Chemogenetic Dissection of Neuronal and Astrocytic Compartment of the BOLD Signal

项目摘要 血氧水平依赖性功能磁共振成像（BOLD fMRI）广泛应用于 用于研究人类大脑功能;然而，BOLD背后的细胞和分子机制 对信号仍然知之甚少。BOLD信号是高度复杂的，因为它代表不成比例的 脑血流量（CBF）、脑血容量（CBV）和脑氧代谢率的相互作用 （CMRO 2）在神经元激活期间。在细胞水平上，虽然使用从星形胶质细胞产生的乳酸 为了维持神经元的活动，星形胶质细胞信号也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。分析了 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， CMRO 2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， 主要通过G蛋白偶联受体（GPCR）介导其活性。实质性 药理学证据表明，星形胶质细胞GPCR是其控制细胞凋亡的关键分子。 CBF通过其结合神经元释放的各种旁分泌化合物。有趣的是， 我对这一结论提出了质疑，证明星形胶质细胞Gq-GPCR的激活对CBF并不重要 调变此外，仍不清楚其他GPCR亚家族（即，Gs和Gi）影响BOLD。这些 争议和缺失的数据促使我们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调节 2）神经元或星形胶质细胞是否能够独立地引起血液动力学和BOLD反应， BOLD反应没有参与的其他，和3）什么分子机制有助于 在发生星形胶质细胞增生和神经元重塑的疾病状态中BOLD信号中断。 我们会使用最先进的化学基因工具。设计师专用接收器 设计药物（DREADD），以选择性地调节神经元中的Gq-，Gs-和Gi-信号级联， 星形胶质细胞我们还将利用多模态功能磁共振成像工具，允许测量BOLD，CBV，CBF， CMRO 2在单个设置中发生变化。此外，我们将在所有受试者中进行免疫组织化学， 受试者内活化/抑制细胞的数量或比率与观察到的血流动力学比较 应答在目标1中，我们建议使用DREADD直接激活每个主要的信号传导。 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， 这是对BOLD变化的贡献。在Aim 2a中，我们将采用一种新的方法同时抑制星形胶质细胞增殖， 在神经元激活期间使用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映射期间“去除”星形胶质细胞。在目标2b中，我们将沉默 神经元使用Gi-DREADD，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能的旁分泌因子，这些因子可以直接调节血管张力。在 目的3，我们将采用内毒素诱导的慢性神经炎症模型使用脂多糖 (LPS)因此产生了良好表征的区域和时间特异性病理分布。我们将扫描这些 与目标2中所述相同，但处于两个神经炎症阶段的动物：1）急性期（3 LPS暴露后30天），其包括星形胶质细胞增生的峰值存在， 重塑，和2）慢性期（LPS暴露后90天），包括中度至轻度的 具有大量神经元重塑的星形胶质细胞增生。我们预计，我们的结果将揭示各自的作用， 神经元、星形胶质细胞和特异性GPCR信号级联在BOLD产生中的作用。我们也期待我们的 一项研究揭示了BOLD信号在疾病中被破坏的机制 涉及神经炎症的状态。最后，我们将使用唯一的数据集执行BOLD建模， 最终希望为人类大脑绘图建立更坚实的基础。

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.