Ocular Symptoms Associated with COVID-19 Are Correlated with the Expression Profile of Mouse SARS-CoV-2 Binding Sites.

The COVID-19 pandemic has engendered significant scientific efforts in the understanding of its infectious agent SARS-CoV-2 and of its associated symptoms. A peculiar characteristic of this virus lies in its ability to challenge our senses, as its infection can lead to anosmia and ageusia. While ocular symptoms, such as conjunctivitis, optic neuritis or dry eyes, are also reported after viral infection, they have lower frequencies and severities, and their functional development is still elusive. Here, using combined technical approaches based on histological and gene profiling methods, we characterized the expression of SARS-CoV-2 binding sites (Ace2/Tmprss2) in the mouse eye. We found that ACE2 was ectopically expressed in subtissular ocular regions, such as in the optic nerve and in the Harderian/intraorbital lacrimal glands. Moreover, we observed an important variation of Ace2/Tmprss2 expression that is not only dependent on the age and sex of the animal, but also highly heterogenous between individuals. Our results thus give new insight into the expression of SARS-CoV-2 binding sites in the mouse eye and propose an interpretation of the human ocular-associated symptoms linked to SARS-CoV-2.

Food preference acquired by social transmission is altered by the absence of the olfactory marker protein in mice.

Food preference is conserved from the most primitive organisms to social animals including humans. A continuous integration of olfactory cues present both in food and in the different environmental and physiological contexts favors the intake of a given source of food or its avoidance. Remarkably, in mice, food preference can also be acquired by olfactory communication in-between conspecifics, a behavior known as the social transmission of food preference (STFP). STFP occurs when a mouse sniffs the breath of a conspecific who has previously eaten a novel food emitting specific odorants and will then develop a preference for this never encountered food. The efficient discrimination of odorants is performed by olfactory sensory neurons (OSNs). It is essential and supports many of the decision-making processes. Here, we found that the olfactory marker protein (OMP), an enigmatic protein ubiquitously expressed in all mature olfactory neurons, is involved in the fine regulation of OSNs basal activity that directly impacts the odorant discrimination ability. Using a previously described Omp null mouse model, we noticed that although odorants and their hedonic-associated values were still perceived by these mice, compensatory behaviors such as a higher number of sniffing events were displayed both in the discrimination of complex odorant signatures and in social-related contexts. As a consequence, we found that the ability to differentiate the olfactory messages carried by individuals such as those implicated in the social transmission of food preference were significantly compromised in Omp null mice. Thus, our results not only give new insights into the role of OMP in the fine discrimination of odorants but also reinforce the fundamental implication of a functional olfactory system for food decision-making.

From In Vitro Data to In Vivo Interspecies Danger Communication: A Study of Chemosensing via the Mouse Grueneberg Ganglion.

In the wild, mice have developed survival strategies to detect volatile cues that warn them of potential danger. Specific olfactory neurons found in the Grueneberg ganglion olfactory subsystem can detect alarm pheromones emitted by stressed conspecifics, as well as kairomones involuntarily released by their predators. These volatile chemical cues allow intra- and interspecies communication of danger, respectively. Alarm pheromones, kairomones and bitter taste ligands share a common chemical motif containing sulfur or nitrogen. Interestingly, three specific bitter taste receptors (TAS2Rs) have been found in the Grueneberg ganglion neurons that are implicated in danger signalling pathways. We have recently developed a TAS2R-expressing heterologous system that mimics the Grueneberg ganglion neuron responses after kairomone stimulation. Here, we demonstrated by in vitro, ex vivo and in vivo experiments that the biological secretions from the raccoon (Procyon lotor) and the skunk (Mephitis mephitis) were acting as potent sources of kairomones. They activated the Grueneberg ganglion neurons and induced fear-related behaviours in mice. Identification of new sources of semiochemicals is a first step towards an understanding of the interspecies danger communication that takes place in the Grueneberg ganglion.

Concentrations of Seven Phthalate Monoesters in Infants and Toddlers Quantified in Urine Extracted from Diapers.

Carrying out exposure studies on children who are not toilet trained is challenging because of the difficulty of urine sampling. In this study, we optimized a protocol for urine collection from disposable diapers for the analysis of phthalate metabolites. The exposure of Swiss children (n = 113) between 6 months and 3 years of life to seven phthalates was assessed by gas chromatography-mass spectrometry measurements. The study showed limited exposures to phthalates, with only 22% of the samples containing some of the metabolites investigated. The three most frequently detected metabolites were monoethyl phthalate, mono-cyclohexyl phthalate, and mono-benzyl phthalate. We also detected mono-n-octyl phthalate and mono(3,5,5-trimethylhexyl) phthalate, which have rarely been observed in urine from infants and toddlers; therefore, di-n-octyl phthalate and bis(3,5,5-trimethylhexyl) phthalate can be considered as potentially new emerging phthalates. This study presents an initial snapshot of the Swiss children's exposure to phthalates and provides a promising approach for further phthalate biomonitoring studies on young children using disposable diapers as urine sampling technique.

Age-dependent appearance of SARS-CoV-2 entry sites in mouse chemosensory systems reflects COVID-19 anosmia-ageusia symptoms.

COVID-19 pandemic has given rise to a collective scientific effort to study its viral causing agent SARS-CoV-2. Research is focusing in particular on its infection mechanisms and on the associated-disease symptoms. Interestingly, this environmental pathogen directly affects the human chemosensory systems leading to anosmia and ageusia. Evidence for the presence of the cellular entry sites of the virus, the ACE2/TMPRSS2 proteins, has been reported in non-chemosensory cells in the rodent's nose and mouth, missing a direct correlation between the symptoms reported in patients and the observed direct viral infection in human sensory cells. Here, mapping the gene and protein expression of ACE2/TMPRSS2 in the mouse olfactory and gustatory cells, we precisely identify the virus target cells to be of basal and sensory origin and reveal the age-dependent appearance of viral entry-sites. Our results propose an alternative interpretation of the human viral-induced sensory symptoms and give investigative perspectives on animal models.

The Grueneberg ganglion controls odor-driven food choices in mice under threat.

The ability to efficiently search for food is fundamental for animal survival. Olfactory messages are used to find food while being aware of the impending risk of predation. How these different olfactory clues are combined to optimize decision-making concerning food selection remains elusive. Here, we find that chemical danger cues drive the food selection in mice via the activation of a specific olfactory subsystem, the Grueneberg ganglion (GG). We show that a functional GG is required to decipher the threatening quality of an unfamiliar food. We also find that the increase in corticosterone, which is GG-dependent, enhances safe food preference acquired during social transmission. Moreover, we demonstrate that memory retrieval for food preference can be extinguished by activation of the GG circuitry. Our findings reveal a key function played by the GG in controlling contextual food responses and illustrate how mammalian organisms integrate environmental chemical stress to optimize decision-making.

Exposure to New Emerging Bisphenols Among Young Children in Switzerland.

Restrictions on the use of bisphenol A (BPA) in consumer products led to its replacement by various bisphenol (BP) analogues, yet young children's exposure to these analogues has been poorly characterized so far. This study aimed to characterize infants' and toddlers' exposure to BPA and 14 emerging BP analogues (i.e., bisphenol AF, bisphenol AP, bisphenol B, bisphenol BP, bisphenol C (BPC), bisphenol E, bisphenol F (BPF), bisphenol G, bisphenol M (BPM), bisphenol P, bisphenol PH, bisphenol S (BPS), bisphenol TMC, and bisphenol Z). We extracted infants' and toddlers' urine from diapers (n = 109) collected in Swiss daycare centers as a practical and noninvasive alternative approach to urinary biomonitoring. Bisphenols were present in 47% of the samples, with BPC and BPM being the most frequently detected (23% and 25% of all samples, respectively). The mean concentrations of urinary BPS and BPF were greater than that of BPA. This contrasts with data reported previously. Furthermore, statistical analysis revealed a significant and negative correlation between urinary BPM concentration and the population's age. Our results provide a first characterization of infants' and toddlers' exposure to bisphenols in Switzerland. This knowledge can be used to support ongoing biomonitoring studies and to prioritize exposure reduction and prevention strategies.

Alarm pheromone and kairomone detection via bitter taste receptors in the mouse Grueneberg ganglion.

BACKGROUND: The mouse Grueneberg ganglion (GG) is an olfactory subsystem specialized in the detection of volatile heterocyclic compounds signalling danger. The signalling pathways transducing the danger signals are only beginning to be characterized. RESULTS: Screening chemical libraries for compounds structurally resembling the already-identified GG ligands, we found a new category of chemicals previously identified as bitter tastants that initiated fear-related behaviours in mice depending on their volatility and evoked neuronal responses in mouse GG neurons. Screening for the expression of signalling receptors of these compounds in the mouse GG yielded transcripts of the taste receptors Tas2r115, Tas2r131, Tas2r143 and their associated G protein α-gustducin (Gnat3). We were further able to confirm their expression at the protein level. Challenging these three G protein-coupled receptors in a heterologous system with the known GG ligands, we identified TAS2R143 as a chemical danger receptor transducing both alarm pheromone and predator-derived kairomone signals. CONCLUSIONS: These results demonstrate that similar molecular elements might be used by the GG and by the taste system to detect chemical danger signals present in the environment.

Identification of pyridine analogs as new predator-derived kairomones.

In the wild, animals have developed survival strategies relying on their senses. The individual ability to identify threatening situations is crucial and leads to increase in the overall fitness of the species. Rodents, for example have developed in their nasal cavities specialized olfactory neurons implicated in the detection of volatile cues encoding for impending danger such as predator scents or alarm pheromones. In particular, the neurons of the Grueneberg ganglion (GG), an olfactory subsystem, are implicated in the detection of danger cues sharing a similar chemical signature, a heterocyclic sulfur- or nitrogen-containing motif. Here we used a "from the wild to the lab" approach to identify new molecules that are involuntarily emitted by predators and that initiate fear-related responses in the recipient animal, the putative prey. We collected urines from carnivores as sources of predator scents and first verified their impact on the blood pressure of the mice. With this approach, the urine of the mountain lion emerged as the most potent source of chemical stress. We then identified in this biological fluid, new volatile cues with characteristic GG-related fingerprints, in particular the methylated pyridine structures, 2,4-lutidine and its analogs. We finally verified their encoded danger quality and demonstrated their ability to mimic the effects of the predator urine on GG neurons, on mice blood pressure and in behavioral experiments. In summary, we were able to identify here, with the use of an integrative approach, new relevant molecules, the pyridine analogs, implicated in interspecies danger communication.

Morphological and physiological species-dependent characteristics of the rodent Grueneberg ganglion.

In the mouse, the Grueneberg ganglion (GG) is an olfactory subsystem implicated both in chemo- and thermo-sensing. It is specifically involved in the recognition of volatile danger cues such as alarm pheromones and structurally-related predator scents. No evidence for these GG sensory functions has been reported yet in other rodent species. In this study, we used a combination of histological and physiological techniques to verify the presence of a GG and investigate its function in the rat, hamster, and gerbil comparing with the mouse. By scanning electron microscopy (SEM) and transmitted electron microscopy (TEM), we found isolated or groups of large GG cells of different shapes that in spite of their gross anatomical similarities, display important structural differences between species. We performed a comparative and morphological study focusing on the conserved olfactory features of these cells. We found fine ciliary processes, mostly wrapped in ensheating glial cells, in variable number of clusters deeply invaginated in the neuronal soma. Interestingly, the glial wrapping, the amount of microtubules and their distribution in the ciliary processes were different between rodents. Using immunohistochemistry, we were able to detect the expression of known GG proteins, such as the membrane guanylyl cyclase G and the cyclic nucleotide-gated channel A3. Both the expression and the subcellular localization of these signaling proteins were found to be species-dependent. Calcium imaging experiments on acute tissue slice preparations from rodent GG demonstrated that the chemo- and thermo-evoked neuronal responses were different between species. Thus, GG neurons from mice and rats displayed both chemo- and thermo-sensing, while hamsters and gerbils showed profound differences in their sensitivities. We suggest that the integrative comparison between the structural morphologies, the sensory properties, and the ethological contexts supports species-dependent GG features prompted by the environmental pressure.

Mouse Grueneberg ganglion neurons share molecular and functional features with C. elegans amphid neurons.

The mouse Grueneberg ganglion (GG) is an olfactory subsystem located at the tip of the nose close to the entry of the naris. It comprises neurons that are both sensitive to cold temperature and play an important role in the detection of alarm pheromones (APs). This chemical modality may be essential for species survival. Interestingly, GG neurons display an atypical mammalian olfactory morphology with neurons bearing deeply invaginated cilia mostly covered by ensheathing glial cells. We had previously noticed their morphological resemblance with the chemosensory amphid neurons found in the anterior region of the head of Caenorhabditis elegans (C. elegans). We demonstrate here further molecular and functional similarities. Thus, we found an orthologous expression of molecular signaling elements that was furthermore restricted to similar specific subcellular localizations. Calcium imaging also revealed a ligand selectivity for the methylated thiazole odorants that amphid neurons are known to detect. Cellular responses from GG neurons evoked by chemical or temperature stimuli were also partially cGMP-dependent. In addition, we found that, although behaviors depending on temperature sensing in the mouse, such as huddling and thermotaxis did not implicate the GG, the thermosensitivity modulated the chemosensitivity at the level of single GG neurons. Thus, the striking similarities with the chemosensory amphid neurons of C. elegans conferred to the mouse GG neurons unique multimodal sensory properties.

Mouse alarm pheromone shares structural similarity with predator scents.

Sensing the chemical warnings present in the environment is essential for species survival. In mammals, this form of danger communication occurs via the release of natural predator scents that can involuntarily warn the prey or by the production of alarm pheromones by the stressed prey alerting its conspecifics. Although we previously identified the olfactory Grueneberg ganglion as the sensory organ through which mammalian alarm pheromones signal a threatening situation, the chemical nature of these cues remains elusive. We here identify, through chemical analysis in combination with a series of physiological and behavioral tests, the chemical structure of a mouse alarm pheromone. To successfully recognize the volatile cues that signal danger, we based our selection on their activation of the mouse olfactory Grueneberg ganglion and the concomitant display of innate fear reactions. Interestingly, we found that the chemical structure of the identified mouse alarm pheromone has similar features as the sulfur-containing volatiles that are released by predating carnivores. Our findings thus not only reveal a chemical Leitmotiv that underlies signaling of fear, but also point to a double role for the olfactory Grueneberg ganglion in intraspecies as well as interspecies communication of danger.

Imaging pheromone sensing in a mouse vomeronasal acute tissue slice preparation.

Peter Karlson and Martin Lüscher used the term pheromone for the first time in 1959 to describe chemicals used for intra-species communication. Pheromones are volatile or non-volatile short-lived molecules secreted and/or contained in biological fluids, such as urine, a liquid known to be a main source of pheromones. Pheromonal communication is implicated in a variety of key animal modalities such as kin interactions, hierarchical organisations and sexual interactions and are consequently directly correlated with the survival of a given species. In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO), a paired structure located at the base of the nasal cavity, and enclosed in a cartilaginous capsule. Each VNO has a tubular shape with a lumen allowing the contact with the external chemical world. The sensory neuroepithelium is principally composed of vomeronasal bipolar sensory neurons (VSNs). Each VSN extends a single dendrite to the lumen ending in a large dendritic knob bearing up to 100 microvilli implicated in chemical detection. Numerous subpopulations of VSNs are present. They are differentiated by the chemoreceptor they express and thus possibly by the ligand(s) they recognize. Two main vomeronasal receptor families, V1Rs and V2Rs, are composed respectively by 240 and 120 members and are expressed in separate layers of the neuroepithelium. Olfactory receptors (ORs) and formyl peptide receptors (FPRs) are also expressed in VSNs. Whether or not these neuronal subpopulations use the same downstream signalling pathway for sensing pheromones is unknown. Despite a major role played by a calcium-permeable channel (TRPC2) present in the microvilli of mature neurons TRPC2 independent transduction channels have been suggested. Due to the high number of neuronal subpopulations and the peculiar morphology of the organ, pharmacological and physiological investigations of the signalling elements present in the VNO are complex. Here, we present an acute tissue slice preparation of the mouse VNO for performing calcium imaging investigations. This physiological approach allows observations, in the natural environment of a living tissue, of general or individual subpopulations of VSNs previously loaded with Fura-2AM, a calcium dye. This method is also convenient for studying any GFP-tagged pheromone receptor and is adaptable for the use of other fluorescent calcium probes. As an example, we use here a VG mouse line, in which the translation of the pheromone V1rb2 receptor is linked to the expression of GFP by a polycistronic strategy.

Grueneberg ganglion cells mediate alarm pheromone detection in mice.

Alarm pheromones (APs) are widely used throughout the plant and animal kingdoms. Species such as fish, insects, and mammals signal danger to conspecifics by releasing volatile alarm molecules. Thus far, neither the chemicals, their bodily source, nor the sensory system involved in their detection have been isolated or identified in mammals. We found that APs are recognized by the Grueneberg ganglion (GG), a recently discovered olfactory subsystem. We showed with electron microscopy that GG neurons bear primary cilia, with cell bodies ensheathed by glial cells. APs evoked calcium responses in GG neurons in vitro and induced freezing behavior in vivo, which completely disappeared when the GG degenerated after axotomy. We conclude that mice detect APs through the activation of olfactory GG neurons.

Gene cluster lock after pheromone receptor gene choice.

In mammals, perception of pheromones is based on the expression in each vomeronasal sensory neuron of a limited set of receptor genes, chosen among a large repertoire. Here, we report an extremely tight control of the monogenic and monoallelic transcription of the V1rb2 receptor gene. Combining genetic and electrophysiological approaches, we show that the transcription of a non-functional V1r allele leads to the coexpression of another, functional V1r gene. The choice of this coexpressed gene surprisingly includes genes located on the cluster homologous to the one from which the mutant allele is transcribed. However, V1r genes located in cis relative to the transcribed mutant allele are excluded from the coexpression choice. Our observations strongly suggest a monogenic regulatory mechanism acting (a) at a general level, via the expression of the V1r receptor itself, and (b) at a more local level, defined by the V1r gene cluster.