Merkel cells transduce and encode tactile stimuli to drive Aß-afferent impulses.

Sensory systems for detecting tactile stimuli have evolved from touch-sensing nerves in invertebrates to complicated tactile end organs in mammals. Merkel discs are tactile end organs consisting of Merkel cells and Aß-afferent nerve endings and are localized in fingertips, whisker hair follicles, and other touch-sensitive spots. Merkel discs transduce touch into slowly adapting impulses to enable tactile discrimination, but their transduction and encoding mechanisms remain unknown. Using rat whisker hair follicles, we show that Merkel cells rather than Aß-afferent nerve endings are primary sites of tactile transduction and identify the Piezo2 ion channel as the Merkel cell mechanical transducer. Piezo2 transduces tactile stimuli into Ca(2+)-action potentials in Merkel cells, which drive Aß-afferent nerve endings to fire slowly adapting impulses. We further demonstrate that Piezo2 and Ca(2+)-action potentials in Merkel cells are required for behavioral tactile responses. Our findings provide insights into how tactile end-organs function and have clinical implications for tactile dysfunctions.

Effects of inflammation on the properties of Nav1.8-ChR2-positive and Nav1.8-ChR2-negative afferent mechanoreceptors in the hindpaw glabrous skin of mice.

We recently used Nav1.8-ChR2 mice in which Nav1.8-expressing afferents were optogenetically tagged to classify mechanosensitive afferents into Nav1.8-ChR2-positive and Nav1.8-ChR2-negative mechanoreceptors. We found that the former were mainly high threshold mechanoreceptors (HTMRs), while the latter were low threshold mechanoreceptors (LTMRs). In the present study, we further investigated whether the properties of these mechanoreceptors were altered following tissue inflammation. Nav1.8-ChR2 mice received a subcutaneous injection of saline or Complete Freund's Adjuvant (CFA) in the hindpaws. Using the hind paw glabrous skin-tibial nerve preparation and the pressure-clamped single-fiber recordings, we found that CFA-induced hind paw inflammation lowered the mechanical threshold of many Nav1.8-ChR2-positive Aß-fiber mechanoreceptors but heightened the mechanical threshold of many Nav1.8-ChR2-negative Aß-fiber mechanoreceptors. Spontaneous action potential impulses were not observed in Nav1.8-ChR2-positive Aß-fiber mechanoreceptors but occurred in Nav1.8-ChR2-negative Aß-fiber mechanoreceptors with a lower mechanical threshold in the saline goup, and a higher mechanical threshold in the CFA group. No significant change was observed in the mechanical sensitivity of Nav1.8-ChR2-positive and Nav1.8-ChR2-negative Aδ-fiber mechanoreceptors and Nav1.8-ChR2-positive C-fiber mechanoreceptors following hind paw inflammation. Collectively, inflammation significantly altered the functional properties of both Nav1.8-ChR2-positive and Nav1.8-ChR2-negative Aß-fiber mechanoreceptors, which may contribute to mechanical allodynia during inflammation.

Role of Voltage-Gated K+ Channels and K2P Channels in Intrinsic Electrophysiological Properties and Saltatory Conduction at Nodes of Ranvier of Rat Lumbar Spinal Ventral Nerves.

Ion channels at the nodes of Ranvier (NRs) are believed to play essential roles in intrinsic electrophysiological properties and saltatory conduction of action potentials (AP) at the NRs of myelinated nerves. While we have recently shown that two-pore domain potassium (K2P) channels play a key role at the NRs of Aß-afferent nerves, K+ channels and their functions at the NRs of mammalian motor nerves remain elusive. Here we addressed this issue by using ex vivo preparations of lumbar spinal ventral nerves from both male and female rats and the pressure-patch-clamp recordings at their NRs. We found that depolarizing voltages evoked large noninactivating outward currents at NRs. The outward currents could be partially inhibited by voltage-gated K+ channel blockers, largely inhibited by K2P blockers and cooling temperatures. Inhibition of the outward currents by voltage-gated K+ channel blockers, K2P blockers, or cooling temperatures significantly altered electrophysiological properties measured at the NRs, including resting membrane potential, input resistance, AP width, AP amplitude, AP threshold, and AP rheobase. Furthermore, K2P blockers and cooling temperatures significantly reduced saltatory conduction velocity and success rates of APs in response to high-frequency stimulation. Voltage-gated K+ channel blockers reduced AP success rates at high-frequency stimulation without significantly affecting saltatory conduction velocity. Collectively, both K2P and voltage-gated K+ channels play significant roles in intrinsic electrophysiological properties and saltatory conduction at NRs of motor nerve fibers of rats. The effects of cooling temperatures on saltatory conduction are at least partially mediated by K2P channels at the NRs.SIGNIFICANCE STATEMENT Ion channels localized at the NRs are believed to be key determinants of saltatory conduction on myelinated nerves. However, ion channels and their functions at the NRs have not been fully studied in different types of mammalian myelinated nerves. Here we use the pressure-patch-clamp recordings to show that both K2P and voltage-gated K+ channels play significant roles in intrinsic electrophysiological properties and saltatory conduction at NRs of lumbar spinal ventral nerves of rats. Furthermore, cooling temperatures exert effects on saltatory conduction via inhibition of ion channels at the NRs. Our results provide new insights into saltatory conduction on myelinated nerves and may have physiological as well as pathologic implications.

Non-nociceptive and nociceptive-like trigeminal Aß-afferent neurons of rats: Distinct electrophysiological properties, mechanical and chemical sensitivity.

The role of Aß-afferents in somatosensory function is often oversimplified as low threshold mechanoreceptors (LTMRs) with large omission of Aß-afferent involvement in nociception. Recently, we have characterized Aß-afferent neurons which have large diameter somas in the trigeminal ganglion (TG) and classified them into non-nociceptive and nociceptive-like TG afferent neurons based on their electrophysiological properties. Here, we extend our previous observations to further characterize electrophysiological properties of trigeminal Aß-afferent neurons and investigate their mechanical and chemical sensitivity by patch-clamp recordings from large-diameter TG neurons in ex vivo TG preparations of adult male and female rats. Based on cluster analysis of electrophysiological properties, trigeminal Aß-afferent neurons can be classified into five discrete types (type I, IIa, IIb, IIIa, and IIIb), which responded differentially to mechanical stimulation and sensory mediators including serotonin (5-HT), acetylcholine (ACh) and adenosine triphosphate (ATP). Notably, type I neuron action potential (AP) was small in amplitude, width was narrow in duration, and peak dV/dt repolarization was great with no deflection observed, whereas discretely graded differences were observed for type IIa, IIb, IIIa, and IIIb, as AP increased in amplitude, width broadened in duration, and peak dV/dt repolarization reduced with the emergence of increasing deflection. Type I, IIa, and IIb neurons were mostly mechanically sensitive, displaying robust and rapidly adapting mechanically activated current (IMA) in response to membrane displacement, while IIIa and IIIb, conversely, were almost all mechanically insensitive. Interestingly, mechanical insensitivity coincided with increased sensitivity to 5-HT and ACh. Together, type I, IIa and IIb display features of LTMR Aß-afferent neurons while type IIIa and type IIIb show properties of nociceptive Aß-afferent neurons.

Saltatory conduction and intrinsic electrophysiological properties at the nodes of ranvier of Aα/ß-afferent fibers and Aα-efferent fibers in rat sciatic nerves.

Large-diameter myelinated fibers in sciatic nerves are composed of both Aα/ß-afferent fibers and Aα-efferent fibers to convey sensory and motor impulses, respectively, via saltatory conduction for rapid leg responses. Saltatory conduction and electrophysiological properties at the nodes of Ranvier (NRs) of these sciatic nerve fibers have not been directly studied. We used ex vivo sciatic nerve preparations from rats and applied patch-clamp recordings at the NRs of both Aα/ß-afferent fibers and Aα-efferent fibers in the sciatic nerves to characterize their saltatory conduction and intrinsic electrophysiological properties. The velocity and frequency of saltatory conduction in both types of fibers were similar. Resting membrane potentials (RMPs), input resistance, action potential (AP) threshold, and AP rheobase were also not significantly different at the NRs of the two types of fibers in the sciatic nerves. In comparison with Aα/ß-afferent fibers, Aα-efferent fibers in the sciatic nerves show higher amplitude and broader width of APs at their NRs. At the NRs of both types of fibers, depolarizing voltages evoked transient inward currents followed by non-inactivating outward currents, and the inward currents and non-inactivating outward currents at the NRs were not significantly different between the two types of fibers. Using AP-clamp, inward currents during AP upstroke were found to be insignificant difference, but amplitudes of non-inactivating outward currents during AP repolarization were significantly lower at the NRs of Aα-efferent fibers than at the NRs of Aα/ß-afferent fibers in the sciatic nerves. Collectively, saltatory conduction, ionic currents, and intrinsic electrophysiological properties at the NRs of Aα/ß-afferent fibers and Aα-efferent fibers in the sciatic nerves are generally similar, but some differences were also observed.

Kv4.3 Channel Dysfunction Contributes to Trigeminal Neuropathic Pain Manifested with Orofacial Cold Hypersensitivity in Rats.

Trigeminal neuropathic pain is the most debilitating pain disorder but current treatments including opiates are not effective. A common symptom of trigeminal neuropathic pain is cold allodynia/hyperalgesia or cold hypersensitivity in orofacial area, a region where exposure to cooling temperatures are inevitable in daily life. Mechanisms underlying trigeminal neuropathic pain manifested with cold hypersensitivity are not fully understood. In this study, we investigated trigeminal neuropathic pain in male rats following infraorbital nerve chronic constrictive injury (ION-CCI). Assessed by the orofacial operant behavioral test, ION-CCI animals displayed orofacial cold hypersensitivity. The cold hypersensitivity was associated with the hyperexcitability of small-sized trigeminal ganglion (TG) neurons that innervated orofacial regions. Furthermore, ION-CCI resulted in a reduction of A-type voltage-gated K+ currents (IA currents) in these TG neurons. We further showed that these small-sized TG neurons expressed Kv4.3 voltage-gated K+ channels, and Kv4.3 expression in these cells was significantly downregulated following ION-CCI. Pharmacological inhibition of Kv4.3 channels with phrixotoxin-2 inhibited IA-currents in these TG neurons and induced orofacial cold hypersensitivity. On the other hand, pharmacological potentiation of Kv4.3 channels amplified IA currents in these TG neurons and alleviated orofacial cold hypersensitivity in ION-CCI rats. Collectively, Kv4.3 downregulation in nociceptive trigeminal afferent fibers may contribute to peripheral cold hypersensitivity following trigeminal nerve injury, and Kv4.3 activators may be clinically useful to alleviate trigeminal neuropathic pain.SIGNIFICANCE STATEMENT Trigeminal neuropathic pain, the most debilitating pain disorder, is often triggered and exacerbated by cooling temperatures. Here, we created infraorbital nerve chronic constrictive injury (ION-CCI) in rats, an animal model of trigeminal neuropathic pain to show that dysfunction of Kv4.3 voltage-gated K+ channels in nociceptive-like trigeminal ganglion (TG) neurons underlies the trigeminal neuropathic pain manifested with cold hypersensitivity in orofacial regions. Furthermore, we demonstrate that pharmacological potentiation of Kv4.3 channels can alleviate orofacial cold hypersensitivity in ION-CCI rats. Our results may have clinical implications in trigeminal neuropathic pain in human patients, and Kv4.3 channels may be an effective therapeutic target for this devastating pain disorder.

Effects on low threshold mechanoreceptors in whisker hair follicles by 5-HT, Cd2+, tetraethylammonium, 4-aminopyridine, and Ba2.

Low threshold mechanoreceptors (LTMRs) are important for environmental exploration, social interaction, and tactile discrimination. Whisker hair follicles are mechanical sensory organs in non-primate mammals that are functionally equivalent to human fingertips. Several functional types of LTMRs have been identified in rodent whisker hair follicles, including rapidly adapting (RA), slow adapting type 1 (SA1), and slowly adapting type 2 (SA2) LTMRs. Properties of these LTMRs have not been fully characterized. In the present study, we have used pressure-clamped single-fiber recording technique to record impulses of RA, SA1, and SA2 LTMRs in mouse whisker hair follicles, and tested effects of 5-HT, Cd2+, tetraethylammonium (TEA), 4-aminopyridine (4-AP), and Ba2+ on the LTMR impulses. We show that 5-HT at 2 mM suppresses SA1 impulses but has no effects on RA and SA2 impulses. Cd2+ at 100 µM suppresses both SA1 and SA2 impulses but has no effects on RA impulses. TEA at 10 mM has no effects on RA and SA1 impulses but increased SA2 impulses. However, TEA at 1 mM and 200 µM decreases SA2 impulses. 4-AP at 1 mM suppresses both SA1 and SA2 impulses but has no effects on RA impulses. Ba2+ at 5 mM increases both RA and SA1 impulses but suppresses SA2 impulses. Collectively, RA, SA1, and SA2 LTMRs show distinct pharmacological properties, suggesting that these LTMRs may use different mechanisms to tune their mechanical signaling.

Effects of GABAB receptor activation on excitability of IB4-positive maxillary trigeminal ganglion neurons: Possible involvement of TREK2 activation.

IB4-positive maxillary trigeminal ganglion (TG) neurons are a subtype of afferent neurons involving nociception in orofacial regions, and excitability of these neurons is associated with orofacial nociceptive sensitivity. TREK-2 channel is a member of two-pore domain potassium (K2P) channel family mediating leak K+ currents. It has been shown previously that TREK-2 channel activity can be enhanced following GABAB receptor activation, leading to a reduction of cortical neuron excitability. In the present study, we have characterized TREK-2 channel expression on maxillary TG neurons and investigated the effect of the GABAB agonist baclofen on electrophysiological properties of small-sized maxillary TG neurons of rats. We show with immunohistochemistry that TREK-2 channels are predominantly expressed in small-sized IB4-positive maxillary TG neurons. Patch-clamp recordings on neurons in ex vivo TG preparations show that baclofen hyperpolarizes resting membrane potentials, increases outward leak currents, and decreases input resistances in IB4-positive maxillary TG neurons. Moreover, baclofen significantly reduces action potential (AP) firing in IB4-positive maxillary TG neurons. In contrast, baclofen shows no significant effect on electrophysiological properties of small-sized nociceptive-like and non-nociceptive-like maxillary trigeminal neurons that are IB4-negatve. Our results suggest that TREK-2 channel activity can be enhanced by baclofen, leading to reduced excitability of IB4-positive maxillary TG neurons. This finding provides new insights into the role of TREK-2 and GABAB receptors in controlling nociceptive sensitivity in orofacial regions, which may have therapeutic implications.

Electrophysiological properties of maxillary trigeminal Aß-afferent neurons of rats.

Aß-afferents in maxillary or V2 trigeminal ganglion (TG) neurons are somatosensory neurons that may be involved in both non-nociceptive and nociceptive functions in orofacial regions. However, electrophysiological properties of these V2 trigeminal Aß-afferent neurons have not been well characterized so far. Here, we used rat ex vivo trigeminal nerve preparations and applied patch-clamp recordings to large-sized V2 TG neurons to characterize their electrophysiological properties. All the cells recorded had afferent conduction velocities in the range of Aß-afferent conduction speeds. However, these V2 trigeminal Aß-afferent neurons displayed different action potential (AP) properties. APs showed fast kinetics in some cells but slow kinetics with shoulders in repolarization phases in other cells. Based on the derivatives of voltages in AP repolarization with time (dV/dt), we classified V2 trigeminal Aß-afferent neurons into four types: type I, type II, type IIIa and type IIIb. Type I V2 trigeminal Aß-afferent neurons had the largest dV/dt of repolarization, the fastest AP conduction velocities, the shortest AP and afterhyperpolarization (AHP) durations, and the highest AP success rates. In contrast, type IIIb V2 trigeminal Aß-afferent neurons had the smallest dV/dt of AP repolarization, the slowest AP conduction velocities, the longest AP and AHP durations, and the lowest AP success rates. The type IIIb cells also had significantly lower voltage-activated K+ currents. For type II and type IIIa V2 trigeminal Aß-afferent neurons, AP parameters were in the range between those of type I and type IIIb V2 trigeminal Aß-afferent neurons. Our electrophysiological classification of V2 trigeminal Aß-afferent neurons may be useful in future to study their non-nociceptive and nociceptive functions in orofacial regions.

Role of microtubules in Piezo2 mechanotransduction of mouse Merkel cells.

Piezo2 channels are expressed in Merkel cells and somatosensory neurons to mediate mechanotransduction leading to the sense of touch. Components of the cytoskeleton including microtubules are key intracellular structures that maintain cellular membrane mechanics and thereby may be important in mechanotransduction. In the present study, we have explored, with microtubule-targeting agents, the potential role of microtubules in Piezo2-mediated mechanotransduction in Merkel cells of mouse whisker hair follicles. Applying patch-clamp recordings to Merkel cells in situ in whisker hair follicles, we show that Piezo2-mediated mechanically activated (MA) currents in Merkel cells are significantly potentiated by the microtubule stabilizer paclitaxel but reduced by the microtubule destabilizer vincristine. Furthermore, electrophysiological recordings made from whisker hair follicle afferent nerves show that mechanically evoked whisker afferent impulses are significantly enhanced by paclitaxel and its analog docetaxel but significantly suppressed by vincristine and its analog vinblastine. Our findings suggest that microtubules play an essential role in Piezo2 mechanotransduction in Merkel cells.NEW & NOTEWORTHY Piezo2 channels are expressed in Merkel cells to mediate mechanotransduction leading to the sense of touch. Here we determined the role of microtubules in regulating Piezo2-mediated mechanotransduction in Merkel cells. Piezo2-mediated currents in Merkel cells are potentiated by microtubule stabilizer paclitaxel but reduced by microtubule destabilizer vincristine. Mechanically evoked afferent impulses are also enhanced by microtubule stabilizers and suppressed by microtubule destabilizers. Microtubules may play an essential role in Piezo2 mechanotransduction in Merkel cells.

Effects on tactile transmission by serotonin transporter inhibitors at Merkel discs of mouse whisker hair follicles.

The Merkel disc is a main type of tactile end organs formed by Merkel cells and Aß-afferent endings as first tactile sensory synapses. They are highly abundant in fingertips, touch domes, and whisker hair follicles of mammals and are essential for sensory tasks including social interaction, environmental exploration, and tactile discrimination. We have recently shown that Merkel discs use serotonin to transmit tactile signals from Merkel cells to Aß-afferent endings to drive slowly adapting type 1 impulses on the Aß-afferent nerves. This raises a question as whether the serotoninergic transmission at Merkel discs may be regulated by serotonin transporters and whether serotonin transporter inhibitors may affect the tactile transmission. Here, we made recordings from whisker afferent nerves of mouse whisker hair follicles and tested the effects of monoamine transporter inhibitors on slowly adapting type 1 impulses. We show that methamphetamine, a monoamine releasing facilitator and reuptake inhibitor, elicited spontaneous impulses as well as increased the numbers of slowly adapting type 1 impulses elicited by whisker hair deflections. S-duloxetine, a potent inhibitor of transporters of serotonin and norepinephrine, and fluoxetine, a selective inhibitor of serotonin transporters, both also increased the numbers of slowly adapting type 1 impulses. Prolonged treatment of whisker hair follicles with methamphetamine abolished most of slowly adapting type 1 impulses. Furthermore, the treatment of whisker hair follicles with methamphetamine resulted in serotonin release from whisker hair follicles. Taken together, our results suggest that serotonin transporters play a role in regulating tactile transmission at Merkel discs.

Pressure-clamped single-fiber recording technique: A new recording method for studying sensory receptors.

An electrophysiological technique that can record nerve impulses from a single nerve fiber is indispensable for studying modality-specific sensory receptors such as low threshold mechanoreceptors, thermal receptors, and nociceptors. The teased-fiber single-unit recording technique has long been used to resolve impulses that are likely to be from a single nerve fiber. The teased-fiber single-unit recording technique involves tedious nerve separation procedures, causes nerve fiber impairment, and is not a true single-fiber recording method. In the present study, we describe a new and true single-fiber recording technique, the pressure-clamped single-fiber recording method. We have applied this recording technique to mouse whisker hair follicle preparations with attached whisker afferents as well as to skin-nerve preparations made from mouse hindpaw skin and saphenous nerves. This new approach can record impulses from rapidly adapting mechanoreceptors (RA), slowly adapting type 1 mechanoreceptors (SA1), and slowly adapting type 2 mechanoreceptors (SA2) in these tissue preparations. We have also applied the pressure-clamped single-fiber recordings to record impulses on Aß-fibers, Aδ-fibers, and C-fibers. The pressure-clamped single-fiber recording technique provides a new tool for sensory physiology and pain research.

Heterogeneity of adult masseter muscle satellite cells with cardiomyocyte differentiation potential.

Although resident cardiac stem cells have been reported, regeneration of functional cardiomyocytes (CMs) remains a challenge. The present study identifies an alternative progenitor source for CM regeneration without the need for genetic manipulation or invasive heart biopsy procedures. Unlike limb skeletal muscles, masseter muscles (MM) in the mouse head are developed from Nkx2-5 mesodermal progenitors. Adult masseter muscle satellite cells (MMSCs) display heterogeneity in developmental origin and cell phenotypes. The heterogeneous MMSCs that can be characterized by cell sorting based on stem cell antigen-1 (Sca1) show different lineage potential. While cardiogenic potential is preserved in Sca1+ MMSCs as shown by expression of cardiac progenitor genes (including Nkx2-5), skeletal myogenic capacity is maintained in Sca1- MMSCs with Pax7 expression. Sca1+ MMSC-derived beating cells express cardiac genes and exhibit CM-like morphology. Electrophysiological properties of MMSC-derived CMs are demonstrated by calcium transients and action potentials. These findings show that MMSCs could serve as a novel cell source for cardiomyocyte replacement.

Merkel disc is a serotonergic synapse in the epidermis for transmitting tactile signals in mammals.

The evolution of sensory systems has let mammals develop complicated tactile end organs to enable sophisticated sensory tasks, including social interaction, environmental exploration, and tactile discrimination. The Merkel disc, a main type of tactile end organ consisting of Merkel cells (MCs) and Aß-afferent endings, are highly abundant in fingertips, touch domes, and whisker hair follicles of mammals. The Merkel disc has high tactile acuity for an object's physical features, such as texture, shape, and edges. Mechanisms underlying the tactile function of Merkel discs are obscured as to how MCs transmit tactile signals to Aß-afferent endings leading to tactile sensations. Using mouse whisker hair follicles, we show herein that tactile stimuli are transduced by MCs into excitatory signals that trigger vesicular serotonin release from MCs. We identify that both ionotropic and metabotropic 5-hydroxytryptamine (5-HT) receptors are expressed on whisker Aß-afferent endings and that their activation by serotonin released from MCs initiates Aß-afferent impulses. Moreover, we demonstrate that these ionotropic and metabotropic 5-HT receptors have a synergistic effect that is critical to both electrophysiological and behavioral tactile responses. These findings elucidate that the Merkel disc is a unique serotonergic synapse located in the epidermis and plays a key role in tactile transmission. The epidermal serotonergic synapse may have important clinical implications in sensory dysfunctions, such as the loss of tactile sensitivity and tactile allodynia seen in patients who have diabetes, inflammatory diseases, and undergo chemotherapy. It may also have implications in the exaggerated tactile sensations induced by recreational drugs that act on serotoninergic synapses.

Effects of cooling temperatures and low pH on membrane properties and voltage-dependent currents of rat nociceptive-like trigeminal ganglion neurons.

Cooling temperatures and low pH have profound effects on somatosensory functions including nociception. The effects not only can be mediated by cooling temperature transducers and proton transducers expressed in subpopulations of somatosensory neurons but may also be mediated by ion channels involving membrane excitability such as voltage-dependent K+ channels in somatosensory neurons. In the present study, we performed the in situ patch-clamp recordings from nociceptive-like trigeminal ganglion neurons in ex vivo trigeminal ganglion preparations of adult rats. We determined effects of cooling temperatures and low pH on membrane properties and voltage-dependent currents in nociceptive-like trigeminal ganglion neurons. Action potential rheobase levels were decreased when nociceptive trigeminal ganglion neurons were cooled from 24°C down to 12°C or when extracellular pH levels were reduced from 7.3 to 6. This indicates that the excitability of nociceptive-like trigeminal ganglion neurons was increased at the cooling temperatures and low pH. The decreases of action potential rheobase levels were accompanied by increases of trigeminal ganglion neuron input resistances at cooling temperatures and low pH, suggesting a possible involvement of background K+ channels. Cooling temperatures and low pH suppressed voltage-activated inward Na+ currents and also voltage-dependent outward K+ currents in nociceptive-like trigeminal ganglion neurons. Voltage-dependent outward K+ currents in nociceptive-like trigeminal ganglion neurons consist of inactivating A-type K+ currents and non-inactivating type K+ currents, and the former were more sensitive to cooling temperatures and low pH. Collectively, suppressing multiple types of K+ channels may be associated with the enhanced excitability of nociceptive trigeminal ganglion neurons by cooling temperatures and low pH.

Down-regulation of Kv4.3 channels and a-type K+ currents in V2 trigeminal ganglion neurons of rats following oxaliplatin treatment.

Chemotherapy drugs such as oxaliplatin can increase nociceptive neuron excitability to result in neuropathic pain in orofacial and other regions in patients following chemotherapy. However, mechanisms underlying chemotherapy-induced increases of nociceptive neuron excitability are not fully understood. Kv4.3 channels are voltage-gated K+ channels mediating A-type K+ (IA) currents to control neuronal excitability. In the present study, we examined Kv4.3 channel expression on trigeminal neurons that innervate orofacial regions (V2 TG neurons) of rats using immunostaining method. We showed that strong Kv4.3 immunoreactivity (Kv4.3-ir) was present mainly in small-sized V2 TG neurons. The numbers of Kv4.3-ir positive V2 TG neurons were significantly reduced in oxaliplatin-treated rats, suggesting down-regulation of Kv4.3 channel expression on V2 TG neurons by the chemotherapy drug. Patch-clamp recordings from acutely dissociated rat V2 TG neurons showed that almost all nociceptive-like V2 TG neurons displayed IA currents with slow inactivation kinetics. The amplitudes of IA currents were significantly reduced in these nociceptive-like V2 TG neurons of oxaliplatin-treated group. Furthermore, we found that the excitability of nociceptive-like V2 TG neurons was significantly higher in the oxaliplatin-treated group than in the control group. These findings raise a possibility that down-regulation of Kv4.3 channels and IA currents in nociceptive V2 TG neurons is an underlying mechanism of oxaliplatin-induced orofacial neuropathic pain.

Abstracts of the 7th Asian Pain Symposium.

The Asian Pain Symposium (APS) is a main pain research meeting in Asia. Since established in 2000 in Kyoto, five other APSs have been held in different Asian regions including Seoul of Korea in 2004, Fukuoka of Japan in 2008, Shanghai of China in 2011, Okazaki of Japan in 2013, and Suzhou of China in 2015. The 7th Asian Pain Symposium (APS 2017) was held in Taipei of Taiwan during October 26th to October 29th, 2017. The APS 2017 was sponsored by The Ministry of Science and Technology of Taiwan and Institute of Biomedical Science and Neuroscience Program of Academia Sinica and Taiwan Pain Society. The president of the APS 2017 was Dr. Bai Chuang Shyu, Institute of Biomedical Sciences, Academia Sinica, Taiwan. Local organizing committee also include Dr. Jen-Chuen Hsieh, Institute of Brain Science, National Yang-Ming University and Veteran General Hospital, Taiwan, Dr. Wei-Zen Sun, Department of Anesthesiology, National Taiwan University Hospital, Taiwan, and Dr. Chih-Cheng Chen, Institute of Biomedical Sciences, Academia Sinica, Taiwan. Main topics of the APS 2017 included the latest progress of pain research and novel strategies of pain treatments. Symposium attendees presented their interesting and exciting research findings in the areas of 1) basic sensory and nociceptive functions, 2) ion channels and their functions in somatosensory physiology and pain, 3) brain functions and regulations in pain, 4) spinal cord mechanisms of nociception and pain, 5) analgesia and pain regulations, 6) chronic pain mechanisms and treatment, and 7) brain circuits underlying the physiological and pathological pain. There were a total of 29 oral presentations and 23 poster presentations at the 7th APS. A council meeting was held during the 7th APS, and at this council meeting Dr. Seog Bae OH (Seoul National University) was elected as the president of 8th Asian Pain Symposium to organize the next symposium in Seoul, Korea in 2019. In order to keep a permanent record and to help promote pain research in Asia, we have collected abstracts of oral presentations and posted them below in the order when the presentations were given at the 7th Asian Pain Symposium.

Molecular Mechanisms of the Sense of Touch: An Overview of Mechanical Transduction and Transmission in Merkel Discs of Whisker Hair Follicles and Some Clinical Perspectives.

The Merkel disc is a main type of tactile end organs for sensing gentle touch and is essential for sophisticated sensory tasks including social interaction, environmental exploration, and tactile discrimination. Recent studies have shown that Merkel cells are primary sites of mechanotransduction using Piezo2 channels as a molecular transducer in Merkel discs. Furthermore, tactile stimuli trigger serotonin release from Merkel cells to excite their associated whisker Aß-afferent endings and transmit tactile signals. The tactile transduction and transmission at Merkel discs may have important clinical implications in sensory dysfunctions such as the loss of tactile sensitivity and tactile allodynia seen in patients who have diabetes and inflammatory diseases and undergo chemotherapy.

Membrane Mechanics of Primary Afferent Neurons in the Dorsal Root Ganglia of Rats.

Membrane mechanics is an important biological factor regulating many cellular functions including cell motility, intercellular and intracellular signaling, gene expression, and membrane ion channel activity. Primary afferent neurons transduce sensory information about temperature, touch, and pain. These sensory functions may be profoundly affected by the states of primary afferent neuron mechanics. However, membrane mechanics of primary afferent neurons is largely unknown. In this study, we established the optical trapping technique for determining membrane mechanics of cultured primary afferent neurons of the dorsal root ganglia (DRG). We further determined the roles of cytoskeleton and membrane lipids in DRG neuron mechanics. We found that DRG neurons had a plasma membrane tension of ∼54 pN/µm, and the tension was significantly decreased to ∼29 pN/µm by cytochalasin D treatment to disrupt actin cytoskeleton and increased to ∼79 pN/µm by methyl-ß-cyclodextrin treatment to sequester membrane cholesterol. DRG neuron membrane stiffness was not significantly affected by the cytoskeleton disruption but was significantly increased after cholesterol sequestration. Our findings elucidate membrane mechanical properties of primary afferent neurons, which provide, to our knowledge, a new perspective on their sensory functions.

Regulation of Piezo2 Mechanotransduction by Static Plasma Membrane Tension in Primary Afferent Neurons.

The Piezo2 channel is a newly identified mammalian mechanical transducer that confers rapidly adapting mechanically activated (RA-MA) currents in primary afferent neurons. The Piezo2 channels sense rapid membrane displacement, but it is not clear whether they are sensitive to osmotic swelling, which slowly increases static plasma membrane tension (SPMT). Here, we show that SPMT exerts a profound impact on the mechanical sensitivity of RA-MA channels in primary afferent neurons. RA-MA currents are greatly enhanced, and the mechanical threshold was reduced in both primary afferent neurons of rat dorsal root ganglia (DRG) and HEK293 cells heterologously expressing Piezo2 when these cells undergo osmotic swelling to increase SPMT. Osmotic swelling switches the kinetics of RA-MA currents to the slowly adapting type in both cultured DRG neurons and HEK293 cells heterologously expressing Piezo2. The potentiation of RA-MA currents is abolished when cultured DRG neurons are treated with cytochalasin D, an actin filament disruptor that prevents SPMT of cultured DRG neurons from an increase by osmotic swelling. Osmotic swelling significantly increases DRG neuron mechano-excitability such that a subthreshold mechanical stimulus can result in action potential firing. Behaviorally, the mechanical hind paw withdrawal threshold in rats is reduced following the injection of a hypotonic solution, but this osmotic effect is abolished when cytochalasin D or Gd(3+) is co-administered with the hypo-osmotic solution. Taken together, our findings suggest that Piezo2-mediated mechanotransduction is regulated by SPMT in primary afferent neurons. Because SPMT can be changed by multiple biological factors, our findings may have broad implications in mechanical sensitivity under physiological and pathological conditions.