作者要感谢史蒂夫·西格尔鲍姆的支持。资金由5R01NS106983-02以及1 F31 NS113466-01提供。

这里描述的所有方法都得到了哥伦比亚大学机构动物护理和使用委员会（AC-AAU9451）的批准。
1. 准备解决方案
<ol><li>准备蔗糖切割溶液，用于切片，如 表 1中所述。 注:在准备了1升蔗糖溶液后，在冰盘中冷冻少量（约100-200毫升）。这些冷冻蔗糖冰块将被混合成冰浆（见步骤4.3）。</li>
	<li>准备人工脑脊液（ACSF）进行记录，如 表2所述。</li>
	<li>将 0.05844 克 NaCl 溶解在经过过滤的 1 mL 纯净水中，以去除微量金属和其他杂质，从而准备 1 M NaCl 刺激移液器。 注:蔗糖切割解决方案和ACSF应为每个实验准备新鲜。</li>
</ol>2. 准备阿加坡道
<ol><li>通过将琼脂粉溶解到经过过滤以去除微量金属和其他杂质的纯净水中，准备 4% 琼脂（例如，50 mL 水中的 2 克琼脂）。将混合物加热到微波炉中，直到它开始沸腾（大约30-60 s）。将熔融的琼脂倒入模具中，使其在大约 12° 倾斜的冰箱中以 4 °C 冷却和凝固。 注:对于模具，可以使用任何玻璃或塑料容器设置在一个角度，有足够的熔融琼脂倒入形成一个坡道4厘米长，约0.8厘米的高度。</li>
</ol>3. 阶段切片区域
<ol><li>用大约 250 mL 的 ACSF 填充一个包含切片回收室的 400 mL 烧杯;将其放在加热到32°C的水浴中，开始用碳水化合物冒泡（95%的氧气/5%的二氧化碳气体）。 注:在回收烧杯中填充足够的液体，将夹子的尼龙网放在溶液的表面，以便将切片放在温溶液混合物和空气的接口处（图1）。将小片镜头纸放在尼龙网上。切片将放在镜头纸的顶部，后者以后可用于将切片单独转移到录音室（见步骤 6.6）。</li>
	<li>将带蔗糖溶液的烧瓶放入冰桶中冷却，并开始用碳水化合物冒泡。 注:回收烧杯和蔗糖溶液应分别加热和冷却，在将鼠标放入异氟室之前，碳水化合物冒泡至少30分钟。</li>
	<li>从冰柜中取出预制的冷冻蔗糖溶液冰块托盘，放在长凳上部分解冻。</li>
	<li>将清洁的双刃剃须刀刀片的一半放入微切口中，并在必要时校准。将剃须刀刀片的另一半放在一边，用于解剖。</li>
	<li>运行一个碳水化合物线和温度探头到切片室和周围的冰冷却室。</li>
	<li>准备一个干净的长凳或桌子覆盖着两个一次性吸水垫。将所有解剖工具和三块实验室胶带放在左垫上，在那里进行心电图灌注。 注:解剖工具包括小波恩剪刀、解剖剪刀、大铲/勺、微孢子、带新刀片的手术刀、铲子、锋利/钝化手术剪刀、大组织钳和小组织钳（见 材料表）。</li>
	<li>在灌注区右侧的另一个吸水垫上，将一张圆形滤纸放入直径为 100 mm 的玻璃培养皿的盖子中。将培养皿的底部放在盖子旁边，将碳水化合物线放入菜的两块中。</li>
	<li>使用剃须刀刀片或手术刀切割一小坡道（沿倾斜表面约4厘米，高度0.8厘米，宽2厘米）。从高端切断一块坡道，并将其倒车，以创建一个阿加的后背块。使用青花胶粘剂将琼脂坡道和后背块贴在切片平台上并放在一边。 注:agar的后背块有助于在切片过程中稳定大脑，并提供一个表面，从每片中解剖不必要的组织（图1）。</li>
</ol>4. 心肌输液
<ol><li>将 60 mL 容量的注射器作为蔗糖切割溶液的储液池，在台面上方约 18 英寸处（例如，在垂直柱子上使用三管齐下旋转夹）。将管子连接到储层底部，通过滚筒夹运行管子，并将管的另一端连接到干净的 20 G 针。</li>
	<li>用大约 30 mL 的冰镇蔗糖溶液填充 60 mL 水库，并将碳水化合物线引导到蔗糖储层中，以持续冒泡香水。 注意:确保蔗糖流过管子和针头，没有任何捕获的气泡。流速应该足够快，有一个稳定的，持续的滴蔗糖从针。</li>
	<li>使用搅拌机，将冷冻蔗糖粉碎并混合到冰冷的泥浆中，然后使用大铲子/勺子分配浆料。<ol>
<li>将少量蔗糖浆放在玻璃培养皿盖的边缘。向培养皿底部添加少量蔗糖浆。</li>
		<li>将大约 20-30 mL 的蔗糖浆添加到注入液库中，将其与 30 mL 的冰镇液体蔗糖很好地混合，直到储液库包含非常冷的、主要是液体的蔗糖与一些残留的冷冻溶液的混合物。</li>
	</ol></li>
	<li>将鼠标放入连接到异氟蒸发器的室内。当氧气在大约 2 L / min 动时，转动蒸发器的表盘以 5% 的浓度提供异氟化液，并启动一个测时器。 注意:在整个切片过程中保持此计时器，以测量切片的获取速度。手术应尽快完成，以便切片完成，组织在动物进入异氟室的15-20分钟内在接口室恢复。观察鼠标，以确保达到深度麻醉状态。至少5分钟后，鼠标应深度麻醉，对脚趾捏无反应。</li>
	<li>在将鼠标从异物室中取出之前，立即将 Petri 菜盖填充，将冷冻蔗糖溶液填充至约 3-5 mm 的深度，并在 Petri 菜底填充冰镇蔗糖溶液，深度约为 1.0 厘米。</li>
	<li>快速将鼠标转移到左吸水垫，并使用 3 块胶带固定前肢和尾部。执行后肢脚趾捏合，以确保鼠标在执行任何切口之前无响应。使用大组织钳和手术剪刀，帐篷的皮肤，使从胸骨底部到胸部顶部的长切口。用钳子拉起胸骨，用剪刀切开隔膜。 注:应尽快执行鼠标的初始定位和切口以切断隔膜的前几个切口，以确保鼠标不会恢复知觉。为了确保在整个过程中麻醉的足够深度，可以在灭鼠器上放置一个鼻锥，以便在注入过程中提供异丙苯丙。</li>
	<li>使用剪刀切开两侧的肋骨笼，向前肢与身体交汇处切开一个大动作。用钳子，将肋骨笼的前部向左摆动，朝头部向上摆动，然后用大剪刀用水平切口将其完全取出。使用大钳子将心脏保持到位，并将 20 G 注水针插入左心室。一旦针头定位正确，心脏的左侧应迅速苍白，因为冰镇蔗糖溶液填充心室。</li>
	<li>使用小解剖部门剪刀切入右中庭，让血液从循环系统流出。如果切口执行得当，心脏的损伤应该最小，并且应该在整个灌注过程中继续泵送。 注意:卷起的纸巾可用于从心脏吸走血液和蔗糖溶液，并在手术过程中保持输液针的可见性。</li>
	<li>确保香水针保持原位，不要从左心室脱落。随着适当的流速和放置，肝脏将开始苍白到浅褐色/米色与20-30s。</li>
	<li>30-45岁以后，用大剪刀斩首鼠标。将头部握在左手上，将皮肤从头骨上推开或剥落。在一个精心注入的动物，头骨应该非常苍白，大脑应该出现一个非常浅的粉红色（接近白色）通过头骨没有清晰可见的血管。</li>
</ol>5. 提取大脑并切片
<ol><li>使用小波恩剪刀，使两个横向切口通过头骨朝中线在头骨的前面，靠近眼睛。在头骨底部的两侧再做两个切口。</li>
	<li>将头部转移到玻璃培养皿的底部，在那里它应该主要浸泡在冰镇蔗糖溶液中。使用小波恩剪刀沿着头骨的长度切下中线，用剪刀拉起，以尽量减少对底层脑组织的损伤。使用小组织钳，牢牢抓住头骨的每一侧，摆动它向上和远离大脑，像打开一本书。</li>
	<li>用左手的手指把头骨的皮瓣打开，在嗅灯泡附近的大脑下插入微孢子。将大脑从头骨中翻转到蔗糖中，然后用微孢子切断脑干。洗脑以去除任何残留的血液、毛皮或其他组织。</li>
	<li>使用大铲子/勺子将大脑转移到玻璃培养皿的盖子上。与双刃剃须刀刀片的另一半以前预留，使两个日冕切割，首先删除小脑，然后大脑的最前部分，包括嗅灯泡（图1）。</li>
	<li>将青亚酸胶粘剂涂抹在琼脂坡道上。非常短暂地将大脑放在一块干燥的滤纸上，使用大组织钳，然后立即将其转移到琼脂坡道，将大脑的腹腔表面放在胶粘剂上。 注:对于中间海马的切片，大脑应面向前方朝上坡道的斜坡，后部更接近刀片。对于腹海马的切片，用前端定向大脑指向坡道的斜坡，后端在坡道顶部，远离刀片。无论哪种情况，大脑都应该位于坡道的顶部，这样大脑的冠状切口表面就会接触到琼脂背块（图1）。</li>
	<li>将切片平台与琼脂和大脑放入微切片的切片室，并完全浸入冰镇蔗糖溶液。使用大铲子/勺子将一些蔗糖浆转移到腔室，搅拌融化任何冷冻蔗糖，并迅速将混合物的温度降低到 ±1-2 °C。 注:在整个切片过程中观看温度计。如果蔗糖溶液加热到3°C以上，添加更多的泥浆和混合，使温度回落。</li>
	<li>切片厚度为 450μm，微原子速度设置为 0.07 mm/s。当每个切片被释放时，使用小组织钳和锋利的手术刀首先分离两个半球，然后切开组织，直到切片主要由海马和准希波坎帕尔区域组成（图1）。</li>
	<li>使用塑料转移移液器将切片单独转移到接口恢复室，确保切片位于ACSF和空气的接口处，只有薄薄的ACSF半月板覆盖切片。紧紧地关闭房间的盖子，让切片在32°C下恢复30分钟。</li>
	<li>在 32 °C 的初始恢复后，将接口恢复室从水浴中取出，并放置在搅拌器上，使其速度变慢，使磁搅拌杆促进室内ACSF的循环。允许它逐渐冷却到室温，因为切片恢复额外的90分钟。确保回收室不断用碳水化合物冒泡，不要让大气泡困在切片下面。</li>
</ol>6. 执行自发活动的本地现场潜力 （LFP） 录音
<ol><li>通过打开所有必要的设备（包括运行采集软件的计算机、连接到计算机的监视器、刺激器、微操纵器、温度控制器、显微镜光源、显微镜连接摄像头、微电子放大器、数字化器和永久泵）来准备 LFP 录制。如果使用中央真空系统，请打开墙壁阀，准备真空线，将ACSF从录音室中取出。</li>
	<li>用ACSF填充加热的储层，然后将管子的一端放入含有冒泡ACSF的400 mL烧杯中。打开永久泵，将ACSF从400 mL烧杯引向加热的储层，然后从水库进入录音室。点击或捏住管子以释放任何被困的气泡。</li>
	<li>调整腹腔泵，以确保通过录音室的ACSF流速快（~8-10 mL/min）。使用温度探针确保记录室中心 ACSF 为 32 °C。 注:如果ACSF使用长息泵输送到录音室，高流量可能导致流速显著波动。使用简单的脉动阻尼器（图 2），可以实现一致的流速，该阻尼器由集成到管中的一系列空注射器组成。</li>
	<li>使用加热灯丝拉拔器准备来自波罗西拉特玻璃毛细血管的刺激和记录移液器。应配置拉拔器协议，以产生电阻为 2-3 MOhm 的移液器，用于刺激或局部场电位记录电极。</li>
	<li>用 ACSF 填充 1 M NaCl 和 LFP 移液器的刺激移液器。</li>
	<li>短暂地夹住油管并关闭泵以暂停ACSF的流量。使用细钳将切片转移到录音室，以抓住镜头纸的一角，切片放在镜头纸上。将镜头纸放入录音室，朝下切片，然后"剥"掉镜头纸，将切片浸入录音室。使用竖琴固定切片。 注:竖琴可以使用U形不锈钢或铂金和细尼龙灯丝在实验室内购买预制或制造。</li>
	<li>将充满 NaCl 的刺激移液器放入手动微操纵器中，以大约 30-45° 的角度缓慢地将移液器尖端推进到切片表面（例如 ，在地层散热 层中）。一旦移液器的尖端进入组织，缓慢地向前推进移液器，避免在横向或垂直方向进行大运动，这些运动可能会不必要地损坏切片内的轴突。将移液器的尖端至少 50-100μm 深放入切片中，以避免在切片过程中损坏的表面附近的细胞。</li>
	<li>将充满ACSF的LFP移液器放入连接到电动微操纵器的移液器支架中。使用口腔压力或 1 mL 注射器施加非常轻的正压力，该注射器通过塞式阀门连接，并将短长度的管子连接到移液器支架上。 注:将LFP移液器放置在切片中以记录兴趣信号:在剧烈波纹的情况下，应将一个LFP移液器放置在地层金字塔（SP）中，将第二个移液器放置在地层辐射（SR）中。此配置允许同时录制 SR 中的负锐波和 SP 中的高频波纹振荡（图 3）。</li>
	<li>使用微操纵器将LFP移液器的尖端以大约30-45°的角度缓慢地推进到感兴趣的区域（例如CA1金字塔细胞层）。将移液器的尖端至少 50-100μm 深放入切片中，以避免在切片过程中损坏的表面附近的细胞。在推进LFP移液器的同时，使用采集软件不断提供小电压测试脉冲，并观察电极电阻的突然增加。这可能表明移液器已堵塞或压在细胞上。</li>
	<li>一旦LFP移液器放置在感兴趣的区域，通过打开连接到移液器支架的管子上的阀门，小心释放正压力。</li>
	<li>要同时在第二个位置记录 LFP，需要用第二个移液器和微操纵器重复步骤 6.8-6.10。</li>
	<li>使用当前夹子配置中的微电子放大器，在采集软件中使用桥平衡功能纠正移液器的系列电阻后，记录本地场潜力中的自发活动。自发 SWR 将显示为 SP 层（图 3）的细胞外电位的正偏转。</li>
	<li>为了记录唤起的场电位，使用连接到数字化器的刺激器通过充满 NaCl 的刺激移液器传递短（200 我们）方形电压脉冲。调整刺激电压表盘以唤起一系列响应振幅。</li>
</ol>

提交人没有什么可透露的。

这个切片协议有几个步骤，旨在促进组织健康，有利于自发的自然网络活动的出现:小鼠在心电图上注入冰镇蔗糖切割溶液:水平内皮质 （HEC） 切片的厚度为 450μm，从中间或腹海马;切片在加热的ACSF和加湿，富含碳水化合物的空气的界面恢复:在录制过程中，切片被超级注入ACSF加热到32°C，并以快速流速交付，双表面超级输液在水下录音室。
切片健康对于 体外网络振荡?...

体外活海马片 的 电生理测量是一种强大的实验方法，具有诸多优点。实验者可以使用显微镜、微操纵器和记录系统直接可视化和收集组织中单个神经元的测量结果。组织切片也非常容易用于光刺激或药物输送，用于光遗传学、化学遗传学或药理学实验。
海马网络在体内产生高度同步的人动，可见于细胞外局部场的振荡潜力1 ， 2 ， 3 ， 4 ， 5 。脑切片方法已被利用来深入了解这些神经元网络振荡背后的细胞和回路机制。迈尔等人的基础工作表明，尖锐的波纹复合物（SWR）可以自发地出现在腹海马6，7的切片中。来自多个研究者的后续研究已经逐渐阐明了SWR的许多方面，包括神经调节器在调节海马8、9、10的网络状态方面的作用，以及推动神经元合奏在体内活动期间体外重新激活的突触机制。脑切片实验也提供了对伽马范围振荡（30-100赫兹）的洞察，这是一种独特的海马网络状态，被认为支持内存编码和回忆12，13。最后，认识到海马和相关结构在时间叶癫痫病理生理学中的核心作用14、15，研究人员利用海马切片制剂研究癫痫活性的生成和繁殖。Carter等人证明，从慢性癫痫动物身上制备的海马-内皮层组合片可以在体外16中自发产生癫痫放电。随后，Karlécai等人利用经过改良的人工脑脊液（ACSF）改变离子浓度（降低Mg2+或升高K+）或添加药物（4AP或加巴津）17，探索了海马片中癫痫放电的机制。
研究者已经开发出许多海马切片方法，这些方法在关键方面有所不同:（1） 切片中海马的区域（多孔、中间或腹腔）:（二）内皮层等外皮组织的存在或者不存在的:（3） 用于切割切片（冠状、下垂、水平或倾斜）的方向:（4） 切片后组织维持的条件（完全浸入ACSF或保存在ACSF的接口和加湿，富含碳水化合物的空气）。
使用哪种切片方法的选择应由实验目标决定。例如，在水下条件下保存的背海马的横向或日冕切片已非常有效地用于研究海马内电路和突触可塑性18、19、20。然而，这种准备不会像21、22、23号海马的叶片那样自发地产生网络振荡。虽然从多佛和心室海马24的横向切片中，破伤风刺激可以诱发持续的SWR活动状态，但自发性SWR更容易在腹腔切片7、25中观察到。
在体内和体外26进行的研究支持了多骨和心室海马之间固有的生理和解剖学区别。大鼠的录音显示，整个脊椎和中海马的节奏非常连贯，但腹腔区域与海马27号其余部分之间的连贯性较差。体内的 SWR 很容易在多拉尔和中海马之间传播，而源自腹海马体的 SWR 通常仍然是本地的 28。来自位于背心和中海马体的CA3金字塔神经元的关联投影沿着海马的纵轴进行长距离投射。来自腹地的CA3预测仍然相对局部，因此在切片过程中不太可能被切断。因此，心室切片可以更好地保存生成人口同步所需的经常性网络。室室切片在体外产生自发网络活动的倾向也可能反映金字塔神经元的内在兴奋性更高，或与更多的背部区域31相比，心室海马体中较弱的GABAErgic抑制。事实上，腹海马片更容易受到癫痫活性32，33。因此，许多关于自发生理8、9、11、24或病理学16、34、35、36网络振荡的研究传统上都采用水平切片方法，有时在前脑方向有轻微的角度，产生与心室海马横向平面平行的组织切片。
网络连接不可避免地受到切片过程的影响，因为切片中的许多单元格将被切断。应考虑切片和制备中保留的组织的角度和厚度，以优化感兴趣的电路中的连接性。许多研究利用水平组合海马-内皮层切片（HEC）来探索两种结构在生理或病理网络振荡背景下的相互作用。Roth等人从海马的CA1子场和中腹皮层的V层进行了双重录音，以证明通过 HEC 切片37传播 SWR 活动。许多有关癫痫活性的研究都利用 HEC 切片制剂来研究癫痫菌体如何通过皮质皮质细胞网络 16、35、36、38 传播。需要注意的是，保存完好的皮质皮质环不是自发 SWR、癫痫放电或伽马振荡的先决条件:网络振荡可以在圆周或心室海马的横向切片中产生，没有附加的寄生虫组织21，22，23，25，39，40，41。 海马片中自发生成网络振荡的一个更重要的因素可能是每片的厚度，因为较厚的切片（400-550μm）将保留CA2/CA3复发网络21、22、25的更多连接。
虽然角水平 HEC 切片（在前部方向的切口角约为 12°角）已用于研究皮质希波坎帕尔环 11、16、34、35、42 的功能连接，但这种角度准备对于自发网络活动43、44、45并不需要。但是，使用倾斜的切片平面确实允许研究者选择性地制作切片，以最好地保存腹腔或中海马体的横向偏斜，具体取决于是否应用向下或向上的角度（图 1）。这种方法在概念上与2002年帕帕西奥多罗普洛斯等人所使用的方法相似，他们免费解剖了每个海马，然后使用组织斩波器沿着整个正交轴21创建横向切片。根据上述腹腔和正向中间海马之间的功能差异，研究人员在设计实验或解释结果时应考虑切片的解剖起源。在切片过程中使用琼脂坡道是一种从中间或心室海马体中优先生产切片的简单方法。
海马切片可以保存在水下室（组织完全浸入ACSF），或界面风格的腔室（如奥斯陆或哈斯室，切片只覆盖由流动介质薄膜覆盖）。接口维护可增强组织的氧化，促进神经元存活，并允许持续高水平的内分活动。传统上，水下记录条件使用较慢的ACSF流速，不能提供足够的组织氧合，以稳定地表达网络水平振荡。在水下海马片中，只观察到卡巴乔尔引起的伽马振荡，瞬时只有46，47，而它们可以稳定地维持在界面记录室10，48，49。因此，许多关于体外复杂自发活动的研究都依赖于界面记录室来研究剧烈波纹复合体6、7、8、9、10、25、37、伽马振荡10、13和癫痫活性16、38、45、47。
在水下风格的录音室中，浸入式显微镜目标可用于可视化单个细胞，并选择性地瞄准健康外观的细胞进行录制。水下制剂还允许对细胞环境进行精细控制，因为潜水有助于药物或其他化合物快速扩散到组织。因此，在水下条件下保持稳定的网络振荡的修改方法是一种强有力的实验方法。Höjos等人的工作就是例证，海马切片在简化的界面式支架室中恢复数小时，然后转移到具有高流量ACSF（约6 mL/min）的经过改造的水下录音室，以增强对组织12、48、49的氧气供应。在这种情况下，在水下录音室中可以保持高水平的自相残杀活动和稳定的自发网络振荡。这种修改的方法允许调查人员执行视觉引导的全细胞贴片夹子录音，并描述形态识别的细胞类型对卡巴乔尔诱导的伽马振荡12的贡献。SWR也可以自发地出现在水下海马片中，其快速流速为ACSF 11、48、49。迈尔等人证明，海马片在转移到水下录音室之前在接口室中恢复，可靠地表现出自发的SWR，而在转移到水下录音室之前在烧杯中恢复的片状物则显示出较小的引起场反应，自发突触电流水平较低，而且很少显示自发的SWR43。施林洛夫等人利用这种改进的方法，展示了帕瓦尔布明表达篮细胞在产生自发SWR44中的作用。
以下协议提出了一种切片方法，通过这种方法，可以在接口条件下恢复水平海马切片中的自发活跃神经元，随后保存在适合药理学或光遗传学操作和视觉引导记录的水下录音室中。

<table><tbody><tr><td>3D printer</td><td>Lulzbot</td><td>LulzBot TAZ 6</td><td></td></tr><tr><td>Acute brain slice incubation holder</td><td>NIH 3D Print Exchange</td><td>3DPX-001623</td><td>Designed by ChiaMing Lee, available at https://3dprint.nih.gov/discover/3dpx-001623</td></tr><tr><td>Adenosine 5&amp;prime;-triphosphate magnesium salt</td><td>Sigma Aldrich</td><td>A9187-500MG</td><td></td></tr><tr><td>Ag-Cl ground pellets</td><td>Warner</td><td>64-1309, (E205)</td><td></td></tr><tr><td>agar</td><td>Becton, Dickinson</td><td>214530-500g</td><td></td></tr><tr><td>ascorbic acid</td><td>Alfa Aesar</td><td>36237</td><td></td></tr><tr><td>beaker (250 mL)</td><td>Kimax</td><td>14000-250</td><td></td></tr><tr><td>beaker (400 mL)</td><td>Kimax</td><td>14000-400</td><td></td></tr><tr><td>biocytin</td><td>Sigma Aldrich</td><td>B4261</td><td></td></tr><tr><td>blender</td><td>Oster</td><td>BRLY07-B00-NP0</td><td></td></tr><tr><td>Bonn scissors, small</td><td>becton, Dickinson</td><td>14184-09</td><td></td></tr><tr><td>borosilicate glass capillaries with filament (O.D. 1.5 mm, I.D. 0.86 mm, length 10 cm)</td><td>Sutter Instruments</td><td>BF150-86-10HP</td><td>Fire polished capillaries are preferable.</td></tr><tr><td>calcium chloride solution (1 M)</td><td>G-Biosciences</td><td>R040</td><td></td></tr><tr><td>camera</td><td>Olympus</td><td>OLY-150</td><td></td></tr><tr><td>compressed carbogen gas (95% oxygen / 5% carbon dioxide)</td><td>Airgas</td><td>X02OX95C2003102</td><td></td></tr><tr><td>compressed oxygen</td><td>Airgas</td><td>OX 200</td><td></td></tr><tr><td>constant voltage isolated stimulator</td><td>Digitimer Ltd.</td><td>DS2A-Mk.II</td><td></td></tr><tr><td>coverslips (22x50 mm)</td><td>VWR</td><td>16004-314</td><td></td></tr><tr><td>cyanoacrylate adhesive</td><td>Krazy Glue</td><td>KG925</td><td>Ideally use the brush-on form for precision</td></tr><tr><td>data acquisition software</td><td>Axograph</td><td>N/A</td><td>Any equivalent software (e.g. pClamp) would work.</td></tr><tr><td>Dell Precision T1500 Tower Workstation Desktop</td><td>Dell</td><td>N/A</td><td>Catalog number will depend on specific computer - any computer will work as long as it can run electrophysiology acquisition software.</td></tr><tr><td>Digidata 1440A</td><td>Molecular Devices</td><td>1-2950-0367</td><td></td></tr><tr><td>digital timer</td><td>VWR</td><td>62344-641</td><td>4-channel Traceable timer</td></tr><tr><td>disposable absorbant pads</td><td>VWR</td><td>56616-018</td><td></td></tr><tr><td>dissector scissors</td><td>Fine Science Tools</td><td>14082-09</td><td></td></tr><tr><td>double-edge razor blades</td><td>Personna</td><td>BP9020</td><td></td></tr><tr><td>dual automatic temperature controller</td><td>Warner Instrument Corporation</td><td>TC-344B</td><td></td></tr><tr><td>dual-surface or laminar-flow optimized recording chamber</td><td>N/A</td><td>N/A</td><td>The chamber presented in this protocol is custom made. A commercial equivalent would be the RC-27L from Warner Instruments.</td></tr><tr><td>equipment rack</td><td>Automate Scientific</td><td>FR-EQ70&quot;</td><td>A rack is not strictly necessary but useful for organizing electrophysiology</td></tr><tr><td>Ethylene glycol-bis(2-aminoethyiether)- N,N,N&#039;,N&#039;-teetraacetic acid (EGTA)</td><td>Sigma Aldrich</td><td>324626-25GM</td><td></td></tr><tr><td>filter paper</td><td>Whatman</td><td>1004 070</td><td></td></tr><tr><td>fine scale</td><td>Mettler Toledo</td><td>XS204DR</td><td></td></tr><tr><td>Flaming/Brown micropipette puller</td><td>Sutter Instruments</td><td>P-97</td><td></td></tr><tr><td>glass petri dish (100 x 15 mm)</td><td>Corning</td><td>3160-101</td><td></td></tr><tr><td>glucose</td><td>Fisher Scientific</td><td>D16-1</td><td></td></tr><tr><td>Guanosine 5&amp;prime;-triphosphate sodium salt hydrate</td><td>Sigma Aldrich</td><td>G8877-250MG</td><td></td></tr><tr><td>ice buckets</td><td>Sigma Aldrich</td><td>BAM168072002-1EA</td><td></td></tr><tr><td>isoflurane vaporizer</td><td>General Anesthetic Services</td><td>Tec 3</td><td></td></tr><tr><td>lab tape</td><td>Fisher Scientific</td><td>15-901-10R</td><td></td></tr><tr><td>lens paper</td><td>Fisher Scientific</td><td>11-996</td><td></td></tr><tr><td>light source</td><td>Olympus</td><td>TH4-100</td><td></td></tr><tr><td>magnesium chloride solution (1 M)</td><td>Quality Biological</td><td>351-033-721EA</td><td></td></tr><tr><td>magnetic stir bars</td><td>Fisher Scientific</td><td>14-513-56</td><td>Catalog number will be dependent on the size of the stir bar.</td></tr><tr><td>micromanipulator</td><td>Luigs &amp;amp; Neumann</td><td>SM-5</td><td></td></tr><tr><td>micromanipulator (manual)</td><td>Scientifica</td><td>LBM-2000-00</td><td></td></tr><tr><td>microscope</td><td>Olympus</td><td>BX51WI</td><td></td></tr><tr><td>microspatula</td><td>Fine Science Tools</td><td>10089-11</td><td></td></tr><tr><td>monitor</td><td>Dell</td><td>2007FPb</td><td></td></tr><tr><td>MultiClamp 700B Microelectrode Amplifier</td><td>Molecular Devices</td><td>MULTICLAMP 700B</td><td>The MultiClamp 700B should include headstages, pipette holders, and a model cell.</td></tr><tr><td>N-(2-Hydroxyethyl)piperazine-N&amp;prime;-(2-ethanesulfonic acid), (HEPES)</td><td>Sigma Aldrich</td><td>H3375-25G</td><td></td></tr><tr><td>needle (20 gauge, 1.5 in length)</td><td>Becton, Dickinson</td><td>305176</td><td></td></tr><tr><td>nylon filament</td><td>YLI Wonder Invisible Thread</td><td>212-15-004</td><td>size 0.004. This cat. # is from Amazon.com</td></tr><tr><td>nylon mesh</td><td>Warner Instruments Corporation</td><td>64-0198</td><td></td></tr><tr><td>perstaltic pump</td><td>Harvard Apparatus</td><td>70-2027</td><td></td></tr><tr><td>Phosphocreatine di(tris) salt</td><td>Sigma Aldrich</td><td>P1937-1G</td><td></td></tr><tr><td>pipette holders</td><td>Molecular Devices</td><td>1-HL-U</td><td></td></tr><tr><td>platinum wire</td><td>World Precision</td><td>PT0203</td><td></td></tr><tr><td>polylactic acid (PLA) filament</td><td>Ultimaker</td><td>RAL 9010</td><td></td></tr><tr><td>potassium chloride</td><td>Sigma Aldrich</td><td>P3911-500G</td><td></td></tr><tr><td>potassium gluconate</td><td>Sigma Aldrich</td><td>1550001-200MG</td><td></td></tr><tr><td>potassium hydroxide</td><td>Sigma Aldrich</td><td>60377-1KG</td><td></td></tr><tr><td>razor blades</td><td>VWR</td><td>55411-050</td><td></td></tr><tr><td>roller clamp</td><td>World Precision Instruments</td><td>14041</td><td></td></tr><tr><td>scale</td><td>Mettler Toledo</td><td>PM2000</td><td></td></tr><tr><td>scalpel handle</td><td>Fine Science Tools</td><td>10004-13</td><td></td></tr><tr><td>slice harp</td><td>Warner</td><td>SHD-26GH/2</td><td></td></tr><tr><td>sodium bicarbonate</td><td>Fisher Chemical</td><td>S233-500</td><td></td></tr><tr><td>sodium chloride</td><td>Sigma Aldrich</td><td>S9888-1KG</td><td></td></tr><tr><td>sodium phosphate monobasic anhydrous</td><td>Fisher Chemical</td><td>S369-500</td><td></td></tr><tr><td>sodium pyruvate</td><td>Fisher Chemical</td><td>BP356-100</td><td></td></tr><tr><td>spatula</td><td>VWR</td><td>82027-520</td><td></td></tr><tr><td>spatula/spoon, large</td><td>VWR</td><td>470149-442</td><td></td></tr><tr><td>sterile scalpel blades</td><td>Feather</td><td>72044-10</td><td></td></tr><tr><td>stirrer / hot plate</td><td>Corning</td><td>6795-220</td><td></td></tr><tr><td>stopcock valves, 1-way</td><td>World Precision Instruments</td><td>14054</td><td></td></tr><tr><td>stopcock valves, 3-way</td><td>World Precision Instruments</td><td>14036</td><td></td></tr><tr><td>sucrose</td><td>Acros Organics</td><td>AC177142500</td><td></td></tr><tr><td>support for swivel clamps</td><td>Fisher Scientific</td><td>14-679Q</td><td></td></tr><tr><td>surgical scissors, sharp/blunt</td><td>Fine Science Tools</td><td>14001-12</td><td></td></tr><tr><td>syringe (1 mL)</td><td>Becton, Dickinson</td><td>309659</td><td></td></tr><tr><td>syringe (60 mL with Luer-Lok tip)</td><td>Becton, Dickinson</td><td>309653</td><td></td></tr><tr><td>three-pronged clamp</td><td>Fisher Scientific</td><td>05-769-8Q</td><td></td></tr><tr><td>tissue forceps, large</td><td>Fine Science Tools</td><td>11021-15</td><td></td></tr><tr><td>tissue forceps, small</td><td>Fine Science Tools</td><td>11023-10</td><td></td></tr><tr><td>transfer pipettes</td><td>Fisher Scientific</td><td>13-711-7M</td><td></td></tr><tr><td>tubing</td><td>Tygon</td><td>E-3603</td><td>ID 1/16 inch, OD 3/16 inch</td></tr><tr><td>tubing</td><td>Tygon</td><td>R-3603</td><td>ID 1/8 inch, OD 1/4 inch</td></tr><tr><td>vacuum grease</td><td>Dow Corning</td><td>14-635-5D</td><td></td></tr><tr><td>vibrating blade microtome</td><td>Leica</td><td>VT 1200S</td><td></td></tr><tr><td>vibration-dampening table with faraday cage</td><td>Micro-G / TMC-ametek</td><td>2536-516-4-30PE</td><td></td></tr><tr><td>volumetric flask (1 L)</td><td>Kimax</td><td>KIM-28014-1000</td><td></td></tr><tr><td>volumetric flask (2 L)</td><td>PYREX</td><td>65640-2000</td><td></td></tr><tr><td>warm water bath</td><td>VWR</td><td>1209&lt;br/&gt; &amp;nbsp;</td><td></td></tr></tbody></table>

acute mouse brain slicing to investigate spontaneous hippocampal network activity

急性啮齿动物脑切片提供了一种可伸缩的实验方法，利用电生理学、显微镜和药理学，通过单细胞分辨率深入了解神经回路的组织和功能。然而， 体外 实验设计的一个主要考虑因素是，不同的切片制剂在多大程度上回顾了 在体内 观察到的神经活动的自然形态。在完好无损的大脑中，海马网络产生反映动物行为状态的高度同步的种群活动，在清醒的完善状态或非REM睡眠期间发生的剧烈波纹复合物（SWR）就是例证。SWR 和其他形式的网络活动可以在适当的条件下在孤立的海马片中自发出现。为了将强大的脑切片工具包应用于海马网络活动的调查，有必要采用一种优化海马网络内组织健康和保持功能连接的方法。小鼠通过心电图注入冷蔗糖为基础的人工脑脊液。含有海马的水平切片被切成450μm的厚度，以保持突触连接。切片在接口式腔室中恢复，并转移到水下腔室进行录制。录音室设计用于以高流速对人造脑脊液进行双表面超级输液，以改善切片的氧合。此协议产生适合体 外复杂和自发的网络活动研究的健康组织。

此处介绍的是本协议中描述的 HEC 切片中的代表性录音。在接口保持室（图1C）中恢复后，切片被单独转移到水下录音室（图2B）。录音室使用长眠泵（图2A）提供碳水化合物饱和的ACSF。泵首先将ACSF从手持烧杯中拉入加热储层。碳水化合物线被放置在手持烧杯和加热储层中，以提供介质的连续氧合。脉动阻尼器由一系列充满空气的...

Watch this Scientific Journal Video about Acute Mouse Brain Slicing to Investigate Spontaneous Hippocampal Network Activity at JoVE.com

Acute Mouse Brain Slicing to Investigate Spontaneous Hippocampal Network Activity

此协议描述了从表现出自发锐波波纹活动的小鼠身上制作水平海马-内皮层 （HEC） 切片的准备工作。切片在简化的接口夹腔中孵育，并在水下条件下用快速流动的人工脑脊液进行录音，以促进组织氧合和网络水平活动的自发出现。

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 ...

acute-mouse-brain-slicing-to-investigate-spontaneous-hippocampal

Research

JoVE Journal

Neuroscience

8.9K 次观看.  Columbia University. 此协议描述了从表现出自发锐波波纹活动的小鼠身上制作水平海马-内皮层 （HEC） 切片的准备工作。切片在简化的接口夹腔中孵育，并在水下条件下用快速流动的人工脑脊液进行录音，以促进组织氧合和网络水平活动的自发出现。

视频: Acute Mouse Brain Slicing to Investigate Spontaneous Hippocampal Network Activity

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

Investigations on Alterations of Hippocampal Circuit Function Following Mild Traumatic Brain Injury

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological impairments 1. Two pervasive and disabling symptoms experienced by TBI survivors, memory deficits and a reduction in seizure threshold, are thought to be mediated by TBI-induced hippocampal dysfunction 2,3. In order to demonstrate how altered hippocampal circuit function adversely affects behavior after TBI in mice, we employ lateral fluid percussion injury, a commonly used animal model of TBI that recreates many features of human TBI including neuronal cell loss, gliosis, and ionic perturbation 4-6.
	Here we demonstrate a combinatorial method for investigating TBI-induced hippocampal dysfunction. Our approach incorporates multiple ex vivo physiological techniques together with animal behavior and biochemical analysis, in order to analyze post-TBI changes in the hippocampus. We begin with the experimental injury paradigm along with behavioral analysis to assess cognitive disability following TBI. Next, we feature three distinct ex vivo recording techniques: extracellular field potential recording, visualized whole-cell patch-clamping, and voltage sensitive dye recording. Finally, we demonstrate a method for regionally dissecting subregions of the hippocampus that can be useful for detailed analysis of neurochemical and metabolic alterations post-TBI.
	These methods have been used to examine the alterations in hippocampal circuitry following TBI and to probe the opposing changes in network circuit function that occur in the dentate gyrus and CA1 subregions of the hippocampus (see Figure 1). The ability to analyze the post-TBI changes in each subregion is essential to understanding the underlying mechanisms contributing to TBI-induced behavioral and cognitive deficits. 
	The multi-faceted system outlined here allows investigators to push past characterization of phenomenology induced by a disease state (in this case TBI) and determine the mechanisms responsible for the observed pathology associated with TBI.

A multi-faceted approach to investigating functional changes to hippocampal circuitry is explained. Electrophysiological techniques are described along with the injury protocol, behavioral testing and regional dissection method. The combination of these techniques can be applied in similar fashion for other brain regions and scientific questions.

调查轻度创伤性脑损伤海马电路功能的改变

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological ...

Neural Activity Propagation in an Unfolded Hippocampal Preparation with a Penetrating Micro-electrode Array

This protocol describes a method for preparing a new in vitro flat hippocampus preparation combined with a micro-machined array to map neural activity in the hippocampus. The transverse hippocampal slice preparation is the most common tissue preparation to study hippocampus electrophysiology. A longitudinal hippocampal slice was also developed in order to investigate longitudinal connections in the hippocampus. The intact mouse hippocampus can also be maintained in vitro because its thickness allows adequate oxygen diffusion. However, these three preparations do not provide direct access to neural propagation since some of the tissue is either missing or folded. The unfolded intact hippocampus provides both transverse and longitudinal connections in a flat configuration for direct access to the tissue to analyze the full extent of signal propagation in the hippocampus in vitro. In order to effectively monitor the neural activity from the cell layer, a custom made penetrating micro-electrode array (PMEA) was fabricated and applied to the unfolded hippocampus. The PMEA with 64 electrodes of 200 &#181;m in height could record neural activity deep inside the mouse hippocampus. The unique combination of an unfolded hippocampal preparation and the PMEA provides a new in-vitro tool to study the speed and direction of propagation of neural activity in the two-dimensional CA1-CA3 regions of the hippocampus with a high signal to noise ratio.

We have developed an in vitro unfolded hippocampus which preserves CA1-CA3 array of neurons. Combined with the penetrating micro-electrode array, neural activity can be monitored in both the longitudinal and transverse orientations. This method provides advantages over hippocampal slice preparations as the propagation in the entire hippocampus can be recorded simultaneously.

神经活动的传播的展开海马准备具有穿透微电极阵列

This protocol describes a method for preparing a new in vitro flat hippocampus preparation combined with a micro-machined array to map neural activity in ...

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is tightly regulated during development and neuronal maturation. Importantly, deficits in synapse number can lead to cognitive dysfunction. Therefore, the evaluation of synapse number is an integral part of neurobiology. However, as synapses are small and highly compact in the intact brain, the assessment of absolute number is challenging. This protocol describes a method to easily identify and evaluate synapses in hippocampal rodent slices using immunofluorescence microscopy. It includes a three-step procedure to evaluate synapses in high-quality confocal microscopy images by analyzing the co-localization of pre- and postsynaptic proteins in hippocampal slices. It also explains how the analysis is performed and gives representative examples from both excitatory and inhibitory synapses. This protocol provides a solid foundation for the analysis of synapses and can be applied to any research investigating the structure and function of the brain.

A protocol for accurately identifying and analyzing synapses in hippocampal slices using immunofluorescence is outlined in this article.

海马鼠脑切片的突触密度评价

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is ...

Recording and Modulation of Epileptiform Activity in Rodent Brain Slices Coupled to Microelectrode Arrays

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) is a promising approach when pharmacological treatment fails or neurosurgery is not recommended. Acute brain slices coupled to microelectrode arrays (MEAs) represent a valuable tool to study neuronal network interactions and their modulation by electrical stimulation. As compared to conventional extracellular recording techniques, they provide the added advantages of a greater number of observation points and a known inter-electrode distance, which allow studying the propagation path and speed of electrophysiological signals. However, tissue oxygenation may be greatly impaired during MEA recording, requiring a high perfusion rate, which comes at the cost of decreased signal-to-noise ratio and higher oscillations in the experimental temperature. Electrical stimulation further stresses the brain tissue, making it difficult to pursue prolonged recording/stimulation epochs. Moreover, electrical modulation of brain slice activity needs to target specific structures/pathways within the brain slice, requiring that electrode mapping be easily and quickly performed live during the experiment. Here, we illustrate how to perform the recording and electrical modulation of 4-aminopyridine (4AP)-induced epileptiform activity in rodent brain slices using planar MEAs. We show that the brain tissue obtained from mice outperforms rat brain tissue and is thus better suited for MEA experiments. This protocol guarantees the generation and maintenance of a stable epileptiform pattern that faithfully reproduces the electrophysiological features observed with conventional field potential recording, persists for several hours, and outlasts sustained electrical stimulation for prolonged epochs. Tissue viability throughout the experiment is achieved thanks to the use of a small-volume custom recording chamber allowing for laminar flow and quick solution exchange even at low (1 mL/min) perfusion rates. Quick MEA mapping for real-time monitoring and selection of stimulating electrodes is performed by a custom graphic user interface (GUI).

We illustrate how to perform recording and electrical modulation of 4-aminopyridine-induced epileptiform activity in rodent brain slices using microelectrode arrays. A custom recording chamber maintains tissue viability throughout prolonged experimental sessions. Live electrode mapping and selection of stimulating pairs are performed by a custom graphical user interface.

鼠脑切片偶联微电极阵列癫痫活性的记录与调控

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) ...

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

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

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

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

Horizontal Hippocampal Slices of the Mouse Brain

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as well as the manifestation of severe brain disorders, including Alzheimer&#8217;s disease and epilepsy. The hippocampus receives a high degree of intra- and inter-connectivity, securing a proper communication with internal and external brain structures. This connectivity is accomplished via different informational flows in the form of fiber pathways. Brain slices are a frequently used methodology when exploring neurophysiological functions of the hippocampus. Hippocampal brain slices can be used for several different applications, including electrophysiological recordings, light microscopic measurements as well as several molecular biological and histochemical techniques. Therefore, brain slices represent an ideal model system to assess protein functions, to investigate pathophysiological processes involved in neurological disorders as well as for drug discovery purposes.
There exist several different ways of slice preparations. Brain slice preparations with a vibratome allow a better preservation of the tissue structure and guarantee a sufficient oxygen supply during slicing, which present advantages over the traditional use of a tissue chopper. Moreover, different cutting planes can be applied for vibratome brain slice preparations. Here, a detailed protocol for a successful preparation of vibratome-cut horizontal hippocampal slices of mouse brains is provided. In contrast to other slice preparations, horizontal slicing allows to keep the fibers of the hippocampal input path (perforant path) in a fully intact state within a slice, which facilitates the investigation of entorhinal-hippocampal interactions. Here, we provide a thorough protocol for the dissection, extraction, and acute horizontal slicing of the murine brain, and discuss challenges and potential pitfalls of this technique. Finally, we will show some examples for the use of brain slices in further applications.

This article aims to describe a systematic protocol to obtain horizontal hippocampal brain slices in mice. The objective of this methodology is to preserve the integrity of hippocampal fiber pathways, such as the perforant path and the mossy fiber tract to assess dentate gyrus related neurological processes.

鼠脑水平海马片

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as ...

Preparation of Acute Slices from Dorsal Hippocampus for Whole-Cell Recording and Neuronal Reconstruction in the Dentate Gyrus of Adult Mice

Although the general architecture of the hippocampus is similar along its longitudinal axis, recent studies have revealed prominent differences in molecular, anatomical and functional criteria suggesting a division into different sub-circuits along its rostro-caudal extent. Owing to differential connectivity and function the most fundamental distinction is made between the dorsal and the ventral hippocampus, which are preferentially involved in spatial and emotional processing, respectively. Accordingly, in vivo work regarding spatial memory formation has focused on the dorsal hippocampus.
In contrast, electro-physiological in vitro recordings have been preferentially performed on intermediate-ventral hippocampus, largely motivated by factors like slice viability and circuit integrity. To allow for direct correlation of in vivo data on spatial processing with in vitro data we have adapted previous sectioning methods to obtain highly viable transverse brain slices from the dorsal-intermediate hippocampus for long-term recordings of principal cells and interneurons in the dentate gyrus. As spatial behavior is routinely analyzed in adult mice, we have combined this transversal slicing procedure with the use of protective solutions to enhance viability of brain tissue from mature animals. We use this approach for mice of about 3 months of age. The method offers a good alternative to the coronal preparation which is frequently used for in vitro studies on dorsal hippocampus. We compare these two preparations in terms of quality of recordings and preservation of morphological features of recorded neurons.

We present a protocol to prepare acute-slices from the dorsal-intermediate hippocampus of mice. We compare this transversal preparation with coronal slicing in terms of quality of recordings and preservation of morphological features of recorded neurons.

从多萨尔海马的急性切片准备全细胞记录和神经元重建在成人小鼠的登特陀螺

Although the general architecture of the hippocampus is similar along its longitudinal axis, recent studies have revealed prominent differences in ...

Craniotomy Procedure for Visualizing Neuronal Activities in Hippocampus of Behaving Mice

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit functions in systems neuroscience. However, high absorbance and scattering of light in mammalian tissue limit intravital imaging mostly to superficial brain regions, leaving deep-brain areas, such as the hippocampus, out of reach for optical microscopy. In this video, we show the preparation and implantation of the custom-made imaging window to enable chronic in vivo imaging of the dorsal hippocampal CA1 region in head-fixed behaving mice. The custom-made window is supplemented with an infusion cannula that allows targeted delivery of viral vectors and drugs to the imaging area. By combining this preparation with wide-field imaging, we performed a long-term recording of neuronal activity using a fluorescent calcium indicator from large subsets of neurons in behaving mice over several weeks. We also demonstrated the applicability of this preparation for voltage imaging with single-spike resolution. High-performance genetically encoded indicators of neuronal activity and scientific CMOS cameras allowed the recurrent visualization of subcellular morphological details of single neurons at high temporal resolution. We also discuss the advantages and potential limitations of the described method and its compatibility with other imaging techniques.

This article demonstrates the preparation of a custom-made imaging window supplemented with infusion cannula and its implantation onto the CA1 region of the hippocampus in mice.

行为小鼠希波坎帕的神经元活动可视化的颅骨切除术

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit ...

Recording of Local Field Potential in Mouse Hippocampal-Entorhinal Cortex Slices

Source: Whitebirch, A. C. <a href="https://app.jove.com/v/61704">Acute Mouse Brain Slicing to Investigate Spontaneous Hippocampal Network Activity.</a> J. Vis. Exp. (2020)
The video demonstrates a procedure to record the local field potential (LFP) in mouse hippocampal-entorhinal cortex slices. Electrical stimulation is provided at the CA3 stratum radiatum of the hippocampus, which causes changes in the postsynaptic potential at the CA1 stratum pyramidale. The combined change in the membrane potential of CA1 neurons, termed the local field potential, is recorded.

小鼠海马-内嗅皮层切片中局部场电位的记录

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Transcardial perfusion

	Suspend ...