Abstract
Recent advances combining two-photon calcium imaging and two-photon optogenetics with computer-generated holography now allow us to read and write the activity of large populations of neurons in vivo at cellular resolution and with high temporal resolution. Such ‘all-optical’ techniques enable experimenters to probe the effects of functionally defined neurons on neural circuit function and behavioral output with new levels of precision. This greatly increases flexibility, resolution, targeting specificity and throughput compared with alternative approaches based on electrophysiology and/or one-photon optogenetics and can interrogate larger and more densely labeled populations of neurons than current voltage imaging-based implementations. This protocol describes the experimental workflow for all-optical interrogation experiments in awake, behaving head-fixed mice. We describe modular procedures for the setup and calibration of an all-optical system (~3 h), the preparation of an indicator and opsin-expressing and task-performing animal (~3–6 weeks), the characterization of functional and photostimulation responses (~2 h per field of view) and the design and implementation of an all-optical experiment (achievable within the timescale of a normal behavioral experiment; ~3–5 h per field of view). We discuss optimizations for efficiently selecting and targeting neuronal ensembles for photostimulation sequences, as well as generating photostimulation response maps from the imaging data that can be used to examine the impact of photostimulation on the local circuit. We demonstrate the utility of this strategy in three brain areas by using different experimental setups. This approach can in principle be adapted to any brain area to probe functional connectivity in neural circuits and investigate the relationship between neural circuit activity and behavior.
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Code availability
Custom codes used for experimental control, data acquisition, behavioral training and analysis have been deposited at Naparm (https://github.com/llerussell/Naparm), PyBehaviour (https://github.com/llerussell/PyBehaviour), 3D SLM calibration (https://github.com/llerussell/SLMTransformMaker3D), STAMovieMaker (https://github.com/llerussell/STAMovieMaker), RawDataStream (https://github.com/llerussell/Bruker_PrairieLink), Objective rotation (https://github.com/llerussell/MONPangle) and TPBS (https://github.com/hwpdalgleish/TPBS). The code in this protocol has been peer reviewed.
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Acknowledgements
We thank Hillel Adesnik, Isaac Bianco, Spencer Brown, Jacques Carolan, Valentina Emiliani, Darcy Peterka, Evelyn Wong and Zihui Zhang for comments on the manuscript; Theshika Jeyaratnam for CA1/CA3 behavioral training and surgeries; Soyon Chun, Agnieszka Jucht and Olivia Houghton for mouse breeding; Selmaan Chettih and Christopher Harvey for developing and sharing the somatically restricted C1V1 opsin; and Bruker Corporation for technical support. This work was supported by grants from the Wellcome Trust, Gatsby Charitable Foundation, ERC, MRC and the BBSRC.
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L.E.R., H.W.P.D., R.N., O.M.G., D.H., M.F. and A.M.P. built experimental apparatus, wrote software, performed surgeries, trained animals, carried out experiments and analyzed data. L.E.R. and H.W.P.D. wrote the manuscript, with input from A.M.P. and M.H., and all authors jointly edited and revised the protocol.
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Nature Protocols thanks Andrew Hires, Jinho Kim, Luis Carrillo-Reid, Weijian Yang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Key references using this protocol
Dalgleish, H. W. et al. eLife 9, e58889 (2020): https://doi.org/10.7554/eLife.58889
Packer, A. M. et al. Nat. Methods 12, 140–146 (2015): https://doi.org/10.1038/nmeth.3217
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Russell, L.E., Dalgleish, H.W.P., Nutbrown, R. et al. All-optical interrogation of neural circuits in behaving mice. Nat Protoc 17, 1579–1620 (2022). https://doi.org/10.1038/s41596-022-00691-w
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DOI: https://doi.org/10.1038/s41596-022-00691-w
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