Neuronal network imaging in acute slices using Ca2+ sensitive bioluminescent reporter.

Area of Science:

• Neuroscience
• Molecular Biology
• Biochemistry

Background:

• Genetically encoded indicators are crucial for real-time intracellular signaling studies.
• Aequorin, a natural bioluminescent calcium (Ca2+) sensor, offers advantages over fluorescent reporters, including minimal background signal and no need for light excitation.
• Expressing genetically encoded sensors like aequorin in intact brain tissue is challenging compared to dissociated cells.

Purpose of the Study:

• To develop and present a method for expressing the GFP-aequorin (GA) fusion protein in pyramidal cells of neocortical acute slices.
• To enable bioluminescence imaging of Ca2+ transients in neurons within intact brain tissue.

Main Methods:

• Utilized recombinant Sindbis virus for efficient gene delivery.
• Expressed a GFP-aequorin (GA) fusion protein in pyramidal neurons.
• Employed bioluminescence imaging techniques to record Ca2+ transients.

Main Results:

• Successfully expressed the GA fusion protein in hundreds of neurons within neocortical acute slices.
• Enabled simultaneous bioluminescence recording of Ca2+ transients in single or multiple neurons.
• Demonstrated the feasibility of using aequorin-based indicators in complex neural tissue.

Conclusions:

• Recombinant Sindbis virus is an effective tool for expressing genetically encoded Ca2+ indicators in intact brain slices.
• This method facilitates the study of neuronal activity and intracellular signaling in complex neural circuits.
• The developed technique enhances the utility of bioluminescent Ca2+ imaging in neuroscience research.