UC Berkeley

Fluorescent reporters of membrane potential are important tools in neuroscience, offering a non-invasive, real-time optical readout of neuronal activity. Voltage sensitive dyes of the VoltageFluor family report neuronal activity with high sensitivity and sub-millisecond response times, but lack the cell-type specific targetability of genetically encoded tools, placing certain constraints on their applicability in complex environments (i.e. tissue slice, in vivo, sub-cellular localization).

The work presented in this thesis addresses these problems through two distinct strategies. In Chapters 1 and 2 I describe the development of weakly-fluorescent, photoactivatable “caged” VoltageFluors. Spatial control of illumination allows for release of VoltageFluor at the single cell and subcellular levels in cultured mammalian neurons, and subsequent recording of evoked action potentials. This caging strategy has proven to be generalizable, and Chapter 3 and Appendix 1 detail the synthesis of enzymatically-activatable probes. In Chapter 4 I present a strategy for the selective labeling of cells with a VoltageFluor derivative, which harnesses the known covalent interaction between a short peptide, SpyTag, and its cognate binding protein, SpyCatcher. I synthesized a series of VoltageFluor-SpyTag conjugates, or VoltageSpy dyes, which specifically label the outer membranes of cells expressing SpyCatcher at the cell surface, with minimal off-target labeling, and without requiring a wash step. I demonstrate the application of this system to imaging spontaneous activity in cultured mammalian neurons, as well as imaging evoked activity in axons and dendrites. Appendix 2 describes efforts to apply VoltageSpy dyes in mouse brain slice. Finally, Chapter 5 presents a general strategy for minimizing phototoxic effects sometimes encountered when imaging with VoltageFluor dyes, using either bath applied triplet state quenchers or an intramolecularly-stabilized VoltageFluor derivative. Cyclooctatetraene was found to be an effective protective agent – a result which may prove broadly useful in imaging VoltageFluor dyes, improving the applicability of the fluorogenic and covalently-targeted tools presented in Chapters 1-4.