Neuron
Volume 94, Issue 4, 17 May 2017, Pages 774-789.e5
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Linking Neurons to Network Function and Behavior by Two-Photon Holographic Optogenetics and Volumetric Imaging

https://doi.org/10.1016/j.neuron.2017.04.034Get rights and content
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Highlights

  • Simultaneous two-photon 3D imaging and 3D photostimulation

  • Holographic optogenetics to explore behavior-related neuronal circuits

  • Iterative approach plus analysis framework to generate/validate circuit models

  • Morphological reconstruction of functionally identified neurons

Summary

We introduce a flexible method for high-resolution interrogation of circuit function, which combines simultaneous 3D two-photon stimulation of multiple targeted neurons, volumetric functional imaging, and quantitative behavioral tracking. This integrated approach was applied to dissect how an ensemble of premotor neurons in the larval zebrafish brain drives a basic motor program, the bending of the tail. We developed an iterative photostimulation strategy to identify minimal subsets of channelrhodopsin (ChR2)-expressing neurons that are sufficient to initiate tail movements. At the same time, the induced network activity was recorded by multiplane GCaMP6 imaging across the brain. From this dataset, we computationally identified activity patterns associated with distinct components of the elicited behavior and characterized the contributions of individual neurons. Using photoactivatable GFP (paGFP), we extended our protocol to visualize single functionally identified neurons and reconstruct their morphologies. Together, this toolkit enables linking behavior to circuit activity with unprecedented resolution.

Keywords

3D targeted two-photon optogenetics
computer-generated holography
multiplane 2P calcium imaging
circuit exploration
behavior
motor control
zebrafish
nMLF

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These authors contributed equally

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