Serotonergic network mechanisms in postictal generalized EEG suppression

项目摘要 每26个美国人中就有一个会在一生中患上癫痫。不幸的是，三分之一的癫痫患者 患者将无法通过常规疗法实现癫痫发作的自由。这些患者的风险最大， 癫痫猝死（SUDEP）是难治性癫痫患者死亡的主要原因。 虽然SUDEP的确切病因尚不清楚，但认为心肺功能障碍和觉醒 有损伤。癫痫发作后EEG活动抑制，或发作后全身EEG 抑制（PGES）可能与SUDEP风险相关。PGES的起源尚不清楚，但在PGES期间 患者会出现昏迷和反应迟钝。5-羟色胺（5-HT）广泛参与SUDEP，由于其 角色呼吸、睡眠/觉醒和觉醒。中缝背核是5-HT的重要来源 预测。DRN 5-HT活性被癫痫发作抑制。我们假设PGES可能代表一种 电图标记物，其是由电刺激诱导的DRN失调引起的觉醒受损的结果。我们的初步 小鼠中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 在诱导癫痫发作之前对DRN 5-HT神经元进行化学或光遗传学刺激缩短了PGES持续时间。 然而，PGES的具体网络和受体机制尚不清楚。我们的目标是 鉴定DRN网络和5-HT受体机制，其可被操纵以减少PGES并预防 SUDEP。一个潜在的下游靶点是脚桥被盖核（PPT）， 参与睡眠-觉醒调节、注意力、脑电图调节和觉醒。癫痫发作诱导的5- HT信号可能干扰皮层下唤醒网络，如涉及PPT的那些，并产生 PGES。在目标1中，为了确定DRN的PPT电路在PGES中的作用，我们将光遗传学刺激和 在杏仁核点燃前抑制PPT中的DRN 5-HT终末 观察小鼠清醒/NREM/REM期间PGES持续时间的变化。几种5-HT受体已被 与SUDEP有关，可能在PPT中的胆碱能神经元上发现。在目标2中，我们将利用 溶剂清除器官的免疫标记三维成像，原位荧光RNAscope 杂交和免疫组织化学以确定PPT神经元和5-HT受体的身份 与DRN 5-HT终端联系然后，我们将给予颅内5-HT拮抗剂到PPT中， 在没有选择性5-羟色胺再摄取抑制剂的情况下，诱导杏仁核点燃的癫痫发作，并观察 对PGES的影响参加拟议的培训计划和完成拟议的实验将 推进申请人的神经科学培训。它还将阐明DRN-PPT电路，并确定其作用 在PGES。通过操纵5-HT回路，我们可能会发现消除PGES的方法， 预防高风险癫痫患者的死亡。

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.