Optimized design and in vivo application of optogenetically modified Drosophila dopamine receptors to elucidate endogenous functions

Abstract

Precise control over G-protein-coupled receptors (GPCRs) signaling is key to understanding their role in physiology and potential as drug targets. Light-activated chimeric GPCRs (optoXRs) combining naturally occurring opsins with the desired signaling properties have been developed to study the function of target GPCRs. Despite extensive efforts however, achieving functional optoXRs has remained a challenge. In this thesis, I investigated newly designed optogenetically modified dopamine receptors (optoDopRs) to study Dopaminergic signaling and receptor-specific function in Drosophila. In vitro, I characterized optoDopR signaling and found that the optimized design resulted in improved signaling specificity and light sensitivity. These optimized optoDopRs offer a broad range of activation wavelengths, making them compatible with other optogenetic tools, such as the cation channelrhodopsin Chrimson. In vivo, I observed that optimized optoDopRs exhibited a localization pattern and signaling responses similar to the endogenous dopamine receptors. Second, I showed that the optoDopR variants could functionally replace endogenous DopRs in various behavioral experiments, including odor preference, locomotion, and odor-reward learning. Furthermore, specific behaviors such as arousal and feeding were influenced by cell type-specific optoDopR activation. Taken together, I demonstrated that optoDopRs display high light sensitivity, cell type specificity, and endogenous-like dopaminergic signaling. Future strategies like structure-based design and the use of spectrally tuned or bistable rhodopsin backbones could provide further strategies to extend the optogenetic toolbox. The improved design of optoDopRs as shown here should thus offer a valuable tool for studying DA signaling in vitro and in vivo.