Activity‐dependent modulation of intracellular ATP in cultured cortical astrocytes

Winkler, Ulrike; Seim, Pauline; Enzbrenner, Yvonne; Köhler, Susanne; Sicker, Marit; Hirrlinger, Johannes

doi:10.1002/jnr.24020

Cited by 29 publications

(39 citation statements)

References 75 publications

(123 reference statements)

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“…(Glt1), the increase in the NADH/NAD + redox ratio was strongly alleviated (Figure 5f ), implying that glutamate uptake plays a major role in modulating astrocytic metabolism as also recently shown for the regulation of ATP levels (Winkler et al, 2017). (Glt1), the increase in the NADH/NAD + redox ratio was strongly alleviated (Figure 5f ), implying that glutamate uptake plays a major role in modulating astrocytic metabolism as also recently shown for the regulation of ATP levels (Winkler et al, 2017).…”

Section: Resultssupporting

confidence: 74%

“…Primary cultures of cortical astrocytes were prepared from newborn mice of either sex as described (Hirrlinger, Schulz, & Dringen, 2002;Requardt et al, 2010;Winkler et al, 2017). In brief, 1.2 mio cells isolated from cerebral cortices from mice (C57BL/6 background; p0 to p1) were seeded in 6-well plates onto glass coverslips (30 mm in diameter …”

Section: Astroglia-rich Primary Culturementioning

confidence: 99%

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NBCe1 mediates the regulation of the NADH/NAD⁺redox state in cortical astrocytes by neuronal signals

Köhler

Winkler

Sicker

et al. 2018

Glia

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Astrocytes are a glial cell type, which is indispensable for brain energy metabolism. Within cells, the NADH/NAD redox state is a crucial node in metabolism connecting catabolic pathways to oxidative phosphorylation and ATP production in mitochondria. To characterize the dynamics of the intracellular NADH/NAD redox state in cortical astrocytes Peredox, a genetically encoded sensor for the NADH/NAD redox state, was expressed in cultured cortical astrocytes as well as in cortical astrocytes in acutely isolated brain slices. Calibration of the sensor in cultured astrocytes revealed a mean basal cytosolic NADH/NAD redox ratio of about 0.01; however, with a broad distribution and heterogeneity in the cell population, which was mirrored by a heterogeneous basal cellular concentration of lactate. Inhibition of glucose uptake decreased the NADH/NAD redox state while inhibition of lactate dehydrogenase or of lactate release resulted in an increase in the NADH/NAD redox ratio. Furthermore, the NADH/NAD redox state was regulated by the extracellular concentration of K , and application of the neurotransmitters ATP or glutamate increased the NADH/NAD redox state dependent on purinergic receptors and glutamate uptake, respectively. This regulation by K , ATP, and glutamate involved NBCe1 mediated sodium-bicarbonate transport. These results demonstrate that the NADH/NAD redox state in astrocytes is a metabolic node regulated by neuronal signals reflecting physiological activity, most likely contributing to adjust astrocytic metabolism to energy demand of the brain.

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Section: Resultssupporting

confidence: 74%

Section: Astroglia-rich Primary Culturementioning

confidence: 99%

NBCe1 mediates the regulation of the NADH/NAD⁺redox state in cortical astrocytes by neuronal signals

Köhler

Winkler

Sicker

et al. 2018

Glia

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“…; Winkler et al . ; Lerchundi et al . b ), ATeam was employed to monitor relative changes in cellular ATP levels, we aimed to establish a more quantitative approach by performing an intracellular calibration.…”

Section: Resultsmentioning

confidence: 99%

“…A similar phenomenon was reported from astrocytes in culture upon bath application of glutamate (Winkler et al . ), suggesting that with strong stimulation (and Na + influx, respectively), cells establish a new steady‐state.…”

Section: Discussionmentioning

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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“…While elevations in [K + ] e result in a decrease in astrocyte sodium concentrations, application of glutamate or D‐aspartate increases astrocyte sodium due to activation of sodium‐dependent glutamate transport (Karus et al, ; Langer & Rose, ; Rose & Ransom, ). This evokes a decrease in cytosolic ATP levels as shown following expression of Ateam in astrocytes in culture (Winkler et al, ), in organotypic hippocampal slice cultures (Fernandez‐Moncada et al, ) and also using Magnesium Green as a sensor for changes in ATP levels (Fernandez‐Moncada & Barros, ; Langer et al, ; Magistretti & Chatton, ). A concomitant increase in [K + ] e as well as in extracellular glutamate at excitatory synapses will exert opposing effects on astrocyte ATP levels.…”

Section: Discussionmentioning

confidence: 98%

FRET‐based imaging of intracellular ATP in organotypic brain slices

Lerchundi

Kafitz

Winkler

et al. 2018

J of Neuroscience Research

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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 ATeam1.03YEMK (“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.

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Activity‐dependent modulation of intracellular ATP in cultured cortical astrocytes

Cited by 29 publications

References 75 publications

NBCe1 mediates the regulation of the NADH/NAD⁺redox state in cortical astrocytes by neuronal signals

NBCe1 mediates the regulation of the NADH/NAD⁺redox state in cortical astrocytes by neuronal signals

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

FRET‐based imaging of intracellular ATP in organotypic brain slices

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Activity‐dependent modulation of intracellular ATP in cultured cortical astrocytes

Cited by 29 publications

References 75 publications

NBCe1 mediates the regulation of the NADH/NAD+redox state in cortical astrocytes by neuronal signals

NBCe1 mediates the regulation of the NADH/NAD+redox state in cortical astrocytes by neuronal signals

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

FRET‐based imaging of intracellular ATP in organotypic brain slices

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NBCe1 mediates the regulation of the NADH/NAD⁺redox state in cortical astrocytes by neuronal signals

NBCe1 mediates the regulation of the NADH/NAD⁺redox state in cortical astrocytes by neuronal signals