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|Title:||DEVELOPMENT OF FAST-GCAMP INDICATORS FOR NEURONAL SPIKE COUNTING|
|Authors:||Applegate, Marissa Claire|
|Advisors:||Wang, Samuel S.|
|Abstract:||Optical imaging of calcium transients is a promising technique in neuroscience to track neuronal activity with high spatial and temporal resolution. Previous work has made progress in optimizing GFP Calmodulin Fusion Proteins (GCaMP), a popular genetically encodable fluorescent calcium indicator, for use in experiments in the nervous system. However, the best performing GCaMP, GCaMP6f, still suffers from slow binding and release kinetics. These introduce temporal delays into the probe’s fluorescent readouts and limit the ability of GCaMP indicators to serve as accurate detectors of neuronal activity. This work sought to develop fast-GCaMP indicators capable of accurately detecting single action potential-triggered calcium transients in rapidly-firing neurons. Structure-guided site-directed mutagenesis pursuing several overarching strategies was used to generate 40 GCaMP variants. Variants were assayed using UV/Vis spectroscopy, fluorimetry, and stopped-flow photometry to determine their relevant biophysical parameters and kinetic behavior. Selected variants were monitored using slice physiology to ascertain their responses to induced calcium transients in cerebellar parallel fiber boutons. Up to six-fold acceleration of decay kinetics and four-fold acceleration of rise kinetics over those of GCaMP6f were achieved in the best performing variants. These data point towards several sites that are of interest for pursuing further mutations to improve the kinetics of fast-GCaMP indicators while increasing the indicator’s affinity for calcium, dynamic range of brightness, and maximum fluorescence.|
|Type of Material:||Princeton University Senior Theses|
|Appears in Collections:||Chemistry, 1926-2017|
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