Real-Time Fluorescent Measurement of Synaptic Functions in Models of Amyotrophic Lateral Sclerosis

Karthik Krishnamurthy; Davide Trotti; Piera Pasinelli; Brigid Jensen

doi:10.3791/62813

Mesure fluorescente en temps réel des fonctions synaptiques dans les modèles de sclérose latérale...

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Neuroscience

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JoVE Journal Neuroscience

Real-Time Fluorescent Measurement of Synaptic Functions in Models of Amyotrophic Lateral Sclerosis

Mesure fluorescente en temps réel des fonctions synaptiques dans les modèles de sclérose latérale amyotrophique

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08:59 min

July 16, 2021

DOI: 10.3791/62813-v

Karthik Krishnamurthy*¹, Davide Trotti¹, Piera Pasinelli¹, Brigid Jensen*¹

¹Jefferson Weinberg ALS Center,Thomas Jefferson University

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Overview

This study discusses two methods for visualizing subcellular events pertinent to synaptic transmission, focusing on real-time monitoring of presynaptic calcium influx and synaptic vesicle membrane fusion in cultured neurons. The protocols presented can be applied to assess experimental manipulations related to synaptic processes and investigate potential therapeutic interventions.

Key Study Components

Area of Science

Neuroscience
Synaptic Transmission
Live-Cell Imaging

Background

Understanding synaptic processes is crucial for elucidating mechanisms of neuronal development and degeneration.
Real-time imaging enables monitoring of calcium dynamics and synaptic vesicle release.
Techniques can be adapted for various neuronal models, including rodent primary cultures and iPSC-derived neurons.

Purpose of Study

To develop reliable methods for monitoring synaptic function and evaluating the impact of experimental or therapeutic compounds.
To investigate the effects of disease-related proteins or RNAs on synaptic transmission.
To offer a rapid alternative to traditional electrophysiological assessments.

Methods Used

Live-cell imaging in in vitro cultured neurons implemented using confocal microscopy.
Primary rodent cortical neurons and neurons derived from induced pluripotent stem cells were the biological models used.
Timelines included phase-based imaging over intervals of 3-5 minutes for data collection.
Specific protocols for dye loading and imaging settings were detailed, enhancing imaging quality and data reliability.

Main Results

Successful visualization of synaptic vesicle release showed that calcium dynamics relate to synaptic function.
Impaired synaptic transmission was indicated by retained dye fluorescence in certain experimental conditions.
Fluorescence intensity measurements provided insight into synaptic activity pre- and post-depolarization.
Findings support the potential therapeutic application of the methods for understanding and treating synaptic dysfunction.

Conclusions

The study enables the visualization of critical events in synaptic transmission, aiding in the assessment of neuronal health.
Methods developed can facilitate the exploration of therapeutic compounds targeting synaptic processes.
Overall, these approaches contribute fundamentally to our understanding of synaptic mechanisms and potential disease interventions.

Frequently Asked Questions

What are the advantages of using live-cell imaging in these studies?

Live-cell imaging allows researchers to monitor synaptic events in real-time, providing insights into dynamic processes that are often lost with static methods.

How are the primary rodent neurons prepared for imaging?

The primary cortical neurons are transfected and incubated with specific buffers and dyes to facilitate visualization under confocal microscopy.

What types of data are obtained from these imaging techniques?

Researchers obtain fluorescence intensity measurements related to calcium dynamics and synaptic vesicle release, enabling the assessment of synaptic function.

How can these methods be adapted for other neuronal types?

The protocols can be customized for various neuronal cultures, including those derived from induced pluripotent stem cells, by modifying dye loading and imaging settings.

What limitations should be considered when using these methods?

Key limitations may include issues related to dye specificity and the need for optimized imaging conditions to avoid artifacts in the data.

What implications do the findings of this study have for therapeutic research?

The findings provide a framework for evaluating potential therapeutic compounds that can alter synaptic processes, relevant for conditions impacting neuronal function.

Deux méthodes connexes sont décrites pour visualiser les événements subcellulaires nécessaires à la transmission synaptique. Ces protocoles permettent de surveiller en temps réel la dynamique de l’afflux de calcium présynaptique et de la fusion de la membrane des vésicules synaptiques à l’aide de l’imagerie cellulaire vivante de neurones cultivés in vitro .

Ces méthodes permettent d’évaluer rapidement si une manipulation expérimentale, une protéine responsable de la maladie ou de l’ARN peut avoir un impact sur les processus synaptiques et si les composés thérapeutiques peuvent restaurer la fonction. Ces techniques fournissent une lecture fiable de la fonction synaptique d’un groupe de neurones dans un laps de temps relativement court par rapport à l’électrophysiologie. Ce protocole peut être appliqué à toute maladie de développement neuronal ou de dégénérescence.

Cela fonctionne bien pour les cultures neuronales primaires de rongeurs et pour les neurones dérivés de cellules souches pluripotentes induites. Pour commencer, optimisez les paramètres d’acquisition d’image à l’aide d’un logiciel d’acquisition d’imagerie confocale. Maintenez le temps d’exposition de la puissance d’excitation, le gain du détecteur et la fréquence d’images constants sur tous les échantillons.

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