The Journal of Neuroscience, December 20, 2006, ():
c-Fos Facilitates the Acquisition and Extinction of Cocaine-Induced Persistent Changes
J. Neurosci. Zhang et al.
26: 13287
Supplemental Data
Files in this Data Supplement:
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Supplemental table
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Supplemental Figure 1. LacZ gene activation in various brain regions of DCi94 (120) reporter mice. X-Gal staining data were obtained from 7 each age-matched DCi94 (120) reporter and wild-type mice and 5 coronal brain sections from each region per mouse were used. Representative parts of the following brain regions are shown: dorsal lateral caudoputamen (CPu); CA3 subfield of the hippocampus (HIP); basal lateral nucleus of the amygdala (AMG); prefrontal cortex (PFC). i indicates reporter mouse. Scale bar represents 25 μm.
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Supplemental Figure 2. Normal brain anatomy in f/f-Fos-D1-Cre mice compared to wild-type mice. Both groups of mice (n=4-7 each) were perfused and their brains sectioned at 40 μm thickness. Nissl staining was performed using brain sections covering (A) cortex, (B) nucleus accumbens and caudate putamen and (C) hippocampus from the two groups of mice. Scale bars indicate 100 μm.
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Supplemental Figure 3. c-Fos regulates cocaine-induced IEG expression. We isolated nuclear extracts from the NAc and CPu together from individual control (+/+) and f/f-Fos-D1-Cre (-/-) mouse (n=4-7 mice for each time point) after saline (0hr), and 2 hours (2hr, 30 mg/kg) or 7 days (7d, 20 mg/kg) after cocaine injections. (A) Western blotting for the indicated gene products using extracts from each mouse. (B) Quantification of gene expression levels with saline-treated wild-type as 100% controls. Filled columns and open columns represent data from wild-type and mutant mice respectively. Data show mean+SEM. *p < 0.05 between genotypes.
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Supplemental Figure 4. c-Fos regulates AP-1 transcription complex formation in vivo. We isolated nuclear extracts from the NAc and CPu together from individual wild-type (+/+) and f/f-Fos-D1-Cre mouse either before or after cocaine injections. We performed gel-shift and antibody supershift analyses using extracts from before (A, n=3 mice each), or after 2 hours (B, n=5 mice each, 30 mg/kg, acute) and 7 consecutive days (C, n=5 mice each, 20 mg/kg, repeated) of cocaine treatment. As measured by densitometry, there was a 1.4 fold reduction in the level of AP-1 transcription complexes in the f/f-Fos-D1-Cre mice compared to wild-type mice after the acute cocaine injections. Moreover, as measured by the ratio between the supershifts and regular shifts, there were relatively more FosB/ΔFosB participating in AP-1 transcription complexes in wild-type mice than in f/f-Fos-D1-Cre mice after both acute (0.8:1 versus 0.2:1) and repeated (4:1 versus 1:1) cocaine injections. Individual mouse gave parallel results. Competition using an increasing amount of un-labeled AP-1 sequences gradually reduced the AP-1 binding whereas AP-1 sequences with a point mutation within the binding site failed to do so, indicating the specificity of the protein/DNA interaction.
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