Name: acute mouse brain slicing to investigate spontaneous hippocampal network activity
Uploaded: 2020-08-28T14:00:00
Description: Watch this Scientific Journal Video about Acute Mouse Brain Slicing to Investigate Spontaneous Hippocampal Network Activity at JoVE.com

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...

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?...

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>
		</tr>
		<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&#8242;-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&#34;</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&#39;,N&#39;-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&#8242;-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 &#38; 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&#8242;-(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 
			&#160;</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 ...

Using this protocol, researchers can investigate the mechanisms underlying hippocampal network oscillations in acute mouse brain slice preparations. In this preparation, recordings are performed under submerged conditions in slices generating spontaneous network oscillations, allowing pharmacological and optogenetic techniques to be performed, and single-cell recordings to be visualized. For transcardial perfusion, immediately before removing the mouse from the isoflurane chamber, fill a Petri dish lid with three to five millimeters of chilled sucrose solution, and fill the Petri dish bottom with approximately one centimeter of chilled sucrose solution.Quickly transfer the anesthetized mouse to the left absorbent pad, and use three pieces of tape to secure the forelimbs and tail. Using large tissue forceps and surgical scissors, tent the skin and make a lengthwise incision from the bottom of the sternum to the top of the chest. Use the forceps to pull up on the sternum and use the scissors to cut through the diaphragm.Use the scissors to cut through the rib cage on each side in one large motion toward the point at which the forelimb meets the body, and use the forceps to position the front of the rib cage toward the head. Use the scissors to make a horizontal cut to completely remove the ribs, and use the forceps to hold the heart in place while inserting a 20-gauge perfusion needle into the left ventricle. When the needle is in place, use small dissection scissors to make an incision in the right atrium, and allow the blood to be flushed out of the circulatory system.To extract the brain, after decapitation, use small bond scissors to make two lateral cuts through the skull toward the midline at the front of the skull near the eyes, and make two additional cuts on either side of the base of the skull. Immerse the head in the glass Petri dish, and use the scissors to cut along the midline across the entire length of the skull, while pulling up with the scissors to minimize damage to the underlying brain tissue. Use small tissue forceps to firmly grasp each side of the skull, and to lift the bone up and away from the brain to open the cranium like a book.Using the fingers of the left hand to hold the flaps of the skull open, insert the microspatula under the brain near the olfactory bulbs, and flip the brain out of the skull into the sucrose. Use the microspatula to sever the brainstem, and wash the brain to remove any residual blood, fur, or tissues. Use the large spatula to transfer the brain to the glass Petri dish lid, and use half of a double-edged razor blade to make a coronal cut in the most anterior portion of the brain, including the olfactory bulbs.Next, make a coronal cut to remove the cerebellum, and apply cyanoacrylate adhesive to an agar ramp. Use forceps to briefly dry the brain on a piece of filter paper, before placing the tissue into the adhesive on the agar ramp, ventral-side down. Place the slicing platform into the slicing chamber of a microtome, and completely cover the setup with chilled sucrose solution.Use the large spatula to stir some sucrose slurry into the chamber, melting any frozen sucrose and rapidly bringing down the mixture temperature to one to two degrees Celsius. Cut slices to a thickness of 450 microns at 0.07 millimeters per second slicing speed. As each slice is freed, use the small-tissue forceps and a sharp scalpel to separate the two hemispheres, and to cut away tissue until the slice consists primarily of the hippocampus and perihippocampal regions.Use a plastic transfer pipette to transfer the slices individually to an interface recovery chamber containing warmed aCSF. With the slices positioned at the interface of the aCSF and air, and with only a thin meniscus of aCSF covering the slices. When all of the slices have been acquired, tightly close the chamber to allow the slices to recover at 32 degrees Celsius for 30 minutes.At the end of the recovery period, place the chamber on a stirrer set to a slow speed to promote aCSF circulation within the chamber. To record local field potentials, fill a 400 milliliter beaker with carbogen-bubbled aCSF, and place one end of the pump tubing into the beaker. Turn on a peristaltic pump at 8 to 10 milliliters per minute to direct aCSF from the 400 milliliter beaker to a heated reservoir, and from the reservoir to the recording chamber at 32 degrees Celsius.Next, briefly clamp the tubing and turn off the pump to pause the flow. Using fine forceps, transfer a brain tissue slice to the recording chamber by the corner of the lens paper the tissue is resting on, slice down. Peel away the lens paper, leaving the slice emerged in the recording chamber, and use a harp to secure the slice.Using a manual micromanipulator, slowly advance the tip of a sodium chloride-filled stimulation pipette into the surface of the slice at a 30 to 45 degree angle, then use a second micromanipulator to slowly advance the tip of an aCSf-filled local field potential pipette into the region of interest at a 30 to 45 degree angle, and record the local field potential of the sample according to standard protocols. In this figure, representative recordings from hippocampal entorhinal cortex slices, prepared according to the protocol as demonstrated, can be observed. In healthy slices, electrical stimulation should produce a field post-synaptic potential with a small pre-synaptic fiber volley and a large post-synaptic potential with a rapid initial descent.Spontaneous sharp-wave ripples should also be visible as positive deflections in the local field potential in the stratum pyramidale. In suboptimal slices, evoked field post-synaptic potentials demonstrate a large fiber volley, and a relatively small post-synaptic potential, and such slices do not exhibit spontaneous sharp-wave ripples. In vitro sharp-wave ripples demonstrate a positive field potential in the stratum pyramidale layer, with an overlaid high-frequency oscillation paired with a negative field potential in the stratum radiatum layer.As illustrated, sharp-wave ripples in hippocampal entorhinal cortex slices originate within CA2/CA3 recurrent circuits, and propagate to CA1. It is essential to bubble carbogen into the solutions throughout the procedure, to ensure that the sucrose solution is chilled, and to perform each step as quickly as possible. After preparing these slices, extracellular or intracellular recordings can be performed, along with optogenetic or pharmacological experiments to determine how different cell types contribute to the function of neural networks.

Research

JoVE Journal

Neuroscience

Combining Transcranial Magnetic Stimulation and fMRI to Examine the Default Mode Network

结合经颅磁刺激和功能磁共振成像检查默认模式网络

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful ...

科研

神经科学

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

在自发活动的测定<em&gt;果蝇</em

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

在器官型海马脑片搭配全细胞记录

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

Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature

切片热门：成人急性脑切片在生理温度

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments ...

Immunohistochemical Visualization of Hippocampal Neuron Activity After Spatial Learning in a Mouse Model of Neurodevelopmental Disorders

海马神经元活动的可视化免疫组化空间后学习神经发育障碍的小鼠模型

Induction of phosphorylated extracellular-regulated kinase (pERK) is a reliable molecular readout of learning-dependent neuronal activation. Here, we ...

Induction of an Isoelectric Brain State to Investigate the Impact of Endogenous Synaptic Activity on Neuronal Excitability In Vivo

Induction of an Isoelectric Brain State to Investigate the Impact of Endogenous Synaptic Activity on Neuronal Excitability In Vivo

等电脑国家的诱导研究内源性突触活动对神经元兴奋性影响<em&gt;在体内</em

The way neurons process information depends both on their intrinsic membrane properties and on the dynamics of the afferent synaptic network. In ...

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

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

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

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 ...

A Preclinical Model to Assess Brain Recovery After Acute Stroke in Rats

评估大鼠急性中风后脑恢复的临床前模型

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery ...

Preparation of Acute Human Hippocampal Slices for Electrophysiological Recordings

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 ...