Physiological Dissection of the SCN

描述（由申请人提供）：哺乳动物视交叉上核（SCN）是调节生理和行为节律的“昼夜节律钟”所在地。虽然这种功能已经很好地建立起来，但目前还不知道组成细胞核的20，000个神经元是如何同步的，从而产生相干的输出信号。这个谜团始于这样一个事实，即昼夜节律是一种细胞特性，但单个SCN细胞具有不同的内源性周期和阶段。SCN神经元之间的异质性也见于输入和输出、神经元形态、肽和“时钟”基因表达。我们的研究重点是了解SCN组织。我们已经发现，一些SCN神经元，包括GRP细胞的“核心”在小鼠SCN是不可检测的节奏（评估时钟基因表达和电活动），但响应于光输入。其他SCN神经元，包括“壳”SCN中含有加压素的细胞是有节律的。由于仓鼠和小鼠SCN都具有这些特征，我们认为这是哺乳动物脑时钟的一般特征。我们现在建议分析SCN在生物学上重要的准备。在小鼠中，睾丸激素在调节运动活动中起主要作用;性腺切除导致自由奔跑时间延长，精确性丧失和完全丧失日常活动开始时间。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），具有特征性的电活动模式，以及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，紧张性雄激素依赖性输入的GRP细胞到Per 1轴承振荡器是必要的行为反应中看到的完整的小鼠，性腺切除术产生的起搏器输出的损失，从而导致行为的变化。为了确定睾丸激素作用的机制，我们将使用我们的3个转基因小鼠克隆中可用的强大分析工具，这些转基因小鼠携带Per 1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）的神经元和核标记的报告基因。将检查5个具体目标：1）雄激素在行为昼夜节律调节中的作用; 2）SCN中含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和节律性过表达细胞的电生理反应。这项工作的广泛意义在于理解SCN如何使用雄激素介导的变化作为分析工具来调节节律性。这项工作也有助于更好地了解与青春期，男性更年期，睡眠调节和性别差异的某些方面有关的昼夜节律变化。

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.