Excitability Of Dorsal Root Ganglion Neurons Research Articles

The natural product argentatin C attenuates postoperative pain via inhibition of voltage-gated sodium and T-type voltage-gated calcium channels.

Postoperative pain occurs in as many as 70% of surgeries performed worldwide. Postoperative pain management still relies on opioids despite their negative consequences, resulting in a public health crisis. Therefore, it is important to develop alternative therapies to treat chronic pain. Natural products derived from medicinal plants are potential sources of novel biologically active compounds for development of safe analgesics. In this study, we screened a library of natural products to identify small molecules that target the activity of voltage-gated sodium and calcium channels that have important roles in nociceptive sensory processing. Fractions derived from the Native American medicinal plant, Parthenium incanum, were assessed using depolarization-evoked calcium influx in rat dorsal root ganglion (DRG) neurons. Further separation of these fractions yielded a cycloartane-type triterpene identified as argentatin C, which was additionally evaluated using whole-cell voltage and current-clamp electrophysiology, and behavioural analysis in a mouse model of postsurgical pain. Argentatin C blocked the activity of both voltage-gated sodium and low-voltage-activated (LVA) calcium channels in calcium imaging assays. Docking analysis predicted that argentatin C may bind to NaV 1.7-1.9 and CaV 3.1-3.3 channels. Furthermore, argentatin C decreased Na+ and T-type Ca2+ currents as well as excitability in rat and macaque DRG neurons, and reversed mechanical allodynia in a mouse model of postsurgical pain. These results suggest that the dual effect of argentatin C on voltage-gated sodium and calcium channels supports its potential as a novel treatment for painful conditions.

Positive interaction between GPER and β-alanine in the dorsal root ganglion uncovers potential mechanisms: mediating continuous neuronal sensitization and neuroinflammation responses in neuropathic pain

BackgroundThe pathogenesis of neuropathic pain and the reasons for the prolonged unhealing remain unknown. Increasing evidence suggests that sex oestrogen differences play a role in pain sensitivity, but few studies have focused on the oestrogen receptor which may be an important molecular component contributing to peripheral pain transduction. We aimed to investigate the impact of oestrogen receptors on the nociceptive neuronal response in the dorsal root ganglion (DRG) and spinal dorsal horn using a spared nerve injury (SNI) rat model of chronic pain.MethodsWe intrathecally (i.t.) administered a class of oestrogen receptor antagonists and agonists intrathecal (i.t.) administrated to male rats with SNI or normal rats to identify the main receptor. Moreover, we assessed genes identified through genomic metabolic analysis to determine the key metabolism point and elucidate potential mechanisms mediating continuous neuronal sensitization and neuroinflammatory responses in neuropathic pain. The excitability of DRG neurons was detected using the patch-clamp technique. Primary culture was used to extract microglia and DRG neurons, and siRNA transfection was used to silence receptor protein expression. Immunofluorescence, Western blotting, RT-PCR and behavioural testing were used to assess the expression, cellular distribution, and actions of the main receptor and its related signalling molecules.ResultsIncreasing the expression and function of G protein-coupled oestrogen receptor (GPER), but not oestrogen receptor-α (ERα) and oestrogen receptor-β (ERβ), in the DRG neuron and microglia, but not the dorsal spinal cord, contributed to SNI-induced neuronal sensitization. Inhibiting GPER expression in the DRG alleviated SNI-induced pain behaviours and neuroinflammation by simultaneously downregulating iNOS, IL-1β and IL-6 expression and restoring GABAα2 expression. Additionally, the positive interaction between GPER and β-alanine and subsequent β-alanine accumulation enhances pain sensation and promotes chronic pain development.ConclusionGPER activation in the DRG induces a positive association between β-alanine with iNOS, IL-1β and IL-6 expression and represses GABAα2 involved in post-SNI neuropathic pain development. Blocking GPER and eliminating β-alanine in the DRG neurons and microglia may prevent neuropathic pain development.

Protease‐Induced Excitation of Dorsal Root Ganglion Neurons in Response to Acute Perturbation of the Gut Microbiota is Associated with Visceral Hypersensitivity

Abdominal pain is a major symptom of diseases associated with microbial dysbiosis. Disruption of the gut microbiota with antibiotics increases visceral pain, and germ‐free mice are more prone to pain than conventionally‐raised mice. However, the mechanisms underlying microbial modulation of pain remain elusive. We hypothesized that disruption of the intestinal microbiota modulates the excitability of peripheral nociceptive neurons. Patch clamp electrophysiological recordings of dorsal root ganglion (DRG) neuron excitability were obtained from control mice and mice treated with the non‐absorbable antibiotic vancomycin (50 µg/ml in drinking water) for one week. Ten days prior to recording visceromotor response (VMR) telemetric transmitters were placed into the abdominal cavity of the mice and allowed to recover. VMR was measured by insertion of balloon catheter into the rectum under light anesthetization in both control and vancomycin treated mice, then distended to 80 mmHg and VMR recorded. Bacterial dysbiosis was verified by metagenomic analysis of stool microbial composition. Mice treated with vancomycin were more sensitive to colorectal distension in vivo (VMR increased by 70% at 80 mmHg compared to control), and DRG neurons from vancomycin‐treated mice were hyperexcitable in vitro compared to water‐treated controls (rheobase decreased by 30% relative to control). Interestingly, hyperexcitability of DRG neurons was not restricted to gut projecting neurons, suggesting a widespread effect of gut dysbiosis on pain pathways. Incubation of DRG neurons from naïve mice in serum from vancomycin‐treated mice increased neuron excitability (rheobase decreased by 30% relative to control), suggesting that microbial dysbiosis alters circulating mediators that influence nociception. Multiplex ELISA measurements did not detect any significant changes in serum cytokines or chemokines between vancomycin‐treated and control mice. The cysteine protease inhibitor E64 (30 nM) and the protease‐activated receptor 2 (PAR2) antagonist GB‐83 (10 µM) each blocked the increase in DRG neuron excitability in response to serum from vancomycin‐treated mice. Naïve DRG neurons incubated with fecal supernatants from vancomycin‐treated mice also exhibited increased excitability (rheobase decreased by 40% relative to control), but supernatants derived from colonic tissue failed to cause hyperexcitability. Overall, this data suggests that microbial dysbiosis within the gut alters pain sensitivity. This effect is not caused by inflammation or host derived factors, rather bacterially‐derived cysteine proteases activating PAR2 on DRG neurons.

Sustained Depolarization Induces Homeostatic Plasticity in Mouse and Human Sensory Neurons

Remodeling of the nervous system serves as a protective mechanism during abrupt disturbance of neuronal homeostasis. This offers adequate maintenance of overall network activity that includes both synaptic scaling and regulation of intrinsic neuronal excitability in an adaptive process known as homeostatic plasticity. Although alterations in neuronal excitability of nociceptors have been associated with the development of different forms of chronic pain, the involvement of homeostatic plasticity in the excitability of nociceptors, in the context of prolonged excitation, and in the pathophysiology of chronic pain is poorly understood. Using a combination of pharmacological, optogenetic and electrophysiological approaches, we show that sustained depolarization of cultured dorsal root ganglia (DRG) neurons, especially nociceptors, strongly reduces intrinsic excitability of mouse and human DRG neurons. These changes include decreases in input resistance, action potential (AP) fall time and AP half width, and an increase in rheobase associated with an overall decrease in AP firing, thus suggesting that mechanisms of homeostatic plasticity are engaged in mouse and human nociceptors. Unexpectedly, RNA sequencing studies show that there are not significant changes in the global mouse and human DRG neuronal gene expression after sustained depolarization, suggesting that transcriptional changes are not the underlying cause of homeostatic plasticity in nociceptors. Our data reveal the presence of intrinsic homeostatic mechanisms that regulate neuronal excitability in response to sustained depolarization of mouse and human nociceptors. Therefore, dysregulation of these homeostatic mechanisms could contribute to the understanding of the pathophysiology of chronic pain syndromes. Grant support from NS042595; The Dr. Seymour and Rose T. Brown Professorship in Anesthesiology K00NS113422 American Neuromuscular Foundation Career Development Award F32DA051160-01.

Inhibition of sodium conductance by cannabigerol contributes to a reduction of dorsal root ganglion neuron excitability.

Cannabigerol (CBG), a non-psychotropic phytocannabinoid and a precursor of ∆9 -tetrahydrocannabinol and cannabidiol, has been suggested to act as an analgesic. A previous study reported that CBG (10μM) blocks voltage-gated sodium (Nav ) currents in CNS neurons, although the underlying mechanism is not well understood. Genetic and functional studies have validated Nav 1.7 channels as an opportune target for analgesic drug development. The effects of CBG on Nav 1.7 channels, which may contribute to its analgesic properties, have not been previously investigated. To determine the effects of CBG on Nav channels, we used stably transfected HEK cells and primary dorsal root ganglion (DRG) neurons to characterize compound effects using experimental and computational techniques. These included patch-clamp, multielectrode array, and action potential modelling. CBG is a ~10-fold state-dependent Nav channel inhibitor (KI -KR : ~2-20 μM) with an average Hill-slope of ~2. We determined that, at lower concentrations, CBG predominantly blocks sodium Gmax and slows recovery from inactivation. However, as the concentration is increased, CBG also induces a hyperpolarizing shift in the half-voltage of inactivation. Our modelling and multielectrode array recordings suggest that CBG attenuates DRG excitability. Inhibition of Nav 1.7 channels in DRG neurons may underlie CBG-induced neuronal hypoexcitability. As most Nav 1.7 channels are inactivated at the resting membrane potential of DRG neurons, they are more likely to be inhibited by lower CBG concentrations, suggesting functional selectivity against Nav 1.7 channels, compared with other Nav channels (via Gmax block).

A240 OPIOID-INDUCED HYPEREXCITABILITY IN DRG NEURONS IS MEDIATED BY DOR-DEPENDENT ENDOCYTOSIS

Abstract Background Opioids are effective for the treatment of abdominal pain but are also associated with tolerance, and increased dosing leads to severe side effects. We previously showed that prolonged exposure to high doses of opioids evoked paradoxical hyperexcitability of colonic afferent nerves that was mediated by δ-opioid receptor (DOR) signaling. Recent studies suggest that DOR-dependent analgesia is mediated not only by G proteins but also via receptor endocytosis and downstream signaling, but it is unclear what intracellular signaling mechanisms are underlying opioid-induced hyperexcitability. Aims To examine the mechanisms underlying DOR-mediated hyperexcitability of dorsal root ganglia (DRG) neurons. Methods We assessed the excitability of DRG neurons isolated from C57BL/6 mice by measuring the rheobase (minimal current to elicit an action potential, i.e. lower rheobase=increased excitability) using perforated patch-clamp recordings. Dissociated neurons were exposed to a high concentration (10 µM) of the μ-opioid receptor agonist DAMGO, the DOR agonist DADLE, or the weakly internalizing DOR agonist ARM390 overnight. To examine the role of receptor endocytosis and intracellular receptor activation underlying the excitatory effect by opioids, DRG neurons exposed to DAMGO or DADLE were preincubated with the membrane-permeable opioid receptor antagonist naloxone or the endocytosis inhibitor Pitstop2. To further understand the mechanisms involved in the hyperexcitability evoked by opioid re-exposure, following overnight incubation with high concentrations of DAMGO or DADLE, neurons were washed for 1 hr and treated either with the PKA inhibitor H89 or the PKC inhibitor GFX before re-exposure to DAMGO or DADLE at a low concentration (10 nM). Results Neurons exposed to 10 µM DAMGO or DADLE were hyperexcitable (rheobase decreased 25 % and 26 % compared to controls respectively; p≤0.05, 2-way ANOVA). Naloxone and Pitstop2 blocked the increased excitability of DRG neurons induced by overnight incubations with either DAMGO or DADLE. In contrast to the hyperexcitability induced by DAMGO and DADLE, overnight incubation with 10 µM ARM390 decreased excitability (rheobase increased 31%, p≤0.05, unpaired t-test). The hyperexcitability induced by DAMGO and DADLE was reversed after a 1 hr washout but acute reapplication of a low concentration of DAMGO or DADLE (10nM) now evoked hyperexcitability (rheobase decreased 34 and 35 % respectively, p≤0.05, 2-way ANOVA). This effect was prevented by inhibiting PKC but not PKA. Conclusions Our data suggest that the DOR-dependent hyperexcitability evoked by prolonged exposure to high concentrations of opioids is dependent on receptor endocytosis and downstream PKC signaling. Targeting these pathways could mitigate the hyperexcitability of pain signaling neurons caused by high doses of opioids. Funding Agencies CCC

A243 HISTAMINE H1-RECEPTOR ANTAGONISTS SUPPRESS THE HYPEREXCITABILITY OF NOCICEPTIVE NEURONS EXPOSED TO IBS-C PATIENT STOOL SUPERNATANTS

Abstract Background Abdominal pain is the primary cause of morbidity in many chronic GI disorders such as IBS, but the molecular mechanisms contributing to pain signaling are unclear. Stool supernatants from patients with diarrhea-predominant IBS increase the excitability of DRG neurons compared to supernatants from healthy controls, suggesting that mediators in the stool may sensitize nociceptors. Additionally, histamine has been implicated as a mediator of hypersensitivity in IBS patients. However, it is unclear if stool supernatants from constipation-predominant IBS (IBS-C) patients affect the excitability of DRG neurons. Furthermore, it is unknown if specific neuro-mediators in stool such as histamine impact the excitability of nociceptive neurons in this subgroup of IBS patients. Aims To evaluate whether IBS-C stool supernatants induce nociceptive signaling in DRG neurons compared to healthy control (HC) stool supernatants. If so, to evaluate the role of histamine 1 (H1) receptors in DRG neuron nociceptive signaling initiated by mediators in IBS-C stool supernatants. Methods IBS-C (n=5) and HC (n=2) patient stool was collected, filtered, and dissolved with Krebs solution in a 1:8 (g/v) dilution. Dorsal root ganglion (DRG) neurons from C57BL/6 mice were incubated with HC or IBS-C stool supernatant for 30 minutes. To evaluate whether histamine in stool supernatants can sensitize H1 receptors on nociceptors, DRG neurons were pre-incubated with the H1-receptor antagonist pyrilamine (1μM, 30 min) before stool supernatants. Changes in DRG neuronal excitability were recorded using perforated patch-clamp techniques to measure the rheobase (minimum input current needed to elicit an action potential) and the resting membrane potential (RMP). Results In neurons incubated with IBS-C stool supernatants (n=28) the rheobase decreased (63%) compared to healthy controls (78 ± 13.7 pA; n=6). This effect was reversed in DRG neurons pre-incubated with the H1 receptor antagonist, pyrilamine, (n=26) (77 ± 5.9 pA, p<0.001) compared to neurons incubated with IBS-C supernatant alone (50 ± 5.13 pA; n=28). No changes were found in the RMP. The data were analyzed with the non-parametric Kruskal-Wallis test. Conclusions IBS-C stool supernatants increase the excitability of DRG neurons compared to HC. Furthermore, the H1-receptor antagonist pyrilamine inhibits the neuronal hyperexcitability evoked by mediators in IBS-C patient stool. These findings suggest that the neuroactive metabolite histamine may contribute to visceral pain experienced by patients with IBS-C. Further studies are needed to examine whether similar signaling to nociceptive neurons by stool supernatants occurs in other subtypes of IBS. Funding Agencies CAG, CIHR

A population of in silico models identifies the interplay between Nav 1.8 conductance and potassium currents as key in regulating human dorsal root ganglion neuron excitability

Background: The Nav 1.8 sodium channel has a key role in generating repetitive action potentials in nociceptive human dorsal root ganglion neurons. Nav 1.8 is differentiated from other voltage-gated sodium channels by its unusually slow inactivation kinetics and depolarised voltage-dependence of activation. These features are particularly pronounced in the human Nav 1.8 channel and allow the channel to remain active during repolarisation. Gain-of-function mutations in Nav 1.8 have been linked to neuropathic pain and selective blockers of Nav 1.8 have been developed as potential new analgesics. However, it is not well understood how modulating the Nav 1.8 conductance alters neuronal excitability and how this depends on the balance of other ion channels expressed by nociceptive neurons. Methods: To investigate this, we developed a novel computational model of the human dorsal root ganglion neuron and used it to construct a population of models that mimicked inter-neuronal heterogeneity in ionic conductances and action potential morphology Results: By simulating changes to the Nav 1.8 conductance in the population of models, we found that moderately increasing the Nav 1.8 conductance led to increased firing rate, as expected, but increasing Nav 1.8 conductance beyond an inflection point caused firing rate to decrease. We found that the delayed rectifier and M-type potassium conductances were also critical for determining neuronal excitability. In particular, altering the delayed rectifier potassium conductance shifted the position of the Nav 1.8 inflection point and therefore the relationship between Nav 1.8 conductance and firing rate. Conclusions: Our results suggest that the effects of modulating Nav 1.8 in a nociceptive neuron can depend significantly on other conductances, particularly potassium conductances.

Upregulation of acid sensing ion channels is associated with esophageal hypersensitivity in GERD

Proton pump inhibitors (PPIs) are the mainstay of therapy for gastroesophageal reflux disease (GERD) but up to 60% of patients have inadequate response to therapy. Acid sensing ion channels (ASICs) play important roles in nociception. This study aimed to investigate whether the increased expression of ASICs results in neuronal hyperexcitability in GERD. Esophageal biopsies were taken from GERD patients and healthy subjects to compare expression of ASIC1 and 3. Next, gene and protein expression of ASIC1 and 3 from esophageal mucosa and dorsal root ganglia (DRG) neurons were measured by qPCR, Western‐blot and immunofluorescence in rodent models of reflux esophagitis (RE), non‐erosive reflux disease (NERD), and sham operated groups. Excitability of DRG neurons in the GERD and sham groups were also tested by whole‐cell patch‐clamp recordings. We demonstrated that ASIC1 and 3 expression were significantly increased in patients with RE compared with healthy controls. This correlated positively with symptom severity of heartburn and regurgitation (p < .001). Next, ASIC1 and 3 gene and protein expression in rodent models of RE and NERD were similarly increased in esophageal mucosa as well as T3–T5 DRG neurons compared with sham operation. DRG neurons from RE animals showed hyperexcitability compared with sham group. However, intrathecal injection of ASIC specific inhibitors, PcTx1 and APTEx‐2, as well as silencing ASIC1 and 3 genes with specific siRNAs prevented visceral hypersensitivity. Overall, upregulation of ASIC1 and 3 may lead to visceral hypersensitivity in RE and NERD and may be a potential therapeutic target for PPI non‐responsive patients.

Effect of sympathetic sprouting on the excitability of dorsal root ganglion neurons and afferents in a rat model of neuropathic pain

Increased sympathetic nerve excitability has been reported to aggravate a variety of chronic pain conditions, and an increase in the number of sympathetic nerve fibers in the dorsal root ganglion (DRG) has been found in neuropathic pain (NP) models. However, the mechanism of the neurotransmitter norepinephrine (NE) released by sympathetic nerve fiber endings on the excitability of DRG neurons is still controversial, and the adrenergic receptor subtypes involved in this biological process are also controversial. In our study, we have two objectives: (1) To determine the effect of the neurotransmitter NE on the excitability of different neurons in DRG; (2) To determine which adrenergic receptors are involved in the excitability of DRG neurons by NE released by sprouting sympathetic fibers. In this experiment, a unique field potential recording method of spinal cord dorsal horn was innovatively adopted, which can be used for electrophysiological study in vivo. The results showed that： Forty days after SNI, patch clamp and field potential recording methods confirmed that NE enhanced the excitability of ipsilateral DRG large neurons, and then our in vivo electrophysiological results showed that the α2 receptor blocker Yohimbine could block the excitatory effect of NE on A-fiber and the inhibitory effect on C-fiber, while the α2A-adrenergic receptor agonist guanfacine (100 μM) had the same biological effect as NE. Finally, we concluded that NE from sympathetic fiber endings is involved in the regulation of pain signaling by acting on α2A-adrenergic receptors in DRG.

ɑO-Conotoxin GeXIVA isomers modulate N-type calcium (CaV 2.2) channels and inwardly-rectifying potassium (GIRK) channels via GABAB receptor activation.

αO-Conotoxin GeXIVA is a 28 amino acid peptide derived from the venom of the marine snail Conus generalis. The presence of four cysteine residues in the structure of GeXIVA allows it to have three different disulfide isomers, that is, the globular, ribbon or bead isomer. All three isomers are active at α9α10 nicotinic acetylcholine receptors, with the bead isomer, GeXIVA[1,2], being the most potent and exhibiting analgesic activity in animal models of neuropathic pain. The original report of GeXIVA activity failed to observe any effect of the isomers on high voltage-activated (HVA) calcium channel currents in rat dorsal root ganglion (DRG) neurons. In this study, we report, for the first time, the activity of globular GeXIVA[1,3] at G protein-coupled GABAB receptors (GABAB R) inhibiting HVA N-type calcium (Cav2.2) channels and reducing membrane excitability in mouse DRG neurons. The inhibition of HVA Ba2+ currents and neuroexcitability by GeXIVA[1,3] was partially reversed by the selective GABAB R antagonist CGP 55845. In transfected HEK293T cells co-expressing human GABAB R1 and R2subunits and Cav2.2 channels, both GeXIVA[1,3] and GeXIVA[1,4] inhibited depolarization-activated Ba2+ currents mediated by Cav2.2 channels, whereas GeXIVA[1,2] had no effect. The effects of three cyclized GeXIVA[1,4] ribbon isomers were also tested, with cGeXIVA GAG being the most potent at human GABAB R-coupled Cav2.2 channels. Interestingly, globular GeXIVA[1,3] also reversibly potentiated inwardly-rectifying K+ currents mediated by human GIRK1/2 channels co-expressed with GABAB R in HEK293T cells. This study highlights GABAB R as a potentially important receptor target for the activity of αO-conotoxin GeXIVA to mediate analgesia.

Peripheral Inflammation Results in Increased Excitability of Capsaicin-Insensitive Nociceptive DRG Neurons Mediated by Upregulation of ASICs and Voltage-Gated Ion Channels.

Previously, we have characterized the capsaicin-insensitive low pH-sensitive (caps−lpH+) subtype of small-sized nociceptive dorsal root ganglion (DRG) neurons that express acid-sensing ion channels, T-type Ca2+ channels, and have isolectin B4-negative phenotype. These neurons demonstrated increased excitability in a model of long-term diabetes, contributing to chronic pain sensation. Here we studied changes in the excitability of the caps−lpH+ neurons and underlying changes in the functional expression and gating properties of ion channels under complete Freund's adjuvant (CFA)-induced peripheral inflammation. We have found that, under these pathological conditions, the functional expression of the acid-sensing ion channels (ASICs) and voltage-gated Na+ channels, was increased. In addition, T-type Ca2+ current was significantly increased in the neurons at the membrane potentials close to its resting value. Altogether, the observed changes in the channel functioning shifted a pH level evoking an action potential (AP) toward its physiological value and led to an increase of evoked and spontaneous excitability of the caps−lpH+ neurons that may contribute to hyperalgesia and chronic inflammatory pain.

Protein kinase G signaling pathway is involved in sympathetically maintained pain by modulating ATP-sensitive potassium channels

BackgroundSympathetically maintained pain (SMP) involves an increased excitability of dorsal root ganglion (DRG) neurons to sympathetic nerve stimulation and circulating norepinephrine. The current treatment of SMP has limited efficacy, and...

Functional Characterization of Ovine Dorsal Root Ganglion Neurons Reveal Peripheral Sensitization after Osteochondral Defect.

Knee joint trauma can cause an osteochondral defect (OD), a risk factor for osteoarthritis (OA) and cause of debilitating pain in patients. Rodent OD models are less translatable because of their smaller joint size and open growth plate. This study proposes sheep as a translationally relevant model to understand the neuronal basis of OD pain. A unilateral 6-mm deep OD was induced in adult female sheep. Two to six weeks after operation, lumbar dorsal root ganglia (DRG) neurons were collected from the contralateral (Ctrl) and OD side of operated sheep. Functional assessment of neuronal excitability and activity of the pain-related ion channels transient receptor potential vanilloid receptor 1 (TRPV1) and P2X3 was conducted using electrophysiology and Ca2+ imaging. Immunohistochemistry was used to verify expression of pain-related proteins. We observed that an increased proportion of OD DRG neurons (sheep, N = 3; Ctrl neurons, n = 15, OD neurons, n = 16) showed spontaneous electrical excitability (Ctrl: 20.33 ± 4.5%; OD: 50 ± 10%; p = 0.009, unpaired t test) and an increased proportion fired a greater number of spikes above baseline in response to application of a TRPV1 agonist (capsaicin) application (Ctrl: 40%; OD: 75%; p = 0.04, χ2 test). Capsaicin also produced Ca2+ influx in an increased proportion of isolated OD DRG neurons (Ctrl: 25%; OD: 44%; p = 0.001, χ2 test). Neither protein expression, nor functionality of the P2X3 ion channel were altered in OD neurons. Overall, we provide evidence of increased excitability of DRG neurons (an important neural correlate of pain) and TRPV1 function in an OD sheep model. Our data show that functional assessment of sheep DRG neurons can provide important insights into the neural basis of OD pain and thus potentially prevent its progression into arthritic pain.

Estrogen augmented visceral pain and colonic neuron modulation in a double-hit model of prenatal and adult stress.

BACKGROUNDChronic stress during pregnancy may increase visceral hyperalgesia of offspring in a sex-dependent way. Combining adult stress in offspring will increase this sensitivity. Based on the evidence implicating estrogen in exacerbating visceral hypersensitivity in female rodents in preclinical models, we predicted that chronic prenatal stress (CPS) + chronic adult stress (CAS) will maximize visceral hyperalgesia; and that estrogen plays an important role in colonic hyperalgesia.AIMThe aim was to illuminate the role of estrogen in colonic hyperalgesia and its underlying mechanisms.METHODSWe established a CPS plus CAS rodent model in which the balloon was used to distend the colorectum. The single-fiber recording in vivo and patch clamp experiments in vitro were used to monitor the colonic neuron’s activity. The reverse transcription-polymerase chain reaction, western blot, and immunofluorescence were used to study the effects of CPS and CAS on colon primary afferent sensitivity. We used ovariectomy and letrozole to reduce estrogen levels of female rats respectively in order to assess the role of estrogen in female-specific enhanced primary afferent sensitization.RESULTSSpontaneous activity and single fiber activity were significantly greater in females than in males. The enhanced sensitization in female rats mainly came from low-threshold neurons. CPS significantly increased single-unit afferent fiber activity in L6-S2 dorsal roots in response. Activity was further enhanced by CAS. In addition, the excitability of colon-projecting dorsal root ganglion (DRG) neurons increased in CPS + CAS rats and was associated with a decrease in transient A-type K+ currents. Compared with ovariectomy, treatment with the aromatase inhibitor letrozole significantly reduced estrogen levels in female rats, confirming the gender difference. Moreover, mice treated with letrozole had decreased colonic DRG neuron excitability. The intrathecal infusion of estrogen increased brain-derived neurotrophic factor (BDNF) protein levels and contributed to the response to visceral pain. Western blotting showed that nerve growth factor protein was upregulated in CPS + CAS mice.CONCLUSIONThis study adds to the evidence that estrogen-dependent sensitization of primary afferent colon neurons is involved in the development of chronic stress-induced visceral hypersensitivity in female rats.

Improving NKCC1 Function Increases the Excitability of DRG Neurons Exacerbating Pain Induced After TRPV1 Activation of Primary Sensory Neurons.

Background Our aim was to investigate the effects of the protein expression and the function of sodium, potassium, and chloride co-transporter (NKCC1) in the dorsal root ganglion (DRG) after activation of transient receptor potential vanilloid 1 receptor (TRPV1) in capsaicin-induced acute inflammatory pain and the possible mechanism of action.Methods Male Sprague–Dawley rats were randomly divided into control, capsaicin, and inhibitor groups. The expression and distribution of TRPV1 and NKCC1 in rat DRG were observed by immunofluorescence. Thermal radiation and acetone test were used to detect the pain threshold of heat and cold noxious stimulation in each group. The expressions of NKCC1 mRNA, NKCC1 protein, and p-NKCC1 in the DRG were detected by PCR and western blotting (WB). Patch clamp and chloride fluorescent probe were used to observe the changes of GABA activation current and intracellular chloride concentration. After intrathecal injection of protein kinase C (PKC) inhibitor (GF109203X) or MEK/extracellular signal-regulated kinase (ERK) inhibitor (U0126), the behavioral changes and the expression of NKCC1 and p-ERK protein in L4–6 DRG were observed.Result: TRPV1 and NKCC1 were co-expressed in the DRG. Compared with the control group, the immunofluorescence intensity of NKCC1 and p-NKCC1 in the capsaicin group was significantly higher, and the expression of NKCC1 in the nuclear membrane was significantly higher than that in the control group. The expression of NKCC1 mRNA and protein of NKCC1 and p-NKCC1 in the capsaicin group were higher than those in the control group. After capsaicin injection, GF109203X inhibited the protein expression of NKCC1 and p-ERK, while U0126 inhibited the protein expression of NKCC1. In the capsaicin group, paw withdrawal thermal latency (WTL) was decreased, while cold withdrawal latency (CWL) was prolonged. Bumetanide, GF109203X, or U0126 could reverse the effect. GABA activation current significantly increased in the DRG cells of the capsaicin group, which could be reversed by bumetanide. The concentration of chloride in the DRG cells of the capsaicin group increased, but decreased after bumetanide, GF109203X, and U0126 were administered.Conclusion Activation of TRPV1 by exogenous agonists can increase the expression and function of NKCC1 protein in DRG, which is mediated by activation of PKC/p-ERK signaling pathway. These results suggest that DRG NKCC1 may participate in the inflammatory pain induced by TRPV1.

Sevoflurane excites nociceptive sensory neurons by inhibiting K+ conductances in rats

Sevoflurane, which is preferentially used as a day-case anesthetic based on its low blood solubility, acts on the central nervous system and exerts analgesic effects. However, it still remains unknown whether sevoflurane affects the excitability of nociceptive sensory neurons. Therefore, we conducted this study to examine the effects of sevoflurane on the excitability of small-sized dorsal root ganglion (DRG) neurons of rats using the whole-cell patch-clamp technique. In a voltage-clamp condition, sevoflurane elicited the membrane current in a concentration-dependent manner, in which the reversal potential was similar to the equilibrium potential of K+. In a current-clamp condition, sevoflurane directly depolarized the membrane potentials in a concentration-dependent manner. Moreover, at a clinically relevant concentration, sevoflurane decreased the threshold for action potential generation. These findings suggest that sevoflurane acts on the leak K+ channels to increase the excitability of DRG neurons. Sevoflurane increased the half-width of single action potentials, which resulted from the inhibition of voltage-gated K+ currents, including the fast inactivating A-type and non-inactivating delayed rectifier K+ currents. Our study indicates that sevoflurane could exhibit pronociceptive effects on nociceptive sensory neurons by inhibiting K+ conductances.

Oxidative stress induced by NOX2 contributes to neuropathic pain via plasma membrane translocation of PKC\u03b5 in rat dorsal root ganglion neurons

BackgroundNicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2)-induced oxidative stress, including the production of reactive oxygen species (ROS) and hydrogen peroxide, plays a pivotal role in neuropathic pain. Although the activation and plasma membrane translocation of protein kinase C (PKC) isoforms in dorsal root ganglion (DRG) neurons have been implicated in multiple pain models, the interactions between NOX2-induced oxidative stress and PKC remain unknown.MethodsA spared nerve injury (SNI) model was established in adult male rats. Pharmacologic intervention and AAV-shRNA were applied locally to DRGs. Pain behavior was evaluated by Von Frey tests. Western blotting and immunohistochemistry were performed to examine the underlying mechanisms. The excitability of DRG neurons was recorded by whole-cell patch clamping.ResultsSNI induced persistent NOX2 upregulation in DRGs for up to 2 weeks and increased the excitability of DRG neurons, and these effects were suppressed by local application of gp91-tat (a NOX2-blocking peptide) or NOX2-shRNA to DRGs. Of note, the SNI-induced upregulated expression of PKCε but not PKC was decreased by gp91-tat in DRGs. Mechanical allodynia and DRG excitability were increased by ψεRACK (a PKCε activator) and reduced by εV1-2 (a PKCε-specific inhibitor). Importantly, εV1-2 failed to inhibit SNI-induced NOX2 upregulation. Moreover, the SNI-induced increase in PKCε protein expression in both the plasma membrane and cytosol in DRGs was attenuated by gp91-tat pretreatment, and the enhanced translocation of PKCε was recapitulated by H2O2 administration. SNI-induced upregulation of PKCε was blunted by phenyl-N-tert-butylnitrone (PBN, an ROS scavenger) and the hydrogen peroxide catalyst catalase. Furthermore, εV1-2 attenuated the mechanical allodynia induced by H2O2ConclusionsNOX2-induced oxidative stress promotes the sensitization of DRGs and persistent pain by increasing the plasma membrane translocation of PKCε.

Adenosine A3 agonists reverse neuropathic pain via T cell-mediated production of IL-10.

The A3 adenosine receptor (A3AR) has emerged as a therapeutic target with A3AR agonists to tackle the global challenge of neuropathic pain, and investigation into its mode of action is essential for ongoing clinical development. Immune cell A3ARs, and their activation during pathology, modulate cytokine release. Thus, the use of immune cells as a cellular substrate for the pharmacological action of A3AR agonists is enticing, but unknown. The present study discovered that Rag-KO mice lacking T and B cells, as compared with WT mice, are insensitive to the anti-allodynic effects of A3AR agonists. Similar findings were observed in interleukin-10 and interleukin-10 receptor knockout mice. Adoptive transfer of CD4+ T cells from WT mice infiltrated the dorsal root ganglion (DRG) and restored A3AR agonist-mediated anti-allodynia in Rag-KO mice. CD4+ T cells from Adora3-KO or Il10-KO mice did not. Transfer of CD4+ T cells from WT mice, but not Il10-KO mice, into Il10-KO mice or Adora3-KO mice fully reinstated the anti-allodynic effects of A3AR activation. Notably, A3AR agonism reduced DRG neuron excitability when cocultured with CD4+ T cells in an IL-10-dependent manner. A3AR action on CD4+ T cells infiltrated in the DRG decreased phosphorylation of GluN2B-containing N-methyl-D-aspartate receptors at Tyr1472, a modification associated with regulating neuronal hypersensitivity. Our findings establish that activation of A3AR on CD4+ T cells to release IL-10 is required and sufficient evidence for the use of A3AR agonists as therapeutics.

A10 FOOD ANTIGEN-STRESS INTERACTION LEADS TO INCREASE PAIN SIGNALING IN ILEUM AND COLON VIA STAT6 IN AN IBS MODEL

Abstract Background Abdominal pain can be triggered by food ingestion in IBS patients. Previously we have shown that a food antigen induces local release of immune mediators in the colon that increase dorsal root ganglion (DRG) neuron excitability when there is previous antigen exposure in the presence of psychological stress. However, it is unknown if this effect is limited to the colon. Furthermore, the involvement of histamine in the neuronal hyperexcitability suggests that the stress-food antigen interaction evokes a Th2 immune response. Thus, we sought to investigate the role of STAT6, a transcription factor downstream of Th2 cytokines and important for IgE production. Aims 1) Determine if stress-food antigen interaction leads to release of mediators within the small intestine that increase DRG neuron excitability. 2) Determine the involvement of STAT6 on neuronal hyperexcitability induced by the stress-food antigen interaction. Methods BALB/c mice were exposed to water avoidance stress (WAS) or sham stress (SHAM) for 1 hr daily for 10 days. On day 2–10, mice were exposed to ovalbumin (OVA) or saline (SAL). Seven days later, mice were re-exposed to either OVA or SAL every 2 days for 2 weeks yielding 3 groups: WAS/OVA+OVA, WAS/SAL+OVA, and SHAM/OVA+OVA. STAT6 deficient mice were also exposed to WAS/OVA+OVA protocol. Ileum or colonic supernatants were obtained 4 hours after tissue collection. DRG neurons were incubated overnight with supernatants prior to perforated patch clamp recordings. Neuronal excitability was evaluated by measuring the rheobase (minimum current to elicit an action potential, decreased rheobase indicates increased excitability). Mechanosensitivity of extrinsic afferent nerves innervating distal ileum was examined using ex vivo extracellular afferent nerve recordings. Data was analyzed by one or two-way ANOVA with Bonferroni post-hoc test. Results Ileum supernatants from WAS/OVA+OVA mice increased DRG neuron excitability compared to WAS/SAL+OVA and SHAM/OVA+OVA mice (63.3 ± 6.2 pA vs 83.2 ± 5.4 pA, 86.7 ± 4.5 pA, p&amp;lt;0.05). Ileum afferent nerve response to distention was significantly augmented in WAS/OVA+OVA mice compared to WAS/SAL+OVA and SHAM/OVA+OVA (P&amp;lt;0.05, n=4–7). DRG neurons incubated with WAS/OVA+OVA supernatant from STAT6 deficient mice were less excitable compared to neurons incubated with colonic supernatants from wild type mice (86.5 ± 4.1 pA vs 67.6 ± 4.8 pA, p&amp;lt;0.05). Conclusions Stress-food antigen interaction releases mediators in both the small intestine and colon to increase nociceptive signaling, an important finding as IBS can involve both areas. The release of excitatory mediators within the gut appears to involve STAT6. Thus, a stress-food antigen interaction evoking a Th2 immune response in the gut may be a mechanism underlying food induced symptoms in IBS. Funding Agencies Queen’s University, Department of Medicine