Induction of deltaFosB in reward-related brain structures after chronic stress

Abstract

Acute and chronic stress differentially regulate immediate-early gene (IEG) expression in the brain. Although acute stress induces c-Fos and FosB, repeated exposure to stress desensitizes the c-Fos response, but FosB-like immunoreactivity remains high. Several other treatments differentially regulate IEG expression in a similar manner after acute versus chronic exposure. The form of FosB that persists after these chronic treatments has been identified as DeltaFosB, a splice variant of the fosB gene. This study was designed to determine whether the FosB form induced after chronic stress is also DeltaFosB and to map the brain regions and identify the cell populations that exhibit this effect. Western blotting, using an antibody that recognizes all Fos family members, revealed that acute restraint stress caused robust induction of c-Fos and full-length FosB, as well as a small induction of DeltaFosB, in the frontal cortex (fCTX) and nucleus accumbens (NAc). The induction of c-Fos (and to some extent full-length FosB) was desensitized after 10 d of restraint stress, at which point levels of DeltaFosB were high. A similar pattern was observed after chronic unpredictable stress. By use of immunohistochemistry, we found that chronic restraint stress induced DeltaFosB expression predominantly in the fCTX, NAc, and basolateral amygdala, with lower levels of induction seen elsewhere. These findings establish that chronic stress induces DeltaFosB in several discrete regions of the brain. Such induction could contribute to the long-term effects of stress on the brain.

Figure 2.

Induction of ΔFosB in brain after 10 d of chronic restraint stress. Levels of FosB-like immunoreactivity were analyzed by immunohistochemistry using an anti-FosB(N-terminus) antibody in control (A, C, E, G, I) and chronic stressed (B, D, F, H, J) animals. Note that chronic restraint stress increased FosB-like immunoreactivity in the medial prefrontal cortex (mPFC; A, B), NAc core (C, D), and basolateral amygdala (BLA; E, F). In contrast, chronic restraint stress did not induce FosB-like immunoreactivity in the bed nucleus of stria terminalis (BNST; G, H) or paraventricular nucleus of the hypothalamus (PVH; I, J), areas in which c-Fos and full-length FosB are induced after acute stress (see Fig. 5).

Figure 1.

Induction of Fos family proteins in brain after acute and chronic stress. A, Representative Western blot, using a pan-Fos antibody, showing induction of Fos family proteins in the frontal cortex under four conditions: control (Ctl), non-handled controls; acute (Ac), single restraint stress with animals used 2 hr later; chronic (Chr), daily restraint stress for 10 d with animals used 24 hr after the last stress; chronic plus acute (Chr+Ac), daily restraint stress for 11 d with animals used 2 hr after the last stress. B, Levels of Fos family protein induction in the frontal cortex revealed by Western blotting were quantified and expressed as the mean percentage of change from control ± SEM (n = 4-10 animals in each group). The M_r values labeled on the x-axis correspond to the bands shown in A. ^*p < 0.05 by ANOVA and by t test compared with control. C, Similar analysis of ΔFosB (35-37 kDa) induction by acute and chronic stress in the NAc. D, Time course of ΔFosB (35-35 kDa) expression in the NAc, dorsal striatum [caudate-putamen (CPu)], frontal cortex, and lateral septum 24 hr after 1, 5, or 10 d of restraint stress. E, Levels of 35-37 kDa ΔFosB isoforms in the NAc and frontal cortex after 1 or 7 d of withdrawal from chronic (10 d) restraint stress. F, Levels of 35-37 kDa ΔFosB isoforms in the NAc, CPu, frontal cortex, and lateral septum after 10 d of chronic unpredictable stress.

Figure 3.

Specificity of anti-FosB(N-terminus) and anti-FosB(C-terminus) antibodies. A, Extracts of PC12 cells overexpressing FosB or ΔFosB, by use of HSV vectors, were subjected to Western blotting using an anti-FosB(N-terminus) or anti-FosB(C-terminus) antibody raised against the indicated portions of the proteins. B, Double-labeling immunohistochemistry using the anti-FosB(N-terminus) or anti-FosB(C-terminus) antibody through the NAc of animals injected with HSV-FosB and HSV-ΔFosB in this brain region. In A and B, the N-terminus antibody stains both ΔFosB and FosB, whereas the C-terminus antibody stains FosB only.

Figure 4.

Quantification of ΔFosB induction in brain after 10 d of chronic restraint stress. The bar graphs show the mean density of FosB+ cells in control animals and in animals subjected to chronic restraint stress in the NAc core (A), NAc shell (B), medial prefrontal cortex (C), basolateral amygdala (D), basomedial amygdala (E), and central medial amygdala (F). Data are expressed as mean ± SEM (n = 3-4 animals in each group). ^*p < 0.05 by t test.

Figure 5.

Induction of c-Fos in brain after acute restraint stress. Levels of c-Fos immunoreactivity were analyzed by immunohistochemistry using a specific anti-c-Fos antibody in control animals (A, C, E) and in animals subjected to acute restraint stress 2 hr earlier (B, D, F). Note that acute stress increased c-Fos expression in the NAc core (A, B), paraventricular nucleus of the hypothalamus (PVH; C, D), and bed nucleus of stria terminalis (BNST; E, F).

Figure 6.

Cellular specificity of ΔFosB induction by chronic restraint stress in the NAc. Representative photomicrographs demonstrating colocalization of ΔFosB with dynorphin mRNA (A) or enkephalin mRNA (B) are shown. The arrows mark examples of double-labeled cells; arrowheads indicate ΔFosB⁺, peptide^- cells. Also shown is the lack of colocalization of ΔFosB with GFAP (a marker of astrocytes) (C), with parvalbumin (Parv), calretinin, or calbindin (markers of GABAergic interneurons) (D, E), or with choline acetyltransferase (ChAT; a marker of cholinergic interneurons) (C). The results shown are representative of the analysis of multiple sections from at least three chronically stressed animals.

Figure 7.

Cellular specificity of ΔFosB induction by chronic restraint stress in the medial prefrontal cortex. A representative photomicrograph demonstrating colocalization of ΔFosB with vGluT1, a marker of glutamatergic pyramidal neurons (A), is shown. The arrows mark examples of double-labeled cells. Virtually no colocalization was observed with calbindin or parvalbumin (Parv), markers of GABAergic interneurons (B, C). The results shown are representative of the analysis of multiple sections from at least three chronically stressed animals.