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JoVE Journal
Neuroscience
Ex útero Electroporación y culturas Organotypic rebanada de cerebros embrionarios de rat...
Ex útero Electroporación y culturas Organotypic rebanada de cerebros embrionarios de rat...
JoVE Journal
Neuroscience
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JoVE Journal Neuroscience
Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Ex útero Electroporación y culturas Organotypic rebanada de cerebros embrionarios de ratón para vivir-la proyección de imagen de interneuronas GABAérgicas de migrando

Full Text
10,481 Views
09:50 min
April 20, 2018

DOI: 10.3791/57526-v

Lara Eid1,2, Mathieu Lachance1, Gilles Hickson1,3, Elsa Rossignol1,2,4

1Centre de recherche du CHU Sainte-Justine, 2Department of Neuroscience,Université de Montréal, 3Department of pathology and cellular biology,Université de Montréal, 4Department of Pediatrics,Université de Montréal

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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 study presents a low-cost and effective technique for generating electroporated brain organotypic slice cultures from mouse embryos, enabling visualization of genetically modified cortical GABAergic interneurons during their tangential migration. The method aims to investigate the effects of specific gene mutations on brain development, offering potential insights into neurodevelopmental disorders.

Key Study Components

Area of Science

  • Neuroscience
  • Developmental Biology
  • Neurogenetics

Background

  • Understanding GABAergic interneuron migration is crucial for developmental neuroscience.
  • Gene function loss during brain development raises questions about neurodevelopmental disorders.
  • Low-cost techniques can enhance diagnostic capabilities for disorders such as autism and epilepsy.
  • Electroporation methods allow targeted manipulation of gene expression in vivo.

Purpose of Study

  • To visualize the migration and behavior of genetically modified GABAergic interneurons in cultured environments.
  • To identify the impacts of gene mutations on neuronal development.
  • To develop a more accessible method for studying developmental neuroscience.

Methods Used

  • The method employs organotypic slice cultures derived from mouse embryos.
  • The biological model involves cortical GABAergic interneurons, specifically focusing on their migration.
  • Electroporation is used to introduce plasmid solutions into the developing brain.
  • Key steps include careful embryo dissection, electroporation, and culture setup for imaging.
  • Incubation and sectioning for imaging are critically timed and monitored.

Main Results

  • The study enables the observation of internal migration patterns of GABAergic interneurons.
  • Insights garnered may clarify mutation impacts on developmental processes.
  • Mechanical and environmental conditions during dissection and culture significantly affect viability and observation.
  • Results will help elucidate mechanisms underlying neurodevelopmental disorders.

Conclusions

  • This technique provides a reliable platform for studying neuronal migration and gene function.
  • The method stands to advance understanding of genetic influences on brain development.
  • Insights may enhance approaches to treating neurodevelopmental disorders.

Frequently Asked Questions

What are the advantages of this electroporation method?
The electroporation method is low-cost and relatively straightforward compared to traditional techniques, enabling broader accessibility for research in developmental neuroscience.
How is the biological model of GABAergic interneurons implemented?
The model involves isolating and electroporating embryonic mouse brains to study the migration and development of cortical GABAergic interneurons in a controlled culture environment.
What types of outcomes can be observed using this method?
Key outcomes include the behavior and migration patterns of interneurons, as well as the effects of specific gene mutations during brain development.
How can this method be adapted for other studies?
The technique is adaptable for various genetic manipulations and can be applied to different neural cell types for diverse developmental studies.
What are some limitations of this study?
Limitations may include the potential variability in results due to the precise timing of interventions and environmental conditions during the procedure.
How might findings from this study impact understanding of neurodevelopmental disorders?
By clarifying the pathogenic effects of specific gene mutations, the findings could lead to improved diagnostic and therapeutic strategies for disorders like autism and epilepsy.

Aquí, nos proporcionan un método confiable y de bajo costo para generar culturas de electroporated cerebro organotypic rebanada de embriones de ratón adecuados para microscopía confocal y técnicas de imagen en vivo.

El objetivo general de este experimento es visualizar interneuronas GABAérgicas corticales modificadas genéticamente durante la migración tangencial en el entorno más naturalista posible. Este método puede ayudar a responder preguntas clave en las neurociencias del desarrollo al detectar el impacto de la pérdida de función de genes específicos de proteínas durante el desarrollo del cerebro. La principal ventaja de esta técnica es que es de bajo coste y relativamente fácil de realizar en comparación con otros métodos similares como la electroporación intrauterina.

Por lo tanto, esta técnica puede, en última instancia, mejorar el diagnóstico de los pacientes con trastornos genéticos del neurodesarrollo, como el autismo y la epilepsia, al aclarar la patogenicidad de las mutaciones específicas identificadas en los pacientes, así como el papel de los nuevos genes de la enfermedad durante el desarrollo del cerebro. Comience por configurar el gabinete de bioseguridad con todos los instrumentos necesarios para la disección y el cultivo. Rocíe generosamente todos los instrumentos de la cabina de bioseguridad con etanol al 70% y esterilice los instrumentos y el interior del armario con luz ultravioleta durante 15 a 20 minutos.

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Neurociencia número 134 desarrollo cerebral microscopia confocal interneuronas GABAérgicas corticales e13.5 embrión electroporación represión de genes ratón migración neuronal cultura organotypic ARN de interferencia migración tangencial Time-lapse la proyección de imagen

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