Modulation of Nicotine-Associated Behaviour in Rats By µ-Opioid Signals from the Medial Prefrontal Cortex to the Nucleus Accumbens Shell.

Nicotine addiction is a concern worldwide. Most mechanistic investigations are on nicotine substance dependence properties based on its pharmacological effects. However, no effective therapeutic treatment has been established. Nicotine addiction is reinforced by environments or habits. We demonstrate the neurobiological basis of the behavioural aspect of nicotine addiction. We utilized the conditioned place preference to establish nicotine-associated behavioural preferences (NABP) in rats. Brain-wide neuroimaging analysis revealed that the medial prefrontal cortex (mPFC) was activated and contributed to NABP. Chemogenetic manipulation of µ-opioid receptor positive (MOR+) neurons in the mPFC or the excitatory outflow to the nucleus accumbens shell (NAcShell) modulated the NABP. Electrophysiological recording confirmed that the MOR+ neurons directly regulate the mPFC-NAcShell circuit via GABAA receptors. Thus, the MOR+ neurons in the mPFC modulate the formation of behavioural aspects of nicotine addiction via direct excitatory innervation to the NAcShell, which may provide new insight for the development of effective therapeutic strategies.

Axonal membrane stretch suppresses neuronal excitability by activating mechanosensitive K2P channels at the node of Ranvier.

Saltatory conduction is the propagation of action potentials along myelinated nerves, which enables fast propagation through the node of Ranvier. Recently, we demonstrated that K2P channels, TWIK-related K+ channel-1 (TREK-1), and TWIK-related arachidonic acid-activated K + channel (TRAAK), are highly expressed in the mammalian node of Ranvier of sensory nerves and have an important role in action potential repolarization instead of voltage-gated K+ channels. TREK-1/TRAAK channels are activated by membrane depolarization as well as various stimuli, such as temperature, pH, arachidonic acid, and mechanical membrane stretch. Although membrane mechanical stretch has been suggested to modulate action potential conduction, how membrane stretching modulates intrinsic electrophysiological properties at the node of Ranvier remains unclear. In the present study, we examined the effects of membrane stretch on neuronal membranes at the node of Ranvier in rat sciatic nerves. The single-channel conductance was approximately 90 pS at 80 mV. Membrane stretch increased the single-channel event numbers and open probability in a pressure-dependent manner. Consistent with single-channel activity, intra-pipette positive pressure increased outward leak currents and decreased membrane excitability in a whole-cell configuration. Furthermore, blockage of TREK-1/TRAAK channels by Ba2+ reversed the changes in the intrinsic electrophysiological properties induced by intra-pipette pressure. These results indicate that the activation of mechanosensitive TREK-1/TRAAK channels may suppress neuronal excitability following axonal stretch. Our findings suggest that TREK-1/TRAAK channels may play an important role in the prevention of ectopic action potential discharge at the axon by intense mechanical nerve stretch under physiological conditions.

Yokukansan Suppresses Gastric Hypersensitivity and Eosinophil-associated Microinflammation in Rats With Functional Dyspepsia.

Background/Aims: Herbal medicine is an important complementary therapy for functional dyspepsia (FD). However, its effect against gastric hypersensitivity in patients with FD has rarely been evaluated. Yokukansan (YKS), a traditional Japanese herbal medicine, is effective against neuropathic and inflammatory pain. This study aims to use a maternal separation (MS) stress-induced FD model to investigate the effects of YKS against gastric hypersensitivity, gastric motility, and duodenal micro-inflammation. Methods: The MS stress model was established by separating newborn Sprague-Dawley rats from their mothers for 2 hours a day from postnatal days 1 to 10. At the age of 7-8 weeks, the rats were treated with YKS at a dose of 5 mL/kg (1 g/kg) for 7 consecutive days. After YKS treatment, electromyographic activity in the acromiotrapezius muscle by gastric distention and the gastric-emptying rate were assessed. Immunohistochemical analysis of eosinophils in the duodenum and phosphorylated extracellular signal-regulated kinase (p-ERK) 1/2 in the spinal cord was performed. Results: YKS treatment suppressed MS stress-induced gastric hypersensitivity and decreased the elevated levels of p-ERK1/2 in the spinal cord. In the gastroduodenal tract, YKS inhibited eosinophil-associated micro-inflammation but did not improve gastric dysmotility. Conclusions: YKS treatment improved gastric hypersensitivity by alleviating eosinophil-associated micro-inflammation in the gastroduodenal tract. This treatment may be considered an effective therapeutic option for epigastric pain and micro-inflammation in patients with FD.

Reactive Oxygen Species Cause Exercise-Induced Angina in a Myocardial Ischaemia-Reperfusion Injury Model.

Percutaneous coronary intervention (PCI) effectively treats obstructive coronary artery syndrome. However, 30-40% patients continue to have angina after a successful PCI, thereby reducing patient satisfaction. The mechanisms underlying persistent angina after revascularisation therapy are still poorly understood; hence, the treatment or guideline for post-PCI angina remains unestablished. Thus, this study aimed to investigate the mechanisms underlying effort angina in animals following myocardial ischaemia-reperfusion (I/R) injury. Phosphorylated extracellular signal-regulated kinase (p-ERK), a marker for painful stimulation-induced neuronal activation, was used for the investigation. After a forced treadmill exercise (FTE), the number of p-ERK-expressing neurons increased in the superficial dorsal horn of the I/R model animals. Moreover, FTE evoked hydrogen peroxide (H2O2) production in the I/R-injured heart, inducing angina through TRPA1 activation on cardiac sensory fibres. Notably, the treatment of a TEMPOL, a reactive oxygen species scavenger, or TRPA1-/- mice successfully alleviated the FTE-induced p-ERK expression in the dorsal horn. The production of H2O2, a reactive oxygen species, through physical exercise contributes to angina development following I/R. Hence, our findings may be useful for understanding and treating angina following revascularisation therapy.

Acid increases PGE2 in the duodenal mucosa in rats.

Attention has recently been paid to the duodenum as the pathophysiologic center of functional dyspepsia. However, the precise mechanisms of symptom generation remain unknown. We here investigated the effect of acid on duodenal prostaglandin E2 and localization of prostaglandin E2 related receptors. Sprague-Dawley rats were used for this study. Hydrochloric acid was administered in the duodenum, then prostaglandin E2 levels in the duodenum were measured using the ELISA. The expression and localization of prostaglandin receptors (EP1-4) and the mRNAs of prostaglandin synthases were investigated using in situ hybridization histochemistry in duodenal tissue. After acid perfusion, prostaglandin E2 levels in the duodenum significantly increased. EP3 was expressed mainly at the myenteric plexus in the duodenal mucosa, and EP4 at both the epithelial surface and myenteric plexus. Contrary, EP2 was sparsely distributed in the villi and EP1 were not clearly seen on in situ hybridization histochemistry. Prostaglandin-synthetic enzymes were also distributed in the duodenal mucosa. The prostaglandin E2 levels in the duodenum increased after acidification. Prostaglandin E2 receptors and prostaglandin E2-producing enzymes were both observed in rat duodenum. These observations suggest that duodenal prostaglandin E2 possibly play a role in the symptom generation of functional dyspepsia.

Effects of Cooling Temperatures via Thermal K2P Channels on Regeneration of High-Frequency Action Potentials at Nodes of Ranvier of Rat Aß-Afferent Nerves.

Temperature-sensitive two-pore domain potassium channels (thermal K2P) are recently shown to cluster at nodes of Ranvier (NRs) and play a key role in action potential (AP) regeneration and conduction on Aß-afferent nerves. Cooling temperatures affect AP regeneration and conduction on Aß-afferent nerves but the underlying mechanisms are not completely understood. Here, we have performed patch-clamp recordings directly at the NR in an ex vivo trigeminal nerve preparation. We have characterized the effects of cooling temperatures on intrinsic electrophysiological properties and AP regeneration at the NR on rat Aß-afferent nerves, and determined whether and how thermal K2P channels may be involved in the effects of cooling temperatures. We show that cooling temperatures from 35°C to 15°C decrease outward leak currents, increase input resistance, depolarize resting membrane potential (RMP), broaden AP width and increase latency of AP threshold at the NR. We further demonstrate that cooling temperatures impair regeneration of high-frequency AP trains at the NR. The effects of cooling temperatures on the intrinsic electrophysiological properties and regeneration of high-frequency AP trains at the NR can be partially reversed by BL-1249 (BL), arachidonic acid (AA), and intra-axonal protons, three thermal K2P activators, indicating the involvement of thermal K2P channels. Moreover, we show that at cooling temperatures there are interplays among thermal K2P channels, RMPs, and voltage-gated Na+ channels, which together limit regeneration of high-frequency AP trains at the NR. Our findings demonstrate a new role of thermal K2P channels in temperature-dependent conduction of high-frequency sensory signals.

Atractylodin Produces Antinociceptive Effect through a Long-Lasting TRPA1 Channel Activation.

Atractylodin (ATR) is a bioactive component found in dried rhizomes of Atractylodes lancea (AL) De Candolle. Although AL has accumulated empirical evidence for the treatment of pain, the molecular mechanism underlying the anti-pain effect of ATR remains unclear. In this study, we found that ATR increases transient receptor potential ankyrin-1 (TRPA1) single-channel activity in hTRPA1 expressing HEK293 cells. A bath application of ATR produced a long-lasting calcium response, and the response was completely diminished in the dorsal root ganglion neurons of TRPA1 knockout mice. Intraplantar injection of ATR evoked moderate and prolonged nociceptive behavior compared to the injection of allyl isothiocyanate (AITC). Systemic application of ATR inhibited AITC-induced nociceptive responses in a dose-dependent manner. Co-application of ATR and QX-314 increased the noxious heat threshold compared with AITC in vivo. Collectively, we concluded that ATR is a unique agonist of TRPA1 channels, which produces long-lasting channel activation. Our results indicated ATR-mediated anti-nociceptive effect through the desensitization of TRPA1-expressing nociceptors.

Kv4.3 Channel Dysfunction Contributes to Trigeminal Neuropathic Pain Manifested with Orofacial Cold Hypersensitivity in Rats.

Trigeminal neuropathic pain is the most debilitating pain disorder but current treatments including opiates are not effective. A common symptom of trigeminal neuropathic pain is cold allodynia/hyperalgesia or cold hypersensitivity in orofacial area, a region where exposure to cooling temperatures are inevitable in daily life. Mechanisms underlying trigeminal neuropathic pain manifested with cold hypersensitivity are not fully understood. In this study, we investigated trigeminal neuropathic pain in male rats following infraorbital nerve chronic constrictive injury (ION-CCI). Assessed by the orofacial operant behavioral test, ION-CCI animals displayed orofacial cold hypersensitivity. The cold hypersensitivity was associated with the hyperexcitability of small-sized trigeminal ganglion (TG) neurons that innervated orofacial regions. Furthermore, ION-CCI resulted in a reduction of A-type voltage-gated K+ currents (IA currents) in these TG neurons. We further showed that these small-sized TG neurons expressed Kv4.3 voltage-gated K+ channels, and Kv4.3 expression in these cells was significantly downregulated following ION-CCI. Pharmacological inhibition of Kv4.3 channels with phrixotoxin-2 inhibited IA-currents in these TG neurons and induced orofacial cold hypersensitivity. On the other hand, pharmacological potentiation of Kv4.3 channels amplified IA currents in these TG neurons and alleviated orofacial cold hypersensitivity in ION-CCI rats. Collectively, Kv4.3 downregulation in nociceptive trigeminal afferent fibers may contribute to peripheral cold hypersensitivity following trigeminal nerve injury, and Kv4.3 activators may be clinically useful to alleviate trigeminal neuropathic pain.SIGNIFICANCE STATEMENT Trigeminal neuropathic pain, the most debilitating pain disorder, is often triggered and exacerbated by cooling temperatures. Here, we created infraorbital nerve chronic constrictive injury (ION-CCI) in rats, an animal model of trigeminal neuropathic pain to show that dysfunction of Kv4.3 voltage-gated K+ channels in nociceptive-like trigeminal ganglion (TG) neurons underlies the trigeminal neuropathic pain manifested with cold hypersensitivity in orofacial regions. Furthermore, we demonstrate that pharmacological potentiation of Kv4.3 channels can alleviate orofacial cold hypersensitivity in ION-CCI rats. Our results may have clinical implications in trigeminal neuropathic pain in human patients, and Kv4.3 channels may be an effective therapeutic target for this devastating pain disorder.

Protocol for pressure-clamped patch-clamp recording at the node of Ranvier of rat myelinated nerves.

The patch-clamp recording technique is indispensable for studying ion channel functions of cells but is challenging to apply to the node of Ranvier, a key site where action potentials are conducted along myelinated nerves. We have developed a pressure-clamped patch-clamp recording method applying to the node of Ranvier of rat myelinated nerves. The step-by-step protocol described here allows researchers to apply this approach to study mechanisms underlying saltatory conduction and information processing in myelinated nerves of mammals. For complete information on the generation and use of this protocol, please refer to Kanda et al. (2019).

The ubiquitin E3 ligase Nedd4-2 relieves mechanical allodynia through the ubiquitination of TRPA1 channel in db/db mice.

Neural precursor cell-expressed developmentally downregulated protein 4-2 (Nedd4-2) is a member of the E3 ubiquitin ligase family that is highly expressed in sensory neurons and involved in pain modulation via downregulation of ion channels in excitable membranes. Ubiquitination involving Nedd4-2 is regulated by adenosine monophosphate-activated protein kinase (AMPK), which is impaired in the dorsal root ganglion (DRG) neurons of db/db mice. AMPK negatively regulates the expression of transient receptor potential ankyrin 1 (TRPA1), a recognised pain sensor expressed on the membrane of DRG neurons, consequently relieving mechanical allodynia in db/db mice. Herein, we studied the involvement of Nedd4-2 in painful diabetic neuropathy and observed that Nedd4-2 negatively regulated diabetic mechanical allodynia. Nedd4-2 was co-expressed with TRPA1 in mouse DRG neurons. Nedd4-2 was involved in TRPA1 ubiquitination, this ubiquitination, as well as Nedd4-2-TRPA1 interaction, was decreased in db/db mice. Moreover, Nedd4-2 levels were decreased in db/db mice, while an abnormal intracellular distribution was observed in short-term high glucose-cultured DRG neurons. AMPK activators not only restored Nedd4-2 distribution but also increased Nedd4-2 expression. These findings demonstrate that Nedd4-2 is a potent regulator of TRPA1 and that the abnormal expression of Nedd4-2 in DRG neurons contributes to diabetic neuropathic pain.

Localization of prostaglandin E2 synthases and E-prostanoid receptors in the spinal cord in a rat model of neuropathic pain.

Prostaglandin E2 (PGE2) is a lipid mediator which plays a role in the generation of inflammatory and neuropathic pain. In the peripheral nervous system, PGE2 sensitizes nociceptive afferent neurons through E-prostanoid (EP) receptors. In the central nervous system, PGE2 modulates pain sensitivity and contributes to the development of neuropathic pain. However, the distribution of PGE2 and EP receptors in the spinal cord remains unclear. In the present study, we examined the expression of PGE2 synthases (microsomal PGE synthase [mPGES]-1, mPGES-2, and cytosolic PGE synthase [cPGES]) and EP receptors (EP1-4) in a rat model of neuropathic pain. We identified that mPGES-1 mRNA was upregulated in spinal endothelial cells after nerve injury and exhibited co-localization with cyclooxygenase-2 (COX-2). We detected that mPGES-2 mRNA and cPGES mRNA were expressed in spinal neurons and noted that their expression level was not affected by nerve injury. With respect to EP receptors, EP2 mRNA and EP4 mRNA were expressed in spinal neurons in the dorsal horn. EP3 mRNA was expressed in motor neurons, whereas EP1 mRNA was not detected in the spinal cord. Intrathecal injection of tumor necrosis factor alpha (TNFα) upregulated mPGES-1 mRNA in blood vessels in the spinal cord. Intrathecal injection of a TNFα-neutralizing antibody partially inhibited the upregulation of mPGES-1 mRNA after nerve injury. These results indicate that PGE2 is synthesized by COX-2/mPGES-1 in spinal endothelial cells after nerve injury. These results suggest that in neuropathic pain condition, endothelial cell-derived PGE2 may act on EP2 and EP4 receptors on spinal neurons and modulate pain sensitivity.

Eosinophil-associated microinflammation in the gastroduodenal tract contributes to gastric hypersensitivity in a rat model of early-life adversity.

Gastric hypersensitivity is a major pathophysiological feature of functional dyspepsia (FD). Recent clinical studies have shown that a large number of patients with FD present with gastroduodenal microinflammation, which may be involved in the pathophysiology of FD. However, no animal model reflecting this clinical characteristic has been established. The underlying mechanism between microinflammation and FD remains unknown. In this study, using a maternal separation (MS)-induced FD model, we aimed to reproduce the gastroduodenal microinflammation and reveal the interaction between gastroduodenal microinflammation and gastric hypersensitivity. The MS model was established by separating newborn Sprague-Dawley rats for 2 h a day from postnatal day 1 to day 10. At 7-8 wk of age, electromyography was used to determine the visceromotor response to gastric distention (GD) and immunohistochemistry was performed to detect distension-associated neuronal activation as well as immunohistological changes. Our results demonstrated that MS-induced FD rats underwent gastric hypersensitivity with GD at 60 and 80 mmHg, which are related to increased p-ERK1/2 expression in the dorsal horn of T9-T10 spinal cords. Eosinophils, but not mast cells, were significantly increased in the gastroduodenal tract, and the coexpression rate of CD11b and major basic protein significantly increased in MS rats. Treatment with dexamethasone reversed gastric hypersensitivity in MS-induced FD rats by inhibiting eosinophil infiltration. These findings indicated that neonatal MS stress induces eosinophil-associated gastroduodenal microinflammation and gastric hypersensitivity in adulthood in rats. Microinflammation contributes to gastric hypersensitivity; therefore, anti-inflammatory therapy may be effective in treating patients with FD with gastroduodenal microinflammation.NEW & NOTEWORTHY We showed for the first time that neonatal MS stress-induced FD rats undergo gastroduodenal eosinophil-associated microinflammation in adulthood. Suppression of microinflammation attenuated gastric hypersensitivity in MS rats. These findings established a functional link between microinflammation and gastric hypersensitivity, which may provide a potential clue for the clinical treatment of FD.

Daikenchuto attenuates visceral pain and suppresses eosinophil infiltration in inflammatory bowel disease in murine models.

BACKGROUND AND AIM: Daikenchuto (DKT), a traditional Japanese formula, comprises four herbal medicines and is used for abdominal pain. Inflammatory bowel disease (IBD) includes ulcerative colitis (UC) and Crohn's disease (CD) and is characterized by colonic inflammation and chronic abdominal pain. The present study aimed to investigate whether DKT suppresses colonic hypersensitivity and inflammation associated with IBD in animal models. METHODS: Sprague-Dawley rats were administered 4% sodium dextran sulfate (DSS) or trinitrobenzene sulfate (TNBS) in the colon to establish UC or CD models, respectively. DKT and 5-aminosalicylic acid (5-ASA) were administered orally once a day from Days 3 to 7 after induction of colitis. On Day 7, visceral pain and inflammation were evaluated by measuring the visceromotor response (VMR) to colorectal distention (CRD) and inflammatory indicators, including histological score, length of leukocyte infiltration, MPO activity, and eosinophil count. RESULTS: DSS and TNBS increased VMR to CRD and the inflammation indicators. DKT, but not 5-ASA, suppressed the VMR to CRD in DSS- and TNBS-treated rats. DKT and 5-ASA decreased the eosinophil count in both IBD models. In DSS-treated rats, 5-ASA, but not DKT, suppressed the MPO activity. In TNBS-treated rats, neither 5-ASA nor DKT suppressed MPO activity. CONCLUSION: These results suggest that DKT is beneficial for abdominal pain associated with IBD. The anti-inflammatory effect of DKT on IBD may involve inhibition of eosinophils. The mechanism of anti-inflammatory effect of DKT partially differs from that of 5-ASA. Coapplication of DKT and conventional medicine may produce a positive synergy effect for IBD treatment.

Increased TRPV4 expression in non-myelinating Schwann cells is associated with demyelination after sciatic nerve injury.

Transient receptor potential vanilloid 4 (TRPV4) is a non-selective calcium-permeable cation channel that is widely expressed and activated in various neurons and glial cells in the nervous system. Schwann cells (SCs) are primary glia cells that wrap around axons to form the myelin sheath in the peripheral nervous system. However, whether TRPV4 is expressed and functions in SCs is unclear. Here, we demonstrate functional expression of TRPV4 in mouse SCs and investigated its physiological significance. Deletion of TRPV4 did not affect normal myelin development for SCs in sciatic nerves in mice. However, after sciatic nerve cut injury, TRPV4 expression levels were remarkably increased in SCs following nerve demyelination. Ablation of TRPV4 expression impaired the demyelinating process after nerve injury, resulting in delayed remyelination and functional recovery of sciatic nerves. These results suggest that local activation of TRPV4 could be an attractive pharmacological target for therapeutic intervention after peripheral nerve injury.

TREK-1 and TRAAK Are Principal K+ Channels at the Nodes of Ranvier for Rapid Action Potential Conduction on Mammalian Myelinated Afferent Nerves.

Rapid conduction of nerve impulses is critical in life and relies on action potential (AP) leaps through the nodes of Ranvier (NRs) along myelinated nerves. While NRs are the only sites where APs can be regenerated during nerve conduction on myelinated nerves, ion channel mechanisms underlying the regeneration and conduction of APs at mammalian NRs remain incompletely understood. Here, we show that TREK-1 and TRAAK, the thermosensitive and mechanosensitive two-pore-domain potassium (K2P) channels, are clustered at NRs of rat trigeminal Aß-afferent nerves with a density over 3,000-fold higher than that on their somas. These K2P channels, but not voltage-gated K+ channels as in other parts of nerves, are required for rapid AP repolarization at the NRs. Furthermore, these channels permit high-speed and high-frequency AP conduction along the myelinated afferent nerves, and loss of function of these channels at NRs retards nerve conduction and impairs sensory behavioral responses in animals.

TRPA1-expressing lamina propria mesenchymal cells regulate colonic motility.

The physiological process of defecation is directly controlled by colorectal motility. The transient receptor potential ankyrin 1 (TRPA1) channel is expressed in small intestine enterochromaffin cells and is involved in gastrointestinal motility via serotonin release. In the colorectum, however, enterochromaffin cell localization is largely distinct from that in the small intestine. Here, we investigated the role of lower gastrointestinal tract TRPA1 in modulating colorectal motility. We found that in colonic tissue, TRPA1 is predominantly expressed in mesenchymal cells of the lamina propria, which are clearly distinct from those in the small intestine. These cells coexpressed COX1 and microsomal prostaglandin E synthase-1. Intracolonic administration of TRPA1 agonists induced colonic contraction, which was suppressed by a prostaglandin E2 (PGE2) receptor 1 antagonist. TRPA1 activation induced calcium influx and PGE2 release from cultured human fibroblastic cells. In dextran sulfate sodium-treated animals, both TRPA1 and its endogenous agonist were dramatically increased in the colonic lamina propria, accompanied by abnormal colorectal contractions. Abnormal colorectal contractions were significantly prevented by pharmacological and genetic inhibition of TRPA1. In conclusion, in the lower gastrointestinal tract, mesenchymal TRPA1 activation results in PGE2 release and consequently promotes colorectal contraction, representing what we believe is a novel physiological and inflammatory bowel disease-associated mechanism of gastrointestinal motility.

Effects of norepinephrine and ß2 receptor antagonist ICI 118,551 on whisker hair follicle mechanoreceptors dissatisfy Merkel discs being adrenergic synapses.

Merkel discs, located in skin touch domes and whisker hair follicles, are tactile end organs essential for environmental exploration, social interaction, and tactile discrimination. Recent studies from our group and two others have shown that mechanical stimulation excites Merkel cells via Piezo2 channel activation to subsequently activate sensory neural pathways. We have further shown that mechanical stimulation leads to the release of 5-HT from Merkel cells to synaptically transmit tactile signals to whisker afferent nerves. However, a more recent study using skin touch domes has raised the possibility that Merkel discs are adrenergic synapses. It was proposed that norepinephrine is released from Merkel cells upon mechanical stimulation to subsequently activate ß2 adrenergic receptors on Merkel disc nerve endings leading to nerve impulses. In the present study, we examined effects of norepinephrine and ß2 adrenergic receptor antagonist ICI 118,551 on Merkel disc mechanoreceptors in mouse whisker hair follicles. We show that norepinephrine did not directly induce impulses from Merkel disc mechanoreceptors. Furthermore, we found that ICI 118,551 at 50 µM inhibited voltage-gated Na+ channels and suppressed impulses of Merkel disc mechanoreceptors, but ICI 118,551 at 1 µM had no effects on the impulse. These findings challenge the hypothesis of Merkel discs being adrenergic synapses.

Urothelial bladder afferent neurons in the rat are anatomically and neurochemically distinct from non-urothelial afferents.

There is mounting evidence underscoring a role for the urothelium in urinary bladder sensation. Previous functional studies have identified bladder primary afferents with mechanosensitive properties suggesting urothelial innervation and/or communication. The current study identifies a group of urothelium-innervating afferent neurons in rat, and characterizes and compares the properties of these and non-urothelial afferent neuron populations. Lumbosacral (LS) primary afferent neurons were retrogradely labeled using intraparenchymal (IPar) microinjection or intravesical (IVes) infusion of tracer into the bladder. Using these techniques, separate populations of neurons were differentiated by dorsal root ganglion (DRG) somata labeling and dye distribution within the bladder. IPar- and IVes-labeled neurons accounted for 85.0% and 14.4% of labeled L6-S1 neurons (P < .001), respectively, with only 0.6% of neurons labeled by both techniques. Following IVes labeling, dye was contained only within the periurothelial bladder region in contrast to non-urothelial distribution of dye after IPar labeling. Electrophysiological characterization by in situ patch-clamp recordings from whole-mount DRG preparations indicated no significant difference in passive or active membrane properties of IPar and IVes DRG neurons. However, calcium imaging of isolated neurons indicates that a greater proportion of IPar- than IVes-labeled neurons express functional TRPA1 (45.7% versus 25.6%, respectively; P < .05). This study demonstrates that two anatomically distinct groups of LS bladder afferents can be identified in rat. Further studies of urothelial afferents and the phenotypic differences between non-/urothelial afferents may have important implications for normal and pathophysiological bladder sensory processing.

Orofacial neuropathic pain induced by oxaliplatin: downregulation of KCNQ2 channels in V2 trigeminal ganglion neurons and treatment by the KCNQ2 channel potentiator retigabine.

Abstract: Neuropathic pain induced by chemotherapy drugs such as oxaliplatin is a dose-limiting side effect in cancer treatment. The mechanisms underlying chemotherapy-induced neuropathic pain are not fully understood. KCNQ2 channels are low-threshold voltage-gated K+ channels that play a role in controlling neuronal excitability. Downregulation of KCNQ2 channels has been proposed to be an underlying mechanism of sensory hypersensitivity that leads to neuropathic pain. However, it is currently unknown whether KCNQ channels may be downregulated by chemotherapy drugs in trigeminal ganglion neurons to contribute to the pathogenesis of chemotherapy-induced orofacial neuropathic pain. In the present study, mechanical sensitivity in orofacial regions is measured using the operant behavioral test in rats treated with oxaliplatin. Operant behaviors in these animals show the gradual development of orofacial neuropathic pain that manifests with orofacial mechanical allodynia. Immunostaining shows strong KCNQ2 immunoreactivity in small-sized V2 trigeminal ganglion neurons in controls, and the numbers of KCNQ2 immunoreactivity positive V2 trigeminal ganglion neurons are significantly reduced in oxaliplatin-treated animals. Immunostaining is also performed in brainstem and shows strong KCNQ2 immunoreactivity at the trigeminal afferent central terminals innervating the caudal spinal trigeminal nucleus (Vc) in controls, but the KCNQ2 immunoreactivity intensity is significantly reduced in oxaliplatin-treated animals. We further show with the operant behavioral test that oxaliplatin-induced orofacial mechanical allodynia can be alleviated by the KCNQ2 potentiator retigabine. Taken together, these findings suggest that KCNQ2 downregulation may be a cause of oxaliplatin-induced orofacial neuropathic pain and KCNQ2 potentiators may be useful for alleviating the neuropathic pain.

Microglial TNFα Induces COX2 and PGI2 Synthase Expression in Spinal Endothelial Cells during Neuropathic Pain.

Prostaglandins (PGs) are typical lipid mediators that play a role in homeostasis and disease. They are synthesized from arachidonic acid by cyclooxygenase 1 (COX1) and COX2. Although COX2 has been reported to be upregulated in the spinal cord after nerve injury, its expression and functional roles in neuropathic pain remain unclear. In this study, we investigated the expression of Cox2, PGI2 synthase (Pgis), and prostaglandin I2 receptor (IP receptor) mRNA in the rat spinal cord after spared nerve injury (SNI). Levels of Cox2 and Pgis mRNA increased in endothelial cells from 24 to 48 h after nerve injury. IP receptor mRNA was constitutively expressed in dorsal horn neurons. A COX2 inhibitor and IP receptor antagonists attenuated pain behavior in the early phase of neuropathic pain. Furthermore, we examined the relationship between COX2 and tumor necrosis factor-α (TNFα) in the spinal cord of a rat SNI model. Levels of TNFα mRNA transiently increased in the spinal microglia 24 h after SNI. The TNF receptors Tnfr1 and Tnfr2 mRNA were colocalized with COX2. Intrathecal injection of TNFα induced Cox2 and Pgis mRNA expression in endothelial cells. These results revealed that microglia-derived TNFα induced COX2 and PGIS expression in spinal endothelial cells and that endothelial PGI2 played a critical role in neuropathic pain via neuronal IP receptor. These findings further suggest that the glia-endothelial cell interaction of the neurovascular unit via transient TNFα is involved in the generation of neuropathic pain.