Mechanisms of Synaptic Homeostasis Governing Pre-Sympathetic Neurons in the Hypothalamic Paraventricular Nucleus

下丘脑室旁核前交感神经元突触稳态的调控机制

• 批准号: 10400957
• 负责人: GLENN M TONEY
• 金额: $ 48.7万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10400957
• 关键词: Acute; Angiotensin II; Animals; Antihypertensive Agents; Automobile Driving; Autonomic Dysfunction; Bathing; Behavior; Brain; CRISPR/Cas technology; Cardiovascular Diseases; Cell model; Chronic; Dependovirus; Disease; Ensure; Equilibrium; Experimental Water Deprivation; Exposure to; Functional disorder; GABA-A Receptor; Generations; Genes; Glutamate Transporter; Glutamates; Goals; Heart failure; Homeostasis; Hypertension; Individual; Injections; Interruption; Kinetics; Knowledge; Label; Lamina Terminalis; Link; Mediating; Metabolic Diseases; Modeling; Mus; Nerve; Neurons; Nuclear; Pathogenicity; Pathologic; Physiologic pulse; Physiological; Play; Preparation; Probability; Process; Property; Prosencephalon; Recovery; Rest; Role; Rose Bengal; Saline; Slice; Synapses; Synaptic Vesicles; Time; Transgenic Mice; Vesicle; Viral; Virulence Factors; Virus; acute stress; clinical efficacy; density; excitatory amino acid transporter 3; functional outcomes; gamma-Aminobutyric Acid; improved; in vivo; insight; knock-down; mouse Cre recombinase; mouse model; novel; optogenetics; overexpression; paraventricular nucleus; photoactivation; postsynaptic; preoptic nucleus; preservation; presynaptic; prevent; quantum; response; restoration; retrograde transport; salt intake; stressor; synaptic depression; synaptic inhibition; uptake; vesicular release

Project Summary Pre-sympathetic neurons (PSNs) of the hypothalamic paraventricular nucleus (PVN) are essential drivers of physiological and pathological increases of sympathetic nerve activity (SNA). Perhaps their most robust property is their resting state of discharge quiescence. Early studies linked quiescence to the dominance of synaptic inhibition, but mechanisms that establish and defend GABAergic inhibitory tonus in the PVN are understood only on a rudimentary level. This is an important knowledge gap because pathogenic factors that increase PVN-driven SNA must ultimately subvert or overwhelm mechanisms that regulate the quiescent resting state of PSNs. In preliminary studies, we uncovered a presynaptic mechanism that is novel to the PVN, referred to as “Glutamate-GABA strengthening (GGS)”, that increases GABAergic inhibition in pace with synaptic glutamate (Glu) spillover. To do so, GGS regulates the amplitude of GABA-A receptor-mediated inhibitory postsynaptic currents (IPSCs) through uptake of synaptically released Glu, ostensibly into local GABA terminals, by the neuronal excitatory amino acid transporter 3 (EAAT3). Once internalized, Glu is converted to GABA and GABA molecules are packaged into synaptic vesicles at greater than normal density. Stressors that acutely increase PVN-driven SNA are hypothesized to increase synaptic Glu release without changing extrinsic GABAergic input. As a result, “over-filled” GABA vesicles are released that dampen excitation and aid restoration of PSN quiescence. During chronic sympathoexcitation challenges accompanied by reduced GABA input, GGS is subverted (due to low GABA release) and can therefore provide little opposition to synaptic excitation. Proposed studies will use state-of-the-art transgenic mouse models, optogenetics and virus-mediated gene over-expression and CRISPR-Cas9 knockdown to assess mechanisms and functional outcomes of GGS. Kinetics, sensitivity and efficacy of GGS will be established at the single PSN level using a novel horizontal brain slice preparation that preserves Glu input from the forebrain median preoptic nucleus (MnPO) as well as GABA input from the PVN peri-nuclear zone (PNZ). Retrogradely transported AAV will be injected into the PVN of vGlut2-Cre mice to express channelrhodopsin (ChR2) in glutamatergic MnPO-PVN neurons. Optogenetic activation will determine the capacity of MnPO inputs to drive GGS amongst RVLM-projecting PVN PSNs. Using vGlut2fl/fl mice, we will determine functional effects of GGS on GABA-A receptor inhibitory tone and SNA responses to forebrain angiotensin II (AngII) and hyperosmolality when glutamatergic MnPO neurons have normal (vGlut2 intact) or diminished (vGlut2 knockdown) capacity to release Glu from PVN synapses. To further illuminate in vivo mechanisms and efficacy of GGS, EAAT3 on PNZ GABA inputs to the PVN will be increased and decreased to grade PVN GABAergic tonus and the magnitude of PVN-driven SNA responses to (1) acute forebrain AngII and hyperosmolality as well as (2) sub-acute water deprivation and high salt intake. Proposed studies will provide unprecedented mechanistic insight into the physiological role GGS plays in generating and defending PVN PSN quiescence, and are essential for advancing the goal of preventing and reversing disease-promoting sympathoexcitation.

项目摘要 下丘脑旁脑核（PVN）的交感前神经元（PSN）是必不可少的驱动因素 交感神经活动（SNA）的生理和病理增加。也许他们最强大的财产是 它们的静止静止状态。早期研究将静止与突触抑制的优势联系在一起， 但是，仅在A中理解并捍卫PVN中建立和捍卫Gabaergic抑制性Tonus的机制 基本水平。这是一个重要的知识差距，因为增加了PVN驱动的SNA的病原因子 必须最终颠覆或压倒调节PSN静止状态的机制。在初步 研究，我们发现了一种新型PVN的突触前机制，称为“谷氨酸-GABA 加强（GGS）”，这可以增加合成谷氨酸（GLU）Spilover空间中的GABA能抑制作用。为此， GGS通过摄取来调节GABA-A受体介导的抑制性突触后电流（IPSC）的放大器 神经元兴奋性氨基酸转运蛋白的合成释放的GLU，表面上是局部GABA终端 3（eaat3）。一旦内部化，GLU将转换为GABA，然后将GABA分子包装成突触蔬菜 在密度高于高度时。假设急性增加PVN驱动的SNA的压力源增加 突触GLU释放而不改变外部GABA能输入。结果，“过度填充”的GABA蔬菜是 释放了Dancen兴奋和辅助PSN静止的恢复。在慢性交感神经期间 通过减少的GABA输入而实现的挑战，GGS被颠覆（由于GABA释放较低），因此可以 几乎没有反对突触兴奋。拟议的研究将使用最先进的转基因小鼠模型， 光遗传学和病毒介导的基因过表达和CRISPR-CAS9敲低，以评估机制和 GGS的功能结果。 GGS的动力学，灵敏度和效率将在单个PSN水平上建立 使用一种新型的水平脑切片制剂，该制剂可保留前脑前核的GLU输入 （MNPO）以及PVN核核管（PNZ）的GABA输入。将注入逆运运行的AAV 进入VGLUT2-CRE小鼠的PVN，以在谷氨酸能MNPO-PVN神经元中表达通道Rhopoptin（ChR2）。 光遗传激活将确定MNPO输入在RVLM-PROXTING PVN中驱动GGS的能力 PSN。使用vglut2fl/fl小鼠，我们将确定GGS对GABA-A受体抑制性张力和 当谷氨酸能MNPO神经元具有谷氨酸血管紧张素II（ANGII）和高胶质的SNA反应 正常（VGLUT2完整）或减小（VGLUT2敲低）从PVN突触中释放GLU的能力。进一步 照明GGS的体内机制和效率，PNZ GABA输入到PVN上的EAAT3将增加，并且 降低到PVN级GABA能TONUS和PVN驱动的SNA对（1）急性前脑的响应的大小 Angii和高温性以及（2）亚急性水剥夺和高盐摄入量。拟议的研究将 提供对GGS在生成和捍卫中发挥的身体作用的前所未有的机械洞察力 PVN PSN静止 同情兴趣。