The vomeronasal system (VNS) mediates pheromonal communication in mammals. From the vomeronasal organ, two populations of sensory neurons, expressing either Gαi2 or Gαo proteins, send projections that end in glomeruli distributed either at the rostral or caudal half of the accessory olfactory bulb (AOB), respectively. Neurons at the AOB contact glomeruli of a single subpopulation. The dichotomic segregation of AOB glomeruli has been described in opossums, rodents and rabbits, while Primates and Laurasiatheres present the Gαi2-pathway only, or none at all (such as apes, some bats and aquatic species). We studied the AOB of the Madagascan lesser tenrec Echinops telfairi (Afrotheria: Afrosoricida) and found that Gαi2 and Gαo proteins are expressed in rostral and caudal glomeruli, respectively. However, the segregation of vomeronasal glomeruli at the AOB is not exclusive, as both pathways contained some glomeruli transposed into the adjoining subdomain. Moreover, some glomeruli seem to contain intermingled afferences from both pathways. Both the transposition and heterogeneity of vomeronasal afferences are features, to our knowledge, never reported before. The organization of AOB glomeruli suggests that synaptic integration might occur at the glomerular layer. Whether intrinsic AOB neurons may make synaptic contact with axon terminals of both subpopulations is an interesting possibility that would expand our understanding about the integration of vomeronasal pathways.
Citation: Suárez R, Villalón A, Künzle H, Mpodozis J (2009) Transposition and Intermingling of Gαi2 and Gαo Afferences into Single Vomeronasal Glomeruli in the Madagascan Lesser Tenrec Echinops telfairi. PLoS ONE 4(11): e8005. doi:10.1371/journal.pone.0008005
Editor: Andrew Iwaniuk, University of Lethbridge, Canada
Received: July 6, 2009; Accepted: November 2, 2009; Published: November 24, 2009
Copyright: © 2009 Suárez et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors acknowledge financial support from FEBA (Fundación para Estudios Biomédicos Avanzados), Mecesup UCH 0306, Conicyt (Término de tesis doctoral), Fondecyt (1080094) and Deutsche Forschungsgemeinschaft (Ku 624/3-3). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
In most mammals, the establishment and maintenance of social and sexual behaviours depend on the detection of semiochemicals by the vomeronasal system (VNS). The sensory surface of the VNS is the vomeronasal organ, a blind-ended tubular structure located bilaterally at the base of the nasal septum. Its neuroepithelium contains two spatially segregated populations of sensory neurons, each co-expressing either V1R receptors and Gαi2 protein or V2R receptors and Gαo protein, that send projections to distinct portions of the accessory olfactory bulb (AOB). Gαi2-expressing axons end in glomeruli located exclusively in the rostral half of the AOB, while Gαo-axons end in glomeruli of the caudal half of the AOB , , , , , , . The dichotomic segregation of vomeronasal pathways into the AOB has been described in opossums , rabbits  and rodents , , , and was initially thought to represent a common feature of the mammalian VNS . However, later reports showed that the V2R-Gαo pathway was absent in primates, shrews, goats, cows, horses, and dogs , , , .
In rodents, each pathway has been related to different functional specializations. Small, volatile ligands activate V1R-Gαi2 neurons , , , while responses to larger molecules, such as major urinary proteins, exocrine-gland secreted peptides and MHC class I peptides, have been recorded at V2R-Gαo neurons , . In addition, stimulation with opposite-sex semiochemicals, in both males and females, activates more neurons of the rostral than the caudal AOB , , , , .
Synaptic integration of the segregated pathways has been shown to occur at two levels in the VNS. First, although mitral/tufted (M/T) cells contact 2–5 glomeruli of the same subdomain only , , , , they extend lateral dendrites that span through both subdomains , , . Second, rostral and caudal M/T cells send projections that overlap, depending on the species, in the majority , , or the totality  of their recipient nuclei. As far as we know, the exclusive segregation of vomeronasal afferences occurs in all species with a two-pathway VNS, and no study has suggested that glomeruli of both pathways may undergo synaptic integration by intrinsic AOB neurons.
The objective of this study was to gain insights about the organization of vomeronasal afferences into the AOB of an early-branched placental mammal. We studied cellular organization and glomerular segregation at the AOB of the Madagascan lesser tenrec Echinops telfairi, (Afrotheria: Afrosoricida), a small species with insectivorous, nocturnal and solitary habits. This is the first time the vomeronasal pathways are investigated in the other great branch at the split of placental mammals. All previous studies in placentals have been done in Boreoeutherian species (superorders Euarchontoglires and Laurasiatheria). The ancestors of tenrecs branched off from the Boreoeutheria more than 100 million years ago , , . We found several unprecedented traits that expand our knowledge about the neurobiology and evolution of the mammalian VNS.
The AOB of the Lesser Tenrec, E. telfairi
As previously described , the olfactory bulbs of the tenrec are relatively large structures located at the rostralmost telencephalon (Figure 1). They contain a prominent olfactory ventricle (OV), surrounded by small and dense periventricular cells that may represent newly born neurons . The AOB is located at the dorso-caudal extent of the main olfactory bulb (MOB) (Figure 1A). The anterior olfactory nuclear complex (AON), composed of an external (AONe) and a central (AONc) portion , partially encircles the deep MOB from its lateral aspect and can be observed lying below the AOB (Figure 1A). The AONe is a narrow band of densely packed cells, caudal to AOB granular cells, while the AONc prolongs caudally into the frontal cortex (FrCx), in a transition zone that has been referred to as the sulcal cortex . There is a clear boundary between the MOB and AOB (dotted line; Figure 1B, 1C and 1D), marked by an abrupt discontinuity of MOB granular cells. Granular cells of the AOB lie below the lateral olfactory tract (lot; Figure 1C) and may be distinguished from AONe cells by their dense laminar packing and a more rostral distribution (dotted areas in Figure 1B).
Sagittal sections of the tenrec olfactory bulb immunolabelled against Gαi2 (A–C) or Gαo (D–E) proteins and counterstained with cresyl violet. (A) The relative sizes of the AOB and the MOB, and the continuity of the AONc towards the FrCx can be appreciated. Gαi2-expressing glomeruli are located at the rostral aspect of the AOB (A–C), however some glomeruli are displaced to caudal territories (arrowhead in C). The margin between the AOB and MOB is depicted by a discontinuous line (B–D). The Gr and AONe are ventral to the lot, and differ in their clustering (B). (C) Higher magnification of (B) showing the rostro-caudal asymmetry of M/T. (D) Gαo-expressing glomeruli are located at the caudal AOB, but not all caudal glomeruli seem to show full expression. (E) The cellular layers of the MOB are compactly stratified. All MOB glomeruli express Gαo protein, and they are larger and better defined than those of the AOB (compare with C, same magnification). AONc, anterior olfactory nucleus, central aspect; AONe, anterior olfactory nucleus external aspect; EPi, external plexiform layer, inner sublayer; EPo, external plexiform layer, outer sublayer; Gl, glomerular layer; Gr, granular cell layer; IP, internal plexiform layer; lot, lateral olfactory tract; M/T, mitral/tufted cell layer. Scale bar: 500 µm in A, 200 µm in B and D, and 100 µm in C and E.
Vomeronasal glomeruli expressing Gαi2 or Gαo proteins are arranged in rostral and caudal territories, respectively (Figure 1B and 1D). However, some Gαi2 glomeruli can be seen in caudal territories with biotinylated immunostaining (arrowhead in Figure 1C). The density and distribution of the mitral/tufted cell layer (M/T) is not homogeneous across the rostro-caudal axis of the AOB. It seemed more abundant and wider at its rostral extent, and consisted in 8–20 cells in depth (Figure 1C). In contrast, the M/T of the MOB is much narrower, containing up to 3 cells in depth (Figure 1E). The AOB seem to lack plexiform spaces, while the MOB has internal (IP) and external (EP) plexiform layers. The latter is divided in outer (EPo) and inner (EPi) sublayers that differ in cell density (Figure 1A and 1E). Glomeruli of the MOB are Gαo-positive, as also described in other mammals , , ,  and are distributed in a compact layer of 1–2 glomeruli in depth (Figure 1E and Supporting information S1). They are larger than AOB glomeruli, and are individually surrounded by a large number of periglomerular cells (compare Figures 1C and 1E).
Nonexclusive Segregation and Heterogeneity of Vomeronasal Glomeruli
All mammals with a two-pathway VNS studied so far show a clear-cut segregation of Gαi2 and Gαo positive glomeruli , , , . In rodents, the vomeronasal nerve arrives from either the medial  or lateral  aspect of the olfactory bulbs, and bifurcates entirely into rostral and caudal territories before ending in glomerular neuropil . In the tenrec, however, Gαi2-expressing axons arrive at the AOB passing through Gαo-expressing glomeruli to occupy a rostral position in more lateral sections (Figure 2A). Although vomeronasal glomeruli show a rostro-caudal segregation, some glomeruli of both populations locate within the adjacent subdomain (Figure 2A and 2B). The transposition of Gαi2 and Gαo-positive glomeruli into the adjacent subdomain was observed in all animals examined (n = 5). The relative position of transposed glomeruli was conserved across individuals (Supporting information S1).
Confocal 3D reconstructions of Gαi2 and Gαo afferences into the AOB of a representative specimen. (A) Serial sagittal sections 50 µm width, separated 300 µm each, showing Gαi2-expressing axons (green) passing through Gαo-expressing glomeruli (red), to occupy an anterior position at the AOB. Sections are arranged from medial (left) to lateral (right). (B) Three-dimensional confocal reconstruction of the last picture of (A) represented as seen from one side (top row), from a ventral transverse view (middle row) and from the opposite side (bottom row). Each row shows Gαi2, Gαo and both labels. Arrows depict transposed glomeruli. Fibers that cross to the opposite subdivision are indicated with arrowheads. Interrupted lines contour glomeruli containing both Gαi2- and Gαo-expressing elements. Glomeruli presumably containing axonal boutons and/or neuropil of the adjoined subpopulation are indicated with asteriks. Scale bar: 200 µm in A, 50 µm in B.
A 50 µm three-dimensional confocal reconstruction of the last section of Figure 2A is shown at higher magnification in Figure 2B. The glomerular layer is displayed as if seen from one side (upper row), from ventral (middle row) and from the opposite side (lower row). Afferent axons from both populations, some of them with apparent varicosities, cross to the adjacent subdomain (arrowheads, Figure 2B) to end in transposed glomeruli (arrows, Figure 2B). Furthermore, and to our surprise, some glomeruli contained both Gαi2 and Gαo intermingled afferences (dotted regions, Figure 2B). Although decussating axons often passed between glomeruli, some entered into converse glomeruli and terminated in what seemed to be axonal boutons and/or glomerular neuropil (asterisks, Figure 2B). Fluorescent labeling of Gαo at MOB glomeruli also revealed corpuscular structures that may represent axonal endings (Supporting information S1). The organization and intermingling of afferences within single vomeronasal glomeruli can be better appreciated in an animated three-dimensional reconstruction of the same preparation (Video S1).
Diversity in the Synaptic Organization of Chemosensory Systems
The organization of vomeronasal sensory representation at the AOB has revealed several differences when compared to the more-studied MOB. Main olfactory neurons expressing the same receptor converge their terminations into a single glomerulus, which is innervated by neuropil from a single apical dendrite of a M/T neuron . Thus, a one-to-one topography is established between glomeruli expressing the same receptor and M/T cells, resembling a labeled line of sensory processing (Figure 3). In contrast, the synaptic organization between sensory afferences and M/T efferences at the AOB is integrative. While axons expressing the same vomeronasal receptor project to several glomeruli, each M/T cell may contact up to 5 glomeruli expressing the same , , or similar receptors , within a single subdomain. Moreover, each glomeruli is contacted by 1–3 M/T cells . Thus, a high combination of synaptically integrated elements may converge onto individual M/T cells (Figure 3). The integration of glomeruli expressing different receptors occurs within each subdomain only and no study has reported, to our knowledge, evidence that any AOB neuron -projection or interneuron- would make synaptic contact with terminals from both subpopulations. Indeed, a space rich in glial cells and void of axonal connections, named linea alba, has been described at the margin between both AOB glomerular subpopulations of the rat . Similarly, in the Caviomorph rodent Octodon degus, we described an invagination spanning all cellular layers at the margin between rostral and caudal territories , further supporting the notion of a structural and functional independence of both subdomains.
The organization of the main olfactory system has been regarded to as a labeled line. Sensory neurons expressing the same receptor converge their afferences into single glomeruli, where synaptic contact is established with only one M/T cell, in a one-to-one topology. Thus, each olfactory receptor is represented by a single M/T cell. The accessory olfactory system, however, is integrative in the sense that each sensory neuron sends projections to several glomeruli and each M/T cell contacts several glomeruli. Although M/T cells contact glomeruli receiving afferences from different receptors, they integrate glomeruli from the same V1R or V2R subpopulation, most probably from closely related receptors. This dichotomic segregation suggests that no M/T cell contact glomeruli from both subpopulations. The non-exclusive segregation of vomeronasal afferences of the tenrec is characterized by the integration of afferences from both subpopulations into single glomeruli, suggesting that AOB neurons (such as M/T or periglomerular) may also make synaptic contact with both subpopulations.
In the tenrec, however, we found that the segregation is not exclusive, as not only were some glomeruli located within the adjoining subdomain, but also some seem to receive mixed afferences from both populations (Figure 2 and Figure 3). These results suggest that individual AOB neurons may integrate synaptic activity from both vomeronasal pathways. To elucidate this, additional experiments, such as cell filling of AOB neurons combined with differential sensory immunolabeling, would be required.
Targeting of sensory axons, in both main and accessory olfactory systems, is directed by the specific expression of chemorepulsive peptides, mostly of the Semaphorin and Slit families, and their membrane receptors , . Whether particular patterns of chemorepulsive molecules and their receptors are present in transposed and/or intermingled glomeruli deserves further investigation.
We have shown that, although not fully segregated, both vomeronasal pathways are present in the AOB of the tenrec. Results from comparative studies of Gα-protein expression at the VNS ,  and genomic enquiries of functional V2R gene sequences ,  lead to the parsimonious assumption that at least two events of deterioration of the V2R-Gαo pathway have occurred, independently, in the lineages leading to Primates and Laurasiatheria (Fig. 4). A possible scenario is that the Gαi2 pathway, which is conserved in all species with a functional VNS, would play a similar role in pheromonal communication across species, perhaps mediating the assessment of reproductive status between the sexes.
Chronogram of placental mammals showing the presence of both vomeronasal pathways (black lines), the V1R-Gαi pathway only (red lines), or the complete loss of both pathways (interrupted line). Two independent events of degeneration of the V2R-Gαo pathway might have occurred in the lineages leading to Primates and Laurasiatheria. Estimated times of divergence are based on refs. , ,  for nodes 1–4, 6, 7, and on ref.  for node 5.
Whether alternative configurations to the dichotomical segregation of the vomeronasal pathways were present in early mammalian species is an interesting possibility that deserves further comparative analysis in early-branched placental and non-placental mammals.
Materials and Methods
All experimental procedures followed the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80–23, 1996) and were approved by the faculty ethics committee (Comité de Etica de la Facultad de Ciencias, Universidad de Chile). All efforts were made to minimize the number of animals used and their suffering.
We employed eight (3 females and 5 males) Madagascan lesser tenrecs (Echinops telfairi) raised in a breeding colony in Munich , weighing 66–153 g (110.5 g mean weight). The animals were deeply anesthetized (tribromoethanol, 1 ml/100 g body weight, ip.) and perfused as previously described . We obtained 50 µm thick sagittal and coronal sections of the olfactory bulbs with a freezing microtome. Slices were either mounted for cresyl violet staining or collected in vials for further immunohistochemical processing.
Sagittal sections were incubated overnight in 3% normal goat serum (NGS) in PBS with 0.05% Triton X-100 (PBST) at 25°C. Then, they were incubated in primary immunoglobulins against Gαi2 (1∶200, mouse monoclonal, cat no. sc-13534, Santa Cruz Biotechnology, Santa Cruz, CA) and Gαo (1 µg/ml, rabbit polyclonal, cat no. 551, Medical and Biological Laboratories, Nagoya, Japan or 1∶200 mouse monoclonal, cat no. sc-13532, Santa Cruz Biotechnology, Santa Cruz, CA, for single or double reactions, respectively) with 3% NGS in PBST for 3 days at 25°C. The sections were then rinsed in PBS and incubated in a mixture of fluorescent goat anti-mouse and anti-rabbit secondary antibodies (1∶200; Alexa fluor 568 and 633 nm, respectively, Invitrogen), or in biotinylated goat anti-mouse antibodies (1∶200, cat no. sc-2039, Santa Cruz Biotechnology, Santa Cruz, CA) for 2 hours. Biotinylated sections were processed as described before . They were mounted on gelatine-coated slides, counterstained with cresyl violet, observed under light microscopy (BX60; Olympus Optical, Thornwood, NY) and photographed with SPOT camera and software (Spot Advanced; Diagnostic instrument, Sterling Heights, MI). Fluorescent sections were examined with a confocal laser-scanning microscope (Zeiss LSM 510 Meta; Jena, Germany) using laser beams of 488 and 633 nm for excitation. Three-dimensional reconstructions and analyses were made with the LSM 510 software (version 3.2). All figures were prepared for presentation purposes with Adobe Photoshop CS3 (Adobe Systems, San Jose, CA).
(0.05 MB PDF)
Confocal reconstruction of Gαi2-positive axons (green) and Gαo-axons (red) in the AOB of the tenrec.
(9.67 MB MOV)
We thank Miguel Concha and Pablo Sabat for their hospitality, Elisa Sentis, Solano Henríquez and Lorena Sarragoni for technical assistance, and Alexander Vargas and the reviewers for helpful comments on the manuscript.
Conceived and designed the experiments: RS JM. Performed the experiments: RS AV. Analyzed the data: RS AV JM. Contributed reagents/materials/analysis tools: RS HK JM. Wrote the paper: RS JM.
- 1. Halpern M, Shapiro LS, Jia CP (1995) Differential localization of G proteins in the opossum vomeronasal system. Brain Research 677: 157–161.
- 2. Dulac C, Axel R (1995) A novel family of genes encoding putative pheromone receptors in mammals. Cell 83: 195–206.
- 3. Berghard A, Buck LB (1996) Sensory transduction in vomeronasal neurons: evidence for Gαo, Gαi2, and adenylyl cyclase II as a major components of a pheromone signaling cascade. Journal of Neuroscience 16: 909–918.
- 4. Jia CP, Halpern M (1996) Subclasses of vomeronasal receptor neurons: Differential expression of G proteins (Giα2 and Goα) and segregated projections to the accessory olfactory bulb. Brain Research 719: 117–128.
- 5. 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.
- 6. Matsunami H, Buck LB (1997) A multigene family encoding a diverse array of putative pheromone receptors in mammals. Cell 90: 775–784.
- 7. Ryba NJP, Tirindelli R (1997) A new multigene family of putative pheromone receptors. Neuron 19: 371–379.
- 8. Imamura K, Mori K, Fujita SC, Obata K (1985) Immunochemical identification of subgroups of vomeronasal nerve fibers and their segregated terminations in the accessory olfactory bulb. Brain Research 328: 362–366.
- 9. Sugai T, Sugitani M, Onoda N (1997) Subdivisions of the Guinea-pig Accessory Olfactory Bulb revealed by the combined method with immunohistochemistry, electrophysiological and optical recordings. Neuroscience 79: 871–885.
- 10. Suárez R, Mpodozis J (2009) Heterogeneities of size and sexual dimorphism between the subdomains of the lateral-innervated accessory olfactory bulb (AOB) of Octodon degus (Rodentia: Hystricognathi). Behavioural Brain Research 198: 306–312.
- 11. Halpern M, Jia CP, Shapiro LS (1998) Segregated pathways in the vomeronasal system. Microscopy Research and Technique 41: 519–529.
- 12. Takigami S, Mori Y, Tanioka Y, Ichikawa M (2004) Morphological evidence for two types of mammalian vomeronasal system. Chemical Senses 29: 301–310.
- 13. Takigami S, Mori Y, Ichikawa M (2000) Projection pattern of vomeronasal neurons to the accessory olfactory bulb in goats. Chemical Senses 25: 387–393.
- 14. Shi P, Zhang J (2007) Comparative genomic analysis identifies an evolutionary shift of vomeronasal receptor gene repertoires in the vertebrate transition from water to land. Genome Research 17: 166–174.
- 15. Young JM, Trask BJ (2007) V2R gene families degenerated in primates, dog and cow, but expanded in opossum. Trends in Genetics 23: 212–215.
- 16. Sugai T, Yoshimura H, Kato N, Onoda N (2006) Component-dependent urine responses in the rat accessory olfactory bulb. Neuroreport 17: 1663–1667.
- 17. Leinders-Zufall T, Lane AP, Puche AC, Ma W, Novotny MV, et al. (2000) Ultrasensitive pheromone detection by mammalian vomeronasal neurons. Nature 405: 792–796.
- 18. Peele P, Salazar I, Mimmack M, Keverne EB, Brennan PA (2003) Low molecular weight constituents of male mouse urine mediate the pregnancy block effect and convey information about the identity of the mating male. European Journal of Neuroscience 18: 622–628.
- 19. Leinders-Zufall T, Brennan P, Widmayer P, Chandramani PS, Maul-Pavicic A, et al. (2004) MHC class I peptides as chemosensory signals in the vomeronasal organ. Science 306: 1033–1037.
- 20. Kimoto H, Haga S, Sato K, Touhara K (2005) Sex-specific peptides from exocrine glands stimulate mouse vomeronasal sensory neurons. Nature 437: 898–901.
- 21. Dudley CA, Moss RL (1999) Activation of an anatomically distinct subpopulation of accessory olfactory bulb neurons by chemosensory stimulation. Neuroscience 91: 1549–1556.
- 22. Halem HA, Cherry JA, Baum MJ (2001) Central forebrain Fos responses to familiar male odours are attenuated in recently mated female mice. European Journal of Neuroscience 13: 389–399.
- 23. Inamura K, Kashiwayanagi M, Kurihara K (1999) Regionalization of Fos immunostaining in rat accessory olfactory bulb when the vomeronasal organ was exposed to urine. European Journal of Neuroscience 11: 2254–2260.
- 24. Kumar A, Dudley CA, Moss RL (1999) Functional dichotomy within the vomeronasal system: Distinct zones of neuronal activity in the accessory olfactory bulb correlate with sex-specific behaviors. Journal of Neuroscience 19: 1–6.
- 25. Matsuoka M, Yokosuka M, Mori Y, Ichikawa M (1999) Specific expression pattern of Fos in the accessory olfactory bulb of male mice after exposure to soiled bedding of females. Neuroscience Research 35: 189–195.
- 26. Jia CP, Halpern M (1997) Segregated populations of mitral/tufted cells in the accessory olfactory bulb. Neuroreport 8: 1887–1890.
- 27. Larriva-Sahd J (2008) The accessory olfactory bulb in the adult rat: a cytological study of its cell types, neuropil, neuronal modules, and interactions with the main olfactory system. The Journal of Comparative Neurology 510: 309–350.
- 28. Wagner S, Gresser AL, Torello AT, Dulac C (2006) A multireceptor genetic approach uncovers an ordered integration of VNO sensory inputs in the accessory olfactory bulb. Neuron 50: 697–709.
- 29. Yonekura J, Yokoi M (2008) Conditional genetic labeling of mitral cells of the mouse accessory olfactory bulb to visualize the organization of their apical dendritic tufts. Mol Cell Neurosci 37: 708–718.
- 30. Martinez-Marcos A, Halpern M (1999) Differential projections from the anterior and posterior divisions of the accessory olfactory bulb to the medial amygdala in the opossum, Monodelphis domestica. European Journal of Neuroscience 11: 3789–3799.
- 31. Mohedano-Moriano A, Pro-Sistiaga P, Úbeda-Bañón I, Crespo C, Insausti R, et al. (2007) Segregated pathways to the vomeronasal amygdala: differential projections from the anterior and posterior divisions of the accessory olfactory bulb. European Journal of Neuroscience 25: 2065–2080.
- 32. von Campenhausen H, Mori K (2000) Convergence of segregated pheromonal pathways from the accessory olfactory bulb to the cortex in the mouse. European Journal of Neuroscience 12: 33–46.
- 33. Hallström BM, Janke A (2008) Resolution among major placental mammal interordinal relationships with genome data imply that speciation influenced their earliest radiations. BMC Evolutionary Biology 8: 162.
- 34. Murphy WJ, Pringle TH, Crider TA, Springer MS, Miller W (2007) Using genomic data to unravel the root of the placental mammal phylogeny. Genome Research 17: 413–421.
- 35. Springer MS, Murphy WJ, Eizirik E, O'Brien SJ (2003) Placental mammal diversification and the Cretaceous–Tertiary boundary. Proceedings of the National Academy of Sciences of the United States of America 100: 1056–1061.
- 36. Radtke-Schuller S, Künzle H (2000) Olfactory bulb and retrobulbar regions in the hedgehog tenrec: Organization and interconnections. The Journal of Comparative Neurology 423: 687–705.
- 37. Alvarez-Buylla A, Garcia-Verdugo JM (2002) Neurogenesis in Adult Subventricular Zone. J Neurosci 22: 629–634.
- 38. Shinohara H, Asano T, Kato K (1992) Differential localization of G-proteins Gi and Go in the accessory olfactory bulb of the rat. The journal of neuroscience 12: 1275–1279.
- 39. Wekesa KS, Anholt R (1999) Differential expression of G proteins in the mouse olfactory system. Brain Res 837: 117–126.
- 40. Halpern M, Martinez-Marcos A (2003) Structure and function of the vomeronasal system: an update. Progress in Neurobiology 70: 245–318.
- 41. Dulac C, Wagner S (2006) Genetic analysis of brain circuits underlying pheromone signaling. Annual Review of Genetics 40: 449–467.
- 42. Del Punta K, Puche A, Adams NC, Rodriguez I, Mombaerts P (2002) A divergent pattern of sensory axonal projections is rendered convergent by second-order neurons in the accessory olfactory bulb. Neuron 35: 1057–1066.
- 43. Rodriguez I, Feinstein P, Mombaerts P (1999) Variable patterns of axonal projections of sensory neurons in the mouse vomeronasal system. Cell 97: 199–208.
- 44. Cloutier JF, Sahay A, Chang EC, Tessier-Lavigne M, Dulac C, et al. (2004) Differential requirements for semaphorin 3F and Slit-1 in axonal targeting, fasciculation, and segregation of olfactory sensory neuron projections. Journal of Neuroscience 24: 9087–9096.
- 45. Knoll B, Schmidt H, Andrews W, Guthrie S, Pini A, et al. (2003) On the topographic targeting of basal vomeronasal axons through Slit-mediated chemorepulsion. Development 130: 5073–5082.
- 46. Künzle H, Poulsen-Nautrup C, Schwarzenberger F (2007) High inter-individual variation in the gestation length of the hedgehog tenrec, Echinops telfairi (Afrotheria). Animal Reproduction Science 97: 364–374.
- 47. Kosaka K, Künzle H, Kosaka T (2005) Organization of the main olfactory bulb of lesser hedgehog tenrecs. Neuroscience Research 53: 353–362.
- 48. Glazko GV, Nei M (2003) Estimation of Divergence Times for Major Lineages of Primate Species. Mol Biol Evol 20: 424–434.