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Implantation of Neuropixels probes for chronic recording of neuronal activity in freely behaving mice and rats


How dynamic activity in neural circuits gives rise to behavior is a major area of interest in neuroscience. A key experimental approach for addressing this question involves measuring extracellular neuronal activity in awake, behaving animals. Recently developed Neuropixels probes have provided a step change in recording neural activity in large tissue volumes with high spatiotemporal resolution. This protocol describes the chronic implantation of Neuropixels probes in mice and rats using compact and reusable 3D-printed fixtures. The fixtures facilitate stable chronic in vivo recordings in freely behaving rats and mice. They consist of two parts: a covered main body and a skull connector. Single-, dual- and movable-probe fixture variants are available. After completing an experiment, probes are safely recovered for reimplantation by a dedicated retrieval mechanism. Fixture assembly and surgical implantation typically take 4–5 h, and probe retrieval takes ~30 min, followed by 12 h of incubation in probe cleaning agent. The duration of data acquisition depends on the type of behavioral experiment. Since our protocol enables stable, chronic recordings over weeks, it enables longitudinal large-scale single-unit data to be routinely obtained in a cost-efficient manner, which will facilitate many studies in systems neuroscience.

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Fig. 1
Fig. 2: Exploded view of the three fixture variants for chronic Neuropixels probe implantation.
Fig. 3: Assembly of single-probe fixture.
Fig. 4: Assembly of dual-probe fixture.
Fig. 5: Assembly of movable-probe fixture.
Fig. 6: Assembly of LED holder.
Fig. 7: Preparation of the implant.
Fig. 8: Probe implantation in mouse.
Fig. 9: Probe implantation in rat.
Fig. 10: Probe recovery and cleaning.
Fig. 11: Anticipated results.

Data availability

Source data for Fig. 11 are available in our GitHub repository (


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We thank H. den Bakker, G. Vincente Ortiz and M. D’Andola for providing their electrophysiological data recorded with the Neuropixels probes and the fixtures. We thank M. Broux for performing the histology. This work was supported by Cohen Veterans Bioscience through a generous grant COH-0011 from S. A. Cohen. R.J.J.v.D. is supported by the Hermesfonds (VLAIO Baekeland mandate, HBC.2018.2114). C.A. is supported by KU Leuven Postdoctoral Mandate (PDM/19/173). S.H. is funded by Research Foundation Flanders (FWO) grant G096219N and KU Leuven C1 grant C14/17/109. F.K. is funded through Flemish Research Project FWO G0D7516N, KU Leuven C1 grant C14/17/042 and European FET-Open grant STARDUST (agreement 767092).

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Authors and Affiliations



R.J.J.v.D.: conceptualization of the fixtures; design, prototyping and fabrication of the fixtures; preparing the figures; writing of the original manuscript. C.A.: conceptualization of the fixtures; design and prototyping of the fixture; implantations to animals; data acquisition and analysis; preparing the figures; writing of the original manuscript. F.M.: conceptualization of the fixtures; design and prototyping of the fixtures; implantations to animals; data acquisition and analysis; preparing the figures; writing of the original manuscript. A.A.A.A.: conceptualization of the fixtures; revising the manuscript; funding acquisition. M.K.: conceptualization of the fixtures; revising the manuscript; funding acquisition. F.K.: conceptualization of the fixtures; writing of the original manuscript; funding acquisition. S.H.: conceptualization of the project; writing of the original manuscript; funding acquisition. All authors revised the manuscript.

Corresponding authors

Correspondence to Fabian Kloosterman or Sebastian Haesler.

Ethics declarations

Competing interests

R.J.J.v.D. is an employee of ATLAS Neuroengineering BV, Leuven, Belgium. A.A.A.A. is managing director of ATLAS Neuroengineering BV, Leuven, Belgium. The remaining authors declare no conflict of interests.

Additional information

Peer review information Nature Protocols thanks Ying Fang, Bozhi Tian and the other, anonymous reviewer(s) for their contribution to the peer review of this work

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Key reference using this protocol

Steinmetz, N. A. et al. Science 372, eabf4588 (2021):

Supplementary information

Reporting Summary

Supplementary Video 1

Freely behaving male C57BL/6J mouse (16 weeks old) in box

Supplementary Video 2

Freely behaving male C57BL/6J mouse (20 weeks old) in open field

Supplementary Data 1

CAD files single probe fixture

Supplementary Data 2

CAD files dual-probe fixture

Supplementary Data 3

CAD files movable-probe fixture

Supplementary Data 4

CAD files stereotaxic adapters

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van Daal, R.J.J., Aydin, Ç., Michon, F. et al. Implantation of Neuropixels probes for chronic recording of neuronal activity in freely behaving mice and rats. Nat Protoc 16, 3322–3347 (2021).

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