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.

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.