FM Dye Cycling at the Synapse: Comparing High Potassium Depolarization, Electrical and Channelrhodopsin Stimulation

Danielle L. Kopke; Kendal Broadie

doi:10.3791/57765

FM sinaps Bisiklete binme boya: yüksek potasyum depolarizasyon, elektrik ve Channelrhodopsin stim...

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Neuroscience

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JoVE Journal Neuroscience

FM Dye Cycling at the Synapse: Comparing High Potassium Depolarization, Electrical and Channelrhodopsin Stimulation

FM sinaps Bisiklete binme boya: yüksek potasyum depolarizasyon, elektrik ve Channelrhodopsin stimülasyon karşılaştırma

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08:31 min

May 24, 2018

DOI: 10.3791/57765-v

Danielle L. Kopke¹, Kendal Broadie²

¹Department of Biological Sciences,Vanderbilt University, ²Departments of Biological Sciences, Pharmacology, Cell and Developmental Biology, Kennedy Center for Research on Human Development,Vanderbilt University and Medical Center

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Overview

This study investigates the cycling of synaptic vesicles (SV) at the Drosophila larval neuromuscular junction using fluorescent FM dyes. It explores various stimulation methods, including high potassium depolarization, electrical stimulation, and optogenetics, to quantitatively assess SV endo- and exocytosis.

Key Study Components

Area of Science

Neuroscience
Synaptic transmission
Fluorescent imaging

Background

SV cycling is essential for intercellular communication at neuronal synapses.
FM dye uptake and release serve as primary methods to assay SV cycling.
This study utilizes the Drosophila NMJ as a model system.
Understanding synaptic mechanisms can address fundamental questions in neuroscience.

Purpose of Study

To compare different stimulation techniques for driving FM1-43 cycling.
To identify critical genes involved in SV cycling.
To optically track vesicle cycling in live preparations.

Methods Used

Live preparation of Drosophila larval NMJ.
Fluorescent FM dye tracking for assessing SV cycling.
Comparison of three stimulation methods: electrical, optogenetic, and potassium depolarization.
Key steps include dissection for preparation and application of calcium-free saline to stop SV cycling.
Image analysis using ImageJ for quantification of fluorescence measurements.

Main Results

The study confirms the effectiveness of each stimulation method for analyzing SV cycling.
Differences in fluorescence intensity highlight activity-dependent vesicle behavior.
FM dye can be photoconverted for enhanced visualizations via TEM.
Results provide insights into the mechanistic processes underlying synaptic transmission.

Conclusions

This study enables a deeper understanding of SV cycling mechanisms at NMJs.
The results pave the way for further exploration of genetic influences on synaptic behavior.
Implications extend to neuronal plasticity and underlying mechanisms in synaptic transmission.

Frequently Asked Questions

What are the advantages of using the Drosophila NMJ model?

The Drosophila NMJ model provides a well-defined and accessible system to study synaptic mechanisms in live preparations, allowing for detailed analysis of SV cycling.

How is the live preparation of Drosophila larval NMJ implemented?

The preparation involves careful dissection to expose the NMJ while maintaining the integrity of the sample for accurate imaging and stimulation.

What types of data are obtained from this study?

The study yields quantitative data on fluorescence intensity during endo- and exocytosis, providing insights into SV cycling dynamics.

Can this method of SV cycling analysis be adapted for other models?

Yes, though adaptations are needed, the principles of FM dye uptake and the stimulation techniques can be applied to other synaptic models.

What are the limitations of using FM dyes in this study?

One limitation is the potential for phototoxicity or dye leakage, which can affect the accuracy of results and the interpretation of synaptic activity.

Sinaptik vezikül (SV) Bisiklete binme nöronal sinapslarda, hücreler arası iletişim çekirdek mekanizması vardır. FM boya alımı ve yayın kantitatif SV endo - ve ekzositozu raporlaması birincil yoludur. Burada, biz FM1-43 Drosophila nöromüsküler kavşak (NMJ) modeli sinaps Bisiklet sürmeyi tüm uyarım yöntemleri karşılaştırın.

Bu deneyin genel amacı, floresan FM boyaları kullanılarak Drosophila larva nöromüsküler kavşağının canlı hazırlığı üzerinde sinaptik veziküllerin endo ve ekzositoz döngüsünü gözlemlemektir. Bu yöntem, sinaptik vezikül döngüsünün işlevi için hangi genlerin kritik olduğu gibi sinirbilim alanındaki temel soruları yanıtlamaya yardımcı olabilir. Bu tekniğin temel avantajı, üç farklı uyaran türü, yüksek potasyum depolarizasyonu, elektrik stimülasyonu veya optogenetik kullanarak canlı bir preparatta vezikül döngüsünü optik olarak izleyebilmenizdir.

Bu prosedür için, diseksiyon yapan saline bir lexoflor 647'ye konjuge edilmiş anti-HRP antikorunu ekleyin. Daha sonra, diseksiyon odasına elastomer kaplı bir kapak fişi ekleyin ve ardından diseksiyon sırasında NMJ presinaptik terminali etiketlemek için HRP antikoru ile diseksiyon solüsyonunu ekleyin. İnce bir boya fırçası kullanarak, gıda şişesinden dolaşan üçüncü bir yıldızı elastomer kaplı bir kapak camına aktarın.

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