Visualizing Shifts on Neuron-Glia Circuit with the Calcium Imaging Technique

Matheus H. Tempone; Hercules R. Freitas; Clarissa S. Schitine; Ricardo A. de Melo Reis

doi:10.3791/63338

Visualizando mudanças no Circuito Neuron-Glia com a Técnica de Imagem de Cálcio

JoVE Journal
Neuroscience
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JoVE Journal Neuroscience

Visualizing Shifts on Neuron-Glia Circuit with the Calcium Imaging Technique

Visualizando mudanças no Circuito Neuron-Glia com a Técnica de Imagem de Cálcio

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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.

A imagem celular de cálcio é uma metodologia versátil para estudar a sinalização dinâmica de células individuais, em populações mistas na cultura ou mesmo em animais despertados, com base na expressão de canais/receptores permeáveis de cálcio que dão assinaturas funcionais únicas.

O objetivo geral deste procedimento é monitorar mudanças nas concentrações citosóicas de cálcio em células vivas para diferenciar respostas neuronais ou glia com base em cloreto de potássio, ou estímulos ATP. O cálcio é um importante segundo mensageiro envolvido em vários processos celulares, incluindo neurotransmissão, plasticidade e apoptose. O advento do corante de cálcio fluorescente de alta resolução, como o Fura-2 AM, associado ao desenvolvimento de melhores microscópios fluorescentes e métodos de computação, produziu dados ópticos de alta resolução com alto grau de resolução espacial e temporal para a sinalização de cálcio de imagem em células e organismos vivos.

Este protocolo é adaptável a neurônios enriquecidos, glia purificada ou culturas populacionais mistas. Ele rastreia quais tipos de células responderam a estímulos determinantes com base na resposta diferencial ao cloreto de potássio e ATP. O potencial de membrana da célula de alteração do cloreto de potássio.

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