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.

Single-nucleus transcriptomic analysis of human dorsal root ganglion neurons.

Somatosensory neurons with cell bodies in the dorsal root ganglia (DRG) project to the skin, muscles, bones, and viscera to detect touch and temperature as well as to mediate proprioception and many types of interoception. In addition, the somatosensory system conveys the clinically relevant noxious sensations of pain and itch. Here, we used single nuclear transcriptomics to characterize transcriptomic classes of human DRG neurons that detect these diverse types of stimuli. Notably, multiple types of human DRG neurons have transcriptomic features that resemble their mouse counterparts although expression of genes considered important for sensory function often differed between species. More unexpectedly, we identified several transcriptomic classes with no clear equivalent in the other species. This dataset should serve as a valuable resource for the community, for example as means of focusing translational efforts on molecules with conserved expression across species.

Decoding Cellular Mechanisms for Mechanosensory Discrimination.

Single-cell RNA-sequencing and in vivo functional imaging provide expansive but disconnected views of neuronal diversity. Here, we developed a strategy for linking these modes of classification to explore molecular and cellular mechanisms responsible for detecting and encoding touch. By broadly mapping function to neuronal class, we uncovered a clear transcriptomic logic responsible for the sensitivity and selectivity of mammalian mechanosensory neurons. Notably, cell types with divergent gene-expression profiles often shared very similar properties, but we also discovered transcriptomically related neurons with specialized and divergent functions. Applying our approach to knockout mice revealed that Piezo2 differentially tunes all types of mechanosensory neurons with marked cell-class dependence. Together, our data demonstrate how mechanical stimuli recruit characteristic ensembles of transcriptomically defined neurons, providing rules to help explain the discriminatory power of touch. We anticipate a similar approach could expose fundamental principles governing representation of information throughout the nervous system.

Assigning transcriptomic class in the trigeminal ganglion using multiplex in situ hybridization and machine learning.

ABSTRACT: Single cell sequencing has provided unprecedented information about the transcriptomic diversity of somatosensory systems. Here, we describe a simple and versatile in situ hybridization (ISH)-based approach for mapping this information back to the tissue. We illustrate the power of this approach by demonstrating that ISH localization with just 8 probes is sufficient to distinguish all major classes of neurons in sections of the trigeminal ganglion. To further simplify the approach and make transcriptomic class assignment and cell segmentation automatic, we developed a machine learning approach for analyzing images from multiprobe ISH experiments. We demonstrate the power of in situ class assignment by examining the expression patterns of voltage-gated sodium channels that play roles in distinct somatosensory processes and pain. Specifically, this analysis resolves intrinsic problems with single cell sequencing related to the sparseness of data leading to ambiguity about gene expression patterns. We also used the multiplex in situ approach to study the projection fields of the different neuronal classes. Our results demonstrate that the surface of the eye and meninges are targeted by broad arrays of neural classes despite their very different sensory properties but exhibit idiotypic patterns of innervation at a quantitative level. Very surprisingly, itch-related neurons extensively innervated the meninges, indicating that these transcriptomic cell classes are not simply labeled lines for triggering itch. Together, these results substantiate the importance of a sensory neuron's peripheral and central connections as well as its transcriptomic class in determining its role in sensation.

Diversity amongst trigeminal neurons revealed by high throughput single cell sequencing.

The trigeminal ganglion contains somatosensory neurons that detect a range of thermal, mechanical and chemical cues and innervate unique sensory compartments in the head and neck including the eyes, nose, mouth, meninges and vibrissae. We used single-cell sequencing and in situ hybridization to examine the cellular diversity of the trigeminal ganglion in mice, defining thirteen clusters of neurons. We show that clusters are well conserved in dorsal root ganglia suggesting they represent distinct functional classes of somatosensory neurons and not specialization associated with their sensory targets. Notably, functionally important genes (e.g. the mechanosensory channel Piezo2 and the capsaicin gated ion channel Trpv1) segregate into multiple clusters and often are expressed in subsets of cells within a cluster. Therefore, the 13 genetically-defined classes are likely to be physiologically heterogeneous rather than highly parallel (i.e., redundant) lines of sensory input. Our analysis harnesses the power of single-cell sequencing to provide a unique platform for in silico expression profiling that complements other approaches linking gene-expression with function and exposes unexpected diversity in the somatosensory system.

High salt recruits aversive taste pathways.

In the tongue, distinct classes of taste receptor cells detect the five basic tastes; sweet, sour, bitter, sodium salt and umami. Among these qualities, bitter and sour stimuli are innately aversive, whereas sweet and umami are appetitive and generally attractive to animals. By contrast, salty taste is unique in that increasing salt concentration fundamentally transforms an innately appetitive stimulus into a powerfully aversive one. This appetitive-aversive balance helps to maintain appropriate salt consumption, and represents an important part of fluid and electrolyte homeostasis. We have shown previously that the appetitive responses to NaCl are mediated by taste receptor cells expressing the epithelial sodium channel, ENaC, but the cellular substrate for salt aversion was unknown. Here we examine the cellular and molecular basis for the rejection of high concentrations of salts. We show that high salt recruits the two primary aversive taste pathways by activating the sour- and bitter-taste-sensing cells. We also demonstrate that genetic silencing of these pathways abolishes behavioural aversion to concentrated salt, without impairing salt attraction. Notably, mice devoid of salt-aversion pathways show unimpeded, continuous attraction even to very high concentrations of NaCl. We propose that the 'co-opting' of sour and bitter neural pathways evolved as a means to ensure that high levels of salt reliably trigger robust behavioural rejection, thus preventing its potentially detrimental effects on health.

The taste of carbonation.

Carbonated beverages are commonly available and immensely popular, but little is known about the cellular and molecular mechanisms underlying the perception of carbonation in the mouth. In mammals, carbonation elicits both somatosensory and chemosensory responses, including activation of taste neurons. We have identified the cellular and molecular substrates for the taste of carbonation. By targeted genetic ablation and the silencing of synapses in defined populations of taste receptor cells, we demonstrated that the sour-sensing cells act as the taste sensors for carbonation, and showed that carbonic anhydrase 4, a glycosylphosphatidylinositol-anchored enzyme, functions as the principal CO2 taste sensor. Together, these studies reveal the basis of the taste of carbonation as well as the contribution of taste cells in the orosensory response to CO2.

RGS21 is a novel regulator of G protein signalling selectively expressed in subpopulations of taste bud cells.

Abstract G-protein-mediated signalling processes are involved in sweet and bitter taste transduction. In particular, the G protein alpha-subunit gustducin has been implicated in these processes. One of the limiting factors for the time-course of cellular responses induced by tastants is therefore the intrinsic GTPase activity of alpha-gustducin, which determines the lifetime of the active G protein complex. In several signalling systems specific 'regulator of G protein signalling' (RGS) proteins accelerate the GTPase activity of G protein alpha-subunits. Using differential screening approaches, we have identified a novel RGS protein termed RGS21, which represents the smallest known member of this protein family. Reverse transcription polymerase chain reaction and in situ hybridization experiments demonstrated that RGS21 is expressed selectively in taste tissue where it is found in a subpopulation of sensory cells. Furthermore, it is coexpressed in individual taste cells with bitter and sweet transduction components including alpha-gustducin, phospholipase Cbeta2, T1R2/T1R3 sweet taste receptors and T2R bitter taste receptors. In vitro binding assays demonstrate that RGS21 binds alpha-gustducin in a conformation-dependent manner and has the potential to interact with the same Galpha subtypes as T1R receptors. These results suggest that RGS21 could play a regulatory role in bitter as well as sweet taste transduction processes.