GDNF对原代培养脊髓神经元的影响

本文研究了GDNF对体外培养各个时期的脊髓神经元的作用。通过MTT法检测GDNF对脊髓神经元存活率的影响,发现GDNF能促进培养7天及14天的神经元存活。 通过活体观察、尼氏染色、NSE免疫细胞化学染色观察GDNF对脊髓神经元生长锥数目、胞体大小、突起长度及分枝、侧棘形成的影响,发现GDNF对体外培养1—3周的脊髓神经元有明显的营养作用。     

GDNF对多巴胺能神经元作用机制的研究进展

胶质细胞源性神经营养因子（glial cell line-derived neurotrophic factor,GDNF）是神经保护治疗帕金森病（Parkinson＇s disease,PD）的一种神经营养因子,越来越多的在体和离体实验研究显示GDNF是中脑多巴胺（dopaminergic neuron,DA）能神经元的有效存活因子。GDNF受体是由结合在细胞质膜外的糖基化磷酯酰基（glycosyl-phosphatidylinositol,GPI）和GDNF功能性孤儿受体酪氨酸激酶Ret蛋白质组成。特异性的GDNF与其受体结合后,激活其胞内部分c-Ret,经由不同的第二信使来传递信号发挥作用。主要可能的机制有顺式作用和反式作用。而探索GDNF促进中脑黑质DA能神经元再生修复的可能机制,为进一步深入研究GDNF的作用机制提供科学依据。     

周围神经损伤后外源性GDNF对神经元的保护作用

采用硅管套接大鼠切断的坐骨神经模型 ,局部给予胶质细胞源性神经营养因子 (GDNF) ,应用尼氏染色、酶组织化学染色方法 ,观察到外源性GDNF能减少脊髓修复侧前角运动神经元死亡的数目 ,降低脊髓前角运动神经元及脊神经节感觉神经元中胆碱酯酶 (CHE)及酸性磷酸酶 (ACP)变化的幅度。这表明外源性GDNF能保护周围神经切断后引起的神经元损伤。     

GDNF及BDNF对受损运动神经元的长期修复

为了研究胶质细胞源神经营养因子（ＧＤＮＦ） 及脑源神经营养因子（ＢＤＮＦ） 对切断轴突的新生运动神经元的长期维持存活及促进神经再生的作用, 我们选用出生时单侧切断坐骨神经的雏鸡模型, 用裸ＤＮＡ 转染方法, 在损伤神经附近的肌肉中转染ＧＤＮＦ ｃＤＮＡ 和ＢＤＮＦ ｃＤＮＡ 的真核表达载体,观察在体表达的神经营养因子对损伤的修复作用。结果显示,在体表达的ＧＤＮＦ 在８ 周内能使切断坐骨神经的腰脊髓运动神经元近９０ ％ 维持存活。切断的坐骨神经从断端向远体端再生,最长再生达９ ．５ｍ ｍ 。表达两个因子比单独表达ＧＤＮＦ 对运动神经元的存活无显著性差异。而两个因子协同作用对坐骨神经的再生更为有效,坐骨神经再生最长的可达１５ ．４ｍ ｍ 。     

体外培养的鸡胚神经元迁移的光镜和扫描电镜的研究

用鸡胚神经细胞为材料,建立神经细胞体外培养技术,相差显微镜观察到鸡胚神经元可以沿着神经胶质细胞纤维运动。扫描电子显微镜揭示体外培养神经元与胶质细胞的关系。     

调控RET依赖性GDNF信号通路对大鼠癫痫模型的影响

目的:探究双侧海马CA1区立体定向注射anti-GDNF抗体对匹鲁卡品诱导的大鼠癫痫模型的影响。方法:选择成年雄性SD大鼠60只,并随机分为3组,即假手术组(sham组,n=20)、癫痫模型组(model组,n=20)和GDNF抑制剂组(anti-GDNF组,n=20)。使用氯化锂-匹鲁卡品腹腔注射诱导癫痫模型,sham组只给予氯化锂,anti-GDNF组在造模前2 h给予大鼠双侧海马CA1区立体定向注射anti-GDNF抗体。在造模后1、3、7 d观察大鼠癫痫的发作频率,7 d后采用脑电图监测(EEG)测定脑电波的变化情况,通过免疫组化方法测定海马CA1区域神经元数量变化(Neu N表达水平),造模后1 d时使用western blot方法测定海马CA1区GDNF、RET和P53蛋白的表达。结果:Model组大鼠棘-慢波数量明显高于Sham组,anti-GDNF组以上指标较model组显著减少(P0.05);Model组海马CA1区神经元大量凋亡,但anti-GDNF组凋亡较model组显著减少(P0.05)。与Sham组比较,在癫痫发作后1 d,model组的GDNF、RET表达水平上调,P53表达水平下降(P0.05),而anti-GDNF组大鼠海马CA1区GDNF、RET表达较model组明显下调,P53表达水平显著上降(P0.05)。结论:双侧海马CA1区立体定向注射anti-GDNF抗体能够减少癫痫发作,并对海马神经元起到保护作用,可能与其抑制GDNF/RET/P53信号通路有关。     

胶质细胞源神经营养因子

胶质细胞源神经营养因子陈哲宇何成王成海（第二军医大学神经生物教研室,上海２００４３３）关键词胶质细胞源神经营养因子多巴胺能神经元运动神经元神经营养因子是指能够促进神经细胞存活、生长和分化的一类蛋白质。胶质细胞源神经营养因子（ＧＤＮＦ）,因其最初从大鼠...     

GDAF对原代培养脊髓神经元的影响

The effect of GDNF on long-term cultured spinal cord neurons was studied. GDNF could promote spinal cord neurons survival after 7 d or 14 d culture by MTT assay. The effect of GDNF on growth cones, neuron soma magnitude, neurite length and spines formulation of spinal cord neurons in cell culture was observed by phase microscopy, Nissl stain and NSE immunocytochemistry stain. The results indicated that GDNF had significant trophic effects on long-term cultured spinal cord neurons.     

胶质源性神经营养因子对不同组织细胞的影响

GDNF来自于小胶质神经元,首先作为中脑多巴胺能神经元的复活因子被发现,可促进细胞存活,并有增加多巴胺神经元细胞大小及轴突长度的作用。GDNF通过与锚定蛋白细胞表面受体糖基磷脂酰肌醇的相互作用来调节细胞活性。GDNF家族a-1受体,通过跨膜酪氨酸受体或者神经元细胞黏附分子,来促进细胞存活,神经突生长,以及突触发育。后续的研究提示,无论未成年还是成体大脑,GDNF对多种神经细胞都有复活的作用,并与一些周围神经复活、迁移、分化相关。不同的脑缺血实验模型均证实了外源性GDNF对于病灶部位及全脑的神经保护作用,包括局部应用营养因子,利用病毒载体运载GDNF基因以及移植表达GDNF的细胞。近来研究还证实,GDNF不仅对多巴胺能神经元,中枢和周围神经系统的运动、感觉神经元,以及自主神经元有营养和保护作用,对于非神经系统也有不同调节作用。本文将重点讨论这些GDNF作用的不同策略以及机制。     

GDNF家族成员及其受体的研究进展

胶质细胞源性神经营养因子(GDNF)家族是一类结构上属于转化生长因子-β（TGF-β）超家族的神经营养因子,目前包括GDNF,neurturin(NTN)和persephin(PSP)三种因子,它们在体内有着广泛的作用.近年来发现GDNF和NTN的受体均为多组分结构,由不同的糖基磷脂酰肌醇（GPI）蛋白和共享的跨膜酪氨酸激酶受体Ret蛋白构成.有关这一家族的因子及其受体的研究正在不断深入.     

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.     

Inositol trisphosphate-induced hyperpolarization in rat dorsal root ganglion neurons.

Inositol 1,4,5-trisphosphate (1,4,5-InsP3) was perfused into rat dorsal root ganglion (DRG) neurons by whole-cell patch-clamp electrodes, while measuring the membrane potential. This operation evoked a transient (2-3 min) membrane hyperpolarization of about -15 mV (from -42 mV) followed by a depolarization. The membrane hyperpolarization was abolished when 30 mM EGTA was perfused together with 1,4,5-InsP3 or when 0.2 mM quinine was added to the bath solution. The hyperpolarizing response was enhanced when a low-Ca2+ EGTA-free intracellular solution was used. Two InsP2 isomers induced a different response. Our results suggest that the hyperpolarization is due to 1,4,5-InsP3-induced Ca2+ release which may trigger Ca-sensitive K+ channels to open. Present results show that cultured DRG neurons are able to respond to 1,4,5-InsP3 perfusion in the whole-cell configuration.     

Effects of levobupivacaine and bupivacaine on intracellular calcium signaling in cultured rat dorsal root ganglion neurons

Bupivacaine and levobupivacaine have been shown to be effective in the treatment of pain as local anesthetics, although the mechanisms mediating their antinociceptive actions are still not well understood. The aim of this study was to investigate the effects of bupivacaine and levobupivacaine on intracellular calcium ([Ca²⁺]_i) signaling in cultured rat dorsal root ganglion (DRG) neurons. DRG neuronal cultures loaded with 5?μM Fura-2/AM and [Ca²⁺]_i transients for stimulation with 30?mM KCl (Hi K⁺) were assessed by using fluorescent ratiometry. DRGs were excited at 340 and 380?nm, emission was recorded at 510?nm, and responses were determined from the change in the 340/380 ratio (basal-peak) for individual DRG neurons. Data were analyzed by using Student’s t-test. Levobupivacaine and bupivacaine attenuated the KCl-evoked [Ca²⁺]_i transients in a reversible manner. [Ca²⁺]_i increase evoked by Hi K⁺ was significantly reduced to 99.9?±?5.1% (n?=?18) and 62.5?±?4.2% (n?=?15, P?<?0.05) after the application of 5 and 50?µM levobupivacaine, respectively. Bupivacaine also inhibited Hi K⁺-induced [Ca²⁺]_i responses, reduced to 98.7?±?4.8% (n?=?10) and 69.5?±?4.5% (n?=?9, P?<?0.05) inhibition of fluorescence ratio values of Hi K⁺-induced responses at 5 and 50?μM, respectively. Our results indicate that bupivacaine and levobupivacaine, with no significant differences between both agents, attenuated KCl-evoked calcium transients in a reversible manner. The inhibition of calcium signals in DRG neurons by levobupivacaine and bupivacaine might contribute to the antinociceptive effects of these local anesthetics.     

Effects of pentobarbital on purinergic P2X receptors of rat dorsal root ganglion neurons

Purinergic P2X receptors are ligand-gated ion channels that are activated by extracellular adenosine triphosphate (ATP) and are widely expressed not only in the central and peripheral nervous system but also in tissues throughout the body, playing an important role in the transfer of nociceptive information. Since the influence of barbiturates on P2X receptor subtypes is not known, we studied the effects of pentobarbital sodium (PB) on ATP responses in dorsal root ganglion (DRG) neurons. DRG neurons were dissected from 10- to 14-day-old rats and dissociated after enzyme treatment. Electrical measurements were performed using the nystatin-perforated patch recording mode under voltage-clamp conditions. Drugs were applied using the Y-tube method. ATP evoked three types of inward current at -60 mV: fast desensitizing, slow desensitizing, and mixed. The fast-type current was attributed to activation of P2X3 subtype and the slow type to the P2X2 subtype. PB suppressed the fast-type current in a concentration-dependent manner, while the slow type was slightly reduced. A noncompetitive inhibition was suggested by a downward shift of the ATP concentration-response curves. The current-voltage relationships showed inward rectification, and the extent of suppression was not affected by the holding potential. The reduction was greater in external solutions of higher pH. PB had subtype-specific effects on P2X receptors. The ionized form is likely to be responsible for the suppression of the P2X3 receptor current, which may result in a reduction of the excitability of central and peripheral neurons and may contribute to the anesthetic and analgesic actions of the agent.     

Radiation-induced apoptosis in dorsal root ganglion neurons

Ionizing radiation (IR) results in apoptosis in a number of actively proliferating or immature cell types. The effect of IR on rat dorsal root ganglion (DRG) neurons was examined in dissociated cell cultures. After exposure to IR, embryonic DRG neurons, established in cell culture for six days, underwent cell death in a manner that was dose-dependent, requiring a minimum of 8 to 16 Gy. Twenty-five per cent cell loss occurred in embryonic day 15 (E-15) neurons, grown in cell culture for 6 days (“immature”), and then treated with 24 Gy IR. In contrast, only 2% cell loss occurred in E-15 neurons maintained in culture for 21 days ("mature") and then treated with 24 Gy IR. Staining with a fluorescent DNA-binding dye demonstrated clumping of the nuclear chromatin typical of apoptosis. Initiation of the apoptosis occurred within 24 h after exposure to IR. Apoptosis was prevented by inhibition of protein synthesis with cycloheximide. Apoptosis induced by IR occurred more frequently in immature than in mature neurons. Immature DRG neurons have a lower concentration of intracellular calcium ([Ca2+]i) than mature neurons. Elevation of [Ca2+]i by exposure to a high extracellular potassium ion concentration (35 μM) depolarizes the cell membrane with a resultant influx of calcium ions. The activation of programmed cell death after nerve growth factor (NGF) withdrawal is inversely correlated with [Ca2+]i in immature DRG neurons. When treated with high extracellular potassium, these immature neurons were resistant to IR exposure in a manner similar to that observed in mature neurons. These data suggest that [Ca2+]i modulates the apoptotic response of neurons after exposure to IR in a similar manner to that proposed by the “Ca2+ setpoint hypothesis” for control of NGF withdrawal-induced apoptosis.     

The role of GDNF family ligand signalling in the differentiation of sympathetic and dorsal root ganglion neurons

The diversity of neurons in sympathetic ganglia and dorsal root ganglia (DRG) provides intriguing systems for the analysis of neuronal differentiation. Cell surface receptors for the GDNF family ligands (GFLs) glial cell-line-derived neurotrophic factor (GDNF), neurturin and artemin, are expressed in subpopulations of these neurons prompting the question regarding their involvement in neuronal subtype specification. Mutational analysis in mice has demonstrated the requirement for GFL signalling during embryonic development of cholinergic sympathetic neurons as shown by the loss of expression from the cholinergic gene locus in ganglia from mice deficient for ret, the signal transducing subunit of the GFL receptor complex. Analysis in mutant animals and transgenic mice overexpressing GFLs demonstrates an effect on sensitivity to thermal and mechanical stimuli in DRG neurons correlating at least partially with the altered expression of transient receptor potential ion channels and acid-sensitive cation channels. Persistence of targeted cells in mutant ganglia suggests that the alterations are caused by differentiation effects and not by cell loss. Because of the massive effect of GFLs on neurite outgrowth, it remains to be determined whether GFL signalling acts directly on neuronal specification or indirectly via altered target innervation and access to other growth factors. The data show that GFL signalling is required for the specification of subpopulations of sensory and autonomic neurons. In order to comprehend this process fully, the role of individual GFLs, the transduction of the GFL signals, and the interplay of GFL signalling with other regulatory pathways need to be deciphered.     

A few axonal proteins distinguish ventral spinal cord neurons from dorsal root ganglion neurons

A series of proteins putatively involved in the generation of axonal diversity was identified. Neurons from ventral spinal cord and dorsal root ganglia were grown in a compartmented cell-culture system which offers separate access to cell somas and axons. The proteins synthesized in the neuronal cell somas and subsequently transported into the axons were selectively analyzed by 2-dimensional gel electrophoresis. The patterns of axonal proteins were substantially less complex than those derived from the proteins of neuronal cell bodies. The structural and functional similarity of axons from different neurons was reflected in a high degree of similarity of the gel pattern of the axonal proteins from sensory ganglia and spinal cord neurons. Each axonal type, however, had several proteins that were markedly less abundant or absent in the other. These neuron-population enriched proteins may be involved in the implementation of neuronal diversity. One of the proteins enriched in dorsal root ganglia axons had previously been found to be expressed with decreased abundance when dorsal root ganglia axons were co-cultured with ventral spinal cord cells under conditions in which synapse formation occurs (P. Sonderegger, M. C. Fishman, M. Bokoum, H. C. Bauer, and P.G. Nelson, 1983, Science [Wash. DC], 221:1294-1297). This protein may be a candidate for a role in growth cone functions, specific for neuronal subsets, such as pathfinding and selective axon fasciculation or the initiation of specific synapses. The methodology presented is thus capable of demonstrating patterns of protein synthesis that distinguish different neuronal subsets. The accessibility of these proteins for structural and functional studies may contribute to the elucidation of neuron-specific functions at the molecular level.     

Effects of ion channel activity on development of dorsal root ganglion neurons

Studies of mouse dorsal root ganglion neurons in vitro demonstrate that ion channel function and regulation can influence a wide range of developmental processes. The work suggests that much as exposure to different trophic factors, the pattern of impulse activity a neuron experiences can have significant structural and functional effects during development. Studies concerning effects of ion channel activity on growth cone motility, axon fasciculation, synaptic plasticity, myelination, and intracellular signaling pathways regulating gene expression are presented in the context of changes in endogenous firing patterns during development. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 158–170, 1998     

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     

GPR35 is a functional receptor in rat dorsal root ganglion neurons

GPR35, previously an orphan G-protein coupled receptor, is a receptor for kynurenic acid. Here we examine the distribution of GPR35 in the rat dorsal root ganglion (DRG) and the effects of its selective activation. GPR35 was expressed predominantly by small- to medium-diameter neurons of the DRG. Many of these same neurons also expressed the transient receptor potential vanilloid 1 channel, a nociceptive neuronal marker. The GPR35 agonists kynurenic acid and zaprinast inhibited forskolin-stimulated cAMP production by cultured rat DRG neurons. Inhibition required G_i/o proteins as the effect was completely abolished by pretreatment with pertussis toxin. This is the first study to report the expression and function of GPR35 in rat nociceptive DRG neurons. We propose that GPR35 modulates nociception and that continued study of this receptor will provide additional insight into the role of kynurenic acid in pain perception.