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Effect of Ovarian Hormone on Gene Expression in Rhesus Dorsal Raphe Nucleus with Rhesus Affymetrix Chip

ABSTRACT: A small block of the midbrain containing the dorsal raphe nucleus was obtained from ovariectomized monkeys treated with placebo, estrogen, progesterone or estrogen plus progesterone for one month. The RNA was extracted and hybridized to the Rhesus Affymetrix chip. The results were analyzed with GCOS 1.0 Each chip represents one monkey. N=3 monkeys per treatment.

ORGANISM(S): Macaca mulatta

SUBMITTER: Arubala P. Reddy Arubala P Reddy Cynthia L Bethea

PROVIDER: E-GEOD-16172 | ArrayExpress | 2010-04-09

SECONDARY ACCESSION(S): GSE16172PRJNA115057

REPOSITORIES: GEO, ArrayExpress

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Project description:CART (cocaine and amphetamine regulated transcript) is a neuropeptide involved in the control of several physiological processes, such as response to psychostimulants, food intake, depressive diseases and neuroprotection. It is robustly expressed in the brain, mainly in regions that control emotional and stress responses and it is regulated by estrogen in the hypothalamus. There is a distinct population of CART neurons located in the vicinity of the Edinger-Westphal nucleus of the midbrain that also colocalize urocortin-1. The aims of this study were 1) to determine the distribution of CART immunoreactive neurons in the monkey midbrain, 2) to examine the effects of estrogen (E) and progesterone (P) on midbrain CART mRNA and peptide expression and 3) to determine whether midbrain CART neurons contain steroid receptors. Adult female rhesus monkeys (Macaca mulatta) were spayed and either treated with placebo (OVX), estrogen alone (E), progesterone alone (P) or E+P. Animals were prepared (a) for RNA extraction followed by microarray analysis and quantitative (q) RT-PCR (n=3/group); (b) for immunohistochemical analysis of CART and CART+tryptophan hydroxylase (TPH), CART+estrogen receptors (ER) or CART+progesterone receptors (n=5/group) and (c) for Western blots (n=3/group). Both E- and E+P-administration decreased CART gene expression on the microarray and with qRT-PCR. Stereological analysis of CART immunostaining at five levels of the Edinger-Westphal nucleus indicated little effect of E or E+P administration on the area of CART immunostaining. However, P administration increased CART-immunopositive area in comparison to the OVX control group with Student's t-test, but not with ANOVA. CART 55-102 detection on Western blot was unchanged by hormone administration. ERbeta and PR were detected in CART neurons and CART fibers appeared to innervate TPH-positive serotonin neurons in the dorsal raphe. In summary, E decreased CART mRNA, but this effect did not translate to the protein level. Moreover, P administration alone had a variable effect on CART mRNA, but it caused an increase in CART immunostaining. Together, the data suggest that CART neurons in the midbrain have a unique steroid response, which may be mediated by nuclear receptors, neuroactive steroids or interneurons.

Project description: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.

			Action	DRS
	E-GEOD-16172.README.txt	Txt
	E-GEOD-16172.eSet.r	Other
	E-GEOD-16172.idf.txt	Idf
	E-GEOD-16172.processed.1.zip	Processed
	E-GEOD-16172.raw.1.zip	Raw

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Effect of Ovarian Hormone on Gene Expression in Rhesus Dorsal Raphe Nucleus with Rhesus Affymetrix Chip

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