The neural circuitry of seizure-induced apnea and SUDEP

癫痫猝死(SUDEP)占所有癫痫相关死亡的17%，以及 治疗难治者50%。越来越多的证据表明，呼吸暂停(呼吸停止)是 主要死因是SUDEP。我们已经在许多小鼠模型中展示了这一点--包括注射PTZ， 快速点火(预演。数据)，以及携带从SUDEP患者身上发现的Scn8a突变的小鼠模型- 猝死是由于强直性发作期间发生的惊厥性呼吸暂停(SIA)，几分钟前 到终末期停搏。与这项建议有关，我们还提出了另外四点重要意见： 1)人工呼吸可以预防死亡，抑制心脏活动不会增加死亡的可能性； 2)呼吸肌紧张性收缩是SIA的可能机制；3)抑制皮质癫痫发作 活动不会影响SIA；4)抑制吸气振荡器不会减少呼吸暂停(初步。数据)、 表明癫痫发作扩散绕过了呼吸控制回路的稳态元件。中区 假设：[目标1]增加中脑和桥脑的兴奋性既是必要的，也是充分的 桥脑臂旁核/Kölliker融合核(PB/KF)是一个重要的结节 SIA产生的时间点，以及脑干核团，如PB/KF和PAG被招募 特别是在SIA期间，我们将使用电生理学和基因作图进行探索。目标1：我们 以前曾使用化学遗传学证明SIA不需要皮质癫痫活动，我们现在 认为脑桥和中脑的超兴奋性驱动SIA和SUDEP。我们将使用化学遗传学来 抑制Scn8a突变小鼠脑干的不同部位，以测试其对SIA和SUDEP的必要性。 此外，我们将通过选择性地表达Scn8a突变来测试这些区域的充分性 神经元兴奋性。使用快速点燃模型，我们还将进行膜片钳记录，以测试 这些区域神经元兴奋性的增加与SIA的发展同步发生。目标2： 桥脑臂旁/Kölliker融合核PB/KF是前脑传入下行的关键结点。 通过吸气振荡器投射，并由迷走神经调节。在这个目标中，我们将光抑制 Foxp2阳性PB/KF神经元在SIA期间恢复正常呼吸模式，光刺激P2ry1阳性 迷走神经传入纤维阻断SIA期间的吸气，并确定PB/KF和NPY2R是否阳性迷走神经 神经元光刺激挽救了我们的PTZ SUDEP小鼠模型的后遗症呼吸。目标3：我们有 证明了有SIA的强直性发作和没有SIA的克隆性发作可以发生在同一只小鼠身上， 在Scn8a突变体和快速点燃模型中(Prelim.数据)，以及某些中脑和后脑结构 在SIA期间激活。我们将使用以下方法确定在SIA和SUDEP期间特定激活的神经元回路 激活神经元的在体电生理学和TRAP2鉴定。

Sudden Unexpected Death in Epilepsy (SUDEP) accounts for up to 17% of all epilepsy-related deaths, and 50% for those refractory to treatment. There is increasing evidence that apnea (respiratory arrest) is the primary cause of death from SUDEP. We have shown in a number of mouse models - including PTZ injection, rapid kindling (prelim. data), and mouse models harboring Scn8a mutations identified from SUDEP patients - that sudden death is due to seizure-induced apnea (SIA) that occurs during tonic seizures, minutes prior to terminal asystole. As it pertains to this proposal, we have also made four other important observations: 1) artificial ventilation can prevent death and suppressed cardiac activity does not increase likelihood of fatality; 2) tonic contraction of breathing muscles represents a likely mechanism of SIA; 3) inhibition of cortical seizure activity does not impact SIA; and 4) inhibition of the inspiratory oscillator does not reduce apnea (prelim. data), suggesting seizure spread bypasses homeostatic elements of respiratory control circuitry. CENTRAL HYPOTHESIS: [AIM 1] Increased midbrain and pontine excitability is both necessary and sufficient to produce SIA, [AIM 2] the parabrachial/kölliker fuse (PB/KF) nucleus in the pons represents a key nodal point for SIA generation, and [AIM 3] that brainstem nuclei, like the PB/KF and PAG are recruited specifically during SIA, which we will explore using electrophysiology and genetic mapping. AIM 1: We have previously used chemogenetics to demonstrate that cortical ictal activity is not required for SIA and we now propose that hyperexcitability of the pons and midbrain drive SIA and SUDEP. We will use chemogenetics to inhibit different parts of the brainstem of Scn8a mutant mice to test their necessity for SIA and SUDEP. Additionally, we will test sufficiency of these regions by selectively expressing an Scn8a mutation that increases neuronal excitability. Using the rapid kindling model, we will also perform patch clamp recordings to test whether increased neuronal excitability in these regions occurs concurrently with the development of SIA. AIM 2: The parabrachial/kölliker fuse nucleus PB/KF in the Pons is a key nodal point for descending forebrain input and projects past the inspiratory oscillator, and is modulated by the vagus nerve. In this aim, we will photoinhibit the Foxp2-positive PB/KF neurons to regain normal breathing pattern during SIA, photostimulate P2ry1-positive vagal afferent nerve fibers to interrupt inspiration during SIA, and determine if PB/KF and Npy2r-positive vagal neuron photostimulation rescues postictal breathing in our PTZ SUDEP mouse model. AIM 3: We have demonstrated that tonic seizures with SIA and clonic seizures without SIA can occur in the very same mouse, both in Scn8a mutant and rapid kindling models (prelim. data) and that certain mid- and hindbrain structures are activated during SIA. We will determine the neuronal circuitry activated specifically during SIA and SUDEP using in vivo electrophysiology and TRAP2 identification of activated neurons.