Understanding nervous system function will require the simultaneous monitoring of the electrical activity of a large number of functioning neurons. It will be important to understand the temporal interactions between large groups in networked neurons to fully understand how the central system processes information. Optical recording methods offer advantages in that they provide the spatial and temporal resolution necessary to record salient neuronal events with limited perturbations of the system. Currently available voltage-sensitive dyes are very difficult to apply experimentally, produce small changes in fluorescence for a given potential change and cannot be targeted to a specific subset of neurons, thereby reducing the complexity of the signal obtained. The present application seeks to produce a protein-based, voltage-sensitive, optical probe for use in neurons. We have developed a probe (termed SPARC), which is based on the fusion of the rat mu1 skeletal muscle sodium channel with a fluorescent protein (i.e., GFP). This construct embodies many of the features we are seeking in an optical probe of membrane potential. Xenopus laevis oocytes, injected with the cRNA for SPARC, exhibit a fluorescent signal that undergoes a rapid (<1 ms), reproducible (>1000 pulses/hour tested) and reversible change (0.1-0.5% delta F/F/100mV) in intensity during depolarization of the cell membrane. This change can be easily recorded during action potential sized depolarization pulses (<2 ms). The present application seeks to increase the delta F/F and signal-to-noise ratio of the fluorescent signal arising from this probe and validate the use of this probe in neurons. This will entail the following studies: (I) fully characterize the presently developed SPARC probe. (II) Modify the nature and location of the fluorescent protein insertion site. (III) Alter the type and number of fluorescent proteins inserted at these sites. (IV) Finally, we will attempt to develop a FRET version of the probe to enhance the fluorescence change seen with voltage steps.
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