Viral Vectors to Study Synaptic Function - Viral Vector Approaches in Neurobiology and Brain Diseases - page 127

The main variable is in successful transfection of the RNAs.
Electroporation of the BHK21 cells should yield more than 80 %
transfection (as visualized by GFP expression at 36 h posttransfec-
tion). This can be achieved by obtaining high-quality RNA and
optimizing the electroporation conditions. Also, fresh and low-
passage BHK21 cells should be used. We have noticed that freeze-
thaw cycles do not signifi cantly affect the virus titer if performed
within a few days of each other, but prolonged storage (>1 year)
could reduce infection potential. Hence, date of virus production
should be kept in records in case loss of successful infection is
noticed over time.
The main variable during in vivo stereotaxic injection is the
rate of success for suffi cient injection of viral solution in the appro-
priate brain area. If you plan to use an injection pump, we have
found the following trick helps optimize the chances of obtaining
a successful injection. During the lowering of the cannula, it is
important to monitor the fl ow of the viral solution. We have often
noticed that the fl ow of the viral solution is greatly reduced or
stops during the lowering of the cannula and resumes suddenly at
the site of injection with a gush damaging the surrounding tissue.
If that is the case, we suggest fi rst lowering the cannula at 0.5 mm
into the brain while injecting sterile PBS to avoid infection else-
where in the brain and wait at this height until enough pressure has
built in the pump to resume the fl ow. Then, slowly lower the can-
nula to the fi nal destination. This trick ensures that you do not
damage the area to be injected by the sudden gush of viral solu-
tion, which often occurs when the fl ow of solution resumes.
Also, accurately targeting the region of interest can be a diffi -
cult variable to master. We have found that keeping the weight of
the animal constant yields more accurate injections than solely
choosing the animal by age. Also, systematically using the coordi-
nates by carefully locating bregma for each injection is crucial to
obtain reliable injections at the desired location. Judging the best
place of injection by eye never works reliably.
Although diffi cult to obtain, it is best to record from infected
and closed-by uninfected neurons within the same slices using the
same placement and stimulation intensity of the stimulating elec-
trode whenever possible (e.g., for analysis of current amplitude or
short-term plasticity). This ensures the most accurate comparison
of parameters of synaptic function between the two types of cells
by minimizing slice to slice variability.
Healthy GFP-labeled neurons should have preserved the shape
of the neuron (e.g., CA1 pyramidal neurons should be visualized
as pyramidal in shape under epifl uorescence). Patching round, sur-
face GFP-labeled cells will not work as they represent dying cells
due to the dissection. It is best to aim at patching GFP-labeled cells
with the appropriate shape that are deeper within the slice to
increase the chances of obtaining good recordings.