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: 10618815
• 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/10618815
• 关键词: Acute; Angiotensin II; Animals; Antihypertensive Agents; Automobile Driving; Autonomic Dysfunction; Bathing; Behavior; Brain; CRISPR/Cas technology; Cardiovascular Diseases; Cell Nucleus; 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; Transgenic Mice; Vesicle; Viral; Virulence Factors; Virus; acute stress; clinical efficacy; density; excitatory amino acid transporter 3; excitatory neuron; 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; 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.

项目概要 下丘脑室旁核 (PVN) 的前交感神经元 (PSN) 是 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 它们的放电静止状态。早期研究将静止与突触抑制的主导地位联系起来， 但在 PVN 中建立和保护 GABA 能抑制张力的机制仅在以下方面被了解： 初级水平。这是一个重要的知识差距，因为增加 PVN 驱动的 SNA 的致病因素 最终必须颠覆或压倒调节 PSN 静态静息状态的机制。在初步 研究中，我们发现了一种 PVN 新颖的突触前机制，称为“谷氨酸-GABA 强化（GGS）”，这会随着突触谷氨酸（Glu）溢出的速度增加 GABA 能抑制。为此， GGS 通过摄取调节 GABA-A 受体介导的抑制性突触后电流 (IPSC) 的幅度 突触释放的 Glu，表面上通过神经元兴奋性氨基酸转运蛋白进入局部 GABA 末端 3（EAAT3）。一旦内化，Glu 就会转化为 GABA，GABA 分子会被包装到突触小泡中 大于正常密度。假设急剧增加 PVN 驱动的 SNA 的压力源会增加 突触 Glu 释放而不改变外在 GABA 能输入。结果，“过度填充”的 GABA 囊泡 释放抑制激发并帮助恢复 PSN 静止。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 释放量低），因此可以 对突触兴奋几乎没有抵抗作用。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表达和 CRISPR-Cas9 敲除来评估机制和 GGS 的功能结果。 GGS 的动力学、敏感性和功效将在单一 PSN 水平上建立 使用一种新颖的水平脑切片制备方法，保留来自前脑正中视前核的 Glu 输入 (MnPO) 以及来自 PVN 核周区 (PNZ) 的 GABA 输入。逆行运输的 AAV 将被注射 进入 vGlut2-Cre 小鼠的 PVN，以在谷氨酸能 MnPO-PVN 神经元中表达视紫红质通道 (ChR2)。 光遗传学激活将决定 MnPO 输入在 RVLM 投射的 PVN 中驱动 GGS 的能力 PSN。使用 vGlut2fl/fl 小鼠，我们将确定 GGS 对 GABA-A 受体抑制音的功能影响和 当谷氨酸能 MnPO 神经元具有时 SNA 对前脑血管紧张素 II (AngII) 和高渗透压的反应 从 PVN 突触释放 Glu 的能力正常（vGlut2 完整）或减弱（vGlut2 敲低）。为了进一步 阐明 GGS、EAAT3 对 PNZ GABA 对 PVN 输入的体内机制和功效将增加， 降低至PVN GABA能紧张度和PVN驱动的SNA对(1)急性前脑反应的强度 AngII 和高渗透压以及 (2) 亚急性缺水和高盐摄入。拟议的研究将 为 GGS 在生成和防御中发挥的生理作用提供前所未有的机制见解 PVN PSN 静止，对于推进预防和逆转疾病促进的目标至关重要 交感神经兴奋。

项目成果

High dietary salt amplifies osmoresponsiveness in vasopressin-releasing neurons.

• DOI: 10.1016/j.celrep.2021.108866
• 发表时间: 2021-03-16
• 期刊: Cell reports
• 影响因子: 8.8
• 作者: Levi DI;Wyrosdic JC;Hicks AI;Andrade MA;Toney GM;Prager-Khoutorsky M;Bourque CW
• 通讯作者: Bourque CW

Anterior basolateral amygdala neurons comprise a remote fear memory engram.

• DOI: 10.3389/fncir.2023.1167825
• 发表时间: 2023
• 期刊: FRONTIERS IN NEURAL CIRCUITS
• 影响因子: 3.5
• 作者: Hammack, Robert J.;Fischer, Victoria E.;Andrade, Mary Ann;Toney, Glenn M.
• 通讯作者: Toney, Glenn M.