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

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.The present study was funded by grants from the Deutsche Forschungsgemeinshaft (grant nos. SFB665-B6 to G.R.L., SFB1315 to J.F.A.P. and SFB1158-A01 to S.G.L.) and grants from the European Research Council (grant nos. 789128 to G.R.L. and ERC-2015-CoG-682422 to J.F.A.P.). Additional funding was from the Institute of Health Carlos III (Spanish Ministry of Science and Innovation, grant no. FIS PI16/00539 to J.M.).Peer reviewe

Die Effekte von Acid-Sensing Ionkanaele ASIC3 und Stomatin-like Proteinen an Mechanosensation und Noziception

Titelblatt und Inhaltsverzeichnis Introduction Materials and Methods Results Discussion Zusammenfassung ReferencesTransformation of mechanical energy into electrical signals in mechanosensory neurons is essential for mechanosensation and nociception. This transformation occurs via sensory transduction channels that are activated by external force. Recent genetic and electrophysiological studies in Caenorhabditis elegans have directly shown that the degenerin/epithelial sodium channel (DEG/ENaC) ion channel subunits, MEC-4 and MEC-10, and the accessory ion channel subunits MEC-2 and MEC-6 form a sensory transduction ion channel within a mechanotransduction complex that also includes intra- and extracellular proteins. In mammals DEG/ENaC ion channel subunits are also proposed to function as mechanotransducers. Consistent with a function in mechanosensation, the mammalian acid-sensing ion channel subunit ASIC3 belongs to the DEG/ENaC family of ion channels; it is highly expressed in mechanosensory neurons including their peripheral structures; and it has been shown to be required for normal mechanosensation in mice. MEC-2 protein, which contains a stomatin-like domain in its central region, interacts and modulates MEC-4 ion channel activity. Mammalian stomatin-like proteins, like stomatin and stomatin-like protein (SLP3), might have similar roles. Here we show that ASIC3 coimmunoprecipitates with stomatin and SLP3 in a heterologous system. We asked whether the physical interaction between ASIC3 and stomatin proteins has any effects on mechanotransduction in mechanosensory neurons innervating skin. To look for a functional interaction between ASIC3 and stomatin in mechanosensory neurons single fiber analysis of mechanosensitivity in ASIC3/stomatin double mutant mice in the in vitro skin nerve preparation were used. The loss of ASIC3 function specifically increases mechanosensitivity in rapidly adapting mechanoreceptors (RAM) and reduces the sensitivity of nociceptors, including A-mechanonociceptors (AM) and C-fibers. In comparison, the additional loss of stomatin does not alter the increased mechanosensitivity in RAM; however, it slightly decreases the speed of response (mechanical latency). In addition, AM and C-fibers in ASIC3/stomatin double mutants show reduced mechanosensitivity that is not significantly different from the alterations due to loss of ASIC3 alone. However, polymodal nociceptors (C-MH) in ASIC3/stomatin double mutants show significant decrease in mechanosensitivity to suprathreshold stimuli compared to C-MH in ASIC3 single mutants. Therefore, the loss of stomatin produced additional alteration in mechanoreceptor function already altered by loss of ASIC3. The data suggest that ASIC3 is required for normal mechanoreceptor function and that a weak functional interaction exists between ASIC3 and stomatin.Die Transformation eines mechanischen Stimulus in einen Nervenimpuls in sensorischen Neuronen geschieht durch das Aktivieren von Transduktionsionkanälen in der Zellmembran von Nervenendungen in der Haut. Dieser Prozess wird Mechanotransduktion genannt. Er spielt eine wichtige Rolle für den Tastsinn und bei der Entstehung von Schmerz. In den letzten Jahren wurde durch genetische und electrophysiologische Untersuchungen am Caenorhabditis elegans (C.elegans) Wurm festgestellt dass die Ionenkanaluntereinheiten (MEC-4 und MEC-10) der Degenerinen/Epithelialen Natriumkanäle (DEG/ENaC) und die akzessorischen Ionenkanaluntereinheiten (MEC-2 und MEC-6) einen sensorischen Transduktionkanal bzw. einen mechanosensitiven Ionenkanal formen. Dieser mechanosensitive Ionenkanal ist mit Proteinen aus der extrazellularen Matrix und dem intrazellularen Cytoskelett verbunden. In Säugetieren sind diese molekularem Grundlagen der Mechanotransduktion bisher nicht bekannt. Diese Dissertation untersucht, ob orthologe Moleküle in Säugetieren eine ähnliche Rolle bei der Mechanotranduktion spielen, wie die Mechanotransduktionsproteine in C. elegans. Ein orthologes Molekül ist die Ionenkanaluntereinheit acid-sensing ion channel (ASIC3) der DEG/ENaC Ionenkanäle in Säugern, denn sie ist mit den Ionenkanal MEC-4 der C. elegans verwandt. Deshalb ist anzunehmen, dass ASIC3 eine ähnliche Funktion bei der Mechanotransduktion hat, wie MEC-4. Hinzu kommt, dass ähnlich MEC-4 auch ASIC3 in Nervenzellen sowie deren peripheren Nervenendungen hochexpremiert ist und für eine normale Mechanotransduktion in Mäusen erforderlich ist. MEC-2 Proteine, die eine Stomatindomäne beinhalten, interagieren und modulieren die Aktivität des MEC-4 Ionenkanals. Aufgrund der Homologie von MEC-2 und stomatinähnlichen Proteinen könnten diese ebenfalls eine solche Rolle spielen. In der vorliegenden Arbeit wird die Rolle von ASIC3 und stomatinähnlichen Proteinen (SLP) bei der Mechanotransduktion in Mäusen untersucht. Es wird gezeigt, dass eine physikalische Interaktion zwischen ASIC3 und Stomatin sowie ASIC3 und dem stomatinähnlichen Protein 3 (SLP3) in einem heterologen System vorliegt. Um zu testen, ob diese Interaktion für die Mechanotransduktion in sensorischen Neuronen wichtig ist, wurde die in vitro Haut-Nerv Preparation, in der die Einzelfasermechanosensitivität analysiert wird, angewendet. Die Mechanosensitivität der verschiedenen Haut- mechanorezeptoren von normalen Wildtypmäusen wird mit Mäusen verglichen, denen ASIC3 sowie ASIC3 und Stomatin fehlt. Der Verlust von ASIC3 in Mäusen führt zu einer Zunahme der Mechanosensitivität von schnelladaptierenden Mechanorezeptoren (RAM) und zu eine Abnahme der Mechanosensitivität von A-Mechanonociceptoren (AM) und C-Fasern. Im Vergleich dazu hat der zusätzliche Verlust von Stomatin keinen Effekt an der Zunahme der Mechanosensitivität in RAM allerdings wird deren Antwort auf mechanische Reize etwas verlangsamt. Weiterhin ist die Mechanosensitivität von den AM und C-Fasern in ASIC3 /Stomatin-Mutanten Mäuse vermindert. Diese Verminderung ist jedoch nicht Signifikant im Vergleich zu ASIC3-Mutaten Mäuse. Andererseits zeigen polymodale Nociceptoren (C-MH) in ASIC3/Stomatin-Mutanten Mäuse unter starkem mechanischem Stimulus eine signifikante Verminderung der Mechanosensitivität im Vergleich zu C-MH in ASIC3-Mutanten. Die Ergebnisse dieser Arbeit zeigen, dass ASIC3 für eine normale Mechanorezeptorenfunktion in Mäusen notwendig ist. Die Analyse von Mechanorezeptoren in ASIC3/Stomatin-Mutanten zeigte eine schwache funktionelle Interaktion zwischen ASIC3 und Stomatin

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(1). It is postulated that mechanically activated (MA) cation channels initiate touch sensation, but the identity of these molecules in mammals has been elusive(2). Piezo2 is a rapidly adapting (RA) MA ion channel expressed in a subset of sensory neurons of the dorsal root ganglion (DRG) and in cutaneous mechanoreceptors known as Merkel cell-neurite complexes(3,4). Merkel cells have been demonstrated to play a role in vertebrate mechanosensation using Piezo2, particularly in shaping the type of current sent by its innervating sensory neuron(4-6). However, major aspects of touch sensation remain intact without Merkel cell activity(4,7). 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 (LTMRs) that innervate both hairy and glabrous skin. Most RA MA currents in DRG neuronal cultures are absent in Piezo2(CKO) mice, and ex vivo skin nerve preparation studies show that mechanosensitivity of LTMRs strongly depends on Piezo2. This striking cellular phenotype correlates with an unprecedented behavioral phenotype: an almost complete deficit in light touch sensation in multiple behavioral assays, without affecting other somatosensory functions. Our results highlight that a single ion channel that displays RA MA currents in vitro is responsible for the mechanosensitivity of most LTMR subtypes involved in innocuous touch sensation. Interestingly, we find that touch and pain sensation are separable, suggesting that yet-unknown MA ion channel(s) must account for noxious (painful) mechanosensation