Name: recording synaptic plasticity in acute hippocampal slices maintained in a small-volume recycling-, perfusion-, and submersion-type chamber system
Uploaded: 2018-01-01T13:00:00.000+00:00
Duration: 9 min 51 s
Description: Watch this Scientific Journal Video about Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System at JoVE.com

. 进行、分析、设计了实验并写了手稿。D.X. 和中共协助图准备和进行实验。这项工作得到了自然科学基金 (31320103906) 和111项目 (B16013) 对肺结核

这些动物是按照中国上海复旦大学脑科学研究所和医学神经生物学国家重点实验室的既定动物保育标准和程序进行维护的。
1. 溶液制备
注意: 请参见材料表。
<ol>
	<li>准备切片缓冲区 (修改后的 Gey 的解决方案):92 毫米氯化钠, 2.5 毫米氯化钾, 1.25 毫米 NaH2PO4, 30 mm NaHCO3, 25 mm 葡萄糖, 20 mm HEPES, 3 mm Na+-丙酮酸, 10 mm MgSO4, 0.5 mm CaCl2。</li>
	<li>将 pH 值调整到 7.6, 使用1米氢氧化钠而不 carbogenation。</li>
	<li>将缓冲区存储在4° c。 注: 滴定澄清混浊液体。渗透是 305-310 mOsm。卡波金21包含5% 的二氧化碳和95% 的氧气。</li>
	<li>通过稀释不含碳酸氢盐和葡萄糖的 aCSF 的10x 储存液来制备 aCSF, 给出最后的成分: 124 毫米氯化钠, 2.5 毫米氯化钾, 2.5 mm CaCl2, 2 mm 氯化镁2, 和 1.25 mm 的2PO4。</li>
	<li>添加碳酸氢钠和葡萄糖达到10毫米葡萄糖和 26 mm NaHCO3。</li>
	<li>Carbogenate 的 aCSF 通过一个穿孔的硅管, 并在添加 NaHCO3后立即阻止其结束。</li>
	<li>测量 ph 值而 carbogenating (ph 值 7.3-7.4)。 注意: 通过更改 NaHCO3的数量, 可以稍微调整缓冲区容量。由于所产生的 ph 值是温度依赖性的, 因此必须在工作温度 (例如, 30 ° c) 时检查所产生的 ph 值而 carbogenating。</li>
</ol>
2. 急性海马切片的制备
注意: 请参见材料表。
<ol>
	<li>将切片保持网格放入一个用于急性脑切片的孵化室中, 用 aCSF 填充腔, carbogenate (4-6 升/小时), 在28-30 ° c 时至少为 30 min22 。</li>
	<li>把手术器械冷却到2-4 ° c。</li>
	<li>放置一个玻璃烧杯充满冷和 carbogenated 切片缓冲 (2-4 ° c), 保持在一个冰/水混合物, 附近的 vibratome。</li>
	<li>麻醉鼠或小鼠, 直到角膜反射消失 (30-50 秒) 使用异氟醚, 采取500µL 到2升玻璃罐或12.5 µL 到50毫升管。</li>
	<li>使用在《马修斯et al.的出版物中详细描述和可视化的方法隔离大脑(2011)23和远et al.(2014)24。</li>
	<li>在解剖小脑和分离半球之前, 将大脑降温至2-4 ° c, ice-cold 切片缓冲 (至少2分钟)。</li>
	<li>使用冷手术刀片解剖在每个半球的背缘25的一个70角度的背部皮质块, 从腹侧和海马的中间部分获得横向切片, 如图 1C所示和E。</li>
	<li>用一个冷的牙科水泥刮刀, 转移一个半球到一块滤纸, 以简要干燥创建的表面 (1-2 s)。</li>
	<li>在 ice-cold 切片平台上用快作用的胶粘胶将两个半球用新的切割表面装入;让半球尾端面对刀片式服务器 (图 1D)。</li>
	<li>将切片平台放入 vibratome 的冷却室中, 并将该腔室与切片缓冲 (2-4 ° c) 填充。Carbogenate 使用穿孔硅管。</li>
	<li>使用适当的 vibratome 设置 (例如,刀片前进速度: 1.2 毫米/秒, 刀片振幅: 1 mm) 切割350µm 切片。</li>
	<li>用两个注射套管 (#62; 28 克) 将海马的形成从 subiculum 和嗅皮层分离出来, 作为剪刀。</li>
	<li>使用巴斯德吸管从先前准备的孵化室的切片保持网格中除去气泡。</li>
	<li>将具有一定透明度的切片从孵化室的网格上的切片平台转移到 pyramidale 层。避免任何折叠, 伸展, 重叠, 和浮动的吸 aCSF 和切片到一个大嘴巴斯德吸管。 注: 整个过程 (步骤 2.5-2.14) 可以采取5-10 分钟;因此, 在4° c 的情况下, 检查缓冲液的温度以保持它的时间是很重要的。</li>
	<li>孵育在30° c 的切片在孵化室至少 1.5-2 小时, 并提供卡波金在4-6 升/小时。 注意: 调整卡波金的流速以在孵化器中循环缓冲, 但防止切片漂浮。</li>
</ol>
3. Carbogenation 对小型水库 aCSF 循环的改造
<ol>
	<li>将3路管道连接器添加到回收系统的流出 (图 2B)。</li>
	<li>将3路管接头的中间臂与卡波金的电源管连接起来, 并使用气流指示器 (3-4 升/小时、图 2D和图 4C) 调节流量。</li>
	<li>添加第二个3路管接头到回收系统的流入。将中间臂与内圈加热器的管子相连, 将上臂与一根薄薄的硅管 (10 厘米长) 和下臂连接到流入管 (低通过滤器;图 4C)。</li>
	<li>将管机放在一个薄的硅管上 (外径: 2 毫米), 在该管的位置上, 由蠕动泵产生的 aCSF 在切片室中的脉动最小。 注: 流出-carbogenation (流出-碳水化合物) 的改性降低了 carbogenation 水平对 aCSF 储层容积、aCSF 回收率和小 aCSF 油藏内相对管位的敏感性 (#60; 50 毫升;图 3)。对于低通滤波器, 在稀薄的硅管中一定量的空气减少了 aCSF 流的脉动;因此, 该管应填补大部分与空气。</li>
</ol>
4。在浸没的切片室中记录突触反应
注意: 请参见材料表。
<ol>
	<li>预热 aCSF 溶液在水浴到大约28-30 ° c (这将防止气泡形成在记录室)。</li>
	<li>启动回收系统 (4-5 毫升/分钟), 并激活内联加热器, 平衡系统至少1小时。</li>
	<li>打一小块镜片清洁纸和一个尼龙网格固定在 U 形的铂丝在浸没的切片室几分钟 (图 4)。</li>
	<li>取下 U 形铂丝。</li>
	<li>关闭回收, 并在镜头清洁纸上放置一个切片。</li>
	<li>立即将切片保持网格放在切片的顶部, 打开泵, 让切片平衡30分钟, 而不将目标浸入 aCSF。</li>
	<li>填充硼硅酸盐微 (即,记录电极) 与 aCSF (尖端电阻: 1-2 ω, 填充1/3 与 aCSF), 并将其装入吸管支架。</li>
	<li>通过将金属刺激电极放入 NaHCO3溶液 (和 #62; 40 mM) 来检查其绝缘性。将电极连接到直流电压发生器的负极 (1-2 V, 正极: 银或铂线), 并观察在解剖显微镜 (和 #62; 60x 放大倍率) 下, 在尖端形成气泡。</li>
	<li>将经测试的环氧绝缘钨刺激电极放在机械手支架和切片室中的参考线上。</li>
	<li>将第二个参考电极添加到腔室, 并将其连接到记录电极的 headstage 的参考插座 (图 4a)。</li>
	<li>在放大器控制软件, 点击 "零钳模式" 框, 并通过双击 "输出增益列表" 框进行。选择增益 "100", 双击 "高通滤波器列表框 (贝塞尔)", 选择 "0.1 Hz", 然后双击 "低通滤波器列表框 (AC)" 以选择 "3 赫"。</li>
	<li>在录制软件中, 单击 "获取 |开放的协议。选择一个协议, 允许在10-20 赫50-100 毫秒的场景刺激和放大电位的数字化的设置, 并自动触发了一个在一幕式录音开始后, 一个激励隔离10毫秒。</li>
	<li>将刺激和记录电极与地层 pyramidale 平行, 例如图 4B和图 5)。</li>
	<li>点击 "获取 |编辑协议 ", 然后选择" 触发器 "选项卡 (在弹出窗口中)。点击 "触发源" 框, 选择 "空格键" 作为触发源。单击 "确定" 按钮。单击 "记录" 按钮开始获取, 并注意带有触发器按钮的弹出窗口。</li>
	<li>在刺激隔离软件中, 点击 "电压控制" 框, 进入 "0"。单击 "下载" 按钮。单击 "录制软件" 窗口并按空格键。重复此循环, 按顺序输入值从1到 8, 例如, 在 "电压控制" 框中的 1 mV 步骤。</li>
	<li>将激发强度与 fEPSP 斜率值进行关联, 并确定获得40% 的 fEPSP 斜率最大的激发强度。单击 "电压控制箱" 并输入确定的值。单击 "下载" 按钮。 注: 刺激隔离器用于将短电压或电流脉冲应用于脑组织。双相脉冲可以有100µs 每个阶段。</li>
	<li>在录制软件中, 单击 "停止" 按钮然后 "获取" 菜单。单击 "编辑协议", 然后在弹出窗口中选择 "触发器" 选项卡。单击 "触发器源" 框, 然后选择 "内部计时器" 作为触发器源。单击 "确定" 按钮。例如, 单击 "记录" 按钮, 每个三十年代开始自动记录字段电位。30-60 分钟后单击 "停止" 按钮。</li>
	<li>点击 "获取" 菜单, 点击 "打开协议", 选择所需的突触可塑性感应协议, 然后点击 "OK" 按钮。点击 "记录" 按钮, 自动激活高频刺激和录音。单击 "停止" 按钮。 注: 在与突触可塑性相关的研究中, 在长时间的基线记录之后, 应用一种标准的归纳范式来长期增强或长期抑郁。在图 7和图 8中描述了由此产生的突触传输调制的典型示例。</li>
	<li>在录制软件中, 单击 "获取 |打开协议 "并选择与步骤4.12 中相同的协议。单击 "确定" 按钮, 然后单击 "录制"。例如, 保持自动运行2-4 小时的现场潜在录音。单击 "停止" 按钮以终止录制。</li>
	<li>在药物研究的情况下, 如果需要永久性复合管理 (图 6), 直接将化合物应用于 aCSF 油藏。</li>
</ol>
5. 清理设置和提示
注: 请参阅下面的一般提示。
<ol>
	<li>每周通过回收 10% H2O2来清洁系统, 不超过20分钟, 然后用去离子 H2o 进行清洗。 注: 不推荐使用乙醇清洗。同样不建议在 H2O2中浸入参考线和目标。</li>
	<li>在物镜或室周围的表面上用0.1 的 HCl 和水除去沉淀的盐。</li>
	<li>在蒸馏水或0.1 的 HCl 中清洗卡波金起泡。</li>
	<li>将切片孵化室浸泡在10% 小时2O2中, 每周5分钟, 然后用去离子 H2O 彻底冲洗孵化室。</li>
	<li>在金属刺激电极的情况下, 每次实验后用去离子水冲洗刺激电极尖端, 用乙醇浸泡的棉球轻轻擦拭刺激电极尖端, 除去残留的组织。</li>
	<li>通过对砂纸进行仔细的滑动, 去除尖端的绝缘层, 从而降低商业金属刺激电极的电阻。</li>
	<li>通过用聚四氟乙烯管取代硅管, 减少从 aCSF 中回收卡波金的损失。</li>
	<li>将记录电极和参考电极的银线保持在漂白剂中数天, 以形成氯化在金属丝表面上。</li>
</ol>

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L., 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=Synaptic+tagging+and+capture:+differential+role+of+distinct+calcium/calmodulin+kinases+in+protein+synthesis-dependent+long-term+potentiation.">Synaptic tagging and capture: differential role of distinct calcium/calmodulin kinases in protein synthesis-dependent long-term potentiation.</a> J Neurosci. 30 (14), 4981-4989 (2010).</li><li>Stopps, 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=Design+and+application+of+a+novel+brain+slice+system+that+permits+independent+electrophysiological+recordings+from+multiple+slices.">Design and application of a novel brain slice system that permits independent electrophysiological recordings from multiple slices.</a> J Neurosci Methods. 132 (2), 137-148 (2004).</li><li>Behnisch, T., Matsushita, S., Knopfel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+gene+expression+during+long-term+potentiation.">Imaging of gene expression during long-term potentiation.</a> Neuroreport. 15 (13), 2039-2043 (2004).</li><li>Karpova, A., 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=Encoding+and+transducing+the+synaptic+or+extrasynaptic+origin+of+NMDA+receptor+signals+to+the+nucleus.">Encoding and transducing the synaptic or extrasynaptic origin of NMDA receptor signals to the nucleus.</a> Cell. 152 (5), 1119-1133 (2013).</li><li>Karpova, A., Mikhaylova, M., Thomas, U., Knopfel, T., Behnisch, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+protein+synthesis+and+degradation+in+long-term+potentiation+of+Schaffer+collateral+CA1+synapses.">Involvement of protein synthesis and degradation in long-term potentiation of Schaffer collateral CA1 synapses.</a> J Neurosci. 26 (18), 4949-4955 (2006).</li><li>Weng, W., Chen, Y., Wang, M., Zhuang, Y., Behnisch, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potentiation+of+Schaffer-Collateral+CA1+Synaptic+Transmission+by+eEF2K+and+p38+MAPK+Mediated+Mechanisms.">Potentiation of Schaffer-Collateral CA1 Synaptic Transmission by eEF2K and p38 MAPK Mediated Mechanisms.</a> Front Cell Neurosci. 10 (247), (2016).</li><li>Meduna, L. J., Jackman, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbon+dioxide+inhalation+therapy.">Carbon dioxide inhalation therapy.</a> Res Publ Assoc Res Nerv Ment Dis. 31, 280-286 (1953).</li><li>Edwards, F. A., Konnerth, A., Sakmann, B., Takahashi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+thin+slice+preparation+for+patch+clamp+recordings+from+neurones+of+the+mammalian+central+nervous+system.">A thin slice preparation for patch clamp recordings from neurones of the mammalian central nervous system.</a> Pflugers Arch. 414 (5), 600-612 (1989).</li><li>Mathis, D. M., Furman, J. L., Norris, C. 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(90), e51310 (2014).</li><li>Leutgeb, J. K., Frey, J. U., Behnisch, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LTP+in+cultured+hippocampal-entorhinal+cortex+slices+from+young+adult+(P25-30)+rats.">LTP in cultured hippocampal-entorhinal cortex slices from young adult (P25-30) rats.</a> J Neurosci Meth. 130 (1), 19-32 (2003).</li><li>Kloosterman, F., Peloquin, P., Leung, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apical+and+basal+orthodromic+population+spikes+in+hippocampal+CA1+in+vivo+show+different+origins+and+patterns+of+propagation.">Apical and basal orthodromic population spikes in hippocampal CA1 in vivo show different origins and patterns of propagation.</a> J Neurophysiol. 86 (5), 2435-2444 (2001).</li><li>Thiemann, W., Malisch, R., Reymann, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+microcirculation+chamber+for+inexpensive+long-term+investigations+of+nervous+tissue+in+vitro.">A new microcirculation chamber for inexpensive long-term investigations of nervous tissue in vitro.</a> Brain Res Bull. 17 (1), 1-4 (1986).</li><li>Shetty, M. S., 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=Investigation+of+Synaptic+Tagging/Capture+and+Cross-capture+using+Acute+Hippocampal+Slices+from+Rodents.">Investigation of Synaptic Tagging/Capture and Cross-capture using Acute Hippocampal Slices from Rodents.</a> J Vis Exp. (103), (2015).</li><li>Du, H., Lin, J., Zuercher, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Higher+efficiency+of+CO2+injection+into+seawater+by+a+venturi+than+a+conventional+diffuser+system.">Higher efficiency of CO2 injection into seawater by a venturi than a conventional diffuser system.</a> Bioresour Technol. 107, 131-134 (2012).</li><li>Weinman, J., Mahler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Analysis+of+Electrical+Properties+of+Metal+Electrodes.">An Analysis of Electrical Properties of Metal Electrodes.</a> Med Electron Biol Eng. 2, 299-310 (1964).</li><li>Fanselow, M. S., Dong, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+the+dorsal+and+ventral+hippocampus+functionally+distinct+structures.">Are the dorsal and ventral hippocampus functionally distinct structures.</a> Neuron. 65 (1), 7-19 (2010).</li><li>Wang, 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=Translation+of+BDNF-gene+transcripts+with+short+3'+UTR+in+hippocampal+CA1+neurons+improves+memory+formation+and+enhances+synaptic+plasticity-relevant+signaling+pathways.">Translation of BDNF-gene transcripts with short 3' UTR in hippocampal CA1 neurons improves memory formation and enhances synaptic plasticity-relevant signaling pathways.</a> Neurobiol Learn Mem. , (2016).</li></ol>

提交人声明, 这项研究是在没有任何商业或财务关系的情况下进行的, 可被理解为潜在的利益冲突。

虽然界面切片室表现出更强大的突触响应25,26,27,28, 淹没腔室为膜片钳记录和荧光提供了额外的便利成像.因此, 我们已经描述了几个方面的现场电位记录的急性海马切片使用商业浸没切片室, 可以很容易地扩展到成像的荧光探针在神经元17,18, <sup class=...

在 1951年, 第一次报告的急性脑切片实验进行了1。在 1971年, 在成功的体外录音从梨皮层2,3和发现海马神经元是相互连接横向沿 septotemporal 轴海马4, 其中一个第一个体外海马神经元活动的记录实现了5。神经或 neurostructural 参数的相似性神经元在体内和体外条件仍然是一些辩论的主题6, 但在 1975年, Schwartzkroin7表示, 基神经元的性质是保持在体外和高频刺激 (即,后背) 的传入在海马形成诱导一个长期的促进突触潜能8。神经元活动的电生理记录在体外极大地扩展了活动依赖性突触可塑性的细胞机制的研究910在1973年被极乐发现et al.11在体内实验中用家兔。
研究神经元活动或信号通路的脑切片, 特别是在急性海马切片, 现在是一个标准的工具。然而, 令人惊讶的是,在试管中的实验还没有标准化, 这证明了在急性海马切片的制备和维持方面仍然存在着多种方法。里德et al.(1988)12回顾了在不同类型的切片室中维持急性脑切片的方法学挑战, 以及沐浴培养基、pH 值、温度和氧水平的选择。由于体外切片记录设置的 custom-made 元素, 这些参数在记录室中仍然难以控制。可以找到的出版物, 可能有助于克服一些方法上的挑战, 并描述了新类型的淹没切片室, 如间质 3D microperfusion 系统13, 一个室的层流和氧气增强提供14, 一个具有计算机温度控制的系统15, 以及一个多记录系统16。由于这些房间不易建造, 大多数科学家依赖于商业上可用的切片室。这些室可以安装在显微镜系统, 从而允许的电生理和荧光成像的结合17,18,19。由于这些腔室保持脑切片淹没在人工脑脊液 (aCSF), 高流速的缓冲液的解决方案需要保持, 增加了药物应用的费用。为此, 我们已经采用了一个循环再灌注系统与流出-carbogenation, 提供足够的稳定性, 以长期记录的现场电位在一个浸没的切片室使用相对较小的 aCSF 体积。此外, 我们总结了如何使用这个实验 carbogenation/灌注系统影响的结果, 活动相关的突触可塑性10和如何抑制真核伸长 factor-2 激酶 (eEF2K) 调节突触传输20。

<table><tbody><tr><td>&lt;strong&gt;Reagents required&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>NaCl</td><td>Sinopharm Chemical Reagent, China</td><td>10019318</td><td></td></tr><tr><td>KCl</td><td>Sinopharm Chemical Reagent, China</td><td>10016318</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sinopharm Chemical Reagent, China</td><td>10017618</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sinopharm Chemical Reagent, China</td><td>10012818</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sinopharm Chemical Reagent, China</td><td>10005861</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sinopharm Chemical Reagent, China</td><td>10018960</td><td></td></tr><tr><td>Glucose</td><td>Sinopharm Chemical Reagent, China</td><td>10010518</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sinopharm Chemical Reagent, China</td><td>20040718</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Sigma</td><td>A4043</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sinopharm Chemical Reagent, China</td><td>20025118</td><td></td></tr><tr><td>NaOH</td><td>Sinopharm Chemical Reagent, China</td><td>10019718</td><td></td></tr><tr><td>&lt;strong&gt;Tools and materials for dissection&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Decapitators</td><td>Harvard apparatus</td><td>55-0012</td><td>for rat decapitation</td></tr><tr><td>Bandage Scissors</td><td>SCHREIBER</td><td>12-4227</td><td>for mouse decapitation</td></tr><tr><td>double-edge blade</td><td>Flying Eagle, China</td><td>74-C</td><td></td></tr><tr><td>IRIS Scissors</td><td>RWD, China</td><td>S12003-09</td><td></td></tr><tr><td>Bone Rongeurs</td><td>RWD, China</td><td>S22002-14</td><td></td></tr><tr><td>Spoon</td><td>Hammacher&amp;nbsp;</td><td>HSN 152-13</td><td></td></tr><tr><td>dental cement spatula</td><td>Hammacher&amp;nbsp;</td><td>HSN 016-15</td><td></td></tr><tr><td>dental double end excavator</td><td>Blacksmith Surgical, USA</td><td>BS-415-017</td><td></td></tr><tr><td>Vibrating Microtome</td><td>Leica, Germany</td><td>VT1200S</td><td></td></tr><tr><td>surgical blade&amp;nbsp;</td><td>RWD, China</td><td>S31023-02</td><td></td></tr><tr><td>surgical holder</td><td>RWD, China</td><td>S32007-14</td><td></td></tr><tr><td>&lt;strong&gt;Electrophysiology equipment and materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Vertical Pipette Puller</td><td>Narishige, Japan</td><td>PC-10</td><td></td></tr><tr><td>Vibration isolation table</td><td>Meirits, Japan</td><td>ADZ-A0806</td><td></td></tr><tr><td>submerged type recording chamber</td><td>Warner Instruments</td><td>RC-26GLP</td><td></td></tr><tr><td>4 Axis Micromanipulator</td><td>Sutter, USA</td><td>MP-285, MP-225</td><td></td></tr><tr><td>Platinum Wire</td><td>World Precision Instruments</td><td>PTP406</td><td></td></tr><tr><td>Amplifier</td><td>Molecular Devices, USA</td><td>Multiclamp 700B</td><td></td></tr><tr><td>Data Acquisition System</td><td>Molecular Devices, USA</td><td>Digidata 1440A</td><td></td></tr><tr><td>Anaysis software</td><td>Molecular Devices, USA</td><td>Clampex 10.2</td><td></td></tr><tr><td>Fluorescence Microscope</td><td>Nikon, Japan</td><td>FN1</td><td></td></tr><tr><td>LED light source</td><td>Lumen Dynamics Group, Canada</td><td>X-cite 120LED</td><td></td></tr><tr><td>micropipettes</td><td>Harvard apparatus</td><td>GC150TF</td><td>extracelluar recording</td></tr><tr><td>borosilicate micropipettes</td><td>Sutter, USA</td><td>BF150-86</td><td>patch clamp</td></tr><tr><td>tungsten electrode</td><td>A-M Systems, USA</td><td>575500</td><td></td></tr><tr><td>peristaltic pump</td><td>Longer, China</td><td>BT00-300T</td><td></td></tr><tr><td>tubes for peristaltic pump</td><td>ISMATEC, Wertheim, Germany</td><td>SC0309</td><td>1x inflow, ID: 1.02 mm</td></tr><tr><td>tubes for peristaltic pump</td><td>ISMATEC, Wertheim, Germany</td><td>SC0319</td><td>2x tubes for outflow, ID: 2.79 mm</td></tr><tr><td>CCD camera</td><td>PCO, Germany</td><td>pco.edge sCMOS</td><td></td></tr><tr><td>lens cleaning paper</td><td>Kodak</td><td></td><td></td></tr><tr><td>50 mL conical centrifuge tube</td><td>Thermo scientific</td><td>339652</td><td></td></tr><tr><td>Prechamber</td><td>Warner Instruments</td><td>BSC-PC</td><td></td></tr><tr><td>Inline heater</td><td>Warner Instruments</td><td>SF-28</td><td></td></tr><tr><td>Temperature Controller</td><td>Warner Instruments</td><td>TC-324B</td><td></td></tr></tbody></table>

recording synaptic plasticity in acute hippocampal slices maintained in a small-volume recycling-, perfusion-, and submersion-type chamber system

尽管自1951年以来, 脑切片的实验已经开始使用, 但问题仍然存在, 这就降低了在执行场电位或胞内记录时对突触传输调制进行稳定和成功分析的可能性。这篇手稿描述的方法学方面, 可能有助于改善实验条件的维护急性脑切片和记录场兴奋性突触后电位在一个商业可用的淹没室与流出-carbogenation 单位。外流 carbogenation 有助于稳定氧气水平的实验, 依靠回收的小缓冲水库, 以提高成本效益的药物实验。此外, 该手稿还有代表性的实验, 考察不同的 carbogenation 模式和刺激范式对突触传递的活动依赖性突触可塑性的影响。

在 "协议" 部分, 我们描述了由雄性 C57BL/6 小鼠和雄性大鼠 (5-8 周) 的海马形成 (图 1) 的腹侧和中间部分的急性海马切片的制备。半球在切片机平台上的位置有助于保持它们的稳定, 并消除琼脂或琼脂糖对稳定的需要。灌注系统本身是基于在高转速下运行的蠕动泵, 以提供所需的液体流速。因此, 在泵的标准管迅速老化造成了一个很大的问题, 我们克服了...

Watch this Scientific Journal Video about Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System at JoVE.com

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

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

本协议描述了在少量回收的缓冲液中的氧水平的稳定, 以及在淹没的急性海马切片中记录活动依赖性突触可塑性的方法学方面。

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

recording-synaptic-plasticity-acute-hippocampal-slices-maintained

Research

JoVE Journal

Neuroscience

11.5K 次观看.  Fudan University. 本协议描述了在少量回收的缓冲液中的氧水平的稳定, 以及在淹没的急性海马切片中记录活动依赖性突触可塑性的方法学方面。

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

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 ...

科研

神经科学

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 ...

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

Long-term potentiation (LTP) is a type of synaptic plasticity characterized by an increase in synaptic strength and believed to be involved in memory encoding. LTP elicited in the CA1 region of acute hippocampal slices has been extensively studied. However the molecular mechanisms underlying the maintenance phase of this phenomenon are still poorly understood. This could be partly due to the various experimental conditions used by different laboratories. Indeed, the maintenance phase of LTP is strongly dependent on external parameters like oxygenation, temperature and humidity. It is also dependent on internal parameters like orientation of the slicing plane and slice viability after dissection.
The optimization of all these parameters enables the induction of a very reproducible and very stable long-term potentiation. This methodology offers the possibility to further explore the molecular mechanisms involved in the stable increase in synaptic strength in hippocampal slices. It also highlights the importance of experimental conditions in in vitro investigation of neurophysiological phenomena.

This paper presents a complete methodology to prepare and preserve in vitro acute hippocampal slices from adult mice. This protocol allows the recording of very stable long-lasting long-term potentiation (LTP) for more than 8 hours with a success rate of 95%.

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 ...

Investigation of Synaptic Tagging/Capture and Cross-capture using Acute Hippocampal Slices from Rodents

Synaptic tagging and capture (STC) and cross-tagging are two important mechanisms at cellular level that explain how synapse-specificity and associativity is achieved in neurons within a specific time frame. These long-term plasticity-related processes are the leading candidate models to study the basis of memory formation and persistence at the cellular level. Both STC and cross-tagging involve two serial processes: (1) setting of the synaptic tag as triggered by a specific pattern of stimulation, and (2) synaptic capture, whereby the synaptic tag interacts with newly synthesized plasticity-related proteins (PRPs). Much of the understanding about the concepts of STC and cross-tagging arises from the studies done in CA1 region of the hippocampus and because of the technical complexity many of the laboratories are still unable to study these processes. Experimental conditions for the preparation of hippocampal slices and the recording of stable late-LTP/LTD are extremely important to study synaptic tagging/cross-tagging. This video article describes the experimental procedures to study long-term plasticity processes such as STC and cross-tagging in the CA1 pyramidal neurons using stable, long-term field-potential recordings from acute hippocampal slices of rats.

This video article describes experimental procedures to study long-term plasticity and its associative processes such as synaptic tagging, capture and cross-tagging in the CA1 pyramidal neurons using acute hippocampal slices from rodents.

使用急性海马脑片啮齿动物突触标记/捕获和交叉捕获的研究

Synaptic tagging and capture (STC) and cross-tagging are two important mechanisms at cellular level that explain how synapse-specificity and associativity ...

Tuning in the Hippocampal Theta Band In Vitro: Methodologies for Recording from the Isolated Rodent Septohippocampal Circuit

This protocol outlines the procedures for preparing and recording from the isolated whole hippocampus, of WT and transgenic mice, along with recent improvements in methodologies and applications for the study of theta oscillations. A simple characterization of the isolated hippocampal preparation is presented whereby the relationship between internal hippocampal theta oscillators is examined together with the activity of pyramidal cells, and GABAergic interneurons, of the cornu ammonis-1 (CA1) and subiculum (SUB) areas. Overall, we show that the isolated hippocampus is capable of generating intrinsic theta oscillations in vitro and that rhythmicity generated within the hippocampus can be precisely manipulated by optogenetic stimulation of parvalbumin-positive (PV) interneurons. The in vitro isolated hippocampal preparation offers a unique opportunity to use simultaneous field and intracellular patch-clamp recordings from visually-identified neurons to better understand the mechanisms underlying theta rhythm generation.

Tuning in the Hippocampal Theta Band In Vitro: Methodologies for Recording from the Isolated Rodent Septohippocampal Circuit

Here, we present a protocol for recording rhythmic neuronal network theta and gamma oscillations from an isolated whole hippocampal preparation. We describe the experimental steps from extraction of the hippocampus to details of field, unitary and whole-cell patch clamp recordings as well as optogenetic pacing of the theta rhythm.

调整在海马Theta乐队<em&gt;体外</em&gt;:从孤立的啮齿动物皮下注射电路记录的方法

This protocol outlines the procedures for preparing and recording from the isolated whole hippocampus, of WT and transgenic mice, along with recent ...

Investigating Long-term Synaptic Plasticity in Interlamellar Hippocampus CA1 by Electrophysiological Field Recording

The study of synaptic plasticity in the hippocampus has focused on the use of the CA3-CA1 lamellar network. Less attention has been given to the longitudinal interlamellar CA1-CA1 network. Recently however, an associational connection between CA1-CA1 pyramidal neurons has been shown. Therefore, there is the need to investigate whether the longitudinal interlamellar CA1-CA1 network of the hippocampus supports synaptic plasticity.
We designed a protocol to investigate the presence or absence of long-term synaptic plasticity in the interlamellar hippocampal CA1 network using electrophysiological field recordings both in vivo and in vitro. For in vivo extracellular field recordings, the recording and stimulation electrodes were placed in a septal-temporal axis of the dorsal hippocampus at a longitudinal angle, to evoke field excitatory postsynaptic potentials. For in vitro extracellular field recordings, hippocampal longitudinal slices were cut parallel to the septal-temporal plane. Recording and stimulation electrodes were placed in the stratum oriens (S.O) and the stratum radiatum (S.R) of the hippocampus along the longitudinal axis. This enabled us to investigate the directional and layer specificity of evoked excitatory postsynaptic potentials. Already established protocols were used to induce long-term potentiation (LTP) and long-term depression (LTD) both in vivo and in vitro. Our results demonstrated that the longitudinal interlamellar CA1 network supports N-methyl-D-aspartate (NMDA) receptor-dependent long-term potentiation (LTP) with no directional or layer specificity. The interlamellar network, however, in contrast to the transverse lamellar network, did not present with any significant long-term depression (LTD).

We used recording and stimulation electrodes in longitudinal hippocampal brain slices and longitudinally positioned recording and stimulation electrodes in the dorsal hippocampus in vivo to evoke extracellular postsynaptic potentials and demonstrate long-term synaptic plasticity along the longitudinal interlamellar CA1.

通过电生理场记录研究跨体海马CA1的长期突触可塑性

The study of synaptic plasticity in the hippocampus has focused on the use of the CA3-CA1 lamellar network. Less attention has been given to the ...

Preparation of Acute Human Hippocampal Slices for Electrophysiological Recordings

Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for sudden death. Despite an abundance of available treatment options, about 30% of patients are drug-resistant. Several novel therapeutics have been developed using animal models, though the rate of drug-resistant patients remains unaltered. One of probable reasons is the lack of translation between rodent models and humans, such as a weak representation of human pharmacoresistance in animal models. Resected human brain tissue as a preclinical evaluation tool has the advantage to bridge this translational gap. Described here is a method for high quality preparation of human hippocampal brain slices and subsequent stable induction of epileptiform activity. The protocol describes the induction of burst activity during application of 8 mM KCl and 4-aminopyridin. This activity is sensitive to established AED lacosamide or novel antiepileptic candidates, such as dimethylethanolamine (DMEA). In addition, the method describes induction of seizure-like events in CA1 of human hippocampal brain slices by reduction of extracellular Mg2+ and application of bicuculline, a GABAA receptor blocker. The experimental set-up can be used to screen potential antiepileptic substances for their effects on epileptiform activity. Furthermore, mechanisms of action postulated for specific compounds can be validated using this approach in human tissue (e.g., using patch-clamp recordings). To conclude, investigation of vital human brain tissue ex vivo (here, resected hippocampus from patients suffering from temporal lobe epilepsy) will improve the current knowledge of physiological and pathological mechanisms in the human brain.

The presented protocol describes the transport and preparation of resected human hippocampal tissue with the ultimate goal to use vital brain slices as a preclinical evaluation tool for potential antiepileptic substances.

为电子生理记录准备急性人类河马切片

Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for ...

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 ...

Recording Neuronal Field Excitatory Postsynaptic Potentials in Acute Hippocampal Slices

Source: Weng, W., et al. <a href="https://app.jove.com/v/55936">Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System.</a> J. Vis. Exp. (2018).
This video demonstrates the procedure for setting up a brain slice in a submersion chamber to record synaptic activity. The process involves securing the slice on lens paper, maintaining a continuous flow of artificial cerebrospinal fluid (aCSF) to ensure neuronal viability, and accurately positioning the stimulation and recording electrodes relative to a specific brain region of interest. This setup allows for precise monitoring of baseline synaptic activity and the effects of electrical stimulation.

记录急性海马切片中的神经元场兴奋性突触后电位

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee. 
1. Recording Synaptic ...

JoVE Encyclopedia of Experiments

Neurophysiology

Electrophysiology Techniques

In Vitro Recording of Theta Oscillations in the Mouse Hippocampus

Source: Manseau, F., et.al. <a href="https://app.jove.com/v/55851">Tuning in the Hippocampal Theta Band In Vitro: Methodologies for Recording from the Isolated Rodent Septohippocampal Circuit.</a> J. Vis. Exp. (2017).
This video demonstrates the recording of rhythmic neuronal network activity, also known as theta oscillations, in an isolated hippocampus. Electrodes are inserted into various layers and spatial positions to detect and analyze theta oscillations. This shows the unique neuronal compositions and the connectivity of the neuronal network across different layers of the hippocampus.

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

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