TITLE: Assembly: Chronic recoverable Neuropixels in mice
AUTHORS: Emily A Aery Jones

[DESCRIPTION]
This protocol collection explains how to build a low-cost, lightweight system to implant 1 Neuropixels 1.0 probe or 2 2.0 beta probes into mice, record during freely moving behavior, then recover the probe for future use. This protocol explains how to 3D print components, sharpen, solder, and test the probe, and prepare components for surgery.

Chronic recoverable implant based off of Juavinett et al, 2019 (https://elifesciences.org/articles/47188), then modified by Janna Aarse, then modified by Emily Aery Jones.

See also other chronic recoverable designs:
for rats: Luo & Bondy et al, 2020 (https://elifesciences.org/articles/59716)
Neuropixels 2.0: Steinmetz, Aydin, Lebedeva, Okun, & Pachitariu et al, 2021 (https://www.science.org/doi/10.1126/science.abf4588) and van Daal, Aydin, & Michon et al, 2021 (https://www.nature.com/articles/s41596-021-00539-9)
overview: Neuropixels 2021 course lecture 3.7 by Yoh Isogai (https://www.youtube.com/watch?v=7cZqYIiGvBQ&list=PLfhWmWntvjl64ti_a-MzHlwqwEU0ZlALb&index=18) 

Advantages of this design:
Lightweight (entire assembly, including headbar, dental cement, and tape, weighs <3g)
Allows headfixed or freely moving recordings
Quickly attaches to a headstage holder, which provides LEDs for tracking and a counterweight to encourage running
Position and angle of internal mount on the probe adjustable before gluing, allowing implantation into any brain region, but custom adjustment is not necessary, allowing the same probe to be re-inserted into a variety of sites
Unlike completely enclosed designs, the shank is uncovered during insertion for better visualization, yet doesn't require delicately surrounding the shank with a glue column
Dual 2.0 probes: probes are independently insertable, so can set custom positions, depths, and angles for each

Disadvantages of this design:
Not suitable for larger animals (lightweight design likely can't withstand larger forces, flex cable remains exposed, moisture known to wick up shank into PCB in rats)
External components are assembled around the probe during surgery rather than during assembly, so surgeries take slightly longer
Less elegant than completely enclosed designs, and requires a larger skull surface area for gluing
Dual 2.0 probes: how close together the probes can get is limited by some of the components

Assembly preview:
Single 1.0 probe
 

Dual 2.0 beta probes:

 


[BEFORE_START]
If your probes do not have a metal cap, you can 3D print or machine the file provided at https://github.com/emilyasterjones/chronic_NPX_mouse/tree/main/probe_holders (or https://github.com/emilyasterjones/chronic_NPX_mouse/tree/main/2.0_probes for 2.0 probes). To attach the cap, secure the probe in the modeling clay, apply superglue to dovetail cap, position, press together for 30 s , and allow to cure   .
The grindstone only accepts thin metal rods, not those compatible with stereotaxes. Machine a compatible rod using the file provided at https://github.com/emilyasterjones/chronic_NPX_mouse/tree/main/probe_holders.

[GUIDELINES]
These steps can be done in any order and simultaneously, except that printing and sharpening must happen before gluing, and soldering must happen before testing.
The probe shank is fragile and hard to see. Secure the probe in a closed container whenever not in use, or secure in the Sensapex holder to a clamp or other immobilizing device with the shank pointed well clear of any items the experimenter might reach for to prevent accidental breakage.

[STEPS]
SECTION: 3D print components
1. Single 1.0 probe: Build a print file for the following pieces per mouse: 1 each of body piece, back and front flex cable holders, and dome, plus 2 wings. 

Dual 2.0 probes: Build a print file for the following pieces per mouse: 1 each of left body, right body, dovetail, flex holder 1, flex holder 2 with dovetail, and dome, plus 2 flex holder 2s.

To re-use explanted probes, print everything except for the body pieces, which are permanently affixed to the probe. Print 1 headstage holder per recording rig. Files located at https://github.com/emilyasterjones/chronic_NPX_mouse.
 

 

 


SECTION: 3D print components
1.1. Place all build files into a single print file. Orient each component so it is well-supported, with supports attaching to non-interface points. These are: rounded hooks of wings, top of body piece (where hex nut slot is), flat backs of flex cable holders and headstage holder, and any side of dome. See single_mouse_print.form file for example using Formlabs system.
SECTION: 3D print components
2. Print the file.
SECTION: 3D print components
3. Remove prints from the build platform. Remove liquid resin from prints and cure according to manufacturer instructions for your printer and resin.
 

SECTION: 3D print components
4. Remove supports with wire cutters for fine surfaces & twisting print & raft apart for larger surfaces.
SECTION: Build probe box
6. Along the base inside the plastic case, place a thick (>1cm) piece of modeling clay in a ~3cm strip about one-quarter of the way from the top of the box. The clay should be thick enough so that you can press a probe into it without worrying about the shank hitting the box and wide enough to securely hold the PCB board of the probe.
SECTION: Build probe box
7. From an empty probe box, remove the foam interior. Cut the probe holding foam strip out of this. Tape this strip to the top of the box. This will grip any metal rod attached to the Sensapex holder to secure the probe during soldering, gluing, or waiting to be mounted to the stereotax or clamp.
  

SECTION: Sharpen the probe
8. Load the probe into the Sensapex holder. Screw the holder into the thin metal rod.
SECTION: Sharpen the probe
9. Sharpening probes can improve cell yield and allow you to puncture dura without removing it. Sharpen probe according to this protocol: https://github.com/cortex-lab/neuropixels/wiki/Sharpening
    

SECTION: Solder probe and ground screw
10. Thread silver wire between the ground and reference pads on the back of the PCB board, then down to a few mm below the PCB. Solder. Keep the iron cool (~315C/600F) and don’t heat for longer than 4s. Optional: trim the ground and reference pad flexes above the PCB board as these aren't necessary.

 

 

 

 

SECTION: Solder probe and ground screw
11. Solder gold male pin to the end of wire.
SECTION: Solder probe and ground screw
12. Apply flux just under the head of the ground screw. Wrap a loop of wire around this & twist to tighten. Solder closed.

2.0 probes: Solder a second loop of wire around the ground screw for the second probe.
 

SECTION: Solder probe and ground screw
13. Solder gold female pin to the end of this wire.
SECTION: Test signal
14. Mount the probe in a clamp or micromanipulator and submerge shank into saline. Clip the ground wire into the saline on the side of the beaker.
SECTION: Test signal
15. Plug in the headstage. Run BIST tests. Observe the signal and noise level on SpikeGLX to confirm your soldering is good and the probe is functional.
SECTION: Assemble
16. Add nuts into slots on body piece. Attach wings and screw to affix
SECTION: Assemble
17. 1.0 probes: Superglue the back of the probe to the 3D printed body piece. The  wings extend 6mm beyond the body piece and you'll need some room for  dental cement, so set the piece at [your target depth]-3mm away from the  base of the shank. 

  
2.0 probes: Superglue the front of the first probe to the right body piece. This will be your anterior probe. Superglue the back (dovetail-side) of the second probe to the left body piece. This will be your posterior probe. Position so that the body pieces are at the top of the metal cap and interior edge aligned with the edge of the probe.

 
Press together for 30 s . Allow to cure   .
 

SECTION: Assemble
19. Mark the center of each headplate (1" long) with a lab marker.
SECTION: Assemble
20. Slice the pipette tips into 1mm diameter, 0.5mm tall circles to create wells for the craniotomy.
  
  

SECTION: Build the headstage holder
21. Insert the LEDs through the ends of the headstage holder arms. Wire: LED short lead => resistor => ground, LED long lead => switched power. Insert a coin cell battery and flip the switch to check the connection.

 

SECTION: Build the headstage holder
22. Cover any exposed wire with epoxy. Epoxy the battery breakout to the back of the headstage holder. Mount headstage into slot and optionally secure with tape. Plug into Omnetics connector and secure this connection with tape (this connection easily comes loose). 
   
  
  
  
  

SECTION: Machine components
5. Machine 1 headbar per mouse from stainless steel.

Dual 2.0 probes: machine 2 wings per mouse from stainless steel.
 

SECTION: Assemble
18. 2.0 probes: Superglue a 3D printed dovetail to the front (metal cap side) of the second probe. Superglue a flex holder 2 with dovetail to the front (metal cap side) of the first probe, just above the body piece. These 3D printed dovetail substitutes will be used to insert the probes during surgery.

  
Press together for 30 s . Allow to cure   .