Bioluminescent Genetically Encoded Glutamate Indicators for Molecular Imaging of Neuronal Activity

Abstract

Genetically encoded optical sensors and advancements in microscopy instrumentation and techniques have revolutionized the scientific toolbox available for probing complex biological processes such as release of specific neurotransmitters. Most genetically encoded optical sensors currently used are based on fluorescence and have been highly successful tools for single-cell imaging in superficial brain regions. However, there remains a need to develop new tools for reporting neuronal activity in vivo within deeper structures without the need for hardware such as lenses or fibers to be implanted within the brain. Our approach to this problem is to replace the fluorescent elements of the existing biosensors with bioluminescent elements. This eliminates the need of external light sources to illuminate the sensor, thus allowing deeper brain regions to be imaged noninvasively. Here, we report the development of the first genetically encoded neurotransmitter indicators based on bioluminescent light emission. These probes were optimized by high-throughput screening of linker libraries. The selected probes exhibit robust changes in light output in response to the extracellular presence of the excitatory neurotransmitter glutamate. We expect this new approach to neurotransmitter indicator design to enable the engineering of specific bioluminescent probes for multiple additional neurotransmitters in the future, ultimately allowing neuroscientists to monitor activity associated with a specific neurotransmitter as it relates to behavior in a variety of neuronal and psychiatric disorders, among many other applications.

Theranostics - a sure cure for cancer after 100 years?

“…Recently, split NanoLuc was used to create a sensor for the neurotransmitter glutamate. Upon binding of glutamate, a marked increase in luminescence was observed 182 , 183 . Bioluminescence has the potential to non-invasively stimulate nearby optogenetics for therapeutic purposes.…”

Bioluminescence Imaging of Potassium Ion Using a Sensory Luciferin and an Engineered Luciferase

“…This strategy has led to the development of several bioluminescent sensors for calcium ions (Ca 2+ ) and neurotransmitters. 21,[27][28][29][30] However, this approach necessitates the identification of appropriate protein-based sensory domains for specific analytes of interest, and the engineering process can be tedious with unpredictable outcomes.…”

Bioluminescence Imaging of Potassium Ion Using a Sensory Luciferin and an Engineered Luciferase

“…In certain cases, the luciferases are further fused with red-emitting FPs to achieve red-shifted emission for better tissue penetration. This strategy has led to the development of several bioluminescent sensors for calcium ions (Ca 2+ ) and neurotransmitters. ,− However, this approach necessitates the identification of appropriate protein-based sensory domains for specific analytes of interest, and the engineering process can be tedious with unpredictable outcomes.…”