Monocyte chemoattractant protein-1 functions as a neuromodulator in dorsal root ganglia neurons

It has previously been observed that expression of chemokine monocyte chemoattractant protein-1 (MCP-1/CC chemokine ligand 2 (CCL2)) and its receptor CC chemokine receptor 2 (CCR2) is up-regulated by dorsal root ganglion (DRG) neurons in association with rodent models of neuropathic pain. MCP-1 increases the excitability of nociceptive neurons after a peripheral nerve injury, while disruption of MCP-1/CCR2 signaling blocks the development of neuropathic pain, suggesting MCP-1 signaling is responsible for heightened pain sensitivity. To define the mechanisms of MCP-1 signaling in DRG, we studied intracellular processing, release, and receptor-mediated signaling of MCP-1 in DRG neurons. We found that in a focal demyelination model of neuropathic pain both MCP-1 and CCR2 were up-regulated by the same neurons including transient receptor potential vanilloid receptor subtype 1 (TRPV1) expressing nociceptors. MCP-1 expressed by DRG neurons was packaged into large dense-core vesicles whose release could be induced from the soma by depolarization in a Ca²⁺-dependent manner. Activation of CCR2 by MCP-1 could sensitize nociceptors via transactivation of transient receptor potential channels. Our results suggest that MCP-1 and CCR2, up-regulated by sensory neurons following peripheral nerve injury, might participate in neural signal processing which contributes to sustained excitability of primary afferent neurons.     

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.     

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。     

Expression of a unique globo-series glycolipid in cultured rat dorsal root ganglion neurons: Relationship with neuronal development

Previous studies from this laboratory demonstrated the presence of a UDP-galactose:Gb3Cer α1-3galactosyltansferase activity responsible for the synthesis of a unique glycosphingolipid (GSL), Galα1-3Gb3Cer, in cultured PC12 pheochromocytoma cells (21). In this investigation, we examined the presence of this enzyme activity in isolated rat embryonic dorsal root ganglion neurons (DRGN), which, like pheochromocytoma cells, originate from the neural crest cells. DRGN exhibited the α-galactosyltransferase activity and the activity was comparable to that of the PC12 cells while several other rat tissues, with the exception of kidney, showed minimal activity. In order to define the spatial and temporal expression of Galα1-3Gb3Cer in DRGN, we examined the expression of Galα1-3Gb3Cer in cultured DRGN derived from embryonic day 16 rat embryos. Using a polyclonal antibody raised against Galα1-3Gb3Cer, we examined the localization of this glycolipid in DRGN cells after, 5, 8, 12, and 15 days in culture. Immunostaining was restricted to the neurons while Schwann cells were negative. At day 5, the immunostaining was weak and confined to the cell body of the DRGN, though neurites were present at this stage. The period between days 5 and 15 represented a period of rapid neuritic growth and continued enlargement of the cell bodies. Immunoreactivity in the cell bodies increased dramatically by day 8. By day 12, immunoreactivity was present in neurites, and by day 15, was strong in both cell bodies and neurites. The expression of Galα1-3Gb3Cer in vivo was confirmed by immunostaining of frozen sections of dorsal root ganglia. Our present studies which demonstrate neuron-specific expression of Galα1-3Gb3Cer during neurotigenesis combined with previous observations for its expression in PC12 cells, strongly implicates this GSL in neuronal development. This paper is dedicated to Dr. Marion Smith.     

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     

Fibronectin stimulates TRPV1 translocation in primary sensory neurons

Extracellular matrix (ECM) molecules are highly variable in their composition and receptor recognition. Their ubiquitous expression profile has been linked to roles in cell growth, differentiation, and survival. Recent work has identified certain ECM molecules that serve as dynamic signal modulators, versus the more-recognized role of chronic modulation of signal transduction. In this study, we investigated the role that fibronectin (FN) plays in the dynamic modulation of transient receptor potential family V type 1 receptor (TRPV1) translocation to the plasma membrane in trigeminal ganglia (TG) sensory neurons. Confocal immunofluorescence analyses identify co-expression of the TRPV1 receptor with integrin subunits that bind FN. TG neurons cultured upon or treated with FN experienced a leftward shift in the EC₅₀ of capsaicin-stimulated neuropeptide release. This FN-induced increase in TRPV1 sensitivity to activation is coupled by an increase in plasma membrane expression of TRPV1, as well as an increase in tyrosine phosphorylation of TRPV1 in TG neurons. Furthermore, TG neurons cultured on FN demonstrated an increase in capsaicin-mediated Ca²⁺ accumulation relative to neurons cultured on poly- d -lysine. Data presented from these studies indicate that FN stimulates tyrosine-phosphorylation-dependent translocation of the TRPV1 receptor to the plasma membrane, identifying FN as a critical component of the ECM capable of sensory neuron sensitization.     

Substance P release evoked by capsaicin or potassium from rat cultured dorsal root ganglion neurons is conversely modulated with bradykinin

To clarify the molecular mechanism of substance P (SP) release from dorsal root ganglion (DRG) neurons, we investigated the involvement of several intracellular effectors in the regulation of SP release evoked by capsaicin, potassium or/and bradykinin. Bradykinin-evoked SP release from cultured adult rat DRG neurons was attenuated by either the mitogen-activated protein kinase kinase (MEK) inhibitor (U0126) or cycloheximide. As the long-term exposure of DRG neurons to bradykinin (3 h) resulted in extracellular signal-regulated kinase (ERK) phosphorylation at an early stage and thereafter induced cyclooxygenase-2 (COX-2) protein expression, which both contribute to the SP release triggered by bradykinin B2 receptor. The long-term exposure of DRG neurons to bradykinin enhanced the SP release by capsaicin, but attenuated that by potassium. Interestingly, the inositol 1,4,5-triphosphate (IP3)-induced calcium release blocker [2-aminoethyl diphenylborinate (2-APB)] not only inhibited the potassium-evoked SP release, but also completely abolished the enhancement of capsaicin-induced SP release by bradykinin from cultured DRG neurons. Together, these findings suggest that the molecular mechanisms of SP release by bradykinin involve the activation of MEK, and also require the de novo protein synthesis of COX-2 in DRG neurons. The IP3-dependent calcium release could be involved in the processes of the regulation by bradykinin of capsaicin-triggered SP release.     

The effect of acute dissociation on the electrophysiological properties of rat dorsal root ganglion neurons

The acutely dissociated neurons from the dorsal root ganglia (DRGs) are extensively used. The effects of acute dissociation on the properties of these neurons are, however, not clear. In this study, the action potentials (APs) were recorded from both acutely dissociated and in vivo identified DRG neurons with patch clamp and sharp electrode recording techniques, respectively. We found that acute dissociation slowed both the depolarizing and repolarizing rate of APs, and elongated the AP duration (APD). The lower recording temperature presented in the acutely dissociated neurons contributed to about 10% of these differences. The major contributor of these differences was possibly modulation of the mRNA expression especially those of the ion channels, as suggested by our observation that acute dissociation significantly reduced the mRNA abundance of Nav1.6–1.9. In conclusion, acute dissociation altered the electrophysiological properties of the DRG neurons; the disrupted gene-expression pattern may contribute to this effect.     

实验性结肠炎对脊髓传入神经元中降钙素基因相关肽和香草酸受体1表达的调节

本文旨在探讨肠道局部炎症对脊髓肠道感觉传入神经通路的近期及远期效应,应用三硝基苯磺酸(trinitrobenzenesulfonic acid,TNBS)建立大鼠结肠炎动物模型,用DiI(3)逆行神经标记法识别支配肠道炎症部位的脊髓背根神经节(dorsalrootganglia,DRG)神经元,通过肉眼观察、平均组织损伤评分及髓过氧化物酶活性测定等方法评价肠道组织的炎症反应状态,用免疫组织化学法测定香草酸受体l(vanilloid receptor 1,VRl)和降钙素基因相关肽(calcitonin gene-related peptide,CGRP)在支配结肠炎症部位的DRG神经元中的表达,比较炎症不同阶段(给予TNBS后7、21、42 d)CGRP和VRI阳性神经元的数目.结果显示,炎症急性期(即给予TNBS后7 d)结肠黏膜肉眼可见明显损伤,同时相应DRG中表达CGRP及VRl的神经元增加近2倍[(95.38±9.45)%VS(42.86±5.02)%,(89.23±8.21)%VS(32.54±4.58)%].给予TNBS后21、42 d,肠道炎症反应已完全消退,但表达CGRP及VRl的DRG神经元数目仍明显高于对照组[(86.25±8.21)%,(68.28±7.12)%VS(42.86±5.02)%;(67.22±6.52)%,(56.25±4.86)%VS(32.54±4.58)%].结果提示,肠道局部炎症可以上调支配肠道的脊髓传入神经元中CGRP和VRl的表达,这种异常表达可以持续至肠道炎症反应消退后的一定时间.     

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     

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。     

Expression of tachykinin receptors in Xenopus oocytes injected with poly(A)~+RNA from cat dorsal root ganglion

ＴａｃｈｙｋｉｎｉｎｆａｍｉｌｙｉｓａｇｒｏｕｐｏｆｎｅｕｒｏｐｅｐｔｉｄｅｓｗｉｔｈｓｉｍｉｌａｒＣｔｅｒｍｉｎａｌｓｅｑｕｅｎｃｅｓａｎｄｒｅｌａｔｅｄｂｉｏａｃｔｉｖｉｔｉｅｓ.ＴｈｅｍａｊｏｒｔａｃｈｙｋｉｎｉｎｓｉｎｍａｍｍａｌｉａｎａｒｅｓｕｂｓｔａｎｃｅＰ(ＳＰ),ｎｅｕｒｏｋｉｎｉｎＡ(ＮＫＡ)ａｎｄｎｅｕｒｏｋｉｎｉｎＢ(ＮＫＢ).Ｃｏｒｒｅｓｐｏｎｄｉｎｇｔｏｔｈｅｓｅｐｅｐｔｉｄｅｓ,ｔｈｒｅｅｄｉｓｔｉｎｃｔｔａｃｈｙｋｉｎｉｎｒｅｃｅｐｔｏｒｓｗｅｒｅｄｉｓｃｏｖｅｒｅｄａｎｄｎ…     

The cross channel activities of spider neurotoxin huwentoxin-I on rat dorsal root ganglion neurons

In this paper, we investigated the action of huwentoxin-I (HWTX-I) purified from the venom of the Chinese bird spider Ornithoctonus huwena on Ca(2+), Na(+) channels of adult rat dorsal root ganglion (DRG) neurons. The results showed that huwentoxin-I could reduce the peak currents of N-type Ca(2+) channels (IC(50) approximately 100 nM) and TTX-S Na(+) channels (IC(50) approximately 55 nM), whereas no effect was detected on TTX-R Na(+) channels. The comparative studies indicated that the selectivity of HWTX-I on Ca(2+) channels was higher that of MVIIA and approximately the same as that of GVIA. HWTX-I is the first discovered toxin with the cross channel activities from the spider O. huwena venom similar to micro O-conotoxins MrVIA and MrVIB.     

Activation of transient receptor potential ankyrin 1 evokes nociception through substance P release from primary sensory neurons

To examine mechanisms underlying substance P (SP) release from primary sensory neurons in response to activation of the non-selective cation channel transient receptor potential ankyrin 1 (TRPA1), SP release from cultured rat dorsal root ganglion neurons was measured, using radioimmunoassay, by stimulating TRPA1 with allyl isothiocyanate (AITC), a TRPA1 agonist. AITC-evoked SP release occurred in a concentration- and time-dependent manner. Interestingly, p38 mitogen-activated protein kinase (p38) inhibitor SB203580 significantly attenuated AITC-evoked SP release. The in vivo effect of AITC-evoked SP release from primary sensory neurons in mice was evaluated. Hind paw intraplantar injection of AITC induced nociceptive behaviors and inflammation (edema, thermal hyperalgesia). AITC-induced thermal hyperalgesia and edema were inhibited by intraplantar pre-treatment with either SB203580 or neurokinin-1 receptor antagonist CP96345. Moreover, intrathecal pre-treatment with either CP96345 or SB203580 inhibited AITC-induced nociceptive behaviors and thermal hyperalgesia. Immunohistochemical studies demonstrated that intraplantar AITC injection induced the phosphorylation of p38 in mouse dorsal root ganglion neurons containing SP. These findings suggest that activation of TRPA1 evokes SP release from the primary sensory neurons through phosphorylation of p38, subsequent nociceptive behaviors and inflammatory responses. Furthermore, the data also indicate that blocking the effects of TRPA1 activation at the periphery leads to significant antinociception.     

Chronic pregabalin inhibits synaptic transmission between rat dorsal root ganglion and dorsal horn neurons in culture

In this study, we have examined the properties of synaptic transmission between dorsal root ganglion (DRG) and dorsal horn (DH) neurons, placed in co-culture. We also examined the effect of the anti-hyperalgesic gabapentinoid drug pregabalin (PGB) at this pharmacologically relevant synapse. The main method used was electrophysiological recording of excitatory post synaptic currents (EPSCs) in DH neurons. Synaptic transmission between DRG and DH neurons was stimulated by capsaicin, which activates transient receptor potential vanilloid-1 (TRPV1) receptors on small diameter DRG neurons. Capsaicin (1 μM) application increased the frequency of EPSCs recorded in DH neurons in DRG-DH co-cultures, by about 3-fold, but had no effect on other measured properties of the EPSCs. There was also no effect of capsaicin in the absence of co-cultured DRGs. Application of PGB (100 μM) for 40-48 h caused a reduction in the capsaicin-induced increase in EPSC frequency by 57%. In contrast, brief preincubation of PGB had no significant effect on the capsaicin-induced increase in EPSC frequency. In conclusion, this study shows that PGB applied for 40-48 h, but not acute application inhibits excitatory synaptic transmission at DRG-DH synapses, in response to nociceptive stimulation, most likely by a presynaptic effect on neurotransmitter release from DRG presynaptic terminals.     

Microarray analyses of lncRNAs and mRNAs expression profiling associated with diabetic peripheral neuropathy in rats

Diabetic peripheral neuropathy (DPN) is considered to be the most frequent neuropathic complication of diabetes, and severely affects the quality of life of patients. Long noncoding RNAs (lncRNAs) participate in various pathophysiological processes and associate with many diseases. However, the exact impact of lncRNAs on DPN remains obscure. To discover a potential connection, a microarray study was conducted to analyze the expression profiling of lncRNAs and messenger RNAs (mRNAs) in dorsal root ganglia (DRG) from streptozotocin-induced diabetic rats with DPN. As a result, 983 lncRNAs and 1357 mRNAs were aberrantly expressed compared with control samples. Using bioinformatics analyses, we identified 558 Gene Ontology terms and 94 Kyoto Encyclopedia of Genes and Genomes pathways to be significantly enriched. Additionally, the signal-net analysis indicated that integrin receptors, including Itgb3, Itgb1, Itgb8, and Itga6, might be important players in network regulation. Furthermore, the lncRNA-mRNA network analysis showed dynamic interactions between the dysregulated lncRNAs and mRNAs. This is the first study to present an overview of lncRNA and mRNA expressions in DRG tissues from DPN rats. Our results indicate that these differentially expressed lncRNAs may have crucial roles in pathological processes of DPN by regulating their coexpressed mRNAs. The data may provide novel targets for future studies, which should focus on validating their roles in the progression of DPN.     

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.     

Melittin activates TRPV1 receptors in primary nociceptive sensory neurons via the phospholipase A2 cascade pathways

Previous studies demonstrated that melittin, the main peptide in bee venom, could cause persistent spontaneous pain, primary heat and mechanical hyperalgesia, and enhance the excitability of spinal nociceptive neurons. However, the underlying mechanism of melittin-induced cutaneous hypersensitivity is unknown. Effects of melittin applied topically to acutely dissociated rat dorsal root ganglion neurons were studied using whole-cell patch clamp and calcium imaging techniques. Melittin induced intracellular calcium increases in 60% of small (<25 μm) and medium (<40 μm) diameter sensory neurons. In current clamp, topical application of melittin evoked long-lasting firing in 55% of small and medium-sized neurons tested. In voltage clamp, melittin evoked inward currents in sensory neurons in a concentration-dependent manner. Repeated application of melittin caused increased amplitude of the inward currents. Most melittin-sensitive neurons were capsaicin-sensitive, and 65% were isolectin B4 positive. Capsazepine, the TRPV1 receptor inhibitor, completely abolished the melittin-induced inward currents and intracellular calcium transients. Inhibitions of signaling pathways showed that phospholipase A₂, but not phospholipase C, was involved in producing the melittin-induced inward currents. Inhibitors of cyclooxygenases (COX) and lipoxygenases (LOX), two key components of the arachidonic acid metabolism pathway, each partially suppressed the inward current evoked by melittin. Inhibitors of protein kinase A (PKA), but not of PKC, also abolished the melittin-induced inward currents. These results indicate that melittin can directly excite small and medium-sized sensory neurons at least in part by activating TRPV1 receptors via PLA2-COXs/LOXs cascade pathways.