UC San Diego

Neuronal networks are responsible for complicated responses to stimuli and behavior. Understanding these networks involves recording the changes in transmembrane electrical potential from small subcellular locations such as dendritic spines, to large networks of neurons. This dissertation represents a concerted effort toward the development of small molecule probes with fluorescent intensities responsive to the transmembrane electrical potential of neurons.

Fluorescent voltage sensitive molecules termed VoltageFluors (VF) are capable of undergoing photo-induced electron transfer (PeT). Previous work demonstrated that when embedded within a membrane of interest, the local electric field modulates the extent of PeT, and thus the fluorescence intensity as well. This thesis presents efforts toward rationally improving the voltage sensitivity of VF dyes through tuning the driving force for electron transfer and optimizing the structure of bridges. Optimization led to a significant improvement in voltage sensitivity of newly developed dyes epitomized by VF2.1OMe.H with a 48% ∆F/F per 100 mV demonstrated in HEK cells. This voltage sensor was to record voltage changes across neuronal systems from leech ganglia to mouse brain slices with high fidelity as well.

In addition to sensitivity improvements, VF dyes were developed with a Cy5-like acceptor for far red fluorescence imaging in biology, The most sensitive dye in this class, VF Red, showed a 25% ∆F/F per 100 mV in HEK cells and could effectively be used with optical activity activators for all optical stimulation and recording of electrical activity in neurons.

Further improvements to recording voltage changes in biological systems included work toward reducing background fluorescence and generating a higher signal to noise ratio by generating non-fluorescent VF dyes by chemical reduction. These molecules were then subjected to selective reoxidation back to fluorescent VF dyes solely where oxidizing protein was present. Both fluorescein and cy5-based VF dyes were shown to undergo a degree of oxidation in vitro upon addition of oxidizing proteins. These efforts represent significant improvements to VF dyes toward improved optical sensor of voltage across many neuronal systems.