SN performed the experiments described above, performed the data analysis and drafted the manuscript. AD and JDM implemented the technique at our research facilities and aided in the data analysis. HC aided in performing the experiments. WJ, CK and DG assisted in implementing the afferent measurement technique in our lab, the data analysis and interpretation of the results. SF, JDM and BDW designed the study. All authors critically read and approved the final manuscript. SN is an aspirant of the Fund for Scientific Research (FWO), Flanders (11G7415N). This work was supported financially by the FWO (G028615N and G034113N).

下面描述所有的动物实验是由委员会医学伦理和安特卫普大学（文件编号2012-42）使用实验动物的批准。 1.空肠的组织准备和结肠传入神经<ol><li>空肠传入神经的制备<ol><li>执行已经由当地伦理委员会的实验之前批准的青少年或成年啮齿类啮齿类安乐死（ 例如 ，终端镇静随后通过心脏穿刺，颈椎脱臼等 ）。 注意:我们用颈脱位牺牲从而导致实验动物，而不需要进一步的麻醉或手术后护理如组织被体外进一步处理。 注:年龄已经显示出减弱肠系膜传入mechanosensory功能30，因此，我们建议研究者坚持到特定年龄组的持续时间一个单一的实验。 </li><li>放置在仰卧位处死实验动物和用手术刀通过皮肤和腹部肌肉层执行腹部正中切口，从xyphoid过程延伸直到耻骨。 </li><li>洗澡用冷Krebs溶液腹腔中以防止腹内组织变干（克雷布斯组合物:120.03毫摩尔NaCl，6.22毫摩尔的KCl，1.57毫的NaH 2 PO 4，15.43毫碳酸氢钠 ，1.21毫硫酸镁 ，11.52毫D-葡萄糖和1.52毫米氯化钙2）。 </li><li>通过切除约20厘米小肠开始立即远端的十二指肠空肠弯曲的，注意不要损伤周围结构，保持肠道的肠系膜，其中包含空肠血管和传入神经，完整的快速切除用锋利的剪刀整个空肠。 注意:对于在腹腔仅仅空肠解剖，一个不需要使用立体显微镜，因为这可以用肉眼很容易地可视化。 </li><li>放置在冰冷的Krebs溶液的切空肠，并保持在冰上，而具有卡波金（95％O 2，5％的CO 2）连续充氧的Krebs溶液。 </li><li>切约3厘米长的循环用锋利的剪刀空肠。观察含有邻近的各环的中心某处的血管和内脏神经肠系膜束。 </li><li>使用钝的导管以除去管腔内容和食糜作为这些含有消化酶，这将加速体外组织样品的劣化冲洗用Krebs溶液各段。 注:请注意不要损坏冲洗过程中循环的管腔，随着绒毛的破坏将导致可以改变传入神经活性介质的释放。 </li><li>识别段来衡量传入神经活动（ 例如 ，第一空肠segment的远侧特赖茨的涂有硅氧烷弹性体层的专用记录室中的韧带或十二指肠空肠弯曲），和地方这一点。 注:空肠的开头解剖学定义为小肠，其中屈氏韧带穿过小肠的一部分，也被称为十二指肠空肠弯曲。 注意:覆盖具有薄硅氧烷弹性体层的记录室的底部以及预先在实验开始前。此弹性体层的制备应根据制造商的说明1来执行。 </li><li>灌注不断用温室中，carbogenated以10毫升/分钟的速率的Krebs溶液中并在34℃下保持的Krebs&#39;温度在录音室常数。 </li><li>装载在器官浴中的空肠段使口服端被连接到注射器驾驶员提供管腔流和反口端连接到流出。 ST略干呕段，但注意不要施加过大的张力。连接两端牢牢地使用4/0丝线结扎的入口和流出口。 </li><li>附加注射器驱动到口腔端，并灌注以10ml / hr的速率空肠段intraluminally用Krebs溶液（非氧化，环境温度）。 </li><li>针对有机硅弹性体底层安装的肠段平的肠系膜，用昆虫针。舒展肠系膜出来，以优化肠系膜束的可视化;不要捆绑或空肠施加压力。 </li><li>通过关闭输出端口执行测试斜坡腹胀（ 见下文） 直到肠段的腔内的压力达到60毫米汞柱，以验证没有腔内的Krebs溶液从安装段泄漏。观察腔内压力平稳上升不中断。 </li><li>观察段的小收缩（蠕动WAVES）在初始膨胀阶段。如果需要的话，通过添加L-型钙通道阻滞剂硝苯地平为1μM到Krebs溶液方框蠕动活性。 注意:添加1μM阿托品去除了硝苯地平克雷布斯解决方案，将彻底瘫痪肠段。然而，我们必须用阿托品对传入神经记录的使用和效果没有亲身经历。 </li><li>在立体显微镜，开始轻轻地用两个小镊子轻轻揪着它，注意不要损坏埋藏在脂肪组织中的血管和传入神经剥离从肠系膜脂肪组织。 </li><li>开始在从空肠一个遥远的距离，并在肠系膜束露出两个血管。 </li><li>观察两艘船为封装在脂肪组织薄，白线之间的传入空肠神经。只有轻轻地剥离脂肪组织远用镊子在解剖更向近端空肠既肠系膜血管和/或传入神经的初始识别是困难的。 </li><li>通过去除脂肪组织附着在神经解剖自由在几个毫米的距离，段的空肠肠系膜神经。 </li><li>样使用锋利的剪刀组织神经。如果需要的话，通过用小镊子轻轻拉着它剥离剩余的脂肪和结缔组织，以及epineuronal鞘。 </li><li>用显微操作，降低了抽吸电极的前端，连接到具有柱塞的注射器，放入器官浴然后，通过操纵柱塞，轻轻吸从器官浴，使电极的尖端在Krebs溶液（ 图1）被浸没一些Krebs溶液。确保该Krebs溶液覆盖所述抽吸电极内的线电极。 注:准备包含在实验前我们开始时的线电极的硼硅玻璃吸入毛细管ING吸管拉马。 </li><li>抽吸电极的尖端紧靠横断传入神经链定位和绘制横断神经链到毛细管在其整个长度。 </li><li>操纵朝一些脂肪组织的电极的前端和吸入玻璃毛细管此而与柱塞牢牢吸移，从而机械地"密封"在从该器官浴中的内容物的毛细管的神经。 </li><li>验证用数据采集系统传入神经活动， 例如的记录时，通过执行在传入斜坡腹胀引起的增加烧成（ 见下文 ）。继神经入抽吸电极的分离，稳定为了执行实际实验之前，以获得一个稳定的状态自发传入神经活动15分钟制备。 </li><li>执行坡道腹胀，通过关闭输出口扩张的肠段，导致GRADUAL上升压力在肠道段（不超过60毫米汞柱）。仅执行所需的实验方案，当三个连续斜坡distensions（用15分钟的时间间隔）产生一个可重复的多单元放电（ 图2）。 </li></ol></li><li>腰内脏传入的制备（结肠传入神经） 注:结肠传入神经的解剖需要更详细的解剖。来自前&#39;空肠&#39;协议偏差如下: <ol><li>通过人性化的方法来安乐死的动物，将其放置在仰卧位，进行剖腹探查中线和过分倒在腹腔冰冷的克雷布斯解决方案。注:克雷布斯组合物:118 mM氯化钠，4.75毫米氯化钾，1mM的的NaH 2 PO 4，22的NaHCO 3，1.2mM的MgSO 4上，11毫D-葡萄糖和2.5mM 氯化钙 ，为3μM吲哚美辛。 注意:注意Krebs溶液的组成改变;消炎痛加入到如此lution为了防止传入神经活性改变前列腺素。 </li><li>丢弃脂肪组织，膀胱和内生殖器，轻轻小肠在腹腔移位到一侧。执行与从腹部完好肠系膜神经结肠的远端部分的一个扩展prelevation。 注意:可以被包括在这个夹层，以帮助组织的进一步制备重要的地标包括腹主动脉和腔静脉的左肾和盆底肌肉。 注:在解剖，注意不要施加冒号和腹主动脉之间的连接组织牵拉，因为这个区域包含腰部内脏神经。 </li><li>转移组织成段两旁录音室的有机硅弹性体。使用左肾动脉，从腹主动脉起源，作为起点。按照在反口方向的腹主动脉，遇到上级mesenteriÇ动脉腹腔及肠系膜神经节在一起。最后，到达结肠和主动脉，其中感兴趣的神经位于的远端部分之间的连接组织。 </li><li>确定肠系膜下动脉与腹主动脉起源。腰内脏传入神经可以在肠系膜下动脉的基础上确定;它平行于动脉（ 图3）。 </li><li>继神经横断，轻轻剥离周围结缔组织鞘和挑逗神经分成几个细线。请一定要保持从结肠的安全距离。 </li><li>绘制这些单个线程到吸入电极，&#39;密封&#39;与如先前所描述，并执行所需的实验方案周围脂肪组织的毛细管中的一个。 </li><li>丢弃任何多余的组织存在于器官浴（ 例如，肾，腹部血管，肌肉组织），一个这帮可能会干扰传入信号。 注:硝苯地平（1μM）可以被添加到在壳体的Krebs溶液的传入神经信号由自发肠运动不安由于平滑肌细胞收缩。 注:1）serosally由所需化合物进入克雷布斯该灌注到录音室，2）直接放入器官浴而暂时停止灌注或溶解3）intraluminally由感兴趣化合物溶解:药物可以在三个不同的部位给予成在注射器驱动的Krebs溶液。当化合物的累积剂量太快连续服用可出现传入神经的脱敏。 </li></ol></li></ol><img alt="图1" src="/files/ftp_upload/54576/54576fig1.jpg" /> 图1:特制的录音室和吸电极的示意图的TECHNI的详细概述。<html> CAL设置与抽吸电极和到位的录音室。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54576/54576fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54576/54576fig2.jpg" /> 图2:空肠传入神经活动的体外拍摄的代表性示踪在基线和响应于2斜坡distensions直至60毫米汞柱空肠多单位传入神经活动（imp.sec -1）（上图）的典型记录。 （下图），以及不同的单单位的神经信号（第三组）的后续识别（wavemark分析）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54576/54576fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="&lt;/ HTML">"图3"SRC ="/文件/ ftp_upload / 54576 / 54576fig3.jpg"/&gt; 图3:对结肠的神经解剖学 ）从结肠感官信息经由腰部结肠神经（LCN）向中枢神经系统输送时，与LCN靠近运行的肠系膜下动脉（IMA）。纤维从这个腰神经结肠的部分将沿intermesenteric神经（IMN）当然，形成腰内脏神经（LSN）。肠系膜下神经节（IMG）位于所述IMA的从腹主动脉的原点。录制远端的IMG是强制性的研究者应该希望学习viscerofugal传入神经的活动。B）的实验装置的示意图。的LCN的传入记录在器官都通过连接到数据采集系统中的抽吸电极的装置执行。斜坡腹胀可以经出口端口的关闭，同时继续流入来进行克雷布斯解决方案。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54576/54576fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 2.数据采集<ol><li>记录通过连接到探头抽吸电极的神经活性。放大（增益10K）和过滤信号（带通500 - 5000赫兹）20。 注:50/60 Hz的噪声消除应包括在实验装置，以消除50/60赫兹电磁干扰噪声信号。该信号是自动数字化，并在20千赫用于分析取样。 </li></ol> 3.分析5,20 <ol><li>报告传入神经放电作为脉冲的总数目/秒为整个神经或使用专门的软件来执行进一步的分析，作为整体神经活动的多单元记录包含不同的形状，幅度和宽度的动作电位，对应于不同的神经单位在每个传入纤维（ 图2）。 </li><li>匹配各个wavemarks到预定义模板，允许单个单位之间的歧视。 1应该执行:一个尖峰的分配到一定波标记，信噪比&gt; 2之前。 注意:在wavemark分析，我们构建了一个新的模板时，至少10个类似的尖峰由分析软件确定。没有模板构建了形状，50比峰值1罕见。的道钉被匹配到使用时它的点中的至少80％是相同的模板尖峰主成分分析的模板。模板匹配是取决于运营商，并始终由同一研究人员以不知情方式进行。 </li><li>减去基线的自发活动由在固定的时间点腹胀（从0到20毫米汞柱腔内压力为5mmHg增量期间的响应（0毫米汞柱腔内压力）计算动作电位的响应，以10为增量从毫米汞柱毫米汞柱20起）。用10秒的仓尺寸测量基线传入放电。 </li><li>使用单单元分析，以分类基于斜坡腹胀（ 图4）在其放电曲线的纤维。此外，单一单位分析可用于研究不同类型的单元的化学敏感信息，因为不是所有类型的单元将响应于药物或化合物显示相同的改变的电活动。 </li><li>分类纤维作为门槛低纤维（"LT"，在较低的扩张压力通常发挥排出增多），门槛高纤维（"HT"，发挥最高扩张压力增加放电），宽动态范围的纤维（"世界发展报告"，证明整个斜坡腹胀期间增加射击）或机械不敏感的传入（&#39;MIA&#39;，神经纤维通常不响应斜坡distensions）5; 20。 </li><li>表达在20毫米汞柱作为p神经击发响应腹胀（LT％），为它反映在低膨胀压力烧成的程度时的最高烧制响应的ercentage。 注:因此，LT纤维由LT％&gt; 55％的特点，而HT通过的&lt;15％的值定义。对于WDR单位值的范围在15和55％（20）之间。一个MIA显示自发传入发射由distensions不受影响。 </li></ol><img alt="图4" src="/files/ftp_upload/54576/54576fig4.jpg" /> 图4:在20毫米汞柱压力扩张其燃烧速率相比腹胀时的最高烧制响应的基于百分比（LT％） 基于它们的机械敏感资料的不同传入光纤单元的示意图单位进行分类。门槛低纤维（左上图），在低扩张压力主要显示增加神经活性，导致超过55％的LT％。高门槛单位（右上面板），相反仅显示在有害压力焙烧率（％LT &lt;15）的增加。宽动态范围纤维（左下图），显示整个腹胀过程中神经活动逐渐增加（％LT 15和55之间不等），而机械不敏感光纤（右下图），不增加扩张压力作出回应。 LT％（传入发射20毫米汞柱/最大传入射击） <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54576/54576fig4large.jpg" target="_blank">点击此处查看该图的放大版本。</a>

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The authors have nothing to disclose.

本文的协议描述一个可重复的实验技术13759我们组和其他3,4,7,8,12,20,21,31研究啮齿动物的肠系膜传入神经活动。在协议中的关键步骤包括组织的快速分离，神经链的抽吸进入抽吸电极和通过吸周围脂肪组织进入毛细管从器官浴中的玻璃毛细管的适当"密封"。玻璃毛细管的孔径应当精确确定:太小的孔将神经链的抽吸复杂化到电极，而太宽孔径会阻碍与脂肪组织的毛细血管的"密封"，从而导致冗余这将妨碍分析（低信号对噪声录音）的背景噪音。以允许可靠的单单元分类，内脏传入应当在不同的绳股以减少分割在记录单元的数目。通常情况下，我们将建议旨在具有最大的4 - 5单位在每个记录。因此研究人员应该调整基于感兴趣的纤维的孔，并且所应用的实验室动物。 另一个关键点包括实验装置的接地充足。抽吸电极和记录室应适当地接地，以最大限度地减少干扰，阻碍神经元活动的分析电场，而所有其他设备，包括记录装置，注射器驱动器等等应安装在笼子外由一个法拉第笼覆盖。 通过记录在靠近空肠或结肠传入神经活动，可以隔离传入信号传导链的第一部分和容易学习从中心NERV发生在鞋底传入水平无干扰的贡献和变更OU的体系。一个这种技术的局限性是， 在体外观察不能总是费力外推到体内设置，如在体外设置仅在复杂的神经信号级联包含一个中继站的事实。因此，更宽阔的视野必须进行整合所有其它站，如背根神经节，中枢神经系统（ 例如 ，脑功能成像）和下行（抑制）传出通路。 这种方法的另一个优点构成了相当简单的技术过程，因为一个不再具有监测实验室动物提供胃肠检体的健康。而另一方面是神经元活动的体外测量不适合在阐明传入神经放电全身给药药物的效果，但研究人员可以通过全身给药药物Ø理论上克服这一障碍˚F感兴趣的动物，随后传入神经活动的体外体外记录。然而，应注意到这样的事实:任何存在于录音室药物将由于解剖和随后的记录过程中稀释至浴灌注。最后， 在体外进行用遗传工程动物内脏神经录音可以允许研究人员充分阐明不同频道和传入纤维表达的受体的作用。 研究人员试图实现这种技术也必须记住，肠系膜传入和盆腔传入的识别和隔离显然需要基本的解剖和技术培训的知识承担，研究人员应该与神经电生理的基本原则相识。 体外设置还使研究人员能够轻松地识别可能的药理焦油得到，并在若干疾病过程提供神经元活动以及改变感官信号传导的生理作用的见解。 在空肠传入测量情况下，单一的动物的几个组织片段可以同时研究，这一特点是相当困难的使用体内设置。不过研究人员谨慎地应该解释从不同的细分得到的结果，因为区域差异可能偏差结果。因此，我们会建议始终来自同一站点（从远端屈氏韧带或十二指肠空肠弯曲如第一段）测量传入神经活动。 这种技术的典型当前和未来的应用包括可病理学为特征的内脏过敏及疼痛过程中改变肠系膜传入致敏药理化合物的筛选。如前面已经提到的，目标SE化合物可以某处复杂的神经系统，从肠道内在神经系统大脑会遇到;因此，表征和调制传入神经活性有助于更广泛的画面，还包括本征肠神经和背根神经节的钙成像的内脏运动响应作为内脏高敏感性的体内的指标，脑功能成像的测定中，等等。

感官信令和疼痛知觉是一个复杂的过程，从传入神经，脊髓神经元，升序和降序易化和抑制途径和几种不同的脑区域之间的错综复杂的相互作用的结果。正因为如此，在一个或多个这些水平的变化可能导致改变的感觉的信号和在疾病状态内脏疼痛。研究的感官信令多种技术所有这些不同的方面已经开发了从单细胞实验（ 例如，在神经元的钙成像）到整个动物模型（ 例如，行为反应，如内脏运动响应）。本文描述的技术可以让研究人员专门从啮齿动物的小肠或结肠的一隔离段评估体外传入神经活动。总之，分离的胃肠道段（通常空肠或结肠）安装在用生理ķ灌注一个专用记录室篮板的解决方案。内脏神经解剖自由和连接到允许传入的神经元活动的登记内脏或盆腔传入神经的电极。神经活性可以基部或响应于，或者通过管腔内灌洗液（粘膜）提高腔内的压力和/或药理学化合物可直接施加到记录室（serosally），以评估其对传入放电1-6效应被记录。值得注意的是，内脏神经还含有传出纤维和除了感觉传入viscerofugal传入。一种体外内脏神经记录的一个主要优点是，研究人员可以量化神经活动而不脱离中枢神经系统调制或输入，使一个以研究局部施加化合物对神经活动的直接作用的事实。此外，重要的参数的监控，如使用体内方法是必要的（见下文），为正Ø不再相关。 体外内脏记录终于要少得多耗时比其在体内的对应。 响应于其他刺激，如粘膜划线，使用von Frey毛探测或段的拉伸传入神经元活动，可以在修改的实验装置，其中肠组织被牵制和纵向打开（这是相对于被研究我们的设置使用一个完整的片段），如在先前的问题7,8进行了说明。此外，仅在最近，这种技术被描述来研究通过钙成像结肠壁本身结肠传入神经激活，再次使用牵制，纵向打开段9。 这种体内技术的另一种版本包括了测量传入的进入脊髓邻近神经元激活的。总之，镇静动物置于俯卧位，例如xposing腰骶脊髓通过椎板切除术的手段，构建感兴趣的项目的该传入神经石蜡填充孔用切口的皮肤和悬垂在铂双极电极10,11背侧支根。这种技术还使研究人员能够从薄髓Aδ纤维表征基于其传导速度纤维和无髓鞘区分C纤维。此外，背侧支根只包含感觉传入纤维，而相比之下，前面提到的混合传入和传出内脏神经。 从孤立的肠道段录音在体外传入神经放电，也可以使用人体标本进行，如独立出版首次在男子手稿记录在人类结肠切除术传入纤维的活动标本12,13两个研究小组。该技术的实施可能会导致更容易translati对小鼠的数据，以人性化的状态，并且可以让研究人员能够轻松识别靶向药物致敏的感觉神经。表征传入神经活动，以及为针对过高的传入神经活动的新治疗剂的发现的临床重要性，一直精心许多专家在现场讨论14-19。 上述体外技术补充了在传入神经活动的体内测量更多的俗称。期间在体内神经元活性的测量，神经活性可以直接在其间感兴趣的段被识别，并随后插管的镇静动物测定，和液体石蜡填充井使用啮齿类20的腹壁和皮肤构成。然后感兴趣的传入神经被识别，切片并放置于双极铂电极，使神经元活动measuremen吨。这种技术允许研究人员来调节生活虽然镇静动物传入纤维的活动;因此，可以研究神经元活动响应干扰诸如鲁米腹胀或化合物的静脉内给药。 转化研究现在主要集中在人衍生的上清液中的应用（ 例如 ，从结肠活检，栽培外周血单核细胞等 ）上空肠和/或结肠鼠标传入21,22。研究人员可以直接申请上清要么进机关浴缸或成灌注到段肠管腔内的解决方案，因此浆膜与粘膜应用的不同影响可以传入神经放电进行研究。因此，它表现出从患者肠易激综合症结肠黏膜活检supernatans可导致小鼠结肠传入，豚鼠粘膜神经元和小鼠背根超敏反应神经节神经元21,23,24。 最后，记录神经元的活动并不限于肠系膜和/或骨盆神经支配胃肠道。他人已经证明，神经录音可以在传入供给膝关节25来执行，而另一些特征膀胱传入神经活性以及26-28，并表明从膀胱骨盆传入以及胃肠道汇合，从而可能导致神经元串扰29。

<table border="0" cellpadding="0" cellspacing="0" width="788">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">sodium chloride (NaCl)</td>
			<td style="width:247px;">VWR Chemicals</td>
			<td align="right" style="width:119px;">27,810,295</td>
			<td style="width:167px;">compound Krebs solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">potassium chloride (KCl)</td>
			<td style="width:247px;">Acros organics</td>
			<td align="right" style="width:119px;">196770010</td>
			<td>compound Krebs solution</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:255px;">sodium dihydrogen phosphate (NaH2PO4)</td>
			<td style="width:247px;">VWR Chemicals</td>
			<td align="right" style="width:119px;">1,063,461,000</td>
			<td>compound Krebs solution</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:255px;">sodium bicarbonate (NaHCO3)</td>
			<td style="width:247px;">Merck</td>
			<td align="right" style="width:119px;">1,063,291,000</td>
			<td>compound Krebs solution</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:255px;">magnesium sulfate (MgSO4)</td>
			<td style="width:247px;">Merck</td>
			<td align="right" style="width:119px;">1,058,861,000</td>
			<td>compound Krebs solution</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:255px;">calcium chloride (CaCl2)</td>
			<td style="width:247px;">Merck</td>
			<td align="right" style="width:119px;">23,811,000</td>
			<td>compound Krebs solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">D-glucose</td>
			<td style="width:247px;">VWR Chemicals</td>
			<td style="width:119px;">1011175P</td>
			<td>compound Krebs solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Distilled water</td>
			<td style="width:247px;"></td>
			<td style="width:119px;"></td>
			<td>compound Krebs solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">PVC tubing</td>
			<td style="width:247px;">Scientific Laboratory Supplies</td>
			<td style="width:119px;"></td>
			<td>The intestinal segment should be mounted over PVC tubing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Silicone tubing</td>
			<td style="width:247px;">Scientific Laboratory Supplies</td>
			<td style="width:119px;"></td>
			<td>The rest of the tubing, ideally silicone-based - more easily dislodging of debris in the tubing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Silk thread</td>
			<td style="width:247px;">Pearsall Limited</td>
			<td style="width:119px;">10B15S220</td>
			<td>Attachment of the segment over the PVC tubing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Syringe driver</td>
			<td style="width:247px;">Harvard Apparatus</td>
			<td style="width:119px;">55-2222</td>
			<td>Intraluminal infusion of Krebs</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Binocular - including 10x magnification in oculair</td>
			<td style="width:247px;">Zeiss STEMI 2000</td>
			<td style="width:119px;"></td>
			<td>Optimal visualization for the dissection of the afferent nerve</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Homeothermic Blanket Control Unit</td>
			<td style="width:247px;">Harvard Apparatus</td>
			<td align="right" style="width:119px;">507214</td>
			<td>Heating of the organ chamber</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Custom made organ bath with Sylgard covered bottom</td>
			<td style="width:247px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Spike2 software</td>
			<td style="width:247px;"></td>
			<td style="width:119px;"></td>
			<td>Recording and analysis of the data</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Insect pins, 500 pieces, stainless steel, diameter 0.2 mm</td>
			<td style="width:247px;">Austerlitz insect pins minutiens</td>
			<td style="width:119px;"></td>
			<td>Dissection of the afferent nerve</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Tweezer Dumont #5 inox 11cm</td>
			<td style="width:247px;">World Precision Instrument</td>
			<td align="right" style="width:119px;">500341</td>
			<td>Dissection of the afferent nerve</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Scissors, spring, 14 cm</td>
			<td style="width:247px;">World Precision Instrument</td>
			<td align="right" style="width:119px;">15905</td>
			<td>Dissection of the afferent nerve</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">DB digitimer&#160;</td>
			<td style="width:247px;">NL 108T2/10</td>
			<td style="width:119px;"></td>
			<td>pressure transducer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Micromanipulator</td>
			<td style="width:247px;">Narishige</td>
			<td style="width:119px;">M-3333</td>
			<td>3D manipulation of the suction electrode</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Micromanipulator</td>
			<td style="width:247px;">X-4 rotating block</td>
			<td style="width:119px;"></td>
			<td>3D manipulation of the suction electrode</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Micromanipulator</td>
			<td style="width:247px;">GJ-8 magnetic stand</td>
			<td style="width:119px;"></td>
			<td>3D manipulation of the suction electrode</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">LightSource</td>
			<td style="width:247px;">Euromex Microscopes Holland EK-1</td>
			<td style="width:119px;"></td>
			<td>Optimal visualization for the dissection of the afferent nerve</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">CED 1401 Recording Apparatus</td>
			<td style="width:247px;"></td>
			<td style="width:119px;"></td>
			<td>Recording of afferent nerve activity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Humbug 50/60Hz Noise Eliminator</td>
			<td style="width:247px;">Quest Scientific Instruments</td>
			<td style="width:119px;"></td>
			<td>Elimination of background noise</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Infusion Pump</td>
			<td style="width:247px;">Gibson Minipuls 2</td>
			<td style="width:119px;"></td>
			<td>Infusion of the organ chamber in which the segment is mounted</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Microelectrode Holder Half Cells 1.5 mm</td>
			<td style="width:247px;">World Precision Instrument</td>
			<td style="width:119px;">MEH2SW</td>
			<td>Suction electrode for isolation of the afferent fiber</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Borosilicate Glass Capillaries, 300 pc; 1.5/0.84 OD/ID</td>
			<td style="width:247px;">World Precision Instrument</td>
			<td style="width:119px;">1B150-4</td>
			<td>Capillary for the isolation of the afferent nerve</td>
		</tr>
	</tbody>
</table>

in vitro recording of mesenteric afferent nerve activity in mouse jejunal and colonic segments

传入神经不仅传达信息有关正常生理，而且信号干扰稳态和从向中枢神经系统周围的不同器官系统的病理生理过程。这样，增加的活性或肠系膜传入神经的"敏化"已被分配在内脏高敏感性和腹痛综合征的病理生理学中起重要作用。 肠系膜传入神经活性可以在体外 ，其安装在一个特制的器官浴中，并从该内脏神经是隔离的，使研究人员能够直接评估邻近胃肠段神经活性的分离的肠段来测量。活性可在标准条件基线段的膨胀或增加intraluminally或serosally递送药理化合物以下期间被记录。这种技术允许研究者容易学习的药物靶向于对照样品外周神经系统的影响;此外，它提供了对疾病的过程中是如何的神经元活动改变的关键信息。但是，应该注意的是，测量传入的神经元放电活动只构成了复杂的神经元一个中继站的信号级联反应，研究人员应该牢记没有其他级别（ 例如，背根神经节，脊髓或中枢神经系统忽视的神经元活动）为了充分阐明在健康和疾病的复杂的神经生理学。 常用的应用包括神经元活动的响应于脂多糖给药研究中，和传入神经活性的肠易激综合症的动物模型的研究。在一个更平的方法中，分离的小鼠肠道段可暴露于来自IBS患者结肠上清。此外，修改这种技术的最近已经证明是适用于人结肠标本。

空肠传入神经活动在基线测量和响应在9 OF-1小鼠八周大的雄性坡道腹胀。动物饲养在在标准条件组（每笼6只，20 - 22°C，湿度40 - 50％，12小时明暗周期）无限制地使用自来水和常规食物。小鼠空肠段在0毫米汞柱的腔内压力显示在基线不规则自发传入神经放电（平均自发活动11.47±3.31小鬼/秒）。 空肠传入神经活动一旦执行斜坡distensions直到60毫米汞柱增加。通常，下面的在腔内压力的上升增加传入神经活动的特点是双相反应（ 图5），由在焙烧活性的初始迅速增加，直到管腔内的压力达到20毫米汞柱，whicH可主要是由于门槛低纤维的增加射速。这随后是一个平台期，在这之后可以从40毫米汞柱观察烧成活性的第二上升起，代表主要高阈值的纤维的活化。 根据他们的波形，单单元可以在每个多单元记录被辨别和分类在上述四类中的一个。在9只小鼠中，我们区分40种不同的单位（4.44±1.01单位/空肠传入神经），用LT单元是最普遍的那些，随后WDR和HT纤维（ 图6）。不同单元的响应电活动坡道腹胀可以在图7中可以观察到。 <img alt="图5" src="/files/ftp_upload/54576/54576fig5.jpg" /> 图5: <stron<html> g&gt;的肠系膜传入神经电（imp.sec - 1）。斜坡腹胀整个神经过程中加减速时腹胀野生型小鼠肠系膜多单元传入神经放电（IMP /秒-1）在野生型小鼠。数值代表平均放电±SEM，N = 9只小鼠。 imp.sec -1:每秒脉冲<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54576/54576fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/54576/54576fig6.jpg" /> 图6:从9野生型小鼠空肠传入神经确定了40台单机分布 HT:门槛高纤维，LT:门槛低纤维，MIA:机械不敏感光纤，宽动态:宽动态范围的光纤。_blank"&gt;请点击此处查看该图的放大版本。 <img alt="图7" src="/files/ftp_upload/54576/54576fig7.jpg" /> 图7: 压力-响应曲线为亚基的野生型小鼠的不同类型的单单元传入神经放电（imp.sec -1）从可斜坡期间被识别，在野生型小鼠的四个不同的单位distensions。低门槛纤维（LT，左上图）的特点是烧成活动distensions在初始迅速增加，而高门槛纤维（HT，左下图）只显示在增加有毒腔内的压力射击。宽动态范围纤维（WDR，右上图），显示整个过程腹胀烧成活动也在稳步增加，机械性刺激不敏感的传入纤维（MIA，右下图）不增加腔内的压力作出回应。值代表怨恨平均放电±SEM imp.sec -1:每秒脉冲<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54576/54576fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about In Vitro Recording of Mesenteric Afferent Nerve Activity in Mouse Jejunal and Colonic Segments at JoVE.com

In Vitro Recording of Mesenteric Afferent Nerve Activity in Mouse Jejunal and Colonic Segments

Mesenteric afferent nerves convey information from the gastrointestinal tract towards the brain regarding normal homeostasis as well as pathophysiology. Gastrointestinal afferent nerve activity can be assessed by mounting isolated intestinal segments with attached afferent nerves into an organ bath, isolating the nerve, and assessing basal as well as stimulated activity.

在鼠标空肠和结肠段肠系膜传入神经活性的体外记录

Afferent nerves not only convey information concerning normal physiology, but also signal disturbed homeostasis and pathophysiological processes of the ...

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Nanyang Technological University

Research

JoVE Journal

Neuroscience

In Vitro Recording of Mesenteric Afferent Nerve Activity in Mouse Jejunal and Colonic Segments

11.4K 次观看.  University of Antwerp. Mesenteric afferent nerves convey information from the gastrointestinal tract towards the brain regarding normal homeostasis as well as pathophysiology. Gastrointestinal afferent nerve activity can be assessed by mounting isolated intestinal segments with attached afferent nerves into an organ bath, isolating the nerve, and assessing basal as well as stimulated activity.

视频: In Vitro Recording of Mesenteric Afferent Nerve Activity in Mouse Jejunal and Colonic Segments

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

逆行荧光染料标记允许有针对性的外单细胞记录技术鉴定神经元<em&gt;在体内</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

科研

神经科学

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time1. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.
First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode3. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings8. We provide details of our experimental setup and present representative recording traces for each of these techniques.

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as well.

多单位记录方法来描述蝗虫的神经活动（<em&gt; Schistocerca美洲</em&gt;）嗅觉电路

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT).
Although circadian rhythms in running-wheel activity are typically linked to the SCN clock6-8, circadian oscillators in many other regions of the brain and body9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators17-20. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.

Circadian rhythms in voluntary wheel-running activity in mammals are tightly coupled to the molecular oscillations of a master clock in the brain. As such, these daily rhythms in behavior can be used to study the influence of genetic, pharmacological, and environmental factors on the functioning of this circadian clock.

运行轮活动在啮齿类动物的昼夜节律的记录和分析

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, ...

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional afferent fibers monitor other characteristics of the muscle environment, including metabolite buildup, temperature, and nociceptive stimuli. Overall, abnormal activation of sensory neurons can lead to movement disorders or chronic pain syndromes. We describe the isolation of the extensor digitorum longus (EDL) muscle and nerve for in vitro study of stretch-evoked afferent responses in the adult mouse. Sensory activity is recorded from the nerve with a suction electrode and individual afferents can be analyzed using spike sorting software. In vitro preparations allow for well controlled studies on sensory afferents without the potential confounds of anesthesia or altered muscle perfusion. Here we describe a protocol to identify and test the response of muscle spindle afferents to stretch. Importantly, this preparation also supports the study of other subtypes of muscle afferents, response properties following drug application and the incorporation of powerful genetic approaches and disease models in mice.

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

Muscle sensory neurons are involved in proprioceptor signaling and also report on metabolic state and injury related events. We describe an adult mouse in vitro muscle-nerve preparation for studies on stretch-activated muscle afferents.

一<em&gt;在体外</em&gt;成年小鼠肌肉，神经准备留学肌肉传入放电的放电特性

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional ...

Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to monitor the disease course in the follow-up period. Changes in the VEP parameters closely correlate with pathological damage in the optic nerve. This protocol provides a detailed description about the rodent model of optic nerve microinjection, in which a partial demyelination lesion is produced in the optic nerve. VEP recording techniques are also discussed. Using skull implanted electrodes, we are able to acquire reproducible intra-session and between-session VEP traces. VEPs can be recorded on individual animals over a period of time to assess the functional changes in the optic nerve longitudinally. The optic nerve demyelination model, in conjunction with the VEP recording protocol, provides a tool to investigate the disease processes associated with demyelination and remyelination, and can potentially be employed to evaluate the effects of new remyelinating drugs or neuroprotective therapies.

Focal demyelination is induced in the optic nerve using lysolecithin microinjection. Visual evoked potentials are recorded via skull electrodes implanted over the visual cortex to examine the signal conduction along the visual pathway in vivo. This protocol details the surgical procedures underlying electrode implantation and optic nerve microinjection.

在实验视神经脱髓鞘模型大鼠视觉诱发电位记录

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to ...

Combined Recording of Mechanically Stimulated Afferent Output and Nerve Terminal Labelling in Mouse Hair Follicle Lanceolate Endings

A novel dissection and recording technique is described for monitoring afferent firing evoked by mechanical displacement of hairs in the mouse pinna. The technique is very cost-effective and easily undertaken with materials commonly found in most electrophysiology laboratories, or easily purchased. The dissection is simple and fast, with the mechanical displacement provided by a generic electroceramic wafer controlled by proprietary software. The same software also records and analyses the electroneurogram output. The recording of the evoked nerve activity is through a commercial differential amplifier connected to fire-polished standard glass microelectrodes. Helpful tips are given for improving the quality of the preparation, the stimulation and the recording conditions to optimize recording quality. The system is suitable for assaying the electrophysiological and optical properties of lanceolate terminals of palisade endings of hair follicles, as well as the outcomes from their pharmacological and/or genetic manipulation. An example of combining electrical recording with mechanical stimulation and labeling with a styryl pyridinium vital dye is given.

A simple and novel technique for recording afferent discharge due to mechanical stimulation of lanceolate terminals of palisade endings innervating mouse ear skin hair follicles is presented.

机械刺激传入输出和神经末梢标签联合录制在小鼠毛囊披针形结局

A novel dissection and recording technique is described for monitoring afferent firing evoked by mechanical displacement of hairs in the mouse pinna. The ...

In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats

Dysfunction of the colonic sensory nerves has been implicated in the pathophysiology of several common conditions, including functional and inflammatory bowel diseases and diabetes. Here, we describe a protocol for the in vitro characterization of the electrophysiological properties of colonic afferents in rats. The colorectum, with the intact pelvic ganglion (PG) attached, is removed from the rat; superfused with carbogenated Krebs solution in the recording chamber; and cannulated at the oral and anal ends to allow for distension. A fine nerve bundle emanating from the PG is identified, and the multiunit afferent nerve activity is recorded using a suction electrode. Distension of the colonic segment elicits gradual increases in multiunit discharge. A principal component analysis is conducted to differentiate the low-threshold, the high-threshold, and the wide-dynamic range afferent fibers. Chemical sensitivity of colonic afferents can be studied through the bath or intraluminal administration of test compounds. This protocol can be modified for application to other species, such as mice and guinea pigs, and to study the differences in the electrophysiological properties of thoracolumbar/hypogastric and lumbosacral/pelvic afferents of the descending colon in normal and pathological conditions.

In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats

Abnormal sensory function underlies visceral pain and other symptoms of functional and inflammatory bowel diseases. A protocol for the electrophysiological recording of the colonic afferent nerves in an ex vivo rat colorectum preparation is presented here.

体外大鼠结肠传入纤维的电生理特性

Dysfunction of the colonic sensory nerves has been implicated in the pathophysiology of several common conditions, including functional and inflammatory ...

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 ...

Recording and Modulation of Epileptiform Activity in Rodent Brain Slices Coupled to Microelectrode Arrays

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) is a promising approach when pharmacological treatment fails or neurosurgery is not recommended. Acute brain slices coupled to microelectrode arrays (MEAs) represent a valuable tool to study neuronal network interactions and their modulation by electrical stimulation. As compared to conventional extracellular recording techniques, they provide the added advantages of a greater number of observation points and a known inter-electrode distance, which allow studying the propagation path and speed of electrophysiological signals. However, tissue oxygenation may be greatly impaired during MEA recording, requiring a high perfusion rate, which comes at the cost of decreased signal-to-noise ratio and higher oscillations in the experimental temperature. Electrical stimulation further stresses the brain tissue, making it difficult to pursue prolonged recording/stimulation epochs. Moreover, electrical modulation of brain slice activity needs to target specific structures/pathways within the brain slice, requiring that electrode mapping be easily and quickly performed live during the experiment. Here, we illustrate how to perform the recording and electrical modulation of 4-aminopyridine (4AP)-induced epileptiform activity in rodent brain slices using planar MEAs. We show that the brain tissue obtained from mice outperforms rat brain tissue and is thus better suited for MEA experiments. This protocol guarantees the generation and maintenance of a stable epileptiform pattern that faithfully reproduces the electrophysiological features observed with conventional field potential recording, persists for several hours, and outlasts sustained electrical stimulation for prolonged epochs. Tissue viability throughout the experiment is achieved thanks to the use of a small-volume custom recording chamber allowing for laminar flow and quick solution exchange even at low (1 mL/min) perfusion rates. Quick MEA mapping for real-time monitoring and selection of stimulating electrodes is performed by a custom graphic user interface (GUI).

We illustrate how to perform recording and electrical modulation of 4-aminopyridine-induced epileptiform activity in rodent brain slices using microelectrode arrays. A custom recording chamber maintains tissue viability throughout prolonged experimental sessions. Live electrode mapping and selection of stimulating pairs are performed by a custom graphical user interface.

鼠脑切片偶联微电极阵列癫痫活性的记录与调控

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) ...

Purification of Fibroblasts and Schwann Cells from Sensory and Motor Nerves in Vitro

The principal cells in the peripheral nervous system are the Schwann cells (SCs) and the fibroblasts. Both these cells distinctly express the sensory and motor phenotypes involved in different patterns of neurotrophic factor gene expression and other biological processes, affecting nerve regeneration. The present study has established a protocol to obtain highly purified rat sensory and motor SCs and fibroblasts more rapidly. The ventral root (motor nerve) and the dorsal root (sensory nerve) of neonatal rats (7-days-old) were dissociated and the cells were cultured for 4-5 days, followed by isolation of sensory and motor fibroblasts and SCs by combining differential digestion and differential adherence methods sequentially. The results of immunocytochemistry and flow cytometry analyses showed that the purity of the sensory and motor SCs and fibroblasts were &#62;90%. This protocol can be used to obtain a large number of sensory and motor fibroblasts/SCs more rapidly, contributing to the exploration of sensory and motor nerve regeneration.

Here, we present a method to purify fibroblasts and Schwann cells from sensory and motor nerves in vitro.

从体外感觉和运动神经中纯化成纤维细胞和施万细胞

The principal cells in the peripheral nervous system are the Schwann cells (SCs) and the fibroblasts. Both these cells distinctly express the sensory and ...