Retinal Neurons Afferent to the Circadian System

描述(申请人提供)：光携带在视交叉上核(SCN)，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。固有光敏性视网膜神经节细胞(IpRGC)表达黑素，一种新的脊椎动物视蛋白和垂体腺苷环化酶激活肽(PACAP)。这些单元向SCN传送信号，提供有关环境照明条件的信息。然而，这些ipRGC所使用的光传导系统(S)以及与它们突触前的视网膜神经元尚不清楚。此外，ipRGCs在调节视网膜内生理方面的潜在作用尚未被探索。这项应用的长期目标是提供对ipRGC所使用的光转导级联的了解，描述这一神经元系统的视网膜内电路，以更好地了解它们的功能，并探索黑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，使用免疫泛屏程序产生了高度浓缩的ipRGC群体。分离的细胞用体外钙成像观察光刺激的钙内流。IpRGC使用黑素作为发色团，并使用伪狂犬病病毒(PRV)作为跨神经元示踪剂来识别黑素表达细胞的传入神经元。在Cre-重组酶受黑素蛋白启动子控制的小鼠中，将感染一种条件性PRV，这种病毒只在表达Cre-重组酶的神经元和与最初感染细胞突触接触的神经元中复制。将使用电子显微镜和钙成像来探索ipRGC和视网膜的多巴胺能系统之间的关系。最后，利用钙成像和PACAP缺失小鼠的行为研究，研究了PACAP在调节光对ipRGCs和昼夜节律系统的影响中所起的作用。我们生物钟的携带或分相的干扰是睡眠节律异常分相的原因，并可能是严重的情感障碍的基础。了解SCN传入的视网膜神经元将有助于我们理解和治疗这些相位障碍。

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.