Comparative Analysis of the optogenetic Cal-Light and FLARE Systems: A context-dependent Efficacy and Reliability Evaluation
1 Functional Neuroconnectomics Group, Institute for Experimental Epileptology and Cognition Research, University of Bonn Medical Faculty
Calcium-dependent optogenetic systems such as Cal-Light and FLARE provide powerful tools to establish direct relationships between active neuronal ensembles during select behaviors by translating neuronal activity into gene expression within a time window defined by light exposure. Mechanistically, both systems consist of synthetic proteins that, in response to an increase in intracellular calcium concentration and simulatenous blue light illumination, release the transcriptional activator tTa into the nucleus to initiate transcription of a tet-dependent reporter. Despite a similar use case and significant improvements since their first documented use, a direct comparative analysis is lacking. Cal-Light and its variant, ST-Cal-Light, utilize a three-component system, whereas scFLARE operates with an optimized two-component system. Although scFLARE's design mitigates stoichiometric imbalance by requiring only one protein plus the fluorescent reporter gene, the dynamics of the complex multimodular scFLARE protein compared to the split Cal-Light proteins require further investigation. In this study, we systematically compare the scFLARE- with the Cal-Light systems. Our findings provide a guideline for the contexts in which each system performs optimally, thus providing critical insights for their application in neuroscience research.