USH2A is a Meissner's corpuscle protein necessary for normal vibration sensing in mice and humans.

Fingertip mechanoreceptors comprise sensory neuron endings together with specialized skin cells that form the end-organ. Exquisitely sensitive, vibration-sensing neurons are associated with Meissner's corpuscles in the skin. In the present study, we found that USH2A, a transmembrane protein with a very large extracellular domain, was found in terminal Schwann cells within Meissner's corpuscles. Pathogenic USH2A mutations cause Usher's syndrome, associated with hearing loss and visual impairment. We show that patients with biallelic pathogenic USH2A mutations also have clear and specific impairments in vibrotactile touch perception, as do mutant mice lacking USH2A. Forepaw rapidly adapting mechanoreceptors innervating Meissner's corpuscles, recorded from Ush2a-/- mice, showed large reductions in vibration sensitivity. However, the USH2A protein was not found in sensory neurons. Thus, loss of USH2A in corpuscular end-organs reduced mechanoreceptor sensitivity as well as vibration perception. Thus, a tether-like protein is required to facilitate detection of small-amplitude vibrations essential for the perception of fine-grained tactile surfaces.

Incidence and linguistic quality of speech errors: a comparison of preoperative transcranial magnetic stimulation and intraoperative direct cortex stimulation.

OBJECTIVE: Given the interindividual variance of functional language anatomy, risk prediction based merely on anatomical data is insufficient in language area-related brain tumor surgery, suggesting the need for direct cortical and subcortical mapping during awake surgery. Reliable, noninvasive preoperative methods of language localization hold the potential for reducing the necessity for awake procedures and may improve patient counseling and surgical planning. Repetitive navigated transcranial magnetic stimulation (rnTMS) is an evolving tool for localizing language-eloquent areas. The aim of this study was to investigate the reliability of rnTMS in locating cortical language sites. METHODS: Twenty-five patients with brain tumors in speech-related areas were prospectively evaluated with preoperative rnTMS (5 Hz, train of five, average 105% resting motor threshold) and navigated direct cortical stimulation (DCS; bipolar, 50 Hz, 6-8 mA, 200-µsec pulse width) during awake surgeries employing a picture-naming task. Positive and negative stimulation spots within the craniotomy were documented in the same MRI data set. TMS and DCS language-positive areas were compared with regard to their spatial overlap, their allocation in a cortical parcellation system, and their linguistic qualities. RESULTS: There were over twofold more positive language spots within the exposed area on rnTMS than on DCS. The comparison of positive rnTMS and DCS (ground truth) overlaps revealed low sensitivity (35%) and low positive predictive value (16%) but high specificity (90%) and high negative predictive value (96%). Within the overlaps, there was no correlation in error quality. On DCS, 73% of language-positive spots were located in the pars opercularis and pars triangularis of the frontal operculum and 24% within the supramarginal gyrus and dorsal portion of the superior temporal gyrus, while on rnTMS language positivity was distributed more evenly over a large number of gyri. CONCLUSIONS: The current protocol for rnTMS for language mapping identified language-negative sites with good dependability but was unable to reliably detect language-positive spots. Further refinements of the technique will be needed to establish rnTMS language mapping as a useful clinical tool.

Congenital deafness is associated with specific somatosensory deficits in adolescents.

Hearing and touch represent two distinct sensory systems that both rely on the transformation of mechanical force into electrical signals. Here we used a battery of quantitative sensory tests to probe touch, thermal and pain sensitivity in a young control population (14-20 years old) compared to age-matched individuals with congenital hearing loss. Sensory testing was performed on the dominant hand of 111 individuals with normal hearing and 36 with congenital hearing loss. Subjects with congenital deafness were characterized by significantly higher vibration detection thresholds at 10 Hz (2-fold increase, P < 0.001) and 125 Hz (P < 0.05) compared to controls. These sensory changes were not accompanied by any major change in measures of pain perception. We also observed a highly significant reduction (30% compared to controls p < 0.001) in the ability of hearing impaired individual's ability to detect cooling which was not accompanied by changes in warm detection. At least 60% of children with non-syndromic hearing loss showed very significant loss of vibration detection ability (at 10 Hz) compared to age-matched controls. We thus propose that many pathogenic mutations that cause childhood onset deafness may also play a role in the development or functional maintenance of somatic mechanoreceptors.

Kasuistik: Late-onset Small-Fiber-Neuropathie nach kritischer Erkrankung.

A 42-year-old patient presented with acute allodynia and hyperalgesia in her distal limbs, most severe in the innervation area of the ulnar nerve. The patient developed critical illness myopathy/polyneuropathy after septic shock 5 months prior to her presentation. After exclusion of differential diagnosis, "late onset small fiber neuropathy" after critical illness was diagnosed. Recent studies showed small fiber lesions during critical illness and in follow-up exams, where additionally neuropathic pain were proved. Dysfunction of voltage-gated Sodium channels related to severe insulin resistance during critical illness might explain the pathophysiology, as seen in critical illness myopathy/polyneuropathy. Therefore we recommend cooling, pharmacotherapy with carbamazepin/oxcarbazepine, tricyclic antidepressive agents and peripheral nerve blocks to treat patients with "late onset small fiber neuropathy" after critical illness.

Measurement of Vibration Detection Threshold and Tactile Spatial Acuity in Human Subjects.

Tests that allow the precise determination of psychophysical thresholds for vibration and grating orientation provide valuable information about mechanosensory function that are relevant for clinical diagnosis as well as for basic research. Here, we describe two psychophysical tests designed to determine the vibration detection threshold (automated system) and tactile spatial acuity (handheld device). Both procedures implement a two-interval forced-choice and a transformed-rule up and down experimental paradigm. These tests have been used to obtain mechanosensory profiles for individuals from distinct human cohorts such as twins or people with sensorineural deafness.

A Molecular Signature of Myalgia in Myotonic Dystrophy 2.

BACKGROUND: Chronic muscle pain affects close to 20% of the population and is a major health burden. The underlying mechanisms of muscle pain are difficult to investigate as pain presents in patients with very diverse histories. Treatment options are therefore limited and not tailored to underlying mechanisms. To gain insight into the pathophysiology of myalgia we investigated a homogeneous group of patients suffering from myotonic dystrophy type 2 (DM2), a monogenic disorder presenting with myalgia in at least 50% of affected patients. METHODS: After IRB approval we performed an observational cross-sectional cohort study and recruited 42 patients with genetically confirmed DM2 plus 20 healthy age and gender matched control subjects. All participants were subjected to an extensive sensory-testing protocol. In addition, RNA sequencing was performed from 12 muscle biopsy specimens obtained from DM2 patients. FINDINGS: Clinical sensory testing as well as RNA sequencing clearly separated DM2 myalgic from non-myalgia patients and also from healthy controls. In particular pressure pain thresholds were significantly lowered for all muscles tested in myalgic DM2 patients but were not significantly different between non-myalgic patients and healthy controls. The expression of fourteen muscle expressed genes in myalgic patients was significantly up or down-regulated in myalgic compared to non-myalgic DM2 patients. INTERPRETATION: Our data support the idea that molecular changes in the muscles of DM2 patients are associated with muscle pain. Further studies should address whether muscle-specific molecular pathways play a significant role in myalgia in order to facilitate the development of mechanism-based therapeutic strategies to treat musculoskeletal pain. FUNDING: This study was funded by the German Research Society (DFG, GK1631), KAP programme of Charité Universitätsmedizin Berlin and Max Delbrück Center for Molecular Medicine.

A Probabilistic Model for Estimating the Depth and Threshold Temperature of C-fiber Nociceptors.

The subjective experience of thermal pain follows the detection and encoding of noxious stimuli by primary afferent neurons called nociceptors. However, nociceptor morphology has been hard to access and the mechanisms of signal transduction remain unresolved. In order to understand how heat transducers in nociceptors are activated in vivo, it is important to estimate the temperatures that directly activate the skin-embedded nociceptor membrane. Hence, the nociceptor's temperature threshold must be estimated, which in turn will depend on the depth at which transduction happens in the skin. Since the temperature at the receptor cannot be accessed experimentally, such an estimation can currently only be achieved through modeling. However, the current state-of-the-art model to estimate temperature at the receptor suffers from the fact that it cannot account for the natural stochastic variability of neuronal responses. We improve this model using a probabilistic approach which accounts for uncertainties and potential noise in system. Using a data set of 24 C-fibers recorded in vitro, we show that, even without detailed knowledge of the bio-thermal properties of the system, the probabilistic model that we propose here is capable of providing estimates of threshold and depth in cases where the classical method fails.

Photoswitchable fatty acids enable optical control of TRPV1.

Fatty acids (FAs) are not only essential components of cellular energy storage and structure, but play crucial roles in signalling. Here we present a toolkit of photoswitchable FA analogues (FAAzos) that incorporate an azobenzene photoswitch along the FA chain. By modifying the FAAzos to resemble capsaicin, we prepare a series of photolipids targeting the Vanilloid Receptor 1 (TRPV1), a non-selective cation channel known for its role in nociception. Several azo-capsaicin derivatives (AzCAs) emerge as photoswitchable agonists of TRPV1 that are relatively inactive in the dark and become active on irradiation with ultraviolet-A light. This effect can be rapidly reversed by irradiation with blue light and permits the robust optical control of dorsal root ganglion neurons and C-fibre nociceptors with precision timing and kinetics not available with any other technique. More generally, we expect that photolipids will find many applications in controlling biological pathways that rely on protein-lipid interactions.

Skin-nerve preparation to assay the function of opioid receptors in peripheral endings of sensory neurons.

This chapter describes the methodology of the in vitro skin-saphenous nerve preparation and its application to test for the modulatory effects of opioids on the function of cutaneous sensory neurons in experimental models of pain. We detail the skin-nerve setup requirements and the technique to record action potentials from single sensory fibers. We address how to test for inhibitory effects of opioid receptor activation on mechanical and thermal sensitivity of nociceptors and mechanoreceptors in the complete Freund's adjuvant-induced inflammation and the chronic constriction injury model of neuropathic pain.

Piezo2 is the major transducer of mechanical forces for touch sensation in mice.

The sense of touch provides critical information about our physical environment by transforming mechanical energy into electrical signals. It is postulated that mechanically activated cation channels initiate touch sensation, but the identity of these molecules in mammals has been elusive. Piezo2 is a rapidly adapting, mechanically activated ion channel expressed in a subset of sensory neurons of the dorsal root ganglion and in cutaneous mechanoreceptors known as Merkel-cell-neurite complexes. It has been demonstrated that Merkel cells have a role in vertebrate mechanosensation using Piezo2, particularly in shaping the type of current sent by the innervating sensory neuron; however, major aspects of touch sensation remain intact without Merkel cell activity. Here we show that mice lacking Piezo2 in both adult sensory neurons and Merkel cells exhibit a profound loss of touch sensation. We precisely localize Piezo2 to the peripheral endings of a broad range of low-threshold mechanoreceptors that innervate both hairy and glabrous skin. Most rapidly adapting, mechanically activated currents in dorsal root ganglion neuronal cultures are absent in Piezo2 conditional knockout mice, and ex vivo skin nerve preparation studies show that the mechanosensitivity of low-threshold mechanoreceptors strongly depends on Piezo2. This cellular phenotype correlates with an unprecedented behavioural phenotype: an almost complete deficit in light-touch sensation in multiple behavioural assays, without affecting other somatosensory functions. Our results highlight that a single ion channel that displays rapidly adapting, mechanically activated currents in vitro is responsible for the mechanosensitivity of most low-threshold mechanoreceptor subtypes involved in innocuous touch sensation. Notably, we find that touch and pain sensation are separable, suggesting that as-yet-unknown mechanically activated ion channel(s) must account for noxious (painful) mechanosensation.

Stomatin-domain protein interactions with acid-sensing ion channels modulate nociceptor mechanosensitivity.

Acid-sensing ion channels (ASICs) and their interaction partners of the stomatin family have all been implicated in sensory transduction. Single gene deletion of asic3, asic2, stomatin, or stoml3 all result in deficits in the mechanosensitivity of distinct cutaneous afferents in the mouse. Here, we generated asic3(-/-):stomatin(-/-), asic3(-/-):stoml3(-/-) and asic2(-/-):stomatin(-/-) double mutant mice to characterize the functional consequences of stomatin-ASIC protein interactions on sensory afferent mechanosensitivity. The absence of ASIC3 led to a clear increase in mechanosensitivity in rapidly adapting mechanoreceptors (RAMs) and a decrease in the mechanosensitivity in both Aδ- and C-fibre nociceptors. The increased mechanosensitivity of RAMs could be accounted for by a loss of adaptation which could be mimicked by local application of APETx2 a toxin that specifically blocks ASIC3. There is a substantial loss of mechanosensitivity in stoml3(-/-) mice in which ∼35% of the myelinated fibres lack a mechanosensitive receptive field and this phenotype was found to be identical in asic3(-/-):stoml3(-/-) mutant mice. However, Aδ-nociceptors showed much reduced mechanosensitivity in asic3(-/-):stoml3(-/-) mutant mice compared to asic3(-/)(-) controls. Interestingly, in asic2(-/-):stomatin(-/-) mutant mice many Aδ-nociceptors completely lost their mechanosensitivity which was not observed in asic2(-/-) or stomatin(-/-) mice. Examination of stomatin(-/-):stoml3(-/-) mutant mice indicated that a stomatin/STOML3 interaction is unlikely to account for the greater Aδ-nociceptor deficits in double mutant mice. A key finding from these studies is that the loss of stomatin or STOML3 in asic3(-/-) or asic2(-/-) mutant mice markedly exacerbates deficits in the mechanosensitivity of nociceptors without affecting mechanoreceptor function.

Fentanyl decreases discharges of C and A nociceptors to suprathreshold mechanical stimulation in chronic inflammation.

An essential component of mechanical hyperalgesia resulting from tissue injury is an enhanced excitability of nociceptive neurons, termed mechanical sensitization. Local application of opioids to inflamed rat paws attenuates mechanical hyperalgesia and reduces electrical excitability of C-fiber nociceptors in acute injury. Here, we examined the effects of the opioid receptor agonist fentanyl on the mechanical coding properties of not only C- but also A-fiber nociceptors innervating the rat hind paw in a model of chronic pain, i.e., 4 days after Freund's complete adjuvant-induced inflammation. The peripheral mechanosensitive terminals of C-fibers (n = 143), A-fibers (n = 79), and low-threshold mechanoreceptors (n = 25) were characterized using the in vitro skin-nerve preparation from the saphenous nerve. Although mechanical activation thresholds were not changed, discharges to suprathreshold mechanical stimuli were elevated significantly in both A- and C-fiber nociceptors from inflamed tissue. In addition, the proportion of nociceptors as well as the frequency of spontaneous discharges in A (14% vs. 0%)- and C (28% vs. 8%)-fibers were increased in inflamed compared with normal tissue. Fentanyl inhibited responses to suprathreshold stimuli in a significantly higher proportion of not only C (36% vs. 7%)- but also A (41% vs. 8%)-fibers in inflamed tissue in a naloxone-reversible and concentration-dependent manner. Our results demonstrate that mechanical sensitization persists in chronic inflammation, in correlation with behavioral hyperalgesia. Opioid sensitivity of both A- and C-fibers is markedly augmented. This is consistent with an upregulation or enhanced functionality of opioid receptors located at the peripheral terminals of sensitized nociceptors.

Modulation of tight junction proteins in the perineurium to facilitate peripheral opioid analgesia.

BACKGROUND: Peripheral application of opioids reduces inflammatory pain but is less effective in noninflamed tissue of rats and human patients. Hypertonic solutions can facilitate the antinociceptive activity of hydrophilic opioids in noninflamed tissue in vivo. However, the underlying mechanisms are not well understood. We hypothesized that the enhanced efficacy of opioids may be because of opening of the perineurial barrier formed by tight junction-proteins like claudin-1. METHODS: Male Wistar rats were treated intraplantarly with 10% NaCl. Pain behavior (n = 6) and electrophysiological recordings (n = 9 or more) from skin-nerve preparations after local application of the opioid [d-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAMGO) were explored. Tight junction-proteins as well as permeability of the barrier were examined by immunohistochemistry and Western blot (n = 3 or more). RESULTS: Local administration of 10% NaCl facilitated increased mechanical nociceptive thresholds in response to DAMGO, penetration of horseradish peroxidase into the nerve, as well as a reduced response of C- but not Aδ-nociceptors to mechanical stimulation after application of DAMGO in the skin-nerve preparation. In noninflamed paw tissue, claudin-1 was expressed in the epidermis, blood vessels, and the perineurium, surrounding neurons immunoreactive for calcitonin gene-related peptide or protein gene product 9.5. Claudin-1 but not claudin-5 or occludin was significantly reduced after pretreatment with 10% NaCl. Intraplantar application of a metalloproteinase inhibitor (GM6001) completely reversed these effects. CONCLUSION: Hypertonic saline opens the perineurial barrier via metalloproteinase activation and claudin-1 regulation, thereby allowing access of hydrophilic drugs to peripheral opioid receptors. This principle may be used to specifically target hydrophilic drugs to peripheral neurons.

Speed and temperature dependences of mechanotransduction in afferent fibers recorded from the mouse saphenous nerve.

Here we have systematically characterized the stimulus response properties of mechanosensitive sensory fibers in the mouse saphenous nerve. We tested mechanoreceptors and nociceptors with defined displacement stimuli of varying amplitude and velocity. For each sensory afferent investigated we measured the mechanical latency, which is the delay between the onset of a ramp displacement and the first evoked spike, corrected for conduction delay. Mechanical latency plotted as a function of stimulus strength was very characteristic for each receptor type and was very short for rapidly adapting mechanoreceptors (<11 ms) but very long in myelinated and unmyelinated nociceptors (49-114 ms). Increasing the stimulus speed decreased mechanical latency in all receptor types with the notable exception of C-fiber nociceptors, in which mean mechanical latency was not reduced less, similar100 ms, even with very fast ramp stimuli (2,945 microm/s). We examined stimulus response functions and mechanical latency at two different temperatures (24 and 32 degrees C) and found that stimulus response properties of almost all mechanoreceptors were not altered in this range. A notable exception to this rule was found for C-fibers in which mechanical latency was substantially increased and stimulus response functions decreased at lower temperatures. We calculated Q(10) values for mechanical latency in C-fibers to be 5.1; in contrast, the Q(10) value for conduction velocity for the same fibers was 1.4. Finally, we examined the effects of short-term inflammation (2-6 h) induced by carrageenan on nociceptor and mechanoreceptor sensitivity. We did not detect robust changes in mechanical latency or stimulus response functions after inflammation that might have reflected mechanical sensitization under the conditions tested.

A stomatin-domain protein essential for touch sensation in the mouse.

Touch and mechanical pain are first detected at our largest sensory surface, the skin. The cell bodies of sensory neurons that detect such stimuli are located in the dorsal root ganglia, and subtypes of these neurons are specialized to detect specific modalities of mechanical stimuli. Molecules have been identified that are necessary for mechanosensation in invertebrates but so far not in mammals. In Caenorhabditis elegans, mec-2 is one of several genes identified in a screen for touch insensitivity and encodes an integral membrane protein with a stomatin homology domain. Here we show that about 35% of skin mechanoreceptors do not respond to mechanical stimuli in mice with a mutation in stomatin-like protein 3 (SLP3, also called Stoml3), a mammalian mec-2 homologue that is expressed in sensory neurons. In addition, mechanosensitive ion channels found in many sensory neurons do not function without SLP3. Tactile-driven behaviours are also impaired in SLP3 mutant mice, including touch-evoked pain caused by neuropathic injury. SLP3 is therefore indispensable for the function of a subset of cutaneous mechanoreceptors, and our data support the idea that this protein is an essential subunit of a mammalian mechanotransducer.

Role of T-type calcium current in identified D-hair mechanoreceptor neurons studied in vitro.

Different subsets of dorsal root ganglion (DRG) mechanoreceptors transduce low- and high-intensity mechanical stimuli. It was shown recently that, in vivo, neurotrophin-4 (NT-4)-dependent D-hair mechanoreceptors specifically express a voltage-activated T-type calcium channel (Ca(v)3.2) that may be required for their mechanoreceptive function. Here we show that D-hair mechanoreceptors can be identified in vitro by a rosette-like morphology in the presence of NT-4 and that these rosette neurons are almost all absent in DRG cultures taken from NT-4 knock-out mice. In vitro identification of the D-hair mechanoreceptor allowed us to explore the electrophysiological properties of these cells. We demonstrate that the T-type Ca(v)3.2 channel induced slow membrane depolarization that contributes to lower the voltage threshold for action potential generation and controls spike latency after stimulation of D-hair mechanoreceptors. Indeed, the properties of the T-type amplifier are particularly well suited to explain the high sensitivity of D-hair mechanoreceptors to slowly moving stimuli.

Mechanosensation and pain.

The ability of cells to detect and transduce mechanical stimuli impinging on them is a fundamental process that underlies normal cell growth, hearing, balance, touch, and pain. Surprisingly, little research has focused on mechanotransduction as it relates to the sensations of somatic touch and pain. In this article we will review data on the wealth of different mechanosensitive sensory neurons that innervate our main somatic sense organ the skin. The role of different types of mechanosensitive sensory neurons in pain under physiological and pathophysiological conditions (allodynia and hyperalgesia) will also be reviewed. Finally, recent work on the cellular and molecular mechanisms by which mechanoreceptive sensory neurons signal both innocuous and noxious sensation is evaluated in the context of pain.