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Neuroscience
カルシウムイメージング技術によるニューロン-グリア回路の変化の可視化
カルシウムイメージング技術によるニューロン-グリア回路の変化の可視化
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Neuroscience
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JoVE Journal Neuroscience
Visualizing Shifts on Neuron-Glia Circuit with the Calcium Imaging Technique

カルシウムイメージング技術によるニューロン-グリア回路の変化の可視化

Full Text
5,121 Views
11:41 min
April 8, 2022

DOI: 10.3791/63338-v

Matheus H. Tempone1, Hercules R. Freitas2, Clarissa S. Schitine3, Ricardo A. de Melo Reis1

1Laboratory of Neurochemistry, Institute of Biophysics Carlos Chagas Filho,Universidade Federal do Rio de Janeiro, 2Laboratory of Neuroenergetics and Inborn Errors of Metabolism, Institute of Medical Biochemistry Leopoldo de Meis,Universidade Federal do Rio de Janeiro, 3Laboratory of Neurochemistry and Cell Biology, Institute of Life Sciences,Universidade Federal da Bahia

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Overview

This study focuses on cell calcium imaging as a method to monitor cytosolic calcium concentration changes in live cells. Using a chicken retina culture, the work differentiates responses of neurons and glial cells to potassium chloride and ATP stimuli, highlighting the involvement of calcium dynamics in signaling processes.

Key Study Components

Area of Science

  • Neuroscience
  • Cell Biology
  • Imaging Techniques

Background

  • Calcium is a crucial second messenger in various cellular processes.
  • High selective fluorescent calcium dyes enable advanced imaging techniques.
  • The response of different cell types to specific stimuli can provide insight into their functional roles.
  • This procedure allows for the investigation of neuronal and glial signaling in cultured cells.

Purpose of Study

  • To monitor changes in cytosolic calcium concentrations in live cells.
  • To differentiate the calcium responses of neurons and glia based on specific stimuli.
  • To elucidate the functional roles of these cell types in calcium signaling.

Methods Used

  • The platform used is a cell culture of chicken retina cells.
  • This study primarily focuses on enriched neuron cultures and glial cells.
  • The method involves the use of Fura-2 AM for calcium imaging.
  • Cells are treated with potassium chloride and ATP to measure intracellular calcium shifts.
  • Data is analyzed using Metafluor software and presented in Excel tables.

Main Results

  • The findings reveal distinct calcium responses in neurons when stimulated with potassium chloride.
  • Glial cells primarily respond to ATP stimuli, underscoring their role in calcium signaling.
  • The study provides insights into the spatial and temporal dynamics of calcium signaling in mixed cultures.
  • Results indicate that the method effectively differentiates cellular responses based on treatment.

Conclusions

  • This study demonstrates the efficacy of calcium imaging in understanding cell-specific responses.
  • The methodology enables detailed tracking of calcium dynamics, enhancing understanding of neuronal mechanisms.
  • These insights can inform future research on plasticity and cellular communication in the nervous system.

Frequently Asked Questions

What are the advantages of using chicken retina cells?
Chicken retina cells are accessible for dissection and provide a well-defined model for studying neuronal and glial interactions in a controlled environment.
How are neuronal and glial responses differentiated?
Responses are differentiated based on their distinct reaction to potassium chloride and ATP, enabling specific insights into calcium dynamics in each cell type.
What types of data are obtained from calcium imaging?
Calcium imaging provides quantifiable data on intracellular calcium concentration changes, allowing analysis of cell excitability and signaling mechanisms.
Can this method be adapted for other cell types?
Yes, the procedure can be adapted to other neuronal or glial cultures based on the specific calcium imaging needs and targeted stimuli.
What are some key considerations when performing this procedure?
It is essential to maintain sterile conditions, accurately prepare reagents, and carefully monitor incubation times to ensure reliable results.

細胞カルシウムイメージングは、ユニークな機能的シグネチャを与えるカルシウム透過性チャネル/受容体の発現に基づいて、培養中の混合集団、または覚醒した動物でさえも、個々の細胞の動的シグナル伝達を研究するための汎用性の高い方法論です。

この手順の全体的な目標は、塩化カリウムまたはATP刺激に基づいてニューロンまたはグリア応答を分化させるために、生細胞における細胞質ゾルカルシウム濃度の変化を監視することである。カルシウムは、神経伝達、可塑性、およびアポトーシスを含むいくつかの細胞プロセスに関与する重要なセカンドメッセンジャーである。Fura-2 AMなどの高選択的蛍光カルシウム色素の出現は、より優れた蛍光顕微鏡および計算方法の開発に関連して、生きた細胞および生物上のカルシウムシグナル伝達を画像化するために、高度の空間的および時間的分解能を有する高解像度光学データをもたらした。

このプロトコルは、富化ニューロン、精製グリア、または混合集団培養物に適応可能である。これは、塩化カリウムおよびATPに対する差動応答に基づいて、どの細胞型が決定的な刺激に応答したかを追跡する。塩化カリウムは細胞の膜電位を変化させます。

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