The Eye-Attached SCN Slice Preparation

描述（由申请人提供）：视交叉上核（SCN）通过视网膜下丘脑束接收有关环境光水平的信息。这些信息被用来重置单个SCN神经元的分子钟，导致明显的动物行为和生理学的夹带。最近，发现了一类利用黑视素作为其色素的新的固有光敏视网膜神经节细胞（ipRGC），并发现其向SCN提供大部分视网膜输入。然而，关于这些黑视素神经节细胞如何与单个SCN神经元通信仍然存在许多问题。一个复杂的因素是，经典的杆和锥途径也提供输入到SCN，由黑素神经节细胞和传统的神经节细胞类型中继。由于在SCN中记录体内光诱发反应的困难，人们对这些不同输入途径的组织和功能以及它们如何导致生物钟重置知之甚少。为了规避这个问题，我们已经开发了一种新的体外SCN脑切片制备物，其保持与两个视网膜的功能连接，使得能够从视觉识别的能够对光做出响应的SCN神经元进行膜片钳记录。这使我们能够进行有关SCN中光处理的实验，并对我们记录的细胞进行前所未有的控制和了解。主要优势之一是我们能够靶向SCN的背壳或腹外侧核心中的细胞进行记录，这两个子区域被认为在处理视网膜输入中发挥不同的作用。利用这种新的切片制备，所提出的研究的目标是使用膜片钳记录技术结合药理学来描绘视杆/视锥和基于黑素的SCN输入的功能组织，并了解这些各种输入如何塑造来自单个SCN细胞的光响应。此外，内源性PACAP，黑视素神经节细胞中发现的神经肽的功能，将在SCN中的视觉信息的处理过程中进行表征。由于大鼠和人类昼夜节律系统之间的相似性，研究SCN如何在我们的新型切片制备中处理视网膜输入将提供有关昼夜节律，光诱导和人类睡眠调节的细胞基础的有价值的信息。这将有望为数百万轮班工人制定工作条件和时间表标准，在这些工人中，昼夜节律的破坏和睡眠剥夺是一种主要的职业危害，在医院中，这对公共健康构成威胁。

DESCRIPTION (provided by applicant): The suprachiasmatic nucleus (SCN) receives information about ambient light levels through the retinohypothalamic tract. This information is used to reset the molecular clock of individual SCN neurons, leading to entrainment of overt animal behavior and physiology. Recently, a new class of intrinsically photosensitive retinal ganglion cells (ipRGC's) utilizing melanopsin as their photopigment was discovered and found to provide the majority of retinal input to the SCN. However, many questions remain about how these melanopsin ganglion cells communicate with individual SCN neurons. One complicating factor is that classic rod and cone pathways also provide input to the SCN, relayed by melansopin ganglion cells and conventional ganglion cell types. Due to difficulties in recording in vivo light-evoked responses in the SCN, little is known about the organization and function of these various input pathways and how they lead to resetting of the biological clock. To circumvent this issue, we have developed a novel in vitro SCN brain- slice preparation that maintains functional connectivity to both retinas, enabling patch-clamp recordings from visually identified SCN neurons that are capable of responding to light. This allows us to conduct experiments concerning light processing in the SCN with unprecedented control and knowledge of the cells we record from. One of the main advantages is our ability to target cells for recording in the dorsal shell or ventrolateral core of the SCN, two sub-regions thought to play different roles in processing retinal input. Utilizing this novel slice preparation, the goal of the proposed study is to delineate the functional organization of rod/cone and melansopin based input to the SCN using patch-clamp recording techniques in combination with pharmacology, and to understand how these various inputs shape light responses from individual SCN cells. In addition, the function of endogenous PACAP, a neuropeptide found in melanopsin ganglion cells, will be characterized during processing of visual information in the SCN. Because of the similarities between rat and human circadian systems, studying how the SCN processes retinal input in our novel slice preparation will provide valuable information about the cellular basis of circadian rhythms, photic entrainment, and sleep regulation in humans. This will hopefully lead to standards of work conditions and schedules for millions of shift workers where disruption of circadian rhythms and sleep deprivation is a major occupational hazard, and in the case of hospitals, a threat to public health.