全细胞膜片钳技术检测化学损伤致痫大鼠海马脑片电生理特性

目的应用全细胞膜片钳技术检测化学损伤致痫大鼠海马脑片神经元基本电生理特性。方法将成年SD大鼠建立氯化锂-匹罗卡品模型后,急性分离获得海马脑片,应用全细胞膜片钳电流钳技术实时观察化学损伤致痫大鼠海马脑片CA1区锥体神经元膜电位及其单位时间内动作电位频率的变化情况;通过全细胞膜片钳电压钳技术,检测化学损伤后大鼠海马脑片CA1区锥体神经元的诱发兴奋性突触后电流的变化情况。结果急性分离所得海马脑片CA1区锥体神经元的胞体饱满并可见突起。可在大鼠海马脑片CA1区锥体神经元记录到自发的动作电位及刺激诱发兴奋性突触后电流,且化学损伤后神经元膜电位绝对值降低,动作电位频率加快,诱发的兴奋性突触后电流幅值升高。结论全细胞膜片钳技术可以用于癫痫大鼠急性分离所得海马脑片的电生理研究,化学损伤后神经元兴奋性明显升高,为开展癫痫相关的功能研究提供了新的途径。     

大鼠前脑Meynert基底核伤害性神经元电生理特征的衰老性变化

目的研究前脑Meynert基底核神经元的自发电活动和对皮下注射福尔马林伤害性刺激的反应特性及其衰老性变化。方法采用在体细胞外记录技术观察麻醉大鼠Meynert基底核神经元的自发放电频率和模式以及诱发放电的潜伏期、反应性质和反应时程等电生理学特性。结果大鼠Meynert基底核96％以上的神经元有自发电活动,不同月龄大鼠自发放电频率分别为（16．14±5．74）Hz（3～5）月龄组、（14．44±8．91）Hz（10～12）月龄组和20．23±9．67Hz（18-24）月龄组,其间的差异具有统计学意义（P〈0．01）;65．7％1以上Meynert基底核神经元对伤害性刺激发生反应,主要表现为兴奋性反应,18-24月龄大鼠该脑区神经元对福尔马林伤害性刺激诱发的兴奋性反应程度和时程有明显减小和缩短。结论衰老引起的Meynert基底核神经元自发电活动的改变可能损害其对外周伤害性刺激反应的能力。     

单个人心房肌细胞的分离、鉴定及影响因素分析

目的 探讨分离、鉴定单个人心房肌细胞的最佳方法及其影响因素.方法 人心房肌组织取自5例先天性心脏病(PCHD)、8例冠心病(CHD)、15例瓣膜性心脏病(VHD)手术患者,采用两步酶解法分离制备心房肌细胞,光学显微镜下观察细胞形态,全细胞膜片钳技术记录乙酰胆碱敏感钾电流(I K,Ach)情况.结果 两步酶解法分离所得杆状、横纹清楚、折光性好、静止、贴壁良好的心肌细胞产率为30%～50%.PCHD记录I K,Ach成功率最高(9/10个细胞),其次为CHD(6/10个细胞),VHD最低(3/10个细胞).结论 两步酶解法可用于人心房肌细胞的分离,全细胞膜片钳技术有助于细胞活性的鉴定,其分离数量和活性受酶解情况、年龄、病种等因素影响.     

不同年龄组抑郁症模型大鼠Meynert基底核神经元凋亡

目的探讨不同年龄组抑郁症模型鼠Meynert基底核神经元凋亡情况。方法清洁级Sprague-Dawley大鼠,雄性,4-5周龄32只,4-5月龄32只,14-15月龄32只,不同年龄组随机平均分为对照组与模型组,应用慢性轻度不可预见性刺激制作大鼠抑郁症模型,采用流式细胞仪与透射电镜观察神经元凋亡情况。结果流式细胞仪检测Meynert基底核神经元凋亡情况可见模型组较对照组明显增加(P<0.01),且随年龄增长神经元凋亡增加显著,14-15月龄模型组与4-5周龄模型组和4-5月龄模型组对比有统计学意义(P<0.01);透射电镜观察Meynert基底核神经元凋亡情况可见模型组与对照组比较,Meynert基底核神经元细胞凋亡明显,且随年龄增长细胞器变化明显,凋亡逐渐增加,以14-15月龄模型组为最重。结论抑郁症模型鼠Meynert基底核神经元存在明显凋亡,且随年龄增长细胞凋亡增加。     

小鼠心室肌细胞分离方法的改良及钾电流的记录

目的报道一种改良的小鼠心室肌细胞分离方法,并观察小鼠心室肌细胞动作电位以及钾电流的电生理特性。方法采用双酶消化法分离单个心室肌细胞,应用全细胞膜片钳技术记录动作电位和钾电流。先记录外向钾电流(Ipeak),用低浓度4-氨基吡啶(100μmol/L)使延迟整流钾电流(IKur)失活后记录瞬时外向钾电流(Ito),用Ipeak减去Ito即可得到IKur,在完全失活IKur及Ito后可记录到稳态钾电流(Iss)。结果本法分离所得心室肌细胞横纹清晰,具有正常电生理活性,细胞池中加入层粘连蛋白后有助于细胞贴壁,从而易于形成高阻封接,并记录出小鼠心室肌细胞特征性的动作电位和钾电流。结论本实验所采用的分离方法简便,获得的小鼠心室肌细胞易于封接,且具有正常的电生理活性。     

无名质核与阿尔茨海默病

Meynert底核 (nucleusbasalisofmeynert,nbM)为无名质最主要的神经元 ,又称无名质核。nbM形态学上分为前、中、后3个亚区 (nbMa、nbMi、nbMp) ,其细胞在整个基底前脑中最大并高度嗜铬 ,易于辨认。nbM神经元系中等大小多极细胞 ,直径为 2 0～ 4 0 μm ,胞浆尼氏体丰富 ,核周体浓染 ,核仁清晰可见。nbM接受来自杏仁核、丘脑下部、中脑脚间核以及其他脑干核团发生的多种纤维 ,然后弥漫的向某些大脑皮质区域 (特别是额、顶叶 )、脑干核团、杏仁核发出投射纤维。nbM神经元是投射到全部大脑皮质胆碱能神经支配的主要发源地 ,多数学者认为nbM与…     

兔心室肌细胞的急性分离及电生理记录

目的建立一种简单、稳定的兔心室肌细胞分离方法并进行多种电生理记录。方法采用Langendorff灌流装置及急性酶解分离技术获取单个兔心室肌细胞,并利用全细胞膜片钳技术记录动作电位及多种膜电流。结果耐钙心室肌细胞的存活率在50%～60%,其静息膜电位在-80～90mV,并成功地记录到典型的动作电位及钾、钠、钙等电流。结论该方法简便、稳定,细胞存活率高且易于进行各种电生理实验。     

成年家兔心房肌细胞的分离和电生理记录

目的建立一种简单稳定的成年家兔心房肌细胞的分离、培养方法并进行电生理记录。方法麻醉后取出成年家兔心脏,采用Langendorff灌流装置及急性酶裂解法分离心房肌细胞,差速贴壁法进行纯化后培养于DMEM培养基。在倒置显微镜下观察细胞形态,利用透射电镜观察细胞超微结构,用免疫荧光染色法对心房肌细胞进行鉴定,利用全细胞膜片钳技术记录动作电位和内向钙电流和外向钾电流。结果本方法分离的心房肌细胞纯度和细胞存活率较高,并使用膜电钳技术成功记录了L-型钙电流和瞬时外向钾电流。结论该方法简便有效,细胞存活率高且为进一步进行各种电生理实验打下了基础。     

乳鼠心室肌起搏电流的特点及其基因的表达

目的研究乳鼠心室肌细胞起搏电流(If)的特点,并运用实时定量聚合酶链式反应(PCR)分析乳鼠心室肌If基因的表达。方法通过酶消化法分离乳鼠心室肌细胞,用光镜、透射电镜、免疫组化鉴定心肌细胞;用全细胞膜片钳技术记录If并研究其特性;采用SYBR-GreenI染料进行实时定量PCR,测定乳鼠心室肌If基因即超极化激活的环核苷酸门控通道(mHCN)亚型2和mHCN4的表达。结果光电显微镜下见搏动频率50~100次/分的细胞团;透射电镜看到丰富的线粒体、肌丝明显;用抗肌动蛋白α-actin的单克隆抗体鉴定心肌细胞,95%以上呈现阳性。全细胞膜片钳记录到心室肌细胞的If,并得到电流密度-电压曲线,其激活电压约为-75mV;实时定量PCR检测mHCN2∶mHCN4为(5.15±0.19)∶1。结论乳鼠心室肌细胞有超极化激活、可被Cs+阻断的If,其基因表达以mHCN2为主。     

何首乌二苯乙烯苷对大鼠海马神经元L型钙离子通道的影响

目的 研究二苯乙烯苷对大鼠海马神经元L型钙离子通道的影响.方法 用全细胞膜片钳技术记录大鼠海马神经元钙通道电流.结果 ①细胞外给予2.0和6.0μnol/L二苯乙烯苷对正常的高电压激活钙通道电流的抑制差异不具有统计学意义(P＞0.05).②细胞外给予1 μnol/L的Aβ后再分别给予2.0和6.0μmol/L二苯乙烯苷,显示L型钙电流/电压(Ⅰ-V)曲线逐渐上移,但对钙离子的抑制作用与Aβ组差异无统计学意义(P＞0.05).结论 二苯乙烯苷对大鼠海马神经元L型钙离子通道可能不具有影响.     

Neurotensin excites basal forebrain cholinergic neurons: ionic and signal-transduction mechanisms.

The whole-cell patch-clamp technique was used to investigate the effect of neurotensin on cholinergic neurons cultured from the rat nucleus basalis of Meynert. Neurotensin excited the neurons by inducing an initial inward current carried, at least in part, by Na+ and by reducing inwardly rectifying K+ conductance. Reduction of the inwardly rectifying K+ conductance was mediated by a pertussis toxin-insensitive G protein.     

Wistar大鼠乳鼠与成年鼠心室肌细胞的分离与性质鉴定

目的 探索和优化稳定的适于电生理实验研究的乳鼠及成年大鼠心室肌细胞分离方法。方法 切碎乳鼠心室肌,胰蛋白酶消化,差速贴壁2 h纯化心室肌细胞,台盼蓝染色判定心肌细胞活力,体外培养48 h后分别行倒置显微镜观察细胞形态,免疫组化鉴定,微电极阵列记录细胞搏动频率和场电位。采用Langendorff灌流成年大鼠心脏,主动脉逆行插管,胶原酶域反复灌流消化约30 min,无钙台氏液冲洗心脏5 min,剪下心室肌组织,台氏液中室温下剪碎,吹打,孵育5 min后,用200目筛网过滤,将细胞悬液用逐步复钙法复钙后,室温静置1 h,用于膜片钳记录。结果 经4 -6次消化后,乳鼠心室组织消化完全,细胞存活率大于80%。倒置显微镜下观察,细胞呈梭形、多角形。 12 h有少部分细胞搏动,48 h细胞交织成网,搏动呈同步性,搏动频率30 - 80次/分。 琢鄄辅肌动蛋白（琢鄄actin）经免疫组化检测,纯度达96%。 Langendorff灌流酶解法可获得形态呈杆状、横纹清晰、膜周边光滑完整、立体感强的单个成年鼠心肌细胞,存活率85%,复钙后存活率50%,可用于膜片钳记录。结论 采用本方法可以获得高产量与高质量的用于电生理检测的心室肌细胞。     

Neuropeptide W influences the excitability of neurons in the rat hypothalamic arcuate nucleus

Neuropeptide W (NPW) is a ligand of the recently deorphaned receptor GPR7. Intracerebroventricular injection of this peptide results in reduced serum growth hormone concentration. Using whole-cell patch clamp recordings from somatostatin (SS) neurons in the hypothalamic arcuate nucleus, identified post-hoc using single-cell RT-PCR, we investigated the effects of NPW on membrane excitability. NPW application in acute slices of the arcuate nucleus resulted in the depolarization of the majority (62.5%) of the SS neurons tested, while smaller proportions of cells showed hyperpolarization or no response. Both the depolarization and hyperpolarization of arcuate SS neurons were preserved during recordings where voltage-gated sodium channels were blocked with tetrodotoxin, suggesting direct effects of NPW on the excitability of SS neurons. The observed depolarization of the majority of the SS neurons tested suggests that the central effects of NPW to inhibit growth hormone release results from activation of arcuate SS neurons, which could result in an inhibition of GHRH-releasing neurons.     

Nuclear hourglass technique: an approach that detects electrically open nuclear pores in Xenopus laevis oocyte

Nuclear pore complexes (NPCs) mediate both active transport and passive diffusion across the nuclear envelope (NE). Determination of NE electrical conductance, however, has been confounded by the lack of an appropriate technical approach. The nuclear patch clamp technique is restricted to preparations with electrically closed NPCs, and microelectrode techniques fail to resolve the extremely low input resistance of large oocyte nuclei. To address the problem, we have developed an approach for measuring the NE electrical conductance of Xenopus laevis oocyte nuclei. The method uses a tapered glass tube, which narrows in its middle part to 2/3 of the diameter of the nucleus. The isolated nucleus is sucked into the narrow part of the capillary by gentle fluid movement, while the resulting change in electrical resistance is monitored. NE electrical conductance was unexpectedly large (7.9 +/- 0.34 S/cm(2)). Evaluation of NPC density by atomic force microscopy showed that this conductance corresponded to 3.7 x 10(6) NPCs. In contrast to earlier conclusions drawn from nuclear patch clamp experiments, NPCs were in an electrically "open" state with a mean single NPC electrical conductance of 1.7 +/- 0.07 nS. Enabling or blocking of active NPC transport (accomplished by the addition of cytosolic extracts or gp62-directed antibodies) revealed this large NPC conductance to be independent of the activation state of the transport machinery located in the center of NPCs. We conclude that peripheral channels, which are presumed to reside in the NPC subunits, establish a high ionic permeability that is virtually independent of the active protein transport mechanism.     

Dissociated cell culture of cholinergic neurons from nucleus basalis of Meynert and other basal forebrain nuclei.

Degeneration of cholinergic neurons from the basal forebrain nuclei is suspected to be the cause of Alzheimer disease. We have developed dissociated cultures of cholinergic neurons from these nuclei (the nucleus basalis of Meynert, the medial septal nucleus, and the diagonal band nuclei). Brain slices of the forebrains were made by a vibratome, and the basal forebrain nuclei were dissected out, dissociated, and cultured. Choline acetyltransferase immunocytochemistry and acetylcholinesterase cytochemistry revealed large cholinergic cells (average diameter, 20-25 micron) in these cultures. About 75% of large neurons (20 micron or larger in diameter) were cholinergic. Electrophysiological experiments were performed on these large neurons. The neurons usually did not show spontaneous firing, but steady depolarizations produced trains of action potentials, which adapted quickly. The neurons responded with depolarization to the application of L-glutamic acid. Substance P produced depolarization (sometimes hyperpolarization), and during the depolarization membrane resistance was increased.     

Effect of nociceptin in acid-evoked cough and airway sensory nerve activation in guinea pigs

RATIONALE: Nociceptin/orphanin FQ has been reported to inhibit capsaicin- and mechanically provoked cough in animal models, but the mechanism of this effect has not been elucidated. OBJECTIVES: The objectives of this study were to determine whether nociceptin inhibits acid-evoked cough in conscious animals and to evaluate the mechanism of this effect. METHODS: We tested the effect of nociceptin on acid-induced cough in conscious guinea pigs and acid-induced nerve activation in airway-specific vagal sensory neurons using calcium imaging techniques and the gramicidin-perforated patch clamp technique. MEASUREMENTS AND MAIN RESULTS: Nociceptin (3 mg/kg, intraperitoneal) effectively inhibited acid-evoked cough in guinea pigs by nearly 70%. Acid (pH 5) increased intracellular free calcium in acutely dissociated vagal jugular ganglionic neurons. The acid-induced increase in intracellular calcium was inhibited by a selective transient receptor potential vanilloid-1 antagonist, 5-iodo-resiniferatoxin (1 microM, approximately 80% reduction). The inhibitory effect of 5-iodo-resiniferatoxin on acid-induced increases in calcium was mimicked by nociceptin (0.1 microM). In gramicidin-perforated patch clamp recordings on airway-specific capsaicin-sensitive jugular ganglion neurons, acid (pH 5) induced two distinct inward currents. A transient current was evoked that was inhibited by amiloride and a sustained current was evoked that was inhibited by 5-iodo-resiniferatoxin. Nociceptin selectively inhibited only the sustained component of acid-induced inward current. CONCLUSION: These results indicate that the inhibitory effect of nociceptin on acid-induced cough may result from a direct inhibitory effect on peripheral C-fiber activity caused by the selective inhibition of acid-induced transient receptor potential vanilloid-1 activation.     

Aggravated decrease in the activity of nucleus basalis neurons in Alzheimer’s disease is apolipoprotein E-type dependent

As reported before, the metabolic activity of nucleus basalis neurons is reduced significantly in Alzheimer patients. Because the apolipoprotein E (ApoE) 4 genotype is a major risk factor for Alzheimer’s disease (AD), we determined whether the decrease in metabolic activity in nucleus basalis neurons in AD is ApoE-type dependent. The size of the Golgi apparatus (GA) was determined as a measure of neuronal metabolic activity in 30 controls and 41 AD patients with a known ApoE genotype by using an image analysis system in the nucleus basalis of Meynert. A polyclonal antibody directed against MG-160, a sialoglycoprotein of the GA, was used to visualize this organelle. There was a very strong reduction in the size of the GA in the nucleus basalis of AD patients. Furthermore, a strong and significant extra reduction in the size of the GA was found in the nucleus basalis neurons of AD patients with either one or two ApoE 4 alleles compared with Alzheimer patients without ApoE 4 alleles. Our data show that the decreased activity of nucleus basalis neurons in AD is ApoE 4 dependent and suggest that ApoE 4 participates in the pathogenesis of AD by decreasing neuronal metabolism.     

Ion channels in synaptic vesicles from Torpedo electric organ.

A simple method has been developed for fusing synaptic vesicles into spherical structures 20-50 micron in diameter. The method has been applied to purified cholinergic synaptic vesicles from Torpedo electric organ, and the membrane properties of these fused structures have been studied by the "cell"-attached version of the patch clamp technique. A large conductance potassium-preferring channel, termed the P channel, was consistently observed in preparations of fused synaptic vesicles. The selectivity of the channel for potassium over sodium was approximately equal to 2.8-fold. Two major conductance levels were observed during P-channel activity, and their relative proportion was dependent on the voltage applied to the membrane through the patch pipette. P channels were not seen in fused preparations of purified Torpedo lipids, nor was the frequency of their occurrence increased in preparations enriched with plasma membrane or nonvesicular membranes. We suggest, therefore, that the P channels are components of the synaptic vesicle membrane. Their function in synaptic transmission physiology is still unknown.