The Plasticity of the DRG Neurons Belonging to Different Subpopulations After Dorsal Rhizotomy

SUMMARY 1. The plasticity of sensory neurons following the injury to their axons is very important for prognosis of recovery of afferent fibers with different modality. It is evident that the response of dorsal root ganglion (DRG) neurons after peripheral axotomy is different depending on the deficiency in neurotrophic factors from peripheral region. The loss of cells appears earlier and is more severe in B-cells (small, dark cells with unmyelinated axons) than in A-cells (large, light cells with myelinated axons).2. We studied using immunohistochemical methods the response of DRG neurons to dorsal rhizotomy and combined injury of central and peripheral neuronal processes. A quantitative analysis of DRG neurons tagged by the selective markers isolectin B4 (IB4) and the heavy molecular component of the neurofilament triplet (NF200) antibody, selective for subpopulations of small and large/medium DRG neurons, respectively, was performed after dorsal rhizotomy, peripheral axotomy, and their combination.3. The number of NF200⁺-neurons is reduced substantially after both dorsal rhizotomy and peripheral axotomy, while the decrease of IB4⁺-neurons is observed only in combined injury, i.e., dorsal rhizotomy accompanied with sciatic nerve injury.4. Our results show that distinct subpopulations of DRG neurons respond differently to the injury of their central processes. The number of NF200⁺-neurons decreases to greater degree following dorsal rhizotomy in comparison to IB4⁺-neurons.     

鸡胚脊髓背角中神经营养活性物质的初步分离和检测

从9d龄鸡胚脊髓背、腹角组织提取液中,用SephadexC-75凝胶层析法分离出DⅠ和DⅡ,VⅠ和VⅡ各组蛋白质组分,体外培养检测各组分对鸡胚背根节的神经营养活性,结果表明,DⅡ组分具有明显的促背根节神经突起生长的作用,而DⅠ组分无此作用;经SDS-PAGE电泳检测,DⅡ组分中蛋白质的分子量范围是61-15kD。而腹角的分离组分VⅠ和VⅡ对背根神经节神经突起的生长并无明显作用。     

Hyperexcitable neurons and altered non-neuronal cells in the compressed spinal ganglion

The cell body or soma in the dosal root ganglion (DRG) is normally excitable and this excitability can increase and persist after an injury of peripheral sensory neurons. In a rat model of radicular pain, an intraforaminal implantation of a rod that chronically compressed the lumbar DRG ("CCD" model) resulted in neuronal somal hyperexcitability and spontaneous activity that was accom-panied by hyperalgesia in the ipsilateral hind paw. By the 5th day after onset of CCD, there was a novel upregulation in neuronal expression of the chemokine, monocyte chemoattractant protein-1 (MCP- 1 or CCL2) and also its receptor, CCR2. The neurons developed, in response to topically applied MCP-1, an excitatory response that they normally do not have. CCD also activated non-neuronal cells including, for example, the endothelial cells as evidenced by angiogenesis in the form of an increased number of capillaries in the DRG after 7 days. A working hypothesis is that the CCD induced changes in neurons and non-neuronal cells that may act together to promote the survival of the injured tissue. The release of ligands such as CCL2, in addition to possibly activating nociceptive neurons (maintaining the pain), may also act to preserve injured cells in the face of ischemia and hypoxia, for example, by promoting angiogenesis. Thus, somal hyperexcitability, as often said of inflammation, may represent a double edged sword.     

Segmental independence and age dependence of neurite outgrowth from embryonic chick sensory neurons

Targets in limb regions of the chick embryo are further removed from the dorsal root ganglia that innervate them compared with thoracic ganglion-to-target distances. It has been inferred that axons grow into the limb regions two to three times faster than into nonlimb regions. We tested whether the differences were due to intrinsic properties of the neurons located at different segmental levels. Dorsal root ganglia (DRG) were isolated from the forelimb, trunk, and hind limb regions of stage 25–30 embryos. Neurite outgrowth was measured in dissociated cell culture and in cultures of DRG explants. Although there was considerable variability in the amount of neurite outgrowth, there were no substantive differences in the amount or the rate of outgrowth comparing brachial, thoracic, or lumbosacral neurons. The amount of neurite outgrowth in dissociated cell cultures increased with the stage of development. Overall, our data suggest that DRG neurons express a basal amount of outgrowth, which is initially independent of target-derived neurotrophic influences; the magnitude of this intrinsic growth potential increases with stage of development; and the neurons of the DRG are not intrinsically specified to grow neurites at rates that are matched to the distance they are required to grow to make contact with their peripheral targets in vivo. We present a speculative model based on Poisson statistics, which attempts to account for the variability in the amount of neurite outgrowth from dissociated neurons. © 1995 John Wiley & Sons, Inc.     

蟾蜍离体脊神经节神经元对其外周突与中枢突重复刺激的反应

本工作用细胞内记录技术记录了18个蟾蜍离体背根节神经元对其外周突与中枢突刺激的反应。1-2Hz刺激背根和坐骨神经引起的背根节细胞的动作电位参数相同。随着刺激频率增高,30％的细胞对背根和坐骨神经刺激同步地发生动作电位振幅降低、波形分解和脱失;70％的细胞也产生不同程度的变化。背根节细胞对背根和坐骨神经重复刺激不能跟随的频率分别平均为126 Hz和323Hz。结果表明,本工作所记录的背根节细胞为A型神经元,其中枢突的直径小于外周突。对上述变化的可能机制,文中也做了讨论。     

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。     

Voltage and Use Dependence of Sodium Channel Blockade by a New Analgesic,D57, in Rat Dorsal Root Ganglion Neurons

We studied the voltage- and use-dependent action of pyrrolo-imidazole derivative, D57, on sodium currents in different dorsal root ganglion neurons of rats. At the level of 50% of maximum tonic block, which corresponded to a concentration of 0.44 mM, the use-dependent block of tetrodotoxin resistant (TTXr) sodium currents reached 59 ± 12% of the remaining currents when neurons were stimulated by 6-msec-long impulses up to -10 mV with a 20 sec^-1 frequency, whereas for TTX sensitive (TTXs) currents this value was equal to 38 ± 9%. This block was dependent on the holding potential, and for cells with only TTXr currents the dependence was shifted to more positive potentials compared with that for neurons with only TTXs currents or with both of them.     

Effects of Gabapentin on Calcium Transients in Neurons of the Rat Dorsal Root Ganglia

In our experiments on rat dorsal root ganglia (DRG) neurons, we studied the effects of an antiepileptic agent, gabapentin, on calcium transients evoked by depolarization of the membrane using the fluorescence calciumsensitive dye Fura-2/AM. Application of gabapentin to neurons with large-diameter somata practically did not change the characteristics of calcium transients. In mid-sized neurons, the amplitude of transients decreased, on average, by 27% with respect to the control, while in small-sized neurons the transients changed insignificantly (on average, less than by 7%). The mid-sized neurons were additionally subjected to the capsaicin test, which allowed us to differentiate primary nociceptive neurons of this group where TRPV1-type channels are expressed. In capsaicin-sensitive neurons, application of gabapentin led to a decrease in the amplitude of calcium transients, on average, by 37%, while such a decrease was only 16% in capsaicininsensitive neurons. Based on our own data and findings of other researchers on the ability of gabapentin to demonstrate affine binding with the accessory α₂δ subunit of voltage-dependent calcium channels and also on the peculiarities of expression of these channels in somatosensory neurons of the corresponding types, we discuss the probable pattern of expression of subunits of the α₂δ-1 subtype in DRG cells of different sizes. We demonstrated that the effects of gabapentin on calcium transients in nociceptive and hypothetically nonnociceptive mid-sized DRG neurons are selective (the effects in neurons involved in the sensation of acute pain are probably more intense). Neirofiziologiya/Neurophysiology, Vol. 40, No. 4, pp. 281–287, July–August, 2008.     

缓激肽对大鼠背根神经节分离神经元ATP激活电流的调制作用

在新鲜分离大鼠背根神经节（ＤＲＧ）的５６个细胞标本上,应用全细胞膜片箝技术进行记录。胞外加缓激肽（ＢＫ,１０＾－６￣１０＾－４ｍｏｌ／Ｌ）引坊的ＤＲＧ细胞膜反应结果如下：（１）７１．４％的细胞为内向电流,其电流反应的幅值具有明显的浓度信赖性;（２）１２．５％的细胞为外向电流;（３）１６．１％的细胞未引起可检测的膜反应,单独给予ＡＴＰ（１０＾－６￣１０＾－３ｍｏｌ／Ｌ）在大多数受栓细胞（５４／５６）     

Changes in skin levels of two neutotrophins （glial cell line derived neurotrohic factor and neurotrophin-3） cause alterations in cutaneous neuron responses to mechanical stimuli

Neurotrophins are important for the development and maintenance of both high and low threshold mechanoreceptors (HTMRs and LTMRs). In this series of studies, the effects of constitutive overexpression of two different neurotrophins, neurotrophin-3 (NT-3) and glial cell line derived neurotrohic factor (GDNF), were examined. Previous studies indicated that both of them may be implicated in the normal development of mouse dorsal root ganglion (DRG) neurons. Neurons from mice transgenically altered to overexpress NT-3 or GDNF (NT-3-OE or GDNF-OE mice) in the skin were examined using several physiological, immunohistochemi-cal and molecular techniques. Ex vivo skin/nerve/DRG/spinal cord and skin/nerve preparations were used to determine the response characteristics of the cutaneous neurons; immunohistochemistry was used to examine the biochemical phenotype of DRG cells and the skin; RT-PCR was used to examine the levels of candidate ion channels in skin and DRG that may correlate with changes in physiologi-cal responses. In GDNF-OE mice, I-isolectin B4 (IB4)-immunopositive C-HTMRs (nociceptors), a large percentage of which are sensitive to GDNF, had significantly lower mechanical thresholds than wildtype (WT) neurons. Heat thresholds for the same cells were not different. Mechanical sensitivity changes in GDNF-OE mice were correlated with significant increases in acid sensing ion channels 2a (ASIC2a) and 2b (ASIC2b) and transient receptor potential channel AI (TRPAI), all of which are putative mechanosensitive ion channels. Overexpression of NT-3 affected the responses of A-LTMRs and A-HTMRs, hut had no effect on C-HTMRs. Slowly adapting type 1 (SA1) LTMRs and A-HTMRs had increased mechanical sensitivity compared to WT. Mechanical sensitivity was correlated with significant increases in acid-sensing ion channels ASIC1 and ASIC3. This data indicates that both neurotrophins play roles in determining mechanical thresholds of cutaneous HTMRs and LTMRs and that sensitivity changes involve the ASIC family of putative mechanoreceptive ion channels.     

The Effect of Nimodipine on Calcium Homeostasis and Pain Sensitivity in Diabetic Rats

Summary 1. The pathogenesis of diabetic neuropathy is a complex phenomenon, the mechanisms of which are not fully understood. Our previous studies have shown that the intracellular calcium signaling is impaired in primary and secondary nociceptive neurons in rats with streptozotocin (STZ)-induced diabetes. Here, we investigated the effect of prolonged treatment with the L-type calcium channel blocker nimodipine on diabetes-induced changes in neuronal calcium signaling and pain sensitivity.2. Diabetes was induced in young rats (21 p.d.) by a streptozotocin injection. After 3 weeks of diabetes development, the rats were treated with nimodipine for another 3 weeks. The effect of nimodipine treatment on calcium homeostasis in nociceptive dorsal root ganglion neurons (DRG) and substantia gelatinosa (SG) neurons of the spinal cord slices was examined with fluorescent imaging technique.3. Nimodipine treatment was not able to normalize elevated resting intracellular calcium ([Ca²⁺] _i ) levels in small DRG neurons. However, it was able to restore impaired Ca²⁺ release from the ER, induced by either activation of ryanodine receptors or by receptor-independent mechanism in both DRG and SG neurons.4. The beneficiary effects of nimodipine treatment on [Ca²⁺] _i signaling were paralleled with the reversal of diabetes-induced thermal hypoalgesia and normalization of the acute phase of the response to formalin injection. Nimodipine treatment was also able to shorten the duration of the tonic phase of formalin response to the control values.5. To separate vasodilating effect of nimodipine Biessels et al., (Brain Res. 1035:86–93) from its effect on neuronal Ca²⁺ channels, a group of STZ-diabetic rats was treated with vasodilator – enalapril. Enalapril treatment also have some beneficial effect on normalizing Ca²⁺ release from the ER, however, it was far less explicit than the normalizing effect of nimodipine. Effect of enalapril treatment on nociceptive behavioral responses was also much less pronounced. It partially reversed diabetes-induced thermal hypoalgesia, but did not change the characteristics of the response to formalin injection.6. The results of this study suggest that chronic nimodipine treatment may be effective in restoring diabetes-impaired neuronal calcium homeostasis as well as reduction of diabetes-induced thermal hypoalgesia and noxious stimuli responses. The nimodipine effect is mediated through a direct neuronal action combined with some vascular mechanism.     

Amygdala, an important regulator for food intake

Amygdala plays a critical role in the regulation of emotional behavior and food intake. Neuropeptides are short chains of amino acids secreted by neurons as intercellular messengers, which regulate different functions such as emotion, food intake, learning and memory. In this review, we summarize the recent progress on the regulation of food intake by amygadala, which is mediated by those neuropeptides known to be critical in the regulation of this process.     

咖啡因对大鼠背根神经节急性分离神经元GABA-激活电流的抑制作用

应用全细胞膜片钳记录技术,在大鼠新鲜分离背根神经节(dorsal root ganglion,DRG)神经元上,观察预加咖啡因对GABA-激活电流(IGABA)的调制作用。实验中,大部分受检细胞(97．4％,l13／116)对外加GABA敏感。1-1000μmol／L GABA引起一剂量依赖性、有明显上敏感作用的内向电流。在受检的108个DRG细胞中,约有半数(53．7％,58／108)对胞外加咖啡因(0．1-100μmol／L)敏感．产生一幅值很小的内向电流。倾加咖啡因(0．1～100μmol／L)30s后再加GABA能明显抑制GABA(100μmol／L)激活电流的幅值。预加咖啡因后GABA量效曲线明显下移;GABA-激活电流的最人值较之对照下降约57％;而Kd值(30μmol／L)几乎不变,表示此种抑制为非竞争性的。预加安定(diazepam,1μmol／L)对GABA(100μmol／L)激活电流有增强作用,而预加咖啡因(10μmol／L)有拈抗安定增强IGABA的作用。胞内透析H-8后,几乎可以完全消除咖啡因对,IGABA的抑制作用。已知GABA作用于初级感觉神经元能引起初级传入去极化,因而实验结果提示,咖啡因有可能在初级传入末梢产生对抗突触前抑制的效应。     

Target specificity and size of avian sensory neurons supported in vitro by nerve growth factor,brain-derived neurotrophic factor,and neurotrophin-3

To obtain insight into which subpopulations of sensory neurons in dorsal root ganglia are supported by different neurotrophins, we retrogradely labeled cutaneous and muscle afferents in embryonic day 9 chick embryos and followed their survival in neuron-enriched cultures supplemented with either nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), or neurotrophin-3 (NT-3). We found that NGF is a wide survival factor for subpopulations of both cutaneous and muscle afferents, whereas the survival effects of BDNF and NT-3 are restricted primarily to muscle afferents. We also measured soma size in each neurotrophic factor. These new data show that BDNF- and NT-3–dependent cells appear to be a mixture of two populations of neurons: one small diameter and the other large diameter. In contrast, based on size alone, NGF-dependent cells appear to be a single population of only small-diameter neurons. Thus, BDNF and NT-3 may have some new, previously unreported effects on small-diameter afferent neurons. © 1994 John Wiley & Sons, Inc. 1994 John Wiley & Sons, Inc.