Retinal Neurons Afferent to the Circadian System

描述（由申请人提供）：光携带视交叉上核（SCN）、昼夜节律振荡器和哺乳动物昼夜节律系统的主要组成部分。一个子集的内在光敏视网膜神经节细胞（ipRGC）表达黑视蛋白，一种新的脊椎动物视蛋白，和垂体腺苷酸环化酶激活肽（PACAP）。这些单元将信号传送到SCN，提供关于环境照明条件的信息。然而，这些ipRGC使用的光转导系统和与它们突触前的视网膜神经元是未知的。此外，ipRGC在调节视网膜内生理学中的潜在作用尚未探索。本申请的长期目标是提供对ipRGC使用的光转导级联的理解，描述该神经元系统的视网膜内电路以更好地理解其功能，并探索黑视素视网膜神经节细胞在视网膜内多巴胺调节中的作用。为了研究光转导过程，使用免疫淘选程序产生高度富集的ipRGC群体。使用体外钙成像检查分离的细胞以观察光刺激的钙内流。ipRGC使用黑视蛋白作为发色团，并且将使用伪狂犬病病毒（PRV）作为跨神经元示踪剂来鉴定传入表达黑视蛋白的细胞的神经元。其中Cre重组酶在黑视蛋白启动子控制下的小鼠将被条件PRV感染，所述条件PRV仅在表达Cre重组酶的神经元中和在与最初感染的细胞突触接触的神经元中复制。电子显微镜和钙成像将用于探索ipRGC和视网膜多巴胺能系统之间的关系。最后，使用钙成像和在PACAP无效小鼠的行为研究中检查PACAP在调节光对ipRGC和昼夜节律系统的影响中的作用。我们生物钟的干扰或相位的干扰是睡眠节律异常相位的原因，可能是严重情感障碍的基础。了解传入SCN的视网膜神经元将有助于我们理解和治疗这些相位紊乱的能力。

DESCRIPTION (provided by applicant): Light entrains the suprachiasmatic nucleus (SCN), a circadian oscillator and a primary component of the mammalian circadian system. A subset of intrinsically photosensitive retinal ganglion cells (ipRGCs) express melanopsin, a novel vertebrate opsin, and pituitary adenylate cyclase-activating peptide (PACAP). These cells convey signals to the SCN providing information about ambient lighting conditions. However, the phototransduction system(s) used by these ipRGCs and the retinal neurons that are presynaptic to them are unknown. Moreover, the potential role of ipRGCs in regulating intra-retinal physiology is unexplored. The long term goals of this application are to provide an understanding of the phototransduction cascade used by ipRGCs, to describe the intra-retinal circuitry of this system of neurons to better understand their function, and to explore the role of melanopsin retinal ganglion cells in the regulation of intra-retinal dopamine. To study the phototransduction process, a highly enriched population of ipRGCs is generated using an immunopanning procedure. Isolated cells are examined using in vitro calcium imaging to observe light- stimulated calcium influx. The ipRGCs use melanopsin as a chromophore and neurons afferent to melanopsin-expressing cells will be identified using pseudorabies virus (PRV) as a transneuronal tracer. Mice in which Cre-recombinase is under the control of the melanopsin promoter will be infected with a conditional PRV that replicates only in neurons that express Cre-recombinase and in neurons in synaptic contact with the originally infected cells. Electron microscopy and calcium imaging will be used to explore the relationship between ipRGCs and the dopaminergic system of the retina. Finally, the role PACAP in modulating the effects of light on ipRGCs and the circadian system is examined using calcium imaging and in behavioral studies with PACAP null mice. Disturbances in the entrainment or phasing of our biological clock are responsible for abnormal phasing of sleep rhythms and may underlie serious affective disorders. Understanding the retinal neurons afferent to the SCN will aid in our ability to understand and treat these disturbances of phase.