided into two regions, the rostromedial (RM) and the caudolateral (CL) regions. The RM region is positive for Gαs/olf, whereas the CL region and the AOB are not. The CL region and the AOB are positive for Gαo, whereas the RM region is not. VN vomeronasal nerve. Bars 500 μm
RM and CL regions, by the G-proteins they express. The former was Golf-positive and Go-negative, whereas the latter was Go-positive and Golf-negative. These data suggest that the Golf-positive and the Go-positive RNs, located in the upper and lower layers of the MCE, send axons to the RM and CL regions of the v-MOB,
Cell Tissue Res (2011) 346:27–33
respectively. Due to the structure of the nasal cavity in Xenopus laevis, the principal chamber is filled with air and the middle chamber with water (Reiss and Eisthen 2008). Thus, the OE and MCE are thought to detect odorants in the air and in water, respectively. The OE of Xenopus laevis, like that of mammals, possesses the Bowman’s gland and contains the ciliated RNs exclusively, whereas the MCE, like the OE of fish, lacks Bowman’s gland and contains both the ciliated and microvillous RNs (Hansen et al. 1998; Oikawa et al. 1998). In addition, the OE and MCE of Xenopus laevis express mammalian-type (Class II) ORs and fish-type (Class I) ORs, respectively (Freitag et al. 1995, 1998). Thus, based on both the ultrastructural properties and the OR type expressed, the OE of Xenopus laevis can be referred to as a mammalian-type OE, whereas the MCE has a fish-type OE. The present data suggest that the somata of the ciliated RNs and microvillous RNs are located in the upper and lower layers of the MCE, respectively, and that they send axons to the RM and CL regions of the v-MOB. To date, such characteristics have been found only in the OE of fish; i.e., both ciliated RNs and microvillous RNs are contained in a single sensory epithelium, have somata separately located in the upper or lower layers and send their axons to the discrete regions of the olfactory bulb (Morita and Finger 1998; Hamdani et al. 2001; Hamdani and Døving 2002; Hansen et al. 2003; Sato et al. 2005). This evidence further supports the idea that the MCE of Xenopus laevis is a fish-type OE. Vertebrates changed their lifestyles from aquatic to terrestrial and acquired VNO during the evolution from fish to tetrapods. By comparison of the olfactory systems among various vertebrates, it has been supposed that the Golf-expressing ciliated RNs and Go-expressing microvillous RNs, intermingled in the fish OE, are the precursors of the olfactory RNs and the vomeronasal RNs in tetrapods, respectively (Bertmar 1981; Eisthen 2004; Halpern 2006). As revealed in the present study, the OE and the VNE in Xenopus laevis contain Golf-positive ciliated RNs and Gopositive microvillous RNs, respectively, whereas the MCE contains both Golf-positive ciliated RNs and Go-positive microvillous RNs. The presence of three olfactory sensory epithelia in Xenopus laevis may imply that the Golfpositive ciliated RNs and Go-positive microvillous RNs were derived from a fish-type OE and separately distributed in the OE and VNE, whereas the fish-type OE has not been completely lost and remained as the MCE. The middle chamber is lined with the non-sensory epithelium in frogs, which adapt to the terrestrial environment after metamorphosis, whereas it is lined with the sensory epithelium in the Pipidae, which spend their entire life in water (Reiss and Eisthen 2008). Thus, Xenopus laevis is a very interesting model to understand
Author's personal copy Cell Tissue Res (2011) 346:27–33
the evolution of vertebrate olfactory systems because they have both fish-type olfactory system and mammalian-type olfactory system.
References Bertmar G (1981) Evolution of vomeronasal organs in vertebrates. Evolution 35:359–366 Buck L, Axel R (1991) A novel multigene family may encode odorant receptors: a molecular basis for odor recognition. Cell 65:175–187 Date-Ito A, Ohara H, Ichikawa M, Mori Y, Hagino-Yamagishi K (2008) Xenopus V1R vomeronasal receptor family is expressed in the main olfactory system. Chem Senses 33:339–346 Døving KB, Trotier D (1998) Structure and function of the vomeronasal organ. J Exp Biol 201:2913–2925 Dulac C, Axel R (1995) A novel family of genes encoding putative pheromone receptors in mammals. Cell 83:195–206 Eisthen HL (2004) The goldfish knows: olfactory receptor cell morphology predicts receptor gene expression. J Comp Neurol 477:341–346 Franceschini V, Giorgi PP, Ciani F (1992) Primary olfactory terminations in the forebrain of amphibia: a comparative study with soybean agglutinin. J Hirnforsch 33:627–635 Freitag J, Krieger J, Strotmann J, Breer H (1995) Two classes of olfactory receptors in Xenopus laevis. Neuron 15:1383–1392 Freitag J, Ludwig G, Andreini I, Rössler P, Breer H (1998) Olfactory receptors in aquatic and terrestrial vertebrates. J Comp Physiol A 183:635–650 Hagino-Yamagishi K, Nakazawa H (2011) Involvement of Gαolf expressing neurons in the vomeronasal system of Bufo japonicus. J Comp Neurol, in press Halpern M (2006) The evolution of the vomeronasal system. In: Kaas JH, Striedter GF, Bullock TH, Preuss TM, Rubenstein J, Krubitzer LA (eds) Evolution of nervous systems, vol 2. Academic, New York, pp 407–415 Hamdani EH, Døving KB (2002) The alarm reaction in crucian carp is mediated by olfactory neurons with long dendrites. Chem Senses 27:395–398 Hamdani EH, Alexander G, Døving KB (2001) Projection of sensory neurons with microvilli to the lateral olfactory tract indicates their participation in feeding behaviour in crucian carp. Chem Senses 26:1139–1144
33 Hansen A, Reiss JO, Gentry CL, Burd GD (1998) Ultrastructure of the olfactory organ in the clawed frog, Xenopus laevis, during larval development and metamorphosis. J Comp Neurol 398:273–288 Hansen A, Rolen SH, Anderson K, Morita Y, Caprio J, Finger TE (2003) Correlation between olfactory receptor cell type and function in the channel catfish. J Neurosci 23:9328–9339 Hansen A, Anderson KT, Finger TE (2004) Differential distribution of olfactory receptor neurons in goldfish: structural and molecular correlates. J Comp Neurol 477:347–359 Herrada G, Dulac C (1997) A novel family of putative pheromone receptors in mammals with a topographically organized and sexually dimorphic distribution. Cell 90:763–773 Hofmann MH, Meyer DL (1991) Functional subdivisions of the olfactory system correlate with lectin-binding properties in Xenopus. Brain Res 564:344–347 Keverne EB (1999) The vomeronasal organ. Science 286:716–720 Mori K, Nagao H, Yoshihara Y (1999) The olfactory bulb: coding and processing of odor molecule information. Science 286:711–715 Morita Y, Finger TE (1998) Differential projections of ciliated and microvillous olfactory receptor cells in the catfish, Ictalurus punctatus. J Comp Neurol 398:539–550 Oikawa T, Suzuki K, Saito TR, Takahashi KW, Taniguchi K (1998) Fine structure of three types of olfactory organs in Xenopus laevis. Anat Rec 252:301–310 Reiss JO, Burd GD (1997) Metamorphic remodeling of the primary olfactory projection in Xenopus: developmental independence of projections from olfactory neuron subclasses. J Neurobiol 32:213–222 Reiss JO, Eisthen HL (2008) Comparative anatomy and physiology of chemical senses in amphibians. In: Thewissen JGM, Naummela S (eds) Sensory evolution on the threshold: adaptation in secondary aquatic vertebrates. University of California Press, California, pp 43–63 Saito S, Taniguchi K (2000) Expression patterns of glycoconjugates in the three distinctive olfactory pathways of the clawed frog, Xenopus laevis. J Vet Med Sci 62:153–159 Sato Y, Miyasaka N, Yoshihara Y (2005) Mutually exclusive glomerular innervation by two distinct types of olfactory sensory neurons revealed in transgenic zebrafish. J Neurosci 25:4889–4897 Taniguchi K, Saito S, Oikawa T, Taniguchi K (2008) Phylogenic aspects of the amphibian dual olfactory system. J Vet Med Sci 70:1–9
