Neuromodulators play a crucial role in regulating neural activity and brain functions. Understanding their effects on target cells and circuits is vital for neuroscience. Genetically encoded fluorescent dopamine indicators revolutionized the field by allowing high-resolution imaging of neuromodulators.

This study emerged from our efforts to enhance and streamline the engineering process for G-protein-coupled receptor (GPCR)-based indicators in our laboratory. Our approach leverages on previously-optimized fluorescent protein modules from a green fluorescent and a red fluorescent dopamine indicator (dLight1.3b and RdLight1) to develop new indicators for other neuromodulators in an efficient and resource-effective manner. To identify optimal positions for inserting the fluorescent protein module into GPCRs, we started by examining sequence alignments and structural models generated by AlphaFold. We reasoned that the incorporation of the fluorescent protein modules should follow a sequence alignment guided by the Ballesteros-Weinstein (BW) numbering scheme for GPCRs. This scheme utilizes highly conserved residues in each of the seven transmembrane helices as reference points. Our hypothesis was that aligning the module insertion based on the BW numbering scheme is vital to account for variations in length among transmembrane alpha-helices of different GPCRs. This alignment strategy is considered to effectively prevent distortions of the sensing module and allow for the successful integration of a chromophore microenvironment optimized for sensing GPCR activation.

To evaluate the versatility of the chosen strategy for developing GPCR-based indicators, we grafted the fluorescent protein modules onto ten different GPCRs. The modules, composed of two building blocks, were inserted in two cloning steps, replacing specific regions within the second and third intracellular loops of the receptor. Most indicators successfully expressed and localized at the cell membrane. Seven green fluorescent indicators showed ligand-induced responses above 100% ΔF/F, indicating potential for in vivo use. Five red fluorescent indicators showed detectable responses, comparable to previously characterized red fluorescent GPCR-based indicators.

First-generation indicators for norepinephrine (a major neuromodulator with broad physiological and behavioral regulation) were recently introduced, but suffered from limitations in ligand-selectivity, sensitivity, kinetics, and pharmacology. To address these issues, we used our new approach to develop a new family of multicolor norepinephrine indicators by leveraging on a previously unexplored receptor subtype (adrenergic receptor Alpha-1a  (ADRA1A)).

We explored norepinephrine receptors from various animal species, and interestingly, the receptor derived from the sperm whale, known for being the animal with the largest brain on our planet, yielded the most sensitive  indicators (Figure 1a). To optimize the dynamic range of the generated indicators we varied the size of the grafted modules from dLight1.3b and RdLight1 (Figure 1b). Our breakthrough came when we discovered that grafting both, the fluorescent protein domain and the second intracellular loop (ICL2) of the receptor, simultaneously enhanced dynamic range and ligand selectivity (Figure 1c).

The resulting indicators, nLightG (green fluorescent) and nLightR (red fluorescent), showed exceptional sensitivity compared to previous tools. We extensively compared them to the existing GRAB_NE indicator in various experimental settings. The kinetics of nLightG were significantly faster, allowing precise comparison of norepinephrine reuptake kinetics in different brain areas. Notably, we observed slower norepinephrine clearance in the hippocampus compared to the locus coeruleus.

Another crucial aspect is ligand selectivity. Our indicators exhibited superior selectivity (28-fold) for norepinephrine over dopamine compared to GRAB_NE (8-fold). Additionally, our indicators displayed distinct pharmacological properties and were able to detect norepinephrine release blocked by Alpha-2 adrenergic receptor antagonists during acute stress.

Our findings shed light on the diverse efficacy of neuromodulator reuptake across brain regions, impacting mechanistic studies of reuptake blockers used for psychiatric disorders. In vivo two-photon imaging validated the nLightG indicators effectiveness, enabling detection of norepinephrine transients in the hippocampus during specific behaviors. Future work will focus on developing variants with higher affinity for better sensitivity in areas with low norepinephrine release. Efforts to enhance the dynamic range of the red fluorescent norepinephrine indicator are well underway, with the goal of matching the sensitivity of its green counterpart. Combining these indicators with miniaturized microscopes (both one-photon and two-photon) will allow us to explore norepinephrine signals at high-resolution directly in freely-moving animals, offering new insights into the influence of this neuromodulator on animal behavior and brain function.

Check out our work here: https://www.nature.com/articles/s41592-023-01959-z