This proposal aims to develop better tools for analyzing brain cells and circuits and for large-scale recordings of brain activity. The currently available tools are relatively primitive in terms of sensitivity and speed. One major function of a neuron is to process electrical signals. Thus a tool that is of particular significance is high speed membrane potential imaging. Genetically encoded fluorescent protein voltage indicators (GEVI's) are a obvious strategic approach for ?visualizing the brain in action?. Genetically encoded sensors are especially interesting to neuroscientists because, as proteins, they can be expressed in individual cell types in the mammalian brain. Because each brain region has up to 100 different cell types, sensor expression in a specific cell type is essential for imaging the activity of that cell type. Recently, there has been a dramatic improvement in the signal size of GEVIs (i.e. ArcLight, 40%/100 mv) but ArcLight has a relatively slow response time constant (?=10 msec). There are now several faster GEVIs (?=0.3 to 2.0 msec) but they have smaller signal sizes (~10%/100mv). One goal of this proposal is developing a GEVI with both large and fast responses to membrane potential changes. Several probe characteristics other than size and speed are also critically important. One is the wavelength range of excitation and emission. At present many sensors and activators are based on GFP and its analogues. Thus, probes with red excitation and emission spectra would allow simultaneous dual function measurements.
One aim i s to develop useful red GEVIs. Second, many GEVIs have a sigmoidal fluorescence-voltage relationship. The position of this relationship along the voltage axis can be adjusted via mutations in the voltage sensitive domain of the GEVI. Thus GEVIs can be selective reporters of different ranges of the neuron membrane potential and thereby selective for action potential activity versus subthreshold activity. This selectivity depends on both the voltage at half-maximal activation as well as the steepness of the sigmoidal curve. Lastly, it will be important to target the GEVIs to specific regions of the neuron including cell body, dendritic post-synaptic zones, and presynaptic terminals. All of the probes in the proposal are based on the voltage sensitive domain of a membrane protein (a phosphatase from Ciona or zebrafish) with one or two fluorescent proteins inserted into the N- or C-terminal region. The development of improved GEVIs will involve molecular biology for generating novel probes (bigger, faster, red, targeted anatomically and physiologically) followed by testing in cultured HEK293 cells, acutely dissociated neurons, and zebrafish embryos. Probes that function well in these initial screens will then be incorporated into virus particles for in vivo transfection and measurements in the mammalian brain. Improved and selective GEVIs would be useful to the community carrying out optical measurements of brain activity.

Public Health Relevance

Voltage sensitive dyes have been used extensively in the study of epilepsy and action potential propagation during cardiac arhythmias (e.g. Matiukas et al, 2006; Mironov et al, 2006). Both kinds of measurements would benefit from the cell-type specificity provided by GEVIs.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project--Cooperative Agreements (U01)
Project #
5U01NS099691-03
Application #
9552934
Study Section
Special Emphasis Panel (ZNS1)
Program Officer
Talley, Edmund M
Project Start
2016-09-30
Project End
2019-08-31
Budget Start
2018-09-01
Budget End
2019-08-31
Support Year
3
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Yale University
Department
Physiology
Type
Schools of Medicine
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
Sepehri Rad, Masoud; Cohen, Lawrence B; Braubach, Oliver et al. (2018) Monitoring voltage fluctuations of intracellular membranes. Sci Rep 8:6911
Piatkevich, Kiryl D; Jung, Erica E; Straub, Christoph et al. (2018) A robotic multidimensional directed evolution approach applied to fluorescent voltage reporters. Nat Chem Biol 14:352-360
Eum Kang, Bok; Lee, Sungmoo; Baker, Bradley J (2018) Optical consequences of a genetically-encoded voltage indicator with a pH sensitive fluorescent protein. Neurosci Res :
Yi, Bumjun; Kang, Bok Eum; Lee, Sungmoo et al. (2018) A dimeric fluorescent protein yields a bright, red-shifted GEVI capable of population signals in brain slice. Sci Rep 8:15199
Sepehri Rad, Masoud; Choi, Yunsook; Cohen, Lawrence B et al. (2017) Voltage and Calcium Imaging of Brain Activity. Biophys J 113:2160-2167
Jung, Arong; Rajakumar, Dhanarajan; Yoon, Bong-June et al. (2017) Modulating the Voltage-sensitivity of a Genetically Encoded Voltage Indicator. Exp Neurobiol 26:241-251
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Tanese, Dimitrii; Weng, Ju-Yun; Zampini, Valeria et al. (2017) Imaging membrane potential changes from dendritic spines using computer-generated holography. Neurophotonics 4:031211
Storace, Douglas; Sepehri Rad, Masoud; Kang, BokEum et al. (2016) Toward Better Genetically Encoded Sensors of Membrane Potential. Trends Neurosci 39:277-289