Recombinant Adenovirus in Neurobiology - Viral Vector Approaches in Neurobiology and Brain Diseases - page 23

of the pressure from injector on the brain should be consid-
ered. During injection, the pressure of the needle can push the
brain moving to a more ventral and lateral location, resulting
in a change of the coordinates. This particularly occurs when
injecting regions are in the ventral region of the brain such as
the hypothalamus and amygdala. Thus, the actual dorsal/ven-
tral coordinates are usually greater than those in the atlas (more
ventral). Similarly, the injector may enlarge the ventricle when
passing through it. Thus, the dorsal-ventral coordinate
becomes larger than that in the atlas. Therefore, it is necessary
to test the coordinates prior to experiments.
8. To evaluate the effects of recombinant adenovirus, it is essential
to collect the tissue with viral infection, not the brain region
intended for injection, because it is possible that the actual
injection site is different from the desired injection site. We
punch the infected tissues out of 300- m coronal brain sec-

tions. It is diffi cult to observe the fl uorescent areas in such
thick sections even with dissection of the tissue under a micro-
scope (unless dissecting microscope has a UV light source
available). We have used thin sections (20-30

μ

m) to estimate
the position of GFP (RFP) in the brain and dissected the cor-
related regions from thick sections. The fl uorescence on the
thick sections is then checked under fl uorescent microscope to
ensure the GFP (RFP)-positive area is dissected. On the other
hand, after the effects of recombinant adenovirus is confi rmed,
the protein levels in the specifi c brain regions should be mea-
sured to determine the manipulation of target protein in the
specifi c brain regions. In this case, the location of GFP expres-
sion should be observed and recorded in brain sections (20-
30

μ

m) before, in the middle of, and after the sections used for
collecting the tissues. The animals should be excluded from
the study if the GFP expression is not located the desired brain
region in sites of the injection.
9. The alterations in protein levels after injection of recombinant
adenovirus not only depends on the effi ciency of the virus but
also depends on the protein turnover rate and feedback regula-
tion. The effect of knockdown of a recombinant adenovirus
may not be observed until existing proteins are degraded.
On the other hand, the feedback regulation may increase the
synthesis of the protein, once the protein level is reduced by
the viral treatment. Thus, a time-course study is necessary to
determine the optimal period for knockdown. From our expe-
rience, most of antisense and siRNAs produce maximal knock-
down 5-7 days after the viral treatment.

μ