Probing epileptic circuits with novel Cre- and drug-regulated genetic approaches

 描述(由申请人提供)：癫痫是一种主要的神经疾病，具有巨大的经济和人类负担。最常见的形式之一是内侧颞叶癫痫(TLE)。不幸的是，TLE的药物治疗在许多情况下都失败了，留下了大量未得到满足的临床需求。因此，深入了解癫痫发作的启动和传播所涉及的神经元回路将推动新疗法的发展。对许多患者来说，内侧颞叶癫痫的一个特征是海马体硬化。海马体特别容易受到兴奋性毒性的影响。神经元死亡的关键部位是齿状回的门部，在那里许多抑制性GABA能中间神经元丢失。反复癫痫发作会增加神经发生和剩余神经元的发育。齿状回尤其如此，其反应包括：新颗粒细胞的诞生和迁移，新的基础树突的出现，以及它们的轴突投射增加，包括兴奋性侧支返回到邻近的颗粒细胞(苔藓纤维发芽)。在所有这些反应中，哪一种是导致自发性癫痫发作的最主要原因？这一提议将检验抑制性中间神经元活性降低是癫痫发生的关键驱动因素的假设。这一假说的提出是为了解释偶然发现的一个偶然发现，即大鼠齿状回神经元中修饰的泄漏K+通道(Trek-M)的表达引发了类似于TLE患者的边缘癫痫发作。这项研究将使用一种新的化学遗传学方法，基于腺相关病毒递送Trek-M，其表达依赖于Cre重组酶的作用，并受多西环素调节。这使我们能够利用NIH神经科学蓝图开发的CRE驱动小鼠。将首先在所有GABA能中间神经元表达CRE的小鼠身上测试GABA能神经元化学发生沉默的能力。第一个目标将验证交付，记录癫痫发作活动，并检查结果的病理，重点是苔藓纤维发芽。第二个目标将开始剖析许多GABA能亚型中的哪一种对癫痫的发生至关重要。这种创新的化学遗传学方法可能会对神经科学领域产生重大影响，因为它提供了比光遗传学更长的时间来探索复杂的行为。这些研究还可能提供第一个没有神经元死亡的自发性复发性癫痫的动物模型，这种模型将模仿没有表现出海马体硬化的人类TLE患者。

 DESCRIPTION (provided by applicant): Epilepsy is a major neurological disorder with significant economic and human burdens. One of the most common forms is mesial temporal lobe epilepsy (TLE). Unfortunately, medical treatment of TLE fails in many cases, leaving a large unmet clinical need. Therefore, a thorough understanding of the neuronal circuits involved in seizure initiation and propagation will drive the development of novel therapies. A hallmark of mesial temporal lobe epilepsy for many patients is hippocampal sclerosis. The hippocampus is particularly susceptible to excitotoxicity. Key sites of neuronal death are the hilus of the dentat gyrus, where many inhibitory GABAergic interneurons are lost. Recurrent seizures trigger an increase in both neurogenesis and the development of remaining neurons. This is particularly true for the dentate gyrus, whose responses include: birth and migration of new granule cells, appearance of novel basal dendrites, and an increase in their axonal projections including excitatory collaterals back onto neighboring granule cells (mossy fiber sprouting). Which of all these responses is most responsible for the development of spontaneous seizures? This proposal will test the hypothesis that reduced activity of inhibitory interneurons is a key driver f epileptogenesis. This hypothesis was developed to explain the serendipitous finding that expression of a modified leak K+ channel (TREK-M) in rat dentate hilar neurons triggered limbic seizures similar to those that occur in TLE patients. The research will use a novel chemogenetic approach based on adeno-associated viral delivery of TREK-M whose expression is dependent on the action of Cre recombinase and regulated by doxycycline. This allows us to exploit Cre-driver mice that have been developed as a result of the NIH Neuroscience Blueprint. The ability of chemogenetic silencing of GABAergic neurons will be first tested in mice where Cre is expressed in all GABAergic interneurons. The first aim will validate delivery, record seizure activity, and examine the pathology that results, focusing on mossy fiber sprouting. The second aim will begin to dissect which of the many GABAergic subtypes are critical for seizure generation. This innovative chemogenetic approach will likely have a major impact on the field of neuroscience, as it provides a longer time scale to probe complex behaviors than is possible with optogenetics. The studies may also provide the first animal model of spontaneous recurrent seizures without neuronal death, which would mimic human TLE patients who do not show hippocampal sclerosis.