Viral-Mediated Gene Transfer to Study the Behavioral Correlates of CREB Function in the Nucleus Accumbens of Rats - Drugs of Abuse: Neurological Reviews and Protocols - page 332

encodes a control protein (e.g., E. coli -galactosidase, encoded by the LacZ

gene) and, typically, a “mock” infection (10% sucrose, the vehicle in which the
vector is resuspended after packaging). We do not proceed to in vivo studies
unless it is clear that the vector causes substantial up-regulation of the protein
encoded by the transgene. General procedures for infection of primary neuronal
cultures with HSV vectors and subsequent processing of the cultures for
immunoblots have been described (4) . The following are details specifi c to our
Western blotting procedures for CREB (or mCREB, expression of which is
indistinguishable from that of CREB using the antibodies described here):

1. Load 20 g of protein (in homogenate containing 1% sodium dodecyl sulfate

[SDS]) per lane.

µ

2. Block nonspecifi c protein binding to membranes for 2 h at room temperature with
a 5% milk in PBS containing 0.1% Tween 20 (PBS-T) blocking solution.

3. Incubate membrane in primary antibody (anti-CREB, 1 4000 in PBS-T) for

2 h at RT.

4. Wash membranes three times for 15 min each in PBS-T at room temperature.

5. Incubate membrane in secondary antibody (anti-rabbit, 1 5000 in 5% milk in

PBS-T) for 2 h at room temperature.

6. Wash membranes three times for 15 min each in PBS-T at room temperature.

7. Detect CREB protein on the membrane using chemiluminescence ( Fig. 2 ).

3.4. Surgery
The fi nal step before initiating behavioral studies is to confi rm that the
vector expresses in vivo. We unilaterally microinject the vector at full strength
titer into the NAc, perfuse the rat on day 3 (when we normally begin the
behavioral studies, a time at which transgene expression is maximal), and
perform within-animal immunohistochemical comparisons (gene expression
within the injected vs noninjected hemisphere) to ensure protein overexpression
( see Notes 2-4 ).

1. Establish an aseptic surgical fi eld if necessary. Attach the Hamilton syringes to
the stereotaxic apparatus, and angle the arms at 10° from the midline ( see Note 5 ).
Thoroughly clean the Hamilton syringes with alternating washes of 95% ethanol
and distilled water. The fi nal wash should be distilled water, such that the vector
does not come into contact with ethanol.

2. Anesthetize each rat with intraperitoneal (i.p.) pentobarbital (65 mg/kg sodium
pentobarbital) accompanied by subcutaneous (s.c.) atropine (0.25 mg/kg) to
minimize bronchial secretions ( see Note 6 ). Once the rat is anesthetized, shave
the scalp with an electric trimmer.

3. Place the rat (300-325 g) in a stereotaxic instrument. Use a scalpel incision to
expose the skull, and clean the area with sterile saline.