Mouse stereotaxic surgery

Turn on the heating pad to and set to 36.9°C.

Weigh mouse and record weight before starting surgery.

Place mouse in isoflurane chamber (3% isoflurane).

Record time of anesthesia.

After mouse is fully anesthetized, use hair clippers to shave head.

Place mouse back into isoflurane chamber (3% isoflurane) until fully anesthetized.

Transfer mouse onto stereotaxic frame and place the mouth / nose into nose cone with isoflurane.

Turn down isoflurane to 2%.

Apply sterile ophthalmic ointment to eyes using a sterile swab (this will prevent desiccation).

Use ear bars to secure mouse in stereotaxic frame ensuring head is level in all directions.

Using a sterile swab apply 70% EtOH to the scalp.

Using a sterile swab, apply povidone-iodine solution to the scalp.

Wait for povidone-iodine solution to dry before making surgical incision.

Perform a subcutaneous injection of Ethiqa XR (buprenorphine extended-release) into the leg.

Use 0.05mL per 20g mouse.

Inject 30µL of Marcaine in 2-3 locations underneath the scalp near the incision site. This is a local analgesic.

Wait 30 seconds - 0h 1m 0s for the Marcaine to diffuse before performing incision.

Using a sterile scalpel, make a surgical incision to expose the skull.

Minimize the size of the incision as much as possible.

Position your injection needle at bregma and save location in the AP and XY axis.

Enter and save injection coordinates into the motorized stereotaxic frame.

Coordinates for right substantia nigra

• Anterior-posterior (AP): -2.9 mm
• Medial-lateral (ML): -1.3 mm
• Dorso-ventral (DV): -4.2

Raise the needle away from the skull slightly and move to AP and ML injection coordinates.

Slowly lower the needle to touch the skull. Raise the needle and drill a small hole where the needle touched the skull.

Slowly drill through the skull keeping the drill shallow enough to not damage brain tissue.

Once the hole is drilled lower the needle to the surface of the brain ensuring that the needle is not deflected by the skull.

Raise the needle 30 mm - 40 mm providing space to flush the needle and load with virus.

Flush the needle with sterile Room temperature H₂O.

• Cover the mouse’s head with sterile gauze to absorb the H₂O.

Draw up a 1µL air bubble.

Load needle with 2.5µL of virus.

• Slowly draw up virus and watch the air / liquid interface to determine volume.
• Needle / tubing should be marked with 1µL intervals using a sharpie to help with this step.

Move the prepared needle down to the surface of the brain. Move the needle to the desired DV coordinate at a slow speed (250µm / sec).

Wait 0h 0m 30s and then start the virus injections at 0.2 µL / min.

After the injection is finished wait for 0h 5m 0s, leaving the needle in place and allowing the virus to diffuse away from injection site.

Remove the needle at slow speed (250 µm / sec).

Move the needle up away from the skull and then release it and rotate it out of the way.

Close the incision site using sterile forceps and surgical glue.

Administer 0.5mL sterile saline solution subcutaneously to avoid dehydration.

Place the mouse back into the home cage and monitor recovery.

Record time at recovery.

Monitor post operation recover for 72h 0m 0s and record any observations of pain or distress onto surgery cards.