Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics

Area of Science:

• Neuroscience
• Biochemistry
• Molecular Biology

Background:

• Genetically encoded calcium indicators (GECIs) are crucial for systems neuroscience.
• Red fluorescent proteins like mRuby are key components in developing novel GECIs.
• Understanding calcium dynamics is essential for deciphering neural circuit function.

Purpose of the Study:

• To engineer and characterize novel red, single-wavelength GECIs, termed RCaMPs.
• To evaluate the performance of RCaMP sensors in vitro and in vivo across multiple model organisms.
• To demonstrate the utility of RCaMPs in advanced applications like 2-color imaging and integrated optogenetics.

Main Methods:

• Engineering of RCaMP sensors through circular permutation of mRuby.
• Biophysical characterization and in vivo performance assessment in *C. elegans*, *Drosophila*, and zebrafish.
• Demonstration of 2-color calcium imaging within single cells and between cell populations.
• Integration with optogenetics for simultaneous neural activation and activity imaging.

Main Results:

• RCaMPs were successfully engineered and characterized for their calcium-sensing properties.
• In vivo experiments demonstrated effective calcium imaging in multiple model organisms.
• Successful 2-color imaging and simultaneous optogenetics with RCaMP were achieved.
• Comparison with R-GECO1 revealed RCaMP's suitability for optogenetic applications due to lower photoactivation.

Conclusions:

• RCaMP sensors represent a valuable addition to the GECI toolkit for neuroscience research.
• The developed sensors facilitate advanced imaging and optogenetic experiments, enabling new discoveries.
• Engineered chromatic variants of GECIs expand experimental possibilities in functional imaging and optogenetics.