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

• 批准号: 10438528
• 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
• 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
• 来源: https://reporter.nih.gov/project-details/10438528
• 关键词: Accounting; Address; Arousal; Attenuated; Award; Basic Science; Behavior; Binding; Brain; Brain region; CRISPR/Cas technology; Calcium-Binding Proteins; Caring; Cell Nucleus; Cerebral cortex; Characteristics; Clustered Regularly Interspaced Short Palindromic Repeats; Cognitive; Cognitive deficits; Data; Development; Dissection; Dose; Electroencephalography; Electrophysiology (science); Eszopiclone; Excision; Foundations; GABA-A Receptor; General Population; Genes; Genetic; Genetic Engineering; Glutamates; Homeostasis; Hypothalamic structure; In Vitro; Injections; Intervention; Lateral; Light; Link; Mediating; Medicine; Memory; Mental Health; Microdialysis; Mind; Modeling; Modernization; Molecular; Moods; Mus; Neocortex; Neurons; Neurotransmitters; Pacemakers; Parvalbumins; Patient Care; Patients; Periodicity; Pharmaceutical Preparations; Population; Post-Traumatic Stress Disorders; Prevalence; Proteins; Psyche structure; Regulation; Reporting; Reproducibility; Research; Research Proposals; Rest; Role; Sensory Receptors; Sleep; Sleep Apnea Syndromes; Sleep Disorders; Sleep disturbances; Sleeplessness; Slow-Wave Sleep; Speed; Stress; Structure; Structure of paraventricular nucleus of thalamus; Synapses; Techniques; Testing; Thalamic structure; Therapeutic; Therapeutic Effect; Thinking; Time; Training; Transgenic Mice; Traumatic Brain Injury; United States; Veterans; Woman; Work; adeno-associated viral vector; attenuation; basal forebrain; base; cognitive performance; gamma-Aminobutyric Acid; genetic approach; genetic manipulation; improved; in vivo; intraperitoneal; knock-down; men; mood regulation; mouse genetics; neural circuit; neuropsychiatry; next generation; non rapid eye movement; overexpression; patch clamp; positive allosteric modulator; pre-clinical; receptor; response; side effect; suicidal risk; tool; transmission process; vesicular glutamate transporter 2; zolpidem

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.

这项以小鼠为研究对象的基础研究计划，剖析了治疗vs的神经回路和受体基础