We would like to thank the members of the Montgomery and Madison labs for helpful discussion. We acknowledge the funding received from the following sources in this research: NFNZ, AMRF, Marsden Fund, HRC, and NIH.

动物伦理声明： 在这个手稿中描述的协议，遵循由奥克兰大学和斯坦福大学的大学建立了动物护理指引。 P7的幼鼠被迅速砍头处死。海马解剖，然后立即进行，如下所述。 1，海马器官切片文化<ol><li>准备<ol><li>准备夹层中（仅用于解剖的大脑）。结合（在0.85％的NaCl 10,000单位的每一个，），将5ml的HEPES缓冲溶液，加入2ml的1M Tris溶液（pH 7.2）中200毫升最小必需培养基，加入2ml青霉素 - 链霉素溶液，并过滤消毒用0.22微米的过滤器。开展在冰冷的夹层中海马解剖。 </li><li>冷却夹层介质。放置在冷冻机的清扫介质开始的夹层，直到液体是非常冷之前约1小时。不要让大冰CRystals形成。冰储存备用。 </li><li>准备培养基（用于一切，除了海马解剖）。结合百毫升最小必需培养基（1X浓度，液）重量/ Hank氏盐，W / L-谷氨酰胺，2个ml的青霉素 - 链霉素溶液（液，10,000单位的每一个，在0.85％NaCl）中，2.5毫升HEPES 1M的缓冲溶液中加入50ml Hank氏平衡盐溶液，将50ml马血清（定义，热灭活的），并过滤除菌用0.22微米的过滤器。 </li><li>准备培养皿。将1毫升每35毫米培养皿中的培养基，并添加一个膜插入到每道菜。把多达七个的这些菜入1个150毫米的培养皿（以下称为一个“板”）。将板在CO 2培养箱中培养至少一个小时前开始清扫，这样的菜培养基达到适当的温度和pH值。 </li></ol></li><li>海马从幼鼠在出生后第7天的夹层（P7） <ol><li>预消毒紫外光下的所有清扫工具，在手术前。 </li><li>快速断头后，取出大脑并放入冷冻介质在一个盘中，然后将其转移到一片湿润滤纸的清扫。用钝光滑的塑料涂层的微型小铲，露出挑逗海马皮层远离脑。切开穹窿，然后轻轻地锅铲海马下方翻转出来（ 图1A）。 注：成功的切片培养可以使用动物多达10页准备。 </li><li>修剪所述分离海马远离大脑的其余部分。转移到海马用湿润的软画笔含冷冻切除媒体一道新菜（ 如白色貂皮＃4）。 </li><li>清洁脉络丛的解剖中，海马的下侧（ 即平坦的侧面），因为这些海绵，meninge状组织，使其难以分离海马彼此稍后切片。 注：留在原地这些组织，如果他们不能轻轻逗了。 </li><li>尽可能快地执行整个剥离而不损伤海马。 注：小心剥离比快多了一个重要的，只要实验条件冷藏。切三只同时海马。切片健康不受影响，只要从开始的总时间，当镀片进入培养箱是下30分钟。 </li></ol></li><li>切片<ol><li>切片海马横向成使用手动组织切片机400微米的横截面。通过该切片中进行的方法并不重要，只要它是不是太有害的组织，并产生一致的厚度与容易看到层状结构的切片（ 即阿蒙的角是很容易看到，在组织被保留）。 </li><li>躺下的海马组织上的阶段菜刀在＃2滤纸厚度三重顶。海马是完全切片而不个别地除去任何切片（ 即整个海马留在斩波器的阶段，直到所有的削减已经作出，而是像在这一点上切面包; 图1B）。 </li><li>使用软画笔奠定相邻的海马（白色貂皮＃2）转移到海马夹层介质。轻轻地推每个海马到侧打破海马和底层的滤纸之间的粘附力。每卷海马下方刷把它捡起来扫尾阶段。将所有海马进入冷冻夹层介质相同35mm平皿。通过手动搅拌该盘以交替的顺时针方向和逆时针方向运动分离海马组织成单个的片。 </li><li>检查切片解剖显微镜下，并丢弃任何的损坏，小，或不具备清晰可见单元格体层。 注意：它常常是，并不是所有的条带被成功地从他们的兄弟姐妹以这种方式分离的情况。在这种情况下，它们可以通过把它们的边缘，并与细钳提示催促他们分开。 “好”的片，然后转移到含有冷冻夹层介质使用切断和火抛光的巴斯德吸液管（ 图1C-E）的另一个干净的培养皿。 </li></ol></li><li>存储<ol><li>将各个片上的膜过滤器插入。使用巴斯德吸管切断和火抛光，以得到一个较大的孔，单独的切片转移到培养插入。 注：切割和火抛光的吸管是重要的，当创建的孔的大小。过小，因而切片不会轻易离开吸管的膜。过大，过量的液体被放置在膜的表面上沿着与片。最好孔直径通常大约一半的直径ETER移液器的枪管。当切割，该直径可以通过在移液管的锥形切割在适当的地方来选择。 </li><li>切片转移<ol><li>第一填充介质中的吸管，然后吸了吸小力单片。这样可以使切片的移液管的开口部附近，可以防止过多介质排出到插入放置的片。 </li><li>适用于灯泡有轻微的压力，形成一个小吊滴，让片安定成液滴，然后轻触液滴膜，让片“落”到膜上。一般三片被放置到每个膜插入。 注：如果在流体液滴过大，从三个切片的液滴趋向于对膜表面进行合并，并在片将因此所有收集到的膜的中央，使得它更难以给他们后来分离为电生理记录。 </li></ol></li><li>流体祛瘀人<ol><li>吸出多余的培养基从培养板插入在该盘的所有的切片（每个刀片3片=每板21片）的顶部，以使片不浸入或通过夹层介质池所包围。 </li><li>执行此是用未切割的，但火抛光的巴斯德吸液管。允许切片定居，并坚持以膜吸取前一分钟。小心不要吸片成吸管。进行流体去除该先镀的插入，以允许足够的时间片来解决。 </li></ol></li><li>切片存储<ol><li>商店切片文化在5.0％CO 2的培养箱中37℃。观察图1E文化的最终安排。各个切片搁置在培养板上的插入物具有一底部的多孔膜，并在培养皿填充有少量培养基内该插入配合;通过这种方式，该带被不沉浸在我dium，但仅通过该膜在培养板上的插入件的底部可以访问它。 </li><li>可选地，通过去除培养板的插入或通过切割含有一个切片插入膜的部分取出切片可用于研究两种。 </li></ol></li></ol></li><li>保养<ol><li>当天使文化后交换介质的培养皿。将培养板插入转移到含有1ml新鲜培养基是平衡的培养箱中至少在传输之前一小时的新培养皿。 </li><li>使培养后的第三天再以相同的方式改变它的介质。到了第三天，文化转移到孵化器设定在34℃。每星期（每3-4天）改变介质的两倍。 </li><li>识别健康的文化。选择健康的培养成对全细胞记录。 注：健康文化有一个明确的优势和明确的锥体细胞层。文化智慧小时黑暗（可能坏死）区域存在或空泡外观扁平的边框将被拒绝。采用这些标准，通常有三分之二切片文化有足够的健康记录。 </li><li>在规定时间内相当小窗口，利用这些文化。不要用过的纸巾是在两个多星期以来培养的神经元开始显示稍癫痫行为，慢慢地随着时间的推移恶化。没有已知的神经元存活的确切上限，但也可以从在培养正常锥体细胞迟16周记录。 </li></ol></li></ol> 2，配对全细胞记录<ol><li>学联和细胞内的解决方案<ol><li>通过混合（以mM计）120葡糖酸钾，40 HEPES，5的MgCl 2，2 NaATP和0.3 NaGTP（;：290毫渗透摩尔渗透压pH为7.2，用KOH）制备突触前电极溶液。而同一电极的溶液用于突触后神经元，铯葡糖（120毫）通常被用作在突触后电极的主要的盐，加5毫QX314。 注意：这使稳定的电压钳位在正电位记录突触后NMDA受体介导的电流8-10,13。这也防止了突触前神经元的钾引起过度兴奋由突触后记录电极流过千兆欧密封是由之前的内部解决方案。 </li><li>制备突触后电极溶液组成（以mM计）120葡糖酸铯，40 HEPES，5的MgCl 2，5 QX314，2 NaATP和0.3 NaGTP（pH值7.2用CsOH;渗透压：290毫渗透摩尔浓度）。拉玻璃微电极在5-10MΩ电阻和充满过滤后的内部解决方案。 </li><li>制备人工脑脊液（ACSF）组成（以mM计）119氯化钠，氯化钾2.4，1.3 MgSO 4干燥，2.4的CaCl 2，1的Na 2 HPO 4，26.2的NaHCO 3，11葡萄糖，pH为7.4，用95％O 2，5饱和％的CO 2。注：如果AMPAR介导Řesponses需要被封锁检查NMDA受体EPSCS的，包括10微米CNQX或NBQX的学联。 </li></ol></li><li>获得配对全细胞记录<ol><li>为了检验两个独立的神经元之间的突触传递，指定一个神经元的突触前神经元，并持有电流钳诱发动作电位，并启动突触传递。 </li><li>指定所述第二神经元，作为突触后神经元，并根据由所述研究者所需要的信息保持它在电流或电压钳。通过保持突触后神经元的电压钳位得到谷氨酸电流检测用在-65毫伏AMPAR介导EPSCS和NMDA受体介导的EPSCS在30 mV的检测6-16详细检查</li><li>建立配对记录。要建立配对记录，突触前的全细胞记录总是第一个得到允许多个连续的突触后神经元，直到一个是突触connecte获得获得Ð。如果执行的突触可塑性的实验中，它是特别重要的，以先获得突触前神经元的诱导LTP的突触后神经元内必须获得全细胞记录，以防止所需的LTP 8,19胞质因子的洗脱10分钟开始。 </li><li>避免运动引起的细胞损失。执行成对全细胞记录时的一个主要挑战是避免振动和第一全细胞记录，而获得所述第二记录的运动引起的破坏。 <ol><li>获得第一全细胞记录，然后将显微镜透镜为10〜200μm，在x轴上，以使建立的记录位于该区域上的监视器（ 图2A）可见的边缘。 </li><li>提高显微镜的镜头〜5-10毫米，同时确保学联仍然保持与镜片接触。安装在第二电极和引导到流体弯月面（ 图2B </s仲&gt;）。 </li><li>移动电极的xy平面内，直到它是直接的光路下通过透镜，但仍显著大于建立的记录电极。一旦电极在显微镜下可见，确保尖端处于显示器远离所述第一电极的远边缘。 </li><li>移动所述第二电极向下依次与显微镜聚焦到两个电极都在同一焦平面上。重要的是正压的水平是足够强，以避免尖堵塞，但不能太强烈以破坏所述第一全细胞记录。这转化为正压力诱导的运动在从电极2-3的神经元时，它首先进入切片。 </li><li>定位在一个类似的焦平面突触后伙伴到第一突触前记录（ 图2C）。记录一般是获得来自第一R的0-200毫米的神经元配对CA3锥体神经元，第二全细胞记录之间ecording（ 图2C，D）。 注：神经元对之间的突触传递是稳定在长达3-4个小时的时间内使用这些标准的全细胞记录技术时。 </li></ol></li></ol></li><li>突触可塑性：黄飞鸿获得成功的突触连接的锥体细胞对（ 图3A，B），研究突触传递和可塑性的特点。 <ol><li> LTP的诱导。 <ol><li>诱导LTP通过配对突触前动作电位（1赫兹）与突触后去极化到-10到0毫伏，持续1分钟（ 图3C）7-9,12-14。在磨合到突触后神经元的10分钟开始配对。 注：LTP也可诱发与配对的突触前和突触后动作电位在1赫兹为1分钟，用突触后动作电位在电流钳保持两个神经元引起10毫秒以下注入的电流进入突触前神经元8 </s起来&gt;。 </li></ol></li><li>在1赫兹结合突触后神经元的轻微的去极化至-55毫伏5-10分钟（ 图3C）9进一步诱导LTD由低频率刺激（LFS）。 </li></ol></li></ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paired-pulse+facilitation+and+depression+at+unitary+synapses+in+rat+hippocampus:+quantal+fluctuation+affects+subsequent+release.">Paired-pulse facilitation and depression at unitary synapses in rat hippocampus: quantal fluctuation affects subsequent release.</a> Journal of Physiology. 491, 163-176 (1996).</li><li>Debanne, D., G&#228;hwiler, B. H., Thompson, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cooperative+interactions+in+the+induction+of+long-term+potentiation+and+depression+of+synaptic+excitation+between+hippocampal+CA3-CA1+cell+pairs+in+vitro.">Cooperative interactions in the induction of long-term potentiation and depression of synaptic excitation between hippocampal CA3-CA1 cell pairs in vitro.</a> Proceedings of the National Academy of Sciences U S A. 93 (20), 11225-11230 (1996).</li><li>Malinow, R., Tsien, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+enhancement+shown+by+whole+cell+recordings+of+long-term+potentiation+in+hippocampal+slices.">Presynaptic enhancement shown by whole cell recordings of long-term potentiation in hippocampal slices.</a> Nature. 346 (6280), 177-180 (1990).</li><li>DeBello, W. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SNAP-mediated+protein-protein+interactions+essential+for+neurotransmitter+release.">SNAP-mediated protein-protein interactions essential for neurotransmitter release.</a> Nature. 373 (6515), 626-630 (1995).</li><li>Bekkers, J. M., Stevens, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+of+variability+in+quantal+size+in+cultured+hippocampal+neurons+and+hippocampal+slices.">Origin of variability in quantal size in cultured hippocampal neurons and hippocampal slices.</a> Proceedings of the National Academy of Sciences U S A. 87, 5359-5362 (1990).</li><li>Malinow, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmission+between+pairs+of+hippocampal+slice+neurons:+quantal+levels,+oscillations,+and+LTP.">Transmission between pairs of hippocampal slice neurons: quantal levels, oscillations, and LTP.</a> Science. 252, 722-724 (1991).</li><li>Debanne, D., Gu&#233;rineau, N. C., G&#228;hwiler, B. H., Thompson, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiology+and+pharmacology+of+unitary+synaptic+connections+between+pairs+of+cells+in+areas+CA3+and+CA1+of+rat+hippocampal+slice+cultures.">Physiology and pharmacology of unitary synaptic connections between pairs of cells in areas CA3 and CA1 of rat hippocampal slice cultures.</a> Journal of Neurophysiology. 73 (3), 1282-1294 (1995).</li><li>Mitra, A., Blank, M., Madison, D. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmentally+altered+inhibition+in+Ts65Dn,+a+mouse+model+of+Down+syndrome.">Developmentally altered inhibition in Ts65Dn, a mouse model of Down syndrome.</a> Brain Research. 1440, 1-8 (2012).</li><li>Hanson, J. E., Madison, D. 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The authors have nothing to disclose

在这里，我们描述了在海马脑片培养建立成功配对的全细胞记录的要求。成对的记录，也可以在多个制剂中，包括急性切片和分离培养系统26,27进行。虽然这里的重点一直是在较长形式的突触可塑性（即LTP和LTD）的诱导，它强调的是配对的全细胞记录的器官，急性切片是重要的，分离细胞的准备工作提供了重要的见解量子大小，短-term可塑性，突触功能，以及LTP和LTD 2,4,8,26,27。 ...

谷氨酸受体介导的大部分兴奋性突触传递的中枢神经系统突触。的局部存在于突触后膜的棘头离子型谷氨酸受体2亚型主要是N-甲基-D-天冬氨酸（NMDA），α-氨基-3 - 羟基-5 - 甲基异恶唑-4 - 丙酸（AMPA）受体。在静息膜电位，AMPA受体进行最突触后电流的突触传递过程中。在海马中，NMDA受体在触发变化AMPA受体在突触后膜的数量关键作用：通过作为“符合探测器”1来启动改变的突触强度1，NMDA受体参与突触机制这被认为是巩固学习和记忆在亚细胞水平。响应于在与突触前递质释放平行突触后神经元的去极化，钙经由NMDA进入受体启动AMPA受体插入或除去2。这些受体动力学背后突触可塑性：增加突触强度是长时程增强2,3（LTP），而在突触强度的降低是长期抑郁4（LTD）。因此，AMPA受体的运动被认为是负责突触可塑性的表达，而NMDA受体被认为是控制其诱导。 确定突触传递和可塑性背后的确切机制，需要研究突触小种群，最好的单突触。虽然有些突触非常适合于研究在这个水平上， 如对持有5的花萼，对于大多数突触的人口，这是非常困难的，因为突触连接的小型和分散性。两个主要的电生理技术，已经开发了检查单突触连接：首先是最小的刺激，wherê1突触前纤维推测刺激细胞外。第二种技术是成对的记录，其中，来自突触连接的神经元的两个同时全细胞记录的处理。最小的刺激的一个主要优点是，它是快速和相对简单的执行，涉及到细胞外的刺激电极放置到轴突道同时从突触后神经元的记录。使用这种技术时，主要关注的是一个单细胞的可靠的刺激很少能保证审讯后审。 在过去的十五年中，我们经常使用的两个突触连接的锥体神经元6-17配对的全细胞记录。该技术的主要优点是，只有一个突触前神经元持续和可靠地刺激。它还允许不仅电特性，而且药理操纵突触前神经元6,18 &lt;中/ SUP&gt;。然而，突触连接的神经元之间的概率是低的，使得连接对难以得到19。使用器官型脑切片培养物绕过这个障碍如突触连接可以重新建立体外而且所得到的连接的性质是在天然脑组织20的类似。此外，器官型培养物表达的LTP，LTD 7-10,12-15,21和短期突触可塑性的另外的形式，包括双脉冲便利（PPF）和抑郁症（PPD）6,22,23，使可塑性的机制来要研究对神经元。在这里，我们描述了参与成功地实现这个体外系统配对录音的详细方法。这个信息可以很容易地适用于其它的实验系统，包括急性切片和其他脑区域。

<table border="0" cellpadding="0" cellspacing="0" width="686">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:256px;">Organotypic cultures</td>
			<td style="width:111px;"></td>
			<td style="width:255px;"></td>
			<td style="width:64px;">Paired recordings</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Minimum Essential Medium</td>
			<td></td>
			<td></td>
			<td>Stable motorized micromanipulators&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin solution&#160;</td>
			<td></td>
			<td></td>
			<td>Shallow tissue bath</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES buffer solution</td>
			<td></td>
			<td></td>
			<td>DIC camera</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1M Tris stock solution&#160;</td>
			<td></td>
			<td></td>
			<td>Amplifier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hank&#8217;s Balanced Salt Solution</td>
			<td></td>
			<td></td>
			<td>Computer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Horse Serum&#160;</td>
			<td></td>
			<td></td>
			<td>Vibration isolation table</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">plastic-coated miniature spatulas&#160;</td>
			<td></td>
			<td></td>
			<td>Upright microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">soft paintbrush&#160;</td>
			<td></td>
			<td></td>
			<td>Data acquistion and analysis software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">manual tissue chopper</td>
			<td></td>
			<td></td>
			<td>Electrode puller</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">#2 filter paper</td>
			<td></td>
			<td></td>
			<td>Faraday cage</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">#5&#160; forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Membrane inserts</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">CO2 incubator&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissection hood</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Class II hood</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

突触连接是通过刺激突触前神经元被通过记录电极传递去极化电流脉冲（通常是20〜50 pA的20毫秒），以触发一个动作电位明显。突触后电流迹线，然后检查了突触EPSC的存在突触前动作电位（ 图3A）的峰值后诱发的短（&lt;5毫秒）和一致的延迟。在大多数实验中多个突触后神经元进行测试可以得到突触连接的一对前。总体而言，突触前CA3细胞〜1/3的monosynaptically耦合到突触后CA3细胞通过?...

Watch this Scientific Journal Video about Paired Whole Cell Recordings in Organotypic Hippocampal Slices at JoVE.com

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

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 ...

paired-whole-cell-recordings-in-organotypic-hippocampal-slices

Research

JoVE Journal

Neuroscience

14.6K 次观看.  University of Auckland. 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.

视频: Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle. This is a direct consequence of the accessibility to both cell types and ability to visually distinguish the single segmental CaP motor-neuron on the basis of morphology and location. This video demonstrates the microscopic methods used to identify a CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type. Identification of the CaP motor-neuron type is confirmed by either dye filling or by the biophysical features such as action potential waveform and cell input resistance. Motor-neuron recordings routinely last for one hour permitting long-term recordings from multiple different target muscle cells. Control over the motor-neuron firing pattern enables measurements of the frequency-dependence of synaptic transmission at the neuromuscular junction. Owing to a large quantal size and the low noise provided by whole cell voltage clamp, all of the unitary events can be resolved in muscle. This feature permits study of basic synaptic properties such as release properties, vesicle recycling, as well as synaptic depression and facilitation. The advantages offered by this in vivo preparation eclipse previous neuromuscular model systems studied wherein the motor-neurons are usually stimulated by extracellular electrodes and the muscles are too large for whole cell patch clamp. The zebrafish preparation is amenable to combining electrophysiological analysis with a wide range of approaches including transgenic lines, morpholino knockdown, pharmacological intervention and in vivo imaging. These approaches, coupled with the growing number of neuromuscular disease models provided by mutant lines of zebrafish, open the door for new understanding of human neuromuscular disorders.

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target skeletal muscle. This video demonstrates the microscopic methods used to identify a segmental CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type.

配对补丁钳从电机神经元和斑马鱼的目标骨骼肌

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle.  ...

科研

神经科学

Organotypic Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

器官型海马切片培养

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

Whole Cell Recording from an Organotypic Slice Preparation of Neocortex

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex. Because of the lack of specific pharmacological agents for these channels, we have taken a genetic approach to manipulating channel expression. We use an organotypic culture preparation (16) in order to maintain cell morphology and the laminar pattern of cortex. We typically isolate acute neocortical slices at postnatal days 8-10 and maintain the slices in culture for 3-7 days. This allows us to study neurons at a similar age to those in our work with acute slices and minimizes the development of exuberant excitatory connections in the slice. We record from visually-identified pyramidal neurons in layers II/III or V using infrared illumination (IR-) and differential interference contrast microscopy (DIC) with whole cell patch clamp in current- or voltage-clamp. We use biolistic (Gene gun) transfection of wild type or mutant potassium channel DNA to manipulate expression of the channels to study their function. The transfected cells are easily identified by epifluorescence microscopy after co-transfection with cDNA for green fluorescent protein (GFP). We compare recordings of transfected cells to adjacent, untransfected neurons in the same layer from the same slice.

This is a protocol to prepare and maintain a neocortical slice preparation in organotypic culture for the purpose of making electrical recordings from pyramidal neurons.

从大脑皮层的器官型切片制备全细胞记录

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex.  Because of the ...

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

The cortex is spontaneously active, even in the absence of any particular input or motor output. During development, this activity is important for the migration and differentiation of cortex cell types and the formation of neuronal connections1. In the mature animal, ongoing activity reflects the past and the present state of an animal into which sensory stimuli are seamlessly integrated to compute future actions. Thus, a clear understanding of the organization of ongoing i.e. spontaneous activity is a prerequisite to understand cortex function. 
Numerous recording techniques revealed that ongoing activity in cortex is comprised of many neurons whose individual activities transiently sum to larger events that can be detected in the local field potential (LFP) with extracellular microelectrodes, or in the electroencephalogram (EEG), the magnetoencephalogram (MEG), and the BOLD signal from functional magnetic resonance imaging (fMRI). The LFP is currently the method of choice when studying neuronal population activity with high temporal and spatial resolution at the mesoscopic scale (several thousands of neurons). At the extracellular microelectrode, locally synchronized activities of spatially neighbored neurons result in rapid deflections in the LFP up to several hundreds of microvolts. When using an array of microelectrodes, the organizations of such deflections can be conveniently monitored in space and time. 
Neuronal avalanches describe the scale-invariant spatiotemporal organization of ongoing neuronal activity in the brain2,3. They are specific to the superficial layers of cortex as established in vitro4,5, in vivo in the anesthetized rat 6, and in the awake monkey7. Importantly, both theoretical and empirical studies2,8-10 suggest that neuronal avalanches indicate an exquisitely balanced critical state dynamics of cortex that optimizes information transfer and information processing.
In order to study the mechanisms of neuronal avalanche development, maintenance, and regulation, in vitro preparations are highly beneficial, as they allow for stable recordings of avalanche activity under precisely controlled conditions. The current protocol describes how to study neuronal avalanches in vitro by taking advantage of superficial layer development in organotypic cortex cultures, i.e. slice cultures, grown on planar, integrated microelectrode arrays (MEA; see also 11-14).

A robust way to study neuronal avalanches, i.e. scale-invariant spatio-temporal activity bursts, indicative of critical state dynamics in cortex. Avalanches emerge spontaneously in developing superficial layers of cultured cortex which allows for long-term measurements of the activity with planar integrated multi-electrode arrays (MEA) under precisely controlled conditions.

器官型培养的神经元雪崩多电极阵列记录

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

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

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

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

双突触诱发的星形胶质细胞和神经元的反应，在急性海马脑片电生理记录

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

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons.

Cortical networks are controlled by a small, but diverse set of inhibitory interneurons. Functional investigation of interneurons therefore requires targeted recording and rigorous identification. Described here is a combined approach involving whole-cell recordings from single or synaptically-coupled pairs of neurons with intracellular labeling, post-hoc morphological and immunocytochemical analysis.

从Morphologically-和确定的Neurochemically海马的interneurons全细胞膜片钳记录

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

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

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

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

神经微电路急性脑片电生理和形态特征采用配对膜片钳记录

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

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices,&#160;a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular,&#160;when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals.

This protocol describes basic procedural steps for performing whole-cell patch-clamp recordings. This technique allows the study of&#160;the electrical behavior of neurons, and when performed in brain slices, allows the assessment of various neuronal functions from neurons that are still integrated in relatively well preserved brain circuits.

在脑片全细胞膜片钳记录

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

器官海马切片培养模型研究肿瘤细胞的神经保护和侵袭性

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...

Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins

Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate electrical processes in target cells. In this context, ion selectivity of a channelrhodopsin is of particular importance. This article describes the investigation of chloride selectivity for a recently identified anion-conducting channelrhodopsin of Proteomonas sulcata via electrophysiological patch-clamp recordings on HEK293 cells. The experimental procedure for measuring light-gated photocurrents demands a fast switchable &#8211; ideally monochromatic &#8211; light source coupled into the microscope of an otherwise conventional patch-clamp setup. Preparative procedures prior to the experiment are outlined involving preparation of buffered solutions, considerations on liquid junction potentials, seeding and transfection of cells, and pulling of patch pipettes. The actual recording of current-voltage relations to determine the reversal potentials for different chloride concentrations takes place 24 h to 48 h after transfection. Finally, electrophysiological data are analyzed with respect to theoretical considerations of chloride conduction.

This article describes how the ion selectivity of channelrhodopsin is determined with electrophysiological whole-cell patch-clamp recordings using HEK293 cells. Here, the experimental procedure for investigating chloride selectivity of an anion-selective channelrhodopsin is demonstrated. However, the procedure is transferable to other channelrhodopsins of distinct selectivity.