Fig. 1.

Cocaine dynamically regulates heterochromatin in NAc. (A) H3K9me3 levels are altered by cocaine administration. Mice were treated with saline or acute or repeated cocaine, and NAc samples were collected at 0.5, 1, 24, or 168 h (n = 5–8/group). Western blot analysis reveals a significant main effect (two-way ANOVA) of drug treatment (F_2,68 = 6.439, P = 0.003), time after cessation of drug treatment (F_3,68 = 11.21, P < 0.0001), and interaction between drug treatment and time (F_6,68 = 3.891, P = 0.002) on H3K9me3 levels. All samples were normalized to actin, and data are represented as fold change from saline controls (–). Bonferroni post hoc tests indicate significant differences from saline controls (*P < 0.05, **P < 0.01) at 0.5 and 1 h. A posteriori Student t tests indicate significant differences from saline controls (^#P < 0.05) at 24 and 168 h. The 0 and 24 h groups represent the same animals; 0 h is included to depict basal levels of H3K9me3 preceding cocaine administration for both the acute and repeated treatment groups. (B) Immunohistochemical analysis of mouse NAc qualitatively confirms increased H3K9me3 at 1 h and decreased H3K9me3 at 24 h after repeated cocaine administration. DAPI stains were used as a nuclear marker. All images were taken at 10× objective. (C) Altered heterochromatin domain numbers are consistent with altered levels of H3K9me3 after repeated cocaine at 1 and 24 h. Heterochromatin domains (puncta chromocenters) were quantified from DAPI-stained NAc sections at 1 h (n = 8/group) and 24 h (n = 10 to 11/group) after either saline or repeated cocaine. Approximately 112 H3K9me3/HP1⁺ neuronal nuclei were quantified from each animal at 40× objective. Statistical significance was determined using Student t tests (1 h-t₁₆ = 2.159, *P < 0.05; 24 h-t₂₁ = 3.730, **P < 0.01). Saline controls between 1 and 24 h were not significantly different (t₁₉ = 0.6596, P > 0.05). (D) Repeated cocaine administration promotes increased nuclear volumes in NAc neurons after repeated drug administration. Nuclear volumes were quantified from DAPI⁺ nuclei obtained from NAc sections at 1 h (n = 6/group) and 24 h (n = 7–9/group) after saline or repeated cocaine. Approximately 20 nuclei were examined from each animal at 40× objective. Statistical significance was determined using Student t tests (1 h-t₁₀ = 1.688, P > 0.05; 24 h-t₁₄ = 3.610, **P < 0.01). Saline controls between 1 h and 24 h were not significantly different (t₁₁ = 1.563, P > 0.05). All data are presented as average ± SEM.