Using CRISPR-Cas9 genetic abscission in vivo to study the role of GABA-A receptors of the thalamic reticular nucleus in regulating non-rapid-eye-movement sleep and drug induced sleep

这项在小鼠身上进行的基础研究计划剖析了治疗性与非治疗性脑损伤的神经回路和受体。 用于治疗退伍军人的睡眠药物的副作用，使用最先进的基因编辑方法， 成簇规则间隔短回文重复序列（CRISPR）。该CDA 2奖项的支持将 允许申请人接受体内反向微透析和体外电生理学培训， 全面验证了他的基因操作和神经回路的解剖，并允许他 成为退伍军人事务部临床前睡眠研究的领导者。 睡眠障碍发生在神经精神疾病，如失眠，睡眠呼吸暂停，创伤后 应激障碍和创伤性脑损伤。美国退伍军人的睡眠时间是美国退伍军人的两倍多。 与其他人群相比，这是一种干扰。因此，睡眠药物，如唑吡坦（安必恩）和 右佐匹克隆（Lunesta）被广泛用于退伍军人。2005年至2014年，唑吡坦的VA处方 增加了近7倍，右佐匹克隆的VA处方增加了100倍以上，男性和50 女性退伍军人的时代然而，这些药物并不能促进自然睡眠，而且有副作用。 因此，需要更好地了解其作用机制，以开发更好的治疗方法。 δ波是以每秒0.5至4波的速度缓慢的脑节律，并且大量的 这些波是“深”NREM睡眠的定义特征。三角洲波与 深度睡眠（情绪调节、突触稳态、细胞能量调节和有毒蛋白质清除）。 有问题的是，唑吡坦和右佐匹克隆诱导“轻度”睡眠，并大幅降低NREM δ波。所以 也许不足为奇的是，这些药物与自杀风险和认知问题有关。三角洲波是 从大脑皮层通过脑电图记录下来，但它们是在大脑的深处产生的。 核心结构丘脑兴奋性“丘脑皮质（TC）”神经元形成来自 从丘脑到大脑皮层，它们就像是δ波起搏器。但它们需要一种抑制性的驱动力来执行 这个函数这种抑制驱动力是由神经递质GABA提供的，它来自外壳- 就像丘脑的一部分，叫做丘脑网状核（TRN）。最近的发现表明， 刺激TRN促进δ波。TRN神经元本身，接收来自唤醒活动神经元的GABA， 基底前脑和外侧下丘脑。考虑到这一点，我们将测试一个假设， 对TRN的抑制通过对TC神经元的GABA能抑制调节δ波。这将是第一次研究 在这个主题中，解剖分子，细胞和脑区域的具体机制，同时在体内。 α3亚基是TRN天然的GABAA受体类型的主要结构组分。在 我们使用CRISPR-Cas9局部消融TRN神经元亚组内的α3亚基， 由称为小清蛋白（PV）的钙结合蛋白的存在来定义。我们的初步数据显示 破坏这种GABA传递会增加NREM δ波并促进体内NREM;以及 降低体外自发抑制性突触后电流（sIPSC）。为了增加严谨性和可重复性，我们使用 另一种在PV+ TRN神经元中过表达α3亚基的小鼠遗传方法。培训将使 体外实验在SA 2中，我们使用CRISPR-Cas9局部消融α1亚基，其形成GABAA型 TC神经元的天然受体。在这里，我们集中在室旁丘脑，这是参与 压力引起的兴奋我们还将在TC神经元中过表达α1。体外数据将在 训练在SA 3中，Uygun博士将接受培训，使用体内反向微透析局部给予艾司唑匹克隆， 唑吡坦对TRN和TC神经元的作用。这将检查睡眠药物的δ抑制成分。 这项工作将指导下一代GABA能睡眠药物的开发， 改善退伍军人患者护理，降低自杀风险，改善情绪和认知表现。

This basic research proposal in mice dissects the neural circuitry and receptors that underlie therapeutic vs side effects of the sleep medicines used to treat Veterans, using a state-of-the-art gene editing approach called clustered regularly interspaced short palindromic repeats (CRISPR). The support of this CDA2 award would allow the applicant to be trained in in vivo reverse microdialysis and in vitro electrophysiology to allow him to comprehensively validate his genetic manipulations and dissection of neurocircuits, and would allow him to become a leader in pre-clinical sleep research within the VA. Disturbed sleep occurs in neuro-psychiatric illnesses such as insomnia, sleep apnea, post-traumatic stress disorder and traumatic brain injury. United States Veterans have more than double the amount of sleep disturbance compared to the rest of the population. As a result, sleep medicines like zolpidem (Ambien) and eszopiclone (Lunesta) are prescribed widely to Veterans. From 2005 to 2014, VA prescriptions of zolpidem increased nearly 7 times, and VA prescriptions of eszopiclone increased over 100 times for men and over 50 times for women Veterans. However, these medications do not promote a natural sleep and have side effects. Thus, a better understanding of their mechanism of action is needed to develop better treatments. Delta waves are slow brain rhythms at the speed of 0.5 to 4 waves per second, and large amounts of these waves are a defining feature of `deep' NREM sleep. Delta waves are linked to the restorative aspects of deep sleep (mood regulation, synaptic homeostasis, cellular energy regulation and clearance of toxic proteins). Problematically, zolpidem and eszopiclone induce `light' sleep and drastically reduce NREM delta waves. So perhaps unsurprisingly, these drugs are linked to suicide risk and cognitive problems. Delta waves are recorded from the cerebral cortex by electroencephalography, but they are generated deep within the brain's core structure, the thalamus. Excitatory “Thalamocortical (TC)” neurons form the connections from the thalamus to the cortex, and they act as delta wave pacemakers. But they require an inhibitory drive to perform this function. This inhibitory drive is provided by the neurotransmitter GABA, which comes from an outer shell- like part of the thalamus called the thalamic reticular nucleus (TRN). Recent discoveries have shown that a stimulated TRN promotes delta waves. TRN neurons themselves, receive GABA from wake active neurons in the basal forebrain and lateral hypothalamus. With this in mind, we will test a hypothesis that GABAergic inhibition onto TRN regulates delta waves via GABAergic inhibition onto TC neurons. This will be the 1st study in this topic that dissects molecular, cellular, and brain-region specific mechanisms simultaneously in vivo. α3 subunits are a major structural component of the type of GABAA receptors that are native to TRN. In Specific Aim (SA) 1 we use CRISPR-Cas9 to locally ablate α3 subunits within a subset of TRN neurons that are defined by the presence of a calcium-binding protein called parvalbumin (PV). Our preliminary data shows that disrupting this GABA transmission increases NREM delta waves and promotes NREM in vivo; and reduces spontaneous inhibitory post synaptic currents (sIPSC) in vitro. To add rigor and reproducibility, we use an alternative mouse genetic approach to overexpress α3 subunits in PV+ TRN neurons. Training will enable the in vitro work. In SA2 we use CRISPR-Cas9 to locally ablate α1 subunits, which form the type of GABAA receptors that are native to TC neurons. Here we focus on the paraventricular thalamus, which is involved in stress-induced arousal. We will also overexpress α1 in the TC neurons. In vitro data will be collected during the training. In SA3 Dr Uygun will train to use in vivo reverse microdialysis to locally administer eszolpiclone and zolpidem to TRN and TC neurons. This will examine the delta suppressing component of sleep medicines. This work will guide the development of next-generation GABAergic sleep medicines, leading to improved Veteran patient care with lower suicide risk and better mood and cognitive performance.