Using Viral Vectors to Study the Memory Trace in Mice - Viral Vector Approaches in Neurobiology and Brain Diseases - page 138

2. Immobilize the head of the animal with ear bars of the
stereotactic frame. If the ear bars are placed correctly (behind
the jawbone), you should be able to move the head up and
down without lateral displacement. Then place a platform
under the torso and insert the mouthpiece with the animal's
front teeth in groove, with a slight amount of tension pulling
the mouth. Gently lower the nose bar so it is holding down the
snout with minimal pressure.
3. Place drops (e.g., lacrimol) in the mouse eye. It is important to
keep the eyes moist throughout the procedure.
4. Shave and clean incision area with ethanol.
5. Use scalpel to cut and gently open the skin above the region of
interest. Insert scissors and cut anteriorly until Bregma can be
visualized. Clean the skull (dilute H 2 O 2 may be gently wiped
on and off the skull to better visualize Bregma and Lambda).
Bregma and Lambda may be marked on the skull using a fi ne-
tip marker ( see Note 2).
6. Ensure a level head confi guration (such that Bregma and
Lambda are on the same horizontal plane). If the skull is not
level, adjust the animal's position.
7. Measure and mark fi nal coordinates on the skull relative to
Bregma. Gently drill holes in the skull (ideally 1-1.5 mm in
diameter) ( see Note 3).
8. Clean any blood from the area with a moistened cotton-tipped
applicator (e.g., Q-tip). It is important that this area be free of
blood so the infusion pipette maintains patency.

1. Thoroughly clean the Hamilton gastight syringe with ethanol
and clamp in the infusion pump.
2. Using a 1-ml syringe, fi ll the pipette/tubing with water, run
through once, and then fi ll with water again.
3. Attach the tubing to the Hamilton syringe and introduce a
small (typically 0.5

3.2.2 Virus Injection

l) air bubble into the pipette. This air bub-
ble will separate the water from the virus solution. Draw up an
appropriate volume of virus (typically 5.0

μ

l to be
injected on each side) and gently touch tip of pipette with a
kimwipe to remove any excess virus solution. Mark the begin-
ning and end of the air bubble with a marker to aid in the
visualization of viral infusion.
4. Position the pipette above the hole in the skull. Slowly lower
the pipette into the brain to the depth coordinate correspond-
ing to the tip of the LA.
5. Allow at least 2 min before beginning infusion (rate of 0.1

μ l, with 2.0 μ

l/
min). The movement of the bubble can be monitored to
ensure virus is being infused into the brain.

μ