Fluorescent biosensors offer an innovative approach to studying axonal metabolism, providing real-time, subcellular insights into neuronal energy dynamics. Dr. Julien Courchet’s work emphasizes their potential in elucidating mitochondrial distribution and metabolic flux, advancing our understanding of neuronal function and circuitry.

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1. Why use this method?

Mitochondria are critical for many cellular functions, particularly in neurons, where high energy demands require precise distribution of these organelles throughout the extensive cytoplasmic volume. This distribution is of particular importance along the axon, as it ensures the efficient transport of mitochondria to the presynaptic sites, in order to support the formation and maintenance of neuronal circuits. While several techniques exist to study mitochondrial function, these are often limited by a lack of single-cell resolution. This limitation can be overcome using plasmid-based fluorescent biosensors, which provide real-time, subcellular insights into changes in cellular content, enabling detailed characterization of metabolic activity and flux within developing axons. These biosensors rely on distinct technology, such as FRET, or conformation change, which need adapted microscopy. However, the shared principle is that, upon binding of the molecule of interest, changes in fluorescence can be measured and converted into changes in the absolute or relative concentration of a cellular metabolite.

2.   What you’ll need

Neuronal primary cell culture and electroporation

• Mice embryos (E 15.5)
• Plasmid DNA of interest (endotoxin free)
• Fast green
• Microinjector (e.g. MicroInject 1000, BTX)
• Electroporator (e.g. ECM 830, BTX)
• HBSS supplemented with Hepes, CaCl₂, MgSO₄. NaHCO₃, D-glucose 
• Papain, DNAse
• 24 well cell culture plate, glass bottom dish coated with poly D-lysine
• Neurobasal medium supplemented with B27, N2, penicillin-streptomycin, L-glutamate

Image acquisition 

• Confocal or epifluorescence microscope (e.g. Nikon C2 or CMOS Flash4)
• Optional: light beam splitter with adapter filters for CFP/YFP simultaneous imaging (e.g. FRET gemini module).
• CO₂ ­5% v/v and temperature control (all images should be acquired at 37°C)
• HBSS supplemented with Hepes, CaCl₂, MgSO₄. NaHCO₃, D-glucose.

Image analysis 

• FIJI software 

3.   Step-by-step instructions 

• Neuronal primary cell culture and electroporation 

Isolate the brain from the embryo at E15.5 and transfer it on ice cold HBSS supplemented with Hepes, CaCl2, MgSO4. NaHCO3, D-glucose. Using microinjector, inject the plasmid DNA with fast green to the lateral ventricles, and perform an electroporation. Dissect the somatosensory cortices from both sides of the brain. Incubate the tissue with papain, DNAse, and seed the cells into 24-well plate with glass bottom dishes. Culture the neurons until 5^th day in vitro.

• Image acquisition 

To perform live imaging, use the microscope with adapted settings corresponding to the biosensor. As an example, FRET-based biosensors can be imaged with a CMOS Flash4 camera using the FRET gemini module in 1024×1024 mode. During live imaging, cells should be kept at 37°C, with CO₂ v/v in a humidified atmosphere. If needed, generate kymographs using the time-lapse movies to analyze mitochondria trafficking. Analyze the data with FIJI or an equivalent image-analysis software.

4.   Practical tips 

Cells in primary neuronal culture are sensitive and require gentle manipulation. There are several biosensors that can be used when studying mitochondrial trafficking in axons: mito-dsRed (mitochondria), PercevalHR (ATP:ADP ratio), ATeam (ATP), Pyronic (pyruvate).

5.  Critical appraisal & implications for future research 

The limitations of using metabolic biosensors are closely related to the challenges associated with live imaging.  One of the major challenges is the accuracy of measurements, which depends on the correlation between the fluorescence signal and the parameter being measured. Moreover, the imaging frequency is limited by the risk of long-term phototoxicity. Environmental factors, such as pH can affect the fluorescence signal of biosensors. Similarly, binding interactions can affect ligand availability and, consequently, cell signaling. Furthermore, inter-cellular variability can complicate data analysis. Future research should aim to improve the subcellular precision of mitochondrial tracking to achieve high-resolution imaging within the desired time windows.

This protocol is part of the Braining project, co-founded by the European Union under the project “Collaborative learning and innovative teaching in brain drug screening” (2023-1-PL01-KA220-HED-000160284). It is licensed under a Creative Commons Attribution-NonCommercial (CC BY-NC) license, allowing sharing and adaptation for non-commercial purposes with proper attribution.