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Neuroscience
脑切片和原细胞培养中金字塔神经元的弹道标记
脑切片和原细胞培养中金字塔神经元的弹道标记
JoVE Journal
Neuroscience
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JoVE Journal Neuroscience
Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

脑切片和原细胞培养中金字塔神经元的弹道标记

Full Text
7,485 Views
09:40 min
April 2, 2020

DOI: 10.3791/60989-v

Hailong Li1, Kristen A. McLaurin1, Charles F. Mactutus1, Rosemarie M. Booze1

1Program in Behavioral Neuroscience, Department of Psychology,University of South Carolina

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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 labeling and analyzing pyramidal neurons, focusing on morphological alterations in neurons and dendritic spines. The method combines visualizations of brain regions in rats with advanced reconstruction software, aiding in the understanding of neurochemical and behavioral abnormalities.

Key Study Components

Area of Science

  • Neuroscience
  • Neuronal Morphology
  • Dendritic Spine Analysis

Background

  • Pyramidal neurons are crucial for brain function and are involved in various cognitive processes.
  • Dendritic spines serve as the sites for synaptic connections and are important in learning and memory.
  • Assessing morphological changes can provide insights into neurodegenerative conditions and behavioral dysfunctions.
  • The protocol aims to facilitate the study of these neurons in different brain regions of rats.

Purpose of Study

  • To present a reliable method for labeling and analyzing pyramidal neurons and their dendritic spines.
  • To understand potential changes that may reflect underlying neurochemical and behavioral issues.
  • To implement a protocol that combines visuals with computational analysis for better insight into neuron morphology.

Methods Used

  • The study involved in vivo labeling of pyramidal neurons in rats.
  • Pyramidal neurons in specific brain regions were targeted for analysis of morphology and dendritic spines.
  • No multiomics workflows were employed in this analysis.
  • The protocol included critical steps such as tissue perfusion, slicing, and advanced imaging techniques.
  • The use of automated software for neuron reconstruction and analysis was emphasized.

Main Results

  • Key findings indicate that the protocol effectively allows for the visualization and quantification of dendritic spines.
  • Morphological assessments included measurements of dendritic branching and spine characteristics.
  • Results from the software highlighted variations in dendritic complexity and spine classifications.
  • The approach validated accuracy in labeling and assessing primary cortical neurons in cell cultures.

Conclusions

  • The study provides a robust protocol for investigating pyramidal neurons and spine dynamics, important for understanding neural mechanisms.
  • This methodology enhances the potential for exploring neuronal plasticity and related behavioral outcomes.
  • Implications of this study extend to potential applications in neurodegenerative research and cognitive disorder models.

Frequently Asked Questions

What are the advantages of the protocol presented?
The protocol allows for precise labeling and visualization of neurons, facilitating detailed morphological analysis in various brain regions.
How are pyramidal neurons targeted for analysis?
Pyramidal neurons are identified based on their unique structural characteristics and are subjected to a comprehensive imaging protocol.
What types of data are obtained from this study?
Data includes morphological details of dendritic spines and neuron structure, assessed through quantitative analysis software.
Can the method be adapted for different species or models?
While the protocol focuses on rats, it may be adjusted for other species with suitable anatomical considerations.
What are the key limitations of the protocol?
Limitations include the specificity of the technique to certain brain regions and the need for precise execution of each step for reliable results.
How does this study enable further research in neuroscience?
The findings can pave the way for better understanding of neuronal alterations related to various cognitive dysfunctions, guiding future studies.

我们提出了一种用于标记和分析金字塔神经元的协议,这对于评估神经元和树突脊柱的潜在形态变化至关重要,这些变化可能是神经化学和行为异常的基础。

该协议使得评估神经元和树突脊柱的潜在形态变化成为可能,这些改变可能突出神经化学和行为异常。它对于可视化大鼠不同大脑区域的神经元是独特和有用的,它结合复杂的重建软件,使研究人员能够阐明神经认知功能障碍背后的可能机制。首先根据手稿说明准备 PVP 解决方案。

用 PVP 填充油管,并离开 20 分钟。用10毫升注射器将它通过另一端排出。将170毫克钨微载体珠与250微升的氯化甲基汞混合。

然后彻底涡流悬浮。接下来,加入6毫克的脂亲D二钙18(3)染料到300微升的二氯甲烷和涡流。将钨珠悬浮在玻璃幻灯片上的 250 微升的管道。

等待悬浮液风干,并加入300微升的染料溶液。干燥后,使用剃须刀将混合物分成两个1.5毫升的离心管,并加注水管。在水浴中对混合物进行声波化,直到均匀。

确保声波尖端直接位于管子上。将两种混合物混合在15毫升锥形管中。再声化三分钟,确保没有大面积的染料涂层珠子。

声化后,用10毫升注射器将混合物抽入PVP涂层管中。将油管喂入制备站。旋转油管一分钟。

使用注射器小心地取出所有水。打开氮气,将氮气流量调整到每分钟约 0.5 升。在制备站中旋转油管,用氮气干燥 30 分钟。

从站中拆下油管,用管切割机将其切割成 13 毫米段。确保大鼠对有毒刺激没有反应,并且没有反射。然后把它固定在上位。

沿着胸腔中线切开。将隔膜分开,用剪刀打开胸部。然后将 20 量表 25 毫米针插入左心室。

立即用剪刀切割右中庭,并用每分钟 5 毫米的流量对 15 毫升 100 毫升 PBS 进行测。然后在 PBS 中缓冲 100 毫升 4%PFA。灌注后,取出整个大鼠大脑。

并后修复它与4%PFA10分钟。使用大鼠大脑矩阵切割500微米厚的日冕部分。做一个拳头切割,并保持刀片到位。

然后用第二个刀片进行第二次切割,并垂直取出第一个刀片,将组织放在刀片表面上。将大脑切片放在24井板中,每个井中有1毫升PBS。并重复此过程,直到所有切片都已切割。

从每个目标井中删除 PBS。用迪尔/钨管将软骨装入施用器中。在两个网屏之间放一张滤纸。

将施用器连接到氦软管。然后将氦气的输出压力调整到每平方英寸90磅。将施用器垂直放置在样品和网格屏幕之间 1.5 厘米的目标井的中心。

然后发射迪尔/钨管。用下一个管子加载软骨,并在剩余切片上连续从管子上发射珠子。用 100 毫摩尔 PBS 填充 24 井板。

用500微升新鲜PBS洗三次切片。确保切片在洗涤过程中不会翻转。完成后,向切片中添加 500 微升新鲜 PBS。

在黑暗中四摄氏度下孵育它们三个小时。孵育后,使用细刷将大脑切片转移到玻璃幻灯片上。并立即添加一毫升的防淡安装介质到每个部分。

将 22 到 15 毫米的盖玻片放在各部分上,在黑暗中擦干滑梯。打开共体显微镜系统并切换到 60 倍目标。根据手稿方向调整成像设置。

根据神经元的脑区边界和形态特征,获取靶向神经元类型的Z-堆栈图像。在产后第一天从F344/N大鼠身上分离出原发性皮质神经元,并在35毫米玻璃底盘培养它们一周。隔离后的第三天刷新一半的介质。

用1毫升100毫摩尔PBS洗两次。在室温下用4%PFA固定细胞15分钟。如前面所述,以弹道标记细胞。

用1毫升PBS洗三次。将500微升PBS加入细胞,在黑暗中4摄氏度下孵育3小时。然后添加 200 微升防淡安装介质。

并获取每个目标神经元的 Z 堆栈图像。大鼠大脑部分海马区域的典型金字塔神经元被识别为弹道标记技术,其特点是一个大的皮毛树突和周围几个较小的基底树突。神经元重建定量分析软件用于追踪树突状分支和检测脊柱。

随后,该软件被用来评估树突分支的复杂性和神经元树干的复杂性。用长度、体积和头部直径评估树突脊柱的形态变化。然后脊柱被分为薄,存根,蘑菇脊柱。

并检查了每个半径之间的脊柱数的相对频率。此外,在细胞培养的初级金字塔神经元上验证了弹道标记技术。根据索玛和大的脂肪树突的三角形来识别金字塔神经元。

然后使用神经元重建软件来分析细树突脊柱和树突状脊柱长度的分布。在尝试此协议时,重要的是避免大团团或弹道染料涂层钨珠开始制备。团块不允许区分单个神经元。

结合神经元重建软件,这种方法使我们能够检查海马金字塔神经元的神经元和树突状脊柱形态。神经元重建软件利用一种算法提供树突状脊柱的自动辅助分类。多个神经元参数的量化提供了一个机会,以更好地了解神经元认知功能障碍背后的机制。

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