Biomedical Engineering Reference
In-Depth Information
Fig. 2. Western immunoblot confi rming that HSV-CREB elevates expression of
CREB in cell culture. For comparison, HSV-LacZ (encoding
E. coli
-galactosidase)
was used as a viral vector control, and mock (10% sucrose) was used as a control for
the vehicle into which the vector constructs are resuspended after packaging.
4. Set the stereotaxic arms to perpendicular (90°) at their base. Once set, the arms
should not be pivoted, or all measurements should be reestablished. Ensure that
the skull is fl at by using one of the Hamilton syringes to obtain measurements
for the height of the skull surface within the dorsal-ventral plane at lambda and
bregma. If the measurements are not within 0.1 mm, adjust the height of the
head at the bite-bar on the stereotaxic instrument. After any adjustment, always
re-measure both lambda and bregma.
5. The stereotaxic coordinates that we use for the NAc are 1.7 mm anterior to
bregma and ± 2.3 mm from the midline
(5)
. Using a 10° angle from the midline
for the Hamilton syringes, bilateral microinjections can proceed in parallel. Raise
the stereotaxic arms to their maximal height, and mark the appropriate points
on the skull with a pen if necessary. Do not pivot the arms, or the stereotaxic
coordinates should be reestablished.
6. Drill burr holes (1 mm in diameter) into the skull, to the level of dura. We aim
our microinjections into the NAc at 6.5 mm below dura. If dura is damaged
during drilling, assume that the skull of a 300-325 g rat is 0.5 mm thick, and
subtract this thickness from the dorsal-ventral measurements used to ensure that
the skull was fl at. Without rupturing dura (or penetrating the brain if the dura
was damaged), ensure that the trajectory of each injection needle will not be
affected by the size or the shape of the burr holes.
7. Load the Hamilton syringes with vector. The vectors should be thawed as close
to the time of microinjections as possible (
see
Subheading 3.1.
). Lower the
injection syringes to dura, but use a sterile 26-gauge needle to puncture dura.
Slowly lower the injection needle to the appropriate depth, over 20-30 s. Once
at the desired depth, retract the needle 0.1 mm.
8. Inject the vectors over a period of 10 min. We perform the microinjections
manually, at a rate of 0.1
L/30 s. After the fi rst microinjection, we start a timer;
as such, the last microinjection is performed at 9 min 30 s. We then wait an
additional 5 min before removing the injection needle from the brain, to allow
µ
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