The Eye-Attached SCN Slice Preparation

描述(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线水平的信息。这些信息被用来重置单个SCN神经元的分子时钟，导致公开的动物行为和生理的缠绕。最近发现了一类新的固有光敏视网膜神经节细胞(ipRGC‘s)，它利用黑素作为其光色素，为SCN提供了大部分的视网膜输入。然而，关于这些黑素神经节细胞如何与单个SCN神经元沟通仍有许多问题。一个复杂的因素是，经典的视杆和视锥通路也为SCN提供输入，由Melansopin神经节细胞和传统的神经节细胞类型传递。由于在SCN中记录在体的光诱发反应的困难，人们对这些不同的输入通路的组织和功能以及它们如何导致生物钟的重置知之甚少。为了绕过这个问题，我们开发了一种新型的体外SCN脑片制剂，它保持了与两个视网膜的功能连接，能够从视觉上识别的能够对光做出反应的SCN神经元进行膜片钳记录。这使我们能够在对我们记录的细胞进行前所未有的控制和了解的情况下，在SCN中进行关于光处理的实验。其中一个主要优势是我们有能力在SCN的背壳或腹外侧核定位细胞进行记录，这两个亚区被认为在处理视网膜输入方面发挥不同的作用。利用这种新的切片制备方法，拟议研究的目标是利用膜片钳记录技术结合药理学描绘基于视杆/锥体和黑索平的SCN输入的功能组织，并了解这些不同的输入如何塑造单个SCN细胞的光反应。此外，内源性PACAP是黑素神经节细胞中发现的一种神经肽，其功能将在SCN的视觉信息处理过程中进行表征。由于大鼠和人类昼夜节律系统的相似之处，在我们的新型切片制备中，研究SCN如何处理视网膜输入将提供关于人类昼夜节律、光携带和睡眠调节的细胞基础的有价值的信息。这有望为数百万倒班工人带来工作条件和时间表的标准，在这些工人中，昼夜节律中断和睡眠剥夺是一种主要的职业危害，就医院而言，也是对公共健康的威胁。

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.