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Related Experiment Video

Updated: May 7, 2026

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Published on: April 3, 2014

Biosensor imaging in brain slice preparations.

Marina Polito¹, Pierre Vincent, Elvire Guiot

¹Centre National de la Recherche Scientifique, Unité Mixe de Recherche and Université Pierre et Marie Curie, Paris, France.

Methods in Molecular Biology (Clifton, N.J.)

|September 21, 2013

Summary

Researchers developed a new method to measure cyclic-AMP dependent protein kinase (PKA) activation in real-time using FRET biosensors. This technique allows for high-resolution monitoring of PKA activity in living neurons, advancing our understanding of cellular signaling.

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Last Updated: May 7, 2026

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Area of Science:

Neuroscience
Cell Biology
Biochemistry

Background:

Cyclic-AMP dependent protein kinase (PKA) is a key regulator in the nervous system, integrating neuromodulator effects.
Measuring PKA activation in living cells has been challenging due to limitations in temporal and spatial resolution.

Purpose of the Study:

To present a novel protocol for real-time monitoring of PKA activation in intact neurons.
To demonstrate the utility of FRET-based biosensors for studying kinase signaling dynamics.

Main Methods:

Utilized FRET-based biosensors (e.g., AKAR) to measure PKA activation via fluorescence emission ratios (CFP/YFP).
Employed wide-field and two-photon imaging techniques for real-time monitoring in rodent brain slices.
Optimized protocol for morphologically intact mature neurons, including pyramidal cortical neurons.

Main Results:

Achieved high temporal resolution (seconds) for PKA activation measurements at cellular and subcellular levels.
Successfully monitored PKA activity in real-time within intact neurons using advanced imaging modalities.
Provided practical details applicable to various cellular preparations and FRET biosensors.

Conclusions:

The developed FRET-based protocol offers a powerful tool for studying PKA signaling dynamics in living neurons.
This approach significantly enhances the understanding of spatiotemporal integration processes in cellular signaling.
The methodology is adaptable for other kinase FRET biosensors and broadly applicable to future cellular assays.