Name: preparation of rhythmically-active in vitro neonatal rodent brainstem-spinal cord and thin slice
Uploaded: 2019-03-23T13:00:00
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s. b. p. 是洛玛琳达大学暑期本科生研究奖学金的获得者。

以下协议已被洛玛·琳达大学动物护理和使用机构委员会 (IACUC) 接受并批准。在实验室进行的所有动物实验都遵循国家卫生研究院关于动物伦理治疗的准则。执行本协议的个人维护了所有道德标准。
1. 解决方案
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准备人工脑脊液 (aCSF)。
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<li>在1升的实验前的晚上, 使用以下配方准备新鲜的 aCSF: 7.250 g Nocl (124 mM), 0.224 g KCl (3 mM), 2.100 g NaHCO3 (25 mm), 0.0...

<ol>
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将这里介绍的协议改编成一个整体或切片工作流程, 对于希望利用整体脑脊髓和薄片制剂进行电生理记录的实验室和研究是有利的。所提出的解剖和切片方法, 加上其他人先前报告的方法 17, 18,19, 将允许可重复地制备坚固和可行的组织, 广泛适应一系列使用啮齿类动物后脑或脊髓的实验。解剖是非常详细的, 包括背侧和腹侧椎板?...

脑干呼吸神经网络为了解节律神经网络的一般特征提供了一个肥沃的领域。特别是, 兴趣是新生儿啮齿类动物呼吸的发展和了解呼吸节律是如何发展的。这可以使用多级方法, 包括体内整体动物胸腔造影, 体外的群内神经记录, 以及包含呼吸节律发生器的体外切片记录。体外还原法和切片记录是在询问发育中啮齿类动物脑脊髓区呼吸节律发生和神经回路背后的机制时使用的一种有利方法。发育中的呼吸系统包括大约40种细胞类型, 其特点是射击模式, 包括中央呼吸1,2的细胞类型。中央呼吸网络包括一组有节奏的活跃神经元,位于南边外侧髓质 1,3。哺乳动物呼吸节律发生是由一个被称为前 Bötzinger 复合体 (pBC) 的自心律失常网络产生的, 该网络通过新生儿哺乳动物的切片和整体制剂进行了局部实验脑干脊髓 3,4,5, 6,7,8。这个区域的功能类似于心脏的窦房结 (SA), 并产生一个吸气计时系统来驱动呼吸。从 pBC, 吸气节律被带到脑干的其他区域 (包括舌下运动核) 和脊髓运动池 (如驱动隔膜的肾上腺运动神经元)9.
节律活动可通过脑髓整体制备或切片从各种细胞群, 包括 C3-c5 神经根, 十二神经根, 下运动核 (XII mn), 下腔运动神经元 (XII pMN), 以及pbc3,10,11,12。虽然这些数据收集方法在少数实验室都是成功的, 但许多协议的提出方式并不能完全再现进入该领域的新研究人员。要获得可行的和有节奏的活跃的整体和切片准备工作, 需要通过解剖和切片方案的所有步骤, 非常注意细节。以前的协议广泛地描述了各种记录程序和电生理学, 但缺乏细节, 在获得一个可行的组织准备最关键的部分: 执行脑干脊髓解剖和切片程序。
有效地获得节奏活跃和可行的整体或切片准备脑干脊髓电生理记录要求正确、仔细和快速地执行所有步骤 (通常情况下, 这里相关的整个过程可以是在大约30分钟内完成)。脑干-脊髓电生理协议的临界点, 以前没有很好地描述包括神经根子的解剖和在振动体上的切片过程。该方案是第一个逐步视觉沟通脑脊髓解剖为新的研究人员和专家在该领域。该协议还详细解释了手术技术、地标和其他程序, 以帮助未来的研究人员标准化切片和整体准备, 以包含每个实验所需的确切电路。这里介绍的程序可以在老鼠和老鼠新生幼崽。

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	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">NaCl</td>
			<td style="width:288px;">Fisher Scientific</td>
			<td style="width:320px;">S271-500</td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td>Sigma Aldrich</td>
			<td>P5405-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaHCO3</td>
			<td>Fisher Scientific</td>
			<td>BP328-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaH2PO4 &#8226;H2O</td>
			<td>Sigma Aldrch</td>
			<td>S9638-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CaCl2&#8226;2H2O</td>
			<td>Sigma Aldrich</td>
			<td>C7902-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgSO4&#8226;7H2O</td>
			<td>Sigma Aldrich</td>
			<td>M7774-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-Glucose</td>
			<td>Sigma Aldrich</td>
			<td>G8270-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cold-Light source Halogen lamp 150W</td>
			<td>AmScope</td>
			<td>H2L50-AY</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissection Microscope</td>
			<td>Leica</td>
			<td>M-60</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vibratome 1000 Plus</td>
			<td>Vibratome</td>
			<td>W3 69-0353</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnetic Base</td>
			<td>Kanetic</td>
			<td>MB-B-DG6C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoflurane, USP</td>
			<td>Patterson Veterinary</td>
			<td>NDC 14043-704-06</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sword Classic Double Edge Blades</td>
			<td>Wilkinson</td>
			<td>97573</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Histoclear</td>
			<td>Sigma-Aldrich</td>
			<td>H2779</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont #5 Fine Forceps</td>
			<td>Fine Science Tools</td>
			<td>11254-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont #5/45 Forcep</td>
			<td>Fine Science Tools</td>
			<td>11251-35</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scalpel Blades #10</td>
			<td>Fine Science Tools</td>
			<td>10010-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scalpel Handel #3</td>
			<td>Fine Science Tools</td>
			<td>10003-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spring Scissors Straight&#160;</td>
			<td>Fine Science Tools</td>
			<td>15024-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Narrow Pattern Forcep Serrated/straight</td>
			<td>Fine Science Tools</td>
			<td>11002-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Castroviejo Micro Dissecting Spring Scissors; Straight</td>
			<td>Roboz</td>
			<td>RS-5650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vannas Scissors 3&#34; Curved</td>
			<td>Roboz</td>
			<td>RS-5621</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insect pins, 0</td>
			<td>Fine Science Tools/8840604</td>
			<td>26000-35&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insect pins, 0, SS</td>
			<td>Fine Science Tools</td>
			<td>26001-35</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insect pins, 00</td>
			<td>Fine Science Tools</td>
			<td>26000-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insect pins, 00, SS</td>
			<td>Fine Science Tools</td>
			<td>26001-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insect pins, 000</td>
			<td>Fine Science Tools</td>
			<td>26000-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insect pins, 000, SS</td>
			<td>Fine Science Tools</td>
			<td>26001-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Minutien pins, 0.10 mm</td>
			<td>Fine Science Tools</td>
			<td>26002-10</td>
			<td></td>
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			<td height="21" style="height:21px;">Minutien pins, 0.15 mm</td>
			<td>Fine Science Tools</td>
			<td>26002-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Minutien pins, 0.2 mm</td>
			<td>Fine Science Tools</td>
			<td>26002-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fisher Tissue prep Parafin&#160;</td>
			<td>fisher</td>
			<td>T56-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Graphite&#160;</td>
			<td>fisher&#160;</td>
			<td>G67-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Delrin Plastic&#160;</td>
			<td>Grainger</td>
			<td>3HMT2</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">18 Gauge Hypodermic Needle</td>
			<td>BD</td>
			<td>305195</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of rhythmically-active in vitro neonatal rodent brainstem-spinal cord and thin slice

哺乳动物的吸气节律是由髓质区域的神经元网络产生的, 称为前 Bötzinger 复合体 (pBC), 它产生一个信号, 推动吸气肌肉的节律收缩。在 pBC 中产生并携带到其他神经元池以驱动呼吸肌肉组织的艺术神经活动可以使用各种方法进行研究, 包括整块神经记录和横向切片记录。然而, 以前公布的方法并没有广泛地描述脑干细胞-脊髓解剖过程以透明和可重复的方式为未来的研究。在这里, 我们提出了一个全面的概述的方法, 用于可重现切割节奏活跃脑干切片包含必要和足够的神经元电路产生和传输吸气驱动器。这项工作建立在以前的脑干-脊髓电生理方案的基础上, 以提高可靠地获得可行和有节奏活性的切片记录 pBC 的神经元输出, 舌下前运动神经元 (XII pMN),低性腺运动神经元 (XII MN)。所介绍的工作扩展了以前公布的方法, 提供了详细的, 逐步的插图解剖, 从整个老鼠小狗, 体外切片含有十二根。

这里介绍的方法允许研究人员有兴趣获得有节奏的活跃的脑干切片可重现和可靠地削减一个可行的, 可靠的切片, 将允许记录虚构的电机输出数小时。使用这种方法, 可以在薄片中捕获产生和传输吸气节律所需的最低限度的神经电路元件。这些元素包括: 前 Bötzinger 复合体, 投射到舌下运动神经元 (pXII MNs) 和舌下运动神经元 (XII Mn), 和舌下神经根子。pBC、Xin 和 C4 神经根常用?...

Watch this Scientific Journal Video about Preparation of Rhythmically-active In Vitro Neonatal Rodent Brainstem-spinal Cord and Thin Slice at JoVE.com

Preparation of Rhythmically-active In Vitro Neonatal Rodent Brainstem-spinal Cord and Thin Slice

该方案既直观地传达脑干脊髓的准备, 并阐明脑干横向切片的准备工作, 一个全面的逐步的方式。它的目的是提高重现性, 并提高获得可行的, 持久的, 有节奏的活性切片记录神经输出从脑干的呼吸区域的可能性。

头脊髓和薄片体外制备心律失常活性

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a ...

这种方法有助于对啮齿动物脑干进行可重复的解剖和切片，以便对米里亚三头呼吸道网络进行捕获的分析。此技术提供了极大的实验灵活性，使得可以准备体外解除阻止或切片准备进行录制。该方法可用于电生理学实验，以探索产生呼吸节奏的神经控制回路，并了解转基因啮齿动物呼吸的病理学和失调。解剖后，迅速将动物的分离躯干转移到解剖显微镜下加的解剖室，将组织侧侧向上放置，将组织端朝室前部。将组织固定在肩部和最肺脊髓的一端，在骨缝合线下通过头骨进行中下半切切口，以避免损伤头骨下面的皮层和脑干。从下垂缝合开始，横向工作，剪断头骨的腹骨缝合线，形成可能反射并固定固定到骨骼的上腹，并稳定头骨的上腹部分。在反射两个头骨瓣后，切除大脑皮层的余下部分，使小脑的骨部相对完整，以进行后侧拉明切除术。使用微型弹簧剪刀和钳子去除头骨和椎柱周围的肌肉。沿着肋骨的后侧取出组织，保持肋骨完好无损，并小心地剪掉椎骨的侧侧过程。切开任何覆盖庞和梅杜拉的组织后，紫光、小脑、肺和脊髓的开始将清晰可见。要进行腹体腹骨切除术，请将组织支点侧向下放置，并固定在肋骨和脊柱最腹的端。使用头骨皮瓣固定组织的左侧，并切除肋骨的腹腔一半，包括胸骨和所有腹部器官。解剖附着在肋骨上的软组织，露出肋骨和脊髓，并切除舌头、食道、气管、喉和所有其他软组织和肌肉，覆盖头骨和脊柱的基座。为了识别硬托盘，解剖在头骨底部覆盖这个矩形骨板的组织，其中包括一个V形缩进。然后沿着托盘的中线切割，小心地向上抬起组织，并执行横向切割以将其移除。要开始腹腔切除术，请取出脑膜，将脑干和脊髓的腹腔表面从第一颈椎暴露在大约胸椎七，沿着脑筋的脊柱两侧切动五到十毫米。当脊髓暴露后，将根块双边地截断约 20 至 25 毫米，沿着脊柱到大约 T7，并小心地抬起 C1、C2 和 C3 的左边缘，用钩钩或弯曲的钳子在骨骼下进行剪断，以去除三根椎骨。当所需的脊髓长度从椎柱中分离时，进行横向切割以去除脊髓组织。取出杜拉后，将脑干放在石蜡平台的中心，放在塑料切割块上，并使用精细的昆虫针脚，修剪到不超过一厘米长，通过端骨脊髓固定脑干的脑干。然后将石蜡覆盖的切割块与振动块支架中的固定脑干对齐，使刀片垂直于脑干的卷面切割。接下来，做一个初始切片，去除200至300微米的不均匀无关组织在组织最端，作出必要的小调整，以确保PARP线性和小切口，以去除任何不均匀的组织。术语咽根将在脑干的侧缘可见，从这些左体神经解剖学标记中切割300至500微米的脑干片，以捕获博辛格前复合物和相关传输电路。所有切片过程步骤对于创建可重复的可行切片至关重要，所有必要的神经元电路都以正确的方向进入，从而产生健壮的节奏。使用此方法，可以在薄片中捕获用于生成和传输吸入节律的所有最起码的神经电路元素。包括预博辛格复合体，运动前神经元投射到低牙运动神经元和低牙神经根。然后，Botzinger 前复合体、低牙或第 12 颅神经根和 C4 神经根可用于吸气记录。此过程的关键方面是小心分离根茎，并在切入最终切片之前确认脑干没有受损。按照此过程，可以使用贴片夹法、额外的细胞记录或吸取电极进行电生理学记录，以记录网络产生的电活动。以前公布的协议提供了一些循序渐进的细节，使得新研究人员很难使用这种方法，而不前往另一个实验室，并花时间与经验丰富的研究人员在一起。

Research

JoVE Journal

Neuroscience

Whole Cell Recording from an Organotypic Slice Preparation of Neocortex

从大脑皮层的器官型切片制备全细胞记录

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex.  Because of the ...

科研

神经科学

Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation

成像遥感信息素在小鼠犁鼻急性组织切片的制备

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. ...

Chicken Embryo Spinal Cord Slice Culture Protocol

鸡胚胎脊髓片培养协议

Slice cultures can facilitate the manipulation of  embryo development both pharmacologically and through gene manipulations. In  this reduced system, ...

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

鼠神经母细胞核转染到研究神经细胞迁移<em&gt;在体外</em

The subventricular zone (SVZ) located in the lateral wall of the lateral ventricles plays a fundamental role in adult neurogenesis. In this restricted ...

Electrophysiology on Isolated Brainstem-spinal Cord Preparations from Newborn Rodents Allows Neural Respiratory Network Output Recording

电从新生儿鼠害隔离脑干，脊髓准备允许呼吸中枢网络输出记录

While it is well known that the central respiratory drive is located in the brainstem, several aspects of its basic function, development, and response to ...

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

脊髓神经元从新生小鼠中分离和培养

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on ...

Preparation and Immunostaining of Myelinating Organotypic Cerebellar Slice Cultures

髓鞘有机小脑片培养的制备及免疫染色

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical ...

In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity

体外楔形切片准备模拟在Vivvo神经元电路连接

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively ...

Uptake of Fluorescent Labeled Small Extracellular Vesicles In Vitro and in Spinal Cord

Uptake of Fluorescent Labeled Small Extracellular Vesicles In Vitro and in Spinal Cord

吸收荧光标记的小细胞外囊泡 在体外 和脊髓

Small extracellular vesicles (sEVs) are 50-150 nm vesicles secreted by all cells and present in bodily fluids. sEVs transfer biomolecules such as RNA, ...

Whole Neonatal Cochlear Explants as an In vitro Model

Whole Neonatal Cochlear Explants as an In vitro Model

整个新生儿耳蜗外植体作为 体外 模型

Untreated hearing loss imposes significant costs on the global healthcare system and impairs individuals' quality of life. Sensorineural hearing loss is ...