All-Optical Neurophysiology in the 1-Photon Regime

Abstract

Simultaneous optical recording and optical stimulation of neuronal activity could enable faster and more comprehensive investigation of neuronal function but is hampered by underdeveloped tools. Pairs of molecular transducers between light and neuronal function are necessary to enable independent perturbation and measurement of the system. These molecular tools must be further complemented by optical systems and light delivery protocols to minimize cross-talk between color channels and between cells. Here I present three all-optical neurophysiology (AON) systems. Each combines a red-shifted fluorescent reporter of voltage or calcium combined with spectrally orthogonal blue-shifted channelrhodopsins to enable independent control and readout of neuronal function in different contexts. AON was first shown with a near-infrared fluorescent microbial rhodopsin-based voltage sensor. This system, Optopatch, can initiate and monitor synaptic inputs, action potential propagation, and neuronal excitability in cultured rodent neurons and human induced pluripotent stem-cell derived neurons over large fields of view. A subsequent iteration used FlicR1, a bright and fast red-fluorescent voltage indicator. FlicR1’s brightness is compatible with imaging voltage over larger areas of cultured brain slices, but the indicator is challenging to pair with channelrhodopsins in a crosstalk free manner. Finally, we identified a channelrhodopsin blue-shifted enough to be combined with existing red-shifted calcium indicators. We developed a form of structured illumination microscopy based on Hadamard matrices which enables calcium based AON in thousands of cells in parallel in acute brain slices. We used this system to map pharmacological perturbations of excitability and synaptic connections on millimeter-length scales across brain slices.