这项工作是由澳大利亚研究理事会根据联动项目赠款LP120100264支持。

1。螺旋神经节神经元的文化<ol><li>消毒小轮（ 如 10毫米直径）盖玻片，在高压釜中弯钳。无菌盖玻片转移到无菌的4 - 环35毫米陪替氏培养皿或4 - 孔板的各井，用无菌镊子。应用150μl的聚-L-鸟氨酸（500微克/毫升），小鼠层粘连蛋白（0.01毫克/毫升）在盖玻片和地点的培养箱中（37℃）的顶表面48小时。确保盖玻片不浮离井底。 </li><li>准备50毫升无菌的Neurobasal培养基（NBM）的每个神经培养：47.5毫升的neurobasal甲，0.5毫升N 2补充B27的补充，将1ml，0.5毫升L-谷氨酰胺，0.5 ml青霉素-链霉素。注：在-20°C补充剂可冻结，储存，并添加到媒体所需的日子。 </li><li>游离产后4-7天幼鼠如前所述20,21螺旋神经节神经元，我们ING两个酶（0.025％胰蛋白酶和DNA酶I 0.001％）和机械技术。请参阅等 Whitlon 22和维埃拉等人 23 螺旋神经节神经元培养，帕克等人24的详细过程，示范蜗轴隔离。 </li><li>吸剩余poly-L-ornithine/laminin的解决方案，从盖玻片和洗简要NBM。 </li><li>加入150-200微升分离的螺旋神经节细胞悬液盖玻片，并放入培养箱（37℃，10％CO 2）。注：盖玻片可以准备多达20平均产仔8幼鼠。 </li><li>电镀神经元四小时后，吸出的解决方案，去除细胞碎片，并更换与150-200微升温暖的新鲜NBM。注：媒体可能需要每天补充，以避免脱水。 </li><li>返回盖玻片的孵化器，直到所需的电生理记录。注：游离螺旋神经节细胞培养可用于电生理实验，4个小时后，分离和其后两天。 在体外的时间应加以考虑，在分析过程中的结果。补充NBM每24-48小时。 </li></ol> 2。膜片钳记录的准备<ol><li>准备解决方案<ol><li>细胞内（微量）解决方案：115毫米K-葡萄糖酸，氯化钾7毫米，10毫米HEPES，EGTA 0.05毫米，2毫米的Na 2 ATP MgATP，2毫米，0.5毫米的Na 2 GTP（KOH调节pH至7.3，调整至295 mOsmol /公斤蔗糖）。通过该溶液通过无菌过滤器（0.2微米），并分成200微升的等份储存在-20℃下，直到有一天记录。 </li><li>外（浴）：137毫米氯化钠，氯化钾5毫米，2毫米氯化钙2，1毫米氯化镁2，10毫米HEPES，10mM葡萄糖（用NaOH调节pH值至7.4，调整为300-310毫渗透摩尔/千克蔗糖） 。该解决方案是由天记录。 </li></ol></li><li>准备记录微量2-6MΩ的电阻。我们使用的CO 2激光器拉出器（P-2000;萨特仪器）和硼硅酸盐玻璃（1.0毫米外径，0.58毫米内径75毫米的长度）。 </li><li>准备的激光。该协议的目的是用于与光纤耦合激光1,870 nm的红外神经刺激，如光磊P / L在我们的实验中用于光传输的光纤是一个200/220微米的纤芯/包层直径的石英光纤的数值孔径为0.22，和FC-PC连接器两端（AFW技术MM1-FC2-200/220-5-C -0.22）。跳线被削减了一半，产生两个光纤尾纤（ 即连接器的一端，并在其他裂解）。光纤的纤芯直径和数值孔径激光引起的温度变化的影响，已详细讨论了由Thompson 等人25 <ol><li>用标准技术的光传输光纤的前端劈斩并确保所得的小费是高质量的观察用光学显微镜（ 即尖端应垂直于纤维轴的目视检查时显示为平面）。光传输光纤连接的光纤耦合输出的刺激激光通过接口（ 例如 Thorlabs公司ADAFC2）使用适当的。 </li><li>测量裂尖使用适当的工具（ 例如，的相干FieldMate与LM-3探测器头）的光传输光纤输出的激光功率。建议检查激光功率，光传输光纤切割或作出重大调整的激光（ 如运输从一个实验室到另一个）每次。 </li><li>将光传输光纤，光纤夹头或同等器件以及吸盘贴上适当的微定位。重要的是能够精确地确定角度θ，使光纤与盖玻片。此ANGLe可以被测量的实验安排拍摄照片，使用图像处理软件（ 例如 ImageJ的）取得角度。主要受制于空间相对有限的实验安排的 - 特别是在显微镜的角度θ（或可能的角度范围）。在我们的实验中（基于直立显微镜）的典型值θ是36°左右，但在最佳角度可能显着不同的替代的设置（ 例如，那些使用倒置显微镜）。 </li><li>之间的激光和膜片钳数据采集系统（ 例如 Digidata 1440A，Molecular Devices公司），如在图2中所示进行连接。膜片钳数据采集系统的数字输出应连接到激光通过一个外部的函数发生器，使得可以指定独立的数据采集系统的激光脉冲参数。或者该输出可以直接连接到激光驱动器（需要由数据采集软件设置的脉冲宽度和重复率）。在这两种情况下，所使用的信号来触发激光应该连接到数据采集系统的一个输入端，以确保激光脉冲的时序和长度的可记录，可同时进行的电生理信号。 </li></ol></li></ol> 3。膜片钳记录调查INS <ol><li>填充合适的容器内，细胞外液的灌注系统和调节流量在1-2毫升/分钟的速率提供灌流浴槽。我们使用重力给料系统（吸气器瓶，夹管阀，PE管材）在线加热器，使快速加热该溶液中，并通过抽吸除去使用了的溶液的蠕动泵。 </li><li>放置盖玻片培养细胞转化为一个堂堂正正的显微镜录音室（洗澡）。使用高倍率水浸泡的目的（E克40X）和相位对比（ 如微分干涉对比或梯度对比DODT），视觉定位螺旋神经节神经元内的文化。一个典型的螺旋神经节神经元相亮，圆，直径约15微米，一个突出的髓核， 如图1a所示。 </li><li>一旦神经元已经找到，切换到一个低倍物镜（ 如 10倍）视野内神经元的目标定位。 </li><li>移动位置使用下列程序（或同等学历）的光传输光纤到： <ol><li>使用微定位器移动到它的输出光纤，直到针尖靠近在水平和垂直平面上的目标神经元。纤维笔尖的垂直位置的向上和向下移动的目标（扫描焦点），可以验证通过。 </li><li>切换回高倍率的目标和定位光纤的尖端在其预期的位置到t他神经元（ 见图2插图）。纤维的垂直位置调整时，重要的是，纤维的底部边缘放置盖玻片上（ 即只接触），以尽量减少在纤维的位置不确定性。将取决于在水平平面内的最优对应的角度θ，使纤维与盖玻片的纤维r的半径。为了定位，使纤维沿纤维轴向的目标神经元的中心在于，纤维的上边缘之间的水平距离和目标小区应该是 Δ 目标 = R（。余割θ - 2罪θ） 其中负数值表示的光学纤维悬细胞。对齐纤维时，纤维的顶部边缘和神经元的中心之间的距离Δ 目标可以通过视觉检查来近似实现。然而，Δ 目标被设计为作为一个粗略的指引。纤维，使得纤维的上边缘和神经元的中心之间的实际距离ΔΔ 目标 （如在图1中），可测量不同的位置，可以提供从中心的径向位移（ 即洞察的效果束）和轴向位移（ 即沿着纤维轴）的光束相对于目标神经元。例如，设置Δ≈0将有望增加当地的气温在附近的细胞- 即典型的多模光纤由于光束轮廓很好地近似一顶帽子，减少局部吸收的能量（温度分布26）由于从光束的中心的位移是最小的移动光纤端面更接近细胞相比，吸收的能量的增加。 虽然应小心，纤维的位置，尽可能准确和可重复婆ssible使用的视觉提示，精确测量的光纤的相对位置的目标神经元（ 例如，Δ）是更重要的，比它定位一个预定的距离相差。 Δ（估计的最大误差为±3微米），通过图像分析来确定，如步骤3.8.2中所述的协议。在一些实验中5,8，白色光源通过光纤连接，以目标所需的细胞。这种方法被认为是无效的的直立显微镜设置，从目标区域散射光的强度相对较低，这些浅层角度（θ）。 </li><li>一旦纤维是在位置，公知的量将其移出，使微量的简单的定位。的纤维，然后可以返回到原来的位置，当一个神经记录实现。 </li></ol></li><li>填充的微细胞内的解决方案，并牢固地融入地方的头上踏歌放大器（ 如 Multiclamp 700B，分子器件）。使用微电极保持器的侧管连接，适用于正压，以防止堵塞的微少量。 </li><li>利用显微操作，将微量的正上方目标神经元的位置。 </li><li>协议的全细胞记录： <ol><li>应用以10毫秒的方波脉冲，+10 mV的（ 如密封试验）在电压钳模式的放大器和调整的基准信号的微电位（吸管偏移）0 NA。应该也可以用密封试验，以确定是否在所需范围内（ 例如，2 - 6MΩ）的微电极的电阻。 </li><li>同时监测的电阻，使用的显微操作器的微调的目标神经元的表面上轻轻地定位微量。的微管是与神经元接触时，电阻会增加（约0.5〜1MΩ）。我mmediately的阻力增加，除去正面的流体压力和施加一个小的负压。一旦阻力已经过去了10 - 20MΩ，修复膜电位控股水平（ 例如 -60毫伏左右）。 </li><li>电极电阻应继续增加，直到超过1GΩ，标志着一个有效的密封（称为&#39;gigaseal&#39;）之间已经形成细胞膜和微量。在这一点上，删除所有的微流体压力。 </li><li>一旦gigaseal已经形成，使用短电流脉冲（25至100微秒，+1 V）或负的流体压力破裂细胞膜的简要脉冲和中实现全细胞配置。 </li><li>记录膜电容，串联电阻和输入电阻为从密封试验脉冲期间的电流的指数曲线拟合确定。尽量减少电容瞬态，通过放大器上调整CpFast和CpSlow控制，然后SWITCh的放大器进入全细胞模式，并赔偿一台电流的电容和电阻，直到观察到在密封试验。应用串联电阻补偿（〜70％的调整，70％的预测），电容和电阻调整控制手段来保持一个平面密封试验响应。 </li><li>切换到电流钳模式放大器。注意到的静息膜电位（电流注入的情况下）。设置的保持电流，膜电位稳定在所需的电平（ 例如 -60毫伏）。中和该移液管的电容，并调节电桥平衡，平衡的电压降。 </li><li>检查烧成性能受刺激的神经元去极化电流（+10〜+200 PA在+10 PA步骤;持续300毫秒）。 </li></ol></li><li><ol><li>移动光纤回到原来的位置，对神经元的旁边。使用连接的CCD照相机的图像处理软件，相对于目标神经纤维的位置捕捉​​图像为n，聚焦在飞机上的最初的神经元，然后该光纤的顶部边缘上（参见图1）。 </li><li>通过随后的分析所产生的图像（ 例如， 图1b和1c）中，有可能精确地确定Δ（目标神经元的中心相对于光纤的上边缘的位置）。一旦Δ是已知的，从光纤端面的目标神经元（沿纤维轴向）z和神经元的径向位移，束δŗ的从中心的距离参数，例如使用简单的三角关系，可以计算： Ž= R罪2θ：+ΔCOSθ δR =（（COS2θ -单Δθ）2 +ΔY2）1/2 ΔY是与纤维轴和从上面看的神经元的中心之间的距离（ 例如，本身e 图1）。 分析应该考虑到从细胞到细胞的位置变化，可能需要这些位置参数的精确的知识来解决可能存在的差异刺激过程25。 </li></ol></li></ol> 4。 INS实验<ol><li>记录电生理数据，无论是电流钳或电压钳位配置，运行所需的参数（ 如功率，脉冲宽度，重复率等）的刺激激光。使用我们的激光，光功率控制通过直接输入到激光驱动器，每次录音前手动指定。脉冲宽度和重复频率可以控制的外部信号产生器或数据采集软件（步骤2.3.4中所描述的）。确保膜片钳通道和激光触发通道数据记录。 </li></ol>激光脉冲长度范围从大约500微秒至15毫秒〜0.25-5 MJ每个脉冲的能量通常产生可测量的电反应。激光脉冲的重复速率设置为1赫兹或更低时可能是有用的，因为它最初的实验中，此参数的影响减到最小。典型的结果，示出所记录的信号的变化，都在下面的分类。

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Opt. 12, 021008 (2007).</li><li>Richter, C. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+stimulation+of+auditory+neurons:+Effects+of+acute+and+chronic+deafening.">Optical stimulation of auditory neurons: Effects of acute and chronic deafening.</a> Hearing Research. 242, 42-51 (2008).</li><li>Littlefield, P. D., Vujanovic, I., Mundi, J., Matic, A. I., Richter, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+stimulation+of+single+auditory+nerve+fibers.">Laser stimulation of single auditory nerve fibers.</a> Laryngoscope. 120, 2071-2082 (2010).</li><li>Izzo, A. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+parameter+variability+in+laser+nerve+stimulation:+A+study+of+pulse+duration,+repetition+rate,+and+wavelength.">Optical parameter variability in laser nerve stimulation: A study of pulse duration, repetition rate, and wavelength.</a> IEEE Transactions on Biomedical Engineering. 54, 1108-1114 (2007).</li><li>Sakmann, B., Neher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch+clamp+techniques+for+studying+ionic+channels+in+excitable-membranes.">Patch clamp techniques for studying ionic channels in excitable-membranes.</a> Annual Review of Physiology. 46, 455-472 (1984).</li><li>Needham, K., Nayagam, B. A., Minter, R. L., O'Leary, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+application+of+brain-derived+neurotrophic+factor+and+neurotrophin-3+and+its+impact+on+spiral+ganglion+neuron+firing+properties+and+hyperpolarization-activated+currents.">Combined application of brain-derived neurotrophic factor and neurotrophin-3 and its impact on spiral ganglion neuron firing properties and hyperpolarization-activated currents.</a> Hearing Research. 291, 1-14 (2012).</li><li>Coleman, B., Fallon, J. B., Pettingill, L. N., de Silva, M. G., Shepherd, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auditory+hair+cell+explant+co-cultures+promote+the+differentiation+of+stem+cells+into+bipolar+neurons.">Auditory hair cell explant co-cultures promote the differentiation of stem cells into bipolar neurons.</a> Experimental Cell Research. 313, 232-243 (2007).</li><li>Whitlon, D. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+and+morphology+of+auditory+neurons+in+dissociated+cultures+of+newborn+mouse+spiral+ganglion.">Survival and morphology of auditory neurons in dissociated cultures of newborn mouse spiral ganglion.</a> Neuroscience. 138, 653-662 (1016).</li><li>Vieira, M., Christensen, B. L., Wheeler, B. C., Feng, A. S., Kollmar, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+and+stimulation+of+neurite+outgrowth+in+a+serum-free+culture+of+spiral+ganglion+neurons+from+adult+mice.">Survival and stimulation of neurite outgrowth in a serum-free culture of spiral ganglion neurons from adult mice.</a> Hearing Research. 230, 17-23 (2007).</li><li>Parker, M., Brugeaud, A., Edge, A. S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+culture+and+plasmid+electroporation+of+the+murine+organ+of+corti.">Primary culture and plasmid electroporation of the murine organ of corti.</a> J. Vis. Exp. (36), e1685 (2010).</li><li>Thompson, A. C., Wade, S. A., Brown, W. G. A., Stoddart, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+of+light+absorption+in+tissue+during+infrared+neural+stimulation.">Modeling of light absorption in tissue during infrared neural stimulation.</a> Journal of Biomedical Optics. 17, 075002 (2012).</li><li>Snyder, A. W., Love, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Optical Waveguide Theory. , (1983).</li><li>Thompson, A. C., Wade, S. A., Cadusch, P. J., Brown, W. G. A., Stoddart, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modelling+of+the+temporal+effects+of+heating+during+infrared+neural+stimulation.">Modelling of the temporal effects of heating during infrared neural stimulation.</a> J. Biomed. Opt. 18, 035004-0310 (2013).</li><li>Yao, J., Liu, B. Y., Qin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+temperature+jump+by+infrared+diode+laser+irradiation+for+patch-clamp+studies.">Rapid temperature jump by infrared diode laser irradiation for patch-clamp studies.</a> Biophysical Journal. 96, 3611-3619 (2009).</li></ol>

什么都没有透露。

使用本文中列出的协议，它可以提取和培养螺旋神经节神经元，并探讨激光诱发的电活动进行全细胞膜片钳实验。 在体外使用时，膜片钳技术提供实验参数的控制，是无法实现在体内的水平。激光刺激参数，如波长，脉冲能量，脉冲长度，脉冲形状和脉冲重复序列，可以研究在一个可重复的设置。此外，维持神经元所处的环境（ 例如，溶液的温度，化学因素）可以系统地改?...

神经生理学和医学仿生学领域倚重允许可控刺激的电反应神经组织的技术的。虽然电刺激神经 ​​兴奋的金标准，它受到来自一些缺点，如刺激工件的存在下录音时的神经反应，和缺乏刺激特异性的电流的扩散到周围组织1。 过去二十年的发展已经看到光学介导的刺激技巧2。这些技术中需要几个靶组织的变形，可通过另外一个特定的分子（ 如笼分子）3或某种形式的遗传操作（ 例如，光遗传学）4，这两者都不容易申请以外的研究设置。因此特别令人感兴趣的是红外神经刺激（INS），whereb的Ÿ神经组织兴奋红外激光脉冲。 INS有可能克服许多电刺激的缺点，通过使具有高度特异性，非接触刺激神经 ​​组织2。然而，虽然INS已成功地展示了在体内的各种设置，激励的准确机理仍然不明朗。 最近的一些出版物揭露背后的机制INS 5-7所示的进展。由于吸收的激光水快速加热，似乎发挥了关键作用。然而，除了尚未达成共识。 Shapiro 等人 7提出了一种高度的通用性的机制，从而快速加热导致的扰动，在细胞膜上相邻的带电粒子的分布，导致细胞膜和随后的去极化的电容的变化。此外，阿尔伯特等 5断言LASER明显加热温度敏感离子通道（瞬时受体电位香草频道）激活一个特定的类，允许离子通过细胞膜。在这个阶段，目前还不清楚如何将这些机制相结合，或什至是否有进一步的因素，这些因素尚未确定。 调查虽然数量不多的出版物（5,7-9引用）INS 体外发表在这一领域的工作，绝大多数已进行了体内 （ 如引用1,6,10-18）。红外刺激听觉神经元已经特别感兴趣的区域，由于人工耳蜗植入10,14-18中的潜在应用。 在体内实验中是很重要的验证技术在各种设置中， 在体外研究中所提供的控制水平增加，预计将导致一个更详细的了解机甲的有效性负责INS anism。这份报告介绍了编制的螺旋神经节神经元膜片钳调查，因为这些可以用来研究的基本机制，同时也连接到现有的数据从听觉系统的庞大的身躯。 膜片钳技术电现象的调查是一个很好的工具，它提供了一种在单细胞记录电活动和研究的贡献，个人的基本电流19。 在体外制备，如螺旋神经节神经元的初级神经元，当这项技术被应用到一个稳定的，它提供了学习的机会，在深度神经活动的机制，控制和操纵。 在这项工作中轮廓的方法调查激光刺激螺旋神经节神经元对电性能的影响，通过膜片钳指定的协议录音。该方法是基于在光纤耦合激光，而不是一个自由空间激光，允许更安全的操作，以及更容易，更可重复的对齐，而不需要修改标准的显微镜配置。这些协议的基础上，它应该是可能的，为了更清楚地确定的机制或机制背后INS进行了广泛的实验。

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Cell culture materials and equipment </td>
 </tr>
 <tr>
 <td>Glass coverslips</td>
 <td>Lomb Scientific</td>
 <td>CSC 10 1 GP</td>
 <td></td>
 </tr>
 <tr>
 <td>4-ring cell culture dish </td>
 <td>VWR International</td>
 <td>82050-542</td>
 <td></td>
 </tr>
 <tr>
 <td>Poly-L-ornithine solution</td>
 <td>Sigma-Aldrich</td>
 <td>P4957</td>
 <td></td>
 </tr>
 <tr>
 <td>Laminin</td>
 <td>Invitrogen</td>
 <td>23017-015</td>
 <td></td>
 </tr>
 <tr>
 <td>Curved forceps</td>
 <td>WPI</td>
 <td>14101</td>
 <td>Dumont #5 tweezers (45&deg; angle tip)</td>
 </tr>
 <tr>
 <td>CO2 Incubator</td>
 <td>ThermoScientific</td>
 <td>Heracell 150i</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Table 1. Cell culture materials and equipment.</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Neurobasal media</td>
 </tr>
 <tr>
 <td>Neurobasal A</td>
 <td>Gibco</td>
 <td>10888-022</td>
 <td></td>
 </tr>
 <tr>
 <td>N-2 supplement</td>
 <td>Invitrogen</td>
 <td>17502-048</td>
 <td></td>
 </tr>
 <tr>
 <td>B27 serum-free supplement</td>
 <td>Invitrogen</td>
 <td>17504-044</td>
 <td></td>
 </tr>
 <tr>
 <td>Penicillin-Streptomycin</td>
 <td>Invitrogen</td>
 <td>15140-148</td>
 <td></td>
 </tr>
 <tr>
 <td>L-Glutamine</td>
 <td>Invitrogen</td>
 <td>25030-149</td>
 <td></td>
 </tr>
 <tr>
 <td>Intracellular solution</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Potassium chloride</td>
 <td>Sigma-Aldrich</td>
 <td>P4504</td>
 <td></td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>Sigma-Aldrich</td>
 <td>H4034</td>
 <td></td>
 </tr>
 <tr>
 <td>Potassium D-gluconate</td>
 <td>Sigma-Aldrich</td>
 <td>G4500</td>
 <td></td>
 </tr>
 <tr>
 <td>EGTA</td>
 <td>Sigma-Aldrich</td>
 <td>E3889</td>
 <td></td>
 </tr>
 <tr>
 <td>Na2ATP</td>
 <td>Sigma-Aldrich</td>
 <td>A2383</td>
 <td></td>
 </tr>
 <tr>
 <td>MgATP</td>
 <td>Sigma-Aldrich</td>
 <td>A9187</td>
 <td></td>
 </tr>
 <tr>
 <td>NaGTP</td>
 <td>Sigma-Aldrich</td>
 <td>G8877</td>
 <td></td>
 </tr>
 <tr>
 <td>Potassium hydroxide</td>
 <td>LabServ</td>
 <td>BSPPL738.500</td>
 <td></td>
 </tr>
 <tr>
 <td>Sucrose</td>
 <td>Sigma-Aldrich</td>
 <td>S8501</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Extracellular solution </td>
 </tr>
 <tr>
 <td>Sodium chloride</td>
 <td>Sigma-Aldrich</td>
 <td>310166</td>
 <td></td>
 </tr>
 <tr>
 <td>Potassium chloride</td>
 <td>Sigma-Aldrich</td>
 <td>P4504</td>
 <td></td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>Sigma-Aldrich</td>
 <td>H4034</td>
 <td></td>
 </tr>
 <tr>
 <td>Calcium chloride</td>
 <td>Sigma-Aldrich</td>
 <td>383147</td>
 <td></td>
 </tr>
 <tr>
 <td>Magnesium chloride</td>
 <td>Sigma-Aldrich</td>
 <td>M8266</td>
 <td></td>
 </tr>
 <tr>
 <td>D-Glucose</td>
 <td>Sigma-Aldrich</td>
 <td>G8270</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium hydroxide</td>
 <td>LabServ</td>
 <td>BSPSL740.500</td>
 <td></td>
 </tr>
 <tr>
 <td>Sucrose</td>
 <td>Sigma-Aldrich</td>
 <td>S8501</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Table 2. Solutions for cell culture and patch clamp. a) Neurobasal media. b) Intracellular solution. c) Extracellular solution.</td>
 </tr>
 <tr>
 <td>Upright microscope</td>
 <td>Zeiss </td>
 <td>AxioExaminerD1</td>
 <td>Equipped with Dodt contrast </td>
 </tr>
 <tr>
 <td>Water-immersion objective</td>
 <td>Zeiss</td>
 <td>W Plan-APOCHROMAT 40x/0.75</td>
 <td></td>
 </tr>
 <tr>
 <td>Platform and X-Y stage </td>
 <td>ThorLabs</td>
 <td>Burleigh Gibraltar</td>
 <td></td>
 </tr>
 <tr>
 <td>Recording chamber</td>
 <td>Warner Instruments</td>
 <td>RC-26G</td>
 <td></td>
 </tr>
 <tr>
 <td>Vibration isolation table</td>
 <td>TMC</td>
 <td>Micro-g 63-532</td>
 <td></td>
 </tr>
 <tr>
 <td>CCD Camera</td>
 <td>Diagnostic Instruments</td>
 <td>RT1200</td>
 <td></td>
 </tr>
 <tr>
 <td>Camera software</td>
 <td>Diagnostic Instruments</td>
 <td>SPOT Basic</td>
 <td></td>
 </tr>
 <tr>
 <td>In-line solution heater</td>
 <td>Warner</td>
 <td>SH-27B</td>
 <td></td>
 </tr>
 <tr>
 <td>Temperature controller</td>
 <td>Warner</td>
 <td>TC-324B</td>
 <td></td>
 </tr>
 <tr>
 <td>Patch clamp amplifier</td>
 <td>Molecular Devices</td>
 <td>Multiclamp 700B</td>
 <td></td>
 </tr>
 <tr>
 <td>Patch clamp data acquisition system</td>
 <td>Molecular Devices</td>
 <td>Digidata 1440A</td>
 <td></td>
 </tr>
 <tr>
 <td>Micromanipulator</td>
 <td>Sutter Instruments</td>
 <td>MPC-325</td>
 <td></td>
 </tr>
 <tr>
 <td>Micropipette glass</td>
 <td>Sutter Instruments</td>
 <td>GBF100-58-15</td>
 <td>Borosilicate glass with filament</td>
 </tr>
 <tr>
 <td>Micropipette Puller</td>
 <td>Sutter Instruments</td>
 <td>P2000</td>
 <td></td>
 </tr>
 <tr>
 <td>Recording Software</td>
 <td>AxoGraph</td>
 <td></td>
 <td>Lab pack and electrophysiology tools</td>
 </tr>
 <tr>
 <td>Aspirator bottle</td>
 <td>Sigma-Aldrich</td>
 <td>CLS12201L</td>
 <td>1 L Pyrex aspirator bottle, with outlet for tubing</td>
 </tr>
 <tr>
 <td>PE Tubing</td>
 <td>Harvard</td>
 <td>PolyE #340</td>
 <td></td>
 </tr>
 <tr>
 <td>Masterflex peristaltic pump</td>
 <td>Cole-Parmer</td>
 <td>HV-07554-85</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Table 3.Patch clamp equipment.</td>
 </tr>
 <tr>
 <td>1,870 nm laser diode</td>
 <td>Optotech</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>200/220 &mu;m diameter multimode optical fiber patch cord (FC/PC)</td>
 <td>AFW Technologies</td>
 <td>MM1-FC2-200/220-5-C-0.22</td>
 <td>Light delivery optical fiber, silica core and cladding, 0.22 NA</td>
 </tr>
 <tr>
 <td>Optical fiber through connector (FC/PC)</td>
 <td>Thorlabs</td>
 <td>ADAFC2</td>
 <td></td>
 </tr>
 <tr>
 <td>Optical fiber cleaver</td>
 <td>EREM</td>
 <td>FO1 </td>
 <td></td>
 </tr>
 <tr>
 <td>Optical fiber stripping tool (0.25 - 0.6 mm) </td>
 <td>Siemens</td>
 <td></td>
 <td>For removing optical fiber jacket</td>
 </tr>
 <tr>
 <td>Optical fiber stripping tool (0.6 - 1.0 mm)</td>
 <td>Siemens</td>
 <td></td>
 <td>For removing outer coating of patch cord</td>
 </tr>
 <tr>
 <td>Signal generator</td>
 <td></td>
 <td></td>
 <td>Any signal generator that can output the necessary pulse shapes and is capable of being externally triggered</td>
 </tr>
 <tr>
 <td>Optical fiber positioner</td>
 <td></td>
 <td></td>
 <td>Custom made positioner. Could substitute with standard micropositioner used for patch clamp experiments</td>
 </tr>
 <tr>
 <td>Optical fiber chuck</td>
 <td>Newport</td>
 <td>FPH-DJ</td>
 <td></td>
 </tr>
 <tr>
 <td>Laser power meter and detector head</td>
 <td>Coherent</td>
 <td>FieldMate (power meter) with LM-3 (detector head)</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Table 4. Laser equipment.</td>
 </tr>
 </tbody>
</table>

whole cell patch clamp for investigating the mechanisms of infrared neural stimulation

近年来已被证明在脉冲红外激光可用于诱发电反应在神经组织​​中，独立于目标组织的任何进一步的修改。红外神经刺激已报道在体内的各种外设和感觉神经组织中，特别令人感兴趣的听觉神经的神经元的刺激中所示。但是，而INS已被证明在这些设置，由红外光引起的神经兴奋的机制（或机制）目前没有得到很好的理解。这里介绍的协议描述了全细胞膜片钳的方法设计，以方便调查红外培养初级听觉神经元的神经刺激。彻底红外激光照射在受控条件下在体外 ，这些细胞的特征的响应，它可能是未能获得的基本物理之后更好地理解l和相关的生化过程红外神经刺激。

螺旋神经节神经元激光照射可重复波形电压钳和电流钳记录配置中， 图3a示出典型的电流的变化，响应穿过细胞膜的一个2.5毫秒，0.8毫焦耳的激光脉冲（平均响应于6激光脉冲，在1秒的间隔）与膜电位在-70mV，-60 mV和-50 mV的重复。净内向电流一贯诱发在激光脉冲响应，停止光照后返回到初始值。可以看出，激光诱导的电流的形状，以改变膜电位改变，这表明它可能是重要的，在钳制电?...

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Whole Cell Patch Clamp for Investigating the Mechanisms of Infrared Neural Stimulation

红外神经刺激已经提出作为一种替代电刺激神经类型，包括那些与听觉系统的范围内。本协议描述了膜片钳技术，在初级听觉神经元的文化学习机制的红外神经刺激。

全细胞膜片钳红外神经刺激机制的调查

It has been  demonstrated in recent years that pulsed, infrared laser light can be used to  elicit electrical responses in neural tissue, independent of ...

whole-cell-patch-clamp-for-investigating-mechanisms-infrared-neural

Research

JoVE Journal

Neuroscience

20.8K 次观看.  Swinburne University of Technology. 红外神经刺激已经提出作为一种替代电刺激神经类型，包括那些与听觉系统的范围内。本协议描述了膜片钳技术，在初级听觉神经元的文化学习机制的红外神经刺激。

视频: Whole Cell Patch Clamp for Investigating the Mechanisms of Infrared Neural Stimulation

Investigating the Neural Mechanisms of Aware and Unaware Fear Memory with fMRI

Pavlovian fear conditioning is often used in combination with functional magnetic resonance imaging (fMRI) in humans to investigate the neural substrates of associative learning 1-5. In these studies, it is important to provide behavioral evidence of conditioning to verify that differences in brain activity are learning-related and correlated with human behavior. 
Fear conditioning studies often monitor autonomic responses (e.g. skin conductance response; SCR) as an index of learning and memory 6-8. In addition, other behavioral measures can provide valuable information about the learning process and/or other cognitive functions that influence conditioning. For example, the impact unconditioned stimulus (UCS) expectancies have on the expression of the conditioned response (CR) and unconditioned response (UCR) has been a topic of interest in several recent studies 9-14. SCR and UCS expectancy measures have recently been used in conjunction with fMRI to investigate the neural substrates of aware and unaware fear learning and memory processes 15. Although these cognitive processes can be evaluated to some degree following the conditioning session, post-conditioning assessments cannot measure expectations on a trial-to-trial basis and are susceptible to interference and forgetting, as well as other factors that may distort results 16,17 .
Monitoring autonomic and behavioral responses simultaneously with fMRI provides a mechanism by which the neural substrates that mediate complex relationships between cognitive processes and behavioral/autonomic responses can be assessed. However, monitoring autonomic and behavioral responses in the MRI environment poses a number of practical problems. Specifically, 1) standard behavioral and physiological monitoring equipment is constructed of ferrous material that cannot be safely used near the MRI scanner, 2) when this equipment is placed outside of the MRI scanning chamber, the cables projecting to the subject can carry RF noise that produces artifacts in brain images, 3) artifacts can be produced within the skin conductance signal by switching gradients during scanning, 4) the fMRI signal produced by the motor demands of behavioral responses may need to be distinguished from activity related to the cognitive processes of interest. Each of these issues can be resolved with modifications to the setup of physiological monitoring equipment and additional data analysis procedures. Here we present a methodology to simultaneously monitor autonomic and behavioral responses during fMRI, and demonstrate the use of these methods to investigate aware and unaware memory processes during fear conditioning.

A methodology to investigate the neural mechanisms that support aware and unaware memory processes during fear conditioning is described. This method monitors blood oxygen level dependent (BOLD) functional magnetic resonance imaging, skin conductance response, and unconditioned stimulus expectancy during Pavlovian fear conditioning to assess the neural correlates of distinct memory processes.

调查的意识到，不知道用功能磁共振成像的恐惧记忆的神经机制

Pavlovian fear conditioning is often used in combination with functional magnetic resonance imaging (fMRI) in humans to investigate the neural substrates ...

科研

神经科学

A Computer-assisted Multi-electrode Patch-clamp System

The patch-clamp technique is today the most well-established method for recording electrical activity from individual neurons or their subcellular compartments. Nevertheless, achieving stable recordings, even from individual cells, remains a time-consuming procedure of considerable complexity. Automation of many steps in conjunction with efficient information display can greatly assist experimentalists in performing a larger number of recordings with greater reliability and in less time. In order to achieve large-scale recordings we concluded the most efficient approach is not to fully automatize the process but to simplify the experimental steps and reduce the chances of human error while efficiently incorporating the experimenter's experience and visual feedback. With these goals in mind we developed a computer-assisted system which centralizes all the controls necessary for a multi-electrode patch-clamp experiment in a single interface, a commercially available wireless gamepad, while displaying experiment related information and guidance cues on the computer screen. Here we describe the different components of the system which allowed us to reduce the time required for achieving the recording configuration and substantially increase the chances of successfully recording large numbers of neurons simultaneously.

Multi-electrode patch-clamp recordings constitute a complex task. Here we show how, by automating of many of the experimental steps, it is possible to accelerate the process leading to qualitative improvement in performance and number of recordings.

计算机辅助多电极膜片钳系统

The patch-clamp technique is today the most  well-established method for recording electrical activity from individual  neurons or their subcellular ...

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons.

Cortical networks are controlled by a small, but diverse set of inhibitory interneurons. Functional investigation of interneurons therefore requires targeted recording and rigorous identification. Described here is a combined approach involving whole-cell recordings from single or synaptically-coupled pairs of neurons with intracellular labeling, post-hoc morphological and immunocytochemical analysis.

从Morphologically-和确定的Neurochemically海马的interneurons全细胞膜片钳记录

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices,&#160;a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular,&#160;when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals.

This protocol describes basic procedural steps for performing whole-cell patch-clamp recordings. This technique allows the study of&#160;the electrical behavior of neurons, and when performed in brain slices, allows the assessment of various neuronal functions from neurons that are still integrated in relatively well preserved brain circuits.

在脑片全细胞膜片钳记录

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...

Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins

Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate electrical processes in target cells. In this context, ion selectivity of a channelrhodopsin is of particular importance. This article describes the investigation of chloride selectivity for a recently identified anion-conducting channelrhodopsin of Proteomonas sulcata via electrophysiological patch-clamp recordings on HEK293 cells. The experimental procedure for measuring light-gated photocurrents demands a fast switchable &#8211; ideally monochromatic &#8211; light source coupled into the microscope of an otherwise conventional patch-clamp setup. Preparative procedures prior to the experiment are outlined involving preparation of buffered solutions, considerations on liquid junction potentials, seeding and transfection of cells, and pulling of patch pipettes. The actual recording of current-voltage relations to determine the reversal potentials for different chloride concentrations takes place 24 h to 48 h after transfection. Finally, electrophysiological data are analyzed with respect to theoretical considerations of chloride conduction.

This article describes how the ion selectivity of channelrhodopsin is determined with electrophysiological whole-cell patch-clamp recordings using HEK293 cells. Here, the experimental procedure for investigating chloride selectivity of an anion-selective channelrhodopsin is demonstrated. However, the procedure is transferable to other channelrhodopsins of distinct selectivity.

全细胞贴片记录用于电泳生理测定通道视紫质中的离子选择性

Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

时间依赖性增加对微电极阵列刺激神经元细胞培养物的网络响应

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

Auditory Brainstem Response and Outer Hair Cell Whole-cell Patch Clamp Recording in Postnatal Rats

The outer hair cell is one of the two types of sensory hair cells in the mammalian cochlea. They alter their cell length with the receptor potential to amplify the weak vibration of low-level sound signal. The morphology and electrophysiological property of outer hair cells (OHCs) develop in early postnatal ages. The maturation of outer hair cell may contribute to the development of the auditory system. However, the process of OHCs development is not well studied. This is partly because of the difficulty to measure their function by an electrophysiological approach. With the purpose of developing a simple method to address the above issue, here we describe a step-by-step protocol to study the function of OHCs in acutely dissociated cochlea from postnatal rats. With this method, we can evaluate the cochlear response to pure tone stimuli and examine the expression level and function of the motor protein prestin in OHCs. This method can also be used to investigate the inner hair cells (IHCs).

This protocol describes methods to record the auditory brainstem response from postnatal rat pups. To examine the functional development of outer hair cells, the experimental procedure of whole-cell patch clamp recording in isolated outer hair cells is described step-by-step.

产后大鼠听觉脑干反应及外毛细胞全细胞贴片钳记录

The outer hair cell is one of the two types of sensory hair cells in the mammalian cochlea. They alter their cell length with the receptor potential to ...

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic tectum, is a popular model to study how neural circuits self-assemble. The ability to carry out whole cell patch clamp recordings from tectal neurons and to record RGC-evoked responses, either in vivo or using a whole brain preparation, has generated a large body of high-resolution data about the mechanisms underlying normal, and abnormal, circuit formation and function. Here we describe how to perform the in vivo preparation, the original whole brain preparation, and a more recently developed horizontal brain slice preparation for obtaining whole cell patch clamp recordings from tectal neurons. Each preparation has unique experimental advantages. The in vivo preparation enables the recording of the direct response of tectal neurons to visual stimuli projected onto the eye. The whole brain preparation allows for the RGC axons to be activated in a highly controlled manner, and the horizontal brain slice preparation allows recording from across all layers of the tectum.

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

In this paper, we discuss three brain preparations used for whole cell patch clamp recording to study the retinotectal circuit of Xenopus laevis tadpoles. Each preparation, with its own specific advantages, contributes to the experimental tractability of the Xenopus tadpole as a model to study neural circuit function.

爪蟾Tectal 神经元全细胞膜片钳记录的制备与协议

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic ...

Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms underlying sensory transmission, nociceptive regulation, and chronic pain or itch development. Implementations of electrophysiological recordings together with morphological studies based on the utility of acute spinal cord slices have further improved our understanding of neuronal properties and the composition of local circuitry in SG. Here, we present a detailed and practical guide for the preparation of spinal cord slices and show representative whole-cell recording and morphological results. This protocol permits ideal neuronal preservation and can mimic in vivo conditions to a certain extent. In summary, the ability to obtain an in vitro preparation of spinal cord slices enables stable current- and voltage-clamp recordings and could thus facilitate detailed investigations into the intrinsic membrane properties, local circuitry and neuronal structure using diverse experimental approaches.

Here, we describe the essential steps for whole-cell patch-clamp recordings made from substantia gelatinosa (SG) neurons in the in vitro spinal cord slice. This method allows the intrinsic membrane properties, synaptic transmission and morphological characterization of SG neurons to be studied.

凝胶神经元全细胞斑块夹的急性脊髓切片的制备

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms ...

Evaluation of Synaptic Multiplicity Using Whole-cell Patch-clamp Electrophysiology

In the central nervous system, a pair of neurons often form&#160;multiple synaptic contacts and/or functional neurotransmitter release sites (synaptic multiplicity). Synaptic multiplicity is plastic and changes throughout development and in different physiological conditions, being an important determinant for the efficacy of synaptic transmission. Here, we outline experiments for estimating the degree of multiplicity of synapses terminating onto a given postsynaptic neuron using whole-cell patch clamp electrophysiology in acute brain slices. Specifically, voltage-clamp recording is used to compare the difference between the amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) and miniature excitatory postsynaptic currents (mEPSCs). The theory behind this method is that afferent inputs that exhibit multiplicity will show large, action potential-dependent sEPSCs due to the synchronous release that occurs at each synaptic contact. In contrast, action potential-independent release (which is asynchronous) will generate smaller amplitude mEPSCs. This article outlines a set of experiments and analyses to characterize the existence of synaptic multiplicity and discusses the requirements and limitations of the technique. This technique can be applied to investigate how different behavioral, pharmacological or environmental interventions in vivo affect the organization of synaptic contacts in different brain areas.

Here, we present a protocol for evaluating the functional synaptic multiplicity using whole-cell patch clamp electrophysiology in acute brain slices.

全细胞膜片电生理方法对突触倍数的评价

In the central nervous system, a pair of neurons often form&#160;multiple synaptic contacts and/or functional neurotransmitter release sites (synaptic ...