Staphylococcus aureus: The Bug Behind the Itch in Atopic Dermatitis.

Pruritus or itch is a defining symptom of atopic dermatitis (AD). The origins of itch are complex, and it is considered both a defense mechanism and a cause of disease that leads to inflammation and psychological stress. Considerable progress has been made in understanding the processes that trigger itch, particularly the pruritoceptive origins that are generated in the skin. This perspective review discusses the implications of a recent observation that the V8 protease expressed by Staphylococcus aureus can directly trigger sensory neurons in the skin through activation of protease-activated receptor 1. This may be a key to understanding why itch is so common in AD because S. aureus commonly overgrows in this disease owing to deficient antimicrobial defense from both the epidermis and the cutaneous microbiome. Increased understanding of the role of microbes in AD provides increased opportunities for safely improving the treatment of this disorder.

Distinct cellular expression and subcellular localization of Kv2 voltage-gated K+ channel subtypes in dorsal root ganglion neurons conserved between mice and humans.

The distinct organization of Kv2 voltage-gated potassium channels on and near the cell body of brain neurons enables their regulation of action potentials and specialized membrane contact sites. Somatosensory neurons have a pseudounipolar morphology and transmit action potentials from peripheral nerve endings through axons that bifurcate to the spinal cord and the cell body within ganglia including the dorsal root ganglia (DRG). Kv2 channels regulate action potentials in somatosensory neurons, yet little is known about where Kv2 channels are located. Here, we define the cellular and subcellular localization of the Kv2 paralogs, Kv2.1 and Kv2.2, in DRG somatosensory neurons with a panel of antibodies, cell markers, and genetically modified mice. We find that relative to spinal cord neurons, DRG neurons have similar levels of detectable Kv2.1 and higher levels of Kv2.2. In older mice, detectable Kv2.2 remains similar, while detectable Kv2.1 decreases. Both Kv2 subtypes adopt clustered subcellular patterns that are distinct from central neurons. Most DRG neurons co-express Kv2.1 and Kv2.2, although neuron subpopulations show preferential expression of Kv2.1 or Kv2.2. We find that Kv2 protein expression and subcellular localization are similar between mouse and human DRG neurons. We conclude that the organization of both Kv2 channels is consistent with physiological roles in the somata and stem axons of DRG neurons. The general prevalence of Kv2.2 in DRG as compared to central neurons and the enrichment of Kv2.2 relative to detectable Kv2.1 in older mice, proprioceptors, and axons suggest more widespread roles for Kv2.2 in DRG neurons.

Single-Cell Analysis of Rohon-Beard Neurons Implicates Fgf Signaling in Axon Maintenance and Cell Survival.

Peripheral sensory neurons are a critical part of the nervous system that transmit a multitude of sensory stimuli to the central nervous system. During larval and juvenile stages in zebrafish, this function is mediated by Rohon-Beard somatosensory neurons (RBs). RBs are optically accessible and amenable to experimental manipulation, making them a powerful system for mechanistic investigation of sensory neurons. Previous studies provided evidence that RBs fall into multiple subclasses; however, the number and molecular makeup of these potential RB subtypes have not been well defined. Using a single-cell RNA sequencing (scRNA-seq) approach, we demonstrate that larval RBs in zebrafish fall into three, largely nonoverlapping classes of neurons. We also show that RBs are molecularly distinct from trigeminal neurons in zebrafish. Cross-species transcriptional analysis indicates that one RB subclass is similar to a mammalian group of A-fiber sensory neurons. Another RB subclass is predicted to sense multiple modalities, including mechanical stimulation and chemical irritants. We leveraged our scRNA-seq data to determine that the fibroblast growth factor (Fgf) pathway is active in RBs. Pharmacological and genetic inhibition of this pathway led to defects in axon maintenance and RB cell death. Moreover, this can be phenocopied by treatment with dovitinib, an FDA-approved Fgf inhibitor with a common side effect of peripheral neuropathy. Importantly, dovitinib-mediated axon loss can be suppressed by loss of Sarm1, a positive regulator of neuronal cell death and axonal injury. This offers a molecular target for future clinical intervention to fight neurotoxic effects of this drug.

G protein-coupled receptor-based thermosensation determines temperature acclimatization of Caenorhabditis elegans.

Animals must sense and acclimatize to environmental temperatures for survival, yet their thermosensing mechanisms other than transient receptor potential (TRP) channels remain poorly understood. We identify a trimeric G protein-coupled receptor (GPCR), SRH-40, which confers thermosensitivity in sensory neurons regulating temperature acclimatization in Caenorhabditis elegans. Systematic knockdown of 1000 GPCRs by RNAi reveals GPCRs involved in temperature acclimatization, among which srh-40 is highly expressed in the ADL sensory neuron, a temperature-responsive chemosensory neuron, where TRP channels act as accessorial thermoreceptors. In vivo Ca2+ imaging demonstrates that an srh-40 mutation reduced the temperature sensitivity of ADL, resulting in supranormal temperature acclimatization. Ectopically expressing SRH-40 in a non-warmth-sensing gustatory neuron confers temperature responses. Moreover, temperature-dependent SRH-40 activation is reconstituted in Drosophila S2R+ cells. Overall, SRH-40 may be involved in thermosensory signaling underlying temperature acclimatization. We propose a dual thermosensing machinery through a GPCR and TRP channels in a single sensory neuron.

Better than being aPARt: S. aureus itches to get close to sensory neurons.

In a recent issue of Cell, Deng et al. show that S. aureus serine protease V8 triggers itch, independent of inflammation, by activating sensory neurons through PAR1. This study presents mechanistic insights into pruritogenic bacteria and their interactions with sensory neurons while providing a possible approach for treating itch-related diseases.

Making sense of proprioception.

Proprioception - the sense of body position in space - is intimately linked to motor control. Here, we briefly review the current knowledge of the proprioceptive system and how advances in the genetic characterisation of proprioceptive sensory neurons in mice promise to dissect its role in health and disease.

Role of proprioceptors in chronic musculoskeletal pain.

Proprioceptors are non-nociceptive low-threshold mechanoreceptors. However, recent studies have shown that proprioceptors are acid-sensitive and express a variety of proton-sensing ion channels and receptors. Accordingly, although proprioceptors are commonly known as mechanosensing neurons that monitor muscle contraction status and body position, they may have a role in the development of pain associated with tissue acidosis. In clinical practice, proprioception training is beneficial for pain relief. Here we summarize the current evidence to sketch a different role of proprioceptors in 'non-nociceptive pain' with a focus on their acid-sensing properties.

Proprioceptive innervation of the human lips.

The objective of this study was to analyze the proprioceptive innervation of human lips, especially of the orbicularis oris muscle, since it is classically accepted that facial muscles lack typical proprioceptors, that is, muscle spindles, but recently this has been doubted. Upper and lower human lips (n = 5) from non-embalmed frozen cadavers were immunostained for detection of S100 protein (to identify nerves and sensory nerve formations), myosin heavy chain (to label muscle fibers within muscle spindles), and the mechano-gated ion channel PIEZO2. No muscle spindles were found, but there was a high density of sensory nerve formations, which were morphologically heterogeneous, and in some cases resemble Ruffini-like and Pacinian sensory corpuscles. The axons of these sensory formations displayed immunoreactivity for PIEZO2. Human lip muscles lack typical proprioceptors but possess a dense sensory innervation which can serve the lip proprioception.

Assessing spontaneous sensory neuron activity using in vivo calcium imaging.

ABSTRACT: Heightened spontaneous activity in sensory neurons is often reported in individuals living with chronic pain. It is possible to study this activity in rodents using electrophysiology, but these experiments require great skill and can be prone to bias. Here, we have examined whether in vivo calcium imaging with GCaMP6s can be used as an alternative approach. We show that spontaneously active calcium transients can be visualised in the fourth lumbar dorsal root ganglion (L4 DRG) through in vivo imaging in a mouse model of inflammatory pain. Application of lidocaine to the nerve, between the inflamed site and the DRG, silenced spontaneous firing and revealed the true baseline level of calcium for spontaneously active neurons. We used these data to train a machine learning algorithm to predict when a neuron is spontaneously active. We show that our algorithm is accurate in 2 different models of pain: intraplantar complete Freund adjuvant and antigen-induced arthritis, with accuracies of 90.0% ±1.2 and 85.9% ±2.1, respectively, assessed against visual inspection by an experienced observer. The algorithm can also detect neuronal activity in imaging experiments generated in a different laboratory using a different microscope configuration (accuracy = 94.0% ±2.2). We conclude that in vivo calcium imaging can be used to assess spontaneous activity in sensory neurons and provide a Google Colaboratory Notebook to allow anyone easy access to our novel analysis tool, for the assessment of spontaneous neuronal activity in their own imaging setups.

Peptide toxins that target vertebrate voltage-gated sodium channels underly the painful stings of harvester ants.

Harvester ants (genus Pogonomyrmex) are renowned for their stings which cause intense, long-lasting pain, and other neurotoxic symptoms in vertebrates. Here, we show that harvester ant venoms are relatively simple and composed largely of peptide toxins. One class of peptides is primarily responsible for the long-lasting local pain of envenomation via activation of peripheral sensory neurons. These hydrophobic, cysteine-free peptides potently modulate mammalian voltage-gated sodium (NaV) channels, reducing the voltage threshold for activation and inhibiting channel inactivation. These toxins appear to have evolved specifically to deter vertebrates.

Opposing chemosensory functions of closely related gustatory receptors.

In the fruit fly Drosophila melanogaster, gustatory sensory neurons express taste receptors that are tuned to distinct groups of chemicals, thereby activating neural ensembles that elicit either feeding or avoidance behavior. Members of a family of ligand -gated receptor channels, the Gustatory receptors (Grs), play a central role in these behaviors. In general, closely related, evolutionarily conserved Gr proteins are co-expressed in the same type of taste neurons, tuned to chemically related compounds, and therefore triggering the same behavioral response. Here, we report that members of the Gr28 subfamily are expressed in largely non-overlapping sets of taste neurons in Drosophila larvae, detect chemicals of different valence, and trigger opposing feeding behaviors. We determined the intrinsic properties of Gr28 neurons by expressing the mammalian Vanilloid Receptor 1 (VR1), which is activated by capsaicin, a chemical to which wild-type Drosophila larvae do not respond. When VR1 is expressed in Gr28a neurons, larvae become attracted to capsaicin, consistent with reports showing that Gr28a itself encodes a receptor for nutritious RNA. In contrast, expression of VR1 in two pairs of Gr28b.c neurons triggers avoidance to capsaicin. Moreover, neuronal inactivation experiments show that the Gr28b.c neurons are necessary for avoidance of several bitter compounds. Lastly, behavioral experiments of Gr28 deficient larvae and live Ca2+ imaging studies of Gr28b.c neurons revealed that denatonium benzoate, a synthetic bitter compound that shares structural similarities with natural bitter chemicals, is a ligand for a receptor complex containing a Gr28b.c or Gr28b.a subunit. Thus, the Gr28 proteins, which have been evolutionarily conserved over 260 million years in insects, represent the first taste receptor subfamily in which specific members mediate behavior with opposite valence.

Development of a Hypersensitivity Evaluation Method for Cultured Sensory Neurons Using Electrical Activity Recording.

Investigation of hypersensitivity caused by peripheral sensitization progression is important for developing novel pain treatments. Existing methods cannot record plastic changes in neuronal activity because they occur over a few days. We aimed to establish an efficient method to evaluate neuronal activity alterations caused by peripheral sensitization on high-density microelectrode arrays (HD-MEAs) which can record neuronal activity for a long time. Rat dorsal root ganglion (DRG) neurons were dissected from rat embryos and cultured on HD-MEAs. DRG neurons were labeled with NeuO, live staining dye. Neurons were detected with the fluorescence signal and electrodes were selected with the fluorescence images. The number of DRG neurons, whose activity were recorded, detected based on fluorescence observation was five times greater than that based on neuronal activity. Analysis of changes in neuronal activity observed in pharmacological stimulation experiments suggested that substance P induced peripheral sensitization and enhanced capsaicin sensitivity. In addition, results of immunofluorescence staining suggested that peripheral sensitization occurred mostly in neurons that co-expressed transient receptor potential vanilloid 1 (TRPV1) and neurokinin 1 receptor (NK1R). In conclusion, we established an efficient method for assessing the effects of peripheral sensitization on DRG neurons cultured on HD-MEAs.

Vagal sensory neurons mediate the Bezold-Jarisch reflex and induce syncope.

Visceral sensory pathways mediate homeostatic reflexes, the dysfunction of which leads to many neurological disorders1. The Bezold-Jarisch reflex (BJR), first described2,3 in 1867, is a cardioinhibitory reflex that is speculated to be mediated by vagal sensory neurons (VSNs) that also triggers syncope. However, the molecular identity, anatomical organization, physiological characteristics and behavioural influence of cardiac VSNs remain mostly unknown. Here we leveraged single-cell RNA-sequencing data and HYBRiD tissue clearing4 to show that VSNs that express neuropeptide Y receptor Y2 (NPY2R) predominately connect the heart ventricular wall to the area postrema. Optogenetic activation of NPY2R VSNs elicits the classic triad of BJR responses-hypotension, bradycardia and suppressed respiration-and causes an animal to faint. Photostimulation during high-resolution echocardiography and laser Doppler flowmetry with behavioural observation revealed a range of phenotypes reflected in clinical syncope, including reduced cardiac output, cerebral hypoperfusion, pupil dilation and eye-roll. Large-scale Neuropixels brain recordings and machine-learning-based modelling showed that this manipulation causes the suppression of activity across a large distributed neuronal population that is not explained by changes in spontaneous behavioural movements. Additionally, bidirectional manipulation of the periventricular zone had a push-pull effect, with inhibition leading to longer syncope periods and activation inducing arousal. Finally, ablating NPY2R VSNs specifically abolished the BJR. Combined, these results demonstrate a genetically defined cardiac reflex that recapitulates characteristics of human syncope at physiological, behavioural and neural network levels.

Specific sensory neurons and insulin-like peptides modulate food type-dependent oogenesis and fertilization in Caenorhabditis elegans.

An animal's responses to environmental cues are critical for its reproductive program. Thus, a mechanism that allows the animal to sense and adjust to its environment should make for a more efficient reproductive physiology. Here, we demonstrate that in Caenorhabditis elegans specific sensory neurons influence onset of oogenesis through insulin signaling in response to food-derived cues. The chemosensory neurons ASJ modulate oogenesis onset through the insulin-like peptide (ILP) INS-6. In contrast, other sensory neurons, the olfactory neurons AWA, regulate food type-dependent differences in C. elegans fertilization rates, but not onset of oogenesis. AWA modulates fertilization rates at least partly in parallel to insulin receptor signaling, since the insulin receptor DAF-2 regulates fertilization independently of food type, which requires ILPs other than INS-6. Together our findings suggest that optimal reproduction requires the integration of diverse food-derived inputs through multiple neuronal signals acting on the C. elegans germline.

Cold-Temperature Coding with Bursting and Spiking Based on TRP Channel Dynamics in Drosophila Larva Sensory Neurons.

Temperature sensation involves thermosensitive TRP (thermoTRP) and non-TRP channels. Drosophila larval Class III (CIII) neurons serve as the primary cold nociceptors and express a suite of thermoTRP channels implicated in noxious cold sensation. How CIII neurons code temperature remains unclear. We combined computational and electrophysiological methods to address this question. In electrophysiological experiments, we identified two basic cold-evoked patterns of CIII neurons: bursting and spiking. In response to a fast temperature drop to noxious cold, CIII neurons distinctly mark different phases of the stimulus. Bursts frequently occurred along with the fast temperature drop, forming a peak in the spiking rate and likely coding the high rate of the temperature change. Single spikes dominated at a steady temperature and exhibited frequency adaptation following the peak. When temperature decreased slowly to the same value, mainly spiking activity was observed, with bursts occurring sporadically throughout the stimulation. The spike and the burst frequencies positively correlated with the rate of the temperature drop. Using a computational model, we explain the distinction in the occurrence of the two CIII cold-evoked patterns bursting and spiking using the dynamics of a thermoTRP current. A two-parameter activity map (Temperature, constant TRP current conductance) marks parameters that support silent, spiking, and bursting regimes. Projecting on the map the instantaneous TRP conductance, governed by activation and inactivation processes, reflects temperature coding responses as a path across silent, spiking, or bursting domains on the map. The map sheds light on how various parameter sets for TRP kinetics represent various types of cold-evoked responses. Together, our results indicate that bursting detects the high rate of temperature change, whereas tonic spiking could reflect both the rate of change and magnitude of steady cold temperature.

The making of a proprioceptor: a tale of two identities.

Proprioception, the sense of body position in space, has a critical role in the control of posture and movement. Aside from skin and joint receptors, the main sources of proprioceptive information in tetrapods are mechanoreceptive end organs in skeletal muscle: muscle spindles (MSs) and Golgi tendon organs (GTOs). The sensory neurons that innervate these receptors are divided into subtypes that detect discrete aspects of sensory information from muscles with different biomechanical functions. Despite the importance of proprioceptive neurons in motor control, the developmental mechanisms that control the acquisition of their distinct functional properties and positional identity are not yet clear. In this review, we discuss recent findings on the development of mouse proprioceptor subtypes and challenges in defining them at the molecular and functional level.

Pain and itch coding mechanisms of polymodal sensory neurons.

Pain and itch coding mechanisms in polymodal sensory neurons remain elusive. MrgprD+ neurons represent a major polymodal population and mediate both mechanical pain and nonhistaminergic itch. Here, we show that chemogenetic activation of MrgprD+ neurons elicited both pain- and itch-related behavior in a dose-dependent manner, revealing an unanticipated compatibility between pain and itch in polymodal neurons. While VGlut2-dependent glutamate release is required for both pain and itch transmission from MrgprD+ neurons, the neuropeptide neuromedin B (NMB) is selectively required for itch signaling. Electrophysiological recordings further demonstrated that glutamate synergizes with NMB to excite NMB-sensitive postsynaptic neurons. Ablation of these spinal neurons selectively abolished itch signals from MrgprD+ neurons, without affecting pain signals, suggesting a dedicated itch-processing central circuit. These findings reveal distinct neurotransmitters and neural circuit requirements for pain and itch signaling from MrgprD+ polymodal sensory neurons, providing new insights on coding and processing of pain and itch.

Chemogenetic Silencing of NaV1.8-Positive Sensory Neurons Reverses Chronic Neuropathic and Bone Cancer Pain in FLEx PSAM4-GlyR Mice.

Drive from peripheral neurons is essential in almost all pain states, but pharmacological silencing of these neurons to effect analgesia has proved problematic. Reversible gene therapy using long-lived chemogenetic approaches is an appealing option. We used the genetically activated chloride channel PSAM4-GlyR to examine pain pathways in mice. Using recombinant AAV9-based delivery to sensory neurons, we found a reversal of acute pain behavior and diminished neuronal activity using in vitro and in vivo GCaMP imaging on activation of PSAM4-GlyR with varenicline. A significant reduction in inflammatory heat hyperalgesia and oxaliplatin-induced cold allodynia was also observed. Importantly, there was no impairment of motor coordination, but innocuous von Frey sensation was inhibited. We generated a transgenic mouse that expresses a CAG-driven FLExed PSAM4-GlyR downstream of the Rosa26 locus that requires Cre recombinase to enable the expression of PSAM4-GlyR and tdTomato. We used NaV1.8 Cre to examine the role of predominantly nociceptive NaV1.8+ neurons in cancer-induced bone pain (CIBP) and neuropathic pain caused by chronic constriction injury (CCI). Varenicline activation of PSAM4-GlyR in NaV1.8-positive neurons reversed CCI-driven mechanical, thermal, and cold sensitivity. Additionally, varenicline treatment of mice with CIBP expressing PSAM4-GlyR in NaV1.8+ sensory neurons reversed cancer pain as assessed by weight-bearing. Moreover, when these mice were subjected to acute pain assays, an elevation in withdrawal thresholds to noxious mechanical and thermal stimuli was detected, but innocuous mechanical sensations remained unaffected. These studies confirm the utility of PSAM4-GlyR chemogenetic silencing in chronic pain states for mechanistic analysis and potential future therapeutic use.

Distinctive features of the central synaptic organization of Drosophila larval proprioceptors.

Proprioceptive feedback is critically needed for locomotor control, but how this information is incorporated into central proprioceptive processing circuits remains poorly understood. Circuit organization emerges from the spatial distribution of synaptic connections between neurons. This distribution is difficult to discern in model systems where only a few cells can be probed simultaneously. Therefore, we turned to a relatively simple and accessible nervous system to ask: how are proprioceptors' input and output synapses organized in space, and what principles underlie this organization? Using the Drosophila larval connectome, we generated a map of the input and output synapses of 34 proprioceptors in several adjacent body segments (5-6 left-right pairs per segment). We characterized the spatial organization of these synapses, and compared this organization to that of other somatosensory neurons' synapses. We found three distinguishing features of larval proprioceptor synapses: (1) Generally, individual proprioceptor types display segmental somatotopy. (2) Proprioceptor output synapses both converge and diverge in space; they are organized into six spatial domains, each containing a unique set of one or more proprioceptors. Proprioceptors form output synapses along the proximal axonal entry pathway into the neuropil. (3) Proprioceptors receive few inhibitory input synapses. Further, we find that these three features do not apply to other larval somatosensory neurons. Thus, we have generated the most comprehensive map to date of how proprioceptor synapses are centrally organized. This map documents previously undescribed features of proprioceptors, raises questions about underlying developmental mechanisms, and has implications for downstream proprioceptive processing circuits.