Structure and function of the vomeronasal system: an update

Progress in Neurobiology 70 (2003) 245–318

Structure and function of the vomeronasal system: an update Mimi Halpern a,∗ , Alino Mart´ınez-Marcos b a

Department of Anatomy and Cell Biology, SUNY Downstate Medical Center, Box 5, 450 Clarkson Avenue, Brooklyn, NY 11203, USA b Departamento de CC. Médicas, Facultad de Medicina, Centro Regional de Investigaciones Biomédicas, Universidad de Castilla-La Mancha, 02071 Albacete, Spain Received 9 January 2003; accepted 24 June 2003

Abstract Several developments during the past 15 years have profoundly affected our understanding of the vomeronasal system (VNS) of vertebrates. In the mid 1990s, the vomeronasal epithelium of mammals was found to contain two populations of receptor cells, based on their expression of G-proteins. These two populations of neurons were subsequently found to project their axons to different parts of the accessory olfactory bulb (AOB), forming the basis of segregated pathways with possibly heterogeneous functions. A related discovery was the cloning of members of at least two gene families of putative vomeronasal G-protein-coupled receptors (GPRs) in the vomeronasal epithelium. Ligand binding to these receptors was found to activate a phospholipase C (PLC)-dependent signal transduction pathway that primarily involves an increase in intracellular inositol-tris-phosphate and intracellular calcium. In contrast to what was previously believed, neuron replacement in the vomeronasal epithelium appears to occur through a process of vertical migration in most mammals. New anatomical studies of the central pathways of the olfactory and vomeronasal systems indicated that these two systems converge on neurons in the telencephalon, providing an anatomical substrate for functional interactions. Combined anatomical, physiological and behavioral studies in mice provided new information that furthered our understanding of one of the most striking pheromonal phenomena, the Bruce effect. Finally, contrary to prior observations, new anatomical studies indicated that a vomeronasal organ (VNO) was present in human adults and reports were published indicating that this system might be functional. These latter observations are still controversial and require confirmation from independent laboratories. © 2003 Elsevier Ltd. All rights reserved.

Contents 1. 2. 3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . General reviews. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Heterogeneity in the VNS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Signal transduction molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.1. Receptor proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.2. G-proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.3. Phosphodiesterase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.4. NADPH–diaphorase staining and GABA-immunoreactivity . . . . . . . . . . . . . . . . 3.2. Developmental markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.1. Glycoconjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: AA, arachidonic acid; AC, adenylyl cyclase; ACvn , vomeronasal adenylyl cyclase; AOB, accessory olfactory bulb; BAOT, bed nucleus of the accessory olfactory tract; BNST, bed nucleus of the stria terminalis; BrdU, bromodeoxyuridine; CICR, calcium induced calcium release; DAG, diacylglycerol; DHB, 2,3-dihydro-exo-brevicomin; EPL, external plexiform layer; ESS, earthworm shock secretion; ES20, purified earthworm shock secretion; EVG, electrovomeronasogram; FS, follicle stimulating hormone; GL, glomerular layer of the AOB; GR, granule cell layer of the AOB; IP3 , inositol-1,4,5-tris-phosphate; ir, immunoreactivity; LH, luteinizing hormone; LHRH, luteinizing hormone releasing hormone; Mabs, monoclonal antibodies; MOB, main olfactory bulb; M/T, mitral/tufted; MUP, major urinary protein; NCAM, neural cell adhesion molecule; NL, nerve layer of the AOB; NOS, nitric oxide synthase; NSE, neuron-specific enolase; OCAM, olfactory cell adhesion molecule; OMP, olfactory marker protein; PDD, pregna-4,20-diene-3,6-dione; PDE, phosphodiesterase; PGP 9.5, protein gene product 9.5; PLC, phospholipase C; SBT, 2-sec-butyl-4,5-dihydro-thiazole; SER, smooth endoplasmic reticulum; SNP, sodium nitroprusside; TRP, transient receptor potential; VVA, Vicia villosa agglutinin; VN, vomeronasal; VNO, vomeronasal organ; VNS, vomeronasal system; VNX, vomeronasal organ removal ∗ Corresponding author. Tel.: +1-718-270-2416; fax: +1-718-270-3378. E-mail address: [email protected] (M. Halpern). 0301-0082/$ – see front matter © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0301-0082(03)00103-5

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4.

5. 6.

7.

8.

3.2.2. Neurestin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.3. Ephrins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Markers of unknown function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.1. Olfactory marker protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.2. Lectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Central projections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Physiological responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6. Immediate early gene expression. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Discovery of multiple families of putative VN receptors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Olfactory receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. First family of putative vomeronasal receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Second family of putative vomeronasal receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Additional families of putative vomeronasal receptors? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. Implications of the discovery of multiple families of putative vomeronasal receptors . . . 4.5.1. Receptors functionality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5.2. Evolutionary origin of receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5.3. Receptors map onto the accessory olfactory bulb . . . . . . . . . . . . . . . . . . . . . . . . . . . New information on signal transduction in the VNS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Developmental issues in the VNS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Ontogenetic development of VNS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.1. Developmental markers and factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Re-evaluation of neurogenesis in the VNS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2.1. Vomeronasal epithelium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2.2. Degeneration and reconstitution of the VN sensory epithelium . . . . . . . . . . . . . . . 6.2.3. Accessory olfactory bulb . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . New information on the anatomy of the VNS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1. Descriptive anatomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1.1. Chemoarchitecture: presence of functionally defined peptides and proteins . . . . 7.1.2. Sexual dimorphism in the VNS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2. Re-evaluation of the anatomical connections of the vomeronasal system. . . . . . . . . . . . . . . 7.2.1. Amphibians . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.2. Reptiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.3. Mammals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . New functional data on the role of the VNS in species-typical behaviors . . . . . . . . . . . . . . . . . . . . 8.1. Reviews . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2. Pheromonal effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2.1. Bruce effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2.2. Whitten effect and induced estrus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2.3. Vandenbergh effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2.4. Delay of puberty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3. Mammalian sexual behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3.1. Hamster . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3.2. Mouse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3.3. Rat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3.4. Vole . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3.5. Lemur . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3.6. Elephant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3.7. Ferret . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3.8. Opossum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3.9. Guinea pig . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3.10. Salamanders and newts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.4. Parental behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.5. Aggressive behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.6. Marking behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.7. Individual odor discrimination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.8. Aggregation in reptiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.9. Non-pheromonal functions of the VNS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.10. Behaviors not dependent on a functional VNS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.11. Odorant access to the VNO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.11.1. Behavioral and structural specializations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

251 251 251 251 252 252 254 255 256 256 257 257 258 259 259 259 259 260 265 265 266 268 268 270 270 271 271 273 274 275 276 276 278 280 280 281 282 283 284 285 285 285 286 287 288 288 288 289 289 289 289 289 290 290 291 291 291 292 292 292

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8.11.2. Physiological mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.11.3. Ligand binding proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.12. Reinforcing effects of VN stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9. Is there a functional human vomeronasal organ? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1. Reviews . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2. Morphological observations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3. Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4. Putative human pheromones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5. Physiological and behavioral responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10. Electrophysiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction It has been more than 15 years since the last comprehensive reviews of the structure and function of the vomeronasal system (VNS) (Halpern, 1987; Wysocki and Meredith, 1987). At that time, the main interest was testing the dual olfactory hypothesis, i.e. elucidating anatomical, behavioral and physiological differences between the olfactory and the VNS, which still are not completely understood. Since then, and focusing on the VNS, a number of dramatic observations have been reported that have had a major influence on our understanding of this relatively poorly understood

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sensory system. Among these findings was the observation that, in mammals, the VNS is dichotomous. This duality can be appreciated by the heterogeneous chemoarchitecture of apical and basal cells in the VN epithelium and it is correlated to the projections of these cells to anterior and posterior portions of the accessory olfactory bulb (AOB) (Figs. 1 and 2). These two populations of VN cells express different putative pheromone receptors, which apparently are activated by different ligands and trigger distinct behaviors. The most outstanding question in the field has been, therefore, to try to characterize these two subsystems within the VNS. Apart from anatomical and behavioral

Fig. 1. General organization of the vomeronasal system (VNS) in mammals. Upper figures illustrate the relationships between the vomeronasal organ (VNO), olfactory epithelium (OE), and main (MOB) and accessory olfactory (AOB) bulbs. Lower figures represent coronal (VNO and cerebral hemispheres) and parasagittal (AOB) sections showing apical and basal populations of vomeronasal receptor cells projecting to the anterior and posterior portions of the AOB, respectively. The AOB in turn projects to the amygdaloid complex (A). Abbreviations: NL, nerve; GL, glomerular; M/T, mitral/tufted; GR, granule cell layers of the AOB; H, hippocampus; IC, internal capsule; T, thalamus; HY, hypothalamus; OT, optic tract.

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Fig. 2. Coronal sections of the vomeronasal organ (VNO, panels A, B, D, E, G, H) and parasagittal sections of the accessory olfactory bulb (AOB, panels C, F, I) of opossum illustrating the heterogeneity in the vomeronasal system (VNS). Nissl-stained sections of the VNO (A, B) and AOB (C) show histological organization. Gi2␣ is preferentially expressed in the apical population of vomeronasal receptor cells (D, E) and in the anterior portion of the AOB (F). Go␣ -protein-expression is restricted to the basal population of vomeronasal receptor cells (G, H) and to the posterior portion of the AOB (I). Abbreviations: SE, sensory epithelia; NSE, non-sensory epithelia; L, lumen; V, vessel; SC, supporting cells; RC, receptor cells; BC, basal cells; GL, glomerular; M/T, mitral/tufted; GR, granule cell layers of the AOB.

approaches, cloning of families of putative VN receptors has allowed the use of a number of molecular techniques which have greatly expanded our insight on the dual perspective of pheromone detection and behavioral responses. Major advances have been also made in understanding the signal transduction mechanisms in the VN epithelium, indicating that, in contrast to the olfactory system, the principal second messenger system in the vomeronasal organ (VNO) is probably a phospholipase C (PLC)-dependent pathway. New studies, investigating the turnover of VN bipolar neurons, have resulted in a re-evaluation of the accepted dogma that in mammals neurons are generated at the margins of the epithelium and these neurons migrate toward the center

of the epithelium as they mature. Finally, recent anatomical studies have caused us to rethink the parcellation of the amygdala and its functional implications. These new findings have resulted, in recent years, in a remarkable increase in published reports on this system. These studies have advanced our understanding of the functional significance of the VNS as well as raised interesting questions about the functional integrity of the human VNS. The present review takes a broad approach to recent developments concerning the VNS. We have endeavored to include published studies using a variety of approaches: anatomy, electrophysiology, biochemistry, molecular biology and behavior in all vertebrates studied, not just mammals.

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2. General reviews Broad ranging reviews of the VNS by Døving and Trotier (1998) and Trotier et al. (1996) cover the history of the discovery by Jacobson of the VNO, the structure of the VNO, the distribution of the VNO in vertebrates, functions of the VNO, stimulus access, stimulus composition, cell turnover, subclasses and physiological properties of VN receptor neurons. Eisthen (1992, 1997) reviews the literature on the peripheral anatomy of the VN and main olfactory systems from a phylogenetic perspective and concludes that the VNS, which is present only in tetrapods, appeared after the development of the olfactory system. In addition, she concludes that the sensory epithelium of the nasal cavity of fishes is exclusively olfactory and the development of a separate VNS was not a response to selective pressure associated with a terrestrial life style. In tetrapods, the presence of a discrete VNS is a derived character and the presence of microvillar receptor cells, supporting cells and basal cells is an ancestral condition. Reviews by Meredith (1991, 1998a,b) are concerned primarily with mammals and include functional comparisons between the olfactory and vomeronasal systems, a discussion of stimuli that excite the VNS, stimulus access, properties of receptor cells, sensory coding and analysis, central connections, in particular comparison of odor maps in the main olfactory and VN systems, accessory olfactory bulb (AOB) memory and hormonal consequences of VN stimulation. The VNS of mammals has most recently been reviewed by Brennan (2001), Brennan and Keverne (2003) and Doty (2001). The reviews by Brennan, and Brennan and Keverne cover a broad range of topics including the nature of VN stimuli, receptors, transduction mechanisms, the structure of the VNO, information coding, synaptic plasticity, a comparison of olfactory and VN central connections, and behavioral and physiological responses mediated by the VNS. The review by Doty is more limited in scope. Mason (1992) and Halpern (1992) provide extensive reviews of the VNS of reptiles, approaching the system from both functional and structural perspectives. In a review of the evolution of chemoreception in squamate reptiles, Schwenk (1993) discusses chemoreception in a phylogenetic context, tabulating different characters in different taxa. He calls attention to missing data as well as proposing general phylogenetic patterns. In a review of the goldfish olfactory system, Dulka (1993) argues that the medial olfactory nerve, medial olfactory bulb and medial olfactory tract, all of which respond to and transmit information about sex pheromones, may be considered analogous to the tetrapod VNS, and that the lateral olfactory nerve, lateral olfactory bulb and lateral olfactory tract may be considered analogous to the tetrapod main olfactory system. The author, however, stops short of claiming homology and suggests that further phylogenetic analysis and more sophisticated experimental techniques may permit

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re-examination of the question of whether teleosts possess a VNS. However, one must consider the cogent arguments made by Eisthen (1992, 1997) that a VNS is not present in fishes.

3. Heterogeneity in the VNS The VNS in mammals is heterogeneous with respect to a number of molecular, anatomical and physiological parameters, e.g. types of receptors, chemoarchitecture (G-protein-expression, NADPH–diaphorase staining, presence of glycoconjugates, expression of olfactory marker protein, lectin staining and other markers), connections and response to stimulating substances (Figs. 1, 2 and 7). This section tries to emphasize that morphological duality observed in the VNS should have a functional correlate that is not always obvious. This topic was reviewed most recently by Halpern et al. (1998a,b) and Mori et al. (2000). 3.1. Signal transduction molecules 3.1.1. Receptor proteins Two families of candidate VN receptor genes have been identified in the VNO (see Section 4) and their expression is differentially distributed. The genes encode proteins with seven predicted membrane-spanning domains. The first gene family to be identified (Dulac and Axel, 1995) is expressed in apically situated receptor neurons, those co-expressing Gi2␣ -proteins. The second gene family (Herrada and Dulac, 1997; Matsunami and Buck, 1997; Ryba and Tirindelli, 1997) is expressed in more basally situated receptor neurons, those that co-express Go␣ -proteins. This latter family of genes consists of many pseudogenes which probably do not code for functional receptors. The presence of at least two families of putative receptor genes adds credence to the idea that the VNS is heterogeneous and its different parts are likely to respond to different stimuli (see Sections 3.1.2 and 4). 3.1.2. G-proteins Differential G-protein-expression in the VN epithelium and AOB has been observed in a broad group of mammals including opossum (Halpern et al., 1995; Jia and Halpern, 1995; Jia et al., 1997), guinea pig (Sugai et al., 1997), mouse (Berghard and Buck, 1996; Jia and Halpern, 1996; Rünnenberger et al., 2002; Wekesa and Anholt, 1999) and rat (Jia and Halpern, 1996; Shinohara et al., 1992). In all cases examined, the anterior AOB and the receptor cells in the apical sublayer of the VN epithelium were Gi2␣ -positive and the posterior AOB and receptor cells in the basal sublayer of the VN epithelium were Go␣ -positive (Fig. 2). Electron microscopic examination of immunoreactivity (ir) to G-proteins, Gi2␣ and Go␣ on the epithelial surface of the rat VNO (Matsuoka et al., 2001) indicated that the immunoreactivity was located on the microvilli and knob-like

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surface structures of receptor cells. Receptor cells appeared to be either Gi2␣ -, Go␣ -immunoreactive or neither, but not immunoreactive to both classes of G-proteins. At the surface of the epithelium the luminal terminations of the two classes of cells appear to be distributed randomly. The ␥-subunits of G-proteins are also differentially expressed in the mouse VNO. G␥8 is expressed in the basal region of the receptor cell layer whereas G␥2 is expressed in its more apical region (Rünnenberger et al., 2002). In some mammals (goat, dog, horse, musk shrew, marmoset) no differential expression of G-proteins is observed (Takigami et al., 2000a,b). In goats, Gi2␣ -ir was found on the luminal surface of the sensory epithelium of the VNO and in the VN nerve (NL) and glomerular (GL) layers throughout the AOB (Takigami et al., 2000a). Go␣ -ir was not found in the VNO or AOB. In dog, horse, musk shrew and marmoset Gi2␣ -ir was found throughout the NL and GL of the AOB, whereas Go␣ -ir was present throughout the glomerular, mitral/tufted cell (M/T) and granule cell layers, but was not present in the NL (Takigami et al., 2000b). G-proteins are expressed very early in the VN epithelium, earlier than other markers that differentiate the two populations of neurons. In the opossum VNO, G-proteins are expressed as early as postnatal day 0 (P0, the day of birth) and are differentially localized in their respective sublayers by P3 (Jia et al., 1997). In the AOB, G-protein-ir is observed at P10 and at this time the segregated projections of the receptor cell axons is evident (Jia et al., 1997). The Gi2␣ -expressing neurons in the apical sublayer of the VN epithelium project their axons to the anterior AOB and the Go␣ -expressing neurons in the basal sublayer of the VN epithelium project their axons to the posterior AOB (Jia and Halpern, 1996; Shapiro et al., 1995a). The differential expression of G-proteins in the AOB reflects differences in the projections of axons from the two subpopulations of neurons in the VNO and not a molecular difference in AOB neurons themselves. Mice with a targeted deletion of the Go␣ gene have normal VNSs at birth, indicating that in utero Go␣ -protein does not have a role in receptor selection, cell generation or cell survival (Tanaka et al., 1999). However, shortly after birth apoptosis increases in the Go␣ -positive layer of the epithelium. The number of cells in the Go␣ -positive layer is reduced by one-half and the posterior AOB is also reduced in size, as are the number of c-Fos-immunoreactive M/T cells. Several additional studies examine the location and ontogenetic expression in the VNO of the G-protein ␣-gustucin in mouse (Zancanaro et al., 1999) and rat (Menco et al., 2001), the GTP-binding protein subunit, G␥8 , in rat (Ryba and Tirindelli, 1995; Tirindelli and Ryba, 1996) and the G-protein, Gq␣ /G11␣ in rat (Menco et al., 1994). To summarize, in many, but not all mammals, G-proteins are differentially expressed in the VN sensory epithelium and AOB. The pattern is the same in all species that show this differential expression, Gi2␣ being expressed in the apical sublayer of the VN sensory epithelium and anterior AOB,

Go␣ being expressed in the basal sublayer of the VN sensory epithelium and posterior AOB. 3.1.3. Phosphodiesterase Heterogeneity in the VNS of the mouse has also been reported using antibodies to the cAMP-specific phosphodiesterase isoforms, PDE4A and PDE4D (Cherry and Pho, 2002; Lau and Cherry, 2000). PDE4A-ir was found in apically situated receptor cells in the VN sensory epithelium and in the GL of the rostral AOB (Lau and Cherry, 2000); PDE4D was found in basally situated receptor cells and in the GL of the posterior AOB Cherry and Pho (2002). These observations, in conjunction with others on G-protein-expression and NADPH–diaphorase staining (see Section 3.1.4), suggest that different subdivisions may use somewhat different second messenger system cascades. 3.1.4. NADPH–diaphorase staining and GABA-immunoreactivity The heterogeneity in the VNS can be demonstrated in some mammals using NADPH–diaphorase histochemistry— a marker for oxido-reductive enzymes that require NADPH as a co-factor. Nitric oxide synthase is such an enzyme. In opossum (Shapiro and Halpern, 1998) and mouse (Halpern and Jia, 1995; Halpern et al., 1998a) there is a heterogeneity in NADPH–diaphorase activity in the VNO and AOB. The apical VN sensory epithelium and GL of the anterior AOB are more reactive than the basal epithelium and GL of the posterior AOB. In contrast, no such differential NADPH–diaphorase activity is observed in the VNO or AOB of the rat (Dantzer, 1998; Halpern et al., 1998a; Porteros et al., 1994) or hamster (Davis, 1991). The significance of differential activation of an activity marker such as NADPH–diaphorase is presently unknown. It is possible that areas of high enzyme activity, e.g. nitric oxide synthase, are localized with NADPH–diaphorase, reflecting high functional activity similar to neurotransmitter release. As mentioned above, it is also possible, in the animals showing differential expression, that different transduction and/or transmission pathways are utilized by the different subdivisions and this is reflected in the heterogeneity in the presence of the marker. In the opossum, differential staining with NADPH– diaphorase in the AOB is developmentally regulated. At 30 days of age this staining is homogeneous, by 45 days of age it has attained its differential pattern (Shapiro and Halpern, 1998). Development of differential NADPH–diaphorase staining patterns has not been studied in other mammals. Using NADPH–diaphorase histochemistry, NOS-ir or NOS in situ hybridization the VNS has been examined as well in amphibians (González et al., 2002; Porteros et al., 1996), reptiles (Jiang and Terashima, 1996) and mammals (Alonso et al., 1995; Kishimoto et al., 1993b; Kulkarni et al., 1994; Matsuda et al., 1996; Nakajima et al., 1998b; Nasu and Haneji, 2002; Zancanaro et al., 2002).

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Interspecies differences have been observed in all groups studied and none of these studies report heterogeneity in NADPH–diaphorase activity. There has been considerable speculation about the relation of NADPH–diaphorase activity to function in the VN and olfactory systems, however no reliable data are currently available on this issue. In the rat, which does not show differential expression of NADPH–diaphorase activity, there is a differential expression of GABA-ir in the AOB (Takami et al., 1992b). There are more GABA-immunoreactive periglomerular neurons in the posterior AOB than in the anterior AOB. The significance and generality of this finding have yet to be determined. 3.2. Developmental markers 3.2.1. Glycoconjugates Monoclonal antibodies generated from the VNO or olfactory bulb have been used to visualize a variety of structures or substructures in the VNS and to examine ontogenetic development of the peripheral VNS of the African clawed frog, Xenopus laevis (Petti et al., 1999), rabbit (Onoda, 1988) and rat (Allen and Akeson, 1985; Carr et al., 1989; Osada et al., 1994; Yoshida et al., 1995; Yoshida-Matsuoka et al., 1999). These monoclonal antibodies have distinct staining patterns within the VNO. The heterogeneity in the VNS has also been demonstrated using antibodies to glycoconjugates (for earlier review see Mori, 1993). Monoclonal antibodies CC1 and CC6, raised against different glycolipids, stain the rostral and caudal AOB of the rat, respectively (Schwarting and Crandall, 1991; Schwarting et al., 1994). As with VVA lectin in opossums, the differential staining is developmentally regulated and may be important in targeting of axons to their appropriate termination site in the AOB (Schwarting et al., 1992). In rabbits, monoclonal antibodies (Mabs) generated in mouse from rabbit olfactory bulb differentially stain the rostrolateral AOB (Mab4C9 and MabR4B12) (Mori, 1987a; Mori et al., 1987) or the caudomedial AOB (MabR5A10) (Imamura et al., 1985; Mori et al., 1987). The glycoprotein identified by MabR4B12 is a member of the neural cell adhesion molecule (NCAM) family and was renamed (olfactory cell adhesion molecule (OCAM)) (Yoshihara et al., 1997). In the mouse, OCAM mRNA is expressed exclusively in the apical zone of the epithelium and rostral zone of the AOB. OCAM is differentially expressed in two subsets of M/T cells during development. Although OCAM-ir in the NL and GL of the AOB are expressed in the rostral subdivision of the bulb, M/T cells in the caudal AOB are OCAM-immunoreactive during early development (E13–P24) when the axons of VN receptor cells are making synaptic contact with the M/T cell dendrites in the glomerular layer. Thus, OCAM-positive M/T cells in the caudal zone receive inputs from OCAM-negative VN axons and vice versa for the M/T cells in the rostral zone. From P24 on, there is a decrease in OCAM-ir in M/T cells. During development the boundary between the OCAM-positive

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and OCAM-negative zones sharpens, becoming distinct by P15. These results suggest that OCAM may be involved in the development of zonal projections between the VN epithelium and the AOB (von Campenhausen et al., 1997). Rb-8 neural cell adhesion molecule (RNCAM), a novel mouse protein, has structural features and overall sequence identity that place it in a subgroup of cell adhesion molecules to which neural cell adhesion molecule, Aplesia cell adhesion molecule and Drosophila fasciculin II belong. RNCAM expression in the VNO is restricted to neurons in the Gi2␣ -expressing zone (Alenius and Bohm, 1997). It thus appears, as in other systems, that carbohydrate moities are likely to be important in cell–cell recognition and adhesion and cell–substrate attraction and repulsion (see also Section 6). 3.2.2. Neurestin The expression of neurestin, a putative transmembrane molecule, is spatially segregated in the AOB. In situ hybridization in developing Sprague–Dawley rats demonstrated expression as early as embryonic day 17 (E17) in the far caudal AOB. The expression level increased to P1 and declined by P3. The neurestin-positive area of the AOB was not co-extensive with the Go␣ -immunoreactive portion of the AOB, being more restricted (Otaki and Firestein, 1999). 3.2.3. Ephrins Investigation of the ephrin-A (Eph-A) family of receptor kinases during development of the mouse VNO has revealed that the ligand ephrin-A5 (Eph-A5) is differentially expressed in the VNO and the receptor Eph-A6 has a graded expression in the AOB (Knöll et al., 2001). Ephrin-A5 is expressed in high concentrations in the apical neurons of the sensory epithelium while the anterior AOB expresses high levels of the Eph-A6 receptor. In a stripe assay, VN axons preferentially extend along lanes with Eph-A7-Fc/laminin as compared to lanes with Fc/laminin. In ephrin-A5−/− knockout mice, ephrin-A molecules are not expressed on VN axons and the preference of axons originating from apical VN receptor cells for the anterior AOB is lost. Thus, ephrin-As appear to act as axon guidance molecules in the projection from VNO to AOB. 3.3. Markers of unknown function 3.3.1. Olfactory marker protein Olfactory marker protein (OMP), a 19 kDa cytoplasmic protein, is present in mature olfactory and VN receptor cells and in the main olfactory bulb (MOB) and AOB of most species studied to date. The function of OMP is, at present, unknown. In mammals OMP is usually present in all VN receptor neurons, however, in the ferret, Mustel putorius, not all receptor cells are OMP-immunoreactive, and those that are immunoreactive are weakly stained (Weiler et al., 1999b). Antibodies to OMP stain the opossum anterior AOB more intensely than the posterior AOB. This differential

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staining is reflected in the VN epithelium where apically located VN receptor cells are more intensely stained than the more basally situated cells (Shapiro et al., 1997; Shnayder et al., 1993). As with VVA lectin, this differential staining is developmentally regulated, appearing most clearly at 2 months of age (Shapiro et al., 1997). Interestingly, in no other mammal has differential OMP-ir been reported in the VNS. Although the role of OMP in chemosensory transduction is currently unknown, the heterogeneity of its expression in the opossum VNS could lead to the design of studies that will further our understanding of OMPs function. 3.3.2. Lectins Lectin staining has been used to identify the sugar moities present in the VNO and AOB. A heterogeneity has been demonstrated in the presence of carbohydrate epitopes in different parts of the VNS as revealed by use of lectins and carbohydrate-binding proteins. In different species, the same lectin stains the anterior or posterior AOB differently. For example, in the rat, Takami et al. (1992a) found that the lectin V. villosa agglutinin (VVA) stained the posterior two-thirds of the NL and GL strongly, but the anterior AOB weakly. In contrast, in the hamster (Taniguchi et al., 1993a) and opossum (Shapiro et al., 1995b), VVA staining was strong anteriorly in the AOB, but weak posteriorly. Additional studies have demonstrated differential lectin staining in rat (Sugai et al., 2000), hamster (Taniguchi et al., 1993a) and mouse (Salazar et al., 2001) using a variety of lectins. The lectins used in these studies differed as did the distribution of the staining. In most cases lectin staining revealed further subdivisions in the posterior and, occasionally, in the anterior AOB than was originally described. It now seems clear that a division of the AOB into anterior and posterior divisions may be too simple and further subdivision, at least of the posterior portion, will be necessary. Lectin staining patterns appear at different developmental stages. The differential staining pattern of VVA in the opossum appears at about 45 days of age (Shapiro et al., 1995a). In the rat, VVA binding in the AOB is first observed at embryonic day 18 and achieves its position-specific pattern, staining the posterior two-thirds of the NL and GL by the day of birth (P0) (Ichikawa et al., 1994a). Bandeiraea simplicifolia lectin-I (BSL-I) stains the posterior half of the NL and GL at P0, but this staining expands to include the entire NL and GL by P28 when the staining resembles the adult pattern. Not all studies of lectin staining in the VNO–AOB system have revealed a heterogeneity in the system. Table 1 lists the studies reported since 1987, including the species studied and the lectins examined (see abbreviation in Table 1). It is beyond the scope of this review to summarize all of the reported findings. However, several general statements can be made concerning these studies. There is a surprising consistency in staining patterns between divergent species, yet also interesting species variation. For example, although SBA stains the NL and GL of the AOB in rat (Barber, 1989;

Salazar et al., 1998), mink (Salazar et al., 1998), Xenopus borealis (Key and Giorgi, 1986), Xenopus laevis (Hofmann and Meyer, 1991) and eight species of frog (Meyer et al., 1996), it does not stain these structures in the dog (Salazar et al., 1992). Many lectins with different sugar affinities stain the NL and GL of the AOB. Thus, UEA1, LEA, SBA and BSL-I-B4 all stain these structures in rat (Salazar and Sánchez Quinteiro, 1998), LEA and UEA1 stain these structures in pig (Salazar et al., 2000), LEA and DBA stain these structures in sheep (Salazar et al., 2000), UEA1 stains these structures in the dog (Salazar et al., 1992) and PCT, PNA, GS-I-B4 , VVA, HA, SBA and DBA stain these structures in the opossum (Shapiro et al., 1995b). Some lectins selectively stain the AOB, but not the MOB, e.g. in mouse (Salazar et al., 2001), DBA, BS-I-B4 and UEA1 stain the AOB but not the MOB. In opossum (Shapiro et al., 1995a) VVA and GS-I-B4 stain the AOB but not the MOB. In the rat VN sensory epithelium, the mucomicrovillar complex can be visualized with a number of lectins— sWGA, DSA, BS-I-B4 , VVB4 (V. villosa isolectin B4 ) and DBA (Takami et al., 1994). Using confocal laser scanning microscopy, two components of the mucomicrovillar complex can be resolved by double labeling with different lectins, a sensory and a mucoid component (Takami et al., 1995). Thus, with respect to lectin staining and carbohydrate localization, among mammals there are distinct species differences. On occasion lectins that recognize the same carbohydrate epitopes yield different patterns in the same animal. Furthermore, although a carbohydrate moiety may be present early in development, its differential expression may be developmentally controlled and may not be evident prior to maturation. Since at present the function of lectins is unknown, further comments on the functional significance of both the heterogeneous expression in the VNO and species differences in that expression serves little purpose. 3.4. Central projections The dichotomous nature of the VNS extends to the secondary neurons in the system, the M/T cells. In opossums, the dendrites of M/T cells situated in the anterior AOB project their primary dendrites exclusively to glomeruli in the anterior AOB; conversely, dendrites of M/T cells situated in the posterior AOB project their primary dendrites exclusively to glomeruli in the posterior AOB (Jia and Halpern, 1997) (Fig. 1). The lateral dendrites of these M/T cells, however, cross the anterior/posterior boundary providing, in addition to the processes of periglomerular cells, an avenue for “cross-talk” between the two subdivisions. The further projections of this system into the telencephalon show an element of differentiation in opossum but not in mouse. Although the axons of M/T cells from the anterior and posterior AOB overlap considerably throughout

Species (reference) Salamander (Franceschini et al., 2003) Frog-8 species (Meyer et al., 1996) Xenopus borealis (Key and Giorgi, 1986) Xenopus laevis (Hofmann and Meyer, 1991) Pseudemys scripta (Franceschini et al., 1996) Monodelphis domestica (Shapiro et al., 1995b) Pig (Salazar et al., 2000) Sheep (Salazar et al., 2000) Mink (Salazar et al., 1998) Rat (Moreno et al., 1998, 1999) Rat (Salazar et al., 1998) Rat (Barber, 1989) Rat (Franceschini et al., 1994) Rat (Ichikawa et al., 1992) Rat (Takami et al., 1992a, 1994, 1995) Rat (Ichikawa et al., 1994b) Mouse (Lundh et al., 1989) Mouse (Plendl and Schmahl, 1988) Mouse (Salazar et al., 2001) Dog (Salazar et al., 1992) Marmoset (Nakajima et al., 1998c)

BS-I-B4 BSA (BSA-1-B4 )

BSL-I CON A DBA DSA ECA ECL HA LCA LEA LEL LTA PCT PNA PSA RCA1 SBA ST or STL UEA1 VVA WGA (9S-WGA)

×

× × × ×

×

× × ×

× × ×

×

×

×

×

×

×

× × ×

× × × × ×

×

×

× ×

×

× ×

× × × ×

×

×

× ×

×

×

×

×

×

×

×

× ×

×

× ×

×

× × × × × ×

× ×

× × ×

×

×

×

×

× ×

×

×

× × ×

×

×

×

× × ×

×

×

Abbreviations: BS-I-B4 , Bandeiraea simlliciforia (also known as Griffonia simplicilofia lectin-I-isolectin B4 ); BSA (BSA-1-B4 ), Bandeiraea simplicifolia (its B4 isomer); BSL-I Bandeiraea simlliciforia lectin-I; CONA, conconavalin A; DBA, Dolichos biflorus agglutinin; DSA, Datura stratonium; ECA, Erythrian crista galli agglutinin; ECL, Erythrina cristagali lectin; HA, Helix aspersa; LCA, Lens cunicularis; LEA, Lycopersicon esculentum; LEL, Lycopersicum esculentum lectin; LTA, Lotus tetragonolobus agglutinin; PCT, Psophocarpus tetragonolobus; PNA, Arachis hypogaea (peanut agglutinin); PSA, Pisum sativum; RCA1, Ricinus communis agglutinin; SBA, soybean agglutinin (from Glycine max); ST or STL, Solanum tuberosum; UEA1, Ulex europeus agglutinin; VVA, V. villosa agglutinin; WGA (9S-WGA) wheat germ agglutinin or the succinylated form.

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Table 1 Lectins used in studies of the VN system

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the VN-recipient amygdala, in the ventral division of the medial amygdala of opossum, axons of posteriorly situated M/T cells terminate more deeply than those of anteriorly situated M/T cells (Mart´ınez-Marcos and Halpern, 1999a). It is difficult to determine at this point how important these distinctions are for the functioning of the system, although it has been suggested that different physiological effects could result from differential input to the cell layers (layers 2 and 3) of the ventral division of the medial amygdala as compared to inputs reaching only distal dendrites in layer 1 (Mart´ınez-Marcos and Halpern, 1999a) (Fig. 7). In a similar vein, most of the telencephalic cell groups that send centrifugal efferents to the opossum AOB project to both its anterior and posterior divisions; however, neurons in the posteromedial cortical amygdaloid nucleus project to the posterior division exclusively (Mart´ınez-Marcos and Halpern, 1999b) (Fig. 7). In contrast to the situation in opossum, the distribution of axonal terminations of rostrally situated M/T cells is indistinguishable from the distribution of axonal terminations of caudally situated M/T cells in the mouse. Using biotin-, rhodamine-, and fluorescein-conjugated dextrans, von Campenhausen and Mori (2000) found that injections into the anterior or posterior AOB resulted in an identical pattern or labeling in the bed nucleus of the accessory olfactory tract (BAOT), medial amygdaloid nucleus, posteromedial cortical amygdaloid nucleus and bed nucleus of the stria terminalis (BNST). This latter finding has been confirmed in mice using the lipophillic tracer DiI (Salazar and Brennan, 2001). As expected, local injections of DiI into the antero-dorsal and postero-ventral divisions of the medial amygdala and into the postero-medial cortical amygdala produced no evidence for differential projections from the anterior and posterior subdivisions of the AOB in mice. Injections affecting the deeper layers of the medial amygdala do not show any labeling in the AOB. In rats, however, injections affecting layers 2 and 3 of the medial amygdala give rise to retrogradely labeled cells in the AOB (Luiten et al., 1985). Thus, it would appear that there is a true species difference in this projection pattern that should be explored further (see Section 7). 3.5. Physiological responses Physiologically, a heterogeneity in the anterior and posterior divisions of the AOB has been demonstrated in the guinea pig (Sugai et al., 1997) and rat (Sugai et al., 2000). Field potentials generated by stimulation of the NL of the anterior AOB are restricted to the external plexiform layer of the anterior AOB. Conversely, field potentials generated by stimulation of the NL of the posterior AOB are restricted to the external plexiform layer of the posterior AOB. Using real-time optical imaging, it was possible to demonstrate that the spread of neural activity was restricted to each subdivision of the AOB but extended within each subdivision to all of its layers. In the rat, the posterior AOB can be subdivided

into a rostral two-thirds and caudal one-third on the basis of lectin (R. communis agglutinin) histochemistry. Using electrophysiological and optical recordings, this subdivision can be demonstrated to be a functional subdivision (Sugai et al., 2000). The different subdivisions of the VNS have been shown to be sensitive to different aspects of pheromonal signals in mammals. Urine contains a number of components that act as pheromonal signals that activate the VNS. These studies include examination of electrophysiological activity in the different classes of receptor cells in response to urine from different types of donors or various urine components (see Sections 5 and 8), as well as differential activity in the subdivisions of the AOB. In many of these studies the activity in the subdivisions of the AOB was monitored using expression of the immediate early genes fos and egr1. As observed using an on-cell patch-clamp, cells in different sublayers of the VN sensory epithelium respond differentially to urine derived from conspecifics of the same sex, opposite sex, congenerics and animals of different genera. Urine from Wistar male rats causes an increase in impulse frequency in VN receptor cells located in the apical sublayer of the sensory epithelium of female Wistar rats, i.e. the Gi2␣ -protein expressing zone (Inamura et al., 1999a). In contrast, urine derived from male Donryu rats, Sprague–Dawley rats, C57BL/6 mice, Syrian hamsters or female Wistar rats cause an increase in impulse frequency in the basal sublayer of the sensory epithelium of female Wistar rats. Furthermore, most VN receptor cells responded to only one type of urine. These results suggest that cells are responding to something in the urine that signals strain, species and sex (Inamura et al., 1999a). Inward currents were recorded in VN sensory neurons of female Wistar rats in response to urine. Male Wistar rat urine activated cells in the apical sublayer, whereas urine from Donryu males or female Wistar rats activated cells in the basal sublayer (Inamura and Kashiwayanagi, 2000a). Thus, it would appear that bipolar neurons in the apical sublayer respond to conspecific signals from the opposite gender and those in the basal layer to signals from heterospecifics and same-sex conspecifics. However, different urine fractions taken from the same animal source may differentially activate the two classes of VN sensory neurons. Different fractions of male urine cause activation of Gi or Go in membrane preparations taken from female rat VNO. Lipophilic volatile odorants activate Gi -proteins whereas a major urinary protein, ␣2u -globulin, activates Go -proteins (Krieger et al., 1999). As noted above, G␥8 is localized to the basal region of the VN sensory epithelium. G␥8 -specific antibodies attenuate IP3 generation elicited by ␣2u -globulin, whereas antibodies to G␥2 , which is localized to the apical zone of the VN sensory epithelium, attenuates IP3 generation elicited by lipophilic volatile urinary odorants (Rünnenberger et al., 2002) (see also Section 5). These varying findings suggest caution in interpreting the differential activation of VN sensory neurons with urine from different sexes or different urine fractions.

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3.6. Immediate early gene expression Nuclear proto-oncogenes such as fos, jun and egr1 are referred to as immediate early genes because their transient induction is one of the first changes in gene activity that occurs in response to extracellular stimulation. Immunohistochemistry for the Fos, Jun and Egr-1 proteins or in-situ hybridization for their respective mRNAs has been used to evaluate the efficacy of sensory stimuli in a broad range of sensory systems, including the olfactory and VN systems. In the VNS, this technique has been used by a number of investigators to determine if VN stimulants differentially activate Gi - or Go -expressing receptor cells, or the anterior or posterior AOB. However, it has become clear that results of experiments using immediate early gene expression to identify structures or substructures involved in particular behaviors are often contradictory. Problems of interpretation of results may be due to experimental design faults, lack of sensitivity of the method, differences in the time course of early gene activation in different classes of neurons and a general absence of understanding why some cells show early gene expression and others do not. Bedding soiled by intact male mice induces c-fos expression in the AOB of female mice. The number of c-Fos-expressing mitral cells is significantly higher in the anterior AOB than in the posterior, as it is for male and female mice that are mated (Halem et al., 1999). Similarly, male mice of the ICR strain exposed to bedding soiled by ICR females exhibit induction of c-fos expression in the anterior AOB primarily (Dudley and Moss, 1999). In a more recent study (Halem et al., 2001a), gonadectomized, steroid-treated mice were exposed to male- and female-soiled bedding and the resulting increases in immediate early gene (c-Fos and Egr-1) expression analyzed in the VN epithelium and AOB. Soiled male bedding increased Egr-1-ir to a greater extent in receptor cells in the basal sublayer of the sensory epithelium than in the apical sublayer in estradiol-treated males and females, but not in testosterone-treated males. Although soiled female bedding increased Egr-1-ir in receptor cells in the VN sensory epithelium in testosterone-treated males, there was no differential response in the apical and basal sublayers of the epithelium. Both soiled male bedding and soiled female bedding increased c-Fos-ir in the mitral cell and granule cell layers of the AOB, with a greater increase being observed in the rostral AOB. Since the basal cell layer of the sensory epithelium projects to the posterior AOB, these results are difficult to interpret. Confirmation of these results and further studies on the mechanisms of induction of immediate early genes will help in future interpretations of these findings. Male ICR mice exposed to conspecific and heterospecific female bedding exhibit increased c-Fos-ir in the AOB (Kumar et al., 1999; Matsuoka et al., 1999). The increased c-Fos-ir is greater when the male mice are exposed to conspecific than to heterospecific (BALB) female bedding. However, the difference between conspecific and heterospe-

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cific activation in the AOB is primarily in the rostral AOB. Males exposed to conspecfic female bedding have more c-Fos-immunoreactive cells in the rostral than the caudal GL and granule cell layer. No such difference between c-Fos-ir in the rostral and caudal AOB laminae is seen when the ICR males are exposed to female BALB bedding (Matsuoka et al., 1999). ICR male mice involved in aggressive encounters with male BALB/c mice demonstrate a greater increase in c-Fos-ir in the posterior AOB compared to the anterior AOB (Kumar et al., 1999). The c-Fos activation does not require displays of aggression, as it can be demonstrated even when the stimulus animal is anesthetized. These results suggest that different parts of the VNS are involved in interstrain recognition and intrastrain sexual signal detection. Different components of urine such as the small volatile molecules, 2,3-dihydro-exo-brevicomin (DHB) and 2-secbutyl-4,5-dihydro-thiazole (SBT), activate parts of the AOB different from those activated by the major urinary proteins (MUPs) to which these small volatiles are normally bound (Brennan et al., 1999). Stimulation of female mice with DHB and SBT results in Egr-1 activation in clusters of presumed mitral cells in the medial and lateral margins of the posterior AOB, whereas stimulation with whole urine or MUPs results in activation of mitral cells in the anterior AOB and posterior AOB clusters. These results suggest that anterior and posterior AOB divisions are processing different aspects of pheromonal signals. Brennan et al. (1999) suggest that the anterior AOB is likely to process signals concerned with strain recognition, information for which could be imparted by the MUPs, citing evidence that male pheromone recognition is determined by the pattern of activity across the population of mitral and granule cells. They state that it is probable that the MUPs to which volatiles are bound carry the code for individual recognition, the volatiles themselves being incapable of coding for individual characteristics (Brennan et al., 1990; Keverne, 1998). See Section 8 for further discussion of urine components and their effects on vomeronasally-mediated behaviors. From these studies, one can conclude that a number of factors are involved in determining the cellular response to chemical signals in neurons of the VNS. These include the gender, strain, hormonal status of the odor donor and the odor recipient as well as the chemical composition of the odor stimulus. Fewer studies on the effects of urine on activation of immediate early genes have been conducted in rats. However, the results of these few studies confirm a heterogeneous response in the AOB to conspecific urine. Male Wistar rat urine activates twice as many cells in the M/T layer of the female anterior AOB as in the posterior AOB (Inamura et al., 1999b). Female urine does not produce such an obvious differentiation between anterior and posterior activation. Overall there are fewer cells activated by female urine and an approximately equal number activated in anterior and posterior subdivisions.

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Male rat urine treated with papain induces c-fos expression only in the anterior AOB, while urine treated with pronase abolishes all c-Fos induction. These results suggest that there are at least two proteins in male rat urine that activate the VNS, a papain-sensitive protein that activates the posterior AOB and a papain-insensitive protein that activates the anterior AOB (Tsujikawa and Kashiwayanagi, 1999). Regardless of the source of the odor stimulus, in most studies of odor-induced Fos-ir in the rodent AOB which compare rostral versus caudal labeling, significantly more Fos-ir cells are seen in the rostral than in the caudal AOB. This is most true for Fos-ir cells in the granule cell layer, but also in the mitral cell layer. This observation may be due to activity of lateral dendrites that cross the anterior/posterior boundary or, as mentioned above, may reflect our lack of understanding of the mechanisms of immediate early gene induction. These findings in mouse and rat leave unresolved the issue of the nature of the stimulus that produces differential responding in the neurons of the AOB. Is it the source of the urine? Or is it the urine component that determines which portion of the AOB will be activated? Further studies are needed to resolve these issues. The studies reviewed here concerning heterogeneity in the VNS have used a variety of approaches yet all point to similar conclusions. From anatomical, molecular or physiological points of view, the VNS can, in most mammalian species, be dichotomized or parceled into multiple subdivisions. Receptors, signal transduction systems and functional distinctions all confirm that the VNS is heterogeneous. Despite much progress, many questions remain to be investigated including identifying the characteristics of stimuli that activate the subdivisions of the VNS and how they are similarly or differently transduced and eventually result in distinguishing behavioral responses by intact animals.

4. Discovery of multiple families of putative VN receptors As a result of the recent identification of two families of putative VN receptors, new physiological issues have emerged and new molecular approaches have been applied to study this system. The cloning of the genes encoding VN receptor proteins, the implication of these findings for the field of chemosensory research and the questions raised by these discoveries have been reviewed extensively (Bargmann, 1997, 1999; Buck, 1995, 2000; Dulac, 1997, 2000a,b; Dulac and Axel, 1998; Keverne, 1999; Liman, 1996, 2001; Mombaerts, 1999a,b; Ryba, 1999; Sullivan, 2002; Tirindelli et al., 1998). The characterization of candidate VN receptors followed the identification of olfactory receptors. Therefore, we first discuss the identification of olfactory receptors to provide the scientific context that led to the isolation of putative VN receptors.

4.1. Olfactory receptors Unlike the visual or auditory systems, whose basic sensory codes have been known for decades, the principles of chemosensory coding remained elusive until just a decade ago (e.g. Farbman, 1992; Reed, 1990). A vast literature on sensory coding in the olfactory system existed prior to the discovery of the odorant receptor gene family (e.g. Kauer, 1991; Kauer et al., 1987; Mori, 1987b; Stewart et al., 1979). However, as recently as the early nineties, the existence of olfactory receptors was suspected, but none had been identified. Based on previous data, three premises led to the characterization of such receptors: olfactory receptors were likely to be coupled to G-proteins; the large number of discriminable odor molecules suggested a large number of different odorant receptor types, likely to be encoded by a multigene family; and the expression of such receptors would be restricted to the olfactory epithelium. In 1991, a paradigmatic discovery resulted in a new perspective in the understanding of olfactory perception. The cloning of a multigene family of olfactory G-protein-coupled receptors (GPCRs) provided a molecular basis for odor recognition (Buck and Axel, 1991). The importance of this discovery was immediately recognized (Dodd and Castellucci, 1991). Whereas color vision is achieved by three different classes of cones, each containing a different photopigment, olfactory recognition is based on the existence of a family of approximately 1000 genes coding for seven transmembrane domain olfactory receptors. Each olfactory receptor appears to show high specificity for certain molecular features, but high tolerance for others (Araneda et al., 2000), which suggests a new complex system of sensory coding. The characterization of the multigene family of olfactory receptors (Ben-Arie et al., 1994; Breer et al., 1998; Buck and Axel, 1991; Parmentier et al., 1992; Raming et al., 1993; Touhara et al., 1999) together with analysis of expression patterns of these genes (e.g. Ressler et al., 1993; Vassar et al., 1993) provided a model for olfactory coding. Each neuron in the olfactory epithelium is likely to express only one or very few of the 1000 receptor genes (Chess et al., 1994; Kishimoto et al., 1994). Different receptor genes are rarely expressed simultaneously in individual olfactory neurons (Rawson et al., 2000; Serizawa et al., 2000). Neurons expressing the same receptor are generally distributed within one of four zones in the olfactory epithelium (Conzelmann et al., 2000; Ressler et al., 1993; Strotmann et al., 1992, 1994a,b, 1995, 1999; Vassar et al., 1993) and project their axons to a few glomeruli in the olfactory bulb (Mombaerts et al., 1996; Ressler et al., 1994; Strotmann et al., 2000; Vassar et al., 1994; Wang et al., 1998). Although there are exceptions to this general rule, this arrangement provided, for the first time, a topographic organization for olfactory perception (see Shepherd, 1994; Axel, 1995; Mombaerts, 1996, 1999b and Buck, 1996 for reviews). There has been only limited progress, however, linking specific ligands with their putative olfactory receptors. Zhao

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et al. (1998) transfected odorant receptor I7 to rat olfactory sensory neurons and found elevated electrophysiological responses to n-octanal (reviewed by Reed, 1998). Subsequently, three additional receptors were linked to specific odorants (Krautwurst et al., 1998). The relation between odorants and olfactory receptors, however, appears not to be one-to-one but follows a combinatorial coding pattern (Buck, 2000; Malnic et al., 1999). Recently, calcium imaging experiments coupled with gene-targeting have permitted the simultaneous demonstration of functionally expressed receptors, odorant response properties and the pattern of connections in the MOB, thus defining functional units in the olfactory system (Bozza et al., 2002).

4.3. Second family of putative vomeronasal receptors In 1997, three research groups simultaneously cloned a second family of putative VN receptors (Herrada and Dulac, 1997; Matsunami and Buck, 1997; Ryba and Tirindelli, 1997). This second family of putative VN receptors (V2R) did not have significant homology to either olfactory receptors or the first family of putative VN receptors (V1R). They were, however, related to the Ca2+ -sensing receptors and metabotropic glutamate receptors. The Buck and Dulac groups looked for genes in Go␣ -protein expressing VN neurons, whereas Ryba’s group probed a VNO cDNA library with a mixed probe of olfactory receptors to identify the second family of putative VN receptor genes

S

Lumen

V1R V2R Microvilli

4.2. First family of putative vomeronasal receptors The VNS shares a number of characteristics with the olfactory system since both are chemosensory systems with similar receptor cells and parallel pathways to the brain. However, the detailed organization of the olfactory and VN systems is quite different. In fact, attempts to identify genes encoding VN receptors by homology to genes encoding olfactory receptors were unsuccessful (Dulac and Axel, 1995). Therefore, a different strategy for isolating VN receptors was applied, making two assumptions: expression of VN receptors should be restricted to the VN epithelium; and individual VN neurons should express different receptors. Comparing cDNA libraries from single VN neurons (cDNAs present in one library but not in another) allowed the characterization of a novel family of 30–100 genes encoding mammalian putative VN receptors (VN1, later called V1R) (Dulac and Axel, 1995). These genes encoded seven transmembrane domain protein receptors, which were likely to be coupled to G-proteins, and distantly related to genes of olfactory receptors. The expression of mRNAs for those genes characterized by in situ hybridization using V1R probes, revealed that these receptors were expressed in a subpopulation of VN neurons more or less restricted to the apical portion of the receptor cell layer of the VN sensory epithelium (see Buck, 1995; Dulac, 1997; Dulac and Axel, 1995 for reviews) (Fig. 3) (see Section 3).

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Luminal Surface Membrane Intracellular Space Microvilli

V1R/Gi2α

Luminal Surface Membrane Intracellular Space

V2R/Goα

Fig. 3. Schematic diagrams showing the location in the vomeronasal epithelium and structure of V1R and V2R putative vomeronasal receptors. Abbreviation: S, supporting cells.

(see Bargmann, 1997) (Fig. 3). The three groups used different nomenclatures, but V1R and V2R have been used largely for the first and second families of putative VN receptors, respectively (Bargmann, 1997). By comparing gene expression in mouse single VN neurons, a novel multigene family of about 140 members encoding candidate VN receptors located in Go␣ -expressing VN neurons was identified (Matsunami and Buck, 1997). Each V2R gene was found only in a small percentage of Go␣ -protein expressing VN neurons. Although all three families of olfactory and VN receptors were members of the GPCR superfamily, olfactory receptors and the V1R family of VN receptors had a predicted structure consisting of seven membrane-spanning domains with a relatively short N-terminal extracellular domain, whereas members of the V2R family had an extremely long N-terminal domain. In V2Rs, by analogy to calcium-sensing and metabotropic glutamate receptors, this domain could interact with ligands, whereas in olfactory and V1R receptors

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ligand binding would be predicted to occur in the pocket formed by the transmembrane domains. Accordingly, these two classes of receptors could recognize different types of VN stimuli (Fig. 3). This second family of putative VN receptors (V2R) was identified also in rats by using a similar approach (Herrada and Dulac, 1997). This family was composed of about 100 genes. One of these receptors showed a sexually dimorphic distribution. In females, Go␣ -VN2-positive cells were mostly located in the center of the epithelium, whereas in males cells expressing such receptor were apically situated, probably intermingled with Gi2␣ -protein-expressing cells. This sexually dimorphic distribution could constitute a molecular basis for differential sensitivity to reproduction-related pheromones in male and female VNSs and this possibility should be explored in the future. Since the probe used in this experiment could detect several receptors genes, these results should, however, be interpreted with caution (Bargmann, 1997). One surprising observation by two previous reports was that about two-thirds of identified genes corresponded to pseudogenes. Finally, Ryba and Tirindelli (1997) succeeded in isolating, also in rats, this second family of 30–100 putative VN receptors by homology screening using an olfactory receptor-based probe. Paradoxically, the V2R family has no significant homology to known olfactory or V1R receptors. The V2Rs are expressed in neurons in the basal portion of VN sensory epithelium corresponding to Go␣ -protein expressing cells. The patterns of hybridization, however, revealed an undulating border, with some V2R receptors intermingled with Gi2␣ -protein-expressing cells. Furthermore, Ryba and Tirindelli (1997), by performing double in situ hybridization and observing V2R-expressing neurons surrounded by cells expressing Gi2␣ -protein, suggested the possibility that some V2R receptors were present in Gi2␣ -protein expressing neurons. Comparison between cDNA from VN neurons with probes representing different putative VN receptors of the V2R family, however, do not support expression of V2R receptors in Gi2␣ -protein expressing neurons (Herrada and Dulac, 1997). In situ hybridization studies using probes of V1R and Gi2␣ -protein indicate that V1R receptors are exclusively expressed in Gi2␣ -protein expressing neurons (Saito et al., 1998). Immunocytochemical localization of G-proteins demonstrates a complementary undulating border between Gi2␣ - and Go␣ -protein expressing cells, in which both layers interdigitate in rats and mice. Further double labeling studies should be performed in order to resolve the discrepancies of the complementary distribution of these VN receptors and G-proteins. Although sensory neurons in the olfactory epithelium express only a single receptor, it has recently been reported that VN sensory neurons in the basal sublayer of the epithelium express more than one receptor. Using antibodies raised to several V2Rs, V2R2—a very particular subfamily among V2Rs genes—was found to be broadly expressed in the Go␣ -layer and co-expressed in the same cells as other V2Rs (Martini et al., 2001).

Two families (M10 and M1) of genes encoding molecules of the class 1b major histocompatibility complex (MHC) are co-expressed with V2Rs putative pheromone receptors in the basal zone of the VN sensory epithelium of mice (Ishii et al., 2003; Loconto et al., 2003). The M10s form multimolecular complexes with V2Rs and ␤2-microglobulin (␤2m) (Loconto et al., 2003). Cotransfection of M10 with ␤2m leads to M10 surface expression, and, apparently, interaction between V2Rs and M10 is essential for V2Rs to gain access to the plasma membrane. Thus, M10 molecules may act as escorts of V2R receptors to the cell surface (Loconto et al., 2003). ␤2m−/− mutant mice have a defect in receptor transport such that no V2Rs are present in dentritic terminals. Behaviorally, B2m−/− mutant males fail to demonstrate normal aggressiveness to other males. However, unlike males with non-functional transient receptor potential (TRP) channels, TRP2−/− males, they do not attempt to mate with other males (Loconto et al., 2003) (see Section 5). The implications of these findings are extensively discussed by Thorne and Amrein (2003). 4.4. Additional families of putative vomeronasal receptors? The dual perspective of apical and basal populations of VN neurons expressing two different G-proteins and two different families of putative VN receptors appeared to become more complex since the characterization of a putative third class of candidate VN receptor (Pantages and Dulac, 2000). Comparison of transcripts expressed by olfactory neurons and by apical and basal VN neurons, subsequent construction of cDNA libraries from individual neurons, and differential screening of cDNAs led to the characterization of several related transcripts proposed to constitute a new family of VN receptors named V3Rs. The proposed novel family would encode over 100 putative VN receptors, whose expression would be restricted to the apical portion of the VN sensory epithelium, and whose sequence would be distantly related to the V1R or V2R families of VN receptors. Global analysis of the mouse V1R genomic repertoire, however, indicates that this family is very divergent and it comprises a total of 12 extremely isolated subfamilies, 1 of which would include V3Rs (Del Punta et al., 2000; Lane et al., 2002; Rodriguez et al., 2002). Thus, V3R would not constitute a separate family type of VN receptor. The dichotomous nature of the VNS appears to be preserved at the receptor level as well. The human genome contains several V1R-like genes. Most of these sequences, however, show interrupted reading frames, indicating that they represent non-functional pseudogenes and that the human VNO may have lost the V1R-mediated sensory function of rodents (Giorgi et al., 2000; Kouros-Mehr et al., 2001; Lane et al., 2002). An exception is V1RL1 that possesses an open reading frame (Pantages and Dulac, 2000; Rodriguez et al., 2000) that

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encodes a polypeptide of 313 amino acids, which, interestingly, is expressed in the human olfactory mucosa, raising the possibility of pheromonal detection by the olfactory system (Rodriguez et al., 2000). Attempts to identify and compare V1R functional sequences in different species including humans (Rodriguez and Mombaerts, 2002), New World monkeys (Giorgi and Rouquier, 2002) and goats (Wakavayashi et al., 2002) have shown that these sequences are functional in some species but not in others. This raises the possibility that the V1R family has evolved in such a manner that every species having a functional VNO possesses its own set of functional VN genes.

4.5. Implications of the discovery of multiple families of putative vomeronasal receptors The characterization of the two families of putative VN receptors has raised issues such as the demonstration of functional VN receptors and identification of biologically active ligands of given receptors, evolutionary origin of such families of receptors, and organization of projections from VN neurons to the AOB (Buck, 2000; Dulac and Axel, 1995; Tirindelli et al., 1998). 4.5.1. Receptors functionality Antibodies raised against a synthetic peptide deduced from the VN6 receptor (V1R) bind to the dendritic knobs and microvilli of VN neurons further supporting the idea of functional VN receptors (Takigami et al., 1999). This idea is also supported by a report of activation of a putative mouse VN receptor by male urine (Hagino-Yamagishi et al., 2001). The mouse V1R gene was isolated and introduced into an adenovirus expression vector. Primary culture of VNO neurons prepared from rat embryos on embryonic day 19 were infected and the response of the cells to mouse urine analyzed by calcium imaging. The cells responded to male urine, but not to female urine. The response was attenuated when infected VNO cells were treated with pertussis toxin, a specific inhibitor of Gi /Go receptor coupling, suggesting that Gi2␣ and/or Go␣ are functionally coupled to the receptors. Finally, deletion of a genomic region containing several V1R genes have resulted in deficits in a number of VNO-dependent behaviors. The VNO epithelium of such mice does not respond electrophysiologically to specific pheromonal ligands either (Del Punta et al., 2002a). These latter results also support a role for V1R receptors as pheromone receptors. The most recent support for the idea that V1R genes code for functional VN receptors comes from a study using single mouse VN neurons that express the V1Rb2 receptor. A mouse pheromone, 2-heptone, induces calcium transients and a reversible inward current in these neurons (Boschat et al., 2002). None of the other mouse pheromones or odorants tested stimulated these cells indicating that they were quite specifically activated by 2-heptone (see also Section 5).

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4.5.2. Evolutionary origin of receptors To date, five distinct multigene families of mammalian chemosensory receptors have been reported including olfactory receptors (ORs), VN receptors (V1Rs and V2Rs) and taste receptors (T1Rs and T2Rs). These families encode receptors that belong to the GPCR superfamily, a large and diverse superfamily of receptors that share a core domain consisting of seven transmembrane spanning helices (Sullivan, 2002). Despite a common embryonic origin in the olfactory placode, the lack of homologies between olfactory and VN receptor genes supports the idea of early independent evolution of these two chemosensory systems. Further, the VN receptor subfamily V3R is distantly related to taste receptors T2R, which suggests that all of these genes could have originated from a common ancestor possessing cells detecting water-soluble substances (Pantages and Dulac, 2000). Interestingly, a family of olfactory GPCRs, related to the Ca2+ -sensing receptors in fishes has been characterized, which closely resembles the V2R family of mammalian VN receptors (Cao et al., 1998; Naito et al., 1998; Speca et al., 1999). Collectively, these data question the largely accepted ideas that the VNS originates as an adaptation to terrestrial life and is specialized for detection of non-volatile compounds. This challenge is supported by the recent identification of a VNO in aquatic amphibians, the group in which the VNS is thought to first appear (Eisthen, 1997, 2000). 4.5.3. Receptors map onto the accessory olfactory bulb A topographical organization in the projections of different classes of VN neurons to the AOB was originally demonstrated using immunocytochemical techniques (see Section 3.1.1). This topographical organization was later refined, using gene targeting, by tracing axons of neurons expressing a single putative VN receptor to glomeruli in the AOB (e.g. Belluscio et al., 1999; Rodriguez et al., 1999). In the olfactory system, receptor neurons are believed to express only one or a few of a thousand receptor genes (Rawson et al., 2000; Serizawa et al., 2000). The projections from neurons expressing a specific receptor converge upon 2 of 1800 glomeruli in the olfactory bulb (Mombaerts et al., 1996; Ressler et al., 1994; Vassar et al., 1994; Wang et al., 1998). Each mature mitral cell sends a single apical dendrite to one glomerulus in the olfactory bulb (Mori, 1987b; Shepherd, 1972). Thus, the bulb provides a spatial map reflecting receptors that have been activated by a given odorant (reviewed in Mori et al., 1999). The topography is somewhat more diffuse in the VNS. Axonal projections from both VN2- and VN12-expressing neurons (belonging to the V1R family) were traced to 10–30 glomeruli at various depths within the glomerular layer of the AOB Belluscio et al. (1999). With some exceptions, all of these glomeruli were located in the anterior portion of the AOB, and the pattern was similar across individuals and on both sides of the brain. Using a similar technique with another gene of the V1R family (Vri 2) and two different markers, Rodriguez et al.

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(1999) were able to demonstrate that VN receptors exhibited monoallelic expression. The pattern of projections consisted of axons terminating in multiple glomeruli, exclusively in the anterior portion of the AOB. The pattern, however, was not conserved across individuals and no bilateral symmetry was observed (Rodriguez et al., 1999). Moreover, individual glomeruli in the AOB may receive input from more than one type of VN neuron (Belluscio et al., 1999). Individual glomeruli in the AOB can not always be discriminated and mitral cells possess more than one apical dendrite which can synapse in multiple glomeruli, and therefore with the axon terminals of multiple classes of sensory neurons (Takami and Graziadei, 1991). Thus, integration of information from different receptor types could already occur within glomeruli of the AOB (Belluscio et al., 1999; Rodriguez et al., 1999) (reviewed in Keverne, 1999). Gene deletion experiments have demonstrated that receptor expression is not only necessary for establishment of connections between VN receptor neurons and AOB mitral cells (Belluscio et al., 1999; Gogos et al., 2000; Rodriguez et al., 1999; Wang et al., 1998), but also for cell survival (Belluscio et al., 1999). In contrast to other sensory systems, the absence of odorant-evoked neuronal activity does not appear to perturb the pattern of axonal olfactory projections to the olfactory bulb (Lin et al., 2000). Very recently, it has been demonstrated that the divergent pattern of axonal projections to multiple glomeruli in the AOB is rendered convergent by mitral/tufted cells. Second-order neurons in the AOB send their several apical dendrites to glomeruli innervated by axons of neurons expressing the same V1R or V2R gene. Thus, convergence of a given receptor type occurs within the AOB (Del Punta et al., 2002b). This is supported by electrophysiological experiments in which responses of AOB mitral cells were recorded in behaving mice (Luo et al., 2003). The divergent pattern of projections from VN neurons to the AOB initially predicted that mitral cells would respond to a broad range of stimuli, the responses were, however, exquisitely specific regarding sex and strain of conspecifics (Luo et al., 2003). Recordings, unfortunately, were not restricted to the anterior or posterior portions of the AOB. In some ways, sensory coding in the VNS is more complex than in the olfactory system and several questions remain unanswered (see Bargmann, 1999; Buck, 2000; Dulac, 2000a,b; Keverne, 1999; Ryba, 1999 for reviews). (1) Recently, the posterior portion of the AOB has been subdivided into rostral and caudal functional compartments (Sugai et al., 2000). While it is expected that neurons expressing the V2R family of putative VN receptors project their axons to the posterior portion of the AOB, how are these axons distributed within the rostral and caudal compartments of the posterior portion of the AOB? (2) Recent data have introduced new levels of complexity in the organization of primary VN projections. Based on the patterns of expression of individual genes it was thought that a single VN neuron expressed exclusively one receptor type (e.g. Bargmann, 1997; Pantages and Dulac, 2000). However, co-expression of different V2Rs

in individual VN neurons has recently been demonstrated (Martini et al., 2001), thus complicating the pattern of primary VN projections. (3) Despite these new data, the idea of two segregated VN pathways still appears valid, but raises the intriguing question of how this segregated information in the AOB is relayed to VN recipient centers in the basal telencephalon (see Section 7).

5. New information on signal transduction in the VNS The major issues addressed in the many recent studies on signal transduction in the VNO are the role of different receptor protein families, the differential activation of Gi , Go and Gq proteins, the involvement of the PLC and cyclic AMP (cAMP) second messenger cascades and the importance of the transient receptor potential (TRP) ion channel in the initial activation of calcium signaling. These issues have been reviewed recently by Liman (1996, 2001), Tirindelli et al. (1998), Zufall and Leinders-Zufall (2000) and Zufall and Munger (2001). Arguably, the most completely characterized VNS with respect to signal transduction comes from work on garter snake response to prey-derived chemicals. The ability of garter snakes to respond to chemicals isolated from earthworms, a favorite prey, is dependent on a functional VNS (reviewed previously in (Halpern, 1987)). Application to the VN sensory epithelium of a 20 kDa chemoattractant (Jiang et al., 1990; Liu et al., 1997) purified from earthworm shock secretion (ES20) results in generation of an inward current in VN receptor cells (Taniguchi et al., 2000) and increased firing of mitral cells in the AOB (Jiang et al., 1990). Binding of ES20 to its GPCR results in increased intracellular inositol-1,4,5-tris-phosphate (IP3 ) and decreased cAMP (both basal and forskolin- or GTP␥S-stimulated levels) (Luo et al., 1994). In addition, ES20 receptor binding regulates phosphorylation of membrane-bound proteins with molecular masses of 42 and 44 kDa (p42/44) (Liu et al., 1999). ES20s chemoattractive activity and receptor binding require Ca2+ (Luo et al., 1994) and the decrease in cAMP can be mimicked by increases in Ca2+ (Wang et al., 1997). Adenylate cyclase (AC) type VI (ACvn ), which is sensitive to Ca2+ , has been cloned from a garter snake VNO cDNA library and shows high homology with type VI AC of rat, mouse and human (Liu et al., 1998). Protein phosphatase 2C has been similarly cloned (Wang et al., 2002b). Most recently, using calcium imaging, a release of Ca2+ from IP3 -sensitive intracellular stores as well as an influx of Ca2+ from the extracellular compartment have been demonstrated following ligand binding to VN receptor cells (Cinelli et al., 2002). In addition, calcium-induced calcium release (CICR) has been demonstrated from ryanodine-sensitive stores, as has return of intracellular Ca2+ to prestimulus levels involving a Na+ /Ca2+ exchanger mechanism (Cinelli et al., 2002). Using immunohistochemical techniques, IP3

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Fig. 4. Model of signal transduction in the garter snake (based on data reported in Cinelli et al., 2002). Ligand binding to GPCRs activates phospholipase C increasing intracellular IP3 , resulting in increased cytosolic Ca2+ levels through two different mechanisms: Ca2+ release from internal stores and Ca2+ influx through a putative IP3 -gated conductance in the plasma membrane. There is a further Ca2+ release through a CICR mechanism mediated by a ryanodine-sensitive pool and Ca2+ influx through the activation of VSCC following membrane depolarization. A Na+ /Ca2+ exchanger contributes in limiting the magnitude and duration of these cytosolic Ca2+ elevations by Ca2+ efflux.

receptors, ryanodine receptors and Na+ /Ca2+ exchanger proteins have been localized respectively to dendritic regions, somata regions and throughout VN receptor neurons (Wang et al., 2002a). Thus, the following elements in the VN signal transduction pathway of garter snakes include: G-proteins, AC and PLC, changes in second messengers (IP3 and cAMP), CICR from ryanodine-sensitive stores and a Na+ /Ca2+ exchanger (Fig. 4). Many of these elements have been found in the VNSs of other vertebrates as well (Fig. 5). For example, G-proteins have been found in all VNOs examined, including, for example, porcine (Wekesa and Anholt, 1997), musk turtle (Murphy et al., 2001) and mouse (Berghard and Buck, 1996; Wekesa et al., 2003) VNOs. Although not observed in snakes, in many mammals there is a segregation of receptor cells based on Go␣ - and Gi2␣ -ir or expression of Go␣ and

Gi2␣ mRNAs (see Section 3 and Halpern et al., 1998a,b). Regulators of G-protein-expression, RGS3, RGS9, RGS1 and RG21 are expressed in VN neurons of mice (Norlin and Berghard, 2001). Whereas RGS9 and RGS21 are present in both Gi2␣ - and Go -expressing neurons, RGS3 is co-expressed with Gi2␣ and RGS11 is present in immature VN neurons. Type III IP3 receptors are expressed throughout the receptor cell layer and in the microvillar region of the rat VNO (Brann et al., 2002). Ion channels of the TRP family mediate cyclic nucleotideindependent responses to sensory stimuli, e.g. light-induced depolarization in Drosophila photoreceptors (Pak and Leung, 2003). This is a well-characterized system that could help in the understanding of TRP functions in mammals. cDNA encoding a VNO-specific transient receptor potential (TRP) ion channel (rTRP2) was isolated from rat VNO.

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Fig. 5. Model of signal transduction in mammals (based on data reported in multiple references cited in the text). Ligand binding to GPCRs activates phospholipase C increasing intracellular IP3 and DAG. TRP channels are thought to open allowing influx of Ca2+ into the receptor cell. The increase in intracellular Ca2+ results in release of Ca2+ from ryanodine-sensitive stores. The primary transduction current is dependent on PLC activation.

In situ hybridization with rTRP2 antisense RNA probe revealed a strong signal in VN receptor cells throughout the rat VN sensory epithelium (Liman et al., 1999). Antibodies generated against rTRP2 intensely stain the luminal surface of the VNO (Liman et al., 1999; Menco et al., 2001; Murphy et al., 2001) and only lightly stain the receptor cell body layer, suggesting that they are associated with transduction processes occurring in the microvillar compartment of the sensory epithelium. cDNA of mouse TRPC2 was cloned and expressed in two splice variants, mTRPC2␣ and mTRPC2␤, 886 and 890 amino acids, respectively, in length (Hofmann et al., 2000). TRPC2␤ transcripts were found exclusively in the VNO. TRPC2 and Type III IP3 receptor co-immunoprecipitate from lysates of rat VNO (Brann et al., 2002). The protein–protein interactions thus suggested support the idea that these events initiate calcium signaling in VN receptor neurons (Brann et al., 2002). Genetic ablation

of the TRP2 channel in male mice results in an absence of or reduction in electrophysiologically recorded responses to urine in the VN epithelium as well as a failure to display male–male aggression and inappropriate attempts to mate with male conspecifics (Stowers et al., 2002; Leypold et al., 2002; see Keverne, 2002b; McCarthy and Auger, 2002 for discussion). These striking abnormalities suggest that activation of this cation channel may be an essential early event in VNO transduction of gender-related chemosignals. Adenylate cyclase II is highly expressed in VN neurons expressing both Go␣ and Gi2␣ (Berghard and Buck, 1996). Adenylate cyclase III (ACIII) and an olfactory cyclic nucleotide-gated channel subunit (oCNC2 also known as oCNC␤) have been identified in the VN sensory epithelium of mice. The ACIII is associated with supporting cells and the oCNC2 is associated with receptor cells (Berghard et al., 1996). In the hamster VNO a Ca2+ -activated non-selective

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cation channel has been identified (Liman, 2003). This channel is blocked by the adenine nucleotides ATP and cAMP. Whether this channel is involved directly in sensory transduction or in amplifying a primary sensory response is currently unknown. Other signal transduction elements identified in the olfactory epithelium such as Golf and oCNC1 (also known as oCNC␣) have not been observed in the VN epithelium suggesting that signal transduction in the VN epithelium is distinct from that in the olfactory epithelium (Berghard et al., 1996). Similar findings have been reported in the rat (Wu et al., 1996) in which no mRNA for Go␣ , ACIII or oCNC1 was observed. It would thus appear that only the ␤-subunit of the olfactory cyclic nucleotide-gated channel is generally expressed in the VN sensory epithelium. Is this ␤-subunit of the olfactory cyclic nucleotide-gated channel functional? Membrane patches excised from the soma of VN bipolar neurons are unaffected by application of cAMP or cGMP. However, application of the nitric oxide donor S-nitrosocystein causes immediate channel opening and long bursts of channel openings (Broillet and Firestein, 1997). Thus, the ␤-subunits of the oCNC-channels appear to form functional channels in VN neurons that are activated by nitric oxide, but insensitive to cyclic nucleotides. The presence of cAMP-regulating proteins, e.g. PDE4A and PDE4D (Cherry and Pho, 2002; Lau and Cherry, 2000) and isoforms of AC, ACII (Berghard and Buck, 1996) and ACIV (Liu et al., 1998) in the VNO suggest that cAMP may play a role in VN sensory transduction. As suggested by Lau and Cherry (2000) the lack of PDE4A in microvilli of receptor neurons indicates that cAMP changes may act “downstream” of the initial signal transduction events, modulating activity by either desensitizing neurons or changing the time course of the response. A recent study by Cherry and Pho (2002), using inhibitors of cAMP hydrolysis, provides evidence for the idea that PDE1 and PDE4 isoforms are the primary source for degradation of cAMP in the VNO. Whereas prey chemicals have been shown to activate receptor cells in the snake, in mammals urine, urine products and components of reproductive system secretions have been demonstrated to act on these neurons. The source of the urine may affect the population of VN neurons that are activated (see Section 3). Bipolar neurons in the apical (Gi2␣ -expressing) sublayer of female Wistar rat VN sensory epithelium respond to urine from conspecific males, whereas neurons in the basal (Go␣ -expressing) sublayer respond to urine from heterospecific males and conspecific females (Inamura et al., 1999a). Stimulation of the Wistar female rat VNO with urine from male conspecifics and decreases adenosine diphosphate (ADP) ribosylation of Gi -proteins with pertussis toxin (PTX), whereas urine from male Donryu rats decreases ADP ribosylation of Go -proteins with PTX (Sasaki et al., 1999). These results suggest that responses of females to urine of male conspecifics is via the Gi pathway, while responses of females to urine from male heterospecifics is via the Go pathway. However, recent studies

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(see further and Krieger et al., 1999) suggest that different components within male urine may differentially activate different populations of VN receptor cells. One of the frequently observed effects of VN stimulants on VN receptor cells is an increase in IP3 . Urine from male Wistar rat, female Wistar rat and male Donryu rat all induce increases in IP3 production in membrane preparations from female Wistar rat VNO (Sasaki et al., 1999). Increases in IP3 production have been observed as well in microvillar preparations from the VNO of prepubertal female pigs incubated with boar seminal fluid or urine (Wekesa and Anholt, 1997). Purified aphrodisin, a vomeronasally-mediated mounting pheromone isolated from female hamster vaginal secretions, increases IP3 accumulation in male hamster VN membrane preparations in a concentration-dependent manner (Kroner et al., 1996). In mice, Gq␣ /G11␣ has been implicated in the response of female VN microvillar membranes to male urine (Wekesa et al., 2003). The increase in IP3 observed following incubation with male urine is blocked by the PLC inhibitor U-73122, but not blocked by pertussis toxin, indicating that neither Go nor Gi2␣ are involved in the increase in IP3 . The authors conclude therefore that urine-induced increases in IP3 levels occurs via activation of PLC by the alpha subunit of Gq /G11 . The role of gamma and beta subunits of G-proteins has been recently investigated by Rünnenberger et al. (2002) who found that urine-induced IP3 formation in the mouse VNO was reduced when G␤-subunits were neutralized using a scavenger for beta-gamma dimmers (GST-GRK3ct). It would thus appear that the beta and gamma subunits of G-proteins are important in the generation of IP3 in response to pheromonal substances in mice (see Section 3.1.1). In mammals, the increase in IP3 resulting from stimulation with different VN stimulants is not uniform. Different subsets of cells respond differently depending on the source or nature of the stimulating substance. For example, male urine elicits dose-dependent increases in IP3 in female rat VN membrane preparations (Krieger et al., 1999). Volatile ligands from urine appear to activate neurons that co-express Gi2␣ and V1Rs and non-volatile MUPs, such as ␣2u -globulin, appear to activate neurons that co-express Go␣ and V2Rs (Krieger et al., 1999). VN neurons are known to respond to general, non-biological, odorants. Shoji et al. (1993) recorded large summed responses in the AOB of turtles (Geoclemys reevesii) when the VN epithelium was stimulated with amyl acetate, hedione, citralva and geraniol. Irrigation of the VN epithelium with forskolin also produced large summed responses in the AOB. Desensitizing the cAMP pathway by pretreatment with forskolin did not significantly diminish the response to amyl acetate, suggesting that the cAMP pathway is not necessary for this response. In contrast, pretreatment of the VN epithelium with the IP3 channel blocker ruthenium red significantly reduced the response to amyl acetate, indicating that the IP3 pathway contributes significantly to the response (Taniguchi et al., 1996a).

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Supporting the idea that IP3 is a major component of the signal transduction pathway for the VNS, is the observation that dialysis of IP3 into VN neurons of turtles (Taniguchi et al., 1995), rats (Inamura et al., 1997a) and garter snakes (Taniguchi et al., 2000) under patch-clamp conditions results in transient inward currents. These responses to IP3 are blocked by ruthenium red (Inamura et al., 1997b; Taniguchi et al., 2000). The increases in spike activity of VN receptor cells observed when VNO slice preparations from female rats are treated with male rat urine can be blocked by the PLC inhibitors U-73122 and neomycin as well as ruthenium red, indicating an IP3 -mediated response (Inamura et al., 1997a). Similarly, in mice the response of VN receptor cells to male or female urine is blocked by U-73122 (Holy et al., 2000). However, in addition to IP3 , arachidonic acid (AA) may also be a critical member of the signal transduction pathway for VN response to urine. Using calcium imaging and whole-cell voltage clamp recordings of freshly dissociated rat VNO neurons, Spehr et al. (2002) demonstrated that increases in intracellular calcium in response to urine is dependent on PLC, diacylglycerol (DAG) and AA. Interestingly, in this preparation inhibition of IP3 receptors did not block the response to urine, nor did the depletion of intracellular stores of Ca2+ . Removing Ca2+ from the extracellular fluid did, however, block the response. Increases in intracellular concentrations of IP3 are frequently accompanied by increases in intracellular calcium caused by release of calcium from intracellular stores or influx from the extracellular milieu. Leinders-Zufall et al. (2000) report that six male mouse pheromones activate the VN epithelium of female mice causing increases in intracellular calcium that are dose-dependent. Two of their findings are of particular interest. First, although increasing concentration of the ligand caused an increased in the strength of the calcium transient, they did not observe an increase in the number of responding cells or a change in the distribution of those cells. Second, they found a total loss of responding to the pheromones when extracellular calcium levels were reduced to 0.6 ␮M, which they interpret as indicating that influx of extracellular Ca2+ is required for increased intracellular Ca2+ response (see also Zufall et al., 2002). These results are consistent with those reported by Spehr et al. (2002) in rat (see above) and contrast with those of Cinelli et al. (2002) who found, in the snake VN epithelium, that ligand-induced increases in intracellular calcium occur even in the absence of extracellular calcium. These appear to be species differences which should be investigated in a number of different families. Cyclic AMP levels are reduced in epithelial preparations stimulated with known VN stimulants. For example, cAMP is reduced in the VN sensory epithelium of snakes stimulated with the earthworm-derived chemoattractant, ESS (Luo et al., 1994). Urine or urine-derived stimuli reduce cAMP in the mouse VNO (Moss et al., 1998; Zhou and Moss, 1997). Dehydro-exo-brevicomin (DHB) and

2-(sec-butyl)-dihydrothiazole (SBT), volatiles in mouse urine, reduce cAMP accumulations in a concentrationdependent manner. Lipophilic urinary components and ␣2u -globulin, obtained from male rat urine, cause a decrease in cAMP in a concentration dependent manner in the VN epithelium of female rats (Rossler et al., 2000). The mechanism by which cAMP is reduced following VN stimulation has yet to be completely understood. Using microvillar preparations from female rat VN sensory epithelium, Rossler et al. (2000) observed increases in IP3 and decreases in cAMP when the epithelium was stimulated with male urine. They present excellent evidence that the ligand-induced decrease in cAMP level does not result from enhanced phosphodiesterase (PDE) activity, nor is it due to inhibition of AC by Gi2␣ or Go␣ . It would appear that the reduction in cAMP is a result of activating the phosphatidylinositol cascade which results in increases in Ca2+ . The increase in Ca2+ could then modulate calcium-sensitive ACVI resulting in decreases in cAMP. These findings are very similar to those reported for the snake VNO (Wang et al., 1997). Nonetheless, it should be noted that ligand-stimulated decreases in cAMP have not been reported universally. In the male hamster (Kroner et al., 1996) and female rat (Sasaki et al., 1999) no reduction in baseline cAMP was observed when the VN epithelia were stimulated with aphrodisin and male rat urine, respectively. However, no attempt was made in these two studies to examine the effect of VN stimulants on forskolin-elevated levels of cAMP. Although forskolin and GTP both induce accumulation of cAMP in turtle VN neurons, odorants that produce electrophysiological responses in these neurons fail to induce cAMP increases (Okamoto et al., 1996). Only one study, using patch-clamped turtle VN neurons, has reported activation of VN neurons with injection of cAMP (Taniguchi et al., 1996b). Injection of cAMP into VN neurons in garter snakes evoked no detectable response (Taniguchi et al., 2000). Vomeronasal receptor cells have unusually high input resistances and are activated at very low currents. Examined in patch-clamp mode, VN neurons respond with action potentials to current injections of as little as 1 pA (Inamura et al., 1997a; Liman and Corey, 1996; Taniguchi et al., 1996b, 2000; Trotier et al., 1993). The high input resistance (as great as 3 G) may account for their remarkable sensitivity. This high input resistance is in line with the observations of Trotier and Døving (1996) on the maintenance of the resting membrane potential in VN receptor cells. Unlike most neurons, the resting membrane potential of VN receptor cells does not appear to be due to a potassium diffusion potential. Instead it is produced by the electrogenic activity of the Na+ -, K+ -ATPase. Inhibition of Na+ -, K+ -ATPase with ouabain blocks the outward sodium pump current and results in depolarization of the membrane. Addition of external K+ activates the pump current and results in a rapid hyperpolarization of the membrane (Trotier and Døving, 1996). This Na+ , K+ pump may thus be responsible for maintaining a high input resistance.

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In sum, the preponderance of evidence from a large number of laboratories, using a broad range of animals, is that the signal transduction pathway for the VNO involves ligand binding to GPCRs that activate PLC leading to generation of IP3 and DAG. Increases in IP3 are linked to an increase in intracellular Ca2+ resulting from release of Ca2+ from intracellular stores and/or influx from extracellular sources. DAG has been reported to activate TRP cation channels, allowing influx of Ca2+ . The link between the second messenger systems and electrical activation of VN receptor cells has yet to be elucidated.

6. Developmental issues in the VNS The heterogeneity in the VNS and issues related to how vomeronasal information is accurately transmitted from the VN epithelium to the AOB raises several questions related to development. How do VN axons locate the appropriate glomeruli in the AOB during ontogeny? Since there is continuous turnover in the VN epithelium, how do newly generated neurons connect with appropriate targets in the AOB. The VN system is known to regenerate following nerve section. How do the axons in the regenerated vomeronasal nerve locate their appropriate targets. The major known function of the VNS is to detect chemical signals arising from conspecifics. At what stage in ontogenetic development is the VN apparatus sufficiently mature to transmit information about chemical signals to the brain? This section emphasizes findings related to maturation and plasticity throughout life. 6.1. Ontogenetic development of VNS A number of issues have emerged concerning development of the VNS. Among these is the question of when the VNS becomes functional during ontogeny, whether major changes occur during metamorphosis and how axons of VN receptor cells find their targets in the AOB. Development in the mammalian (Garrosa et al., 1998; Mori, 1993) and snake (Holtzman, 1993, 1998) VNSs has been reviewed recently. Mori (1993) addresses the issue of how receptor cell axonal projections to the olfactory bulbs form during development, discussing development of VN and olfactory receptor cell axons, axonal guidance, glomerular formation, projection patterns and molecular properties of axons of chemosensory receptor cells. Garrosa et al. (1998) are particularly concerned with prenatal development of the mammalian VNO, emphasizing morphogenesis during different stages of development. Holtzman (1998) concentrates on the issues of cell dynamics in the VN epithelium of developing snakes. One issue concerning development in rodents is when, during ontogeny, the VNS becomes functional. In mice (Coppola and O’Connell, 1989) fluorescent beads injected into the amniotic fluid surrounding E18 fetuses, do not enter the VNO, although they are found in all other regions of the nasal cavity. Histological studies indicated that the VN duct

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was not open in E19 mice, opening only at P5. In contrast, fluorescent beads injected into the amniotic sac of pregnant rats can be found in the VNO of E20 rat embryos, 1 day before birth (Coppola and Millar, 1994; Coppola et al., 1993), indicating that the VN ducts are patent prenatally. However, since injection of epinephrine does not increase the number of beads found in the VNO, the pumping mechanism may not be functional at this time. Therefore, in rats, but not mice, it is possible that amniotic fluid could reach the VN sensory epithelium. However, Coppola (2001) found no evidence that the rat VNO is functional before birth. Several studies have examined general morphological development of the VNO and AOB of the rat during ontogeny (Garrosa and Coca, 1991; Garrosa et al., 1986, 1992, 1998; Mendoza and Szabo, 1988; Szabo and Mendoza, 1988; Yoshida-Matsuoka et al., 1999). The anlage of the VNO is observed in the embryo between E12 and E13, and a clearly distinguished VNO is present between E15 and E18, reaching adult size between P42 and 49, just before puberty. Using the lipophillic dye, DiI, in E16 rat embryos, Marchand and Belanger (1991) describe VN receptor cell axons extending to the primordium of the AOB. Megachiropteran bats appear to lack a functional VNS. In the rousette fruit bat, Rousettus leschenaulti, a VN primordium is present in 7–14 mm crown-rump length specimens, but there is no evidence of a VNO or associated structures in older specimens (18.5–34.1 mm crown-rump length) (Bhatnagar et al., 1996). The development of the VNO and AOB of garter snakes was studied using routine histological techniques (Holtzman and Halpern, 1990) as well as thymidine autoradiography (Holtzman and Halpern, 1991a,b). The cell dynamics in the VNO and AOB are described in detail and compared to the olfactory system. Although most neurogenesis in the central nervous system occurs prenatally, some postnatal neurogenesis appears to occur in the MOB, AOB and in the nucleus sphericus. An interesting developmental question arises when considering the changes that may occur in the VNS during amphibian metamorphosis since these animals move from a purely aquatic environment to a terrestrial or aquatic and terrestrial environment. In Xenopus laevis, the VNO does not appear to change in structure, function or innervation during metamorphosis (Hansen et al., 1998). Furthermore, as compared to the olfactory system, which undergoes almost total reorganization through extensive cell turnover, the VNO appears to exhibit little cell turnover during metamorphosis (Higgs and Burd, 2001). In the Japanese reddishfrog, Rana japonica, the VNO appears 4 days after hatch, remains immature 24–36 days after hatch and is still not completely mature 60 days after hatch (Taniguchi et al., 1996). At the end of metamorphosis, there are fewer microvilli per receptor cell than in the adult. A fundamental problem in chemical sense developmental research is determining when and how synaptogenesis occurs in the olfactory bulbs. A related issue is whether synaptogenesis in the AOB occurs before, after, or synchronously

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with that in the MOB. A number of different approaches have been used to investigate different aspects of these questions. Based on evidence that transneuronal transfer of barley lectin (BL), expressed under the control of an olfactoryspecific promoter in transgenic mice, occurs prenatally in the MOB, but only postnatally in the AOB, it is likely that VN sensory neurons connect to AOB neurons after main olfactory sensory neurons (Horowitz et al., 1999). Scattered BL-positive neurons were present in the VNO and main olfactory epithelium at E11.5. Between E17.5 and E18.5 BL was detected in dendrites of mitral cells of the MOB, whereas mitral cells in the AOB were unstained at P0, only faintly stained at P8 and strongly stained only at 3 weeks of age. Immunoreactivity for synaptophysin and SV2 (synaptic vesicle proteins) was present in the MOB at E17.5 and in the AOB at P0. This study is a forceful demonstration of the fact that, in the mouse, synaptic contact between receptor cells and the olfactory bulb in the main olfactory system precedes that in the VNS. It also strongly supports the notion (see above) that the mouse VNS is not functional prior to birth. In snakes, the lateral cortex, the main recipient of MOB efferents, matures prior to the nucleus sphericus, the main recipient of AOB efferents (Holtzman and Halpern, 1990). Thus, the VNS of snakes, may develop later than the main olfactory system. How important is the target in axon-bulbar connectivity? When 15-day-old embryonic rat VNOs were cocultured with olfactory bulbs from siblings for more than 10 days in vitro, VN axons were observed leaving the organ explants and making contact with the olfactory bulb explants (Ichikawa et al., 1995). However, the presence of the normal target was not necessary. Neonatal rat VNOs transplanted into the parietal cortex of littermates developed in the parietal cortex, forming vesicles and canals lined with respiratory and sensory epithelium (Morrison and Graziadei, 1995, 1996). Axon bundles were observed leaving the epithelium and forming plexuses with “distinct gomerular-like structures”. The authors suggest “that glomerular formation is not dependent on specific target or length of axon development, but rather on a set of complementary axons that display mutual recognition” (Morrison and Graziadei, 1995). However, a somewhat different conclusion can be drawn from more recent work by Muramoto et al. (2003). Partially dissociated VN epithelial cells co-cultured with AOB of rats form spherical structures called vomeronasal pockets (Muramoto et al., 2003). Over time the number of tyrosine hydroxylase-containing neurons in the AOBs co-cultured with vomeronasal pockets increases. However, this increase in tyrosine hydroxlase-containing neurons does not occur when the AOB is co-cultured with olfactory epithelial explants, suggesting that the effect of VN explants on tyrosine hydroxylase expression in the AOB is through specific interactions between the receptor cells and their target.

6.1.1. Developmental markers and factors A number of developmental markers appear at different maturational stages in the VNS of vertebrates. Some of these molecules disappear during ontogeny and others remain. In most cases their function is still poorly understood. Olfactory marker protein is present in mature VN receptor neurons. An ultrastructural study of the supranuclear region of the rat VN sensory epithelium found OMP-ir within dendrites of receptor cells and the bases of microvili, but not in the distal portions of microvilli or in supporting cells (Johnson et al., 1993). OMP is expressed at E14 in the rat olfactory epithelium but not until P2 in the VN epithelium (Kulkarni-Narla et al., 1997). In the mouse VN sensory epithelium OMP is expressed during the last week of gestation (Tarozzo et al., 1998). Thus, based on data from OMP-ir studies, it would appear that in the rat receptor cells in the VN sensory epithelium are not mature at birth, whereas at least some mouse receptor cells are already mature prior to birth. Interestingly, whereas OMP-ir is typically found in the sensory epithelium but not the non-sensory epithelium of adults, during fetal development OMP-immunoreactive cells are also located in the non-sensory epithelium. This latter staining diminishes after E18, disappearing by P7. Camoletto et al. (2001) examined the distribution of stathmin and SCG10 proteins during neurogenesis in the mouse olfactory and VN epithelia. These proteins are believed to be important for protein phosphorylation–dephospohorylation events involved in control of neuronal differentiation and proliferation. Both proteins were present at E12 in VN and olfactory neurons, and the number of immunoreactive cells increased with increasing age. During postnatal development stathmin and SCG10 became restricted to immature neurons. In the VN epithelium of adult mice the immunoreactive cells were largely located at the margins of the sensory epithelium, but significant numbers were also observed in the intermediate layers of the central portions of the sensory epithelium. PGP 9.5 is a ubiquitin-associated hydrolase that has been immunolocalized to the rat VN epithelium (Johnson et al., 1994; Soler and Suburo, 1998; Taniguchi et al., 1993b) and dog AOB (Nakajima et al., 1998a). Developmentally, PGP 9.5 is expressed in hamster VNO at E13 (Nakajima et al., 1998b), in mouse between E14 and birth (Tarozzo et al., 1998) and in rat at E16 (Kulkarni-Narla et al., 1997). Interestingly, PGP 9.5 is expressed earlier in the rat olfactory epithelium, E14, than in the VNO (Kulkarni-Narla et al., 1997). The hamster AOB is PGP 9.5-immunoreactive at P15 (Nakajima et al., 1998b). In the mouse, GAP-44/B50 phosphoprotein, a marker of immature chemosensory neurons, and PGP 9.5 are expressed in the developing VN sensory and non-sensory epithelia between E14 and birth (Tarozzo et al., 1998). Antibodies to these proteins intensely stain several layers of neurons in the basal portion of the sensory epithelium. Oxidative metabolic activity has been investigated in the MOB and AOB of embryonic and newly born garter snakes

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using cytochrome oxidase histochemistry (Holtzman et al., 1993). Cytochrome oxidase activity was greater in the AOB than in the MOB of embryonic snakes, but equal in neonates. Since no stimuli were used to activate these systems, it is not possible to conclude whether these results indicate changes in activity from chemical stimuli at birth. 6.1.1.1. Expression of guidance molecules during development. The netrins are a family of secreted molecules involved in axon guidance. Mutations in netrin genes result in defects in axon guidance. A novel netrin, ␤-netrin, has been identified, its expression pattern described and functionally characterized (Koch et al., 2000). In the VN nerve of mice and rats, ␤-netrin is expressed in the basement membrane of the glial ensheathment. Functional studies using E15 rat olfactory bulb explants indicate that the presence of ␤-netrin increases neurite outgrowth. Thus, this molecule may be important for guidance of VN sensory neurons to the AOB. The Eph family of receptor tyrosine kinases has been implicated in axonal guidance in a number of systems. Examination of the expression patterns of the Eph-A class of the Eph family on VN axons and their receptors in the AOB suggest that they play an important role in axonal guidance during development (Knöll et al., 2001; St. John and Key, 2001). Eph-A ligands are localized on mouse and rat VN receptor cell axons throughout development, appearing as early as E12.5 (mouse) or E18.5 (rat), when VN receptor cell axons are beginning to emerge from the epithelium, and throughout development. In stripe assays, axons emerging from VN explants prefer a substrate coated with Eph-A-Fc/laminin compared to Fc/laminin (Knöll et al., 2001). The semaphorins, a large family of glycoproteins, have been implicated in guidance of growing axons (e.g. Cloutier et al., 2002; Pasterkamp et al., 1999). Neuropilins (Npn-1 and Npn-2) are transmembrane proteins that bind to semaphorin haloreceptors (Giger et al., 1998). Npn-2 is expressed at high levels in the developing VNS of rats and mice (Chen et al., 1997; Cloutier et al., 2002) and in basal V2R-expressing neurons (Giger et al., 1998). Npn-null mice lack semaphorin binding in the AOB (Giger et al., 2000) and demonstrate defasciculation in the VN nerve and misguided VN axons terminating in the MOB (Walz et al., 2002). Additionally, in rats, Npn-2 ligand is secreted by the posterior AOB, repelling axons that normally terminate in the anterior AOB (Cloutier et al., 2002). Reelin, an extracellular matrix protein, is present in the VNO and AOB of mice. This protein, which has been implicated in neuronal migration, may also function in axonal guidance. By crossing mice with the reeler gene mutated, i.e. reeler mice with VN12-IRES-tau-lacZ mice, Teillon et al. (2003) were able to visualize a subpopulation of VNO neurons in the brains of reeler mice. Since the projections of the visualized axons were indistinguishable from normal, Teillon et al. (2003) concluded that reelin does not provide guidance cues for vomeronasal nerves.

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6.1.1.2. Glycoproteins/cell surface proteins. Cell surface and substrate molecules, such as glycoproteins, are important for cell–cell interactions and cell–substrate interactions. These relationships are particularly relevant when studying systems, such as the olfactory system and VNS, that undergo continuous neurogenesis and therefore must reestablish axon–target contact throughout the lifetime of the animal. Of particular interest is the differential localization of these molecules and their appearance or disappearance during development. A 240 kDa glycoprotein, VNO240 , has been identified in mouse VN receptor cells, but not in the main olfactory epithelium (Clarris and Key, 2001). The pattern of developmental expression of this glycoprotein is unusual, appearing earliest in perikarya in the central portion of the VN epithelium at E20.5, but not in axons until P3.5. As mice mature, expression spreads from central to peripheral regions of the VN epithelium. VNO240 does not cross-react with NCAM isoforms, suggesting that it is not a member of the NCAM family. Another glycoprotein, NOC1, is associated with NCAM and is expressed in both VN and olfactory epithelia (Clarris and Key, 2001). At E16.5, it is not expressed in the VN sensory epithelium, but is present throughout the epithelium at E20.5. Immunoreactivity for chromagranin A (CgA), a member of the granin family of acidic glycoproteins normally found in secretory granules of endocrine and neuroendocrine cells, has been observed in the VN epithelium, VN nerve and AOB of tadpoles of the American toad, Bufo americanus and the green frog, Rana clamitans (Wittle et al., 2000). Interestingly, in the toad CgA-ir was not observed after hindlimb buds formed or later, but was present in adult green frog VN epithelium and nerve. NCAM is expressed homogeneously in the VNO and AOB of the opossum (Shapiro et al., 1997). Developmentally, NCAM is expressed in the VNO at the end of the first postnatal week and in the AOB by the end of the second postnatal week. BIG-2, a mouse cDNA related to axon-associated cell adhesion molecules, is maximally expressed in mature VN sensory neurons but not in regions containing immature receptor cells (Mimmack et al., 1997). The cell surface protein, plexin, has been immunohistochemically localized to VN axons of Xenopus laevis using a monoclonal antibody B2 (Satoda et al., 1995). Anosmin-1, a protein encoded by KAL-1, the gene responsible for the X-linked form of Kallman’s syndrome, is expressed in the VNO at ages E18–E23 of the Asian musk shrew, Suncus murinus (Dellovade et al., 2003). Anosmin-1 immunoreactivity diminishes after E-23 and is quite faint by E29, an age when few GnRH-immunoreactive cells are observed in the nose. Since at the earlier stages GnRH-immunoreative cells are observed in the VNO and in close association with anosmin-1 immunoreactive VN axons, it is possible that anosmin-1 is directly involved in migration of GnRH neurons from the olfactory placode to the brain in Asian musk shrews (Dellovade et al., 2003). The topic of the role

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of the VNO and guidance molecules in the migration of GnRH neurons from the nose to the forebrain has been the subject of intensive investigation and beyond the scope of the present review. For a recent review of this topic see Schwanzel-Fukuda and Pfaff (2003). 6.1.1.3. Transcription factors. Mash1, a basic helix–loop– helix transcription factor is expressed in neuronal progenitor cells including cells in the basal region of the sensory epithelium of the VNO. Transgenic mice lacking Mash1 function exhibit severe reductions in the number of cells in the VNO, have thinned VN sensory epithelia and small VN nerves (Murray et al., 2003). The AOB of Mash1−/− mice is decreased in size. Thus, Mash1 function appears to be necessary for normal development of VN sensory neurons. The CREB/ATF transcription factors are known to be important in developmental processes of diverse species. Atf5, a novel member of the ATF4 (activating transcription factor) family, is expressed during embryological development in the mouse VNO (Hansen et al., 2002). In situ hybridization revealed strong expression in VN receptor cells at E12.5–E18.5; however, after birth expression of Atf5 RNA was low to undetectable. A few of these studies suggest that the VNS of mammals is not functional prenatally and develops during the postnatal period becoming fully functional close to puberty. The significance of the several markers that appear and disappear in the VNO during this developmental period at present is not understood. 6.2. Re-evaluation of neurogenesis in the VNS Compared to other sensory systems, the nasal chemical sense systems are particularly plastic during adulthood. Olfactory and VN epithelia undergo continuous cell turnover, and newly generated interneurons arising from the subventricular zone of the telencephalon are constantly added to the MOB and AOB. Important advances in our understanding of both phenomena have taken place during the last few years. The major issues addressed in recent years have been the migration patterns of newly generated neurons in the vomeronasal epithelium and AOB. 6.2.1. Vomeronasal epithelium A number of characteristics differentiate olfactory and VN receptor neurons from other mammalian neurons and sensory cells (see Farbman, 1990, 1992 for reviews). For example, chemosensory cells are both peripheral sensory receptors and true neurons, possessing axons that terminate in the central nervous system. Furthermore, olfactory and VN neurons regularly undergo cell death and are continuously replaced, even in adults. The process of epithelial reconstitution apparently provides for tissue repair following environmental insult. In the early seventies, neuronal replacement was demonstrated in the mammalian olfactory epithelium (Moulton

et al., 1970). Basal cells located along the basal lamina of the olfactory epithelium were found to generate cells that migrated vertically, from basal to apical regions, as they became neurons and replaced apoptotic sensory cells (Graziadei, 1977; Graziadei and Monti Graziadei, 1979; Moulton et al., 1970) (Fig. 6). At that time, cell replacement in the VN epithelium had not yet been demonstrated. The paucity of [3 H]thymidine-labeled cells (Moulton et al., 1970) and the absence of a clearly identifiable basal cell layer in the mammalian VN epithelium (Cuschieri and Bannister, 1975), led to the assumption that cell turnover might not occur in the VN sensory epithelium. This issue was reinvestigated in mice using [3 H]thymidine autoradiography (Barber and Raisman, 1978a,b; Graziadei, 1977). In C-shaped frontal sections, newly generated cells were found almost exclusively in the margins of the VN sensory epithelium, at the junction between the sensory and non-sensory epithelia. These cells were reported to migrate slowly from the margins of the VN sensory epithelium to its center as they matured into receptor cells (Barber and Raisman, 1978a) (Fig. 6). It was not possible to determine from the data presented whether this process represented a mechanism for neuronal turnover or a mechanism for postnatal growth of the VN sensory epithelium by accretion of cells at its margins (Addison and Rademaker, 1927). Two sets of data demonstrated that neuronal replacement took place in the VNO: the VN epithelium was repopulated after VN nerve section, which virtually kills all receptor neurons Barber and Raisman (1978b); and neurogenesis was observed in mice at 7 months of age when postnatal growth appears to be complete (Wilson and Raisman, 1980). Since neuronal turnover occurred in the VNO and, in these early studies, the only detected dividing cells were situated at the margins of the VN sensory epithelium, it was assumed that newly generated receptor cells migrated horizontally from the margins to the center of the epithelium to replace apoptotic neurons. This pattern of horizontal migration contrasts with the vertical pattern of cell migration observed in the olfactory epithelium of mammals (Graziadei, 1977; Graziadei and Monti Graziadei, 1979; Moulton et al., 1970) and the VN epithelium of snakes (Wang and Halpern, 1980, 1982a,b) (reviewed in (Halpern, 1987; Takami, 2002)) (Fig. 6). Recent studies, using bromodeoxyuridine (BrdU), have shed additional light on this issue. BrdU-labeled cells have been detected not only at the margins of the VN sensory epithelium of different mammals, but adjacent to the entire basal lamina of opossums (Jia and Halpern, 1998; Mart´ınez-Marcos et al., 2000a), rats (Inamura et al., 2000; Mart´ınez-Marcos et al., 2000b; Weiler et al., 1999a), ferrets (Weiler et al., 1999b), hamsters (Ichikawa et al., 1998) and mice (Cappello et al., 1999; Giacobini et al., 2000). Excluding BrdU-labeled cells situated in the supporting cell layer, at least two different pools of dividing cells have been reported, one at the margins and one adjacent to the basal lamina in the central portions of the VN sensory epithelium. It should be noted that the basal lamina frequently

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Garter Snake vomeronasal epithelium

Olfactory epithelium

Opossum vomeronasal epithelium. Vertical migration of Goα- and Gi2α-expressing cells takes 5 and 7 days, respectively. Horizontal migration was no detected during the first 11 days.

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Rat vomeronasal epithelium.. Vertical migration takes about 7 days. Horizontal migration is slow and stops after 14 days.

MOB

AOB

Mouse vomeronasal epithelium. Vertical migration has been demonstrated. Horizontal migration is slow and stops after 56 days. Fig. 6. Neurogenesis in the vomeronasal system. Neurogenesis in the olfactory epithelium occurs through a process of vertical migration from basal cells (B, dark gray circles) that become immature neurons (1, 2, light gray circles) and finally become mature neurons (3, open circles). The process is similar in the snake vomeronasal epithelium. In opossums, rats and mice cell turnover also occurs through a process of vertical migration. In opossums, differential migration of subclasses of vomeronasal neurons have been demonstrated as well. In rats and mice, basal cells situated at the edges of the epithelium move slowly horizontally and it is unlikely that never reach the central regions of the epithelium, although these cells also could participate in cell renewal. New cells are added to the accessory olfactory bulb (AOB) arising from the subventricular zone (SVZ) and migrating through the rostral migratory stream (RMS). Main olfactory bulb (MOB), supporting cells (S).

accompanies blood vessels intruding into the sensory epithelium (Breipohl et al., 1981). Accordingly, at short survival periods, a few BrdU-positive cells have been observed situated apically in the epithelium adjacent to intruding capillaries (e.g. Mart´ınez-Marcos et al., 2000a,b; Weiler et al., 1999b). Both central and marginal populations of di-

viding cells are able to repopulate the VN epithelium after experimentally induced degeneration (Barber and Raisman, 1978b; Ichikawa, 1999; Ichikawa et al., 1998; Matsuoka et al., 2002; Suzuki, 1998; Suzuki et al., 1998). Although the number of dividing cells at the margins of the epithelium is greater than the number observed

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centrally, cells originating in the margins migrate centrally very slowly and stop migrating after a few weeks. There is no evidence, at present, that cells generated at the margins ever reach the center of the epithelium (Giacobini et al., 2000; Mart´ınez-Marcos et al., 2000a,b; Wilson and Raisman, 1980), and the possibility that these cells die while migrating can not be excluded. It is difficult, therefore, to reconcile these data with the previous hypothesis that cell turnover in the VN epithelium occurs by horizontal migration of newly generated cells from the margins to the central regions of the epithelium. It should be recalled, however, that this hypothesis was generated from data obtained exclusively in mice and the new data is derived exclusively from non-murine species. No one has recently replicated the Barber and Raisman study in mice, although a population of dividing cells, presumably participating in cell turnover, has been reported in the central regions of the VN epithelium of adult mice (Cappello et al., 1999; Giacobini et al., 2000). One interpretation of the current data, which is compatible with the original results reported by Barber and Raisman (1978a), is that mitotic cells at the edges of the VN sensory epithelium participate in cell turnover in these marginal regions and constitute either a precursor pool for postnatal growth or undergo apoptosis (Giacobini et al., 2000; Mart´ınez-Marcos et al., 2000a,b; Weiler et al., 1999b; Wilson and Raisman, 1980). The progeny of dividing cells in the central regions of the epithelium appear to migrate vertically from the basal lamina to more apical locations in the epithelium and express markers of neuronal maturity shortly after BrdU administration. This suggests that basal cells in the center of the epithelium participate in cell turnover (Giacobini et al., 2000; Jia and Halpern, 1998; Mart´ınez-Marcos et al., 2000a,b). Therefore, in the mammalian species that have been investigated recently, cell turnover in VN sensory epithelium appears to occur through a process of vertical migration common to other tetrapods (Dawley et al., 2000; Wang and Halpern, 1988), and to that observed in the olfactory epithelium. As discussed elsewhere in this review (Section 3), the mammalian VN sensory epithelium is not homogeneous, but can be partitioned into basal and apical cell populations. In opossums neurogenesis of these two populations is somewhat different. By injecting BrdU and dividing the epithelium into basal (Go␣ ) and apical (Gi2␣ -protein-expression) zones, it has been demonstrated that most daughter cells arising from the base of the epithelium enter the Go␣ -protein-expression zone by day 5, while most daughter cells fated to become Gi2␣ -protein cells do not reach the Gi2␣ -protein-expressing zone until day 7 (Mart´ınez-Marcos et al., 2000a). Therefore, these two populations of VN neurons appear to migrate and mature at slightly different rates. Since newly generated neurons expressing Go␣ - and Gi2␣ -proteins have been simultaneously found shortly after BrdU administration (Mart´ınez-Marcos et al., 2000a,b), it is unlikely that these cells switch their expression of G-proteins. Instead, all neurons appear to arise from the

same pool of progenitor cells and differentiate into Go␣ or Gi2␣ -protein-expressing neuronal populations under the influence of environmental factors (Mart´ınez-Marcos et al., 2000a,b; Weiler et al., 1999a). 6.2.2. Degeneration and reconstitution of the VN sensory epithelium Degeneration and reconstitution of the VN sensory epithelium was reviewed previously (Halpern, 1987). A few additional studies have been published recently, all of which demonstrate some regeneration following VN nerve transection, but not necessarily complete reconstitution (Ichikawa, 1999; Ichikawa et al., 1998; Matusokoa et al., 2002; Yoshida-Matsuoka et al., 2000). Raisman (1985) has discussed the morphological properties of VN axons and their glial ensheathments as they might relate to regeneration in this system. In the rat, three types of neuroglia are associated with the VN nerves and AOB: (1) sheathing glia of the VN nerve fibers; (2) superficial glia of the fiber layer of the AOB, and (3) astrocytes of the GL, external plexiform layer (EPL) and mitral cell layer (MCL). As the VN axons enter the AOB they carry with them the superficial glia into the glomerular regions. In the glomeruli, an inner glial layer derived from superficial glia encapsulates the synapses between VN axons and postsynaptic elements. An outer glial layer is formed by astrocytes. Raisman (1985) suggests that this “peculiar glial arrangement may be important for the unique regenerative capacity of this system”. 6.2.3. Accessory olfactory bulb A second source of neural plasticity during adulthood occurs at the level of the olfactory bulbs. Newly-generated cells arising from the subventricular zone of the telencephalon are postnatally incorporated as interneurons in the MOB and AOB of amphibians (Fritz et al., 1996), reptiles (Garcia-Verdugo et al., 1989; Perez-Canellas and Garcia-Verdugo, 1996; Perez-Canellas et al., 1997) and mammals (Altman, 1969; Altman and Das, 1966; Bayer, 1983; Hinds, 1968a,b; Kaplan and Hinds, 1977; Kaplan et al., 1985; Kishi, 1987) (Fig. 6). In mammals, these cells arise specifically from the anterior subventricular zone (Luskin, 1993; Luskin and Boone, 1994). Dividing cells migrate tangentially several millimeters through the rostral migratory stream. In the olfactory bulbs, these cells turn and migrate radially, becoming periglomerular and granule cells (see Peretto et al., 1999a for a review) (Fig. 6). In the AOB, addition of new cells was previously thought to be restricted to the early postnatal period (Bayer, 1983). Recently, however, arrival of new neurons to the AOB has been also demonstrated during adulthood (Bonfanti et al., 1997; Mart´ınez-Marcos et al., 2001b). Little is known about how these newly added cells are incorporated into the neural circuitry of the olfactory bulbs or what functions they subserve (e.g. Petreneau and Alvarez-Buylla, 2002; Pomeroy et al., 1990). In the

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MOB, the addition of new interneurons may be crucial for odor discrimination (Gheusi et al., 2000), whereas in the AOB, Bonfanti et al. (1997) suggest that newly added cells may play a role in the formation of memories for sexual pheromones (see Brennan et al., 1990 and Kaba and Nakanishi, 1995 for reviews). One could hypothesize that pheromone memories, at the level of the AOB, should have a sexually dimorphic neural substrate. Interestingly, cell addition to the AOB has been demonstrated to be sexually dimorphic in adult rats (Peretto et al., 1999b, 2001). On the other hand, although the anterior and posterior divisions of the AOB may be considered independent functional entities, no differences were found in the pattern of cell migration to these two divisions (Mart´ınez-Marcos et al., 2001a).

7. New information on the anatomy of the VNS The functional domains of the VNS include neuroendocrine and behavioral responses to chemosignals, particularly those arising from conspecifics. To understand how different chemical signals are able to generate appropriate responses attention must be directed to the anatomical substrates for VN functions. Integrating information about the heterogeneity of receptors, signal transduction mechanisms, and patterns of stimulus activation in the VNS with the central connections of the system will aid in our eventual understanding of how detection of different social signals result in distinctive behavior patterns. The vast majority of recent studies on the anatomy of the VNS are focused on the VN epithelium or AOB. Knowledge of connections beyond the AOB is critical to understanding the natural flow of VN information and to characterize this system in all its complexity. 7.1. Descriptive anatomy A relatively large number of reports describing the anatomy of the VNO or AOB in a variety of species have been published in recent years (see Table 2). The reader is referred to these studies for details. We concentrate on aspects of the morphology of the VNO and AOB that are either surprising or contribute to the understanding of functional aspects of these structures (see below). Two reviews of the anatomy of the AOB are of particular interest. Mori’s (1987b) review of the anatomy of the AOB includes illustrations of M/T cell primary dendrites with multiple glomerular tufts and also describes the relatively poorly developed secondary dendrites as compared to MOB mitral cells. The presence of multiple primary dendrites on M/T cells is particularly important in light of the heterogeneity of the VNS and the fact that the terminal tufts of these dendrites end in multiple glomeruli. Meisami and Bhatnagar (1998) review the distribution, morphology and development of the AOB in vertebrates. They discuss, in the context of vertebrate evolution, the

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fact that the AOB is not universally present and that its development may vary considerably even within a taxon. An extensive and detailed description of AOB morphology is included, as is a discussion of the apparent loss of a functional AOB in higher primates. There continues to be great interest in charting the presence and development of the VNS in various species and using this information to understand under what circumstances VNSs develop. Bhatnagar and Meisami (1998) review the appearance of the VNO in bats and primates. In only 1 of the 18 bat families, the Phyllostomidae, does a well developed, functional VNO exist. Primates have not been well studied. In all prosimians investigated, a VNO is present and well enough developed in adults to be functional (Evans and Schilling, 1995). Some variation in the degree of development of the sensory epithelium was noted among different species, but these were not clearly associated with activity pattern or other ecological factors. In general, New World monkeys possess VNOs and adult Old World monkeys do not appear to have well developed VNOs. Chimpanzees, Old World primates that are thought to be devoid of a VNO, possess bilateral epithelial tubes resembling, in position and histological structure, the VNOs found in humans (Smith et al., 2001b). The putative VNO of chimpanzees differs from the VNO of prosimians. The latter have well differentiated sensory and non-sensory epithelia whereas the chimpanzee VN epithelium is ciliated throughout. According to Smith et al. (2001a) macaques have no structure resembling the VNO of prosimians or humans, although Price (1990) includes the AOB in a drawing of the macaque MOB. Within group comparisons can often provide information on either the origins or functions of the VNS. Comparisons among alcelaphine antelopes reveal that those lacking the connection between the VNO and the oral cavity (topi and Coke’s hartebeest) do not exhibit flehmen behavior and are less interested in female urine during reproductive encounters compared to ruminants that have patent connections between the VNO and oral cavity (Hart et al., 1988). A VNO and AOB have been described recently in several species of salamanders: A. mexicanum (Eisthen et al., 1994), Amphiuma tridactylum and Siren intermedia (Eisthen, 2000). In proteids, which are thought to be basal to the ambystomid family, no VNO or AOB is present. Eisthen addresses the evolutionary question of whether the VNS develops at metamorphosis as an adaptation to terrestrial life and concludes that since Amphiumidae and Sirenidae are fully aquatic and non-metamorphosing salamanders, it is not possible that the VNS developed in response to terrestrial demands. Regarding the structure of the VNO, interesting advances in our knowledge have been achieved at the histological, cellular and ultrastructural levels. Although a discernable basal cell layer is difficult to appreciate in most mammalian VNOs, a basal cell layer is clearly identifiable in New World monkeys, including the saddle-back tamarin (Mendoza et al., 1994). Snakes and frogs have well-developed basal

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Table 2 Anatomical studies of the vomeronasal system Animal

Structure

Techniques

Comments

Reference

Axolotl, Ambystoma mexicanum

VNO

LM/TEM

No non-sensory epithelium in the VNO; ciliated supporting cells but also non-ciliated supporting cells

Eisthen et al., 1994

Salamander, Plethodon cinereus

VNO

LM

No cilia in VNO

Dawley and Bass, 1988

Frog, Rana esculenta

VNO

LM/TEM/SEM

Supporting cells ciliated

Franceschini et al., 1991

Bullfrog, Rana catesbeiana

VN nerves and AOB

Dissection/LM/TEM

Very detailed description and quantitative analysis

Burton et al., 1990; Burton, 1990

Japanese reddish frog, Rana japonica

VNO

LM/EM

Developmental study; supporting cells ciliated

Taniguchi et al., 1996

Clawed frog, Xenopus laevis

VNO

SEM/TEM

Hansen et al., 1998

VNO

LM/TEM

Developmental study; supporting cells ciliated Supporting cells ciliated

Frog

VNO

MRI

Good correlation between MRI image and light microscopy

Sbarbati et al., 1991

Habu (snake), Trimeresurus flavoviridis

VNO

LM

Reconstruction; 0.5 ␮m sections; quite complete description

Takami and Hirosawa, 1987

Snake, Elaphe quadrivirgata

AOB

Golgi

Detailed analysis of lamination and cell types

Iwahori et al., 1989

Opossum, Monodelphis domestica

VNO

LM/SEM

Emphasis on structures associated with nuzzling

Poran, 1998

Ferret

VNO and AOB

LM

Analysis of size in adult males and females and effects of hormone treatment

Kelliher et al., 2001

House musk shrew, Suncus murinus

VNO

TEM

In Japanese; ultrastructure of sensory and non-sensory epithelium and VN glands

Oikawa et al., 1993

Elephant shrews, Elephantulus brachyrhynchus and E. myurus

VNO

LM/EM

Drawings and description of rostral nasal cavity

Kratzing and Woodall, 1988

Chinchilla, Chinchilla laniger

VNO

LM/EM

Structure of sensory and non-sensory epithelium and VN glands;histochemistry

Oikawa et al., 1994

Mink, Mustela vison

VNO

LM

Mole rat, Spalax ehrenbergi

VNO

LM/TEM/SEM

Juvenile and adult specimens

Zuri et al., 1998

NMRI mouse

VNO

LM/TEM/SEM

Quite complete description of VNSE

Mendoza, 1993

Mouse

VNO

MRI

Sbarbati et al., 1991

Rat

VNO

Osada et al., 1998

VNO VNO VNO

TEM/atomic force microscopy LM LM/immunohistochemistry EM/freeze fracture

In Spanish; sparsely illustrated Variety of antibodies used Cilia during development

Moreno et al., 1999 Soler and Suburo, 1998 Menco, 1988

Guinea pig

VNO

LM/TEM

New born (in German)

Mendoza and Kühnel, 1989b

Cat

VNO

LM

Late embryonic specimens; ducts between mouth and VNO (nasopalatine) not yet patent. Immunohistochemistry; basal cells in sensory epithelium

Salazar et al., 1996

Ferret, Mustel putorius

VNO

LM/immunohistochemistry for OMP and BrdU

BrdU+ cells along basal lamina of VNSE one hour after injection, not concentrated in marginal area. OMP+ cells not strongly ir and not all receptor cells OMP-ir

Weiler et al., 1999b

Oikawa et al., 1998

Salazar et al., 1994

M. Halpern, A. Mart´ınez-Marcos / Progress in Neurobiology 70 (2003) 245–318

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Table 2 (Continued ) Animal

Structure

Techniques

Comments

Pig

VNO VNO

LM TEM

Calves

VNO

LM/SEM/TEM

Cow

VNO

LM

Horse

VNO

LM

Elephant, Elephas maximus

VNO

LM

Colugo (flying lemur), Cynocedphalus

VNO

LM

Marmoset, Callithrix jacchus

VNO

LM/TEM

Basal cells are scattered over the basement membrane. Jacobson’s glands penetrate the sensory epithelium

Taniguchi et al., 1992a

Saddle-back tamarins (New World monkeys), Saguinus fuscicollis

VNO

LM/TEM

Basal cell nuclear region; smooth ER not present in receptor cells; no intraepithelial blood vessels

Mendoza et al., 1994

Chimpanzee, Pan troglodytes

?VNO

LM

No recognizable neuroepithelium or VN nerve

Smith et al., 2002

Well illustrated, detailed ultrastructure Well illustrated, detailed ultrastructure

Reference Salazar et al., 1997 Adams, 1992 Adams, 1986 Salazar et al., 1997 Salazar et al., 1997

Ciliated supporting cells

Johnson and Rasmussen, 2002 Bhatnagar and Wible, 1994

Abbreviations: EM, electron microscopy; ER, endoplasmic reticulum; ir, immunoreactive; LM, light microscopy; MRI, magnetic resonance imaging; SEM, scanning electron microscopy; TEM, transmission electron microscopy.

cell layers in their VNOs. The significance of the presence or absence of an identifiable basal layer is not obvious although it is likely related to the mechanisms of neuronal renewal in the epithelium. Initially, it was thought that in mammals neurogenesis occurred exclusively at the margins of the epithelium and that differentiating cells migrated to the central portions of the epithelium. In non-mammalian vertebrates such as the snake, neurogenesis occurs in the basal layer of the epithelium and migration of newly differentiating neurons is vertical, toward the lumen of the epithelium (Wang and Halpern, 1988). It is now known (see Section 6) that in some mammals, e.g. opossums, migration is vertical (Jia and Halpern, 1998; Mart´ınez-Marcos et al., 2000a), as in snakes, and in others, e.g. rats there is both vertical and horizontal migration through the epithelium (Mart´ınez-Marcos et al., 2000b). In species in which most of the newly generated cells are found in the margins of the epithelium, it may be very difficult to identify a basal cell layer. It has been known for some time that in most species the receptor cells of the VNO have microvillar endings, in contrast to the ciliated endings of olfactory receptor cells. In the marmoset, C. jacchus, occasional VN receptor cells have been observed with cilia protruding from the distal ends of the dendrite (Taniguchi et al., 1992a). Furthermore, ciliated supporting cells have been reported in the VNO of several amphibian species (Eisthen et al., 1994; Franceschini et al., 1991; Hansen et al., 1998; Oikawa et al., 1998; Taniguchi et al., 1996). The significance of cilia versus microvilli on receptor cell dendrites or supporting cells is unknown. It is possible to obtain interesting insights to functional issues by examining structure using transmission electron

microscopy. For example, VNO receptor cells have a considerable amount of smooth endoplasmic reticulum (SER). In the frog there is an enlargement of the dendrite near the cell body caused by hypertrophy of the SER (Trotier et al., 1994). In some species (e.g. the marmoset, Taniguchi et al., 1992a), the SER is very close to the luminal surface of the epithelium. This is of particular interest because there is considerable evidence that calcium release or influx may be a critical step in signal transduction (see Section 5) and the SER is a repository of intracellular calcium. However, it should be noted that in saddle-back tamarins (Mendoza et al., 1994) an absence of SER in VNO receptor cells has been reported. Bannister and Dodson (1992) compare the morphology of receptor cells in the mouse main olfactory epithelium and VN epithelium. Larger amounts of SER were observed in the receptor cells of the VN epithelium than in receptor cells of the olfactory epithelium. In the olfactory epithelium SER is mainly in supporting cells. These differences may be related to removal of exogenous substances from the epithelium as suggested by the authors, or, reflective of differences in signal transduction mechanisms. 7.1.1. Chemoarchitecture: presence of functionally defined peptides and proteins 7.1.1.1. Calcium binding proteins. Intracellular calcium is an important second messenger in olfactory and VN neurons. Neurons contain several calcium-binding proteins including calmodulin, calbindin D28K, calretinin, parvalbumin and neurocalcin. These proteins are thought to

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modulate the actions of cytosolic calcium and, therefore, their distribution in the cytoplasm of VNS neurons may be important in the signal transduction pathway. VN receptor cells are immunoreactive to calretinin, parvalbumin and calbindin D28 in the mouse (Kishimoto et al., 1993a), to neurocalcin (Iino et al., 1995) and calbindin (Abe et al., 1992; Johnson et al., 1992) in the rat, and to calretinin in the tree shrew, Tupaia belangeri (Malz et al., 2000). AOB neurons in the rat (Celio, 1990; Porteros et al., 1995) and hedgehog, Erinaceus europaeus (Alonso et al., 1995), are calbindin-immunoreactive and in the tree shrew, T. belangeri, are calretinin-immunoreactive (Malz et al., 2000, 2002). Since the precise role of calcium binding proteins is not understood, it is difficult to generalize from the limited studies listed above. In addition to understanding how the different proteins function in signal transduction, comparative studies using multiple species and multiple calcium binding proteins would help determine what similarities and differences exist between species in the presence and localization of the different proteins. 7.1.1.2. Neurotransmitters. Although it is generally accepted, based on electrophysiological evidence, that the neurotransmitter released at axonal terminals of VN receptor cells is glutamate (Dudley and Moss, 1995) and that GABA, norepinephrine and acetylcholine are released at other synapses in the AOB, relatively little is known about the precise localization of these transmitters and their associated enzymes. The distribution of glutamate- and GABA-like ir (Quaglino et al., 1999), choline acelyltransferase (ChAT)-ir and acetylcholinesterase (AChE) activity have been described in the rat (Ojima et al., 1988; Phelps et al., 1992) and hedgehog (Erinaceus europaeus) (Crespo et al., 1999) AOBs. Interestingly, GAD mRNA, GABA- and GAD-ir are transiently present in the mouse olfactory pit/VNO during embryonic development, being maximally expressed at E12.5 and reduced by E16.5 (Wray et al., 1996). 7.1.1.3. Peptides. A few recent studies describe the presence of peptides in the VNO or AOB. It is difficult, at this point to ascribe to these findings any significance due to the lack of comparative data. Antibodies to carnosine, a dipeptide found in nasal chemosensitive neurons, stains the NL and GL of the AOB of rats (Sakai et al., 1988). Galanin and substance P fibers are restricted mostly to the “receptor free” epithelium of the mouse VNO (Matsuda et al., 1994; Nagahara et al., 1995), although a few are seen in the sensory epithelium (Matsuda et al., 1994). The rat VNO exhibits calcitonin gene-related peptide (CGRP)-like ir. However, it is not the receptor cells, but the axons subjacent to the VN sensory epithelium, surrounding VN glands, blood vessels and the non-sensory epithelium of the VNO that are CGRP-immunoreactive (Silverman and Kruger, 1989).

Zancanaro et al. (1999) investigated the expression of regulatory neuorpeptides (substance P, CGRP, neuropeptide Y and atrial natriuretic peptide) in the mouse VNO from postnatal days 1 to 2 months of age. None of the regulatory neuropeptides were present in the sensory epithelium of the VNO. Fibers in the vascular pump were immunoreactive for atrial natriuretic peptide initially at postnatal day 8. The staining became intense at 21 days and 2 months of age. Messenger RNA for connexin 36 (CX36), a gap junction channel-forming protein subunit, is expressed in the VN sensory epithelium and AOB of mice (Zhang and Restrepo, 2003). In the VNO the expression is most robust in the microvillar region, although also sparsely expressed in receptor cells with nuclei located in its apical region. In the AOB, CX36 mRNA expression is observed in the M/T cell layer. Immunoreactivity to CX36 protein was also observed in the GL and VN nerve layer of the AOB (Zhang and Restrepo, 2003). 7.1.1.4. Markers for metabolic activity. Using a battery of enzymes, Taniguchi et al. (1992b) examined the epithelia of the olfactory and VNOs of the golden hamster. In the VN sensory epithelium they found intense adenosine triphosphatase, lactate dehydrogenase, succinate dehydrogenase, acid phosphatase and non-specific esterase activity. Most of the intense activity was located in apical portions of the epithelium. The authors discuss the functional implications of the observed similarities and differences between the olfactory and VN epithelia as well as between sensory and respiratory (or non-sensory) epithelia in enzymatic activity. 7.1.2. Sexual dimorphism in the VNS This area of research has been reviewed repeatedly by Guillamón and Segovia (1993, 1996, 1997), Segovia and Guillamón (1993, 1996). First described in the early 1980s, the sexual dimorphism in the VNS is now well established. Although most structures in this system are larger in males than females, i.e. the AOB, BAOT (Collado et al., 1990), posteromedial cortical nucleus of the amygdala (Vinader-Caerols et al., 1998) and most of the BNST, some structures, such as the medial anterior and lateral anterior portions of the BNST, are larger in females than males. Gonadal steroids during the perinatal period appear to be responsible for these dimorphisms (Guillamón et al., 1988). For those structures that are larger in males than in females, the aromatization of testosterone to estrogen is thought to be the causative factor resulting in a greater number of neurons and a consequent increase in volume. Note, however, that administration of dihydrotestosterone to gonadally intact male rats during the early postnatal period results in a reduction in the size of the AOB and BAOT and administration of the antiandrogen cyproterone acetate to females during the same period results in an increase in the size of the BAOT (Collado et al., 1992). As mentioned in Section 3, in the gray short-tailed opossum, the anterior AOB is larger in males than in females, but the posterior

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AOB is not sexually dimorphic in volume (Mansfield et al., 1996). Interestingly, in wild voles (Microtus pennsylvanicus and M. ochrogaster) there is no sexual dimorphism in the size of the VNO (Maico et al., 2001). For those structures that are larger in females than males, testicular androgens are thought to have an inhibitory influence on the male brain. Lesion studies of sexually dimorphic brain nuclei suggest that those structures which are larger in males suppress female-appropriate behavior, such as maternal behavior and lordosis. It is beyond the scope of this review to summarize the large number of studies in this area. The reader is referred to the excellent reviews mentioned above. In addition to sexual dimorphism in the structures of the VNS, Simerly (1990) discusses the complementary increases in sensitivity to sex-related odors that accompany cyclic changes in levels of circulating hormones. Segovia et al. (1999) develop a model of the control of reproductive physiology and behavior involving complex neural networks such as the VNS. They emphasize the importance of considering brain sex differences when developing theories of motivated behavior in the reproductive sphere. There is a sexual dimorphism in the rate of neuronal generation in the AOB of rats (Peretto et al., 2001). Using BrdU to mark newly generated neurons, male rats were found to have more BrdU-positive cells in the anterior AOB than females. Recently, Miranda et al. (2000) have investigated whether sex-related changes in biosynthetic activity occur in the mitral cell population of the AOB of rats. The authors argue that since there are clear morphological differences in the VNS of males and females, one should be able to observe functional differences. Using silver impregnation of the nucleolus of AOB mitral cells they compared the size of the silver-stained nucleolar organizer regions (Ag-NOR), the number of Ag-NORs per cell and the percentage of neurons having different numbers of Ag-NORs in males and females under different hormonal conditions. Significant differences were observed in all three measures. Diestrus females had a larger area of Ag-NOR than any of the other groups and the greatest number of Ag-NORs. Castrated males and estrus females had the largest number of neurons with only one Ag-NOR whereas diestrus females and intact males had the largest number of cells with three or more Ag-NORs. Thus, sex and hormonal status appear to modify protein synthetic activity in AOB mitral cells as measured by size and number of Ag-NORs. Few studies of sexual dimorphism in the VNS of non-mammalian vertebrates have been reported. Male red-backed salamanders (P. cinereus) have larger VNOs than females at all times of the year (Dawley and Crowder, 1995). Both males and females have larger VNOs during the summer months, a period of intense foraging and gametogenesis, than at any other time of the year. In musk turtles females have a greater amount of Gi␣1–3 in the VN epithelium than males (Murphy et al., 2001). The levels of biogenic amines present in the VNO may differ in males and females. In adult frog VNOs, females have greater

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amounts of epinephrine and norepinephrine than males (Zancanaro et al., 1997). There are no sex differences in serotonin levels in the VNO of frogs. In mice, prepubertal males have higher titers of serotonin, adrenalin and noradrenalin than pubertal or adult mice. Pubertal female mice have greater titers of epinephrine than either prepubertal or adult female mice and adult females have higher concentrations of serotonin than prepubertal or pubertal female mice. Female mice exposed to male urine exhibit decreased amine concentrations in the VNO. 7.2. Re-evaluation of the anatomical connections of the vomeronasal system The discoveries in the late 1960s and early 1970s of segregated and parallel projections of the olfactory and VN systems in vertebrates led to the enunciation of the dual olfactory system hypothesis (Raisman, 1972; Scalia and Winans, 1975; Winans and Scalia, 1970). This hypothesis stated that there would be two parallel pathways from the olfactory and VN sensory epithelia through the limbic system to the diencephalon and that these pathways would subserve different functions. Many of the predictions arising from this hypothesis have been confirmed, but more importantly, the dual olfactory hypothesis led to a wealth of new data from a variety of animal species that clarified much of what we know about the structure and function of the olfactory and VN systems. The original dual olfactory hypothesis was based on data obtained in rabbit (Winans and Scalia, 1970), showing that the MOB and AOB projected to adjacent but mostly separate terminal fields in the basal telencephalon. Subsequently, it was confirmed in a variety of mammals, reptiles and amphibians, that the MOB and AOB essentially project, in a parallel, non-overlapping manner, to a number of limbic system structures (see Halpern, 1987 for review). Their segregated pattern of projections within one of these structures, the amygdala, suggests that the differences in projections could define functional axes that run from the olfactory and VN epithelia through the limbic system. These segregated pathways, present in most tetrapods studied to date, further suggest that these functional axes may be characteristic of tetrapod vertebrate limbic systems in general. Although details of the projections may differ, the broad outlines of the parallel projections are held in common by amphibians, reptiles and mammals (Bruce and Neary, 1995). Functional studies in reptiles and mammals have supported the hypothesis that the main olfactory and VN systems subserve different functions. As predicted by Raisman (1972), most of the pheromonal effects on neuroendocrine states in mice and other mammals have been found to depend primarily on the VNS (see Halpern, 1987; Wysocki and Lepri, 1991; Wysocki and Meredith, 1987 for reviews). The situation is similar in many tetrapod vertebrates, suggesting the functional conservatism of this system.

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To what extent are the VN and olfactory system segregated? The amygdala is the common recipient area of the VN secondary projections in amphibians, reptiles and mammals (see Eisthen, 1997; Gloor, 1997; Halpern, 1987 for reviews). Since 1987, important advances have been made in elucidating additional secondary and tertiary connections of the VNS, i.e. efferent projections from the olfactory bulbs and further projections from olfactory- and VN-recipient structures in the basal telencephalon, respectively. These new observations continue to support the overall notion of a dual olfactory system, while at the same time revealing additional areas of overlap and integration between the two subsystems. With few exceptions, studies on the VNSs of all vertebrates studied have yielded similar results. The anatomy of the peripheral organs, AOBs and the targets of projection neurons from the AOB are virtually identical. VN central anatomy is generally very conservative, with differences being observed primarily in level of development, which typically reflects the importance of the system to the behavior and ecological niche of the animal. In recent years, however, new evidence, especially from reptiles and mammals, has produced increasing examples of convergence of olfactory and VN information in telencephalic structures. In reptiles, the lateral cortex, previously believed to receive exclusively olfactory information, has been found in both Podarcis hispanica (Spanish wall lizard) and Thamnophis sirtalis (garter snake) to be an associative center integrating main olfactory and VN information (Lanuza and Halpern, 1997; Mart´ınez-Garc´ıa et al., 1993). In addition, evidence is accumulating that the posterior–dorsal ventricular ridge of reptiles, a part of the amygdala, receives convergent information from multiple sensory systems including the olfactory system and, perhaps, the VNS. The posterior–dorsal ventricular ridge is the major source of amygdalar efferents to the ventromedial hypothalamus (Lanuza et al., 1997, 1998; Mart´ınez-Marcos et al., 1999). Also in mammals, there is physiological evidence for considerable interaction between the VN and olfactory systems at an early stage in transmission through the telencephalon (e.g. Licht and Meredith, 1987). Thus, anatomical evidence from mammals and reptiles strongly supports the view that interactions between these two systems occur in the telencephalon (Gomez and Newman, 1992; Kevetter and Winans, 1981; Krettek and Price, 1978a,b; Lanuza et al., 1998; Lanuza and Halpern, 1997; Lohman and Smeets, 1993; Mart´ınez-Garc´ıa et al., 1993; Mart´ınez-Marcos et al., 1999). Accordingly, the anatomical assumptions of the dual olfactory hypothesis that the projections of the olfactory and VNSs mostly remain parallel and non-overlapping in the telencephalon, requires re-evaluation and reformulation. 7.2.1. Amphibians The VNS is thought to first appear in amphibians (Bertmar, 1981; Eisthen, 1997). Two classes of chemosensory receptors “fish-like” and “mammalian-like”, have been isolated from the lateral and main diverticula of the nasal

cavity of Xenopus, respectively (Freitag et al., 1995). The receptor neurons of the epithelia of the lateral (VN) and main (olfactory) diverticula project to the AOB and MOB, respectively (e.g. Eisthen et al., 1994). The efferent projections of the AOB were first described in amphibians by Herrick (1921) and later, using degeneration methods and neural tracers such as HRP (Kemali and Guglielmotti, 1987; Scalia et al., 1991; Schmidt and Roth, 1990) as running via the lateral olfactory tract to reach the lateral amygdala (see Halpern, 1987 for review of earlier studies). Secondary olfactory and VN projections are mostly segregated in the telencephalon of R. pipiens as well as in salamanders thus supporting the dual olfactory hypothesis. Secondary VN projections reach the lateral amygdala of salamanders (Schmidt and Roth, 1990) and the medial and cortical amygdaloid nuclei (two subdivision of the lateral amygdala) of R. pipiens (Scalia et al., 1991). Interestingly, the cortical amygdaloid nucleus also receives olfactory inputs and the possibility that olfactory and VN information could converge at this level has already been considered (Scalia et al., 1991). The consequences for the dual olfactory hypothesis and its reformulation remain to be addressed. Tertiary connections of the amphibian VN amygdala have been traced to the hypothalamus (reviewed in Bruce and Neary, 1995) thus indicating a common pathway for VN information in tetrapods. 7.2.2. Reptiles Efferent projections of the MOB and AOB have been described as running separately in squamate reptiles (lizards, snakes and amphisbaenians) as well (Halpern, 1976, 1980; Lanuza and Halpern, 1998; Lohman et al., 1988; Mart´ınez-Garc´ıa et al., 1991; see Halpern, 1992; Lohman and Smeets, 1993 for reviews) (Fig. 7). Early studies on these projections in turtles (Reiner and Karten, 1985) and crocodiles (Scalia et al., 1969) were made assuming that the VNS was not present in these groups. Projections to the amygdala of turtles and crocodiles were more extensive than expected (see Lohman et al., 1988). Since subsequently it was demonstrated that many turtles possess a functional VNS (e.g. Hatanaka and Matsuzaki, 1993), the possibility arises that some of the areas identified in turtles as olfactory recipient structures, were, instead, VN-recipient structures (Eisthen, 1997). This issue needs to be investigated further in different species of turtles. Secondary VN projections in squamate reptiles were first described as terminating in a broad manner in the amygdaloid complex (Gamble, 1952; Goldby, 1937; Halpern, 1976; Heimer, 1969; Lanuza and Halpern, 1998; Lohman et al., 1988; Lohman and Smeets, 1993; Mart´ınez-Garc´ıa et al., 1991). Efferent projections of the AOB are reciprocated by centrifugal afferent connections mainly arising from VN-recipient areas (Lanuza and Halpern, 1998; Mart´ınez-Garc´ıa et al., 1993). Important advances in our understanding of the secondary and tertiary olfactory and VN projections in lizards

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Fig. 7. Some of the main connections in the vomeronasal system of snakes (left) and opssums (right). In snakes, the vomeronasal organ (VNO) projects to the accessory olfactory bulb (AOB) which in turn mainly projects to the medial amygdala (Me) and nucleus sphericus (NS). The NS also projects to the Me and the lateral cortex (LC), which is the main olfactory recipient structure. The Me projects to the lateral posterior hypothalamic nucleus (LHN), which in turn projects to the hypoglossal nucleus (NXII) constituting a presumptive circuit controlling tongue-flicking behavior. In opossums, apical and basal receptor cells of the vomeronasal epithelium project to the anterior and posterior portions of the AOB, respectively. The AOB projects to layer 1 of the Me. The posterior portion of the Me alone projects to layers 2 and 3 of the Me. Centrifugal projections arising from the posteromedial cortical amygdaloid nucleus (PMCo) reaches exclusively the posterior portion of the AOB. Abbreviations: NL, nerve; GL, glomerular; M/T, mitral/tufted; GR, granule cell layers of the AOB.

and snakes have occurred recently. In snakes, the AOB projects mainly to the nucleus sphericus and medial amygdala (Fig. 7), whereas the MOB mainly projects to the lateral cortex, the external and ventral anterior amygdaloid nuclei, retrobulbar formation and olfactory tubercle (Lanuza and Halpern, 1998). Until recently, it was thought that

the tertiary projections arising from the nucleus sphericus projected mainly or exclusively to the ventromedial hypothalamic nucleus, thus relaying VN information directly to the hypothalamus (Halpern, 1980; Mart´ınez-Garc´ıa et al., 1993; Voneida and Sligar, 1979). A recent study in snakes has demonstrated that the nucleus sphericus gives rise to

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only a very small projection to the hypothalamus, but does project to other telencephalic structures such as the medial amygdala (Lanuza and Halpern, 1997). The medial amygdala receives VN information from the AOB (Lanuza and Halpern, 1998) and from the nucleus sphericus (Lanuza and Halpern, 1997). In addition, the medial amygdala receives an olfactory input from the external and ventral anterior amygdaloid nuclei and projects to the lateral posterior hypothalamic nucleus (Mart´ınez-Marcos et al., 1999). Since the lateral posterior hypothalamic nucleus projects to areas controlling the tongue musculature, i.e. the hypoglossal nucleus (Fig. 7), it has been suggested that this pathway could mediate chemosensory influences on tongue-flicking behavior in snakes (Mart´ınez-Marcos et al., 2001b). Vomeronasal–olfactory interactions may occur as well in olfactory recipient structures. The nucleus sphericus of snakes projects to the lateral cortex and to the external and ventral anterior amygdaloid nuclei (Fig. 7). Thus, convergence of olfactory and VN information could occur in these areas (Lanuza and Halpern, 1997). Furthermore, the rostroventral lateral cortex (Hoogland and Vermeulen-VanderZee, 1995; Mart´ınez-Marcos et al., 1999) and the external and ventral anterior amygdaloid nuclei (Mart´ınez-Marcos et al., 1999) project to other areas of the amygdala such as the posterior–dorsal ventricular ridge and the dorsolateral amygdaloid nucleus, both of which, in turn, project to the hypothalamus. The posterior–dorsal ventricular ridge projects massively to the ventromedial hypothalamic nucleus (Bruce and Neary, 1995; Lanuza et al., 1997; Mart´ınez-Marcos et al., 1999), a role previously attributed to the nucleus sphericus, whereas the dorsolateral amygdaloid nucleus projects to the periventricular and mammillary hypothalamus (Mart´ınez-Marcos et al., 1999). In summary, VN information is mostly relayed to the hypothalamus, not via a direct relay in the nucleus sphericus, but through other telencephalic structures. Convergence of olfactory and VN information appears to occur at different levels in the telencephalon of reptiles (reviewed in Mart´ınez-Marcos et al., 2002). Accordingly, interaction of olfactory and VN information could occur at different levels in the telencephalon of reptiles. 7.2.3. Mammals Earlier studies of the efferent projections of the AOB of mammals (reviewed in Halpern, 1987; Price, 1987; Wysocki and Meredith, 1987) considered the AOB a single entity. The efferent projections of the AOB were described reaching the bed nuclei of the accessory tract and stria terminalis and, particularly, layer 1 of the medial and posteromedial cortical amygdaloid nuclei. These connections were reinvestigated and confirmed using transneuronal transport of WGA-HRP placed in the VNO (Itaya, 1987). Later, projections from the AOB were extended to the supraoptic nucleus in rats (Smithson et al., 1992), although this single observation needs replication. Similarly, AOB projections have been

observed in deep cell layers (layers 2 and 3) of some nuclei of the medial amygdala in opossums (Mart´ınez-Marcos and Halpern, 1999a) (Fig. 7) and mice (von Campenhausen and Mori, 2000). A direct projection by-passing the amygdala from the VN epithelium to the hypothalamus has been reported in rats (Larriva-Sahd et al., 1993), however this report is based on electron microscopic examination of hypothalamic tissue 72 h after purported VN nerve transection. Since this study reports findings contrary to a large body of anatomical data it must be evaluated very critically and acceptance of its conclusions should await independent confirmation. The VN amygdala projects reciprocally to the AOB, thus forming a feed-back circuit. These projections have been demonstrated using anterograde methods (reviewed in Canteras et al., 1992, 1995; Coolen and Wood, 1998; Gomez and Newman, 1992; Halpern, 1987) and retrograde tracers (Luiten et al., 1985 and reviewed in Halpern, 1987) (Fig. 7). Other important afferents for AOB function arise from noradrenergic and serotonergic structures in the brainstem (reviewed in Brennan and Keverne, 1997; Halpern, 1987; Keverne, 1983). Most previous reports did not consider the AOB as a dichotomous structure. As discussed in Section 3, anterior and posterior divisions of the AOB have differential afferent connections from the VNO (Fig. 8) and, in some species, differential afferent and efferent connections with the amygdala (Fig. 7). VN-recipient structures themselves, and VN- and olfactory-recipient areas in the basal telencephalon are largely interconnected prior to relaying chemosensory information to the hypothalamus. Thus, VN and olfactory information could converge at the level of the amygdala. This was already noted by Scalia and Winans (1975), who described a small number of axons arising from the MOB reaching the anteroventromedial portion of the medial amygdaloid nucleus in the rat, a VN-recipient structure. Regarding interconnectivity of structures in the basal telencephalon, the piriform cortex, which is the major target of the MOB, projects to the endopiriform nucleus, which in turn projects to the medial and posteromedial cortical amygdaloid nuclei (Krettek and Price, 1978a,b). Further, the posterolateral cortical and anterior cortical nuclei, both of which are olfactory-recipient structures, project to the posteromedial cortical and medial amygdaloid nuclei (e.g. Canteras et al., 1992; Coolen and Wood, 1998). Convergence of olfactory and vomeronasal projections was demonstrated to be real integration at this level after electrical stimulation of the main or accessory olfactory system which activated some of the same neurons in the posteromedial cortical amygdala (Licht and Meredith, 1987). An interesting corollary is that integration appears to occur in one direction, i.e. olfactory information appears to flow to VN areas, but not conversely. Apart from other projections, tertiary connections from the VN-recipient amygdala primarily reach hypothalamic areas, namely, the preoptic area, ventromedial hypothalamic

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Fig. 8. Injections of dextran amines into the accessory olfactory bulb (AOB) (A, C, E, and parasagittally sectioned) and the resulting retrograde labeling in the vomeronasal organ (VNO) (B, D, F, and coronally sectioned) of opossums. Injections in the anterior (A) or posterior (C) AOB result in retrograde labeling in the apical (B) or basal (D) vomeronasal neurons, respectively. Double labeling experiments with injections in the whole AOB (red) or restricted to the posterior portion of the AOB (green) (E) results in cells labeled in red in both layers or cells labeled in green restricted to the basal layer of the VNO (F). Abbreviations: FC, frontal cortex; MOB, main olfactory bulb. Calibration bar for (A), (C), and (E) is 200 ␮m and for (B), (D), and (F) is 25 ␮m.

nucleus and premammillary nucleus, through the stria terminalis (e.g. Canteras et al., 1992, 1995). Before reaching the hypothalamus, the output pathway through the stria terminalis sends branches to areas such as the BNST, a part of the so-called extended amygdala (e.g. Alheid et al., 1995; de Olmos and Heimer, 1999; Swanson and Petrovich, 1998), which appears to play a major role in executing behaviors triggered by VN cues (reviewed in Newman, 1999). The medial amygdala is not a single homogeneous nucleus, but a complex structure composed of different divisions (Canteras et al., 1995; Coolen et al., 1997; Gomez and Newman, 1991, 1992; Risold et al., 1997). In rats and hamsters the medial amygdaloid complex has been partitioned into four nuclei (anterodorsal, anteroventral, posterodorsal and posteroventral) that differ in their cytoarchitecture, cell morphology, neurotransmitter content, distribution of sex steroid hormone receptors, connections and functions (Canteras et al., 1995; Coolen and Wood, 1998; de Olmos et al., 1985; Gomez and Newman, 1991, 1992; Wood and Newman, 1995). Furthermore, two broad functional divisions have been proposed for the medial amygdaloid complex. In rats, the dorsal division (posterodorsal

medial amygdaloid nucleus) appears to be involved in autonomic functions (Canteras et al., 1995; Risold et al., 1997), whereas, in hamsters, it has been reported to participate in consummation of mating behavior (male ejaculation) (Coolen et al., 1997; Kollack-Walker and Newman, 1997) and the timing of the behavior sequence involved in mating (Parfitt and Newman, 1998). In contrast, the ventral division (anterodorsal, anteroventral and posteroventral medial amygdaloid nuclei) appears to participate in sexual and agonistic behaviors in rats (Canteras et al., 1995; Risold et al., 1997). It is interesting to note that the deep cell layers of the medial amygdaloid complex described in opossums (Mart´ınez-Marcos and Halpern, 1999a), which receive inputs from the posterior portion of the AOB, correspond to the ventral division of the medial amygdala as defined in rats. The significance of this additional projection system in the opossum remains obscure (discussed in Mart´ınez-Marcos and Halpern, 1999a). Regarding behavioral correlates, functional activation along the VN pathway has been recently reviewed (Keverne, 1999; Newman, 1999; Newman et al., 1997; Numan and Sheehan, 1997; see also Section 8). This issue, however, is

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quite complex since it covers a large number of different behaviors, including a vast body of literature. In addition, the VN versus olfactory contribution to each one of these functions is far from being elucidated. Finally, there are remarkable differences between sexes and across species in VN activation. Collectively, data from amphibians, reptiles and mammals show that olfactory and VN anatomical pathways run parallel in the brain, thus supporting the dual olfactory hypothesis. It is also clear, however, that important interconnections between these pathways occur at telencephalic levels prior to relaying information to other regions such as the hypothalamus. Interactions between these two chemosensory systems are probably responsible for some of the difficulty in distinguishing olfactory from VN contributions to given behaviors. Finally, in mammals, the two subdivisions of the VNS are well established to the AOB, although the partial segregation of these pathways within the medial amygdala is still controversial.

8. New functional data on the role of the VNS in species-typical behaviors Prior to 1987, it was widely accepted that the VNS was importantly involved in chemosensory-mediated pheromonal effects on endocrine regulation and sexual behavior. In addition, it had been demonstrated in snakes that the VNS was critical for response to prey-derived chemicals and conspecific odors used in aggregation and courtship (Halpern, 1987; Wysocki and Meredith, 1987). In the ensuing years, few additional functions for the VNS have been described, although there has been important additional information on the details of the effects, mechanisms of VN involvement and physiological bases for some of the effects. In the following survey of the literature on functional aspects of VN stimulation we begin with a summary of recent reviews and then discuss pheromonal effects, reproductive, parental, aggressive and marking behavior, individual odor discrimination and other responses to chemosignals in mammals. In addition, pheromonal communication in non-mammalian vertebrates and non-pheromonal functions of the VNS are reviewed. Studies demonstrating that responses to some pheromones do not depend on a functional VNS are also described. Finally, we report on studies concerning odorant access to the VNO and the reinforcing effects of VN stimulation. 8.1. Reviews Although no global reviews of the functional aspects of the VNS have been published since 1987, several critical and comprehensive reviews of specific aspects of VN involvement in reproductive behavior have appeared. Extensive discussions of the nature of pheromones and their roles in

chemical communication are presented by Preti and Wysocki (1999), Meredith (2001) and McClintock (2002). Meredith (1998a) discusses a variety of issues concerning the VNO as pheromone detector, odorant access to the VNO, signal transduction, afferent coding and the role of the AOB in memory. Most recently Johnston (1998, 2000, 2001) has reviewed the literature on functional aspects of the VNS, emphasizing the need to be quite clear about what is meant by the term “pheromone” and cautioning the reader about thinking of the VNO as exclusively a “pheromone” receptor organ. Johnston (2000, 2001) is concerned about the widespread use, in the literature, of the term “pheromone” without proper definition. He introduces a novel categorization of types of chemical signals, which should aid in understanding different types of chemical communication. Johnston defines “chemical signals” as chemical compounds or mixtures released by one individual that affect a second individual of the same species, and suggest that this term should be used as a generic term for odors involved in communication. Subcategories of chemical signals include: (1) pheromones: single chemical compounds that have the effects of chemical signals; (2) pheromone blends: mixtures of small numbers of compounds that are effective only in relatively precise ratios; (3) mosaic groups or odor mosaics: mixtures of a large number of compounds that have the effects of chemical signals. A number of animal models, such as hamsters, voles, mice, rats, farm animals and reptiles, have proved especially useful for elucidating the functional roles of the VNS. Hamsters were the first animals in which VN deficits were demonstrated to affect reproductive behavior (Powers and Winans, 1975) and they continue to be particularly useful animal subjects for examination of the roles of the olfactory and VN systems in mating behavior. This literature was recently reviewed by Johnston (1998), Meredith (1998b) and Newman (1999). Johnston (1998) discusses primarily hamster chemical communication, with particular attention to pheromones, and makes several important points, among which are the following. The great diversity of signals used in chemical communication by vertebrates indicates that this communication is not mediated exclusively by pheromones. A multiplicity of signals may be present in the same secretion and it is unlikely that response to this diversity of signals will be mediated exclusively by a single sensory system. Although the VNS is known to mediate some behavioral and endocrinological responses to pheromones, even in these cases experience and context may alter the dependence of the response on a functional VNS. A number of pheromonal responses are clearly not dependent on the VNS, but are dependent on the olfactory system. For example, in the presence of estrous females, male rats deprived of direct access to the females display penile erection (Sachs, 1997; Sachs et al., 1994). Olfactory cues, but not VN input, are necessary for this response (Kondo et al., 1999). Although bedding from estrous females does not elicit penile erection (Kelliher et al., 1999), immediate early gene expression (c-Fos) in rats

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displaying non-contact penile erection is absent in the main olfactory bulb, but is present in several central structures (nucleus accumbens, medial amygdala, bed nucleus of the stria terminalis and medial preoptic area) that are part of the projection circuit that is activated by pheromones present in estrous bedding (Kelliher et al., 1999). In many behaviors, it is therefore likely that the VN and olfactory systems interact to provide appropriate context for behaviors. In his review of the role of VN, olfactory and hormonal systems in mating behavior of male golden hamsters, Meredith (1998b) emphasizes the convergence of VN and olfactory information onto a common neural circuit controlling reproductive behavior and the role of luteinizing hormone releasing hormone (LHRH) in activating this circuit. Focusing on these central circuits, Newman (1999) reviews the literature on the role of the medial extended amygdala in a variety of social behaviors regulated by hormonal and chemosensory signals including male and female mating behavior, aggression, territorial marking and maternal behavior. The review is not primarily oriented to chemosensory modulation of these behavior, but to the shared circuitry involved in these behaviors. In recent years, voles have become particularly interesting subjects for studies of the role of the VNS in reproductive functions because the females of different vole species use different reproductive strategies, e.g. spontaneous ovulation as compared to induced or reflex ovulation. Wysocki and Lepri (1991) review the literature on VNO removal (VNX) in house mice and prairie voles (M. orchragaster). Removal of the VNO of male mice or voles results in deficits in female odor-induced testosterone increases, ultrasonic vocalization to females, sexual behavior, territorial marking and inter-male aggression. Removal of the VNO in group-housed female mice results in the loss of secretion of puberty-delaying substances. In female voles male-induced activation of reproduction is decreased or eliminated by VNX. Maternal aggression is also reduced in female mice following VNX. Rats and mice have, of course, been the staple of laboratory studies of vomeronasally-mediated hormonal control of reproductive behavior. Dudley et al. (1996) provide an overview of rodent reproductive behavior as it is guided and facilitated by the VNS. In particular, they emphasize the role of vomeronasally-mediated signals on release of LHRH in female rats. Keverne (2002a) discusses gender-specific behaviors, particularly in mice in light of new findings that mice deficient in the TRP2 ion channel engage in behaviors that suggest that the mice are unable to discriminate males from females (Stowers et al., 2002; Leypold et al., 2002). Farm animals have offered interesting insights into the importance or lack of importance of the VNS in reproductive behavior. In addition, due to the size of the animals and their VNOs, they have served well as subjects for studying odorant access to the organ. Reviewing the roles of the olfactory and VN systems in farm animal behavior, Hart (1987) provides a particularly interesting discussion of the role of

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flehmen in reproductive communication. In a review of the roles of the olfactory and VN systems in reproductive behavior in sheep, Signoret (1991) concludes that neither is necessary for normal behavior. The reptilian VNS provided some of the earliest demonstrations of the role of the VNS in both reproductive and appetitive behaviors. This literature was reviewed by Halpern (1992) and Mason (1992) who emphasize the critical importance of chemical signals in prey detection and intraspecific communication. 8.2. Pheromonal effects Behavioral changes that depend on chemosignals to modify activity of the hypothalamic–pituitary axis have been termed, primer pheromone effects. As normally understood, primer pheromones are chemical substances secreted or excreted by an individual that cause endocrinological changes in other members of the same species. Most of these primer pheromone effects were initially described in mice. The role of the VNO and primer pheromones in mammals, with particular attention to the effects of VN stimuli on female sexual behavior and development has been reviewed by Vandenbergh (1989). Although mainly concerned with mice, the review also discusses the implications of this research on domestic farm animals. Female mouse urine contains a vomeronasally-mediated pheromone that causes increases in circulating LH in male mice. Urine retains its biological activity after dialysis, suggesting that the activity is associated with a urinary protein; however, it loses its activity after absorption chromatography on a neutral polystyrene column, suggesting that the protein is a carrier, but not the pheromone itself. Proteolytic degradation does not destroy pheromonal activity, therefore the protein is not necessary for male response. A low molecular weight fraction, depleted of protein has high biological activity. This pheromone thus appears to be a low molecular weight substance associated with a major urinary protein (MUP) (Singer et al., 1988). Presently there is a controversy over the issue of what components of male urine are the active stimulants for a number of other vomeronasally-mediated endocrine effects in females. A distinctive component of male urine is the MUP to which small, volatile molecules bind. MUPs have a molecular mass of approximately 20 kDa, have a ␤-barrel structure enclosing a hydrophobic ligand-binding site and are members of the lipocalin family. The MUPs secreted in the urine of male mice bind a number of volatiles. Is it the MUPs or is it the volatiles that are responsible for the endocrine effects? This issue was recently reviewed by Cavaggioni et al. (1999) and Cavaggioni and Mucignat-Caretta (2000) who take the strong position that MUPs alone are capable of inducing puberty acceleration and estrus synchrony. A very different point of view has been expressed by Novotny et al. (1999a,b). Since the groups report contradictory results, it is not possible at this point to

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resolve this issue. Data in other mammals could help to design experiments to resolve this controversy. In rats, for instance, lipophilic volatiles of urine activate Gi2␣ -expressing neurons, whereas some MUPs activate Go␣ -expressing neurons (Krieger et al., 1999; see also Section 3). It is now well established that pheromonal control of mouse reproduction, particularly that controlling many of the “primer pheromonal effects”, is mediated via the VNS. Many of these primer effects appear to use the same neural pathway from VNO to the AOB to amygdala to dopaminergic neurons in the arcuate nucleus of the hypothalamus. These latter neurons, in turn, regulate prolactin secretion from the anterior pituitary. Among the murine primer effects, the Bruce effect (Bruce, 1959) has been of particular interest because of its reliance on development of memories for chemical signals emitted by the stud male. 8.2.1. Bruce effect In the Bruce effect (Bruce, 1959), newly mated female mice return to estrus if exposed to strange males prior to embryo implantation (<72 h). The physiological result of exposure to the strange male is a decrease in prolactin which causes implantation failure. The VNS is essential to this effect (Bellringer et al., 1980) and its control is effected through its connections to dopaminergic tuberoinfundibular neurons in the arcuate nucleus (see below). The neural pathways involved in the Bruce effect have been described. VN bipolar receptor cells make glutaminergic synapses onto the dendrites of M/T cells in the GL of the AOB. Glutamate released by mitral cells causes depolarization of granule cells (Brennan et al., 1990) which, in turn, hyperpolarize mitral cells via release of GABA (Mori, 1987b). Chemical information from the AOB is transmitted to the amygdala via glutamate-related substances released in the amygdala. Subsequent transmission to the tuberoinfundibular region of the hypothalamus is via the stria terminalis, the fibers of which terminate on dopaminergic cells in the tuberoinfundibular region (Li et al., 1990a). Inhibition of GABAergic transmission in the AOB, by local infusion of the GABA antagonist bicuculline, results in increased spontaneous firing of tuberoinfundibular neurons (Li et al., 1990b), suggesting that decreased inhibition in the AOB mitral/granule cell reciprocal synapses may account for the Bruce effect. Furthermore, in newly-mated female mice, electrical stimulation of the AOB, appropriately timed to coincide with a prolactin surge, is capable of producing pregnancy block (Li et al., 1994). Stimulation of the mouse AOB has been shown to activate excitatory amino acid receptors in the amygdala and subsequent cholecystokinin-␤ receptors in the preoptic area which results in excitation of tuberoinfundibular dopaminergic arcuate neurons (Li et al., 1989, 1990a, 1992a). Estradiol injected into the amygdala of ovariectomized female mice increases the excitatory responses of tuberinfundibular arcuate neurons to AOB stimulation, suggesting that estrogen modifies vomeronasally-mediated responsiveness

by acting directly on the amygdaloid neurons (Li et al., 1992b). Exposure of a newly-mated female to the odor of an unfamiliar male is the critical event causing implantation failure in the Bruce effect. The odor of the stud male, however, has no such effect. Thus, the memory of the odor of the stud male is critical for this discriminated endocrine response. One “location” for this “memory” could be the AOB. Memory formation (of the stud male) occurs after mating and is dependent on noradrenergic innervation of the AOB (Keverne and de la Riva, 1982). Synaptic changes indicative of plasticity have been observed in the dendrodendritic synapses between mitral cells and granule cells that accompany memory formation (Matsuoka et al., 1997). Mated females have significantly longer synaptic densities in asymmetric synapses between mitral and granule cells in the mitral/tufted cell layer of the AOBs compared to unmated females. These results suggest that synaptic plasticity in the AOB could be part of the anatomical substrate for the chemosensory memory underlying the Bruce effect. High levels of nitric oxide synthase (NOS) are present in the AOB of mice (Bredt et al., 1991) and therefore, nitric oxide generation could participate in memory formation. The NOS inhibitor N ␻ -nitro-l-arginine infused into the AOB, however, does not prevent olfactory recognition memory formation of the stud male (Brennan and Kishimoto, 1993; Okere et al., 1995), indicating that NOS activity is not required for memory formation in the Bruce effect. Nonetheless, nitric oxide may be involved in memory formation for the odor of the stud male (Okere et al., 1996). Typically, a memory for the stud male forms only during mating, and will not form on simple exposure to the male’s odor. Using the nitric oxide generator sodium nitroprusside (SNP), Okere et al. (1996) demonstrated that exposure of females to male pheromone without mating during two infusions of SNP results in memory formation for that pheromone, i.e. the pheromone does not cause a block to a subsequent pregnancy resulting from a mating with a different stud male. Infusion of the ␣-adrenergic antagonist, phentolamine during SNP infusion prevents SNP-mediated memory formation as does injection of 6-hydroxydopamine into the AOB. Thus, it appears that nitric oxide activation may be sufficient, but is not necessary for formation of an olfactory recognition memory. Since NMDA receptor activation has been implicated in many forms of memory formation, it is important to determine whether memory for the stud male is dependent on NMDA receptor activation. Infusion of non-selective excitatory amino acid receptor blockers (e.g. d-glutamylglycine) into the AOB blocks memory of the stud male (Brennan and Keverne, 1989). However, local infusion of selective NMDA antagonists fail to block memory of the stud male, suggesting that the memory of the stud male may depend on non-NMDA excitatory amino acid receptors. There is an increase in cells expressing immediate early genes during formation of memory for the stud male.

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Fig. 9. Female mouse brain (dorsal view) illustrating location of Fos-ir locations following exposure to male bedding. Based on data from Halem et al. (1999). Structures that are Fos-ir include the: vomeronasal organ (VNO), accessory olfactory bulb (AOB), medial preoptic area (MPA), bed nucleus of the stria terminalis (BNST), medial amygdala (MeA and MePD).

Brennan et al. (1992) reported that the number of mitral and granule cells in the female AOB expressing the immediate early genes c-fos and egr1, but not c-jun, increase following mating. Pheromonal stimulation alone or mating without pheromonal stimulation did not increase the number of cells expressing immediate early genes. Thus, an association appeared to be necessary between pheromonal stimulation and mating for the increased expression. However, note that this is not universally the case. Infusion of the GABAA antagonist, bicuculline, in the absence of mating resulted in a large increase in c-fos and egr1 expression. Increases in immediate early gene expression are also observed in neurons in the VN amygdala and preoptic area following mating (Halem et al., 2001b) (Fig. 9). However, when female mice are re-exposed to the odor of the stud female following mating, that odor fails to activate immediate early gene expression in the medial amygdala and medial preoptic area. This result suggests that the mating event modifies the VNS in such a manner that the memory for the stud male’s odor results in a subsequent failure to activate portions of the VNS central to the AOB when the female is re-exposed to that odor. This failure of response to the stud male’s odor may account for the absence of pregnancy block in response to stud male chemosignals. How long do pheromonal memories last and what governs formation of memories for new stud males? The results of a study investigating the time course of memory for the chemosignals from a stud male indicate that memory is retained for at least 30 days (Kaba et al., 1988). However, by 50 days the memory of the odor of the stud male is no longer capable of preventing implantation failure. In addition, dur-

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ing pregnancy, a time when estradiol levels are high, the memory trace fades more rapidly. Pregnancy or treatment of female mice with estradiol enhances neurogenesis in the VN epithelium. The authors suggest that this enhanced neurogenesis results in fading of the memory trace for the stud male. However, according to this scenario memory formation would take place in the VNO or in the glomerular layer of the AOB, which is not likely. Not all aspects of pheromonal memory are mediated by the VNS. Food deprivation of newly inseminated female mice during the pre-implantation period induces implantation failure. This implantation failure is prevented if the stud male remains with the female. Interestingly, food-deprived VNX females housed with stud males do not demonstrate implantation failure. In contrast, food-deprived females made anosmic by zinc sulfate (ZnSO4 ) irrigation of the nasal cavity exhibit high rates of implantation failure even, when housed with the stud male. These results suggest that recognition of the chemosignals emitted by the stud male that protect the newly inseminated, food-deprived females from implantation failure are detected by the main olfactory system and not by the VNS (Archunan and Dominic, 1990). 8.2.2. Whitten effect and induced estrus The Whitten effect (Whitten, 1959) is the induction of estrus in female mice made anestrus by group housing. This induction is caused by stimuli from males or their urine and is known to be dependent on a functional VNS. Jemiolo et al. (1986) identified DHB and SBT, volatiles normally bound to MUPs as androgen-dependent urinary metabolites that promote estrus synchrony. The ␣- and ␤-farnesenes are capable of inducing estrus in female mice, as well as signaling dominance in male mice (Novotny et al., 1985). Female rats undergo a spontaneous estrus every 4–6 days. A functional VNS is not required for this cycling, as VNX females cycle normally. However, female rats reported to be made anestrus by injections of estradiol benzoate, group housing or constant light, will enter estrus when exposed to male pheromones (Mora and Cabrera, 1997; Whitten, 1959) and this effect was previously demonstrated to depend on a functional VNS (Mora and Cabrera, 1997; Sánchez-Criado, 1982; Sánchez-Criado and Gallego, 1979). Voles are a particularly interesting group in which to study the roles of the VNS in reproduction since females within this group may be reflex or spontaneous ovulators. Female prairie voles (M. ochrogaster) are reflex ovulators, i.e. isolated female prairie voles do not normally exhibit spontaneous estrus, but normally require tactile stimuli and chemosignals from males to activate their reproductive system. Female prairie voles exposed to urine from male prairie voles have increased Fos-ir in the AOB compared to controls (Tubbiola and Wysocki, 1997) and removal of their VNOs results in a disruption of male-induced reproductive activation (Lepri and Wysocki, 1987). It would thus appear that exposure to male prairie vole urine activates the female VNS and that functionality of this system is

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critical to urine-induced activation of the female reproductive system. Furthermore, there is species specificity in the chemical cues that activate this process. Female pine voles (M. pinetorum) are also reflex ovulators, however, exposure of these females to male urine or male-soiled bedding is not adequate to induce reproductive activation (Solomon et al., 1996). Female pine voles require contact with males to exhibit such reproductive activation. Interestingly, females with VNX do not exhibit reproductive activation when permitted contact with males, suggesting that contact alone is an insufficient stimulus for reproductive activation. In contrast to female prairie and pine voles, VNX in female meadow voles (M. pennsylvanicus), which are spontaneous ovulators, does not suppress mating. Remarkably, VNX in female meadow voles maintained on long photoperiods (the period of normally intense breeding) facilitates mating behavior (Meek et al., 1994). The differences in the results reported for VNX in female prairie and pine voles, compared to meadow voles, indicates that caution must be exercised in generalizing among species, even those of the same genera. Female gray short-tailed opossums, M. domestica, will remain anestrous when isolated, but within 4–6 days of exposure to a male, his scent-marked cage or secretions from his suprasternal gland will evidence signs of estrus (Fadem, 1987, 1989; Jackson and Harder, 1996, 2000). Females with VNXs are not induced to go into estrus when exposed to male pheromones, whereas sham-operated animals and opossums with partial ablations can be induced with male pheromones to enter estrus (Jackson and Harder, 1996). Thus, the signals inducing estrus in female opossums appear to be vomeronasally-mediated. It appears from the studies in rats, voles and opossums that the VNS is not essential for normal cycling in species that display spontaneous estrus, but is important for some species in which reflex ovulation has a strong chemosensory component. 8.2.3. Vandenbergh effect Puberty acceleration (Vandenbergh, 1983), caused by exposure to male chemosignals during female development, occurs in several mammalian species and has been shown previously to depend on a functional VNS (Lomas and Keverne, 1982). Female mice exposed to male urine will enter puberty earlier than those not exposed to male urine. It has been reported that puberty acceleration can be induced with the MUPs stripped of their bound ligands by organic extraction or competitive displacement with a high-affinity ligand (Mucignat-Caretta et al., 1995). According to these authors, odorants bound to MUPs do not themselves induce puberty acceleration. A MUP-related hexapeptide was reported to induce puberty acceleration. The authors proposed that VNO receptors recognize an N-terminal consensus sequence, N-Glu-Glu-Ala-X-Ser (where X is a polar

residue), a sequence common to both MUP and the hexapeptide (Mucignat-Caretta et al., 1995). In contrast to these results, Novotny et al. (1999b) report that neither the recombinant MUP nor the hexapeptide described by Mucignat-Caretta et al. (1995) have biological activity in the bioassay used to demonstrate puberty acceleration, i.e. increased uterine weight in juvenile female mice. Instead, Novotny and collaborators have identified several different urine constituents, all of which are normally bound to MUPs, that independently are capable of accelerating puberty in female mice (Novotny et al., 1999b). These are: 2-sec-butyldihydrothiazole (SBT), 3,4-dehydro-exo-brevicomin (DHB), 6-hydroxy-6methyl-3-heptanone, and ␣- and ␤-farnesenes (Novotny et al., 1999a). However, it should be noted that SBT and DHB were previously reported by this group to be ineffective in promoting growth in prepubertal females when added to plain water or added to castrate male urine (Novotny et al., 1985). Improved techniques, no doubt, contributed to the later finding that both of these compounds, independently, do contribute to puberty acceleration. In a more recent publication (Novotny et al., 1999b), the synthetic analogues of DHB, SBT and ␣- and ␤-farnesene all exhibited biological activity and strong affinity to MUPs. These compounds were also implicated in estrus synchronization (see above). In an early study, immature females exposed to synthetic analogues of urinary volatiles and mixtures of synthetic volatiles exhibited puberty acceleration or delayed puberty depending on the quantitative ratios of a number of urinary compounds. High concentrations of volatile ketones appeared to be essential for puberty acceleration and estrus extension in young female mice (Jemiolo et al., 1989). Juvenile female mice exposed to male-soiled bedding exhibit increased c-fos expression in the AOB, whereas females exposed to peppermint odor exhibit an increase in c-fos expression in the MOB (Schellinck et al., 1993). The authors conclude that the AOB is stimulated by social odors and the MOB is stimulated by non-social odors. Female Djungarian hamsters, Phodopus campbelli, whose puberty has been accelerated after having been housed with adult males have a smaller lateral division of the anterior BNST than females housed alone or females matched for sexual maturity but housed alone, suggesting a morphological consequence to environmental signals that induce accelerated puberty (Reasner et al., 1993). In female prairie voles (M. ochrogaster) uterine growth in preparation for breeding is activated by VN-mediated chemosignals from males (Lepri and Wysocki, 1987; Wysocki et al., 1991). As compared to sham operated females, females with VNX have lighter uterine and ovarian weights after exposure to male chemosignals (Tubbiola and Wysocki, 1997). Furthermore, females with VNX are less likely than sham operated voles to exhibit lordosis or copulatory behavior and this difference persists regardless of prior sexual experience (Wysocki et al., 1991). Thus, it would appear that the induction of estrus in prairie voles

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by male chemosignals is mediated by the VNS and experience is unlikely to obviate the need for a functional VNS. 8.2.4. Delay of puberty Juvenile female house mice treated with the urine of intact females that have been housed together exhibit a delayed onset of puberty. The urine of adrenalectomized group-housed females, however, does not produce puberty delay in immature female mice. Addition of several urinary volatiles, 2,5-dimethylpyrazine, n-pentyl acetate and cis-2-penten-l-yl acetate, to the urine of adrenalectomized female mice restores its puberty delaying properties (Novotny et al., 1986). Jemiolo and Novotny (1994) identified the adrenal-mediated metabolite 2,5-dimethylpyrazine as the pheromone responsible for female–female delay of puberty. This latter pheromone is also involved in adult female estrus suppression. 8.3. Mammalian sexual behavior Whereas primer pheromone effects usually require a relatively long interval (e.g. hours or days) between stimulation and response, the so-called “releaser pheromone” effects are observed almost immediately. For example, ultrasound vocalizations to conspecific odors and male mating behavior in the presence of appropriate chemosignals are immediate responses to pheromones. Similarly, activation of brain regions by these chemical signals occurs within a relatively short interval. VNS involvement in reproductive behavior has been reported in a number of mammalian species using a variety of techniques. Ablation of the VNO frequently leads to alterations in reproductive behavior, usually in a deficit. We review the recent literature on VN involvement in sexual behavior and the concomitant increase in the expression of immediate early genes (Section 8.3.1). 8.3.1. Hamster In rodents and some other mammals, estrus females stimulated by a male will exhibit a characteristic posture called lordosis, involving dorsiflexion of the spine and elevation of the rump. Typically, lordosis occurs in the presence of the male. Female hamsters display lordosis in response to mounts by males and hold the lordosis posture for a prolonged period while the male dismounts and remounts the female several times. However, shortly after the male is removed, female hamsters respond to lumbosacral stimulation with a reinstatement of lordosis. VNX or closure of the nasopalatine ducts results in a significant increase in the latency to reinstatement of lordosis (Mackay-Sim and Rose, 1986). Injection of LHRH reverses the disruptive effects of VNX or duct closure. These results suggest that facilitation of somatically-induced lordosis is mediated by chemical signals from the male hamster and that these signals are conveyed to the brain by the VNS and involve a hormone(LHRH) priming mechanism.

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VNS involvement in male hamster mating behavior was first reported by Powers and Winans (1975). They found that approximately one-third of male hamsters with VN nerve cuts developed a severe mating deficit, whereas 100% of male hamsters with combined VN nerve cuts and ZnSO4 lavage of the main olfactory epithelium were sexually deficient. Subsequently, Meredith (1986) demonstrated that sexually inexperienced male hamsters were severely deficient following VNX, but that sexual experience prior to VN deafferentation could mitigate the lesion effects. Intraventricular delivery of LHRH significantly reduced the deficit in sexually inexperienced male hamsters following VNX (Fernandez-Fewell and Meredith, 1995; Meredith and Howard, 1992). This finding supports the idea that the facilitatory influence of VN stimulation acts through release of LHRH (Meredith and Howard, 1992). However, an LHRH analog, AcLHRH5–10 , that does not induce luteinizing hormone (LH) release, is also effective in mitigating the effect of VNX on mating behavior in inexperienced hamsters, suggesting that there may be an extra-pituitary mode of action of LHRH in this behavior (Fernandez-Fewell and Meredith, 1995). These studies and others related to LHRH release in response to VN stimulation are reviewed by Meredith and Fernandez-Fewell (1994). Aphordisin, isolated from female hamster vaginal secretions, is a powerful VN stimulant (Singer et al., 1986, 1987). This protein is a member of the ␣2u -globulin superfamily of extracellular transport proteins. Members of this family are known to bind smaller molecules. Male hamster response to the high molecular weight fraction of female vaginal secretion is entirely dependent on the VNS (Singer et al., 1987). Aphrodisin (17,600 MW), loses activity with proteolytic digestion. However, there remains a question concerning whether the protein itself is the pheromone or if the protein binds the pheromone. Molecular cloning of aphrodisin in E. coli indicates that the polypeptide backbone is only partially active. Full activity may depend on post-translational modification of the protein or on the presence of a tightly bound ligand that has not yet been identified (Singer and Macrides, 1990). In a more recent experiment, male hamsters were exposed to aphrodisin or its cloned protein backbone (rAPH). Although rAPH was not biologically active in the hamster mating assay, both rAPH and aphrodisin increased the number of c-Fos-immunoreactive cells in the M/T cell layer of the AOB as compared to a control substance, ␤-lactoglobulin (Jang et al., 2001), suggesting that both substances activate VN neurons. When stimulated with aphrodisin, more c-Fos immunoreactive cells were found in the caudal half of the M/T cell body layer and EPL than in the rostral half (see Section 3). Stimulation with rAPH did not show a difference in spatial distribution. Male hamsters exposed to female vaginal secretions exhibit an androgen surge. Vomeronasal organ ablation, but not olfactory epithelial destruction with ZnSO4 , results in a loss of this pheromone-induced hormonal response in sexually experienced and inexperienced male hamsters (Pfeiffer and

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Johnston, 1994). However, both VNX and ZnSO4 treatments result in decreases in the behavioral response to vaginal secretions. Interestingly, neither lesion has an influence on the androgen surge following interaction with an estrus female, suggesting that experience with the female can compensate for the absence of chemosensory information. Combined VNX and ZnSO4 treatment results in a loss of the androgen surge in inexperienced, but not experienced males, and mating behavior is lost in these males regardless of prior experience. These results suggest that, in hamsters, hormonal response to chemical stimuli is mediated by the VNS regardless of prior sexual experience, but that the olfactory system is capable of mediating hormonal responses to contact with a female, provided the male has had prior sexual experience. Immediate early gene expression patterns have been used to examine the neural pathways involved in mating behavior, particular those aspects related to chemosensory stimulation (Fernandez-Fewell and Meredith, 1994; Fewell and Meredith, 2002; Fiber and Swann, 1996; Fiber et al., 1993; Meredith and Fernandez-Fewell, 1994, 2001; Newman et al., 1997; Swann et al., 2001). Vomeronasal organ removal reduces this immediate early gene expression (Fernandez-Fewell and Meredith, 1994; Fiber et al., 1993; Meredith and Fernandez-Fewell, 1994; Newman et al., 1997). The pattern of mating-induced immediate early gene expression in male Syrian hamsters has been reviewed by Newman et al. (1997). Based on work from their laboratory and others they conclude that both olfactory and VN systems are important for maintenance of male mating behavior. Interestingly, Fos-ir stimulated by mating and agonistic behaviors is observed in many of the same structures of the chemosensory pathways (Kollack-Walker and Newman, 1995). However, the distribution and number of Fos-immunoreactive cells differs under these different conditions. For example, mating alone induces Fos-immunoreactive cells at the posteroventral level of the posteromedial BNST whereas agonistic behavior induces Fos-ir in the anterolateral BNST. Supporting the idea that the olfactory system is involved in hamster reproductive behavior are the results of a study in which male hamsters were made anosmic with ZnSO4 irrigation. These hamsters mate with receptive females as do control, intact males; however the anosmic males show little or no Fos expression in the main olfactory pathways. Compared to intact mating males, there is a significant decrease in Fos expression in the rostral anterior medial amygdala, a structure known to receive main olfactory input (Fernandez-Fewell and Meredith, 1998). Whereas VNO removal in sexually experienced male hamsters decreases female vaginal secretion-induced Fos-ir in the AOB it does not affect Fos-ir in the preoptic area (Fewell and Meredith, 2002; Swann et al., 2001). These results further reinforce the importance of the main olfactory system in the response of central structures to reproductive odors. Studies by Wood and Newman (1993, 1995) are particularly interesting in stressing the importance of integration of

chemosensory cues and hormonal cues in mating behavior of male Syrian hamsters. The role of the chemical senses in male hamster sexual behavior is one of the best studied areas. These studies have demonstrated the interaction of experience, hormones and chemical sense system in producing deficits in reproductive behavior. The wealth of data on functional pathways derived from immediate early gene studies has contributed to our understanding of both the pathways involved and the participation of both olfactory and VN systems in this behavior. Excitation of the VNS by relevant chemical cues can also be demonstrated using ultrastructural analysis of synaptic regions in the VN pathway. Development of the AOB appears to be modified by different social rearing conditions (Matsuoka et al., 1994). Male hamsters, reared under conditions where they have sensory exposure to conspecifics have larger AOB M/T cells than those reared in isolated conditions. The size of glomerular synapses also vary as a function of rearing condition, with larger synaptic contact zones observed in animals raised under social conditions compared to those raised in isolation or neighbor-only conditions. In a similar study, synapses in the granular layer of the AOB of adult male hamsters and rats reared for 2 months in a social environment were longer than similar synapses in the AOB of males reared in isolation (Matsuoka et al., 1996). These results suggest that plasticity in the AOB extends to adulthood and that chemical stimulation by conspecifics may affect synaptic efficacy. However, it is not possible to determine from these reports the extent to which these effects relate to the availability of chemical cues as compared to stimulation from other sensory systems. More direct evidence that chemical cues are responsible for this plasticity comes from a study in which male hamsters were exposed to urine of female hamsters and increases in the length of synaptic contacts between receptor cell terminals and M/T cell dendrites in the GL of the AOB were observed (Matsuoka et al., 1998). In contrast, however, exposure of male hamsters to female hamster urine, reduced the length of asymmetric synaptic contacts in the M/T cell layer, but had no effect on symmetric synaptic length (Matsuoka et al., 1998). The significance of this apparent plasticity in the AOB awaits further investigation. 8.3.2. Mouse Virgin female mice with bilateral ablation of the VNO or bilateral transection of the VN nerves exhibit normal estrous cycles and mating patterns, indicating that the VNS is not critical for normal female reproductive function in these animals (Rajendren and Dominic, 1986). Immediate early gene expression (egr1 or c-fos) is induced in the VNO and AOBs of male and female mice by exposure to soiled bedding (Guo et al., 1997; Halem et al., 1999, 2001b; Dominguez-Salazar et al., 2002). Increase in immediate early gene expression also results from stimulation with urine from intact males or a combination of the MUP and the volatiles normally bound to the MUP, DHB and SBT (Guo et al., 1997). A functional

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VNO is necessary to demonstrate this increase in immediate early gene expression. Neither DHB and SBT together, nor castrate male urine increase c-sfos mRNA in the AOB of mice with intact VNOs, suggesting that the volatiles alone are insufficient to activate the VNS. However, note that these results are at variance with those reported above by Brennan et al. (1999) (Section 3.6) and the issue of the efficacy of the volatiles versus the MUPs in mouse urine as VN stimulants is still controversial. As noted above (Section 5), the TRP2 ion channel is a critical part of the VN signal transduction pathway. Male mice with a genetic ablation of the trp2 gene engage in sexual behavior with conspecifics of both sexes and fail to display male–male aggression (Leypold et al., 2002; Stowers et al., 2002). trp2−/− males mount and emit ultrasounds to castrated male mice scented with intact male urine, a response that would be expected toward normal females (Fig. 10). Since TRP2 is found exclusively in the VN epithelium, these results strongly support the idea that the VNS is critical for male-specific behavior in response to sensory cues for sex discrimination (Leypold et al., 2002; Stowers et al., 2002). However, note that female transgenic mice, in which the expression of a bacterial nitroreductase gene is linked to the promoter for olfactory marker protein, made anosmic by administration of the pro-drug CB1954 delivered directly to the olfactory epithelium, are unable to discriminate and respond to male mouse urine (Ma et al., 2002). In these animals, the VNS was functional indicating the VNS alone is insufficient to mediate female responses to male mouse urine and to mediate discrimination between urine from different individuals. Thus, caution must be exercised in interpreting the findings of studies purporting to demonstrate that the VN system, but not the main olfactory system, is critical for sex discrimination.

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8.3.3. Rat Numerous studies have demonstrated immediate early gene (fos) expression in the VNS of male and female rats after mating, exposure to bedding, or urine of conspecifics (Baum and Everitt, 1992; Bressler and Baum, 1996; Dudley et al., 1992; Inamura and Kashiwayanagi, 2000b; Oboh et al., 1995; Paredes et al., 1998; Rajendren et al., 1993; Tsujikawa and Kashiwayanagi, 1999; Wersinger et al., 1993; Yamaguchi et al., 2000). The early gene product Arc is also expressed in male rat AOB after mating (Matsuoka et al., 2002). Vomeronasal organ removal reduces stimulus induced fos expression in the rat (Rajendren et al., 1993) as does administration of the serotonin antagonist ketanserin or the noradrenergic antagonist propanolol (Inamura and Kashiwayanagi, 2000b). Activation of immediate early gene expression has been used to determine the chemical nature of the exciting stimuli to the VNS. Exposure of female rats to male urine or to a preparation of urine processed by ultrafiltration (less than 5000 Da) induce Fos expression in the AOB, but not a preparation of the substances remaining after ultrafiltration, i.e. greater than 5000 Da (Tsujikawa and Kashiwayanagi, 1999). Dialyzed male rat urine (less than 500 Da) or the remaining substances (greater than 500 Da) are also ineffective in inducing Fos expression in female rat AOB, but combining these two fractions restores their effectiveness, suggesting that the effective chemosignal is a combination of high and low molecular weight elements (Yamaguchi et al., 2000). Proestrous female rats will spend more time in proximity to intact rats as compared to castrate rats, and that preference can be demonstrated using only chemosignals obtained from bedding of these animals. Rats with bilateral olfactory nerve transection, VNX or posteromedial cortical

Fig. 10. TRP2 knockout male mice have difficulty discriminating between male and female mice. Male knockout mice emit ultrasounds to and mate normally with female mice. They also emit ultrasounds to and attempt to mate with castrated male mice coated with urine of intact male mice (left). Wild-type male mice mount and emit ultrasounds to females (center), but attack male mice (right).

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amygdaloid nucleus lesions all fail to demonstrate this preference (Romero et al., 1990). In this study, it is not clear whether the bilateral olfactory nerve transections also involved the VN nerves, so it is not possible to determine whether the olfactory system itself is necessary for the discriminative response to male odors. Deficits in female rat reproductive behavior caused by VNX are apparently ameliorated by appropriate hormone treatment. Although VNX does not interfere with normal estrous cycling in virgin female Wistar rats (Saito and Moltz, 1986a), these animals are severely deficient in exhibiting lordosis and other proceptive behaviors such as “darting” and “hopping” in response to male stimulation (Saito and Moltz, 1986a). This deficit can be overcome partially by treatment with estrogen and progesterone and completely reversed by treatment with estrogen and LHRH (Saito and Moltz, 1986a). Ovariectomized estrogen-primed female rats stimulated by conspecific males demonstrate an increase in sexual receptivity and an elevation in LH levels. As compared to sham-operated females, VNX significantly reduces the enhancement of receptivity as well as reducing the LH elevation produced by mating with conspecific males (Rajendren et al., 1990). It thus appears that VN stimulation in female rats leads to LH release and, in the absence of this stimulation, treatment with other hormones, e.g. LHRH, can compensate for the absence of chemosensory activation of the pituitary–gonadal axis. Additional support for this idea comes from studies of co-localization of LHRH-ir and c-fos expression. In the brain of female rats, LHRH neurons increase c-fos expression after mating as compared to female rats exposed to male-soiled bedding or clean bedding. VNX reduces enhancement of lordosis and Fos staining of LHRH neurons (Rajendren et al., 1993; Rajendren and Moss, 1994). Mating also stimulates LH release and VNX suppresses such LH release. Male-soiled bedding is apparently an insufficient stimulus to produce the LH surge in female rats (Rajendren et al., 1993). Male rats, sexually na¨ıve or experienced, do not appear to be dependent on a VNS for sexual arousal or copulation although the system does contribute to both. Bilateral VNX depresses sexual activity as measured by intramission latency, latency to first ejaculation, number of mounts to ejaculation, number of ejaculations and mating efficiency. With sufficient time, however, in the presence of a receptive female, all males mate. Inexperienced males are more severely effected by VNX than experienced males (Saito and Moltz, 1986b). Sexually experienced male rats with their VNOs removed mate with stimulus females, mounting with similar frequency as sham-operated females, but with longer latencies (Kondo et al., 2003). Mating-induced c-fos expression is diminished in the granule cell layer of the AOB and medial amygdala of the VNX animals, but is unchanged in the mitral cell layer of the AOB. Thus, it would appear that mating-induced c-fos expression in the medial

amygdala is not dependent on activation of AOB mitral cells. 8.3.4. Vole Female prairie voles are induced ovulators and pair-bond after mating Curtis et al. (2001). Immediate early gene expression, c-Fos, has been induced in female prairie voles by male urine (Hairston et al., 2003; Tubbiola and Wysocki, 1997). Although male odors normally induce estrus, VNO removal prevents this induction. However, VNX females treated with estrogen mate normally, but fail to pair-bond after mating (Curtis et al., 2001) suggesting that the VNO of female prairie voles is critical not only for male odor-induced estrus, but for establishment of pair- bonding. Removal of the VNOs of sexually inexperienced male prairie voles (M. ochrogaster) results in reduced reproductive performance, fewer offspring sired and less aggressiveness as compared to sham-operated males (Wekesa and Lepri, 1994; Wysocki and Lepri, 1991). However, after initial testing all VNX males sired offspring and became equally aggressive as sham operates. Thus, these animals are still capable of mating with females and therefore do not require a VNS for this function. Similar to the findings in hamsters, experience appears to be a major factor in determining the effectiveness of VNX in producing mating deficits. 8.3.5. Lemur Removal of the VNO of the male lesser mouse lemur, a prosimian, decreases sniffing, mounting and anogenital investigation of proestrous females (Aujard, 1997). However, it has no effect on successful mating or testosterone levels. Since the animals deprived of a VNO are also less active, the authors question whether the effects of VNO removal are a result of a sensory deficit or a general disturbance to the central nervous system. 8.3.6. Elephant Female Asian elephants secrete a compound in their urine during estrus and before ovulation that acts as a signal to males of their readiness to mate. Rasmussen et al. (1996, 1997) isolated this compound and identified it as (Z)-7-dodecenyl acetate, a pheromone used by female insects to attract male insects. The pattern of release of (Z)-7-dodecenyl acetate has been described (Rasmussen, 2001). During the follicular stage of estrus the pheromone is elevated, reaching a maximum concentration just prior to ovulation. Adult male elephants secrete a bicyclic ketal, frontalin (1,5-dimethyl-6,8-dioxabicyclo[3.2.1]octane) during musth. Both young males and females respond to this substance; the response of females varies with the stage of the estrous cycle (Rasmussen et al., 2003). Although this data is most interesting, it should be noted that to date no study has demonstrated that the VNS is involved in the responses to these odors.

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8.3.7. Ferret Interestingly, exposure to soiled female bedding in the ferret increases neuronal Fos-ir in the MOB, but not in the AOB of conspecific males and females (Kelliher et al., 1998). Maternal odors also induce fos expression in the MOB but not the AOB of neonatal ferrets (Chang et al., 2001). Similarly, in ferrets, detection of airborne odorants by the main olfactory system appears to be necessary for heterosexual mate choice. Ferrets, both male and female, with permanently occluded nares, which block access of odors to the main olfactory epithelium, fail to exhibit heterosexual partner preferences (Kelliher and Baum, 2001). These surprising finding suggests that there are species differences in the types of signals that activate the VN and olfactory systems, but the subject requires further investigation using methods similar to those used in other species before concluding that these findings represents true species differences. 8.3.8. Opossum Nuzzling in the gray short-tailed opossum, M. domestica, delivers chemicals to the VNO (Poran et al., 1993a). Males nuzzle unfamiliar male odors significantly more than familiar odors (Poran et al., 1993b), however section of the VN nerves does not disrupt this differential nuzzling (Shapiro et al., 1996). These findings suggest that the differential nuzzling response to conspecific novel odors may be mediated by the olfactory system and not the VNS. As noted in the section on marking behavior, these same lesions do produce a deficit in discriminated marking behavior in response to chemosignals. 8.3.9. Guinea pig Neonatal VNX of male guinea pigs (Cavia porcellus) has no effect on vocalization to chemosignals from home cage bedding or strange male bedding when the guinea pigs are tested as pups. In adulthood, these same animals exhibit a diminished vocalization and a rapid extinction of head bobbing to vaginal secretions from female guinea pigs (Eisthen et al., 1987). 8.3.10. Salamanders and newts The submandibular mental gland of the male terrestrial salamander, P. jordani contains mainly two proteins Pj-22 and Pj-10 (22 and 10 kDa, respectively) (Feldhoff et al., 1999). A solution enriched with these proteins increases female receptivity (Houck and Reagan, 1990; Houck et al., 1998). The proteinacious secretions from the male mental gland activate VN receptor cells as demonstrated using uptake of a guanidinium analog, agmatine (Wirsig-Wiechmann et al., 2002). The 22 kDa protein, termed “plethodontid receptivity factor (PRF)” has been shown to increase female receptivity when applied to the female nares. DNA sequences for four PRF isoforms were determined and the derived

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amino acid sequences revealed homology to members of the interleukin-6 cytokine family. The authors state that pheromonal activity is a previously unnoted function for cytokines (Rollmann et al., 1999). A female-attracting pheromone from the abdominal glands of Cynops pyrrhogaster, the red-bellied newt, has been isolated, purified, sequenced and synthesized. This molecule, sodephrin, is a novel decapeptide that increases electrical activity in the VNO of sexually mature females. Castration or hypohysectomy diminishes the amount of sodephrin in the abdominal gland, but these levels can be restored by treatment with testosterone and prolactin. Sodephrin appears to be species-specific, as synthetic sodefrin cloned from C. ensicauda abdominal glands which attracts C. ensicauda females, fails to attract C. pyrrhogaster females (Kikuyama and Toyoda, 1999; Kikuyama et al., 1995, 1999, 2002). In conclusion, the VNS appears to be important, but not necessarily essential, for reproductive behavior in many of the vertebrate species studied to date. The precise role of this system, however, varies among species, between genders, as a function of experience and reproductive strategy. Chemosignal activation of the VNS of hamsters, rats, mice and voles has been demonstrated using the increased expression of immediate early genes and, in rats, ultrastructural changes in the VN pathway. Lesion studies indicate that a variety of responses to conspecific chemosignals are dependent on a functional VNS. In addition, the ability of the olfactory system to compensate for the loss of VN stimulation similarly varies. There is an extensive literature on the effects of lesions of central structures to which the AOB projects, e.g. BNST and medial amygdala, on reproductive behavior, however, a review of this literature is beyond the scope of the present publication. 8.4. Parental behavior In a review of the role of the VNS in rat parental behavior, Del Cerro (1998) stresses that the system is sexually dimorphic and that the response to rat pups is quite different for males and females. Induction of parental behavior in nulliparous females and virgin males have a very different time course. Earlier studies indicated that olfactory and VN systems participated in pup avoidance in nulliparous females (Fleming et al., 1979, 1992). VN ablation accelerates maternal behavior in virgin female rats exposed continuously to young foster pups (Saito et al., 1988). Virgin male rats are more likely to kill newborn pups than females and this infanticide is markedly reduced following VNX (Mennella and Moltz, 1988). Interestingly, not only is there a reduction in infanticide, but males lacking a VNO are more likely to exhibit parental behaviors such as grooming, retrieval and nest-building than rats subjected to sham surgery that do not kill the young. Lesions of the BAOT, a major component of the telencephalic VNS, reduce latencies to parental behavior in virgin female rats (Del Cerro et al., 1991), as well as

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in male rats with no prior pup-care experience (Izquierdo et al., 1992). There has been some speculation about the mechanism by which VNS lesions affect parental behavior. Initially it was thought that some aversive odors, both volatile and of low volatility, emanated from the pups leading to aversion by females and, perhaps, triggering infanticide by males. More recently, Del Cerro (1998) has proposed that the VNS chronically suppresses parental behavior via inhibition of the medial preoptic area by the BAOT. In support of this idea, virgin female rats induced to become maternal by exposing them to newborn pups over a period of days show decreased levels of 2-deoxyglucose uptake in several structures associated with the VNS including the AOB, BAOT and medial amygdala (Del Cerro et al., 1995). Somewhat contradictory results have been reported on the importance of the VNS in maternal behavior of lactating female rats. Saito et al. (1990) report that lactating female rats with VNX are deficient in nursing behavior, whereas Kolunie and Stern (1995) report that this same procedure does not disturb maternal behavior of lactating female rats or maternal aggression. It is not possible at this time to reconcile these differences. Licking the anogenital region of newborn rat pups is a maternal behavior necessary for the survival of pups. A stimulus for this licking behavior is dodecyl propionate, a secretion of the pup’s preputial gland. Removal of the VNO of dams reduces licking behavior and other responses to dodecyl propionate (Brouette-Lahlou et al., 1999). Thus, in at least this case, the effect of deafferenting the VNS on maternal behavior appears to be primarily a sensory deficit. Further evidence of VN involvement in parental behavior comes from a study of the response of ewes to alien lambs (Booth and Katz, 2000). In this study, Dorsett ewes were subjected to ZnSO4 or procaine irrigation of their nasal cavities, cauterization of the nasoincisive duct to prevent odorant access to the VNO or no intervention (control). Control and anosmic ewes rejected the alien lambs and did not permit them to suckle. Ewes with cauterized nasoincisive ducts allowed the alien lambs to suckle, sniffed the alien lambs more and were less aggressive to the alien lambs than the control and anosmic ewes. Ewes with non-functional VNOs were not able to discriminate between their own ewes and the alien ewes. These results strongly imply that the VN but not the olfactory system is critical for recognition by sheep of newly born young. However, contradictory results were reported by Levy et al. (1995) who found that VN nerve section in primiparous or multiparous ewes before parturition did not disturb maternal selectivity at suckling. Anosmia caused by ZnSO4 treatment did eliminate selectivity of maternal care in both primarous and multiparous ewes and delayed onset of licking and suckling and reduced licking times and maternal bleats in primparous ewes, but not multiparous ewes. Thus, it would appear from this study that, in ewes,

the VNS is not critical for maternal care, the olfactory system may be important in ewes with no prior maternal experience, and that selective response to a ewe’s own offspring may require a functional olfactory system regardless of prior experience with parity (Levy et al., 1995). At this point, reconciling the results of these two studies is not possible. 8.5. Aggressive behavior Social interactions in many species include aggressive displays. The appropriateness of this behavior is largely dependent on recognition of one’s adversary as a potential threat, sexual competitor or territorial intruder. This recognition frequently involves detection of chemical cues sensed by the olfactory and/or VN systems. The involvement of the VNS in such recognition is clearly demonstrated by several studies. Genetic ablation of the TRP2 channel results in a failure of pheromones to induce action potentials in the VN primary pathway. Male mice with this genetic defect fail to display aggression toward other male mice (Leypold et al., 2002; Stowers et al., 2002) (Fig. 10). Vomeronasal organ removal markedly reduces aggressive behavior in male mice (Maruniak et al., 1986) and eliminates aggressive behavior of lactating female mice (Bean and Wysocki, 1989). This latter effect is not mitigated by extensive social or sexual experience. In contrast, VNX does not reduce maternal aggression of lactating female rats (Kolunie and Stern, 1995). In the male lesser mouse lemur, a prosimian, VNX decreases aggressive behavior (Aujard, 1997). Apparently, VNS involvement in aggressive behavior varies among species, between the sexes, under different reproductive conditions, and, probably, under different testing conditions. Furthermore, VNX may affect aggressive behavior indirectly by altering hormonal levels; therefore one should be cautious in interpreting these results as purely indicative of a sensory system disruption. Using synthetic pheromones, Novotny et al. (1990a) identified DHB and SBT as urinary components that elicit aggression in mice. Later, ␣- and ␤-farnesene were found to be elevated in the voided urine of dominant male mice several days following the establishment of dominance. These compounds are absent in bladder urine, but abundant in the preputial gland. The ␣- and ␤-farnesene added to water or bladder urine significantly decrease the time subordinate males investigate urine deposits (Novotny et al., 1990b). These findings indicate that four different urinary constituents contribute to male–male interactions. 8.6. Marking behavior Urine marking, feces marking and marking with glandular secretions is a common mechanism used by many species to delineate a territory. The frequency of marking may be

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affected by the hormonal status of the marker, detection of an intruder or the presence of a conspecific of the same or different sex. Recent studies on the role of the nasal chemical senses in this behavior are few. In male mice VNX markedly reduces urine marking rates (Labov and Wysocki, 1989; Maruniak et al., 1986). However, prior experience with females appears to mitigate the effect of VNX. Female golden hamsters emit ultrasonic calls and scent mark in response to conspecific odors (Johnston, 1992; Johnston and Mueller, 1990). VNX and olfactory epithelium destruction by ZnSO4 irrigation reduce the frequency of ultrasonic calls and may reduce scent marking in response to these odors depending on the sex of the odor donor, the sex of the marker and the type of scent marking (Johnston, 1992; Johnston and Mueller, 1990; Petrulis and Johnston, 1999). The results of these studies indicate that, in hamsters, both olfactory and VN systems mediate female ultrasonic calls to male odors, and both may be involved in differential scent marking to male versus female odors (Johnston, 1992; Johnston and Mueller, 1990). Lesions of the medial amygdala, an area of convergence of olfactory and VN information, impair scent marking and sex odor recognition in female golden hamsters, but do not impair discrimination of individual conspecific odors (Petrulis and Johnston, 1999). Male gray short-tailed opossums, M. domestica, preferentially mark objects covered with novel odors, in comparison to objects covered with familiar odors (Shapiro et al., 1996). VN nerve section disrupts this discriminated marking behavior, but, paradoxically, does not effect differential nuzzling, a behavior that brings odorants to the VNO (see Section 8.12). It would appear, from the above, that the importance of the VNS in marking behavior varies depending on the species, the sex of the marker and the sex of the odor donor. 8.7. Individual odor discrimination Whereas female golden hamsters do not require the VNS to discriminate between individuals on the basis of male flank gland odor or female vaginal secretions or urine (Johnston and Peng, 2000; Petrulis et al., 1999), male hamsters without a functional VNO are unable, when tested in a single-stimulus paradigm, to discriminate between individuals based on flank gland secretions, vaginal secretions or feces odors (Johnston and Peng, 2000). Interestingly, males are able to discriminate between individuals based on urine in a single-stimulus paradigm and, in two-choice testing, between individual flank gland odors and vaginal secretions. These results indicate that one must be vigilant in drawing conclusions about the role of the VNS when using a single testing paradigm. Social recognition in male rats is based on chemical cues. VNO ablation temporarily impairs formation of these social memories (Bluthe and Dantzer, 1993). This social recogni-

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tion is modulated by vasopressin, i.e. the duration of the memory can be prolonged by injections of arginine vasopressin (AVP). However, the action of vasopressin in this behavior is dependent on circulating androgens. Interestingly, similar to castrated rats, animals with their VNOs removed are no longer sensitive to AAVP, an AVP antagonist. Since circulating androgen levels are not reduced in rats lacking a VNO, these findings suggest that androgen-dependent vasopressinergic neurons are involved in chemosignal memory formation mediated by the VNS in male rats (see Dantzer, 1998 for review on this subject). 8.8. Aggregation in reptiles Ribbon snakes are attracted to shelters marked with skin lipid extracts from conspecifics. This preference is not manifest in snakes whose VN ducts have been sutured closed, preventing chemical access to the VNO (Graves et al., 1991). 8.9. Non-pheromonal functions of the VNS Garter snakes respond to chemicals derived from prey with increased tongue-flicking. When these chemicals are delivered as an airborne stream, the snakes increase their tongue flicks during the delivery of worm odors, fish odors or non-biological odors such as limonene or amyl acetate (Halpern et al., 1997). Main olfactory nerve section as well as VN nerve section significantly diminish this response. Snakes with main olfactory nerve section fail to respond to any airborne odors whereas snakes with VN nerve section respond to non-biological odors, but not to prey odors (Zuri and Halpern, 2003). Snakes can also be trained to go to the source of a prey-derived airborne odor in a Y maze. Section of the main olfactory nerve results in immediate loss of discrimination of airborne odorants whereas section of the VN nerve only gradually results in a loss of discriminated responding (Halpern et al., 1997). The authors suggest that the airborne odors are probably detected primarily by the main olfactory system and that the loss of response following VN nerve section is related to the loss of reward value of the prey. Earthworms are a favorite prey of several species of garter snakes and response by snakes to extracts of earthworms requires a functional VNS. Eight proteins, all chemoattractants to garter snakes, have been isolated from two different earthworm preparations, earthworm wash (EWW) and earthworm shock secretion (ESS). Six of the proteins have been isolated from EWW and two purified to homogeneity (Wang et al., 1993). One, a sulfhydryl-containing 20 kDa protein, was found to require free sulfhydryls for its chemoattractant activity (Wang et al., 1988). The other protein (LMW) was of relatively low molecular weight, 3000 Da, and consisted of a single polypeptide chain with a blocked amino acid terminal (Wang et al., 1993). Another proteinaceous snake chemoattractant was isolated and purified from ESS and shown to bind saturably and reversibly to VN sensory

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epithelial membranes with a Kd of about 0.3 ␮M and a Bmax of 0.4 nmol. The 20 kDa chemoattractant (ES20) evoked concentration-dependent increased firing in AOB mitral cells when delivered as an aqueous solution to the VN sensory epithelium (Jiang et al., 1990). The ES20 gene has been cloned and the fusion protein, expressed in E. coli, was found to be an active chemoattractant for garter snakes (Liu et al., 1997). Immunological analysis, using polyclonal antibodies raised in rabbit against LMW and ES20 indicated that the proteins isolated from ESS and EWW could be divided into three distinct groups: those related to ESS, those related to LMW and those related to neither. Thus, although all of these proteins isolated from earthworms share the property of being chemoattractants to snakes, they differ considerably in their primary structure (Wang et al., 1992). Rattlesnakes (Crotalus viridis oreganus) with VN nerves sectioned, exhibit fewer strikes at prey compared to controls, cease trailing following strikes and do not ingest envenomated prey (Alving and Kardong, 1996). Those deafferented snakes that do strike, show a significant reduction in poststrike tongue-flick rate and chemosensory searching. The VNS of lizards and salamanders may also be important for feeding behavior. Unlike snakes, the lizard Calcides ocellatus deprived of a functional VNS will attack prey, but will not ingest it (Graves and Halpern, 1990). Plethodontid salamanders without a functional VNS appear to be deficient in response to stationary prey (Placyk and Graves, 2002), but not deficient in response to moving prey. The VNS appears to be critical in snakes for the detection of and response to potential predators. Rattlesnakes and copperheads with their VN ducts sutured closed fail to make defensive responses when encountering a king snake, a common predator (Miller and Gutzke, 1999). Prior to duct suture and following suture removal, these same animals exhibit typical defense responses when exposed to the predator. These studies indicate that in snakes appropriate responding to prey chemicals and predator odors is mediated by the VNS and, in some cases, by the olfactory system, as well. In lizards and salamanders the role of the VNS is less clear. 8.10. Behaviors not dependent on a functional VNS In a variety of mammalian species responses to some pheromones have been found to be independent of VN stimulation, e.g. pheromone release of suckling behavior in rabbits (Hudson and Distel, 1986). More recent studies are summarized below. Male gray mouse lemurs (M. murinus) emit ultrasounds directed at proestrous and estrous females during the breeding season. VNX had no significant effect on ultrasound production in the presence of a proestrous female (Zimmermann, 1996). However, it should be noted that no attempt was made in this study to compare ultrasound vocalization to diestrous females. It is possible that the VNS may be necessary for such discrimination.

In the domestic pig (Sus scrofa) the steroid androstenone, a component of boar saliva, acts as a pheromone facilitating attraction of males and the assumption of a receptive mating posture in estrous females. Sensitivity to androstenone was not affected by blocking access to the VNO with surgical cement, nor was there an effect on attraction to the odor or androstenone-mediated receptive standing behavior. The authors conclude that androstenone access to the VNO is not necessary for expression of attraction to the steroid or for androstenone-mediated mating stance behavior (Dorries et al., 1997). Female ewes respond to male chemosignals with an increase in luteinizing hormone secretion which eventually results in ovulation. The VNO is not required for this response in sexually experienced ewes (Cohen-Tannoudji et al., 1989). Subordinate female marmoset monkeys are anovulatory in the presence of the dominant female of their group. The presence of the scent from the dominant female is sufficient to maintain reproductive suppression in the subordinates (Barrett et al., 1993), however neither main olfactory epithelium ablation nor VNX or both of these procedures is sufficient to overcome the suppression of ovulation in subordinate females when the dominant female is present (Barrett et al., 1993). The fresh urine of female house mice elicits ultrasonic vocalizations from male mice. A functional VNO does not appear to be essential for this vocalization, although males with a VNO vocalize more than those whose organs have been removed (Sipos et al., 1995). The studies summarized above give clear evidence for the observation that pheromonal communication is not necessarily dependent on a functional VNS. 8.11. Odorant access to the VNO 8.11.1. Behavioral and structural specializations The VNO of most vertebrates is sequestered from the external environment and therefore specialized behaviors have evolved to deliver chemicals to the organs. In amphibians, the VN epithelium is usually contiguous with the main olfactory epithelium, and is not in a separate organ. In plethodontid salamanders a nasolabial groove extends from the upper lip to the lateral-most corner of the nares, immediately adjacent to the anterior portion of the VN epithelium. Nose-tapping transfers odorants to the nasolabial groove which acts as a capillary tube for chemicals to access the nasal cavity. Tritiated proline applied to the base of the nasolabial groove accumulates in the VNO, but not in the olfactory epithelium of P. cinereus, a plethodontid salamander (Dawley and Bass, 1989). Trotier et al. (1994) placed the dye cresyl violet into a solution bathing frogs and found that the VNO, but not the olfactory organ took up the dye, confirming the notion that water-borne stimuli enter the VNO but not the olfactory epithelium. Snakes and lizards tongue-flick to deliver odorants to the VNO (Graves and Halpern, 1989; Halpern and Kubie, 1980

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reviewed by Graves, 1993). Young (1993) evaluated varying hypotheses concerning the transfer of odorants from the tongue to the VNO of snakes, concluding that transfer involves a suction mechanism in the VN duct and organ. The source of this suction could be either a vascular pumping mechanism, or pressure produced by occluding the duct opening with the tongue itself or the anterior lingual processes. Young preferred the latter mechanism because it was previously demonstrated that elevation of the anterior processes occurred during tongue-flicking (Gillingham and Clarke, 1981) and that the tongue was not necessary for delivery of odorants to the VNO (Halpern and Kubie, 1980). Gillingham and Clarke (1981) suggested, based on cinematographic recordings and behavioral observations, that the anterior processes, connective tissue masses just below the opening to the VNO in snakes, transferred chemicals from the tongue to the VN duct. A subsequent study (Halpern and Borghjid, 1997) found that the anterior processes were not necessary for either delivery of odorants to the VNO or for discriminated responding to prey odors, a behavior known to depend on a functional VNS. Young (1993) too readily dismisses the vascular hypothesis, that changes in blood flow result in changes in the size of the VN lumen facilitating fluid ingress to or egress from the organ. The VNO of snakes is highly vascularized, with an extensive vascular plexus below the supporting cell layer, between adjacent columns and at the base of the epithelium Wang and Halpern (1980). This vascular plexus could function similarly to the vascular pump of mammals, however no evidence is presently available to support or disprove this hypothesis. In the lizard, Anguis fragilis, the ventral surface of the tongue, which exhibits microridges, microfacets and micropores, makes contact with the substrate during tongueflicking. Using light and scanning electron microscopy and high speed cinematography, Toubeau et al. (1994) demonstrate that the anterior processes, which bear parallel oblique ridges, come in contact with the roof of the mouth as the floor of the buccal cavity is elevated once the tongue is retracted. This sequence of events and morphological specializations suggests that the tongue may transfer molecules to the anterior processes which in turn transfer the molecules to the openings of the VN ducts (Toubeau et al., 1994). Additional studies are needed to verify this mechanism for stimulus access to the VNO of lizards. Nuzzling, which consists of forward rubbing motions with the ventral aspect of the snout interspersed with rapid snout tapping motions on an odor-containing substrate, delivers odorants to the VNOs of the gray short-tailed opossums, M. domestica (Poran et al., 1993a). This phenomenon was demonstrated using conspecific odor deposits laced with [3 H]proline and autoradiography. Using 2-deoxyglucose to monitor metabolic activity in the MOB and AOB of a prosimian, the mouse lemur M. murinus, Schilling et al. (1990) demonstrate activation of the MOB by urine vapors and activation of the AOB by liquid urine delivered to the incisive foramen of the palatine

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papilla, suggesting that this may be a route for normal access of chemicals to the VNO. Thus, there appear to be a number of behavioral adaptations that facilitate odorant access to the VNO. Some of these were previously reviewed and vary among different species (Halpern, 1987). 8.11.2. Physiological mechanisms Previous studies (Meredith and O’Connell, 1979; Meredith et al., 1980) demonstrated that large blood vessels surrounding the hamster VN epithelium act as a pump to bring chemicals into the VN lumen. Recently, Meredith (1994), using chronic recording of VNO movements that correlate with pump activity, reported that the pump operates under novel stimulus conditions; it does not operate solely in a reproductive context, as one might have expected based on the role of the VNS in reproductive activity. The newborn guinea pig VNO is vascularized and capable of activating a pumping action that permits suction of stimuli from the nasal cavity (Mendoza and Kühnel, 1989a), however no evidence is presented in this report concerning the functionality of the vasculature of the VNO at birth. Stimulation of the cervical sympathetic nerve of the ram activates a mechanism resulting in negative intraluminal pressure, as compared to atmospheric pressure, in the VNO (Bland and Cottrell, 1989). This negative pressure draws fluids into the lumen of the organ. The decrease in intraluminal pressure is under ␣-adrenergic control. Thus, the mechanism of fluid entry in the VNS of the ram is similar to those described previously for hamster (Meredith and O’Connell, 1979; Meredith et al., 1980) and cat (Eccles, 1982). Increases in intraluminal pressure in the ram, and the consequent expulsion of fluid from the organ appears to be controlled by the trigeminal nerve, and a non-␣adrenergic sympathetic component in the cervical sympathetic trunk. Based on the evidence gathered to date it appears that a pumping mechanism for chemical delivery to the VNO is a common feature of mammalian VNSs. 8.11.3. Ligand binding proteins In the olfactory epithelium, olfactant binding proteins are thought to act as carriers of odorants to and through the mucus covering the sensory epithelium. In recent years glandular products have been identified associated with the VNO that are thought to act as carrier molecules to the VN sensory epithelium. In squamate reptiles, the secretions of the Harderian gland, a large retro-orbital gland travel through the VN duct to reach the oral cavity (Rehorek, 1997). These secretions enter the VNO, as demonstrated by the fact that [3 H]proline injections into the gland results in radioactivity in the VNO (Rehorek et al., 2000); however, there has been no demonstration that these secretions are necessary for transporting or mediating access of stimulating substances to the microvillar surface of VN receptor neurons.

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Similarly in mammals a number of molecules have been identified that bear a functional or structural resemblance to olfactant binding protein (Khew-Goodall et al., 1991; Matsushita et al., 2000; Mechref et al., 1999; Miyawaki et al., 1994; Ohno et al., 1996; Rama Krishna et al., 1994, 1995) however, although they are localized to glands associated with the VNO, none of these studies demonstrate that the proteins act as transport molecules. As a result, it is not possible to determine the functional significance of these findings. 8.12. Reinforcing effects of VN stimulation Previous studies had suggested that VN stimulation was reinforcing (see Halpern, 1987 for review). Coppola and O’Connell (1988) were unable to train male hamsters to make an operant response for access to hamster vaginal discharge, a vomeronasally-mediated pheromone in hamsters. The authors interpret their results as challenging the notion that pheromones are intrinsically rewarding. However, Moncho-Bogani et al. (2002) demonstrate that female mice with no prior experience with male odors prefer male odors to female odors when they can have direct contact with the odor sources. When direct contact is prevented by a perforated platform placed between the odor source and the female mouse, no preference is observed. After direct contact experience with male odors, the volatiles emanating through the platform are preferred. The authors conclude that this study “represents a Pavlovian-like associative learning in which previously neutral volatiles (very likely odorants) acquire attractive properties by association with the non-volatile innately attractive pheromone(s)” (p. 167). It should be noted that the authors have not independently demonstrated that the VNS is required for this learning, although response to male bedding odors, the stimuli used here, has been shown to activate both olfactory and vomeronasal systems (see above for extensive discussion).

9. Is there a functional human vomeronasal organ? 9.1. Reviews The history of the discovery of the human VNO has recently been revisited (Bhatnagar and Reid, 1996; Bhatnagar and Smith, 2003) and the status of the human VNS has been extensively reviewed (Greene and Kipen, 2002; Mart´ınez-Marcos, 2001; McClintock, 1998; Meredith, 2001; Monti-Bloch et al., 1998a; Preti and Wysocki, 1999; Trotier et al., 2000; Wysocki and Preti, 2000). Interestingly, these reviewers come to very different conclusions on the functionality of the human VNS. Although over the years, the presence of a human VNO in adults has been controversial, most recent studies report the presence of a discernable vomeronasal pit and, presumably, a VNO in a position

comparable to other mammalian VNOs (Fig. 11). However, the histological structure of that organ, the presence or absence of neuron-like cells and the absence of demonstrable neural connections to the brain remain at issue. Physiological and behavioral effects of stimulating the presumptive VNO of humans have been reported from a single research group. These studies await independent confirmation. The most positive position on a functional VNS in humans is taken by Monti-Bloch et al. (1998a) who conclude that the human VNS is functional in adults. This assertion is based on a number of observations, namely, that the VNO does not atrophy during development (Smith et al., 1996), the organ appears to have receptor-like cells that express neuron-specific enolase (NSE) (Takami et al., 1993), and their own physiological and behavioral data (see below) indicating that “vomeropherins” (their term for chemicals that stimulate the VNS) delivered to the VNO cause changes in electrical activity in the VNO, autonomic and endocrine activity and behavior (see below). Based on a thorough and thoughtful review of the literature, Meredith (2001) takes a more moderate position concluding that there is a VNO in adult humans, but questioning whether there are sensory neurons in the VN epithelium. He also questions whether a newly discovered human gene (Giorgi et al., 2000; Pantages and Dulac, 2000; Rodriguez et al., 2000) expressed in the human main olfactory epithelium is informative about human VN function. This gene is closely related to the V1R family of “putative pheromone receptor genes”, and is considered by Pantages and Dulac to be a member of a third VN receptor family, V3R. Furthermore, Meredith states that the absence of demonstrable neural connections between the VNO and the brain remains a major obstacle to concluding that the organ is functional. Interestingly, he considers evidence of a selective electrovomeronasogram (EVG) response to putative human pheromones the best evidence for a functional VNO in humans. However, the absence of solid data concerning the origin of this response raises the question of the role of this response in chemical communication. Two recent reviews of the literature on the functionality of the human VNS (Preti and Wysocki, 1999; Wysocki and Preti, 2000) conclude that current evidence is suggestive of the presence, in adults, of structures similar to VN pits on the nasal septum and an epithelial sac or tube, but question whether this is a verifiable VNO. Trotier et al. (2000) and Mart´ınez-Marcos (2001) are equally skeptical concerning the functionality of the VNO in humans. Doty and Mishra (2001) in an otherwise thoughtful review of human olfaction, claim, with little evidence, that the human VNS is non-functional. 9.2. Morphological observations There has been a relatively large number of recent studies reporting the presence of a VN pit in humans. The proportion of humans exhibiting VN pits in the expected position,

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Fig. 11. Schematic diagram showing the approximate location of the human vomeronasal organ (VNO) at the base of the nasal septum. Abbreviations: MOB, main olfactory bulb; OE, olfactory epithelium. The accessory olfactory bulb (AOB) has not been demonstrated in humans.

along the nasal septum, approximately 1 cm dorsal to the columella and 1 mm above the floor of the nose (although see Bhatnagar et al., 2002 concerning variability) varies with the method of examination (Gaafar et al., 1998; Moran et al., 1991). As few as 6% of subjects examined were found to have VN pits in one study (Zbar et al., 2000). In general, fewer pits are observed in living patients (28.2%) than in cadavers (59.1%) (Won et al., 2000) and fewer observed with the naked eye than by endoscopic examination (Gaafar et al., 1998; Garcia-Velasco and Garcia-Casas, 1995). However, in other studies, VN pits have been observed in virtually all patients free of pathological nasal conditions (Garcia-Velasco and Mondragon, 1991; Moran et al., 1991). Knecht et al. (2001), using a speculum and 30◦ endoscope, detected a putative VNO in about two-thirds of the population, bilaterally in 40% of the subjects. However, Trotier et al. (2000) found that the presence of VN pits was not a constant feature. In repeated observations of the same subjects, only 65.3% of the initial observations could be confirmed on a second examination. Thus, although several studies report a high percentage of patients/subjects with VN pits, in at least one carefully conducted study (Trotier et al., 2000) the percentage is lower. In their subjects with no pathology of the nasal region, 41% of the subjects had no visible pit. High variability in the occurrence of the VN duct has been reported as well

using magnetic resonance imaging (Abolmaali et al., 2001). Possible explanations for the high variability of occurrence in different studies could be due to misidentification of the nasopalatine duct (Jacob et al., 2000) or fossa (Bhatnagar et al., 2002) for the VN pit. Finally, as argued by Bhatnagar et al. (2002), in the absence of serial section histology, it is probably impossible to identify, with certainty, the opening to the VNO since the organ itself is a deep mucosal structure. Histological examination of presumptive VNOs have described a pseudostratified columnar epithelium composed of three cell types: dark-staining columnar cells, light-staining columnar cells, and basal cells. The luminal surface is typically described as covered with microvilli (Gaafar et al., 1998; Moran et al., 1991; Stensaas et al., 1991). However, Smith et al. (1998) describe a ciliated epithelium on both medial and lateral surfaces (sensory and non-sensory, respectively) of the organ and Johnson et al. (1985) and Smith et al. (2002) report no evidence of a neuroepithelium or nerve endings in relation to any VN pit or VN duct examined from autopsy-derived nasal septa. The human VN duct examined histologically by Trotier et al. (2000) lacked the appearance of a typical chemosensory epithelium. The broad discrepancies in the appearance of the human VN epithelium, as described in these different reports and by Witt et al. (2002), is disturbing and requires careful evaluation. At present, we

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are left with very different views on the development of this epithelium. Immunohistochemical studies of the human VN epithelium have revealed the presence of a few, widely separated NSE-positive cells as well as a few cells that express PGP 9.5, but no expression of OMP has been found (Takami et al., 1993). Trotier et al. (2000) also observed a few NSE-positive cells in the lining of the presumptive VNO but failed to observe OMP or S-100 expression as would be expected in a chemosensory neuroepithelium. Although Johnson (1998), Johnson et al. (1994) observed calbindin D28K expression in epithelial cells of the human VNO, they concluded that these cells are not neurons since none had basal processes extending into the subepithelial mucosa and they did not resemble cells described by others as NSE- or PGP-positive. The results of these studies are quite consistent: a few NSEand PGP-positive cells are seen in the human VNO, but no OMP-positive cells, suggesting that “typical” chemosensory neurons are not present. Three electron microscopic studies confirm the presence of two types of columnar cells, dark and light, with microvillar luminal surfaces, and basal cells (Jahnke and Merker, 2000; Moran et al., 1991; Stensaas et al., 1991). Stensaas et al. (1991) estimate that 60–70% of the cells are of the light variety and 10–20% of the dark. The dark cells appear to conform to the standard description of supporting cells in other mammalian VN epithelia. In addition, ciliated and globlet cells are described, as are intraepithelial unmyelinated axons. The source or termination of these axons is unknown. Aggregates of unmyelinated axons in the subepithelial mucosa are described but neither the cells of origin nor their terminations are indicated (Jahnke and Merker, 2000; Stensaas et al., 1991). If the human VNO contains receptor neurons (bipolar neurons), one would expect these neurons to possess axons that terminate in an AOB. However, in adult humans no AOB has been found (Meisami and Bhatnagar, 1998; Meisami et al., 1998). In an extensive review of the anatomy of the AOB of vertebrates, Meisami and Bhatnagar (1998) site numerous reports that humans do not possess an AOB.

8 to 30 weeks of postmenstrual age; however they note (Smith et al., 2001b) that the growth of the human VNO is quite variable. Similarly, Sherwood et al. (1999) observed an increase in organ volume and epithelial volume as a function of developmental age. Smith et al. (1999) describe the appearance of the human VNO during 8–26 weeks of postmenstrual age as compared to adults. Between 8 and 14.5 weeks, the VNO appeared to increase in complexity, whereas older fetuses and adult VNOs appeared simplified (Smith and Bhatnagar, 2000). It is difficult to reconcile the different results from these and earlier studies. Perhaps better methods of tissue preservation and more information on the location and appearance of the VNO have permitted more accurate identification in recent studies of the prenatal VNO (Smith and Bhatnagar, 2000; Smith et al., 1997). Histological examination of human fetal material has revealed silver-stained “receptor cells” in specimens 11–18 weeks of age (Ortmann, 1989). In the VNO of human fetuses younger than 23 weeks, NSE-immunoreactive cells, similar to olfactory receptor cells, have been described. Conversely, the VNOs of fetuses older than 23 weeks of age appeared to contain no NSE-positive receptor-like cells and the few NSE-positive cells observed were pear-shaped and lacking axons that extended into the subepithelial mucosa (Boehm and Gasser, 1993; Wang et al., 1994). Interestingly, the lumens of the VNOs of older fetuses were lined by ciliated columnar epithelium (Boehm and Gasser, 1993; Smith and Bhatnagar, 2000; Wang et al., 1994), similar to the respiratory-like epithelium reported in adult VNOs by Johnson et al. (1985). Most recently, Gi2␣ - and Go␣ -immunoreactive cells have been observed in the VN epithelium of 5-month-old human fetuses (Takami et al., 2001). These cells are also PGP 9.5-immunoreactive. Although the authors state that the presence of microvilli on the surfaces of these cells would indicate that they are functional chemosensory neurons, the presence of microvilli could occur in non-chemosensory cells.

9.3. Development

It is common to describe the VNO of vertebrates as a pheromone-detecting organ, implying that its sole function is pheromone detection and, perhaps, that it is the only pheromone-detecting sensory organ. Neither of these implications is warranted, as discussed in detail by Meredith (2001). Not only are there examples of intraspecific chemical communication mediated by the main olfactory system, but there are examples, in reptiles, of non-pheromonal chemosensory functions of the VNO (see Section 8.9 and Halpern, 1987 for review). Nonetheless, for researchers interested in demonstrating the functionality of the human VNO, identification of putative human pheromones and evidence of putative pheromonal communication between humans has been important. Berliner et al. (1991) and Wysocki and Preti (2000)

Prior to the current resurgence of interest in the human VNO, it was generally accepted (see Halpern, 1987; Wysocki, 1979) that the human VNO was present in early fetal life, but degenerated during gestation and was not present at birth. O’Rahilly et al. (1988) noted the presence of a VNO in human embryos at stages 18 and 19 (postovulatory day 135). Kjaer and Fischer Hansen (1996) observed VNOs in 8–16-week-old specimens, saw apparently diminishing VNOs in some fetuses at 11–16 weeks, and were no longer able to distinguish VNOs at 17–19 weeks of gestational age. In contrast, Smith et al. (1996) observed a linear increase in the length of the VNO and a logarithmic increase in the volume of the VNO and VN epithelium from

9.4. Putative human pheromones

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discuss the skin and other organs as sources and carriers of putative pheromones. One recent finding used to support the idea that humans have a functional VNO is the report by Stern and McClintock (1998) that axillary compounds from female donors in the follicular phase of the menstrual cycle delivered to the upper lip shorten the menstrual cycle of recipients. Conversely, axillary compounds from donors in the ovulatory phase of the cycle lengthen the cycle of the recipient. Subjects did not report sensing the putative pheromones in the samples, suggesting that they were not consciously perceived. McClintock (2002) has proposed the use of the term “vasana” for these social chemosignals that are not consciously perceived. Urine analysis indicated that only the length of the follicular phase of the cycle was affected by application of the axillary compounds. However, note that not all agree with this interpretation of the data (see exchange of letters between Whitten (1999) and McClintock (1999)). Additional reports have provided evidence of the effects of axillary secretions on the neuroendocrine system that influences menstrual cycle length and frequency (Cutler et al., 1986; Preti et al., 1986; Russell et al., 1980) and sexual activity (McCoy and Pitino, 2002). However, as convincingly argued by Wysocki and Preti (2000), there is nothing in these reports that suggest that the VNS, and not the olfactory system, is instrumental in the detection of these presumptive pheromones. Functional magnetic imaging has been utilized to determine if one of the compounds isolated by the EROX corporation, oestra-1,3,5(10),16-tetraen-3yl acetate, presumably a skin-derived “pheromone” evokes detectable brain activity (Sobel et al., 1999). Subjects reported that they could not detect any odor, but in a forced-choice task they responded to a high concentration stimulus at greater than chance levels. Significant brain activation was observed in the anterior medial thalamus, inferior frontal gyrus, amygdala, hippocampus and hypothalamus. This study is of particular interest since it uses one of the compounds delivered to the VNO in the experiments of Monti-Bloch and coworkers. However, since in the present experiment the stimulus was delivered in a manner not restricted to the VNO, it is not possible to conclude that VNS stimulation resulted in brain activation. Most recently, using regional cerebral blood flow with positron emission tomography, Savic et al. (2001) found that in females stimulated with 4,16-androstadien-3-one, a derivative of testosterone, a portion of the hypothalamus was activated whereas in males stimulated with oestra-1,3,5(10),16-traen-3-ol, a substance resembling naturally occurring estrogens, a different portion of the hypothalamus was activated. No attempt was made in this study to selectively stimulate the VNO and since the stimulating apparatus was placed 10 mm from the nose, it is likely that the olfactory system was stimulated. Thus, although this study provides no direct information about the functionality of the human VNS, it does suggest a sexually dimorphic activation of the hypothalamus by putative human pheromones.

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The finding of a sexual dimorphism in hypothalamic response to presumptive pheromones in humans is similar to findings reported previously in the rat (Bressler and Baum, 1996), hamster (Fiber and Swann, 1996), ferret (Wersinger and Baum, 1997) and mouse (Halem et al., 1999). These studies, not surprisingly, support the idea of chemical communication between humans, but do not resolve the issue of which sensory system is responsible for the detection of the presumptive pheromones. 9.5. Physiological and behavioral responses Monti-Bloch and coworkers have reported a series of studies using a standard protocol with varying dependent variables. The basic paradigm is to introduce “vomeropherins”, substances secreted in human skin, to the region of the VNO of human subjects using a multifunctional miniprobe (Berliner et al., 1996; Monti-Bloch and Grosser, 1991; Monti-Bloch et al., 1994). Control stimulations with non-pheromonal substances, control recordings and stimulations in the olfactory epithelium with pheromonal and non-pheromonal stimuli typically, but not invariably, parallel the experimental VN stimulation. It should be noted that these studies have been criticized, e.g. Zbar et al. (2000), for failing to precisely describe the position of the stimulating electrode with respect to nasal landmarks. In several studies (Berliner et al., 1996; Monti-Bloch and Grosser, 1991; Monti-Bloch et al., 1994), human subjects were tested for response to putative human pheromones delivered to the region of the VNO. Negative potentials were elicited and the response was sexually dimorphic. Subjects did not report smelling the putative pheromones. Olfactory stimuli, such as clove oil, produced responses in the olfactory epithelium but no significant response in the VNO. No responses were evoked by putative pheromones delivered directly to the olfactory epithelium. Galvanic skin responses (GSR) were decreased, and ␣ EEG activity was increased in response to the stimuli that produced responses in the VNO (Monti-Bloch and Grosser, 1991). Skin temperatures increased or decreased depending on the stimulus and sex of the subject (Berliner et al., 1996; Monti-Bloch et al., 1994). In addition to the effects on autonomic function, Berliner et al. (1996) examined the effect of the steroidal vomeropherin pregna-4,20-diene-3,6-dione (PDD) on gonadotropin release. Delivery of PDD to the VN lumen of male subjects resulted in a reduction in pulsatility in follicle-stimulating hormone (FSH) and LH and a reduction in basal FSH and LH plasma levels. There was no effect of PDD when administered to female subjects. In another study, PDD delivered to male volunteers yielded changes in gonadotropin levels, EVG responses and autonomic activity as previously observed, but also a decrease in serum testosterone levels as a function of time (Diaz-Sánchez et al., 1997; Monti-Bloch et al., 1998b). This effect was not seen in males with hypogoandotropic-hypogonadism, suggesting to the authors

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that the VN/terminalis system was non-functional in these patients (Monti-Bloch and Berliner, 1997). Cells isolated from the VNO of adult male volunteers were studied using whole cell and patch-clamp recording techniques (Monti-Bloch, 1997). PDD stimulation resulted in a dose-dependent transient inward current in the cells. Interestingly, cAMP activated opening of clusters of channels in the patch-clamp configuration and PDD enhanced this response. This finding is surprising since in most species cAMP is an ineffective modulator of VN neuron membrane conductance (see Section 5). Grosser et al. (2000), Monti-Bloch et al. (1998c) tested the effect of androstadienone delivered to the presumptive VNO of female subjects. They report a significant reduction in negative affect, and an increase in relaxation, sense of well-being and contentment compared to pretreatment. They also observed a significant decrease in respiration, cardiac frequency, and skin conductance, and an increase in body temperature and cortical alpha activity. In a similar study (Jacob and McClintock, 2000), two steroids obtained commercially, 4,16-androstadien-3-one and 1,3,5(10),16-estratetraen-3-ol, were reported to have sexually dimorphic effects on mood states. In women the steroids increased positive mood states and in men they decreased positive mood states. Unfortunately, little is known about the stimuli used in these studies, therefore interpreting the significance of these results is difficult. This series of physiological and behavioral experiments yield consistent results: presumptive skin-derived human pheromones produce sexually dimorphic responses on electrical activity in the VNO, autonomic and neuroendocrine changes and alterations in behavioral affect. Since virtually all the experiments were conducted by the same research group, this consistency is not surprising. However, in the absence of an understanding of the possible anatomical substrate that subserves these effects, we must await replication from independent laboratories before we can conclude that the adult human VNO is functional. Furthermore, the findings that the gene encoding the TRP2 channel in mice is a pseudogene in humans (Vannier et al., 1999), that relaxed selective pressure on this gene in primates probably led to the loss of function of this essential signal transduction gene (Liman and Innan, 2003), and the failure to find functional VNO receptor genes in the human genome (Kouros-Mehr et al., 2001) suggest that the organ in humans may be vestigial, having no mechanism for initial signal transduction.

10. Electrophysiology The literature prior to 1987 on electrophysiological characterization of the VNS was reviewed previously (Halpern, 1987; Mori, 1987b). Since 1987 there have been relatively few studies using electrophysiological techniques to examine the response of neurons to external stimuli or to investigate the interactions of neurons in the VNS. Some of these

studies have been discussed elsewhere in this review (see Sections 3, 5 and 8). Recordings from the periphery address a number of issues: what are the membrane properties and signal transduction mechanisms of VN receptor cells? What are the substances that induce conductance changes in peripheral neurons? Single unit and field recordings in the AOB frequently address different issues, such as the spread of effects, efficacy of stimuli, cell–cell interactions, excitatory and inhibitory influences and the role of neurotransmitters. Whole cell patch-clamp and perforated patch recordings of VN receptor cells have been made in frog (Trotier et al., 1996, 1993), snake (Taniguchi et al., 2000), turtle (Fadool et al., 2001; Taniguchi et al., 1996b), rat (Trotier et al., 1998) and mouse (Liman and Corey, 1996; Moss et al., 1998). The findings from these diverse species are remarkably similar. VN receptor cells have resting potentials between −54 and −85 mV; high input resistance (2–6 G), very low spontaneous firing rates and surprisingly low thresholds for firing (1–10 pA, depending on the resting potential). Receptor cells are capable of responding to current injections of 1–2 pA with multiple action potentials. Thus, VN receptor neurons are very sensitive to small depolarizing inward currents. In VN neurons fast transient TTX-sensitive sodium currents and sustained outward potassium currents are activated by membrane depolarization. Trotier and Døving (1996) found that 66% of VN frog neurons examined had stable resting potentials more negative than the calculated equilibrium potentials for K+ (−82 mV). Their data indicate that the membrane potential is not generated by passive diffusion of K+ ions, but by a hyperpolarizing current created by N+ - and K+ -ATPase. Liman and Corey (1996) found no evidence for cyclic nucleotide-gated channels in dissociated mouse neurons. Thus, VN neurons appear to be quite different from olfactory receptor cells. Electro-olfactograms (EOGs) and electrovomeronasograms (EVGs) are volume recordings made from the surface of the olfactory or VN sensory epithelia, respectively, to determine what substances stimulate populations of receptor cells. As measured by EOGs and EVGs, the olfactory epithelium of garter snakes (T. sirtalis) is more sensitive to the vapor of amyl acetate, butanol and earthworm wash than the VN epithelium (Inouchi et al., 1993). The VN epithelium was only sensitive to vapor of amyl acetate as compared to the olfactory epithelium which was sensitive to all three vapor stimuli. In a later study (Taniguchi et al., 1998), an EVG was recorded in garter snakes to earthworm electric shock secretion, amyl acetate and glutamate. The earthworm product evoked responses in a concentration-dependent manner. Bilateral VN nerve sectioning caused a loss of the EVG response (Taniguchi et al., 1998). EVG responses to urine have been observed in mice. Homozygous trp2-deficient mice demonstrate a severe diminution or loss in the EVG normally evoked by urine administered to the VN epithelium (Leypold et al., 2002).

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Similarly, extracellular, multielectrode recordings of the response of VNO receptor cells to urine revealed increased firing in wild-type mice, but no stimulus-driven firing in trp2−/− mice (Stowers et al., 2002). The studies on mouse urinary products have provided us with considerable information on which components are active in various pheromonal phenomena. Unfortunately, they are not coupled with studies that discriminate whether the olfactory or VNS is responsible for detection of these particular constituents. Although the pheromonal effects are known to be mediated largely by the VNS, it still is necessary to demonstrate that these chemical compounds are stimulating the VNS. Using dissociated mouse VN receptor cells, Moss et al. (1997, 1998) found that DHB causes an outward current at negative holding potentials in approximately 26% of tested neurons. In current-clamp mode DHB causes a hyperpolarization of the neurons or a reduction in the frequency of action potentials. This response to a putative VN stimulus is unusual and awaits confirmation in vivo, or at least in intact slice preparations. According to the authors, this was the first demonstration that a compound from male urine directly affected membrane currents in VN neurons. Hatanaka and Matsuzaki (1993) recorded from individual VN receptor cells in Reeve’s turtle, G. reevesii and found that general odorants (e.g. amyl acetate, geraniol, propionic acid), salines (e.g. ammonium chloride; calcium chloride), sugars (e.g. sucrose), acids (acetic acid), amino acids (l-arginine, sodium glutamate) were effective stimuli. Similar results were reported by Sam et al. (2001) using calcium imaging of mouse VN neurons and Trinh and Storm (2003) using type-3 adenylyl cyclase knockout mice. From these studies it is clear that VN sensory neurons are sensitive to a broad range of stimuli, not solely putative pheromones (Rodriguez, 2003). Recording from supporting cells in the mouse VNO, Ghiaroni et al. (2003) found that these cells manifest unique membrane properties. For example, the membranes are permeable to K+ , Na+ and Cl− with ratios peculiar to VNO supporting cells (PK /PNa ∼ 4; PK /PCl ∼ 0.6) compared to other glial cells. The authors suggest that supporting cells may contribute to pheromone signal transduction by removing excess inorganic ions present in pheromone-containing fluids. Summed AOB responses to general odorants were recorded in Reeve’s turtle, G. reevesii (Shoji and Kurihara, 1991). Concentration-dependent AOB responses were obtained from VNO stimulation with amyl acetate, ionone, cineole, menthone and citral. The responses of AOB neurons to “standard chemicals” further supports the idea that the VNS is responsive to a broader range of substances than putative pheromones. Similarly, single unit recording from mitral cells in the AOB of garter snakes during liquid delivery of odorants to the VN sensory epithelium revealed that classical odorants, amino acids (alanine, arginine, glutamate) and proteins de-

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rived from prey (earthworm, goldfish) all altered unit firing in the AOB (Inouchi et al., 1993). Excitatory and inhibitory responses were observed to these stimuli; the direction of the response differed for different neurons. Individual neurons responded to multiple classes of stimuli, suggesting that they are broadly tuned. Only one report has been published in which single unit activity in the AOB has been recorded from behaving mice. Luo et al. (2003) observed robust firing from presumed mitral cells in the AOB several seconds after snout contact with lightly anesthetized male and female mice of different strains. The responses were long lasting (several seconds), specific with respect to sex and strain of the stimulus animal, could be excitatory or inhibitory and were reproducible for a given unit. Interestingly, purified putative phermones (e.g. 2-heptanone; 2,5-demethylpyrazine-E,E-␣-farnesene and E-␤-farnesene) did not produce responses in the AOB. Direct contact with the source of the stimulus was required for a response (Luo et al., 2003). The failure of the purified putative pheromones to activate AOB neurons in this study suggests that they may require MUPs to transport them to the VNO, since in vitro studies (e.g. Leinders-Zufall et al., 2000) indicate that they stimulate the VN sensory epithelium when delivered directly to the epithelium. Volume recordings in the AOB permit analysis of integration and processing of signals from the VNO. Field potentials in the AOB from electrical stimulation of the VN nerve or VNO have been recorded in rabbit (van Groen et al., 1986), mouse (Kaba and Kawasaki, 1996), and rat (Jia et al., 1999). Current source-density analysis indicated that the main activity in the rabbit AOB is generated by synapses between mitral cells and granule cells (van Groen et al., 1986). Current sinks in mice were primarily located in the GL and EPL (Kaba and Kawasaki, 1996; Kaba and Keverne, 1992). Field potential recordings combined with current source-density analysis and patch-clamp recordings in the AOB of rats permitted clarification of the functional connections in the AOB (Jia et al., 1999). Following VN nerve stimulation field potentials were recorded with three identifiable periods. The first represents a presynaptic component generated by action potentials in the NL, the second is indicative of depolarization of mitral cells, and the third is probably generated by granule cells. Whole cell patch recording from mitral cells revealed a long lasting excitatory postsynaptic potential (EPSP) (>100 ms) following VN stimulation as did recordings from granule cells. In slice preparations of the AOB of guinea pig, damped oscillatory potentials are evoked by single shocks to the NL (Sugai et al., 1995). The effects of GABA agonists and antagonists on these AOB responses, recorded electrophysiologically and optically, was investigated (Sugai et al., 1999). Application of the GABAergic agonists GABA, muscimol and baclofen indicated that GABAB action was primarily in the GL whereas GABAA action was in both the GL and EPL. Bicuculline, a GABAA antagonist, produced long-lasting excitation in the EPL and MCL. These results suggest that

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GABAA action participates in the generation of the oscillatory potentials observed in the AOB to VN nerve stimulation. Stimulation of the VNO of rats leads to excitation in the AOB frequently followed by inhibition. The non-NMDA antagonist CNQX reduces this excitation, as does the NMDA antagonist AP5 (Dudley and Moss, 1995). Although the authors claim that the excitatory response is glutaminergic, there is no direct evidence in this report that glutamate is the neurotransmitter between VN receptor cell terminals and mitral cell dendrites. In the rat, CNQX, a non-NMDA receptor antagonist decreases the amplitude of VN nerve-evoked EPSP in mitral cells except for a small slow component which is blocked by the NMDA receptor antagonist APV (Jia et al., 1999). The reciprocal interactions between mitral cells and granule cells in the rat AOB are through dendrodendritic reciprocal synapses in which glutamate acts as the mitral cell to granule cell transmitter and GABA acts as the granule cell to mitral cell transmitter. Thus, taken together these studies suggest that the basic synaptic organization of the AOB is quite similar to that of the MOB (see Mori, 1987b). In the mouse AOB NMDA receptors are reported to play an important role in generating the inhibitory response in mitral cells resulting from activation of reciprocal synapses between dendrites of mitral cells and granule cells (Taniguchi and Kaba, 2001). In Section 8, we discuss studies dealing with activation of the amygdala as related to the pathways that mediate various pheromonal effects. Here, we limit our review to a single study dealing with AOB electrical stimulation and the responses in the medial amygdala. Wong et al. (1993) examined synaptic processing between different loci in the VN pathway. Extracellular single unit recordings were made in the medial amygdala. Electrical stimulation of the AOB produced excitatory (88%) or inhibitory responses (67%) in the medial amygdala which could be mimicked by glutamate or GABA, respectively, applied to the medial amygdala. Ventromedial hypothalamic stimulation also produced excitatory (29%) or inhibitory (59%) responses in the medial amygdala. A very large number of reports characterize electrophysiological responses in the amygdala. A review of those studies is beyond the scope of this publication.

11. Conclusion During the past 15 years, there have been significant advances in our understanding of the VNS. We now accept that the system is heterogeneous based on a variety of criteria, although we still do not know how its different parts contribute to vomeronasally-mediated behaviors. Multiple families of VN receptors have been identified, and molecular approaches using this information are providing insights into receptor–ligand interactions and connectivity between the VNO and the central nervous system. Major participants in the signal transduction pathway of VN receptor

neurons have been identified, and it is clear that this pathway differs significantly from that described for olfactory receptor cells. However, in vomeronasal receptor neurons, the specific links between the second messengers, specifically inositol-1,4,5-tris-phosphate and calcium, and changes in membrane conductance have yet to be elucidated. In contrast to what was previously believed, it is now apparent that neuronal turnover in some mammals occurs through a process of vertical migration from the basal lamina of the epithelium to the receptor cell layer. Additional experiments are needed to determine whether the horizontal migration described previously in mice and observed in rats is primarily related to growth or is also involved in neuronal turnover. New information on the anatomical connections of the accessory and MOBs indicate that there is considerably more convergence in the telencephalon of olfactory and VN information than was previously believed. Attempts have been made to investigate segregation of the VNS at the level of the amygdala. At present, there is conflicting data in opossum and mouse. Solving this controversy in different species could aid in understanding the functional significance of the heterogeneity in the VNS. Behavioral studies have provided interesting insights on pheromonal effects, although in many cases involvement of the vomeronasal versus the olfactory system in these behaviors is still unclear. A structure similar to the VNO appears to exist in some adult humans, however, it may be rudimentary. The functionality of a human VNS continues to be controversial, since no connection between the presumptive vomeronasal organ and brain has been described and no AOB is present in humans. Acknowledgements During the preparation of this manuscript the authors received support from the following grants: NIH Grant nos. DC02531, DC03735 and DC02745 (M.H.) and Spanish MEC postdoctoral fellowship EX99573688 (A.M.M.). We are grateful to Isabel Ubeda-Bañón, Takisha Galaor and Wei Su for their help with the references and Frank Fasano for help with the illustrations. The following colleagues read portions of earlier drafts of this manuscript for which we are very grateful: Heather Eisthen, Albert Farbman, Masumi Ichikawa, Changping Jia, Robert E. Johnston, Frank Margolis, Michael Meredith, Kensaku Mori, Sarah Winans Newman, Mutsuo Taniguchi, Dalton Wang, Ido Zuri. They are not responsible for any errors that may have occurred. References Abe, H., Watanabe, M., Kondo, H., 1992. Developmental changes in expression of a calcium-binding protein (spot 35-calbindin) in the nervus terminalis and the vomeronasal and olfactory receptor cells. Acta Otolaryngol. 112, 862–871. Abolmaali, N.D., Kuhnau, D., Knecht, M., Kohler, K., Huttenbrink, K.B., Hummel, T., 2001. Imaging of the human vomeronasal duct. Chem. Senses 26, 35–39.

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