Transcriptional profiling of dental sensory and proprioceptive trigeminal neurons using single-cell RNA sequencing / 国际口腔科学杂志·英文版

Dental primary afferent (DPA) neurons and proprioceptive mesencephalic trigeminal nucleus (MTN) neurons, located in the trigeminal ganglion and the brainstem, respectively, are essential for controlling masticatory functions. Despite extensive transcriptomic studies on various somatosensory neurons, there is still a lack of knowledge about the molecular identities of these populations due to technical challenges in their circuit-validated isolation. Here, we employed high-depth single-cell RNA sequencing (scRNA-seq) in combination with retrograde tracing in mice to identify intrinsic transcriptional features of DPA and MTN neurons. Our transcriptome analysis revealed five major types of DPA neurons with cell type-specific gene enrichment, some of which exhibit unique mechano-nociceptive properties capable of transmitting nociception in response to innocuous mechanical stimuli in the teeth. Furthermore, we discovered cellular heterogeneity within MTN neurons that potentially contribute to their responsiveness to mechanical stretch in the masseter muscle spindles. Additionally, DPA and MTN neurons represented sensory compartments with distinct molecular profiles characterized by various ion channels, receptors, neuropeptides, and mechanoreceptors. Together, our study provides new biological insights regarding the highly specialized mechanosensory functions of DPA and MTN neurons in pain and proprioception.

A Critical Involvement of Glutamatergic Neurons in the Anterior Insular Cortex for Subdiaphragmatic Vagotomy-induced Analgesia

Subdiaphragmatic vagotomy (SDV) is known to produce analgesic effect in various pain conditions including not only visceral pain but also somatic pain. We aimed to determine brain mechanisms by which SDV induces analgesic effect in somatic pain condition by using formalin-induced acute inflammatory pain model. We identified brain regions that mediate SDV-induced analgesic effect on acute inflammatory pain by analyzing cFos expression in the whole brain. We found that c-Fos expression was specifically increased in the anterior insular cortex (aIC) among subregions of the insular cortex in acute inflammatory pain, which was reversed by SDV. These results were not mimicked in female mice, indicating sexualdimorphism in SDV-induced analgesia. SDV decreased c-Fos expressions more preferentially in glutamatergic neurons rather than GABAergic neurons in the aIC, and pharmacological activation of glutamatergic neurons with NMDA in the aIC inhibited SDV-induced analgesic effect. Furthermore, chemogenetic activation of glutamatergic neurons in the aIC reversed SDV-induced analgesia. Taken together, our results suggest that the decrease in the neuronal activity of glutamatergic neurons in the aIC mediates SDV-induced analgesic effect, potentially serving as an important therapeutic target to treat inflammatory pain.

Mitochondrial Reactive Oxygen Species Elicit Acute and Chronic Itch via Transient Receptor Potential Canonical 3 Activation in Mice / 神经科学通报·英文版

Mitochondrial reactive oxygen species (mROS) that are overproduced by mitochondrial dysfunction are linked to pathological conditions including sensory abnormalities. Here, we explored whether mROS overproduction induces itch through transient receptor potential canonical 3 (TRPC3), which is sensitive to ROS. Intradermal injection of antimycin A (AA), a selective inhibitor of mitochondrial electron transport chain complex III for mROS overproduction, produced robust scratching behavior in naïve mice, which was suppressed by MitoTEMPO, a mitochondria-selective ROS scavenger, and Pyr10, a TRPC3-specific blocker, but not by blockers of TRPA1 or TRPV1. AA activated subsets of trigeminal ganglion neurons and also induced inward currents, which were blocked by MitoTEMPO and Pyr10. Besides, dry skin-induced chronic scratching was relieved by MitoTEMPO and Pyr10, and also by resveratrol, an antioxidant. Taken together, our results suggest that mROS elicit itch through TRPC3, which may underlie chronic itch, representing a potential therapeutic target for chronic itch.

Upregulation of Toll-like Receptor 2 in Dental Primary Afferents Following Pulp Injury

Pulpitis (toothache) is a painful inflammation of the dental pulp and is a prevalent problem throughout the world. This pulpal inflammation occurs in the cells inside the dental pulp, which have host defense mechanisms to combat oral microorganisms invading the pulp space of exposed teeth.This innate immunity has been well studied, with a focus on Toll-like receptors (TLRs). The function of TLR4, activated by Gram-negative bacteria, has been demonstrated in trigeminal ganglion (TG) neurons for dental pain. Although Gram-positive bacteria predominate in the teeth of patients with caries and pulpitis, the role of TLR2, which is activated by Gram-positive bacteria, is poorly understood in dental primary afferent (DPA) neurons that densely innervate the dental pulp. Using Fura-2 based Ca2+ imaging, we observed reproducible intracellular Ca2+ responses induced by Pam3 CSK 4 and Pam2 CSK 4 (TLR2-specific agonists) in TG neurons of adult wild-type (WT) mice. The response was completely abolished in TLR2 knock-out (KO) mice. Single-cell RT-PCR detected Tlr2 mRNA in DPA neurons labeled with fluorescent retrograde tracers from the upper molars. Using the mouse pulpitis model, real-time RT-PCR revealed that Tlr2 and inflammatory-related molecules were upregulated in injured TG, compared to non-injured TG, from WT mice, but not from TLR2 KO mice. TLR2 protein expression was also upregulated in injured DPA neurons, and the change was corresponded with a significant increase in calcitonin gene-related peptide (CGRP) expression. Our results provide a better molecular understanding of pulpitis by revealing the potential contribution of TLR2 to pulpal inflammatory pain.

Update on dentin hypersensitivity: with the focus on hydrodynamic theory and mechanosensitive ion channels

Dentin hypersensitivity is an abrupt intense pain caused by innocuous stimuli to exposed dentinal tubules. Mechanosensitive ion channels have been assessed in dental primary afferent neurons and odontoblasts to explain dentin hypersensitivity. Dentinal fluid dynamics evoked by various stimuli to exposed dentin cause mechanical stress to the structures underlying dentin. This review briefly discusses three hypotheses regarding dentin hypersensitivity and introduces recent findings on mechanosensitive ion channels expressed in the dental sensory system and discusses how the activation of these ion channels is involved in dentin hypersensitivity.

Oxytocin produces thermal analgesia via vasopressin-1a receptor by modulating TRPV1 and potassium conductance in the dorsal root ganglion neurons

Recent studies have provided several lines of evidence that peripheral administration of oxytocin induces analgesia in human and rodents. However, the exact underlying mechanism of analgesia still remains elusive. In the present study, we aimed to identify which receptor could mediate the analgesic effect of intraperitoneal injection of oxytocin and its cellular mechanisms in thermal pain behavior. We found that oxytocin-induced analgesia could be reversed by d(CH₂)₅[Tyr(Me)²,Dab⁵] AVP, a vasopressin-1a (V1a) receptor antagonist, but not by desGly-NH₂-d(CH₂)₅[DTyr², Thr⁴]OVT, an oxytocin receptor antagonist. Single cell RT-PCR analysis revealed that V1a receptor, compared to oxytocin, vasopressin-1b and vasopressin-2 receptors, was more profoundly expressed in dorsal root ganglion (DRG) neurons and the expression of V1a receptor was predominant in transient receptor potential vanilloid 1 (TRPV1)-expressing DRG neurons. Fura-2 based calcium imaging experiments showed that capsaicin-induced calcium transient was significantly inhibited by oxytocin and that such inhibition was reversed by V1a receptor antagonist. Additionally, whole cell patch clamp recording demonstrated that oxytocin significantly increased potassium conductance via V1a receptor in DRG neurons. Taken together, our findings suggest that analgesic effects produced by peripheral administration of oxytocin were attributable to the activation of V1a receptor, resulting in reduction of TRPV1 activity and enhancement of potassium conductance in DRG neurons.

Neurochemical Properties of Dental Primary Afferent Neurons

The long belief that dental primary afferent (DPA) neurons are entirely composed of nociceptive neurons has been challenged by several anatomical and functional investigations. In order to characterize non-nociceptivepopulation among DPA neurons, retrograde transport fluorescent dye was placed in upper molars of rats and immunohistochemical detection of peripherin and neurofilament 200 in the labeled trigeminal ganglia was performed. As the results, majority ofDPA neurons were peripherin-expressing small-sized neurons, showing characteristic ofnociceptive C-fibers. However, 25.7% of DPA were stained with antibody against neurofilament 200, indicating significant portion of DPA neurons are related to large myelinated Abeta fibers. There were a small number of neurons thatexpressed both peripherin and neurofilament 200, suggestive of Adelta fibers. The possible transition of neurochemical properties by neuronal injury induced by retrograde labeling technique was ruled out by detection of minimal expression of neuronal injury marker, ATF-3. These results suggest that in addition to the large population of C-fiber-related nociceptive neurons, a subset of DPA neurons is myelinated large neurons, which is related to low-threshold mechanosensitive Abeta fibers. We suggest that these Abeta fiber-related neurons might play a role as mechanotransducers of fluid movement within dentinal tubules.

Retraction: A Novel Carbamoyloxy Arylalkanoyl Arylpiperazine Compound (SKL-NP) Inhibits Hyperpolarization-Activated Cyclic Nucleotide-Gated (HCN) Channel Currents in Rat Dorsal Root Ganglion Neurons

No abstract available.

A Novel Carbamoyloxy Arylalkanoyl Arylpiperazine Compound (SKL-NP) Inhibits Hyperpolarization-Activated Cyclic Nucleotide-Gated (HCN) Channel Currents in Rat Dorsal Root Ganglion Neurons

In this study, we determined mode of action of a novel carbamoyloxy arylalkanoyl arylpiperazine compound (SKL-NP) on hyperpolarization-activated cyclic nucleotide-gated (HCN) channel currents (Ih) that plays important roles in neuropathic pain. In small or medium-sized dorsal root ganglion (DRG) neurons (<40 microm in diameter) exhibiting tonic firing and prominent Ih, SKL-NP inhibited Ih and spike firings in a concentration dependent manner (IC50=7.85 microM). SKL-NP-induced inhibition of Ih was blocked by pretreatment of pertussis toxin (PTX) and N-ethylmaleimide (NEM) as well as 8-Br-cAMP, a membrane permeable cAMP analogue. These results suggest that SKL-NP modulates Ih in indirect manner by the activation of a Gi-protein coupled receptor that decreases intracellular cAMP concentration. Taken together, SKL-NP has the inhibitory effect on HCN channel currents (I h) in DRG neurons of rats.

Involvement of Crosstalk Between cAMP and cGMP in Synaptic Plasticity in the Substantia Gelatinosa Neurons

Substantia gelatinosa (SG) neurons receive synaptic inputs from primary afferent Adelta- and C-fibers, where nociceptive information is integrated and modulated by numerous neurotransmitters or neuromodulators. A number of studies were dedicated to the molecular mechanism underlying the modulation of excitability or synaptic plasticity in SG neurons and revealed that second messengers, such as cAMP and cGMP, play an important role. Recently, cAMP and cGMP were shown to downregulate each other in heart muscle cells. However, involvement of the crosstalk between cAMP and cGMP in neurons is yet to be addressed. Therefore, we investigated whether interaction between cAMP and cGMP modulates synaptic plasticity in SG neurons using slice patch clamp recording from rats. Synaptic activity was measured by excitatory post-synaptic currents (EPSCs) elicited by stimulation onto dorsal root entry zone. Application of 1 mM of 8-bromoadenosine 3,5-cyclic monophosphate (8-Br-cAMP) or 8-bromoguanosine 3,5-cyclic monophosphate (8-Br-cGMP) for 15 minutes increased EPSCs, which were maintained for 30 minutes. However, simultaneous application of 8-Br-cAMP and 8-Br-cGMP failed to increase EPSCs, which suggested antagonistic cross-talk between two second messengers. Application of 3-isobutyl-1-methylxanthine (IBMX) that prevents degradation of cAMP and cGMP by blocking phosphodiesterase (PDE) increased EPSCs. Co-application of cAMP/cGMP along with IBMX induced additional increase in EPSCs. These results suggest that second messengers, cAMP and cGMP, might contribute to development of chronic pain through the mutual regulation of the signal transduction.

R-type Calcium Channel Isoform in Rat Dorsal Root Ganglion Neurons

R-type Cav2.3 high voltage-activated Ca2+ channels in peripheral sensory neurons contribute to pain transmission. Recently we have demonstrated that, among the six Cav2.3 isoforms (Cav2.3a~Cav2.3e), the Cav2.3e isoform is primarily expressed in trigeminal ganglion (TG) nociceptive neurons. In the present study, we further investigated expression patterns of Cav2.3 isoforms in the dorsal root ganglion (DRG) neurons. As in TG neurons, whole tissue RT-PCR analyses revealed the presence of two isoforms, Cav2.3a and Cav2.3e, in DRG neurons. Single-cell RT-PCR detected the expression of Cav2.3e mRNA in 20% (n=14/70) of DRG neurons, relative to Cav2.3a expression in 2.8% (n=2/70) of DRG neurons. Cav2.3e mRNA was mainly detected in small-sized neurons (n=12/14), but in only a few medium-sized neurons (n=2/14) and not in large-sized neurons, indicating the prominence of Cav2.3e in nociceptive DRG neurons. Moreover, Cav2.3e was preferentially expressed in tyrosine-kinase A (trkA)-positive, isolectin B4 (IB4)-negative and transient receptor potential vanilloid 1 (TRPV1)-positive neurons. These results suggest that Cav2.3e may be the main R-type Ca2+ channel isoform in nociceptive DRG neurons and thereby a potential target for pain treatment, not only in the trigeminal system but also in the spinal system.

Effects of Saccharin Intake on Hippocampal and Cortical Plasticity in Juvenile and Adolescent Rats

The sensory system is developed and optimized by experiences given in the early phase of life in association with other regions of the nervous system. To date, many studies have revealed that deprivation of specific sensory experiences can modify the structure and function of the central nervous system; however, the effects of sensory overload remains unclear. Here we studied the effect of overloading the taste sense in the early period of life on the synaptic plasticity of rat hippocampus and somatosensory cortex. We prepared male and female Sprague Dawley rats with ad libitum access to a 0.1% saccharin solution for 2 hrs per day for three weeks after weaning on postnatal day 22. Saccharin consumption was slightly increased in males compared with females; however, saccharin intake did not affect chow intake or weight gain either in male or in female rats. We examined the effect of saccharin-intake on long term potentiation (LTP) formation in hippocampal Schaffer collateral pathway and somatosensory cortex layer IV - II/III pathways in the 6-week old saccharin-fed rats. There was no significant difference in LTP formation in the hippocampus between the control group and saccharin-treated group in both male and female rats. Also in the somatosensory cortex, we did not see a significant difference in LTP among the groups. Therefore, we conclude that saccharin-intake during 3~6 weeks may not affect the development of physiological function of the cortical and hippocampal synapses in rats.

Eugenol Inhibits ATP-induced P2X Currents in Trigeminal Ganglion Neurons

Eugenol is widely used in dentistry to relieve pain. We have recently demonstrated voltage-gated Na+ and Ca2+ channels as molecular targets for its analgesic effects, and hypothesized that eugenol acts on P2X3, another pain receptor expressed in trigeminal ganglion (TG), and tested the effects of eugenol by whole-cell patch clamp and Ca2+ imaging techniques. In the present study, we investigated whether eugenol would modulate 5'-triphosphate (ATP)-induced currents in rat TG neurons and P2X3-expressing human embryonic kidney (HEK) 293 cells. ATP-induced currents in TG neurons exhibited electrophysiological properties similar to those in HEK293 cells, and both ATP- and alpha,beta-meATP-induced currents in TG neurons were effectively blocked by TNP-ATP, suggesting that P2X3 mediates the majority of ATP-induced currents in TG neurons. Eugenol inhibited ATP-induced currents in both capsaicin-sensitive and capsaicin-insensitive TG neurons with similar extent, and most ATP-responsive neurons were IB4-positive. Eugenol inhibited not only Ca2+ transients evoked by alpha,beta-meATP, the selective P2X3 agonist, in capsaicin-insensitive TG neurons, but also ATP-induced currents in P2X3-expressing HEK293 cells without co-expression of transient receptor potential vanilloid 1 (TRPV1). We suggest, therefore, that eugenol inhibits P2X3 currents in a TRPV1-independent manner, which contributes to its analgesic effect.

Effects of Somatostatin on the Responses of Rostrally Projecting Spinal Dorsal Horn Neurons to Noxious Stimuli in Cats

Somatostatin (SOM) is a widely distributed peptide in the central nervous system and exerts a variety of hormonal and neural actions. Although SOM is assumed to play an important role in spinal nociceptive processing, its exact function remains unclear. In fact, earlier pharmacological studies have provided results that support either a facilitatory or inhibitory role for SOM in nociception. In the current study, the effects of SOM were investigated using anesthetized cats. Specifically, the responses of rostrally projecting spinal dorsal horn neurons (RPSDH neurons) to different kinds of noxious stimuli (i.e., heat, mechanical and cold stimuli) and to the A delta-and C-fiber activation of the sciatic nerve were studied. Iontophoretically applied SOM suppressed the responses of RPSDH neurons to noxious heat and mechanical stimuli as well as to C-fiber activation. Conversely, it enhanced these responses to noxious cold stimulus and A delta-fiber activation. In addition, SOM suppressed glutamate-evoked activities of RPSDH neurons. The effects of SOM were blocked by the SOM receptor antagonist cyclo-SOM. These findings suggest that SOM has a dual effect on the activities of RPSDH neurons; that is, facilitation and inhibition, depending on the modality of pain signaled through them and its action site.

Double-stranded RNA Induces Inflammatory Gene Expression in Schwann Cells: Implication in the Wallerian Degeneration

Schwann cells play an important role in peripheral nerve regeneration. Upon neuronal injury, activated Schwann cells clean up the myelin debris by phagocytosis, and promote neuronal survival and axon outgrowth by secreting various neurotrophic factors. However, it is unclear how the nerve injury induces Schwann cell activation. Recently, it was reported that certain cytoplasmic molecules, which are secreted by cells undergoing necrotic cell death, induce immune cell activation via the toll-like receptors (TLRs). This suggests that the TLRs expressed on Schwann cells may recognize nerve damage by binding to the endogenous ligands secreted by the damaged nerve, thereby inducing Schwann cell activation. Accordingly, this study was undertaken to examine the expression and the function of the TLRs on primary Schwann cells and iSC, a rat Schwann cell line. The transcripts of TLR2, 3, 4, and 9 were detected on the primary Schwann cells as well as on iSC. The stimulation of iSC with poly (I: C), a synthetic ligand for the TLR3, induced the expression of TNF-alpha and RANTES. In addition, poly (I: C) stimulation induced the iNOS expression and nitric oxide secretion in iSC. These results suggest that the TLRs may be involved in the inflammatory activation of Schwann cells, which is observed during Wallerian degeneration after a peripheral nerve injury.

Nimodipine as a potential pharmacological tool for characterizing R-type calcium currents

Nimopidine, one of dihydropyridine derivatives, has been widely used to pharmacologically identify L-type Ca currents. In this study, it was tested if nimodipine is a selective blocker for L-type Ca currents in sensory neurons and heterologous system. In mouse dorsal root ganglion neurons (DRG), low concentrations of nimodipine (10 muM) further reduced the "residual" currents in DRG neurons from alpha1E knock-out mice, after blocking L-, N- and P/Q-type Ca currents with 10 muM nimodipine, 1 muM omega-conotoxin GVIA and 200 nM omega-agatoxin IVA, indicating inhibitory effects of nimodipine on R-type Ca currents. Nimodipine (>10 muM) also produced the inhibition of both low-voltage-activated calcium channel currents in DRG neurons and alpha1B and alpha1E subunit based Ca channel currents in heterologous system. These results suggest that higher nimodipine (>10 muM) is not necessarily selective for L-type Ca currents. While care should be taken in using nimodipine for pharmacologically defining L-type Ca currents from native macroscopic Ca currents, nimodipine (>10 muM) could be a useful pharmacological tool for characterizing R-type Ca currents when combined with toxins blocking other types of Ca channels.