AMP (cAMP) second-messenger system is an important element in the 
induction of c -fos gene expression in these cells. 
The antibody used in the immunocytochemistry studies was directed 
against a c -fos sequence KVEQLSPEEEEKRRIRRERNKMAAA, 
which is highly conserved between species and among the four distinct 
members of the Fos/Fos-related antigen (Fra) family, which includes 
Fos-B (REETLTPEEEEKRRVRRERNKLAAA) (Zerial et al. 1989), Fra-1 
(PCEQISPEEEERRRVRRERNKLAAA) (Cohen and Curran 1988), and 
Fra-2 (RDEQLSPEEEEKRRIRRERNKLAAA) (Nishina et al. 1990; Matsui et 
al. 1990). This suggested that more than one protein species may be elevated 
in the Fos-immunoreactive striatal nuclei as seen with immunocytochemistry. 
If multiple proteins were detected via Western blot methodology, they might 
correspond to one or more of the multiple Fos/Fra proteins or fragments thereof. 
To investigate this possibility, we performed Western blot analysis of striatal 
nuclear extracts (figure 10). After isolation of the nuclei by centrifugation 
through a 2.1 -M sucrose solution, the nuclear pellet was homogenized directly 
in SDS sample buffer, assayed for protein content, and electrophoresed in a 4- 
percent stacking/1 0-percent separating polyacrylamide gel along with molecular 
weight markers. We observed a temporally distinct cascade of immunoreactive 
protein bands with the immunoblot technique (figure 10). The basal state 
contained a small but detectable amount of 55-kDa Fos protein (barely visible 
on the autoradiogram) as well as several, more distinct, lower molecular weight 
Fra proteins, especially one at 41 kDa. Within 2 hours an abundant amount of 
the 55-kDa Fos protein was seen and was followed thereafter by increases 
in the lower molecular weight Fra proteins (figures 10 and 11). We have 
confirmed the identity of the 55-kDa protein by probing with an antibody raised 
against an n-terminal peptide (DYEASSSRCSSASPAGDSLS) from the rat Fos 
protein. In these blots (not shown), an increase in the 55-kDa nuclear protein 
was detected as soon as 1 hour after a 30-mg/kg IP dose of cocaine. By 2 
hours after cocaine treatment, and even more conspicuously after 4 hours, 
increases were seen in the lower molecular weight Fra proteins (figure 11). 
An analogous temporal pattern of Fos and Fra expression has been seen in 
hippocampus with convulsant stimulation (Sonnenberg et al. 1989). 
One of the hallmarks of lEGs is that they do not require protein synthesis to 
undergo an increase in transcription. All the mechanisms and proteins needed 
to activate IEG transcription preexist in the cell’s cytosol or nucleus. The 
activation of c -fos expression by cocaine fits this criterion. We observed that 
the potent protein synthesis inhibitor anisomycin, which crosses the blood-brain 
barrier, did not prevent the increase in c -fos mRNA (figure 12). In fact, we 
saw the phenomenon of superinduction, in which the stimulus produces an 
exaggerated elevation in mRNA levels in the presence of a protein synthesis 
195 
