Red-shifted voltage indicators for two-photon all-optical physiology and multi-spectral imaging - PROJECT SUMMARY/ABSTRACT Membrane voltage plays a vital role in transmitting information in the brain: neurons regulate their membrane voltage to communicate and process information, while irregular neuronal voltage dynamics are associated with several neurological disorders. Therefore, tools for recording voltage signals are crucial for studying brain computations in both healthy and diseased states. Genetically Encoded Voltage Indicators (GEVIs) —fluorescent proteins whose brightness is modulated by voltage— have been recognized for many years as promising tools with enabling features for neuroscience applications, including high spatiotemporal resolution and cell type specificity. Particularly sought after are GEVIs that perform well under two-photon (2P) microscopy, the method of choice for imaging neural activity in highly scattering tissue such as the rodent brain. We have recently demonstrated that the GFP-based indicator JEDI-2P enables sustained (> 30 min), fast (> 1 kHz), and deep-tissue (400 µm or deeper) monitoring of voltage dynamics in individual neuronal somas in awake behaving mice. However, JEDI-2P and other green GEVIs are spectrally incompatible with optogenetic tools and the best-performing calcium and neurotransmitter indicators. Red GEVIs have been developed but cannot report spikes under two-photon microscopy in vivo. The goal of this proposal is thus to address this technology gap and develop red-shifted voltage indicators for robust 2P imaging in vivo in combination with optogenetics or green indicators of voltage or other modalities. We propose several complementary but independent approaches to achieve our goal. In Aims 1 and 2, we propose to conduct two-photon high-throughput screening of indicators repurposing two different classes of fluorescent domains. In Aim 3, we propose experimental (higher-throughput screening) and computational approaches to accelerate screening and potentiate the other Aims. We anticipate that this project will produce red GEVIs that enable robust two-photon recordings of spikes and subthreshold depolarizations in awake behaving animals. We will also optimize and demonstrate approaches to perform two-photon all-optical physiology and multi-spectral imaging. These new sensors and methods will allow the neuroscience community to ask questions that are currently technically infeasible, paving the way for a more detailed understanding of brain computations and how neuronal functional connections are established and modulated during development, learning, and aging.
