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time course of the vector. This should be checked with each indi-
vidual promoter construct. In our hands, HSV promoter-driven
gene expression peaks at around 4 days and is essentially gone by
7-10 days [ 13 ]. We have not closely examined how early the dyn-
orphin, enkephalin, or serotonin transporter promoters initiate
gene expression, but we have found that expression seems stable
after about 1 week and lasts for several more weeks at least [ 12 ].
4.2 Stereotaxic
Surgery
There are many ways to do stereotaxic injections, but we have
found several procedures that enhance the success of transgene
expression. For example, a simple one-time injection may be used
with viruses that are stable expressers. For our injections, we use
hubless dental needles attached with Tygon tubing to a
microprocessor-driven pump. The smooth infusion delivered by
such a pump is absolutely essential, as virus preparations can be
sticky and clog the line without constant pressure. We observe less
clogging with beveled needles than blunt infusion cannula. In
some cases, we implant a guide cannula, especially if it becomes
necessary to make subsequent drug infusions. Then, the viral vec-
tor can be infused at the ideal interval prior to the planned experi-
ment, usually while the animal is lightly anesthetized to facilitate
the infusion and minimize movement and distress.
The type of behavioral procedure can also infl uence the infu-
sion method. For example, in the resident intruder paradigm in
which animals scuffl e, there is the potential for a guide cannula
assembly to either hurt the aggressor male, become loosened or
damaged, or for the skin around the cannula to be injured during a
confrontation (however it is not impossible; for example, Covington
and Miczek have successfully conducted social defeats with indwell-
ing intravenous catheters [ 14 ]). Guide cannula sites may also be
more vulnerable to contamination during procedures such as the
forced swim test. Other relevant behaviors that could be easily con-
ducted with cannula implants would be the open fi eld test, elevated
plus maze, probe burying, and saccharin preference test.
As per any stereotaxic injection, the infusion coordinates should be
established empirically. We confi rm the accuracy of injections in
every animal by examining GFP expression whenever possible. In
a few cases, we complete behavioral testing after transgene expres-
sion has been allowed to dissipate; in this case, we have used gliosis
around needle tracks or cannula placement, but this is less reliable.
The histological analysis should be performed in a blinded fashion
and the sites and extent of infection documented on brain atlas
illustrations. In our lab, we consider an injection to be accurate
when more than 50 % of the transgene expression (usually indi-
cated by GFP) is within the target region, and “hits” must be bilat-
eral when both sides of the brain are targeted.
4.3 Evaluating the
Accuracy of Injection
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