Name: minimizing hypoxia in hippocampal slices from adult and aging mice
Uploaded: 2020-07-02T14:00:00
Description: Watch this Scientific Journal Video about Minimizing Hypoxia in Hippocampal Slices from Adult and Aging Mice at JoVE.com

我感谢卡拉·沙茨博士的建议和支持，感谢芭芭拉·布罗特博士和米歇尔·德鲁斯博士认真阅读手稿。这项工作得到了NIH EY02858和马瑟斯慈善基金会对CJS的资助。

该协议是根据国家卫生研究院实验室动物护理和使用指南执行的，并经斯坦福大学机构动物护理和使用委员会批准。方法也符合神经科学学会关于在神经科学研究中使用动物和人类的政策。
注：所有小鼠都保持在无病原体的环境中。除非另有说明，否则此处使用混合C57Bl/6 x SV/129J遗传背景的野生小鼠。
1. 设置
<ol><li>准备 1 L 的 1x aCSF （以 mM）： 125 NaCl，...

<ol>
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此处描述的协议表明，从成年和老化的小鼠获得的海马切片在切割后可以保持健康和存活数小时。使用此协议准备的切片适用于修补夹录制以及 CA1 区域中的持久现场录制。
此协议中有两个关键步骤。第一步是使用冰冷的溶液的跨心灌注步骤。快速清除血液是由肝脏颜色的快速变化发出的信号。提取的大脑应该是非白色的颜色。如果大脑保持粉红色，这意味着全身血液没有被...

哺乳动物急性脑切片制剂的出现促进了细胞和突触水平的实验，这些实验以前只能在无脊椎动物制剂（如Aplysia1）中实现。急性海马切片的发展具有特殊意义，因为它是一种负责工作记忆和上下文形成的结构，并且具有一种专门的三突触电路，易于生理操作。然而，绝大多数急性脑片仍然由相对年轻的小鼠和大鼠组成，因为它更容易保存健康的神经元和回路，而且这些切片在较长时间2、3、43期间仍然存活。2在这里，我们引入对标准切片协议的修改，提高成年和老化小鼠急性海马切片的生存能力。
哺乳动物大脑白症的长期外生力的主要障碍是，一旦血液在斩首后停止流向大脑，就迅速发生最初的缺氧损伤。缺氧导致大脑中主要能量资源的快速代谢消耗，磷肌酸（P-肌酸）损失速度最快，其次是葡萄糖、三磷酸腺苷（ATP）和糖原4。保存ATP对于大脑切片的长期健康特别重要，因为ATP需要通过Na-K ATPase保持膜电位，从而维持神经活动5，6。,6成年啮齿动物大脑中的ATP水平为±2.5 mM，在斩首后约1分钟、7、8时，在斩首后20秒内急剧下降，达到基底稳定状态（±0.5 mM）。4,7,8在幼年动物中，观察ATP相同下降需要更长的时间（±2分钟）;与苯巴比他麻醉，它进一步放缓到4分钟4。这些考虑表明，防止ATP和其他能量资源的丧失是防止大脑缺氧损伤的必要策略，反过来又可以长期维持大脑切片的健康，特别是在成年动物中。
低温减慢新陈代谢。因此，已经证明适度的体温过低可以保护大脑能量储备：在幼年动物中，将体温从37°C降低6度至31°C，将ATP水平控制在4小时以上的控制缺氧9中，将ATP水平控制在正常水平的80%左右。P-肌酸水平同样保留，以及整体磷酸化电位9。这表明，在斩首前降低体温可能是神经保护，因为ATP的接近正常水平可以通过切片切割和切片恢复期保持。
由于在斩首时无法完全防止ATP下降，预计Na-K ATPase的功能会部分受损，然后通过被动钠流入去极化。由于被动钠的涌入后，水进入细胞，它导致细胞毒性水肿，并最终皮刀病。在成年大鼠中，用蔗糖代替蔗糖是减轻细胞毒性水肿10、11,负担的成功策略。最近，减少钠通道渗透性12的甲基化有机化合物已经证明比蔗糖提供更有效的保护，特别是在成年小鼠的切片中，N-甲基-D-葡萄糖,胺（NMDG）在不同年龄和大脑区域,13、14、15、16中13,14应用得最广泛。15
许多大脑切片方案涉及仅在切片切割步骤期间使用低温，有时与 Na+离子替换策略16，,17 相结合。在幼年动物中，这些协议似乎提供了足够的神经保护，因为大脑可以在斩首后迅速提取，因为头骨仍然薄，易于切除3。然而，这种策略并不能从成年动物身上产生健康的切片。随着时间的推移，一些研究成年啮齿动物的实验室已经引进了转心灌注与冰冷的溶液，以降低动物的体温，因此在斩首之前对大脑的缺氧损伤。该程序被成功地应用于产生小脑片18，中脑切片19，新皮质切片11，20，腹皮层11,2021，大鼠海马体10，22，23，嗅球24，腹状盘,22,23状体25，视觉皮层26。
尽管跨心灌注和Na+离子替代在准备大鼠切片和小鼠某些大脑区域提供了优势，但小鼠海马仍然是保护13，20,缺氧的最具挑战性的区域之一。迄今为止，从老化的小鼠和神经退化的小鼠模型中切分海马体的最常见方法之一涉及分离的海马27的经典快速切片。在此描述的协议中，我们通过将脱毛前引入体温过低，通过用冰冷的 Na+- 基于 NMDG 的免费人工脑脊髓液 （NMDG） 来将动物转行，从而最大限度地减少成人大脑中 ATP 的损失。然后切成冰片在冰冷的 Na+ 无Nmdg - acsf。通过这种增强的协议，我们从成年和老化的小鼠获得急性海马切片，切片后健康长达10小时，适合长期现场记录和贴片夹研究。

<table>
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			<td>&#8220;60 degree&#8221; tool</td>
			<td>made in-house</td>
			<td></td>
			<td></td>
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			<td>#10 scalpel blade</td>
			<td>Bard-Parker (Aspen Surgical)</td>
			<td>371110</td>
			<td></td>
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			<td>1M CaCl2</td>
			<td>Fluka Analytical</td>
			<td>21114</td>
			<td></td>
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			<td>95%O2/5%CO2</td>
			<td>Praxiar or another local supplier</td>
			<td></td>
			<td></td>
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			<td>Acepromazine maleate (AceproJect)</td>
			<td>Henry Schein</td>
			<td>5700850</td>
			<td></td>
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			<td>Agar</td>
			<td>Fisher</td>
			<td>BP1423-500</td>
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			<td>Beakers, measuring cylinders, reagent bottles</td>
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			<td>Brushes size 00-2</td>
			<td>Ted Pella</td>
			<td></td>
			<td>Crafts stores are another source of soft brushes, with larger selection and better quality than Ted Pella.</td>
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			<td>CCD camera</td>
			<td>Olympus</td>
			<td>XM10</td>
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			<td>Choline bicarbonate</td>
			<td>Pfalz &#38; Bauer</td>
			<td>C21240</td>
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			<td>Cyanoacrilate glue</td>
			<td>Krazy glue</td>
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			<td>Singles</td>
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			<td>Decapitation scissors</td>
			<td>FST</td>
			<td>14130-17</td>
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			<td>Feather blades</td>
			<td>Feather</td>
			<td>FA-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper #2</td>
			<td>Whatman</td>
			<td></td>
			<td>Either rounds or pieces cut from a bigger sheet work well.</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>A. Dumont &#38; Fils</td>
			<td>Inox 3c</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass bubblers (Robu glass borosillicate microfilter candles) - porosity 3</td>
			<td>Robuglas.com</td>
			<td>18103 or 18113</td>
			<td>Glass bubblers are more expensive than bubbling stones used in aquaria. However, they are easy to clean and sterilize, and can last a long time.</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Fisher</td>
			<td>A144SI-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Ice buckets</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P4504</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine HCl (KetaVed)</td>
			<td>VEDCO</td>
			<td>NDC 50989-996-06</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>P0662</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica Tissue slicer VT1000S</td>
			<td></td>
			<td></td>
			<td>The cutting settings are 1 mm horizontal blade amplitude, frequency dial at 9, and speed setting at 2</td>
		</tr>
		<tr>
			<td>Magnetic stirrers and stir bars</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mg2SO4 x 7H2O</td>
			<td>Sigma-Aldrich</td>
			<td>230391</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>M9272</td>
			<td></td>
		</tr>
		<tr>
			<td>MilliQ water machine</td>
			<td>Millipore</td>
			<td></td>
			<td>Source for 18 Mohm water</td>
		</tr>
		<tr>
			<td>Na-ascorbate</td>
			<td>Sigma-Aldrich</td>
			<td>A4035</td>
			<td></td>
		</tr>
		<tr>
			<td>Na-pyruvate</td>
			<td>Sigma-Aldrich</td>
			<td>P8574</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>EMD</td>
			<td>SX0320-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle 27G1/2</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NMDG</td>
			<td>Sigma-Aldrich</td>
			<td>M2004</td>
			<td></td>
		</tr>
		<tr>
			<td>Paper tape</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Cole-Parmer</td>
			<td>#7553-70</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic pump head</td>
			<td>Cole-Parmer</td>
			<td>Masterflex #7518-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Personna blades</td>
			<td></td>
			<td>Personna double edge</td>
			<td>Amazon</td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Recovery chamber</td>
			<td>in-house made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel blade handle size 3</td>
			<td>Bard-Parker (Aspen Surgical)</td>
			<td>371030</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors angled blade</td>
			<td>FST</td>
			<td>14081-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Single edge industrial razor blade #9</td>
			<td>VWR</td>
			<td>55411</td>
			<td></td>
		</tr>
		<tr>
			<td>Spatulas</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer pipettes</td>
			<td>Samco Scientific</td>
			<td>225</td>
			<td></td>
		</tr>
		<tr>
			<td>Upright microscope</td>
			<td>Olympus BX51WI</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine HCl (XylaMed)</td>
			<td>VetOne</td>
			<td>510650</td>
			<td></td>
		</tr>
	</tbody>
</table>

minimizing hypoxia in hippocampal slices from adult and aging mice

急性海马切片使几代神经科学家能够详细探索突触、神经元和电路特性，并具有高保真度。没有这种经典的准备，不可能探索LTP和LTC机制、单神经元树突计算以及电路中依赖于经验的变化。然而，除了少数例外，大多数使用急性海马切片的基础研究都是使用相对年轻的啮齿动物的切片进行，+P20-P40，即使突触和内在兴奋机制有一个长长的发育尾巴，达到过去的P60。使用年轻的海马切片的主要吸引力是维持神经元健康，辅之以对低氧损伤的耐受性。然而，有必要了解神经功能在更成熟的发展阶段，进一步突出了各种动物模型的神经退行性疾病，需要老化的大脑准备。在这里，我们描述了对急性海马切片制剂的修改，该制剂可靠地从成年和老化的小鼠海马中提供健康的切片。该协议的关键步骤是转心灌注和切割与无冰钠无NMDG-aSCF。总之，这些步骤一起减轻脱毛时 ATP 中缺氧引起的下降，以及被动钠通量引起的细胞毒性水肿。我们演示如何使用振动微原子切割海马体和皮层的横切片。用这种方式获得的急性海马切片在数小时的录音中可靠健康，适用于现场记录和靶向贴片记录，包括荧光标记神经元的目标。

我们应用上述协议，从CamKIIa-Cre+生成海马切片;混合遗传背景的WT小鼠C57Bl/ 6 x SV/ 129J，在P> 120。在CA1场（图2A）和亚分细胞（图2B）中，当在红外差分对比度显微镜（IR-DIC）下观察到时，会出现低对比度，这是切片制备中健康细胞的标志。通过这种制备，可高千兆欧姆密封件（>90%）当目标为表面以下约 20 微米的最健康细胞时，通常实现。对于此成功率，...

Watch this Scientific Journal Video about Minimizing Hypoxia in Hippocampal Slices from Adult and Aging Mice at JoVE.com

Minimizing Hypoxia in Hippocampal Slices from Adult and Aging Mice

这是一个急性切片制备从成人和老化的小鼠海马，利用转心灌注和切片切割与冰冷的NMDG-aCSF，以减少对组织的低氧损伤。生成的切片在几个小时内保持健康，并适用于长期的贴片夹和现场记录。

从成人和老化小鼠的河马切片中尽量减少低氧

Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high ...

这里提出的协议减少了成人和老化小鼠海马切片制备过程中的过度缺氧损伤，从而消除了研究成熟和老化神经回路功能的主要障碍。在无钠溶液中引入低温，可产生在切割后长达 10 小时的海马切片，适用于长期现场记录和贴片夹研究。这种制备海马切片的方法对于神经退行性疾病的动物模型特别相关，根据定义，这些动物模型需要老化的大脑准备。该协议中最关键的一步是通过心电图通过心电图大量引入低温，并采用冰冷的溶液。通过一些实践，研究人员将能够可靠地执行此步骤。首先为恢复室和后续录音准备一升的 aCSF 解决方案。然后准备300毫升的NMDG-aCSF的转心和切割步骤。将振动微切板切割托盘和安装盘放在零下 20 摄氏度的冰柜中。要准备恢复室，请将回收室填充到切片保持网格的上方，然后启动气泡器，将室室保持室温。将整个 300 毫升 NMDG-aCSF 冷藏在冰柜中，直到冰晶开始在瓶子的表面和墙壁上形成。将瓶子与冷藏和NMDG-aCSF放在冰上，然后泡上，将溶液保持零至两摄氏度。将组织安装盘从冰柜中取出，并根据需要擦干。切出一块5%的阿加大小，鼠标大脑，并粘在光盘的中心使用薄层的氰丙烯酸酯胶水。将带粘胶的盘放在冰上，用纸巾盖住，直到准备好使用。将切割托盘从冰柜中取出，放入显微原子中，然后用冰包围，然后装上刀片。提前准备所有工具进行大脑解剖。设置用于转心灌注的心气吸管泵。用冰的 NMDG-aCSF 将泵管的一侧插入瓶子中，用 27 表针将另一侧安装。将泵速设置为每分钟约 3.5 毫升。以这个速度，NMDG-aCSF 的流出是快速行程，而不是连续流。将鼠标放在尿布的背上。在继续之前，通过执行一个手趾捏，确认小鼠在麻醉的手术平面上。鼠标应无响应，然后贴在其前腿和后腿上，使胸部和腹部暴露。切掉胸骨下到喉咙的一大片皮肤。用钳子抓住胸骨，轻轻抬起胸骨，开始切开两侧的肋骨笼，直到胸腔暴露。切开隔膜，留下肋骨笼的皮瓣通过一块薄薄的肌肉连接。应该可以把它放在一边，而不会让它掉回暴露的胸腔上。检查心脏是否仍在跳动，并确保大部分肝脏可见。将针头插入左心室，其颜色看起来比右侧浅。要稳定针头，请将针头穿过身体左侧的剩余肋骨。找到深红色的右中庭，用小剪刀切开。血液应该开始流出。启动泵并观察肝脏，肝脏的颜色会从红色更改为棕色。监测肝脏颜色并继续灌注，直到肝脏变成淡棕色。运行泵几分钟。动物的体温应该降至28至29摄氏度，鼻子应该冷到触摸。用大斩首剪刀斩首鼠标，然后用10号刀片的手术刀切开头骨顶部的皮肤。用小角度剪刀，在中线切头骨。接下来，使用三号钳子撬开头骨的左右半部分，小心地用它带走杜拉。取出大脑，用铲子挖出大脑，然后放入冰上NMDG-aCSF溶液中，将其取出。留在那里长达一分钟。将大脑从 NMDG-aCSF 中拿出来，放在一张滤纸上。用 60 度工具从前脑角端的中线剪切并移除 60 度组织楔块。使用安装表面的切割边，因为它为海马切片提供了适当的角度。用手术刀将半球向下半球分离，并粘在安装盘上。从冰上拿去安装盘，如果需要，擦干。然后将每个半球粘在阿加块前面，切面向下。确保两个半球的腹侧接触美洲狮块，并确保两个半球的后侧朝向刀片。当粘在切割侧时，每个半球应相对于刀片的方向，以确保在原位位置的侧海马体的横向切片。将圆盘与半球一起淹没到含有冰冷碳化 NMDG-aCSF 的切割室中。切割 400 微米部分，然后使用带切断尖端的一次性转移移液器将切片转移到室温下含有碳化 NMDG-aCSF 的回收室。继续切割其余部分，并将它们转移到恢复室，不超过 10 分钟，从后海马区总共切割 8 到 10 片。在室温下孵育切片约两小时，然后进行记录。该协议用于从成年小鼠生成海马切片。在红外差分对比度显微镜下观察到，CA1 场和亚分细胞中的大量金字塔细胞以低对比度出现，这是健康细胞的标志。从6个月大的小鼠的CA1神经元中可以获取贴片夹记录。在下面的示例实验中，在NMDA-LTD相对于控制条件进行诱导后，测量了微型兴奋柱突触电流的频率。NMDA-LTD 诱导后神经元中微缩兴奋后电流的频率较低，表明CA1中概要的活性依赖性修剪。未检测到振幅变化。在mEPSC记录期间，CA1细胞也充满了生物细胞素和完整的树突状，以及金字塔神经元的健康细胞习性在这里可以看到。荧光染料在整个细胞中的强劲分布允许分析树突和树突脊柱在不同条件下。观察到CA3-CA1突触的长期强效，或LTP约170%，这表明，在成人小鼠准备的切片中，存在LTP所需的信号级联的维持。一个强大的现场兴奋后突触电位信号表明，网络连接也得到保留。该协议的两个关键方面是通过冰冷的溶液进行转心，诱导体温过低，并在防止细胞毒性水肿的解决方案中引入NMDG作为钠离子替代品。使用这些预防措施，可以从任何大脑区域准备急性切片。此外，以这种方式准备的切片可用于使用双光子或宽场显微镜进行钙和电压成像。该协议可以作为对来自老化动物的急性海马切片进行标准化准备的基础，从而促进神经退行性疾病机制背景下研究的比较。

Research

JoVE Journal

Neuroscience

Preparation of Oligomeric &beta;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

Preparation of Oligomeric ÃƒÅ½Ã‚Â²-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

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

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 ...

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

从产前小鼠海马神经元的分离和培养

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

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

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 ...

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 ...

Investigation of Synaptic Tagging/Capture and Cross-capture 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 ...

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 ...

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Optogenetic 在行为小鼠海马θ振荡中的作用

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools ...

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 ...

Continuous Video Electroencephalogram during Hypoxia-Ischemia in Neonatal Mice

新生小鼠缺氧缺血期间的连续视频脑电图

Hypoxia ischemia is the most common cause of neonatal seizures. Animal models are crucial for understanding the mechanisms and physiology underlying ...