Apr 01, 2024
Duragel Application for Acute Electrophysiology Recordings
- 1Allen Institute for Neural Dynamics
ProtocolĀ Citation:Ā Anna Lakunina 2024. Duragel Application for Acute Electrophysiology Recordings. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn2dwqg5d/v1
License: This is an open accessĀ protocolĀ distributed under the terms of theĀ Creative Commons Attribution License, Ā which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 05, 2023
Last Modified: June 18, 2024
ProtocolĀ Integer ID: 81475
Disclaimer
TheĀ protocols.ioĀ team notes that research involving animals and humans must be conducted according to internationally-accepted standards and should always have prior approval from an Institutional Ethics Committee or Board.
Abstract
This protocol describes the process of coating an implant with duragel in preparation for in vivo electrophysiology experiments. Duragel seals and protects the surface of the brain while also being soft enough for silicon probes to penetrate through, allowing recordings to take place without disturbing or damaging the brain. It is possible to perform acute recordings for up to two weeks after the duragel layer has been added.
Materials
Anesthesia and related:
- Isoflurane 1-3%Patterson VeterinaryCatalog #07-893-1389
Tool Kit:
Equipment
Iris forceps
NAME
Surgical tools
TYPE
Fine Science Tools
BRAND
11064-07
SKU
Consumable supplies:
- DuragelEllsworth AdhesivesCatalog #DOW DOWSILā¢ 3-4680
- Alcohol prep padVWR InternationalCatalog #15648-907
- Pressn Seal Food WrapAmazonCatalog #B001RHUP4Q
- Luer-Lock Syringe, 1ml (VWR BD-309628 or equivalent)
- 20G x 1 needle (VWR BD305176 or equivalent)
- Small weigh boats
- Double Cotton Tipped ApplicatorVWR InternationalCatalog #89133-810
- KimWipesFischer Scientific
- Sugi mounted sterile absorption spears (Fine Science Tools 18105-01)
Equipment:
Equipment
Model 1900 Stereotaxic Alignment Instrument
NAME
Surgical equipment
TYPE
Kopf
BRAND
Model 1900
SKU
Equipment
Leica M80 Stereo Microscope
NAME
Surgical equipment
TYPE
Leica
BRAND
M80
SKU
Equipment
Isoflurane with oxygen delivery system
NAME
Surgical equipment
TYPE
Patterson
BRAND
78914722
SKU
Equipment
Isoflurane induction chamber
NAME
Surgical equipment
TYPE
Patterson
BRAND
78933385
SKU
Equipment
Small Animal Temperature Controller
NAME
Surgical equipment
TYPE
CWE
BRAND
08-13000
SKU
- Fiber optic illuminator
- Ear bar Headframe clamp (0155-100-00 or equivalent)
Safety:
- Non-Sterile Gloves
- Disposable lab coat
- Face mask; 0.6 micron filter (optional)
- Biohazard sharps disposal container
- Biohazard waste disposal container
Before start
Wear appropriate PPE including gloves, lab coat and optional face mask. Have biohazard waste and sharps disposal accessible.
Setup
Setup
Draw up the white and blue duragel components into separate 1 mL Luer lock syringes. Attach 20G needles.
Note
The duragel components should be dispensed into separate containers when the duragel first arrives. This makes it easier to draw up the two components separately without cross-contamination!
3m
Turn on microscope lights and heating pad.
10s
Wrap heating pad in press ānā seal.
30s
Anesthetize the mouse (see section Prepare the anesthesia system and anesthetize the mouse (all procedures) in protocol General Setup and Takedown Procedures for Rodent Neurosurgery).
3m
Transfer mouse to stereotax.
2m
If the mouse does not currently have a well and well cap, attach a well to the headframe at this time.
15m
Procedure
Procedure
Clean the surface of the implant and the ground wire with an alcohol wipe.
1m
Using forceps, peel away the elastomer covering the implant.
10s
If the holes in the implant fill with CSF, gently dab it away with a Sugi or Kimwipe.
Using forceps, insert the ground wire into the designated grounding hole in the implant.
Note
The ground wire should touch the surface of the brain, but should not penetrate or compress the brain.
Elastomer removed and ground wire inserted into grounding hole.
1m
Prepare the duragel mixture.
5m
Add the white and blue components to a clean weigh boat, using a ratio of 35:50 white to blue.
Note
This ratio can be achieved by counting drops as you dispense the components from the syringes. More blue will ensure the mixture cures a little tougher, and will not turn sticky in a way that can damage recording electrodes.
1m
Use the broken end of a cotton swab to mix the two components until the mixture is uniform in color.
1m
Apply the duragel to the surface of the implant.
Note
Depending on the implant, you may wish to tilt the mouse's head to ensure even coverage. For instance, for the dual hemisphere implant, turn the mouse's head 10 degrees to the right prior to application.
5m
Apply a few drops at a time with the broken end of a cotton swab (you can use the same swab you used to mix the components). Do not apply directly to the holes in the implant as this can cause bubbles. Try to apply near the holes and let the mixture flow into the holes.
2m
If bubbles do form in the holes, try to push them out with the cotton swab.
Continue applying until all parts of the implant are covered with a layer of duragel at least 1 mm thick.
Implant covered with layer of duragel.
3m
Wait to allow the duragel to cure. You can test this by checking the unused mixture in the weigh boat: tilt the boat to see if the mixture is still flowing. If it's not, the duragel is cured enough. This usually takes around ten minutes.
10m
Replace well cap and return mouse to its cage.
Note
Wait at least 24 hours before performing electrophysiological recordings, to allow sufficient time for the duragel to cure completely. Otherwise, there is a risk that the duragel will stick to the probes used for recording.
1m
Take Down
Take Down
3m
Dispose of the syringes containing the duragel components in the sharps container.
10s
Dispose of the elastomer implant cover and any other consumable that came in contact with the mouse in the biohazard waste container.
30s
Turn off oxygen and isoflurane flow.
10s
Turn off heating pad and microscope lights.
10s
Clean and disinfect stereotax, surfaces, and induction chamber with 70% ethanol.
2m