我们感谢巴纳德Foucart的技术援助。这项工作是由比利时科学研究基金（FRS-FNRS）和伊丽莎白女王医学研究基金的支持。艾格尼丝维莱是研究员在比利时科学研究基金。

按照与国立卫生法规的护理和使用动物的研究，并与当地伦理委员会的协议，所有动物的程序进行。 1。人工脑脊髓液的制备同一介质，则用于解剖，切割和灌注片（1毫升/分钟），在休息期间和电生理记录。这种介质组成的124 mM氯化钠，氯化钾4.4毫米，26毫米的NaHCO 3，1毫米的NaH 2 PO 4，1.3毫米，2.5毫米的 CaCl 2，用MgSO 4和10mM D-葡萄糖。 <ol><li>称取的不同组成部分，在2 L的ACSF MgSO 4干燥，这是已在溶液（1M）除外。使用标准的解决方案，可以肯定的准确浓度的Mg 2 +，因为硫酸镁粉是高吸湿性。的Ca 2 +：Mg 2 +的比例大大影响LTP的诱导和维持14岁 ，因此他们的比例必须始终得到尊重。 </li><li>把氯化钙在玻璃以外的所有元件量筒带来2升蒸馏水H 2 O </li><li>大力搅拌。当一切都溶解后，加卡波通过水族馆鼓泡管连接到所述容器（95％O 2，5％CO 2）。等待几分钟添加氯化钙，当pH值固定在7.4的碳酸氢盐缓冲液的作用。 </li><li>长方形玻璃盘在冷藏，并保持600毫升，冷却至4℃，周围的冰盘。这的媒体将用于海马解剖。 </li><li>过滤其它1400毫升的，并保持在一个锥形烧瓶中。 </li></ol> 2。制备电钻机和脑切片录音室灌注电路由2个蠕动泵，一个泵送新鲜的流体从储备罐和其他用泵送流体从吨他在录音室的垃圾桶。新鲜的流体被添加到重新充氧并温热才到达的接口的录音室，通过重力（ 图1A）的自制的灌注系统。接口录音室的设计是由FST（精细的科学工具，温哥华，加拿大， 图1B）。组织切片被放置在连接到一个可移动的以及环，并且可以连续地保持在接口条件下通过调节在抽吸的高度以及针机织网式过滤器。自制挡在其端和横向穿孔（0.5毫米）的塑料管吸针固定室水平稳定提高。在记录头安装在一个水浴含有气体分散石头这有助于保持潮湿的环境中，通过潮湿的空气中，通过在记录头中的偏转端口（减少水滴的形成）。巴斯和介质温度控制连续变化的水平直流电流日粗糙的硅橡胶嵌入在水浴加热器元件。的水浴中，并记录井的热敏电阻在允许的腔室温度设置和精确监测站点。 的腔室的加热系统是一个全球性的加热系统的温度控制整个钻机完成。开发的软件由爱丁堡大学（ <a href="http://www.etcsystem.com" target="_blank">www.etcsystem.com</a> ）的研究人员在监控和均衡的含氧空气的温度，脑片，电极，其余钻机提供一个稳定的环境，长期的录音（ 图1C） 。用于小鼠海马脑片的温度是28°C <ol><li>最少20分钟，用蒸馏水H 2 O冲洗灌注电路，启动加热系统。 </li><li>启动卡波在电路中的鼓泡。 CARBOGEN到达自制灌注管到fILTER蜡烛和在水浴低于录音室。水浴中的流速控制由流量计。如果流速太慢，则可能会成为缺氧组织的。反之，如果流速太高，气体可能不会有足够的加热的和滋润的，导致组织的干燥。高的气体的流速也可以增加从水浴中水喷洒在组织下面，导致渗透压冲击的机会。这可以防止通过增加比特尼龙网（100微米）的出口处的偏转端口。 </li><li>沥干电路和填补过滤学联。 </li><li>放环将支持片控股室。甲织网式过滤器具有范围从500到750微米的网孔被拉长，并连接到该环由两部分组成的树脂环氧树脂胶。无纺布滤净87％锦纶13％氨纶（内裤15旦）。胶，必须小心，以避免形成浮雕的打印在表面克。 </li><li>仔细清除所有气泡从电路。 </li><li>调节电平，在与螺钉的录音室的吸入针ACSF。蠕动泵的速度调整为1毫升/分钟的入口泵出口泵以5毫升/分钟。 </li></ol>灌注系统被放置在一个刚性的耐振表，周围环绕着一个法拉第笼。 3。准备解剖区手术器械：手术刀＃11刀片，小型标准解剖剪刀，弹簧剪刀，弯钳，两个海德曼铲刀和勺子。 补充材料：铡刀，焊，McIlwain组织斩波器，用刀片，滤纸，玻璃巴斯德吸管与橡胶奶头，2个塑料巴斯德移液管，用广口，陪替氏培养皿中，一个金属圆筒直径7毫米（ 图2A）。 <ol><l&gt;在填充用冷的人工脑脊液（在步骤1中制备）的解剖盘，冷冻玻璃盖从染色菜，包在滤纸浸入。小心地取出气泡和氧化学联的通过一个水族馆起泡（ 图2A）。 </li><li>布局工具的使用顺序。添加3层滤纸斩波器板和在纸巾上一个新的刀片，清洗用蒸馏水和乙醚（ 图2B）。接触纸张时，刀片必须是水平的。剑锋调整的最大价值和速度最大值约三分之一到一半。 </li><li>用心检查，如果一切准备就绪（乐器，温度...），因为你没有足够的时间之后。 </li><li>请同学们用巴斯德吸管充满冷学联的喷解剖。 </li><li>麻醉鼠标斩首前与的戊巴比妥IP（100毫克/千克）。也可以用异氟烷麻醉。我们比较了这两种方法，要想获取独立非执行董事相同的结果。断头必须在麻醉下进行，但也可以用颈椎脱位。然而，颈椎脱位，需要一个非常好的做法，以避免动物的痛苦。 </li><li>解剖上焊下尽可能快冷学联连续淋浴。 </li></ol> 4。脑去除<ol><li>按住枪口上，用食指和一只手的拇指头，做一个切口，解剖剪刀沿着中间的顶部开始的头铡切的边缘和运行头侧额骨。 </li><li>切透皮肤的每一侧的头部充分暴露颅骨板和删除头部上面的尾侧的肌肉的肌肉。 </li><li>解剖剪刀，切断颞肌，每侧沿颞板，然后切额叶板夹在中间，横向。然后，做一个小切口枕骨上，两者之间的排位阿泰。 </li><li>的每一侧，切枕部板上面的尾鳍基。 </li><li>切沿矢状缝与春风似剪刀。 </li><li>随着镊子，取出头骨半散布他们远离对方。 </li><li>用手术刀，切开后横向只是嗅球和小脑前然后投身提取的大脑解剖盘用冷学联。 </li></ol> 5。海马解剖<ol><li>一旦大脑被浸泡在解剖盘，切断两半球互相用手术刀插在中间。 </li><li>海马解剖用铲刀进行视觉控制下，通过双目手术显微镜（X25， 图2A）。在一个半球，仔细涂抹结构看侧脑室。取出脑干和间脑。这是通过应用在一侧额叶皮质上的铲刀上的间脑另一边。小心不要触摸海马铲刀和不舒展期间组织切片。 </li><li>断绝穹窿，然后轻轻一推海马皮层（滚开），插入脑室锅铲。 </li><li>当海马提取，去除多余的皮层组织和剩余血管。 </li></ol> 6。切割片<ol><li>随着广口塑料巴斯德吸管，勺子海马转移其的阿尔韦亚尔表面向上（凸侧）。 </li><li>一个标准的塑料巴斯德吸管取出多余的液体。 </li><li>提示勺子几乎垂直接触滤纸的菜刀（ 图2B）和落海马勺子，迅速 ​​接触滤纸，然后搬回勺子。 </li><li>定向海 ​​马切片横向到400微米的菜刀（ 见图2C取向海马相对于刀片的脓）。尽快行动。 </li><li>取出滤纸切片海马和它周围的金属圆柱体包裹，一点点蔓延切片。然后，片使用一个标准的塑料的巴斯德吸管和收集他们在培养皿中充满冷学联，学联喷雾剂。 </li></ol> 7。孵化片接口<ol><li>选择一个标准的塑料巴斯德吸管片，并迅速将它们放置在录音室。片恢复解剖创伤直接在录音室，在28°C接口</li><li>切片将最有可能下沉。降低流体水平达到限幅电平，然后抬起它，使它浮。 </li><li>将切片在正确的位置，同时降低的水平。切片总是以同样的方式，以方便定位的电极（2刺激和一个记录电极）在CA1区取向。 </li><li>时停止该流体是在接口。 A“弯月”媒体围绕切片表明有足够的媒体。净过滤器必须与媒体饱和，但不会完全被淹没。 </li><li>保持腔室与穿孔的盖子放置在滤纸覆盖。 </li><li>让切片休息至少1.5小时，在28°C。 </li></ol> 8。突触反应的记录<ol><li>记录电极：玻璃毛细管拉到尖取得2-5MΩ电阻时充满了学联。一小片滤纸周围放置的电极的前端和骨蜡是适用于肢体以减少结露。刺激电极：铂 - 铱的的双极群集电极与12.5微米直径的FHC（美国）购买。有了适当的照顾，每个电极可使用至少30倍（ 图1B）。 </li><li> CA1区辐射层中放置电极，电极排队（ 图3A）。降低电极成茂显微操作器中的切片的表面下75〜150微米。两个刺激电极置于刺激两种截然不同的束谢弗抵押品。当电极降低到切片，小心地放在滤纸各地关闭室。 </li><li>双相刺激（0.08毫秒脉冲持续时间每半波）进行恒定电压连接到SIU-V隔离单位与基层刺激。最大的反应是通过增加刺激强度从2 V最大12五场EPSPS一个WPI ISO-80放大器，放大1000倍，在10 Hz和10 kHz滤波检查。通过国家仪器A / D转换的信号，然后发送到PC。使用WinLTP程序（ <a href="http://www.winltp.com">www.winltp.com</a> ）进行刺激，数据采集和分析。得到的最大振幅的40％的场兴奋性突触后的记录，可在输入把曲线。对于每一个切片，fEPSP的斜坡LTP诱导前30分钟对平均坡度超过归。 </li><li> LTP是通过施加刺激（100赫兹）的一个单一的列车在测试强度的一个通路，而第二通路作为控制触发。 </li></ol> 9。清洁的安装<ol><li>冲洗所有的电路，用3％的过氧化氢 ​​（H 2 O 2）溶液至少10分钟，然后沥干。电路将被小心地用蒸馏水​​冲洗在任何实验开始前。其他实验室只用蒸馏水进行清洗，但我们注意到深色残余物的沉积，管道系统中，仅使用水时，H 2 O 2的使用不是绝对必需的。 </li><li>更换水水浴中下方的录音室。 </li><li>浴的刺激电极提示酒精5分钟。 </li></ol>

没有利益冲突的声明。

我们在我们的实验室已经开发相结合的方法开发和使用的其他实验室有一个大的专业知识在LTP录音11,17的协议。此协议适合于成年小鼠海马，并可以用在任何年龄段的任何背景基因型的动物。它还允许在转基因小鼠的神经退行性疾病如阿尔茨海默病18,19分析LTP。 利用大鼠海马脑片这个协议必须适应。例如，大多数的大鼠进行研究，使用温度为32°C，而不是28°C?...

如今，有多么复杂的存储和调用记忆在神经回路水平的了解有限。然而，记忆存储是一个统一的假设和广泛接受的记忆存储在中枢神经系统的神经元之间的突触连接强度的变化。就其本身而言，对突触可塑性的研究已经在很大程度上得益于两个突破性的发现。 （1）在一个开创性的实验，极乐和Lomo 1，使用完整的麻醉兔，结果发现提供一个短暂的高频率（1秒，100赫兹）刺激海马穿路径造成一个长期持久的（几个小时）增加相关的突触连接。这迷人的现象被称为“长时程增强”或LTP由道格拉斯和戈达德在1975年2。 （2）随后，我们发现了类似的现象可能会触发在脑切片（0.4毫米）的人为保持的活体外 </eM&gt;。研究得最广泛的LTP是通过提供一个或多个破伤风记录产生的场兴奋性突触电位在所谓的CA1区锥体细胞诱发的成捆的轴突（所谓的谢弗络），而在体外观察到。诱导LTP的机制在很大程度上被显露出来。基本上，Ca 2 +的涌入通过NMDA受体激活酶有两个后果：AMPA受体的磷酸化（提高效率）和加入额外的AMPA受体在突触后膜3。 LTP的维持阶段的机制，与此相反，在很大程度上是未知的，特别是因为它是实验更难以维持健康的许多小时，比30至60分钟切片。 大量的研究，一直致力于为LTP机制的理解和有趣的理论已经阐述多年来4-11。但联合国直到现在，精确的分子突触强度的稳定增长机制尚未阐明。这可能是部分原因是由于难以再现先前结果在不同实验室使用不同的技术来制备和海马切片的维护。在他们的方法纸，Sajikumar 等12强调的实验条件下对大鼠海马脑片的制备和记录LTP稳定的重要性。在这段视频中，我们介绍了多年来在我们的实验室开发的，能够记录一个非常稳定的小鼠海马脑片LTP的优化步骤。 这种优化已经开发并成功使用小鼠13只大鼠和11其他实验室研究LTP机制的协议。它允许有经验的研究人员，以诱导和记录一个很长的持久LTP具有很高的成功率，在成年小鼠。在p诱导LTP hysiological基础进行了仔细的检查，并展示了14。在这种方法中纸，我们展示的任何修改的实验条件下，可以产生深远的影响，如温度或氧LTP维护解剖过程，同时可以深入修改切片兴奋。还必须强调的是，所有这些参数的精确控制需要几个月的培训新手同学。

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Reagent/Material</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma - Aldrich</td>
			<td>S7653</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma - Aldrich</td>
			<td>S8875</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma - Aldrich</td>
			<td>P9333</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>Sigma - Aldrich</td>
			<td>G7528</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Sigma - Aldrich</td>
			<td>S9638</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MgSO4 1M</td>
			<td>Sigma - Aldrich</td>
			<td>63126</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma - Aldrich</td>
			<td>C4901</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Carbogen</td>
			<td>Air Liquide (Belgium)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Capillaries</td>
			<td>WPI, Inc. (UK)</td>
			<td>TW150-4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Stimulating Electrodes</td>
			<td>FHC (USA)</td>
			<td>CE2B30</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Surgical tools</td>
			<td>FST (Germany)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Filter paper 84 g/m2</td>
			<td>Sartorius</td>
			<td>FT-3-105-110</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mesh</td>
			<td>Lycra</td>
			<td>15 den</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glue</td>
			<td>UHU</td>
			<td>plus endfest300</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Instrument</td>
		</tr>
		<tr>
			<td>Amplifier</td>
			<td>WPI, Inc. (UK)</td>
			<td>ISO-80</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Interface recording chamber</td>
			<td>FST (Germany)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Peristaltic pumps</td>
			<td>Gilson (USA)</td>
			<td>Minipuls 3</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Temperature controller</td>
			<td>University of Edinburgh</td>
			<td><a href="http://www.etcsystem.com" target="_blank">www.etcsystem.com</a></td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tissue Chopper</td>
			<td>Mcllwain</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Stimulators</td>
			<td>Grass (USA)</td>
			<td>S88X + SIU-V</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Program analysis</td>
			<td>WinLTP</td>
			<td><a href="http://www.winltp.com" target="_blank">www.winltp.com</a></td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Micromanipulators</td>
			<td>Narishige</td>
			<td>MM-3 and MMO-220A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Surgical microscope</td>
			<td>Leica Microsystem</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>A/D converter</td>
			<td>National Instruments</td>
			<td>NIPCI-6229 M-series</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

improved preparation and preservation of hippocampal mouse slices for a very stable and reproducible recording of long-term potentiation

长时程增强（LTP）是一种类型的神经元突触可塑性，其特征在于由突触强度的增加，被认为参与记忆编码。 LTP诱导急性海马切片的CA1区中已被广泛研究。然而，维护阶段这种现象背后的分子机制仍然知之甚少。这可能是部分原因是由于不同的实验室所使用的各种实验条件。事实上，对LTP的维持阶段是强烈地依赖于充氧，温度和湿度等外部参数。它也是依赖于内部参数，如解剖后切片平面和切片活力的方向。 所有这些参数的优化使一个高度可再现性和非常稳定的长时程增强的诱导。这种方法提供了可能的分子机制，进一步探索参与稳定增长海马脑片突触强度。这也凸显了在体外研究的神经生理现象的实验条件的重要性。

此方法已被用来分析性能的持久的成年C57BL/6J小鼠（SAS JANVIER，法国）14在急性海马脑片长时程增强诱导。令人惊讶的是，实验条件的改善已导致了一种新的方式寻找在LTP。我们发现，持久的突触强度的增加并不需要新的蛋白质合成。 在这里，我们将展示，诱导LTP取决于片上的生存能力和兴奋。当夹层的海马速度太慢或太有害，切片兴奋性增加，可观察到多突触反应LTP...

Watch this Scientific Journal Video about Improved Preparation and Preservation of Hippocampal Mouse Slices for a Very Stable and Reproducible Recording of Long-term Potentiation at JoVE.com

Improved Preparation and Preservation of Hippocampal Mouse Slices for a Very Stable and Reproducible Recording of Long-term Potentiation

本文提出了一种完整的方法来准备和保存<em&gt;在体外</em&gt;急性成年小鼠海马脑片。该协议允许非常稳定持久的长时程增强（LTP）的记录超过8小时，95％的成功率。

长时程增强一个非常稳定的和可重复的记录和保存海马鼠标片制备工艺的改进

Long-term potentiation (LTP) is a type of synaptic plasticity characterized by an increase in synaptic strength and believed to be involved in memory ...

improved-preparation-preservation-hippocampal-mouse-slices-for-very

Research

JoVE Journal

Neuroscience

26.6K 次观看.  University of Mons. 本文提出了一种完整的方法来准备和保存在体外急性成年小鼠海马脑片。该协议允许非常稳定持久的长时程增强（LTP）的记录超过8小时，95％的成功率。

视频: Improved Preparation and Preservation of Hippocampal Mouse Slices for a Very Stable and Reproducible Recording of Long-term Potentiation

Preparation of Oligomeric &beta;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). &beta;-amyloid (A&beta;), a peptide produced in high amounts in AD, is known to reduce Long-Term Potentiation (LTP), a cellular correlate of learning and memory. Indeed, LTP impairment caused by A&beta; is a useful experimental paradigm for studying synaptic dysfunctions in AD models and for screening drugs capable of mitigating or reverting such synaptic impairments. Studies have shown that A&beta; produces the LTP disruption preferentially via its oligomeric form. Here we provide a detailed protocol for impairing LTP by perfusion of oligomerized synthetic A&beta;1-42 peptide onto acute hippocampal slices. In this video, we outline a step-by-step procedure for the preparation of oligomeric A&beta;1-42. Then, we follow an individual experiment in which LTP is reduced in hippocampal slices exposed to oligomerized A&beta;1-42 compared to slices in a control experiment where no A&beta;1-42 exposure had occurred.

Preparation of Oligomeric &amp;#946;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

One feature of Alzheimer's Disease is the elevation of A&beta;1-42 peptide. Here we provide a protocol for preparing synthetic A&beta;1-42 oligomers, which impairs hippocampal Long-Term Potentiation, a cellular correlate of memory. This procedure is useful for investigating mechanisms of A&beta;-induced pathology and drug screening.

低聚β-淀粉样蛋白的制备 1-42和突触可塑性损伤诱导海马脑片

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). ...

科研

神经科学

Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The hippocampus can be extracted quickly and easily from rats and mice and slices remain viable for hours in oxygenated artificial cerebrospinal fluid. Moreover, basic electrophysisologic techniques are easily applied to the investigation of synaptic function in hippocampal slices and have provided some of the best biomarkers for cognitive impairments. The hippocampal slice is especially popular for the study of synaptic plasticity mechanisms involved in learning and memory. Changes in the induction of long-term potentiation and depression (LTP and LTD) of synaptic efficacy in hippocampal slices (or lack thereof) are frequently used to describe the neurologic phenotype of cognitively-impaired animals and/or to evaluate the mechanism of action of nootropic compounds. This article outlines the procedures we use for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and Alzheimer's disease (AD)1-3. Use of aged rats and AD model mice can present a unique set of challenges to researchers accustomed to using younger rats and/or mice in their research. Aged rats have thicker skulls and tougher connective tissue than younger rats and mice, which can delay brain extraction and/or dissection and consequently negate or exaggerate real age-differences in synaptic function and plasticity. Aging and amyloid pathology may also exacerbate hippocampal damage sustained during the dissection procedure, again complicating any inferences drawn from physiologic assessment. Here, we discuss the steps taken during the dissection procedure to minimize these problems. Examples of synaptic responses acquired in "healthy" and "unhealthy" slices from rats and mice are provided, as well as representative synaptic plasticity experiments. The possible impact of other methodological factors on synaptic function in these animal models (e.g. recording solution components, stimulation parameters) are also discussed. While the focus of this article is on the use of aged rats and transgenic mice, novices to slice physiology should find enough detail here to get started on their own studies, using a variety of rodent models.

This article outlines procedures for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and age-related neurodegenerative diseases, such as Alzheimer&#x2019;s disease.

突触改变老化过程中淀粉样蛋白病理研究从大鼠和转基因小鼠海马急性片的制备

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

重复性小鼠坐骨神经挤压和再生的后续评估整装肌肉分析

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form a polished and reinforced thinned skull (PoRTS) window in the mouse skull that spans several millimeters in diameter and is stable for months. The skull is thinned to 10 to 15 &mu;m in thickness with a hand held drill to achieve optical clarity, and is then overlaid with cyanoacrylate glue and a cover glass to: 1) provide rigidity, 2) inhibit bone regrowth and 3) reduce light scattering from irregularities on the bone surface. Since the skull is not breached, any inflammation that could affect the process being studied is greatly reduced. Imaging depths of up to 250 &mu;m below the cortical surface can be achieved using two-photon laser scanning microscopy. This window is well suited to study cerebral blood flow and cellular function in both anesthetized and awake preparations. It further offers the opportunity to manipulate cell activity using optogenetics or to disrupt blood flow in targeted vessels by irradiation of circulating photosensitizers.

We present a method to form an imaging window in the mouse skull that spans millimeters and is stable for months without inflammation of the brain. This method is well suited for longitudinal studies of blood flow, cellular dynamics, and cell/vascular structure using two-photon microscopy.

抛光和钢筋，颅骨变薄窗口，为长期的小鼠脑成像

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form ...

Preparation of an Awake Mouse for Recording Neural Responses and Injecting Tracers

It is well known that anesthesia alters neural response properties in various regions of the brain.13. In the auditory system, fundamental response properties of brainstem neurons including threshold, frequency specificity, and inhibitory sidebands are altered in significant ways under anesthesia1-2. These observations prompted physiologists to seek ways to record from single neurons without the contaminating effects of anesthesia. One result was a decerebrate preparation, where the brainstem was completely transected at the level of the midbrain4. The drawbacks of this preparation are a formidable surgery, the elimination of descending projections from the forebrain, and an inability to use sensory stimulation to examine structures above the midbrain. A different strategy has been to implant electrode arrays chronically to record from single neurons and multiunit clusters while the animal is awake and/or behaving5,6. These techniques however are not compatible with injecting tracer dyes after first electrophysiologically characterizing a brain structure. To avoid altering neural response properties with anesthetics while recording electrophysiological response properties from single neurons, we have adapted a head restraint technique long used in bats7-9 to mouse10-12. Using this method, we are able to conduct electrophysiological recordings over several days in the unanesthetized mouse. At the end of the recording sessions, we can then inject a dye to reconstruct electrode positions and recording sites or inject a tracer so that pathways to and from the recording loci can be determined. This method allows for well isolated single neuron recordings over multiple days without the use anesthetics.

Electrophysiological characterization of neuronal responses is important for understanding brain function and for guiding the placement of dyes for pathway tracing. However, many studies are performed in anesthetized animals. To understand brain function without anesthetics, we developed a method to record neuronal response properties and inject dyes in awake mouse.

记录神经反应和注射示踪剂一个清醒小鼠的制备

It is well known that anesthesia alters neural response properties in various regions of the brain.13.  In the auditory system, fundamental response ...

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps shape attention and also protects cells from over-stimulation. Adaptation within the olfactory circuit of C. elegans was first described by Colbert and Bargmann1,2. Here, the authors defined parameters of the olfactory adaptation paradigm, which they used to design a genetic screen to isolate mutants defective in their ability to adapt to volatile odors sensed by the Amphid Wing cells type C (AWC) sensory neurons. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor3 for 30 min they will adapt their responsiveness to the odor and will then ignore the adapting odor in a chemotaxis behavioral assay for ~1 hr. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor for ~1 hr they will then ignore the adapting odor in a chemotaxis behavioral assay for ~3 hr. These two phases of olfactory adaptation in C. elegans were described as short-term olfactory adaptation (induced after 30 min odor exposure), and long-term olfactory adaptation (induced after 60 min odor exposure). Later work from L'Etoile et al.,4 uncovered a Protein Kinase G (PKG) called EGL-4 that is required for both the short-term and long-term olfactory adaptation in AWC neurons. The EGL-4 protein contains a nuclear localization sequence that is necessary for long-term olfactory adaptation responses but dispensable for short-term olfactory adaptation responses in the AWC4. By tagging EGL-4 with a green fluorescent protein, it was possible to visualize the localization of EGL-4 in the AWC during prolonged odor exposure. Using this fully functional GFP-tagged EGL-4 (GFP::EGL-4) molecule we have been able to develop a molecular readout of long-term olfactory adaptation in the AWC5. Using this molecular readout of olfactory adaptation we have been able to perform both forward and reverse genetic screens to identify mutant animals that exhibit defective subcellular localization patterns of GFP::EGL-4 in the AWC6,7. Here we describe: 1) the construction of GFP::EGL-4 expressing animals; 2) the protocol for cultivation of animals for long-term odor-induced nuclear translocation assays; and 3) the scoring of the long-term odor-induced nuclear translocation event and recovery (re-sensitization) from the nuclear GFP::EGL-4 state.

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

Here we describe a molecular readout of long-term olfactory adaptation in Caenorhabditis elegans. The Protein Kinase G, EGL-4, is necessary for stable adaptation responses in the primary sensory neuron pair called AWC. During prolonged odor exposure EGL-4 translocates from the cytosol to nucleus of the AWC.

长期的嗅觉适应性的分子读数<em&gt; C.线虫</em

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

在器官型海马脑片搭配全细胞记录

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

Long-term Time Lapse Imaging of Mouse Cochlear Explants

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building the highly ordered, complex structure of the mammalian cochlea proceeds for around ten days. In order to study changes in gene expression over this period and their response to pharmaceutical or genetic manipulation, long-term imaging is necessary. Previously, live imaging has typically been limited by the viability of explanted tissue in a humidified chamber atop a standard microscope. Difficulty in maintaining optimal conditions for culture growth with regard to humidity and temperature has placed limits on the length of imaging experiments. A microscope integrated into a modified tissue culture incubator provides an excellent environment for long term-live imaging. In this method we demonstrate how to establish embryonic mouse cochlear explants and how to use an incubator microscope to conduct time lapse imaging using both bright field and fluorescent microscopy to examine the behavior of a typical embryonic day (E) 13 cochlear explant and Sox2, a marker of the prosensory cells of the cochlea, over 5 days.

Live imaging of the embryonic mammalian cochlea is challenging because the developmental processes at hand operate on a temporal gradient over ten days. Here we present a method for culturing and then imaging embryonic cochlear explant tissue taken from a fluorescent reporter mouse over five days.

老鼠耳蜗外植体的长期时间推移成像

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building ...

A Simplified Method for Ultra-Low Density, Long-Term Primary Hippocampal Neuron Culture

Culturing primary hippocampal neurons in vitro facilitates mechanistic interrogation of many aspects of neuronal development. Dissociated embryonic hippocampal neurons can often grow successfully on glass coverslips at high density under serum-free conditions, but low density cultures typically require a supply of trophic factors by co-culturing them with a glia feeder layer, preparation of which can be time-consuming and laborious. In addition, the presence of glia may confound interpretation of results and preclude studies on neuron-specific mechanisms. Here, a simplified method is presented for ultra-low density (~2,000 neurons/cm2), long-term (&#62;3 months) primary hippocampal neuron culture that is under serum free conditions and without glia cell support. Low density neurons are grown on poly-D-lysine coated coverslips, and flipped on high density neurons grown in a 24-well plate. Instead of using paraffin dots to create a space between the two neuronal layers, the experimenters can simply etch the plastic bottom of the well, on which the high density neurons reside, to create a microspace conducive to low density neuron growth. The co-culture can be easily maintained for &#62;3 months without significant loss of low density neurons, thus facilitating the morphological and physiological study of these neurons. To illustrate this successful culture condition, data are provided to show profuse synapse formation in low density cells after prolonged culture. This co-culture system also facilitates the survival of sparse individual neurons grown in islands of poly-D-lysine substrates and thus the formation of autaptic connections.

Low density cultures of primary hippocampal neurons usually require glia feeder layer to supply neurotrophic factors and sustain longevity. We describe here a simplified method to culture ultra-low density neurons on glass coverslips in the presence of a high density neuronal feeder layer, which facilitates investigation of specific neuronal-autonomous mechanisms.

的简化方法超低密度，长期主要海马神经元文化

Culturing primary hippocampal neurons in vitro facilitates mechanistic interrogation of many aspects of neuronal development. Dissociated embryonic ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

小容积回收-灌注-浸没式室系统中维持的急性海马切片的突触可塑性记录

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...