Plug Removal for acute in vivo Electrophysiology Experiments
Ryan Gillis, Mikayla Carlson, Severine Durand
Abstract
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.
Steps
Graphical overview of procedure
Prepare the surgical rig and anesthesia system
Cover heating pad with Press'n Seal and ensure that the heating pad is turned on to 36-37°C
Obtain a container of ice and place the Metabond tray and a 10mL syringe of ACSF on top of the ice
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.
Connect induction chamber to the isoflurane and oxygen (if not already connected).
Ensure the oxygen regulator is set to 0.8-1 L/min.
Double check all the gas tubing to ensure the system is connected correctly.
Anesthetize the mouse
Open the vacuum valve and isoflurane valves. Direct the flow to the induction chamber.
Remove the animal from its experimental cage and place the mouse into the induction chamber
Turn the isoflurane regulator to setting 5% (v/v)
Once the mouse is fully unconscious, turn off the isoflurane. Leave the oxygen and vacuum lines open.
Transfer and position the mouse on the heating pad and attach the headpost into the clamp.
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.
Redirect isoflurane flow from the induction chamber to the surgical rig.
Turn the isoflurane regulator to 1-2% (v/v)
Turn off the vacuum line of the induction chamber and close the lid.
Continue to monitor the mouse’s breathing throughout the procedure
Cover the mouse’s eyes with a drop of I-DROP on each eye to keep them moist.
Place plastic surgical drape over mouse and screw the drape into place with an Allen key
Paint the inside of the well with white Metabond to facilitate probe visibility during insertion
Remove the plastic snap-on cap from the well
Gently swab the inside of the well three times with ethanol wipes to remove debris and dust
While the ethanol evaporates, prepare the white Metabond
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
Stir well with a toothpick
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.
Wait for the Metabond to dry completely. Use a toothpick to poke the Metabond - advance if it feels hard to the touch
Remove the SORTA-clear plug
Ensure the well is clear of excess debris
Fill the well with enough ACSF to cover the plug, around 3-4 drops.
With forceps, find the anterior or posterior edge of the SORTA-clear plug
Gently peel the SORTA-clear plug off the implant, generally from the posterior or anterior edge.
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
After SORTA-Clear plug has been removed
When all implant holes are clear of any debris or SORTA-clear, remove the ACSF from the well with 1-2 Sugi spears
Fill the well with Kwik-Cast
Wait for the Kwik-Cast to dry, then replace the snap-on cap on the well
Surgery takedown
Unscrew the plastic hand rest
Turn off the isoflurane and oxygen
Remove the mouse and recover it in its cage
Clean surgery station with 70% ethanol
