Biology Reference
In-Depth Information
experimenter should begin handling the animals for brief peri-
ods of time (1-5 min/handling session) in order to habituate
the animals to being handled; this minimizes stress, which
can be an important source of undesired variability. Animals
should be handled at least three times prior to the start of a
behavioral assay.
2. In order to deliver the viral vectors into discrete brain regions,
stereotaxic surgery will be performed on the animals. For our
protocol, Sprague Dawley or Long-Evans rats are fi rst anes-
thetized in an induction chamber with 3-4 % isofl urane gas
and given buprenorphine analgesia (0.1 mg/kg, s.c.). Animals
are monitored for absence of withdrawal response to mild paw
pinch prior to incision. The animal is placed on a heating pad
covered by a sterile drape to maintain body temperature.
Lacrilube is applied to eyes. The top of the head is shaved,
scrubbed with betadine and alcohol, and a 1-cm incision is
made using standard sterile procedures. The skull surface anat-
omy is used to identify the site of cranial drilling based on
injection coordinates identifi ed from an anatomical atlas. A
small bore hole is drilled in the skull above the site of microin-
jection. We typically use a 27-gauge hubless dental needle
connected by polyethylene tubing to a 10
l Hamilton syringe
and controlled by a microprocessor driven pump; we backfi ll
the injection needle immediately prior to infusion and confi rm
free fl ow of the viral vector. The syringe and tubing is fi lled
with sterile water, and we backfi ll the viral vector into the
injection needle (preceded by a small air bubble). We prefer
these injection needles to blunt infusion catheters because the
beveled tip is less likely to become plugged by the viscous viral
vector. If this is the fi rst time targeting a particular brain
region, it is recommended to pilot the injection coordinates
using a small amount of India ink (0.5
μ
l or less) to confi rm
accuracy. For microinjection of the viral vectors, the injection
needles are slowly lowered to the targeted region and 0.5-3
μ
l
of the viral vector (depending on the vector type and the size
of the target region) is slowly infused at a fl ow rate of 0.2
μ
l/
min. As discussed in Chap.
9
of this topic, each viral vector has
different characteristics regarding the area of infection and the
density of infected cells that depends upon the titer and the
affi nity of the viral particles for binding sites on cells. For
example, with HSV, 2
μ
l will produce excellent infections even
in small targets because the viral particles bind with such high
affi nity to neurons, although we do use smaller volumes for
very small targets. We have found that infusing larger volumes
of vector is rarely productive, and instead we prefer to make
multiple injections if a large region is being targeted.
The injector is left in place an additional 5 min to minimize
diffusion up the injector tract and then slowly removed; if the
μ
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