We would like to acknowledge the significant contribution Dr. Shiwei Huang (Australian National University) in validating the method. Furthermore, we would like to thank Ms. Kasia Pietrajtis for helpful comments regarding Golgi cells and Mr. Vitaly Lerner for the cortex experimental data. This work was supported by PITN-GA-2009-238686 (CEREBNET), FP7-ICT (REALNET), ELSC and ISF.

在本协议中所述的所有实验程序批准了希伯来大学的动物护理和使用委员会。 1.准备的解决方案和工具切片<ol><li>制备1升标准生理溶液（SPS）的含有表1中记载的离子。 <ol><li>制备含有该盐的最终浓度10倍的原液中预先存储玻璃瓶填充有1000毫升去离子水（0.055μS/厘米电导）。添加的盐（氯化钠，氯化钾，KH 2 PO 4，和硫酸镁 ），并搅拌至完全溶解。存储原液在4℃。 </li><li>使在实验当天最后的SPS溶液。加入100 ml的储备液至700毫升纯水中，用磁力搅拌棒溶解葡萄糖（3.6克）和碳酸氢钠 （2.18克）和然后添加精制水至1L。 </li><li>平衡的soluti在通过用95％氧气脱气它4/10％CO 2的〜20分钟。在此之后，加入2毫升的1M的CaCl 2溶液。 </li></ol></li><li>装配以下工具（ 图1）。 <ol><li>制备大剪刀或者其他工具，断头，小外科剪刀用于打开头骨，细尖镊子用于提升颅骨以暴露脑，手术刀用刀片解剖脑的所需部分被切下的小抹刀，用于操纵解剖的大脑部位。 </li><li>此外，制备小片滤纸，用于从大脑除去过量的液体，两个小的培养皿为大脑中两个玻璃500毫升烧杯被解剖用于容纳加热的纯净水和SPS，及氰基丙烯酸酯胶胶合脑到切片的阶段。 </li><li>准备一个小刷子用于切片和一个广口的玻璃巴斯德移液管，用于移动切片从切片浴到恢复茶时的处理的切片MBER。消毒工具，以防止细菌生长在温暖的切片浴场。此外，使用清洁的实验室手套。 </li></ol></li><li>所描述的吉布和爱德华兹15准备淹没切片复苏室等。连续气SPS它与95％O 2/5％CO 2，并保持在36℃。确保该冲气不会导致被困在可能损坏切片浴的小气泡。 </li><li>暖〜300毫升的SPS至36℃，在加热板有磁搅拌器，或在水浴中。如果一个加热板的情况下，要小心防止过热的溶液中，这可能导致离子沉淀。如果它发生，温暖清新的SPS，而不是冷却旧的。 </li><li>带来〜500毫升的纯净水通过一个电热水壶煮沸并且在一个烧杯中，将结合300毫升它用冷水，得到稍暖的水比切片温度。用断头术在此温热纯水并使用remai宁开水后维持切片沐浴的生理温度。 </li><li>通过注入0.1毫升戊巴比妥（60毫克/毫升）腹腔内麻醉的小鼠。几分钟后检查该动物不强脚趾或尾巴捏响应，以确定缺少感觉。 </li></ol> 2.解剖大脑<ol><li>迅速斩首鼠标通过切割颈部附近与头骨后部的大剪刀，让头部落入与温热纯水冲洗掉多余的血液的烧杯中。 </li><li>暴露在颅骨的枕骨大孔下颈部肌肉和皮肤，去除可能更颈部的小剪刀。 </li><li>拉颅骨顶部的皮肤能够清楚地看到颅骨缝合，以引导开放的头骨。 </li><li>通过枕骨大孔中插入的小剪刀下尖，马上把他们往切开颅骨开放侧面。沿顶骨到眼后和额顶缝合位置的横向边缘轻轻地切割;然后，把对颅骨的中心，跨越中线的头骨的另一边（见绿色虚线图2A）。 </li><li>用细尖镊子，解除从额顶角落颅骨和拉对角线向上和向侧面露出的脑（参见图2A中的黄色虚线箭头）。确保该头骨不附着到头部的任何骨或皮肤，使大脑将留在原地，当颅骨被提起。的情况下，大脑是移动与颅骨，使用小剪刀除去头骨和大脑之间的任何结缔组织。 </li><li>当大脑暴露出来，不要让大脑保持干燥。因而，执行下一个步骤，在保持头骨和大脑在培养皿中填充有温热，充气的SPS。 </li><li>分离的大脑用于电子零件从大脑的使用手术刀和刮勺其余xperiment。在使用小脑，来自前脑分离，通过中脑和脑桥单切（沿红线在图2B-D）和它移动到一个小的培养皿中，包含新鲜，充气的SPS。 </li><li>除去脑干以形成直链和宽基胶合大脑切片在水平面小脑当切割阶段。简要地放置小脑在湿滤纸，以便使被来自前脑切的面朝下。 <ol><li>切脑干用手术刀以形成基极（与蓝线在图2B中所示），和小脑返回到所述SPS。当需要切割的其他飞机，修剪小脑相应的块（ 图2C＆D蓝线）。 </li></ol></li></ol> 3.切片脑<ol><li>通过确定它是d准备切割阶段RY，将一小滴强力胶在舞台中间前。 </li><li>解除从所述SPS使用具有正确的一面朝上抹刀脑块，并使用小片滤纸大脑除去任何过量的液体。然后，用一个单一的运动，滑动距离锅铲大脑上的胶水在舞台上跌落。 </li><li>为了防止干燥的大脑，并从上所述组织的侧面跑上胶水，申请几滴的SPS用巴斯德移液管，并放置在切割阶段中的切片室。对准脑块，使得所感兴趣的区域（ 例如，小脑皮层）所面对的叶片，从而将经受最少的机械压力被切片之前。 </li><li>根据制造商的说明削减每个切片的同时，不断脱气所述SPS在切割室以及保持在34-37℃的温度。保持切割室的温度保持在该范围内通过填充的extern用温热的纯净水并更换水时的温度下降得太低的限幅器的人的腔室中。 注:该限幅参数必须通过实验发现，对于每种类型的组织和神经元的正在审查。万一平卡姆登SMZ 700切片机用陶瓷刀片和小脑高尔基细胞，用0.75毫米振幅在65赫兹和推进为0.05毫米/秒的速度;对于小脑核神经元更高的振幅和频率（1毫米和90赫兹，分别地）和较慢的前进速度（0.01-0.02毫米/秒）被推荐。为高尔基体和小脑核神经元，切片厚度应为300微米。 </li><li>在切片，不允许片折叠自己。用软毛刷轻轻支持他们，最好是触摸只有在那些对实验不感兴趣区域的切片防止这种情况。请注意，以防止刷接触到的vibratome刀片;特别是在壳体的陶瓷刀片，即使是光接触可能损坏叶片。 </li><li>移动从切片室每个切片，以回收/储存室用广口，火抛光的玻璃巴斯德吸管。 </li><li>让在开始实验前的切片休息在室中至少1小时。这允许损坏或垂死的组织和细胞的退化，从而导致清洁片表面。在第一个小时内，保持所述SPS的温度在34-36℃的范围内的腔室;确保切片不被气泡，会损坏片或使其浮触摸。 </li></ol> 4.实验<ol><li> 1小时后，让该回收室的温度冷却至室温（17-25℃）。 注意:这可能会延长，在此期间片可用于通过减缓细菌的生长以及细胞代谢的时间段。这个周期取决于大脑区域和细胞类型。例如，某些类型的高尔基体细胞可以被认为是健康的切片切而其他类型后8小时仅持续6小时。 </li><li> 1小时后，由限幅传递，使用切片关于各种电生理实验。 </li></ol>

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The authors declare that they have no competing financial interests.

我们证明了在生理，而不是冰冷的温度筹备急性脑切片的小鼠的方法。 已经显示11，当与制备的那些与冷的条件下相比在温暖条件下获得的切片的质量是优异的，其前提是该切片机叶片具有最小的垂直振动。切片在生理温度可以防止生理伪影引起的低温，例如那些涉及到改变在代谢过程17-19引起可在单细胞的像差以及网络行为。此外，即使在切片过程显然应避免不必要的延迟完成的，没有必要用切片快点（因为它是在冰冷却条件下，以完成切割之前，溶液变得太温暖切片时，并防止从持久冷冻的组织的损伤）。因此，慢的切割速度可我们ED。这可能是有益的情况下，分片非常微妙的结构。最后，省略了大脑的两个解和冷却步骤显著降低所需的时间的方法，以及其复杂性的量。该溶液于冰冷的温度的冷却需要大约40分钟内比溶液至生理温度的升温更多，留下更多的时间进行实验本身。 已经显示20检查切成20℃的切片时与切断0℃，相比荧光在GAD67-GFP小鼠的皮质神经元的强度更强。这进一步支持了切片在冰冷的温度下施加一个有害于佛罗里达州uence对神经元的生存力的概念。另一方面，在相同的纸张它表明，荧光神经元的密度降低，在37℃的切片得到的相比，削减在20℃。对结果的争议可能ARI本身从Z轴的偏转没有下考虑采取的事实。如上所述，在z偏转在生理温度切片时，除了叶片的质量和速度，限幅器的其它设置在组织的质量有很大的影响。 一个过程的关键部分涉及微调切片机刀片的前切片。用冰冷却的溶液，其中所述限幅可能既包括横向切割以及垂直"裂化"刚性脂膜时不同的是，在生理条件下，在z方向上的切片机刀片的任何运动将导致组织损伤。为了最大限度地减少这种情况，刀片应为硬和直接的;根据我们的经验，传统的刀片有太多的缺陷是可用的。专门针对的vibratome使用设计的高品质不锈钢刀片是更好的，但为了达到最佳效果，我们建议单斜CERAMIC刀片。此外，我们强烈建议您花费显著的时间为叶片的完美水平对齐方式，因为这直接影响切片质量。我们使用坎普登700SMZ限幅器，它允许在叶片取向的调谐，使得在z平面上的振动是小于0.5微米。事实上，热切断方法中最重要的限制是其上切片机刀片的质量和稳定性依赖。如果不能满足这些要求，最好是用传统的冰冷的切割方法来代替，尽管缺点在冰冷的切片，如从被部分地冷冻不同的渗透压。 证明该方法是用于获取小脑水平片段;用简单的修改同样的方法可以用来得到切片中任一冠状或矢状面，从前面的许多其他区域 - 或脑以及脑干。为前脑和olfact的最前部分的切片储器系统，颅骨应该削减更前方，以防止损坏这些部件。我们刻意保持方法简单，越短越好，不使用预灌注的动物，我们也不为切割的生理溶液替换任何部件。这使得该方法是根据感兴趣的特定的神经元亚型进行修改。它是可能的切割方案（如在引言中的参考文献中描述）的修饰可以进一步提高片的结果的可行性和鲁棒性。在一般情况下，该方法应当与大多数其他常见的生理溶液通常用于电生理记录兼容。 在这里所示的方法中，条带被切割300微米厚。最佳切片厚度取决于切片大脑区域和所关注的细胞。对于网络的完整性的考虑，切片不应薄于250微米时，由于扩散限制，并且向上每片厚度的上限为〜400-450微米。 在此方法的一个关键步骤对于胶合大脑切割阶段，因为即使很小的不稳定组织可导致不均匀的或不可用的条带。因此，应小心以确保在脑表面没有过量的SPS当它被降低到胶水降;还，过多胶水将导致脑躺在不均匀的舞台上和从台切割时可能脱落。另外，过量的胶水可能蠕变对组织两侧和中，除了引入不均匀性成片，可能损坏叶片。 有迹象表明，从冰冷的方法来表现出的热法改变切片协议时可能会上升一些困难。困难之一是细菌在片的外观。由于这个原因，消毒工具，该腔室，并且在切片浴用乙醇步骤之前是重要的。此外，转关闭加热系统在1小时后的恢复浴减缓细菌的生长。 最后，应当强调的是，在切割中生理温度切片的神经元的改善健康也可能导致改变其固有的属性。因此，最好是执行从与两个冷暖方法的切片所获得的结果的初始比较，特别是当实验是与早期的作品利用冷方法做了较长的项目的一部分。

该方法的目的是为了得到高质量的急性脑切片的体外电生理实验，尤其是当使用成人或甚至老的动物。 急性脑切片的方法，因为两个优雅的句子描述Skrede和Westgaard 1，已成为现代神经科学研究的基础之一，并在全球范围内采用无数的变化。切片的质量反映在活每片神经元的数目，该时间周期，在此期间细胞保持它们的电生理学和形态学特性，以及在组织中的完整性。此外，最大持续时间为稳定记录依赖于切片的质量。因此，沿几十年来，原来切断方法有了进一步的发展由个体研究团体切割2-10，常常通过切割的组合物的复杂的修改后，提升片恢复öR恢复解决方案（例如，添加抗坏血酸，硫脲或甚至H 2 O 2），以及心内预灌注动物用冷却的生理溶液。 正如最近的研究显示11，切片过程中的生理温度似乎比冷却到神经元的健康更有益;与成年（2-8月）的啮齿动物工作时的改善是最引人注目的。避免剧烈的温度变化时防止工件因温度依赖性过程中的细胞，如可塑性13和离子通道动力学13,14。这些变化可能会影响膜电位和细胞内钙信号，穗门槛，穗形。 "热"急性切片制备这里介绍的方法是用于获得高品质的急性脑切片从任何脑区域，包括小脑，皮层，海马，脑干晶核16的一般步骤&lt;/ SUP&gt;以及嗅球，无论是在大鼠和小鼠。 值得注意的是，在生理温度下的切片过程要求的切削刀片的振动几乎完全水平，是没有任何结构上的缺陷。这样的精确度可能无法实现与老款切片;在这种情况下，我们建议在进行切片准备在冷冻 - 寒冷的条件下作为低温度似乎使机械损伤，在代谢异常的费用的组织更耐即使。

<table><tbody><tr><td>Pentobarbital</td><td>CTS</td><td>170066</td><td>Concentration: 60 mg/ml in physiological saline.</td></tr><tr><td>Big scissors&amp;nbsp;</td><td>FST</td><td>14001-16</td><td>Any large scissors or a guillotine with sufficiently sharp edges can be used for decapitation&amp;nbsp;</td></tr><tr><td>Iris scissors&amp;nbsp;</td><td>Prestige medical</td><td>48,148</td><td>Any fine tip scissors can be used, provided the scissor blades are not longer than 1.5&amp;#8211;2 cm</td></tr><tr><td>Fine tip forceps&amp;nbsp;</td><td>FST</td><td>11254-20</td><td></td></tr><tr><td>Scalpel&amp;nbsp;</td><td>FST</td><td>91003-12</td><td></td></tr><tr><td>Scalpel blade #11</td><td>FST</td><td>10011-00</td><td></td></tr><tr><td>Small spatula&amp;nbsp;</td><td>Fisher&amp;nbsp;</td><td>2350</td><td></td></tr><tr><td>Filter paper</td><td></td><td></td><td>Any laboratory brand can be used.</td></tr><tr><td>Petri dishes</td><td>Duroplan</td><td>Z231509-1</td><td></td></tr><tr><td>Glass beakers&amp;nbsp;</td><td>SCHOT</td><td>10022846</td><td></td></tr><tr><td>Pasteur pipette&amp;nbsp;</td><td>Maple Leaf Brand</td><td>14672-029</td><td></td></tr><tr><td>Super glue&amp;nbsp;</td><td>LOCTITE</td><td>4091361/1</td><td></td></tr><tr><td>Slicer</td><td>Campden</td><td>7000-smz</td><td></td></tr><tr><td>Ceramic slicing blade</td><td>Campden</td><td>7550-1-C</td><td></td></tr><tr><td>Magnetic heater/stirrer</td><td></td><td></td><td>For heating up the SPS for the procedure</td></tr><tr><td>Electric kettle</td><td></td><td></td><td>For heating up water for temperature control</td></tr><tr><td>Slice recovery chamber + heating unit</td><td>Warner instruments</td><td>&amp;nbsp;BSC-HT +&amp;nbsp; BSC-BUW</td><td>Home-built models may also be used.</td></tr><tr><td>Thermometer</td><td></td><td></td><td>For monitoring SPS temperature during dissection and slicing</td></tr></tbody></table>

slice it hot: acute adult brain slicing in physiological temperature

在这里，我们提出了一个协议，准备对急性脑片。这个程序是用于电生理膜片钳实验的关键元件，其在很大程度上决定结果的质量。它已经显示，省略了切割过程中的冷却步骤是有利的，获得健康的切片和细胞，特别是当与从成熟动物的高度髓脑结构处理。虽然确切的机制，使高温下支持神经健康时只能推测，它按理说，只要有可能，在切片时的温度应接近生理条件，以防止温度有关的文物。该方法的另一个重要的优点是该过程的简单性，因此，制备时间短。在证明方法成年小鼠进行使用，但相同的过程可以与年轻小鼠以及大鼠施用。此外，下面的补丁CL安培实验是在水平的小脑切片进行的，但同样的过程也可以用在其它的面以及大脑的其他后部区域内使用。

在所描述的方式制备的切片可用于各种电和光遗传学实验。在图3A和图3C，我们显示一个水平小脑切片和冠状的大脑皮质切片，分别根据微分干涉（DIC）的光学观察的一个代表性的例子。在小脑切片，几种类型的小脑的神经元可以容易地由他们的位置和细胞体的形状识别，允许有针对性的电生理记录。在图3B和3D中 ，例如从在电流钳模式全细胞膜片钳记录从自发活性小脑高尔基细胞和皮质的锥体细胞的痕迹被示出。从热分片大脑得到的电生理和光遗传学记录质量的进一步的例子可在 ​​黄和Uusisaari 11和Lefler 等[16]中找到。 <table border="0" cellpadding="0"CELLSPACING ="0"&gt; <tbody><tr height="34"><td height="34" style="height:34px;width:108px;"></td><td style="width:103px;">浓度[MM] </td><td style="width:143px;">重（g / L）的</td></tr><tr height="20"><td height="20" style="height:20px;width:108px;">氯化钠</td><td style="width:103px;"> 124 </td><td style="width:143px;"> 72.5 </td></tr><tr height="20"><td height="20" style="height:20px;width:108px;">氯化钾</td><td style="width:103px;"> 3 </td><td style="width:143px;"> 2.23 </td></tr><tr height="21"><td height="21" style="height:21px;width:108px;"> KH 2 PO 4的</td><td style="width:103px;"> 1.2 </td><td style="width:143px;"> 1.63 </td></tr><tr height="21"><td height="21" style="height:21px;width:108px;"> 硫酸镁 * 6H 2 O </td><td style="width:103px;"> 1.9 </td><td style="width:143px;"> 4.3 </td></tr><tr height="20"><td height="20" style="height:20px;width:108px;">葡萄糖</td><td style="width:103px;"> 20 </td><td style="width:143px;"> 3.6 </td></tr><tr height="21"><td height="21" style="height:21px;width:108px;"> 碳酸氢钠 </td><td style="width:103px;"> 26 </td><td style="width:143px;"> 2.18 </td></tr><tr height="21"><td height="21" style="height:21px;width:108px;"> 氯化钙 </td><td style="width:103px;"> 2 </td><td style="width:143px;"> 1.109 </td></tr></tbody></table> 表1组合物的标准生理溶液。 <img alt="图1" src="/files/ftp_upload/52068/52068fig1highres.jpg" /> 图1安排的工具1。大剪刀，2小的手术剪刀，手术刀3.刀片＃11，4，精尖的镊子，5。小铲，6小培养皿中，7玻璃烧杯中的去离子水，8.玻璃烧杯加热gasseð生理溶液，9强力胶，10薄刷，11.巴斯德吸管，12小注射器，13戊巴比妥，14滤纸，15切割阶段。 <img alt="图2" src="/files/ftp_upload/52068/52068fig2highres.jpg" /> 图2.在颅骨和脑解剖的示意图（A）的图形描绘了头骨的开口以暴露脑（B - D）的原理图，描述切割大脑的三个主要切片平面的平面。蓝线表示其上大脑会粘在飞机上。 <img alt="图3" src="/files/ftp_upload/52068/52068fig3highres.jpg" /> 图3.该方法的代表性成果。（A）水平小脑切片Øbtained的证明方法。小脑皮层的三个主要层（颗粒细胞层，气相色谱层;浦肯野神经元层，PN层;分子层，ML）以及白质（WM）表示。甲膜片钳电极被示意性绘制到高尔基细胞（千兆铜缆）（比例尺为40μm）。（B）的例子，从与一个膜片钳电极得到电流钳记录中A所示的高尔基细胞记录模式。 （在黑色:代表跟踪总分6灰迹线）。（C）的一个冠状的大脑皮质切片得到的证明方法。皮质锥体细胞与膜片钳电极的示意图显示在一起。（D）例如膜片钳记录，在电流钳模式下，从（c）所示的锥体细胞。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52068/52068fig3large.jpg" target="_blank">请点击此处查看这是一个更大的版本图。</a>

Watch this Scientific Journal Video about Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature at JoVE.com

Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature

In this paper we show a method for preparing acute brain slices in physiological temperature, using a conventional physiological solution without special modifications for the cutting (such as adding sucrose) and without intracardial perfusion of the animal before slice preparation.

切片热门:成人急性脑切片在生理温度

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments ...

slice-it-hot-acute-adult-brain-slicing-in-physiological-temperature

Nanyang Technological University

Research

JoVE Journal

Neuroscience

26.2K 次观看.  Hebrew University of Jerusalem. In this paper we show a method for preparing acute brain slices in physiological temperature, using a conventional physiological solution without special modifications for the cutting (such as adding sucrose) and without intracardial perfusion of the animal before slice preparation.

视频: Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

用优化的n-甲基 d-葡乙胺保护恢复法制备急性脑切片

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

科研

神经科学

Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding these fundamental functions will shed light on the information transfer in these neurons. Dendritic patch-clamp recordings provide the possibility to directly examine the electrical properties of dendrites and underlying voltage-gated ion channels. However, these fine structures are not easily accessible to patch pipettes because of their small diameter. This report describes a step-by-step procedure to collect stable and reliable recordings from the dendrites of dopaminergic neurons in acute slices. Electrophysiological measurements are combined with post hoc recovery of cell morphology. Successful experiments rely on improved preparation of slices, solutions and pipettes, adequate adjustment of the optics and stability of the pipette in contact with the recorded structure. Standard principles of somatic patch-clamp recording are applied to dendrites but with a gentler approach of the pipette. These versatile techniques can be implemented to address various questions concerning the excitable properties of dendrites.

Subcellular patch-clamp recordings offer the possibility to investigate the functional properties of dendrites. However these fine structures are not easily accessible due to their small diameter. The protocol described here aims to facilitate the collection of stable and reliable recordings from the dendrites of dopamine neurons in slices in vitro.

从黑质多巴胺神经元的Somatodendritic域亚细胞的膜片钳记录

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding ...

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

利用体外大鼠脑切片研究变性主要事件的一种替代方法

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...

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 ...

Short-Term Free-Floating Slice Cultures from the Adult Human Brain

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of slice cultures in neuroscience, studies using adult nervous tissue to prepare such cultures are still scarce, particularly those from human subjects. The use of adult human tissue to prepare slice cultures is particularly attractive to enhance the understanding of human neuropathologies, as they hold unique properties typical of the mature human brain lacking in slices produced from rodent (usually neonatal) nervous tissue. This protocol describes how to use brain tissue collected from living human donors submitted to resective brain surgery to prepare short-term, free-floating slice cultures. Procedures to maintain and perform biochemical and cell biology assays using these cultures are also presented. Representative results demonstrate that the typical human cortical lamination is preserved in slices after 4 days in vitro (DIV4), with expected presence of the main neural cell types. Moreover, slices at DIV4 undergo robust cell death when challenged with a toxic stimulus (H2O2), indicating the potential of this model to serve as a platform in cell death assays. This method, a simpler and cost-effective alternative to the widely used protocol using membrane inserts, is mainly recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases. Finally, although the protocol is devoted to using cortical tissue collected from patients submitted to surgical treatment of pharmacoresistant temporal lobe epilepsy, it is argued that tissue collected from other brain regions/conditions should also be considered as sources to produce similar free-floating slice cultures.

A protocol to prepare free-floating slice cultures from adult human brain is presented. The protocol is a variation of the widely used slice culture method using membrane inserts. It is simple, cost-effective, and recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases.

来自成人大脑的短期自由浮动切片培养

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of ...

Minimizing Hypoxia in Hippocampal Slices from Adult and Aging Mice

Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high fidelity. Exploration of LTP and LTD mechanisms, single neuron dendritic computation, and experience-dependent changes in circuitry, would not have been possible without this classical preparation. However, with a few exceptions, most basic research using acute hippocampal slices has been performed using slices from rodents of relatively young ages, ~P20-P40, even though synaptic and intrinsic excitability mechanisms have a long developmental tail that reaches past P60. The main appeal of using young hippocampal slices is preservation of neuronal health aided by higher tolerance to hypoxic damage. However, there is a need to understand neuronal function at more mature stages of development, further accentuated by the development of various animal models of neurodegenerative diseases that require an aging brain preparation. Here we describe a modification to an acute hippocampal slice preparation that reliably delivers healthy slices from adult and aging mouse hippocampi. The protocol&#8217;s critical steps are transcardial perfusion and cutting with ice-cold sodium-free NMDG-aSCF. Together, these steps attenuate the hypoxia-induced drop in ATP upon decapitation, as well as cytotoxic edema caused by passive sodium fluxes. We demonstrate how to cut transversal slices of hippocampus plus cortex using a vibrating microtome. Acute hippocampal slices obtained in this way are reliably healthy over many hours of recording, and are appropriate for both field-recordings and targeted patch-clamp recordings, including targeting of fluorescently labeled neurons.

This is a protocol for acute slice preparation from adult and aging mouse hippocampi that takes advantage of transcardial perfusion and slice cutting with ice-cold NMDG-aCSF to reduce hypoxic damage to the tissue. The resulting slices stay healthy over many hours, and are suitable for long-term patch-clamp and field-recordings.

从成人和老化小鼠的河马切片中尽量减少低氧

Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high ...

Preparing Acute Brain Slices from the Dorsal Pole of the Hippocampus from Adult Rodents

Whole-cell patch-clamp recordings from acute rodent brain slices are a mainstay of modern neurophysiological research, allowing precise measurement of cellular and synaptic properties. Nevertheless, there is an ever increasing need to perform correlated analyses between different experimental modes in addition to slice electrophysiology, for example: immunohistochemistry, molecular biology, in vivo imaging or electrophysiological recording; to answer evermore complex questions of brain function. However, making meaningful conclusions from these various experimental approaches is not straightforward, as even within relatively well described brain structures, a high degree of sub-regional variation of cellular function exists. Nowhere is this better exemplified than in the CA1 of the hippocampus, which has well-defined dorso-ventral properties, based on cellular and molecular properties. Nevertheless, many published studies examine protein expression patterns or behaviorally correlated in vivo activity in the dorsal extent of the hippocampus; and explain findings mechanistically with cellular electrophysiology from the ventro-medial region. This is further confounded by the fact that many acute slice electrophysiological experiments are performed in juvenile animals, when other experimental modes are performed in more mature animals. To address these issues, this method incorporates transcardial perfusion of mature (&#62;60 day old rodents) with artificial cerebrospinal fluid followed by preparation of modified coronal slices including the septal pole of the dorsal hippocampus to record from CA1 pyramidal cells. This process leads to the generation of healthy acute slices of dorsal hippocampus allowing for slice-based cellular electrophysiological interrogation matched to other measures.

The purpose of this protocol is to describe a method to produce slices of the dorsal hippocampus for electrophysiological examination. This procedure employs perfusion with chilled ACSF prior to slice preparation with a near-coronal slicing angle which allows for preservation of healthy principal neurons.

Whole-cell patch-clamp recordings from acute rodent brain slices are a mainstay of modern neurophysiological research, allowing precise measurement of ...

Acute Mouse Brain Slicing to Investigate Spontaneous Hippocampal Network Activity

Acute rodent brain slicing offers a tractable experimental approach to gain insight into the organization and function of neural circuits with single-cell resolution using electrophysiology, microscopy, and pharmacology. However, a major consideration in the design of in vitro experiments is the extent to which different slice preparations recapitulate naturalistic patterns of neural activity as observed in vivo. In the intact brain, the hippocampal network generates highly synchronized population activity reflective of the behavioral state of the animal, as exemplified by the sharp-wave ripple complexes (SWRs) that occur during waking consummatory states or non-REM sleep. SWRs and other forms of network activity can emerge spontaneously in isolated hippocampal slices under appropriate conditions. In order to apply the powerful brain slice toolkit to the investigation of hippocampal network activity, it is necessary to utilize an approach that optimizes tissue health and the preservation of functional connectivity within the hippocampal network. Mice are transcardially perfused with cold sucrose-based artificial cerebrospinal fluid. Horizontal slices containing the hippocampus are cut at a thickness of 450 &#956;m to preserve synaptic connectivity. Slices recover in an interface-style chamber and are transferred to a submerged chamber for recordings. The recording chamber is designed for dual surface superfusion of artificial cerebrospinal fluid at a high flow rate to improve oxygenation of the slice. This protocol yields healthy tissue suitable for the investigation of complex and spontaneous network activity in vitro.

This protocol describes the preparation of horizontal hippocampal-entorhinal cortex (HEC) slices from mice exhibiting spontaneous sharp-wave ripple activity. Slices are incubated in a simplified interface holding chamber and recordings are performed under submerged conditions with fast-flowing artificial cerebrospinal fluid to promote tissue oxygenation and the spontaneous emergence of network-level activity.

急性小鼠脑切片研究自发海马网络活动

Acute rodent brain slicing offers a tractable experimental approach to gain insight into the organization and function of neural circuits with single-cell ...

Assessing Changes in Synaptic Plasticity Using an Awake Closed-Head Injury Model of Mild Traumatic Brain Injury

Mild traumatic brain injuries (mTBIs) are a prevalent health issue in North America. There is increasing pressure to utilize ecologically valid models of closed-head mTBI in the preclinical setting to increase translatability to the clinical population. The awake closed-headed injury (ACHI) model uses a modified controlled cortical impactor to deliver closed-headed injury, inducing clinically relevant behavioral deficits without the need for a craniotomy or the use of an anesthetic.
This technique does not normally induce fatalities, skull fractures, or brain bleeds, and is more consistent with being a mild injury. Indeed, the mild nature of the ACHI procedure makes it ideal for studies investigating repetitive mTBI (r-mTBI). Growing evidence indicates that r-mTBI can result in a cumulative injury that produces behavioral symptoms, neuropathological changes, and neurodegeneration. r-mTBI is common in youths playing sports, and these injuries occur during a period of robust synaptic reorganization and myelination, making the younger population particularly vulnerable to the long-term influences of r-mTBI.
Further, r-mTBI occurs in cases of intimate partner violence, a condition for which there are few objective screening measures. In these experiments, synaptic function was assessed in the hippocampus in juvenile rats that had experienced r-mTBI using the ACHI model. Following the injuries, a tissue slicer was utilized to make hippocampal slices to evaluate bidirectional synaptic plasticity in the hippocampus at either 1 or 7 days following the r-mTBI. Overall, the ACHI model provides researchers with an ecologically valid model to study changes in synaptic plasticity following mTBI and r-mTBI.

Here, it is demonstrated how an awake closed-head injury model can be used for examining the effects of repeated mild traumatic brain injury (r-mTBI) on synaptic plasticity in the hippocampus. The model replicates important features of r-mTBI in patients and is used in conjunction with in vitro electrophysiology.

使用轻度创伤性脑损伤的清醒闭合性头部损伤模型评估突触可塑性的变化

Mild traumatic brain injuries (mTBIs) are a prevalent health issue in North America. There is increasing pressure to utilize ecologically valid models of ...

Preparation of Brain Sections Using a NMDG-Based Protective Recovery Method

Source: Ting, J. T. et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/53825">Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method.</a> J. Vis. Exp. (2018).
This video describes a protocol to obtain brain slices using an N-Methyl-D-glucamine (NMDG)-based protective recovery method. A dissected brain is incubated in an aCSF with NMDG and HEPES to minimize neuronal cell death and damage during the sectioning. The section is allowed to recover in the aCSF to restore the cellular activities

使用基于 NMDG 的保护性恢复方法制备脑切片

1. Brain Dissection and Slicing

	Decapitate the animal. Use a scalpel to open the skin on the head and expose the skull cap.
	Use fine super-cut scissors ...