Bioluminescent Genetically Encoded Glutamate Indicators for Molecular Imaging of Neuronal Activity

Genetically encoded optical sensors and advancementsin microscopyinstrumentation and techniques have revolutionized the scientifictoolbox available for probing complex biological processes such asrelease of specific neurotransmitters. Most genetically encoded opticalsensors currently used are based on fluorescence and have been highlysuccessful tools for single-cell imaging in superficial brain regions.However, there remains a need to develop new tools for reporting neuronalactivity in vivo within deeper structures withoutthe need for hardware such as lenses or fibers to be implanted withinthe brain. Our approach to this problem is to replace the fluorescentelements of the existing biosensors with bioluminescent elements.This eliminates the need of external light sources to illuminate thesensor, thus allowing deeper brain regions to be imaged noninvasively.Here, we report the development of the first genetically encoded neurotransmitterindicators based on bioluminescent light emission. These probes wereoptimized by high-throughput screening of linker libraries. The selectedprobes exhibit robust changes in light output in response to the extracellularpresence of the excitatory neurotransmitter glutamate. We expect thisnew approach to neurotransmitter indicator design to enable the engineeringof specific bioluminescent probes for multiple additional neurotransmittersin the future, ultimately allowing neuroscientists to monitor activityassociated with a specific neurotransmitter as it relates to behaviorin a variety of neuronal and psychiatric disorders, among many otherapplications.