Lerchundi Rodrigo, Huang Na, Rose Christine R
Institute of Neurobiology, Faculty of Mathematics and Natural Sciences, Heinrich Heine University Düsseldorf, Düsseldorf, Germany.
Front Cell Neurosci. 2020 Apr 21;14:80. doi: 10.3389/fncel.2020.00080. eCollection 2020.
Genetically encoded nanosensors such as the FRET-based adenosine triphosphate (ATP) sensor ATeam enable the measurement of changes in ATP levels inside cells, promoting our understanding of metabolic interactions between astrocytes and neurons. The sensors are usually well characterized but display altered properties when expressed inside cells, precluding a meaningful conversion of changes in FRET ratios into changes in intracellular ATP concentrations ([ATP]) on the basis of their properties. Here, we present an experimental strategy for the intracellular calibration of two different variants of ATeam in organotypic tissue slice culture of the mouse brain. After cell-type-specific expression of the sensors in astrocytes or neurons, slices were first perfused with a saline containing the saponin β-escin to permeabilize plasma membranes for ATP. Next, cells were depleted of ATP by perfusion with ATP-free saline containing metabolic inhibitors. Finally, ATP was re-added at defined concentrations and resulting changes in the FRET ratio recorded. When employing this protocol, ATeam1.03 expressed in astrocytes reliably responds to changes in [ATP], exhibiting an apparent of 9.4 mM. The high-affinity sensor ATeam1.03 displayed a significantly lower intracellular of 2.7 mM. On the basis of these calibrations, we found that induction of a recurrent neuronal network activity resulted in an initial transient increase in astrocytic [ATP] by ~0.12 mM as detected by ATeam1.03, a result confirmed using ATeam1.03. In neurons, in contrast, [ATP] immediately started to decline upon initiation of a network activity, amounting to a decrease by an average of 0.29 mM after 2 min. Taken together, our results demonstrate that ATeam1.03 and ATeam1.03 display a significant increase in their apparent when expressed inside cells as compared with . Moreover, they show that both ATeam variants enable the quantitative detection of changes of astrocytic and neuronal [ATP] in the physiological range. ATeam1.03, however, seems preferable because its is close to baseline ATP levels. Finally, our data support the idea that synchronized neuronal activity initially stimulates the generation of ATP in astrocytes, presumably through increased glycolysis, whereas ATP levels in neurons decline.
基于荧光共振能量转移（FRET）的三磷酸腺苷（ATP）传感器ATeam等基因编码纳米传感器，能够测量细胞内ATP水平的变化，有助于我们了解星形胶质细胞与神经元之间的代谢相互作用。这些传感器通常已得到充分表征，但在细胞内表达时会表现出不同的特性，这使得无法根据其特性将FRET比值的变化有意义地转化为细胞内ATP浓度（[ATP]）的变化。在此，我们提出了一种在小鼠脑器官型组织切片培养中对两种不同变体的ATeam进行细胞内校准的实验策略。在星形胶质细胞或神经元中进行传感器的细胞类型特异性表达后，首先用含有皂苷β - 七叶皂苷的生理盐水灌注切片，以使质膜对ATP通透。接下来，用含有代谢抑制剂的无ATP生理盐水灌注细胞，使细胞内的ATP耗尽。最后，以确定的浓度重新添加ATP，并记录由此产生的FRET比值变化。采用该方案时，在星形胶质细胞中表达的ATeam1.03能可靠地响应[ATP]的变化，其表观解离常数为9.4 mM。高亲和力传感器ATeam1.03显示出显著更低的细胞内解离常数，为2.7 mM。基于这些校准，我们发现，通过ATeam1.03检测到，反复性神经元网络活动的诱导导致星形胶质细胞[ATP]最初短暂增加约0.12 mM，使用ATeam1.03也证实了这一结果。相比之下，在神经元中，网络活动开始后[ATP]立即开始下降，2分钟后平均下降0.29 mM。综上所述，我们的结果表明，与在体外相比，ATeam1.03和ATeam1.03在细胞内表达时表观解离常数显著增加。此外，它们表明两种ATeam变体都能够在生理范围内定量检测星形胶质细胞和神经元[ATP]的变化。然而，ATeam1.03似乎更可取，因为其解离常数接近基线ATP水平。最后，我们的数据支持这样一种观点，即同步的神经元活动最初会刺激星形胶质细胞中ATP的生成，可能是通过增加糖酵解实现的，而神经元中的ATP水平则下降。
