Linking Neurons to Network Function and Behavior by Two-Photon Holographic Optogenetics and Volumetric Imaging

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 ensemb...

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Bibliographic Details
Published in:Neuron (Cambridge, Mass.) Mass.), 2017-05, Vol.94 (4), p.774-789.e5
Main Authors: dal Maschio, Marco, Donovan, Joseph C., Helmbrecht, Thomas O., Baier, Herwig
Format: Article
Language:English
Subjects:
3D targeted two-photon optogenetics
Activity patterns
Animals
Animals, Genetically Modified
behavior
Behavior, Animal - physiology
Brain - physiology
Channelrhodopsins
circuit exploration
computer-generated holography
Genetics
Green Fluorescent Proteins
High resolution
Holography
Information processing
Interrogation
Methods
motor control
Motor task performance
Movement - physiology
multiplane 2P calcium imaging
Nerve Net - physiology
Neuroimaging
Neurons
Neurons - physiology
nMLF
Optics
Optogenetics
Photic Stimulation
Photons
Stimulation
Swimming
Tail
Zebrafish
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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. •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 Dal Maschio et al. introduce a suite of methods for simultaneous 3D two-photon optogenetics and multiplane functional imaging. This approach, which also includes reconstruction of single neurons, enabled the identification of circuitry driving motor behavior in larval zebrafish.
ISSN:0896-6273
1097-4199
DOI:10.1016/j.neuron.2017.04.034