Thrivikraman-5P50MH058922-050002

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. Keywords: other

有研究表明，应激诱导的下丘脑-垂体-肾上腺（hypothalamic-pituitary-adrenal, HPA）轴激活，以及伴随的血浆促肾上腺皮质激素（adrenocorticotropic hormone, ACTH）和糖皮质激素（glucocorticoids，大鼠中为皮质酮，简称B）水平升高，是多种精神病理发生的重要诱因。然而，HPA轴活性的昼夜节律波动（其特征为在活跃觉醒期开始前血浆ACTH与B水平达到峰值，而在非活动睡眠期处于谷值）贯穿生物体整个生命周期，对正常生理与行为功能至关重要。这种节律的减弱或增强（通过改变低谷或峰值水平实现）是诸多病理状态的典型特征。
在中枢神经系统（central nervous system, CNS）层面，HPA轴活性的昼夜节律波动与结构基因、功能基因及即刻早期基因（immediate early genes）的表达谱变化密切相关。但目前对于B在中枢神经系统基因调控昼夜模式中的具体调控作用尚不清楚。因此，在昼夜周期中，B是影响大量基因家族表达谱的关键因素。
鉴于基因芯片（microarray）技术可同时实现众多基因的表达谱鉴定，是本研究的理想实验手段。由于动物体内的B水平可通过肾上腺切除术（adrenalectomy, ADX）联合或不联合B替代疗法进行精准操控，本研究将采用该动物模型。
本研究拟通过基因芯片技术，探究B环境对大鼠特定脑区昼夜基因表达谱的影响，所选脑区分别为：a.) 受B水平显著调控的脑区：杏仁核（amygdala）、海马体（hippocampus）、下丘脑（hypothalamus）与隔区（septum）；b.) 受昼夜活动显著调控的脑区：中缝背核（dorsal raphe）、额叶皮层（frontal cortex）与蓝斑（locus coeruleus）。
昼夜周期中B的应答梯度表明，睡眠周期开始时盐皮质激素受体（mineralocorticoid receptor, MR）占主导结合状态，而睡眠周期结束时，盐皮质激素受体与糖皮质激素受体（glucocorticoid receptor, GR）均处于结合状态。据此我们推测，这两类核受体的不同激活比例可差异化调控基因表达，并赋予大量基因家族表达谱以时间特异性特征。此外，我们认为这些效应具有脑区特异性，这与GR与MR的脑区分布差异、B摄取能力及神经活动的区域差异相一致。B可通过直接或间接经由中间神经元（interneurons）的方式，对不同脑区的基因表达谱产生昼夜调控效应。
但目前关于B对不同脑区基因表达谱的昼夜调控作用仍知之甚少。阐明该机制对于明确B在中枢神经系统功能昼夜差异中的具体作用，以及潜在的精神病理发生过程，均具有重要意义。
实验动物为雄性Long Evans大鼠，抵达设施后将以12:12小时明暗周期（光照始于0700时）饲养，自由采食饮水。饲养1周后，采用存活麻醉剂进行双侧肾上腺切除术（术后第0天）。本研究设置3组（每组n=10）：(a) ADX组：无肾上腺的大鼠；(b) pADX组：接受皮下植入40% B颗粒（胆固醇与B的融合制剂）的ADX大鼠；(c) 假手术（SHAM）组：仅进行腰部切口但未摘除肾上腺的大鼠。术后大鼠将单笼饲养，ADX组大鼠除正常饮水外，还将提供0.5%生理盐水。
术后第7天，将在光照开始后2小时处死15只大鼠（每组5只），剩余15只将在光照结束后2小时处死。立即取出脑组织，置于干冰上速冻后保存于-80℃。采集躯干血液并加入乙二胺四乙酸（EDTA）抗凝，分离血浆后冻存，用于ACTH与B的放射免疫测定（radioimmunoassay, RIA）。仅对确认成功肾上腺切除的大鼠脑组织进行目标脑区徒手解剖：额叶皮层、隔区、下丘脑、杏仁核、海马体、中缝背核与蓝斑。解剖后的组织将保存于-80℃。
采用Qiagen RNeasy脂质组织提取试剂盒从单个脑区提取总RNA。首先通过吸光度法对RNA的质量与浓度进行初步评估。将总RNA含量≥0.5 μl的冻存样本通过干冰运输至美国国家神经疾病与卒中研究所-国家精神卫生研究所基因芯片联盟（NINDS-NIMH Microarray Consortium），采用安捷伦生物分析仪（Agilent Bioanalyzer）检测28S与18S RNA的完整性。若RNA完整性不佳，则重复实验并重新制备RNA样本。选取28S/18S比值≥1.7的每个脑区重复样本，采用Affymetrix大鼠U34A基因芯片（Affymetrix Rat U34A array）进行检测。同一脑区的所有大鼠RNA样本将统一处理。本研究预计使用84张芯片，但最终芯片数量将取决于总RNA的完整性。关键词：其他