Medium-sized Dorsal Root Ganglion Neurons Research Articles

Peripheral neural mechanism of visceral pain and acupoint sensitization in 2, 4, 6-trinitrobenzene sulfonic acid-induced colitis model mice.

To explore the neural mechanism of visceral pain and related somatic (acupoints) sensitization by using in vivo calcium imaging of dorsal root ganglia (DRG) neurons. Eight BALB/c mice were randomly divided into control and model groups, with 4 mice in each group. The colitis model was induced by colorectal perfusion of 2, 4, 6-trinitrobenzene sulfonic acid (TNBS) once daily for 7 days. Mice of the control group received colorectal perfusion of normal saline once daily for 7 days. The location and area of the somatic neurogenic inflammation (cutaneous exudation of Evans blue ［EB］) of the 2 groups of mice were observed after intravenous injection of EB. For pain behavioral tests, sixteen C57BL/6J mice were randomly divided into control and model groups, with 8 mice in each group, and a Von Frey filament was used to stimulate the referred somatic reactive regions in colitis mice, and the number of avoidance and paw withdraw reaction within 10 tests was recorded. For in vivo DRG calcium imaging tests, 24 Pirt-GCaMP6s transgenic mice were randomly and equally divided into control group and colitis model group. The responses of the neurons in L6 or L4 DRG to colorectal distension (CRD), lower back brushing, or mechanical stimulation at the hindpaw were observed using confocal fluorescence microscope. Compared with the control group, the area of EB exudation spot in the hindpaw and lower back regions was increased in the colitis model group (P<0.05), and the avoidance or paw withdraw numbers induced by Von Frey stimulation at the lower back and hindpaw were increased (P<0.01, P<0.05), indicating that colitis induced regional skin (acupoints) sensitization in the lower back and hindpaw regions. Compared with the control group, the percentage of L6 DRG neurons activated by 60 mm Hg CRD in the colitis model mice were apparently increased (P<0.01), the activated neurons mainly involved the medium-sized DRG neurons (P<0.01). In Pirt-GCaMP6s transgenic mice, following brushing the skin of the receptive field (lower back) of L6 DRG neurons, the fluorescence intensity of the brushing-activated DRG neurons and small, medium and large-sized neurons were significantly higher in the colitis model group than those in the control group (P<0.001, P<0.01, P<0.05). After brushing and clamping the skin of the right hindpaw (receptive field of L4 DRG neurons), the percentages of the activated L4 DRG neurons were obviously higher in the colitis model group than those in the control group (P<0.01, P<0.05), while there were no significant changes in the proportion of small, medium and large-sized neurons between the control and colitis model groups. Colitis may lead to body surface sensitization at the same and adjacent neuro-segments as well as to an increase of the number and activity of the responsive lumbar DRG neurons, among which the L6 DRG neurons at the same neuro-segment as the rectum colon showed an increase in the number of responders and intensity of calcium fluorescence signal while L4 DRG neurons at the level adjacent to the rectum colon showed an increase in the number of responders, suggesting that there may be different mechanisms of peripheral neural sensitization.

Changes in expression of Kv7.5 and Kv7.2 channels in dorsal root ganglion neurons in the streptozotocin rat model of painful diabetic neuropathy

Diabetic peripheral neuropathic pain (DPNP), the most debilitating complication of diabetes mellitus, is resistant to current therapy. The pathogenesis of DPNP is still elusive, but several mechanisms have been proposed including abnormal hyperexcitability of dorsal root ganglion (DRG) neurons. The underlying molecular mechanisms of such aberrant hyperexcitability are incompletely understood. Using the streptozotocin (STZ) rat model of DPNP, we have recently provided evidence implicating neuronal Kv7 channels that normally exert a powerful stabilizing influence on neuronal excitability, in the abnormal hyperexcitability of DRG neurons and in pain hypersensitivity associated with DPNP. In the present immunohistochemical study, we sought to determine whether Kv7.2 and/or Kv7.5 channel expression is altered in DRG neurons in STZ rats. We found 35 days post-STZ: (1) a significant decrease in Kv7.5-immunoreactivity in small (<30 μm) DRG neurons (both IB4 positive and IB4 negative) and medium-sized (30−40 μm) neurons, and (2) a significant increase in Kv7.2-immunoreactivity in small (<30 μm) neurons, and a non-significant increase in medium/large neurons. The decrease in Kv7.5 channel expression in small and medium-sized DRG neurons in STZ rats is likely to contribute to the mechanisms of hyperexcitability of these neurons and thereby to the resulting pain hypersensitivity associated with DPNP. The upregulation of Kv7.2 subunit in small DRG neurons may be an activity dependent compensatory mechanism to limit STZ-induced hyperexcitability of DRG neurons and the associated pain hypersensitivity. The findings support the notion that Kv7 channels may represent a novel target for DPNP treatment.

Functional Expression of Piezo1 in Dorsal Root Ganglion (DRG) Neurons.

Piezo channels are mechanosensitive ion channels. Piezo1 is primarily expressed in nonsensory tissues, whereas Piezo2 is predominantly found in sensory tissues, including dorsal root ganglion (DRG) neurons. However, a recent study demonstrated the intracellular calcium response to Yoda1, a selective Piezo1 agonist, in trigeminal ganglion (TG) neurons. Herein, we investigate the expression of Piezo1 mRNA and protein in mouse and human DRG neurons and the activation of Piezo1 via calcium influx by Yoda1. Yoda1 induces inward currents mainly in small- (<25 μm) and medium-sized (25–35 μm) mouse DRG neurons. The Yoda1-induced Ca2+ response is inhibited by cationic channel blocker, ruthenium red and cationic mechanosensitive channel blocker, GsMTx4. To confirm the specific inhibition of Piezo1, we performed an adeno-associated virus serotype 2/5 (AAV2/5)-mediated delivery of short hairpin RNA (shRNA) into mouse DRG neurons. AAV2/5 transfection downregulates piezo1 mRNA expression and reduces Ca2+ response by Yoda1. Piezo1 also shows physiological functions with transient receptor potential vanilloid 1 (TRPV1) in the same DRG neurons and is regulated by the activation of TRPV1 in mouse DRG sensory neurons. Overall, we found that Piezo1 has physiological functions in DRG neurons and that TRPV1 activation inhibits an inward current induced by Yoda1.

Interleukin-10 resolves pain hypersensitivity induced by cisplatin by reversing sensory neuron hyperexcitability.

Understanding the mechanisms that drive transition from acute to chronic pain is essential to identify new therapeutic targets. The importance of endogenous resolution pathways acting as a "brake" to prevent development of chronic pain has been largely ignored. We examined the role of interleukin-10 (IL-10) in resolution of neuropathic pain induced by cisplatin. In search of an underlying mechanism, we studied the effect of cisplatin and IL-10 on spontaneous activity (SA) in dorsal root ganglia neurons. Cisplatin (2 mg/kg daily for 3 days) induced mechanical hypersensitivity that resolved within 3 weeks. In both sexes, resolution of mechanical hypersensitivity was delayed in Il10 mice, in WT mice treated intrathecally with neutralizing anti-IL-10 antibody, and in mice with cell-targeted deletion of IL-10R1 on advillin-positive sensory neurons. Electrophysiologically, small- to medium-sized dorsal root ganglia neurons from cisplatin-treated mice displayed an increase in the incidence of SA. Cisplatin treatment also depolarized the resting membrane potential, and decreased action potential voltage threshold and rheobase, while increasing ongoing activity at -45 mV and the amplitude of depolarizing spontaneous fluctuations. In vitro addition of IL-10 (10 ng/mL) reversed the effect of cisplatin on SA and on the depolarizing spontaneous fluctuation amplitudes, but unexpectedly had little effect on the other electrophysiological parameters affected by cisplatin. Collectively, our findings challenge the prevailing concept that IL-10 resolves pain solely by dampening neuroinflammation and demonstrate in a model of chemotherapy-induced neuropathic pain that endogenous IL-10 prevents transition to chronic pain by binding to IL-10 receptors on sensory neurons to regulate their activity.

Involvement of Acid-Sensing Ion Channel 1b in the Development of Acid-Induced Chronic Muscle Pain.

Acid-sensing ion channels (ASICs) are important acid sensors involved in neural modulation in the central nervous system and pain-associated tissue acidosis in the peripheral system. Among ASIC subtypes, ASIC1b is the most selectively expressed in peripheral sensory neurons. However, the role of ASIC1b is still elusive in terms of its functions and expression profile. In this study, we probed the role of ASIC1b in acid-induced muscle pain in Asic1b-knockout (Asic1b–/–) and Asic1b-Cre transgenic (Asic1bCre) mice. We tested the effect of ASIC1b knockout in a mouse model of fibromyalgia induced by dual intramuscular acid injections. In this model, a unilateral acid injection to the gastrocnemius muscle induced transient bilateral hyperalgesia in wild-type (Asic1b+/+) but not Asic1b–/– mice; a second acid injection, spaced 1 or 5 days apart, to the same muscle induced chronic hyperalgesia lasting for 4 weeks in Asic1b+/+ mice, but the duration of hyperalgesia was significantly shortened in Asic1b–/– mice. Mambalgin-1, an ASIC1b-containing channel inhibitor that was mixed with acid saline at the first injection, dose-dependently blocked the acid-induced transient and chronic hyperalgesia in Asic1b+/+ mice. In contrast, psalmotoxin 1 (PcTx1), an ASIC1a-selective antagonist, had no effect on acid-induced transient or chronic hyperalgesia. We used whole-cell patch clamp recording to study the properties of acid-induced currents in ASIC1b-expressing dorsal root ganglia (DRG) neurons from Asic1bCre-TdTomato reporter mice. Medium- to large-sized ASIC1b-expressing DRG neurons mainly exhibited an amiloride-sensitive ASIC-like biphasic current (IASIC) in response to acid stimulation, whereas small- to medium-sized ASIC1b-expressing DRG neurons predominantly exhibited an amiloride-insensitive sustained current. Specifically, mambalgin-1 selectively inhibited the IASIC in most ASIC1b-expressing DRG neurons. However, PcTx1 or APETx2 (an ASIC3-selective antagonist) had only a mild inhibitory effect on IASIC in about half of the ASIC1b-expressing DRG neurons. In situ hybridization revealed that ASIC1b-positive DRG neurons co-expressed highly with ASIC1a and ASIC2a mRNA and partially with ASIC3 and ASIC2b. Thus, ASIC1b might form a wide variety of heteromeric channels. ASIC1b-containing heteromeric channels might be promising targets for the therapeutic treatment of acid-induced chronic muscle pain.

Increased Brain-Derived Neurotrophic Factor in Lumbar Dorsal Root Ganglia Contributes to the Enhanced Exercise Pressor Reflex in Heart Failure.

An exaggerated exercise pressor reflex (EPR) is associated with excessive sympatho-excitation and exercise intolerance in the chronic heart failure (CHF) state. We hypothesized that brain-derived neurotrophic factor (BDNF) causes the exaggerated EPR via sensitizing muscle mechanosensitive afferents in CHF. Increased BDNF expression was observed in lumbar dorsal root ganglia (DRGs) from CHF rats compared to sham rats. Immunofluorescence data showed a greater increase in the number of BDNF-positive neurons in medium and large-sized DRG subpopulations from CHF rats. Patch clamp data showed that incubation with BDNF for 4–6 h, significantly decreased the current threshold-inducing action potential (AP), threshold potential and the number of APs during current injection in Dil-labeled isolectin B4 (IB4)-negative medium-sized DRG neurons (mainly mechano-sensitive) from sham rats. Compared to sham rats, CHF rats exhibited an increased number of APs during current injection in the same DRG subpopulation, which was significantly attenuated by 4-h incubation with anti-BDNF. Finally, chronic epidural delivery of anti-BDNF attenuated the exaggerated pressor response to either static contraction or passive stretch in CHF rats whereas this intervention had no effect on the pressor response to hindlimb arterial injection of capsaicin. These data suggest that increased BDNF in lumbar DRGs contributes to the exaggerated EPR in CHF.

Expression pattern of type 3 adenylyl cyclase in rodent dorsal root ganglion and its primary afferent terminals

cAMP (Cyclic Adenosine monophosphate), one of the most highly studied second messengers, is regulated by a family of adenylyl cyclase (AC) enzymes. Type 3 adenylyl cyclase (abbreviated as AC3), a subtype of adenylyl cyclase, is reported to be expressed in cilia in the olfactory and central nervous system and plays an important role in many physiological functions such as olfaction, development. However, expression of AC3 in the dorsal root ganglion (DRG) is not reported. In the present study, using immunohistochemical method, we discovered that AC3 immunoreactivity (IR) is predominantly expressed in the cytoplasm of small to medium sized DRG neurons. Double labelling revealed that the majority of AC3 IR are colocalized with CGRP (a peptidergic nociceptor marker), rarely with NF200 (a myelinated neuronal marker) or IB4 (a nonpeptidergic nociceptor marker). Furthermore, dense AC3 IR exists in the superficial dorsal horn, especially in laminaⅠand dorsal part of lamina II, where CGRP-positive DRG neurons terminate. The expression pattern of AC3 is similar between C57/BL6 J mouse and Sprague Dawley rat. For instance, AC3 is primarily expressed in the cell bodies of small to medium sized DRG neurons and the majority of AC3 IR is also in CGRP-containing neurons in rat. Taken together, our data suggest that AC3 is primarily expressed in the small to medium sized cell bodies and central terminals of CGRP-positive DRG neurons, implying AC3 enzyme might potentially function in nociception.

Increased Deacetylation of Proteins in Sensory Neurons by Sirtuin 1 Protein Overexpression Reverses T2D Peripheral Neuropathy

Sirtuin1 (SIRT1) protein uses NAD+ as a substrate to deacetylate transcription factors, cofactors, and histones to enhance mitochondrial function. We tested the hypothesis whether increased expression of SIRT1 protein in dorsal root ganglion (DRG) neurons would rescue mice from peripheral neuropathy induced by a high fat diet (HFD). Neuron-specific, doxycycline (DOX)-inducible, SIRT1 protein over expressing C57BL6 transgenic mouse (nSIRT1OE Tg) were generated. Expression of SIRT1 protein was observed in small to medium sized DRG neurons. nSIRT1OE was shut off by feeding, weaned Tg mice, with DOX in the diet for 12 weeks. The Tg mice were divided into 3 groups. Group # 1: nSIRT1OE Tg mice fed with standard diet (STD) plus DOX for 4 months; Group # 2: nSIRT1OE Tg mice fed with HFD plus DOX for 4 months; Group # 3: nSIRT1OE Tg mice fed with HFD plus DOX for 2 months until they develop neuropathy and then switched to HFD minus DOX for additional 2 months. Neuropathy was determined by mechanical allodynia thresholds (MAT) and nerve conduction velocity (NCV) at 0, 2 and 4 months. Intraepidermal nerve fiber density (IENFD) was measured at 4 months. MAT, NCV and IENFD were decreased in HFD-fed (Group # 2) mice compared to STD-fed mice (Group # 1) at 2 and 4 months. In Group # 3 mice, 2 months after turning on nSIRT1OE, we observed a reversal of mechanical allodynia, NCV and attenuation of IENFD (Group # 3 compared to Group # 2). Western blot of protein extracts from DRG neurons showed that HFD increased acetylation of proteins and SIRT1OE abolished the HFD-induced acetylation of proteins. The mitochondrial bioenergetics profile of cultured adult DRG neurons showed that hyperglycemia induced a decrease in the spare respiratory capacity in wild type (WT) DRG neurons. This was corrected in nSIRT1OE Tg DRG neurons. In type 2 diabetic neuropathy, altered acetylation of proteins and reduced mitochondrial function via a defective SIRT1 pathway may contribute to developing distal axonopathy. Disclosure K. Chandrasekaran: None. C. Ho: None. M. Salimian: None. P.H. Kumar: None. S. Konduru: None. J.W. Russell: None.

Increased expression of CaV3.2 T-type calcium channels in damaged DRG neurons contributes to neuropathic pain in rats with spared nerve injury.

Ion channels are very important in the peripheral sensitization in neuropathic pain. Our present study aims to investigate the possible contribution of CaV3.2 T-type calcium channels in damaged dorsal root ganglion neurons in neuropathic pain. We established a neuropathic pain model of rats with spared nerve injury. In these model rats, it was easy to distinguish damaged dorsal root ganglion neurons (of tibial nerve and common peroneal nerve) from intact dorsal root ganglion neurons (of sural nerves). Our results showed that CaV3.2 protein expression increased in medium-sized neurons from the damaged dorsal root ganglions but not in the intact ones. With whole cell patch clamp recording technique, it was found that after-depolarizing amplitudes of the damaged medium-sized dorsal root ganglion neurons increased significantly at membrane potentials of −85 mV and −95 mV. These results indicate a functional up-regulation of CaV3.2 T-type calcium channels in the damaged medium-sized neurons after spared nerve injury. Behaviorally, blockade of CaV3.2 with antisense oligodeoxynucleotides could significantly reverse mechanical allodynia. These results suggest that CaV3.2 T-type calcium channels in damaged medium-sized dorsal root ganglion neurons might contribute to neuropathic pain after peripheral nerve injury.

CCR2 upregulation in DRG neurons plays a crucial role in gastric hyperalgesia associated with diabetic gastropathy.

BackgroundDiabetic gastropathy is a complex neuromuscular dysfunction of the stomach that commonly occurs in diabetes mellitus. Diabetic patients often present with upper gastrointestinal symptoms, such as epigastric discomfort or pain. The aim of this study was to assess gastric sensation in streptozocin-induced diabetes mellitus (DM) rats and to determine the contribution of C-C motif chemokine receptor 2 (CCR2) signaling to gastric hyperalgesia.ResultsDM rats showed signs of neuropathy (cutaneous mechanical hyperalgesia) from two weeks after streptozocin administration until the end of the experiment. Accelerated solid gastric emptying was observed at two weeks after streptozocin administration compared to the controls. Intense gastric hyperalgesia also developed in DM rats at two weeks after streptozocin administration, which was significantly reduced after intrathecal administration of the CCR2 antagonist INCB3344. Immunochemical analysis indicated that CCR2 expression was substantially upregulated in small and medium-sized dorsal root ganglia neurons of DM rats, although the protein level of monocyte chemoattractant protein-1, the preferred ligand for CCR2, was not significantly different between the control and DM groups.ConclusionsThese data suggest that CCR2 activation in nociceptive dorsal root ganglia neurons plays a role in the pathogenesis of gastric hyperalgesia associated with diabetic gastropathy and that CCR2 antagonist may be a promising treatment for therapeutic intervention.

Orai1 and Orai3 Mediate Store-Operated Calcium Entry Contributing to Neuronal Excitability in Dorsal Root Ganglion Neurons.

Store-operated calcium channels (SOCs) are highly calcium-selective channels that mediate calcium entry in various cell types. We have previously reported that intraplantar injection of YM-58483 (a SOC inhibitor) attenuates chronic pain. A previous study has reported that the function of SOCs in dorsal root ganglia (DRG) is enhanced after nerve injury, suggesting that SOCs may play a peripheral role in chronic pain. However, the expression, functional distribution and significance of the SOC family in DRG neurons remain elusive and the key components that mediate SOC entry (SOCE) are still controversial. Here, we demonstrated that the SOC family (STIM1, STIM2, Orai1, Orai2, and Orai3) was expressed in DRGs and STIM1 was mainly present in small- and medium-sized DRG neurons. Using confocal live cell imaging, Ca2+ imaging and electrophysiology techniques, we demonstrated that depletion of the endoplasmic reticulum Ca2+ stores induced STIM1 and STIM2 translocation, and that inhibition of STIM1 or blockage of Orai channels with pharmacological tools attenuated SOCE and SOC currents. Using the small inhibitory RNA knockdown approach, we identified STIM1, STIM2, Orai1, and Orai3 as the key components of SOCs mediating SOCE in DRG neurons. Importantly, activation of SOCs by thapsigargin induced plasma membrane depolarization and increased neuronal excitability, which were completely abolished by inhibition of SOCs or double knockdown of Orai1 and Orai3. Our findings suggest that SOCs exert an excitatory action in DRG neurons and provide a potential peripheral mechanism for modulation of pain hypersensitivity by SOC inhibition.

Oxaliplatin-induced changes in expression of transient receptor potential channels in the dorsal root ganglion as a neuropathic mechanism for cold hypersensitivity

Transient receptor potential (TRP) receptors are involved in the development of chemotherapy-induced peripheral neuropathic pain, which is a common side effect of selected chemotherapeutic agents such as oxaliplatin. However, the precise contribution of TRPs to this condition remains unknown. Cold hypersensitivity is the hallmark of oxaliplatin-induced neuropathy, so we used a preclinical model of oxaliplatin-induced cold hypersensitivity in rats to determine the effects of oxaliplatin on TRP channels. To this end, immunohistochemistry was used to examine TRP vanilloid 1 (TRPV1), TRP ankyrin 1 (TRPA1), and TRP melastatin 8 (TRPM8) expression in the rat dorsal root ganglion (DRG) after 4days of oxaliplatin treatment. Behavioral assessment using the acetone spray test showed that oxaliplatin significantly increased acute cold hypersensitivity after 4days of treatment. Double-staining immunohistochemistry showed that 4days after oxaliplatin treatment, there was increased co-expression of TRPA1 and TRPV1 in isolectin B4-positive small-sized DRG neurons, as well as a significant increase in the co-localization of TRPM8 and neurofilament 200 in medium-sized DRG neurons. In addition, in situ hybridization revealed that TRPV1 protein was co-expressed with TRPA1 mRNA on day 4 after oxaliplatin administration. Thus, at an early stage following oxaliplatin treatment there is an increased expression of TRPA1 and TRPV1 in small-sized DRG neurons and of TRPM8 in medium-sized DRG neurons. Collectively, these changes may contribute to the development of oxaliplatin-induced peripheral neuropathic pain.

Visualization of Peripheral Neuron Sensitization in a Surgical Mouse Model of Osteoarthritis by In Vivo Calcium Imaging.

To develop a method for analyzing sensory neuron responses to mechanical stimuli in vivo, and to evaluate whether these neuronal responses change after destabilization of the medial meniscus (DMM). DMM or sham surgery was performed in 10-week-old male C57BL/6 wild-type or Pirt-GCaMP3+/- mice. All experiments were performed 8 weeks after surgery. Knee and hind paw hyperalgesia were assessed in wild-type mice. The retrograde label DiI was injected into the ipsilateral knee to quantify the number of knee-innervating neurons in the L4 dorsal root ganglion (DRG) in wild-type mice. In vivo calcium imaging was performed on the ipsilateral L4 DRG of Pirt-GCaMP3+/- mice as mechanical stimuli (paw pinch, knee pinch, or knee twist) were applied to the ipsilateral hind limb. Eight weeks after surgery, mice subjected to DMM had more hyperalgesia in the knee and hind paw compared to mice subjected to sham surgery. Intraarticular injection of DiI labeled similar numbers of neurons in the L4 DRG of mice subjected to sham surgery and mice subjected to DMM. Increased numbers of sensory neurons responded to all 3 mechanical stimuli in mice subjected to DMM, as assessed by in vivo calcium imaging. The majority of responses in mice subjected to sham surgery and mice subjected to DMM were in small to medium-sized neurons, consistent with the size of nociceptors. The magnitude of responses was similar between mice subjected to sham surgery and mice subjected to DMM. Our findings indicate that increased numbers of small to medium-sized DRG neurons respond to mechanical stimuli 8 weeks after DMM surgery, suggesting that nociceptors have become sensitized by lowering the response threshold.

Increased excitability of medium-sized dorsal root ganglion neurons by prolonged interleukin-1β exposure is K(+) channel dependent and reversible.

Neuropathic pain resulting from peripheral nerve injury is initiated and maintained by persistent ectopic activity in primary afferent neurons. Sciatic nerve injury increases the excitability of medium-sized dorsal root ganglion (DRG) neurons. Levels of the inflammatory cytokine interleukin 1β (IL-1β) increase and peak after 7days. Five to six days of exposure of medium sized DRG neurons to 100pm IL-1β promotes persistent increases in excitability which abate within 3-4days of cytokine removal. This is associated with a profound attenuation of K(+) channel currents but only modest increases in function of cyclic nucleotide-sensitive hyperpolarization-activated channels (HCNs) and of voltage-gated Na(+) and Ca(2+) channel currents. It is unlikely, therefore, that direct interaction of IL-1β with DRG neurons is capable of initiating an enduring phenotypic shift in their electrophysiological properties that follows sciatic nerve injury. The findings also underline the importance of K(+) channel modulation in the actions of inflammatory mediators on peripheral neurons. Chronic constriction injury of rat sciatic nerve promotes signs of neuropathic pain. This is associated with an increase in the level of interleukin 1β (IL-1β) in primary afferents that peaks at 7days. This initial cytokine exposure has been proposed to trigger an enduring alteration in neuronal phenotype that underlies chronic hyper-excitability in sensory nerves, which initiates and maintains chronic neuropathic pain. We have shown previously that 5-6days of exposure of rat dorsal root ganglia (DRGs) to 100pm IL-1β increases the excitability of medium-sized neurons. We have now found using whole-cell recording that this increased excitability reverts to control levels within 3-4days of cytokine removal. The effects of IL-1β were dominated by changes in K(+) currents. Thus, the amplitudes of A-current, delayed rectifier and Ca(2+) -sensitive K(+) currents were reduced by ∼68%, ∼64% and ∼36%, respectively. Effects of IL-1β on other cation currents were modest by comparison. There was thus a slight decrease in availability of high voltage-activated Ca(2+) channel current, a small increase in rates of activation of hyperpolarization-activated cyclic nucleotide-gated channel current (IH ), and a shift in the voltage dependence of activation of tetrodotoxin-sensitive sodium current (TTX-S INa ) to more negative potentials. It is unlikely, therefore, that direct interaction of IL-1β with DRG neurons initiates an enduring phenotypic shift in their electrophysiological properties following sciatic nerve injury. Persistent increases in primary afferent excitability following nerve injury may instead depend on altered K(+) channel function and on the continued presence of slightly elevated levels IL-1β and other cytokines.

Spinorphin inhibits membrane depolarization- and capsaicin-induced intracellular calcium signals in rat primary nociceptive dorsal root ganglion neurons in culture

Objective: Spinorphin is a potential endogenous antinociceptive agent although the mechanism(s) of its analgesic effect remain unknown. We conducted this study to investigate, by considering intracellular calcium concentrations as a key signal for nociceptive transmission, the effects of spinorphin on cytoplasmic Ca2+ ([Ca2+]i) transients, evoked by high-K+ (30 mM) depolariasation or capsaicin, and to determine whether there were any differences in the effects of spinorphin among subpopulation of cultured rat dorsal root ganglion (DRG) neurons. Methods: DRG neurons were cultured on glass coverslips following enzymatic digestion and mechanical agitation, and loaded with the calcium sensitive dye fura-2 AM (1 µM). Intracellular calcium responses in individual DRG neurons were quantified using standard fura-2 based ratiometric calcium imaging technique. All data were analyzed by using unpaired t test, p < 0.05 defining statistical significance. Results: Here we found that spinorphin inhibited cytoplasmic Ca2+ ([Ca2+]i) transients, evoked by depolarization and capsaicin selectively in medium and small cultured rat DRG neurons. Spinorphin (10–300 µM) inhibited the Ca2+ signals in concentration dependant manner in small- and medium diameter DRG neurons. Capsaicin produced [Ca2+]i responses only in small- and medium-sized DRG neurons, and pre-treatment with spinorphin significantly attenuated these [Ca2+]i responses. Conclusion: Results from this study indicates that spinorphin significantly inhibits [Ca2+]i signaling, which are key for the modulation of cell membrane excitability and neurotransmitter release, preferably in nociceptive subtypes of this primary sensory neurons suggesting that peripheral site is involved in the pain modulating effect of this endogenous agent.

Inhibition of Hyperpolarization-Activated Cation Current in Medium-Sized DRG Neurons Contributed to the Antiallodynic Effect of Methylcobalamin in the Rat of a Chronic Compression of the DRG.

Recently several lines of evidence demonstrated that methylcobalamin (MeCbl) might have potential analgesic effect in experimental and clinical studies. However, it was reported that MeCbl had no effect on treating lumbar spinal stenosis induced pain. Thus, the effects of short-term and long-term administration of MeCbl were examined in the chronic compression of dorsal root ganglion (CCD) model. We found that mechanical allodynia was significantly inhibited by a continuous application of high dose and a single treatment of a super high dose of MeCbl. Little is known about mechanisms underlying the analgesia of MeCbl. We examined the effect of MeCbl on the spontaneous activity (SA), the excitability, and hyperpolarization-activated nonselective cation ion current in compressed medium-sized dorsal root ganglion (DRG) neurons using extracellular single fiber recording in vivo and whole-cell patch clamp in vitro. We found that MeCbl significantly inhibited the SA of A-type sensory neurons in a dose-dependent manner and inhibited the excitability of medium-sized DRG neurons. In addition, MeCbl also decreased I h current density in injured medium-sized DRG neurons. Our results proved that MeCbl might exert an analgesic effect through the inhibition I h current and then might inhibit the hyperexcitability of primary sensory neurons under neuropathic pain state.

Expression of NPR-B in neurons of the dorsal root ganglia of the rat

C-type natriuretic peptide (CNP) is an abundant neuropeptide in the central nervous system, which exerts its physiological effects through natriuretic peptide receptor B (NPR-B). Recently, the CNP/NPR-B system has been recognized as an important regulator for the development of sensory axons. The dorsal root ganglion (DRG) contains neurons transmitting several kinds of spinal sensory stimuli to the central nervous system. In this study, we characterized NPR-B receptor expression in the rat DRG, using reverse transcription-polymerase chain reaction, Western blotting and immunohistochemistry. Immunostaining revealed that NPR-B was expressed in neuronal cell bodies and processes of the DRG, with NPR-B immunoreactivity mainly prominent in small and medium-sized DRG neurons. Double-immunolabeling showed that NPR-B was expressed in calcitonin gene-related peptide- and isolectin B4-positive neurons. Furthermore, NPR-B expression was co-localized with calcitonin gene-related peptide in the dorsal horn of the spinal cord. Together, our data suggest that the natriuretic peptides may perform several biological actions on sensory neurons via their binding to NPR-B in the DRG.

Subdiaphragmatic vagotomy increases the sensitivity of lumbar Aδ primary afferent neurons along with voltage‐dependent potassium channels in rats

Subdiaphragmatic vagal dysfunction causes chronic pain. To verify whether this chronic pain is accompanied by enhanced peripheral nociceptive sensitivity, we evaluated primary afferent neuronal excitability in subdiaphragmatic vagotomized (SDV) rats. SDV rats showed a decrease in the electrical stimuli-induced hind limb-flexion threshold at 250 Hz, but showed no similar effect at 5 or 2000 Hz, which indicated that lumbar primary afferent Aδ sensitivity was enhanced in SDV rats. The whole-cell patch-clamp technique also revealed the hyper-excitability of acutely dissociated medium-sized lumbar dorsal root ganglion (DRG) neurons isolated from SDV rats. The contribution of changes in voltage-dependent potassium (Kv) channels was assessed, and transient A-type K(+) (I(A) ) current density was apparently decreased. Moreover, Kv4.3 immunoreactivity in medium-sized DRG neurons was significantly reduced in SDV rats compared to sham. These results indicate that SDV causes hyper-excitability of lumbar primary Aδ afferent neurons, which may be induced along with suppressing I(A) currents via the decreased expression of Kv4.3. Thus, peripheral Aδ neuroplasticity may contribute to the chronic lower limb pain caused by SDV.

Chronic compression of mouse dorsal root ganglion alters voltage-gated sodium and potassium currents in medium-sized dorsal root ganglion neurons

Chronic compression (CCD) of the dorsal root ganglion (DRG) is a model of human radicular pain produced by intraforaminal stenosis and other disorders affecting the DRG, spinal nerve, or root. Previously, we examined electrophysiological changes in small-diameter lumbar level 3 (L3) and L4 DRG neurons treated with CCD; the present study extends these observations to medium-sized DRG neurons, which mediate additional sensory modalities, both nociceptive and non-nociceptive. Whole-cell patch-clamp recordings were obtained from medium-sized somata in the intact DRG in vitro. Compared with neurons from unoperated control animals, CCD neurons exhibited a decrease in the current threshold for action potential generation. In the CCD group, current densities of TTX-resistant and TTX-sensitive Na(+) current were increased, whereas the density of delayed rectifier voltage-dependent K(+) current was decreased. No change was observed in the transient or "A" current after CCD. We conclude that CCD in the mouse produces hyperexcitability in medium-sized DRG neurons, and the hyperexcitability is associated with an increased density of Na(+) current and a decreased density of delayed rectifier voltage-dependent K(+) current.

Subtype-specific reduction of voltage-gated calcium current in medium-sized dorsal root ganglion neurons after painful peripheral nerve injury

Sensory neurons express a variety of voltage-gated Ca 2+ channel subtypes, but reports differ on their proportionate representation, and the effects of painful nerve injury on each subtype are not established. We compared levels of high-voltage activated currents in medium-sized (30–40 μm) dorsal root ganglion neurons dissociated from control animals and those subjected to spinal nerve ligation, using sequential application of semiselective channel blockers (nisoldipine for L-type, SNX-111 or ω-conotoxin GVIA for N-type, agatoxin IVA or ω-conotoxin MVIIC for P/Q-type, and SNX-482 for a component of R-type) during either square wave depolarizations or action potential waveform voltage commands. Using sequential administration of multiple blockers, proportions of total Ca 2+ current attributable to different subtypes and the effect of injury depended on the sequence of blocker administration and type of depolarization command. Overall, however, N-type and L-type currents comprised the dominant components of I Ca in sensory neurons under control conditions, and these subtypes showed the greatest loss of current following injury ( L-type 26–71% loss, N-type 0–51% loss). Further exploration of N-type current identified by its sensitivity to ω-conotoxin GVIA applied alone showed that injury reduced the peak N-type current during step depolarization by 68% and decreased the total charge entry during action potential waveform stimulation by 44%. Isolation of N-type current by blockade of all other subtypes demonstrated a 50% loss with injury, and also revealed an injury-related rightward shift in the activation curve. Non-stationary noise analyses of N-type current in injured neurons revealed unitary channel current and number of channels that were not different from control, which indicates that injury-induced loss of current is due to a decrease in channel open probability. Our findings suggest that diminished Ca 2+ influx through N-type and L-type channels may contribute to sensory neuron dysfunction and pain after nerve injury.