Nuclear factor kappa B signaling regulates neuronal morphology and cocaine reward

Abstract

Although chronic cocaine-induced changes in dendritic spines on nucleus accumbens (NAc) neurons have been correlated with behavioral sensitization, the molecular pathways governing these structural changes, and their resulting behavioral effects, are poorly understood. The transcription factor, nuclear factor kappa B (NFkappaB), is rapidly activated by diverse stimuli and regulates expression of many genes known to maintain cell structure. Therefore, we evaluated the role of NFkappaB in regulating cocaine-induced dendritic spine changes on medium spiny neurons of the NAc and the rewarding effects of cocaine. We show that chronic cocaine induces NFkappaB-dependent transcription in the NAc of NFkappaB-Lac transgenic mice. This induction of NFkappaB activity is accompanied by increased expression of several NFkappaB genes, the promoters of which show chromatin modifications after chronic cocaine exposure consistent with their transcriptional activation. To study the functional significance of this induction, we used viral-mediated gene transfer to express either a constitutively active or dominant-negative mutant of Inhibitor of kappa B kinase (IKKca or IKKdn), which normally activates NFkappaB signaling, in the NAc. We found that activation of NFkappaB by IKKca increases the number of dendritic spines on NAc neurons, whereas inhibition of NFkappaB by IKKdn decreases basal dendritic spine number and blocks the increase in dendritic spines after chronic cocaine. Moreover, inhibition of NFkappaB blocks the rewarding effects of cocaine and the ability of previous cocaine exposure to increase an animal's preference for cocaine. Together, these studies establish a direct role for NFkappaB pathways in the NAc to regulate structural and behavioral plasticity to cocaine.

Figure 1.

Cocaine regulates acetylation of histone H3 (acH3) and trimethylation of Lys9 of H3 (3mK9H3) on NFκB gene promoters (tissue was collected 4–6 h after cocaine administration). A, Increased binding of acH3 at the p105/p50, p65/Rel-A, and IκBβ promoters (*p < 0.05, Student's t test, n = 6–8), and no significant changes on the IκBα or c-rel promoters. B, A general decrease in 3meK9H3 binding, a repressive mark, on all NFκB promoters examined (*p < 0.05, Student's t test, n = 6–8). In contrast, C shows no changes in 3mK4H3 binding at any of the NFκB promoters. All data are expressed as mean fold change ± SEM and are normalized to total input DNA.

Figure 2.

Cocaine regulates NFκB-dependent gene transcription in striatum. A, The NFκB-LacZ reporter construct as well as a 20× image of NFκB activity in the NAc shell and core, and 4× photomicrographs of the relative distribution of activity throughout the anterior to posterior gradient, as marked by β-gal immunohistochemistry (B). NFκB-dependent transcription was measured in the NAc shell 4–6 and 24 h after saline, acute, or chronic cocaine. Representative photomicrographs of β-gal expression after saline or chronic cocaine are shown. The bar graph shows that NFκB-dependent β-gal expression is increased in the NAc shell only by acute or chronic cocaine (*p < 0.05, ANOVA, n = 3–5).

Figure 3.

Chronic cocaine increases protein levels of certain NFκB subunits in the NAc. The figure shows via Western blotting that 4–6 h after chronic cocaine there are increased levels of the NFκB subunits p65/Rel-A (*p < 0.05, Student's t test, n = 7–8) and p105 (*p < 0.05, Student's t test, n = 7–8), with a strong trend for IκBβ as well (^τp = 0.08, Student's t test, n = 7–8), but not the inhibitory IκBα subunit. All data are expressed as the mean optical density percentage control ± SEM.

Figure 4.

Molecular cross-talk between BDNF and NFκB signaling in the NAc. Expression of HSV-IKKca increases BDNF mRNA (*p < 0.05, ANOVA, n = 5–6) and HSV-IKKdn expression produced a trend for decreased BDNF mRNA in the NAc (^τp = 0.08). Neither manipulation altered GDNF mRNA levels in the NAc. Data are expressed as the mean fold change of control ± SEM.

Figure 5.

NFκB signaling is necessary for basal and cocaine-induced changes in the number of dendritic spines on NAc MSNs, as well as growth factor-induced neurite outgrowth in PC12 cells. A–C, Representative confocal scans from MSNs in the adult NAc; as shown in C, the total number of dendritic spines on MSNs (mean ± SEM spines per 10 μm) is reduced by IKKdn and increased by IKKca (*p < 0.05, ANOVA, n = 4–8). IKKdn also blocks the ability of chronic cocaine to increase dendritic spines. D and E show that manipulation of NFκB signaling by IKK mutants similarly regulates the differentiation and outgrowth of PC12 cells by NGF (*p < 0.05, ANOVA, n = 8). Images in D show representative qualitative differences in neurite branching. Data in E are expressed as the mean number of cells with neurite outgrowth ± SEM.

Figure 6.

Regulation of cocaine CPP by NFκB signaling. A, Cocaine CPP (time in cocaine-paired chamber minus time in saline-paired chamber) was measured after intra-NAc injections of HSV-GFP, -IKKdn, or -IKKca in male C57BL/6 mice. Animals infected with HSV-IKKdn had decreased preference for 5 and 10 mg/kg cocaine (*p < 0.05, ANOVA, n = 8–16). B, Enhanced levels of CPP after a prior course of cocaine exposure, as seen in the GFP and IKKca groups (p < 0.05, Student's t test, n = 8–16). In contrast, expression of IKKdn, during chronic cocaine pretreatment, prevents this enhancement of cocaine CPP. Below each graph, in A and B, is the timeline for CPP and CPP sensitization paradigms. For baseline CPP, mice were injected with intra-NAc infusions of HSV-IKKca, -IKKdn, or -GFP and conditioned for cocaine CPP starting 3 d after HSV infusion (A). For CPP sensitization, mice were pretreated with cocaine (20 mg/kg, i.p. for 4 d) at the onset of HSV expression, and after 3 additional days without treatment, animals received CPP training with 5 mg/kg cocaine (B).