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 下丘脑室旁核(PVN)的交感前神经元(PSN)是 生理性和病理性交感神经活动(SNA)增加。也许他们最健壮的属性是 它们静息时的放电状态是静止的。早期的研究将静止与突触抑制的优势联系在一起， 但在下丘脑室旁核建立和防御GABA能抑制张力的机制仅在 初级水平。这是一个重要的知识鸿沟，因为增加PVN驱动的SNA的致病因素 必须最终颠覆或压倒调节PSN静止状态的机制。在预赛中 研究发现，我们发现了一种对下丘脑室旁核来说是新的突触前机制，称为“谷氨酸-GABA” 加强(GGS)“，这增加了GABA能抑制与突触谷氨酸(Glu)溢出的步伐。要做到这一点， GGS通过摄取调节GABA-A受体介导的抑制性突触后电流的幅度 神经元兴奋性氨基酸转运体通过突触释放谷氨酸，表面上进入局部GABA终末 3(EAAT3)。一旦内化，谷氨酸被转化为GABA，GABA分子被包装成突触小泡 比正常密度大。假设急剧增加PVN驱动的SNA的应激源将增加 突触释放谷氨酸而不改变外源性GABA能输入。结果，“过度充满”的GABA囊泡 释放抑制兴奋和帮助恢复PSN静止的物质。在慢性交感神经兴奋期间 伴随着GABA输入减少的挑战，GGS被颠覆(由于GABA释放较少)，因此 对突触兴奋几乎没有抵触作用。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度表达和CRISPR-Cas9敲除以评估机制和 GGS的功能结局。GGS的动力学、敏感性和有效性将建立在单一的PSN水平上 使用一种新的保留前脑正中视前核谷氨酸输入的水平脑片制备方法 (MnPO)以及来自PVN核周带(PNZ)的GABA输入。倒退运输的AAV将被注射 进入vGlut2-CRE小鼠的PVN，在谷氨酸能MnPO-PVN神经元中表达通道视紫红质(ChR2)。 光基因激活将决定MnPO输入在RVLM投射的PVN中驱动GG的能力 PSNS。利用vGlut2fl/fl小鼠，我们将确定GGS对GABA-A受体抑制张力和 谷氨酸能MnPO神经元对前脑血管紧张素II和高渗透压的反应 正常(vGlut2完整)或减弱(vGlut2击倒)从PVN突触释放Glu的能力。为了进一步 阐明GGS、EAAT3对PNZ GABA传入下丘脑室旁核的体内机制和作用 对(1)急性前脑的PVN GABA能张力和PVN驱动的SNA反应的大小 血管紧张素转换酶与高渗血症以及(2)亚急性缺水和高盐摄入。拟议的研究将 对GGS在生成和防御中所起的生理作用提供了前所未有的机械洞察力 PVN和PSN的静止，对于推进预防和逆转致病的目标是必不可少的 交感神经兴奋。