Serotonergic network mechanisms in postictal generalized EEG suppression

项目摘要 二十六个美国人中就有一个会在有生之年患上癫痫。不幸的是，三分之一的癫痫 患者不会用传统的治疗方法获得癫痫的自由。这些患者面临着最大的风险 癫痫猝死(SUDEP)是难治性癫痫患者的主要死亡原因。 虽然SUDEP的确切病因尚不清楚，但人们认为心肺功能障碍和觉醒 都涉及到损害。癫痫发作后的脑电活动抑制或发作后的全身性脑电 抑制(PGES)，可能与SUDEP风险相关。PGES的起源不明，但在pGES期间 患者会出现昏迷和无反应。5-羟色胺(5-羟色胺)被广泛地与SUDEP有关，因为它 扮演呼吸、睡眠/清醒和唤醒的角色。中缝背核是5-羟色胺的重要来源 投射。DRN 5-羟色胺活性因癫痫发作而受到抑制。我们假设pge可能代表一个 惊厥所致DRN失调所致觉醒受损的脑电标记物。我们的预赛 小鼠的数据表明，全身应用选择性5-羟色胺再摄取抑制剂(SSRI)或直接 在诱发癫痫发作前对DRN 5-HT神经元进行化学或光遗传刺激可缩短pGES持续时间。 然而，pGES背后的特定网络和受体机制尚不清楚。我们的目标是 确定可被操纵的DRN网络和5-羟色胺受体机制，以减少PGE并防止 苏德普。一个潜在的下游目标是桥脑脚被盖核(PPT)，这是一个桥脑区域 参与睡眠-觉醒调节、注意力、脑电调节和觉醒。癫痫发作引起的5-羟色胺调节失调 羟色胺信号可能干扰皮质下觉醒网络，如涉及PPT的网络，并产生 太棒了。在目标1中，为了确定DRNppt电路在pGES中的作用，我们将从光基因刺激和 电刺激杏仁核点燃癫痫前抑制PPT内DRN 5-羟色胺终末 小鼠在清醒/非快速眼动/快速眼动过程中，观察PGE持续时间的变化。几种5-羟色胺受体已经被 与SUDEP有关，可能存在于PPT的胆碱能神经元上。在目标2中，我们将利用 免疫标记溶剂清除器官的三维成像，RNAScope原位荧光 杂交和免疫组织化学方法确定PPT神经元和5-羟色胺受体的身份 由DRN 5-HT终端联系。然后我们会将5-羟色胺拮抗剂注入PPT或 在没有选择性5-羟色胺再摄取抑制的情况下，诱导杏仁核点燃的癫痫发作，并观察 对PGE的影响。参加拟议的培训计划和完成拟议的实验将 推进申请者的神经科学培训。它还将阐明DRN-PPT电路并确定其作用 以PGE为单位。通过操纵5-HT电路，我们可能会发现消除PGE和相应地 预防高危癫痫患者死亡。

Project Summary One in twenty-six Americans will develop epilepsy during their lifetime. Unfortunately, one-third of epilepsy patients will not achieve seizure freedom with conventional therapies. These patients are at greatest risk for sudden unexpected death in epilepsy (SUDEP), the leading cause of death in patients with refractory epilepsy. While the exact etiology of SUDEP is unknown, it is thought that cardiorespiratory dysfunction and arousal impairment are involved. Suppression of EEG activity following a seizure, or post-ictal generalized EEG suppression (PGES), may correlate with SUDEP risk. The origin of PGES is unknown, but during PGES patients experience stupor and unresponsiveness. Serotonin (5-HT) is broadly implicated in SUDEP due to its role breathing, sleep/wakefulness, and arousal. The dorsal raphe nucleus (DRN) is a key source of 5-HT projections. DRN 5-HT activity is depressed by seizures. We hypothesize PGES may represent an electrographic marker of impaired arousal consequent to seizure-induced DRN dysregulation. Our preliminary data in mice indicate that systemic application of a selective serotonin reuptake inhibitor (SSRI) or direct chemical or optogenetic stimulation of DRN 5-HT neurons prior to an induced seizure shortens PGES duration. However, the specific network and receptor mechanisms underlying PGES are unknown. Our objective is to identify a DRN network and 5-HT receptor mechanism that could be manipulated to reduce PGES and prevent SUDEP. A potential downstream target is the pedunculopontine tegmental nucleus (PPT), a pontine region involved in sleep-wake regulation, attention, EEG regulation, and arousal. Seizure-induced dysregulation of 5- HT signaling may interfere with subcortical arousal networks, such as those involving the PPT, and produce PGES. In Aim 1, to determine a role for a DRN  PPT circuit in PGES, we will optogenetically stimulate and inhibit DRN 5-HT terminals in the PPT prior to seizures induced by amygdala stimulation in amygdala kindled mice during wake/NREM/REM and observe changes in PGES duration. Several 5-HT receptors have been implicated in SUDEP and may be found on cholinergic neurons in the PPT. In Aim 2, we will utilize immunolabeling-enabled three-dimensional imaging of solvent-cleared organs, RNAscope fluorescent in situ hybridization, and immunohistochemistry to determine the identity of PPT neurons and 5-HT receptors contacted by DRN 5-HT terminals. Then we will administer intracranial 5-HT antagonists into the PPT with or without a selective serotonin reuptake inhibitory onboard, induce an amygdala-kindled seizure, and observe effects on PGES. Participation in the proposed training plan and completion of the proposed experiments will advance the applicant’s neuroscience training. It will also elucidate the DRN-PPT circuit and determine its role in PGES. By manipulating 5-HT circuitry, we may discover ways to eliminate PGES and consequentially prevent death in high risk epilepsy patients.