Viral Vectors to Study Synaptic Function - Viral Vector Approaches in Neurobiology and Brain Diseases - page 121

(b) While the rat is falling asleep, prepare the pump and tubing
that connects the needle placed on the pump to the Plastic
One cannula. This tubing needs to be fi lled from beginning to
end with mineral oil by capillary action. Do not allow pres-
ence of bubbles within the tube. Make sure that the plunger
of the needle is completely inserted into the needle prior to
connecting it to the tubing. Connect the tubing to the needle
and to the cannula, avoiding the spread of oil within the can-
nula or the needle.
(c) Once the animal is asleep, shave the head of the rat with hair
clippers and increase local analgesia by injecting bupivacaine
(2 mg/kg) at site of incision by local subcutaneous injection.
(d) Place rat into stereotaxic frame and insert the ear bars. Make
sure that the bars are well placed on head. Proper placement
can be confi rmed if the rat's head can only move in the for-
ward-backward axis and not sideways. Insert mouth holder
carefully making sure that it is placed between the teeth. Press
the mouth bar onto the head (should be at a level just above
the eyes) until the skin on the skull is not loose anymore. Do
not press too hard as it could injure the animal.
(e) Cut an incision on top of the head so as to expose the skull
from the top of the mouth bar to the end of the skull. Make
sure enough skin is cut to be able to see the Bregma. Place the
hemostats to keep the skin well away exposing the skull. The
rat is now ready for injection.
(f) Fill the cannula and tubing with virus solution using the refi ll
(withdraw) function of the pump. Do not allow for bubbles
to form during that process (except for the air gap between
the mineral oil and the virus solution). For a dual injection
(left and right hemispheres), add about 2.5 μ L of viral solu-

L of PBS may also be added after the viral solution,
avoiding air between the two solutions to help with fl ow (see
Notes below).
(g) Place the cannula above the Bregma. Then, elevate the can-
nula again to avoid contact of cannula with skull that would
bend it while traveling to fi nal destination.
(h) Using Y (vertical/posterior) and X (horizontal/lateral) axes,
travel to fi nal destination:
The coordinates for injection in a 20-22-day-old rat hip-
pocampus CA1 are: X : ±2.5 mm lateral from Bregma; Y:
−4 mm posterior from Bregma.
(i) Drill hole through the skull superfi cially without touching the
meninges at the site where the cannula will be inserted. Open
the dura mater, the outermost of the three layers of the
meninges surrounding the brain, locally using the bent end of

tion. 0.8 μ