Root Ganglion Neurons Of Rats Research Articles

Effect of high-frequency membrane potential alternation between depolarization and hyperpolarization on dorsal root ganglion neurons of rats.

The purpose of this study was to determine how sensory neurons respond to high-frequency membrane potential alternation between depolarization and hyperpolarization. Membrane currents were recorded from dissociated dorsal root ganglion (DRG) neurons of adult rats using the whole cell patch clamp technique in voltage clamp mode. Stepwise depolarization of the membrane was applied first to determine the threshold membrane potential for inducing an action potential (AP) current. Then, membrane potential alternation between depolarization (to +20 mV) and hyperpolarization (to -110 mV) was applied to the neuron for 10s at different frequencies (10 Hz to 1 kHz). The tested DRG neurons had APs of either a long duration (>10ms) or a short duration (<10ms). Membrane potential alternation at ≥500 Hz completely disrupted the AP generation, disabled the ion channel gating function, and produced membrane current alternating symmetrically across zero. Replacing extracellular sodium with potassium increased the amplitude of the membrane current response and caused the membrane current to be larger during hyperpolarization than during depolarization. These results support the hypothesis that high-frequency biphasic stimulation blocks axonal conduction by driving the potassium channel open constantly. Understanding neural membrane response to high-frequency membrane potential alternation is important to reveal the possible mechanisms underlying axonal conduction block induced by high-frequency biphasic stimulation.

KV4 channels in isolectin B4 muscle dorsal root ganglion neurons of rats with experimental peripheral artery disease: effects of bradykinin B1 and B2 receptors.

Muscle afferent nerve-activated reflex sympathetic nervous and blood pressure responses are exaggerated during exercise in peripheral artery diseases (PAD). However, the precise signaling pathways and molecular mediators responsible for these abnormal autonomic responses in PAD are poorly understood. Our previous study suggests that A-type voltage-gated K+ (KV4) channels regulate the excitability in muscle dorsal root ganglion (DRG) neurons of PAD rats; however, it is still lacking regarding the effects of PAD on characteristics of KV4 currents and engagement of bradykinin (BK) subtype receptors. Thus, we examined KV4 currents in two distinct muscle DRG neurons, namely isolectin B4-positive and B4-negative (IB4+ and IB4-) DRG neurons. IB4+ neurons express receptors for glial cell line-derived neurotrophic factor (GDNF), whereas IB4- DRG neurons are depending on nerve growth factors for survival. Our data showed that current density in muscle DRG neurons of PAD rats was decreased and this particularly appeared in IB4+ DRG neurons as compared with IB4- DRG neurons. We also showed that stimulation of BK B1 and B2 receptors led to a greater inhibitory effect on KV4 currents in IB4+ muscle DRG neurons and siRNA knockdown of KV4 subunit KV4.3 decreased the activity of KV4 currents in IB4+ DRG neurons. In conclusion, our data suggest that limb ischemia and/or ischemia-induced BK inhibit activity of KV4 channels in a subpopulation of the thin fiber muscle afferent neurons depending on GDNF, which is likely a part of signaling pathways involved in the exaggerated blood pressure response during activation of muscle afferent nerves in PAD.

Characteristics of Zusanli Dorsal Root Ganglion Neurons in Rats and Their Receptor Mechanisms in Response to Adenosine

Neural systems play important roles in the functions of acupuncture. But the unclear structure and mechanism of acupoints hinder acupuncture standardization and cause the acupuncture effects to be varying or even paradoxical. It has been broadly assumed that the efficacy of acupuncture depends on the biological signals triggered at acupoints and passed up along neural systems. However, as the first station to transmit such signals, the characters of the dorsal root ganglia (DRG) neurons innervating acupoints are still not well elucidated. We adopted Zusanli (ST36) as a representative acupoint and found most DRG neurons innervating ST36 acupoint are middle-size neurons with a single spike firing pattern. This suggests that proprioceptive neurons take on greater possibility than small size nociceptive neurons do to mediate the acupuncture signals. Moreover, we found that adenosine injected into ST36 acupoints could dose- and acupoint-dependently mimic the analgesic effect of acupuncture. However, adenosine could not elicit action potentials in the acutely isolated ST36 DRG neurons, but it inhibited ID currents and increased the areas of overshoots. Further, we found that 4 types of adenosine receptors were all expressed by ST36 DRG neurons, and A1, A2b, and A3 receptors were the principal reactors to adenosine. PerspectiveThis study provides the major characteristics of ST36 DRG neurons, which will help to analyze the neural pathway of acupuncture signals. At the same time, these findings could provide a new possible therapy for pain relief, such as injecting adenosine or corresponding agonists into acupoints.

Sevoflurane modulation of tetrodotoxin-resistant Na+ channels in small-sized dorsal root ganglion neurons of rats.

Volatile anesthetics are widely used for general anesthesia during surgical operations. Voltage-gated Na+ channels expressed in central neurons are major targets for volatile anesthetics; but it is unclear whether these drugs modulate native tetrodotoxin-resistant (TTX-R) Na+ channels, which are involved in the development and maintenance of inflammatory pain. In this study, we examined the effects of sevoflurane on TTX-R Na+ currents (INa) in acutely isolated rat dorsal root ganglion neurons, using a whole-cell patch-clamp technique. Sevoflurane slightly potentiated the peak amplitude of transient TTX-R INa but more potently inhibited slow voltage-ramp-induced persistent INa in a concentration-dependent manner. Sevoflurane (0.86 ± 0.02 mM) (1) slightly shifted the steady-state fast inactivation relationship to hyperpolarizing ranges without affecting the voltage-activation relationship, (2) reduced the extent of use-dependent inhibition of Na+ channels, (3) accelerated the onset of inactivation and (4) delayed the recovery from inactivation of TTX-R Na+ channels. Thus, sevoflurane has diverse effects on TTX-R Na+ channels expressed in nociceptive neurons. The present results suggest that the inhibition of persistent INa and the modulation of the voltage dependence and inactivation might be, at least in part, responsible for the analgesic effects elicited by sevoflurane.

Effects of bradykinin on voltage-gated KV 4 channels in muscle dorsal root ganglion neurons of rats with experimental peripheral artery disease.

During exercise, bradykinin (BK), a muscle metabolite in ischaemic muscles, exaggerates autonomic responses to activation of muscle afferent nerves in peripheral artery disease (PAD). We examined whether BK inhibits activity of KV 4 channels in muscle afferent neurons of PAD rats induced by femoral artery occlusion. We demonstrated that: 1) femoral occlusion attenuates KV 4 currents in dorsal root ganglion (DRG) neurons innervating the hindlimb muscles and decreases the threshold of action potential firing; 2) BK has a greater inhibitory effect on KV 4 currents in muscle DRG neurons of PAD rats; and 3) expression of KV 4.3 is downregulated in DRGs of PAD rats and inhibition of KV 4.3 significantly decreases activity of KV 4 currents in muscle DRG neurons. Femoral artery occlusion-induced limb ischaemia and/or ischaemia-induced metabolites (i.e. BK) inhibit activity of KV 4 channels in muscle afferent neurons and this is likely involved in the exaggerated exercise pressor reflex in PAD. Muscle afferent nerve-activated reflex sympathetic nervous and blood pressure responses are exaggerated during exercise in patients with peripheral artery diseases (PAD) and in PAD rats induced by femoral artery occlusion. However, the precise signalling pathways and molecular mediators responsible for these abnormal autonomic responses in PAD are poorly understood. A-type voltage-gated K+ (KV ) channels are quintessential regulators of cellular excitability in the various tissues. Among KV channels, KV 4 (i.e. KV 4.1 and KV 4.3) in primary sensory neurons mainly participate in physiological functions in regulation of mechanical and chemical sensation. However, little is known about the role of KV 4 in regulating neuronal activity in muscle afferent neurons of PAD. In addition, bradykinin (BK) is considered as a muscle metabolite contributing to the exaggerated exercise pressor reflex in PAD rats with femoral artery occlusion. Our data demonstrated that: 1) KV 4 currents are attenuated in dorsal root ganglion (DRG) neurons innervating the hindlimb muscles of PAD rats, along with a decreasing threshold of action potential firing; 2) KV 4 currents are inhibited by application of BK onto muscle DRG neurons of PAD rats to a greater degree; and 3) expression of KV 4.3 is downregulated in the DRGs of PAD rats and KV 4.3 channel is a major contributor to the activity of KV 4 currents in muscle DRG neurons. In conclusion, data suggest that femoral artery occlusion-induced limb ischaemia and/or ischaemia-induced metabolites (i.e. BK) inhibit the activity of KV 4 channels in muscle afferent neurons likely leading to the exaggerated exercise pressor reflex observed in PAD.

L5 Spinal Nerve Axotomy Induces Distinct Electrophysiological Changes in Axotomized L5- and Adjacent L4-Dorsal Root Ganglion Neurons in Rats In Vivo.

Peripheral neuropathic pain (PNP) is a major health problem for which effective drug treatment is lacking. Its underlying neuronal mechanisms are still illusive, but pre-clinical studies using animal models of PNP including the L5-spinal nerve axotomy (L5-SNA) model, suggest that it is partly caused by excitability changes in dorsal root ganglion (DRG) neurons. L5-SNA results in two DRG neuronal groups: (1) axotomized/damaged neurons in L5- plus some in L4-DRGs, and (2) ipsilateral L4-neurons with intact/uninjured fibers intermingling with degenerating L5-fibers. The axotomized neurons are deprived of peripherally derived trophic factors and degenerate causing neuroinflammation, whereas the uninjured L4-neuorns are subject to increased trophic factors and neuroinflammation associated with Wallerian degeneration of axotomized L5-nerve fibers. Whether these two groups of DRG neurons exhibit similar or distinct electrophysiological changes after L5-SNA remains unresolved. Conflicting evidence for this may result from some studies assuming that all L4-fibers are undamaged. Here, we recorded somatic action potentials (APs) intracellularly from C- and A-fiber L4/L5 DRG neurons in vivo, to examine our hypothesis that L5-SNA would induce distinct electrophysiological changes in the two populations of DRG neurons. Consistent with this hypothesis, we found (7 days post-SNA), in SNA rats with established pain hypersensitivity, slower AP kinetics in axotomized L5-neurons and faster AP kinetics in L4-nociceptive neurons including decreased rise time in Aδ-and Aβ-fiber nociceptors, and after-hyperpolarization duration in Aβ-fiber nociceptors. We also found several changes in axotomized L5-neurons but not in L4-nociceptive neurons, and some changes in L4-nociceptive but not L5-neurons. The faster AP kinetics (decreased refractory period) in L4-nociceptive neurons that are consistent with their reported hyperexcitability may lead to repetitive firing and thus provide enhanced afferent input necessary for initiating and/or maintaining PNP development. The changes in axotomized L5-neurons may contribute to the central mechanisms of PNP via enhanced neurotransmitter release in the central nervous system (CNS).

Effect of oxycodone on function of GABAA receptors in dorsal root ganglion neurons of rats with neuropathic pain

Objective To evaluate the effect of oxycodone on function of GABAA receptors in dorsal root ganglion (DRG) neurons of rats with neuropathic pain (NP). Methods Thirty-six adult male Sprague-Dawley rats, weighing 180-220 g, aged 10 weeks, were allocated into 3 groups (n=12 each) using a random number table method: sham operation group (group S), group NP and oxycodone group (group O). The sciatic nerve was only isolated but not ligated in group S. NP was induced by chronic constriction injury.The sciatic nerve was exposed and 4 loose ligatures were placed on the sciatic nerve at 1 mm intervals with 4-0 chromic catgut.Oxycodone 15 μg/kg was intraperitoneally injected once a day for 14 consecutive days from ligating the sciatic nerve to satisfaction in group O. The thermal paw withdrawal latency (TWL) was measured at 1 day before establishing the model (T0) and 3, 5, 7, 10 and 14 days after establishing the model (T1-5). The rats were sacrificed after measurement of pain threshold at T5, and DRG neurons were acutely isolated for recording the amplitude of GABAA receptors-activated currents using whole-cell patch-clamp technique. Results Compared with group S, the TWL was significantly shortened at T1-5, and the amplitude of GABAA receptors-activated currents in DRG neurons was decreased in NP and O groups (P<0.05). Compared with group NP, the TWL was significantly prolonged at T1-5, and the amplitude of GABAA receptors-activated currents in DRG neurons was increased in group O (P<0.05). Conclusion Oxycodone can enhance the function of GABAA receptors-activated currents in DRG neurons and thus enhance GABAA receptors-mediated presynaptic inhibition, which may be related to the mechanism of oxycodone-induced reduction of NP in rats. Key words: Oxycodone; Neuralgia; Ganglia, spinal; Receptors, GABA-A

Constitutive activity of transient receptor potential vanilloid type 1 triggers spontaneous firing in nerve growth factor-treated dorsal root ganglion neurons of rats

Dorsal root ganglion (DRG) neurons cultured in the presence of nerve growth factor (NGF, 100 ng/ml) often show a spontaneous action potential. Underlying mechanisms of this spontaneous firing were examined using the patch clamp technique. The spontaneous firing in the on-cell configuration was abolished by a decrease in the Na+ concentration and by the TRPV1 antagonists capsazepine (10 μM) and BCTC (1 μM). These responses were accompanied by hyperpolarization of the resting potential. The holding current observed in neurons voltage clamped at –60 mV in the whole-cell configuration was significantly larger in the neurons that fired spontaneously, indicating that these neurons had an additional cation conductance that caused depolarization and triggered action potentials. The holding current in the firing neurons was decreased by extracellular Na+ reduction, capsazepine and BCTC. The amplitudes of the capsazepine- or BCTC-sensitive component of the holding current in the spontaneously firing neurons were ten times as large as those recorded in the other neurons showing no spontaneous firing. However, the amplitudes of the current responses to capsaicin (1 μM) were not different regardless of the presence of spontaneous firing or treatment with NGF. These results indicate that chronic NGF treatment of cultured DRG neurons in rats induces a constitutively active cation conductance through TRPV1, which depolarizes the neurons and triggers spontaneous action potentials in the absence of any stimuli. Since NGF in the DRG is reported to increase after nerve injury, this NGF-mediated regulation of TRPV1 may be a cause of the pathogenesis of neuropathic pain.

Chronic Increases in Daily Neuromuscular Activity Promote Changes in Gene Expression in Small and Large Dorsal Root Ganglion Neurons in Rat

The purpose of this study was to determine the response, in rat, to chronic physical activity in small and large DRG neurons. Rats were cage-confined or underwent 16–18 weeks of daily increased activity, via 2 h of treadmill running per day or free access to voluntary exercise wheels, following which small (≤30 µm) and large (≥40 µm) diameter DRG neurons were harvested by laser capture microdissection from flash-frozen lumbar DRGs. Relative mRNA levels were determined using real-time polymerase chain reaction. Following chronic treadmill and voluntary wheel exercise, gene expression responses in neurons mostly differed between exercise types. Changes in both small and large DRG neurons included increases in opioid receptor mu subunit (MOR), NGF and GAP43, and decreases in 5HT1A, TrkA, TrkB, and delta-type opioid receptor (DOR) mRNAs. In small DRG neurons, treadmill exercise increased the expression of mRNA for 5HT1D and decreased expression for 5HT1F receptors. In large DRG neurons, voluntary wheel exercise decreased the expression for 5HT1D receptors, whereas both treadmill and voluntary wheel exercise decreased the expression of mRNA for TrkC receptors. DRG neurons show slightly more changes in gene expression after voluntary exercise compared to the treadmill exercise group. Small and large lumbar sensory neurons are responsive to chronically increased neuromuscular activity by changing the expression of genes, the products of which could potentially change the sensory processing of nociceptors and proprioceptors, which could in turn alter functions such as pain transmission and locomotor coordination.

Characterization of temperature-sensitive leak K+ currents and expression of TRAAK, TREK-1, and TREK2 channels in dorsal root ganglion neurons of rats

Leak K+ currents are mediated by two-pore domain K+ (K2P) channels and are involved in controlling neuronal excitability. Of 15 members of K2P channels cloned so far, TRAAK, TREK-1, and TREK-2 are temperature sensitive. In the present study, we show that strong immunoreactivity of TRAAK, TREK-1 and TREK-2 channels was present mainly in small-sized dorsal root ganglion (DRG) neurons of rats. The percentages of neurons with strong immunoreactivity of TRAAK, TREK-1 and TREK-2 channels were 27, 23, and 20%, respectively. Patch-clamp recordings were performed to examine isolated leak K+ currents on acutely dissociated small-sized rat DRG neurons at room temperature of 22 °C, cool temperature of 14 °C and warm temperature of 30 °C. In majority of small-sized DRG neurons recorded (76%), large leak K+ currents were observed at 22 °C and were inhibited at 14 °C and potentiated at 30 °C, suggesting the presence of temperature-sensitive K2P channels in these neurons. In a small population (18%) of small-sized DRG neurons, cool temperature of 14 °C evoked a conductance which was consistent with TRPM8 channel activation in cold-sensing DRG neurons. In these DRG neurons, leak K+ currents were very small at 22 °C and were not potentiated at 30 °C, suggesting that few temperature-sensitive K2P channels was present in cold-sensing DRG neurons. For DRG neurons with temperature-sensitive leak K+ currents, riluzole, norfluoxetine and prostaglandin F2α (PGE2α) inhibited the leak K+ currents at both 30 °C and 22 °C degree, and did not have inhibitory effects at 14 °C. Collectively, the observed temperature-sensitive leak K+ currents are consistent with the expression of temperature-sensitive K2P channels in small-sized DRG neurons.

Heteromerization of \u03bc-opioid receptor and cholecystokinin B receptor through the third transmembrane domain of the \u03bc-opioid receptor contributes to the anti-opioid effects of cholecystokinin octapeptide

Activation of the cholecystokinin type B receptor (CCKBR) by cholecystokinin octapeptide (CCK-8) inhibits opioid analgesia. Chronic opiate treatment leads to an increase in the CCK-8 concentration and thus enhances the antagonism of CCK-8 against opioid analgesia; the underlying molecular mechanisms remain of great interest. In the present study, we validated the colocalization of the μ-opioid receptor (MOR) and CCKBR in pain signal transmission-related spinal cord dorsal horn and dorsal root ganglion neurons of rats. Co-immunoprecipitation (Co-IP) and fluorescence lifetime-imaging-microscopy-based fluorescence resonance energy transfer (FLIM-FRET) assays showed that MOR heteromerized with CCKBR directly in transfected HEK293 cells. Combined with MOR mutant construction, the third transmembrane domain of MOR (TM3MOR) was demonstrated to participate in heteromerization with CCKBR. Receptor ligand binding, ERK phosphorylation and cAMP assays showed that MOR heteromerization with CCKBR weakened the activity of MOR. A cell-penetrating interfering peptide consisting of TM3MOR and TAT (a transactivator of HIV-1) sequences from the N terminal to the C terminal disrupted the MOR–CCKBR interaction and restored the activity of MOR in transfected HEK293 cells. Furthermore, intrathecal application of the TM3MOR-TAT peptide alleviated CCK-8-injection-induced antagonism to morphine analgesia in rats. These results suggest a new molecular mechanism for CCK-8 antagonism to opioid analgesia in terms of G-protein-coupled receptor (GPCR) interaction through direct heteromerization. Our study may provide a potential strategy for pain management with opioid analgesics.

Adrenergic receptors inhibit TRPV1 activity in the dorsal root ganglion neurons of rats.

Transient receptor potential vanilloid type 1 (TRPV1) is a polymodal receptor channel that responds to multiple types of stimuli, such as heat, acid, mechanical pressure and some vanilloids. Capsaicin is the most commonly used vanilloid to stimulate TRPV1. TRPV1 channels are expressed in dorsal root ganglion neurons that extend to Aδ- and C-fibers and have a role in the transduction of noxious inputs to the skin into the electrical signals of the sensory nerve. Although noradrenergic nervous systems, including the descending antinociceptive system and the sympathetic nervous system, are known to modulate pain sensation, the functional association between TRPV1 and noradrenaline in primary sensory neurons has rarely been examined. In the present study, we examined the effects of noradrenaline on capsaicin-evoked currents in cultured dorsal root ganglion neurons of the rat by the whole-cell voltage clamp method. Noradrenaline at concentrations higher than 0.1 pM significantly reduced the amplitudes of the inward capsaicin currents recorded at –60 mV holding potential. This inhibitory action was reversed by either yohimbine (an α2 antagonist, 10 nM) or propranolol (a β antagonist, 10 nM). The α2 agonists, clonidine (1 pM) and dexmedetomidine (1 pM) inhibited capsaicin currents, and yohimbine (1 nM) reversed the effects of clonidine. The inhibitory action of noradrenaline was not seen in the neurons pretreated with pertussis toxin (100 μg/ml for 24 h) and the neurons dialyzed intracellularly with guanosine 5’- [β-thio] diphosphate (GDPβS, 200 μM), the catalytic subunit of protein kinase A (250 U/ml) or okadaic acid (1 μM). These results suggest that noradrenaline directly acts on dorsal root ganglion neurons to inhibit the activity of TRPV1 depending on the activation of α2-adrenoceptors followed by the inhibition of the adenylate cyclase/cAMP/protein kinase A pathway.

KCNQ2/3/5 channels in dorsal root ganglion neurons can be therapeutic targets of neuropathic pain in diabetic rats.

BackgroundDiabetic neuropathic pain is poorly controlled by analgesics, and the precise molecular mechanisms underlying hyperalgesia remain unclear. The KCNQ2/3/5 channels expressed in dorsal root ganglion neurons are important in pain transmission. The expression and activity of KCNQ2/3/5 channels in dorsal root ganglion neurons in rats with diabetic neuropathic pain were investigated in this study.MethodsThe mRNA levels of KCNQ2/3/5 channels were analyzed by real-time polymerase chain reaction. The protein levels of KCNQ2/3/5 channels were evaluated by Western blot assay. KCNQ2/3/5 channel expression in situ in dorsal root ganglion neurons was detected by double fluorescent labeling technique. M current (IM) density and neuronal excitability were determined by whole-cell voltage and current clamp recordings. Mechanical allodynia and thermal hyperalgesia were assessed by von Frey filaments and plantar analgesia tester, respectively.ResultsThe mRNA and protein levels of KCNQ2/3/5 channels significantly decreased, followed by the reduction of IM density and elevation of neuronal excitability of dorsal root ganglion neurons from diabetic rats. Activation of KCNQ channels with retigabine reduced the hyperexcitability and inhibition of KCNQ channels with XE991 enhanced the hyperexcitability. Administration of retigabine alleviated both mechanical allodynia and thermal hyperalgesia, while XE991 augmented both mechanical allodynia and thermal hyperalgesia in diabetic neuropathic pain in rats.ConclusionThe findings elucidate the mechanisms by which downregulation of the expression and reduction of the activity of KCNQ2/3/5 channels in diabetic rat dorsal root ganglion neurons contribute to neuronal hyperexcitability, which results in hyperalgesia. These data provide intriguing evidence that activation of KCNQ2/3/5 channels might be the potential new targets for alleviating diabetic neuropathic pain symptoms.

Inhibition of cystathionine β-synthetase suppresses sodium channel activities of dorsal root ganglion neurons of rats with lumbar disc herniation.

The pathogenesis of pain in lumbar disc herniation (LDH) remains poorly understood. We have recently demonstrated that voltage-gated sodium channels (VGSCs) in dorsal root ganglion (DRG) neurons were sensitized in a rat model of LDH. However, the detailed molecular mechanism for sensitization of VGSCs remains largely unknown. This study was designed to examine roles of the endogenous hydrogen sulfide synthesizing enzyme cystathionine β-synthetase (CBS) in sensitization of VGSCs in a previously validated rat model of LDH. Here we showed that inhibition of CBS activity by O-(Carboxymethyl) hydroxylamine hemihydrochloride (AOAA) significantly attenuated pain hypersensitivity in LDH rats. Administration of AOAA also reduced neuronal hyperexcitability, suppressed the sodium current density, and right-shifted the V1/2 of the inactivation curve, of hindpaw innervating DRG neurons, which is retrogradely labeled by DiI. In vitro incubation of AOAA did not alter the excitability of acutely isolated DRG neurons. Furthermore, CBS was colocalized with NaV1.7 and NaV1.8 in hindpaw-innervating DRG neurons. Treatment of AOAA markedly suppressed expression of NaV1.7 and NaV1.8 in DRGs of LDH rats. These data suggest that targeting the CBS-H2S signaling at the DRG level might represent a novel therapeutic strategy for chronic pain relief in patients with LDH.

Mechanical sensitivity and electrophysiological properties of acutely dissociated dorsal root ganglion neurons of rats

Primary afferent fibers use mechanically activated (MA) currents to transduce innocuous and noxious mechanical stimuli. However, it is largely unknown about the differences in MA currents between the afferents for sensing innocuous and noxious stimuli. In the present study, we used dorsal root ganglion (DRG) neurons acutely dissociated from rats and studied their MA currents and also their intrinsic membrane properties. Recorded from small-sized DRG neurons, we found that most of these neurons were mechanically sensitive (MS) showing MA currents. The MS neurons could be classified into nociceptive-like mechanically sensitive (Noci-MS) and non-nociceptive-like mechanically sensitive (nonNoci-MS) neurons based on their action potential shapes. Noci-MS neurons responded to mechanical stimulation with three types of MA currents, rapidly adapting (RA), intermediately adapting (IA), and slowly adapting (SA) currents. In contrast, almost all nonNoci-MS neurons showed RA current type in response to mechanical stimulation. Mechanical thresholds had a broad range for both nonNoci-MS and Noci-MS neurons, and the thresholds were not significantly different between them. However, MA current densities were significantly smaller in Noci-MS than in nonNoci-MS neurons. Noci-MS and nonNoci-MS neurons also showed significant differences in their electrophysiological properties including action potential (AP) thresholds and AP firing patterns. These differences may contribute to the differential sensory encoding for innocuous and noxious mechanical stimuli.

Anti-allodynic effect of intrathecal processed Aconitum jaluense is associated with the inhibition of microglial activation and P2X7 receptor expression in spinal cord

BackgroundFor their analgesic and anti-arthritic effects, Aconitum species have been used in folk medicine in some East Asian countries. Although their analgesic effect is attributed to its action on voltage-dependent sodium channels, they also suppress purinergic receptor expression in dorsal root ganglion neurons in rats with neuropathic pain. In vitro study also demonstrated that the Aconitum suppresses ATP-induced P2X7 receptor (P2X7R)-mediated inflammatory responses in microglial cell lines. Herein, we examined the effect of intrathecal administration of thermally processed Aconitum jaluense (PA) on pain behavior, P2X7R expression and microglial activation in a rat spinal nerve ligation (SNL) model.MethodsMechanical allodynia induced by L5 SNL in Sprague-Dawley rats was measured using the von Frey test to evaluate the effect of intrathecal injection of PA. Changes in the expression of P2X7R in the spinal cord were examined using RT-PCR and Western blot analysis. In addition, the effect of intrathecal PA on microglial activation was evaluated by immunofluorescence.ResultsIntrathecal PA attenuated mechanical allodynia in a dose-dependent manner showing both acute and chronic effects with 65 % of the maximal possible effect. The expression and production of spinal P2X7R was increased five days after SNL, but daily intrathecal PA injection significantly inhibited the increase to the level of naïve animals. Immunofluorescence of the spinal cord revealed a significant increase in P2X7R expression and activation of microglia in the dorsal horn, which was inhibited by intrathecal PA treatment. P2X7R co-localized with microglia marker, but not neurons.ConclusionsIntrathecal PA exerts anti-allodynic effects in neuropathic pain, possibly by suppressing P2X7R production and expression as well as reducing microglial activation in the spinal cord.

Anti-oxidative and anti-inflammatory effects of cinnamaldehyde on protecting high glucose-induced damage in cultured dorsal root ganglion neurons of rats.

To examine the mechanism underlying the beneficial role of cinnamaldehyde on oxidative damage and apoptosis in high glucose (HG)-induced dorsal root ganglion (DRG) neurons in vitro. HG-treated DRG neurons were developed as an in vitro model of diabetic neuropathy. The neurons were randomly divided into five groups: the control group, the HG group and the HG groups treated with 25, 50 and 100 nmol/L cinnamaldehyde, respectively. Cell viability was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and apoptosis rate was evaluated by the in situ TdT-mediated dUTP nick end labeling (TUNEL) assay. The intracellular level of reactive oxygen species (ROS) was measured with flow cytometry. Expression of nuclear factor-kappa B (NF-κB), inhibitor of κB (IκB), phosphorylated IκB (p-IκB), tumor necrosis factor (TNF)-α, interleukin-6 (IL-6) and caspase-3 were determined by western blotting and real-time quantitative reverse transcription polymerase chain reaction (RT-PCR). Expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and hemeoxygenase-1 (HO-1) were also measured by western blotting. Cinnamaldehyde reduced HG-induced loss of viability, apoptosis and intracellular generation of ROS in the DRG neurons via inhibiting NF-κB activity. The western blot assay results showed that the HG-induced elevated expressions of NF-κB, IκB and p-IκB were remarkably reduced by cinnamaldehyde treatment in a dose-dependent manner (P <0.01). The HG-induced over-expression of NF-κB p65 mRNA was remarkably attenuated after cinnamaldehyde treatment in a dose-dependent manner (P <0.01). However, the expressions of Nrf2 and HO-1 were not upregulated. Treatment with cinnamaldehyde not only attenuated caspase-3 activation and the caspase cleavage cascade in DRG neurons, but also lowered the elevated IL-6, TNF-α, cyclo-oxygenase and inducible nitric oxide synthase levels, indicating a reduction in inflammatory damage. Cinnamaldehyde protected DRG neurons from the deleterious effects of HG through inactivation of NF-κB pathway but not through activation of Nrf2/HO-1. And thus cinnamaldehyde may have potential application as a treatment for DPN.

Modulating nitric oxide levels in dorsal root ganglion neurons of rat with low-level laser therapy

Nitric oxide (NO) and nitric oxide synthase (NOS) have an important role in pain signaling transmission in animal models. Low-level laser therapy (LLLT) is known to have an analgesic effect, but the mechanism is unclear. The aim of the study is to investigate the influence of LLLT on NO release and NOS synthesis in dorsal root ganglion (DRG) neurons, in order to find whether LLLI can ameliorate pain through modulating NO production at the cellular level. The results show that in stress conditions, the laser irradiation at 658 nm can modulate NO production in DRG neurons with soma diameter of about 20 μm in a short time after illumination, and affect NOS synthesis in a dose-dependent manner. It is demonstrated that LLLT might treat pain by altering NO release directly and indirectly in DRG neurons.

The role of the GABA-A receptor of the adjacent intact dorsal root ganglion neurons in rats with neuropathic pain.

We aim to investigate the changes of The γ-aminobutyric acid (GABA) signals in the adjacent intact dorsal root ganglion (DRG) and the contribution of these changes to the development and maintenance of neuropathic pain (NPP). After establishment of neuropathic pain model with the lumbar 5 spinal nerve ligation (L5 SNL), the GABA-evoked currents were recorded in the acutely dissociated L4 DRG neurons using whole-cell patch clamp. Moreover, Muscimol or Bicuculline were respectively topically injected to the L4 DRG at the time of nerve injury and post-operative 5 days (POD5). The pain thresholds of hind paw were recorded. We found that the incidence and amplitude of GABA currents significantly decreased in the large and medium L4 DRG neurons after SNL (P<0.05). Furthermore, in the L5 SNL rats, 20 μg Muscimol injected immediately after injury caused a long-lasting attenuation of mechanical allodynia (P<0.05), whereas the thermal hyperalgesia was not alleviated (P>0.05). 0.15 μg Bicuculline further decreased the pain thresholds (P<0.05) and reversed the pain-alleviating effects of Muscimol. However, at POD5, 20 μg Muscimol only exhibited transient alleviating effects (P<0.05). In conclusion, the decrease of inhibition signal mediated by GABA-A receptor in the adjacent intact DRG neurons may be crucial in the development of hyperalgesia in NPP.

The electrophysiology characteristics of dorsal root ganglion neurons in rats with TNBS-induced colitis

The electrophysiology characteristics of dorsal root ganglion neurons in rats with TNBS-induced colitis