Biomedical Engineering Reference
In-Depth Information
to avoid associating the offset of the drug that might be aversive
(6)
with the
drug-assigned compartment. Clean the entire apparatus with isopropyl alcohol
wipes, and leave the tops open to minimize trapped odors.
9.
Day 4
. Repeat exactly the procedures used on d 3. Do not alternate the order of
nondrug and drug pairings (
see
Note 10
).
10.
Day 5
. Test the animals under the conditions used for screening (
items 2-4
).
11. Immediately after testing, collect brain tissue (fresh or perfused, as in
Subhead-
ing 3.6.
) for histological or molecular analyses. In some cases, only one will
be possible (i.e., if Western immunoblotting studies will be conducted, it is
necessary to collect tissue from the area of the microinjection). Generally, we
favor procedures that will allow detailed examination of the microinjection
placements, because rats in which the gene transfer was not targeted to the
appropriate region (i.e., NAc) should be eliminated from statistical analyses.
12. Perform statistical analyses. To maximize statistical power, we typically use
analyses of variance (ANOVA) with repeated measures: we compare the net
differences in time spent in the drug side (i.e., time spent in drug side minus time
spent in saline side, in seconds) before and after treatment. Signifi cant effects
are analyzed further with
post hoc
tests (Fisher's
t
-tests).
4. Notes
1. The viruses should be divided into the smallest aliquots that are practical.
Repeated freeze-thaw cycles can damage the virus particles, effectively decreas-
ing titer. Because we typically use bilateral microinjections of 2.0
µ
L of vector
for each rat, we freeze the vectors in 5.0-
L aliquots. Whenever freezing or
thawing of aliquots is necessary, it should be performed as quickly as possible
(i.e., using a dry ice-ethanol slurry or a 37°C water bath, respectively).
µ
2. Presently, the full-strength titer of HSV vectors is on the order of 10
8
infectious
units (iu)/mL. In our early studies in brain, we examined gene transfer in the
NAc after various combinations of viral titers and injection volumes. We started
with 2.0-
L microinjections (within a range of volumes often used for intra-NAc
microinjections of drugs, for example) and full-strength titer. For comparison,
we also used 1.0-
µ
L
microinjections of titer diluted by 50% (in 10% sucrose). We found that the
most important factor for transgene expression in the VTA was injection volume:
regardless of the vector titer, the sphere of transgene-expressing neurons in
the VTA was substantially larger with 2.0-
µ
L microinjections of full-strength titer, and 2.0-
µ
L or 1.0-
µ
µ
L microinjections than it was with
1.0-
L microinjections. We proceeded with behavioral studies using full-strength
titers because we found more transgene-expressing neurons in rats that received
the undiluted vector than in rats that received the 50% diluted vector, but overall,
the effects attributable to titer were less striking than the effects attributable to
injection volumes. These fi ndings suggest that, at some critical point specifi c
for each type of vector, brain microinjections of higher titers simply cause more
infections per cell rather than more cells infected. Thus the main advantage of
high-titer vectors may be that, eventually, it will be possible to use diluted titer to
µ
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