Antisense Approaches in Analyzing the Functional Role of Proteins in the Central Nervous System - Drugs of Abuse: Neurological Reviews and Protocols - page 361

6. Antisense oligodeoxynucleotides and controls (custom-synthesized, 18-mer
phosphorothioate-modifi ed and HPLC-purifi ed; GIBCO/BRL) ( see Note 1 ):

MGluR5 antisense oligonucleotide complementary to nucleotide -6 through
+12 of the rat mGluR5 mRNA (20) : 5 .

Position +1 corresponds to the A nucleotide of the initiation AUG codon on
mGluR5 mRNA (ATG on DNA) ( see Note 2 ).

-CAGAAGGACCATTTTAGG-3

Three control oligodeoxynucleotides ( see Note 3 ):

a. Sense oligonucleotide: 5 -CCTAAAATGGTCCTTCTG-3 .

b. Mismatched oligonucleotide (3 mismatched bases in the mGluR5 antisense
oligonucleotide): 5 -CAGAAGCACCATATTACG-3 .

c. Scrambled oligonucleotide (scrambled bases in the mGluR5 antisense
oligonucleotide): 5 -GCTGATACAGAGATGACT-3 .

7. Sterile artifi cial cerebrospinal fl uid (aCSF): 123 m M NaCl, 0.86 m M CaCl2,
3.0 m M KCl, 0.89 m M MgCl 2 , 25 m M NaHCO 3 , 0.50 m M NaH 2 PO 4 , and
0.25 m M Na 2 HPO 4 aerated with 95% O 2 -5% CO 2 , pH 7.2-7.4.

Use sterile technique during preparation of all solutions, including the
oligodeoxynucleotide solutions. All surgical instruments must be sterile, and
surgical and oligo infusion procedures should be performed in a sterile manner.

3. Methods
The methods described below outline (1) implantation of guide cannula in
the striatum, (2) intrastriatal infusion of oligonucleotides, and (3) assessment
of effi cacy and selectivity of antisense oligonucleotides.

3.1. Implantation of Guide Cannula
Before surgery, all surgical instruments need to be sterilized by placing them
in the hot glass bead sterilizer. The sterilizer, by allowing rapid sterilization of
instruments, is better adapted than an autoclave for surgery involving multiple
animals.

1. Anesthetize rats by injecting 4% chloral hydrate intraperitoneally at a dose of
400 mg/kg using a 5-mL syringe with a 22-gauge needle.

2. Shave the top of head and place the head in the stereotaxic apparatus. Wipe the
top of head with Povidone-iodine swabsticks before making a midline sagittal
incision with a scalpel and exposing the skull.

3. Scrape the exposed bone area gently with cotton swabs to remove the periosteal
connective tissue and stop any bleeding.

4. Locate the bregma and determine the location for cannula placement in the left
or right hemisphere using the coordinates: 1.0 mm rostral to bregma and 2.0-2.5
lateral to midline (5 mm below the skull surface for targeting the central part of
the dorsal striatum) according to the atlas of Paxinos and Watson (19) . Drill a