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Thermally activated ion channels are known to detect the entire thermal range from extreme heat (TRPV2), painful heat (TRPV1, TRPM3 and ANO1), non-painful warmth (TRPV3 and TRPV4) and non-painful coolness (TRPM8) through to painful cold (TRPA1) 1 , 2 , 3 , 4 , 5 , 6 , 7 . Genetic deletion of each of these ion channels, however, has only modest effects on thermal behaviour in mice 6 , 7 , 8 , 9 , 10 , 11 , 12 , with the exception of TRPM8, the deletion of which has marked effects on the perception of moderate coolness in the range 10–25 °C 13 . The molecular mechanism responsible for detecting non-painful warmth, in particular, is unresolved. Here we used calcium imaging to identify a population of thermally sensitive somatosensory neurons which do not express any of the known thermally activated TRP channels. We then used a combination of calcium imaging, electrophysiology and RNA sequencing to show that the ion channel generating heat sensitivity in these neurons is TRPM2. Autonomic neurons, usually thought of as exclusively motor, also express TRPM2 and respond directly to heat. Mice in which TRPM2 had been genetically deleted showed a striking deficit in their sensation of non-noxious warm temperatures, consistent with the idea that TRPM2 initiates a ‘warm’ signal which drives cool-seeking behaviour. The neuronal mechanism for the detection of non-painful warm stimuli has remained unclear; mammalian TRPM2 ion channel is shown to be required for warmth detection in the non-noxious range of 33–38 °C, and surprisingly to mediate responses to warmth in the autonomic nervous system. An ion channel responding to 'comfortable' warmth Despite the identification of several members of the TRP family of ion channels as heat-sensitive, the neuronal mechanism for the detection of non-painful warm stimuli has remained unclear, because genetic deletion of obvious candidates, such as TRPV3 and TRPV4, has had no effect on thermosensation. Rather than working with cloned channels, as is commonly done, Chun-Hsiang Tan and Peter McNaughton studied the responses of actual sensory neurons to thermal stimuli, then used an RNA-sequencing strategy to identify TRPM2 as genetically required for warmth detection in the non-noxious range of 33–38 °C, and, surprisingly, to mediate warm sensations in the autonomic nervous system.

Neutrophils must navigate accurately towards pathogens in order to destroy invaders and thus defend our bodies against infection. Here we show that hydrogen peroxide, a potent neutrophil chemoattractant, guides chemotaxis by activating calcium-permeable TRPM2 ion channels and generating an intracellular leading-edge calcium “pulse”. The thermal sensitivity of TRPM2 activation means that chemotaxis towards hydrogen peroxide is strongly promoted by small temperature elevations, suggesting that an important function of fever may be to enhance neutrophil chemotaxis by facilitating calcium influx through TRPM2. Chemotaxis towards conventional chemoattractants such as LPS, CXCL2 and C5a does not depend on TRPM2 but is driven in a similar way by leading-edge calcium pulses. Other proposed initiators of neutrophil movement, such as PI3K, Rac and lyn , influence chemotaxis by modulating the amplitude of calcium pulses. We propose that intracellular leading-edge calcium pulses are universal drivers of the motile machinery involved in neutrophil chemotaxis.

Prostaglandin E2 (PGE2) is a major contributor to inflammatory pain hyperalgesia, however, the extent to which it modulates the activity of nociceptive axons is incompletely understood. We developed and characterized a microfluidic cell culture model to investigate sensitisation of the axons of dorsal root ganglia neurons. We show that application of PGE2 to fluidically isolated axons leads to sensitisation of their responses to depolarising stimuli. Interestingly the application of PGE2 to the DRG axons elicited a direct and persistent spiking activity propagated to the soma. Both the persistent activity and the membrane depolarisation in the axons are abolished by the EP4 receptor inhibitor and a blocker of cAMP synthesis. Further investigated into the mechanisms of the spiking activity showed that the PGE2 evoked depolarisation was inhibited by Nav1.8 sodium channel blockers but was refractory to the application of TTX or zatebradine. Interestingly, the depolarisation of axons was blocked by blocking ANO1 channels with T16Ainh-A01. We further show that PGE2-elicited axonal responses are altered by the changes in chloride gradient within the axons following treatment with bumetanide a Na-K-2Cl cotransporter NKCC1 inhibitor, but not by VU01240551 an inhibitor of potassium-chloride transporter KCC2. Our data demonstrate a novel role for PGE2/EP4/cAMP pathway which culminates in a sustained depolarisation of sensory axons mediated by a chloride current through ANO1 channels. Therefore, using a microfluidic culture model, we provide evidence for a potential dual function of PGE2 in inflammatory pain: it sensitises depolarisation-evoked responses in nociceptive axons and directly triggers action potentials by activating ANO1 and Nav1.8 channels.

The rate of action potential firing in nociceptors is a major determinant of the intensity of pain. Possible modulators of action potential firing include the HCN ion channels, which generate an inward current, I h , after hyperpolarization of the membrane. We found that genetic deletion of HCN2 removed the cyclic adenosine monophosphate (cAMP)—sensitive component of I h and abolished action potential firing caused by an elevation of cAMP in nociceptors. Mice in which HCN2 was specifically deleted in nociceptors expressing Na v 1.8 had normal pain thresholds, but inflammation did not cause hyperalgesia to heat stimuli. After a nerve lesion, these mice showed no neuropathic pain in response to thermal or mechanical stimuli. Neuropathic pain is therefore initiated by HCN2-driven action potential firing in Na v 1.8-expressing nociceptors.

Over the past 50 y, behavioral experiments have produced a large body of evidence for the existence of a magnetic sense in a wide range of animals. However, the underlying sensory physiology remains poorly understood due to the elusiveness of the magnetosensory structures. Here we present an effective method for isolating and characterizing potential magnetite-based magnetoreceptor cells. In essence, a rotating magnetic field is employed to visually identify, within a dissociated tissue preparation, cells that contain magnetic material by their rotational behavior. As a tissue of choice, we selected trout olfactory epithelium that has been previously suggested to host candidate magnetoreceptor cells. We were able to reproducibly detect magnetic cells and to determine their magnetic dipole moment. The obtained values (4 to 100 fAm ²) greatly exceed previous estimates (0.5 fAm ²). The magnetism of the cells is due to a μ m -sized intracellular structure of iron-rich crystals, most likely single-domain magnetite. In confocal reflectance imaging, these produce bright reflective spots close to the cell membrane. The magnetic inclusions are found to be firmly coupled to the cell membrane, enabling a direct transduction of mechanical stress produced by magnetic torque acting on the cellular dipole in situ. Our results show that the magnetically identified cells clearly meet the physical requirements for a magnetoreceptor capable of rapidly detecting small changes in the external magnetic field. This would also explain interference of ac powerline magnetic fields with magnetoreception, as reported in cattle.

The TRPA1 ion channel is expressed in nociceptive (pain-sensitive) somatosensory neurons and is activated by a wide variety of chemical irritants, such as acrolein in smoke or isothiocyanates in mustard. Here, we investigate the enhancement of TRPA1 function caused by inflammatory mediators, which is thought to be important in lung conditions such as asthma and COPD. Protein kinase A is an important kinase acting downstream of inflammatory mediators to cause sensitization of TRPA1. By using site-directed mutagenesis, patch-clamp electrophysiology and calcium imaging we identify four amino acid residues, S86, S317, S428, and S972, as the principal targets of PKA-mediated phosphorylation and sensitization of TRPA1.

Immune cell chemotaxis to the sites of pathogen invasion is critical for fighting infection, but in life-threatening conditions such as sepsis and Covid-19, excess activation of the innate immune system is thought to cause a damaging invasion of immune cells into tissues and a consequent excessive release of cytokines, chemokines and neutrophil extracellular traps (NETs). In these circumstances, tempering excessive activation of the innate immune system may, paradoxically, promote recovery. Here we identify the antimalarial compound artemisinin as a potent and selective inhibitor of neutrophil and macrophage chemotaxis induced by a range of chemotactic agents. Artemisinin released calcium from intracellular stores in a similar way to thapsigargin, a known inhibitor of the Sarco/Endoplasmic Reticulum Calcium ATPase pump (SERCA), but unlike thapsigargin, artemisinin blocks only the SERCA3 isoform. Inhibition of SERCA3 by artemisinin was irreversible and was inhibited by iron chelation, suggesting iron-catalysed alkylation of a specific cysteine residue in SERCA3 as the mechanism by which artemisinin inhibits neutrophil motility. In murine infection models, artemisinin potently suppressed neutrophil invasion into both peritoneum and lung in vivo and inhibited the release of cytokines/chemokines and NETs. This work suggests that artemisinin may have value as a therapy in conditions such as sepsis and Covid-19 in which over-activation of the innate immune system causes tissue injury that can lead to death.

Rodent models of tinnitus are commonly used to study its mechanisms and potential treatments. Tinnitus can be identified by changes in the gap-induced prepulse inhibition of the acoustic startle (GPIAS), most commonly by using pressure detectors to measure the whole-body startle (WBS). Unfortunately, the WBS habituates quickly, the measuring system can introduce mechanical oscillations and the response shows considerable variability. We have instead used a motion tracking system to measure the localized motion of small reflective markers in response to an acoustic startle reflex in guinea pigs and mice. For guinea pigs, the pinna had the largest responses both in terms of displacement between pairs of markers and in terms of the speed of the reflex movement. Smaller, but still reliable responses were observed with markers on the thorax, abdomen and back. The peak speed of the pinna reflex was the most sensitive measure for calculating GPIAS in the guinea pig. Recording the pinna reflex in mice proved impractical due to removal of the markers during grooming. However, recordings from their back and tail allowed us to measure the peak speed and the twitch amplitude (area under curve) of reflex responses and both analysis methods showed robust GPIAS. When mice were administered high doses of sodium salicylate, which induces tinnitus in humans, there was a significant reduction in GPIAS, consistent with the presence of tinnitus. Thus, measurement of the peak speed or twitch amplitude of pinna, back and tail markers provides a reliable assessment of tinnitus in rodents.

Background: Breathing rate is affected by physical stressors such as temperature or hypercapnia and by psychosocial stressors such as noise or overcrowding. In behavioral tests for tinnitus, rodents are often presented with trains of startle pulses. We postulated that using these pulses at successively higher sound levels would produce a cumulative increase in stress. Here, we demonstrate a novel means of assessing that increase in stress. Methods: By placing pairs of reflective markers on the abdomen and using a motion tracking system, we were able to remotely measure respiratory movements. A series of 20 startle pulses were presented in sequence at seven increasing sound levels, and changes in respiratory rate were tested with the Wilcoxon matched-pairs signed rank test and the Friedman Test. Results: Markers placed on 20 alert active mice showed evidence of sniffing behavior but no purely respiratory signal. By contrast, in all 18 guinea pigs, abdominal markers did track respiratory movements. The breathing rate in guinea pigs was similar to previous studies: (mean 104 ± 13; range 86–131 bpm). Presenting quiet startle pulses to guinea pigs caused a significant increase in breathing rate (by about 20%), even with pulses at 75–80 dB SPL. Increasing pulse sound levels in the range of 85–105 dB SPL did not reliably produce any further increase in breathing rate. Conclusions: We propose that tracking abdominal movement may allow measurement of psychosocial stress in the guinea pig. Once an animal is startled, increasing the pulse sound level did not produce any further increase in stress levels.

Neutrophils are an essential protective component of the innate immune system. However, in severe bacterial infections, neutrophils are known to mis-localise from the primary site of infection to other organs, where excessive release of cytokines, chemokines, and neutrophil extracellular traps (NETs) can induce organ damage and death. In this study, we use an animal model of bacterial infection originating in the peritoneum to show that hydrogen peroxide (H2O2, a potent neutrophil chemoattractant) is initially released in high concentrations both in the peritoneum and in multiple ‘off-target’ organs (lungs, liver and kidneys). The initial high H2O2 release inhibits neutrophil chemotaxis, but after 24 h concentrations of H2O2 reduce and can promote neutrophil migration to organs, where they release pro-inflammatory cytokines and chemokines along with NETs. The antimalarial compound artesunate potently inhibits neutrophil migration to off-target organs. It also abolishes cytokine, chemokine, and NET production, suggesting that artesunate may be a valuable novel therapy for preventing off-target organ inflammation associated with severe bacterial infections. Finally, the potency of H2O2 as a chemoattractant is shown by in vitro experiments in which, faced with competing gradients of H2O2 and other chemoattractants, neutrophils preferentially migrate towards H2O2.