Retinal Neurons Afferent to the Circadian System

描述（由申请人提供）：光携带视交叉上核（SCN），这是一个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。固有光敏视网膜神经节细胞（ipRGCs）的一个子集表达黑视素，一种新的脊椎动物视蛋白和垂体腺苷酸环化酶激活肽（PACAP）。这些细胞向SCN传递信号，提供有关环境光照条件的信息。然而，这些ipRGCs使用的光导系统和与它们突触前的视网膜神经元是未知的。此外，ipRGCs在调节视网膜内生理方面的潜在作用尚未被探索。该应用的长期目标是提供对ipRGCs使用的光传导级联的理解，描述该神经元系统的视网膜内电路以更好地了解其功能，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。为了研究光导过程，使用免疫计划程序生成了高度富集的ipRGCs群体。用体外钙显像检查离体细胞，观察光刺激下的钙内流。ipRGCs使用黑视素作为发色团，并使用伪狂犬病毒（PRV）作为跨神经元示踪剂来鉴定向表达黑视素的细胞传入的神经元。cre -重组酶受黑视素启动子控制的小鼠将被条件PRV感染，该PRV仅在表达cre -重组酶的神经元和与原始感染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ipRGCs与视网膜多巴胺能系统的关系。最后，通过钙成像和PACAP缺失小鼠的行为研究，研究了PACAP在调节光对iprgc和昼夜节律系统的影响中的作用。对生物钟的干扰会导致睡眠节律的异常相位，并可能导致严重的情感障碍。了解视网膜神经元传入到SCN将有助于我们理解和治疗这些阶段的干扰。

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.