TNF signaling methods initiating in vitro sleep-like states

项目描述： 睡眠和睡眠动态平衡是由睡眠调节物质网络调节的。肿瘤的作用 肿瘤坏死因子α在睡眠调节中的作用已有较好的研究。然而，肿瘤坏死因子-肿瘤坏死因子受体的相互作用是 复杂；参与肿瘤坏死因子调节睡眠的机制尚不清楚。几种电生理学 描述动物睡眠的参数包括动作电位突发性(BI-突发性指数)、 慢波(Sw)(0.5-3.5 Hz)、Sw功率(µV2)和诱发反应电位的同步(SYN) (事件相关电位)波幅。我们使用同源测量来表征神经元/神经胶质细胞联合睡眠状态。 文化。在神经元/神经胶质细胞培养的发展过程中，这些参数作为网络出现 成熟。如果培养的网络受到电刺激，这些参数会降低，这意味着更多的尾流- 就像州政府一样。相反，如果用肿瘤坏死因子处理培养物，这些参数会增加，表明睡眠更深- 就像州政府一样。培养物也表现出睡眠动态平衡；电刺激后BI、SYN反弹增加 就会产生短波功率。肿瘤坏死因子对事件相关电位的影响是惊人的；肿瘤坏死因子大大增强了事件相关电位的波幅和 同步。我们将使用我们的体外睡眠模型来确定肿瘤坏死因子是如何与其受体相互作用的 影响文化状态。在目标1中，提出了四个实验来确定三种已知方法中的哪一种 肿瘤坏死因子-肿瘤坏死因子受体的相互作用负责肿瘤坏死因子-睡眠的作用。肿瘤坏死因子-肿瘤坏死因子受体相互作用 A)可溶性肿瘤坏死因子(17kD)作为其受体之一的配基；b)直接跨膜肿瘤坏死因子(26kD) 与相邻细胞上的肿瘤坏死因子受体结合以启动反应(直接细胞对细胞信号)；以及c)a 可溶性肿瘤坏死因子受体与跨膜肿瘤坏死因子结合启动反应。在目标2中，我们确定哪种肿瘤坏死因子 受体也参与其中。体外系统，由于它的简单和我们控制强度的能力 涌现状态特性，为确定肿瘤坏死因子的作用机制提供了新的实验平台。 睡眠调节和紧急网络特性。我们使用R21机制是因为 用于体外培养的神经胶质细胞/神经元仍处于探索和开发阶段，实验涉及一些风险。 但具有革命性的潜力(例如，局部睡眠状态是直接细胞到细胞的结果 通信)。因此，它们可以改变基础睡眠研究和睡眠临床问题的方法 通过为在实际睡眠中起作用的网络紧急属性提供一种新的、自下而上的方法 药物问题、癫痫、创伤性脑损伤和其他受睡眠影响的中枢神经系统疾病。

Project Description: Sleep and sleep homeostasis are regulated by a network of sleep regulatory substances. A role for tumor necrosis factor α (TNF) in sleep regulation is well characterized. However, TNF-TNF receptor interactions are complex; the mechanism involved in TNF-regulated sleep is unknown. Several electrophysiological parameters characterize sleep in animals; they include action potential burstiness (BI – burstiness index), synchronization (SYN) of slow waves (SW) (0.5-3.5 Hz), SW power (µV2) and evoked response potential (ERP) amplitudes. We use homologous measures to characterize sleep-like states in neuronal/glial co- cultures. Over the course of neuronal/glial culture development these parameters emerge as the networks mature. If cultured networks are stimulated electrically, these parameters decrease suggesting a more wake- like state. In contrast, if cultures are treated with TNF, these parameters increase indicating a deeper sleep- like state. Cultures also exhibit sleep homeostasis; after electrical stimulation a rebound increase in BI, SYN and SW power occurs. The effects of TNF on ERPs are stunning; TNF greatly enhances ERP amplitude and synchronization. We will use our in vitro sleep model to determine how TNF interacts with its receptors to affect culture state. In Aim 1, four experiments are proposed to determine which of the three known methods of TNF-TNF receptor interactions is responsible for the TNF-sleep actions. The TNF-TNF receptor interactions are: a) soluble TNF (17kD) acting as ligand for one of its receptors; b) trans-membrane TNF (26kD) directly binding to a TNF receptor on an adjacent cell to initiate responses (direct cell-to-cell signaling); and c) a soluble TNF receptor binding to trans-membrane TNF to initiate responses. In Aim 2 we determine which TNF receptor is involved. The in vitro system, due to its simplicity and our ability to control the intensity of the emergent state properties, offers a novel experimental platform to determine the mechanisms of action of TNF- sleep regulation and of emergent network properties. We use the R21 mechanism because the approach used, in vitro glial/neuronal cultures, is exploratory and in development and the experiments involve some risk yet have revolutionary potential (e.g. local sleep-like states being a consequence of direct cell-to-cell communication). As such they could transform basic sleep research and approaches to sleep clinical issues by providing a new, bottom-up approach to network emergent properties that have a role in practical sleep medicine problems, epilepsy, traumatic brain injury and other CNS disorders impacted by sleep.