Attaining effective optical modulation and readout of neuronal circuit activities has been a longstanding goal in neuroscience and is a key near-term aim of the BRAIN Initiative. Such neurotechnology is required to decipher how the brain?s electrical signals relate to perceptual, cognitive, emotional and motor functions. The idea to use light to modulate neuronal activities found its first broadly successful realization with the development of caged glutamate, but only since the use of genetically encoded (optogenetic) actuators such as channelrhodopsin, has this approach become overwhelmingly successful. The idea to use light to record electrical signals in the brain was conceptualized with the discovery of the first voltage-sensitive dyes more than half a century ago. Voltage imaging approaches have contributed much to our understanding of brain physiology, both at the cellular and systems levels, but the broad experimental use of these small molecule dyes suffers from several limitations including invasive staining procedures, pharmacological side effects, and blindness towards cellular diversity. These three limitations have been overcome by the recent invention of genetically-encoded voltage indicators (GEVIs). Although in many aspects superior to classical voltage sensitive dyes, GEVIs have not yet been satisfactorily optimized and their combination with optogenetic modulation has been difficult to achieve in practice. One major obstacle is the overlap of the spectral bands of light used to activate opsin-based actuators and at the same time excite and image available GEVIs. What is required to overcome this hurdle are well performing far red GEVIs that can be orthogonally combined with blue light-activated opsin-based actuators. We propose to use novel near-infrared (NIR) phytochrome-based fluorescent proteins (FPs) to generate a new class of GEVIs that are excited and fluoresce in the NIR spectrum, building on our expertise to generate GEVIs using GFP-like FPs. We plan to combine these NIR-GEVIs with blue-light activated excitatory and inhibitory opsins, to enable an optical approach that expands classical microelectrode-based intracellular single cell current-clamp recordings to large numbers of genetically defined neurons in awake mice. Transgenic mice in which this tool can be activated via Cre-recombinase expressing driver mouse lines will be one of our key deliverables.

Public Health Relevance

Understanding the cellular events underlying brain function will require new technologies for modulation and recording of neuronal electrical activity. Having pioneered the development and application of genetically encoded optical voltage-indicators combined with expertise in protein engineering, electrophysiology and optogenetics, here we will develop a new enabling technology for modulation and recording of voltage-signaling in genetically defined ensembles of neurons of awake and behaving mice.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project--Cooperative Agreements (U01)
Project #
1U01NS099573-01
Application #
9229649
Study Section
Special Emphasis Panel (ZNS1)
Program Officer
Talley, Edmund M
Project Start
2016-09-30
Project End
2019-06-30
Budget Start
2016-09-30
Budget End
2017-06-30
Support Year
1
Fiscal Year
2016
Total Cost
Indirect Cost
Name
U of L Imperial Col of Sci/Technlgy/Med
Department
Type
DUNS #
227092590
City
London
State
Country
United Kingdom
Zip Code
SW7 2AZ
Li, Lei; Shemetov, Anton A; Baloban, Mikhail et al. (2018) Small near-infrared photochromic protein for photoacoustic multi-contrast imaging and detection of protein interactions in vivo. Nat Commun 9:2734
van Opbergen, Chantal J M; Koopman, Charlotte D; Kok, Bart J M et al. (2018) Optogenetic sensors in the zebrafish heart: a novel in vivo electrophysiological tool to study cardiac arrhythmogenesis. Theranostics 8:4750-4764
Shcherbakova, Daria M; Cox Cammer, Natasha; Huisman, Tsipora M et al. (2018) Direct multiplex imaging and optogenetics of Rho GTPases enabled by near-infrared FRET. Nat Chem Biol 14:591-600
Redchuk, Taras A; Omelina, Evgeniya S; Chernov, Konstantin G et al. (2017) Near-infrared optogenetic pair for protein regulation and spectral multiplexing. Nat Chem Biol 13:633-639
Piatkevich, Kiryl D; Suk, Ho-Jun; Kodandaramaiah, Suhasa B et al. (2017) Near-Infrared Fluorescent Proteins Engineered from Bacterial Phytochromes in Neuroimaging. Biophys J 113:2299-2309
Quicke, Peter; Barnes, Samuel J; Knöpfel, Thomas (2017) Imaging of Brain Slices with a Genetically Encoded Voltage Indicator. Methods Mol Biol 1563:73-84
Baloban, Mikhail; Shcherbakova, Daria M; Pletnev, Sergei et al. (2017) Designing brighter near-infrared fluorescent proteins: insights from structural and biochemical studies. Chem Sci 8:4546-4557
Shemetov, Anton A; Oliinyk, Olena S; Verkhusha, Vladislav V (2017) How to Increase Brightness of Near-Infrared Fluorescent Proteins in Mammalian Cells. Cell Chem Biol 24:758-766.e3
Stepanenko, Olesya V; Stepanenko, Olga V; Kuznetsova, Irina M et al. (2017) Interaction of Biliverdin Chromophore with Near-Infrared Fluorescent Protein BphP1-FP Engineered from Bacterial Phytochrome. Int J Mol Sci 18:
Oliinyk, Olena S; Chernov, Konstantin G; Verkhusha, Vladislav V (2017) Bacterial Phytochromes, Cyanobacteriochromes and Allophycocyanins as a Source of Near-Infrared Fluorescent Probes. Int J Mol Sci 18:

Showing the most recent 10 out of 14 publications