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Acute head-fixed recordings in awake mice with multiple Neuropixels probes


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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Fig. 1: Overview of a complete experiment.
Fig. 2: Bleeding occurrence after coverslip removal and probe insertion.
Fig. 3: The Allen Brain Observatory Neuropixels recording rig.
Fig. 4: Glass coverslip preparation.
Fig. 5: Headframe and well assembly.
Fig. 6: Headframe and craniotomy implant.
Fig. 7: Probe positioning and insertion window design.
Fig. 8: Coverslip removal and insertion window implant.
Fig. 9: Head-fixation system.
Fig. 10: Fluorescent dye application.
Fig. 11: Probe insertion and effect of brain stabilization.
Fig. 12: Simultaneous recording of spiking activity across many brain regions in head-fixed mice.

Data availability

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 (, the DANDI Archive ( or the AWS Data Exchange (


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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.

Author information

Authors and Affiliations



J.H.S. designed the experiments and the rigs. S.R.O. supervised the project. J.H.S. analyzed the data. S.D., J.H.S, S.R.O., T.K.R. and G.R.H. participated in the writing of the paper. S.D., T.K.R. and G.R.H. performed experiments. S.D., T.K.R. and G.R.H. tested and validated all components of the paper. A.C. and D.T.S. designed custom rig components under the supervision of C.F. J.A.L. co-developed the surgical procedure and trained new surgeons. A.W. and P.A.G. supervised habituation/training and surgery. C.B. developed analysis methods and edited the paper.

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Correspondence to Séverine Durand.

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Key references using this protocol

Siegle, J. H. et al. Nature 592, 86–92 (2021):

Jia, X. et al. Neuron 110, 1585–1598.e9 (2022):

Siegle, J. H. et al. eLife 10, e69068 (2021):

Extended data

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Supplementary Information

Supplementary Tables 1 and 2

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Source Data Fig. 12

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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).

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