Using Viral Vectors to Study the Memory Trace in Mice - Viral Vector Approaches in Neurobiology and Brain Diseases - page 146

performed in a certifi ed biological safety cabinet (tissue
culture hood) when possible and fi ltered pipette tips used.
Used pipette tips and other plasticware can be decontaminated
overnight in 1 % sodium hypochlorite before autoclaving and
appropriate disposal. Similarly, dedicated glassware can be
washed with ethanol or sodium hypochlorite, rinsed with
water, and autoclaved before re-use.
2. This and other dissection steps may be facilitated by the use of
a microscope (e.g., dental lab articulated arm stereomicro-
scope) to better visualize the area to be manipulated.
3. To avoid damage to the underlying tissue, it is possible to
rotate the drill in small concentric circles while opening the
hole in the skull.
4. A quantifi able fear memory in mice can be induced by a single
foot-shock with an intensity ranging from 0.2 to 1.0 mA.
Importantly, the number of activated neurons in the LA (arc+)
remains relatively constant, irrespective of shock intensity.
However, we have found that Arc mRNA visualized, detected,
and counted be more easily following a stronger foot-shocks,
typically 0.4-0.6 mA.
5. In this experiment, we visualized the fear memory trace in
mice infused with GFP-CREB vector and compared to condi-
tioned animals infused with GFP vector alone. Additional con-
trols include mice infused with these viral vectors but not fear
conditioned.
6. The mRNA probes for Arc and GFP correspond to the full-
length coding regions for each of these genes, which are 3.0
and 0.7 kb in length, respectively. In our laboratory, the Arc
mRNA probe is labeled with DIG, whereas the GFP probe is
labeled with fl uorescein. The probes are synthesized by in vitro
transcription from a linearized DNA template, and column-
purifi ed before use.
7. Ideally, a slide contains brain sections from animals that were
infused with different viruses and/or trained with different
behavioral paradigms. This minimizes the infl uence of signal
variability between slides during the fi nal analysis.
8. While it is advisable to check probe size and integrity on a
denaturing gel for RNA electrophoresis the fi rst time the probe
is made, this does not need to be done each time the probe is
used. Rather, it may be quicker to run the probe on a tradi-
tional agarose gel made with 1× TAE buffer to ensure the
probe has not signifi cantly degraded.
9. The beakers, graduated cylinders, and slide racks are auto-
claved prior to use and the bench, glass jars, and slide chamber
can be cleaned with RNase Away reagent (Invitrogen 10328-
011). Solutions should be DEPC treated or made with