Across social species, social touch enhances well-being and reduces pain - two seemingly distinct benefits that enhance survival. Yet where and how the nervous system integrates these functions, and whether a single mechanism could serve both, remains unknown. Here we show that massage triggers oxytocin release, which shapes both pain and touch reward at the earliest stage of central processing - the spinal cord - through a single, state-dependent circuit mechanism. We report that in humans, massage enhances well-being, effects that correlate with endogenous oxytocin release. In mice, gentle touch activates hypothalamic oxytocin neurons that project directly to the spinal dorsal horn. Genetic manipulation of spinal oxytocin circuits alters behavioral responses to both gentle touch and noxious stimuli. Spinal calcium imaging and slice electrophysiology reveal that oxytocin acts on both excitatory and inhibitory spinal neurons to sculpt the relative activity of spinal ascending systems that convey both social touch and pain to the brain. Extending these findings to humans, we show that oxytocin receptors are also expressed on spinal excitatory and inhibitory neurons, and that endogenous oxytocin during massage correlates with altered spinal touch processing. Thus, spinal oxytocin signaling provides an evolutionarily conserved mechanism for the therapeutic benefits of massage.

T-type/Ca(v)3 calcium channels are key in neuronal excitability and pain processing, with Cav3.2 being the prominent isoform in primary sensory neurons of the dorsal root ganglion (DRG). Ca(v)3.2 pharmacological inhibition or gene silencing induces analgesia in several preclinical models of inflammatory and neuropathic pain. However, the presence of Ca(v)3.2, encoded by the CACNA1H gene, in human DRG neurons remains unresolved. Using RNA in situ hybridization and electrophysiological recordings, we show that human DRG neurons express Ca(v)3.2 in a subset of neurons positive for the neurotrophic factor receptor TrkB (NTRK2 gene). The Ca(v)3.2 current exhibits typical biophysical and pharmacological properties, including inhibition by a low concentration of nickel and by Z944, a specific T-type calcium channel blocker in advanced clinical development. Conversely, ABT-639, a T-type calcium channel inhibitor that failed in phase 2 trials for pain relief, does not inhibit Ca(v)3.2 currents in human DRG neurons. Importantly, Ca(v)3.2 currents are prominent in neurons from female organ donors, supporting the presence of sex differences in pain mechanisms in humans. These findings underscore the potential of continued exploration of Ca(v)3.2 as a therapeutic target for pain treatment and highlight a specific subset of human neurons that likely rely on this channel to modulate their excitability.

Acid-sensing ion channels (ASICs) are members of the DEG/ENaC family that includes the only known peptide-gated ion channels. While ASICs are gated by protons, they are also sensitive to peptides and are modulated by the molluscan FMRFamide and other mammalian neuropeptides ending by the RFamide motif. We identified a set of synthetic short amidated hexapeptides, which not only end by the RFamide motif but also by CFamide and FCamide, as potent positive modulators of ASIC3 acid-induced activity. We focused on two of them, a RFamide peptide (FRCC‾RFamide) and a CFamide peptide (FRCRC‾Famide), demonstrating that they have similar specificity for and effects on ASIC3. The potentiating effects of the two peptides are due to a strong slow-down of desensitization, leading to an increase in the amount of current induced by acid pH (≤pH6.6), with apparent affinities ranging from 1 to 5 μM. Surprisingly, the washout kinetic of FRCC‾RFamide peptide was much slower than those of FRCRC‾Famide and other known RFamide peptides, suggesting potential differences in their mechanisms of action. Computational modeling and structure-function analysis reveal interactions of both peptides with the non-proton binding site of ASIC3 as already reported before for other RFamide peptides, but our data also suggest possible additional effects of FRCC‾RFamide involving directly or indirectly the proton binding domain. These findings expand our understanding of ASICs' modulation by peptides, identifying novel short modulators of ASIC3, including peptides with new CFamide and FCamide ending motifs, and showing differences between these peptides using their washout kinetic as a new parameter.

Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder marked by social deficits, repetitive behaviors and atypical sensory perception. The link between ASD and skin abnormalities, inducing itchiness, has never been investigated in depth. This study explores mechanical itch sensitivity in the Shank3ΔC/ΔC mouse model. Key observations include heightened scratching in response to skin deformation and hypersensitivity to mechanical itch (i.e. alloknesis) in Shank3ΔC/ΔC mice. In Shank3ΔC/ΔC mice, ex vivo electrophysiological experiments revealed that C-fiber low-threshold mechanoreceptors (C-LTMRs) were hyporesponsive and transcriptomic analysis showed a downregulation of TAFA4, a protein secreted by C-LMTRs. Interestingly, pharmacologically inhibiting Aβ-LTMR, important in mechanical itch initiation, abolished the itch hypersensitivity. Also, TAFA4 injections reduced the spontaneous scratching response to skin deformation but failed to restore itch sensitivity. Our data suggest that somatosensory deficits in Shank3ΔC/ΔC mice lead to a hypersensitivity to itchiness and indicate that two pathways might be regulating mechanical itchiness, dependent or not on TAFA4.

Acid-sensing ion channels (ASICs) are members of the DEG/ENaC family that includes the only known peptide-gated ion channels. While ASICs are gated by protons, they have kept sensitivity to peptides and are notably modulated by the molluscan FMRFamide and other related mammalian neuropeptides ending by the RFamide motif. Screening efforts have been made to identify and characterize natural peptides able to modulate ASICs’ activity, and some peptides from different species are already known to modulate ASIC1a, ASIC1b and ASIC3. We identified here a set of synthetic short amidated hexapeptides, initially designed thirty years ago for their ability to inhibit the Na/Ca exchanger, as potent and selective positive modulators of the ASIC3 acid-induced activity. We focused on two of them, a RFamide peptide (FRCCRFamide) and a CFamide peptide (FRC̄R̄C̄Famide), demonstrating that they have similar specificity for and effects on ASIC3. The potentiating effects of the two peptides are due to a strong slow-down of the current desensitization, leading to an increase in the amount of current induced by acid pH (≤pH6.6), with apparent affinities ranging from 1 to 5 µM. Surprisingly, the washout kinetic for the FRC̄C̄RFamide peptide was much slower than those of FRC̄R̄C̄Famide and other known RFamide peptides, suggesting potential differences in their mechanisms of action. Computational modeling and structure-function analysis reveal interactions of both peptides with the non-proton binding site of ASIC3 initially identified for the synthetic compound GMQ (2-guanidine-4-methylquinazoline), as already reported before for other RFamide peptides, but our data also suggest possible additional effects of FRC̄C̄RFamide involving directly or indirectly the proton binding domain. These findings expand our understanding of peptide modulation of ASIC channels and identify novel pharmacological tools selective among ASICs for investigating ASIC3 function. ### Competing Interest Statement The authors have declared no competing interest. Agence Nationale de la Recherche, https://ror.org/00rbzpz17, ANR-17-CE16-0018, ANR-22-CE16-0006 Fondation pour la Recherche Médicale, FDT202404018117 Université Côte d’Azur, https://ror.org/019tgvf94, ANR-15-IDEX-01 LabEx ICST, FR, ANR-11-LABX-0015-01 Association Française contre les Myopathies, AFM, FR, 23731

The Ca V 3.2 isoform of T-type voltage-gated calcium channels plays a crucial role in regulating the excitability of nociceptive neurons; the endogenous molecules that modulate its activity, however, remain poorly understood. Here, we used serum proteomics and patch-clamp physiology to discover a novel peptide albumin (1-26) that facilitates channel gating by chelating trace metals that tonically inhibit Ca V 3.2 via H191 residue. Importantly, serum also potently modulated T-currents in human and rodent dorsal root ganglion (DRG) neurons. In vivo pain studies revealed that injections of serum and albumin (1-26) peptide resulted in robust mechanical and heat hypersensitivity. This hypersensitivity was abolished with a T-channel inhibitor, in Ca V 3.2 null mice and in Ca V 3.2 H191Q knock-in mice. The discovery of endogenous chelators of trace metals in the serum deepens our understanding of the role of Ca V 3.2 channels in neuronal hyperexcitability and may facilitate the design of novel analgesics with unique mechanisms of action.

The recent development of single-cell and single-nucleus RNA sequencing has highlighted the extraordinary diversity of dorsal root ganglia neurons. However, the few available genetic tools limit our understanding of the functional significance of this heterogeneity. We generated a new mouse line expressing the flippase recombinase from the scn10a locus. By crossing Nav1.8Ires-FLPo mice with the AdvillinCre and RC::FL-hM3Dq mouse lines in an intersectional genetics approach, we were able to obtain somatodendritic expression of hM3Dq-mCherry selectively in the Nav1.8 lineage. The bath application of clozapine N-oxide triggered strong calcium responses selectively in mCherry+ neurons. The intraplantar injection of CNO caused robust flinching, shaking, and biting responses accompanied by strong cFos activation in the ipsilateral lumbar spinal cord. The Nav1.8Ires-FLPo mouse model will be a valuable tool for extending our understanding of the in vivo functional specialization of neuronal subsets of the Nav1.8 lineage for which inducible Cre lines are available.

T-type/Cav3 calcium channels are key in neuronal excitability and pain processing with Cav3.2 being the prominent isoform in primary sensory neurons of the dorsal root ganglion (DRG). Its pharmacological inhibition or gene silencing induces analgesia in several preclinical models of inflammatory and neuropathic pain. However, the presence of Cav3.2, encoded by the CACNA1H gene, in human DRG neurons remains unresolved. Using RNA in-situ hybridization and electrophysiological recordings, we show that human DRGs express Cav3.2 in a subset of neurons positive for the neurotrophic factor receptor TrkB (NTRK2 gene). The Cav3.2 current exhibits typical biophysical and pharmacological properties, including inhibition by a low concentration of nickel and by Z944, a specific T-type calcium channel blocker in advanced clinical development. Conversely, ABT-639, a T-type calcium channel inhibitor that failed in Phase 2 trials for pain relief, does not inhibit Cav3.2 currents in human DRG neurons. Importantly, Cav3.2 currents are prominent in neurons from female organ donors, supporting the presence of sex differences in pain mechanisms in humans. These findings underscore the potential of continued exploration of Cav3.2 as a therapeutic target for pain treatment and highlight a specific subset of human neurons that likely rely on this channel to modulate their excitability. ### Competing Interest Statement The authors have declared no competing interest.

The disinhibition of the excitatory PKCgamma interneurons plays a central role during mechanical allodynia in the dorsal horn of the spinal cord, routing harmless information to nociceptive pathways. The T-type calcium channel Cav3.2, necessary for mechanical and cold allodynia, is found in most PKCgamma neurons of the spinal cord. In this study, the role of Cav3.2 in PKCgamma neurons was studied after its pharmacological inhibition and its conditional deletion (KO) in Cav3.2GFP-Flox KI x PKCgamma-CreERT2 x Ai14 mice in normal conditions and in the spared-nerve-injury (SNI) model of neuropathic pain. Conditional deletion of Cav3.2 increased the hindpaw basal mechanical sensitivity before surgery, and decreased mechanical pain 7 days, but not 28 days, after surgery. At the cellular level, Cav3.2 participated in the low-threshold currents of PKCgamma neurons and the T-type calcium current of PKCgamma neurons was decreased in KO mice as compared to wild-type (WT). This loss did not convert into proportional alterations in subthreshold properties including rebound potentials, suggesting the involvement of other T-type channels. Action potential kinetics and firing properties seemed similar in WT and KO mice too, but rebound potentials were diminished in the SNI model in WT but not in KO mice. In addition, the modulations of firing properties induced by T-type channel pharmacological blocker Z944 observed in WT mice were absent in KO mice and after SNI. Furthermore, the pairing of action potentials was modified after SNI in WT mice, and not in KO mice. At the synaptic level, excitatory currents were lowered 7- and 28-days after surgery, while inhibitory currents were lowered only at 28 days. These changes were not found in Cav3.2-ablated neurons. Miniature currents analysis indicated that Cav3.2 was involved in both excitatory and inhibitory synaptic transmissions at the level of PKCgamma neurons. Surprisingly, Z944 did not mimic the effects of Cav3.2 ablation in PKCgamma neurons, suggesting distinct and eventually opposite roles of other T-type calcium channels. Altogether, our results show that Cav3.2 is not mandatory for firing of PKCgamma neurons of the dorsal horn of the spinal cord, but that it participates to the SNI-induced changes in their intrinsic and synaptic activity, including changes in their excitatory and inhibitory controls. ### Competing Interest Statement The authors have declared no competing interest.

Venom peptides have evolved to target a wide range of membrane proteins through diverse mechanisms of action and structures, providing promising therapeutic leads for diseases, including pain, epilepsy, and cancer, as well as unique probes of ion channel structure-function. In this work, a high-throughput FLIPR window current screening assay on T-type CaV3.2 guided the isolation of a novel peptide named ω-Buthitoxin-Hf1a from scorpion Hottentotta franzwerneri crude venom. At only 10 amino acid residues with one disulfide bond, it is not only the smallest venom peptide known to target T-type CaVs but also the smallest structured scorpion venom peptide yet discovered. Synthetic Hf1a peptides were prepared with C-terminal amidation (Hf1a-NH2) or a free C-terminus (Hf1a-OH). Electrophysiological characterization revealed Hf1a-NH2 to be a concentration-dependent partial inhibitor of CaV3.2 (IC50 = 1.18 μM) and CaV3.3 (IC50 = 0.49 μM) depolarized currents but was ineffective at CaV3.1. Hf1a-OH did not show activity against any of the three T-type subtypes. Additionally, neither form showed activity against N-type CaV2.2 or L-type calcium channels. The three-dimensional structure of Hf1a-NH2 was determined using NMR spectroscopy and used in docking studies to predict its binding site at CaV3.2 and CaV3.3. As both CaV3.2 and CaV3.3 have been implicated in peripheral pain signaling, the analgesic potential of Hf1a-NH2 was explored in vivo in a mouse model of incision-induced acute post-surgical pain. Consistent with this role, Hf1a-NH2 produced antiallodynia in both mechanical and thermal pain.

Abstract Neuropathic pain remains difficult to treat, with drug development hampered by an incomplete understanding of the pathogenesis of the condition, as well as a lack of biomarkers. The problem is compounded by the scarcity of relevant human peripheral tissues, including skin, nerves, and dorsal root ganglia. Efforts to obtain such samples are accelerating, increasing the need for standardisation across laboratories. In this white paper, we report on a consensus meeting attended by neuropathic pain experts, designed to accelerate protocol alignment and harmonization of studies involving relevant peripheral tissues. The meeting was held in London in March 2024 and attended by 28 networking partners, including industry and patient representatives. We achieved consensus on minimal recommended phenotyping, harmonised wet laboratory protocols, statistical design, reporting, and data sharing. Here, we also share a variety of relevant standard operating procedures as supplementary protocols. We envision that our recommendations will help unify human tissue research in the field and accelerate our understanding of how abnormal interactions between sensory neurons and their local peripheral environment contribute towards neuropathic pain.

Spinal cord dorsal horn inhibition is critical to the processing of sensory inputs, and its impairment leads to mechanical allodynia. How this decreased inhibition occurs and whether its restoration alleviates allodynic pain are poorly understood. Here, we show that a critical step in the loss of inhibitory tone is the change in the firing pattern of inhibitory parvalbumin (PV)-expressing neurons (PVNs). Our results show that PV, a calcium-binding protein, controls the firing activity of PVNs by enabling them to sustain high-frequency tonic firing patterns. Upon nerve injury, PVNs transition to adaptive firing and decrease their PV expression. Interestingly, decreased PV is necessary and sufficient for the development of mechanical allodynia and the transition of PVNs to adaptive firing. This transition of the firing pattern is due to the recruitment of calcium-activated potassium (SK) channels, and blocking them during chronic pain restores normal tonic firing and alleviates chronic pain. Our findings indicate that PV is essential for controlling the firing pattern of PVNs and for preventing allodynia. Developing approaches to manipulate these mechanisms may lead to different strategies for chronic pain relief.

Inflammation and pain are intertwined responses to injury, infection, or chronic diseases. While acute inflammation is essential in determining pain resolution and opioid analgesia, maladaptive processes occurring during resolution can lead to the transition to chronic pain. Here we found that inflammation activates the cytosolic DNA-sensing protein stimulator of IFN genes (STING) in dorsal root ganglion nociceptors. Neuronal activation of STING promotes signaling through TANK-binding kinase 1 (TBK1) and triggers an IFN-β response that mediates pain resolution. Notably, we found that mice expressing a nociceptor-specific gain-of-function mutation in STING exhibited an IFN gene signature that reduced nociceptor excitability and inflammatory hyperalgesia through a KChIP1-Kv4.3 regulation. Our findings reveal a role of IFN-regulated genes and KChIP1 downstream of STING in the resolution of inflammatory pain.

Venom‐derived peptides targeting ion channels involved in pain are regarded as a promising alternative to current, and often ineffective, chronic pain treatments. Many peptide toxins are known to specifically and potently block established therapeutic targets, among which the voltage‐gated sodium and calcium channels are major contributors. Here, we report on the discovery and characterization of a novel spider toxin isolated from the crude venom of Pterinochilus murinus that shows inhibitory activity at both hNaV1.7 and hCaV3.2 channels, two therapeutic targets implicated in pain pathways. Bioassay‐guided HPLC fractionation revealed a 36‐amino acid peptide with three disulfide bridges named μ/ω‐theraphotoxin‐Pmu1a (Pmu1a). Following isolation and characterization, the toxin was chemically synthesized and its biological activity was further assessed using electrophysiology, revealing Pmu1a to be a toxin that potently blocks both hNaV1.7 and hCaV3. Nuclear magnetic resonance structure determination of Pmu1a shows an inhibitor cystine knot fold that is the characteristic of many spider peptides. Combined, these data show the potential of Pmu1a as a basis for the design of compounds with dual activity at the therapeutically relevant hCaV3.2 and hNaV1.7 voltage‐gated channels.

The rhythmical nature of the cardiovascular system constantly generates dynamic mechanical forces. At the centre of this system is the heart, which must detect these changes and adjust its performance accordingly. Mechanoelectric feedback provides a rapid mechanism for detecting even subtle changes in the mechanical environment and transducing these signals into electrical responses, which can adjust a variety of cardiac parameters such as heart rate and contractility. However, pathological conditions can disrupt this intricate mechanosensory system and manifest as potentially life-threatening cardiac arrhythmias. Mechanosensitive ion channels are thought to be the main proponents of mechanoelectric feedback as they provide a rapid response to mechanical stimulation and can directly affect cardiac electrical activity. Here, we demonstrate that the mechanosensitive ion channel PIEZO1 is expressed in zebrafish cardiomyocytes. Furthermore, chemically prolonging PIEZO1 activation in zebrafish results in cardiac arrhythmias. indicating that this ion channel plays an important role in mechanoelectric feedback. This also raises the possibility that PIEZO1 gain of function mutations could be linked to heritable cardiac arrhythmias in humans.

The mammalian spinal cord functions as a community of cell types for sensory processing, autonomic control, and movement. While animal models have advanced our understanding of spinal cellular diversity, characterizing human biology directly is important to uncover specialized features of basic function and human pathology. Here, we present a cellular taxonomy of the adult human spinal cord using single-nucleus RNA sequencing with spatial transcriptomics and antibody validation. We identified 29 glial clusters and 35 neuronal clusters, organized principally by anatomical location. To demonstrate the relevance of this resource to human disease, we analyzed spinal motoneurons, which degenerate in amyotrophic lateral sclerosis (ALS) and other diseases. We found that compared with other spinal neurons, human motoneurons are defined by genes related to cell size, cytoskeletal structure, and ALS, suggesting a specialized molecular repertoire underlying their selective vulnerability. We include a web resource to facilitate further investigations into human spinal cord biology.

Background and Purpose T-type calcium channels, mainly the Ca(v)3.2 subtype, are important contributors to the nociceptive signalling pathway. We investigated their involvement in inflammation and related pain-like symptoms. Experimental Approach The involvement of Ca(v)3.2 and T-type channels was investigated using genetic and pharmacological inhibition to assess mechanical allodynia/hyperalgesia and oedema development in two murine inflammatory pain models. The location of Ca(v)3.2 channels involved in pain-like symptoms was studied in mice with Ca(v)3.2 knocked out in C-low threshold mechanoreceptors (C-LTMR) and the use of ABT-639, a peripherally restricted T-type channel inhibitor. The anti-oedema effect of Ca(v)3.2 channel inhibition was investigated in chimeric mice with immune cells deleted for Ca(v)3.2. Lymphocytes and macrophages from either green fluorescent protein-targeted Ca(v)3.2 or KO mice were used to determine the expression of Ca(v)3.2 protein and the functional status of the cells. Key Results Ca(v)3.2 channels contributed to the development of pain-like symptoms and oedema in the two murine inflammatory pain models. Our results provided evidence of the involvement of Ca(v)3.2 channels located on C-LTMRs and spinal cord in inflammatory pain. Ca(v)3.2 channels located in T cells and macrophages contribute to the inflammatory process. Conclusion and Implications Ca(v)3.2 channels play crucial roles in inflammation and related pain, implying that targeting of Ca(v)3.2 channels with pharmacological agents could be an attractive and readily evaluable strategy in clinical trials, to relieve chronic inflammatory pain in patients.