Using Viral Vectors to Study the Memory Trace in Mice - Viral Vector Approaches in Neurobiology and Brain Diseases - page 139

6. Once infusion is complete on one side, allow the pipette to
remain in place for another 5-10 min to ensure adequate dif-
fusion. Slowly raise the pipette and follow the same procedure
for the other side of the brain ( see Note 4).
7. Inject mice with analgesic (e.g., ketoprofen; 5 mg/kg fi nal
concentration, sc).
8. Remove the animal from the stereotactic frame. Staple/clip the
skin over the incisions and cover with antibiotic/analgesic gel.
9. Place the animal on a paper towel (or similar) placed in a clean
cage. The paper towel prevents ingestion or inhalation of bed-
ding while the animal is still in an anesthetized state. Place the
cage on a heating pad so that the rear half of the body is on the
pad.
10. Monitor the mouse. When fully recovered, return mouse to
homecage. All animals that have undergone surgery should be
monitored daily for general health and to ensure the clip hold-
ing the scalp remains in place.

3.3 Fear
Conditioning and
Tissue Collection

Mice are typically trained for auditory fear conditioning 3 or 4 days
after surgery (when transgene expression is optimal) and brains are
removed for catFISH either shortly after fear conditioning or fear
memory testing. To ensure precise timing between behavioral
manipulation and tissue collection, it is advisable to train or test
each animal individually.

1. Wash the fear conditioning chamber with dH 2 O.
2. Place the animal in the fear conditioning chamber and begin
the conditioning protocol. A typical protocol involves a 2 min
habituation period followed by a 30 s tone (2.8 kHz) that ter-
minates with a 2 s foot-shock (0.4-0.7 mA). Mice are removed
30 s after the foot-shock and placed in the home cage in a quiet
room prior to tissue processing ( see Note 5).
3. The presence of the conditioned fear memory can be tested
24 h later in a novel or modifi ed conditioning chamber.
Modifi cations include changing the scent (70 % ethanol) and
appearance of the chamber (using white plastic sheet to cover
the bars on the fl oor of the chamber as well as two Plexiglas
plates with white backing to change the color and orientation
of the chamber walls) (Fig. 2 ).
4. Place the mouse in the modifi ed chamber and, after a 2-min
habituation period in the new environment, the tone CS is
presented (for 1-3 min). The strength of the fear memory can
be quantifi ed as percent time spent freezing in response to the
tone.
5. After fear conditioning or testing, place the mouse in a quiet
room prior to tissue processing. At the appropriate time (see