Determination of Intracellular Chloride Concentrations by Fluorescence Lifetime Imaging

Gensch, Thomas; Untiet, Verena; Franzen, Arne; Kovermann, Peter; Fahlke, Christoph

doi:10.1007/978-3-319-14929-5_4

Cited by 13 publications

(16 citation statements)

References 92 publications

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“…However, cell‐specific differences in Stern–Volmer constants mean that fluorescence lifetimes cannot be directly compared between different cell types. To determine K SV values in Bergmann glial cells, [Cl − ] int was adjusted by changing extracellular chloride concentrations in the presence of two ionophores, nigericin and tributyltin, and an extracellular [K + ] of 140 mM (Bevensee et al, ; Chao et al, ; Gensch et al, ; Kaneko et al, ). Linear regression analysis of the inverse of the average fluorescence lifetimes at different chloride concentrations provided the K SV for MQAE in Bergmann glial cells (14.97 M −1 ; Fig.…”

Section: Resultsmentioning

confidence: 99%

“…So far, glial [Cl − ] int has been determined in cultured astrocytes with results varying between 29–46 mM (Bekar and Walz, ; Bevensee et al, ; Kettenmann et al, ; Kimelberg, ; Smith et al, ; Walz and Mukerji, ). Here we used fluorescence lifetime imaging microscopy (FLIM) with the chloride‐sensitive dye MQAE (Gensch et al, ; Kovalchuk and Garaschuk, ; Marandi et al, ; Untiet et al, ) in acute cerebellar slices to determine the [Cl − ] int in Bergmann glial cells at different stages of postnatal development and to study pathways involved in its regulation. We found that anion channels associated with two glial glutamate transporters, excitatory amino acid transporter 1 and 2 (EAAT1 and EAAT2), modify the resting [Cl − ] int in adult wild type (WT) glia and dynamically modulate these values in response to cerebellar neuronal activity.…”

Section: Introductionmentioning

confidence: 99%

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Glutamate transporter-associated anion channels adjust intracellular chloride concentrations during glial maturation

Untiet

Kovermann

Gerkau

et al. 2016

Glia

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Astrocytic volume regulation and neurotransmitter uptake are critically dependent on the intracellular anion concentration, but little is known about the mechanisms controlling internal anion homeostasis in these cells. Here we used fluorescence lifetime imaging microscopy (FLIM) with the chloride-sensitive dye MQAE to measure intracellular chloride concentrations in murine Bergmann glial cells in acute cerebellar slices. We found Bergmann glial [Cl ] to be controlled by two opposing transport processes: chloride is actively accumulated by the Na -K -2Cl cotransporter NKCC1, and chloride efflux through anion channels associated with excitatory amino acid transporters (EAATs) reduces [Cl ] to values that vary upon changes in expression levels or activity of these channels. EAATs transiently form anion-selective channels during glutamate transport, and thus represent a class of ligand-gated anion channels. Age-dependent upregulation of EAATs results in a developmental chloride switch from high internal chloride concentrations (51.6 ± 2.2 mM, mean ± 95% confidence interval) during early development to adult levels (35.3 ± 0.3 mM). Simultaneous blockade of EAAT1/GLAST and EAAT2/GLT-1 increased [Cl ] in adult glia to neonatal values. Moreover, EAAT activation by synaptic stimulations rapidly decreased [Cl ] . Other tested chloride channels or chloride transporters do not contribute to [Cl ] under our experimental conditions. Neither genetic removal of ClC-2 nor pharmacological block of K -Cl cotransporter change resting Bergmann glial [Cl ] in acute cerebellar slices. We conclude that EAAT anion channels play an important and unexpected role in adjusting glial intracellular anion concentration during maturation and in response to cerebellar activity. GLIA 2017;65:388-400.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Glutamate transporter-associated anion channels adjust intracellular chloride concentrations during glial maturation

Untiet

Kovermann

Gerkau

et al. 2016

Glia

Self Cite

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“…We sought to confirm that (Fig 1l,m). [Cl -]i was significantly higher in astrocytes (18.48±0.5 mM) visually 196 identified by cell morphology or by co-staining with SR101 (Fig 1l,m), closely matching 197 measurements in cell culture (Gensch et al, 2015). 198 Next, we dialyzed MQAE by whole-cell patch clamp to compare MQAE-FLIM measures 199 using in vitro calibration data in the soma and dendrites.…”

Section: Bulk-and Patch-loaded Cells Report Expected [Cl -]I With Mqamentioning

confidence: 52%

Quantitative fluorescence lifetime imaging uncovers a novel role for KCC2 chloride transport in dendritic microdomains

MacVicar

Weiilnger

LeDue

et al. 2020

Preprint

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27Intracellular chloride ion ([Cl -]i) homeostasis is critical for synaptic neurotransmission yet variations in 28 subcellular domains are poorly understood owing to difficulties in obtaining quantitative, high-resolution 29 measurements of dendritic [Cl -]i. We combined whole-cell patch clamp electrophysiology with 30 simultaneous fluorescence lifetime imaging (FLIM) of the Cldye MQAE to quantitatively map dendritic 31Cllevels in normal or pathological conditions. FLIM-based [Cl -]i estimates were corroborated by Rubi-32 GABA uncaging to measured EGABA. Low baseline [Cl -]i in dendrites required Clefflux via the K + -Cl -33 cotransporter KCC2 (SLC12A5). In contrast, pathological NMDA application generated spatially 34 heterogeneous subdomains of high [Cl -]i that created dendritic blebs, a signature of ischemic stroke. 35 These discrete regions of high [Cl -]i were caused by reversed KCC2 transport. Therefore monitoring [Cl -36 ]i microdomains with a new high resolution FLIM-based technique identified novel roles for KCC2-37 dependent chloride transport to generate dendritic microdomains with implications for disease. 38 91 dendritic pathology. Remarkably, bleb formation was prevented by furosemide, indicating 92 localized Clloading was due to reversed K + /Cltransport. Together, our work highlights the 93 advantages of MQAE-FLIM to measure [Cl -]i shifts with sub-micrometer resolution that would 94 be otherwise undetectable using intensity imaging or electrophysiology alone. Additionally, we 95 5 have identified a novel role for KCC2 in dendritic microdomain Clhomeostasis that critically 96 impacts neuronal structure and function. 97 98 Results 99 MQAE-FLIM calibrations for determination of [Cl -]i 100As a first step we determined the reliability of MQAE-FLIM to report [Cl -]i by 101 calibrating the dye in situ (in bulk-loaded cells) and in vitro (in recording solution in sealed 102 pipettes) by time-correlated single-photon counting using 2-photon microscopy (Fig 1a). In situ 103 calibrations were conducted in acute brain slices bulk-loaded with MQAE using nigericin and 104 tributyltin to equilibrate [Cl -]i = [Cl -]o across neuronal membranes (Gensch et al., 2015; 105 Kovalchuk and Garaschuk, 2012). We then measured MQAE-FLIM lifetimes corresponding to 106 various Clconcentrations (henceforth MQAE-[Cl -]i, Fig 1b,c) in parallel to intensity changes. 107 Bulk-loaded MQAE intensity decreased significantly in response to increasing [Cl -]o but we also 108 observed progressive decline over time due to a combination of dye loss and photobleaching (Fig 109 1a,c). Indeed, intensity changes did not reverse in 0[Cl -]o to wash out Cl - (Fig 1d). In contrast, 110 the MQAE-FLIM channel revealed discrete lifetime changes in response to increasing [Cl -]o that 111 fully recovered in 0[Cl -]o washout despite the reduction in intensity signal (Fig 1d,e), 112 highlighting the advantage and utility of FLIM in overcoming intensity artifacts arising from 113 changes in dye concentration. 114 To combine MQAE-FLIM with whole-cell...

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“…While measuring the extracellular is relatively easy, non-destructive measurements of intracellular poses major challenges. Basically, two methods are available to assess the intracellular chloride activity: (1) micro-photometric techniques, using either chemical or optogenetic Cl-sensors [13,3] and (2) potentiometric techniques with liquid membrane ion-selective micro-electrodes (ISM for short) [31,18]. Photometric techniques allow for specific, high time resolution and space-resolved determination of changes in the activity of physiologically relevant ions, e.g.…”

Section: Introductionmentioning

confidence: 99%

“…However, activitydependent changes in optical properties of sensors are usually difficult to de-convolve into accurate molar units, and in some conditions, sensors can alter the extent and kinetics of the ion concentration changes aimed to be measured. Fluorescence life-time imaging circumvents some of these challenges [13], but requires specialized and expensive detection systems. In principle, an ISM is devoid of these limitations, and is expected to provide a direct determination of intracellular activity.…”

Section: Introductionmentioning

confidence: 99%

A highly-selective chloride microelectrode based on a mercuracarborand anion carrier

DiFranco

Quiñonez

Dziedzic³

et al. 2019

Preprint

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word count: 238 Word count: 5891 Figures: 7 Keywords: ion-selective electrode, liquid membrane microelectrode, skeletal muscle Abbreviations: 9-ACA: 9-anthracenecarboxylic acid, BMT: bumetanide, ISM: ion selective microelectrode, MC3: [9]mercuracarborand-3, NPOE: 2-nitrophenyl octyl ether, TDMAC: tridodecylmethyl ammonium chloride ABSTRACT The chloride gradient plays an important role in regulating cell volume, membrane potential, pH, secretion, and the reversal potential of inhibitory GABAA receptors. Measurement of intracellular chloride activity, , using liquid membrane ion-selective microelectrodes (ISM), however, has been limited by the physiochemical properties of Cl ionophores which have caused poor stability, drift, sluggish response times, and interference from other biologically relevant anions. Most importantly, intracellular HCO3may be up to 4 times more abundant than Cl -(e.g. skeletal muscle) which places severe constraints on the required selectivity of a Clsensing ISM. Previously, a sensitive and highly-selective Cl sensor was developed in a polymeric membrane electrode (Badr et al. 1999) using a trinuclear Hg(II) complex containing carborane-based ligands, [9]mercuracarborand-3, or MC3 for short. Here, we have adapted the use of the MC3 anion carrier in a liquid membrane ion-selective microelectrode and show the MC3-ISM has a linear Nernstian response over a wide range of (0.1 mM to 100 mM), is highly selective for Cl over other biological anions or inhibitors of Cl transport, and has a response time of less than 5 sec. Importantly, over the physiological range of(1 mM to 100 mM) the potentiometric response of the MC3-ISM is insensitive to HCO3or changes in pH.Finally, we demonstrate the biological application of an MC3-ISM by measuring intracellular , and the response to an external Cl-free challenge, for an isolated skeletal muscle fiber.

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Determination of Intracellular Chloride Concentrations by Fluorescence Lifetime Imaging

Cited by 13 publications

References 92 publications

Glutamate transporter-associated anion channels adjust intracellular chloride concentrations during glial maturation

Glutamate transporter-associated anion channels adjust intracellular chloride concentrations during glial maturation

Quantitative fluorescence lifetime imaging uncovers a novel role for KCC2 chloride transport in dendritic microdomains

A highly-selective chloride microelectrode based on a mercuracarborand anion carrier

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