Primary Sensory Neurons Research Articles

Comparative transcriptional analysis of satellite glial cell injury response

Background: Satellite glial cells (SGCs) tightly surround and support primary sensory neurons in the peripheral nervous system and are increasingly recognized for their involvement in the development of neuropathic pain following nerve injury. SGCs are difficult to investigate due to their flattened shape and tight physical connection to neurons in vivo and their rapid changes in phenotype and protein expression when cultured in vitro. Consequently, several aspects of SGC function under normal conditions as well as after a nerve injury remain to be explored. The recent advance in single cell RNA sequencing (scRNAseq) technologies has enabled a new approach to investigate SGCs. Methods: In this study we used scRNAseq to investigate SGCs from mice subjected to sciatic nerve injury. We used a meta-analysis approach to compare the injury response with that found in other published datasets. Furthermore, we also used scRNAseq to investigate how cells from the dorsal root ganglion (DRG) change after 3 days in culture. Results: From our meta-analysis of the injured conditions, we find that SGCs share a common signature of 18 regulated genes following sciatic nerve crush or sciatic nerve ligation, involving transcriptional regulation of cholesterol biosynthesis. We also observed a considerable transcriptional change when culturing SGCs, suggesting that some differentiate into a specialised in vitro state while others start resembling Schwann cell-like precursors. Conclusion: By using integrated analyses of new and previously published scRNAseq datasets, this study provides a consensus view of which genes are most robustly changed in SGCs after injury. Our results are available via the Broad Institute Single Cell Portal, so that readers can explore and search for genes of interest.

Enhancement of P2X3 Receptor-Mediated Currents by Lysophosphatidic Acid in Rat Primary Sensory Neurons.

Lysophosphatidic acid (LPA), a lipid metabolite, plays a role in both neuropathic and inflammatory pain through LPA1 receptors. P2X3 receptor has also been shown to participate in these pathological processes. However, it is still unclear whether there is a link between LPA signaling and P2X3 receptors in pain. Herein, we show that a functional interaction between them in rat dorsal root ganglia (DRG) neurons. Pretreatment of LPA concentration-dependently enhanced α,β-methylene-ATP (α,β-meATP)-induced inward currents mediated by P2X3 receptors. LPA significantly increased the maximal current response of α,β-meATP, showing an upward shift of the concentration-response curve for α,β-meATP. The LPA enhancement was independent on the clamping-voltage. Enhancement of P2X3 receptor-mediated currents by LPA was prevented by the LPA1 receptor antagonist Ki16198, but not by the LPA2 receptor antagonist H2L5185303. The LPA-induced potentiation was also attenuated by intracellular dialysis of either G-protein inhibitor or protein kinase C (PKC) inhibitor, but not by Rho inhibitor. Moreover, LPA significantly changed the membrane potential depolarization and action potential burst induced by α,β-meATP in DRG neurons. Finally, LPA exacerbated α,β-meATP- induced nociceptive behaviors in rats. These results suggested that LPA potentiated the functional activity of P2X3 receptors in rat primary sensory neurons through activation of the LPA1 receptor and its downstream PKC rather than Rho signaling pathway, indicating a novel peripheral mechanism underlying the sensitization of pain.

Elevated 18:0 lysophosphatidylcholine contributes to the development of pain in tissue injury.

Tissue injuries, including burns, are major causes of death and morbidity worldwide. These injuries result in the release of intracellular molecules and subsequent inflammatory reactions, changing the tissues' chemical milieu and leading to the development of persistent pain through activating pain-sensing primary sensory neurons. However, the majority of pain-inducing agents in injured tissues are unknown. Here, we report that, amongst other important metabolite changes, lysophosphatidylcholines (LPCs) including 18:0 LPC exhibit significant and consistent local burn injury-induced changes in concentration. 18:0 LPC induces immediate pain and the development of hypersensitivities to mechanical and heat stimuli through molecules including the transient receptor potential ion channel, vanilloid subfamily, member 1, and member 2 at least partly via increasing lateral pressure in the membrane. As levels of LPCs including 18:0 LPC increase in other tissue injuries, our data reveal a novel role for these lipids in injury-associated pain. These findings have high potential to improve patient care.

Transcriptional Alterations of Mouse Trigeminal Ganglion Neurons Following Orofacial Inflammation Revealed by Single-Cell Analysis.

Orofacial inflammation leads to transcriptional alterations in trigeminal ganglion (TG) neurons. However, diverse alterations and regulatory mechanisms following orofacial inflammatory pain in different types of TG neurons remain unclear. Here, orofacial inflammation was induced by injection of complete Freund’s adjuvant (CFA) in mice. After 7 days, we performed single-cell RNA-sequencing on TG cells of mice from control and treatment groups. We identified primary sensory neurons, Schwann cells, satellite glial cells, oligodendrocyte-like cells, immune cells, fibroblasts, and endothelial cells in TG tissue. After principal component analysis and hierarchical clustering, we identified six TG neuronal subpopulations: peptidergic nociceptors (PEP1 and PEP2), non-peptidergic nociceptors (NP1 and NP2), C-fiber low-threshold mechanoreceptors (cLTMR) and myelinated neurons (Nefh-positive neurons, NF) based on annotated marker gene expression. We also performed differential gene expression analysis among TG neuronal subtypes, identifying several differential genes involved in the inflammatory response, neuronal excitability, neuroprotection, and metabolic processes. Notably, we identified several potential novel targets associated with pain modulation, including Arl6ip1, Gsk3b, Scn7a, and Zbtb20 in PEP1, Rgs7bp in PEP2, and Bhlha9 in cLTMR. The established protein–protein interaction network identified some hub genes, implying their critical involvement in regulating orofacial inflammatory pain. Our study revealed the heterogeneity of TG neurons and their diverse neuronal transcriptomic responses to orofacial inflammation, providing a basis for the development of therapeutic strategies for orofacial inflammatory pain.

Adverse effects of methylene blue in peripheral neurons: An in vitro electrophysiology and cell culture study.

Methylene blue (MB) is an effective treatment for methemoglobinemia, ifosfamide-induced encephalopathy, cyanide poisoning, and refractory vasoplegia. However, clinical case reports and preclinical studies indicate potentially neurotoxic activity of MB at certain concentrations. The exact mechanisms of MB neurotoxicity are not known, and while the effects of MB on neuronal tissue from different brain regions and myenteric ganglia have been examined, its effects on primary afferent neurons from dorsal root ganglia (DRG) have not been studied. Mouse DRG were exposed to MB (0.3-10μM) in vitro to assess neurite outgrowth. Increasing concentrations of MB (0.3-10μM) were associated with neurotoxicity as shown by a substantial loss of cells with neurite formation, particularly at 10μM. In parallel experiments, cultured rat DRG neurons were treated with MB (100μM) to examine how MB affects electrical membrane properties of small-diameter sensory neurons. MB decreased peak inward and outward current densities, decreased action potential amplitude, overshoot, afterhyperpolarization, increased action potential rise time, and decreased action potential firing in response to current stimulation. MB induced dose-dependent toxicity in peripheral neurons, in vitro. These findings are consistent with studies in brain and myenteric ganglion neurons showing increased neuronal loss and altered membrane electrical properties after MB application. Further research is needed to parse out the toxicity profile for MB to minimize damage to neuronal structures and reduce side effects in clinical settings.

Slowly-adapting type II afferents contribute to conscious touch sensation in humans: Evidence from single unit intraneural microstimulation.

Slowly‐adapting type II (SA‐II, Ruffini) mechanoreceptive afferents respond well to pressure and stretch, and are regularly encountered in human microneurography studies. Despite an understanding of SA‐II response properties, their role in touch perception remains unclear. Specific roles of different myelinated Aβ mechanoreceptive afferents in tactile perception have been revealed using single unit intraneural microstimulation (INMS), via microneurography, recording from and then electrically stimulating individual afferents. This method directly links single afferent artificial activation to perception, where INMS produces specific ‘quantal’ touch percepts associated with different mechanoreceptive afferent types. However, SA‐II afferent stimulation has been ambiguous, producing inconsistent, vague sensations, or no clear percept. We physiologically characterized hundreds of individual Aβ mechanoreceptive afferents in the glabrous hand skin and examined the subsequent percepts evoked by trains of low amplitude INMS current pulses (<10 μA). We present 18 SA‐II afferents where INMS resulted in a clear, electrically evoked sensation of large (∼36 mm2) diffuse pressure, which was projected precisely to their physiologically‐defined receptive field in the skin. This sensation was felt as natural, distinctive from other afferents, and showed no indications of multi‐afferent stimulation. Stimulus frequency modulated sensation intensity and even brief stimuli (4 pulses, 60 ms) were perceived. These results suggest that SA‐II afferents contribute to perceived tactile sensations, can signal this rapidly and precisely, and are relevant and important for computational models of touch sensation and artificial prosthetic feedback.Key points Slowly adapting type II mechanoreceptors (SA‐IIs) are primary sensory neurons in humans that respond to pressure and stretch applied to the skin.To date, no specific conscious correlate of touch has been linked to SA‐II activation.Using microneurography and intraneural microstimulation to stimulate single sensory neurons in human subjects, we find a specific sensation linked to the activation of single SA‐II afferents.This sensation of touch was reported as gentle pressure and subjects could detect this with a high degree of accuracy.Methods of artificial tactile sensory feedback and computational models of touch should include SA‐IIs as meaningful contributors to the conscious sensation of touch.

ID:16006 Successful Dorsal Root Ganglion SCS Trial Following Failed Dorsal Column Trial

Targeting the dorsal root ganglion which consists of cell bodies of the primary sensory neurons can play a vital role in reduction of chronic pain conditions (1). The DRG is involved in the transduction of pain signals to the central nervous system (CNS) from the peripheral nervous system (PNS) (2).

Artesunate Alleviates Paclitaxel-Induced Neuropathic Pain in Mice by Decreasing Metabotropic Glutamate Receptor 5 Activity and Neuroinflammation in Primary Sensory Neurons.

Experimental studies on the pathogenetic process of paclitaxel-induced neuropathic pain (PINP) have been initially carried out, but PINP still has no effective therapy. Recently reported studies have highlighted the involvement of glutamate receptors and neuroinflammation in peripheral and central nociceptive transmission in PINP. Artesunate is a first-line antimalarial drug with established efficacy in alleviating pain in a variety of pathologies. The current work assessed whether artesunate inhibits PINP by modulating metabotropic glutamate receptor 5 (mGluR5) and neuroinflammation in mice. The anti-hyperalgesic effect of artesunate was verified by assessing mechanical frequency and thermal latency in the paw withdrawal test as well as spontaneous pain. The expression levels of mGluR5, pain-related receptors and neuroinflammatory markers in dorsal root ganglion (DRG) were examined. In addition, treatment with CHPG and 2-methyl-6-(phenyl ethynyl) pyridine (MPEP) (mGluR5 agonist and antagonist, respectively) was performed to determine mGluR5’s role in the anti-hyperalgesic properties of artesunate. We demonstrated artesunate prevented PINP in a dose-dependent manner, while exerting a clear anti-hyperalgesic effect on already existing PINP. Artesunate normalized paclitaxel-related expression changes in DRG mGluR5, NR1, and GluA2, as well as six paclitaxel related neuroinflammation markers. Intrathecal application of MPEP treated PINP by reversing NR1 and GluA2 expression changes but had no effects on chemokines and inflammatory factors. Furthermore, artesunate treatment reversed acute pain following CHPG application. In conclusion, this study revealed that artesunate alleviates paclitaxel-induced hyperalgesia and spontaneous pain by decreasing DRG mGluR5 expression and neuroinflammation in the mouse model of PINP.

Significantly Delayed Medium-Latency Response of the Stretch Reflex in Delayed-Onset Muscle Soreness of the Quadriceps Femoris Muscles Is Indicative of Sensory Neuronal Microdamage.

Unaccustomed or strenuous eccentric exercise is known to cause delayed-onset muscle soreness. A recent hypothesis postulated that mechano-energetic microinjury of the primary afferent sensory neuron terminals in the muscle spindles, namely a transient Piezo2 channelopathy, could be the critical cause of delayed-onset muscle soreness in the form of a bi-phasic non-contact injury mechanism. This theory includes that this microlesion could delay the medium-latency response of the stretch reflex. Our aim with this study was to investigate this hypothesis. According to our knowledge, no study has examined the effect of delayed-onset muscle soreness on the medium-latency response of the stretch reflex. Our findings demonstrated that a significant delay in the medium-latency stretch reflex could be observed right after a multi-stage fitness test in the quadriceps femoris muscles of Hungarian professional handball players who consequently experienced delayed-onset muscle soreness. The long-latency stretch reflex and most likely short-latency stretch reflex were unaffected by delayed-onset muscle soreness in our study, which is in line with earlier findings. We translate these findings as indicative of proprioceptive Type Ia terminal microdamage in the muscle spindle in line with the aforementioned new acute non-contact compression axonopathy theory of delayed-onset muscles soreness.

The Gain-of-Function R222S Variant in Scn11a Contributes to Visceral Hyperalgesia and Intestinal Dysmotility in Scn11a R222S/R222S Mice.

BackgroundThe SCN11A gene encodes the α-subunit of the Nav1. 9 channel, which is a regulator of primary sensory neuron excitability. Nav1.9 channels play a key role in somatalgia. Humans with the gain-of-function mutation R222S in SCN11A exhibit familial episodic pain. As already known, R222S knock-in mice carrying a mutation orthologous to the human R222S variant demonstrate somatic hyperalgesia. This study investigated whether Scn11aR222S/R222S mice developed visceral hyperalgesia and intestinal dysmotility.MethodsWe generated Scn11aR222S/R222S mice using the CRISPR/Cas9 system. The somatic pain threshold in Scn11aR222S/R222S mice was assessed by Hargreaves' test and formalin test. The excitability of dorsal root ganglia (DRG) neurons was assessed by whole-cell patch-clamp recording. Visceralgia was tested using the abdominal withdrawal reflex (AWR), acetic acid-induced writhing, and formalin-induced visceral nociception tests. Intestinal motility was detected by a mechanical recording of the intestinal segment and a carbon powder propelling test. The excitability of the enteric nervous system (ENS) could influence gut neurotransmitters. Gut neurotransmitters participate in regulating intestinal motility and secretory function. Therefore, vasoactive intestinal peptide (VIP) and substance P (SP) were measured in intestinal tissues.ResultsThe R222S mutation induced hyperexcitability of dorsal root ganglion neurons in Scn11aR222S/R222S mice. Scn11aR222S/R222S mice exhibited somatic hyperalgesia. In addition, Scn11aR222S/R222S mice showed lower visceralgia thresholds and slowed intestinal movements when compared with wild-type controls. Moreover, Scn11aR222S/R222S mice had lower SP and VIP concentrations in intestinal tissues.ConclusionsThese results indicated that Scn11aR222S/R222S mice showed visceral hyperalgesia and intestinal dysmotility.

Arginine-Containing Tripeptides as Analgesic Substances: The Possible Mechanism of Ligand-Receptor Binding to the Slow Sodium Channel.

Two short arginine-containing tripeptides, H-Arg-Arg-Arg-OH (TP1) and Ac-Arg-Arg-Arg-NH2 (TP2), have been shown by the patch-clamp method to modulate the NaV1.8 channels of DRG primary sensory neurons, which are responsible for the generation of nociceptive signals. Conformational analysis of the tripeptides indicates that the key role in the ligand-receptor binding of TP1 and TP2 to the NaV1.8 channel is played by two positively charged guanidinium groups of the arginine side chains located at the characteristic distance of ~9 Å from each other. The tripeptide effect on the NaV1.8 channel activation gating device has been retained when the N- and C-terminal groups of TP1 were structurally modified to TP2 to protect the attacking peptide from proteolytic cleavage by exopeptidases during its delivery to the molecular target, the NaV1.8 channel. As demonstrated by the organotypic tissue culture method, the agents do not affect the DRG neurite growth, which makes it possible to expect the absence of adverse side effects at the tissue level upon administration of TP1 and TP2. The data obtained indicate that both tripeptides can have great therapeutic potential as novel analgesic medicinal substances.

Comparative transcriptional analysis of satellite glial cell injury response

Background: Satellite glial cells (SGCs) tightly surround and support primary sensory neurons in the peripheral nervous system and are increasingly recognized for their involvement in the development of neuropathic pain following nerve injury. SGCs are difficult to investigate due to their flattened shape and tight physical connection to neurons in vivo and their rapid changes in phenotype and protein expression when cultured in vitro. Consequently, several aspects of SGC function under normal conditions as well as after a nerve injury remain to be explored. The recent advance in single cell RNA sequencing (scRNAseq) technologies has enabled a new approach to investigate SGCs. Methods: In this study we used scRNAseq to investigate SGCs from mice subjected to sciatic nerve injury. We used a meta-analysis approach to compare the injury response with that found in other published datasets. Furthermore, we also used scRNAseq to investigate how cells from the dorsal root ganglion (DRG) change after 3 days in culture. Results: From our meta-analysis of the injured conditions, we find that SGCs share a common signature of 18 regulated genes following sciatic nerve crush or sciatic nerve ligation, involving transcriptional regulation of cholesterol biosynthesis. We also observed a considerable transcriptional change when culturing SGCs, suggesting that some differentiate into a specialised in vitro state while others start resembling Schwann cell-like precursors. Conclusion: By using integrated analyses of new and previously published scRNAseq datasets, this study provides a consensus view of which genes are most robustly changed in SGCs after injury. Our results are available via the Broad Institute Single Cell Portal, so that readers can explore and search for genes of interest.

Differential Activation of pERK1/2 and c-Fos Following Injury to Different Regions of Primary Sensory Neuron.

Nerve injury causes hyperexcitability of the dorsal root ganglion (DRG) and spinal dorsal horn (DH) neurons, which results in neuropathic pain. We have previously demonstrated that partial dorsal rhizotomy (PDR) produced less severe pain-like behavior than chronic constriction injury (CCI) or chronic compression of DRG (CCD) and did not enhance DRG neuronal excitability. However, the mechanisms underlying such discrepancy remain unclear. This study was designed to compare the activation of phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) in DRG and DH, and c-Fos in DH following treatments of CCI, CCD, and PDR. We confirmed that thermal hyperalgesia produced by PDR was less severe than that produced by CCI or CCD. We showed that pERK1/2 in DRG and DH was greatly activated by CCI or CCD, whereas PDR produced only transient and mild pERK1/2 activation. CCI, CCD, and PDR induced robust c-Fos expression in DH; nevertheless, c-Fos+ neurons following PDR were much fewer than that following CCI or CCD. Blocking retrograde axonal transport by colchicine proximal to the CCI injury site diminished thermal hyperalgesia and inhibited pERK1/2 and c-Fos activation. These findings demonstrate that less severe pain-like behavior produced by PDR than CCI or CCD attributes to less activation of pERK1/2 and c-Fos. Such neurochemical activation partially relies on retrograde axonal transport of certain “injury signals” from the peripheral injured site to DRG somata.

Yokukansan Inhibits the Development of Morphine Tolerance by Regulating Presynaptic Proteins in DRG Neurons.

Opioids, such as morphine, are used in clinical settings for the management of acute and chronic pain. However, long-term use of morphine leads to antinociceptive tolerance and hypersensitivity. The cellular and molecular mechanisms of morphine tolerance seem to be quite complex, with suggestions including internalization of the μ-opioid receptor (MOR), neuroinflammation with activation of microglia and astrocytes, and changes in synaptic function in the central nervous system. Yokukansan (YKS), a traditional Kampo medicine consisting of seven herbs, has been used to treat emotional instability, neurosis, and insomnia. Interestingly, recent studies have begun to reveal the inhibitory effect of YKS on the development of morphine tolerance. In the present study, we determined the effect of YKS on morphine tolerance formation and its mechanisms in a rat model, focusing on the synapses between primary sensory neurons and spinal dorsal horn secondary neurons. We found that morphine tolerance formation was significantly inhibited by YKS (0.3 or 1.0 g/kg/day) preadministration for 7 days. Repeated administration of morphine (10 mg/kg/day) increased the expression of presynaptic proteins, including synaptotagmin I, in the spinal cord, which was suppressed by YKS. Furthermore, these changes in presynaptic protein expression were more pronounced at isolectin B4 (IB4)-positive excitatory synapses around the lamina II of the dorsal horn. These results suggest that YKS suppresses the development of morphine tolerance by inhibiting the enhancement of presynaptic function of dorsal root ganglia neurons projecting to spinal dorsal horn neurons caused by continuous morphine administration.

Synaptophysin is a selective marker for axons in human cutaneous end organ complexes

BackgroundSmall clear synaptic-like vesicles fill axon terminals of mechanoreceptors. Their functional significance is controversial and probably includes release of neurotransmitters from afferent axon terminals. Synaptophysin, a major protein of the synaptic vesicle membrane, is present in presynaptic endings of the central and peripheral nervous systems. It is also expressed in mechanosensory neurons which extend into skin forming sensory corpuscles. Nevertheless, synaptophysin occurrence in these structures has never been investigated. MethodsHere we used immunohistochemistry to detect synaptophysin in adult human dorsal root ganglia, cutaneous Meissner and Pacinian corpuscles and Merkel cell-neurite complexes from foetal to elderly period. Moreover, we analyzed whether synaptophysin co-localizes with the mechano-gated protein PIEZO2. ResultsSynaptophysin immunoreactivity was observed in primary sensory neurons (36 ± 6%) covering the entire soma size ranges. Axons of Meissner’s and Pacinian corpuscles were positive for synaptophysin from 36 and 12 weeks of estimated gestational age respectively, to 72 years old. Synaptophysin was also detected in Merkel cells (from 14 weeks of estimated gestational age to old age). Additionally in adult skin, synaptophysin and PIEZO2 co-localized in the axon of Meissner and Pacinian corpuscles, Merkel cells as well as in some axons of Merkel cell-neurite complexes. ConclusionPresent results demonstrate that a subpopulation of primary sensory neurons and their axon terminals forming cutaneous sensory corpuscles contain synaptophysin, a typical presynaptic vesicle protein. Although the functional relevance of these findings is unknown it might be related to neurotransmission mechanisms linked to mechanotransduction.

On the effects of diabetes mellitus on the mechanical properties of DRG sensory neurons and their possible relation with diabetic neuropathy

Diabetic neuropathy (DN) is one of the principal complications of diabetes mellitus (DM). Dorsal root ganglion (DRG) neurons are the primary sensory neurons that transduce mechanical, chemical, thermal, and pain stimuli. Diabetes-caused sensitivity alterations and presence of pain are due to cellular damage originated by persistent hyperglycemia, microvascular insufficiency, and oxidative and nitrosative stress. However, the underlying mechanisms have not been fully clarified. The present work addresses this problem by hypothesizing that sensitivity changes in DN result from mechanotransduction-system alterations in sensory neurons; especially, plasma membrane affectations. This hypothesis is tackled by means of elastic-deformation experiments performed on DGR neurons from a murine model for type-1 DM, as well a mathematical model of the cell mechanical structure. The obtained results suggest that the plasma-membrane fluidity of DRG sensory neurons is modified by the induction of DM, and that this alteration may correlate with changes in the cell calcium transient that results from mechanical stimuli.

Distribution of TRPM8-expressing trigeminal nerve fibers in the pons and medulla oblongata of the mouse brain

Transient receptor potential melastatin 8 (TRPM8), a cold-mediated ion channel, is well known to be expressed in primary sensory neurons; however, limited information is currently available on the distribution of TRPM8-expressing trigeminal nerve fibers in the brainstem. The present study showed the distribution of TRPM8-expressing fibers in the pons and medulla oblongata of the TRPM8 KO mice engineered by knocking in EGFP at the frame of the start codon of TRPM8. In addition, TRPM8-expressing fibers were also observed in the brachium pontis, middle cerebellar peduncle, the sensory root of the trigeminal nerve, and spinal trigeminal tract (sp5). Furthermore, TRPM8-expressing nerve fibers surrounded the somata of HuC/D-positive neurons in the sp5. Moreover, the distribution of TRPM8-expressing fibers from rostral to caudal was visualized in sagittal sections of the mouse brain. The present results also revealed that a high number of TRPM8-expressing fibers colocalized with CTB-labeled fibers in the sp5 following an injection of CTB into the whisker compared to mice's eye and ear. These results show the distribution pathway of TRPM8-expressing fibers in the pons and medulla oblongata and possible involvement in peripheral signaling from the trigeminal nerve.

Raf kinase inhibitory protein reduces bradykinin receptor desensitization.

Inflammatory hyperalgesia represents a nociceptive phenotype that can become persistent in nature through dynamic protein modifications. However, a large gap in knowledge exists concerning how the integration of intracellular signaling molecules coordinates a persistent inflammatory phenotype. Herein, we demonstrate that Raf Kinase Anchoring Protein (RKIP) interrupts a vital canonical desensitization pathway to maintain bradykinin (BK) receptor activation in primary afferent neurons. Biochemical analyses of primary neuronal cultures indicate bradykinin‐stimulated PKC phosphorylation of RKIP at Ser153. Furthermore, BK exposure increases G‐protein Receptor Kinase 2 (GRK2) binding to RKIP, inhibiting pharmacological desensitization of the BK receptor. Additional studies found that molecular RKIP down‐regulation increases BK receptor desensitization in real‐time imaging of primary afferent neurons, identifying a key pathway integrator in the desensitization process that controls multiple GRK2‐sensitive G‐protein coupled receptors. Therefore, RKIP serves as an integral scaffolding protein that inhibits BK receptor desensitization.

Sensory neuron-expressed TRPC3 mediates acute and chronic itch.

Chronic pruritus is a prominent symptom of allergic contact dermatitis (ACD) and represents a huge unmet health problem. However, its underlying cellular and molecular mechanisms remain largely unexplored. TRPC3 is highly expressed in primary sensory neurons and has been implicated in peripheral sensitization induced by proinflammatory mediators. Yet, the role of TRPC3 in acute and chronic itch is still not well defined. Here, we show that, among mouse trigeminal ganglion (TG) neurons, Trpc3 mRNA is predominantly expressed in nonpeptidergic small diameter TG neurons of mice. Moreover, Trpc3 mRNA signal was present in most presumptively itch sensing neurons. TRPC3 agonism induced TG neuronal activation and acute nonhistaminergic itch-like and pain-like behaviors in naive mice. In addition, genetic deletion of Trpc3 attenuated acute itch evoked by certain common nonhistaminergic pruritogens, including endothelin-1 and SLIGRL-NH2. In a murine model of contact hypersensitivity (CHS), the Trpc3 mRNA expression level and function were upregulated in the TG after CHS. Pharmacological inhibition and global knockout of Trpc3 significantly alleviated spontaneous scratching behaviors without affecting concurrent cutaneous inflammation in the CHS model. Furthermore, conditional deletion of Trpc3 in primary sensory neurons but not in keratinocytes produced similar antipruritic effects in this model. These findings suggest that TRPC3 expressed in primary sensory neurons may contribute to acute and chronic itch through a histamine independent mechanism and that targeting neuronal TRPC3 might benefit the treatment of chronic itch associated with ACD and other inflammatory skin disorders.

Peptidergic neurons of the Edinger-Westphal nucleus express TRPA1 ion channel that is downregulated both upon chronic variable mild stress in male mice and in humans who died by suicide.

Background:Transient receptor potential ankyrin 1 (TRPA1), a cation channel, is expressed predominantly in primary sensory neurons, but its central distribution and role in mood control are not well understood. We investigated whether TRPA1 is expressed in the urocortin 1 (UCN1)–immunoreactive centrally projecting Edinger–Westphal nucleus (EWcp), and we hypothesized that chronic variable mild stress (CVMS) would reduce its expression in mice. We anticipated that TRPA1 mRNA would be present in the human EWcp, and that it would be downregulated in people who died by suicide.Methods:We exposed Trpa1 knockout and wild-type mice to CVMS or no-stress control conditions. We then performed behavioural tests for depression and anxiety, and we evaluated physical and endocrinological parameters of stress. We assessed EWcp Trpa1 and Ucn1 mRNA expression, as well as UCN1 peptide content, using RNA-scope in situ hybridization and immunofluorescence. We tested human EWcp samples for TRPA1 using reverse transcription polymerase chain reaction.Results:Trpa1 mRNA was colocalized with EWcp/UCN1 neurons. Non-stressed Trpa1 knockout mice expressed higher levels of Ucn1 mRNA, had less body weight gain and showed greater immobility in the forced swim test than wild-type mice. CVMS downregulated EWcp/Trpa1 expression and increased immobility in the forced swim test only in wild-type mice. We confirmed that TRPA1 mRNA expression was downregulated in the human EWcp in people who died by suicide.Limitations:Developmental compensations and the global lack of TRPA1 may have influenced our findings. Because experimental data came from male brains only, we have no evidence for whether findings would be similar in female brains. Because a TRPA1-specific antibody is lacking, we have provided mRNA data only. Limited access to high-quality human tissues restricted sample size.Conclusion:TRPA1 in EWcp/UCN1 neurons might contribute to the regulation of depression-like behaviour and stress adaptation response in mice. In humans, TRPA1 might contribute to mood control via EWcp/UCN1 neurons.