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.

Danger perception and stress response through an olfactory sensor for the bacterial metabolite hydrogen sulfide.

The olfactory system serves a critical function as a danger detection system to trigger defense responses essential for survival. The cellular and molecular mechanisms that drive such defenses in mammals are incompletely understood. Here, we have discovered an ultrasensitive olfactory sensor for the highly poisonous bacterial metabolite hydrogen sulfide (H2S) in mice. An atypical class of sensory neurons in the main olfactory epithelium, the type B cells, is activated by both H2S and low O2. These two stimuli trigger, respectively, Cnga2- and Trpc2-signaling pathways, which operate in separate subcellular compartments, the cilia and the dendritic knob. This activation drives essential defensive responses: elevation of the stress hormone ACTH, stress-related self-grooming behavior, and conditioned place avoidance. Our findings identify a previously unknown signaling paradigm in mammalian olfaction and define type B cells as chemosensory neurons that integrate distinct danger inputs from the external environment with appropriate defense outputs.

A central mechanism of analgesia in mice and humans lacking the sodium channel NaV1.7.

Deletion of SCN9A encoding the voltage-gated sodium channel NaV1.7 in humans leads to profound pain insensitivity and anosmia. Conditional deletion of NaV1.7 in sensory neurons of mice also abolishes pain, suggesting that the locus of analgesia is the nociceptor. Here we demonstrate, using in vivo calcium imaging and extracellular recording, that NaV1.7 knockout mice have essentially normal nociceptor activity. However, synaptic transmission from nociceptor central terminals in the spinal cord is greatly reduced by an opioid-dependent mechanism. Analgesia is also reversed substantially by central but not peripheral application of opioid antagonists. In contrast, the lack of neurotransmitter release from olfactory sensory neurons is opioid independent. Male and female humans with NaV1.7-null mutations show naloxone-reversible analgesia. Thus, inhibition of neurotransmitter release is the principal mechanism of anosmia and analgesia in mouse and human Nav1.7-null mutants.

Cyclic regulation of Trpm4 expression in female vomeronasal neurons driven by ovarian sex hormones.

The vomeronasal organ (VNO), the sensory organ of the mammalian accessory olfactory system, mediates the activation of sexually dimorphic reproductive behavioral and endocrine responses in males and females. It is unclear how sexually dimorphic and state-dependent responses are generated by vomeronasal sensory neurons (VSNs). Here, we report the expression of the transient receptor potential (TRP) channel Trpm4, a Ca2+-activated monovalent cation channel, as a second TRP channel present in mouse VSNs, in addition to the diacylglycerol-sensitive Trpc2 channel. The expression of Trpm4 in the mouse VNO is sexually dimorphic and, in females, is tightly linked to their reproductive cycle. We show that Trpm4 protein expression is upregulated specifically during proestrus and estrus, when female mice are about to ovulate and become sexually active and receptive. The cyclic regulation of Trpm4 expression in female VSNs depends on ovarian sex hormones and is abolished by surgical removal of the ovaries (OVX). Trpm4 upregulation can be restored in OVX mice by systemic treatment with 17ß-estradiol, requires endogenous activity of aromatase enzyme, and is strongly reduced during late pregnancy. This cyclic regulation of Trpm4 offers a neural mechanism by which female mice could regulate the relative strength of sensory signals in their VSNs, depending on hormonal state. Trpm4 is likely to participate in sex-specific, estrous cycle-dependent and sex hormone-regulated functions of the VNO, and may serve as a previously unknown genetic substrate for dissecting mammalian sexually dimorphic cellular and behavioral responses.

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.

Bacterial MgrB peptide activates chemoreceptor Fpr3 in mouse accessory olfactory system and drives avoidance behaviour.

Innate immune chemoreceptors of the formyl peptide receptor (Fpr) family are expressed by vomeronasal sensory neurons (VSNs) in the accessory olfactory system. Their biological function and coding mechanisms remain unknown. We show that mouse Fpr3 (Fpr-rs1) recognizes the core peptide motif f-MKKFRW that is predominantly present in the signal sequence of the bacterial protein MgrB, a highly conserved regulator of virulence and antibiotic resistance in Enterobacteriaceae. MgrB peptide can be produced and secreted by bacteria, and is selectively recognized by a subset of VSNs. Exposure to the peptide also stimulates VSNs in freely behaving mice and drives innate avoidance. Our data shows that Fpr3 is required for neuronal detection and avoidance of peptides derived from a conserved master virulence regulator of enteric bacteria.

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.

P/Q Type Calcium Channel Cav2.1 Defines a Unique Subset of Glomeruli in the Mouse Olfactory Bulb.

Voltage-gated calcium (Cav) channels are a prerequisite for signal transmission at the first olfactory sensory neuron (OSN) synapse within the glomeruli of the main olfactory bulb (MOB). We showed previously that the N-type Cav channel subunit Cav2.2 is present in the vast majority of glomeruli and plays a central role in presynaptic transmitter release. Here, we identify a distinct subset of glomeruli in the MOB of adult mice that is characterized by expression of the P/Q-type channel subunit Cav2.1. Immunolocalization shows that Cav2.1+ glomeruli reside predominantly in the medial and dorsal MOB, and in the vicinity of the necklace glomerular region close to the accessory olfactory bulb. Few glomeruli are detected on the ventral and lateral MOB. Cav2.1 labeling in glomeruli colocalizes with the presynaptic marker vGlut2 in the axon terminals of OSNs. Electron microscopy shows that Cav2.1+ presynaptic boutons establish characteristic asymmetrical synapses with the dendrites of second-order neurons in the glomerular neuropil. Cav2.1+ glomeruli receive axonal input from OSNs that express molecules of canonical OSNs: olfactory marker protein, the ion channel Cnga2, and the phosphodiesterase Pde4a. In the main olfactory epithelium, Cav2.1 labels a distinct subpopulation of OSNs whose distribution mirrors the topography of the MOB glomeruli, that shows the same molecular signature, and is already present at birth. Together, these experiments identify a unique Cav2.1+ multiglomerular domain in the MOB that may form a previously unrecognized olfactory subsystem distinct from other groups of necklace glomeruli that rely on cGMP signaling mechanisms.

Mapping protein interactions of sodium channel NaV1.7 using epitope-tagged gene-targeted mice.

The voltage-gated sodium channel NaV1.7 plays a critical role in pain pathways. We generated an epitope-tagged NaV1.7 mouse that showed normal pain behaviours to identify channel-interacting proteins. Analysis of NaV1.7 complexes affinity-purified under native conditions by mass spectrometry revealed 267 proteins associated with Nav1.7 in vivo The sodium channel ß3 (Scn3b), rather than the ß1 subunit, complexes with Nav1.7, and we demonstrate an interaction between collapsing-response mediator protein (Crmp2) and Nav1.7, through which the analgesic drug lacosamide regulates Nav1.7 current density. Novel NaV1.7 protein interactors including membrane-trafficking protein synaptotagmin-2 (Syt2), L-type amino acid transporter 1 (Lat1) and transmembrane P24-trafficking protein 10 (Tmed10) together with Scn3b and Crmp2 were validated by co-immunoprecipitation (Co-IP) from sensory neuron extract. Nav1.7, known to regulate opioid receptor efficacy, interacts with the G protein-regulated inducer of neurite outgrowth (Gprin1), an opioid receptor-binding protein, demonstrating a physical and functional link between Nav1.7 and opioid signalling. Further information on physiological interactions provided with this normal epitope-tagged mouse should provide useful insights into the many functions now associated with the NaV1.7 channel.

Organization and Plasticity of Sodium Channel Expression in the Mouse Olfactory and Vomeronasal Epithelia.

To understand the molecular basis of neuronal excitation in the mammalian olfactory system, we conducted a systematic analysis of the organization of voltage-gated sodium (Nav) channel subunits in the main olfactory epithelium (MOE) and vomeronasal organ (VNO) of adult mice. We also analyzed changes in Nav channel expression during development in these two systems and during regeneration of the MOE. Quantitative PCR shows that Nav1.7 is the predominant isoform in both adult MOE and VNO. We detected pronounced immunoreactivity for Nav1.7 and Nav1.3 in axons of olfactory and vomeronasal sensory neurons (VSNs). Analysis of Nav1.2 and Nav1.6 revealed an unexpected subsystem-specific distribution. In the MOE, these Nav channels are absent from olfactory sensory neurons (OSNs) but present in non-neuronal olfactory cell types. In the VNO, Nav1.2 and Nav1.6 are confined to VSNs, with Nav1.2-immunoreactive somata solely present in the basal layer of the VNO. The subcellular localization of Nav1.3 and Nav1.7 in OSNs can change dramatically during periods of heightened plasticity in the MOE. During the first weeks of development and during regeneration of the olfactory epithelium following chemical lesion, expression of Nav1.3 and Nav1.7 is transiently enhanced in the somata of mature OSNs. Our results demonstrate a highly complex organization of Nav channel expression in the mouse olfactory system, with specific commonalities but also differences between the MOE and the VNO. On the basis of their subcellular localization, Nav1.3 and Nav1.7 should play major roles in action potential propagation in both MOE and VNO, whereas Nav1.2 and Nav1.6 are specific to the function of VSNs. The plasticity of Nav channel expression in OSNs during early development and recovery from injury could reflect important physiological requirements in a variety of activity-dependent mechanisms.

Trpm5 expression in the olfactory epithelium.

The Ca2+-activated monovalent cation channel Trpm5 is a key element in chemotransduction of taste receptor cells of the tongue, but the extent to which Trpm5 channels are expressed in olfactory sensory neurons (OSNs) of the main olfactory epithelium (MOE) of adult mice as part of a specific pheromonal detection system is debated. Here, we used a novel Trpm5-IRES-Cre knockin strain to drive Cre recombinase expression, employed previously validated Trpm5 antibodies, performed in situ hybridization experiments to localize Trpm5 RNA, and searched extensively for Trpm5 splice variants in genetically-labeled, Trpm5-expressing MOE cells. In contrast to previous reports, we find no evidence for the existence in adult mouse OSNs of the classical Trpm5 channel known from taste cells. We show that Trpm5-expressing adult OSNs express a novel Trpm5 splice variant, Trpm5-9, that is unlikely to form a functional cation channel by itself. We also demonstrate that Trpm5 is transiently expressed in a subpopulation of mature OSNs in the embryonic olfactory epithelium, indicating that Trpm5 channels could play a specific role in utero during a narrow developmental time window. Ca2+ imaging with GCaMP3 under the control of the Trpm5-IRES-Cre allele using a newly developed MOE wholemount preparation of the adult olfactory epithelium reveals that Trpm5-GCaMP3 OSNs comprise a heterogeneous group of sensory neurons many of which can detect general odorants. Together, these studies are essential for understanding the role of transient receptor potential channels in mammalian olfaction.

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 Binary Genetic Approach to Characterize TRPM5 Cells in Mice.

Transient receptor potential channel subfamily M member 5 (TRPM5) is an important downstream signaling component in a subset of taste receptor cells making it a potential target for taste modulation. Interestingly, TRPM5 has been detected in extra-oral tissues; however, the function of extra-gustatory TRPM5-expressing cells is less well understood. To facilitate visualization and manipulation of TRPM5-expressing cells in mice, we generated a Cre knock-in TRPM5 allele by homologous recombination. We then used the novel TRPM5-IRES-Cre mouse strain to report TRPM5 expression by activating a τGFP transgene. To confirm faithful coexpression of τGFP and TRPM5 we generated and validated a new anti-TRPM5 antiserum enabling us to analyze acute TRPM5 protein expression. τGFP cells were found in taste bud cells of the vallate, foliate, and fungiform papillae as well as in the palate. We also detected TRPM5 expression in several other tissues such as in the septal organ of Masera. Interestingly, in the olfactory epithelium of adult mice acute TRPM5 expression was detected in only one (short microvillar cells) of two cell populations previously reported to express TRPM5. The TRPM5-IC mouse strain described here represents a novel genetic tool and will facilitate the study and tissue-specific manipulation of TRPM5-expressing cells in vivo.

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.

Altered synaptic transmission at olfactory and vomeronasal nerve terminals in mice lacking N-type calcium channel Cav2.2.

We investigated the role of voltage-activated calcium (Cav) channels for synaptic transmission at mouse olfactory and vomeronasal nerve terminals at the first synapse of the main and accessory olfactory pathways, respectively. We provided evidence for a central role of the N-type Cav channel subunit Cav2.2 in presynaptic transmitter release at these synapses. Striking Cav2.2 immunoreactivity was localised to the glomerular neuropil of the main olfactory bulb (MOB) and accessory olfactory bulb (AOB), and co-localised with presynaptic molecules such as bassoon. Voltage-clamp recordings of sensory nerve-evoked, excitatory postsynaptic currents (EPSCs) in mitral/tufted (M/T) and superficial tufted cells of the MOB and mitral cells of the AOB, in combination with established subtype-specific Cav channel toxins, indicated a predominant role of N-type channels in transmitter release at these synapses, whereas L-type, P/Q-type, and R-type channels had either no or only relatively minor contributions. In Cacna1b mutant mice lacking the Cav2.2 (α1B) subunit of N-type channels, olfactory nerve-evoked M/T cell EPSCs were not reduced but became blocker-resistant, thus indicating a major reorganisation and compensation of Cav channel subunits as a result of the Cav2.2 deletion at this synapse. Cav2.2-deficient mice also revealed that Cav2.2 was critically required for paired-pulse depression of olfactory nerve-evoked EPSCs in M/T cells of the MOB, and they demonstrated an essential requirement for Cav2.2 in vomeronasal nerve-evoked EPSCs of AOB mitral cells. Thus, Cacna1b loss-of-function mutations are unlikely to cause general anosmia but Cacna1b emerges as a strong candidate in the search for mutations causing altered olfactory perception, such as changes in general olfactory sensitivity and altered social responses to chemostimuli.

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.

Link between pain and olfaction in an inherited sodium channelopathy.

In a major breakthrough in our understanding of human olfaction, a recent study showed that loss-of-function mutations in the voltage-gated sodium channel Nav1.7, encoded by the gene SCN9A, cause a loss of the sense of smell (congenital general anosmia) in mice and humans. These findings are of special clinical relevance because Nav1.7 was previously known for its essential role in the perception of pain; therefore, this channel is being explored as a promising target in the search for novel analgesics. This advance offers a functional understanding of a monogenic human disorder that is characterized by a loss of 2 major senses-nociception and smell-thus providing an unexpected mechanistic link between these 2 sensory modalities.

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.

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.

Grueneberg ganglion neurons are finely tuned cold sensors.

The Grueneberg ganglion is a newly appreciated nasal subsystem with neural connections to the olfactory forebrain, but its functional role has not been well defined. Here, we assess whether Grueneberg ganglion neurons (GGNs) function as thermosensors. By investigating the effect of acute temperature changes on the cytosolic Ca(2+) concentration of genetically labeled mouse GGNs (either gender), we demonstrate that GGNs are thermosensory neurons specialized to detect a temperature decline within a given temperature window. Furthermore, GGNs comprise a relatively homogeneous cell population with respect to temperature sensitivity. GGNs do not respond to ligands of the temperature-sensitive TRP channels TRPM8 and TRPA1, suggesting a novel mechanism for temperature sensing. One possibility is a cGMP-mediated mechanism, as GGNs express the receptor guanylyl cyclase GC-G, the cGMP-sensitive phosphodiesterase PDE2 and the cGMP-sensitive channel CNGA3. Surprisingly, Cnga3-null mice show normal cooling-induced Ca(2+) responses although cGMP-dependent Ca(2+) increases are absent in these mice. Rather, the cooling-induced Ca(2+) response of GGNs depends critically on the activity of a tetrodotoxin-sensitive voltage-gated sodium channel whereas the cGMP-dependent Ca(2+) signal does not. These findings establish the Grueneberg ganglion as a sensory organ mediating cold-evoked neural responses, possibly in conjunction with the sensing of other stress- or fear-related chemical social cues.