Physiological Dissection of the SCN

描述(申请人提供)：哺乳动物视交叉上核(SCN)是调节生理和行为节律的“生物钟”的位置。虽然这一功能已经得到很好的确立，但组成核团的20,000个神经元是如何同步并因此产生连贯的输出信号的，目前尚不清楚。谜团始于这样一个事实，即昼夜节律性是一种细胞属性，但每个SCN细胞有不同的内源性周期和阶段。SCN神经元之间的异质性还表现在输入和输出、神经元形态、多肽和时钟基因表达上。我们的研究重点是了解SCN组织。我们发现，一些SCN神经元，包括小鼠SCN“核心”的GRP细胞，不是可检测到的节律(通过时钟基因表达和电活动来评估)，但对光输入有反应。其他SCN神经元，包括‘壳’SCN中含有加压素的细胞是有节律性的。由于仓鼠和小鼠SCN都有这些共同的特征，我们认为这是哺乳动物脑钟的一个普遍特征。我们现在建议在一种具有重要生物学意义的制剂中分析SCN。在小鼠中，睾丸激素在调节运动活动中起主要作用；性腺切除会导致自由奔跑时间延长，精确度丧失，日常活动开始回合完全丧失。我们将研究这种反应的细胞基础，以了解SCN的网络组织。初步研究表明，GRP细胞含有雄激素受体(AR)，具有电活动的特征模式，并具有可识别的形态特征和投射。我们计划研究性腺激素如何作用于含有AR的细胞，在行为、组织和细胞/分子水平上调节昼夜节律。我们假设，依赖于雄激素的GRP细胞进入PER1振荡器是正常小鼠的行为反应所必需的，性腺切除会导致起搏器输出的损失，从而导致行为的改变。为了确定睾丸激素的作用机制，我们将使用在我们的3个转基因小鼠群体中可用的强大分析工具，这些小鼠携带PER1基因(Per-GFP小鼠)、胃泌素释放肽(GRP-GFP小鼠)或雄激素受体(AR小鼠)的神经元和核标记。5)雄激素在调节行为昼夜节律中的作用；2)SCN内含AR细胞的表型；3)雄激素对SCN神经元可塑性的影响；4)时钟基因表达；5)含AR细胞和节律性高表达细胞的电生理反应。这项工作的广泛意义在于理解SCN如何利用雄激素介导的变化作为分析工具来调节节律性。这项工作还有助于更好地理解与青春期、雄停顿、睡眠调节和性别差异的某些方面相关的昼夜节律变化。

DESCRIPTION (provided by applicant): The mammalian suprachiasmatic nucleus (SCN) is the locus of a "circadian clock" that regulates rhythms in both physiology and behavior. While this function is well established, it is not known how the 20,000 neurons that comprise the nucleus are synchronized and consequently generate a coherent output signal. The mystery starts with the fact that circadian rhythmicity is a cellular property, but that individual SCN cells have different endogenous periods and phases. Heterogeneity among SCN neurons is also seen in inputs and outputs, neuronal morphology, peptides and "clock" gene expression. Our research focuses on understanding SCN organization. We have found that some SCN neurons, including GRP cells of the 'core' in mouse SCN are not detectably rhythmic (assessed by clock gene expression and electrical activity), but respond to photic input. Other SCN neurons, including vasopressin-containing cells of the 'shell' SCN are rhythmic. Because both hamster and mouse SCN share these characteristics, we propose that it is a general feature of mammalian brain clocks. We now propose to analyze the SCN in a biologically important preparation. In mouse, testicular hormones play a major role in regulating locomotor activity; gonadectomy produces a lengthening of free-running period, a loss of precision and complete loss of the daily activity onset bout. We will study the cellular basis of this response to understand the network organization of the SCN. Preliminary studies indicate that GRP cells contain androgen receptors (AR), have a characteristic pattern of electrical activity, and identifiable morphological features and projections. We plan to examine how gonadal hormones act on AR-containing cells to regulate circadian rhythmicity at the behavioral, tissue and cellular/molecular levels. We hypothesize that tonic androgen-dependent input of GRP cells onto Per1 bearing oscillators is necessary for the behavioral responses seen in the intact mouse, and that gonadectomy produces a loss of pacemaker output, with resulting changes in behavior. To determine the mechanisms whereby testicular hormones act, we will use the powerful analytic tools available in our 3 colonies of transgenic mice bearing a reporter for either the Per1 gene (Per-GFP mice), the gastrin releasing peptide (GRP-GFP mice), or a neuronal and nuclear label for androgen receptor (AR mice). 5 specific aims will be examined 1) the role of androgens in the regulation of behavioral circadian rhythms; 2) the phenotype of AR-containing cells in the SCN; 3) androgen effects on SCN neuronal plasticity, 4) clock gene expression and 5) electrophysiological responses of AR-containing and rhythmic Per-expressing cells. The broad significance of this work lies in understanding how the SCN regulates rhythmicity using androgen-mediated changes as an analytic tool. The work also contributes to a better understanding of circadian changes associated with puberty, andropause, sleep regulation and some aspects of sex differences.