TITLE: Plug Removal for acute in vivo Electrophysiology Experiments
AUTHORS: Ryan Gillis, Mikayla Carlson, Severine Durand

[DESCRIPTION]
This protocol describes the procedure for removing the SORTA-clear plug used in acute in vivo electrophysiology experiments in whole-hemisphere craniotomy (WHC) preparation mice with Neuropixels probes. This exact methodology is crucial to these experiments due to the role the SORTA-clear plug plays in preserving the brain prior to the described procedure. After the mice undergo a WHC surgery, the implant used to replace the skull on the left hemisphere is covered with a SORTA-clear plug, which keeps the brain from potentially interacting with air or debris, as the SORTA-clear seals the holes in the implant and thus ensures the brain stays healthy between surgery recovery, training, and subsequent experiments. However, we cannot insert Neuropixels probes through SORTA-clear, meaning we must remove it before the experiment. After the SORTA-clear is removed, we place another temporary seal over the brain - Kwik-Cast. This is a more temporary measure and acts as another easily removable seal to preserve the brain just prior to recording. We recommend doing this procedure 1 day prior to the procedure outlined in Neuropixels Data Collection: Whole Hemisphere Recordings as to not expose the mouse to anesthesia on the day of recording, but if necessary, this procedure can be done on the same day as a recording as long as the mouse is given two hours to recover from the anesthesia. 

[BEFORE_START]
Please note that this protocol is meant for mice that have previously undergone and recovered from a Whole-Hemisphere Craniotomy surgery.

[GUIDELINES]
Only perform this procedure in accordance with IACUC and veterinary requirements.

[STEPS]
SECTION: Prepare the surgical rig and anesthesia system
4. Ensure the vacuum gauge is set to 10-10 pounds per square inch (PSI) and that all vacuum lines are functioning correctly for both the induction chamber and nosecone scoop. 

SECTION: Prepare the surgical rig and anesthesia system
6. Ensure the oxygen regulator is set to 0.8-1 L/min.  
SECTION: Prepare the surgical rig and anesthesia system
7. Double check all the gas tubing to ensure the system is connected correctly.  
SECTION: Anesthetize the mouse
8. Open the vacuum valve and isoflurane valves. Direct the flow to the induction chamber. 
SECTION: Anesthetize the mouse
9. Remove the animal from its experimental cage and place the mouse into the induction chamber
SECTION: Anesthetize the mouse
10. Turn the isoflurane regulator to setting 5 % (v/v)
SECTION: Anesthetize the mouse
11. Once the mouse is fully unconscious, turn off the isoflurane. Leave the oxygen and vacuum lines open.  
SECTION: Anesthetize the mouse
12. Transfer and position the mouse on the heating pad and attach the headpost into the clamp. 
SECTION: Anesthetize the mouse
13. Secure the nose cone over the mouse’s snout. Make sure the body of the mouse is on top of the thermometer on heating pad. 
SECTION: Anesthetize the mouse
14. Redirect isoflurane flow from the induction chamber to the surgical rig. 
SECTION: Anesthetize the mouse
15. Turn the isoflurane regulator to 1-2 % (v/v) 

SECTION: Anesthetize the mouse
16. Turn off the vacuum line of the induction chamber and close the lid. 
SECTION: Anesthetize the mouse
17. Continue to monitor the mouse’s breathing throughout the procedure
 

SECTION: Anesthetize the mouse
18. Cover the mouse’s eyes with a drop of I-DROP on each eye to keep them moist. 
 

SECTION: Anesthetize the mouse
19. Place plastic surgical drape over mouse and screw the drape into place with an Allen key 
SECTION: Paint the inside of the well with white Metabond to facilitate probe visibility during insertion
20. Remove the plastic snap-on cap from the well 
SECTION: Paint the inside of the well with white Metabond to facilitate probe visibility during insertion
21. Gently swab the inside of the well three times with ethanol wipes to remove debris and dust 
SECTION: Paint the inside of the well with white Metabond to facilitate probe visibility during insertion
22. While the ethanol evaporates, prepare the white Metabond 
SECTION: Paint the inside of the well with white Metabond to facilitate probe visibility during insertion
22.1. Mix 4 drops of Quick Base for MetaBond, 1 drop of Universal 4-META Catalyst, and 2 even scoops of Radiopaque L-Powder Metabond in the ceramic plate




SECTION: Paint the inside of the well with white Metabond to facilitate probe visibility during insertion
22.2. Stir well with a toothpick 
SECTION: Paint the inside of the well with white Metabond to facilitate probe visibility during insertion
23. Apply a thin layer of Metabond in a ring around the edge of the implant with a toothpick. The working time of Metabond ranges from 2-3 minutes from the time the powder is mixed with the base and catalyst to the time it begins setting, so it is important to work quickly during this step. The Metabond will thicken during the working time but should remain a liquid. 
 


SECTION: Remove the SORTA-clear plug
25. Ensure the well is clear of excess debris 
 

SECTION: Paint the inside of the well with white Metabond to facilitate probe visibility during insertion
24. Wait for the Metabond to dry completely. Use a toothpick to poke the Metabond - advance if it feels hard to the touch
 


SECTION: Remove the SORTA-clear plug
26. Fill the well with enough ACSF to cover the plug, around 3-4 drops.
SECTION: Remove the SORTA-clear plug
27. With forceps, find the anterior or posterior edge of the SORTA-clear plug  
 

SECTION: Remove the SORTA-clear plug
28. Gently peel the SORTA-clear plug off the implant, generally from the posterior or anterior edge.
SECTION: Remove the SORTA-clear plug
29. Once the plug has completely separated, comb the edge of each hole in the implant with small forceps to ensure that the SORTA-clear plug successfully and cleanly detached from the implant 
SECTION: After SORTA-Clear plug has been removed
30. When all implant holes are clear of any debris or SORTA-clear, remove the ACSF from the well with 1-2 Sugi spears 
SECTION: After SORTA-Clear plug has been removed
31. Fill the well with Kwik-Cast  
 

SECTION: After SORTA-Clear plug has been removed
32. Wait for the Kwik-Cast to dry, then replace the snap-on cap on the well  
SECTION: Prepare the surgical rig and anesthesia system
5. Connect induction chamber to the isoflurane and oxygen (if not already connected). 
SECTION: Prepare the surgical rig and anesthesia system
2. Cover heating pad with Press'n Seal and ensure that the heating pad is turned on to 36-37 °C 

SECTION: Graphical overview of procedure
1. 
 

SECTION: Prepare the surgical rig and anesthesia system
3. Obtain a container of ice and place the Metabond tray and a 10 mL syringe of ACSF on top of the ice

SECTION: Surgery takedown
33. Unscrew the plastic hand rest  
SECTION: Surgery takedown
34. Turn off the isoflurane and oxygen 
SECTION: Surgery takedown
35. Remove the mouse and recover it in its cage 
SECTION: Surgery takedown
36. Clean surgery station with 70% ethanol
SECTION: Anesthetize the mouse
19.1.