Chronically implanted Neuropixels probes enable high-yield recordings in freely moving mice

  1. Ashley L Juavinett
  2. George Bekheet
  3. Anne K Churchland  Is a corresponding author
  1. University of California, San Diego, United States
  2. University of Connecticut School of Medicine, United States
  3. Cold Spring Harbor Laboratory, United States
7 figures, 2 videos, 2 tables and 1 additional file


Schematic of Neuropixels AMIE.

(A) Probe base mounted onto 3D printed internal casing and attached to machined metal stereotax adapter. Inset: Rear view, with screws that attach the internal mount (IM) to the stereotax adapter (SA). (B) Entire assembly in a. within 3D printed external casing. Inset: Rear view. (C) The headstage is positioned on the back of the encasing, with the flex wrapped in an ‘S’ shape. (D) Entire assembly in relation to size of mouse brain and skull. The EC is attached to the skull with cement. Silicon gel is used to as an artificial dura to protect the open craniotomy.
Mounting and implanting the Neuropixels probe.

(A) The internal mount (IM) is attached to the stereotax adapter (SA) with two screws, and probe is attached to the internal mount using an epoxy. (B) Medical-grade silicon is added to the base of the shank to add extra support. (C) The external case (EC) is attached to a breadboard, and the IM+probe assembly is carefully guided into the internal compartment of the EC (top view). (D) After cementing the IM to the EC, the entire assembly is ready to be implanted. (E) During surgery, the the shank is lowered into the brain (here at a ~ 16° angle). The ground wire extends down the side of the implant and is attached to the ground screw. (F) The entire encasing is attached to the headbar and skull using Metabond. Tape is added where necessary to add protection between the encasing and the skull. The stereotax adapter (not shown) is removed after this support structure is dry and secure. (G) Image of a mouse with the implant ~48 hr after surgery. The entire assembly is wrapped in Kapton tape to protect the onboard electronics.
Behavior in implanted mice is comparable to naive mice.

(A) Behavioral testing arena, with a camera to track the position of the mouse and a monitor on top to present visual stimuli. (B) Snapshot of mouse with implant in arena. (C) Sample tracking of 2 min of open field behavior in an implanted mouse. Color of the line indicates the velocity of the mouse. (D) Open field behavior of implanted vs. naive mice. Random 30–180 s exerpts of behavior (N = 8 videos per group, two videos from each mouse) in the open field were used to calculate a percent time moving (>5 cm/s), max velocity, and max acceleration. (E) Visual-looming evoked behavior of implanted vs. naive mice (N = 10 trials, two videos per mouse). A dark dot of linearly increasing diameter (40 cm/s) was presented over the mouse’s head to evoke an escape response. The mean velocity, max velocity, and max acceleration during these responses is presented here. In all panels, orange line indicates the group mean, blue line indicates the median. p-Values (as computed by a two-sided Wilcoxon Rank Sum test) as well as effect sizes (computed by a Cohen’s d) are reported on each panel. Outliers (defined as 1.5*IQR) are marked as light gray points.
Figure 3—source data 1

Behavioral data for implanted and naive mice in an open field and in response to a looming stimulus.
Chronic Neuropixel probe implants in cortex and subcortical regions can record ~20–145 units across multiple days.

(A) Probe location, marked with DiI. Sections from Paxinos and Franklin atlas provided for reference. Mouse #200 was implanted with a probe in visual cortex, hippocampus (subiculum), and the midbrain. (B) Schematic of probe depth in (A). (C) Number of isolated units across recording days for eight different mice. Mouse #3 and #4 were implanted with a probe with fewer recording sites (270 vs. 374). Mouse #2 is featured in the other panels of this figure. Mouse #7 had a probe that was previously implanted in Mouse #5; see Figure 6. (D) Scatter plot of units across days for Mouse #2. Size of circles denotes number of waveforms assigned to that unit. X axis is random for visualization. € Histogram of isolated units across days and brain depth for Mouse #2. (F) Waveforms (n = 200, mean waveform in yellow) recorded from the same four contacts on the probe on day 5 (top) and day 6 (bottom). Units are the same as the yellow filled in circles in (D).
Figure 4—source data 1

Number of isolated units for each probe across post-surgery days.
Implant design enables researchers to further characterize brain regions recorded during freely moving behavior.

(A) Schematic of headfixed setup. The mouse was implanted with a headbar (see Materials and methods) enabling it to be restrained above a wheel. Visual stimuli was presented on a monitor above the mouse’s head (similar to the unrestrained condition). The mouse’s pupil can be tracked with a high resolution IR camera, and movement can be tracked using a rotary encoder on a 3D printed wheel. (B) Eight sample waveforms (n = 200, mean in yellow) from a restrained recording, same mouse as Figure 4D–F (Mouse #2). (C) Distribution of sorted units across the probe, same mouse as in Figure 4D–F (Mouse #2). (D) Peristimulus time histograms for three example neurons from different locations on the probe. The stimuli were a pseudorandomized set of 2 s full contrast sinusoidal drifting gratings in eight different directions. Shaded region is standard error of the mean. Stimuli began at the dotted line. (E) Raster plots for the neurons in (D). Shaded area indicates the duration of the stimulus.
AMIE design allows for probe explantation and subsequent re-implantation with the same Neuropixels probe.

(A) Example successful probe explanation. Cement is drilled away from the wings of the internal casing in order to remove the internal mount from the external casing. (B) Outline of experiment timing. The same probe was used in the first implant and re-implant. (C) Sample mean waveforms (n = 200, mean in yellow) from each mouse. (D) Detected event rate of the first implant versus the re-implanted probe across days of recording. (E) Median signal-to-noise ratio (SNR) for first implant and re-implanted probe across days (see Materials and methods.).
Figure 6—source data 1

Computed event rate and SNR ratio for the initial implant and re-implant of the same probe across post-surgery days.
Author response image 1
Comparison of mean waveforms (n=50) across periods where the mouse was moving (>15 cm/sec) or stationary (<5 cm/sec).


Video 1
3D rendering of the AMIE device demonstrating the configuration of internal mount (IM), external casing (EC), and stereotax adapter.
Video 2
Behavior of mouse implanted with Neuropixels AMIE.

Mouse was free to move around a 16”x16’ arena while implanted and tethered. Video is shown at 2x speed.


Table 1
Overview of experiments, with the Neuropixels probe option used and the outcome of the experiment.

For each of these experiments, even the unsuccessful explants, neural data was obtained from the initial implant and recording sessions. For an explanation of the probe options, see Materials and methods. Starred mice are included in the paper; + sign indicates the experiment was sorted with Kilosort2; M = mean; SD = standard deviation.
MouseProbe optionRecordable channelsM ± SD Isolated unitsSilicone on shankOutcome
NP6* (Figure 4; Mouse #3)427619.8 ± 6.23NoShank broke during explant
NP7* (Figure 4; Mouse #4)427620.0 ± 4.36noShank broke during freely moving recording
NP8* (Figure 4; Mouse #2)138477.2 ± 13.7noShank broke during explant
NP9* (Figure 4; Mouse #1)1384117.4 ± 16.3noShank broke during explant
NP111384-noShank broke during freely moving recording
NP123384-noMouse didn’t recover from surgery, probe successfully explanted and re-implanted in NP13
NP13 (Figure 4; Mouse #8)3384145+yesGround wire issues after surgery; one session successfully recorded. Successful explant
NP14* (Figure 4 and 6; Mouse #5)338480.6 ± 13.6yesSuccessful explant, re-implanted in NP16
NP15* (Figure 4; Mouse #6)338464.3 ± 19.1yesSuccessful explant
NP16* (Figure 4 and 6; Mouse #7)338443.6 ± 17.8yesSuccessful explant
Key resources table
Reagent type/resourceDesignationSource or
Chemical compound/drugMedical-grade clear silicon adhesiveMastersil912MED
Chemical compound/drugLoctite Instant Adhesive 495ULINES-7595
Chemical compound/drugMedigel CPFClear H2074-05-5022
Chemical compound/drugIsofluraneAllivet50562
Chemical compound/drugC and B Metabond 'B' Quick BaseParkellS398
Chemical compound/drugC and B Metabond 'C' Quick BaseParkellS371
Chemical compound/drugC and B Metabond Radiopaque L-PowerParkellS396
Chemical compound/drugOptibond Solo PlusKerr31514
Chemical compound/drugVetbondSanta Cruz Biotechnologysc-361931
Chemical compound/drugCharisma A1 SyringeNet3266000085
Chemical compound/drugEye OintmentRugby370435
Chemical compound/drugDental CementStoelting5217307
Chemical compound/drugDiIThermoFisher ScientificD282
Chemical compound/drugSilicone Gel KitDow Coning3–4860.
Chemical compound/drugBleachAmazonB01K8HT54GAny brand bleach ok
OtherNeuropixel ProbeNeuropixel Stock Center ( 1.0 Probe
Other3D Printed Internal Mount‘this paper’ - Github repositoryIM_Neuropixel1.stlInternal mount design file (.stl) can be downloaded from the following github repository:
Other3D Printed External Casing‘this paper’ - Github repositoryEC_Neuropixel1.stlsame as above
OtherSterotax Adapter‘this paper’ - Github repositorystereotax adapter v4.iptsame as above
Other2-56A ScrewsAmazonB00F34U238
OtherSilver WireWPIAGW1010
Other4’ post holder with thumbscrewThorlabsPH4
OtherSlim right angle bracketThorlabsAB90B
OtherAluminum BreadboardThorlabsMB624
OtherM6 Cap ScrewThorlabsSH6MS20
OtherM6 NutThorlabsHW-KIT2/M
OtherKapton TapeULINES-7595
OtherOxygen CylindersAirgasOX USP300
OtherMouse Anesthesia System with Isoflurance BoxParkland ScientificV3000PK
OtherSmall rodent sterotax fitted with anesthesia maskNarishigeSG-4N
OtherDental DrillOsadaEXL-M40
Other0.9 mm burrs for micro drillFine Science Tools19007–09
OtherT/Pump Warm Water RecirculatorKent ScientificTP-700
OtherWarming Pad for warm water recirculatorKent ScientificTPZ
OtherCotton ApplicatorsFisher Scientific19-062-616
OtherSurgical SpearsBraintree Scientific Inc.SP 40815

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  1. Ashley L Juavinett
  2. George Bekheet
  3. Anne K Churchland
Chronically implanted Neuropixels probes enable high-yield recordings in freely moving mice
eLife 8:e47188.