Dynamics of Cellular Brain Metabolism Using Mass Spectrometry Imaging

项目摘要/摘要 大脑功能需要大量的新陈代谢能量，通常是简单的局部爆发。每个脑细胞的能力 对这种能量需求做出反应的新陈代谢机制对心脏的直接功能特性至关重要 大脑信号和大脑的长期健康。尽管核心代谢途径是由 所有类型的脑细胞，我们假设不同的脑细胞类型可能强调不同的新陈代谢 组件，以应对紧急的能源需求。例如，神经元和星形胶质细胞被认为在 互补的代谢作用；以及几乎不间断地或以非常高的频率间歇性地放电的神经元，可能 管理它们的新陈代谢不同于典型的静止神经元。新陈代谢障碍会导致 疾病和神经退化，细胞类型之间的新陈代谢差异可能是细胞- 神经退行性疾病中出现的脑细胞特定类型的脆弱性。 研究特定细胞类型在完整组织中的独特、动态代谢反应，而不是细胞 培养，我们将在小鼠的急性脑片上进行生理学实验，使用神经刺激， 13C代谢标记物和代谢抑制剂。然后，我们将使用质谱仪成像(MSI)来 定量绘制出这些脑片薄片中众多代谢物的水平。快速散热 脑片在刺激或应用后的特定时间保存(闪热和冷冻) 13C标记的代谢物使我们能够测量代谢变化的精细时间进程，以及成像能力 使我们能够获得特定细胞类型的代谢测量结果。齿状颗粒细胞(DGC)代谢 行为将通过海马体致密颗粒细胞层的MSI来分离；来自 单个星形胶质细胞和快速放电的小白蛋白阳性中间神经元将使用特定的细胞类型 签名，基于与已建立的抗体标记的一致性。 我们将使用这些方法来构建这些单个细胞类型如何利用其核心代谢的丰富图景 糖酵解途径(糖酵解、磷酸戊糖途径、三氯乙酸循环)，在基线和动态响应 神经刺激。我们将测试特定的假设，即在DGC中，神经元糖酵解在以下情况下上调 刺激，然后戊糖磷酸途径开始运作。使用燃料分子的实验 不同的稳定同位素标记将揭示神经元和星形胶质细胞如何灵活地利用混合的能量 消息来源。通过将代谢物水平的数据与单个代谢途径的活动数据结合起来，我们 不仅可以了解代谢的变化是什么，而且还可以了解每条途径上的关键位置 监管方面的变化就会发生。我们将检验这样的假设，即星形胶质细胞、星形胶质细胞和快速突起的中间神经元 以不同的方式使用其核心代谢途径，以响应神经元的刺激。 该项目将揭示不同类型细胞在健康脑组织中的独特新陈代谢，并奠定基础 未来关于新陈代谢如何在衰老或神经退行性疾病中出错(如人们怀疑的那样)的工作。

PROJECT SUMMARY/ABSTRACT Brain function demands a lot of metabolic energy, often in brief, local bursts. The ability of each brain cell’s metabolic machinery to respond to this energy demand is crucial both for the immediate functional properties of brain signaling and for the long-term health of the brain. Although the core metabolic pathways are shared by all types of brain cells, we hypothesize that different brain cell types are likely to emphasize different metabolic components in response to acute energy demand. For instance, neurons and astrocytes are thought to play complementary metabolic roles; and neurons that fire nearly constantly, or episodically at very high rates, may manage their metabolism differently from typically quiescent neurons. Dysfunction in metabolism can lead to disease and neurodegeneration, and the metabolic differences between cell types may underlie the very cell- type-specific vulnerabilities of brain cells seen in neurodegenerative diseases. To study the distinctive, dynamic metabolic responses of specific cell types in intact tissue, rather than cell culture, we will perform physiological experiments on acute brain slices from mice, using neuronal stimulation, 13C metabolic labeling, and metabolic inhibitors. We will then use mass spectrometry imaging (MSI) to quantitatively map the levels of numerous metabolites in thin sections from those brain slices. Fast thermal preservation (flash heating and freezing) of the brain slices at specific times after stimulation or application of 13C-labeled metabolites allows us to measure a fine time course of metabolic changes, and the imaging capability allows us to obtain metabolic measurements from specific cell types. Dentate granule cell (DGC) metabolic behavior will be isolated by MSI of the compact granule cell layer of the hippocampus; the metabolic signals from single astrocytes and fast-spiking parvalbumin-positive interneurons will be isolated using cell-type specific signatures, based on correspondence with labeling by established antibodies. We will use these methods to construct a rich picture of how these individual cell types use their core metabolic pathways (glycolysis, pentose phosphate pathway, TCA cycle), both at baseline and dynamically in response to neuronal stimulation. We will test the specific hypotheses that in DGCs, neuronal glycolysis is upregulated after stimulation, and that the pentose phosphate pathway then becomes engaged. Experiments using fuel molecules with different stable isotope labels will reveal how neurons and astrocytes flexibly utilize a mixture of energy sources. By combining data on metabolite levels with data on the activity of individual metabolic pathways, we can learn not only what the metabolic changes are, but also the positions along each pathway at which key regulatory changes occur. And we will test the hypothesis that DGCs, astrocytes, and fast-spiking interneurons use their core metabolic pathways distinctively in response to neuronal stimulation. This project will reveal the distinctive metabolism of different cell types in healthy brain tissue and lay a foundation for future work on how metabolism may go awry (as is suspected) in aging or in neurodegenerative disease.