Imaging of Intracellular ATP in Organotypic Tissue Slices of the Mouse Brain using the FRET-based Sensor ATeam1.03&lt;sup&gt;YEMK&lt;/sup&gt;

Lerchundi, Rodrigo; Kafitz, Karl W.; Färfers, Marcel; Beyer, Felix; Huang, Na; Rose, Christine R.

doi:10.3791/60294

Cited by 7 publications

(10 citation statements)

References 36 publications

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“…Still, the swelling of neuronal cell bodies was less pronounced than that of astrocytes observed before (N = 5; compare Figures 1B,D). However, when opening the glial scar covering organotypic slices as recently described (Lerchundi et al, 2019b), neurons rounded up more strongly in response to perfusion with β-escin, suggesting a better permeabilization of the cells (N = 5; Figure 3).…”

Section: Atp Sensitivity Of Ateam103 Yemk Expressed In Neuronssupporting

confidence: 53%

“…Activityrelated increases in extracellular K + , which result in a depolarization of astrocytes, activate the electrogenic sodium-bicarbonate-cotransporter NBCe1, inducing a cytosolic alkalization and, as consequence, a stimulation of glycolysis (Bittner et al, 2011;Ruminot et al, 2011;Choi et al, 2012). In line with this, an exposure of astrocytes to a brief period of elevated extracellular K + produces a reversible increase in their cellular ATP level (Fernández-Moncada et al, 2018;Lerchundi et al, 2019b).…”

Section: Differential Metabolic Response Of Neurons and Astrocytes Tomentioning

confidence: 87%

“…About 30 min after switching to the permeabilization saline, cells in the uppermost tissue layers rounded up and showed swollen cell bodies, indicative of a successful permeabilization (Figures 1B,D). The majority of these astrocytes most likely represented cells of the glial scar covering the organotypic tissue slice culture (Lerchundi et al, 2019b). Astrocytes in deeper tissue layers, in contrast, still partially maintained their morphology and ramification, pointing towards an only partial permeabilization of their cell membranes ( Figure 1B).…”

Section: Permeabilization Protocolmentioning

confidence: 99%

“…Two variants of the genetically encoded nanosensor ATeam were employed: ATeam1.03 YEMK and ATeam1.03 (Imamura et al, 2009). Transduction was performed as described in detail recently (Lerchundi et al, 2019b). For transduction of astrocytes, a dilution of 0.5 µl of an adeno-associated vector (AAV) carrying the code for ATeam under the astrocyte-specific promoter glial fibrillary acidic protein (GFAP; AAV5/2 ATeam1.03 YEMK , titer: 2.16 × 10 12 viral genome/ml or AAV5/2 ATeam1.03, titer: 1.43 × 10 12 viral genome/ml) were directly applied to the top of each slice at 1-3 days in vitro (DIV).…”

Section: Transduction Of Organotypic Slicesmentioning

confidence: 99%

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

Lerchundi

Huang

Rose

2020

Front. Cell. Neurosci.

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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.

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Section: Atp Sensitivity Of Ateam103 Yemk Expressed In Neuronssupporting

confidence: 53%

Section: Differential Metabolic Response Of Neurons and Astrocytes Tomentioning

confidence: 87%

Section: Permeabilization Protocolmentioning

confidence: 99%

Section: Transduction Of Organotypic Slicesmentioning

confidence: 99%

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

Lerchundi

Huang

Rose

2020

Front. Cell. Neurosci.

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“…2G). Notably the ATeam ratio does not change under control conditions within this time frame (Lerchundi et al 2019a), indicating that the slow decline observed in the present study was indeed related to the ongoing activity induced by the dis-inhibition.…”

Section: Network [Na + ] Oscillations Induce a Decrease In Neuronal [mentioning

confidence: 99%

Relation between activity‐induced intracellular sodium transients and ATP dynamics in mouse hippocampal neurons

Gerkau

Lerchundi

Nelson

et al. 2019

The Journal of Physiology

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Key points Employing quantitative Na+‐imaging and Förster resonance energy transfer‐based imaging with ATeam1.03YEMK (ATeam), we studied the relation between activity‐induced Na+ influx and intracellular ATP in CA1 pyramidal neurons of the mouse hippocampus. Calibration of ATeam in situ enabled a quantitative estimate of changes in intracellular ATP concentrations. Different paradigms of stimulation that induced global Na+ influx into the entire neuron resulted in decreases in [ATP] in the range of 0.1–0.6 mm in somata and dendrites, while Na+ influx that was locally restricted to parts of dendrites did not evoke a detectable change in dendritic [ATP]. Our data suggest that global Na+ transients require global cellular activation of the Na+/K+‐ATPase resulting in a consumption of ATP that transiently overrides its production. For recovery from locally restricted Na+ influx, ATP production as well as fast intracellular diffusion of ATP and Na+ might prevent a local drop in [ATP]. Abstract Excitatory neuronal activity results in the influx of Na+ through voltage‐ and ligand‐gated channels. Recovery from accompanying increases in intracellular Na+ concentrations ([Na+]i) is mainly mediated by the Na+/K+‐ATPase (NKA) and is one of the major energy‐consuming processes in the brain. Here, we analysed the relation between different patterns of activity‐induced [Na+]i signalling and ATP in mouse hippocampal CA1 pyramidal neurons by Na+ imaging with sodium‐binding benzofurane isophthalate (SBFI) and employing the genetically encoded nanosensor ATeam1.03YEMK (ATeam). In situ calibrations demonstrated a sigmoidal dependence of the ATeam Förster resonance energy transfer ratio on the intracellular ATP concentration ([ATP]i) with an apparent KD of 2.6 mm, indicating its suitability for [ATP]i measurement. Induction of recurrent network activity resulted in global [Na+]i oscillations with amplitudes of ∼10 mm, encompassing somata and dendrites. These were accompanied by a steady decline in [ATP]i by 0.3–0.4 mm in both compartments. Global [Na+]i transients, induced by afferent fibre stimulation or bath application of glutamate, caused delayed, transient decreases in [ATP]i as well. Brief focal glutamate application that evoked transient local Na+ influx into a dendrite, however, did not result in a measurable reduction in [ATP]i. Our results suggest that ATP consumption by the NKA following global [Na+]i transients temporarily overrides its availability, causing a decrease in [ATP]i. Locally restricted Na+ transients, however, do not result in detectable changes in local [ATP]i, suggesting that ATP production, together with rapid intracellular diffusion of both ATP and Na+ from and to unstimulated neighbouring regions, counteracts a local energy shortage under these conditions.

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Cortical brain organoid slices (cBOS) for the study of human neural cells in minimal networks

Petersilie,

Heiduschka,

Nelson

et al. 2024

iScience

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Imaging of Intracellular ATP in Organotypic Tissue Slices of the Mouse Brain using the FRET-based Sensor ATeam1.03<sup>YEMK</sup>

Cited by 7 publications

References 36 publications

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

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

Relation between activity‐induced intracellular sodium transients and ATP dynamics in mouse hippocampal neurons

Cortical brain organoid slices (cBOS) for the study of human neural cells in minimal networks

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