Painspotting.

How do we know an animal is feeling pain? In this issue of Neuron, Bohic et al.1 develop computational methods to detect pain in mice, shining a light on the behavioral changes that occur during pain, its relief, and recovery.

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.

PIEZO2 in somatosensory neurons controls gastrointestinal transit.

The gastrointestinal tract is in a state of constant motion. These movements are tightly regulated by the presence of food and help digestion by mechanically breaking down and propelling gut content. Mechanical sensing in the gut is thought to be essential for regulating motility; however, the identity of the neuronal populations, the molecules involved, and the functional consequences of this sensation are unknown. Here, we show that humans lacking PIEZO2 exhibit impaired bowel sensation and motility. Piezo2 in mouse dorsal root, but not nodose ganglia is required to sense gut content, and this activity slows down food transit rates in the stomach, small intestine, and colon. Indeed, Piezo2 is directly required to detect colon distension in vivo. Our study unveils the mechanosensory mechanisms that regulate the transit of luminal contents throughout the gut, which is a critical process to ensure proper digestion, nutrient absorption, and waste removal.

Wearable Sensory Substitution for Proprioception via Deep Pressure.

We propose a sensory substitution device that communicates one-degree-of-freedom proprioceptive feedback via deep pressure stimulation on the arm. The design is motivated by the need for a feedback modality detectable by individuals with a genetic condition known as PIEZO2 loss of function, which is characterized by absence of both proprioception and sense of light touch. We created a wearable and programmable prototype that applies up to 15 N of deep pressure stimulation to the forearm and includes an embedded force sensor. We conducted a study to evaluate the ability of participants without sensory impairment to control the position of a virtual arm to match a target angle communicated by deep pressure stimulation. A participant-specific calibration resulted in an average minimum detectable force of 0.41 N and maximum comfortable force of 6.42 N. We found that, after training, participants were able to significantly reduce angle error using the deep pressure haptic feedback compared to without it. Angle error increased only slightly with force, indicating that this sensory substitution method is a promising approach for individuals with PIEZO2 loss of function and other forms of sensory loss.

Immunity to the microbiota promotes sensory neuron regeneration.

Tissue immunity and responses to injury depend on the coordinated action and communication among physiological systems. Here, we show that, upon injury, adaptive responses to the microbiota directly promote sensory neuron regeneration. At homeostasis, tissue-resident commensal-specific T cells colocalize with sensory nerve fibers within the dermis, express a transcriptional program associated with neuronal interaction and repair, and promote axon growth and local nerve regeneration following injury. Mechanistically, our data reveal that the cytokine interleukin-17A (IL-17A) released by commensal-specific Th17 cells upon injury directly signals to sensory neurons via IL-17 receptor A, the transcription of which is specifically upregulated in injured neurons. Collectively, our work reveals that in the context of tissue damage, preemptive immunity to the microbiota can rapidly bridge biological systems by directly promoting neuronal repair, while also identifying IL-17A as a major determinant of this fundamental process.

PIEZO2-dependent rapid pain system in humans and mice.

The PIEZO2 ion channel is critical for transducing light touch into neural signals but is not considered necessary for transducing acute pain in humans. Here, we discovered an exception - a form of mechanical pain evoked by hair pulling. Based on observations in a rare group of individuals with PIEZO2 deficiency syndrome, we demonstrated that hair-pull pain is dependent on PIEZO2 transduction. Studies in control participants showed that hair-pull pain triggered a distinct nocifensive response, including a nociceptive reflex. Observations in rare Aß deafferented individuals and nerve conduction block studies in control participants revealed that hair-pull pain perception is dependent on Aß input. Single-unit axonal recordings revealed that a class of cooling-responsive myelinated nociceptors in human skin is selectively tuned to painful hair-pull stimuli. Further, we pharmacologically mapped these nociceptors to a specific transcriptomic class. Finally, using functional imaging in mice, we demonstrated that in a homologous nociceptor, Piezo2 is necessary for high-sensitivity, robust activation by hair-pull stimuli. Together, we have demonstrated that hair-pulling evokes a distinct type of pain with conserved behavioral, neural, and molecular features across humans and mice.

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.

Human Stem Cell-Derived TRPV1-Positive Sensory Neurons: A New Tool to Study Mechanisms of Sensitization.

Somatosensation, the detection and transduction of external and internal stimuli such as temperature or mechanical force, is vital to sustaining our bodily integrity. But still, some of the mechanisms of distinct stimuli detection and transduction are not entirely understood, especially when noxious perception turns into chronic pain. Over the past decade major progress has increased our understanding in areas such as mechanotransduction or sensory neuron classification. However, it is in particular the access to human pluripotent stem cells and the possibility of generating and studying human sensory neurons that has enriched the somatosensory research field. Based on our previous work, we describe here the generation of human stem cell-derived nociceptor-like cells. We show that by varying the differentiation strategy, we can produce different nociceptive subpopulations with different responsiveness to nociceptive stimuli such as capsaicin. Functional as well as deep sequencing analysis demonstrated that one protocol in particular allowed the generation of a mechano-nociceptive sensory neuron population, homogeneously expressing TRPV1. Accordingly, we find the cells to homogenously respond to capsaicin, to become sensitized upon inflammatory stimuli, and to respond to temperature stimulation. The efficient and homogenous generation of these neurons make them an ideal translational tool to study mechanisms of sensitization, also in the context of chronic pain.

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.

The Form and Function of PIEZO2.

Mechanosensation is the ability to detect dynamic mechanical stimuli (e.g., pressure, stretch, and shear stress) and is essential for a wide variety of processes, including our sense of touch on the skin. How touch is detected and transduced at the molecular level has proved to be one of the great mysteries of sensory biology. A major breakthrough occurred in 2010 with the discovery of a family of mechanically gated ion channels that were coined PIEZOs. The last 10 years of investigation have provided a wealth of information about the functional roles and mechanisms of these molecules. Here we focus on PIEZO2, one of the two PIEZO proteins found in humans and other mammals. We review how work at the molecular, cellular, and systems levels over the past decade has transformed our understanding of touch and led to unexpected insights into other types of mechanosensation beyond the skin.

Innocuous pressure sensation requires A-type afferents but not functional ΡΙΕΖΟ2 channels in humans.

The sensation of pressure allows us to feel sustained compression and body strain. While our understanding of cutaneous touch has grown significantly in recent years, how deep tissue sensations are detected remains less clear. Here, we use quantitative sensory evaluations of patients with rare sensory disorders, as well as nerve blocks in typical individuals, to probe the neural and genetic mechanisms for detecting non-painful pressure. We show that the ability to perceive innocuous pressures is lost when myelinated fiber function is experimentally blocked in healthy volunteers and that two patients lacking Aß fibers are strikingly unable to feel innocuous pressures at all. We find that seven individuals with inherited mutations in the mechanoreceptor PIEZO2 gene, who have major deficits in touch and proprioception, are nearly as good at sensing pressure as healthy control subjects. Together, these data support a role for Aß afferents in pressure sensation and suggest the existence of an unknown molecular pathway for its detection.

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.

PIEZO2 in sensory neurons and urothelial cells coordinates urination.

Henry Miller stated that "to relieve a full bladder is one of the great human joys". Urination is critically important in health and ailments of the lower urinary tract cause high pathological burden. Although there have been advances in understanding the central circuitry in the brain that facilitates urination1-3, there is a lack of in-depth mechanistic insight into the process. In addition to central control, micturition reflexes that govern urination are all initiated by peripheral mechanical stimuli such as bladder stretch and urethral flow4. The mechanotransduction molecules and cell types that function as the primary stretch and pressure detectors in the urinary tract mostly remain unknown. Here we identify expression of the mechanosensitive ion channel PIEZO2 in lower urinary tract tissues, where it is required for low-threshold bladder-stretch sensing and urethral micturition reflexes. We show that PIEZO2 acts as a sensor in both the bladder urothelium and innervating sensory neurons. Humans and mice lacking functional PIEZO2 have impaired bladder control, and humans lacking functional PIEZO2 report deficient bladder-filling sensation. This study identifies PIEZO2 as a key mechanosensor in urinary function. These findings set the foundation for future work to identify the interactions between urothelial cells and sensory neurons that control urination.

Publisher Correction: A dietary fatty acid counteracts neuronal mechanical sensitization.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A dietary fatty acid counteracts neuronal mechanical sensitization.

PIEZO2 is the essential transduction channel for touch discrimination, vibration, and proprioception. Mice and humans lacking Piezo2 experience severe mechanosensory and proprioceptive deficits and fail to develop tactile allodynia. Bradykinin, a proalgesic agent released during inflammation, potentiates PIEZO2 activity. Molecules that decrease PIEZO2 function could reduce heightened touch responses during inflammation. Here, we find that the dietary fatty acid margaric acid (MA) decreases PIEZO2 function in a dose-dependent manner. Chimera analyses demonstrate that the PIEZO2 beam is a key region tuning MA-mediated channel inhibition. MA reduces neuronal action potential firing elicited by mechanical stimuli in mice and rat neurons and counteracts PIEZO2 sensitization by bradykinin. Finally, we demonstrate that this saturated fatty acid decreases PIEZO2 currents in touch neurons derived from human induced pluripotent stem cells. Our findings report on a natural product that inhibits PIEZO2 function and counteracts neuronal mechanical sensitization and reveal a key region for channel inhibition.

Parallel Parabrachial Pathways Provide Pieces of the Pain Puzzle.

Noxious stimuli evoke a range of acute and long-lasting sensations, emotions, and behaviors. In this issue of Neuron, Chiang et al. (2020) demonstrate that parallel outputs from the lateral parabrachial nucleus arise from specific cell types with distinct functions in pain.

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.

Cerebellospinal Neurons Regulate Motor Performance and Motor Learning.

To understand the neural basis of behavior, it is important to reveal how movements are planned, executed, and refined by networks of neurons distributed throughout the nervous system. Here, we report the neuroanatomical organization and behavioral roles of cerebellospinal (CeS) neurons. Using intersectional genetic techniques, we find that CeS neurons constitute a small minority of excitatory neurons in the fastigial and interpositus deep cerebellar nuclei, target pre-motor circuits in the ventral spinal cord and the brain, and control distinct aspects of movement. CeS neurons that project to the ipsilateral cervical cord are required for skilled forelimb performance, while CeS neurons that project to the contralateral cervical cord are involved in skilled locomotor learning. Together, this work establishes CeS neurons as a critical component of the neural circuitry for skilled movements and provides insights into the organizational logic of motor networks.

Transcriptional Programming of Human Mechanosensory Neuron Subtypes from Pluripotent Stem Cells.

Efficient and homogeneous in vitro generation of peripheral sensory neurons may provide a framework for novel drug screening platforms and disease models of touch and pain. We discover that, by overexpressing NGN2 and BRN3A, human pluripotent stem cells can be transcriptionally programmed to differentiate into a surprisingly uniform culture of cold- and mechano-sensing neurons. Although such a neuronal subtype is not found in mice, we identify molecular evidence for its existence in human sensory ganglia. Combining NGN2 and BRN3A programming with neural crest patterning, we produce two additional populations of sensory neurons, including a specialized touch receptor neuron subtype. Finally, we apply this system to model a rare inherited sensory disorder of touch and proprioception caused by inactivating mutations in PIEZO2. Together, these findings establish an approach to specify distinct sensory neuron subtypes in vitro, underscoring the utility of stem cell technology to capture human-specific features of physiology and disease.