Name: prolonged incubation of acute neuronal tissue for electrophysiology and calcium-imaging
Uploaded: 2017-02-15T15:00:00.000+00:00
Duration: 10 min 30 s
Description: Watch this Scientific Journal Video about Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging at JoVE.com

We thank Colin Symons and James Wright for technical assistance. This work was supported by seed funding grant (WSU) to Y.B. and the innovation office at Western Sydney University. M.C. is supported by ARC fellowship (DECRA).

下面的协议描述的C57BL / 6和C3H /他（retinally退化）小鼠神经元组织的制备中，但是类似的技术可以应用到其它物种。所有的动物是健康的，并与温度的标准条件，湿度，12小时光照/黑暗周期，自由获得食物和水的处理，并没有任何意图应力刺激。所有实验均获得批准，并按照西悉尼大学的动物护理和伦理委员会，并根据动物的使用和保养指南（动物研究机构＃A9452，＃A10396和＃A8967）进行。 1.脑片制备方法<ol><li>通过异氟烷（5％）的吸入麻醉动物，并使用啮齿类铡斩首。迅速取出大脑，如前所述9，并将其放置到含有（以mM计）的冰冷的生理溶液（ACSF）:125氯化钠，2.5氯化钾，1 MgCl 2的，1.25的NaH 2 PO 4，2的CaCl 2，25的NaHCO 3，25葡萄糖，并用卡波金饱和（95％O 2/5％CO 2的混合物; 310毫渗量; pH 7.4）中。 </li><li>切脑切片，在感兴趣的区域中，300微米厚的用振动切片机。 </li><li>转移切片到定制孵化系统紧密监视和控制pH水平（pH为7.2 - 7.4），卡波金流量和温度如前所述10,11。 </li><li>设定的初始腔室温度至35℃15 - 30分钟，然后慢慢地降低到15 - 如图1C 16℃。直到需要在孵化体系培养，然后切片，无论是电或成像。 注:如果片被用于钙成像，请按照下列步骤操作冷却至低于室温组织之前。 </li></ol> 2.视网膜Wholemount编制和内界膜去除<ol><li>准备在任何正常的视网膜wholemounts实验室照明条件下或昏暗的红/红外光。 </li><li>颈椎脱位，立即去核的眼睛安乐死的动物。使在任一埃姆斯媒体，或脑脊液含有沿锯齿缘一个小切口，和地点（mM计）:125氯化钠，25的NaHCO 3，3的KCl，2的CaCl 2，1 MgCl 2的，10葡萄糖，0.5 L-谷氨酰胺，和与卡波金饱和（95％O 2/5％CO 2的混合物;〜300毫渗量; pH 7.4）中，在RT。 </li><li>立即通过沿与小剪刀锯齿缘切割和除去晶状体和玻璃体用镊子除去角膜，晶状体和玻璃体。将组织在室温孵育体系。 注:视网膜组织可以维持在眼杯，缓慢降低温度至15后 - 16℃下，在孵化系统&gt; 24小时，直到需要。 </li><li>以除去在ILM，包含视网膜眼杯转移到含有30 U / ml的木瓜蛋白酶，1mM的L-胱氨酸，0.5毫摩尔EDTA和0.005％DNA酶在Earl&#39;s-一个小玻璃瓶平衡盐溶液（BSS）在37℃下20分钟。应用95％ 的 O 2/5％CO 2，通过该盖的解决方案，但不起泡。如果使用的是从年轻的动物（&lt;6周）组织，稀释至半强度。 <ol><li>停止酶消化，通过将组织在厄尔氏BSS 10分钟的卵类粘蛋白（10毫克/毫升）与BSA（10毫克/毫升）溶液中。洗净组织温度彻底学联转移到孵化系统之前，减少到15 - 16℃。 </li></ol></li><li>直到需要在眼杯保持视网膜组织。转移到显微镜，隔离从眼杯视网膜和切成4片用剃刀刃。如果需要在整个视网膜上，使在视网膜的周边四个小切口以允许其平放。 注:对于成像实验，以转移到孵化系统之前钙染料负载，见下文。 </li></ol> 3.在孵化器中维持组织<ol><li&gt;在含有用于pH和温度测量（ 图1A，B）的探针主室放置组织。 </li><li>为了辐射细菌漂浮在溶液循环脑脊液通过第二腔室（UVC室，如先前所述12构建）从主室分离并暴露于1.1W¯¯UVC灯（254纳米，5W / 2P杀菌UV灯）（ 图1A）。 30分钟，以避免学联过热 - 通过使用随机的特征，有时15和每15之间的26分钟改变接通可编程定时器控制UVC光的时机。 </li><li>设置在UVC室12毫升/分钟的流速如先前报道12。覆盖UVC室用铝箔以防止该腔室，它可以破坏神经元组织以外的UVC照射。 注:对于暗适应视网膜组织，应用定制的盖主室要排除光。 </li><li>使用蠕动泵以circula德通过两个腔室的溶液（ACSF）和珀耳帖热电冷板冷却器要么冷却或加热所述主腔中的0的范围内所需的温度 - 50℃。为了获得最佳的组织培养，使用15 - 16℃的长期生存能力。 </li></ol> 4.钙染料荷载<ol><li>选择基于个体研究者的优先钙染料。这里我们使用的Fura-2AM，荧光 - 早上8点或荧光 - 凌晨4点。但是，这种方法可以应用到其它染料，以及:在DMSO溶解钙染料1mM溶液和1％的嵌段共聚物（ 例如 ，聚醚酸-127），以50微升的最终体积，并超声10分钟。 </li><li>添加溶液至脑脊液至10μM，对大脑切片0.01％嵌段共聚物和20微米（视网膜），0.02％的嵌段共聚物用于在视网膜的最终浓度。在37℃（的Fura-2 AM）或在RT（的Fluo-4AM视网膜（木瓜蛋白酶处理的）和脑切片从年轻的动物，负载浴45分钟;˚F罗上午8点）。 <ol><li>对于成年动物，（&gt; 12周）吸取染料（50μl），直接到脑切片，并保持75分钟，以允许染料更好地渗透到深层。 </li><li>为了确保染料孵化过程中被淹没切片充足的氧气，具有封闭盖（直径2.5厘米圆形罐子，3.5厘米高）与钙染料在2.5毫升学联稀释制成玻璃装载室。用95％ 的 O 2/5％CO 2的连续充氧。没有泡沫。 </li></ol></li><li>继染料加载，冲洗组织与学联和转移到孵化体系，温度慢慢降低到15 - 16°C，直到实验使用。 </li></ol> 5.电生理记录和影像<ol><li>放置在显微镜下的浸没式录音室组织，并在4流速含氧脑脊液灌注 - 5毫升/分钟或在RT（〜22℃）或生理温度（〜35℃）。霍尔d。通过使用由&#39;&#39;竖琴&#39;定制，制成拉伸并穿过一个U形件的金或铂丝胶尼龙或金线的就位的组织。 </li><li>对于电: <ol><li>使用微量拉马达到5-6MΩ终阻力1.5毫米（1.19毫米）硼硅玻璃，准备记录吸管​​。 </li><li>填充3吸移管-内部溶液的4微升如前所述13和可视化使用CCD照相机下红外微分干涉对比（IR-DIC）细胞。细胞内溶液应仔细针对每个实验来实现的实验结果。 </li><li>位置移液管，同时保持正压力，通过上吸管保持器的吸入口加，使用一个显微细胞的细胞膜。因为在ILM已从视网膜除去，不需要事先膜刮。一旦吸管是对细胞，应用温和的负压向吸移管以实现千兆欧姆密封。然后用负压的简要量破裂细胞膜。 </li><li>使用标准技术酌情全细胞电流或电压钳记录。 </li></ol></li><li>钙成像: <ol><li>对的Fura-2的比例成像，使用超高速波长切换以提供340纳米和380纳米的激发波长。通过510±20nm的发射滤波器通过从各个细胞发出的光，并具有高灵敏度，高速数字相机捕捉。 </li><li>对的Fluo-4，通过一个460过滤激发光的单个激发波长 - 通过一个515 490纳米的带通滤波器和发射的光 - 550 nm的带通滤波器。为了最快，最灵敏的记录使用高速数码相机等。按要求采集图像。 </li><li>测量荧光变化作为时间的函数要么在单个波长（的Fluo-4）或使用波长比方法（的Fura-2），如先前所描述8,14。 </li></ol></li></ol>

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Yossi Buskila and Paul Breen declare that they are co-owners of PAYO Scientific Pty Ltd, a company specialized in building incubation systems for acute neural tissue. All other authors report no financial interests or potential conflicts of interest related to the current study.

本文介绍了培养法延长急性神经组织的可行性进行成像和电生理实验，从而缩短完成实验目标所需的动物数量。两个主要过程支配神经元组织的劣化随时间:ⅰ）增加细菌水平，并在细菌内毒素伴随增加释放，和ii）随后的创伤切片过程10兴奋性中毒。为急性神经组织是环保手无寸铁，通过pH，温度和细菌水平密切监测，组织环境的紧缩调控对维持细胞活性以及网络的完整性至关重要。夹层，和组织治疗（木瓜蛋白酶消化，钙染料加载）可以在2小时内完成，从而导致在实验时间的减少。然而，由于可将组织保持在孵化系统为&gt; 24小时没有可测量的损失我Ñ生存能力7，8，该制剂只需要一次完成，以获得结果的&gt; 24小时。作为3R的战略的一部分（更换，减少，优化）旨在为道德地使用动物16的指导原则，这种方法将会对降低这些类型的实验中使用的动物的数量有很大的影响。 视网膜wholemount组织的ILM的消化允许在GCL用于膜片钳记录和浴应用无处不钙染料加载的细胞容易定向。这些技术的退化视网膜，其中在ILM是厚得多，更难通过用玻璃吸管刮违反特别有用。此外，木瓜蛋白酶制剂提供了一种方式来执行使用两个电极从GCL间隙连接耦合细胞记录配对细胞记录，而这一直没有先前可能的。然而木瓜蛋白酶消化可能对正常的视网膜生理一些效果K +受体表达已经显示出在感光体以下用木瓜蛋白酶6解离而改变。它也可能是视网膜结构受到影响。 Velte和Masland显示，虽然一些RGC胞体和树突通过木瓜蛋白酶处理除去在ILM损坏，它们可以成功地记录从RGC树突远端在大多数的神经元细胞体，表示该细胞的结构完整性和处理5。重要的是，在离开连接于视网膜色素上皮细胞（RPE）和巩膜，如上所述视网膜，隔离木瓜蛋白酶扩散的外视网膜和当该协议被加到WT视网膜可能减少对感光体外段的任何不利影响。在这两种情况下，木瓜蛋白酶处理，或在ILM的刮，有的会发生细胞损伤，实验者必须选择适合于experim方法ental问题。

电生理和功能成像（钙，电压敏感性染料）是两个在神经科学中最常用的实验技术。脑切片制剂和视网膜wholemount，这将在这里检查，提供检查电生理特性和突触连接不脱离麻醉剂或肌肉松弛剂的污染的装置。脑切片和视网膜wholemount保持其结构的完整性，不同文化或细胞匀浆，允许特定电路和大脑网络1的研究。从孤立的组织录制有超过体内录音与心跳和呼吸相关的运动优势被淘汰。此外，直接可视化使细胞的特定的类来进行有针对性的，和药理学工具2，3本地应用程序。 膜片钳和钙鎓染料加载视网膜wholemount由内的界膜（ILM），它涵盖了视网膜神经节细胞（RGC）层，并防止对细胞的直接访问的存在复杂。通常情况下，该膜被刮掉用玻璃吸管，以允许在单个小区中的千兆欧姆密封件的膜片吸管和形成的直接应用。此外，浴施加钙染料不交叉在ILM和必须或者该膜4下方喷射，逆向转移注射后在视神经5或穿过组织6电穿孔。此外，利用视网膜色素变性的啮齿动物模型时，RD / RD鼠标，ILM是更厚，更令人费解。在这里，我们使用的技术与酶消化7删除ILM，同时允许普遍存在钙染料加载，并直接进入视网膜神经节细胞的膜片钳recordiNGS 8。 无论从大脑切片或视网膜wholemount成功的记录取决于解剖和可行的神经组织的培养。通常情况下，组织被提取的实验的早晨，直到它被用于记录在人工脑脊液（ACSF）温育。通常，组织保持生存能力6 - 8小时，用这个时间窗口以下显著降解。但是，这两个脑切片和wholemount视网膜制剂通常产生比可以从该短的时间内被记录更多的组织。因此，组织通常被丢弃在一天结束和解剖再次完成在随后的几天。这意味着其他动物被利用，并建立和解剖/染色反复〜2小时。以下方案描述了一种用于延长神经元组织的寿命为24小时以上，这意味着较少的动物被利用，以及更多的实验时间是可用的。组织活力W¯¯通过录音电生理特性和钙的动态评估，这些特性均&lt;4小时和&gt; 24小时postdissection之间没有什么区别。 这些结果表明，不仅是单细胞特性完整且功能性延长温育后，但网络活动，通过钙成像和电生理记录的评估，是不变的&gt; 24小时postdissection。此外，我们表明，钙染料可以保留在细胞长时间，而不会造成任何有害的影响。演示此协议的应用程序，它在急性神经组织神经元的功能活性可保持长时间，一旦外部环境是高度调节。另外，作为组织的生存力，由于不同的孵育协议实验室之间变化很大，这种方法建立了应该应用，以减少在急性神经健康变性提供了理想的参数的黄金标准Ronal公司薄纸。

<table><tbody><tr><td>Sodium Chloride</td><td>Sigma Aldrich VETEC</td><td>V800372</td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma Aldrich</td><td>P9333</td><td></td></tr><tr><td>N-Methyl-D-glucamine</td><td>Sigma Aldrich</td><td>M2004&amp;nbsp;</td><td></td></tr><tr><td>D-glucose</td><td>Sigma Aldrich</td><td>G5767</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma Aldrich</td><td>S6014</td><td></td></tr><tr><td>Sodium phosphate monobasic</td><td>Sigma Aldrich</td><td>S2554</td><td></td></tr><tr><td>Magnesium chloride</td><td>Sigma Aldrich</td><td>M9272&amp;nbsp;</td><td></td></tr><tr><td>Calcium chloride&amp;nbsp;</td><td>Sigma Aldrich</td><td>223506</td><td></td></tr><tr><td>Papain Dissociation System</td><td>Worthington&amp;nbsp;</td><td>LK003150</td><td></td></tr><tr><td>Dimethyl sulfoxide anhydrous</td><td>Sigma Aldrich</td><td>276855</td><td></td></tr><tr><td>Pluronic F-127 Low UV absorbance*</td><td>Life technologies</td><td>P-6867</td><td>20% solution in DMSO&amp;nbsp;</td></tr><tr><td>Fura-2 AM&amp;nbsp;&amp;nbsp;</td><td>Anaspec</td><td>84017</td><td></td></tr><tr><td>Fluo-4 AM&amp;nbsp;&amp;nbsp;</td><td>Life Technologies</td><td>F23917</td><td></td></tr><tr><td>Fluo-8 AM</td><td>Abcam</td><td>Ab142773</td><td></td></tr><tr><td>Vibrating blade microtome&amp;nbsp;</td><td>Leica Biosystem</td><td>VT1200 S</td><td>Equiped with Leica&amp;rsquo;s Vibrocheck&amp;trade; measurement device&amp;nbsp;</td></tr><tr><td>Antivibration table</td><td>Kinetic Systems Vibraplane&amp;nbsp;</td><td>Vibraplane 9100/9200&amp;nbsp;</td><td></td></tr><tr><td>Fixed Stage Upright Microscope</td><td>Olympus</td><td>BX51WI&amp;nbsp;</td><td></td></tr><tr><td>Microscope Objective</td><td>Olympus</td><td>XLUMPlanFLN 20x/1.00w</td><td>20X&amp;nbsp;</td></tr><tr><td>Microscope Objective</td><td>Olympus</td><td>LUMPlanFLN 60x/1.00w</td><td>60X</td></tr><tr><td>CCD Camera</td><td>Andor</td><td>Ixon +</td><td></td></tr><tr><td>High speed wavelength switcher</td><td>Sutter instrument</td><td>Lambda DG-4&amp;nbsp;</td><td></td></tr><tr><td>Patch clamp amplifier</td><td>Molecular Devices</td><td>MultiClamp 700B</td><td></td></tr><tr><td>Data acquisition system</td><td>Molecular Devices</td><td>Digidata 1440A</td><td>Axon Digidata&amp;reg; System</td></tr><tr><td>Borosilicate glass capillaries</td><td>Sutter Instrument Co</td><td>BF150-86-10</td><td></td></tr><tr><td>Microelectrode puller</td><td>Sutter Instrument Co</td><td>P-97</td><td>Flaming/Brown type micropipette puller</td></tr><tr><td>Recording/perfusion chamber</td><td>Warner Instruments</td><td>RC-40LP</td><td>Low Profile Open Bath Chambers</td></tr><tr><td>Software</td><td>Molecular Devices</td><td>pClamp 10.2</td><td></td></tr><tr><td>Andor iQ Live Cell Imaging Software</td><td>Andor</td><td>Andor iQ3</td><td></td></tr><tr><td>Braincubator</td><td>Payo Scientific</td><td>BR26021976</td><td>www.braincubator.com.au</td></tr><tr><td>Peltier-Thermoelectric Cold Plate Cooler</td><td>TE Technology</td><td>CP-121</td><td></td></tr><tr><td>Temperature Controller</td><td>TE Technology</td><td>TC-36-25 RS232</td><td></td></tr></tbody></table>

prolonged incubation of acute neuronal tissue for electrophysiology and calcium-imaging

急性神经组织准备，脑切片和视网膜wholemount，通常只能维持6 - 解剖之后8小时。这限制了实验时间，并且增加被每研究利用的动物数量。这种限制影响特别协议，如要求延长预孵育浴用的染料钙成像。 3内指数细菌的生长 - 切片后4小时，紧密与组织健康的减少有关。该研究描述了用于限制细菌的增殖急性制剂维持而不需要抗生素，无菌程序，或组织培养基含有生长因子的长时间（&gt; 24小时）的活的神经元组织的方法。通过循环外液通过紫外线照射，并在15保持组织中一个自定义的保持室 - 16℃下，将组织表示在电生理特性，或钙SI没有差别通过细胞内钙染料在&gt; 24小时postdissection gnaling。这些方法不仅会延长实验时间，使用急性神经元组织的那些，但会减少完成实验目标所需动物的数量，并且将设置为急性神经元组织培养的金标准。

孵化过程中学联的细菌负载和温度的严格监管是必要的，保持神经组织活力。 16℃下（ 图1） -这可以通过与UVC光照射和维持脑脊液的温度在15进行优化。此外，脑脊液参数戳（APS; 图1C）提供实验者使用的环境条件下（pH和温度）的记录，从而培养神经元组织，其中，如果随后将减少之间变异性时，为参数的金标准实验。 <img alt="图1" src="/files/ftp_upload/55396/55396fig1.jpg" /> 图1.孵化系统，使组织环境严格监管。冷却珀耳帖板，主室，蠕动泵和UVC室组成的孵化体系的，示意图。 B，示出脑脊液的入口和出口点的主室，以及在卡波金入口，温度和pH探头的侧视图。组织放置在网格所示。所有测量探针连接到盖子，以允许结构稳定性。 C，脑脊液参数邮票描述孵育期间的脑脊液的温度和pH值。 D，示出了用于测量探头的安装孔的主要腔室的顶视图。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55396/55396fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 组织活力可以通过网络活动（ 图2），以及各种成像方法，包括钙的反应（ 图2B，C）的装置来测量。为了监控网络活动，我们采用含氯化钾（KCl; 30毫米）高浓度的脑脊液本地，这导致神经元的去极化和神经胶质所施加的 K +的附近。这种去极化可通过增加钙瞬变（ 图2B，C）和尖峰活性（ 图 2D），这也用作用于细胞活力生理指标进行观察。我们的研究结果表明，在温育在培养系统&gt; 24小时该切片尖峰活动类似于"新鲜"的切片孵育较短（&gt; 4小时; 图2D）。此外，个别神经元存活率，这是通过被动和主动膜特性的细胞内电生理记录，包括静息膜电位，输入电阻，时间常数监测，并且动作电位比得上在文献15（ 图2E，男报告参数）。 <p类="jove_content"&gt; <img alt="图2" src="/files/ftp_upload/55396/55396fig2.jpg" /> 长时间培养后的脑片图2.电生理和钙属性。一个脑切片的A，图像的用于测量网络活动2个胞记录电极切片后取28小时。第三电极（标有星号）用于粉扑氯化钾的30mM。 B，负载Fluo-4AM为&gt; 24小时片时间的推移图像，描绘以下氯化钾浴应用细胞内钙瞬变的增加。 C，以下氯化钾简短应用程序（30毫米; 1秒）细胞内钙瞬变。红-平均信号，灰色-如在B中所示在单个细胞中的钙的痕迹。 D，之前和氯化钾（红色箭头）的申请后，网络活动外生理记录均与"新鲜"的片基本保持不变（&lt;4小时postdissection）和那些孵育在培养箱&gt; 24 h上。注意氯化钾后立即申请扣球活动，表明应对在[K +]与去极化增加神经元存活的增加。 E和F，被动（E），并按照2小时（蓝色的痕迹），或在孵化体系培养26小时（黑色痕迹）锥体神经元的活性（F）膜性能。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55396/55396fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 提供给retinally退化次/ RD小鼠wholemount视网膜神经节细胞层的细胞的直接访问，在ILM被木瓜蛋白酶7,8消化。消化后，视网膜神经节细胞和流离失所的无长突细胞可以清楚地显现UND器DIC照明（ 图3A），并且细胞可以针对膜片钳记录，而不在ILM的任何现有刮。从RGC代表电流钳记录示于图3B。经由膜片吸管的细胞的去极化引起的剂量依赖性动作电位的产生，表示以下木瓜蛋白酶处理的细胞的生存力。 在ILM的去除也允许用的Fura-2AM（ 图3D）和细胞响应的30mM氯化钾的应用与相到F 0三百八十零分之三百四十零纳米比大幅度增加的神经节细胞层（GCL）无处不染色，表示大增加胞内钙浓度（ 图3E）。钙水平回到基线以下的刺激和细胞可以随后刺激产生了类似的幅度响应。此外，这些反应是R的区分etinae录得&lt;4小时和&gt; 24小时postdissection（ 图3F，G）。 <img alt="图3" src="/files/ftp_upload/55396/55396fig3.jpg" /> 图3:视网膜Wholemount电和钙录音。一个膜片钳记录从GCL木瓜蛋白酶消化后视网膜神经节细胞A，微分干涉对比图像。 B，当与50，100和150 pA的分别去极化细胞保留剂量依赖性扣球响应。 C＆D，之前的Fura-2AM的浴应用内界膜的去除允许视网膜神经节细胞的无处不在装载，采取380 nm激发形象。 E，当30毫米氯化钾ASCF施加，染色的细胞的85％的增加三百八十零分之三百四十零比率反应。 F＆G，在三百八十零分之三百四十〇比例恢复到基线后，30毫米氯化钾应用的增加（红色条）和细胞对后续的刺激反应都&lt;4小时和&gt; 24小时以下的夹层。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55396/55396fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging at JoVE.com

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

一旦从主体去除，神经元组织大大受环境条件的影响，从而导致组织的最终降解后6 - 8小时。采用了独特的培养方法，该方法密切监测和调节组织的胞外环境中，组织的生存力可以显著延长&gt; 24小时。

对于电生理和钙成像急性神经组织的长时间培养

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...

prolonged-incubation-acute-neuronal-tissue-for-electrophysiology

Nanyang Technological University

Research

JoVE Journal

Neuroscience

11.9K 次观看.  Western Sydney University. 一旦从主体去除，神经元组织大大受环境条件的影响，从而导致组织的最终降解后6 - 8小时。采用了独特的培养方法，该方法密切监测和调节组织的胞外环境中，组织的生存力可以显著延长> 24小时。 

视频: Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia

It has been suggested that changes in intracellular calcium mediate the induction of a number of important forms of synaptic plasticity (e.g., homosynaptic facilitation) 1. These hypotheses can be tested by simultaneously monitoring changes in intracellular calcium and alterations in synaptic efficacy. We demonstrate how this can be accomplished by combining calcium imaging with intracellular recording techniques. Our experiments are conducted in a buccal ganglion of the mollusc Aplysia californica. This preparation has a number of experimentally advantageous features: Ganglia can be easily removed from Aplysia and experiments use adult neurons that make normal synaptic connections and have a normal ion channel distribution. Due to the low metabolic rate of the animal and the relatively low temperatures (14-16 &deg;C) that are natural for Aplysia, preparations are stable for long periods of time. 
To detect changes in intracellular free calcium we will use the cell impermeant version of Calcium Orange 2 which is easily 'loaded' into a neuron via iontophoresis. When this long wavelength fluorescent dye binds to calcium, fluorescence intensity increases. Calcium Orange has fast kinetic properties 3 and, unlike ratiometric dyes (e.g., Fura 2), requires no filter wheel for imaging. It is fairly photo stable and less phototoxic than other dyes (e.g., fluo-3) 2,4. Like all non-ratiometric dyes, Calcium Orange indicates relative changes in calcium concentration. But, because it is not possible to account for changes in dye concentration due to loading and diffusion, it can not be calibrated to provide absolute calcium concentrations. 
An upright, fixed stage, compound microscope was used to image neurons with a CCD camera capable of recording around 30 frames per second. In Aplysia this temporal resolution is more than adequate to detect even a single spike induced alteration in the intracellular calcium concentration. Sharp electrodes are simultaneously used to induce and record synaptic transmission in identified pre- and postsynaptic neurons. At the conclusion of each trial, a custom script combines electrophysiology and imaging data. To ensure proper synchronization we use a light pulse from a LED mounted in the camera port of the microscope. Manipulation of presynaptic calcium levels (e.g. via intracellular EGTA injection) allows us to test specific hypotheses, concerning the role of intracellular calcium in mediating various forms of plasticity.

Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia

We demonstrate how changes in the intracellular free calcium concentration and synaptic efficacy can be simultaneously monitored in a ganglion preparation of Aplysia. We image intracellular calcium using a fluorescent dye, Calcium Orange, and induce and monitor synaptic transmission with sharp (intracellular) electrodes.

监测变化的细胞内钙离子浓度和突触效能的软体动物<em&gt;海兔</em

It has been suggested that changes in intracellular calcium mediate the induction of a number of important forms of synaptic plasticity (e.g., ...

科研

神经科学

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Multi-photon Intracellular Sodium Imaging Combined with UV-mediated Focal Uncaging of Glutamate in CA1 Pyramidal Neurons

Multi-photon fluorescence microscopy has enabled the analysis of morphological and physiological parameters of brain cells in the intact tissue with high spatial and temporal resolution. Combined with electrophysiology, it is widely used to study activity-related calcium signals in small subcellular compartments such as dendrites and dendritic spines. In addition to calcium transients, synaptic activity also induces postsynaptic sodium signals, the properties of which are only marginally understood. Here, we describe a method for combined whole-cell patch-clamp and multi-photon sodium imaging in cellular micro domains of central neurons. Furthermore, we introduce a modified procedure for ultra-violet (UV)-light-induced uncaging of glutamate, which allows reliable and focal activation of glutamate receptors in the tissue. To this end, whole-cell recordings were performed on Cornu Ammonis subdivision 1 (CA1) pyramidal neurons in acute tissue slices of the mouse hippocampus. Neurons were filled with the sodium-sensitive fluorescent dye SBFI through the patch-pipette, and multi-photon excitation of SBFI enabled the visualization of dendrites and adjacent spines. To establish UV-induced focal uncaging, several parameters including light intensity, volume affected by the UV uncaging beam, positioning of the beam as well as concentration of the caged compound were tested and optimized. Our results show that local perfusion with caged glutamate (MNI-Glutamate) and its focal UV-uncaging result in inward currents and sodium transients in dendrites and spines. Time course and amplitude of both inward currents and sodium signals correlate with the duration of the uncaging pulse. Furthermore, our results show that intracellular sodium signals are blocked in the presence of blockers for ionotropic glutamate receptors, demonstrating that they are mediated by sodium influx though this pathway. In summary, our method provides a reliable tool for the investigation of intracellular sodium signals induced by focal receptor activation in intact brain tissue.

We describe the combination of focal UV-induced photo-activation of neuro-active compounds with whole-cell patch-clamp and multi-photon imaging of intracellular sodium transients in dendrites and spines of hippocampal neurons in acute tissue slices of the mouse brain.

多光子细胞内钠成像结合谷氨酸海马CA1锥体神经元的UV-介导的焦点解笼锁

Multi-photon fluorescence microscopy has enabled the analysis of morphological and physiological parameters of brain cells in the intact tissue with high ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

脑切片神经元树突的双光子钙成像

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

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 ...

Two-photon Imaging of Microglial Processes' Attraction Toward ATP or Serotonin in Acute Brain Slices

Microglial cells are resident innate immune cells of the brain that constantly scan their environment with their long processes and, upon disruption of homeostasis, undergo rapid morphological changes. For example, a laser lesion induces in a few minutes an oriented growth of microglial processes, also called &#34;directional motility&#34;, toward the site of injury. A similar effect can be obtained by delivering locally ATP or serotonin (5-hydroxytryptamine [5-HT]). In this article, we describe a protocol to induce a directional growth of microglial processes toward a local application of ATP or 5-HT in acute brain slices of young and adult mice and to image this attraction over time by multiphoton microscopy. A simple method of quantification with free and open-source image analysis software is proposed. A challenge that still characterizes acute brain slices is the limited time, decreasing with age, during which the cells remain in a physiological state. This protocol, thus, highlights some technical improvements (medium, air-liquid interface chamber, imaging chamber with a double perfusion) aimed at optimizing the viability of microglial cells over several hours, especially in slices from adult mice.

Microglia, the resident immune cells of the brain, respond quickly with morphological changes to modifications of their environment. This protocol describes how to use two-photon microscopy to study the attraction of microglial processes toward serotonin or ATP in acute brain slices of mice.

微胶质过程对急性脑片 atp 或血清素的双光子成像

Microglial cells are resident innate immune cells of the brain that constantly scan their environment with their long processes and, upon disruption of ...

Establishing a Whole-Cell Configuration for Two-Photon Calcium Imaging of Brain Slices

Source: Camir&#233;, O., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/56776">Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices.</a> J. Vis. Exp. (2018).
This video demonstrates a protocol for whole-cell patch-clamp recording and two-photon calcium imaging in brain hippocampal slices. This method allows to study the dynamics of local Ca2+ transients (CaTs) in dendrites of different neuronal types in acute brain slices.

建立脑切片双光子钙成像的全细胞配置

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review.
1. Preliminary Preparation (Optional: ...

JoVE Encyclopedia of Experiments

Neurophysiology

Imaging and Optical Techniques

Measuring Neural Activity in a Mouse Brain Slice after Prolonged Incubation

Source: Cameron, M. A., et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/55396">Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging.</a> J. Vis. Exp. (120), (2017)
This video demonstrates a technique for measuring neuronal activity in mouse brain slices using calcium imaging and electrophysiological recordings. By perfusing the tissue with potassium ions and recording intracellular calcium changes through high-speed digital imaging, this method reveals real-time neuronal responses.

长时间孵育后测量小鼠脑切片中的神经活动

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Calcium Dye Loading

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A Technique for the Prolonged Incubation of Neuronal Tissue of the Retina

Source: Cameron, M. A., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/55396">Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging.</a> J. Vis. Exp. (2017).
This video demonstrates a method for prolonged incubation of neuronal tissue of the retina. Eyecups containing retina are obtained from a mouse eye; the inner limiting membrane (ILM) is enzymatically digested to expose the neuronal cells of the retina, and either the treated eyecup or the isolated retina is transferred to a specialized incubator for prolonged incubation at a low temperature.

一种视网膜神经元组织长期孵育的技术

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Retinal Wholemount Preparation ...