Western blots were prepared with proteins extracted from isolated striatal cell 
nuclei by a modification of the method of Tata (1974). After electrophoresis 
in a 10-percent polyacrylamide gel and transfer to polyvinyldifluoride (PVDF) 
membranes, the membranes were blocked with 1 percent gelatin, 0.25 percent 
Tween 20, 10 mM HEPES buffer pH 7.4, and 0.02 percent sodium azide for 60 
minutes; incubated for 1 hour with rabbit polyclonal antisera; washed 2X times; 
incubated with [ 125 l]-protein A for 1 hour; and washed several times at room 
temperature. Autoradiographs were then obtained. 
Gel shift assays were performed on proteins extracted from the isolated 
nuclei. A synthetic double-stranded 21 -nucleotide oligomer from the gibbon 
ape leukemia virus (GALV) enhancer containing an AP-1 (TGAGTCA) 
consensus sequence at its core was used as a probe. 
Immunocytochemical analysis of Fos-positive neuronal cell nuclei was 
performed with standard peroxidase-antiperoxidase method on 30-^t frozen 
sections after perfusion of rats (under deep pentobarbital anesthesia) with 4 
percent paraformaldehyde. Visualization of immune complexes involved 
using diaminobenzidine as the chromogenic substrate. The anti-Fos antibody 
was employed at a dilution of 1 : 1 0,000 or 1 :20,000. 
RESULTS 
A single injection of cocaine has a powerful effect on c -fos gene expression. 
A dose of 30 mg/kg IP produced a marked, fivefold to sevenfold increase in 
striatal c -fos mRNA within 30 minutes (figure 1). The increase in mRNA content 
was relatively short lived and did not substantially outlast the 30-minute time 
point. The elevation in mRNA content was both dose dependent and regionally 
specific, occurring in regions receiving a strong dopaminergic innervation (e.g., 
striatum) but not in hippocampus (figure 2). Immunocytochemical staining for 
Fos-immunoreactive proteins disclosed nuclear staining in neurons in striatum, 
nucleus accumbens, olfactory tubercle, and the islands of Calleja that was weak 
in saline-injected controls (figure 3) and very intense following treatment with 
cocaine (figure 4) or GBR-12909 (figures 5 and 6). The effect of cocaine was 
dose related in terms of the number of cells that could be counted and in terms 
of the intensity of staining. We quantitated the number of stained cells for the 
dose response at 2 hours postcocaine, a time at which the protein staining was 
at a peak (figure 7, upper panel), using doses of 0.3 to 30 mg/kg (Young et al. 
1991). There was some light staining in control sections, but this varied 
between rats and generally was not of an intensity to be easily quantitated. 
The number of cells was increased significantly at the 3.0 mg/kg dose and 
tended to reach a plateau between 10 and 30 mg/kg (figure 7, lower panel). 
What is not apparent in the cell counts is that at higher doses, although the 
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