We have used gene targeting to generate mice with a homozygous deficiency in trp2, a cation channel expressed in the vomeronasal organ (VNO). Trp2 mutant animals reveal a striking reduction in the electrophysiological response to pheromones in the VNO, suggesting that trp2 plays a central role in mediating the pheromone response. These mutants therefore afford the opportunity to examine the role of the VNO in the generation of innate sexual and social behaviors in mice. Trp2 mutant males and nursing females are docile and fail to initiate aggressive attacks on intruder males. Male-female sexual behavior appears normal, but trp2 mutant males also vigorously mount other males. These results suggest that the cation channel trp2 is required in the VNO to detect male-specific pheromones that elicit aggressive behaviors and dictate the choice of sexual partners.A nimals exhibit behavioral repertoires that are often innate and result in stereotyped sexual and social responses to their environment. Innate behaviors do not require learning or experience and are likely to reflect the activation of developmentally programmed neural circuits. The appropriate expression of an innate behavioral array frequently requires signals from the outside world. Mice rely heavily on olfactory information to sense their environment. In mice, odorants are recognized by two anatomically and functionally distinct sensory organs, the main olfactory epithelium (MOE) and the vomeronasal organ (VNO) (1, 2). The main olfactory epithelium is thought to recognize odors that provide information about the world at large and can result in measured behavioral responses. In contrast, the VNO has traditionally been implicated in the recognition of pheromones, odorants that provide information about the social and sexual status of other individuals within the species (3, 4). Activation of the VNO is thought to result in innate neuroendocrine and behavioral responses.In mammals, the chemical nature of the pheromones that activate the VNO to elicit innate behavioral responses has not been elucidated (5). Moreover, it has been difficult to sort out the relative roles of the MOE and the VNO in mediating specific behaviors. In male hamsters, removal of the olfactory bulb, which receives input from both the MOE and the VNO, abolishes the sexual response (6). Removal of the VNO alone diminishes the robustness of the male mating response, but does not eliminate sexual behavior (7). The consequence of VNO removal is most apparent in sexually naive animals, suggesting that with experience the main olfactory system assumes an increasingly important role in the sexual response (8, 9). Other innate behaviors, including lordosis in female pigs (10) in response to the male hormone, androstenone, or suckling behavior in newborn rabbits (11) in response to mammary secretions, are unaffected by removal of the VNO. These innate behavioral responses are likely to be elicited by pheromones that activate the main olfactory system. Thus, mammals have evolved innate behavioral ...

We have developed a genetic approach to examine the role of spontaneous activity and synaptic release in the establishment and maintenance of an olfactory sensory map. Conditional expression of tetanus toxin light chain, a molecule that inhibits synaptic release, does not perturb targeting during development, but neurons that express this molecule in a competitive environment fail to maintain appropriate synaptic connections and disappear. Overexpression of the inward rectifying potassium channel, Kir2.1, diminishes the excitability of sensory neurons and more severely disrupts the formation of an olfactory map. These studies suggest that spontaneous neural activity is required for the establishment and maintenance of the precise connectivity inherent in an olfactory sensory map.

Ligand-gated ion channels are multi-subunit complexes where each subunit-type is encoded by several related genes. Heterologous expression of any one of the neuronal nicotinic acetylcholine receptors (nAChR) alpha-type subunits, either alone or with any beta-type subunit, typically yields functional nAChR channels. A striking exception is the nAChR alpha5 subunit: although apparently complexed with beta2 and beta4 nAChR subunits in neurons, and expressed in a subset of neurons within the central and peripheral nervous systems, heterologous expression of alpha5, either alone or with any beta-type subunit has failed to yield functional channels. We demonstrate here that alpha5 does participate in nAChRs expressed in hetrologous systems and in primary neurons, and further that alpha5 contributes to the lining of functionally unique nAChR channels, but only if coexpressed with both another alpha- and beta-type subunit. Furthermore, channels containing the alpha5 subunit are potently activated and desensitized by nanomolar concentrations of nicotine.

G-protein-coupled receptors (GPCRs) are known to possess two different conformations, active and inactive, and they spontaneously alternate between the two in the absence of ligands. Here, we analyzed the agonist-independent GPCR activity for its possible role in receptor-instructed axonal projection. We generated transgenic mice expressing activity mutants of the β2-adrenergic receptor, a well-characterized GPCR with the highest homology to odorant receptors (ORs). We found that mutants with altered agonist-independent activity changed the transcription levels of axon-targeting molecules--e.g., Neuropilin-1 and Plexin-A1--but not of glomerular segregation molecules--e.g., Kirrel2 and Kirrel3--thus causing shifts in glomerular locations along the anterior-posterior (A-P) axis. Knockout and in vitro experiments demonstrated that Gs, but not Golf, is responsible for mediating the agonist-independent GPCR activity. We conclude that the equilibrium of conformational transitions set by each OR is the major determinant of expression levels of A-P-targeting molecules.

The mammalian vomeronasal organ detects complex chemical signals that convey information about gender, strain, and the social and reproductive status of an individual. How these signals are encoded is poorly understood. We developed transgenic mice expressing the calcium indicator G-CaMP2 and analyzed population responses of vomeronasal neurons to urine from individual animals. A substantial portion of cells was activated by either male or female urine, but only a small population of cells responded exclusively to gender-specific cues shared across strains and individuals. Female cues activated more cells and were subject to more complex hormonal regulations than male cues. In contrast to gender, strain and individual information was encoded by the combinatorial activation of neurons such that urine from different individuals activated distinctive cell populations.Pheromones are a group of chemicals critical for social communication in many animal species (1,2). Information on sex, strain, social rank, reproductive status, and terrestrial ownership is represented in the complex pheromone components in urine and bodily secretions. In mice, detection of such complex chemical signals by the vomeronasal organ (VNO) and the olfactory epithelium plays an important role in triggering endocrine changes and eliciting innate territorial aggression and mating behaviors (3-5). The rodent VNO expresses more than 250 receptors that detect pheromones and transmit the signals to the brain (6-11). It is not well understood how these neurons encode information about gender and individuals. Urine contains hundreds or even thousands of substances, only a handful of which have been identified as putative pheromones (12-16). The complexity of natural pheromone signals poses a challenge to our understanding of what information is transmitted to the vomeronasal neurons (17,18).Each vomeronasal neuron expresses only one of the ∼250 estimated pheromone receptor genes (6)(7)(8)(9)19,20), and the receptor's activation elevates intracellular calcium (21). To visualize pheromone-induced activity in a large population of neurons, we generated tetO-G-CaMP2 transgenic mouse lines (22)(23)(24). When crossed to animals carrying the OMP-IRES-tTA allele (25), G-CaMP2 expression was restricted to the neurons in the olfactory system (Fig. 1, A and B, and Movie S1). Electrophysiological properties of the G-CaMP2-expressing VNO neurons, as well as their response to pheromones, were indistinguishable from those of the controls ( fig. S1). The projection patterns of the sensory neurons and the innate mating and aggressive behaviors of the G-CaMP2 mice were also indistinguishable from those of wild-type and littermate control animals (figs. S2 to S5). In VNO slices prepared from 2-to 6-month-old male or female animals, application of diluted urine elicited an increase in fluorescence in ∼30 to 40% of G-CaMP2-positive neurons, some of which showed gender-specific responses ( Fig. 1C and Movies S2 and S3). We did not observe significant differences...

In the mammalian brain, similar features of the sensory stimuli are often represented in proximity in the sensory areas. However, how chemical features are represented in the olfactory bulb has been controversial. Questions have been raised as to whether specific chemical features of the odor molecules are represented by spatially clustered olfactory glomeruli. Using a sensitive probe, we have analyzed the glomerular response to large numbers of odorants at single glomerulus resolution. Contrary to the general view, we find that the representation of chemical features is spatially distributed in the olfactory bulb with no discernible chemotopy. Moreover, odor-evoked pattern of activity does not correlate directly with odor structure in general. Despite the lack of spatial clustering or preference with respect to chemical features, some structurally related odors can be similarly represented by ensembles of spatially distributed glomeruli, providing an explanation of their perceptual similarity. Whereas there is no chemotopic organization, and the glomeruli are tuned to odors from multiple classes, we find that the glomeruli are hierarchically arranged into clusters according to their odor-tuning similarity. This tunotopic arrangement provides a framework to understand the spatial organization of the glomeruli that conforms to the organizational principle found in other sensory systems.

The mammalian vomeronasal organ encodes pheromone information about gender, reproductive status, genetic background and individual differences. It remains unknown how pheromone information interacts to trigger innate behaviors. In this study, we identify vomeronasal receptors responsible for detecting female pheromones. A sub-group of V1re clade members recognizes gender-identifying cues in female urine. Multiple members of the V1rj clade are cognate receptors for urinary estrus signals, as well as for sulfated estrogen (SE) compounds. In both cases, the same cue activates multiple homologous receptors, suggesting redundancy in encoding female pheromone cues. Neither gender-specific cues nor SEs alone are sufficient to promote courtship behavior in male mice, whereas robust courtship behavior can be induced when the two cues are applied together. Thus, integrated action of different female cues is required in pheromone-triggered mating behavior. These results suggest a gating mechanism in the vomeronasal circuit in promoting specific innate behavior.DOI: http://dx.doi.org/10.7554/eLife.03025.001

We report here that neuregulin (NRG) isoforms with a conserved cysteine-rich domain (CRD) in their N terminus regulate expression of nicotinic acetylcholine receptors (nAChRs) at developing interneuronal synapses and report the isolation of transmembrane NRG isoforms with this CRD within the N-terminal portion. CRD-NRG mRNA and immunoreactive protein are detected early in developing presynaptic (visceral motor) neurons. The levels of expression of CRD-NRG peak prior to the formation of synapses with their postsynaptic partners, the ganglionic sympathetic neurons. Recombinant CRD-NRG mimics the effects of presynaptic input on target neurons. Functional deletion of CRD-NRG from presynaptic neurons abolishes the upregulation of nAChR expression induced by input-derived soluble material. Thus, CRD-NRG appears to be both a necessary and a sufficient signal for the control of neuronal nAChR expression during synaptogenesis.