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Neuroscience
칼슘 이미징 기술로 뉴런-글리아 회로의 변화 시각화
칼슘 이미징 기술로 뉴런-글리아 회로의 변화 시각화
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Neuroscience
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JoVE Journal Neuroscience
Visualizing Shifts on Neuron-Glia Circuit with the Calcium Imaging Technique

칼슘 이미징 기술로 뉴런-글리아 회로의 변화 시각화

Full Text
5,202 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.

세포 칼슘 이미징은 독특한 기능적 시그니처를 제공하는 칼슘 투과성 채널 / 수용체의 발현을 기반으로 개별 세포, 배양 또는 각성 동물의 혼합 집단에서 동적 신호 전달을 연구하는 다목적 방법론입니다.

이 절차의 전반적인 목표는 염화칼륨 또는 ATP 자극에 따라 신경 세포 또는 아교세포 반응을 분화시키기 위해 살아있는 세포의 세포질 칼슘 농도의 변화를 모니터링하는 것입니다. 칼슘은 신경 전달, 가소성 및 아폽토시스를 포함한 여러 세포 과정에 관여하는 중요한 두 번째 메신저입니다. Fura-2 AM과 같은 높은 선택적 형광 칼슘 염료의 출현은 더 나은 형광 현미경 및 계산 방법의 개발과 관련하여 살아있는 세포 및 유기체에서 칼슘 신호 전달을 이미지화하기 위해 높은 수준의 공간 및 시간 분해능을 가진 고해상도 광학 데이터를 산출했습니다.

이 프로토콜은 농축 뉴런, 정제된 신경교세포 또는 혼합 집단 배양에 적응할 수 있다. 그것은 염화칼륨 및 ATP에 대한 차별적 인 반응을 기반으로 확정 된 자극에 반응 한 세포 유형을 추적합니다. 염화칼륨은 세포의 막 잠재력을 변화시킵니다.

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