Bicarbonate signalling via G protein-coupled receptor regulates ischaemia-reperfusion injury.

Homoeostatic regulation of the acid-base balance is essential for cellular functional integrity. However, little is known about the molecular mechanism through which the acid-base balance regulates cellular responses. Here, we report that bicarbonate ions activate a G protein-coupled receptor (GPCR), i.e., GPR30, which leads to Gq-coupled calcium responses. Gpr30-Venus knock-in mice reveal predominant expression of GPR30 in brain mural cells. Primary culture and fresh isolation of brain mural cells demonstrate bicarbonate-induced, GPR30-dependent calcium responses. GPR30-deficient male mice are protected against ischemia-reperfusion injury by a rapid blood flow recovery. Collectively, we identify a bicarbonate-sensing GPCR in brain mural cells that regulates blood flow and ischemia-reperfusion injury. Our results provide a perspective on the modulation of GPR30 signalling in the development of innovative therapies for ischaemic stroke. Moreover, our findings provide perspectives on acid/base sensing GPCRs, concomitantly modulating cellular responses depending on fluctuating ion concentrations under the acid-base homoeostasis.

Hemoglobin in the blood acts as a chemosensory signal via the mouse vomeronasal system.

The vomeronasal system plays an essential role in sensing various environmental chemical cues. Here we show that mice exposed to blood and, consequently, hemoglobin results in the activation of vomeronasal sensory neurons expressing a specific vomeronasal G protein-coupled receptor, Vmn2r88, which is mediated by the interaction site, Gly17, on hemoglobin. The hemoglobin signal reaches the medial amygdala (MeA) in both male and female mice. However, it activates the dorsal part of ventromedial hypothalamus (VMHd) only in lactating female mice. As a result, in lactating mothers, hemoglobin enhances digging and rearing behavior. Manipulation of steroidogenic factor 1 (SF1)-expressing neurons in the VMHd is sufficient to induce the hemoglobin-mediated behaviors. Our results suggest that the oxygen-carrier hemoglobin plays a role as a chemosensory signal, eliciting behavioral responses in mice in a state-dependent fashion.

Calcitonin receptor signaling in the medial preoptic area enables risk-taking maternal care.

Maternal mammals exhibit heightened motivation to care for offspring, but the underlying neuromolecular mechanisms have yet to be clarified. Here, we report that the calcitonin receptor (Calcr) and its ligand amylin are expressed in distinct neuronal populations in the medial preoptic area (MPOA) and are upregulated in mothers. Calcr+ MPOA neurons activated by parental care project to somatomotor and monoaminergic brainstem nuclei. Retrograde monosynaptic tracing reveals that significant modification of afferents to Calcr+ neurons occurs in mothers. Knockdown of either Calcr or amylin gene expression hampers risk-taking maternal care, and specific silencing of Calcr+ MPOA neurons inhibits nurturing behaviors, while pharmacogenetic activation prevents infanticide in virgin males. These data indicate that Calcr+ MPOA neurons are required for both maternal and allomaternal nurturing behaviors and that upregulation of amylin-Calcr signaling in the MPOA at least partially mediates risk-taking maternal care, possibly via modified connectomics of Calcr+ neurons postpartum.

Transcriptional regulators involved in responses to volatile organic compounds in plants.

Field studies have shown that plants growing next to herbivore-infested plants acquire higher resistance to herbivore damage. This increased resistance is partly due to regulation of plant gene expression by volatile organic compounds (VOCs) released by plants that sense environmental challenges such as herbivores. The molecular basis for VOC sensing in plants, however, is poorly understood. Here, we report the identification of TOPLESS-like proteins (TPLs) that have VOC-binding activity and are involved in VOC sensing in tobacco. While screening for volatiles that induce stress-responsive gene expression in tobacco BY-2 cells and tobacco plants, we found that some sesquiterpenes induce the expression of stress-responsive genes. These results provided evidence that plants sense these VOCs and motivated us to analyze the mechanisms underlying volatile sensing using tobacco as a model system. Using a pulldown assay with caryophyllene derivative-linked beads, we identified TPLs as transcriptional co-repressors that bind volatile caryophyllene analogs. Overexpression of TPLs in cultured BY-2 cells or tobacco leaves reduced caryophyllene-induced gene expression, indicating that TPLs are involved in the responses to caryophyllene analogs in tobacco. We propose that unlike animals, which use membrane receptors for sensing odorants, a transcriptional co-repressor plays a role in sensing and mediating VOC signals in plant cells.

Sexual rejection via a vomeronasal receptor-triggered limbic circuit.

Mating drive is balanced by a need to safeguard resources for offspring, yet the neural basis for negative regulation of mating remains poorly understood. In rodents, pheromones critically regulate sexual behavior. Here, we observe suppression of adult female sexual behavior in mice by exocrine gland-secreting peptide 22 (ESP22), a lacrimal protein from juvenile mice. ESP22 activates a dedicated vomeronasal receptor, V2Rp4, and V2Rp4 knockout eliminates ESP22 effects on sexual behavior. Genetic tracing of ESP22-responsive neural circuits reveals a critical limbic system connection that inhibits reproductive behavior. Furthermore, V2Rp4 counteracts a highly related vomeronasal receptor, V2Rp5, that detects the male sex pheromone ESP1. Interestingly, V2Rp4 and V2Rp5 are encoded by adjacent genes, yet couple to distinct circuits and mediate opposing effects on female sexual behavior. Collectively, our study reveals molecular and neural mechanisms underlying pheromone-mediated sexual rejection, and more generally, how inputs are routed through olfactory circuits to evoke specific behaviors.

G Protein-Coupled Receptor Kinase 3 (GRK3) in Olfaction.

Like in other sensory systems, adaptation is an essential process in the olfactory system, required for its proper functioning. However, the precise molecular mechanism underlying the adaptation process has not been fully understood, especially at the receptor level. Here, we describe methods to evaluate the role of GRK3, one of the members of the GRK family responsible for the desensitization of non-olfactory G-protein-coupled receptor (GPCR), in desensitization of olfactory receptor (OR) using a heterologous expression system. As a parameter to characterize the degree of desensitization, we measure (1) the maximal response to an agonist by either cAMP or Ca2+ imaging assay and (2) the kinetic time course for recovery to basal levels by Ca2+ imaging assay. Differences in the degree of desensitization in the presence or absence of GRK3 can be examined by comparing these parameters, leading to evaluation of GRK3.

Identification of an Intra- and Inter-specific Tear Protein Signal in Rodents.

Rodents use the vomeronasal olfactory system to acquire both inter- and intra-specific information from the external environment and take appropriate actions. For example, urinary proteins from predator species elicit avoidance in mice, while those from male mice attract female mice. In addition to urinary proteins, recent studies have highlighted the importance of lacrimal proteins for intra-specific communications in mice. However, whether the tear fluid of other species also mediates social signals remains unknown. Here, we show that a lacrimal protein in rats (predators of mice), called cystatin-related protein 1 (ratCRP1), activates the vomeronasal system of mice. This protein is specifically produced by adult male rats in a steroid hormone-dependent manner, activates the vomeronasal system of female rats, and enhances stopping behavior. When detected by mice, ratCRP1 activates the medial hypothalamic defensive circuit, resulting in decreased locomotion coupled with lowered body temperature and heart rate. Notably, ratCRP1 is recognized by multiple murine type 2 vomeronasal receptors, including Vmn2r28. CRISPR/Cas9-mediated deletion of vmn2r28 impaired both ratCRP1-induced neural activation of the hypothalamic center and decrease of locomotor activity in mice. Taken together, these data reveal the neural and molecular basis by which a tear fluid compound in rats affects the behavior of mice. Furthermore, our study reveals a case in which a single compound that mediates an intra-specific signal in a predator species also functions as an inter-specific signal in the prey species.

Male mice ultrasonic vocalizations enhance female sexual approach and hypothalamic kisspeptin neuron activity.

Vocal communication in animals is important for ensuring reproductive success. Male mice emit song-like "ultrasonic vocalizations (USVs)" when they encounter female mice, and females show approach to the USVs. However, it is unclear whether USVs of male mice trigger female behavioral and endocrine responses in reproduction. In this study, we first investigated the relationship between the number of deliveries in breeding pairs for 4months and USVs syllables emitted from those paired males during 3min of sexual encounter with unfamiliar female mice. There was a positive correlation between these two indices, which suggests that breeding pairs in which males could emit USVs more frequently had more offspring. Further, we examined the effect of USVs of male mice on female sexual behavior. Female mice showed more approach behavior towards vocalizing males than devocalized males. Finally, to determine whether USVs of male mice could activate the neural system governing reproductive function in female mice, the activation of kisspeptin neurons, key neurons to drive gonadotropin-releasing hormone neurons in the hypothalamus, was examined using dual-label immunocytochemistry with cAMP response element-binding protein phosphorylation (pCREB). In the arcuate nucleus (Arc), the number of kisspeptin neurons expressing pCREB significantly increased after exposure to USVs of male as compared with noise exposure group. In conclusion, our results suggest that USVs of male mice promote fertility in female mice by activating both their approaching behavior and central kisspeptin neurons.

Identification and characterization of the bombykal receptor in the hawkmoth Manduca sexta.

Manduca sexta females attract their mates with the release of a species-specific sex-pheromone blend, with bombykal (E,Z)-10,12-hexadecadienal and (E,E,Z)-10,12,14-hexadecatrienal being the two major components. Here, we searched for the hawkmoth bombykal receptor in heterologous expression systems. The putative pheromone receptor MsexOr1 coexpressed with MsexOrco in Xenopus oocytes elicited dose-dependent inward currents upon bombykal application (10-300 µmol l-1), and coexpressed in HEK293 and CHO cells caused bombykal-dependent increases in the intracellular free Ca2+ concentration. In addition, the bombykal receptor of Bombyx mori BmOr3 coexpressed with MsexOrco responded to bombykal (30-100 µmol l-1) with inward currents. In contrast, MsexOr4 coexpressed with MsexOrco responded neither to bombykal (30-100 µmol l-1) nor to the (E,E,Z)-10,12,14-hexadecatrienal mimic. Thus, MsexOr1, but not MsexOrco and probably not MsexOr4, is the bombykal-binding pheromone receptor in the hawkmoth. Finally, we obtained evidence that phospholipase C and protein kinase C activity are involved in the hawkmoth's bombykal-receptor-mediated Ca2+ signals in HEK293 and CHO cells.

Self-Exposure to the Male Pheromone ESP1 Enhances Male Aggressiveness in Mice.

Exocrine gland-secreting peptide 1 (ESP1) released into male tear fluids is a male pheromone that stimulates sexually receptive behavior in female mice via the vomeronasal sensory system. ESP1 also induces c-Fos expression in male brain regions distinct from those in females. However, behavior in males following ESP1 exposure has not been examined. In the present study, we show that ESP1, in conjunction with unfamiliar male urine, enhances male aggression via the specific vomeronasal receptor V2Rp5. In addition, male mice that secrete ESP1 but lack V2Rp5 exhibit a lower level of aggressiveness than do mice that express V2Rp5. These results suggest that ESP1 not only acts as a male pheromone in both sexes but also serves as an auto-stimulatory factor that enhances male aggressiveness by self-exposure. Finally, re-activation of ESP1-induced c-Fos-positive neurons by using the designer receptor exclusively activated by designer drug (DREADD) approach resulted in enhancement of sexual and aggressive behaviors in female and male mice, respectively, indicating that sexually dimorphic activation in the brain is a neural basis for the sex-specific behavioral responses to ESP1.

Structure and function of a peptide pheromone family that stimulate the vomeronasal sensory system in mice.

Mammals use pheromones to communicate with other animals of the same species. In mice, the VNO (vomeronasal organ) has a pivotal role in pheromone detection. We discovered a 7 kDa peptide, ESP1 (exocrine-gland-secreting peptide 1), in tear fluids from male mice that enhances the sexual behaviour of female mice via the VNO. NMR studies demonstrate that ESP1 adopts a compact structure with a helical fold stabilized by an intramolecular disulfide bridge. Functional analysis in combination with docking simulation indicates that ESP1 is recognized by a specific G-protein-coupled vomeronasal receptor, V2Rp5, via charge-charge interactions in the large extracellular region of the receptor. ESP1 is a member of the ESP family, which comprises 38 homologous genes in mice, and some of these genes are expressed in a sex- or age-dependent manner. Most recently, ESP22 was found to be released specifically in juvenile tear fluids and to inhibit the sexual behaviour of adult male mice. These studies demonstrate that peptide pheromones are used for chemical communication in mice, and they indicate a structural basis for the narrowly tuned perception of mammalian peptide pheromones by vomeronasal receptors.

Olfactory receptor and neural pathway responsible for highly selective sensing of musk odors.

Musk odorants are used widely in cosmetic industries because of their fascinating animalic scent. However, how this aroma is perceived in the mammalian olfactory system remains a great mystery. Here, we show that muscone, one musk odor secreted by various animals from stink glands, activates a few glomeruli clustered in a neuroanatomically unique anteromedial olfactory bulb. The muscone-responsive glomeruli are highly specific to macrocyclic ketones; interestingly, other synthetic musk odorants with nitro or polycyclic moieties or ester bonds activate distinct but nearby glomeruli. Anterodorsal bulbar lesions cause muscone anosmia, suggesting that this region is involved in muscone perception. Finally, we identified the mouse olfactory receptor, MOR215-1, that was a specific muscone receptor expressed by neurons innervating the muscone-responsive anteromedial glomeruli and also the human muscone receptor, OR5AN1. The current study documents the olfactory neural pathway in mice that senses and transmits musk signals from receptor to brain.

Backbone and side-chain ¹H, ¹5N and ¹³C assignments of mouse peptide ESP4.

A peptide or a small protein released from an exocrine gland or in urine is utilized as a chemosignal that elicits social or reproductive behavior in mice. Recently, we identified the male-specific peptide, exocrine gland-secreting peptide 1 (ESP1), in mouse tear fluids that enhanced female sexual receptive behavior, and determined the three dimensional structure. ESP1 appears to be a member of multigene family that consists of 38 genes in mice, which we call the ESP family. ESP4, a member of the ESP family, is expressed in various exocrine glands, and shows the highest sequence similarity with ESP1. Here, we report the NMR assignments of ESP4 which provides a basis for NMR analyses of this protein. Our results will give insight into structural relationships within the ESP family.

Pheromone-induced expression of immediate early genes in the mouse vomeronasal sensory system.

Immediate early genes (IEGs) are powerful tools for visualizing activated neurons and extended circuits that are stimulated by sensory input. Several kinds of IEGs (e.g., c-fos, egr-1) have been utilized for detecting activated receptor neurons in the pheromone sensory organ called the vomeronasal organ (VNO), as well as for mapping the neurons within the central nervous system (CNS) excited by pheromones.In this chapter, we describe the procedure for the detection of pheromone-induced neural activation in the VNO and CNS using the c-Fos immunostaining technique.

Goofy coordinates the acuity of olfactory signaling.

The basic scheme of odor perception and signaling from olfactory cilia to the brain is well understood. However, factors that affect olfactory acuity of an animal, the threshold sensitivity to odorants, are less well studied. Using signal sequence trap screening of a mouse olfactory epithelium cDNA library, we identified a novel molecule, Goofy, that is essential for olfactory acuity in mice. Goofy encodes an integral membrane protein with specific expression in the olfactory and vomeronasal sensory neurons and predominant localization to the Golgi compartment. Goofy-deficient mice display aberrant olfactory phenotypes, including the impaired trafficking of adenylyl cyclase III, stunted olfactory cilia, and a higher threshold for physiological and behavioral responses to odorants. In addition, the expression of dominant-negative form of cAMP-dependent protein kinase results in shortening of olfactory cilia, implying a possible mechanistic link between cAMP and ciliogenesis in the olfactory sensory neurons. These results demonstrate that Goofy plays an important role in establishing the acuity of olfactory sensory signaling.

The scent of disease: volatile organic compounds of the human body related to disease and disorder.

Hundreds of volatile organic compounds (VOCs) are emitted from the human body, and the components of VOCs usually reflect the metabolic condition of an individual. Therefore, contracting an infectious or metabolic disease often results in a change in body odour. Recent progresses in analytical techniques allow rapid analyses of VOCs derived from breath, blood, skin and urine. Disease-specific VOCs can be used as diagnostic olfactory biomarkers of infectious diseases, metabolic diseases, genetic disorders and other kinds of diseases. Elucidation of pathophysiological mechanisms underlying production of disease-specific VOCs may provide novel insights into therapeutic approaches for treatments for various diseases. This review summarizes the current knowledge on chemical and clinical aspects of body-derived VOCs, and provides a brief outlook at the future of olfactory diagnosis.

Chemical identity of a rotting animal-like odor emitted from the inflorescence of the titan arum (Amorphophallus titanum).

The titan arum, Amorphophallus titanum, is a flowering plant with the largest inflorescence in the world. The flower emits a unique rotting animal-like odor that attracts insects for pollination. To determine the chemical identity of this characteristic odor, we performed gas chromatography-mass spectrometry-olfactometry analysis of volatiles derived from the inflorescence. The main odorant causing the smell during the flower-opening phase was identified as dimethyl trisulfide, a compound with a sulfury odor that has been found to be emitted from some vegetables, microorganisms, and cancerous wounds.

The male mouse pheromone ESP1 enhances female sexual receptive behaviour through a specific vomeronasal receptor.

Various social behaviours in mice are regulated by chemical signals called pheromones that act through the vomeronasal system. Exocrine gland-secreting peptide 1 (ESP1) is a 7-kDa peptide that is released into male tear fluids and stimulates vomeronasal sensory neurons in female mice. Here, we describe the molecular and neural mechanisms that are involved in the decoding of ESP1 signals in the vomeronasal system, which leads to behavioural output in female mice. ESP1 is recognized by a specific vomeronasal receptor, V2Rp5, and the ligand-receptor interaction results in sex-specific signal transmission to the amygdaloid and hypothalamic nuclei via the accessory olfactory bulb. Consequently, ESP1 enhances female sexual receptive behaviour upon male mounting (lordosis), allowing successful copulation. In V2Rp5-deficient mice, ESP1 induces neither neural activation nor sexual behaviour. These findings show that ESP1 is a crucial male pheromone that regulates female reproductive behaviour through a specific receptor in the mouse vomeronasal system.

Two chemoreceptors mediate developmental effects of dauer pheromone in C. elegans.

Intraspecific chemical communication is mediated by signals called pheromones. Caenorhabditis elegans secretes a mixture of small molecules (collectively termed dauer pheromone) that regulates entry into the alternate dauer larval stage and also modulates adult behavior via as yet unknown receptors. Here, we identify two heterotrimeric GTP-binding protein (G protein)-coupled receptors (GPCRs) that mediate dauer formation in response to a subset of dauer pheromone components. The SRBC-64 and SRBC-66 GPCRs are members of the large Caenorhabditis-specific SRBC subfamily and are expressed in the ASK chemosensory neurons, which are required for pheromone-induced dauer formation. Expression of both, but not each receptor alone, confers pheromone-mediated effects on heterologous cells. Identification of dauer pheromone receptors will allow a better understanding of the signaling cascades that transduce the context-dependent effects of ecologically important chemical signals.

Dimethyl trisulfide as a characteristic odor associated with fungating cancer wounds.

Some advanced cancer patients suffer from pungent sulfury malodor. To determine the chemical identity of the odorant, we performed gas chromatography-mass spectrometry-olfactometry analysis of volatiles from fungating cancer wounds. We identified the source of the characteristic smell as dimethyl trisulfide, a compound that is known to be emitted from some vegetables and microorganisms. Controlling the production of dimethyl trisulfide should improve quality of life of patients.