An Optimized Intensity-Based Glutamate Sensing Fluorescent Reporter
The major neurotransmitter in the brain, glutamate is an important signaling molecule in biological organisms. The ability to visualize and quantitate glutamate transients is broadly useful to biologists, and particularly valuable in neuroscience research. Glutamate plays a critical role in nearly all aspects of normal brain function, and glutamate dysregulation is associated with stroke and neurodegenerative disorders including Alzheimer’s disease.
Methods for glutamate detection in intact cells are required, but classical tools like microdialysis have suffered from low signal-to-noise ratios, poor localization, and slow kinetics. Recently developed optical sensors are composed of glutamate-binding proteins coupled to fluorescent readouts. These have improved temporal and spatial resolution, but are still hampered by small fluorescence increases upon glutamate binding and limited to proof-of-principle experiments.
Janelia scientists have developed a new intensity-based glutamate sensing fluorescent reporter (iGluSnFR) constructed from E. coli GltI and circularly permutated GFP. An optimized single wavelength glutamate sensor has been engineered in vitro for maximum fluorescence response and then validated in increasingly complex in vivo systems. This sensor is much brighter than existing options, responds rapidly and specifically to glutamate, and correlates with simultaneous electrophysiology. Moreover, iGluSnFR can be used for two-color imaging experiments and long-term in vivo imaging of glutamate signaling in worms, zebrafish, and mice.
The iGluSnFR construct provides an improved means to directly map excitatory synaptic activity in the brain and will complement existing imaging methods for studies of neural activity and signaling events. Glutamate imaging studies in non-neuronal tissues will also benefit from the improved performance of iGluSnFR.
All data from this study are available upon request. All constructs have been deposited at Addgene (#106174–106206; hSynapsin1, FLEX-hSynapsin1, FLEX-CAG, GFAP promoters; some fusions with the red fluorescent protein mRuby3 are available). Sequences have been deposited in GenBank (MH392460, MH392461, MH392462, MH392463, MH392464, and MH392465). AAV is available from Addgene.
- Extremely rapid detection of glutamate transients with high spatial resolution
- Improved signal-to-noise ratio versus existing fluorescent glutamate sensors
- Genetically-encoded and thus can be targeted to specific cellular populations and sub-cellular locations
- Enables direct visualization of synaptic release (as opposed to Ca2+ imaging)
- Neurobiology research using in vitro or in vivo models, including long-term imaging studies
- Glutamate signaling research in non-neuronal tissues
- May increase yield of glutamate in industrial scale fermentation
- Purchase viral constructs from Vigene Biosciences.
- Obtain the plasmid from Addgene for Non-Profit Research.
- Commercial Licenses are available for internal research or sale.
For inquiries, please reference:
Janelia 2013-025 and 2015-038