Peptide Modulation of Physiology and Behavior

描述(申请人提供)：我们的长期目标是在活体内对神经回路中的神经肽信号进行机械解剖。我们使用了多种遗传试剂在体内对三种B1类神经肽进行细胞特异性操作，这些神经肽的G蛋白偶联受体信号通过苍蝇昼夜/睡眠控制电路中的cAMP：PDF(色素分散因子)、DH31(苍蝇降钙素同源物)和DH44(苍蝇促肾上腺皮质激素释放因子同源物)。已经知道PDF在生物钟神经元的一个子集中表达，并且对调节苍蝇的日常节律和睡眠很重要。我们的初步研究表明，DH31和DH44在控制日常睡眠和活动模式中扮演着不同的重要角色。苍蝇使其双峰的休息和活动模式适应普遍的季节性条件：在冬季，大多数活动在晚上(为了避免夜间寒冷)，而在夏天，大多数活动在早上(为了避免白天的炎热)。我们的初步研究表明，分泌PDF的神经元本身所具有的PDF受体(PDFR)的自分泌激活增加，是这种从晚上到早上活动平衡的季节性变化的基础。我们将使用各种遗传工具来检验这一假设，即自分泌PDFR的激活通过调节PDF分泌本身的每日模式而导致昼夜节律网络特性的可塑性变化。最近的研究表明，不分泌PDF的时钟细胞的一个特定子集是这种PDF信号的必需接收者。我们的初步研究表明，这一亚群中的许多神经元表达DH31，并且像缺乏PDF突变的果蝇一样，缺乏DH31的果蝇表现出严重的昼夜早间活动迟钝。此外，分泌PDF的时钟神经元本身具有DH31受体(DH31R)，这表明分泌PDF的时钟神经元和分泌DH31的时钟神经元之间存在一个相互反馈的环路来驱动早晨的活动。我们将使用各种遗传工具来检验这一假设。我们的初步研究表明，CRF的Fly同源物DH44在下丘脑的Fly同源物大脑间部的一部分神经元中高水平表达。此外，我们已经进行了初步的行为遗传学筛查，表明对于哺乳动物CRF-DH44信号发送到中枢脑神经元的信号减少了总睡眠时间，增加了睡眠碎片。基于这些初步发现，我们将使用各种遗传工具来识别负责调节睡眠的特定DH44受体表达神经元。

DESCRIPTION (provided by applicant): Our long-term goal is the mechanistic dissection of neuropeptide signals in neural circuits in vivo. We have used a variety of genetic reagents for in vivo cell-specific manipulation of three Class B1 neuropeptides whose G protein-coupled receptors signal through cAMP in fly circadian/sleep control circuits: PDF (Pigment Dispersing Factor), DH31 (fly calcitonin homologue), and DH44 (fly corticotropin releasing factor (CRF) homologue). PDF is already known to be expressed in a subset of circadian clock neurons and to be important for regulating fly daily rhythms and sleep. Our preliminary studies now suggest distinct important roles for DH31 and DH44 in controlling daily patterns of sleep and activity. Flies adapt their bimodal crepuscular pattern of rest and activity to prevailing seasonal conditions: in the winter most activity is in the evening (to avoid the chill of night), while in the summer most activity is in the morning (to avoid the heat of day). Our preliminary studies suggest that increased autocrine activation of PDF receptors (PDFR) possessed by the PDF-secreting neurons themselves underlies this seasonal shift in the balance of activity from evening to morning. We will use various genetic tools to test the hypothesis that autocrine PDFR activation induces this plastic change in circadian network properties by modulating the daily pattern of PDF secretion itself. Recent studies implicate a particular subset of non-PDF-secreting clock cells as the required recipients of this PDF signal. Our preliminary studies indicate that many of the neurons in this subset express DH31, and that - like flies lacking PDF - mutant flies lacking DH31 exhibit severely blunted circadian morning activity. Furthermore, PDF-secreting clock neurons themselves possess DH31 receptors (DH31R), thus suggesting the hypothesis that a reciprocal feedback loop between PDF-secreting and DH31-secreting clock neurons drives morning activity. We will use various genetic tools to test this hypothesis. Our preliminary studies show that DH44-the fly homologue of CRF-is expressed at high levels in a subset of neurons in the pars intercerebralis, fly homologue of the hypothalamus. Furthermore, we have performed a preliminary behavioral genetic screen suggesting that - as for mammalian CRF - DH44 signaling to central brain neurons decreases total sleep amount and increases sleep fragmentation. Based on these preliminary findings, we will use a variety of genetic tools to identify the specific DH44 receptor-expressing neurons responsible for the regulation of sleep.