Changes in Astroglial K+ upon Brief Periods of Energy Deprivation in the Mouse Neocortex

Abstract

Malfunction of astrocytic K+ regulation contributes to the breakdown of extracellular K+ homeostasis during ischemia and spreading depolarization events. Studying astroglial K+ changes is, however, hampered by a lack of suitable techniques. Here, we combined results from fluorescence imaging, ion-selective microelectrodes, and patch-clamp recordings in murine neocortical slices with the calculation of astrocytic [K+]. Brief chemical ischemia caused a reversible ATP reduction and a transient depolarization of astrocytes. Moreover, astrocytic [Na+] increased by 24 mM and extracellular [Na+] decreased. Extracellular [K+] increased, followed by an undershoot during recovery. Feeding these data into the Goldman–Hodgkin–Katz equation revealed a baseline astroglial [K+] of 146 mM, an initial K+ loss by 43 mM upon chemical ischemia, and a transient K+ overshoot of 16 mM during recovery. It also disclosed a biphasic mismatch in astrocytic Na+/K+ balance, which was initially ameliorated, but later aggravated by accompanying changes in pH and bicarbonate, respectively. Altogether, our study predicts a loss of K+ from astrocytes upon chemical ischemia followed by a net gain. The overshooting K+ uptake will promote low extracellular K+ during recovery, likely exerting a neuroprotective effect. The resulting late cation/anion imbalance requires additional efflux of cations and/or influx of anions, the latter eventually driving delayed astrocyte swelling.

Activation of TRPV4 channels promotes the loss of cellular ATP in organotypic slices of the mouse neocortex exposed to chemical ischemia

“…Our experiments demonstrate that chemical ischemia induces a rapid decrease in ATP levels of neurons and astrocytes, confirming earlier observations (e.g. Eitelmann et al., 2022; Lerchundi et al., 2019; Trevisiol et al., 2017). We also show here that metabolic inhibition for only half a minute already causes a clearly detectable decline in ATeam ratios in both cell types, exemplifying the strong dependence of brain tissue from a steady ATP production (Daniele et al., 2021; Erecinska & Silver, 1994; Hansen, 1985).…”

“…We mimicked the conditions in the penumbra by briefly exposing slices to inhibitors of glycolysis and oxidative phosphorylation (‘chemical ischemia’). Chemical ischemia replicates many key features of SDs, including a reversible increase in extracellular K + , a cellular depolarisation, an increase in [Na + ] i and [Ca 2+ ] i and intracellular acidification (Eitelmann et al., 2022; Gerkau et al., 2018; Hansen, 1985; Meyer, Gerkau et al., 2022; Pietrobon & Moskowitz, 2014).…”

“…The same was true when neuronal bursting activity was induced, upon which astrocytes (but not neurons) showed an initial rise in ATP levels before their ATP slowly declined (Gerkau et al., 2019; Lerchundi et al., 2020; Toloe et al., 2014). The brief increase in astrocytic ATP thus most likely reflects an initial activation of glycolysis and/or glycogenolysis in response to elevation in extracellular K + (Bak et al., 2018; Belanger et al., 2011; Bittner et al., 2011; Eitelmann et al., 2022; Fernandez‐Moncada et al., 2018), happening before the inhibition of ATP production by the pharmacological blockers came into full effect.…”

Ca2+-dependent rapid uncoupling of astrocytes upon brief metabolic stress

“…To address this question, we examined the effects of brief metabolic stress on gap junctional coupling between astrocytes in acute tissue slices of the mouse neocortex in an in situ model mimicking features of the ischemic penumbra, e.g., decrease of ATP levels, [Na + ] i increase, and intracellular acidification ( Pietrobon and Moskowitz, 2014 ; Gerkau et al, 2018 ; Lerchundi et al, 2019 ; Eitelmann et al, 2022 ). Gap junctional coupling was assessed by recording the syncytial isopotentiality, a direct measure of coupling strength ( Stephan et al, 2021 ).…”

Spreading depolarizations pose critical energy challenges in acute brain injury

“…Recent advances in the development of genetically encoded ATP sensors (Koveal et al, 2020) should enhance our ability to characterize ATP dynamics during and after SD. A recent report (Eitelmann et al, 2022) illustrates the utility of such an approach, demonstrating astrocyte ATP dynamics in response to a brief exposure to chemical ischemia (sodium azide and 2‐deoxyglucose). Miniaturization of enzyme‐linked electrochemical probes has recently enabled less invasive methods to detect adenosine, an extracellular surrogate for energy charge (Dale & Frenguelli, 2012; Hinzman, Gibson, et al, 2015; Lindquist & Shuttleworth, 2012).…”