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的干扰。最近，一类新的利用黑视素作为光色素的内在光敏视网膜神经节细胞（ipRGC’s）被发现，并被发现为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神经元交流，仍然存在许多问题。一个复杂的因素是，经典的杆状和锥状通路也向SCN提供输入，由黑素神经节细胞和传统神经节细胞类型传递。由于很难在体内记录SCN中的光诱发反应，人们对这些不同输入通路的组织和功能以及它们如何导致生物钟重置知之甚少。为了解决这个问题，我们开发了一种新的体外SCN脑切片制备方法，该制备方法保持了与两个视网膜的功能连接，使能够对光作出反应的视觉识别的SCN神经元能够进行膜片钳记录。这使我们能够以前所未有的控制和对我们记录的细胞的了解进行关于SCN光处理的实验。其中一个主要优势是我们能够在SCN的背壳或腹外侧核心定位细胞进行记录，这两个亚区域被认为在处理视网膜输入中起着不同的作用。利用这种新颖的切片制备，本研究的目标是利用膜片钳记录技术结合药理学，描述基于视杆/视锥和黑素的SCN输入的功能组织，并了解这些不同的输入如何影响单个SCN细胞的光响应。此外，内源性PACAP（一种在黑视素神经节细胞中发现的神经肽）的功能将在视神经网络的视觉信息处理过程中被表征。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，将为人类昼夜节律、光夹带和睡眠调节的细胞基础提供有价值的信息。这将有望为数百万轮班工人制定工作条件和时间表标准，这些工人的昼夜节律中断和睡眠剥夺是一种主要的职业危害，在医院中，这是对公众健康的威胁。

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.