An Optical Assay for Synaptic Vesicle Recycling in Cultured Neurons Overexpressing Presynaptic Proteins

Donatus Riemann; Andoniya Petkova; Thomas Dresbach; Rebecca Wallrafen

doi:10.3791/58043

Une analyse optique pour le recyclage de vésicule synaptique dans les neurones en culture surexpr...

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JoVE Journal Neuroscience

An Optical Assay for Synaptic Vesicle Recycling in Cultured Neurons Overexpressing Presynaptic Proteins

Une analyse optique pour le recyclage de vésicule synaptique dans les neurones en culture surexprimant protéines présynaptiques

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09:33 min

June 26, 2018

DOI: 10.3791/58043-v

Donatus Riemann¹, Andoniya Petkova¹, Thomas Dresbach¹, Rebecca Wallrafen¹

¹Institute for Anatomy and Embryology,University Medical Centre Göttingen

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Overview

This study presents a simple optical assay for analyzing synaptic vesicle (SV) recycling in cultured neurons, using a combination of transfection to express a presynaptic marker and a protein of interest. Through this method, researchers can identify presynaptic locations and assess the recycling capacity of synaptic vesicles influenced by specific proteins.

Key Study Components

Area of Science

Neuroscience
Cell Biology
Synaptic Physiology

Background

Synaptic vesicle recycling is crucial for neurotransmission.
Understanding the mechanisms can uncover insights into neuronal communication.
Previous studies have utilized antibody specificity to track synaptic components.
This protocol builds upon established methods for studying synaptic dynamics.

Purpose of Study

To develop a reliable technique for assessing synaptic vesicle recycling.
To investigate the localization and function of proteins at presynaptic sites.
To enhance understanding of synaptic resilience and plasticity.

Methods Used

The study employed a cell culture model using primary hippocampal cells.
Neurons were cultured on coverslips and subjected to immunolabeling techniques.
No multiomics workflows were mentioned in the text.
Key steps include preparing transfection mixes, incubating neurons, and performing immunofluorescence.
The method quantitatively assesses synaptic vesicle recycling via Syt1 antibody uptake visualization.

Main Results

Results show colocalization of GFP-Rogdi and Synaptophysin-mOrange at presynaptic sites, indicating functional synaptic components.
Punctate immunofluorescence correlated with active synapse sites supports the method's effectiveness.
The findings demonstrate a straightforward approach to assess synaptic vesicle turnover.
The methodology reveals critical insights into the interaction of specific proteins within the synaptic machinery.

Conclusions

This study establishes a clear protocol to study synaptic vesicle dynamics using simple optical imaging.
Understanding the recycling mechanism contributes to the broader field of synaptic physiology and potential therapeutic avenues.
The findings may have implications for understanding synaptic function and disorders affecting neurotransmission.

Frequently Asked Questions

What are the advantages of this optical assay?

The assay is simple and leverages the specificity of antibodies, allowing for precise localization of synaptic components without complex equipment.

How are the primary hippocampal cells cultured for this assay?

Cells are plated on coverslips in a 24-well plate, cultured for three days under specified conditions to ensure optimal growth and readiness for transfection.

What types of outcomes can be measured with this method?

The assay allows for visualization and quantification of synaptic vesicle recycling and protein localization at active synapses through immunofluorescence readings.

How is the transfection mix prepared?

A specific combination of calcium chloride and endotoxin-free DNA is mixed with a transfection buffer, incubated at room temperature before applying to cultured neurons.

What limitations should be considered when using this method?

Precise timing and handling during washing steps are critical; any deviations may affect results. Additionally, successful transfection relies on optimal cell condition at the time of application.

Nous décrivons une analyse optique des vésicules synaptiques (SV) recyclage dans les neurones en culture. Alliant ce protocole de transfection double pour exprimer un marqueur présynaptique et la protéine d’intérêt permet de localiser les sites présynaptiques, leur Vésicule synaptique capacité de recyclage et déterminer le rôle de la protéine d’intérêt.

Le principal avantage de cette technique est qu’elle est très simple, ne nécessite qu’un équipement standard, et bénéficie du premier avantage des anticorps, qui est leur spécificité. Le protocole a été introduit à l’origine par Michela Matteoli et ses collègues, et s’est avéré très efficace. Je l’ai utilisé pour tester si une certaine protéine cordocti est localisée dans les synapses actives.

Commencez cette procédure en cultivant des cellules primaires de l’hippocampe sur des lamelles dans une plaque de 24 puits pendant trois jours, comme décrit dans le protocole textuel. Préchauffez le milieu sérique réduit, le milieu de culture cellulaire et l’eau distillée à 37 degrés Celsius dans le bain-marie. En travaillant sous la hotte à flux laminaire pour garantir des conditions de travail stériles, préparez le mélange de transfection dans un tube de microcentrifugation de 1,5 millilitre.

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