Chemogenetic Dissection of Neuronal and Astrocytic Compartment of the BOLD Signal

BOLD 信号神经元和星形细胞室的化学遗传学解剖

• 批准号: 9494695
• 负责人: Yen-Yu Ian Shih
• 金额: $ 50.44万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-13 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9494695
• 关键词: Action Potentials; Acute; Address; Affect; Anatomy; Animals; Astrocytes; Binding; Biology; Blood Vessels; Brain; Brain Mapping; Brain region; Cell physiology; Cells; Cerebrovascular Circulation; Cerebrum; Chronic; Chronic Phase; Complex; Cyclic AMP; Data; Data Set; Disease; Dissection; Endotoxins; Ensure; Exclusion; Foundations; Functional Magnetic Resonance Imaging; G Protein-Coupled Receptor Signaling; G alpha q Protein; G-Protein-Coupled Receptors; Generations; Genetic; Human; Imaging Device; Immunohistochemistry; Ion Channel; Light; Link; Lipopolysaccharides; Measurement; Mediating; Mental disorders; Methods; Modeling; Molecular; Neurodegenerative Disorders; Neurons; Oxygen; Pathologic; Pathway interactions; Pharmacology; Phase; Positioning Attribute; Predisposition; Proxy; Role; Scanning; Signal Pathway; Signal Transduction; Solid; Source; Techniques; Time; Transfection; astrogliosis; base; blood oxygen level dependent; blood oxygenation level dependent response; brain cell; cerebral blood volume; designer receptors exclusively activated by designer drugs; experience; experimental study; hemodynamics; improved; in vivo; metabolic rate; multimodality; neuroinflammation; neurotransmission; neurovascular coupling; novel; paracrine; recruit; release factor; response; somatosensory; tool

PROJECT SUMMARY Blood-oxygenation-level-dependent functional magnetic resonance imaging (BOLD fMRI) is widely used in to study human brain function; however the cellular and molecular mechanisms underlying the BOLD signal remain poorly understood. The BOLD signal is highly complex as it represents disproportionate interactions of cerebral blood flow (CBF), cerebral blood volume (CBV), and cerebral metabolic rate of oxygen (CMRO2) during neuronal activation. On the cellular level, while lactate generated from the astrocytes is used to sustain neuronal activity, astrocytic signaling also releases vasoactive compounds, indicating that BOLD could reflect a combined response of both neurons and astrocytes. Dissecting the fractional contribution of neurons, astrocytes, their crosstalk, and specific molecular signaling cascades to BOLD, CBF, CBV, and CMRO2 is crucial to more accurately model and interpret BOLD data. Unlike neurons, astrocytes lack the appropriate ion channels to propagate action potentials but rather mediate their activity predominantly through G-protein-coupled receptors (GPCRs). Substantial pharmacological evidence has suggested that astrocytic GPCRs are key molecular players in their control of CBF through their binding of various paracrine compounds released by neurons. Interestingly, some studies have questioned this conclusion, demonstrating that activation of astrocytic Gq-GPCRs are not critical for CBF modulation. Further, it remains unclear how other GPCR subfamilies (i.e., Gs and Gi) affect BOLD. These controversies and missing data prompted us to systematically investigate the following questions for the first time: 1) whether selective activation of astrocytic Gq-, Gs-, or Gi-GPCR signaling pathways modulate hemodynamic or BOLD responses in vivo, 2) can neurons or astrocytes independently elicit hemodynamic and BOLD responses without the involvement of the other, and 3) what molecular mechanisms contribute to the BOLD signal disruption in disease states where astrogliosis and neuronal remodeling occur. We will employ cutting-edge chemogenetic tools, a.k.a. Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), to selectively modulate Gq-, Gs- and Gi-signaling cascades in neurons and astrocytes. We will also utilize multimodal fMRI tools that allow measurement of BOLD, CBV, CBF, and CMRO2 changes in a single setting. Additionally, we will perform immunohistochemistry in all subjects, allowing within-subject comparison of the number or ratio of activated/suppressed cells and the observed hemodynamic responses. In Aim 1, we propose to use DREADDs to directly activate the signaling of each of the main astrocytic GPCR subfamily during fMRI, allowing precise interrogation of the astrocytic signaling pathways that contribute to changes in BOLD. In Aim 2a, we will employ a novel means to concomitantly suppress astrocytic cyclic-adenosine-monophosphate-related activity using Gi-DREADD during neuronal activation. Conceptually, this will “remove” the astrocytes during fMRI mapping of neuronal activation. In Aim 2b, we will silence neurons using Gi-DREADD while exclusively activating Gq- and Gs-DREADDs in astrocytes. This will ensure the exclusion of potential paracrine factors released from neurons that could directly modulate vascular tone. In Aim 3, we will employ an endotoxin-induced model of chronic neuroinflammation using lipopolysaccharide (LPS), thus creating well-characterized region and time-specific pathological profiles. We will scan these animals identically as described in Aim 2, but under two stages of neuroinflammation: 1) the acute phase (3 days after LPS exposure) which consists of peak presence of astrogliosis with very minimal neuronal remodeling, and 2) the chronic phase (90 days after LPS exposure) which consists of moderate to mild astrogliosis with substantial neuronal remodeling. We anticipate that our results will reveal the respective roles of neurons, astrocytes, and specific GPCR signaling cascades in the generation of BOLD. We also expect our study to shed considerable light on the mechanisms by which the BOLD signal can be disrupted in disease states involving neuroinflammation. Lastly, we will perform BOLD modeling with the unique datasets to be generated in this study, with the ultimate hope of building a more solid foundation for human brain mapping.

项目概要 血氧水平依赖性功能磁共振成像（BOLD fMRI）得到广泛应用 用于研究人类大脑功能；然而，BOLD 背后的细胞和分子机制 信号仍然知之甚少，因为它代表不成比例。 脑血流量（CBF）、脑血容量（CBV）和脑氧代谢率的相互作用 (CMRO2) 在神经元激活过程中，在细胞水平上使用星形胶质细胞产生的乳酸。 为了维持神经元活动，星形胶质细胞信号传导也会释放血管活性化合物，表明 BOLD 可以反映神经元和星形胶质细胞的联合反应。 神经元、星形胶质细胞、它们的串扰以及与 BOLD、CBF、CBV、 CMRO2 对于更准确地建模和解释 BOLD 数据至关重要。 与神经元不同，星形胶质细胞缺乏适当的离子通道来传播动作电位，而是 主要通过 G 蛋白偶联受体 (GPCR) 介导其活性。 药理学证据表明星形胶质细胞 GPCR 是控制 CBF 通过与神经元释放的各种旁分泌化合物结合。 质疑这一结论，证明星形胶质细胞 Gq-GPCR 的激活对于 CBF 并不重要 此外，其他 GPCR 亚家族（即 Gs 和 Gi）如何影响 BOLD 仍不清楚。 争议和数据缺失促使我们首先系统地调查以下问题 时间：1) 星形胶质细胞 Gq-、Gs- 或 Gi-GPCR 信号通路的选择性激活是否会调节 体内血流动力学或 BOLD 反应，2）神经元或星形胶质细胞可以独立引发血流动力学和 无需他人参与的大胆反应，以及 3) 哪些分子机制有助于 在星形胶质细胞增生和神经元重塑发生的疾病状态下，大胆的信号中断。 我们将采用尖端的化学遗传学工具，即由以下人员独家激活的设计受体 设计药物 (DREADD)，选择性调节神经元中的 Gq、Gs 和 Gi 信号级联 我们还将利用多模态 fMRI 工具来测量 BOLD、CBV、CBF 和 此外，我们将对所有受试者进行免疫组织化学分析，从而允许。 受试者内激活/抑制细胞的数量或比率以及观察到的血流动力学的比较 在目标 1 中，我们建议使用 DREADD 直接激活每个主节点的信令。 fMRI 期间的星形胶质细胞 GPCR 亚家族，允许精确询问星形胶质细胞信号通路 在目标 2a 中，我们将采用一种新颖的方法来同时抑制星形胶质细胞。 从概念上讲，在神经元激活过程中使用 Gi-DREADD 进行环腺苷一磷酸相关活动。 这将在神经激活的 fMRI 映射过程中“移除”星形胶质细胞。在目标 2b 中，我们将保持沉默。 使用 Gi-DREADD 的神经元同时专门激活星形胶质细胞中的 Gq- 和 Gs-DREADD，这将确保。 排除神经元释放的可直接调节血管张力的潜在旁分泌因子。 目标 3，我们将采用脂多糖建立内毒素诱导的慢性神经炎症模型 （LPS），从而创建特征明确的区域和特定时间的病理特征，我们将扫描这些。 与目标 2 中描述的相同的动物，但处于神经炎症的两个阶段：1) 急性期 (3 LPS 暴露后几天），其中包括星形胶质细胞增生的高峰期和极少的神经元 重塑，以及 2) 慢性期（LPS 暴露后 90 天），包括中度至轻度 我们预计我们的结果将揭示各自的作用。 我们还期望我们的神经元、星形胶质细胞和特定 GPCR 信号级联在 BOLD 的生成中发挥作用。 研究揭示了疾病中 BOLD 信号被破坏的机制 最后，我们将使用独特的数据集进行大胆建模。 这项研究产生的结果，最终希望为人类大脑绘图奠定更坚实的基础。