Surgery Methods
GRIN lens preparation
Parts list
1. Cut metal sleeve to desired length using Dremel tool. If implant depth is x mm, and GRIN length is ymm, sleeve should be cut to y-x mm. (e.g. our lenses are 7.3mm long, and we implant in the hippocampus at a depth of 1.4mm, so cut sleeves of length at most 5.9 mm).
2. Ensure that sleeve ends are filed flat and center of sleeve is clear of debris.
3. Place lens inside of sleeve. There should be a very small amount of space between the top of the lens and the edges of the sleeve. If any amount of the lens is sticking out from the inside of the sleeve, the mice will find a way to chip it and damage the lens. Careful though, because if too much space is left, the fiber will not be able to reach the working distance of the lens. See picture below for representative lens.
4. Use an insulin needle to apply a very small amount of glue (we use optic glue) to the interface between the lens and the sleeve. Apply this all the way around to ensure a secure connection.
5. Right before implanting, clean with a small amount of ethanol and lens wipes.
6. See “GRIN surgery methods” for implantation.
Note: it is strongly recommended to perform viral injections on the same day as GRIN lens implantation to ensure that coordinates are aligned and increase your likelihood of reaching your desired target.
Viral injection with GRIN lens implant
1. Anesthetize mice and head-fix
2. Apply puralube vet ointment to the eyes.
3. Inject 0.2mg/kg meloxicam intraperitoneally using a 1mL syringe.
4. Trim hair from the scalp and sterilize using povidone-iodine swabs and subsequently ethanol swabs.
5. Make an incision covering the anteroposterior extent to allow access to the skull.
6. Cut away excess skin in arcs laterally to create a full circle of exposed skull.
7. Apply 3% hydrogen peroxide to the skull, and using a scalpel, very carefully clean away all tissue from the top of the skull, leaving only exposed bone.
8. Using stereotaxis, align the skull so that lambda and bregma are even.
9. Using a dental drill, make 1 mm holes through the skull at the desired sites.
10. Cover the site of drilling with chilled 1x PBS, and using a sterile 28 G x 1.2” insulin syringe and low pressure vacuum suction, carefully remove the underlying dura and remaining pieces of skull.
11. Inject desired volume of virus using a 35G beveled needle in a 10ul NanoFil Sub-Microliter Injection syringe (World Precision Instruments) controlled by an injection pump (Harvard Apparatus) at a rate of 100nl/min.
12. After each viral delivery, wait 10-minute before slowly removing the injection needle. This is an important step to avoid backflush.
13. Using a stereotactic cannula holder, slowly lower the GRIN into the brain at a rate of 1mm/min, ending 0.2mm dorsal to the injection site. While doing this, constantly flush the skull with chilled 1x PBS. Every time the lens moves 0.8 mm more ventral, retract it 0.4 mm dorsally at the same rate, before continuing down again. This step crucially improves likelihood of observing cells in the target region.
14. Completely seal the skull-metal sleeve connection with glue before removing the stereotactic cannula holder.
15. Repeat steps 13-14 for as many lenses as needed.
16. Place titanium headplate on top of skull and apply adhesive cement. Make sure to apply enough cement that it moves at least halfway up the height of the GRIN lens (see diagram). The imaging adaptor will be attached directly onto the lenses so it’s important that they are cemented enough so that they don’t move with pressure (but still leave some lens exposed so the adaptor can be attached).
17. Immediately following surgery, inject mouse with 0.2mg/kg dexamethasone subcutaneously, and allow to recover.