缩宫素对大鼠背根神经节神经元胞体膜的作用

应用细胞内记录技术,观察了缩宫素(OXT,10 -8～10-5 mol/L)对2～3周龄大鼠背根神经节神经元的作用.在受检的92个细胞中,有71个神经元滴加缩宫素产生明显的超极化反应.在滴加10-5mol/L缩宫素后, 膜电导由平均的3.68×10-7S增加约21.43%.灌流平衡液中的NaCl以氯化胆碱置代或用C d2+阻断Ca2+通道后,OXT引起超极化反应的幅值无明显变化.当灌流平衡液中含10-2 mol/L四乙基碘化铵后,OXT引起的超极化反应幅值明显减小.     

背根神经节神经元胞体膜可能存在NMDA自身受体

应用经酶和机械消化分离的大鼠背根神经节（ＤＲＧ）神经元进行实验。首先运用全细胞膜片箝技术找到ＮＭＤＡ可引起一内向电流的神经元,此内向电流可被甘氨酸明显增强,并被ＮＭＤＡ受体的特异性拮抗剂ＡＰＶ完全阻断。随后,把经电生理技术证实膜上存在ＮＭＤＡ受体的这一ＤＲＧ神经元用微吸管转移到载玻片上,进行谷氨酸神经递质免疫组织化学检测。１０／１８的ＤＲＧ细胞呈免疫反应阳性,结果为ＤＲＧ神经元膜上存在ＮＭＤＡ自身受体提供了新证据。     

神经生长因子对培养的大鼠背根神经节神经元P物质释放的调节作用

目的观察神经生长因子（nerve growth factor, NGF）对原代培养的背根神经节（dorsal root ganglion, DRG）神经元中P物质（substance P, SP）的基础释放量和辣椒素诱发释放量的调节效应。方法将15 天胚龄的Wistar大鼠DRG神经元培养于含有不同浓度NGF的DMEM/F12培养液中,不含NGF的培养液培养的神经元作为对照。72小时后,用RT-PCR检测神经元中SP mRNA和辣椒素受体（vanilloid receptor 1, VR1）mRNA的表达,用放射免疫分析（radioimmunoassay,RIA）法检测SP的基础释放量和辣椒素（100 nmol/L）刺激10 min后的诱发释放量。结果SPmRNA和VR1 mRNA在NGF孵育的标本中表达增加,并与孵育液中NGF的浓度呈剂量依赖关系。SP的基础释放量和辣椒素诱发释放量在NGF孵育的标本中均增加,而且诱发释放量与NGF的浓度呈剂量依赖关系。结论NGF使DRG神经元SP的基础释放量和诱发释放量增加,表明NGF能增加初级传入神经元感受伤害刺激的敏感性,该效应可能与SP和VR1的mRNA表达增加有关。     

大鼠背根神经节细胞的分离及特性探讨

目的探讨大鼠背根神经节（dorsal root ganglion,DRG）细胞的分离方法以及细胞形态和电生理特征。方法采用显微外科技术获取大鼠DRG体,用双酶法急性分离大鼠DRG获得DRG细胞,全细胞膜片钳技术记录动作电位和钠电流。结果本实验能得到完整圆形或椭圆长条形的大鼠DRG体。正常的单个DRG细胞呈圆形或椭圆形,大小不等,胞膜清晰,折光性好,隐约可见细胞核。在DRG细胞上记录的动作电位都具有从0期到4期,呈正立锐角三角形,静息电位小,动作电位时程短。DRG细胞的钠通道最大电流密度在-30mV左右,几乎能被,TTX完全抑制,具有可逆性恢复。结论本实验采用分离方法简单易行,DRG细胞容易获得和辨认,适合膜片钳技术要求,电生理特征明确可靠,值得推崇。     

离体培养胎鼠背根神经节细胞的细胞内pH

用氢离子选择性玻璃微电极，测量了离体培养纯种胎鼠的背根神经节细胞的细胞内ｐＨ（ｐＨｉ），其值在７．０６～７．１２之间，不同大小的细胞ｐＨｉ值无显著差异，ｐＨｉ随培龄的延长逐渐增加，在培龄为１５ｄ时ｐＨｉ骤然升高，以后趋于稳定，结果表明，细胞的培龄达１４ｄ以上才能测得较稳定的ｐＨｉ值，本文还对如何测得稳定的ｐＨｉ值进行了讨论。     

鸡胚背根神经节神经元在体内培养的研究

目的建立一种易于观察、取材方便、价格低、实验周期短、经济实用的鸡胚背根神经节神经元(dorsal root gan-glions neuron,DRGn)体内培养的方法。方法摘取孵化13～15d间鸡胚的背根神经节,经胰蛋白酶消化分离等方法获得纯化的神经元,将其按一定浓度移植于载体的绒毛尿囊膜层后,继续培养。结果 DRGn在载体的绒毛尿囊膜层培养3～5d时,可观察到神经细胞仍存活;培养6～9d时,可观察到神经细胞的生长;培养11～13d时,可观察到神经网络形成。结论鸡胚背根神经节神经元能在鸡胚的绒毛尿囊膜层生长、分化及再聚集并发育成正常神经网络组织。     

皮质酮对大鼠背根神经节细胞钙电流的快速抑制作用

探讨了皮质酮(Corticosterone)对急性分离的大鼠背根神经节细胞(DRG神经元)上电压依赖性钙通道电流的快速作用及作用机制.实验采用全细胞膜片钳方法,结果显示三种浓度的皮质酮,10-7mol/L,10-9mol/L,10-12mol/L均可以在3～5 s的时间内,快速抑制DRG神经元上的电压敏感钙通道电流,其平均抑制的程度分别为48%,37%和25%.且这种快速抑制作用在吹药15 s后可以达到最大,停药20 s后钙电流基本恢复为原来的大小.在外液中加入500 nmol/L的PKC抑制剂BIS之后,皮质酮的对钙电流的快速抑制作用被阻断,提示这一作用可能与PKC信号转导途径有关.     

皮质酮对大鼠背根神经节细胞钙电流的快速抑制作用

探讨了皮质酮 (Corticosterone)对急性分离的大鼠背根神经节细胞 (DRG神经元 )上电压依赖性钙通道电流的快速作用及作用机制。实验采用全细胞膜片钳方法 ,结果显示三种浓度的皮质酮 ,10 7mol/L ,10 9mol/L ,10 12 mol/L均可以在 3～ 5s的时间内 ,快速抑制DRG神经元上的电压敏感钙通道电流 ,其平均抑制的程度分别为4 8% ,37%和 2 5 %。且这种快速抑制作用在吹药 15s后可以达到最大 ,停药 2 0s后钙电流基本恢复为原来的大小。在外液中加入 5 0 0nmol/L的PKC抑制剂BIS之后 ,皮质酮的对钙电流的快速抑制作用被阻断 ,提示这一作用可能与PKC信号转导途径有关。     

治疗浓度和剂量胶原酶对大鼠脊神经背根神经节安全性的病理研究

目的 从病理学角度阐述胶原酶化学髓核溶解术中治疗浓度和剂量胶原酶对大鼠脊神经背根神经节的安全性. 方法 对57只Sprague-Dawlcy雄性大鼠按随机配伍原则进行分组,分别为正常对照组(n=9);胶原酶手术急性损伤组(n=9)、亚急性损伤组(m=10)和慢性损伤组(n=7);生理盐水手术急性损伤组(n=7)、亚急性损伤组(n=8)和慢性损伤组(n=7).手术暴露大鼠左侧腰5背根神经节,分别在胶原酶手术各组局部滴注胶原酶1 mL(1200 U/4 mL),生理盐水手术各组局部滴注生理盐水1mL.分别于注药后1 h、7d、30d取包含背根神经节的一段神经做病理光镜和透射电子显微镜检测,比较各组的病理改变. 结果 光镜下各组背根神经节的细胞数、胞膜、胞浆、胞核形态和细胞间神经纤维、血管均无明显变化和差别.透射电镜下各组背根神经节的细胞数、细胞大体形态、胞膜和节内神经纤维亦无明显变化和差别,但胶原酶手术各组背根神经节细胞胞内细胞器超微结构与正常对照组和相应生理盐水手术各组比较有差别,表现为核仁部分偏向一侧.大量线粒体肿胀.部分嵴断裂和空泡形成.病理光镜和透射电镜均未见节细胞成群细胞坏死、细胞膜早期破裂和凋亡小体形成. 结论 胶原酶化学髓核溶解术中治疗浓度和剂量胶原酶对大鼠脊神经背根神经节细胞有一定的损伤.应慎重选择胶原酶的用药剂量和浓度,提高胶原酶临床应用的安全性.     

神经生长因子对培养的大鼠背根神经节神经元P物质释放的调节作用(英文)

目的观察神经生长因子(nerve growth factor, NGF)对原代培养的背根神经节(dorsal root ganglion, DRG)神经元中P物质(substance P, SP)的基础释放量和辣椒素诱发释放量的调节效应。方法将15 天胚龄的Wistar大鼠DRG神经元培养于含有不同浓度NGF的DMEM/F12培养液中,不含NGF的培养液培养的神经元作为对照。72小时后,用RT-PCR检测神经元中SP mRNA和辣椒素受体(vanilloid receptor 1, VR1)mRNA的表达,用放射免疫分析(radioimmunoassay,RIA)法检测SP的基础释放量和辣椒素(100 nmol/L)刺激10 min后的诱发释放量。结果SPmRNA和VR1 mRNA在NGF孵育的标本中表达增加,并与孵育液中NGF的浓度呈剂量依赖关系。SP的基础释放量和辣椒素诱发释放量在NGF孵育的标本中均增加,而且诱发释放量与NGF的浓度呈剂量依赖关系。结论NGF使DRG神经元SP的基础释放量和诱发释放量增加,表明NGF能增加初级传入神经元感受伤害刺激的敏感性,该效应可能与SP和VR1的mRNA表达增加有关。     

腺苷对大鼠背根节神经元膜电位及动作电位的影响

在离体灌流的大鼠背根神经节（ＤＲＧ）－脊髓标本上，用微电极进行了胞内记录。在１５７个神经元中，依神经纤维的传导速度将神经元分为Ａ类和Ｃ类，其中Ａ类７５个，Ｃ类８２个。当浴槽中滴加０．５×１０－６ｍｏｌ／Ｌ的２－氯腺苷（ＣＡＤＯ）后，６６．７％（１０／１５）的Ｃ类细胞发生了去极化；而只有３３．３％（４／１２）的Ａ类细胞发生了去极化，且其去极幅值及持续时间均小于Ｃ类细胞的效应。先加入２．５×１０－６ｍｏｌ／Ｌ的８－苯茶碱（８—ＰＴ），再给予ＣＡＤＯ，８５．７％（６／７）的Ｃ类细胞的静息电位无变化，只有１４．３％（１／７）的Ｃ类细胞出现了暂短的去极化效应。另外，给予ＣＡＤＯ后，５８．３％（７／１２）的Ｃ类细胞动作电位幅值减少，有些细胞伴有膜的去极化；但Ａ类细胞的动作电位幅度末见明显变化。结果表明：腺苷主要引起ＤＲＧ中Ｃ类细胞去极化，并减小其动作电位。     

ALK (Anaplastic Lymphoma Kinase) expression in DRG neurons and its involvement in neuron–Schwann cells interaction

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase (RTK) transiently expressed in specific regions of the central and peripheral nervous systems. In this study, we focused on the rat developing dorsal root ganglion (DRG). This ganglion is composed of heterogeneous sensory neurons characterized by the expression of RTK for neurotrophic factors, such as the nerve growth factor receptor TrkA or the glial-derived neurotrophic factor family receptor Ret, which are specifically detected in nociceptive neurons. In DRG, ALK expression reached a maximum around birth. We showed that ALK is specifically present in a subtype of neurons during DRG development, and that the majority of these neurons co-expressed TrkA and Ret. Interestingly, we identified only one form (220 kDa) of ALK in DRG neurons both in vivo and in vitro . On the opposite, in transfected cells as well as in brain extracts, ALK was identified as two forms (220 and 140 kDa). The DRG is composed of neurons and glial cells, principally satellite Schwann cells. Thus, we hypothesized that the presence of satellite Schwann cells was involved in the absence of truncated ALK. Using two different cell types, HEK293 cells stably expressing ALK, and MSC80 cells, a previously described Schwann cell line, we showed that a factor secreted by the Schwann cells is likely involved in the absence of ALK cleavage. All these data hence open new perspectives concerning the role of ALK in the specification of nociceptive DRG neurons and in the neurons–Schwann cells interaction.     

炎症痛引起的猫背根节Aβ神经元自发放电特性的分析

应用角叉菜胶后肢局部致炎作为炎症痛的实验动物模型，通过在细胞内记录技术，观察和分析了猫脊髓背根节Aβ神经元的自发放电特性和诱因。结果表明炎症痛导致猫背根节有自发放电的Aβ神经元数明显增加，并表现为持续高频、簇状和不规则等多种型式的放电；在炎症痛不同时程（4和12h），静息膜电位有一定程度的去极化；大多数有自发放电活动的神经元（91％）对外周机械性刺激表现为兴奋反应，其中有部分神经元（9％）同时具有兴奋性和抑制性感受野。胞体给予TTX能阻断这种自发放电；切断外周神经干能使这种自发放电消失，而切断其背根不影响这种自发放电活动。提示炎症痛猫背根节Aβ神经元的兴奋性增强与炎症部位持续性痛的存在相关，可能是炎症性触诱发痛（Allodynia）形成的基础。     

Passive electrical membrane properties of rat neostriatal neurons in an in vitro slice preparation

The passive electrical membrane properties of rat neostriatal neurons were studied in in vitro slice preparations. The data are only from neurons having stable resting membrane potentials of more than 50 mV and able to generate action potentials of amplitudes greater than 70 mV evoked by local or intracellular stimulation. All neurons measured for current-voltage relationship (n = 52) showed non-linearity of the input resistance in the hyperpolarizing direction. The mean input resistance at the resting membrane potential was 16.6 M omega. Depolarizing postsynaptic potentials evoked by local stimulation were decreased both in their amplitude and half-decay time by inward current injections exceeding more than 1 nA due to the strong membrane rectification at these levels of hyperpolarization. The mean membrane time constant (tau 0) was 5.3 ms, as measured from the semilogarithmic plots of transmembrane potential shift produced by small hyperpolarizing current pulses. In some neurons, the equalizing term (tau 1) could be determined as well and had a mean value of 1.0 ms. Measurement of (tau 0) using the strength-latency relation showed a similar value (5.0 ms) to that measured from the voltage transients. Intracellular labeling of the recorded neurons with horseradish peroxidase suggested that the recordings were obtained from medium spiny neurons.     

牛磺酸对GABA引起的大鼠DRG神经元膜去极化的抑制作用（英文）

目前已普遍认为牛磺酸可能是中枢神经系统中的一种抑制性神经递质或神经调质。本实验的目的在于观察牛酸是否使背根神经节神经元膜产生电位变化及其是否对GABA引起的背根神经节神经元膜去极化有调制作用。应用细胞内记录技术观察，标本灌流液中滴加牛磺酸（0．1～100μmol／L）未记录到可检测的膜电位改变，但牛磺酸对GABA（0．3～1000μmol／L）引起的去极化有明显的抑制作用，并对GABAA受体特异性激动剂muscimol（10～100μmol／L）和isoguvacine（10～100μmol／L）引起的去极化有同样的抑制作用，此作用具有明显的浓度依赖性。如10μmol／L的牛磺酸可使100μmol／LGABA引起的去极化抑制47．2％±13．8％（n=8）。牛磺酸对GABA引起去极化的抑制作用随预加牛磺酸与外加GABA之间间隔时间的延长而逐渐减弱，约间隔8～10min，GABA引起的去极化完全恢复。结果提示，牛磺酸在脊髓背角信息传递中起重要的调制作用。     

Hyperpolarization-activated, cyclic nucleotide-gated cation channels: roles in the differential electrophysiological properties of rat primary afferent neurons

The large, medium-sized, and small neurons of the dorsal root ganglion (DRG) have different functions in the processing of various senses. Hyperpolarization-activated, cyclic nucleotide-gated channels (HCN) contribute greatly to neuronal excitability. In the present study, which used whole-cell patch clamp techniques and immunohistochemical staining methods, the electrophysiological properties of DRG neurons were systematically compared, and the roles of HCN-1, -2, and -4 were examined. The main results were as follows. 1) The large neurons had significantly higher V0.5 values (membrane potential at which the HCN channels were half-activated) and shorter time constants (tau) than small or medium-sized DRG neurons. However, large DRG neurons had higher Ih density (HCN neuron current). 2) HCN-1 was found predominantly, but not exclusively, in large and medium-sized DRG neurons; HCN-2 was found in all DRG neurons; and HCN-4 was poorly visualized in all DRG neurons. HCN-1 and HCN-2 were colocalized in large and medium-sized neurons with immunostaining of adjacent sections. In the dorsal horn of the spinal cord, HCN-1, HCN-2, and HCN-4 were all expressed in laminae I-IV, although HCN-1 was not detectable in lamina II. 3) Blockade of Ih current in DRG neurons caused a significant decrease in V0.5, resting membrane potential, and repetitive firing number of action potential and a significant increase in time of rising phase of action potential. These results suggest that the different HCN channels in the three types of DRG neurons might contribute to their differential electrophysiological properties.     

Opioid regulation of intracellular free calcium in cultured mouse dorsal root ganglion neurons

Opioid agonists induced an increase in the intracellular free calcium concentration ([Ca²⁺]_i) or an inhibition of K⁺ (25 mM)-stimulated increase in [Ca²⁺]_i in different subsets of mouse dorsal root ganglion (DRG) neurons. The total neuronal population was grouped into three classes according to somatic diameter and defined as small (<16 μm), intermediate (16–25 μm), or large (>25 μm) neurons. Substance P-like immunoreactivity was detected mainly in the small and intermediate neurons. The δ, κ, and μ opioid receptor agonists [D-Ser², Leu⁵]enkephalin-Thr (DSLET), U69593, and [D-Ala², MePhe⁴, Gly-ol⁵]enkephalin (DAMGO) each induced a transient increase in [Ca²⁺]_i in a small fraction (<30%) of neurons. The increases in [Ca²⁺]_i were blocked by the opioid antagonist naloxone. The dihydropyridine-sensitive calcium channel blocker nifedipine also blocked the increase in [Ca²⁺]_i induced by 1 μM DSLET. The rank order of potency (percentage of cells responding to each opioid agonist) was DSLET > U69593 > DAMGO. The opioid-induced increase in [Ca²⁺]_i was observed mainly in large neurons, with a low incidence in small and intermediate neurons. Opioid agonists also caused inhibition of K⁺-stimulated increases in [Ca²⁺]_i, which were blocked by naloxone (1 μM). Inhibition of the K⁺-stimulated increase by 1 μM DSLET or U69593 was greater in small and intermediate neurons than in large neurons. © 1996 Wiley-Liss, Inc.     

Morphological and electrophysiological properties of pelvic ganglion cells in the rat

Intracellular recording and dye injection were used to study the morphological and electrophysiological properties of rat pelvic ganglion cells. The dye-injected cells measured on the average 37 micron by 22.5 micron and had a mean number of 1.5 primary processes (axon and dendrites). The cells received unmyelinated preganglionic inputs from either the pelvic (parasympathetic) or the hypogastric (sympathetic) nerves, but no cells received inputs from both nerves. The number of synaptic inputs to each cell varied between 1 and 5 with a mean of 2. Each cell had at least one large amplitude suprathreshold EPSP which always initiated an action potential. These properties, namely, morphological simplicity, small number of inputs, security of synaptic transmission and lack of convergence between sympathetic and parasympathetic inputs, suggest that the capacity for synaptic modulation and integration in this ganglion is minimal. Such a structure should therefore relay preganglionic information to target organs with little or no alteration.     

Electrical membrane properties of rat substantia nigra compacta neurons in an in vitro slice preparation

The electrical membrane properties of rat substantia nigra pars compacta (SNC) neurons were studied in an in vitro slice preparation. Some of the recorded neurons were intracellularly labeled with HRP and were found to have morphological characteristics resembling the presumed SNC dopaminergic neurons, as reported by others. The input resistance of SNC neurons at resting membrane potential ranged between 70 and 250 M omega. The membrane resistance showed strong anomalous rectification when the membrane was hyperpolarized by current injection. The anomalous rectification was decreased by the addition of tetraethylammonium bromide (TEA) to the bathing Ringer solution. Injection of depolarizing current or termination of hyperpolarizing current induced slow depolarizing potentials. Their amplitude was dependent on the membrane potential and the current intensity. In neurons treated with tetrodotoxin (TTX) and TEA, slow action potentials were triggered from the slow depolarizing potentials. Both the slow depolarizing potential and slow action potential were TTX resistant and abolished by superfusion of Ca2+-free medium. Long duration hyperpolarizations were observed following the injection of depolarizing current pulses. The hyperpolarization was abolished by the superfusion of Ca2+-free medium or decreased by addition of TEA to the Ringer solution indicating an involvement of a Ca2+-dependent K+-conductance in generation of the hyperpolarization. The long duration hyperpolarization was also observed following action potentials. The spike after hyperpolarization consisted of an initial short duration fast component and a long lasting component. The amplitude of both components seems to be reduced but not abolished by TEA (up to 10 mM). When hyperpolarizing current pulses were applied to neurons that were held either continuously depolarized or were superfused with Ca2+-free medium, the pattern of the membrane potential after the offset of current pulses consisted of an initial fast and a later slow ramp-shaped phase. The latter was associated with a membrane conductance increase and interpreted to be due to an early K+ current. This early K+ current was relatively resistant to TEA. Injections of strong depolarizing currents triggered action potentials with multiple inflections on their rising phase. The amplitudes of action potentials changed abruptly during current application. These data indicate that SNC neurons have multiple generation sites for action potential.