Semantic context-dependent neural representations of odors in the human piriform cortex revealed by 7T MRI.

Olfactory perception depends not only on olfactory inputs but also on semantic context. Although multi-voxel activity patterns of the piriform cortex, a part of the primary olfactory cortex, have been shown to represent odor perception, it remains unclear whether semantic contexts modulate odor representation in this region. Here, we investigated whether multi-voxel activity patterns in the piriform cortex change when semantic context modulates odor perception and, if so, whether the modulated areas communicate with brain regions involved in semantic and memory processing beyond the piriform cortex. We also explored regional differences within the piriform cortex, which are influenced by olfactory input and semantic context. We used 2 × 2 combinations of word labels and odorants that were perceived as congruent and measured piriform activity with a 1-mm isotropic resolution using 7T MRI. We found that identical odorants labeled with different words were perceived differently. This labeling effect was observed in multi-voxel activity patterns in the piriform cortex, as the searchlight decoding analysis distinguished identical odors with different labels for half of the examined stimulus pairs. Significant functional connectivity was observed between parts of the piriform cortex that were modulated by labels and regions associated with semantic and memory processing. While the piriform multi-voxel patterns evoked by different olfactory inputs were also distinguishable, the decoding accuracy was significant for only one stimulus pair, preventing definitive conclusions regarding the locational differences between areas influenced by word labels and olfactory inputs. These results suggest that multi-voxel patterns of piriform activity can be modulated by semantic context, possibly due to communication between the piriform cortex and the semantic and memory regions.

Synchronized Expansion and Contraction of Olfactory, Vomeronasal, and Taste Receptor Gene Families in Hystricomorph Rodents.

Chemical senses, including olfaction, pheromones, and taste, are crucial for the survival of most animals. There has long been a debate about whether different types of senses might influence each other. For instance, primates with a strong sense of vision are thought to have weakened olfactory abilities, although the oversimplified trade-off theory is now being questioned. It is uncertain whether such interactions between different chemical senses occur during evolution. To address this question, we examined four receptor gene families related to olfaction, pheromones, and taste: olfactory receptor (OR), vomeronasal receptor type 1 and type 2 (V1R and V2R), and bitter taste receptor (T2R) genes in Hystricomorpha, which is morphologically and ecologically the most diverse group of rodents. We also sequenced and assembled the genome of the grasscutter, Thryonomys swinderianus. By examining 16 available genome assemblies alongside the grasscutter genome, we identified orthologous gene groups among hystricomorph rodents for these gene families to separate the gene gain and loss events in each phylogenetic branch of the Hystricomorpha evolutionary tree. Our analysis revealed that the expansion or contraction of the four gene families occurred synchronously, indicating that when one chemical sense develops or deteriorates, the others follow suit. The results also showed that V1R/V2R genes underwent the fastest evolution, followed by OR genes, and T2R genes were the most evolutionarily stable. This variation likely reflects the difference in ligands of V1R/V2Rs, ORs, and T2Rs: species-specific pheromones, environment-based scents, and toxic substances common to many animals, respectively.

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.

Identification of olfactory alarm substances in zebrafish.

Escaping from danger is one of the most fundamental survival behaviors for animals. Most freshwater fishes display olfactory alarm reactions in which an injured fish releases putative alarm substances from the skin to notify its shoaling company about the presence of danger. Here, we identified two small compounds in zebrafish skin extract, designated as ostariopterin and daniol sulfate. Ostariopterin is a pterin derivative commonly produced in many freshwater fishes belonging to the Ostariophysi superorder. Daniol sulfate is a novel sulfated bile alcohol specifically present in the Danio species, including zebrafish. Ostariopterin and daniol sulfate activate distinct glomeruli in the olfactory bulb. Zebrafish display robust alarm reactions, composed of darting, freezing, and bottom dwelling, only when they are concomitantly stimulated with ostariopterin and daniol sulfate. These results demonstrate that the fish alarm reaction is driven through a coincidence detection mechanism of the two compounds along the olfactory neural circuitry.

Axonal mRNA binding of hnRNP A/B is crucial for axon targeting and maturation of olfactory sensory neurons.

Spatiotemporal control of gene expression is important for neural development and function. Here, we show that heterogeneous nuclear ribonucleoprotein (hnRNP) A/B is highly expressed in developing olfactory sensory neurons (OSNs), and its knockout results in reduction in mature OSNs and aberrant targeting of OSN axons to the olfactory bulb. RNA immunoprecipitation analysis reveals that hnRNP A/B binds to a group of mRNAs that are highly related to axon projections and synapse assembly. Approximately 11% of the identified hnRNP A/B targets, including Pcdha and Ncam2, encode cell adhesion molecules. In Hnrnpab knockout mice, PCDHA and NCAM2 levels are significantly reduced at the axon terminals of OSNs. Furthermore, deletion of the hnRNP A/B-recognition motif in the 3' UTR of Pcdha leads to impaired PCDHA expression at the OSN axon terminals. Therefore, we propose that hnRNP A/B facilitates OSN maturation and axon projection by regulating the local expression of its target genes at axon terminals.

Role of a single odorant receptor in the chemotaxis behavior of Bombyx mori.

Silkworm (Bombyx mori), an insect herbivore, is attracted to cis-jasmone released from mulberry leaves. Its olfactory receptor, BmOr56, specifically responds to cis-jasmone. In this study, we constructed a BmOr56 deletion line and found that the attractive behavior of cis-jasmone was completely lost in the mutant, suggesting the involvement of a single receptor in this specific chemoattractive behavior.

Genetic variation in the human olfactory receptor OR5AN1 associates with the perception of musks.

Humans have significant individual variations in odor perception, derived from their experience or sometimes from differences in the olfactory receptor (OR) gene repertoire. In several cases, the genetic variation of a single OR affects the perception of its cognate odor ligand. Musks are widely used for fragrance and are known to demonstrate specific anosmia. It, however, remains to be elucidated whether the OR polymorphism contributes to individual variations in musk odor perception. Previous studies reported that responses of the human musk receptor OR5AN1 to a variety of musks in vitro correlated well with perceptual sensitivity to those odors in humans and that the mouse ortholog, Olfr1440 (MOR215-1), plays a critical role in muscone perception. Here, we took advantage of genetic variation in OR5AN1 to examine how changes in receptor sensitivity are associated with human musk perception. We investigated the functional differences between OR5AN1 variants in an in vitro assay and measured both perceived intensity and detection threshold in human subjects with different OR5AN1 genotypes. Human subjects homozygous for the more sensitive L289F allele had a lower detection threshold for muscone and found macrocyclic musks to be more intense than subjects homozygous for the reference allele. These results demonstrate that the genetic variation in OR5AN1 contributes to perceptual differences for some musks. In addition, we found that the more functional variant of OR5A1, a receptor involved in ß-ionone perception, is associated with the less functional variant of OR5AN1, suggesting that the perceived intensities of macrocyclic musks and ß-ionone are inversely correlated.

Structural model for ligand binding and channel opening of an insect gustatory receptor.

Insect gustatory receptors play roles in sensing tastants, such as sugars and bitter substances. We previously demonstrated that the BmGr9 silkworm gustatory receptor is a d-fructose-gated ion channel receptor. However, the molecular mechanism of how d-fructose could initiate channel opening were unclear. Herein, we present a structural model for a channel pore and a d-fructose-binding site in BmGr9. Since the membrane topology and oligomeric state of BmGr9 appeared to be similar to those of an insect odorant receptor coreceptor, Orco, we constructed a structural model of BmGr9 based on the cryo-EM Orco structure. Our site-directed mutagenesis data suggested that the transmembrane region 7 forms channel pore and controls channel gating. This model also suggested that a pocket formed by transmembrane helices 2 to 4 and 6 binds d-fructose. Using mutagenesis experiments in combination with docking simulations, we were able to determine the potent binding mode of d-fructose. Finally, based on these data, we propose a conformational change that leads to channel opening upon d-fructose binding. Taken together, these findings detail the molecular mechanism by which an insect gustatory receptor can be activated by its ligand molecule.

Prelimbic cortex responds to male ultrasonic vocalizations in the presence of a male pheromone in female mice.

Sensory signals are critical to perform adaptive social behavior. During copulation, male mice emit ultrasonic vocalizations (USVs). Our previous studies have shown that female mice exhibit approach behavior toward sound sources of male USVs and that, after being exposed to a male pheromone, exocrine gland-secreting peptide 1 (ESP1), female mice exhibited a preference toward a particular type of male USVs. These findings suggest that male USVs modulate female courtship behavior. However, it remains unclear which brain regions and what cell types of neurons are involved in neuronal processing of male USVs. To clarify this issue, immediate early gene analysis, behavioral analysis, and neurochemical analysis were performed. The in situ hybridization analysis of c-fos mRNA in multiple brain regions showed that neurons in the prelimbic cortex were responsive to presentation of male USVs in the presence of ESP1. Furthermore, this study found that activity of prelimbic cortex was correlated with the duration of female exploration behavior toward a sound source of the USVs. Finally, by using double immunohistochemistry, the present study showed that the prelimbic neurons responding to the presentation of male USVs were presumably excitatory glutamatergic neurons. These results suggest that the prelimbic cortex may facilitate female courtship behavior in response to male USVs.

A single vomeronasal receptor promotes intermale aggression through dedicated hypothalamic neurons.

The pheromonal information received by the vomeronasal system plays a crucial role in regulating social behaviors such as aggression in mice. Despite accumulating knowledge of the brain regions involved in aggression, the specific vomeronasal receptors and the exact neural circuits responsible for pheromone-mediated aggression remain unknown. Here, we identified one murine vomeronasal receptor, Vmn2r53, that is activated by urine from males of various strains and is responsible for evoking intermale aggression. We prepared a purified pheromonal fraction and Vmn2r53 knockout mice and applied genetic tools for neuronal activity recording, manipulation, and circuit tracing to decipher the neural mechanisms underlying Vmn2r53-mediated aggression. We found that Vmn2r53-mediated aggression is regulated by specific neuronal populations in the ventral premammillary nucleus and the ventromedial hypothalamic nucleus. Together, our results shed light on the hypothalamic regulation of male aggression mediated by a single vomeronasal receptor.

An olfactory self-test effectively screens for COVID-19.

Background: Key to curtailing the COVID-19 pandemic are wide-scale screening strategies. An ideal screen is one that would not rely on transporting, distributing, and collecting physical specimens. Given the olfactory impairment associated with COVID-19, we developed a perceptual measure of olfaction that relies on smelling household odorants and rating them online. Methods: Each participant was instructed to select 5 household items, and rate their perceived odor pleasantness and intensity using an online visual analogue scale. We used this data to assign an olfactory perceptual fingerprint, a value that reflects the perceived difference between odorants. We tested the performance of this real-time tool in a total of 13,484 participants (462 COVID-19 positive) from 134 countries who provided 178,820 perceptual ratings of 60 different household odorants. Results: We observe that olfactory ratings are indicative of COVID-19 status in a country, significantly correlating with national infection rates over time. More importantly, we observe indicative power at the individual level (79% sensitivity and 87% specificity). Critically, this olfactory screen remains effective in participants with COVID-19 but without symptoms, and in participants with symptoms but without COVID-19. Conclusions: The current odorant-based olfactory screen adds a component to online symptom-checkers, to potentially provide an added first line of defense that can help fight disease progression at the population level. The data derived from this tool may allow better understanding of the link between COVID-19 and olfaction.

Spatiotemporal dynamics of odor representations in the human brain revealed by EEG decoding.

How the human brain translates olfactory inputs into diverse perceptions, from pleasurable floral smells to sickening smells of decay, is one of the fundamental questions in olfaction. To examine how different aspects of olfactory perception emerge in space and time in the human brain, we performed time-resolved multivariate pattern analysis of scalp-recorded electroencephalogram responses to 10 perceptually diverse odors and associated the resulting decoding accuracies with perception and source activities. Mean decoding accuracies of odors exceeded the chance level 100 ms after odor onset and reached maxima at 350 ms. The result suggests that the neural representations of individual odors were maximally separated at 350 ms. Perceptual representations emerged following the decoding peak: unipolar unpleasantness (neutral to unpleasant) from 300 ms, and pleasantness (neutral to pleasant) and perceptual quality (applicability to verbal descriptors such as "fruity" or "flowery") from 500 ms after odor onset, with all these perceptual representations reaching their maxima after 600 ms. A source estimation showed that the areas representing the odor information, estimated based on the decoding accuracies, were localized in and around the primary and secondary olfactory areas at 100 to 350 ms after odor onset. Odor representations then expanded into larger areas associated with emotional, semantic, and memory processing, with the activities of these later areas being significantly associated with perception. These results suggest that initial odor information coded in the olfactory areas (<350 ms) evolves into their perceptual realizations (300 to >600 ms) through computations in widely distributed cortical regions, with different perceptual aspects having different spatiotemporal dynamics.

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.

Odorant metabolism of the olfactory cleft mucus in idiopathic olfactory impairment patients and healthy volunteers.

BACKGROUND: It remains unclear whether the metabolic activity of nasal mucus in the olfactory and respiratory areas is different. Moreover, age- and olfaction-related changes may affect metabolism. METHODS: Hexanal, octanal, and 2-methylbutanal were selected for in vitro metabolism analysis and compared between the olfactory cleft and respiratory mucus of participants < 50-year-old with normal olfaction using gas chromatography mass spectrometry. The metabolic activity of hexanal in the olfactory cleft mucus was further compared between three groups, (1) normal olfaction, age < 50 years old, (2) normal olfaction, age ≥50 years old, and (3) idiopathic olfactory impairment. To characterize the enzyme(s) responsible for aldehyde reduction, we also tested if epalr22897estat and 3,5-dichlorosalicylic acid, types of reductase inhibitors, affect metabolism. RESULTS: Conversion of aldehydes to their corresponding alcohols was observed in the olfactory cleft and respiratory mucus. The metabolic production of hexanol, octanol, and 2-methybutanol was significantly higher in the olfactory cleft mucus than in the respiratory mucus (p < 0.01). The metabolic conversion of hexanal to hexanol in the mucus of the idiopathic olfactory impairment group was significantly lower than that in the age-matched normal olfaction group. Excluding the nicotinamide adenine dinucleotide phosphate (NADPH) regenerating system from the reaction mixture inhibited metabolism. The addition of either epalr22897estat or 3,5-dichlorosalicylic acid did not inhibit this metabolic conversion. CONCLUSIONS: The enzymatic metabolism of odorants in the olfactory cleft mucus is markedly higher than in the respiratory mucus and decreases in patients with idiopathic olfactory impairment.

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.

CUBIC-Cloud provides an integrative computational framework toward community-driven whole-mouse-brain mapping.

Recent advancements in tissue clearing technologies have offered unparalleled opportunities for researchers to explore the whole mouse brain at cellular resolution. With the expansion of this experimental technique, however, a scalable and easy-to-use computational tool is in demand to effectively analyze and integrate whole-brain mapping datasets. To that end, here we present CUBIC-Cloud, a cloud-based framework to quantify, visualize, and integrate mouse brain data. CUBIC-Cloud is a fully automated system where users can upload their whole-brain data, run analyses, and publish the results. We demonstrate the generality of CUBIC-Cloud by a variety of applications. First, we investigated the brain-wide distribution of five cell types. Second, we quantified Aß plaque deposition in Alzheimer's disease model mouse brains. Third, we reconstructed a neuronal activity profile under LPS-induced inflammation by c-Fos immunostaining. Last, we show brain-wide connectivity mapping by pseudotyped rabies virus. Together, CUBIC-Cloud provides an integrative platform to advance scalable and collaborative whole-brain mapping.

Origin and Evolution of the Gene Family of Proteinaceous Pheromones, the Exocrine Gland-Secreting Peptides, in Rodents.

The exocrine-gland secreting peptide (ESP)gene family encodes proteinaceous pheromones that are recognized by the vomeronasal organ in mice. For example, ESP1 is a male pheromone secreted in tear fluid that regulates socio-sexual behavior, and ESP22 is a juvenile pheromone that suppresses adult sexual behavior. The family consists of multiple genes and has been identified only in mouse and rat genomes. The coding region of a mouse ESP gene is separated into two exons, each encoding signal and mature sequences. Here, we report the origin and evolution of the ESP gene family. ESP genes were found only in the Muridea and Cricetidae families of rodents, suggesting a recent origin of ESP genes in the common ancestor of murids and cricetids. ESP genes show a great diversity in number, length, and sequence among different species as well as mouse strains. Some ESPs in rats and golden hamsters are expressed in the lacrimal gland and the salivary gland. We also found that a mature sequence of an ESP gene showed overall sequence similarity to the α-globin gene. The ancestral ESP gene seems to be generated by recombination of a retrotransposed α-globin gene with the signal-encoding exon of the CRISP2 gene located adjacent to the ESP gene cluster. This study provides an intriguing example of molecular tinkering in rapidly evolving species-specific proteinaceous pheromone genes.

Response to Drea et al.

We greatly appreciate the critical comments on our paper made by Drea et al. [1]. We would like to emphasize that we are not claiming or giving concrete evidence that the identified compounds are pheromones in our paper. We agree that before we can reasonably conclude that the identified compounds are indeed pheromones, we would at least need to examine whether the responses to the identified compounds are stereotypical and reproducible and exclude the effects of signature differences, such as health, relatedness and genetic quality. To this end, it will be necessary to investigate a broader range of behaviors in the future using a larger number of animals.

Response to Kappeler.

We sincerely appreciate the constructive comments made by Peter Kappeler [1] regarding our paper, "Key male glandular odorants attracting female ring-tailed lemurs" [2]. We largely agree with the points raised in these comments, and believe these should be considered as critical discussion that would enable a more reasonable assessment of our findings.

Comparative genomic analyses illuminate the distinct evolution of megabats within Chiroptera.

The revision of the sub-order Microchiroptera is one of the most intriguing outcomes in recent mammalian molecular phylogeny. The unexpected sister-taxon relationship between rhinolophoid microbats and megabats, with the exclusion of other microbats, suggests that megabats arose in a relatively short period of time from a microbat-like ancestor. In order to understand the genetic mechanism underlying adaptive evolution in megabats, we determined the whole-genome sequences of two rousette megabats, Leschenault's rousette (Rousettus leschenaultia) and the Egyptian fruit bat (R. aegyptiacus). The sequences were compared with those of 22 other mammals, including nine bats, available in the database. We identified that megabat genomes are distinct in that they have extremely low activity of SINE retrotranspositions, expansion of two chemosensory gene families, including the trace amine receptor (TAAR) and olfactory receptor (OR), and elevation of the dN/dS ratio in genes for immunity and protein catabolism. The adaptive signatures discovered in the genomes of megabats may provide crucial insight into their distinct evolution, including key processes such as virus resistance, loss of echolocation, and frugivorous feeding.