Two-photon chloride imaging in neurons of brain slices

Marandi, Nima; Konnerth, Arthur; Garaschuk, Olga

doi:10.1007/s00424-002-0933-7

Cited by 71 publications

(78 citation statements)

References 45 publications

(77 reference statements)

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“…To determine changes of the intracellular Cl Ϫ -concentration, primary hepatocytes grown for 24 h on glass coverslips (diameter, 30 mm) were loaded for 6 -10 h with the chloridesensitive dye MQAE (1 mmol/liter) in phenol red-free culture medium in a humidified 5% CO 2 atmosphere at 37°C (21). Then coverslips were transferred to single cell fluorescence recording using an inverted fluorescence microscope (Zeiss, Axiovert) combined with the QuantiCell 2000 calcium imaging setup (VisiTech).…”

Section: Determination Of Apparent Endosomal Ph (Ph Ves ) and Intracementioning

confidence: 99%

“…By use of regions of interest using the QuantiCell 2000 software, the field of measurement was chosen to be within one single cell. A decrease in intracellular MQAE fluorescence at an excitation wavelength of 350 nm thereby reflects an increase in the intracellular Cl Ϫ concentration (21). Fluorescence is given as relative fluorescence compared with the fluorescence at the beginning of the respective experiment (first second of the recording).…”

Section: Determination Of Apparent Endosomal Ph (Ph Ves ) and Intracementioning

confidence: 99%

“…Evidence has been presented for a direct activation of the H ϩ -ATPase by cytosolic chloride (32,33). To study the effect of CD95L on the cytosolic chloride concentration, rat hepatocytes were loaded for 6 -10 h with the Cl Ϫ -sensitive fluorescent dye MQAE (21). Upon excitation at 350 nm, this dye yields a fluorescence signal, which underlies a diffusion-limited …”

Section: Cd95l Induces Caspase 8-dependent Endosomal Acidification Inmentioning

confidence: 99%

“…Cells were obtained from at least three independent preparations. collisional quenching in presence of Cl Ϫ , whereas the affinity of this dye to other physiologically relevant anions (especially HCO 3 Ϫ ) was shown to be minor (21). However, MQAE fluorescence only gives qualitative information on changes of the intracellular chloride concentration, because no ratio technique can be applied (21).…”

Section: Cd95l-induced Changes Of Apparent Ph Ves In Fitc-dextran-accmentioning

confidence: 99%

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Amplification of CD95 Activation by Caspase 8-induced Endosomal Acidification in Rat Hepatocytes

Reinehr

Sommerfeld

Keitel

et al. 2008

Journal of Biological Chemistry

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Although in rat hepatocytes CD95 is predominantly located inside the cell with almost undetectable immunostaining at the plasma membrane, the addition of CD95-ligand (CD95L) induces hepatocyte apoptosis, which is preceded by a targeting and activation of intracellularly localized CD95 to the plasma membrane including formation of the death-inducing signaling complex. This process involves an NADPH oxidase-dependent generation of reactive oxygen species (ROS) through a ceramide-and protein kinase C-dependent pathway, which leads to an activating phosphorylation of p47 phox . The mechanisms underlying CD95L-induced ceramide formation were addressed in the present study. It was found that CD95L lowered within seconds the apparent vesicular pH from 6.0 to 5.7 in a fluorescein isothiocyanate-dextran-accessible endosomal compartment, which was previously shown to contain acidic sphingomyelinase, and decreased N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide fluorescence, suggestive for an increase of cytosolic [Cl ؊ ]. Bafilomycin or 4,4-diisothiocyanostilbene-2,2-disulfonic acid disodium salt largely abolished the CD95L-induced endosomal acidification, ceramide formation, and downstream events, such as p47 phox phosphorylation, ROS formation, CD95 activation, and apoptosis. These responses were also abolished after knock-down of acidic sphingomyelinase in rat hepatocytes. Interestingly, caspase 8 inhibitors abolished these CD95L-induced signaling events, including the increase in cytosolic [Cl ؊ ], endosomal acidification, ceramide formation, and ROS generation as well as CD95 targeting to the plasma membrane and CD95 activation. The data suggest that CD95L initiates a rapid caspase 8-dependent endosomal acidification, which triggers ceramide-dependent ROS formation as an upstream event of trafficking of intracellularly stored CD95 to the plasma membrane. It is concluded that a rapid caspase 8 activation in response to CD95L signals to intracellularly stored CD95, which becomes activated and targeted to the plasma membrane. This autoamplification of CD95-activation is required for apoptosis induction. CD952 (Apo-1/Fas) belongs to the death receptor family and plays an important role in apoptosis induction in many cell types. In hepatocytes, CD95 is mainly located within the cellular interior and can either be activated after ligation with its natural ligand (CD95L) or in a ligand-independent way by hydrophobic bile salts or hyperosmotic cell shrinkage (Refs. 1-4; for review, see Ref. 5). Ligand-dependent and -independent CD95 activation in hepatocytes is a complex process that finally results in CD95 trafficking from the cellular interior to the plasma membrane, subsequent formation of the death-inducing signaling complex (DISC), and eventually apoptosis (1-12).In hepatocytes, proapoptotic stimuli such as CD95 ligand (CD95L), hydrophobic bile salts, or hyperosmotic cell shrinkage induce a rapid oxidative stress (ROS) response (2, 4), which triggers a Yes-dependent but ligand-independent activation of the epiderm...

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Section: Determination Of Apparent Endosomal Ph (Ph Ves ) and Intracementioning

confidence: 99%

Section: Determination Of Apparent Endosomal Ph (Ph Ves ) and Intracementioning

confidence: 99%

Section: Cd95l Induces Caspase 8-dependent Endosomal Acidification Inmentioning

confidence: 99%

Section: Cd95l-induced Changes Of Apparent Ph Ves In Fitc-dextran-accmentioning

confidence: 99%

See 2 more Smart Citations

Amplification of CD95 Activation by Caspase 8-induced Endosomal Acidification in Rat Hepatocytes

Reinehr

Sommerfeld

Keitel

et al. 2008

Journal of Biological Chemistry

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“…Furthermore, Cl − conductances can shunt concomitant fluxes of cations without affecting membrane potential, making such forms of inhibition virtually invisible to voltage recordings. However, all of these inhibitory actions result from Cl − fluxes that will yield changes in postsynaptic [Cl − ] i (Kaila, 1994;Kuner and Augustine, 2000;Marandi et al, 2002;Isomura et al, 2003;Jedlička and Backus, 2006). Thus, direct measurements of changes in [Cl − ] i seem an ideal means of imaging synaptic inhibition.…”

Section: Introductionmentioning

confidence: 99%

Imaging synaptic inhibition in transgenic mice expressing the chloride indicator, Clomeleon

et al. 2006

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Tuning Neuronal Circuit Formation in 3D Polymeric Scaffolds by Introducing Graphene at the Bio/Material Interface

et al. 2020

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2D cultures are useful platforms allowing studies of the fundamental mechanisms governing neuron and synapse functions. Yet, such models are limited when exploring changes in network dynamics due to 3D‐space topologies. 3D platforms fill this gap and favor investigating topologies closer to the real brain organization. Graphene, an atom‐thick layer of carbon, possesses remarkable properties and since its discovery is considered a highly promising material in neuroscience developments. Here, elastomeric 3D platforms endowed with graphene cues are exploited to modulate neuronal circuits when interfaced to graphene in 3D topology. Ex vivo neuronal networks are successfully reconstructed within 3D scaffolds, with and without graphene, characterized by comparable size and morphology. By confocal microscopy and live imaging, the 3D architecture of synaptic networks is documented to sustain a high rate of bursting in 3D scaffolds, an activity further increased by graphene interfacing. Changes are reported in the excitation/inhibition ratio, potentially following 3D‐graphene interfacing. A hypothesis is thus proposed, where the combination of synapse formation under 3D architecture and graphene interfaces affects the maturation of GABAergic inhibition. This will tune the balance between hyperpolarizing and depolarizing responses, potentially contributing to network synchronization in the absence of changes in GABAergic phenotype expression.

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Two-photon chloride imaging in neurons of brain slices

Cited by 71 publications

References 45 publications

Amplification of CD95 Activation by Caspase 8-induced Endosomal Acidification in Rat Hepatocytes

Amplification of CD95 Activation by Caspase 8-induced Endosomal Acidification in Rat Hepatocytes

Imaging synaptic inhibition in transgenic mice expressing the chloride indicator, Clomeleon

Tuning Neuronal Circuit Formation in 3D Polymeric Scaffolds by Introducing Graphene at the Bio/Material Interface

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