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JoVE Journal
Neuroscience
头脊髓和薄片体外制备心律失常活性
头脊髓和薄片体外制备心律失常活性
JoVE Journal
Neuroscience
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JoVE Journal Neuroscience
Preparation of Rhythmically-active In Vitro Neonatal Rodent Brainstem-spinal Cord and Thin Slice

头脊髓和薄片体外制备心律失常活性

Full Text
12,198 Views
06:32 min
March 23, 2019

DOI: 10.3791/58870-v

Samantha B. Palahnuk1,2, Jonathan A. Abdala1, Vadim V. Gospodarev3, Christopher G. Wilson1

1Center for Perinatal Biology, Department of Basic Sciences,Loma Linda University, 2Department of Biology,The College of New Jersey, 3Department of Neurosurgery,Loma Linda University

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Please note that some of the translations on this page are AI generated. Click here for the English version.

Overview

This article presents a protocol for the dissection and slicing of rodent brainstems to analyze the Medullary Respiratory Network. The method aims to enhance reproducibility in obtaining viable slices suitable for electrophysiological recording of respiratory neural circuits, specifically in understanding breathing regulation.

Key Study Components

Area of Science

  • Neuroscience
  • Electrophysiology
  • Respiratory Network Analysis

Background

  • The brainstem houses critical neurons responsible for breathing.
  • Previous protocols lacked detailed guidance for executing the dissection and slicing methods.
  • This protocol addresses the need for clarity and reproducibility in neural slice preparations.

Purpose of Study

  • To provide a comprehensive methodology for preparing brainstem slices.
  • To facilitate studies on the neural control of breathing.
  • To enhance the understanding of breathing pathologies in genetic models.

Methods Used

  • The main platform is ex vivo brain slice preparations.
  • The biological model involves rodent brainstems, focusing on respiratory regions.
  • No multiomics workflows were mentioned.
  • Critical steps include careful dissection, laminectomy, and slice preparation for electrophysiological recordings.
  • Detailed slicing procedures are outlined to ensure correct neuron orientation and functionality.

Main Results

  • The protocol allows for capturing essential neural circuitry for generating respiratory rhythms.
  • Successful isolation and preparation of the pre-Botzinger complex were achieved.
  • Electrophysiological recordings captured rhythmic neural activity critical for understanding respiration.

Conclusions

  • This study demonstrates an effective strategy for brainstem slice preparation, crucial for respiratory studies.
  • The enhanced methodology supports better reproducibility in recording neural output.
  • Results empower researchers to investigate neuronal mechanisms underpinning respiratory control.

Frequently Asked Questions

What advantages does this brainstem preparation provide?
This method enhances reproducibility and enables the study of critical respiratory circuits in rodent models.
How are brainstem slices prepared for recording?
The preparation involves detailed dissection, laminectomy, and precise slicing to retain neuronal circuitry.
What type of data can researchers obtain from these recordings?
Researchers can capture electrophysiological activity related to the control of breathing rhythms, offering insights into respiratory neurobiology.
Can the method be adapted for other models?
While primarily designed for rodents, the technique can potentially be adapted for other small animal models with appropriate modifications.
What are the key limitations of this technique?
The method requires precision and experience in dissection, which might be a barrier for new researchers without prior training.

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。

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。

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