Astroglial dysfunctions drive aberrant synaptogenesis and social behavioral deficits in mice with neonatal exposure to lengthy general anesthesia.

Lengthy use of general anesthetics (GAs) causes neurobehavioral deficits in the developing brain, which has raised significant clinical concerns such that the United States Food and Drug Administration (FDA) is warning on the use of GAs in children younger than 3 years. However, the molecular and cellular mechanisms for GAs-induced neurotoxicity remain largely unknown. Here, we report that sevoflurane (Sevo), a commonly used GA in pediatrics, caused compromised astrocyte morphogenesis spatiotemporally correlated to synaptic overgrowth, with reduced synaptic function in developing cortex in a regional-, exposure-length-, and age-specific manner. Sevo disrupted astrocyte Ca2+ homeostasis both acutely and chronically, which led to the down-regulation of Ezrin, an actin-binding membrane-bound protein, which we found was critically involved in astrocyte morphogenesis in vivo. Importantly, overexpression of astrocyte Ezrin rescued astrocytic and neuronal dysfunctions and fully corrected deficits in social behaviors in developing mice with lengthy Sevo exposure. Our data uncover that, in addition to neurons, astrocytes may represent important targets for GAs to exert toxic effects and that astrocyte morphological integrity is crucial for synaptogenesis and neurological behaviors.

Activation of the RAS/B-RAF-MEK-ERK pathway in satellite glial cells contributes to substance p-mediated orofacial pain.

The cross talk between trigeminal ganglion (TG) neurons and satellite glial cells (SGCs) is crucial for the regulation of inflammatory orofacial pain. Substance P (SP) plays an important role by activating neurokinin (NK)-I receptors in this cross talk. The activation of extracellular signal-regulated kinase (ERK) 1/2, protein kinase A (PKA) and protein kinase C (PKC) in neurons and SGCs of peripheral ganglions by peripheral inflammation is associated with inflammatory hypersensitivity. This study tested the hypothesis that SP evoked SP-NK-I receptor positive feedback via the Renin-Angiotensin System/B-Protein Kinase A-Rapidly Accelerates Fibrosarcoma-MEK-Extracellular Signal-Regulated Kinase (RAS/PKA-RAF-MEK-ERK) pathway, which is involved in pain hypersensitivity. Inflammatory models were induced in vivo by injecting Complete Freund's adjuvant (CFA) into the whisker pad of rats. SP was administrated to SGCs in vitro for investigating, whether SP regulates the expression of NK-I receptor in the SGC nucleus. The effects of RAS-RAF-MEK, PKA and PKC pathways in this process were measured by co-incubating SGCs with respective Raf, PKA, PKC and MEK inhibitors in vitro and by pre-injecting these inhibitors into the TG in vivo. SP significantly upregulated NK-I receptor, p-ERK1/2, Ras, B-Raf, PKA and PKC in SGCs under inflammatory conditions. In addition, L703,606 (NK-I receptor antagonist), U0126 (MEK inhibitor), Sorafenib (Raf inhibitor) and H892HCL (PKA inhibitor) but not chelerythrine chloride (PKC inhibitor) significantly decreased NK-I mRNA and protein levels induced by SP. The allodynia-related behavior evoked by CFA was inhibited by pre-injection of L703,606, U0126, Sorafenib and H892HCL into the TG. Overall, SP upregulates NK-I receptor in TG SGCs via PKA/RAS-RAF-MEK-ERK pathway activation, contributing to a positive feedback of SP-NK-I receptor in inflammatory orofacial pain.

P2Y14 receptor is functionally expressed in satellite glial cells and mediates interleukin-1ß and chemokine CCL2 secretion.

Satellite glial cells (SGCs) activation in the trigeminal ganglia (TG) is critical in various abnormal orofacial sensation in nerve injury and inflammatory conditions. SGCs express several subtypes of P2 purinergic receptors contributing to the initiation and maintenance of neuropathic pain. The P2Y14 receptor, a G-protein-coupled receptor activated by uridine diphosphate (UDP)-glucose and other UDP sugars, mediates various physiologic events such as immune, inflammation, and pain. However, the expression, distribution, and function of P2Y14 receptor in SGCs remains largely unexplored. Our study reported the expression and functional identification of P2Y14 receptor in SGCs. SGCs were isolated from TG of rat, and the P2Y14 receptor expression was examined using immunofluorescence technique. Cell proliferation and viability were examined via cell counting kit-8 experiment. Immunofluorescence demonstrated the presence of P2Y14 receptor in SGCs. Immunofluorescence and western blot showed that UDP-glucose treatment upregulated glial fibrillary acid protein, a common marker for glial activation. Extracellular UDP-glucose enhanced the phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38, which were both abolished by the P2Y14 receptor inhibitor (PPTN). Furthermore, quantitative reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay demonstrated that extracellular UDP-glucose significantly enhanced interleukin-1ß (IL-1ß) and chemokine CCL2 (CCL2) release, which was abolished by PPTN and significantly decreased by inhibitors of MEK/ERK (U0126) and p38 (SB202190). Our findings directly proved the functional presence of P2Y14 receptor in SGCs. It was also verified that P2Y14 receptor activation was involved in activating SGCs, phosphorylating MAPKs, and promoting the secretion of IL-1ß and CCL2 via ERK and p38 pathway.

Effect of Titanium Particles on the Voltage-Gated Potassium Channel Currents in Trigeminal Root Ganglion Neurons.

PURPOSE: Titanium (Ti) is the key material used in dental implants because of its excellent biocompatibility. But wear and corrosion Ti particles had been widely reported to induce inflammation and promote bone absorption. However, little information is known about the damage of Ti particles on neurons. MATERIALS AND METHODS: Trigeminal root ganglion (TRG) neurons were exposed to Ti particles (<5 µm). The electrophysiological properties of 2 main subtypes of voltage-gated potassium channels (VGPCs) (KA and KV) were examined by whole-cell patch-clamp techniques. RESULT: With the presence of 0.25 mg/mL Ti particles, amplitudes of IK, A and IK, V were both obviously inhibited. For IK, A, the activation V1/2 shifted to the depolarizing direction with an increased k value, whereas the inactivation V1/2 showed obvious hyperdepolarizing shifts. For IK, V, 0.5 mg/mL Ti particles produced a depolarizing shift of activation V1/2 with a slower activation rate. No significant changes of its inactivation kinetics were found. CONCLUSION: Titanium (Ti) particles might alter the electrophysiological properties of VGPCs on TRG neurons, which are likely to further influence the excitability of neurons.

[Xylazine Produced Analgesic Effect via Inhibits Hyperpolarization-activated Cyclic Nucleotide-gated Ion Channels Currents].

OBJECTIVE: To investigate the analgesic mechanism of xylazine by inhibiting the activation of hyperpolarized cyclic nucleotide-gated (HCN) ion channels. METHODS: HCN subchannel 1 (HCN1) knockout mice (HCN1-/-) and HCN1 wild type mice (HCN1+/+) were intraperitoneally injected with physiological saline and xylazine (10, 20, 30, and 40 mg/kg). Mechanical pain test and tail flick test were used to test the analgesic effect of xylazine by using the percentage of the maximal possible effect (%MPE); The control group and test groups of different concentrations of xylazine (12.5, 25, 50, and 100 µmol/L) were set up using HEK 293 cells transfected HCN1 plasmid and HCN subchannel 2 (HCN2) plasmid, respectively. The activated current of hyperpolarized HEK 293 cells expressing HCN1 and HCN2 ion channels and the inhibition rate of xylazine on hyperpolarization-activated currents were recorded using a whole cell patch clamp. RESULTS: The results demonstrated that %MPE of the mechanical pain stimuli test and the thermal radiation stimuli test increased with the higher concentration of xylazine injected for both HCN1+/+ mice and HCN1-/-mice. When injecting xylazine by 30 mg/kg and 40 mg/kg, the %MPE of mechanical pain stimuli test for HCN1-/- mice were %MPE= (62.06±14.72) % and %MPE= (69.92±16.09) %, respectively; and the percentages of tail flick tests were (52.50±1.97) % and %MPE= (64.74±6.34) %, respectively. But for HCN1+/+ mice, the percentages of mechanical pain stimuli test were %MPE= (75.47±8.06) % and %MPE= (86.35±11.31) %; respectively, and the percentage of tail flick tests were %MPE= (57.83±4.82) % and (74.98±9.35) %. The analgesic effect results of the mechanical pain test and tail flick test of HCN1+/+ mice were significantly different from HCN1-/- mice ( P<0.05). Whole-cell patch clamp test results showed that xylazine had inhibitory effects on the currents of HCN1 and HCN2 ion channels, and the hyperpolarization-activated currents inhibition rate of HCN1 by xylazine (12.5-100 µmol/L) was between (24.62±23.62) %- (62.40±15.48) %; V1/2 of HCN1 was between (-79.58±1.56) mV- (-98.95±3.57) mV. The Ih inhibition rate of HCN2 by xylazine (12.5-100 µmol/L) was between (29.19±17.82) %- (80.02±6.64) %; with V1/2 of HCN2 between (-102.17±1.36) mV- (-117.48±2.38) mV. CONCLUSION: Xylazine showed better analgesic effect on HCN1+/+ mice than HCN1-/- mice. Xylazine can produce analgesic effect by inhibiting HCN ion channel currents.

Hyperpolarization-activated cyclic nucleotide-gated channels may contribute to regional anesthetic effects of lidocaine.

BACKGROUND: Local anesthetics (e.g., lidocaine) have been found to inhibit hyperpolarization-activated cyclic nucleotide-gated (HCN) channels besides sodium channels. However, the exact role of HCN channels in regional anesthesia in vivo is still elusive. METHODS: Sciatic nerve block and intrathecal anesthesia were performed using lidocaine in wild-type and HCN1 channel knockout (HCN1) mice. EC50 of lidocaine and durations of 1% lidocaine were determined. In electrophysiologic recordings, effects of lidocaine on HCN channel currents, voltage-gated sodium channel currents, and neural membrane properties were recorded on dorsal root ganglia neurons. RESULTS: In both sciatic nerve block and intrathecal anesthesia, EC50 of lidocaine for tactile sensory blockade (2 g von Frey fiber) was significantly increased in HCN1 mice, whereas EC50 of lidocaine for pinprick blockade was unaffected. Durations of 1% lidocaine were significantly shorter in HCN1 mice for both sciatic nerve block and intrathecal anesthesia (n = 10). ZD7288 (HCN blocker) could significantly prolong durations of 1% lidocaine including pinprick blockade in sciatic nerve block (n = 10). Forskolin (raising cyclic adenosine monophosphate to enhance HCN2) could significantly shorten duration of pinprick blockade of 1% lidocaine in sciatic nerve block (n = 10). In electrophysiologic recordings, lidocaine could nonselectively inhibit HCN channel and sodium channel currents both in large and in small dorsal root ganglia neurons (n = 5 to 6). Meanwhile, lidocaine caused neural membrane hyperpolarization and increased input resistance of dorsal root ganglia neurons but not in large dorsal root ganglia neurons from HCN1 mice (n = 5-7). CONCLUSIONS: These data indicate that HCN channels may contribute to regional anesthetic effects of lidocaine. By inhibiting HCN channels, lidocaine could alter membrane properties of neurons.

Emulsified isoflurane enhances thermal transient receptor potential vanilloid-1 channel activation-mediated sensory/nociceptive blockade by QX-314.

BACKGROUND: QX-314 produces nociceptive blockade, facilitated by permeation through transient receptor potential vanilloid-1 (TRPV1) channels. TRPV1 channel can be activated by noxious heat and sensitized by volatile anesthetics. The authors hypothesized that emulsified isoflurane (EI) could enhance thermal TRPV1 channel activation-mediated sensory/nociceptive blockade by QX-314. METHODS: Rats were perineurally injected with QX-314 (Sigma-Aldrich Co. Ltd. Shanghai, China) alone or QX-314 combined with EI, followed by heat exposure on the injection site. The tail-flick and tail-clamping tests were used to assess sensory and nociceptive blockade, respectively; a sciatic nerve block model was used to assess motor and sensory blockade. Effects of EI on thermal activation of TRPV1 channels were evaluated on rat dorsal root ganglia neurons by whole-cell patch-clamp recordings. RESULTS: Heat exposure enhanced sensory/nociceptive blockade by QX-314 in rat tails, but not motor blockade in sciatic nerve block model. QX-314 alone or QX-314 + 42°C produced no nociceptive blockade. QX-314 + 48°C produced 100% nociceptive blockade with duration of 12.5 ± 2.0 h (mean ± SEM). By adding 2% EI, QX-314 + 42°C produced 80% nociceptive blockade with duration of 8.1 ± 1.9 h, which was similar to the effect of QX-314 + 46°C (7.7 ± 1.1 h; P = 0.781). The enhancement of heat on sensory/nociceptive blockade of QX-314 was prevented by TRPV1 channel antagonist. The temperature thresholds of TRPV1 channel activation on dorsal root ganglia neurons were significantly reduced by EI. CONCLUSIONS: Thermal activation of TRPV1 channels enhanced long-lasting sensory/nociceptive blockade by QX-314 without affecting motor blockade. The addition of EI reduced temperature thresholds for inducing long-lasting sensory/nociceptive blockade due to QX-314.

OECs transplantation results in neuropathic pain associated with BDNF regulating ERK activity in rats following cord hemisection.

BACKGROUND: The olfactory ensheathing cells (OECs) derived from olfactory bulb (OB) may improve motor function after transplantation in injured spinal cord. However, the effects of OEC transplantation on sensory function have not been reported yet. The purpose of this study is to investigate whether OEC transplantation could affect the sensory function and to analyze the underlying mechanism. RESULTS: OEC transplantation into the hemisected spinal cords can result in hyperalgesia, indicated by radiant and mechanical stimuli towards the plantar surface in rats. This could be associated with upregulation of Brain Derived Neurotrophic Factor (BDNF), indicated by RT-PCR. Immunofluorecent staining showed that BDNF was mainly located in the neurons of the laminas I and II of the dorsal horn. Moreover, a notable upregulation on the level of p-ERK (phosphorylation of extracellular signal-regulated kinase), the downstream molecule of BDNF, was detected by using Western Blot. These findings indicate that the increased BDNF level associated with the p-ERK was possibly involved in neuropathic pain in hemisected spinal cord subjected to OEC transplantation. CONCLUSIONS: The transplantation of OECs may induce the noticeable pain hypersensitivity in rats after hemisected spinal cord injury, and the possible mechanism may be associated with the phosphorylation of ERK and the activated BDNF overexpression.

Prolonged exposure of neonatal mice to sevoflurane leads to hyper-ramification in microglia, reduced contacts between microglia and synapses, and defects in adult behavior.

Background: Prolonged exposure to general anesthetics during development is known to cause neurobehavioral abnormalities, but the cellular and molecular mechanisms involved are unclear. Microglia are the resident immune cells in the central nervous system and play essential roles in normal brain development. Materials and methods: In the study, postnatal day 7 (P7) C57BL/6 mice were randomly assigned to two groups. In the sevoflurane (SEVO), mice were exposed to 2.5% sevoflurane for 4 h. In the control group, mice were exposed to carrier gas (30% O2/70% N2) for 4 h. Fixed brain slices from P14 to P21 mice were immunolabeled for ionized calcium-binding adapter molecule 1 (IBA-1) to visualize microglia. The morphological analysis of microglia in the somatosensory cortex was performed using ImageJ and Imaris software. Serial block face scanning electron microscopy (SBF-SEM) was performed to assess the ultrastructure of the microglia and the contacts between microglia and synapse in P14 and P21 mice. The confocal imaging of brain slices was performed to assess microglia surveillance in resting and activated states in P14 and P21 mice. Behavioral tests were used to assess the effect of microglia depletion and repopulation on neurobehavioral abnormalities caused by sevoflurane exposure. Results: The prolonged exposure of neonatal mice to sevoflurane induced microglia hyper-ramification with an increase in total branch length, arborization area, and branch complexity 14 days after exposure. Prolonged neonatal sevoflurane exposure reduced contacts between microglia and synapses, without affecting the surveillance of microglia in the resting state or responding to laser-induced focal brain injury. These neonatal changes in microglia were associated with anxiety-like behaviors in adult mice. Furthermore, microglial depletion before sevoflurane exposure and subsequent repopulation in the neonatal brain mitigated anxiety-like behaviors caused by sevoflurane exposure. Conclusion: Our experiments indicate that general anesthetics may harm the developing brain, and microglia may be an essential target of general anesthetic-related developmental neurotoxicity.

Visualization and Characterization of the Brain Regional Heterogeneity of Astrocyte-Astrocyte Structural Interactions by Using Improved Iontophoresis with Dual-Fluorescent Dyes.

Astrocytes are morphologically intricate cells and actively modulate the function of the brain. Through numerous fine processes, astrocytes come into contact with neurons, blood vessels, and other glia cells. Emerging evidence has shown that astrocytes exhibit brain regional diversity in their morphology, transcriptome, calcium signaling, and functions. However, little is known about the brain regional heterogeneity of astrocyte-astrocyte structural interaction. So far, the visualization and characterization of the morphological features of adjacent astrocytes have been difficult, and as a result, it is still well-accepted that astrocytes in the adult brain share non-overlapped territory. In contrast, employing an approach that combines viral labeling with dual-fluorescent dyes iontophoresis under brightfield and imaging using confocal microscopy allows for the efficient and specific labeling of adjacent astrocytes, enabling a comprehensive visualization of their fine processes and the degree of their territorial overlap. Our study in the hypothalamic regions of the brain revealed a marked spatial overlap among adjacent astrocytes, which differs from the conventional understanding based on more extensively studied regions, like the hippocampus. Additionally, we revealed the heterogeneity of the astrocyte-neuron ratio across brain regions and conducted an assessment of the photostability and labeling efficiency of fluorescent dyes used for labeling adjacent astrocytes. Our study provides new insights for studying the morphological heterogeneity of astrocytes across the central nervous system.

QX-OH/Levobupivacaine: A Structurally Novel, Potent Local Anesthetic Produces Fast-Onset and Long-Lasting Regional Anesthesia in Rats.

PURPOSE: Local anesthetics (LAs) are an important alternative for postoperative analgesia; however, the short duration of LAs limits their use. Thus, we previously developed LL-1, a mixture of QX-OH and levobupivacaine (LB) that produces regional anesthesia for more than 10 h in rats. The aim of this study is to investigate the long-acting mechanism of LL-1 in vivo and in vitro. METHODS: Regional anesthetic effects and local toxicity of the LL-1, QX-OH and LB treatment groups were investigated in a sciatic nerve block rat model. Whole-cell patch-clamping recordings were used to measure the inhibition Nav currents (INa ) in ND7/23 cells. RESULTS: The onset of LL-1 (35mM QX-OH+10mM LB) and 10 mM LB was 10 min, which was much faster than 35 mM QX-OH (27 [18, 60] min, t[12] = -4.535, p = 0.001). The duration of LL-1 (35mM QX-OH+10 mM LB) was significantly longer than 35 mM QX-OH or 10 mM LB alone (F[3, 35] = 191.336, p < 0.0001). No differences in local tissue toxicity were found between LL-1 and LB. In patch-clamping recordings, 5 mM QX-OH produced ~20% inhibition of I Na currents. LB at 40 µM inhibited I Na by 65.51%±3.63%, while QX-OH 2 mM+LB 40 µM inhibited I Na by 77.37%±3.36% (t[14] = 2.358, p = 0.025), and QX-OH 5 mM+LB 40 µM inhibited I Na by 83.88%±1.57% (t[13] = 4.191, p = 0.0003). Furthermore, I Na inhibition by QX-OH+LB was more persistent than that of LB alone during washout. CONCLUSION: LL-1 can produce an additive and stable inhibition of Nagv currents, which can contribute to the long-lasting regional anesthetic action.

Inhibiting Hv1 channel in peripheral sensory neurons attenuates chronic inflammatory pain and opioid side effects.

Both opioids and nonsteroidal anti-inflammatory drugs (NSAIDS) produce deleterious side effects and fail to provide sustained relief in patients with chronic inflammatory pain. Peripheral neuroinflammation (PN) is critical for initiation and development of inflammatory pain. A better understanding of molecular mechanisms underlying PN would facilitate the discovery of new analgesic targets and the development of new therapeutics. Emerging evidence suggests that peripheral sensory neurons are not only responders to painful stimuli, but are also actively engaged in inflammation and immunity, whereas the intrinsic regulatory mechanism is poorly understood. Here we report the expression of proton-selective ion channel Hv1 in peripheral sensory neurons in rodents and humans, which was previously shown as selectively expressed in microglia in mammalian central nervous system. Neuronal Hv1 was up-regulated by PN or depolarizing stimulation, which in turn aggravates inflammation and nociception. Inhibiting neuronal Hv1 genetically or by a newly discovered selective inhibitor YHV98-4 reduced intracellular alkalization and ROS production in inflammatory pain, mitigated the imbalance in downstream SHP-1-pAKT signaling, and also diminished pro-inflammatory chemokine release to alleviate nociception and morphine-induced hyperalgesia and tolerance. Thus, our data reveal neuronal Hv1 as a novel target in analgesia strategy and managing opioids-related side effects.

Sodium leak channel contributes to neuronal sensitization in neuropathic pain.

Neuropathic pain affects up to 10 % of the total population and no specific target is ideal for therapeutic need. The sodium leak channel (NALCN), a non-selective cation channel, mediates the background Na+ leak conductance and controls neuronal excitability and rhythmic behaviors. Here, we show that increases of NALCN expression and function in dorsal root ganglion (DRG) and dorsal spinal cord contribute to chronic constriction injury (CCI)-induced neuropathic pain in rodents. NALCN current and neuronal excitability in acutely isolated DRG neurons and spinal cord slices of rats were increased after CCI which were decreased to normal levels by NALCN-siRNA. Accordingly, pain-related symptoms were significantly alleviated by NALCN-siRNA-mediated NALCN knockdown and completely prevented by NALCN-shRNA-mediated NALCN knockdown in rats or by conditional NALCN knockout in mice. Our results indicate that increases in NALCN expression and function contribute to CCI-induced neuronal sensitization; therefore, NALCN may be a novel molecular target for control of neuropathic pain.

Deficiency of Mitochondrial Functions and Peroxidation of Frontoparietal Cortex Enhance Isoflurane Sensitivity in Aging Mice.

Background: Hypersensitivity to general anesthetics may predict poor postoperative outcomes, especially among the older subjects. Therefore, it is essential to elucidate the mechanism underlying hypersensitivity to volatile anesthetics in the aging population. Given the fact that isoflurane sensitivity increases with aging, we hypothesized that deficiencies of mitochondrial function and elevated oxidative levels in the frontoparietal cortex may contribute to the enhanced sensitivity to isoflurane in aging mice. Methods: Isoflurane sensitivity in aging mice was determined by the concentration of isoflurane that is required for loss of righting reflex (LORR). Mitochondrial bioenergetics of the frontoparietal cortex was measured using a Seahorse XFp analyzer. Protein oxidation and lipid oxidation in the frontoparietal cortex were assessed using the Oxyblot protein oxidation detection kit and thiobarbituric acid reactive substance (TBARS) assay, respectively. Contributions of mitochondrial complex II inhibition by malonate and peroxidation by ozone to isoflurane sensitivity were tested in vivo. Besides, effects of antioxidative therapy on mitochondrial function and isoflurane sensitivity in mice were also measured. Results: The mean concentration of isoflurane that is required for LORR in aging mice (14-16 months old) was 0.83% ± 0.13% (mean ± SD, n = 80). Then, the mice were divided into three groups as sensitive group (S group, mean - SD), medium group (M group), and resistant group (R group, mean + SD) based on individual concentrations of isoflurane required for LORR. Activities of mitochondrial complex II and complex IV in mice of the S group were significantly lower than those of the R group, while frontoparietal cortical malondialdehyde (MDA) levels were higher in the mice of S group. Both inhibition of mitochondrial complexes and peroxidation significantly decreased the concentration of isoflurane that is required for LORR in vivo. After treatment with idebenone, the levels of lipid oxidation were alleviated and mitochondrial function was restored in aging mice. The concentration of isoflurane that required for LORR was also elevated after idebenone treatment. Conclusions: Decreased mitochondrial functions and higher oxidative stress levels in the frontoparietal cortex may contribute to the hypersensitivity to isoflurane in aging mice.

TREK Channel Family Activator with a Well-Defined Structure-Activation Relationship for Pain and Neurogenic Inflammation.

TWIK-related K+ (TREK) channels are potential analgesic targets. However, selective activators for TREK with both defined action mechanism and analgesic ability for chronic pain have been lacking. Here, we report (1S,3R)-3-((4-(6-methylbenzo[d]thiazol-2-yl)phenyl)carbamoyl)cyclopentane-1-carboxylic acid (C3001a), a selective activator for TREK, against other two-pore domain K+ (K2P) channels. C3001a binds to the cryptic binding site formed by P1 and TM4 in TREK-1, as suggested by computational modeling and experimental analysis. Furthermore, we identify the carboxyl group of C3001a as a structural determinant for binding to TREK-1/2 and the key residue that defines the subtype selectivity of C3001a. C3001a targets TREK channels in the peripheral nervous system to reduce the excitability of nociceptive neurons. In neuropathic pain, C3001a alleviated spontaneous pain and cold hyperalgesia. In a mouse model of acute pancreatitis, C3001a alleviated mechanical allodynia and inflammation. Together, C3001a represents a lead compound which could advance the rational design of peripherally acting analgesics targeting K2P channels without opioid-like adverse effects.

Coriaria lactone increased the intracellular level of calcium through the voltage-gated calcium channels in rat hippocampal neurons.

To investigate the effects of Coriaria Lactone (CL), an epileptogenic substance, on intracellular levels of calcium ([Ca(2+)](i)) and physiological properties of voltage-gated calcium channels (VGCCs). Ratiometric calcium imaging using Fura Red and whole-cell voltage patch-clamp technique were explored on freshly isolated rat hippocampal neurons exposed to CL. Coriaria Lactone increased [Ca(2+)](i) from 118 +/- 21 to 440 +/- 35 nM; VGCCs and calcium influx through NMDA receptor served as the main routes of entry. Coriaria Lactone could enhance both Low voltage activated (LVA) and High voltage activated calcium currents in a concentration-dependent way, and its effect on LVA current was more potent (about 60%). The increased calcium currents were accompanied by the shift of voltage-dependent steady-state inactivation to more positive potentials. These effects of CL, especially its impact on LVA current, could activate different calcium-dependent signaling pathways, and influence cellular excitable properties as well, which might play an important role in CL's epileptogenic process.

Activation of mitogen-activated protein kinases in satellite glial cells of the trigeminal ganglion contributes to substance P-mediated inflammatory pain.

Inflammatory orofacial pain, in which substance P (SP) plays an important role, is closely related to the cross-talk between trigeminal ganglion (TG) neurons and satellite glial cells (SGCs). SGC activation is emerging as the key mechanism underlying inflammatory pain through different signalling mechanisms, including glial fibrillary acidic protein (GFAP) activation, phosphorylation of mitogen-activated protein kinase (MAPK) signalling pathways, and cytokine upregulation. However, in the TG, the mechanism underlying SP-mediated orofacial pain generated by SGCs is largely unknown. In this study, we investigated whether SP is involved in inflammatory orofacial pain by upregulating interleukin (IL)-1ß and tumour necrosis factor (TNF)-α from SGCs, and we explored whether MAPK signalling pathways mediate the pain process. In the present study, complete Freund's adjuvant (CFA) was injected into the whisker pad of rats to induce an inflammatory model in vivo. SP was administered to SGC cultures in vitro to confirm the effect of SP. Facial expression analysis showed that pre-injection of L703,606 (an NK-1 receptor antagonist), U0126 (an inhibitor of MAPK/extracellular signal-regulated kinase [ERK] kinase [MEK] 1/2), and SB203580 (an inhibitor of P38) into the TG to induce targeted prevention of the activation of the NK-1 receptor and the phosphorylation of MAPKs significantly suppressed CFA-induced inflammatory allodynia. In addition, SP promoted SGC activation, which was proven by increased GFAP, p-MAPKs, IL-1ß and TNF-α in SGCs under inflammatory conditions. Moreover, the increase in IL-1ß and TNF-α was suppressed by L703, 606, U0126 and SB203580 in vivo and in vitro. These present findings suggested that SP, released from TG neurons, activated SGCs through the ERK1/2 and P38 pathways and promoted the production of IL-1ß and TNF-α from SGCs, contributing to inflammatory orofacial pain associated with peripheral sensitization.

Capsazepine prolongation of the duration of lidocaine block of sensory transmission in mice may be mediated by modulation of HCN channel currents.

BACKGROUND AND OBJECTIVES: Hyperpolarization-activation cyclic nucleotide-gated (HCN) channels contribute to the effects of lidocaine. Capsazepine (CPZ), a competitive inhibitor of capsaicin of transient receptor potential vanilloid-1 channel, has also been found to inhibit HCN channel currents (I h). This study was designed to investigate whether CPZ could prolong durations of lidocaine in regional anesthesia. METHODS: Mouse HCN1 and HCN2 channels were expressed in human embryonic kidney 293 (HEK 293) cells. The effect of CPZ on I h was measured by whole-cell patch-clamping recording. Sciatic nerve block model in mice was used for the study in vivo. The mice were randomly divided into seven groups, respectively, receiving lidocaine, CPZ, ZD7288 (HCN channel blocker), CPZ + lidocaine, ZD7288 + lidocaine, ZD7288 + CPZ + lidocaine, forskolin (an activator of adenylyl cyclase) + CPZ + lidocaine. Regional anesthetic durations of lidocaine were determined. Voltage-gated sodium channel currents (I Na) and I h were recorded in dorsal root ganglion neurons of mice. The effects of CPZ on I Na and I h with or without Cyclic adenosine monophosphate (cAMP) were assessed. Isolated mice sciatic nerve was prepared to evaluate the effect of CPZ on the compound action potentials (CAP). RESULTS: Capsazepine non-selectively inhibited transfected mHCN1 and mHCN2 channel currents in HEK 293 cells. In sciatic nerve block in vivo, compared to lidocaine alone, adding CPZ extended the durations of lidocaine for noxious sensory block (35.1 ± 3.3 vs. 20.3 ± 1.7 min), tactile sensory block (25.5 ± 4.4 vs. 20.0 ± 3.7 min), thermal sensory block (39.6 ± 6.6 vs. 26.8 ± 5.5 min), and motor function block (28.6 ± 4.1 vs. 20.9 ± 4.2 min). Duration of thermal sensory block was longer in CPZ + lidocaine group than that of ZD7288 + lidocaine group (39.6 ± 6.6 vs. 33.4 ± 4.5 min). Forskolin reversed the prolongation by CPZ on lidocaine durations. CPZ or ZD7288 alone did not produce typical regional anesthetic effects. Increased intracellular concentration of cAMP reversed the inhibition of CPZ on I h. Although CPZ alone inhibited I Na at the concentration more than 30 µM, it did not inhibit the CAP amplitudes in isolated sciatic nerves. CPZ dose-dependently enhanced the inhibitory effect of 1% lidocaine on the CAP amplitudes. CONCLUSIONS: Capsazepine may prolong durations of lidocaine in peripheral nerve block by modulation of HCN channel currents.

The P2Y14 receptor in the trigeminal ganglion contributes to the maintenance of inflammatory pain.

P2Y purinergic receptors expressed in neurons and satellite glial cells (SGCs) of the trigeminal ganglion (TG) contribute to inflammatory and neuropathic pain. P2Y14 receptor expression is reported in the spinal cord, dorsal root ganglion (DRG), and TG. In present study, the role of P2Y14 receptor in the TG in inflammatory orofacial pain of Sprague-Dawley (SD) rats was investigated. Peripheral injection of complete Freund's adjuvant (CFA) induced mechanical hyperalgesia with the rapid upregulation of P2Y14 receptor, glial fibrillary acidic protein (GFAP), interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), C-C chemokine CCL2, phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2), and phosphorylated p38 (p-p38) proteins in the TG. Furthermore, immunofluorescence staining confirmed the CFA-induced upregulation of P2Y14 receptor. Double immunostaining showed that P2Y14 receptor colocalized with glutamine synthetase (GS) and neuronal nuclei (NeuN). Finally, trigeminal injection of a selective antagonist (PPTN) of P2Y14 receptor attenuated CFA-induced mechanical hyperalgesia. PPTN also decreased the upregulation of the GFAP, IL-1ß, TNF-α, CCL2, p-ERK1/2, and p-p38 proteins. Our findings showed that P2Y14 receptor in TG may contribute to orofacial inflammatory pain via regulating SGCs activation, releasing cytokines (IL-1ß, TNF-α, and CCL2), and phosphorylating ERK1/2 and p38.

FMRFamide modulates outward potassium currents in mouse olfactory sensory neurons.

1. The olfactory system can detect the presence of low concentrations of odourant molecules and discriminate even slight differences among molecules with a very similar chemical structure. The detection and discrimination of odourants are correlated with the electrophysiology of the olfactory sensory neurons. To get a better understanding of the molecular mechanisms of olfactory transduction, it is therefore of considerable importance to obtain electrophysiological recordings of olfactory sensory neurons. FMRFamide (Phe-Met-Arg-Phe-NH(2)), secreted from the nerve terminals of the nasal cavity, has been suggested to act as a neurotransmitter or neuromodulator, playing an important role in modulating the activity of olfactory receptor neurons. Its effects on voltage-dependent potassium currents in the mouse olfactory sensory neurons were investigated in the present study using the whole-cell patch-clamp technique. 2. Olfactory sensory neurons were isolated from the Kunming Mouse (KM) mouse olfactory epithelium. Different protocols were applied to obtain delayed-rectifier potassium current (I(K)) and fast transient potassium current (I(A)). The effects of FMRFamide on the outward potassium currents, including I(K) and I(A), in mouse olfactory sensory neurons were investigated. 3. We found that FMRFamide (5 micromol/L) increased the magnitude of I(K). However no effect on I(A) was observed. The activation dynamics of both currents were not changed by FMRFamide. 4. In conclusion, FMRFamide may play a role in the modulation of peripheral olfactory signals by regulating I(K). This modulation may shorten the phase of the fast repolarization of the action potential in mouse olfactory sensory neurons and increase the excitability of the neuronal membrane.