Implanting and Recycling Neuropixels Probes for Recordings in Freely Moving Mice

[Abstract] Recording neural activity in unrestricted animals is necessary to unravel the neural basis of ethological behaviors. Recently, Neuropixels probes have made important strides in improving yield and lowering noise, but have limited use cases in freely moving animals. Although there are a number of studies demonstrating the use of these probes in headfixed mice, there are not established protocols for the use and reuse of them in a freely moving mouse. We therefore designed a novel device (the AMIE) that maximizes the potential value of these powerful probes. Here, we provide the technical drawings for the AMIE and detail its preparation, implantation, and explantation. With our approach, researchers can record hundreds of neurons during freely moving behavior across weeks of experiments, and then recycle valuable probes for future use.


Materials and Reagents
For neuropixel casing assembly 1 attach the IM to the SA. B. Entire assembly in a. within 3D printed external casing (EC). 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.

Procedure
Note: We recommend performing the entire process of preparing and implanting the probe using a dummy probe for practice.
A. Making AgCl wire 1. Remove any insulation from silver wire using forceps or a razor blade.
2. Leave silver wire in 100% bleach for 30 min or until black.

B. Assembling Neuropixels holder and storage box (not shown)
Before mounting the probe, ensure that you have a secure location to store it. We've designed a display case for mounted Neuropixels storage: 1. Attach a Thorlabs slim right angle bracket to an aluminum breadboard.  E. After cementing the IM to the EC, the entire assembly is ready to be implanted 4. Mount the Neuropixels to the IM by applying a small drop of cyanoacrylate to the face of the IM.
Using one hand, gently lay the probe into place (Figure 2A).

CRITICAL STEP:
There are two angled spokes on the face of the IM which allows for proper alignment ( Figure 1A). The silver part on the PCB probe is the part that should be aligned with the axis of the shank. Therefore, this silver part is where the probe should be glued to the IM.
The flex should fit snugly between the angled spokes and can be taped to the SA to keep it out of the way. Allow the adhesive to dry. 5. Solder the reference and ground sites on the Neuropixels (see https://github.com/cortexlab/neuropixels/wiki/Referencing for additional information).
6. Solder a 10 cm piece of the prepared AgCl wire to the soldered reference/ground sites.
Note: If you're re-using a previously implanted probe, and need to attach a new ground wire, you may choose to solder it to the PCB board, or to the end of the old ground wire.
7. Using a needle or wire, apply a tiny drop of the MasterSil 912 Med to the interface of the shank and the PCB board. This will reinforce the shank and, in our hands, seemed to improve the chances of a successful explant ( Figure 2B). 8. Place IM/SA/probe assembly in a micromanipulator or stereotax. Secure (with tape or wax) the EC so that it is aligned with the IC/SA/probe assembly. Slowly position the IM/SA assembly so the IM's wings slide into the ECs rail system (Figures 2C-2E). It is imperative to verify the Neuropixels shank isn't coming in contact with the ECs walling. The IC wing should be equidistant from each EC rail (Figures 2C and 2D). 9. Apply a small drop of epoxy or cement to each wing/rail system interface and let dry.
Note: It is perilous applying anything directly to the shank; work with caution.
10. Using a stereotax or micromanipulator, lower the Neuropixels shank in saline solution and test the acquisition.

D. Implanting procedure
Note: Mice should be given an NSAID at least one day prior to surgery. We recommend oral Carprofen (Medigel) to avoid handling mice after the procedure. Injectables such as Metacam (5 mg/kg) also work well.
1. Anesthetize the mouse with isoflurane (2% in oxygen for induction, 1.5% during surgery). 4. Draw a mark on the skull for the desired implant site. It is useful to put a "dummy" pipette into the stereotax in order to identify the target coordinates. 5. Draw a second mark for the ground screw site-we have successfully placed the ground screw over contralateral cortex (Figure 3A), as well as over the cerebellum.
Note: After marking the implant and ground skull sites, one can affix the headbar.   9. Perform a small craniotomy for the ground screw and slowly rotate the screw into place. The skull screw should be lowered 0.2-0.35 mm so it is in contact with the surface of the brain.
CRITICAL STEP: Make sure the ground screw will not interfere with placement of the Neuropixels casing.
10. Carefully drill a 1-2 mm circular craniotomy at the implant site. Gently apply saline to clean the craniotomy area.
11. Attach SA/IM/EC assembly to stereotax and position above the brain ( Figure 3B).
12. Carefully apply DiI solution to the Neuropixels shank.

CRITICAL STEP:
The DiI solution will attract the Neuropixels shank, possibly pulling it away.
We recommend doing this step by pushing out a drop of the DiI solution from a syringe needle, and slowly moving the drop along the length of the shank.
13. Remove excess saline and blood at the craniotomy site using surgical spears and Kimwipes.
14. Zero the "Z" axis on the stereotax (if relevant) and slowly lower the shank into the brain ( Figure   3B). a. CRITICAL STEP: Monitor the shank for bending during implantation. Watch for bleeding and absorb with surgical spears as necessary. Stop lowering at the halfway point so the craniotomy site is still visible. b. TROUBLESHOOTING: If the probe will not enter the brain without significant bending, try a slightly different entry location. You may also try to dry the dura just slightly before insertion.
15. Mix the white and blue silicon gel (1:1) and carefully apply a thin layer to the craniotomy site.
This silicon gel helps to protect the exposed brain after the implant.
16. Slowly lower the second half of the shank and monitor for bending. Watch for bleeding and absorb with surgical spears. The bottom of the external assembly should be a few millimeters from the skull surface.
17. Once the probe is fully inserted to your desired depth, wrap the ground wire around the ground screw.
18. Apply Metabond to EC and skull to build up a support structure that will mount the assembly to the skull (Figures 3C and 3D). You may also need to use tape to protect any remaining openings between the EC and skull.
CRITICAL STEP: It is imperative that no adhesive enter the internal cavity of the EC otherwise this could bind to the Neuropixels shank or the IM, interfering with recording and explantation.
Allow the Metabond to dry for ~15 min.
19. Remove the screws from the SA/IM assembly and slowly raise the SA from the IM/EC assembly.

CRITICAL STEP:
Monitor each component of the assembly to ensure that the SA is the only component pulling away from the assembly. If there is any movement of the EC, pause and identify the source of friction.