Chemosensory Cell-Derived Acetylcholine Drives Tracheal Mucociliary Clearance in Response to Virulence-Associated Formyl Peptides.

Mucociliary clearance through coordinated ciliary beating is a major innate defense removing pathogens from the lower airways, but the pathogen sensing and downstream signaling mechanisms remain unclear. We identified virulence-associated formylated bacterial peptides that potently stimulated ciliary-driven transport in the mouse trachea. This innate response was independent of formyl peptide and taste receptors but depended on key taste transduction genes. Tracheal cholinergic chemosensory cells expressed these genes, and genetic ablation of these cells abrogated peptide-driven stimulation of mucociliary clearance. Trpm5-deficient mice were more susceptible to infection with a natural pathogen, and formylated bacterial peptides were detected in patients with chronic obstructive pulmonary disease. Optogenetics and peptide stimulation revealed that ciliary beating was driven by paracrine cholinergic signaling from chemosensory to ciliated cells operating through muscarinic M3 receptors independently of nerves. We provide a cellular and molecular framework that defines how tracheal chemosensory cells integrate chemosensation with innate defense.

Sensing and avoiding sick conspecifics requires Gαi2+ vomeronasal neurons.

BACKGROUND: Rodents utilize chemical cues to recognize and avoid other conspecifics infected with pathogens. Infection with pathogens and acute inflammation alter the repertoire and signature of olfactory stimuli emitted by a sick individual. These cues are recognized by healthy conspecifics via the vomeronasal or accessory olfactory system, triggering an innate form of avoidance behavior. However, the molecular identity of the sensory neurons and the higher neural circuits involved in the detection of sick conspecifics remain poorly understood. RESULTS: We employed mice that are in an acute state of inflammation induced by systemic administration of lipopolysaccharide (LPS). Through conditional knockout of the G-protein Gαi2 and deletion of other key sensory transduction molecules (Trpc2 and a cluster of 16 vomeronasal type 1 receptors), in combination with behavioral testing, subcellular Ca2+ imaging, and pS6 and c-Fos neuronal activity mapping in freely behaving mice, we show that the Gαi2+ vomeronasal subsystem is required for the detection and avoidance of LPS-treated mice. The active components underlying this avoidance are contained in urine whereas feces extract and two selected bile acids, although detected in a Gαi2-dependent manner, failed to evoke avoidance behavior. Our analyses of dendritic Ca2+ responses in vomeronasal sensory neurons provide insight into the discrimination capabilities of these neurons for urine fractions from LPS-treated mice, and how this discrimination depends on Gαi2. We observed Gαi2-dependent stimulation of multiple brain areas including medial amygdala, ventromedial hypothalamus, and periaqueductal grey. We also identified the lateral habenula, a brain region implicated in negative reward prediction in aversive learning, as a previously unknown target involved in these tasks. CONCLUSIONS: Our physiological and behavioral analyses indicate that the sensing and avoidance of LPS-treated sick conspecifics depend on the Gαi2 vomeronasal subsystem. Our observations point to a central role of brain circuits downstream of the olfactory periphery and in the lateral habenula in the detection and avoidance of sick conspecifics, providing new insights into the neural substrates and circuit logic of the sensing of inflammation in mice.

Central role of G protein Gαi2 and Gαi2+ vomeronasal neurons in balancing territorial and infant-directed aggression of male mice.

Aggression is controlled by the olfactory system in many animal species. In male mice, territorial and infant-directed aggression are tightly regulated by the vomeronasal organ (VNO), but how diverse subsets of sensory neurons convey pheromonal information to limbic centers is not yet known. Here, we employ genetic strategies to show that mouse vomeronasal sensory neurons expressing the G protein subunit Gαi2 regulate male-male and infant-directed aggression through distinct circuit mechanisms. Conditional ablation of Gαi2 enhances male-male aggression and increases neural activity in the medial amygdala (MeA), bed nucleus of the stria terminalis, and lateral septum. By contrast, conditional Gαi2 ablation causes reduced infant-directed aggression and decreased activity in MeA neurons during male-infant interactions. Strikingly, these mice also display enhanced parental behavior and elevated neural activity in the medial preoptic area, whereas sexual behavior remains normal. These results identify Gαi2 as the primary G protein α-subunit mediating the detection of volatile chemosignals in the apical layer of the VNO, and they show that Gαi2+ VSNs and the brain circuits activated by these neurons play a central role in orchestrating and balancing territorial and infant-directed aggression of male mice through bidirectional activation and inhibition of different targets in the limbic system.

Trpc5 deficiency causes hypoprolactinemia and altered function of oscillatory dopamine neurons in the arcuate nucleus.

Dopamine neurons of the hypothalamic arcuate nucleus (ARC) tonically inhibit the release of the protein hormone prolactin from lactotropic cells in the anterior pituitary gland and thus play a central role in prolactin homeostasis of the body. Prolactin, in turn, orchestrates numerous important biological functions such as maternal behavior, reproduction, and sexual arousal. Here, we identify the canonical transient receptor potential channel Trpc5 as an essential requirement for normal function of dopamine ARC neurons and prolactin homeostasis. By analyzing female mice carrying targeted mutations in the Trpc5 gene including a conditional Trpc5 deletion, we show that Trpc5 is required for maintaining highly stereotyped infraslow membrane potential oscillations of dopamine ARC neurons. Trpc5 is also required for eliciting prolactin-evoked tonic plateau potentials in these neurons that are part of a regulatory feedback circuit. Trpc5 mutant females show severe prolactin deficiency or hypoprolactinemia that is associated with irregular reproductive cyclicity, gonadotropin imbalance, and impaired reproductive capabilities. These results reveal a previously unknown role for the cation channel Trpc5 in prolactin homeostasis of female mice and provide strategies to explore the genetic basis of reproductive disorders and other malfunctions associated with defective prolactin regulation in humans.

BTDAzo: A Photoswitchable TRPC5 Channel Activator.

Photoswitchable reagents can be powerful tools for high-precision biological control. TRPC5 is a Ca2+ -permeable cation channel with distinct tissue-specific roles, from synaptic function to hormone regulation. Reagents giving spatiotemporally-resolved control over TRPC5 activity may be key to understanding and harnessing its biology. Here we develop the first photoswitchable TRPC5-modulator, BTDAzo, to address this goal. BTDAzo can photocontrol TRPC5 currents in cell culture, as well as controlling endogenous TRPC5-based neuronal Ca2+ responses in mouse brain slices. BTDAzos are also the first reported azo-benzothiadiazines, an accessible and conveniently derivatised azoheteroarene with strong two-colour photoswitching. BTDAzo's ability to control TRPC5 across relevant channel biology settings makes it suitable for a range of dynamically reversible photoswitching studies in TRP channel biology, with the aim to decipher the various biological roles of this centrally important ion channel.

Ca2+-activated Cl- currents in the murine vomeronasal organ enhance neuronal spiking but are dispensable for male-male aggression.

Ca2+-activated Cl- currents have been observed in many physiological processes, including sensory transduction in mammalian olfaction. The olfactory vomeronasal (or Jacobson's) organ (VNO) detects molecular cues originating from animals of the same species or from predators. It then triggers innate behaviors such as aggression, mating, or flight. In the VNO, Ca2+-activated Cl- channels (CaCCs) are thought to amplify the initial pheromone-evoked receptor potential by mediating a depolarizing Cl- efflux. Here, we confirmed the co-localization of the Ca2+-activated Cl- channels anoctamin 1 (Ano1, also called TMEM16A) and Ano2 (TMEM16B) in microvilli of apically and basally located vomeronasal sensory neurons (VSNs) and their absence in supporting cells of the VNO. Both channels were expressed as functional isoforms capable of giving rise to Ca2+-activated Cl- currents. Although these currents persisted in the VNOs of mice lacking Ano2, they were undetectable in olfactory neuron-specific Ano1 knockout mice irrespective of the presence of Ano2 The loss of Ca2+-activated Cl- currents resulted in diminished spontaneous and drastically reduced pheromone-evoked spiking of VSNs. Although this indicated an important role of anoctamin channels in VNO signal amplification, the lack of this amplification did not alter VNO-dependent male-male territorial aggression in olfactory Ano1/Ano2 double knockout mice. We conclude that Ano1 mediates the bulk of Ca2+-activated Cl- currents in the VNO and that Ano2 plays only a minor role. Furthermore, vomeronasal signal amplification by CaCCs appears to be dispensable for the detection of male-specific pheromones and for near-normal aggressive behavior in mice.

Strain-specific Loss of Formyl Peptide Receptor 3 in the Murine Vomeronasal and Immune Systems.

Formyl peptide receptor 3 (Fpr3, also known as Fpr-rs1) is a G protein-coupled receptor expressed in subsets of sensory neurons of the mouse vomeronasal organ, an olfactory substructure essential for social recognition. Fpr3 has been implicated in the sensing of infection-associated olfactory cues, but its expression pattern and function are incompletely understood. To facilitate visualization of Fpr3-expressing cells, we generated and validated two new anti-Fpr3 antibodies enabling us to analyze acute Fpr3 protein expression. Fpr3 is not only expressed in murine vomeronasal sensory neurons but also in bone marrow cells, the primary source for immune cell renewal, and in mature neutrophils. Consistent with the notion that Fpr3 functions as a pathogen sensor, Fpr3 expression in the immune system is up-regulated after stimulation with a bacterial endotoxin (lipopolysaccharide). These results strongly support a dual role for Fpr3 in both vomeronasal sensory neurons and immune cells. We also identify a large panel of mouse strains with severely altered expression and function of Fpr3, thus establishing the existence of natural Fpr3 knock-out strains. We attribute distinct Fpr3 expression in these strains to the presence or absence of a 12-nucleotide in-frame deletion (Fpr3Δ424-435). In vitro calcium imaging and immunofluorescence analyses demonstrate that the lack of four amino acids leads to an unstable, truncated, and non-functional receptor protein. The genome of at least 19 strains encodes a non-functional Fpr3 variant, whereas at least 13 other strains express an intact receptor. These results provide a foundation for understanding the in vivo function of Fpr3.

Loss-of-function mutations in sodium channel Nav1.7 cause anosmia.

Loss of function of the gene SCN9A, encoding the voltage-gated sodium channel Na(v)1.7, causes a congenital inability to experience pain in humans. Here we show that Na(v)1.7 is not only necessary for pain sensation but is also an essential requirement for odour perception in both mice and humans. We examined human patients with loss-of-function mutations in SCN9A and show that they are unable to sense odours. To establish the essential role of Na(v)1.7 in odour perception, we generated conditional null mice in which Na(v)1.7 was removed from all olfactory sensory neurons. In the absence of Na(v)1.7, these neurons still produce odour-evoked action potentials but fail to initiate synaptic signalling from their axon terminals at the first synapse in the olfactory system. The mutant mice no longer display vital, odour-guided behaviours such as innate odour recognition and avoidance, short-term odour learning, and maternal pup retrieval. Our study creates a mouse model of congenital general anosmia and provides new strategies to explore the genetic basis of the human sense of smell.

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

BACKGROUND: The 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. RESULTS: Herein, 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 Ca(2+) 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. CONCLUSIONS: Peripheral olfactory neurogenesis driven by estrogen may contribute to modulate sensory perception and adaptive VNO-dependent behaviors during pregnancy and early motherhood.

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.

Hypothalamic gonadotropin-releasing hormone (GnRH) receptor neurons fire in synchrony with the female reproductive cycle.

Gonadotropin-releasing hormone (GnRH) controls mammalian reproduction via the hypothalamic-pituitary-gonadal (hpg) axis, acting on gonadotrope cells in the pituitary gland that express the GnRH receptor (GnRHR). Cells expressing the GnRHR have also been identified in the brain. However, the mechanism by which GnRH acts on these potential target cells remains poorly understood due to the difficulty of visualizing and identifying living GnRHR neurons in the central nervous system. We have developed a mouse strain in which GnRHR neurons express a fluorescent marker, enabling the reliable identification of these cells independent of the hormonal status of the animal. In this study, we analyze the GnRHR neurons of the periventricular hypothalamic nucleus in acute brain slices prepared from adult female mice. Strikingly, we find that the action potential firing pattern of these neurons alternates in synchrony with the estrous cycle, with pronounced burst firing during the preovulatory period. We demonstrate that GnRH stimulation is sufficient to trigger the conversion from tonic to burst firing in GnRHR neurons. Furthermore, we show that this switch in the firing pattern is reversed by a potent GnRHR antagonist. These data suggest that endogenous GnRH acts on GnRHR neurons and triggers burst firing in these cells during late proestrus and estrus. Our data have important clinical implications in that they indicate a novel mode of action for GnRHR agonists and antagonists in neurons of the central nervous system that are not part of the classical hpg axis.

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

BACKGROUND: Optimal 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. RESULTS: To 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. CONCLUSIONS: These 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.

TRP channels in reproductive (neuro)endocrinology.

Transient receptor potential (TRP) ion channels have been detected in neurons that are part of the neural network controlling reproductive physiology and behavior. In this chapter we will primarily take a look at the classical/canonical TRP (TRPC) channels but will also examine some other members of the TRP channel superfamily in reproductive (neuro)endocrinology. The referenced data suggest that different TRP proteins could play functional roles at different levels of the reproductive pathway. Still, our understanding of TRP channel involvement in (neuro)endocrinology is quite limited. Due to their mechanism of activation and complex regulation, these channels are however ideally suited to be part of the transduction machinery of hormone-secreting cells.

G protein G(alpha)o 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.

Widespread Distribution of Luteinizing Hormone/Choriogonadotropin Receptor in Human Juvenile Angiofibroma: Implications for a Sex-Specific Nasal Tumor.

Juvenile angiofibroma (JA) is a rare, sex-specific, and highly vascularized nasal tumor that almost exclusively affects male adolescents, but its etiology has been controversial. The G protein-coupled hormone receptor LHCGR [luteinizing hormone (LH)/choriogonadotropin (hCG) receptor] represents a promising new candidate for elucidating the underlying mechanisms of sex specificity, pubertal manifestation, and JA progression. We used highly sensitive RNAscope technology, together with immunohistochemistry, to investigate the cellular expression, localization, and distribution of LHCGR in tissue samples from JA patients. Our results provide evidence for LHCGR expression in subsets of cells throughout JA tissue sections, with the majority of LHCGR+ cells located in close vicinity to blood vessels, rendering them susceptible to endocrine LH/hCG signaling, but LHCGR+ cells were also detected in fibrocollagenous stroma. A majority of LHCGR+ cells located near the vascular lumen co-expressed the neural crest stem cell marker CD271. These results are intriguing as both LH and hCG are produced in a time- and sex-dependent manner, and are known to be capable of inducing cell proliferation and angiogenesis. Our results give rise to a new model that suggests endocrine mechanisms involving LHCGR and its ligands, together with autocrine and paracrine signaling, in JA vascularization and cell proliferation.

Tuft cell IL-17RB restrains IL-25 bioavailability and reveals context-dependent ILC2 hypoproliferation.

The tuft cell-ILC2 circuit orchestrates rapid type 2 responses upon detecting microbe-derived succinate and luminal helminths. Our findings delineate key mechanistic steps, involving IP3R2 engagement and Ca 2+ flux, governing IL-25 production by tuft cells triggered by succinate detection. While IL-17RB plays a pivotal intrinsic role in ILC2 activation, it exerts a regulatory function in tuft cells. Tuft cells exhibit constitutive Il25 expression, placing them in an anticipatory state that facilitates rapid production of IL-25 protein for ILC2 activation. Tuft cell IL-17RB is crucial for restraining IL-25 bioavailability, preventing excessive tonic ILC2 stimulation due to basal Il25 expression. Suboptimal ILC2 stimulation by IL-25 resulting from tuft cell Il17rb -deficiency or prolonged succinate exposure induces a state of hypoproliferation in ILC2s, also observed in chronic helminth infection. Our study offers critical insights into the regulatory dynamics of IL-25 in this circuit, highlighting the delicate tuning required for responses to diverse luminal states.

Subsystem organization of the mammalian sense of smell.

The mammalian olfactory system senses an almost unlimited number of chemical stimuli and initiates a process of neural recognition that influences nearly every aspect of life. This review examines the organizational principles underlying the recognition of olfactory stimuli. The olfactory system is composed of a number of distinct subsystems that can be distinguished by the location of their sensory neurons in the nasal cavity, the receptors they use to detect chemosensory stimuli, the signaling mechanisms they employ to transduce those stimuli, and their axonal projections to specific regions of the olfactory forebrain. An integrative approach that includes gene targeting methods, optical and electrophysiological recording, and behavioral analysis has helped to elucidate the functional significance of this subsystem organization for the sense of smell.

Optical Activation of Photoswitchable TRPC Ligands in the Mammalian Olfactory System Using Laser Scanning Confocal Microscopy.

The transient receptor potential canonical (TRPC) ion channels play important biological roles, but their activation mechanisms are incompletely understood. Here, we describe recent methodological advances using small molecular probes designed for photopharmacology of TRPC channels by focusing on results obtained from the mouse olfactory system. These studies developed and used photoswitchable diacylglycerol (DAG) analogs for ultrarapid activation of native TRPC2 channels in vomeronasal sensory neurons and type B cells of the main olfactory epithelium. Further studies investigated the role of TRPC5 channels in prolactin regulation of dopamine neurons in the arcuate nucleus of the hypothalamus. Here, the first photoswitchable TRPC5 modulator, BTDAzo, was developed and shown to control endogenous TRPC5-based neuronal Ca2+ responses in mouse brain slices. Thus, photoswitchable reagents are rapidly gaining widespread recognition for investigating various types of TRPC channels including TRPC2, TRPC3, TRPC5, and TRPC6, enabling to gain new insights into the gating mechanisms and functions of these channels.

Sensory detection of female olfactory cues as a central regulator of energy metabolism and body weight in male mice.

Olfactory stimuli from food influence energy balance, preparing the body for digestion when food is consumed. Social chemosensory cues predict subsequent energetic changes required for social interactions and could be an additional sensory input influencing energy balance. We show that exposure to female chemostimuli increases metabolic rate in male mice and reduces body weight and adipose tissue expansion when mice are fed a high-fat diet. These responses are linked to detection of female chemostimuli via G-protein Gαo-expressing vomeronasal sensory neurons. Males with Gαo deleted in the olfactory system are fertile but do not show changes in body weight when paired with females and show severely blunted changes in energy expenditure when exposed to female bedding. These results establish that metabolic and reproductive responses to females can be partly uncoupled in male mice and that detection of female chemostimuli is a central regulator of energy metabolism and lipid storage.

A succinate/SUCNR1-brush cell defense program in the tracheal epithelium.

Host-derived succinate accumulates in the airways during bacterial infection. Here, we show that luminal succinate activates murine tracheal brush (tuft) cells through a signaling cascade involving the succinate receptor 1 (SUCNR1), phospholipase Cß2, and the cation channel transient receptor potential channel subfamily M member 5 (TRPM5). Stimulated brush cells then trigger a long-range Ca2+ wave spreading radially over the tracheal epithelium through a sequential signaling process. First, brush cells release acetylcholine, which excites nearby cells via muscarinic acetylcholine receptors. From there, the Ca2+ wave propagates through gap junction signaling, reaching also distant ciliated and secretory cells. These effector cells translate activation into enhanced ciliary activity and Cl- secretion, which are synergistic in boosting mucociliary clearance, the major innate defense mechanism of the airways. Our data establish tracheal brush cells as a central hub in triggering a global epithelial defense program in response to a danger-associated metabolite.