Abstract
The simultaneous imaging and manipulating of neural activity could enable the functional dissection of neural circuits. Here we have combined two-photon optogenetics with simultaneous volumetric two-photon calcium imaging to measure and manipulate neural activity in mouse neocortex in vivo in three-dimensions (3D) with cellular resolution. Using a hybrid holographic approach, we simultaneously photostimulate more than 80 neurons over 150 μm in depth in layer 2/3 of the mouse visual cortex, while simultaneously imaging the activity of the surrounding neurons. We validate the usefulness of the method by photoactivating in 3D selected groups of interneurons, suppressing the response of nearby pyramidal neurons to visual stimuli in awake animals. Our all-optical approach could be used as a general platform to read and write neuronal activity.
Highlights
We built a 3D microscope with two independent two-photon excitation lasers for imaging and photostimulation respectively (Fig. 1a)
C1V1-expressed cells were identified through a co-expressed mCherry fluorophore
Single spikes can be evoked with very low average laser power (~2.25 mW with 20 ms spiral, or ~4.5 mW with 10 ms spiral, 1 MHz pulse train, layer 2/3 in vivo, Fig. 1e), latency and jitter (12.2/7.4 ms latency, and 4.0/2.3 ms jitter for the two conditions, supplementary Fig. S3)
Summary
We built a 3D microscope with two independent two-photon excitation lasers for imaging and photostimulation respectively (Fig. 1a). We employed wavefront shaping strategy with a dual-beam two-photon microscope and simultaneously performed volumetric calcium imaging and 3D patterned photostimulations on mice cortex in vivo. We tested our 3D all-optical system by targeting and photoactivating selected pyramidal cells across layer 2/3 of mouse V1 in vivo (mouse in anesthetized condition), while simultaneously monitoring the neuronal activity in three selected planes (240x240 μm[2] FOV for each plane) at 6.67 vol/s.
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