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

Abstract

Astrocytic gap junctional coupling is a major element in neuron–glia interaction. There is strong evidence that impaired coupling is involved in neurological disorders. Reduced coupling was, e.g., demonstrated for core regions of ischemic stroke that suffer from massive cell death. In the surrounding penumbra, cells may recover, but recovery is hampered by spreading depolarizations, which impose additional metabolic stress onto the tissue. Spreading depolarizations are characterized by transient breakdown of cellular ion homeostasis, including pH and Ca2+, which might directly affect gap junctional coupling. Here, we exposed acute mouse neocortical tissue slices to brief metabolic stress and examined its effects on the coupling strength between astrocytes. Changes in gap junctional coupling were assessed by recordings of the syncytial isopotentiality. Moreover, quantitative ion imaging was performed in astrocytes to analyze the mechanisms triggering the observed changes. Our experiments show that a 2-minute perfusion of tissue slices with blockers of glycolysis and oxidative phosphorylation causes a rapid uncoupling in half of the recorded cells. They further indicate that uncoupling is not mediated by the accompanying (moderate) intracellular acidification. Dampening large astrocytic Ca2+ loads by removal of extracellular Ca2+ or blocking Ca2+ influx pathways as well as a pharmacological inhibition of calmodulin, however, prevent the uncoupling. Taken together, we conclude that astrocytes exposed to brief episodes of metabolic stress can undergo a rapid, Ca2+/calmodulin-dependent uncoupling. Such uncoupling may help to confine and reduce cellular damage in the ischemic penumbra in vivo.

A Deep Learning-Based Segmentation of Cells and Analysis (DL-SCAN)

“…The next part of the validation process was implementing DL-SCAN on previously published experiments [9, 22]. In this work, acute tissue slices prepared from mouse brain were exposed to inhibitors of glycolysis and oxidative respiration for 2 minutes to induce chemical ischemia, resulting in transient increases in intracellular ion concentrations in neurons and astrocytes.…”

“…As a second proof-of-principle, we re-analyzed and compared changes in intracellular Ca 2+ induced by chemical ischemia in astrocytes in cortical brain slices [9]. The changes in astrocytic OGB-1 fluorescence for manual and automatic segmentation are shown in Figure 5A and 5D.…”

“…The imaging experiments shown in this manuscript are taken from previously published work where experimental details and procedures are described in detail [9, 22]. In brief, for the preparation of acute cortical or hippocampal tissue slices, BALB/c mice (both sexes) from postnatal day (P) 14-21 were anaesthetized, decapitated, and their brains removed.…”

“…Wide-field imaging of OGB-1 was performed using an upright microscope (Eclipse FN1, Nikon) equipped with an ORCA FLASH 4.0LT camera (Hamamatsu Photonics Deutschland GmbH, Herrsching am Ammersee, Germany) and a 40x water immersion objective (Fluor 40×/0.8 W, Nikon). Probes were excited using a Polychrome V monochromator (ex: 488 nm, em: >505 nm; Thermo Fisher Scientific/FEI, Planegg, Germany) at a framerate of 1 Hz [9].…”

“…For example, in many organs such as the brain, the concentration of the molecules of interest, e.g. of ions such as Ca 2+ and Na + , undergoes transient changes both in time and space [7-9], while tissue movements additionally shift the exact location of the cells or cellular compartments in the field of view. In such a scenario, the user is often required to draw or adjust the regions of interest (ROIs) in which fluorescence signals are to be analyzed frame by frame.…”