Pablo Chamero

Identification of protein pheromones that promote aggressive behaviour

Mice use pheromones, compounds emitted and detected by members of the same species, as cues to regulate social behaviours such as pup suckling, aggression and mating. Neurons that detect pheromones are thought to reside in at least two separate organs within the nasal cavity: the vomeronasal organ (VNO) and the main olfactory epithelium (MOE). Each pheromone ligand is thought to activate a dedicated subset of these sensory neurons. However, the nature of the pheromone cues and the identity of the responding neurons that regulate specific social behaviours are largely unknown. Here we show, by direct activation of sensory neurons and analysis of behaviour, that at least two chemically distinct ligands are sufficient to promote male–male aggression and stimulate VNO neurons. We have purified and analysed one of these classes of ligand and found its specific aggression-promoting activity to be dependent on the presence of the protein component of the major urinary protein (MUP) complex, which is known to comprise specialized lipocalin proteins bound to small organic molecules. Using calcium imaging of dissociated vomeronasal neurons (VNs), we have determined that the MUP protein activates a sensory neuron subfamily characterized by the expression of the G-protein Gαo subunit (also known as Gnao) and Vmn2r putative pheromone receptors (V2Rs). Genomic analysis indicates species-specific co-expansions of MUPs and V2Rs, as would be expected among pheromone-signalling components. Finally, we show that the aggressive behaviour induced by the MUPs occurs exclusively through VNO neuronal circuits. Our results substantiate the idea of MUP proteins as pheromone ligands that mediate male–male aggression through the accessory olfactory neural pathway.

Impaired maturation of dendritic spines without disorganization of cortical cell layers in mice lacking NRG1/ErbB signaling in the central nervous system

Neuregulin-1 (NRG1) and its ErbB2/B4 receptors are encoded by candidate susceptibility genes for schizophrenia, yet the essential functions of NRG1 signaling in the CNS are still unclear. Using CRE/LOX technology, we have inactivated ErbB2/B4-mediated NRG1 signaling specifically in the CNS. In contrast to expectations, cell layers in the cerebral cortex, hippocampus, and cerebellum develop normally in the mutant mice. Instead, loss of ErbB2/B4 impairs dendritic spine maturation and perturbs interactions of postsynaptic scaffold proteins with glutamate receptors. Conversely, increased NRG1 levels promote spine maturation. ErbB2/B4-deficient mice show increased aggression and reduced prepulse inhibition. Treatment with the antipsychotic drug clozapine reverses the behavioral and spine defects. We conclude that ErbB2/B4-mediated NRG1 signaling modulates dendritic spine maturation, and that defects at glutamatergic synapses likely contribute to the behavioral abnormalities in ErbB2/B4-deficient mice.

G protein Gαo is essential for vomeronasal function and aggressive behavior in mice

The rodent vomeronasal organ (VNO) mediates the regulation of species-specific and interspecies social behaviors. We have used gene targeting to examine the role of the G protein Gαo, encoded by the gene Gnao1, in vomeronasal function. We used the Cre-loxP system to delete Gαo in those cells that express olfactory marker protein, which includes all vomeronasal sensory neurons of the basal layer of the VNO sensory epithelium. Using electrophysiology and calcium imaging, we show that the conditional null mice exhibit strikingly reduced sensory responses in V2R receptor-expressing vomeronasal sensory neurons to specific molecular cues, including MHC1 antigens, major urinary proteins, and exocrine gland-secreting peptide. Gαo is also vital for vomeronasal sensing of two N-formylated mitochondrially encoded peptides derived from NADH dehydrogenase 1. Furthermore, we show that Gαo is an essential requirement for the display of male–male territorial aggression as well as maternal aggression in mice. Finally, we show that Gαo-dependent maternal aggression can be induced by major urinary proteins. These cellular and behavioral phenotypes identify Gαo as the primary G-protein α-subunit mediating the detection of peptide and protein pheromones by sensory neurons of the VNO.

From genes to social communication: molecular sensing by the vomeronasal organ

The ability to distinguish molecular cues emitted by other individuals is a fundamental feature of social interactions such as finding and identifying a mate, establishing social hierarchies, and initiating interspecies defensive behaviors. In rodents, this ability involves the vomeronasal organ (VNO), a distinct chemoreceptive structure that is part of the olfactory system. Recent insights have led to unprecedented progress in identifying ligand and receptor families underlying vomeronasal recognition, characterizing the behavioral consequences caused by VNO activation, and defining higher neural circuits underlying the initiation of instinctive behaviors such as aggression. Here, we review such findings and discuss future areas for investigation, including large-scale mapping studies, immune system-VNO interactions, in vivo recording of neural activity, and optogenetic alteration of sexual and social behaviors.

Innate Predator Odor Aversion Driven by Parallel Olfactory Subsystems that Converge in the Ventromedial Hypothalamus

The existence of innate predator aversion evoked by predator-derived chemostimuli called kairomones offers a strong selective advantage for potential prey animals. However, it is unclear how chemically diverse kairomones can elicit similar avoidance behaviors. Using a combination of behavioral analyses and single-cell Ca(2+) imaging in wild-type and gene-targeted mice, we show that innate predator-evoked avoidance is driven by parallel, non-redundant processing of volatile and nonvolatile kairomones through the activation of multiple olfactory subsystems including the Grueneberg ganglion, the vomeronasal organ, and chemosensory neurons within the main olfactory epithelium. Perturbation of chemosensory responses in specific subsystems through disruption of genes encoding key sensory transduction proteins (Cnga3, Gnao1) or by surgical axotomy abolished avoidance behaviors and/or cellular Ca(2+) responses to different predator odors. Stimulation of these different subsystems resulted in the activation of widely distributed target regions in the olfactory bulb, as assessed by c-Fos expression. However, in each case, this c-Fos increase was observed within the same subnuclei of the medial amygdala and ventromedial hypothalamus, regions implicated in fear, anxiety, and defensive behaviors. Thus, the mammalian olfactory system has evolved multiple, parallel mechanisms for kairomone detection that converge in the brain to facilitate a common behavioral response. Our findings provide significant insights into the genetic substrates and circuit logic of predator-driven innate aversion and may serve as a valuable model for studying instinctive fear and human emotional and panic disorders.

Newborn Interneurons in the Accessory Olfactory Bulb Promote Mate Recognition in Female Mice

In the olfactory bulb of adult rodents, local interneurons are constantly replaced by immature precursors derived from the subventricular zone. Whether any olfactory sensory process specifically relies on this cell renewal remains largely unclear. By using the well known model of mating-induced imprinting to avoid pregnancy block, which requires accessory olfactory bulb (AOB) function, we demonstrate that this olfactory memory formation critically depends on the presence of newborn granule neurons in this brain region. We show that, in adult female mice, exposure to the male urine compounds involved in mate recognition increases the number of new granule cells surviving in the AOB. This process is modulated by male signals sensed through the vomeronasal organ and, in turn, changes the activity of the downstream amygdaloid and hypothalamic nuclei involved in the pregnancy block response. Chemical depletion of newly generated bulbar interneurons causes strong impairment in mate recognition, thus resulting in a high pregnancy failure rate to familiar mating male odors. Taken together, our results indicate that adult neurogenesis is essential for specific brain functions such as persistent odor learning and mate recognition.

A Family of Nonclassical Class I MHC Genes Contributes to Ultrasensitive Chemodetection by Mouse Vomeronasal Sensory Neurons

The mouse vomeronasal organ (VNO) has a pivotal role in chemical communication. The vomeronasal sensory neuroepithelium consists of distinct populations of vomeronasal sensory neurons (VSNs). A subset of VSNs, with cell bodies in the basal part of the basal layer, coexpress Vmn2r G-protein-coupled receptor genes with H2-Mv genes, a family of nine nonclassical class I major histocompatibility complex genes. The in vivo, physiological roles of the H2-Mv gene family remain mysterious more than a decade after the discovery of combinatorial H2-Mv gene expression in VSNs. Here, we have taken a genetic approach and have deleted the 530 kb cluster of H2-Mv genes in the mouse germline by chromosome engineering. Homozygous mutant mice (ΔH2Mv mice) are viable and fertile. There are no major anatomical defects in their VNO and accessory olfactory bulb (AOB). Their VSNs can be stimulated with chemostimuli (peptides and proteins) to the same maximum responses as VSNs of wild-type mice, but require much higher concentrations. This physiological phenotype is displayed at the single-cell level and is cell autonomous: single V2rf2-expressing VSNs, which normally coexpress H2-Mv genes, display a decreased sensitivity to a peptide ligand in ΔH2Mv mice, whereas single V2r1b-expressing VSNs, which do not coexpress H2-Mv genes, show normal sensitivity to a peptide ligand in ΔH2Mv mice. Consistent with the greatly decreased VSN sensitivity, ΔH2Mv mice display pronounced deficits in aggressive and sexual behaviors. Thus, H2-Mv genes are not absolutely essential for the generation of physiological responses, but are required for ultrasensitive chemodetection by a subset of VSNs.

A wide range of pheromone-stimulated sexual and reproductive behaviors in female mice depend on G protein Gαo.

BackgroundOptimal reproductive fitness is essential for the biological success and survival of species. The vomeronasal organ is strongly implicated in the display of sexual and reproductive behaviors in female mice, yet the roles that apical and basal vomeronasal neuron populations play in controlling these gender-specific behaviors remain largely unclear.ResultsTo dissect the neural pathways underlying these functions, we genetically inactivated the basal vomeronasal organ layer using conditional, cell-specific ablation of the G protein Gαo. Female mice mutant for Gαo show severe alterations in sexual and reproductive behaviors, timing of puberty onset, and estrous cycle. These mutant mice are insensitive to reproductive facilitation stimulated by male pheromones that accelerate puberty and induce ovulation. Gαo-mutant females exhibit a striking reduction in sexual receptivity or lordosis behavior to males, but gender discrimination seems to be intact. These mice also show a loss in male scent preference, which requires a learned association for volatile olfactory signals with other nonvolatile ownership signals that are contained in the high molecular weight fraction of male urine. Thus, Gαo impacts on both instinctive and learned social responses to pheromones.ConclusionsThese results highlight that sensory neurons of the Gαo-expressing vomeronasal subsystem, together with the receptors they express and the molecular cues they detect, control a wide range of fundamental mating and reproductive behaviors in female mice.

Signaling mechanisms and behavioral function of the mouse basal vomeronasal neuroepithelium

The vomeronasal organ (VNO) is a sensory organ that is found in most terrestrial vertebrates and that is principally implicated in the detection of pheromones. The VNO contains specialized sensory neurons organized in a pseudostratified neuroepithelium that recognize chemical signals involved in initiating innate behavioral responses. In rodents, the VNO neuroepithelium is segregated into two distinct zones, apical and basal. The molecular mechanisms involved in ligand detection by apical and basal VNO sensory neurons differ extensively. These two VNO subsystems express different subfamilies of vomeronasal receptors and signaling molecules, detect distinct chemosignals, and project to separate regions of the accessory olfactory bulb (AOB). The roles that these olfactory subdivisions play in the control of specific olfactory-mediated behaviors are largely unclear. However, analysis of mutant mouse lines for signal transduction components together with identification of defined chemosensory ligands has revealed a fundamental role of the basal part of the mouse VNO in mediating a wide range of instinctive behaviors, such as aggression, predator avoidance, and sexual attraction. Here we will compare the divergent functions and synergies between the olfactory subsystems and consider new insights in how higher neural circuits are defined for the initiation of instinctive behaviors.

Pregnancy and estrogen enhance neural progenitor-cell proliferation in the vomeronasal sensory epithelium

BackgroundThe hormonal state during the estrus cycle or pregnancy produces alterations on female olfactory perception that are accompanied by specific maternal behaviors, but it is unclear how sex hormones act on the olfactory system to enable these sensory changes.ResultsHerein, we show that the production of neuronal progenitors is stimulated in the vomeronasal organ (VNO) epithelium of female mice during a late phase of pregnancy. Using a wide range of molecular markers that cover the whole VNO cell maturation process in combination with Ca2+ imaging in early postmitotic neurons, we show that newly generated VNO cells adopt morphological and functional properties of mature sensory neurons. A fraction of these newly generated cells project their axons to the olfactory forebrain, extend dendrites that contact the VNO lumen, and can detect peptides and urinary proteins shown to contain pheromone activity. High-throughput RNA-sequencing reveals concomitant differences in gene expression in the VNO transcriptomes of pregnant females. These include relative increases in expression of 20 vomeronasal receptors, of which 17 belong to the V1R subfamily, and may therefore be considered as candidate receptors for mediating maternal behaviors. We identify the expression of several hormone receptors in the VNO of which estrogen receptor α (Esr1) is directly localized to neural progenitors. Administration of sustained high levels of estrogen, but not progesterone, is sufficient to stimulate vomeronasal progenitor cell proliferation in the VNO epithelium.ConclusionsPeripheral olfactory neurogenesis driven by estrogen may contribute to modulate sensory perception and adaptive VNO-dependent behaviors during pregnancy and early motherhood.