We thank those individuals and groups who have used SpikeSorter and who have provided requests for file format support and suggestions and feedback on how to improve it. These include Youping Xiao, Felix Fung, Artak Khachatryan, Eric Kuebler, Curtis Baker, Amol Gharat and Dongsheng Xiao. We thank Adrien Peyrache for the false positive and negative figures given in 'Representative Results'.

1.程序设置<ol><li>转到http://www.swindale.ecc.ubc.ca/SpikeSorter下载程序。所提供的可执行文件复制到您选择的目录。阅读附带的文档。 注:无需正式安装或编译。 </li><li>打开要排序的任何文件之前，请确保有足够的可用内存来容纳录制的整个过程。还要确保有效的通道配置文件，如文档中所描述的，存在于同一目录中的数据文件。 </li><li>启动程序，然后进入"文件 - 打开"，并在所产生的打开文件对话框的右下角然后从下拉列表中录制的文件格式。选择要打开的文件，然后单击"打开"。 </li><li>一旦阅读完成后，检查电压记录显示。在屏幕上双击（或进入"查看 - 电压实录"），弹出一个对话框，允许任何控制杆要观看的记录波形的吨。 注:其他的显示窗口双击往往会带来了相关的对话框。 </li><li>该对话框退出后，将鼠标悬停在波形在显示屏的左上角显示特定的电压值。使用滚轮来放大屏幕的任何部分。按住鼠标左键拖动窗口的内容。 注意:此显示经常更新，以反映增加新检测到的事件，或指示，通过颜色和/或数字，集群后，他们的任务集群的手段。 </li><li>如果记录是未经过滤，包含本地外地的潜力，通过将"前处理 - 变换/过滤器​​"将其删除（或单击工具栏上的过滤器图标）。选择"高通巴特沃斯滤波器"，那么一个合适的截止频率和极数，然后按下"执行它！"。一旦筛选完成后，检查在电压波形新波形窗体窗口。 注:过滤在傅立叶域中进行，是非因果，和不引入波形的相位失真。在很长一段录音，过滤可能需要几分钟。 </li><li>其次，检查渠道可能出现故障，需要屏蔽。进入"前处理 - 通道检查"（或单击通道检查图标），然后检查出现的图形。该图显示作为空间分离5的功能在信道对之间的信号的相关性的变化。违反这种关系渠道可能无法正常工作。要查看任何这样的异常值，点击"单通道净偏差"。 <ol><li>要屏蔽一个边远的渠道要么选择通道号，或从问题列表中选择。当退出此对话框，在提示符下单击"是"保存屏蔽值。 注:此文件将具有相同的名称作为记录数据文件，但扩展.MSK。将要读取automaticall每当相同的数据文件被打开年。 </li></ol></li></ol> 2.事件检测<ol><li>进入"前的过程-事件检测"，弹出事件检测对话框（ 图1）。此对话框还提供了基于其噪音水平掩蔽渠道的选择（尽管这些通常由以前的试验检测到）。例如，已被有意接地的信道可以具有非常低的噪声水平。 </li><li>使用右上角的滑块来考察在特定的频道的噪声水平。电压显示的仔细检查可以发现需要被掩蔽无声或异常噪声信道。 </li><li>选择事件检测阈值方法。使用该组中的帮助按钮有关选项的详细信息。 "变量"阈值，具有4.5倍的阈值- 6X噪音7，值得推荐。使用顶部的控制左选择噪声电平如何为第计算是目的。 </li><li>然后从下拉列表中的检测方法。 "动态多相滤波器"是推荐的方法。这需要一个时间窗口的规范。设置窗口为大致一个典型尖峰的宽度的一半。很窄的值将偏差检测，以窄尖峰虽然效果不是很大。在0.15范围内的值- 0.5毫秒，推荐12。 注:显示的值是在采样间隔（往复采样频率）的整数倍。 </li><li>选择对准方法。选择最能标识正在排序， 例如 ，一个"正峰"尖峰单，暂时局部特征的选项可能是一个糟糕的选择，如果很多尖峰有一个以上的正峰。对于许多录音，一个"负低谷"将是最好的选择。其他选项通常可以在其默认值离开了。按开始&#39;。 注:事件可能检测TA从几秒克到几分钟，取决于记录的长度和信道的数目。 </li><li>按"完成"退出对话框。检查的事件，以灰色显示，在电压波形的窗口。检查已检测到的样子事件的信号。 <ol><li>如果没有，可以考虑用较低的检测阈值重新运行事件检测。然而要注意的是非常低的幅度尖峰可能难以进行排序，并且大量它们可能阻碍较大振幅峰值进行排序。还检查出明显的重复或未能解决峰值附近，并相应调整时空锁定窗口参数。 注:在此阶段事件由发生和信道号的它们的时间标识。通常这是在其上尖峰的峰 - 峰振幅是最大的通道。事件最初非集群，所以每个具有零簇分配。 </li></ol></li></ol> 3.排序</p&gt; 注:不常规排序之前正常地进行下一个步骤，但它是非常有用做排序首次时，或遇到不熟悉的数据时。 <ol><li>去&#39;排序 - 转换渠道集群"。这会为每个未屏蔽电极通道一个集群，假设每个通道都有分配给它的一些事件。通过将研究这些集群"回顾 - 查看清洁和斯普利特集群"。这带来了另一个对话框（ 图2）。使用旋转控制（左上），以选择要观看的集群。 注:固体蓝色（青色）线是集群中的所有的波形的平均值和在下文群集模板提及。集群中的事件的主成分（PC）的分布示于下面的窗口。这些往往会揭示两个或更多子群集的存在。 </li><li>按"重新调整"按钮来改变每个事件的时间（导致在显示波形的小侧向移动），以便更好地匹配到模板的形状，这样做往往使子群集更紧凑的和不同的，有时降低了表观数目（ 图3）。 </li><li>选择具有两个或多个不同子群集，然后按"自动分割"的集群。如果子群集是在PC显示识别的，它们将被着色。作为练习，用小&#39;分裂&#39;的一个按钮来创建一个新的群集和检查。排序可以手动继续这种方式，而是回去使用更快的自动排序过程。 </li><li>去&#39;排序 - 自动排序"（或按工具栏上的自动排序按钮），开始自动排序。所得对话框示于图4。它提出了多种选择。 <ol><li>离开"跳过事件检测"选项，如果事件检测已经完成检查。如果它没有被选中，事件检测将运行用的参数值，并从事件检测对话框继承的选择。由于事件检测已经完成，留下该选项选中。 </li><li>在下面的"集群"面板中，选择一个时间窗口大到足以包含之前和之后的对齐点秒杀波形的全部，但没有更多。如果他们出现在干扰（或贡献不大）排序使用该窗口阻止了尖峰波形， 如长变量afterpotentials的地区。在范围±0.5毫秒是适当的通常值。像其它时间窗口，该窗口是采样点的整数倍，从而使出现的时间值是采样间隔的倍数。 </li><li>接下来，选择聚类期间要使用的调整选项。这将利用模板波形的和工作的详细鲁棒比事件检测的初始情况下的标准已被应用到相对嘈杂的各个S派克波形。推荐的选项是"峰值加权齿轮"，而是"负低谷"可能会更好，如果是这样的尖峰波形的一贯特点。 </li><li>选择的最小群集大小。具有小于该数字的尖峰的集群将被删除，从而防止分选过程中大量的小，可能是伪，簇的积累。 </li><li>决定在PC空间尺寸，这将被用于聚类的数目。二是一般足够的，但稍微好一点的结果可能与3获得，尽管有更长的时间进行排序。 </li><li>在保留其默认设置其他选项。使用帮助按钮来获得的各种选项更详细的解释。 </li></ol></li><li>按"开始"开始自动排序。如步骤3.1中所示首先形成基于信道群集。这些现在处理反过来，通过拆分关闭单个子群集，一次成型的新集群。每当一个新的集群SP点燃时，PC值重新计算并显示出来。这样继续下去，直到没有单独的集群可以进一步分裂。 </li><li>按照显示器，其中红色表示将从父群被剥离的子集群的提示。 注:有时最终的集群是红与不形成明显的子集群无色异常值。这些异常值通常会被删除。在此过程中的簇的数目逐渐增大。当它完成后，集群重叠指数计算每一个符合条件的集群对。有很大的重叠值对的自动合并，而具有中间重合值（默认范围是0.1〜0.5）对合并后resplit。中间值表明，有两个不同的簇，但有些点misassigned。在这一阶段的簇的数目通常降低，并且稳定的簇的数目增加而增加。 </li></ol> 4.自定义<ol><li>如果（在接下来的步骤或可能）使用的程序的第一次，自主的窗口大小和位置。转到"文件 - 首选项"。通过从下拉列表中选择的窗口类型和调整大小，以适应屏幕选择用于各个窗口尺寸。退出对话框，并定位窗口，使屏幕的最佳使用。 </li><li>从该对话框中，选择最适合的记录电极上的通道和尖峰的布局和间距缩放值。有一个自动缩放选项，但这可能并不总是选择最佳值。关闭它，如果它没有。 </li><li>检查粘滞参数选项:如果选择了该选项，在排序参数值（ 例如在事件检测使用）的变化将被保存，继承下一个程序启动时间。这可以是有用的，但还需要的参数值进行检查，以确保他们没有被不小心或改变各种选项进行了探讨，结果阅读在不同的工作文件。改变分簇色彩的选择也可以。 </li><li>运动保健改变处理器的线程数。最佳数目通常为1比身体（未虚拟）的CPU内核的数量较少。增加线程的数量可以不加快处理速度，甚至可能导致一个严重的减速。 </li></ol> 5.合并和拆分<ol><li>自动排序完成后，按"下一步"进入手动引导合并和拆分阶段。产生的对话框显示，在左下角，待检查其余需要暧昧簇对的数目以及稳定簇的数目。 </li><li>按"开始"。另一个对话框出现一起与第一线对要被检查。 </li><li>选择是否合并在一对，resplit它（导致较低的重叠值），来标记对作为"独立的"，意思是该重叠索引的值将被忽略，邻R以标记对作为"含糊"，这意味着它被认为是不确定的尖峰是否从相同或不同的单元。 <ol><li>点击复选框中显示的尖峰参数（峰-峰值的主成分的（PP）的高度，或所述第一（PC1）或第二（PC2）） 与时间的关系 ，和/或自动和交叉的曲线图相关柱状图。 注:聚丙烯高度对时间的显示通常是在决定是否合并两个簇非常有用的。如果尖峰在一个单元的高度平滑地融合到那些在其他的一个单元停止点火，另一个开始它比不更可能是它们是相同的单元和应该合并在同一时间。交叉相关图可以在两个集群透露秒杀时间之间有很强的时间关系。如果交叉相关图具有一个很短的时间间隔一个强有力的，非对称的峰（ 例如约5 - 10毫秒），尤其是如果所述第二尖峰比冷杉小t时，两个单元是最有可能的是烧结尖峰对，其中第二比第一，因为钠离子通道适配的小的单个单元。 </li><li>在合并的决定是不容易的情况下，标记对作为"模棱两可"，并在随后的分析相应地对待集群。 </li></ol></li><li>如果合并和拆分选项是无法找到明确可分离集群，在弹出的对话框中使用滑块来手动相差集群参数（空间带宽，SIGMA），同一套合并按钮组合到一起，找到一个分割看起来令人满意。使用"恢复"按钮，返回到两个集群的原始状态。按"如图所示分割"来完成。注意两个以上的集群可以用这种方法制备。 </li><li>这个过程继续，直到有没有更多的对检查。绝大多数集群现在应该被列为"稳定"。 </li><li>如果某些集群对有很低重叠指数，让他们被引导合并忽略（但仍有证据表明它们合并），进入"回顾 - 比较集群对"菜单选项（或单击工具栏中的相关图标），打开对话框如图5所示。使用旋转控制在对话框的顶部，选择任意一对集群进行比较。 注:由于与引导合并和分割，对被投入的排序列表，但在这种情况下，比较标准附加到群集重叠索引可用。 <ol><li>从下拉列表中的"归一化积"选项。此计算模板值之间的相关性。它是不敏感的乘法缩放变化和非常适合于挑选出那些峰到峰的高度变异性的人为结果簇对。 </li><li>按在对话框中间的"最相似"按钮，显示最相似的对。使用horizo​​nta按钮下面升旋转控制前进或后退通过列表。使用此相关的显示和聚丙烯高度与时间显示，使合并的决定，只作为用户引导合并和拆分。注意，该列表的每个合并操作之后重新计算。这种比较阶段是开放式的，并且它是由用户来决定如何广泛搜索赞成合并的证据。 </li></ol></li></ol> 6.审查 - 后处理<ol><li>现在去"回顾 - 后处理"（或单击相应的工具栏图标）。这个对话框（ 图6）提供的选项中添加或删除群集事件，以及删除与低于阈值信号噪声比（SNR）整个集群的选项。重复事件（在集群中的同时发生的事件）可以通过对准误差排序期间创建。那些很长的路要走从原来的位置删除事件可以sometimES是搬迁;它们也可以被重新安置时不起作用除去。 </li><li>使用对齐清洗按钮从那些不好的匹配模板集群删除的事件。使用"重新群集"按钮做反向， 即重新分配是一个很好的匹配特定模板非集群事件。回收的事件被标记为每个父母群集的子集群，可以使用"查看，清洁和分体式集群"对话框中进行检查。这些活动将继续在集群中（和导出这样），除非它们被删除（用小"删除"按钮，第一个子集群）。返回到后处理对话框中，使用其旁边的"删除"按钮和旋转控制用的SNR小于选择的阈值来删除簇。 </li><li>尽管簇编号从1连续到N，其中N是集群总数，集群中的分选的末端的实际编号方式是靠近一个rbitrary。使用"排序"按钮，根据所选择的标准来重新编号簇， 例如在电极上的垂直位置，或信道编号。需要注意的是，随着重复事件的缺失外，目前还没有客观证据来支持在该对话框中选择特定的作为是比别人做得更好。 </li><li>在过程中的排序的手动过程的任何阶段，可以保存它包含当前参数值，排序选项，事件时间，簇的属性和消息记录文件。通过将创建该文件的"文件 - 保存工作文件"。给文件，这显然是相关的数据文件，然后按"保存"的名字。恢复通过首先打开原始记录文件，接着通过高通滤波（如果最初进行）在稍后的时间排序。然后，打开保存工作文件。然后程序会在相同的一个是，当保存工作文件中的状态。这项工作文件也是一个重的排序是怎么做线 - 使用的参数和排序过程中发出的消息的。 </li><li>最后，出口聚集事件。去&#39;出口 - 排序秒杀文件"（或点击相关按钮的工具栏）。选择从下拉列表&#39;.csv文件"（逗号分隔变量），然后单击"另存为"。选择将包含在排序单位导出CSV数据文件的名称。 注:本文本文件将具有用于容纳每个事件的单个线，为了，事件的时间（以秒到最接近的10微秒），群集数（从1向上），并且被分配信道的数量到该事件。请注意，分配渠道可能不是在集群中的所有事件一样，如果事件是不是在某个特定通道始终较大。 </li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spike+detection+methods+for+polytrodes+and+high+density+microelectrode+arrays.">Spike detection methods for polytrodes and high density microelectrode arrays.</a> J. Comput. Neurosci. 38, 249-261 (2015).</li><li>Swindale, N. V., Spacek, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Veri&#64257;cation+of+multichannel+electrode+array+integrity+by+use+of+cross-channel+correlations.">Veri&#64257;cation of multichannel electrode array integrity by use of cross-channel correlations.</a> J. Neurosci. Meth. 263, 95-102 (2016).</li><li>Fukunaga, K., Hostetler, L. 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Abstr. 598 (10), (2015).</li><li>Hazan, L., Zugaro, M., Buzs&#225;ki, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Klusters,+NeuroScope,+NDManager:+A+free+software+suite+for+neurophysiological+data+processing+and+visualization.">Klusters, NeuroScope, NDManager: A free software suite for neurophysiological data processing and visualization.</a> J. Neurosci. Meth. 155, 207-216 (2006).</li><li>Harris, K. D., Henze, D. A., Csicsvari, J., Hirase, H., Buzs&#225;ki, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accuracy+of+tetrode+spike+separation+as+determined+by+simultaneous+intracellular+and+extracellular+measurements.">Accuracy of tetrode spike separation as determined by simultaneous intracellular and extracellular measurements.</a> J. Neurophysiol. 84, 401-414 (2000).</li><li>Anastassiou, C. A., Perin, R., Buzs&#225;ki, G., Markram, H., Koch, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-type+and+activity+dependent+extracellular+correlates+of+intracellular+spiking.">Cell-type and activity dependent extracellular correlates of intracellular spiking.</a> J. Neurophysiol. 114, 608-623 (2015).</li><li>Wohrer, A., Humphries, M. D., Machens, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Population-wide+distributions+of+neural+activity+during+perceptual+decision-making.">Population-wide distributions of neural activity during perceptual decision-making.</a> Prog. Neurobiol. 103, 156-193 (2013).</li><li>Mizuseki, K., Buzs&#225;ki, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preconfigured,+skewed+distribution+of+firing+rates+in+the+hippocampus+and+entorhinal+cortex.">Preconfigured, skewed distribution of firing rates in the hippocampus and entorhinal cortex.</a> Cell Reports. 4, 1010-1021 (2013).</li><li>Schmitzer-Torbert, N., Jackson, J., Henze, D., Harris, K., Redish, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+measures+of+cluster+quality+for+use+in+extracellular+recordings.">Quantitative measures of cluster quality for use in extracellular recordings.</a> Neuroscience. 131, 1-11 (2005).</li><li>Barnett, A. H., Magland, J. F., Greengard, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+neural+spike+sorting+algorithms+without+ground-truth+information.">Validation of neural spike sorting algorithms without ground-truth information.</a> J. Neurosci. Meth. 264, 65-77 (2016).</li></ol>

The authors have nothing to disclose.

文件格式目前支持的文件格式包括Neuralynx（.ntt和.ncs），Plexon公司（.PLX），Neuroscope（+的.xml .DAT），多通道系统（.mcd），黑石（.nev）和安顺（.rhd）。对于不支持的格式，有两种选择。一是申请加入文件格式为即将到来的释放（电子邮件连结到开发人员在提供"帮助 - 关于"对话框）。另一种是将文件转换为支持的格式。一个简单的选择是使用时间秒杀格式".tsf"。这梗概格式包含?...

人谁使用方法比简单的线上阈值和窗口更复杂的脑记录外信号面临识别和从由电极所记录的噪声电压信号分离不同的神经元的信号的任务。这个任务就是俗称秒杀排序。穗排序的难度是由各种因素复杂。使得由附近的电极从他们记录的信号很可能是相似的，很难区分神经元可以是非常靠近在一起。由一个单一的神经元产生的信号可以在在靠近所述电极，或可能作为树突高燃烧率，不同程度的电压电导的激活期间随时间变化，因为电极，可变钠通道动力学运动也许在大脑状态的变化的结果。 （ - 100微米20）R，这些问题可以通过使用多电极阵列（MEAs）中具有许多紧密间隔来减轻<html> ECORDING通道，它允许从单一神经元的信号的更好的空间定义，因为它们通常在几个通道 1,2摊开。然而，这，与从沿在空间电极重叠的整个长度传播神经元信号的事实相结合，结果在其内对应于唯一的神经元集群一个潜在的非常高维空间需要确定。这个问题变得比少数电极渠道更难以计算。迄今为止，没有通常商定为尖峰分拣最好的方法，虽然许多解决方案已经提出了3，4，5，6，7，8以及从的MEA录音变得越来越普遍9屁股="外部参照"&gt; 10。由于穗排序本身并不是目的，而只是在进一步的数据分析的必要第一步，还需要一个容易使用的包，将原始数据记录文件中读取，并将其转换为有序穗列车用尽可能少的用户输入，并作为快速，可靠地，尽可能。 本文为使用SpikeSorter的教程 - 与满足这些需求为目的开发的程序。该方案是基于先前发表的论文11，12，13所描述的算法。在设计方案的目标是， 一个 ），它应该有一个友好的用户界面需要很少或计算机编程或穗排序方法的先验知识; B）应需要超越标准的Windows或Linux操作系统很少或根本没有其他专门的软件组件; C </em&gt;）范围广泛的记录数据格式的数据导入和导出应予支持; 四 ）排序应该最小化期间分拣需要用户输入，以及e）时间应比例以合理的方式，理想的直线，与记录持续时间和电极上的信道数。在程序中实现的算法包括:a）一组灵活的前处理和事件检测策略; b）一种自动分而降维哪个簇基于该主成分（PC）的从分配给特定的集群信道的子集得到的分布的电压波形的治策略; c）与基于均值漂移算法3,14和d快速聚类程序）部分自动配对合并和簇分裂的PC分布自动聚类，以确保每个尽可能从所有其他截然不同。要THIS，一套程序被加入，允许手动或拆分基于PC的分布，穗列车和尖峰波形的时间幅度图的交叉和自相关图检查集群合并。从四极管，四极管阵列，阵列犹他州以及单和多柄多边环境协定录音可以读取和排序。上的信道数的电流极限是256但这可能在将来被增加。 另一种跨平台的开源实现，"spyke"（http://spyke.github.io），也可用。撰稿（MS）在Python和用Cython自己人，spyke使用相同的总方针为SpikeSorter，有一些差异:减少内存需求，原始数据被加载在小块，只有当绝对必要的;簇专门显示，操作和在3D排序;和主成分和独立成分分析都被用作补充降维的方法。 Spyke需要更多用户teraction，但严重依赖键盘和鼠标快捷键和撤销/重做队列上迅速探索尖峰任何给定的子集的聚集各种因素的影响。这些因素包括秒杀频道和时间范围选择，秒杀调整，群集的尺寸和空间带宽（SIGMA）11。 以下是用于排序的算法和策略的简要说明。更完整的描述可在以前的出版物11，12，13，并在可经由帮助按钮来访问注解内SpikeSorter找到（用标识"？"）。加载原始细胞外电压文件并滤除较低的频率分量，事件检测的结果在一组事件的初始阶段，其中每一个的事件之前的时间后由一个短暂电压快照之后。如果选民骑着网站都足够密集（100微米&lt;），单单元的信号通常出现在几个相邻通道。一个中心通道自动选择对于每个事件，对应于该事件的峰 - 峰电压为最大的信道。自动通过形成每个电极通道的单个初始簇，由该被定位于该通道的所有事件的排序开始。位于中间通道之间的单元可以产生被本地化（也许随机地）到不同的信道尖峰:从这两组尖峰的集群将被标识为类似的，并在稍后的阶段合并。在每个初始簇的事件的平均波形，然后计算。这被称为群集的模板。附属信道分配给基于该振幅和每个通道的模板波形的标准偏差每个群集。主成分值，然后计算每个基于集群ØN于所分配的信道集合的波形。用户可以选择使用主成分的维数:通常为2就足够了。然后每个簇被分成进一步集簇，并且这被重复，直到没有可以通过自动聚类进一步分裂。 在这一点上，一组初始的发言权的，从64通道电极64簇中，可以视其存在于记录单元的数目分成两个或三倍数量。但由于从单一单位不同信道的事件的变量赋值的，在这个阶段中的簇的数目是几乎肯定大于它应该是。分拣的下一阶段是通过比较对簇和合并类似成对或重新分配从一个到另一个事件来纠正oversplitting。排序的这一阶段被称为"合并和拆分"。 融合与分裂 对于N群集，存在N *（N-1）/ 2对和因此对的数目的增长为N 2，这是不希望的。然而，许多对可从比较，因为在一对的两个成员相距很远物理排除。这降低了依赖的东西，更线性关系的信道数。尽管这个快捷方式，合并和拆分阶段仍然可以非常耗时。它工作在以下方式。每个集群对要被进行比较（那些物理上接近在一起，通过在分配给每个信道组的重叠判断）被暂时合并，尽管保持尖峰的身份已知的两个成员的集群。合并后的对的主成分然后计算。在两个集群的点之间的重叠的测量，计算基于所述第一两个主成分的分布。 一路OV<html> erlap量度计算中更详细别处11进行说明。它的值是零，如果群集完全不重叠， 即每个点的近邻是同一群集中。其值接近1，如果簇完全重叠， 即在最近邻同一集群中的存在的概率是相同的，从点的均匀混合的预测。 各种决定是由内搭重叠措施考虑在内。如果重叠大于一定值时，簇可以被合并。如果重叠很小，群集一对可被定义为不同的和单独留下。中间值，表示群集一对的不完全分离，可能预示着所述一对应该合并，然后重新分割，期望的结果是一对簇较少重叠。这些过程是:首先在自动化阶段，然后运行手动引导阶段。 帐篷"&gt;在自动阶段，具有很高的重叠值集群对合并;然后集群对具有中间低的重叠值被合并和再分割在第二，用户引导的阶段，向用户呈现与所有的剩下的暧昧集群对（ 即那些在规定的中间范围的重叠值）的序列，并要求选择是否 ）合并对，b）合并，resplit的对，C）宣布对是不同的（这将覆盖重叠度量的意义），或d）以限定所述一对为"不明确"指示在该对峰值不太可能良好排序，还提供有多种工具，以帮助这些决定，包括汽车之间的关系 - 和交叉相关图和扣球高度和PC值的时间序列图。 理想情况下，在合并和分裂阶段结束时，每个集群应当从所有其他不同，可能是因为它具有很少或没有公共信道与其他集群，或者因为重叠指数小于一个规定值。此值是用户可选择的，但通常为0.1。即通过此测试簇（单位）被定义为"稳定"，那些不（因为与一个或多个其它簇的重叠是大于阈值）被定义为"不稳定"。在实践中，大多数的单元最终被定义为"稳定"在排序的完成，留下其余要么被丢弃或作为潜在的多单元处理。 软件要求 SpikeSorter与Windows 7和Windows 10的64位版本的兼容，也成功地在Linux下使用Wine模拟器运行。数据文件被完全装入存储器（速度），因此可用的RAM需要与记录的大小按比例（允许约2 GB用于节目本身）。电生理学在尺寸上比130 GB的人的数据文件已被成功地分类保存在Windows和Linux环境。选项是通过标准的Windows菜单，工具栏和对话框访问。在菜单上的项目的布局操作的大致顺序相匹配，排序，与左边的数据输入和允许排序的数据进出口权的"导出"菜单中的"文件"菜单开始。工具栏按钮提供快捷键常用的菜单项。 通道配置文件 许多记录的数据格式不存储通道的位置。但是，知道这些是穗排序至关重要。信道也可以在通过采集软件各种方式编号:SpikeSorter要求信道按顺序编号，从信道1开始。因此，一个辅助电极配置文件必须创建，可以重新映射信道号跟随顺序规则，并卖场渠道禄ations。通道配置文件与文本的每个信道的单个行的文本文件。长文件的第一行存储文本名称，最多16个字符，标识了电极。在随后的行中的数字可以通过标签，一个逗号或空格隔开。有每行中的四个数字提供（按顺序）:该文件中的信道号，信道编号，以它是被映射（将由SpikeSorter使用即数），x和的y坐标渠道，微米。 x坐标通常被取为垂直于电极插入的方向和y相应坐标会深入到组织中。配置文件必须放置在相同的目录中记录的文件。这里面如何命名有一定的灵活性。该计划将首先搜索具有相同的名称作为原始数据文件，但有一个.CFG扩展名的文件。如果该文件我s未找到，它将搜索文件"electrode.cfg"。如果未找到反过来该文件中生成一个错误消息以指示缺少信道布局信息。

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="21">
			<td class="xl63" height="21" style="height:15.75pt;width:162pt" width="216">spikesorter.exe</td>
			<td class="xl63" style="width:53pt" width="71">N/A</td>
			<td class="xl63" style="width:89pt" width="119"></td>
			<td class="xl64" style="width:125pt" width="167">program file currently available through invited dropbox link</td>
		</tr>
	</tbody>
</table>

a visual guide to sorting electrophysiological recordings using 'spikesorter'

Few stand-alone software applications are available for sorting spikes from recordings made with multi-electrode arrays. Ideally, an application should be user friendly with a graphical user interface, able to read data files in a variety of formats, and provide users with a flexible set of tools giving them the ability to detect and sort extracellular voltage waveforms from different units with some degree of reliability. Previously published spike sorting methods are now available in a software program, SpikeSorter, intended to provide electrophysiologists with a complete set of tools for sorting, starting from raw recorded data file and ending with the export of sorted spikes times. Procedures are automated to the extent this is currently possible. The article explains and illustrates the use of the program. A representative data file is opened, extracellular traces are filtered, events are detected and then clustered. A number of problems that commonly occur during sorting are illustrated, including the artefactual over-splitting of units due to the tendency of some units to fire spikes in pairs where the second spike is significantly smaller than the first, and over-splitting caused by slow variation in spike height over time encountered in some units. The accuracy of SpikeSorter's performance has been tested with surrogate ground truth data and found to be comparable to that of other algorithms in current development.

图7示出了显示（前往"查看-排序波形的获得）为典型的排序记录。默认视图选项只是显示对每个簇的中心信道的波形。一个常见的经验是，对在相同的信道群集一对波形看起来是相同的，但是当"比较成对&#39;用来对话检查两个簇有在PC投影不同的聚类，最常从相邻通道波形的差异导致。这是事实，例如，在图7上的信道62中的波形。 <p class="jove_c...

Watch this Scientific Journal Video about A Visual Guide to Sorting Electrophysiological Recordings Using 'SpikeSorter' at JoVE.com

A Visual Guide to Sorting Electrophysiological Recordings Using 'SpikeSorter'

The article shows how to use the program SpikeSorter to detect and sort spikes in extracellular recordings made with multi-electrode arrays.

可视化指南排序的电生理记录使用"SpikeSorter"

Few stand-alone software applications are available for sorting spikes from recordings made with multi-electrode arrays. Ideally, an application should be ...

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11.0K 次观看.  University of British Columbia. The article shows how to use the program SpikeSorter to detect and sort spikes in extracellular recordings made with multi-electrode arrays.

视频: A Visual Guide to Sorting Electrophysiological Recordings Using 'SpikeSorter'

Chronic Imaging of Mouse Visual Cortex Using a Thinned-skull Preparation

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The technique presented here allows the imaging of the same area of the brain at several time points (chronic imaging) with microscopic resolution allowing the tracking of dendritic spines which are the small structures that represent the majority of postsynaptic excitatory sites in the CNS. The ability to clearly resolve fine cortical structures over several time points has many advantages, specifically in the study of brain plasticity in which morphological changes at synapses and circuit remodeling may help explain underlying mechanisms. In this video and supplementary material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation. The thinned-skull preparation is a minimally invasive approach, which avoids potential damage to the dura and/or cortex, thus reducing the onset of an inflammatory response. When this protocol is performed correctly, it is possible to clearly monitor changes in dendritic spine characteristics in the intact brain over a prolonged period of time.

In this video and supplemental material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation.

慢性成像小鼠的视觉皮层使用减薄，颅骨的制备

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The ...

科研

神经科学

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" (or "startle") response during the adult stage is mediated by the multi-component neuronal circuit called the Giant Fiber System (GFS). The comparative large size of the neurons, their distinctive morphology and simple connectivity make the GFS an attractive model system for studying neuronal circuitry. The GFS pathway is composed of two bilaterally symmetrical Giant Fiber (GF) interneurons whose axons descend from the brain along the midline into the thoracic ganglion via the cervical connective. In the mesothoracic neuromere (T2) of the ventral ganglia the GFs form electro-chemical synapses with 1) the large medial dendrite of the ipsilateral motorneuron (TTMn) which drives the tergotrochanteral muscle (TTM), the main extensor for the mesothoracic femur/leg, and 2) the contralateral peripherally synapsing interneuron (PSI) which in turn forms chemical (cholinergic) synapses with the motorneurons (DLMns) of the dorsal longitudinal muscles (DLMs), the wing depressors. The neuronal pathway(s) to the dorsovental muscles (DVMs), the wing elevators, has not yet been worked out (the DLMs and DVMs are known jointly as indirect flight muscles - they are not attached directly to the wings, but rather move the wings indirectly by distorting the nearby thoracic cuticle) (King and Wyman, 1980; Allen et al., 2006). The di-synaptic activation of the DLMs (via PSI) causes a small but important delay in the timing of the contraction of these muscles relative to the monosynaptic activation of TTM (~0.5 ms) allowing the TTMs to first extend the femur and propel the fly off the ground. The TTMs simultaneously stretch-activate the DLMs which in turn mutually stretch-activate the DVMs for the duration of the flight. The GF pathway can be activated either indirectly by applying a sensory (e.g."air-puff" or "lights-off") stimulus, or directly by a supra-threshold electrical stimulus to the brain (described here). In both cases, an action potential reaches the TTMs and DLMs solely via the GFs, PSIs, and TTM/DLM motoneurons, although the TTMns and DLMns do have other, as yet unidentified, sensory inputs. Measuring "latency response" (the time between the stimulation and muscle depolarization) and the "following to high frequency stimulation" (the number of successful responses to a certain number of high frequency stimuli) provides a way to reproducibly and quantitatively assess the functional status of the GFS components, including both central synapses (GF-TTMn, GF-PSI, PSI-DLMn) and the chemical (glutamatergic) neuromuscular junctions (TTMn-TTM and DLMn-DLM). It has been used to identify genes involved in central synapse formation and to assess CNS function.

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

The Giant Fiber System is a simple neuronal circuit of adult Drosophila melanogaster containing the largest neurons in the fly. We describe the protocol for monitoring synaptic transmission through this pathway by recording post synaptic potentials in dorsal longitudinal (DLM) and tergotrochanteral (TTM) muscles following direct stimulation of the Giant Fiber interneurons.

从巨纤维通路的电生理记录 D。果蝇</em

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" ...

Transretinal ERG Recordings from Mouse Retina: Rod and Cone Photoresponses

There are two distinct classes of image-forming photoreceptors in the vertebrate retina: rods and cones. Rods are able to detect single photons of light whereas cones operate continuously under rapidly changing bright light conditions. Absorption of light by rod- and cone-specific visual pigments in the outer segments of photoreceptors triggers a phototransduction cascade that eventually leads to closure of cyclic nucleotide-gated channels on the plasma membrane and cell hyperpolarization. This light-induced change in membrane current and potential can be registered as a photoresponse, by either classical suction electrode recording technique1,2 or by transretinal electroretinogram recordings (ERG) from isolated retinas with pharmacologically blocked postsynaptic response components3-5. The latter method allows drug-accessible long-lasting recordings from mouse photoreceptors and is particularly useful for obtaining stable photoresponses from the scarce and fragile mouse cones. In the case of cones, such experiments can be performed both in dark-adapted conditions and following intense illumination that bleaches essentially all visual pigment, to monitor the process of cone photosensitivity recovery during dark adaptation6,7. In this video, we will show how to perform rod- and M/L-cone-driven transretinal recordings from dark-adapted mouse retina. Rod recordings will be carried out using retina of wild type (C57Bl/6) mice. For simplicity, cone recordings will be obtained from genetically modified rod transducin &alpha;-subunit knockout (T&alpha;-/-) mice which lack rod signaling8.

We describe a relatively simple method of transretinal electroretinogram (ERG) recordings for obtaining rod and cone photoresponses from intact mouse retina. This approach takes advantage of the block of synaptic transmission from photoreceptors to isolate their light responses and record them using field electrodes placed across the isolated flat-mounted retina.

transretinal ERG的录音，从小鼠视网膜杆和锥光反应

There are two distinct classes of image-forming photoreceptors in the vertebrate retina: rods and cones. Rods are able to detect single photons of light ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

双突触诱发的星形胶质细胞和神经元的反应，在急性海马脑片电生理记录

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Simultaneous Whole-cell Recordings from Photoreceptors and Second-order Neurons in an Amphibian Retinal Slice Preparation

One of the central tasks in retinal neuroscience is to understand the circuitry of retinal neurons and how those connections are responsible for shaping the signals transmitted to the brain. Photons are detected in the retina by rod and cone photoreceptors, which convert that energy into an electrical signal, transmitting it to other retinal neurons, where it is processed and communicated to central targets in the brain via the optic nerve. Important early insights into retinal circuitry and visual processing came from the histological studies of Cajal1,2 and, later, from electrophysiological recordings of the spiking activity of retinal ganglion cells - the output cells of the retina3,4.
A detailed understanding of visual processing in the retina requires an understanding of the signaling at each step in the pathway from photoreceptor to retinal ganglion cell. However, many retinal cell types are buried deep in the tissue and therefore relatively inaccessible for electrophysiological recording. This limitation can be overcome by working with vertical slices, in which cells residing within each of the retinal layers are clearly visible and accessible for electrophysiological recording.
Here, we describe a method for making vertical sections of retinas from larval tiger salamanders (Ambystoma tigrinum). While this preparation was originally developed for recordings with sharp microelectrodes5,6, we describe a method for dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells in which we manipulate the photoreceptor's membrane potential while simultaneously recording post-synaptic responses in horizontal or bipolar cells. The photoreceptors of the tiger salamander are considerably larger than those of mammalian species, making this an ideal preparation in which to undertake this technically challenging experimental approach. These experiments are described with an eye toward probing the signaling properties of the synaptic ribbon - a specialized synaptic structure found in a only a handful of neurons, including rod and cone photoreceptors, that is well suited for maintaining a high rate of tonic neurotransmitter release7,8 - and how it contributes to the unique signaling properties of this first retinal synapse.

We describe the preparation of thin retinal slices from aquatic tiger salamanders (Ambystoma tigrinum) and explain how we use these slices to study synaptic processing in the retina by obtaining dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells.

同时光感受器和二阶神经元在两栖类动物的视网膜切片制备全细胞记录

One of the central tasks in retinal neuroscience  is to understand the circuitry of retinal neurons and how those connections are  responsible for shaping ...

The Swimmeret System of Crayfish: A Practical Guide for the Dissection of the Nerve Cord and Extracellular Recordings of the Motor Pattern

Here we demonstrate the dissection of the crayfish abdominal nerve cord. The preparation comprises the last two thoracic ganglia (T4, T5) and the chain of abdominal ganglia (A1 to A6). This chain of ganglia includes the part of the central nervous system (CNS) that drives coordinated locomotion of the pleopods (swimmerets): the swimmeret system. It is known for over five decades that in crayfish each swimmeret is driven by its own independent pattern generating kernel that generates rhythmic alternating activity 1-3. The motor neurons innervating the musculature of each swimmeret comprise two anatomically and functionally distinct populations 4. One is responsible for the retraction (power stroke, PS) of the swimmeret. The other drives the protraction (return stroke, RS) of the swimmeret. Motor neurons of the swimmeret system are able to produce spontaneously a fictive motor pattern, which is identical to the pattern recorded in vivo 1.
The aim of this report is to introduce an interesting and convenient model system for studying rhythm generating networks and coordination of independent microcircuits for students&#8217; practical laboratory courses. The protocol provided includes step-by-step instructions for the dissection of the crayfish&#8217;s abdominal nerve cord, pinning of the isolated chain of ganglia, desheathing the ganglia and recording the swimmerets fictive motor pattern extracellularly from the isolated nervous system.
Additionally, we can monitor the activity of swimmeret neurons recorded intracellularly from dendrites. Here we also describe briefly these techniques and provide some examples. Furthermore, the morphology of swimmeret neurons can be assessed using various staining techniques. Here we provide examples of intracellular (by iontophoresis) dye filled neurons and backfills of pools of swimmeret motor neurons. In our lab we use this preparation to study basic functions of fictive locomotion, the effect of sensory feedback on the activity of the CNS, and coordination between microcircuits on a cellular level.

Here we describe the dissection of the crayfish abdominal nerve cord. We also demonstrate an electrophysiological technique to record fictive locomotion from swimmeret motor neurons.

小龙虾的游泳足系统:实用指南为神经线及电机模式外记录的解剖

Here we demonstrate the dissection of the crayfish abdominal nerve cord. The preparation comprises the last two thoracic ganglia (T4, T5) and the chain of ...

Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with simultaneous intracellular biocytin filling allows a correlated analysis of their structural and functional properties. With this method it is possible to identify and characterize both pre- and postsynaptic neurons by their morphology and electrophysiological response pattern. Paired recordings allow studying the connectivity patterns between these neurons as well as the properties of both chemical and electrical synaptic transmission. Here, we give a step-by-step description of the procedures required to obtain reliable paired recordings together with an optimal recovery of the neuron morphology. We will describe how pairs of neurons connected via chemical synapses or gap junctions are identified in brain slice preparations. We will outline how neurons are reconstructed to obtain their 3D morphology of the dendritic and axonal domain and how synaptic contacts are identified and localized. We will also discuss the caveats and limitations of the paired recording technique, in particular those associated with dendritic and axonal truncations during the preparation of brain slices because these strongly affect connectivity estimates. However, because of the versatility of the paired recording approach it will remain a valuable tool in characterizing different aspects of synaptic transmission at identified neuronal microcircuits in the brain.

Patch-clamp recordings and simultaneous intracellular biocytin filling of synaptically coupled neurons in acute brain slices allow a correlated analysis of their structural and functional properties. The aim of this protocol is to describe the essential technical steps of electrophysiological recording from neuronal microcircuits and their subsequent morphological analysis.

神经微电路急性脑片电生理和形态特征采用配对膜片钳记录

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with ...

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 ...

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model.
For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied. Here, we focus on methods that allow the study of an essential property of neurons, electrical excitability. Our preparation for the electrophysiological study of zebrafish spinal neurons involves the use of veterinarian suture glue to secure the preparation to a recording chamber. Alternative methods for recording from zebrafish embryos and larvae involve the attachment of the preparation to the chamber using a fine tungsten pin1,2,3,4,5. A tungsten pin is most often used to mount the preparation in a lateral orientation, although it has been used to mount larvae dorsal-side up4. The suture glue has been used to mount embryos and larvae in both orientations. Using the glue, a minimal dissection can be performed, allowing access to spinal neurons without the use of an enzymatic treatment, thereby avoiding any resultant damage. However, for larvae, it is necessary to apply a brief enzyme treatment to remove the muscle tissue surrounding the spinal cord. The methods described here have been used to study the intrinsic electrical properties of motor neurons, interneurons, and sensory neurons at several developmental stages6,7,8,9.

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

This manuscript describes methods for electrophysiological recordings from spinal neurons of zebrafish embryos and larvae. The preparation maintains neurons in situ and often involves minimum dissection. These methods allow for the electrophysiological study of a variety of spinal neurons, from the initial electrical excitability acquisition through the early larval stages.

斑马鱼<em&gt;原位</em&gt;脊髓制备用于脊髓感觉和运动神经元的电生理记录

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, enabling the use of D. melanogaster molecular genetics to study inositol-lipid signaling and Transient Receptor Potential (TRP) channels at the single-molecule level. A handful of labs have mastered this powerful technique, which enables the analysis of the physiological responses to light under highly controlled conditions. This technique allows control over the intracellular and extracellular media; the membrane voltage; and the fast application of pharmacological compounds, such as a variety of ionic or pH indicators, to the intra- and extracellular media. With an exceptionally high signal-to-noise ratio, this method enables the measurement of dark spontaneous and light-induced unitary currents (i.e.&#160;spontaneous and quantum bumps) and macroscopic Light-induced Currents (LIC) from single D. melanogaster photoreceptors. This protocol outlines, in great detail, all the key steps necessary to perform this technique, which includes both electrophysiological and optical recordings. The fly retina dissection procedure for the attainment of intact and viable ex vivo isolated ommatidia in the bath chamber is described. The equipment needed to perform whole-cell and fluorescence imaging measurements are also detailed. Finally, the pitfalls in using this delicate preparation during extended experiments are explained.

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell recordings from Drosophila melanogaster photoreceptors enable the measurement of spontaneous dark bumps, quantum bumps, macroscopic responses to light, and current-voltage relationships under various conditions. In combination with D. melanogaster genetic manipulation tools, this method enables the study of the ubiquitous inositol-lipid signaling pathway and its target, the TRP channel.

可视化指南排序的电生理记录使用"SpikeSorter"

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