Chemogenetic Dissection of Neuronal and Astrocytic Compartment of the BOLD Signal

项目总结 血氧水平依赖的功能磁共振成像(BOLD FMRI)被广泛应用 用于研究人脑功能；然而，BOLD背后的细胞和分子机制 信号仍然知之甚少。粗体信号非常复杂，因为它代表不成比例的 (CMRO2)在神经元激活过程中。在细胞水平上，而使用从星形胶质细胞产生的乳酸 为了维持神经元的活动，星形细胞信号也会释放血管活性化合物，这表明BOLD 可以反映神经元和星形胶质细胞的联合反应。剖析了小数的贡献 神经元、星形胶质细胞、它们的串扰和特定的分子信号级联到BOLD、CBF、CBV、 而CMRO2对于更准确地建模和解释大胆数据至关重要。 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，而不是 它们的活性主要通过G蛋白偶联受体(GPCRs)来调节。相当可观 药理学证据表明，星形胶质细胞GPCRs是其控制 CBF通过与神经元释放的各种旁分泌化合物结合而发挥作用。有趣的是，一些研究 质疑这一结论，表明星形细胞GQ-GPCRs的激活对CBF并不关键 调制。这些 争议和数据缺失促使我们首次系统地研究了以下问题 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调节 血流动力学或体内大胆反应，2)神经元或星形胶质细胞是否独立地引起血流动力学和 在没有对方参与的情况下做出大胆的反应，以及3)什么分子机制对 在发生星形胶质细胞增生症和神经元重塑的疾病状态下，大胆的信号中断。 我们将使用尖端的化学遗传工具，也就是。专一激活的设计师受体 设计药物(DREADD)，选择性地调节神经元和 星形胶质细胞。 CMRO2在单个设置中变化。此外，我们将对所有受试者进行免疫组织化学检查，允许 受试者内激活/抑制细胞的数量或比率与观察到的血流动力学的比较 回应。在目标1中，我们建议使用DREADD来直接激活每个主要 星形细胞GPCR亚家族在功能磁共振期间，允许精确询问星形细胞信号通路， 大胆地为变革做出贡献。在目标2a中，我们将使用一种新的方法来同时抑制星形细胞 在神经元激活过程中使用GI-DREADD的环腺苷-一磷酸相关活性。从概念上讲， 这将在神经元激活的功能磁共振成像过程中“移除”星形胶质细胞。在目标2b中，我们将保持沉默 神经元使用GI-DREADD，同时独占激活星形胶质细胞中的GQ-和Gs-DREADD。这将确保 排除可能直接调节血管张力的神经元释放的潜在旁分泌因子。在……里面 目的3，我们将采用内毒素内毒素诱导的慢性神经炎模型。 (LP)，从而创建具有良好特征的区域和时间特定的病理特征。我们会扫描这些 动物与目标2相同，但在神经炎症的两个阶段：1)急性期(3 重塑，2)慢性期(脂多糖暴露后90天)，由中度到轻度组成 星形胶质细胞增生症伴实质性神经元重塑。我们预计，我们的结果将揭示各自的角色 神经元、星形胶质细胞和特定的GPCR信号在BOLD的产生中级联。我们也期待我们的 研究揭示了BOLD信号在疾病中可能被干扰的机制 涉及神经炎症的状态。最后，我们将对要使用的唯一数据集执行粗体建模 在这项研究中产生的，最终希望为构建更坚实的人脑图谱奠定基础。

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.