Nuclear factor kappa B in the dorsal raphe of macaques: Anatomical link for steroids, cytokines and serotonin

Abstract

Nuclear factor kappa B (NFkappaB) is a transcription factor that activates gene expression in response to proinflammatory cytokines, and elevated cytokines are associated with depression, which has a serotonergic component. We questioned (1) whether serotonin neurons contain NFkappaB, (2) whether NFkappaB detection with immunocytochemistry is changed in the dorsal raphe nucleus (DRN) by ovarian hormone treatment and (3) whether ovarian hormones regulate midbrain NFkappaB gene or protein expression. Monkeys were spayed and treated with placebo, estrogen (E), progesterone (P) or E+P for 1 month (n = 4 animals/treatment group), and the midbrain was harvested for immunocytochemistry and stereology. An antibody that detects nuclear location-specific (NLS)-NFkappaB p65 was applied, and the numbers of NLS-NFkappaB-immunopositive cells were counted in 9 sections of the DRN. Additional monkeys were used for Western blot analysis and quantitative reverse transcription-polymerase chain reaction (RT-PCR) for NFkappaB p65. In placebo-treated macaques, neurons were double-immunostained for serotonin and nuclear NFkappaB p65 throughout the DRN. The mean total number of NFkappaB-positive cells equalled 2178 (and standard error of the mean [SEM] 129) in the placebo group, 1631 (SEM 221) in the E-treated group, 2314 (SEM 186) in the P-treated group and 1162 (SEM 100) in the E+P-treated group (analysis of variance p = 0.003). The E-treated and E+P-treated groups had a significantly lower density of cells stained positive for NFkappaB than the placebo or P-treated groups (post hoc). Unmasking of NLS-NFkappaB immunostaining in the DRN revealed dense immunostaining in the cytoplasm of large dorsal raphe neurons. There was no difference between treatment groups in the amount of NFkappaB p65 detected by Western blot or in the relative expression of NFkappaB p65 mRNA with quantitative RT-PCR. These observations are consistent with the notion that gene and protein expression of NFkappaB are constitutive but that ovarian hormones can decrease the nuclear location of NFkappaB in dorsal raphe neurons and, thereby, decrease the ability of NFkappaB to drive gene expression in response to cytokines.