Project description:Although glucocorticoids (GCs) are known to exert numerous effects in the hippocampus, their chronic regulatory functions remain poorly understood. Moreover, evidence is inconsistent regarding the longstanding hypothesis that chronic GC exposure promotes brain aging/Alzheimer's disease. Here, we adrenalectomized male F344 rats at 15-months-of-age, maintained them for 3 months with implanted corticosterone (CORT) pellets producing low or intermediate (glucocorticoid-receptor (GR)-activating) blood levels of CORT, and performed microarray/pathway analyses in hippocampal CA1. We defined the chronic GC-dependent transcriptome as 393 genes that exhibited differential expression between Intermediate- and Low-CORT groups. Short-term CORT (4 days) did not recapitulate this transcriptome. Functional processes/pathways overrepresented by chronic CORT-upregulated genes included learning/plasticity, differentiation, glucose metabolism and cholesterol biosynthesis, whereas processes overrepresented by CORT-downregulated genes included inflammatory/immune/glial responses and extracellular structure. These profiles indicate that GCs chronically activate neuronal/metabolic processes while coordinately repressing a glial axis of reactivity/inflammation. We then compared the GC-transcriptome with a previously-defined hippocampal aging transcriptome, revealing a high proportion of common genes. Although CORT and aging moved expression of some common genes in the same-direction, the majority were shifted in opposite directions by CORT and aging (e.g., glial inflammatory genes downregulated by CORT are upregulated with aging). These results contradict the hypothesis that GCs simply promote brain aging, and also suggest that the opposite-direction shifts during aging reflect resistance to CORT regulation. Therefore, we propose a new model in which aging-related GC resistance develops in some target pathways while GC overstimulation develops in others, together generating much of the brain aging phenotype. Forty male Fischer 344 rats aged 14-15 months (late mid-age) were either sham operated (n=10) or adrenalectomized (n=30) at Harlan, Inc. (Indianapolis) and shipped to University of Kentucky 7-10 d after surgery. Animals were allowed to acclimate for 2 weeks after receipt prior to pellet-implant and were provided with food and water ad lib. Adrenalectomized (ADX) animals were maintained on drinking water containing isotonic saline with 1% sucrose, and also given supplemental feed mash (standard chow mixed with water and warmed) and apple chunks, as well as subcutaneous injections of 1.25 mg corticosterone sulfate (Solu-DeltaCort, Pfizer). During pellet-implant surgery, subjects were anesthetized with isoflurane, placed on a warming pad, and a 3 cm2 area on the animal's flank was shaved and sterilized. A 1-cm incision was made through the skin to form a subcutaneous pocket. Each sham animal received an inert pellet. ADX animals were divided into two groups, low-dose animals (n = 20) received one 25 mg CORT pellet and intermediate-dose animals (n = 10) received one 200 mg CORT pellet (slow release pellets, Innovative Research of America, Sarasota, FL). Incisions were closed with wound clips (removed after 7-10 days). Following implant surgery, subjects were housed singly for 3 months prior to euthanasia at age 18 months. Low-dose animals continued to receive saline/ sucrose drinking water, but other supplementation (food mash, Solu-Delta CORT, apple chunks) was discontinued. One ADX animal was removed from the study for declining health. During the twelfth week, just prior to study termination, half of the ADX low-dose CORT subjects were given a short-term, high-dose regimen consisting of 4 daily IP injections of 5mg/d CORT (considered a relatively high dose- Makino et al., 1995). All animals were killed between 8 and 10 AM on the last two days of the study. After deep CO2 anesthesia and decapitation, trunk blood was collected for CORT radioimmunoassay and hippocampal CA1 brain tissue was dissected as described previously (Blalock et al., 2003) for microarray analysis (see below). Final treatment groups consisted of: 1) Sham (intact) control, 2) Low-CORT (ADX), 3) Intermediate-CORT (ADX), and 4) Low-CORT (ADX) + short-term high-dose injected (n=9-10/group).

Project description:Purpose: we sought to search for genes that are transcribed or repressed in a glucocorticoid (GC)-dependent manner in a hypothalamic region containing the paraventricular nucleus of the hypothalamus (PVH), thereby unraveling mechanisms underlying GC-induced neuroendocrine and autonomic regulation. Methods: we employed unbiased quantification of the abundance of DNA transcripts provided by RNA-seq in the PVH region of male rats and identified genes whose expression is enhanced or suppressed by the 12-day exposure to a high dose of corticosterone (Cort) by Cort implant (12-day high Cort) or deprivation of corticoids for 7 days [7-day adrenalectomy (ADX)] (Experiment 1), as well as by acute Cort increase (Experiment 2). Sham-ADX animals served as controls in Experiment 1. In Experiment 2, rats underwent ADX and supplemented with Cort in drinking water. The animals were decapitated at 1 h [acute Cort (1) 1 h] or 3 h [acute Cort (1) 3 h] following injection of HBC Cort (1 mg), a dose shown to elicit plasma Cort surge comparable to that during acute stress. A group of rats without Cort injection served as controls (ADX + Cort suppl). Differential expression was calculated as fold-changes in gene expression [measured as fragments per kilobase of exon per million reads mapped (FPKM)]. The Benjamini & Hochberg method was applied to correct the unavoidable false positive data ensued from the multiple tests, and a q-value was calculated as the false discovery rate-adjusted p-value. The q-value < 0.05 was used as the cut-off criterion. Pearson’s correlation analysis was carried out for grouping genes that showed similar expression patterns. qRT–PCR validation was performed using Power SYBR Green PCR mix. Semi-quantitative in situ hybridization was carried out using 35S-labeled cRNA probes. Results: some canonical GC target genes, among others of a broad functional spectrum, were upregulated prominently by 12-day high Cort. Crh was upregulated or downregulated most prominently by either 7-day ADX or 12-day high Cort, emphasizing the recognized feedback effects of GC on the hypothalamic-pituitary-adrenal (HPA) axis. Concomitant changes in Avp and Aplnr expression are likely to contribute to HPA repression. In keeping with the pleotropic cellular actions of GCs, 7-day ADX downregulated numerous genes of a broad functional spectrum. The transcriptome response signature differed markedly between acute Cort injection and 12-day high Cort. Remarkably, 6 immediate early genes, Bsx, Egr1, Fos, Fosb, Junb, and Nr4a3, were upregulated at 1 h after Cort injection, and the changes were confirmed by qRTPCR and semi-quantitative in situ hybridization. Conclusions: the present study uncovered a broad spectrum of Cort-responsive genes, potentially involved in the regulation of the HPA-axis, as well as other neuroendocrine and pre-autonomic functions of the PVH.

Project description:It is suggested that the stress induced activation of the HPA axis and associated increases in plasma ACTH and glucocorticoids (corticosterone, B in the rat) are contributing factors in the development of many psychopathologies. However, the circadian fluctuation of the HPA axis activity (characterized by a zenith in the plasma ACTH and B, before the beginning of the active awake state and the nadir during the inactive sleeping period) occurs throughout the life and is important for normal physiological and behavioral functioning. A blunting or enhancement of this rhythm through changes in the trough or in the peak is a characteristic feature of many pathological states. At the CNS level, the circadian fluctuation of the HPA axis activity is associated with changes in the expression profile of structural, functional, and immediate early genes. However, little is known about the specific role of B in the modulation of the circadian pattern of gene regulation in the CNS. Thus, during the circadian cycle B is a major factor, which could influence the expression profile of a large family of genes. Accordingly, the microarray technology, because of its characteristic feature that facilitates the identification of the expression profiles of a wide range of genes simultaneously, is an ideal technique. Because the B status of animals can be easily manipulated by adrenalectomy (ADX) surgery with or without B replacement, this model will be used in these studies. Thus, the proposed studies are intended to characterize, by the use of the microarray technique, the effects of the B milieu on diurnal gene expression profiles in selected rat brain regions strongly influenced by: a.) B status: amygdala, hippocampus, hypothalamus, and septum; and b.) Circadian Activity: dorsal raphe, frontal Cortex and locus coeruleus. The gradient of the B response during the circadian cycle implies that at the beginning of the sleep cycle MR are predominantly occupied while at the end of the sleep cycle, both MR and GR are occupied. Thus, we propose that differing ratios of activation of these nuclear receptors could differentially modulate gene expression and impart temporal characteristics in the expression profile of a large family of genes. Furthermore, we propose that these effects may be region specific, reflecting regional differences in GR and MR distribution, B uptake, and neural activity. B may exert these circadian effects on regional gene expression profiles either directly or indirectly via interneurons. However, little is known about the circadian effect of B on gene expression profiles in different brain regions. Such knowledge is important to identify the specific role of B in circadian differences in CNS function and, potentially, the development of psychopathologies. After arrival at the facility the rats (male Long Evans) will be maintained on 12:12 h LD cycle ('lights-on" at 0700 h) with food and water ad libitum. One week after, they will undergo bilateral ADX under survival anesthetics (day-0). The study will include three groups (n=10); (a) ADX: rats with no adrenals, (b) pADX: ADX rats with subcutaneous 40 percent B pellet (fused cholesterol and B), and (c) SHAM: rats undergoing flank incisions without the removal of the adrenals. After surgery, rats will be housed individually. The ADX rats will receive 0.5 percent saline in addition to regular tap water. On post-surgical day-7, 15 rats (five/group) will be sacrificed by decapitation 2h after 'lights-on", and the remaining 15, 2h after "lights-off". The brains will be removed immediately, frozen on dry ice, and stored at -80 C. The trunk blood will be collected with EDTA and plasma frozen for ACTH and B RIA. The brains from rats with confirmed ADX will be used for free hand dissection of brain regions: frontal cortex, septum, hypothalamus, amygdala, hippocampus, dorsal raphe, and locus coeruleus. Tissue will be stored at -80 C. Total RNA will be extracted from individual brain regions using Qiagen RNeasy Lipid Tissue kit. Initial evaluation of quality and quantity of the RNA will be done by the absorbance method. Frozen samples containing 0.5 ul total RNA or higher will be sent to the NINDS-NIMH Microarray Consortium in dry ice for evaluation of the integrity of 28S and 18S RNA on an Agilent Bioanalyzer. If the integrity of RNA is not good, the experiment will be repeated and new RNA samples will be provided. Replicates of each brain region with 28S/18S ratio of 1.7 and higher will be selected for assay using Affymetrix Rat U34A array. The RNA samples from one region from all the brains will be processed together. A total of 84 chips may be utilized in these studies, however the final number will depend upon integrity of the total RNA.

Project description:Although glucocorticoids (GCs) are known to exert numerous effects in the hippocampus, their chronic regulatory functions remain poorly understood. Moreover, evidence is inconsistent regarding the longstanding hypothesis that chronic GC exposure promotes brain aging/Alzheimer's disease. Here, we adrenalectomized male F344 rats at 15-months-of-age, maintained them for 3 months with implanted corticosterone (CORT) pellets producing low or intermediate (glucocorticoid-receptor (GR)-activating) blood levels of CORT, and performed microarray/pathway analyses in hippocampal CA1. We defined the chronic GC-dependent transcriptome as 393 genes that exhibited differential expression between Intermediate- and Low-CORT groups. Short-term CORT (4 days) did not recapitulate this transcriptome. Functional processes/pathways overrepresented by chronic CORT-upregulated genes included learning/plasticity, differentiation, glucose metabolism and cholesterol biosynthesis, whereas processes overrepresented by CORT-downregulated genes included inflammatory/immune/glial responses and extracellular structure. These profiles indicate that GCs chronically activate neuronal/metabolic processes while coordinately repressing a glial axis of reactivity/inflammation. We then compared the GC-transcriptome with a previously-defined hippocampal aging transcriptome, revealing a high proportion of common genes. Although CORT and aging moved expression of some common genes in the same-direction, the majority were shifted in opposite directions by CORT and aging (e.g., glial inflammatory genes downregulated by CORT are upregulated with aging). These results contradict the hypothesis that GCs simply promote brain aging, and also suggest that the opposite-direction shifts during aging reflect resistance to CORT regulation. Therefore, we propose a new model in which aging-related GC resistance develops in some target pathways while GC overstimulation develops in others, together generating much of the brain aging phenotype.

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Corticosteroid responsive genes

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