Dynamics of Cellular Brain Metabolism Using Mass Spectrometry Imaging

项目概要/摘要 大脑功能需要大量的代谢能量，通常是短暂的局部爆发。每个脑细胞的能力 响应这种能量需求的代谢机制对于细胞的直接功能特性和细胞的功能特性都是至关重要的。 大脑信号和大脑的长期健康。虽然核心代谢途径是由 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代谢 以满足对能源的迫切需求。例如，神经元和星形胶质细胞被认为 补充代谢作用;几乎不断地或以非常高的速率间歇性地放电的神经元，可能 它们的代谢方式与通常静止的神经元不同。代谢功能障碍会导致 疾病和神经退行性变，细胞类型之间的代谢差异可能是细胞- 神经退行性疾病中发现的脑细胞类型特异性脆弱性。 研究完整组织中特定细胞类型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切片上进行生理实验，使用神经元刺激， 13 C代谢标记和代谢抑制剂。然后，我们将使用质谱成像（MSI）， 定量绘制脑切片中大量代谢物的水平。快速热 在刺激或应用后的特定时间保存（快速加热和冷冻）脑切片 13 C标记的代谢物使我们能够测量代谢变化的精细时间过程， 使我们能够从特定的细胞类型中获得代谢测量结果。齿状颗粒细胞代谢 行为将通过海马致密颗粒细胞层的MSI分离;来自海马致密颗粒细胞层的代谢信号将被分离。 单个星形胶质细胞和快速尖峰的小清蛋白阳性中间神经元将使用细胞类型特异性免疫荧光法分离。 签名，基于与已建立的抗体标记的对应关系。 我们将使用这些方法来构建这些单个细胞类型如何使用其核心代谢的丰富图片 途径（糖酵解，戊糖磷酸途径，TCA循环），无论是在基线和动态响应 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 刺激，然后戊糖磷酸途径开始参与。使用燃料分子的实验 将揭示神经元和星形胶质细胞如何灵活地利用混合能量， 源通过将代谢物水平的数据与单个代谢途径的活性数据相结合， 不仅可以了解代谢变化是什么，还可以了解沿着每个途径的位置， 监管发生变化。我们将检验这一假设，即DGC，星形胶质细胞和快速尖峰中间神经元 使用它们的核心代谢途径来响应神经元刺激。 该项目将揭示健康脑组织中不同细胞类型的独特代谢， 对于未来的工作如何代谢可能出错（如怀疑）在老化或神经退行性疾病。

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.