Faculty of Mathematics and Natural Sciences, Institute of Neurobiology, Heinrich Heine University Düsseldorf, Düsseldorf, Germany.
Faculty of Medicine, Carl-Ludwig-Institute for Physiology, University of Leipzig, Leipzig, Germany.
J Neurosci Res. 2019 Aug;97(8):933-945. doi: 10.1002/jnr.24361. Epub 2018 Dec 1.
Active neurons require a substantial amount of adenosine triphosphate (ATP) to re-establish ion gradients degraded by ion flux across their plasma membranes. Despite this fact, neurons, in contrast to astrocytes, do not contain any significant stores of energy substrates. Recent work has provided evidence for a neuro-metabolic coupling between both cell types, in which increased glycolysis and lactate production in astrocytes support neuronal metabolism. Here, we established the cell type-specific expression of the Förster resonance energy transfer (FRET) based nanosensor ATeam1.03 ("Ateam") for dynamic measurement of changes in intracellular ATP levels in organotypic brain tissue slices. To this end, adeno-associated viral vectors coding for Ateam, driven by either the synapsin- or glial fibrillary acidic protein (GFAP) promoter were employed for specific transduction of neurons or astrocytes, respectively. Chemical ischemia, induced by perfusion of tissue slices with metabolic inhibitors of cellular glycolysis and mitochondrial respiration, resulted in a rapid decrease in the cellular Ateam signal to a new, low level, indicating nominal depletion of intracellular ATP. Increasing the extracellular potassium concentration to 8 mM, thereby mimicking the release of potassium from active neurons, did not alter ATP levels in neurons. It, however, caused in an increase in ATP levels in astrocytes, a result which was confirmed in acutely isolated tissue slices. In summary, our results demonstrate that organotypic cultured slices are a reliable tool for FRET-based dynamic imaging of ATP in neurons and astrocytes. They moreover provide evidence for an increased ATP synthesis in astrocytes, but not neurons, during periods of elevated extracellular potassium concentrations.
活性神经元需要大量的三磷酸腺苷 (ATP) 来重建其质膜离子流破坏的离子梯度。尽管如此，神经元与星形胶质细胞不同，不含有任何显著的能量底物储存。最近的研究为这两种细胞类型之间的神经代谢偶联提供了证据，其中星形胶质细胞中的糖酵解和乳酸生成增加支持神经元代谢。在这里，我们建立了基于荧光共振能量转移（FRET）的纳米传感器 ATeam1.03（“Ateam”）的细胞类型特异性表达，用于动态测量器官型脑切片中细胞内 ATP 水平的变化。为此，编码 Ateam 的腺相关病毒载体，由突触蛋白或胶质纤维酸性蛋白（GFAP）启动子驱动，分别用于神经元或星形胶质细胞的特异性转导。通过用细胞糖酵解和线粒体呼吸的代谢抑制剂灌注组织切片来诱导化学缺血，导致细胞 Ateam 信号迅速下降到一个新的低水平，表明细胞内 ATP 几乎耗尽。将细胞外钾浓度增加到 8 mM，模拟来自活性神经元的钾释放，不会改变神经元中的 ATP 水平。然而，它会导致星形胶质细胞中 ATP 水平的增加，这一结果在急性分离的组织切片中得到了证实。总之，我们的结果表明器官型培养切片是用于基于 FRET 的神经元和星形胶质细胞中 ATP 动态成像的可靠工具。它们还提供了证据表明，在细胞外钾浓度升高期间，星形胶质细胞而非神经元中 ATP 合成增加。
