A revised conceptual framework for mouse vomeronasal pumping and stimulus sampling.

The physiological performance of any sensory organ is determined by its anatomy and physical properties. Consequently, complex sensory structures with elaborate features have evolved to optimize stimulus detection. Understanding these structures and their physical nature forms the basis for mechanistic insights into sensory function. Despite its crucial role as a sensor for pheromones and other behaviorally instructive chemical cues, the vomeronasal organ (VNO) remains a poorly characterized mammalian sensory structure. Fundamental principles of its physico-mechanical function, including basic aspects of stimulus sampling, remain poorly explored. Here, we revisit the classical vasomotor pump hypothesis of vomeronasal stimulus uptake. Using advanced anatomical, histological, and physiological methods, we demonstrate that large parts of the lateral mouse VNO are composed of smooth muscle. Vomeronasal smooth muscle tissue comprises two subsets of fibers with distinct topography, structure, excitation-contraction coupling, and, ultimately, contractile properties. Specifically, contractions of a large population of noradrenaline-sensitive cells mediate both transverse and longitudinal lumen expansion, whereas cholinergic stimulation targets an adluminal group of smooth muscle fibers. The latter run parallel to the VNO's rostro-caudal axis and are ideally situated to mediate antagonistic longitudinal constriction of the lumen. This newly discovered arrangement implies a novel mode of function. Single-cell transcriptomics and pharmacological profiling reveal the receptor subtypes involved. Finally, 2D/3D tomography provides non-invasive insight into the intact VNO's anatomy and mechanics, enables measurement of luminal fluid volume, and allows an assessment of relative volume change upon noradrenergic stimulation. Together, we propose a revised conceptual framework for mouse vomeronasal pumping and, thus, stimulus sampling.

An airway-to-brain sensory pathway mediates influenza-induced sickness.

Pathogen infection causes a stereotyped state of sickness that involves neuronally orchestrated behavioural and physiological changes1,2. On infection, immune cells release a 'storm' of cytokines and other mediators, many of which are detected by neurons3,4; yet, the responding neural circuits and neuro-immune interaction mechanisms that evoke sickness behaviour during naturalistic infections remain unclear. Over-the-counter medications such as aspirin and ibuprofen are widely used to alleviate sickness and act by blocking prostaglandin E2 (PGE2) synthesis5. A leading model is that PGE2 crosses the blood-brain barrier and directly engages hypothalamic neurons2. Here, using genetic tools that broadly cover a peripheral sensory neuron atlas, we instead identified a small population of PGE2-detecting glossopharyngeal sensory neurons (petrosal GABRA1 neurons) that are essential for influenza-induced sickness behaviour in mice. Ablating petrosal GABRA1 neurons or targeted knockout of PGE2 receptor 3 (EP3) in these neurons eliminates influenza-induced decreases in food intake, water intake and mobility during early-stage infection and improves survival. Genetically guided anatomical mapping revealed that petrosal GABRA1 neurons project to mucosal regions of the nasopharynx with increased expression of cyclooxygenase-2 after infection, and also display a specific axonal targeting pattern in the brainstem. Together, these findings reveal a primary airway-to-brain sensory pathway that detects locally produced prostaglandins and mediates systemic sickness responses to respiratory virus infection.

Hunger enhances food-odour attraction through a neuropeptide Y spotlight.

Internal state controls olfaction through poorly understood mechanisms. Odours that represent food, mates, competitors and predators activate parallel neural circuits that may be flexibly shaped by physiological need to alter behavioural outcome1. Here we identify a neuronal mechanism by which hunger selectively promotes attraction to food odours over other olfactory cues. Optogenetic activation of hypothalamic agouti-related peptide (AGRP) neurons enhances attraction to food odours but not to pheromones, and branch-specific activation and inhibition reveal a key role for projections to the paraventricular thalamus. Mice that lack neuropeptide Y (NPY) or NPY receptor type 5 (NPY5R) fail to prefer food odours over pheromones after fasting, and hunger-dependent food-odour attraction is restored by cell-specific NPY rescue in AGRP neurons. Furthermore, acute NPY injection immediately rescues food-odour preference without additional training, indicating that NPY is required for reading olfactory circuits during behavioural expression rather than writing olfactory circuits during odour learning. Together, these findings show that food-odour-responsive neurons comprise an olfactory subcircuit that listens to hunger state through thalamic NPY release, and more generally, provide mechanistic insights into how internal state regulates behaviour.

Contribution of individual olfactory receptors to odor-induced attractive or aversive behavior in mice.

Odorants are recognized by multiple olfactory receptors (ORs) and induce innate behaviors like attraction or aversion via olfactory system in mice. However, a role of an individual OR is unclear. Muscone is recognized by a few ORs including MOR215-1 and MOR214-3, and attracts male mice. Odor preference tests using MOR215-1 knockout mice revealed that MOR215-1 and other OR(s), possibly including MOR214-3, are involved in the attraction. (Z)-5-tetradecen-1-ol (Z5-14:OH) activates ~3 ORs, including Olfr288, and evokes attraction at low levels but aversion at higher levels. Olfr288 knockout mice show no attraction but aversion, suggesting Olfr288 is involved in preference for Z5-14:OH, whereas activation of other low-affinity Z5-14:OH receptors evokes aversion. Each OR appears to send a signal to a neural circuit that possesses distinct valence, leading to a certain behavior. The final output behavior with multiple ORs stimulation is determined by summation (addition or competition) of valences coded by activated ORs.

Exocrine Gland-Secreting Peptide 1 Is a Key Chemosensory Signal Responsible for the Bruce Effect in Mice.

The Bruce effect refers to pregnancy termination in recently pregnant female rodents upon exposure to unfamiliar males [1]. This event occurs in specific combinations of laboratory mouse strains via the vomeronasal system [2, 3]; however, the responsible chemosensory signals have not been fully identified. Here we demonstrate that the male pheromone exocrine gland-secreting peptide 1 (ESP1) is one of the key factors that causes pregnancy block. Female mice exhibited high pregnancy failure rates upon encountering males that secreted different levels of ESP1 compared to the mated male. The effect was not observed in mice that lacked the ESP1 receptor, V2Rp5, which is expressed in vomeronasal sensory neurons. Prolactin surges in the blood after mating, which are essential for maintaining luteal function, were suppressed by ESP1 exposure, suggesting that a neuroendocrine mechanism underlies ESP1-mediated pregnancy failure. The single peptide pheromone ESP1 conveys not only maleness to promote female receptivity but also the males' characteristics to facilitate memorization of the mating partner.

Ligand Specificity and Evolution of Mammalian Musk Odor Receptors: Effect of Single Receptor Deletion on Odor Detection.

Musk odors have been used widely for fragrance and medicine for >2000 years because of their fascinating scent and physiological effects. Therefore, fragrance manufacturers have been eager to develop high-quality musk compounds that are safe and easily synthesized. We recently identified muscone-responsive olfactory receptors (ORs) MOR215-1 and OR5AN1 in mice and humans, respectively (Shirasu et al., 2014). In this study, we identified musk ORs that are evolutionarily closely related to MOR215-1 or OR5AN1 in various primates and investigated structure-activity relationships for various musk odorants and related compounds. We found that each species has one or two functional musk ORs that exhibit specific ligand spectra to musk compounds. Some of them, including the human OR5AN1, responded to nitro musks with chemical properties distinct from muscone. The ligand specificity of OR5AN1 reflects the perception of musk odors in humans. Genetic deletion of MOR215-1 in mice resulted in drastic reduction of sensitivity to muscone, suggesting that MOR215-1 plays a critical role in muscone perception. Therefore, the current study reveals a clear link between the identified OR and muscone perception. Moreover, the strategy established for screening ligands for the muscone OR may facilitate the development of novel and commercially useful musk odors. SIGNIFICANCE STATEMENT: The long-sought musk odor receptor family in mammals was discovered and found to be well conserved and narrowly tuned to musk odors. In mice, deletion of the most sensitive musk receptor resulted in drastic reduction in sensitivity to muscone, demonstrating a strong link between receptor and odor perception. In humans, we found one musk receptor that recognized both macrocyclic and nitro musks that had distinct chemical structures. The structure-activity relationships were in a good agreement with human sensory perception and therefore may be used to develop novel musk aroma in fragrance fields. Finally, identification of a natural ligand(s) for musk receptors in mammals other than musk deer would reveal an evolutionarily pivotal role in each species in the future.

A juvenile mouse pheromone inhibits sexual behaviour through the vomeronasal system.

Animals display a repertoire of different social behaviours. Appropriate behavioural responses depend on sensory input received during social interactions. In mice, social behaviour is driven by pheromones, chemical signals that encode information related to age, sex and physiological state. However, although mice show different social behaviours towards adults, juveniles and neonates, sensory cues that enable specific recognition of juvenile mice are unknown. Here we describe a juvenile pheromone produced by young mice before puberty, termed exocrine-gland secreting peptide 22 (ESP22). ESP22 is secreted from the lacrimal gland and released into tears of 2- to 3-week-old mice. Upon detection, ESP22 activates high-affinity sensory neurons in the vomeronasal organ, and downstream limbic neurons in the medial amygdala. Recombinant ESP22, painted on mice, exerts a powerful inhibitory effect on adult male mating behaviour, which is abolished in knockout mice lacking TRPC2, a key signalling component of the vomeronasal organ. Furthermore, knockout of TRPC2 or loss of ESP22 production results in increased sexual behaviour of adult males towards juveniles, and sexual responses towards ESP22-deficient juveniles are suppressed by ESP22 painting. Thus, we describe a pheromone of sexually immature mice that controls an innate social behaviour, a response pathway through the accessory olfactory system and a new role for vomeronasal organ signalling in inhibiting sexual behaviour towards young. These findings provide a molecular framework for understanding how a sensory system can regulate behaviour.

Angiotensin II modulates salty and sweet taste sensitivities.

Understanding the mechanisms underlying gustatory detection of dietary sodium is important for the prevention and treatment of hypertension. Here, we show that Angiotensin II (AngII), a major mediator of body fluid and sodium homeostasis, modulates salty and sweet taste sensitivities, and that this modulation critically influences ingestive behaviors in mice. Gustatory nerve recording demonstrated that AngII suppressed amiloride-sensitive taste responses to NaCl. Surprisingly, AngII also enhanced nerve responses to sweeteners, but had no effect on responses to KCl, sour, bitter, or umami tastants. These effects of AngII on nerve responses were blocked by the angiotensin II type 1 receptor (AT1) antagonist CV11974. In behavioral tests, CV11974 treatment reduced the stimulated high licking rate to NaCl and sweeteners in water-restricted mice with elevated plasma AngII levels. In taste cells AT1 proteins were coexpressed with αENaC (epithelial sodium channel α-subunit, an amiloride-sensitive salt taste receptor) or T1r3 (a sweet taste receptor component). These results suggest that the taste organ is a peripheral target of AngII. The specific reduction of amiloride-sensitive salt taste sensitivity by AngII may contribute to increased sodium intake. Furthermore, AngII may contribute to increased energy intake by enhancing sweet responses. The linkage between salty and sweet preferences via AngII signaling may optimize sodium and calorie intakes.

Residual chemoresponsiveness to acids in the superior laryngeal nerve in "taste-blind" (P2X2/P2X3 double-KO) mice.

Mice lacking both the P2X2 and the P2X3 purinergic receptors (P2X-dblKO) exhibit loss of responses to all taste qualities in the taste nerves innervating the tongue. Similarly, these mice exhibit a near total loss of taste-related behaviors in brief access tests except for a near-normal avoidance of acidic stimuli. This persistent avoidance of acids despite the loss of gustatory neural responses to sour was postulated to be due to continued responsiveness of the superior laryngeal (SL) nerve. However, chemoresponses of the larynx are attributable both to taste buds and to free nerve endings. In order to test whether the SL nerve of P2X-dblKO mice remains responsive to acids but not to other tastants, we recorded responses from the SL nerve in wild-type (WT) and P2X-dblKO mice. WT mice showed substantial SL responses to monosodium glutamate, sucrose, urea, and denatonium-all of which were essentially absent in P2X-dblKO animals. In contrast, the SL nerve of P2X-dblKO mice exhibited near-normal responses to citric acid (50 mM) although responsiveness of both the chorda tympani and the glossopharyngeal nerves to this stimulus were absent or greatly reduced. These results are consistent with the hypothesis that the residual avoidance of acidic solutions by P2X-dblKO mice may be attributable to the direct chemosensitivity of nerve fibers innervating the laryngeal epithelium and not to taste.

Sour taste responses in mice lacking PKD channels.

BACKGROUND: The polycystic kidney disease-like ion channel PKD2L1 and its associated partner PKD1L3 are potential candidates for sour taste receptors. PKD2L1 is expressed in type III taste cells that respond to sour stimuli and genetic elimination of cells expressing PKD2L1 substantially reduces chorda tympani nerve responses to sour taste stimuli. However, the contribution of PKD2L1 and PKD1L3 to sour taste responses remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: We made mice lacking PKD2L1 and/or PKD1L3 gene and investigated whole nerve responses to taste stimuli in the chorda tympani or the glossopharyngeal nerve and taste responses in type III taste cells. In mice lacking PKD2L1 gene, chorda tympani nerve responses to sour, but not sweet, salty, bitter, and umami tastants were reduced by 25-45% compared with those in wild type mice. In contrast, chorda tympani nerve responses in PKD1L3 knock-out mice and glossopharyngeal nerve responses in single- and double-knock-out mice were similar to those in wild type mice. Sour taste responses of type III fungiform taste cells (GAD67-expressing taste cells) were also reduced by 25-45% by elimination of PKD2L1. CONCLUSIONS/SIGNIFICANCE: These findings suggest that PKD2L1 partly contributes to sour taste responses in mice and that receptors other than PKDs would be involved in sour detection.

New frontiers in gut nutrient sensor research: nutrient sensors in the gastrointestinal tract: modulation of sweet taste sensitivity by leptin.

The ability to perceive sweet compounds is important for animals to detect an external carbohydrate source of calories and has a critical role in the nutritional status of animals. In mice, a subset of sweet-sensitive taste cells possesses leptin receptors. Increase of plasma leptin with increasing internal energy storage in the adipose tissue suppresses sweet taste responses via this receptor. The data from recent studies indicate that leptin may also act as a modulator of sweet taste sensation in humans with a diurnal variation in sweet sensitivity. The plasma leptin level and sweet taste sensitivity are proposed to link with post-ingestive plasma glucose level. This leptin modulation of sweet taste sensitivity may influence an individual's preference, ingestive behavior, and absorption of nutrients, thereby playing important roles in regulation of energy homeostasis.

Endocannabinoids selectively enhance sweet taste.

Endocannabinoids such as anandamide [N-arachidonoylethanolamine (AEA)] and 2-arachidonoyl glycerol (2-AG) are known orexigenic mediators that act via CB(1) receptors in hypothalamus and limbic forebrain to induce appetite and stimulate food intake. Circulating endocannabinoid levels inversely correlate with plasma levels of leptin, an anorexigenic mediator that reduces food intake by acting on hypothalamic receptors. Recently, taste has been found to be a peripheral target of leptin. Leptin selectively suppresses sweet taste responses in wild-type mice but not in leptin receptor-deficient db/db mice. Here, we show that endocannabinoids oppose the action of leptin to act as enhancers of sweet taste. We found that administration of AEA or 2-AG increases gustatory nerve responses to sweeteners in a concentration-dependent manner without affecting responses to salty, sour, bitter, and umami compounds. The cannabinoids increase behavioral responses to sweet-bitter mixtures and electrophysiological responses of taste receptor cells to sweet compounds. Mice genetically lacking CB(1) receptors show no enhancement by endocannnabinoids of sweet taste responses at cellular, nerve, or behavioral levels. In addition, the effects of endocannabinoids on sweet taste responses of taste cells are diminished by AM251, a CB(1) receptor antagonist, but not by AM630, a CB(2) receptor antagonist. Immunohistochemistry shows that CB(1) receptors are expressed in type II taste cells that also express the T1r3 sweet taste receptor component. Taken together, these observations suggest that the taste organ is a peripheral target of endocannabinoids. Reciprocal regulation of peripheral sweet taste reception by endocannabinoids and leptin may contribute to their opposing actions on food intake and play an important role in regulating energy homeostasis.

Multiple receptor systems for umami taste in mice.

Recent molecular studies proposed that the T1r1/T1r3 heterodimer, mGluR1 and mGluR4 might function as umami taste receptors in mice. However, the roles of each of these receptors in umami taste are not yet clear. In this paper, we summarize recent data for T1r3, mGluR1, and mGluR4 as umami taste receptors and discuss receptor systems responsible for umami detection in mice.

Modulation and transmission of sweet taste information for energy homeostasis.

Perception of sweet taste is important for animals to detect external energy source of calories. In mice, sweet-sensitive cells possess a leptin receptor. Increase of plasma leptin with increasing internal energy storage in the adipose tissue suppresses sweet taste responses via this receptor. Data from our recent studies indicate that leptin may also modulate sweet taste sensation in humans with a diurnal variation in sweet sensitivity. This leptin modulation of sweet taste information to the brain may influence individuals' preference and ingestive behavior, thereby playing important roles in regulation of energy homeostasis.

Multiple receptors underlie glutamate taste responses in mice.

l-Glutamate is known to elicit a unique taste, umami, that is distinct from the tastes of sweet, salty, sour, and bitter. Recent molecular studies have identified several candidate receptors for umami in taste cells, such as the heterodimer T1R1/T1R3 and brain-expressed and taste-expressed type 1 and 4 metabotropic glutamate receptors (brain-mGluR1, brain-mGluR4, taste-mGluR1, and taste-mGluR4). However, the relative contributions of these receptors to umami taste reception remain to be elucidated. We critically discuss data from recent studies in which mouse taste cell, nerve fiber, and behavioral responses to umami stimuli were measured to evaluate whether receptors other than T1R1/T1R3 are involved in umami responses. We particularly emphasized studies of umami responses in T1R3 knockout (KO) mice and studies of potential effects of mGluR antagonists on taste responses. The results of these studies indicate the existence of substantial residual responses to umami compounds in the T1R3-KO model and a significant reduction of umami responsiveness after administration of mGluR antagonists. These findings thus provide evidence of the involvement of mGluRs in addition to T1R1/T1R3 in umami detection in mice and suggest that umami responses, at least in mice, may be mediated by multiple receptors.

Multiple sweet receptors and transduction pathways revealed in knockout mice by temperature dependence and gurmarin sensitivity.

Sweet taste transduction involves taste receptor type 1, member 2 (T1R2), taste receptor type 1, member 3 (T1R3), gustducin, and TRPM5. Because knockout (KO) mice lacking T1R3, gustducin's Galpha subunit (Galphagust), or TRPM5 exhibited greatly reduced, but not abolished responses of the chorda tympani (CT) nerve to sweet compounds, it is likely that multiple sweet transduction pathways exist. That gurmarin (Gur), a sweet taste inhibitor, inhibits some but not all mouse CT responses to sweet compounds supports the existence of multiple sweet pathways. Here, we investigated Gur inhibition of CT responses to sweet compounds as a function of temperature in KO mice lacking T1R3, Galphagust, or TRPM5. In T1R3-KO mice, responses to sucrose and glucose were Gur sensitive (GS) and displayed a temperature-dependent increase (TDI). In Galphagust-KO mice, responses to sucrose and glucose were Gur-insensitive (GI) and showed a TDI. In TRPM5-KO mice, responses to glucose were GS and showed a TDI. All three KO mice exhibited no detectable responses to SC45647, and their responses to saccharin displayed neither GS nor a TDI. For all three KO mice, the lingual application of pronase, another sweet response inhibitor, almost fully abolished responses to sucrose and glucose but did not affect responses to saccharin. These results provide evidence for 1) the existence of multiple transduction pathways underlying responses to sugars: a T1R3-independent GS pathway for sucrose and glucose, and a TRPM5-independent temperature sensitive GS pathway for glucose; 2) the requirement for Galphagust in GS sweet taste responses; and 3) the existence of a sweet independent pathway for saccharin, in mouse taste cells on the anterior tongue.