Visualization of Thalamocortical Axon Branching and Synapse Formation in Organotypic Cocultures

Naoyuki Matsumoto; Nobuhiko Yamamoto

doi:10.3791/56553

Visualizzazione di Thalamocortical Axon ramificazione e Synapse formazione in Cocultures organoti...

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Neuroscience

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JoVE Journal Neuroscience

Visualization of Thalamocortical Axon Branching and Synapse Formation in Organotypic Cocultures

Visualizzazione di Thalamocortical Axon ramificazione e Synapse formazione in Cocultures organotipiche

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06:16 min

March 28, 2018

DOI: 10.3791/56553-v

Naoyuki Matsumoto^1,2, Nobuhiko Yamamoto²

¹Department of Medical Neuroscience, Graduate School of Medical Sciences,Kanazawa University, ²Neuroscience Laboratories, Graduate School of Frontier Biosciences,Osaka University

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Overview

This study presents a protocol for simultaneous imaging of thalamocortical axon branching and synapse formation in organotypic cocultures of the thalamus and cerebral cortex. It focuses on dynamic morphological changes in the thalamocortical projection, providing insights into new neural circuit formation and the relationship between axon growth and synapse development.

Key Study Components

Area of Science

Neuroscience
Neurobiology
Cell Biology

Background

Thalamocortical projections play a critical role in sensory processing.
Understanding axon branching and synapse formation is essential for insights into neural circuit formation.
Current methods have limitations in observing these changes simultaneously at the cellular level.
The study advances techniques for imaging in living cell contexts.

Purpose of Study

To investigate dynamic morphological changes during axon branching and synapse formation.
To provide a method that allows observation of individual living cells in coculture.
To assess the relationship between axon growth and synaptic development in thalamocortical neurons.

Methods Used

Organotypic cocultures of thalamic and cortical slices were utilized.
Rat postnatal day two tissue was used to create the biological model.
Electroporation was performed to introduce fluorescent markers for imaging.
Cultures were maintained at 37 degrees Celsius and observed over specific developmental time points.
Imaging was conducted using a confocal microscope to capture axonal dynamics.

Main Results

Dynamic changes in axon branching and synapse formation were observed, highlighting plasticity in thalamocortical projections.
Discrete accumulation of synaptic markers along axons was noted, indicating ongoing synaptic development.
The technique allowed for real-time observation of morphological changes in individual cells.
Findings emphasize the potential for investigating gene roles in axon and synapse dynamics.

Conclusions

This study demonstrates a novel method for exploring neural circuit dynamics and plasticity.
It provides insights into the coupling of axon growth and synapse formation.
The findings have implications for understanding mechanisms of neural connectivity and plasticity in health and disease.

Frequently Asked Questions

What are the advantages of this imaging protocol?

This protocol allows simultaneous imaging of axon branching and synapse formation, providing a dynamic view of neural development.

How are the cortical and thalamic tissues prepared for the study?

Tissue is obtained from postnatal day two rats, carefully dissected, and cultured in serum-containing medium to maintain viability.

What types of data can be obtained using this method?

Researchers can obtain imaging data showing axonal growth patterns and synaptic development, along with insights into the underlying biological processes.

How can this method be applied to other neural circuits?

The technique can be adapted to study intrinsic connections in the cerebral cortex or other neural networks by altering the biological models utilized.

Are there any limitations to this imaging technique?

While effective, the method relies on the viability of cultured slices, which can vary due to handling or environmental conditions.

Questo protocollo descrive un metodo per l'acquisizione simultanea delle thalamocortical assone ramificazione e sinapsi formazione in organotipiche cocultures del talamo e corteccia cerebrale. Thalamocortical singoli assoni e loro terminali presinaptici sono visualizzati mediante una tecnica di elettroporazione di singola cellula con DsRed e lo synaptophysin GFP-etichettato.

L'obiettivo generale di questo metodo è quello di studiare i cambiamenti morfologici dinamici della ramificazione degli assoni e della formazione delle sinapsi nella proiezione talamocorticale. Questo metodo può aiutare a rispondere a domande chiave nel campo della neurobiologia, come la formazione di nuovi circuiti neurali. Il vantaggio principale di questa tecnica è l'osservazione simultanea della crescita degli assoni e della formazione delle sinapsi nelle singole cellule viventi.

Questo metodo può fornire informazioni sulla relazione tra la ramificazione degli assoni e la formazione delle sinapsi nelle proiezioni talamocorticali. Può anche essere applicato ad altri circuiti neurali, come la connessione intrinseca nella corteccia cerebrale. Per iniziare questa procedura, sterilizzare tutti gli strumenti chirurgici con etanolo al 70% per 10 minuti o più.

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