. 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. 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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。

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			<td height="21" style="width:311px;height:21px;">Reagents required</td>
			<td style="width:244px;"></td>
			<td style="width:179px;"></td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="width:311px;height:27px;">NaCl</td>
			<td style="width:244px;">Sinopharm Chemical Reagent, China</td>
			<td style="width:179px;">10019318</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="width:311px;height:27px;">KCl</td>
			<td style="width:244px;">Sinopharm Chemical Reagent, China</td>
			<td style="width:179px;">10016318</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="width:311px;height:27px;">KH2PO4</td>
			<td style="width:244px;">Sinopharm Chemical Reagent, China</td>
			<td style="width:179px;">10017618</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="width:311px;height:27px;">MgCl2&#183;6H2O</td>
			<td style="width:244px;">Sinopharm Chemical Reagent, China</td>
			<td style="width:179px;">10012818</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="width:311px;height:27px;">CaCl2</td>
			<td style="width:244px;">Sinopharm Chemical Reagent, China</td>
			<td style="width:179px;">10005861</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="width:311px;height:27px;">NaHCO3</td>
			<td style="width:244px;">Sinopharm Chemical Reagent, China</td>
			<td style="width:179px;">10018960</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="width:311px;height:27px;">Glucose</td>
			<td style="width:244px;">Sinopharm Chemical Reagent, China</td>
			<td style="width:179px;">10010518</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="width:311px;height:27px;">NaH2PO4</td>
			<td style="width:244px;">Sinopharm Chemical Reagent, China</td>
			<td style="width:179px;">20040718</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="width:311px;height:27px;">HEPES</td>
			<td style="width:244px;">Sigma</td>
			<td style="width:179px;">H3375</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="width:311px;height:27px;">Sodium pyruvate</td>
			<td style="width:244px;">Sigma</td>
			<td style="width:179px;">A4043</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="width:311px;height:27px;">MgSO4</td>
			<td style="width:244px;">Sinopharm Chemical Reagent, China</td>
			<td style="width:179px;">20025118</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="width:311px;height:27px;">NaOH</td>
			<td style="width:244px;">Sinopharm Chemical Reagent, China</td>
			<td style="width:179px;">10019718</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;"></td>
			<td style="width:244px;"></td>
			<td style="width:179px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="width:311px;height:21px;">Tools and materials for dissection</td>
			<td style="width:244px;"></td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">Decapitators</td>
			<td style="width:244px;">Harvard apparatus</td>
			<td style="width:179px;">55-0012</td>
			<td>for rat decapitation</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">Bandage Scissors</td>
			<td style="width:244px;">SCHREIBER</td>
			<td style="width:179px;">12-4227</td>
			<td>for mouse decapitation</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">double-edge blade</td>
			<td style="width:244px;">Flying Eagle, China</td>
			<td style="width:179px;">74-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">IRIS Scissors</td>
			<td style="width:244px;">RWD, China</td>
			<td style="width:179px;">S12003-09</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">Bone Rongeurs</td>
			<td style="width:244px;">RWD, China</td>
			<td style="width:179px;">S22002-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">Spoon</td>
			<td style="width:244px;">Hammacher&#160;</td>
			<td style="width:179px;">HSN 152-13</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">dental cement spatula</td>
			<td style="width:244px;">Hammacher&#160;</td>
			<td style="width:179px;">HSN 016-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">dental double end excavator</td>
			<td style="width:244px;">Blacksmith Surgical, USA</td>
			<td style="width:179px;">BS-415-017</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">Vibrating Microtome</td>
			<td style="width:244px;">Leica, Germany</td>
			<td style="width:179px;">VT1200S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">surgical blade&#160;</td>
			<td style="width:244px;">RWD, China</td>
			<td style="width:179px;">S31023-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">surgical holder</td>
			<td style="width:244px;">RWD, China</td>
			<td style="width:179px;">S32007-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;"></td>
			<td style="width:244px;"></td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:311px;height:42px;">Electrophysiology equipment and materials</td>
			<td style="width:244px;"></td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">Vertical Pipette Puller</td>
			<td style="width:244px;">Narishige, Japan</td>
			<td style="width:179px;">PC-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">Vibration isolation table</td>
			<td style="width:244px;">Meirits, Japan</td>
			<td style="width:179px;">ADZ-A0806</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">submerged type recording chamber</td>
			<td style="width:244px;">Warner Instruments</td>
			<td style="width:179px;">RC-26GLP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">thermostatic water bath</td>
			<td style="width:244px;">Zhongcheng Yiqi&#65292;China</td>
			<td style="width:179px;">HH-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">4 Axis Micromanipulator</td>
			<td style="width:244px;">Sutter, USA</td>
			<td style="width:179px;">MP-285, MP-225</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">Platinum Wire</td>
			<td style="width:244px;">World Precision Instruments</td>
			<td style="width:179px;">PTP406</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">Amplifier</td>
			<td style="width:244px;">Molecular Devices, USA</td>
			<td style="width:179px;">Multiclamp 700B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">Data Acquisition System</td>
			<td style="width:244px;">Molecular Devices, USA</td>
			<td style="width:179px;">Digidata 1440A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">Anaysis software</td>
			<td style="width:244px;">Molecular Devices, USA</td>
			<td style="width:179px;">Clampex 10.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">Fluorescence Microscope</td>
			<td style="width:244px;">Nikon, Japan</td>
			<td style="width:179px;">FN1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">LED light source</td>
			<td style="width:244px;">Lumen Dynamics Group, Canada</td>
			<td style="width:179px;">X-cite 120LED</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">micropipettes</td>
			<td style="width:244px;">Harvard apparatus</td>
			<td style="width:179px;">GC150TF</td>
			<td>extracelluar recording</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">borosilicate micropipettes</td>
			<td style="width:244px;">Sutter, USA</td>
			<td style="width:179px;">BF150-86</td>
			<td>patch clamp</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">tungsten electrode</td>
			<td style="width:244px;">A-M Systems, USA</td>
			<td style="width:179px;">575500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">peristaltic pump</td>
			<td style="width:244px;">Longer, China</td>
			<td style="width:179px;">BT00-300T</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">tubes for peristaltic pump</td>
			<td style="width:244px;">ISMATEC, Wertheim, Germany</td>
			<td style="width:179px;">SC0309</td>
			<td>1x inflow, ID: 1.02mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">tubes for peristaltic pump</td>
			<td style="width:244px;">ISMATEC, Wertheim, Germany</td>
			<td style="width:179px;">SC0319</td>
			<td>2x tubes for outflow, ID: 2.79 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">CCD camera</td>
			<td style="width:244px;">PCO, Germany</td>
			<td style="width:179px;">pco.edge sCMOS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">lens cleaning paper</td>
			<td style="width:244px;">Kodak</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">50 ml conical centrifuge tube</td>
			<td style="width:244px;">Thermo scientific</td>
			<td style="width:179px;">339652</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">Prechamber</td>
			<td style="width:244px;">Warner Instruments</td>
			<td style="width:179px;">BSC-PC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">Inline heater</td>
			<td style="width:244px;">Warner Instruments</td>
			<td style="width:179px;">SF-28</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">Temperature Controller</td>
			<td style="width:244px;">Warner Instruments</td>
			<td style="width:179px;">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 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 &#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 ...

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This directed trafficking is of fundamental importance to deliver, maintain or remove synaptic proteins.
Super-ecliptic pHluorin (SEP) is a pH-sensitive derivative of eGFP that has been extensively used for live cell imaging of plasma membrane proteins1-2. At low pH, protonation of SEP decreases photon absorption and eliminates fluorescence emission. As most intracellular trafficking events occur in compartments with low pH, where SEP fluorescence is eclipsed, the fluorescence signal from SEP-tagged proteins is predominantly from the plasma membrane where the SEP is exposed to a neutral pH extracellular environment. When illuminated at high intensity SEP, like every fluorescent dye, is irreversibly photodamaged (photobleached)3-5. Importantly, because low pH quenches photon absorption, only surface expressed SEP can be photobleached whereas intracellular SEP is unaffected by the high intensity illumination6-10. 
FRAP (fluorescence recovery after photobleaching) of SEP-tagged proteins is a convenient and powerful technique for assessing protein dynamics at the plasma membrane. When fluorescently tagged proteins are photobleached in a region of interest (ROI) the recovery in fluorescence occurs due to the movement of unbleached SEP-tagged proteins into the bleached region. This can occur via lateral diffusion and/or from exocytosis of non-photobleached receptors supplied either by de novo synthesis or recycling (see Fig. 1). The fraction of immobile and mobile protein can be determined and the mobility and kinetics of the diffusible fraction can be interrogated under basal and stimulated conditions such as agonist application or neuronal activation stimuli such as NMDA or KCl application8,10. 
We describe photobleaching techniques designed to selectively visualize the recovery of fluorescence attributable to exocytosis. Briefly, an ROI is photobleached once as with standard FRAP protocols, followed, after a brief recovery, by repetitive bleaching of the flanking regions. This 'FRAP-FLIP' protocol, developed in our lab, has been used to characterize AMPA receptor trafficking at dendritic spines10, and is applicable to a wide range of trafficking studies to evaluate the intracellular trafficking and exocytosis.

This report describes the use of live cell imaging and photobleach techniques to determine the surface expression, transport pathways and trafficking kinetics of exogenously expressed, pH-sensitive GFP-tagged proteins at the plasma membrane of neurons.

横向扩散和培养的神经元膜蛋白的胞吐评估使用荧光恢复和荧光损失漂白

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

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 ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

诱导星形胶质细胞受体的可塑性由神经元的放电频率操作

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

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 ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.

海马神经元中突触囊泡吞的测量

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic ...

Pentylenetetrazole-Induced Kindling Mouse Model

Pentylenetetrazole (PTZ) is a GABA-A receptor antagonist. An intraperitoneal injection of PTZ into an animal induces an acute, severe seizure at a high dose, whereas sequential injections of a subconvulsive dose have been used for the development of chemical kindling, an epilepsy model. A single low-dose injection of PTZ induces a mild seizure without convulsion. However, repetitive low-dose injections of PTZ decrease the threshold to evoke a convulsive seizure. Finally, continuous low-dose administration of PTZ induces a severe tonic-clonic seizure. This method is simple and widely applicable to investigate the pathophysiology of epilepsy, which is defined as a chronic disease that involves repetitive seizures. This chemical kindling protocol causes repetitive seizures in animals. With this method, vulnerability to PTZ-mediated seizures or the degree of aggravation of epileptic seizures was estimated. These advantages have led to the use of this method for screening anti-epileptic drugs and epilepsy-related genes. In addition, this method has been used to investigate neuronal damage after epileptic seizures because the histological changes observed in the brains of epileptic patients also appear in the brains of chemical-kindled animals. Thus, this protocol is useful for conveniently producing animal models of epilepsy.

This protocol describes a method of chemical kindling with pentylenetetrazole and provides a mouse model of epilepsy. This protocol can also be used to investigate vulnerability to seizure induction and pathogenesis after epileptic seizures in mice.

戊四氮诱导点燃小鼠模型

Pentylenetetrazole (PTZ) is a GABA-A receptor antagonist. An intraperitoneal injection of PTZ into an animal induces an acute, severe seizure at a high ...

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 ...

Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other neurodegenerative disorders, this independence might be compromised due to insufficient glutamate clearance and the resulting spill-in and spill-out effects. Altered astrocytic coverage of the presynaptic terminals and/or dendritic spines as well as a reduced size of glutamate transporter clusters at glutamate release sites have been implicated in the pathogenesis of diseases resulting in symptoms of dys-/hyperkinesia. However, the mechanisms leading to the dysfunction of glutamatergic synapses in HD are not well understood. Improving and applying synapse imaging we have obtained data shedding new light on the mechanisms impeding the initiation of movements. Here, we describe the principle elements of a relatively inexpensive approach to achieve single synapse resolution by using the new genetically encoded ultrafast glutamate sensor iGluu, wide-field optics, a scientific CMOS (sCMOS) camera, a 473 nm laser and a laser positioning system to evaluate the state of corticostriatal synapses in acute slices from age appropriate healthy or diseased mice. Glutamate transients were constructed from single or multiple pixels to obtain estimates of i) glutamate release based on the maximal elevation of the glutamate concentration [Glu] next to the active zone and ii) glutamate uptake as reflected in the time constant of decay (TauD) of the perisynaptic [Glu]. Differences in the resting bouton size and contrasting patterns of short-term plasticity served as criteria for the identification of corticostriatal terminals as belonging to the intratelencephalic (IT) or the pyramidal tract (PT) pathway. Using these methods, we discovered that in symptomatic HD mice ~40% of PT-type corticostriatal synapses exhibited insufficient glutamate clearance, suggesting that these synapses might be at risk to excitotoxic damage. The results underline the usefulness of TauD as a biomarker of dysfunctional synapses in Huntington mice with a hypokinetic phenotype.

We present a protocol to evaluate the balance between glutamate release and clearance at single corticostriatal glutamatergic synapses in acute slices from adult mice. This protocol uses the fluorescent sensor iGluu for glutamate detection, a sCMOS camera for signal acquisition and a device for focal laser illumination.