Multi-electrode arrays such as Neuropixels probes enable electrophysiological recordings from large populations of single neurons with high temporal resolution. By using such probes, the activity from functionally interacting, yet distinct, brain regions can be measured simultaneously by inserting multiple probes into the same subject. However, the use of multiple probes in small animals such as mice requires the removal of a sizable fraction of the skull, while also minimizing tissue damage and keeping the brain stable during the recordings. Here, we describe a step-by-step process designed to facilitate reliable recordings from up to six Neuropixels probes simultaneously in awake, head-fixed mice. The procedure involves four stages: the implantation of a headframe and a removable glass coverslip, the precise positioning of the Neuropixels probes at targeted points on the brain surface, the placement of a perforated plastic imaging window and the insertion of the probes into the brain of an awake mouse. The approach provides access to multiple brain regions and has been successfully applied across hundreds of mice. The procedure has been optimized for dense recordings from the mouse visual system, but it can be adapted for alternative recording configurations to target multiple probes in other brain areas. The protocol is suitable for users with experience in stereotaxic surgery in mice.
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The data used to generate Fig. 12 are publicly available as part of the Allen Brain Observatory Visual Coding–Neuropixels dataset. The data can be accessed via the AllenSDK (https://allensdk.readthedocs.io/en/latest/visual_coding_neuropixels.html), the DANDI Archive (https://dandiarchive.org/dandiset/000021) or the AWS Data Exchange (https://aws.amazon.com/marketplace/pp/prodview-r3vtavkhdgjli).
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We thank the Allen Institute founder, Paul G. Allen, for his vision, encouragement and support. Primary funding for this project was provided by the Allen Institute. We thank the Transgenic Colony Management and Laboratory Animal Services for caring for the mice in this study. We thank the Neurosurgery and Behavior team for performing surgeries and training. We thank the Imaging team for performing OPT imaging.
The authors declare no competing interests.
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Nature Protocols thanks Celian Bimbard, Richard Fiath 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
Siegle, J. H. et al. Nature 592, 86–92 (2021): https://doi.org/10.1038/s41586-020-03171-x
Jia, X. et al. Neuron 110, 1585–1598.e9 (2022): https://doi.org/10.1016/j.neuron.2022.01.027
Siegle, J. H. et al. eLife 10, e69068 (2021): https://doi.org/10.7554/eLife.69068
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Durand, S., Heller, G.R., Ramirez, T.K. et al. Acute head-fixed recordings in awake mice with multiple Neuropixels probes. Nat Protoc (2022). https://doi.org/10.1038/s41596-022-00768-6