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Phospholipase C-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate generates diacylglycerol, inositol 1,4,5-trisphosphate and protons, all of which can regulate TRPV1 activity via different mechanisms. Here we explored the possibility that the diacylglycerol metabolites 2-arachidonoylglycerol and 1-arachidonoylglycerol, and not metabolites of these monoacylglycerols, activate TRPV1 and contribute to this signaling cascade. 2-Arachidonoylglycerol and 1-arachidonoylglycerol activated native TRPV1 on vascular sensory nerve fibers and heterologously expressed TRPV1 in whole cells and inside-out membrane patches. The monoacylglycerol lipase inhibitors methylarachidonoyl-fluorophosphonate and JZL184 prevented the metabolism of deuterium-labeled 2-arachidonoylglycerol and deuterium-labeled 1-arachidonoylglycerol in arterial homogenates, and enhanced TRPV1-mediated vasodilator responses to both monoacylglycerols. In mesenteric arteries from TRPV1 knock-out mice, vasodilator responses to 2-arachidonoylglycerol were minor. Bradykinin and adenosine triphosphate, ligands of phospholipase C-coupled membrane receptors, increased the content of 2-arachidonoylglycerol in dorsal root ganglia. In HEK293 cells expressing the phospholipase C-coupled histamine H1 receptor, exposure to histamine stimulated the formation of 2-AG, and this effect was augmented in the presence of JZL184. These effects were prevented by the diacylglycerol lipase inhibitor tetrahydrolipstatin. Histamine induced large whole cell currents in HEK293 cells co-expressing TRPV1 and the histamine H1 receptor, and the TRPV1 antagonist capsazepine abolished these currents. JZL184 increased the histamine-induced currents and tetrahydrolipstatin prevented this effect. The calcineurin inhibitor ciclosporin and the endogenous \"entourage\" compound palmitoylethanolamide potentiated the vasodilator response to 2-arachidonoylglycerol, disclosing TRPV1 activation of this monoacylglycerol at nanomolar concentrations. Furthermore, intracerebroventricular injection of JZL184 produced TRPV1-dependent antinociception in the mouse formalin test. Our results show that intact 2-arachidonoylglycerol and 1-arachidonoylglycerol are endogenous TRPV1 activators, contributing to phospholipase C-dependent TRPV1 channel activation and TRPV1-mediated antinociceptive signaling in the brain.

Sema3A, a member of the semaphorin family of proteins, is shown to regulate bone remodelling indirectly by modulating sensory nerve development in mice. A role for sensory nerves in bone remodelling The semaphorins are diffusible proteins involved in the development of the nervous system, organs and blood vessels, as well as in immunity. Osteoblasts and osteoclasts express semaphorin family members, and it has been shown recently that semaphorin 3A (Sema3A) can regulate bone remodelling by acting locally. Here Toru Fukuda et al . demonstrate that Sema3A regulates bone remodelling in vivo indirectly by modulating sensory nerve development. Semaphorin 3A (Sema3A) is a diffusible axonal chemorepellent that has an important role in axon guidance 1 , 2 , 3 , 4 , 5 . Previous studies have demonstrated that Sema3a −/− mice have multiple developmental defects due to abnormal neuronal innervations 6 , 7 . Here we show in mice that Sema3A is abundantly expressed in bone, and cell-based assays showed that Sema3A affected osteoblast differentiation in a cell-autonomous fashion. Accordingly, Sema3a −/− mice had a low bone mass due to decreased bone formation. However, osteoblast-specific Sema3A-deficient mice ( Sema3a col1 −/− and Sema3a osx −/− mice) had normal bone mass, even though the expression of Sema3A in bone was substantially decreased. In contrast, mice lacking Sema3A in neurons ( Sema3a synapsin −/− and Sema3a nestin −/− mice) had low bone mass, similar to Sema3a −/− mice, indicating that neuron-derived Sema3A is responsible for the observed bone abnormalities independent of the local effect of Sema3A in bone. Indeed, the number of sensory innervations of trabecular bone was significantly decreased in Sema3a synapsin −/− mice, whereas sympathetic innervations of trabecular bone were unchanged. Moreover, ablating sensory nerves decreased bone mass in wild-type mice, whereas it did not reduce the low bone mass in Sema3a nestin −/− mice further, supporting the essential role of the sensory nervous system in normal bone homeostasis. Finally, neuronal abnormalities in Sema3a −/− mice, such as olfactory development, were identified in Sema3a synasin −/− mice, demonstrating that neuron-derived Sema3A contributes to the abnormal neural development seen in Sema3a −/− mice, and indicating that Sema3A produced in neurons regulates neural development in an autocrine manner. This study demonstrates that Sema3A regulates bone remodelling indirectly by modulating sensory nerve development, but not directly by acting on osteoblasts.

Based on single cell RNA-sequencing of 622 adult mouse sensory neurons, Usoskin et al . performed unbiased classification to identify the cellular and molecular complexity underlying somatic sensation. Eleven different subtypes were identified, including some previously unknown populations such as a new class of neuron which may be sensitive to inflammatory itch. The primary sensory system requires the integrated function of multiple cell types, although its full complexity remains unclear. We used comprehensive transcriptome analysis of 622 single mouse neurons to classify them in an unbiased manner, independent of any a priori knowledge of sensory subtypes. Our results reveal eleven types: three distinct low-threshold mechanoreceptive neurons, two proprioceptive, and six principal types of thermosensitive, itch sensitive, type C low-threshold mechanosensitive and nociceptive neurons with markedly different molecular and operational properties. Confirming previously anticipated major neuronal types, our results also classify and provide markers for new, functionally distinct subtypes. For example, our results suggest that itching during inflammatory skin diseases such as atopic dermatitis is linked to a distinct itch-generating type. We demonstrate single-cell RNA-seq as an effective strategy for dissecting sensory responsive cells into distinct neuronal types. The resulting catalog illustrates the diversity of sensory types and the cellular complexity underlying somatic sensation.

The solid tumour microenvironment includes nerve fibres that arise from the peripheral nervous system 1 , 2 . Recent work indicates that newly formed adrenergic nerve fibres promote tumour growth, but the origin of these nerves and the mechanism of their inception are unknown 1 , 3 . Here, by comparing the transcriptomes of cancer-associated trigeminal sensory neurons with those of endogenous neurons in mouse models of oral cancer, we identified an adrenergic differentiation signature. We show that loss of TP53 leads to adrenergic transdifferentiation of tumour-associated sensory nerves through loss of the microRNA miR-34a. Tumour growth was inhibited by sensory denervation or pharmacological blockade of adrenergic receptors, but not by chemical sympathectomy of pre-existing adrenergic nerves. A retrospective analysis of samples from oral cancer revealed that p53 status was associated with nerve density, which was in turn associated with poor clinical outcomes. This crosstalk between cancer cells and neurons represents mechanism by which tumour-associated neurons are reprogrammed towards an adrenergic phenotype that can stimulate tumour progression, and is a potential target for anticancer therapy. MicroRNAs from head and neck cancer cells, shuttled to sensory neurons by extracellular vesicles, cause a shift to an adrenergic neuronal phenotype that promotes tumour progression.

As in human speech acquisition, songbird vocal learning depends on early auditory experience. During development, juvenile songbirds listen to and form auditory memories of adult tutor songs, which they use to shape their own vocalizations in later sensorimotor learning. The higher-level auditory cortex, called the caudomedial nidopallium (NCM), is a potential storage site for tutor song memory, but no direct electrophysiological evidence of tutor song memory has been found. Here, we identify the neuronal substrate for tutor song memory by recording single-neuron activity in the NCM of behaving juvenile zebra finches. After tutor song experience, a small subset of NCM neurons exhibit highly selective auditory responses to the tutor song. Moreover, blockade of GABAergic inhibition, and sleep decrease their selectivity. Taken together, these results suggest that experience-dependent recruitment of GABA-mediated inhibition shapes auditory cortical circuits, leading to sparse representation of tutor song memory in auditory cortical neurons. Juvenile zebra finches learn to sing by memorizing and imitating their tutor's song, yet neural correlates of the tutor song have not been shown. Here the authors show a small subset of higher-level auditory cortex neurons are sharply tuned to the tutor's song and modulated by inhibition and arousal state.

Chemotherapy-induced peripheral neuropathy (CIPN) and associated neuropathic pain is a debilitating adverse effect of cancer treatment. Current understanding of the mechanisms underpinning CIPN is limited and there are no effective treatment strategies. In this study, we treated male C57BL/6J mice with 4 cycles of either Paclitaxel (PTX) or Oxaliplatin (OXA) over a week and tested pain hypersensitivity and changes in peripheral immune responses and neuroinflammation on days 7 and 13 post 1st injection. We found that both PTX and OXA caused significant mechanical allodynia. In the periphery, PTX and OXA significantly increased circulating CD4+ and CD8+ T-cell populations. OXA caused a significant increase in the percentage of interleukin-4+ lymphocytes in the spleen and significant down-regulation of regulatory T (T-reg) cells in the inguinal lymph nodes. However, conditional depletion of T-reg cells in OXA-treated transgenic DEREG mice had no additional effect on pain sensitivity. Furthermore, there was no leukocyte infiltration into the nervous system of OXA- or PTX-treated mice. In the peripheral nervous system, PTX induced expression of the neuronal injury marker activating transcription factor-3 in IB4+ and NF200+ sensory neurons as well as an increase in the chemokines CCL2 and CCL3 in the lumbar dorsal root ganglion. In the central nervous system, PTX induced significant astrocyte activation in the spinal cord dorsal horn, and both PTX and OXA caused reduction of P2ry12+ homeostatic microglia, with no measurable changes in IBA-1+ microglia/macrophages in the dorsal and ventral horns. We also found that PTX induced up-regulation of several inflammatory cytokines and chemokines (TNF-α, IFN-γ, CCL11, CCL4, CCL3, IL-12p70 and GM-CSF) in the spinal cord. Overall, these findings suggest that PTX and OXA cause distinct pathological changes in the periphery and nervous system, which may contribute to chemotherapy-induced neuropathic pain.

Sensing the odour of acid We are familiar with the unpleasant and often irritating odour associated with acids, but whereas acid receptors are known to underlie the detection of sour tastes, no acid-sensing neurons were known in the olfactory system. Greg Suh and colleagues now report the identification of such neurons in the olfactory system of the fruitfly, Drosophila melanogaster . Acid sensing also requires the transmembrane protein IR64a to be expressed in those neurons. IR64a is not sufficient by itself to determine acid recognition, but its requirement is the first known function for a member of the recently discovered ionotropic receptor family of putative odorant receptors. Acid sensing has so far been demonstrated in the gustatory system only. Now, fruitfly olfactory sensory neurons selectively activated by acidic compounds have been identified. Acid sensing also requires the transmembrane protein IR64a, expressed in those neurons as well as neurons involved in the detection of non acidic odorants. Although the IR64a protein isn't sufficient by itself to determine acid recognition, the requirement for IR64a in acid recognition is the first function for a member of this recently discovered family of putative odorant receptors — the ionotropic receptor family. The odour of acids has a distinct quality that is perceived as sharp, pungent and often irritating 1 . How acidity is sensed and translated into an appropriate behavioural response is poorly understood. Here we describe a functionally segregated population of olfactory sensory neurons in the fruitfly, Drosophila melanogaster , that are highly selective for acidity. These olfactory sensory neurons express IR64a, a member of the recently identified ionotropic receptor (IR) family of putative olfactory receptors 2 . In vivo calcium imaging showed that IR64a+ neurons projecting to the DC4 glomerulus in the antennal lobe are specifically activated by acids. Flies in which the function of IR64a+ neurons or the IR64a gene is disrupted had defects in acid-evoked physiological and behavioural responses, but their responses to non-acidic odorants remained unaffected. Furthermore, artificial stimulation of IR64a+ neurons elicited avoidance responses. Taken together, these results identify cellular and molecular substrates for acid detection in the Drosophila olfactory system and support a labelled-line mode of acidity coding at the periphery.

Joint pain is the defining symptom of osteoarthritis (OA) but its origin and mechanisms remain unclear. Here, we investigated an unprecedented role of osteoclast-initiated subchondral bone remodeling in sensory innervation for OA pain. We show that osteoclasts secrete netrin-1 to induce sensory nerve axonal growth in subchondral bone. Reduction of osteoclast formation by knockout of receptor activator of nuclear factor kappa-B ligand (Rankl) in osteocytes inhibited the growth of sensory nerves into subchondral bone, dorsal root ganglion neuron hyperexcitability, and behavioral measures of pain hypersensitivity in OA mice. Moreover, we demonstrated a possible role for netrin-1 secreted by osteoclasts during aberrant subchondral bone remodeling in inducing sensory innervation and OA pain through its receptor DCC (deleted in colorectal cancer). Importantly, knockout of Netrin1 in tartrate-resistant acid phosphatase-positive (TRAP-positive) osteoclasts or knockdown of Dcc reduces OA pain behavior. In particular, inhibition of osteoclast activity by alendronate modifies aberrant subchondral bone remodeling and reduces innervation and pain behavior at the early stage of OA. These results suggest that intervention of the axonal guidance molecules (e.g., netrin-1) derived from aberrant subchondral bone remodeling may have therapeutic potential for OA pain.

Tumour innervation is associated with worse patient outcomes in multiple cancers 1 , 2 , which suggests that it may regulate metastasis. Here we observed that highly metastatic mouse mammary tumours acquired more innervation than did less-metastatic tumours. This enhanced innervation was driven by expression of the axon-guidance molecule SLIT2 in tumour vasculature. Breast cancer cells induced spontaneous calcium activity in sensory neurons and elicited release of the neuropeptide substance P (SP). Using three-dimensional co-cultures and in vivo models, we found that neuronal SP promoted breast tumour growth, invasion and metastasis. Moreover, patient tumours with elevated SP exhibited enhanced lymph node metastatic spread. SP acted on tumoral tachykinin receptors (TACR1) to drive death of a small population of TACR1 high cancer cells. Single-stranded RNAs (ssRNAs) released from dying cells acted on neighbouring tumoural Toll-like receptor 7 (TLR7) to non-canonically activate a prometastatic gene expression program. This SP- and ssRNA-induced Tlr7 gene expression signature was associated with reduced breast cancer survival outcomes. Therapeutic targeting of this neuro–cancer axis with the TACR1 antagonist aprepitant, an approved anti-nausea drug, suppressed breast cancer growth and metastasis in multiple models. Our findings reveal that tumour-induced hyperactivation of sensory neurons regulates multiple aspects of metastatic progression in breast cancer through a therapeutically targetable neuropeptide/extracellular ssRNA sensing axis. Hyperactivation of sensory neurons regulates multiple aspects of metastatic progression in breast cancer.

In mice, it is possible to induce a psoriasis-like condition by applying imiquimod; here, the production of interleukin-23 that is stimulated by such skin inflammation is shown to depend on the interaction of nociceptors expressing the Na v 1.8 and TRPV1 channels with skin-resident dendritic cells. Peripheral neurons involved in inflammation Repeated topical application of the antiviral immune modifier imiquimod (IMQ) to murine skin provokes interleukin-23-mediated inflammatory lesions that resemble human psoriasis. Ulrich von Andrian and colleagues show that the production of skin inflammation in this disease model depends on the interaction of a subset of sensory neurons expressing the ion channels TRPV1 and Na v 1.8 with skin-resident dendritic cells. Taken together with other recent work, this finding suggests a scenario in which noxious pain fibres integrate environmental signals to modulate local immune responses to a variety of infectious and pro-inflammatory stimuli. The skin has a dual function as a barrier and a sensory interface between the body and the environment. To protect against invading pathogens, the skin harbours specialized immune cells, including dermal dendritic cells (DDCs) and interleukin (IL)-17-producing γδ T (γδT17) cells, the aberrant activation of which by IL-23 can provoke psoriasis-like inflammation 1 , 2 , 3 , 4 . The skin is also innervated by a meshwork of peripheral nerves consisting of relatively sparse autonomic and abundant sensory fibres. Interactions between the autonomic nervous system and immune cells in lymphoid organs are known to contribute to systemic immunity, but how peripheral nerves regulate cutaneous immune responses remains unclear 5 , 6 . We exposed the skin of mice to imiquimod, which induces IL-23-dependent psoriasis-like inflammation 7 , 8 . Here we show that a subset of sensory neurons expressing the ion channels TRPV1 and Na v 1.8 is essential to drive this inflammatory response. Imaging of intact skin revealed that a large fraction of DDCs, the principal source of IL-23, is in close contact with these nociceptors. Upon selective pharmacological or genetic ablation of nociceptors 9 , 10 , 11 , DDCs failed to produce IL-23 in imiquimod-exposed skin. Consequently, the local production of IL-23-dependent inflammatory cytokines by dermal γδT17 cells and the subsequent recruitment of inflammatory cells to the skin were markedly reduced. Intradermal injection of IL-23 bypassed the requirement for nociceptor communication with DDCs and restored the inflammatory response 12 . These findings indicate that TRPV1 + Na v 1.8 + nociceptors, by interacting with DDCs, regulate the IL-23/IL-17 pathway and control cutaneous immune responses.