Quantitative Imaging of Changes in Astrocytic and Neuronal Adenosine Triphosphate Using Two Different Variants of ATeam

Abstract

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 in vitro 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 in vitro 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 K D of 9.4 mM. The high-affinity sensor ATeam1.03 YEMK displayed a significantly lower intracellular K D 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 YEMK , 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 YEMK and ATeam1.03 display a significant increase in their apparent K D when expressed inside cells as compared with in vitro. Moreover, they show that both ATeam variants enable the quantitative detection of changes of astrocytic and neuronal [ATP] in the physiological range. ATeam1.03 YEMK , however, seems preferable because its K D 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.

A Dual Nanosensor Approach to Determine the Cytosolic Concentration of ATP in Astrocytes

“…In the original description of ATP sensors of the ATeam family k D values of 3.3 mM and 1.2 mM (and n H,AT = n H,ATY = 2.1) have been reported for AT and ATY, respectively (Imamura et al, 2009). However, based on calibration of the sensors in astrocytes and neurons in organotypic brain slice cultures, recently k D values of 9.4 mM and 2.7 mM were determined (Gerkau et al, 2019;Lerchundi et al, 2020). The latter studies used a Michaelis-Menten equation for fitting the data, which inherently implies n H = 1.…”

“…As the two sensors were not expressed within the same mice, no pairing of experiments was possible. Based on the two different sets of k D -values (Imamura et al, 2009;Lerchundi et al, 2020), a basal [ATP] 0 of 0.7 mM or 1.3 mM was calculated, respectively ( Figure 6D), showing that the dual nanosensor approach can also be applied to more intact preparations like acute brain slices as well as to other imaging techniques like 2-photon laser scanning microscopy.…”

“…The following sets of parameters were used: k D,AT = 3.3 mM, k D,ATY = 1.2 mM, n H,AT = n H,ATY = 2.1 (Imamura et al, 2009). k D,AT = 9.4 mM, k D,ATY = 2.7 mM, n H,AT = n H,ATY = 1.0 (Lerchundi et al, 2020).…”

“…(C) dR values obtained for AT (blue) and ATY (red) during incubation of the slices with glutamate (100 µM) for 20 min. (D) [ATP] 0 values obtained from the dR AT -dR ATY pair shown in C calculated using the following sets of parameters: k D,AT = 3.3 mM, k D,ATY = 1.2 mM, n H = 2.1 (open circle; Imamura et al, 2009); and k D,AT = 9.4 mM, k D,ATY = 2.7 mM, n H = 1 (filled circle; Lerchundi et al, 2020). n = 80, 92 cells from N = 4, 6 mice were included in the analysis shown in (C and D) for AT and ATY, respectively.…”

“…Values for k D and n H need to be obtained from other experimental systems, e.g., from measurements of the purified sensor protein (Imamura et al, 2009), assuming that these values can be applied to the indicators in the cytosolic environment of the cells of interest ( Table 1). While such transfer of parameters is often used also for other calibration strategies (e.g., Sotelo-Hitschfeld et al, 2012;Fernández-Moncada and Barros, 2014;Arce-Molina et al, 2020), several studies indicate that parameters of various genetically encoded nanosensors can be affected by the cellular environment (Hires et al, 2008;Pérez Koldenkova and Nagai, 2013;Yaginuma et al, 2014;Lerchundi et al, 2019Lerchundi et al, , 2020. Moreover, care has to be taken that these values are determined by appropriate methods including similar or -ideally-identical excitation and emission wavelengths (Pomorski et al, 2013).…”