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.

项目摘要 下丘脑室旁核（PVN）的交感神经前神经元（PSN）是下丘脑-垂体-垂体-肾上腺皮质系统的重要驱动因子。 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 他们的放电静止状态。早期的研究将静止与突触抑制的优势联系起来， 但在室旁核中建立和保护GABA能抑制性紧张的机制仅在 初级水平。这是一个重要的知识差距，因为增加PVN驱动SNA的致病因素 最终必须颠覆或压倒调节PSN静止状态的机制。初步 研究中，我们发现了一种突触前机制，这是新的PVN，被称为"谷氨酸-GABA 增强（GGS）"，其与突触谷氨酸（Glu）溢出同步增加GABA能抑制。为此， GGS调节GABA-A受体介导的抑制性突触后电流（IPSC）的幅度，通过摄取 突触释放的Glu，表面上进入当地的GABA终端，由神经元兴奋性氨基酸转运蛋白 3（EAAT3）。一旦内化，谷氨酸就会转化为GABA，GABA分子被包装到突触小泡中 比正常密度高。假设急剧增加PVN驱动SNA的应激源增加 突触Glu释放而不改变外源性GABA能输入。因此，"过度填充"的GABA囊泡是 释放抑制兴奋和援助恢复PSN静止。在慢性交感神经兴奋过程中 由于GABA输入减少，GGS被破坏（由于GABA释放低），因此可以 对突触兴奋几乎没有抵抗力。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敲低来评估机制， GGS的功能结果。GGS的动力学、灵敏度和有效性将在单一PSN水平上确定 使用一种新的水平脑切片制备，其保留了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。将注射逆行转运的AAV 进入vGlut2-Cre小鼠的PVN以在谷氨酸能MnPO-PVN神经元中表达通道视紫红质（ChR2）。 光遗传激活将决定MnPO输入在RVLM投射PVN中驱动GGS的能力 PSN。使用vGlut2fl/fl小鼠，我们将确定GGS对GABA-A受体抑制性张力的功能作用， SNA对前脑血管紧张素II（AngII）和高渗的反应时， 从PVN突触释放Glu的能力正常（vGlut2完整）或降低（vGlut2敲低）。进一步 阐明GGS、EAAT 3对PNZ GABA输入PVN的体内机制和功效将增加， PVN GABA能紧张度和PVN驱动SNA对（1）急性前脑 AngII和高渗以及（2）亚急性缺水和高盐摄入。拟议的研究将 提供了前所未有的机制洞察GGS在生成和防御中发挥的生理作用 PVN PSN静止，并且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.