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JoVE Journal
Neuroscience
鼠脑水平海马片
鼠脑水平海马片
JoVE Journal
Neuroscience
This content is Free Access.
JoVE Journal Neuroscience
Horizontal Hippocampal Slices of the Mouse Brain

鼠脑水平海马片

Full Text
20,529 Views
08:59 min
September 22, 2020

DOI: 10.3791/61753-v

Evelien Van Hoeymissen1,2, Koenraad Philippaert2, Rudi Vennekens2, Joris Vriens*1, Katharina Held*1,2

1Laboratory of Endometrium, Endometriosis and Reproductive Medicine, Department of Development and Regeneration,KU Leuven, 2Laboratory of Ion Channel Research, VIB-KU Leuven Center for Brain and Disease Research, Leuven, Belgium and Department of Molecular Medicine,KU Leuven

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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 systematic protocol for obtaining horizontal hippocampal brain slices from mice, which aims to maintain the integrity of key hippocampal fiber pathways. This slicing technique facilitates the assessment of neurological processes related to the dentate gyrus.

Key Study Components

Area of Science

  • Neuroscience
  • Electrophysiology
  • Neuroanatomy

Background

  • The hippocampus plays a crucial role in memory and spatial navigation.
  • Preserving hippocampal fiber pathways is essential for studying brain function and pathology.
  • Common pathologies affecting the dentate gyrus include epilepsy and neurodegenerative diseases.
  • Horizontal slices provide clearer access to study specific synaptic activities and pathways.

Purpose of Study

  • To develop an efficient and gentle protocol for preparing hippocampal slices.
  • To facilitate the study of synaptic transmission and plasticity in relation to brain pathologies.
  • To enable reliable electrophysiological recordings from the hippocampal slices.

Methods Used

  • Ex vivo brain slices prepared from mouse hippocampus.
  • Two to six-week-old male mice were used as the biological model.
  • Detailed steps for preparing ACSF and high-sucrose slice solutions were described.
  • Crucial steps include careful dissection, cooling techniques, and using a vibratome for slicing.
  • Electrophysiological recordings were conducted to assess slice viability and synaptic function.

Main Results

  • Successful preparation of viable hippocampal brain slices for electrophysiological studies.
  • Quality assessments of slices were performed using field excitatory post-synaptic potentials (fEPSP).
  • Observations included relationships between fiber volley amplitudes and fEPSP responses.
  • Findings indicated that viable slices maintained stable synaptic transmission, important for studying synaptic plasticity.

Conclusions

  • This study enables efficient and reproducible preparation of hippocampal slices for advanced electrophysiological studies.
  • The protocol's careful balance between speed and gentle handling preserves tissue integrity.
  • Implications include enhanced understanding of synaptic mechanisms and potential applications in studying neurological disorders.

Frequently Asked Questions

What are the advantages of using horizontal hippocampal slices?
Horizontal slices preserve key hippocampal pathways, facilitating targeted electrophysiological assessments of synaptic activity and plasticity.
How is the brain tissue prepared for slicing?
The brain is carefully harvested from a mouse, cleaned, and then cut into hemispheres before being positioned on a specimen plate for slicing with a vibratome.
What types of data can be obtained using this method?
Electrophysiological data, including field excitatory post-synaptic potential (fEPSP) responses, can be recorded to evaluate slice quality and synaptic transmission stability.
Can this protocol be adapted for other types of brain tissues?
While the protocol is optimized for hippocampal slices, similar methods can be adapted for other regions, though specific adjustments may be necessary.
What are the key limitations of this slicing technique?
Careful handling is essential; mishandling can compromise slice integrity, affecting experimental outcomes.
How is slice viability assessed post-preparation?
Viability is evaluated through electrophysiological recordings, particularly examining stable fEPSP baselines and fiber volley relationships.
What biological insights can this method provide?
The method allows for insights into synaptic mechanisms and the impact of various treatments or conditions on synaptic plasticity in the hippocampus.

本文旨在描述一个系统的协议,以获得水平海马脑片在小鼠。这种方法的目的是保持海马纤维通路的完整性,如穿孔路径和青苔纤维道,以评估凹痕陀螺相关的神经过程。

该方案有助于制备水平海马脑切片,用于各种科学应用。该技术保留了单个半球切片内所有海马纤维通路的完整性。该切片方案非常适合评估在齿状回中发展和表现的脑病理学上发生的神经系统变化。

该协议最重要的方面是找到尽可能快速的执行和温和处理脑组织的平衡。要制备一升 ACSF,请按照指示在 800 毫升水中缓慢混合所有固体化学品,并在磁力搅拌棒的不断搅拌下,然后缓慢滴入适当体积的硫酸镁和氯化钙。然后使用蒸气压渗透压计验证 305 至 315 毫摩尔之间的渗透压,并在室温下用碳水化合物连续使 ACSF 溶液鼓泡,以将 pH 值设置在 7.3 至 7.4 之间。

要制备 250 毫升高蔗糖切片溶液,请按照表中所示将化合物添加到 25 毫升预切片溶液中,并验证渗透压在 320 至 325 毫摩尔之间。然后用碳水化合物将高蔗糖切片溶液鼓泡 10 到 15 分钟,使 pH 值保持在 7.3 到 7.4 之间,并将高蔗糖切片溶液在零下 20 摄氏度下存放 30 到 80 分钟,直到部分冻结。为了准备解剖工作区,用碳化 ACSF 溶液填充回收室,并将室置于 32 摄氏度的水浴中。

将振动切片机设置为适当的切割程序,并在支架上装满冰块,将其安装在振动切片机上,然后将刀片连接到振动切片机臂上。用刮刀将部分冷冻的高蔗糖切片溶液粉碎混合,直到获得异质性雪泥。然后用碳化物使溶液起泡,并使用该溶液在冷藏的 90 毫米培养皿上水合一张滤纸,并将 35 毫米培养皿装满冰上。

为了收获大脑,用解剖剪刀切开 2 到 6 周龄雄性小鼠的头皮,并沿着矢状缝打开颅骨。使用弯曲的镊子去除颅骨,直到可以看到整个大脑,包括嗅球,然后用抹刀小心地舀出完整的脑组织。将大脑放入 35 毫米培养皿中,并使用装满高蔗糖切片溶液的巴斯德移液器轻轻去除组织中的任何毛发或血液颗粒。

用抹刀将清洁后的大脑转移到湿透的滤纸上,然后用刀片将大脑纵向切成两半。将两个半球放在新切开的内侧,用刀片在每个半球的背顶上做一个平行的切口,以去除每个大脑的背侧区域。将两个半球放在新切开的背侧,大脑的腹侧部分朝上,然后将一滴强力胶滴在标本板上。

使用移液器吸头将胶水涂抹到一个大区域以容纳两块组织,并将一条滤纸带接触一个半球的腹侧。使用另一条滤纸带小心地将大脑的背面半干燥,并将半球背面向下定位到标本板上的胶水上。如前所述,使用两条额外的滤纸带将第二个半球定位在胶水上,然后将标本板放入切片室中。

然后快速但小心地用冰冷的高蔗糖切片溶液雪泥盖住板子。要获取脑组织切片,请将振动切片机刀片放在半球内侧的前面,然后抬起振动切片机台,使刀片与现在朝上的半球腹侧处于同一高度。使用振动切片机控制器将刀片沿背侧进一步降低 600 微米,然后切片组织,直到前两片与两个半球完全分离。

获得前两个组织切片后,反转切割方向并将刀片再降低 300 微米,然后再次切片。当海马体可见时,使用加宽的塑料巴斯德移液管收集切片并将其转移到水浴中的恢复室中。将切片在装满 ACSF 的回收室中放置 1 小时,然后将回收室在室温下放置 30 分钟,然后对组织样本进行电生理记录。

在这里,可以分别观察到来自低和高质量切片的代表性负场和正场兴奋性突触后电位记录。负示例迹线在受刺激的神经元纤维去极化时表现出较大的纤维凌空振幅,甚至高于实际的 fEPSP 振幅,而高质量的迹线表现出较小的纤维凌空与 fEPSP 比率和高 fEPSP 振幅。脑切片的活力也可以通过绘制场兴奋性突触后电位斜率与纤维凌射振幅来分析。

通常用于

确定切片质量的输入/输出曲线可以通过对脑切片施加增加的电流刺激并监测随后的 fEPSP 反应来获得。由于保存不良的脑组织的次优传导特性,低质量的脑切片表现出降低的输入/输出曲线。可行的脑切片具有稳定的突触传递基线,而基线不稳定的脑切片不能用于进一步的调节方案来研究脑回路的突触可塑性。

fEPSP 基线记录也可用于监测药物对突触传递本身的影响。此外,脑切片可以与钙指示剂结合使用,以获取荧光成像记录并研究不同切片条件或处理下的钙内流。确保在牺牲后不超过 1.5 分钟内进行脑解剖,这仍然可以保证脑组织仅在没有冷却和氧气供应的情况下短暂进行。

海马脑切片有许多不同的应用,从分子生物学技术到电生理学。该程序有助于对海马解剖结构和神经过程进行深入研究。

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