Antisense Approaches in Analyzing the Functional Role of Proteins in the Central Nervous System - Drugs of Abuse: Neurological Reviews and Protocols - page 363

injector/tubing system by slowly pulling the plunger of the syringe. You should
be able to visualize the moving of a small air bubble in the PE10 tubing when
drawing the solution.

5. Remove the stylet and insert the injector cannula. This step can be normally done
without anesthesia. However, prior handling of the animals is recommended to
allow the animals to be accustomed to the experimenter.

6. Start infusion of the oligonucleotide solution at a rate of 0.1 µ L/min. A total

L for a 10-min infusion. Progress of infusion can
be monitored through observing movement of the small air bubble through the
PE-10 tubing.

dose/volume is 2 nmol/1 µ

7. After the termination of the infusion, the injector will be left in the place for
an additional 5 min to reduce any possible backfl ow of the solution along the
injection track.

3.3. Assessment of Effi cacy and Selectivity
Effective and selective properties of antisense oligonucleotides in reducing
target gene expression need to be evaluated before any functional study is
conducted. The mGluR5 mRNA and protein are expressed in both striatonigral
and striatopallidal projection neurons of rat striatum (21-23) . Among all eight
subtypes of mGluRs, mGluR5 represents one of subtypes with the highest level
of expression in postsynaptic striatal neurons. The presynaptic distribution
of mGluR5-immunoreactivity is, however, minimal (24) . These distribution
features make mGluR5 a fi tting target for the antisense inhibition.

3.3.1. Assessment of Protein Levels

The effi cacy and selectivity can be fi rst evaluated at anatomical levels. This
is to detect alterations in basal mGluR5 protein levels following repeated treat-
ments with mGluR5 antisense and their controls. With Western immunoblot,
we found that basal mGluR5 expression in the ipsilateral dorsal striatum was
reduced by 50-60%. The reduction of mGluR5-like immunoreactivity at the
same extent was also seen in the dorsal striatum using immunohistochemistry.
MGluR5 expression returned to normal levels 24-48 h after the termination
of antisense treatment, indicating mGluR5 reduction is reversible and not due
to cell loss ( see Note 5 ). In contrast to mGluR5, other subtypes of mGluRs
surveyed, including mGluR1, mGluR2/3, and mGluR7, did not show signifi cant
changes in their basal expression in the injected striatum. Repeated treatments
with aCSF or any of the three control oligos, i.e., sense, mismatched, and
scrambled oligos, had no signifi cant effects on expression of mGluR5 as well
as other subtypes. Evidently, the mGluR5 antisense oligonucleotides used in
our study possess the ability to suppress effectively and selectively constitutive
mGluR5 synthesis.