Novel algorithms for improved detection and analysis of fluorescent signal fluctuations.

Fluorescent dyes and genetically encoded fluorescence indicators (GEFI) are common tools for visualizing concentration changes of specific ions and messenger molecules during intra- as well as intercellular communication. Using advanced imaging technologies, fluorescence indicators are a prerequisite for the analysis of physiological molecular signaling. Automated detection and analysis of fluorescence signals require to overcome several challenges, including correct estimation of fluorescence fluctuations at basal concentrations of messenger molecules, detection, and extraction of events themselves as well as proper segmentation of neighboring events. Moreover, event detection algorithms need to be sensitive enough to accurately capture localized and low amplitude events exhibiting a limited spatial extent. Here, we present two algorithms (PBasE and CoRoDe) for accurate baseline estimation and automated detection and segmentation of fluorescence fluctuations.

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

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.

Cell-Type Dependent Regulation of the Electrogenic Na+/HCO3- Cotransporter 1 (NBCe1) by Hypoxia and Acidosis in Glioblastoma.

Glioblastoma multiforme (GBM) is the most common and malignant brain tumour. It is characterised by transcriptionally distinct cell populations. In tumour cells, physiological pH gradients between the intracellular and extracellular compartments are reversed, compared to non-cancer cells. Intracellular pH in tumour cells is alkaline, whereas extracellular pH is acidic. Consequently, the function and/or expression of pH regulating transporters might be altered. Here, we investigated protein expression and regulation of the electrogenic sodium/bicarbonate cotransporter 1 (NBCe1) in mesenchymal (MES)-like hypoxia-dependent and -independent cells, as well as in astrocyte-like glioblastoma cells following chemical hypoxia, acidosis and elucidated putative underlying molecular pathways. Immunoblotting, immunocytochemistry, and intracellular pH recording with the H+-sensitive dye 2',7'-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein were applied. The results show NBCe1 protein abundance and active NBCe1 transport. Hypoxia upregulated NBCe1 protein and activity in MES-like hypoxia-dependent GBM cells. This effect was positively correlated with HIF-1α protein levels, was mediated by TGF-ß signalling, and was prevented by extracellular acidosis. In MES-like hypoxia-independent GBM cells, acidosis (but not hypoxia) regulated NBCe1 activity in an HIF-1α-independent manner. These results demonstrate a cell-specific adaptation of NBCe1 expression and activity to the microenvironment challenge of hypoxia and acidosis that depends on their transcriptional signature in GBM.

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

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.

Inward Operation of Sodium-Bicarbonate Cotransporter 1 Promotes Astrocytic Na+ Loading and Loss of ATP in Mouse Neocortex during Brief Chemical Ischemia.

Ischemic conditions cause an increase in the sodium concentration of astrocytes, driving the breakdown of ionic homeostasis and exacerbating cellular damage. Astrocytes express high levels of the electrogenic sodium-bicarbonate cotransporter1 (NBCe1), which couples intracellular Na+ homeostasis to regulation of pH and operates close to its reversal potential under physiological conditions. Here, we analyzed its mode of operation during transient energy deprivation via imaging astrocytic pH, Na+, and ATP in organotypic slice cultures of the mouse neocortex, complemented with patch-clamp and ion-selective microelectrode recordings and computational modeling. We found that a 2 min period of metabolic failure resulted in a transient acidosis accompanied by a Na+ increase in astrocytes. Inhibition of NBCe1 increased the acidosis while decreasing the Na+ load. Similar results were obtained when comparing ion changes in wild-type and Nbce1-deficient mice. Mathematical modeling replicated these findings and further predicted that NBCe1 activation contributes to the loss of cellular ATP under ischemic conditions, a result confirmed experimentally using FRET-based imaging of ATP. Altogether, our data demonstrate that transient energy failure stimulates the inward operation of NBCe1 in astrocytes. This causes a significant amelioration of ischemia-induced astrocytic acidification, albeit at the expense of increased Na+ influx and a decline in cellular ATP.