去甲肾上腺素对蟾蜍背根神经节细胞α-肾上腺素能受体的作用

在蟾蜍离体灌流背根神经节(DRG)标本上,用微电极进行细胞内记录。在51个细胞中A型神经元为46个,C型5个。此两类细胞的静息膜电位为60.06±1.34mV(x±SE)。当灌流液中滴加10~(-4)-10~(-3)mol/L去甲肾上腺素(NA)引起如下的膜电位改变:(1)超极化:幅值8.38±1.12mV(x±SE)(20/48);(2)去极化:幅值9.39±1.24mV(x±SE)(23/48);(3)无反应(5/48)。上述膜电位改变既不能由灌流液中滴加异丙基肾上腺素所拟似,也不能为心得安所阻断,因而排除了β-肾上腺能受体介导的可能性。加苯肾上腺素及可乐宁于灌流液,分别产生膜的去极化和超极化,而应用哌唑唪及育亨宾灌流,则分别阻断NA引起的膜去极化和超极化。因此认为:NA引起的DRG神经元的去极化和超极化反应分别是由胞体膜上之α_1-及α_2-肾上腺素能受体所介导的。     

乙酰胆碱对蟾蜍背根神经节神经元膜电位的影响及离子机制

在离体灌流的蟾蜍背根神经节(DRG)标本上,用微电极进行胞内记录。在73个神经元中,依神经纤维的传导速度将神经元分为 A 型及 C 型,其中 A 型细胞67个,C 型6个,静息膜电位为-67.5±1.3mV((?)±SE)。当加4×10~(-4)—6×10~(-4)mol/L 乙酰胆碱(ACh),可观察到如下四种膜电位变化:1.超极化:幅值9.1±3.0mV((?)±SE,n=23);(2)去极化:幅值12.9±2.2mV((?)+SE,n=20);(3)双相反应(n=24):先超极化,后去极化,超极化幅值8.0±2.4mV((?)+SE),去极化幅值10.9±3.1mV((?)±SE);(4)无反应(n=6)。用阿托品(1.3×10~(-5)mol/L,n=23),或同时应用筒箭毒与六甲双铵(浓度均为1.4×10~(-5)mol/L,n=8)灌流,能分别阻断 ACh 引起的膜的超极化或去极化。ACh 引起超极化反应时膜电导平均增加13.8%,翻转电位值大约-96mV。四乙铵(TEA,20mmol/L)能使 ACh 的去极化幅值增加48.2±3.2%((?)±SE,n=6),超极化幅值减小79.4±4.3%((?)±SE,n=8)。MnCl_2(4mmol/L)使 ACh 的去极化及超极化幅值分别减小54.2±7.2%((?)±SE,n=5)及69.2±6.4%((?)±SE,n=14)。以上结果提示:ACh 引起的 DRG 神经细胞膜去极化反应由 N 型乙酰胆碱受体介导,而超极化反应由 Μ 型乙酰胆碱受体介导,前者可能包含了多种离子电导的改变,后者则可能与钾电导增加有关。     

去甲肾上腺素引起的α1肾上腺素受体三种亚型脱敏过程的差异

将含有α１肾上腺素受体三种亚型的全长ｃＤＮＡ质粒分别转染到人胚肾脏细胞（ＨＥＫ２９３）,α１Ａ－,α１Ｂ－和α１Ｄ－ＡＲ在ＨＥＫ２９３细胞株上得到高水平稳定表达,用＾３Ｈ－ｉｎｏｓｉｔｏｌ标记和柱层析法测定细胞磷酸肌醇积。观察在去甲肾上腺素长期作用下α１三种受体亚型介导磷酸肌醇蓄积敏感性降低的差别。     

去甲肾上腺素通路的研究进展及其在迷走神经电刺激改善难治性癫痫学习记忆中的潜在作用

迷走神经电刺激(vagus nerve stimulation, VNS)对无法手术的耐药难治性癫痫患者能起到较好的抗癫痫效果,目前已被美国FDA批准用于耐药难治性癫痫的临床辅助性治疗。VNS的抗癫痫作用可长期维持,且治疗效果随刺激时间的延长而增加。有研究发现,VNS在减少癫痫发作的同时,还能改善癫痫患者的情绪状态,提高患者的认知能力。去甲肾上腺素能系统参与突触可塑性调节。本文综述了VNS改善难治性癫痫患者学习记忆能力的研究进展,重点讨论了去甲肾上腺素能系统调控突触可塑性变化的相关信号通路,提出VNS激活去甲肾上腺素能系统从而改善患者学习记忆的潜在可能性,为临床癫痫治疗提供新的靶点和思路。     

去甲肾上腺素对骨髓间充质干细胞增殖的影响

目的:观察去甲肾上腺素(norepinephrine,NE)对骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)增殖的影响及其作用途径.方法:分离培养正常大鼠BMSCs,采用3H-TdR掺入实验检测不同浓度的NE(10-7-10-4 M)作用8h及10-5M的NE作用不同时间(0-24h)BMSCs细胞增殖情况,real time RT-PCR检测肾上腺素能受体α1A-AR,α1B-AR和α1D-AR mRNA表达变化情况.结果:10-7-10-4M的NE作用8h后均促进了BMSCs细胞的增殖.并且在10-5M时NE对BMSCs的促增殖效应最为显著;正常组BMSCs细胞的α1A-AR,α1B-AR,α1D-AR mRNA表达维持在较低水平,加入10-5M的NE作用后α1-AR三个亚型mRNA表达水平均有不同程度的升高(P<0.05).结论:NE能够促进BMSCs的增殖,并且这种促增殖作用是通过AR依赖的信号通路来调节的.     

背根神经节神经元阿片受体和离子通道的研究进展

阿片及阿片受体与外周神经系统镇痛机制的研究,随着分子生物学技术的发展,已在受体的分子结构、形态学、分子药理学、离子通道和细胞内信号转导系统等方面取得了显著进展。μ、δ、κ阿片受体分子结构上的部分差异决定了它们各自的功能特征。三种受体在初级感觉神经元分布的比例不同,但都能介导细胞Ｃａ＾２＋通道的抑制和Ｋ＾＋电流增加及减少。阿片受体和通道之间由多种第二信使系统偶联。分子药理学研究表明它们还存在亚型受体     

运动病的去甲肾上腺素能机制的探讨

近年来,拟交感药物防治运动病的作用愈来愈受到再视。有人认为用拟交感药防治运动病比用抗胆碱药有更多的优点。虽然近年来,关于拟交感药物抗运动病的应用研究已有不少报道,但有关机理的实验探讨则很少涉及。     

去甲肾上腺素转运体的研究进展

随着分子克隆技术的发展和应用,已经可以克隆出人的去甲肾上腺素转运体(Norepinephrine transporter,NET)基因转染进哺乳动物细胞内进行体外研究。去甲肾上腺素转运体在神经传递中有着非常重要的作用,许多神经以及精神系统方面的疾病,心血管疾病等都与去甲肾上腺素转运体的功能缺失或紊乱有关。本文主要介绍了去甲肾上腺素转运体的基本概念和近年来国外对去甲肾上腺素转运体的研究概况,综述了与该转运体相关的药物的研究进展以及由于去甲肾上腺素转运体的功能紊乱或丧失而导致的疾病的临床研究。最近几年对于NET基因表达调控以及各种临床疾病的研究对这些疾病的治疗方法的探索有着非常重要的意义。     

可乐定对背根神经节神经元GABA激活电流的抑制作用

本实验在新鲜分离大鼠背根神经节（ＤＲＧ）细胞上应用全细胞膜片的箝记录研究贤上腺素α２－受体激动剂可乐定（ｃｌｏｎｉｄｉｎｅ）对ＧＡＢＡ－激活电流的调制作用。发现缘大多数ＤＲＧ细胞对ＧＡＢＡ（１０＾－６￣１０＾－３ｍｏｌ／Ｌ）敏感（７２／７５）,产生浓度依赖性的内向电流;并且可被ｂｉｃｕｃｕｌｉｎｅ（１０＾－５￣１０＾－４ｍｏｌ／Ｌ）所阻断。在多数细胞中（５１／７２）预加可乐定（１０＾－８￣１０＾－     

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。     

Ionic basis of a mechanotransduction current in adult rat dorsal root ganglion neurons

Activity-dependent synaptic plasticity is known to be important in learning and memory, persistent pain and drug addiction. Glutamate NMDA receptor activation stimulates several protein kinases, which then trigger biochemical cascades that lead to modifications in synaptic efficacy. Genetic and pharmacological techniques have been used to show a role for Ca²⁺/calmodulin-dependent kinase II (CaMKII) in synaptic plasticity and memory formation. However, it is not known if increasing CaMKII activity in forebrain areas affects behavioral responses to tissue injury. Using genetic and pharmacological techniques, we were able to temporally and spatially restrict the over expression of CaMKII in forebrain areas. Here we show that genetic overexpression of CaMKII in the mouse forebrain selectively inhibits tissue injury-induced behavioral sensitization, including allodynia and hyperalgesia, while behavioral responses to acute noxious stimuli remain intact. CaMKII overexpression also inhibited synaptic depression induced by a prolonged repetitive stimulation in the ACC, suggesting an important role for CaMKII in the regulation of cingulate neurons. Our results suggest that neuronal CaMKII activity in the forebrain plays a role in persistent pain.     

Matrix-mediated gene transfer to brain cortex and dorsal root ganglion neurones by retrograde axonal transport after dorsal column lesion

BACKGROUND: In previous studies, we showed that the immobilisation of DNAs encoding basic fibroblast growth factor, neurotrophin-3 and brain-derived neurotrophic factor in a gene-activated matrix (GAM) promotes sustained survival of axotomised retinal ganglion cells after optic nerve injury. Here, we evaluated if the immobilisation of DNAs in a GAM could be an effective approach to deliver genes to axotomised dorsal root ganglion (DRG) neurones after spinal cord injury and if the matrix component of the GAM would modulate the deposition of a dense scar at the injury site. METHODS: We evaluated the expression of the thymidine kinase (TK) reporter gene in brain cortex and DRG after a bilateral T8 dorsal column (DC) lesion using PCR, RT-PCR and in situ hybridisation analyses. Collagen-based GAMs were implanted at the lesion site and the cellular response to the GAM was assessed using cell-specific markers. RESULTS: At 1 week post-injury, PCR analyses confirmed that DNATK was retrogradely transported from the DC lesion where the GAM was implanted to the brain cortex and to caudal DRG neurones, and RT-PCR analyses showed expression of mRNATK. At 7 weeks post-injury, DNATK was still be detected in the GAM and DRG. In situ hybridisation localised DNATK and mRNATK within fibroblasts, glia, endothelial and inflammatory cells invading the GAM and in DRG neurones. Interestingly, the presence of a GAM also reduced secondary cavitation and scar deposition at the lesion site. CONCLUSIONS: These results establish that GAMs act as bridging scaffolds in DC lesions limiting cavitation and scarring and delivering genes both locally to injury-reactive cells and distally to the cerebral cortex and to DRG neuronal somata through retrograde axonal transport.     

Immunoelectronmicroscopical localization of the three neurofilament triplet proteins along neurofilaments of cultured dorsal root ganglion neurones

Correlated immunofluorescence and electron microscopy was used to study neurofilament expression, organization and structure in cultured neurones of newborn rat dorsal root ganglia. The results extend previous immunofluorescent data subdividing the neurones into two main classes: neurones rich in neurofilaments, expressing all three triplet proteins and neurones without noticeable neurofilaments which cannot be stained positively for any of the triplet proteins. The two classes are identified as the large light cells and small dark cells characteristically found in adult dorsal root ganglia in situ. Further ultrastructural characterization identifies the various subclasses of each major class in the cultures used. Cytoskeletons of neurofilament-rich neurones decorated by antibodies specific for each triplet protein lead to the following model. All three triplet proteins are associated with each individual filament, although the antibodies show a different localization. Whereas the 68K protein seems to form the backbone of the filament, the 200K protein is periodically arranged (repeat approx. 100 nm) in a more peripheral position. The 145 K protein is revealed in a nearly continuous manner along the filament.     

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.     

Acute and long-term effects of IL-6 on cultured dorsal root ganglion neurones from adult rat

IL-6 contributes to pain and hyperalgesia in inflamed tissue. We have investigated short- and long-term effects of IL-6 on dorsal root ganglion (DRG) neurones. Glycoprotein 130-like immunoreactivity (the signal transduction receptor subunit) was found in almost all neurones in DRG sections and in cultured DRG neurones from adult rat. In calcium-imaging studies bath application of IL-6 caused an increase of intracellular calcium in about one-third of the DRG neurones suggesting functional IL-6 receptors in a proportion of neurones. Long-term but not short-term exposure of DRG neurones to IL-6 in vitro significantly enhanced the proportion of DRG neurones expressing neurokinin 1 receptor-like immunoreactivity from 10% to up to 40%. This up-regulation was dependent on the activation of mitogen-activated protein kinase kinase (MEK) in the neurones, suggesting that the mitogen-activated protein kinase (MAPK) pathway is important for this effects of IL-6. Calcium-imaging studies demonstrated that previous exposure of DRG neurones to IL-6 enhanced the proportion of neurones that exhibit a substance P-induced rise in intracellular calcium. These data show that IL-6 has short- and long-term effects on a proportion of DRG neurones. These effects are likely to contribute to pro-nociceptive effects of IL-6.     

Phase-dependent changes in dorsal root potential during actual locomotion in rats

Fluctuations in dorsal root potential (DRP) were investigated in trials on white rats during two types of locomotion, differing in the intensity of afferent flow (swimming and walking). Two negative waves of DRP were observed corresponding to the stance (or propulsive) phase and the swing (or transfer) phase within a single locomotor cycle. Whereas DRP had risen primarily during the stroke phase with increased intensity during swimming, it increased during the standing phase in walking. A relationship was revealed between the amplitude of DRP and the intensity of afferent flow apparent during passive displacement of the limb, as well as locomotion. It is concluded that DRP waves are mainly due to influences from peripheral afferents during actual locomotion.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 20, No. 3, pp. 333–340, May–June, 1988.