Engineering and Characterization of Genetically Encoded Fluorescent Indicators for Monitoring Norepinephrine Dynamics In Vivo

The brain is the most complex biological system we currently know. Its high numerical and structural complexity, the fast and massively parallel communication processes and the dynamic nature of the entire network on multiple levels make it extremely challenging to study its function. A central aspect of brain function is the constant exchange of electrical and chemical signals between neurons. In order to understand these signals and find out how they encode and integrate information, scientists need the tools to follow them in vivo and investigate how they respond to various stimuli. Norepinephrine (NE) is one of the most important small molecule neuromodulators in the vertebrate brain and is released broadly along the entire neuroaxis. While it is clear that NE signaling is critically involved in a range of important neurological functions, its exact role in these processes is only partially known. To improve our understanding of noradrenergic signaling it is essential to follow the spatiotemporal dynamics of NE in vivo with high specificity and resolution. A powerful class of tools to track these signals at high spatiotemporal resolution are genetically encoded fluorescent indicators (GEFIs). These protein-based probes can be engineered to report the presence of a specific signal as a fluorescent change, which can be readout using various imaging methods such as fibre photometry, one-photon or two-photon microscopy. By fusing circularly permuted fluorescent proteins (cpFPs) into G-protein coupled receptors (GPCRs) in a conformationally sensitive way, researchers were able to engineer new GEFIs for various neurotransmitters, neuromodulators, and neuropeptides. This thesis describes the development, characterization and in vivo validation of new red and green fluorescent GPCR-based GEFIs for NE. To generate these indicators, we grafted the fluorescent modules of previously published red- and green fluorescent dopamine (DA) indicators onto the sperm whale α1a adrenergic receptor, which we chose due to its increased selectivity for NE over DA. To showcase the utility of our alignment-based grafting approach we tested it on several other GPCRs, which led to the generation of functional but moderately performing indicators for several other ligands. The new NE indicators were tested in mice using fibre photometry or two-photon microscopy, detecting spatiotemporal NE dynamics with high sensitivity in various brain regions. We anticipate that these new tools will enable a more reliable investigation of spatiotemporal NE dynamics by neuroscientists and ultimately help to elucidate the precise role of NE in various neurological processes. $\textit{Full-text embargoed until: 2026-02-17}$