Optical Methods for Simultaneous Imaging and Stimulation of Neural Activity at Cellular Resolution in Awake, Behaving Mice

A central goal in neuroscience is to understand dynamics in the awake mammalian brain on a large scale that is also well-resolved (thousands of individual neurons). Recent optical methods for cellular-resolution fluorescence imaging using two-photon excitation (TPE) microscopy, or population-resolution optogenetic perturbation of activity, have been applied to measure or probe dynamics of neurons in behaving rodents. A technique allowing both fluorescence imaging and optogenetic perturbation at cellular resolution could introduce a new class of experiments to probe functional properties of the brain. Here, an approach is presented allowing all-optical stimulation and imaging of neurons in mice. We show that single, task-modulated neurons may be characterized and then manipulated during a behavior. We first demonstrate that channelrhodopsin-2 (ChR2), a visible-wavelength sensitive optogenetic cation channel, produces photocurrents under infrared TPE. An empirical estimate of ChR2’s two-photon absorption cross-section at λ=920 nm is presented, with a value (260±20 GM) indicating that TPE stimulation of ChR2 is not typically limited by intrinsic molecular excitability. Physiological measurements of ChR2 photocurrents and a ground-state depletion model are used to evaluate how saturation of ChR2’s current-conducting state influences spatial resolution of focused TPE photostimulation, and how photocurrents stimulated with scanning TPE temporally summate. Applying these findings, we illustrate one approach to stimulate action potentials in cultured neurons using TPE of ChR2. An in vivo application of these findings is then presented. We show that transgenic expression of a green calcium sensor (GCaMP3) and viral expression of a red-shifted optogenetic probe (C1V1-2A-EYFP) yields dense dual-labeling in hippocampal CA1 neurons, and spectral unmixing allows identification of neurons expressing both genes despite overlapping emission spectra. Spatially patterned TPE (λ=1064 nm) can stimulate neuronal activity detectable using simultaneous TPE fluorescence imaging iii (λ=920 nm) at cellular resolution in most cases (99%) in awake mice. Combined with a virtual reality environment for mice, we illustrate that endogenous task-modulated activity in hippocampal CA1 neurons (e.g., place cells) may be characterized and then manipulated in patterns mimicking or amplifying natural activity during ongoing behavior. Stimulating activity in place cells also affected activity in anatomically distant place cells with overlapping receptive fields in navigation.