Intradental mechano-nociceptors serve as sentinels that prevent tooth damage.

Pain is the anticipated output of the trigeminal sensory neurons that innervate the tooth's vital interior 1,2 ; however, the contribution of intradental neurons to healthy tooth sensation has yet to be defined. Here, we employ in vivo Ca 2+ imaging to identify and define a population of myelinated high-threshold mechanoreceptors (intradental HTMRs) that detect superficial structural damage of the tooth and initiate jaw opening to protect teeth from damage. Intradental HTMRs remain inactive when direct forces are applied to the intact tooth but become responsive to forces when the structural integrity of the tooth is compromised, and the dentin or pulp is exposed. Their terminals collectively innervate the inner dentin through overlapping receptive fields, allowing them to monitor the superficial structures of the tooth. Indeed, intradental HTMRs detect superficial enamel damage and encode its degree, and their responses persist in the absence of either PIEZO2 or Na v 1.8 3,4 . Optogenetic activation of intradental HTMRs triggers a rapid, jaw opening reflex via contraction of the digastric muscle. Taken together, our data indicate that intradental HTMRs serve as sentinels that guard against mechanical threats to the tooth, and their activation results in physical tooth separation to minimize irreversible structural damage. Our work provides a new perspective on the role of intradental neurons as protective rather than exclusively pain-inducing and illustrates additional diversity in the functions of interoreceptors.

Deciphering the chemical language of inbred and wild mouse conspecific scents.

In most mammals, conspecific chemosensory communication relies on semiochemical release within complex bodily secretions and subsequent stimulus detection by the vomeronasal organ (VNO). Urine, a rich source of ethologically relevant chemosignals, conveys detailed information about sex, social hierarchy, health, and reproductive state, which becomes accessible to a conspecific via vomeronasal sampling. So far, however, numerous aspects of social chemosignaling along the vomeronasal pathway remain unclear. Moreover, since virtually all research on vomeronasal physiology is based on secretions derived from inbred laboratory mice, it remains uncertain whether such stimuli provide a true representation of potentially more relevant cues found in the wild. Here, we combine a robust low-noise VNO activity assay with comparative molecular profiling of sex- and strain-specific mouse urine samples from two inbred laboratory strains as well as from wild mice. With comprehensive molecular portraits of these secretions, VNO activity analysis now enables us to (i) assess whether and, if so, how much sex/strain-selective 'raw' chemical information in urine is accessible via vomeronasal sampling; (ii) identify which chemicals exhibit sufficient discriminatory power to signal an animal's sex, strain, or both; (iii) determine the extent to which wild mouse secretions are unique; and (iv) analyze whether vomeronasal response profiles differ between strains. We report both sex- and, in particular, strain-selective VNO representations of chemical information. Within the urinary 'secretome', both volatile compounds and proteins exhibit sufficient discriminative power to provide sex- and strain-specific molecular fingerprints. While total protein amount is substantially enriched in male urine, females secrete a larger variety at overall comparatively low concentrations. Surprisingly, the molecular spectrum of wild mouse urine does not dramatically exceed that of inbred strains. Finally, vomeronasal response profiles differ between C57BL/6 and BALB/c animals, with particularly disparate representations of female semiochemicals.

The anatomy, neurophysiology, and cellular mechanisms of intradental sensation.

Somatosensory innervation of the oral cavity enables the detection of a range of environmental stimuli including minute and noxious mechanical forces. The trigeminal sensory neurons underlie sensation originating from the tooth. Prior work has provided important physiological and molecular characterization of dental pulp sensory innervation. Clinical dental experiences have informed our conception of the consequence of activating these neurons. However, the biological role of sensory innervation within the tooth is yet to be defined. Recent transcriptomic data, combined with mouse genetic tools, have the capacity to provide important cell-type resolution for the physiological and behavioral function of pulp-innervating sensory neurons. Importantly, these tools can be applied to determine the neuronal origin of acute dental pain that coincides with tooth damage as well as pain stemming from tissue inflammation (i.e., pulpitis) toward developing treatment strategies aimed at relieving these distinct forms of pain.

Trigeminal innervation and tactile responses in mouse tongue.

The mammalian tongue is richly innervated with somatosensory, gustatory and motor fibers. These form the basis of many ethologically important functions such as eating, speaking and social grooming. Despite its high tactile acuity and sensitivity, the neural basis of tongue mechanosensation remains largely mysterious. Here we explored the organization of mechanosensory afferents in the tongue and found that each lingual papilla is innervated by Piezo2 + trigeminal neurons. Notably, each fungiform papilla contained highly specialized ring-like sensory neuron terminations that circumscribe the taste buds. Myelinated lingual afferents in the mouse lingual papillae did not form corpuscular sensory end organs but rather had only free nerve endings. In vivo single-unit recordings from the trigeminal ganglion revealed two types of lingual low-threshold mechanoreceptors (LTMRs) with conduction velocities in the Aδ range or above and distinct response properties: intermediately adapting (IA) units and rapidly adapting (RA) units. IA units were sensitive to static indentation and stroking, while RA units had a preference for tangential forces applied by stroking. Lingual LTMRs were not directly responsive to rapid cooling or chemicals that can induce astringent or numbing sensations. Genetic labeling of lingual afferents in the tongue revealed at least two types of nerve terminal patterns, involving dense innervation of individual fungiform papillae by multiple putatively distinct afferents, and relatively sparse innervation of filiform papillae. Together, our results indicate that fungiform papillae are mechanosensory structures, while suggesting a simple model that links the functional and anatomical properties of tactile sensory neurons in the tongue.

PIEZO2 and perineal mechanosensation are essential for sexual function.

Despite the potential importance of genital mechanosensation for sexual reproduction, little is known about how perineal touch influences mating. We explored how mechanosensation affords exquisite awareness of the genitals and controls reproduction in mice and humans. Using genetic strategies and in vivo functional imaging, we demonstrated that the mechanosensitive ion channel PIEZO2 (piezo-type mechanosensitive ion channel component 2) is necessary for behavioral sensitivity to perineal touch. PIEZO2 function is needed for triggering a touch-evoked erection reflex and successful mating in both male and female mice. Humans with complete loss of PIEZO2 function have genital hyposensitivity and experience no direct pleasure from gentle touch or vibration. Together, our results help explain how perineal mechanoreceptors detect the gentlest of stimuli and trigger physiologically important sexual responses, thus providing a platform for exploring the sensory basis of sexual pleasure and its relationship to affective touch.

Reevaluation of Piezo1 as a gut RNA sensor.

Piezo1 is a stretch-gated ion channel required for mechanosensation in many organ systems. Recent findings point to a new role for Piezo1 in the gut, suggesting that it is a sensor of microbial single-stranded RNA (ssRNA) rather than mechanical force. If true, this would redefine the scope of Piezo biology. Here, we sought to replicate the central finding that fecal ssRNA is a natural agonist of Piezo1. While we observe that fecal extracts and ssRNA can stimulate calcium influx in certain cell lines, this response is independent of Piezo1. Additionally, sterilized dietary extracts devoid of gut biome RNA show similar cell line-specific stimulatory activity to fecal extracts. Together, our data highlight potential confounds inherent to gut-derived extracts, exclude Piezo1 as a receptor for ssRNA in the gut, and support a dedicated role for Piezo channels in mechanosensing.

PIEZO2 ion channels in proprioception.

PIEZO2 is a stretch-gated ion channel that is expressed at high levels in somatosensory neurons. Humans with rare mutations in the PIEZO2 gene have profound mechanosensory deficits that include a loss of the sense of proprioception. These striking phenotypes match those seen in conditional knockout mouse models demonstrating the highly conserved function for this gene. Here, we review the ramifications of loss of PIEZO2 function on normal daily activities and what studies like these have revealed about proprioception at the molecular and cellular level. Additionally, we highlight recent work that has uncovered the surprising functional and molecular diversity of proprioceptors. Together, these findings pioneer a path toward determining how the detection of mechanosensory input from muscles and tendons is used to control posture and refine motor performance.

Do all mice smell the same? Chemosensory cues from inbred and wild mouse strains elicit stereotypic sensory representations in the accessory olfactory bulb.

BACKGROUND: For many animals, chemosensory cues are vital for social and defensive interactions and are primarily detected and processed by the vomeronasal system (VNS). These cues are often inherently associated with ethological meaning, leading to stereotyped behaviors. Thus, one would expect consistent representation of these stimuli across different individuals. However, individuals may express different arrays of vomeronasal sensory receptors and may vary in the pattern of connections between those receptors and projection neurons in the accessory olfactory bulb (AOB). In the first part of this study, we address the ability of individuals to form consistent representations despite these potential sources of variability. The second part of our study is motivated by the fact that the majority of research on VNS physiology involves the use of stimuli derived from inbred animals. Yet, it is unclear whether neuronal representations of inbred-derived stimuli are similar to those of more ethologically relevant wild-derived stimuli. RESULTS: First, we compared sensory representations to inbred, wild-derived, and wild urine stimuli in the AOBs of males from two distinct inbred strains, using them as proxies for individuals. We found a remarkable similarity in stimulus representations across the two strains. Next, we compared AOB neuronal responses to inbred, wild-derived, and wild stimuli, again using male inbred mice as subjects. Employing various measures of neuronal activity, we show that wild-derived and wild stimuli elicit responses that are broadly similar to those from inbred stimuli: they are not considerably stronger or weaker, they show similar levels of sexual dimorphism, and when examining population-level activity, cluster with inbred mouse stimuli. CONCLUSIONS: Despite strain-specific differences and apparently random connectivity, the AOB can maintain stereotypic sensory representations for broad stimulus categories, providing a substrate for common stereotypical behaviors. In addition, despite many generations of inbreeding, AOB representations capture the key ethological features (i.e., species and sex) of wild-derived and wild counterparts. Beyond these broad similarities, representations of stimuli from wild mice are nevertheless distinct from those elicited by inbred mouse stimuli, suggesting that laboratory inbreeding has indeed resulted in marked modifications of urinary secretions.

Synchronous Infra-Slow Oscillations Organize Ensembles of Accessory Olfactory Bulb Projection Neurons into Distinct Microcircuits.

The accessory olfactory system controls social and sexual behavior. In the mouse accessory olfactory bulb, the first central stage of information processing along the accessory olfactory pathway, projection neurons (mitral cells) display infra-slow oscillatory discharge with remarkable periodicity. The physiological mechanisms that underlie this default output state, however, remain controversial. Moreover, whether such rhythmic infra-slow activity patterns exist in awake behaving mice and whether such activity reflects the functional organization of the accessory olfactory bulb circuitry remain unclear. Here, we hypothesize that mitral cell ensembles form synchronized microcircuits that subdivide the accessory olfactory bulb into segregated functional clusters. We use a miniature microscope to image the Ca2+ dynamics within the apical dendritic compartments of large mitral cell ensembles in vivo We show that infra-slow periodic patterns of concerted neural activity, indeed, reflect the idle state of accessory olfactory bulb output in awake male and female mice. Ca2+ activity profiles are distinct and glomerulus-specific. Confocal time-lapse imaging in acute slices reveals that groups of mitral cells assemble into microcircuits that exhibit correlated Ca2+ signals. Moreover, electrophysiological profiling of synaptic connectivity indicates functional coupling between mitral cells. Our results suggest that both intrinsically rhythmogenic neurons and neurons entrained by fast synaptic drive are key elements in organizing the accessory olfactory bulb into functional microcircuits, each characterized by a distinct default pattern of infra-slow rhythmicity.SIGNIFICANCE STATEMENT Information processing in the accessory olfactory bulb (AOB) plays a central role in conspecific chemosensory communication. Surprisingly, many basic physiological principles that underlie neuronal signaling in the AOB remain elusive. Here, we show that AOB projection neurons (mitral cells) form parallel synchronized ensembles both in vitro and in vivo Infra-slow synchronous oscillatory activity within AOB microcircuits thus adds a new dimension to chemosensory coding along the accessory olfactory pathway.

Signal Detection and Coding in the Accessory Olfactory System.

In many mammalian species, the accessory olfactory system plays a central role in guiding behavioral and physiological responses to social and reproductive interactions. Because of its relatively compact structure and its direct access to amygdalar and hypothalamic nuclei, the accessory olfactory pathway provides an ideal system to study sensory control of complex mammalian behavior. During the last several years, many studies employing molecular, behavioral, and physiological approaches have significantly expanded and enhanced our understanding of this system. The purpose of the current review is to integrate older and newer studies to present an updated and comprehensive picture of vomeronasal signaling and coding with an emphasis on early accessory olfactory system processing stages. These include vomeronasal sensory neurons in the vomeronasal organ, and the circuitry of the accessory olfactory bulb. Because the overwhelming majority of studies on accessory olfactory system function employ rodents, this review is largely focused on this phylogenetic order, and on mice in particular. Taken together, the emerging view from both older literature and more recent studies is that the molecular, cellular, and circuit properties of chemosensory signaling along the accessory olfactory pathway are in many ways unique. Yet, it has also become evident that, like the main olfactory system, the accessory olfactory system also has the capacity for adaptive learning, experience, and state-dependent plasticity. In addition to describing what is currently known about accessory olfactory system function and physiology, we highlight what we believe are important gaps in our knowledge, which thus define exciting directions for future investigation.

Sensory Adaptation to Chemical Cues by Vomeronasal Sensory Neurons.

Sensory adaptation is a source of experience-dependent feedback that impacts responses to environmental cues. In the mammalian main olfactory system (MOS), adaptation influences sensory coding at its earliest processing stages. Sensory adaptation in the accessory olfactory system (AOS) remains incompletely explored, leaving many aspects of the phenomenon unclear. We investigated sensory adaptation in vomeronasal sensory neurons (VSNs) using a combination of in situ Ca2+ imaging and electrophysiology. Parallel studies revealed prominent short-term sensory adaptation in VSNs upon repeated stimulation with mouse urine and monomolecular bile acid ligands at interstimulus intervals (ISIs) less than 30 s. In such conditions, Ca2+ signals and spike rates were often reduced by more than 50%, leading to dramatically reduced chemosensory sensitivity. Short-term adaptation was reversible over the course of minutes. Population Ca2+ imaging experiments revealed the presence of a slower form of VSN adaptation that accumulated over dozens of stimulus presentations delivered over tens of minutes. Most VSNs showed strong adaptation, but in a substantial VSN subpopulation adaptation was diminished or absent. Investigation of same- and opposite-sex urine responses in male and female VSNs revealed that adaptation to same-sex cues occurred at ISIs up to 180 s, conditions that did not induce adaptation to opposite-sex cues. This result suggests that VSN sensory adaptation can be modulated by sensory experience. These studies comprehensively establish the presence of VSN sensory adaptation and provide a foundation for future inquiries into the molecular and cellular mechanisms of this phenomenon and its impact on mammalian behavior.

Modulatory Effects of Sex Steroids Progesterone and Estradiol on Odorant Evoked Responses in Olfactory Receptor Neurons.

The influence of the sex steroid hormones progesterone and estradiol on physiology and behavior during menstrual cycles and pregnancy is well known. Several studies indicate that olfactory performance changes with cyclically fluctuating steroid hormone levels in females. Knowledge of the exact mechanisms behind how female sex steroids modulate olfactory signaling is limited. A number of different known genomic and non-genomic actions that are mediated by progesterone and estradiol via interactions with different receptors may be responsible for this modulation. Next generation sequencing-based RNA-Seq transcriptome data from the murine olfactory epithelium (OE) and olfactory receptor neurons (ORNs) revealed the expression of several membrane progestin receptors and the estradiol receptor Gpr30. These receptors are known to mediate rapid non-genomic effects through interactions with G proteins. RT-PCR and immunohistochemical staining results provide evidence for progestin and estradiol receptors in the ORNs. These data support the hypothesis that steroid hormones are capable of modulating the odorant-evoked activity of ORNs. Here, we validated this hypothesis through the investigation of steroid hormone effects by submerged electro-olfactogram and whole cell patch-clamp recordings of ORNs. For the first time, we demonstrate that the sex steroid hormones progesterone and estradiol decrease odorant-evoked signals in the OE and ORNs of mice at low nanomolar concentrations. Thus, both of these sex steroids can rapidly modulate the odor responsiveness of ORNs through membrane progestin receptors and the estradiol receptor Gpr30.

Deep sequencing of the murine olfactory receptor neuron transcriptome.

The ability of animals to sense and differentiate among thousands of odorants relies on a large set of olfactory receptors (OR) and a multitude of accessory proteins within the olfactory epithelium (OE). ORs and related signaling mechanisms have been the subject of intensive studies over the past years, but our knowledge regarding olfactory processing remains limited. The recent development of next generation sequencing (NGS) techniques encouraged us to assess the transcriptome of the murine OE. We analyzed RNA from OEs of female and male adult mice and from fluorescence-activated cell sorting (FACS)-sorted olfactory receptor neurons (ORNs) obtained from transgenic OMP-GFP mice. The Illumina RNA-Seq protocol was utilized to generate up to 86 million reads per transcriptome. In OE samples, nearly all OR and trace amine-associated receptor (TAAR) genes involved in the perception of volatile amines were detectably expressed. Other genes known to participate in olfactory signaling pathways were among the 200 genes with the highest expression levels in the OE. To identify OE-specific genes, we compared olfactory neuron expression profiles with RNA-Seq transcriptome data from different murine tissues. By analyzing different transcript classes, we detected the expression of non-olfactory GPCRs in ORNs and established an expression ranking for GPCRs detected in the OE. We also identified other previously undescribed membrane proteins as potential new players in olfaction. The quantitative and comprehensive transcriptome data provide a virtually complete catalogue of genes expressed in the OE and present a useful tool to uncover candidate genes involved in, for example, olfactory signaling, OR trafficking and recycling, and proliferation.