The TRPV1 receptor is associated with preferential stress in large dorsal root ganglion neurons in early diabetic sensory neuropathy

Chronic diabetic neuropathy is associated with peripheral demyelination and degeneration of nerve fibers. The mechanism(s) underlying neuronal injury in diabetic sensory neuropathy remain poorly understood. Recently, we reported increased expression and function of transient receptor potential vanilloid 1 (TRPV1) in large dorsal root ganglion (DRG) neurons in diabetic sensory neuropathy. In this study, we examined the effects of TRPV1 activation on cell injury pathways in this subpopulation of neurons in the streptozotocin-induced diabetic rat model. Large DRG neurons from diabetic (6–8 weeks) rats displayed increased oxidative stress and activation of cell injury markers compared with healthy controls. Capsaicin (CAP) treatment induced decreased labeling of MitoTracker Red and increased cytosolic cytochrome c and activation of caspase 3 in large neurons isolated from diabetic rats. CAP treatment also induced oxidative stress in large diabetic DRG neurons, which was blocked by pre-treatment with caspase or calpain inhibitor. In addition, both μ-calpain expression and calpain activity were significantly increased in DRG neurons from diabetic rats after CAP treatment. Treatment with capsazepine, a competitive TRPV1 antagonist, markedly reduced these abnormalities in vitro and prevented activation of cell injury in large DRG neurons in diabetic rats in vivo . These results suggest that activation of the TRPV1 receptor activates pathways associated with caspase-dependent and calpain-dependent stress in large DRG neurons in STZ-diabetic rats. Activation of the TRPV1 receptor may contribute to preferential neuronal stress in large DRG neurons relatively early in diabetic sensory neuropathy.     

瞬时外向钾电流的降低介导了慢性压迫背根神经节细胞兴奋性的升高

慢性压迫大鼠背根神经节（chronic compression of the dorsal root,ganglion,CCD）后,背根神经节细胞兴奋性升高,但引起神经元兴奋性改变的离子通道机制还需进一步探索。本实验采用胞内记录以及全细胞膜片钳记录方法,研究急性分离的大鼠背根神经节细胞兴奋性改变与瞬时外向钾电流（A-type potassium current,ⅠA）的关系。结果表明,CCD术后背根神经节细胞兴奋性升高,在急性分离的体外细胞中仍继续存在,表现为对辣椒素敏感的背根神经节细胞产生动作电位的最小电流刺激强度,即阈电流（current threshold）及阈电位（voltage threshold）降低;给予正常对照组神经元（未压迫损伤）瞬时外向钾通道阻断剂4-氨基吡啶,出现了类似CCD术后兴奋性升高的改变。进一步用两步电压钳方法分离ⅠA,研究CCD术后神经元ⅠA的变化,结果表明,CCD组神经元的ⅠA比对照组神经元ⅠA降低,并且与其阈电位的改变一致。以上结果提示,背根神经节压迫受损后,神经节细胞ⅠA降低可能参与介导了神经节细胞兴奋性的升高。     

神经病理痛大鼠DRG神经元GABAA受体激活电流的变化

目的：观察坐骨神经慢性压榨损伤（CCI）致神经病理痛后,大鼠背根节神经元GABAA受体（γ-氨基丁酸A受体）激活电流的变化。方法：运用全细胞膜片钳技术记录CCI模型手术侧、手术对侧及假手术组大鼠背根神经节细胞GABAx受体激活电流,比较坐骨神经慢性压榨损伤后GABAA受体激活电流的变化。结果：①CCI模型组大鼠手术侧DRG神经元在不同浓度（0．1-1000μmol／L）GABAA受体激活电流幅值均显著小于假手术组。②CCI模型组大鼠手术对侧DRG神经元在不同浓度（0．01-1000μmol／L）GABAA受体激活电流幅值均显著大于手术同侧及假手术组。结论：在坐骨神经慢性压榨损伤的过程中,不仅损伤侧的DRG神经元GABAA受体激活电流显著减小,这种损伤同时还引起了手术对侧的DRG神经元GABA激活电流代偿性的增强,GABAA受体功能的改变导致的突触前抑制作用的减弱可能是神经病理痛产生的根本原因之一。     

TRPV1b overexpression negatively regulates TRPV1 responsiveness to capsaicin, heat and low pH in HEK293 cells

Transient receptor potential channel type V (TRPV) 1 is a non-selective cation channel that can be activated by capsaicin, endogenous vanilloids, heat and protons. The human TRPV1 splice variant, TRPV1b, lacking exon 7, was cloned from human dorsal root ganglia (DRG) RNA. The expression profile and relative abundance of TRPV1b and TRPV1 in 35 different human tissues were determined by quantitative RT-PCR using isoform-specific probes. TRPV1b was most abundant in fetal brain, adult cerebellum and DRG. Functional studies using electrophysiological techniques showed that recombinant TRPV1b was not activated by capsaicin (1 microM), protons (pH 5.0) or heat (50 degrees C). However, recombinant TRPV1b did form multimeric complexes and was detected on the plasma membrane of cells, demonstrating that the lack of channel function was not due to defects in complex formation or cell surface expression. These results demonstrate that exon 7, which encodes the third ankyrin domain and 44 amino acids thereafter, is required for normal channel function of human TRPV1. Moreover, when co-expressed with TRPV1, TRPV1b formed complexes with TRPV1, and inhibited TRPV1 channel function in response to capsaicin, acidic pH, heat and endogenous vanilloids, dose-dependently. Taken together, these data support the hypothesis that TRPV1b is a naturally existing inhibitory modulator of TRPV1.     

Slow inactivation of tetrodotoxin-insensitive Na+ channels in neurons of rat dorsal root ganglia

Summary Whole-cell patch-clamp experiments were performed with neurons cultured from rat dorsal root ganglia (DRG). Two types of Na⁺ currents were identified on the basis of sensitivity to tetrodotoxin. One type was blocked by 0.1 nm tetrodotoxin, while the other type was insensitive to 10 m tetrodotoxin. The peak amplitude of the tetrodotoxin-insensitive Na⁺ current gradually decreased after depolarization of the membrane. The steady-state value of the peak amplitude was attained several minutes after the change of holding potential. Such a slow inactivation was not observed in tetrodotoxin-sensitive Na⁺ current. The slow inactivation of the tetrodotoxin-insensitive Na⁺ current was kinetically distinct from the ordinary short-time steady-state inactivation. The voltage dependence of the slow inactivation could be described by a sigmoidal function, and its time course had a double-exponential process. A decrease of external pH partially antagonized the slow inactivation, probably through an increased diffusion potential across the membrane. However, the slow inactivation was not due to change in surface negative charges, since a shift of the kinetic parameters along the voltage axis was not observed during the slow inactivation. Due to the slow inactivation, the inactivation curves for the tetrodotoxininsensitive Na⁺ current were shifted in the negative direction as the prepulse duration was increased. Consequently, the window current activated at potentials close to the resting membrane potential was markedly reduced. Thus, the slow inactivation may be involved in the long-term regulation of the excitability of sensory neurons.We thank Prof. Hirosi Kuriyama for his support and advice and Dr. M. Yoshii for helpful discussions. This study was supported by the Japanese Ministry of Education (Scientific Research 02670090).     

Quercetin relieved diabetic neuropathic pain by inhibiting upregulated P2X4 receptor in dorsal root ganglia

The upregulation of nociceptive ion channels expressed in dorsal root ganglia (DRG) contributes to the development and retaining of diabetic pain symptoms. The flavonoid quercetin (3,3′,4′,5,7-pentahydroxyflavone) is a component extracted from various fruits and vegetables and exerts anti-inflammatory, analgesic, anticarcinogenic, antiulcer, and antihypertensive effects. However, the exact mechanism underlying quercetin's analgesic action remains poorly understood. The aim of this study was to investigate the effects of quercetin on diabetic neuropathic pain related to the P2X₄ receptor in the DRG of type 2 diabetic rat model. Our data showed that both mechanical withdrawal threshold and thermal withdrawal latency in diabetic rats treated with quercetin were higher compared with those in untreated diabetic rats. The expression levels of P2X₄ messenger RNA and protein in the DRG of diabetic rats were increased compared with the control rats, while quercetin treatment significantly inhibited such enhanced P2X₄ expression in diabetic rats. The satellite glial cells (SGCs) enwrap the neuronal soma in the DRG. Quercetin treatment also lowered the elevated coexpression of P2X₄ and glial fibrillary acidic protein (a marker of SGCs) and decreased the upregulation of phosphorylated p38 mitogen-activated protein kinase (p38MAPK) in the DRG of diabetic rats. Quercetin significantly reduced the P2X₄ agonist adenosine triphosphate-activated currents in HEK293 cells transfected with P2X₄ receptors. Thus, our data demonstrate that quercetin may decrease the upregulation of the P2X₄ receptor in DRG SGCs, and consequently inhibit P2X₄ receptor-mediated p38MAPK activation to relieve the mechanical and thermal hyperalgesia in diabetic rats.     

Neurotrophin-3 and TrkC-immunoreactive neurons in rat dorsal root ganglia correlate by distribution and morphology

Previous studies have shown that a subpopulation of large dorsal root ganglion neurons contains neurotrophin-3 (NT3)-like immunoreactivity. It is not known, however, whether these NT3 immunoreactive neurons also express the high affinity receptor for NT3, trkC. In the present study, the distribution and morphology of trkC immunoreactive neurons have been correlated with those of NT3 immunoreactive neurons in the dorsal root ganglia. Size and segmental distributions of both antigens indicate that they are present in the same group of large sensory neurons. Almost twice the number of these neurons are present in the cervical and lumbar spinal ganglia than in the thoracic. Co-localization study indicates that 94% of NT3 immunoreactive neurons express trkC. Our findings support the proposal that NT3 in these neurons is derived from their peripheral targets rather than synthesized in situ. Special issue dedicated to Dr. Hans Thoenen.     

血管升压素对大鼠背根神经节神经元膜的作用

为研究血管升压素(arginine vasopressin,AVP)对大鼠背根神经节(dorsal root ganglion,DRG)神经元的作用及其机制,用细胞内微电极记录技术记录离体灌流DRG神经元的膜电位。结果如下:(1)在受检的120个细胞中,大多数(81.67％)在滴加AVP后产生明显的超极化反应。(2)滴加AVP(10μmol/L)后膜电导增加约19.34％(P<0.05)。(3)灌流平衡液巾的NaCl以氯化胆碱(CH-Cl)置代和用Cd2+阻断Ca2+通道后,AVP引起超极化反应的幅值均无明显变化(P>0.05),而加入K+通道阻断剂四乙铵(TEA)后,AVP引起的超极化反应幅值明显减小(P<0.05)。(4)AVP引起的超极化反应可被AVP V.受体拈抗剂阻断。结果捉示,AVP可使DRG大多数神经元膜产生超极化,DRG神经元膜上存在AVP V,受体,且AVP引起的超极化反应是通过神经元膜上AVP V.受体介导的K+外流所致.AVP可能参与了初级感觉信息传入的调制。     

Roles of extracellular signal-regulated protein kinases 5 in spinal microglia and primary sensory neurons for neuropathic pain

Neuropathic pain that occurs after peripheral nerve injury is poorly controlled by current therapies. Increasing evidence shows that mitogen-activated protein kinase (MAPK) play an important role in the induction and maintenance of neuropathic pain. Here we show that activation of extracellular signal-regulated protein kinases 5 (ERK5), also known as big MAPK1, participates in pain hypersensitivity caused by nerve injury. Nerve injury increased ERK5 phosphorylation in spinal microglia and in both damaged and undamaged dorsal root ganglion (DRG) neurons. Antisense knockdown of ERK5 suppressed nerve injury-induced neuropathic pain and decreased microglial activation. Furthermore, inhibition of ERK5 blocked the induction of transient receptor potential channels and brain-derived neurotrophic factor expression in DRG neurons. Our results show that ERK5 activated in spinal microglia and DRG neurons contributes to the development of neuropathic pain. Thus, blocking ERK5 signaling in the spinal cord and primary afferents has potential for preventing pain after nerve damage.     

催产素对大鼠背根神经节分离细胞GABA激活电流的调制作用

在急性分离的大鼠背根神经节（ｄｏｒｓａｌ ｒｏｏｔ ｇａｎｇｌｉｏｎ,ＤＲＧ）细胞上,应用全细胞膜片箝技术观察了预知催产素（ｏｘｙｔｏｃｉｎ,ＯＴ）对ＧＡＢＡ激活电流的调制作用。结果如下：（１）大多数细胞（４８／５２,９０．５％）对ＧＡＢＡ敏感。（２）ＯＴ可引起５１．３％（２０／３９）的受检细胞出现外向膜电流;４３．６％（１７／３９）无明显膜反应;５．１％（２／３９）出现内向膜电流。（３）预加ＯＴ     

Involvement of hyperpolarization-activated, cyclic nucleotide-gated cation channels in dorsal root ganglion in neuropathic pain

Dorsal root ganglion(DRG)neurons have peripheral terminals in skin,muscle,and other peripheral tissues,andcentral terminals     

电生理学特性得到确定的大鼠脊髓背根神经节细胞内谷氨酸和P物质的共存

在离体灌流带脊髓和坐骨神经经的标本上,对脊髓背根神经节（ＤＲＧ）细胞的电生理学特性、对Ｐ物质（ＳＰ）受体激动剂的反应及谷氨酸（Ｇｌｕ）／ＳＰ共存的特点进行了研究。（１）对１３５个细胞进行了细胞内记录,并依纤维传导速度将其分为Ａα／β（〉１２ｍ／ｓ）和Ｃ（〈１．３ｍ／ｓ）两大类,它们的动作电位的快速后超极化（ｆＡＨＰ）有明显区别,Ｃ类的ｆＡＨＰ幅度小、时程长,Ａα／β类的ｆＡＨＰ幅度大、时程短;（２     

Role of the monocyte chemoattractant protein-1/C-C chemokine receptor 2 signaling pathway in transient receptor potential vanilloid type 1 ablation-induced renal injury in salt-sensitive hypertension

Our recent studies indicate that the transient receptor potential vanilloid type 1 (TRPV1) channel may act as a potential regulator of monocyte/macrophage recruitment to reduce renal injury in salt-sensitive hypertension. This study tests the hypothesis that deletion of TRPV1 exaggerates salt-sensitive hypertension-induced renal injury due to enhanced inflammatory responses via monocyte chemoattractant protein-1 (MCP-1)/C-C chemokine receptor 2 (CCR2)-dependent pathways. Wild type (WT) and TRPV1-null mutant (TRPV1^−/−) mice were subjected to uninephrectomy and deoxycorticosterone acetate (DOCA)-salt treatment for four weeks with or without the selective CCR2 antagonist, RS504393. DOCA-salt treatment increased systolic blood pressure (SBP) to the same degree in both strains, but increased urinary excretion of albumin and 8-isoprostane and decreased creatinine clearance with greater magnitude in TRPV1^−/− mice compared to WT mice. DOCA-salt treatment also caused renal glomerulosclerosis, tubulointerstitial injury, collagen deposition, monocyte/macrophage infiltration, proinflammatory cytokine and chemokine production, and NF-κB activation in greater degree in TRPV1^−/− mice compared to WT mice. Blockade of the CCR2 with RS504393 (4 mg/kg/day) had no effect on SBP in DOCA-salt-treated WT or TRPV1^−/− mice compared to their respective controls. However, treatment with RS504393 ameliorated renal dysfunction and morphological damage, and prevented the increase in monocyte/macrophage infiltration, cytokine/chemokine production, and NF-κB activity in both DOCA-salt hypertensive strains with a greater effect in DOCA-salt-treated TRPV1^−/− mice compared to DOCA-salt-treated WT mice. No differences in CCR2 protein expression in kidney were found between DOCA-salt-treated WT and TRPV1^−/− mice with or without RS504393 treatment. Our studies for the first time indicate that deletion of TRPV1 aggravated renal injury in salt-sensitive hypertension via enhancing MCP-1/CCR2 signaling-dependent inflammatory responses.     

Low maternal licking/grooming stimulation increases pain sensitivity in male mouse offspring

Deprivation of maternal care has been associated with higher pain sensitivity in offspring. In the present study, we hypothesized that the maternal licking/grooming behavior was an important factor for the development of the pain regulatory system. To test this hypothesis, we used male F2 offspring of early-weaned (EW) F1 mother mice that exhibit lower frequency of licking/grooming behavior. The formalin test revealed that F2 offspring of EW F1 dams showed significantly higher pain behavior than F2 offspring of normally-weaned (NW) F1 dams. We found that the mRNA levels of transient receptor potential vanilloid 1 (TRPV1), a nociceptor, were higher in the lumbosacral dorsal root ganglion (DRG) of F2 offspring of EW F1 dams than those of F2 offspring of NW F1 dams, suggesting that the higher pain sensitivity may be attributed to low licking/grooming, which may result in developmental changes in nociceptive neurons. In the DRG, mRNA levels of Mas-related G-protein coupled receptor B4 (MrgprB4), a marker of sensory neurons that detect gentle stroking, was also up-regulated in the F2 offspring of EW F1 dams. Considering that gentle touch alleviates pain, Mrgprb4 up-regulation may reflect a compensatory change. The present findings indicate important implications of maternal licking/grooming behavior in the development of the pain regulatory system.     

Newly Synthesized Oleylgingerol and Oleylshogaol Activate TRPV1 Ion Channels

The oleyl moiety in vanilloids is important in activating vanilloid receptor 1 (TRPV1), but there was no ingredient of ginger containing the oleyl moiety in the natural form. We synthesized oleylgingerol and oleylshogaol and then evaluated their potential to activate a rat TRPV1 channel. Oleylgingerol is a stronger TRPV1 agonist than natural gingerols, but oleylshogaol is a weaker agonist than natural shogaols. The difference in structure between oleylgingerol and oleylshogaol is only the hydroxy group at carbon-5. This hydroxy group might have an important role in activating a TRPV1 channel.     

Okadaic acid reversibly inhibits neurite outgrowth in embryonic dorsal root ganglion neurons

The aim of this study was to assess the effects of low concentrations of okadaic acid (OA) on neurite outgrowth and cellular integrity in cultures of dissociated dorsal root ganglion (DRG) neurons. The complete and fully reversible arrest of neurite outgrowth was achieved at 1 nM OA, thus ruling out the involvement of protein phosphatase 1 in the observed inhibitory effect. OA at 0.5 nM did not completely block neurite outgrowth, although it reduced the rate of growth by about one third. Protein phosphorylation and the integrity of microtubules and neurofilaments in neuron-enriched cultures were unaffected by 1 nM OA. The rate of synthesis of the low-molecular-weight neurofilament subunit (NFL) was also unchanged by OA treatment. Antimitotic agents used to eliminate proliferating cells did not alter the rate of neurite elongation. Since 1 nM OA does not suffice to inhibit neuronal protein phosphatase 2A fully, owing to the high concentration of this enzyme in neurons, we propose that the inhibitor is affecting a neuronal compartment that contains low levels of the phosphatase. This putative compartment is likely to be located in neurites, which were shown to contain levels of protein phosphatase 2A that were two- to threefold lower than in neuronal perikarya. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 193–201, 1997.     

Synaptic connections in a dissociated mixed culture of rat dorsal root ganglion and spinal cord neurons

Postsynaptic currents and action potentials recorded from neurons in a mixed culture of rat dorsal root ganglion and spinal cord cells are described. The existence of mutual synaptic connections between the above two types of neurons is demonstrated. Neirofiziologiya/Neurophysiology, Vol. 38, No. 4, pp. 358–360, July–August, 2006.