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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

利用体内CRISPR-Cas9基因分离研究丘脑网状核GABA-A受体对非快动眼睡眠和药物诱导睡眠的调节作用

基本信息

批准号：
10552611
负责人：
David Samuel Uygun
金额：
--
依托单位：
VA BOSTON HEALTH CARE SYSTEM
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-01-01 至 2024-12-31
项目状态：
已结题

项目摘要

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.

这项对小鼠的基础研究计划剖析了构成治疗性VS的神经回路和受体用于治疗退伍军人的安眠药的副作用，使用最先进的基因编辑方法规则排列的短回文重复序列(CRISPR)。这项CDA2奖的支持将允许申请者接受体内反向微渗析和体外电生理学方面的培训，以使他能够全面验证他的基因操作和神经回路解剖，并将允许他成为退伍军人管理局临床前睡眠研究的领导者。睡眠障碍发生在神经精神疾病中，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增加了一倍多与其他人口相比，这是一种骚乱。因此，像唑吡坦(安必恩)和退伍军人广泛使用埃索匹克隆(LUNESTA)。2005年至2014年，佐吡坦的VA处方增加了近7倍，埃索匹克隆的VA处方增加了100多倍，男性增加了50多倍退伍军人女性时代杂志。然而，这些药物不能促进自然睡眠，而且有副作用。因此，需要更好地了解它们的作用机制，以开发更好的治疗方法。 Delta波是以每秒0.5到4波的速度缓慢的大脑节奏，大量的这些波是“深度”NREM睡眠的一个重要特征。三角洲波与恢复性方面有关深度睡眠(情绪调节、突触动态平衡、细胞能量调节和有毒蛋白质清除)。有问题的是，唑吡坦和埃索匹克隆可引起“轻”睡眠，并显著减少NREM增量波。所以也许并不令人惊讶的是，这些药物与自杀风险和认知问题有关。三角洲波是通过脑电图术从大脑皮层记录下来，但它们是在大脑深处产生的核心结构，丘脑。兴奋性“丘脑皮质(TC)”神经元形成来自丘脑到大脑皮层，它们起着三角波起搏器的作用。但它们需要一种抑制性驱动力才能发挥作用此函数。这种抑制性驱动力是由神经递质GABA提供的，GABA来自一个外壳- 就像丘脑的一部分，称为丘脑网状核(TRN)。最近的发现表明，受刺激的TRN促进三角洲波。TRN神经元本身，从觉醒活动神经元接受GABA 基底前脑和外侧下丘脑。考虑到这一点，我们将检验一个假设，即GABA能对TRN的抑制通过对TC神经元的GABA能抑制来调节Delta波。这将是第一项研究在这个主题中，同时剖析了分子、细胞和脑区域在活体中的特定机制。 GABAA3亚基是α天然受体的主要结构成分。在……里面特定目的(SA)1我们使用CRISPR-CAS9局部消融TRN神经元子集内的α3亚单位，由一种称为小白蛋白(PV)的钙结合蛋白的存在来定义。我们的初步数据显示干扰这种GABA传递会增加NREM增量波，并在体内促进NREM；减少体外自发抑制性突触后电流(SIPSC)。为了增加严密性和重复性，我们使用另一种小鼠遗传学方法在PV+Trn神经元中过表达α3亚单位。培训将使试管工作。在SA2中，我们使用CRISPR-CAS9来局部消融形成GABA AA型的α1亚基 TC神经元固有的受体。在这里，我们关注的是室旁丘脑，它参与了压力诱导的性唤醒。我们还将在TC神经元中过表达α1。体外数据将在实验期间收集训练。在SA3中，Uygan博士将接受培训，使用体内反向微透析局部给药埃索匹克隆和唑吡坦对TRN和TC神经元的作用。这将检查安眠药中抑制三角洲的成分。这项工作将指导下一代GABA能睡眠药物的开发，导致改善退伍军人护理，降低自杀风险，改善情绪和认知表现。