Nerve growth factor maintains potassium conductance after nerve injury in adult cutaneous afferent dorsal root ganglion neurons

Whole-cell patch-clamp techniques were used to study the effects of nerve growth factor on voltage-dependent potassium conductance in normal and axotomized identified large cutaneous afferent dorsal root ganglion neurons (48-50 micrometer diameter) many of which probably give rise to myelinated Abeta fibers. K-currents were isolated by blocking Na- and Ca-currents with appropriate ion replacement and channel blockers. Separation of current components was achieved on the basis of response to variation in conditioning voltage. Cutaneous afferents were labeled by the retrograde marker hydroxy-stilbamide (FluoroGold) which was injected into the skin of the foot. The sciatic nerve was either ligated or crushed with fine forceps five to seven days later. Neurons were dissociated 14-17 days after injury. The cut ends of the sciatic nerves were positioned into polyethylene tubes, which were connected to mini-osmotic pumps filled with either nerve growth factor or sterile saline. Control neurons displayed a prominent sustained K-current and the transient potassium currents "A" and "D". Nerve ligation, which blocks target reconnection resulted in near 50% reduction of total outward current; isolated sustained K-current and transient A-current were reduced by a comparable amount. Nerve crush, which allows regeneration to peripheral targets and exposure of the regenerating nerve to the distal nerve segment, resulted in a small reduction in sustained K-current but no reduction in transient A-current compared to controls. Levels of transient A-current and sustained K-current were maintained at control levels after nerve growth factor treatment.These results indicate that the large reduction in transient A-current, and in sustained K-current, observed in cutaneous afferent cell bodies after nerve ligation is prevented by application of nerve growth factor.     

Calcitonin gene-related peptide enhances calcium current of rat dorsal root ganglion neurons and spinal excitatory synaptic transmission

The actions of calcitonin gene-related peptide (CGRP) were examined on Ca2+-dependent action potentials and voltage-dependent Ca2+ currents in rat dorsal root ganglion (DRG) neurons in vitro. In addition, we tested the effect of CGRP on excitatory synaptic transmission in the rat spinal dorsal horn. CGRP produced a reversible increase in the amplitude and the duration of the Ca2+ spike of DRG neurons and directly increased the voltage-dependent Ca2+ current by enhancing both the transient and the sustained components of the current. The increase in the Ca2+ current is likely to be responsible for the increase in the Ca2+ spike and facilitation of excitatory synaptic transmission.     

Reg-2 expression in dorsal root ganglion neurons after adjuvant-induced monoarthritis

Reg-2 is a secreted protein that is expressed de novo in motoneurons, sympathetic neurons, and dorsal root ganglion (DRG) neurons after nerve injury and which can act as a Schwann cell mitogen. We now show that Reg-2 is also upregulated by DRG neurons in inflammation with a very unusual expression pattern. In a rat model of monoarthritis, Reg-2 immunoreactivity was detected in DRG neurons at 1 day, peaked at 3 days (in 11.6% of DRG neurons), and was still present at 10 days (in 5%). Expression was almost exclusively in the population of DRG neurons that expresses the purinoceptor P2X(3) and binding sites for the lectin Griffonia simplicifolia IB4, and which is known to respond to glial cell line-derived neurotrophic factor (GDNF). Immunoreactivity was present in DRG cell bodies and central terminals in the dorsal horn of the spinal cord. In contrast, very little expression was seen in the nerve growth factor (NGF) responsive and substance P expressing population. However intrathecal delivery of GDNF did not induce Reg-2 expression, but leukemia inhibitory factor (LIF) had a dramatic effect, inducing Reg-2 immunoreactivity in 39% of DRG neurons and 62% of P2X(3) cells. Changes in inflammation have previously been observed predominantly in the neuropeptide expressing, NGF responsive, DRG neurons. Our results show that changes also take place in the IB4 population, possibly driven by members of the LIF family of neuropoietic cytokines. In addition, the presence of Reg-2 in central axon terminals implicates Reg-2 as a possible modulator of second order dorsal horn cells.     

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.     

P物质对大鼠DRG神经元H+门控电流的调制作用

 目的：观察P物质（SP）对神经元H+门控电流的调制作用及其可能存在的机制。方法：采用全细胞膜片钳技术分别记录pH 4.0、pH 5.0、pH 6.0及共加H+ 与SP情况下,急性分离的大鼠背根神经节（DRG)神经元H+门控电流的形式。通过胞内透析技术分析SP对H+门控电流可能的调控机制。结果：H+门控电流可划分为短暂内流型（T型）、持续内向电流型（S型）、反向电流型（O型）及双相内向电流型（B型）。SP对S型和B型中持续成分的H+门控电流有明显增强作用,这种电流幅值增强达（85.53±22.93）%,约81.8%细胞的这种增强效应不能被选择性的SP受体NK1拮抗剂阻断,而非肽类的SP受体NK1拮抗剂对约75%的DRG神经元可明显阻断这种增强效应;SP对S型和B型中持续成分的H+门控电流也有明显抑制作用,抑制的幅值达（48.46±4.45）%,并且约88.9%细胞的这种抑制效应不能被SP受体NK1拮抗剂所阻断。通过胞内透析技术在细胞内液中加入GDP-β-S后不能消除SP对H+门控电流的调制作用。结论：H+门控电流有T型、S型、O型及B型4种电流类型。SP对H+门控电流有增强和抑制的双向调节作用。SP可能通过G蛋白偶联受体通路及与H+门控离子通道的某一位点直接结合来发挥对H+门控电流的双向调节调制作用。     

Morphine treatment induced calcitonin gene-related peptide and substance P increases in cultured dorsal root ganglion neurons

The mechanism of spinal tolerance to the analgesic effects of opiates is unclear at present. We have reported previously that calcitonin gene-related peptide-like immunoreactivity was significantly increased in primary afferents of the spinal dorsal horn during the development of morphine tolerance, suggesting that changes in the level of pain-related neuropeptides in dorsal root ganglion neurons may be involved [Menard D. P. et al. (1996) J. Neurosci. 16, 2342-2351]. In this study, we investigated if in vitro treatment with morphine can mimic the in vivo findings and induce increases in calcitonin gene-related peptide-like immunostaining in cultured dorsal root ganglion neurons from young (three-month-old) and middle-aged (10-month-old) adult rats. Following a repetitive exposure to morphine sulfate (1, 5, 10 microM) for six days, the number of calcitonin gene-related peptide- and substance P-immunoreactive neurons in cultured dorsal root ganglia from three- and 10-month-old rats was significantly increased. A lower concentration (0.5 microM) of morphine induced these increases only in dorsal root ganglion neurons from middle-aged rats. Morphine treatment was also found to increase the number of calcitonin gene-related peptide-immunoreactive neurons possessing multiple, long branches (i.e. with at least one branch >0.5mm). This apparent increase in the number of calcitonin gene-related peptide- and substance P-immunoreactive neurons observed following morphine treatment was blocked by naloxone, an opiate antagonist, indicating the involvement of genuine opioid receptors. No significant change in the number of neuropeptide Y- or galanin-immunoreactive neurons in cultured dorsal root ganglia was detected following any of these treatments.These data suggest that repeated exposure to morphine rather selectively increases calcitonin gene-related peptide- and substance P-like immunoreactivity in cultured dorsal root ganglion neurons. Moreover, the sensitivity to morphine-induced changes is greater in cultured dorsal root ganglion neurons from 10- compared to three-month-old rats. Hence, cultured dorsal root ganglion neurons can provide a model to investigate the cellular and molecular mechanisms underlying alterations in neuropeptide levels following repeated exposure to opiates and their relevance to the development of opioid tolerance.     

Survival and phenotypic characterisctic of axotomized dorsal root ganglion neurons

Using indirect inmunohistochemical method, the expression of Bcl-XL and Bax, anti- and proapoptotic proteins of Bcl-2 family, as well as of cytokine IL-1beta were studied to demonstrate the role of these substances in apoptosis regulation of sensory neurons of different subpopulations after the severance of their peripheral processes. For comparison of the capacity of these neurons to survive after central and peripheral axotomy, the expression of high-molecular component of neurofilament triplet NF200 and isolectin B4 (IB4) binding were studied. At day 30 after central axotomy, the total number of neurons in LIV-LV ganglia of rat was not changed, but the number of NF200+ neurons was decreased. In mouse Lv dorsal root, proapoptotic BaX protein was demonstrated in the nuclei of 46% of small neurons that accounts for 20% of the total neuronal number in this ganglion. By day 30 after the nerve crush separate Bax expression was found in the nuclei of 30% and in the cytoplasm of 20% of neurons. In intact animals, dorsal root ganglion antiapoptotic Bcl-XL protein was expressed in the cytoplasm of 30% of small neurons, as well as in satellite cells surrounding large and intermediate neurons. At day 30 after the nerve crush Bcl-XL expression in LV ganglion was not detected. IL-1beta was present in the cytoplasm of 17% of neurons belonging predominantly to the subpopulations of large and intermediate neurons. By day 30 after the nerve crush IL-1(+-neurons were not found.     

Modulation of action potential firing by iberiotoxin and NS1619 in rat dorsal root ganglion neurons

The present study investigated the effects of iberiotoxin (IbTx), a peptide toxin blocker of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels and NS1619, a BK(Ca) channel opener, on action potential firing of small and medium size afferent neurons from L6 and S1 dorsal root ganglia of adult rats. Application of IbTx (100 nM) reduced whole-cell outward currents in 67% of small and medium size neurons. Analysis of action potential profile revealed that IbTx significantly prolonged the duration of action potential and increased firing frequency of afferent neurons. IbTx did not significantly alter the resting membrane potential, threshold for action potential activation and action potential amplitude. The benzimidazolone NS1619 (10 microM) increased opening activity of a Ca(2+)-dependent channel as assessed by single channel measurements. In contrast to IbTx, NS1619 reversibly suppressed action potential firing, attributable to increases in threshold for evoking action potential, reduction in action potential amplitude and increases in amplitude of afterhyperpolarization. The effect of NS1619 on neuronal firing was sensitive to IbTx, indicating the attenuation of neuronal firing by NS1619 was mediated by opening BK(Ca) channels. NS1619 also reduced neuronal hyperexcitability evoked by 4-aminopyridine (4-AP), a transient-inactivated K(+) channel (A-current) blocker, in an IbTx-sensitive manner.These results indicate that IbTx-sensitive BK(Ca) channels exist in both small and medium diameter dorsal root ganglion (DRG) neurons and play important roles in the repolarization of action potential and firing frequency. NS1619 modulates action potential firing and suppresses 4-AP-evoked hyperexcitability in DRG neurons, in part, by opening BK(Ca) channels. These results suggest that opening BK(Ca) channels might be sufficient to suppress hyperexcitability of afferent neurons as those evoked by stimulants or by disease states.     

P2X receptor-mediated ionic currents in dorsal root ganglion neurons.

Nociceptive neurons in the dorsal root ganglia (DRG) are activated by extracellular ATP, implicating P2X receptors as potential mediators of painful stimuli. However, the P2X receptor subtype(s) underlying this activity remain in question. Using electrophysiological techniques, the effects of P2X receptor agonists and antagonists were examined on acutely dissociated adult rat lumbar DRG neurons. Putative P2X-expressing nociceptors were identified by labeling neurons with the lectin IB4. These neurons could be grouped into three categories based on response kinetics to extracellularly applied ATP. Some DRG responses (slow DRG) were relatively slowly activating, nondesensitizing, and activated by the ATP analogue alpha,beta-meATP. These responses resembled those recorded from 1321N1 cells expressing recombinant heteromultimeric rat P2X2/3 receptors. Other responses (fast DRG) were rapidly activating and desensitized almost completely during agonist application. These responses had properties similar to those recorded from 1321N1 cells expressing recombinant rat P2X3 receptors. A third group (mixed DRG) activated and desensitized rapidly (P2X3-like), but also had a slow, nondesensitizing component that functionally prolonged the current. Like the fast component, the slow component was activated by both ATP and alpha, beta-meATP and was blocked by the P2X antagonist TNP-ATP. But unlike the fast component, the slow component could follow high-frequency activation by agonist, and its amplitude was potentiated under acidic conditions. These characteristics most closely resemble those of rat P2X2/3 receptors. These data suggest that there are at least two populations of P2X receptors present on adult DRG nociceptive neurons, P2X3 and P2X2/3. These receptors are expressed either separately or together on individual neurons and may play a role in the processing of nociceptive information from the periphery to the spinal cord.     

Peripheral nerve injury decreases the expression of metabolic glutamate receptor 7 in dorsal root ganglion neurons

Group II and III metabolic glutamate receptors (mGluRs) are responsible for the glutamate-mediated postsynaptic excitation of neurons. Previous pharmacological evidences show that activation of mGluR7 could inhibit nociceptive reception. However, the distribution and expression patterns of mGluR7 after peripheral injury remain unclear. Herein we found that mGluR7 was expressed in the rat peptidergic dorsal root ganglion (DRG) neurons and large neurons, but rarely in isolectin B4 positive neurons. Sciatic nerve ligation experiment showed that mGluR7 was anterogradely transported from cell body to the peripheral site. Furthermore, after peripheral nerve injury, mGluR7 expression was down-regulated in both peptidergic and large DRG neurons. Our work suggests that mGluR7 might be involved in the regulation of pathological pain after peripheral nerve injury.     

Functional role of prostacyclin receptor in rat dorsal root ganglion neurons

Recent studies on prostanoids showed that some of prostanoid receptors are expressed in rat dorsal root ganglion (DRG) neurons. These facts suggest that prostanoid receptors might be involved in the excitation mechanism of DRG neurons. In the present study, PCR experiments revealed that one of prostanoid receptor, prostacyclin receptor (IP receptor) was expressed in L6 and S1 rat DRG neurons and that the expression of IP receptor was not changed in DRG neurons obtained from the cyclophosphamide (CYP)-induced cystitis rat. We examined the functional role of IP receptor agonist and other prostanoids by measuring cyclic AMP (cAMP) accumulation and substance P (SP) release in primary cultured DRG neurons. The pretreatment of DRG neurons with prostanoid agonists such as iloprost (IP), butaprost (EP(2)), misoprostol (EP(2-4)), PGE(2) (EP(1-4)) or PGD(2) (DP and CRTH2) sensitized the DRG neurons and hence potentiated the lys-bradykinin-induced SP release. The increase of SP release by lys-BK plus prostanoid agonists was proportion to cAMP accumulation. Iloprost was the most potent agonist to induce cAMP accumulation and SP release among prostanoid agonists evaluated in this study and its effect is mediated by IP receptor. Moreover, capsaicin-, ATP- and KCl-induced SP release was also enhanced by iloprost although iloprost did not change intracellular Ca(2+) and membrane depolarization induced by these chemical stimuli. These results strongly indicate that IP receptor play an important role in the sensitization of rat sensory neuron.     

小鼠背根神经节中痒觉神经元的分布特性

目的观察小鼠背根神经节(DRG)中痒觉特异性Mrgpr A3+神经元的分布特征。方法采用遗传学方法将Mrgpr A3+神经元特异性标记强化绿色荧光蛋白(EGFP)和td Tomato;选取3只纯合Mrgpra3EGFP-Cre;ROSA26td Tomato成年转基因小鼠,分离皮肤和背根神经节组织;采用激光扫描共焦成像技术观察Mrgpr A3+神经元的外周神经纤维在小鼠躯体皮肤的投射分布特征;采用双光子成像技术观察Mrgpr A3+神经元在整体背根神经节中的三维空间分布情况。结果脸颊、背部和脚掌皮肤的Mrgpr A3+神经纤维分布密集,粗且长,分布广泛;颈部和腹部皮肤的Mrgpr A3+神经纤维分布稀疏,短且小,呈散点状分布;Mrgpr A3+神经纤维在皮肤的有毛和无毛区域都有投射分布,且不同部位分布特点不同;几乎所有的Mrgpr A3+神经元都为小直径感觉神经元,且在颈、胸、腰、尾段背根神经节中均有分布;颈段、胸段、腰段和尾段的Z轴成像深度分别为350μm、250μm、400μm和200μm;躯体不同部位背根神经节中的Mrgpr A3+神经元的三维空间分布在不同节段存在明显差异。结论小鼠躯体不同部位皮肤的Mrgpr A3+神经纤维的分布特征和整体背根神经节中Mrgpr A3+神经元的三维空间分布特征都存在较大差异,痒觉神经元和末梢分布的差异可能是不同区域存在痒觉生理反应差异的结构基础。     

5HT increases excitability of nociceptor-like rat dorsal root ganglion neurons via cAMP-coupled TTX-resistant Na(+) channels.

The physiological effects of 5HT receptor coupling to TTX-resistant Na(+) current, and the signaling pathway involved, was studied in a nociceptor-like subpopulation of rat dorsal root ganglion (DRG) cells (type 2), which can be identified by expression of a low-threshold, slowly inactivating A-current. The 5HT-mediated increase in TTX-resistant Na(+) current in type 2 DRG cells was mimicked and occluded by 10 microM forskolin. Superfusion of type 2 DRG cells on the outside with 1 mM 8-bromo-cAMP or chlorophenylthio-cAMP (CPT-cAMP) increased the Na(+) current, but less than 5HT itself. However, perfusion of the cells inside with 2 mM CPT-cAMP strongly increased the amplitude of control Na(+) currents and completely occluded the effect of 5HT. Thus it appears that the signaling pathway includes cAMP. The phosphodiesterase inhibitor 3-isobutyl-L-methylxanthine (200 microM) also mimicked the effect of 5HT on Na(+) current, suggesting tonic adenylyl cyclase activity. 5HT reduced the amount of current required to evoke action potentials in type 2 DRG cells, suggesting that 5HT may lower the threshold for activation of nociceptor peripheral receptors. The above data suggest that serotonergic modulation of TTX-resistant Na(+) channels through a cAMP-dependent signaling pathway in nociceptors may participate in the generation of hyperalgesia.     

Pb2+ inhibits the hyperpolarization-activated current in acutely isolated dorsal root ganglion neurons

The hyperpolarization-activated h channel current (Ih) reported to be present in acutely isolated rat dorsal root ganglion (DRG) neurons is inhibited by Cs+ and ZD7288. It was recently reported that lead (Pb2+) inhibits voltage-gated Ca2+ and K+ channels in DRG neurons but the effect of Pb2+ on Ih has so far not been reported. Using whole-cell patch clamp technique we show that Pb2+ specifically inhibited Ih. External application of 0.1, 1 and 10 microM Pb2+ reversibly reduced the amplitude of Ih in a dose-dependent manner, with an IC50 value of 3.7 microM and a Hill coefficient of 1.1. Pb2+ shifted the activation curve of Ih by 9.3 mV but had no effect on the slope factor. Pb2+ inhibited Ih in a voltage-dependent manner and slowed down the activation process, indicating an action of Pb2+ on the activation kinetics of h channels. Our studies thus demonstrated that Pb2+ is a dose-dependent, voltage-dependent and reversible blocker of Ih in DRG neurons.