My work has focused on the development and application of fluorescent voltage-sensitive proteins based on microbial rhodopsins. These probes led to the discovery of electrical activity in the bacterium Escherichia coli, the first robust optical recordings of action potentials (APs) in mammalian neurons using a genetically encoded voltage reporter, and the development of a genetically targetable all-optical electrophysiology system.
I first introduce an engineered fluorescent voltage sensor based on green-absorbing proteorhodopsin. Expression of the proteorhodopsin optical proton sensor (PROPS) in E. coli revealed electrical spiking at up to 1 hertz. Spiking was sensitive to chemical and physical perturbations and coincided with rapid efflux of a small-molecule fluorophore, suggesting that bacterial efflux machinery may be electrically regulated.
I then present another microbial rhodopsin, Archaerhodopsin 3 (Arch), whose endogenous fluorescence exhibited a twofold increase in brightness between -150 mV and +150 mV and a sub-millisecond response time. In rat hippocampal neurons, Arch detected single electrically triggered APs with an optical signal-to-noise ratio > 10. A mutant, Arch(D95N), lacked endogenous proton pumping and had 50% greater sensitivity than the wild type but had a slower response (41 ms). Nonetheless, Arch(D95N) also resolved individual APs.
Finally, I introduce evolved archaerhodopsin-based voltage indicators and a spectrally orthogonal channelrhodopsin actuator, which together enabled all-optical electrophysiology. A directed evolution screen yielded two mutants, QuasAr1 and QuasAr2, that showed improved brightness and voltage sensitivity relative to previous archaerhodopsin-based sensors, and microsecond response times. An engineered channelrhodopsin actuator, CheRiff, showed high light sensitivity and rapid kinetics. A coexpression vector, Optopatch, enabled cross-talk-free genetically targeted all-optical electrophysiology. In cultured neurons, the Optopatch system probed membrane voltage across temporal and spatial scales, from the sub-cellular and sub-millisecond dynamics of AP propagation, to the simultaneous measurement of firing patterns of many neurons in a circuit. In brain slices, Optopatch induced and reported APs and subthreshold events with high signal-to-noise ratios. In human stem cell-derived neurons, Optopatch measurements revealed homeostatic tuning of intrinsic excitability, a subtle form of plasticity that had yet to be observed in human neurons.
The suite of tools and techniques presented here enable high-throughput, genetically targeted, and spatially resolved electrophysiology without the use of conventional electrodes. / Engineering and Applied Sciences
| Identifer | oai:union.ndltd.org:harvard.edu/oai:dash.harvard.edu:1/13064932 |
| Date | 21 October 2014 |
| Creators | Hochbaum, Daniel Ron |
| Contributors | Cohen, Adam Ezra |
| Publisher | Harvard University |
| Source Sets | Harvard University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis or Dissertation |
| Rights | open |
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