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Pheromones, vomeronasal function, minireviewand gender specific behavior


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  • 1. Cell, Vol. 108, 735–738, March 22, 2002, Copyright 2002 by Cell Press Pheromones, Vomeronasal Function, and Gender-Specific Behavior Eric B. Keverne1 Sub-Department of Animal Behaviour University of Cambridge Madingley Cambridge, CB3 8AA United Kingdom The striking behavioral phenotypes of mice lacking the TRP2 ion channel have highlighted the importance of the vomeronasal organ in gender-specific sexual behavior. Terrestrial vertebrates have evolved two anatomically distinct sets of chemosensory neurons that respond to different aspects of an animal’s environment. The main olfactory epithelium lines the surface of the turbinate bones in the nasal cavity and is accessible to volatile odorants. The vomeronasal organ is located within a cartilaginous capsule attached to each side of the nasal septum. Water soluble chemical cues, including urinary proteins, are drawn into the VNO lumen through constriction and dilation of its blood supply, which is controlled by the autonomic nervous system. This active process can be secondary to arousal brought about by internal hormone secretions or environmental factors such as volatile odors stimulating the main olfactory epithelium. Molecular studies have shown that there are two multigene families of G protein-linked vomeronasal receptors, each expressed in a distinct region of the VNO (Tirindelli et al., 1998). These seven transmembrane domain receptors are only distantly related to the main olfactory receptors, and this combined with their location suggests that they may respond to different ligands, collectively referred to as pheromones. These two families of VNO receptors differ in both their proposed linkage to distinct G proteins and the length of their extracellular N-terminal domains. The V1Rs are thought to be linked to G␣i2, have a relatively short N-terminal region/ domain, and have greatest sequence diversity in their transmembrane domains. The V2Rs are linked to G␣o and comprise a family of about 140 genes distinguished by their extensive extracellular NH2 terminal domain, which is thought to bind ligands (Tirindelli et al., 1998). Unlike the main olfactory system, the neurons in the VNO do not use cyclic nucleotides as second messengers for transduction of the pheromone signal. Mammalian pheromone signaling has been proposed to involve a member of the transient receptor potential (TRP) superfamily of ion channels, activated via phospholipase C (Holy et al., 2000). Phototransduction in Drosophila and chemosensation in C. elegans also involves members of this TRP family in a cyclic nucleotide independent G protein-mediated pathway. A specific TRP ion channel, rTRP2, was found to localize to VNO sensory microvilli, the proposed site of pheromone transduction (Liman et al., 1999). Now genetic ablation of TRP2 in the 1 Correspondence: Minireview mouse has allowed Dulac’s group to investigate the requirement of TRP2 in VNO signaling and to assess behavioral impairments which result from its deficiency (Stowers et al., 2002). The striking phenotypes observed in these mice have provided new insights into the role of the VNO and pheromone signaling in gender-specific behavior. Male mice deficient in TRP2 expression fail to display aggression to other males and initiate sexual activity with both males and females. This finding suggests that the VNO neurons provide the essential sensory activity for sexual discrimination of conspecifics to ensure gender-specific behavior. VNO and Sex Signaling Behavioral responses to pheromones, unlike neuroendocrine responses to pheromones, are rarely entirely stereotyped and frequently depend on learning experiences that require associative processing to give an integrated response. For example, ultrasound calling by the male rat in response to female pheromones appears only after males have obtained sexual experience with females, and VNO lesions in hamsters produce severe deficits in mating behavior in sexually naive males, but not in males that are sexually experienced (Keverne et al., 1986). In the neonatal rabbit, nipple search is a stereotyped behavior released by maternal pheromones but occurs independently of the VNO via the main olfactory pathway (Hudson and Distel, 1986). That is not to imply that the VNO has no impact on behavior, and VNO lesion studies have shown that it influences sexual behavior in the hamster, aggressive behavior in the rat (Keverne et al., 1986), and mating-induced pair bonding in the prairie vole (Curtis et al., 2001). Inevitably, lesioned animals require time to recover from surgery, so there has been some debate about the extent to which subsequent behavioral changes are secondary to endocrine changes. This veil of uncertainty as to the primary role of the VNO input has been lifted by the study from Stowers et al. (2002). Pheromonal stimulation of the vomeronasal receptors in these mice fails to induce action potentials. Moreover, the males do not display aggression to intruder males and initiate courtship with both males and females (Figure 1). The same mice have normal levels of testosterone, readily find food, and when they serve as intruders to the territory of other males, the TRP2 mutants are able to retaliate to the aggression of the resident male. On first reading of this paper, it might appear that the vomeronasal receptors, like the main olfactory receptors, can instigate behavioral responses. However, the authors are careful to point out that without a functional VNO receptor system, male mice appear to experience problems in identifying gender cues, but none in the engagement of sex-specific motor programs. This is an important distinction to make and an insightful interpretation of the behavioral data. While it is not surprising to find that VNO receptor neurons with a nonfunctional ion channel fail to signal the communicative nature of pheromones, this study has revealed an essential role of the VNO pathway in ensuring the gender specificity of mating behaviors. TRP2 mutants
  • 2. Cell 736 Figure 1. TRP2Ϫ/Ϫ Behavioral Phenotype This shows two TRPϪ/Ϫ male mice from different cages. If these mice were wild-type, they would be displaying aggression toward each other. Instead, the mutant mice are initiating courtship. Image kindly provided by C. Dulac. are not behaviorally impaired, but the behavior occurs inappropriately to the sexual context in which the male is placed. A similar viewpoint has been expressed for the functioning of the female VNO in the context of pregnancy block, which requires exposure to male urine different from that of the male that mated. Two components of the urine are necessary, one that represents maleness and a second that represent individuality, neither of which are sufficient in themselves to block pregnancy (Brennan et al., 1999). The vomeronasal organ differs from the main olfactory system in that fluids are sucked into the lumen and nonvolatile components like the proteins that are abundant in male mouse urine may also stimulate the vomeronasal receptors. Interestingly, the small volatile molecules that are proposed to represent maleness in the context of pregnancy block bind to these urinary proteins. They have also been implicated in the triggering of aggressiveness in males and from electrophysiological recordings seem to induce action potentials in V1rs in VNO slices (Novotny et al., 1985; Leinders-Zufall et al., 2000). The findings for the TRP2Ϫ/Ϫ mutant suggest that other sensory systems in the mouse are gender blind and that the VNO receptors, and not the main olfactory receptors, are necessary for the discrimination of the sexes. If, as the Stowers paper suggests, the VNO is the only sensory system with the capacity to identify gender state, then it would not be surprising to find this ability is hardwired into the VNO projection pathway. Central Connection of the VNO Receptors The two distinct families of vomeronasal receptors are arranged in two distinct zones of the vomeronasal organ, and these zonal subsets project their axons to converge and form glomeruli in the accessory olfactory bulb (AOB). Studies mapping the convergence of a specific receptor in the TRP2Ϫ/Ϫ VNO to the AOB show that there is no difference between mutant and wild-type mice in the number and pattern of fibers in young postnatal stages, but this is reduced by 2-fold prior to puberty (Stowers et al., 2002). This suggests that the projection map to the AOB is not activity dependent and is set up normally in the absence of TRP2, but that sensory activity of the VNO neurons is required for the maintenance of connections at the first relay. The apical zone V1Rs project their axons exclusively to the anterior region of the AOB while the V2Rs are confined to synapsing in the posterior region of the AOB. This anterior/posterior segregation is also evident in electrophysiological recordings of the AOB (Sugai et al., 2000). However, segregation of these pathways seems to stop at this first relay in the AOB and is not maintained in the downstream neural targets at the medial (MeA) and posterior cortical amygdala (PMCO) or subsequent relays to the bed nucleus of the stria terminals (BNST) and bed nucleus of the accessory olfactory tract (BAOT) (Campenhausen and Mori, 2000) (Figure 1). This convergence of axonal pathways from the anterior and posterior compartments of the AOB results in an integration of the signals originating from the different families of vomeronasal receptors at each nuclear relay. In the context of sexual recognition of an individual mate, this would allow integration of the V1R and V2R component stimuli from the complex urinary signal. Sex Differences in the Rodent VNO Pathway The rodent vomeronasal system exhibits sexual dimorphisms at multiple levels along its projection pathway. The MeA, PMCO, BNST, and MPOA are all larger in the male than female, while subsequent projections to hypothalamic nuclei, particularly those concerned with female endocrine regulation by male pheromones, tend to be larger in the female (Segovia and Guillamon, 1996). Lesions in discrete parts of this projection pathway enhance components of female typical behavior, whereas in the male such lesions inhibit mating behavior. In both sexes, these differences are sensitive to the regulatory effects of gonadal steroids early in development (Segovia and Guillamon, 1996). This sexual dimorphism in the VNO pathway correlates with adult immediate early gene (IEG) expression in response to urinary pheromones. Females but not males show an augmented response to male urinary pheromone exposure in the central (MeA, BNST, and MPOA) portion of the VNO projection pathway, while males show enhancement of IEG expression in these regions when paired with females and allowed to mate (Halem et al., 1999). These central connections of the VNO pathway at the level of the amygdala allow for integration of other sensory stimuli, as shown by infusions of the peptide, ␤-endorphin, which is known to be inhibitory to rodent sexual behavior. At the level of the amygdala, such disruption of the VNO projection pathway impairs the integration of chemosensory stimuli with somatosensory stimuli; males fail to mate and show reduced olfactory investigation of the female. However, after the mating sequence has commenced, this same neurochemical interruption has no effect. Placing the male with a different female requires reengagement of the appetitive sequence before the male is able to recommence mating. Further downstream at the level of the MPOA (see Figure 2) the same neurochemical interference in the pathway locks the male into chasing and increased investigation of the female, but again the male does not engage mating (Herbert, 1993). This suggests that the integration of sensory signals (pheromones, individuality, and somotosensory information) occurs sequentially along the VNO projection pathway to provide for complete engagement of mating behavior. Integration of Gender Recognition with Sex Typical Behavior Over the past 30 years, numerous studies have experimentally examined the effects of VNO lesions on behavior (Wysocki and Lepri, 1991), but none have suggested a role for the VNO in gender recognition. Lesions of the VNO have usually been investigated in the adult, and at
  • 3. Minireview 737 Figure 2. Sexually Dimorphic VNO Projection Pathway The VNO sensory projection pathway (in black) relays at a number of nuclear areas (permitting other sensory integration) prior to terminating in the medial preoptic area (MPOA), which, if lesioned in the adult, abolishes male mating behavior. Neurons in the developing VNO also send axons to the medio-basal hypothalamus, which serve as pathfinders for GnRH neurons (in orange). The hormonal cascade from these GnRH neurons initiates the perinatal testosterone surge in the male (in green) which masculinizes the VNO projection pathway. this stage they have marked impairments on sexual and aggressive behavior. It is possible that these lesions also produce effects on gender recognition, but this is difficult to determine when the behavioral context for this recognition is itself impaired. However, this does raise the question of why there is lack of gender discrimination in the absence of behavioral impairment in TRP2Ϫ/Ϫ mutant. The VNO neurons of TRP2Ϫ/Ϫ mouse have been silent throughout development, including times at which the action of male hormones have determined sex-typical male behavior. A possible explanation for the dissociation of gender recognition and sex-specific behavior may therefore relate to events in development. Sex-typical behavior is determined by the action of hormones on the brain including the VNO projection pathway, while gender recognition requires appropriate connections to be made throughout this pathway to signal the appropriate behavioral response. The critical timing for hormone production in sexual differentiation of the rodent brain is determined by the GnRH neurons, which themselves originate with vomeronasal neurons in the olfactory placode. Indeed, the GnRH neurons migrate from the VNO to the basal forebrain via axons that originate in the developing VNO (Schwarting et al., 2001). Hence, the maturational events in the developing neuroendocrine system are synchronized with the neural development of the VNO projection pathway (Figure 2). Evidence for developmental events of importance to the VNO in the context of behavior is provided by the P73 knockout mouse. P73 is involved in the neurogenesis of the VNO and its central projection pathway (MeA, BNST, and MPOA) and counterbalances the actions of P53 in the regulation of apoptotic cell death (Yang et al., 2000). Behaviorally, the P73 null mutant male mice lack interest in sexually mature females and fail to show aggression to intruder males, i.e., both the behavior and gender signaling are impaired. We have learned from the TRP2Ϫ/Ϫ mouse that specific loss-of-action potentials from these neurons not only results in a subsequent degeneration of this pathway to the AOB, but that this mouse is not able to call up the appropriate gender-specific behavior. This is an important finding because the TRP2Ϫ/Ϫ mouse opens up the possibility of studying the combinatorial code for making appropriate neural connections in the context of hormonal differentiation in the VNO signaling pathway to the brain. Testosterone is well established as important in this context, but the refinement of connection strengths throughout the multiple stages of the VNO pathway may require neural activity in either development or in the context of sexual experience, or both. Further understanding of these issues will be best addressed by conditional mutagenesis of the TRP2 receptor. How Do These Findings Relate to Human Behavior? The Stowers paper considers the significance of these findings on the TRP2Ϫ/Ϫ mutant for human VNO function and the possible evolution of alternative sensory modalities. Kallman’s syndrome in humans (hypogonadal hypogonadism, hyposexuality, and anosmia) was influential for our thinking about functional human pheromones until the discovery of the Kallman gene revealed that the link between anosmia and hypogonadism was not causal, but that both were due to a developmental failure in neural migration. Add to this the finding that a large number of VNO receptor genes have deletions or mutations and are nonfunctional in humans, the absence of a functional VNO specific TRP2 ion channel gene in the human genome, and the vestigial nature of a morphologically intact VNO in most humans (Trotier et al., 2000), and the evidence against a functional pheromone system in humans seems fairly conclusive. It could be argued that pheromone reception along the lines of what is found in other mammals has been taken over by the main olfactory system, but this seems unlikely. A single VNO receptor gene has been identified from the human genome, but since this has only 35% homology with known VNO receptors, it is equally possible that it could be expressed in the testis or serve as a pathfinder for GnRH neurons. In any case, the main olfactory neurons have their primary relay in cortical structures (Savic et al., 2000), and although this might account for Proustian memories of the distant past evoked by familiar odors, it is most unlikely to initiate stereotyped behavior or even influence behavior by acting below the threshold of conscious awareness.
  • 4. Cell 738 The absence of an extensive sexually dimorphic VNO system in humans does not, of course, render humans gender blind, but it does raise important evolutionary questions as to how the common downstream mechanisms for expressing dimorphic behavior are brought into play. Selected Reading Brennan, P.A., Schellinck, H.M., and Keverne, E.B. (1999). Neurosci. 90 1463–1470. Campenhausen, H., and Mori, K. (2000). Eur. J. Neurosci. 12, 33–46. Curtis, J.T., Lui, Y., and Wang, Z. (2001). Brain Res. 901, 167–174. Halem, H.A., Cherry, J.A., and Baum, M.J. (1999). J. Neurobiol. 39, 249–263. Herbert, J. (1993). Prog. Neurobiol. 41, 723–791. Holy, T.E., Dulac, C., and Meister, M. (2000). Science 289, 1569– 1572. Hudson, R., and Distel, H. (1986). Physiol. Behav. 37, 123–128. Keverne, E.B., Murphy, C.L., Silver, W.L., Wysocki, C.J., and Meredith, M. (1986). Chem. Senses 11, 119–133. Leinders-Zufall, T., Lane, A.P., Puche, A.D., Ma, W., Novotny, M.V., Shipley, M.T., and Zufall, F. (2000). Nature 405, 792–796. Liman, E.R., Corey, D.P., and Dulac, C. (1999). Proc. Natl. Acad. Sci. USA 96, 5791–5796. Novotny, M., Harvey, S., Jemiolo, B., and Alberts, J. (1985). Proc. Natl. Acad. Sci. USA 82, 2059–2061. Savic, I., Balazs, G., Larsson, M., and Roland, P. (2000). Neuron 26, 735–745. Segovia, S., and Guillamon, A. (1996). Horm. Behav. 30, 618–626. Schwarting, G.A., Kostek, C., Bless, E.P., Ahmad, N., and Tobet, S.A. (2001). J. Neurosci. 21, 911–919. Stowers, L., Holy, T.E., Markus, M., Dulac, C., and Koentges, G. (2002). Science 295, 1493–1500. Sugai, T., Sugitani, M., and Onoda, N. (2000). Neuroscience 95, 23–32. Tirindelli, R., Mucignat-Caretta, C., and Ryba, N.J.P. (1998). Trends Neurosci. 21, 482–486. Trotier, D., Eliot, C., Wassef, M., Talmain, G., Bensimon, J.L., Doving, K.B., and Ferrand, J. (2000). Chem. Senses 25, 369–380. Wysocki, C.J., and Lepri, J.J. (1991). J Ster. Biochem. Mol. Biol. 39, 661–669. Yang, A., Walker, N., Bronson, R., Kaghad, M., Oosterwegel, M., Bonnin, J., Vagner, C., Bonnet, H., Dikkes, P., Sharpe, A., et al. (2000). Nature 404, 99–103.