The Experts below are selected from a list of 423 Experts worldwide ranked by ideXlab platform

David G Nicholls - One of the best experts on this subject based on the ideXlab platform.

  • Mitochondrial Membrane Potential and Glutamate Excitotoxicity in Cultured
    2013
    Co-Authors: Cerebellar Granule Cells, Manus W. Ward, Cristina A. Rego, Bruno G. Frenguelli, David G Nicholls
    Abstract:

    The relationship between changes in mitochondrial membrane potential (� � m) and the failure of cytoplasmic Ca 2 � homeostasis, delayed Ca 2 � deregulation (DCD), is investigated for cultured rat cerebellar granule cells exposed to glutamate. To interpret the single-cell fluorescence response of cells loaded with TetraMethylrhodamine Methyl Ester (TMRM � ) or rhodamine-123, we devised and validated a mathematical simulation with well characterized effectors of � � m and plasma membrane potential (� � P). Glutamate usually caused an immediate decrease in � � m of �10 mV, attributable to Ca 2 � accumulation rather than enhanced ATP demand, and these cells continued to generate ATP by oxidative phosphorylation until DCD. Cells for which the mitochondria showed a larger initial depolarization deregulated more rapidly. The mitochondria in a subpopulation of glutamate-exposed cells that failed to extrude Ca 2 � that was released from the matri

  • quantitative measurement of mitochondrial membrane potential in cultured cells calcium induced de and hyperpolarization of neuronal mitochondria
    The Journal of Physiology, 2012
    Co-Authors: Akos A Gerencser, Martin Jastroch, David G Nicholls, Christos Chinopoulos, Matthew J Birket, Cathy Vitelli, Martin D Brand
    Abstract:

    Mitochondrial membrane potential (ΔΨM) is a central intermediate in oxidative energy metabolism. Although ΔΨM is routinely measured qualitatively or semi-quantitatively using fluorescent probes, its quantitative assay in intact cells has been limited mostly to slow, bulk-scale radioisotope distribution methods. Here we derive and verify a biophysical model of fluorescent potentiometric probe compartmentation and dynamics using a bis-oxonol-type indicator of plasma membrane potential (ΔΨP) and the ΔΨM probe TetraMethylrhodamine Methyl Ester (TMRM) using fluorescence imaging and voltage clamp. Using this model we introduce a purely fluorescence-based quantitative assay to measure absolute values of ΔΨM in millivolts as they vary in time in individual cells in monolayer culture. The ΔΨP-dependent distribution of the probes is modelled by Eyring rate theory. Solutions of the model are used to deconvolute ΔΨP and ΔΨM in time from the probe fluorescence intensities, taking into account their slow, ΔΨP-dependent redistribution and Nernstian behaviour. The calibration accounts for matrix:cell volume ratio, high- and low-affinity binding, activity coefficients, background fluorescence and optical dilution, allowing comparisons of potentials in cells or cell types differing in these properties. In cultured rat cortical neurons, ΔΨM is −139 mV at rest, and is regulated between −108 mV and −158 mV by concerted increases in ATP demand and Ca2+-dependent metabolic activation. Sensitivity analysis showed that the standard error of the mean in the absolute calibrated values of resting ΔΨM including all biological and systematic measurement errors introduced by the calibration parameters is less than 11 mV. Between samples treated in different ways, the typical equivalent error is ∼5 mV.

  • measurement of instantaneous velocity vectors of organelle transport mitochondrial transport and bioenergetics in hippocampal neurons
    Biophysical Journal, 2008
    Co-Authors: Akos A Gerencser, David G Nicholls
    Abstract:

    Impaired transport of mitochondria, in dendrites and axons of neurons, and bioenergetic deficit are increasingly recognized to be of pathological importance in neurodegenerative diseases. To study the relationship between transport and bioenergetics, we have developed what to our knowledge is a novel technique to quantify organelle velocity in cultured cells. The aim was to combine measurement of motion and bioenergetic parameters while minimizing photodynamic oxidative artifacts evoked by fluorescence excitation. Velocity determination from sequential fluorescence images is not trivial, and here we describe an application of “optical flow”, the flow of gray values in grayscale images, to this problem. Based on the principles of photon shot noise occurring in low light level fluorescence microscopy, we describe and validate here an optical flow-based, robust method to measure velocity vectors for organelles expressing fluorescent proteins. This method features instantaneous velocity determination from a pair of images by detecting motion of edges, with no assumptions about the separation or shapes of the objects in the image. Optical flow was used in combination with single mitochondrion assay of mitochondrial thiol redox status by mitochondrially targeted redox-sensitive green fluorescent protein and measurement of mitochondrial membrane potential by TetraMethylrhodamine Methyl Ester. Mitochondrial populations of resting cultured hippocampal neurons were analyzed. It was found that mitochondria with more oxidized thiol redox status have lower membrane potentials and are smaller in size. These mitochondria are more motile than the average; however, mitochondrial motility is only slightly dependent on the observed bioenergetic parameters and is correlated the best to the size of the mitochondria.

  • mitochondrial and plasma membrane potential of cultured cerebellar neurons during glutamate induced necrosis apoptosis and tolerance
    The Journal of Neuroscience, 2007
    Co-Authors: Manus W. Ward, David G Nicholls, Heinrich J Huber, Petronela Weisova, Heiko Dussmann, Jochen H M Prehn
    Abstract:

    A failure of mitochondrial bioenergetics has been shown to be closely associated with the onset of apoptotic and necrotic neuronal injury. Here, we developed an automated computational model that interprets the single-cell fluorescence for TetraMethylrhodamine Methyl Ester (TMRM) as a consequence of changes in either delta psi(m) or delta psi(p), thus allowing for the characterization of responses for populations of single cells and subsequent statistical analysis. Necrotic injury triggered by prolonged glutamate excitation resulted in a rapid monophasic or biphasic loss of delta psi(m) that was closely associated with a loss of delta psi(p) and a rapid decrease in neuronal NADPH and ATP levels. Delayed apoptotic injury, induced by transient glutamate excitation, resulted in a small, reversible decrease in TMRM fluorescence, followed by a sustained hyperpolarization of delta psi(m) as confirmed using the delta psi(p)-sensitive anionic probe DiBAC2(3). This hyperpolarization of delta psi(m) was closely associated with a significant increase in neuronal glucose uptake, NADPH availability, and ATP levels. Statistical analysis of the changes in delta psi(m) or delta psi(p) at a single-cell level revealed two major correlations; those neurons displaying a more pronounced depolarization of delta psi(p) during the initial phase of glutamate excitation entered apoptosis more rapidly, and neurons that displayed a more pronounced hyperpolarization of delta psi(m) after glutamate excitation survived longer. Indeed, those neurons that were tolerant to transient glutamate excitation (18%) showed the most significant increases in delta psi(m). Our results indicate that a hyperpolarization of delta psi(m) is associated with increased glucose uptake, NADPH availability, and survival responses during excitotoxic injury.

  • mitochondrial membrane potential and glutamate excitotoxicity in cultured cerebellar granule cells
    The Journal of Neuroscience, 2000
    Co-Authors: Manus W. Ward, Cristina A. Rego, Bruno G. Frenguelli, David G Nicholls
    Abstract:

    The relationship between changes in mitochondrial membrane potential (Δψm) and the failure of cytoplasmic Ca2+ homeostasis, delayed Ca2+deregulation (DCD), is investigated for cultured rat cerebellar granule cells exposed to glutamate. To interpret the single-cell fluorescence response of cells loaded with TetraMethylrhodamine Methyl Ester (TMRM+) or rhodamine-123, we devised and validated a mathematical simulation with well characterized effectors of Δψm and plasma membrane potential (ΔψP). Glutamate usually caused an immediate decrease in Δψm of <10 mV, attributable to Ca2+ accumulation rather than enhanced ATP demand, and these cells continued to generate ATP by oxidative phosphorylation until DCD. Cells for which the mitochondria showed a larger initial depolarization deregulated more rapidly. The mitochondria in a subpopulation of glutamate-exposed cells that failed to extrude Ca2+ that was released from the matrix after protonophore addition were bioenergetically competent. The onset of DCD during continuous glutamate exposure in the presence or absence of oligomycin was associated with a slowly developing mitochondrial depolarization, but cause and effect could not be established readily. In contrast, the slowly developing mitochondrial depolarization after transient NMDA receptor activation occurs before cytoplasmic free Ca2+([Ca2+]c) has risen to the set point at which mitochondria retain Ca2+. In the presence of oligomycin no increase in [Ca2+]c occurs during this depolarization. We conclude that transient Ca2+loading of mitochondria as a consequence of NMDA receptor activation initiates oxidative damage to both plasma membrane Ca2+ extrusion pathways and the inhibition of mitochondrial respiration. Depending on experimental conditions, one of these factors becomes rate-limiting and precipitates DCD.

Jochen H M Prehn - One of the best experts on this subject based on the ideXlab platform.

  • toxi sim a simulation tool for the analysis of mitochondrial and plasma membrane potentials
    Journal of Neuroscience Methods, 2009
    Co-Authors: Heinrich J Huber, Manus W. Ward, Martin Plchut, Petronela Weisova, Heiko Dussmann, Jakub Wenus, Markus Rehm, Jochen H M Prehn
    Abstract:

    Abstract Changes in the electrochemical gradients across biological membranes are excellent indicators of pathophysiological processes, drug action, or drug toxicity. Our previous studies have utilized the potentiometric probe TetraMethylrhodamine Methyl Ester (TMRM) to characterize changes in mitochondrial function by monitoring alterations in the mitochondrial membrane potential (Δψm) over time during glutamate excitotoxicity. However, fluorescently charged dyes such as TMRM respond to changes in both Δψm and the plasma membrane (Δψp) potentials making whole cell fluorescence data difficult to interpret. Here we have implemented a mathematical model that exploits the Nernstian behaviour of TMRM and uses automated Newton based root-finding fitting (TOXI-SIM) to model changes in TMRM fluorescence from multiple cells simultaneously, providing output on changes in Δψm and Δψp over time. Based on Ca2+ responses, TOXI-SIM allows for an accurate modelling of TMRM traces for different injury paradigms (necrosis, apoptosis, tolerance). TOXI-SIM is provided as a user friendly public web service for trace analysis, with an additional online data base provided for the storage and retrieval of experimental traces ( http://systemsbiology.rcsi.ie/tmrm/index.html ).

  • mitochondrial and plasma membrane potential of cultured cerebellar neurons during glutamate induced necrosis apoptosis and tolerance
    The Journal of Neuroscience, 2007
    Co-Authors: Manus W. Ward, David G Nicholls, Heinrich J Huber, Petronela Weisova, Heiko Dussmann, Jochen H M Prehn
    Abstract:

    A failure of mitochondrial bioenergetics has been shown to be closely associated with the onset of apoptotic and necrotic neuronal injury. Here, we developed an automated computational model that interprets the single-cell fluorescence for TetraMethylrhodamine Methyl Ester (TMRM) as a consequence of changes in either delta psi(m) or delta psi(p), thus allowing for the characterization of responses for populations of single cells and subsequent statistical analysis. Necrotic injury triggered by prolonged glutamate excitation resulted in a rapid monophasic or biphasic loss of delta psi(m) that was closely associated with a loss of delta psi(p) and a rapid decrease in neuronal NADPH and ATP levels. Delayed apoptotic injury, induced by transient glutamate excitation, resulted in a small, reversible decrease in TMRM fluorescence, followed by a sustained hyperpolarization of delta psi(m) as confirmed using the delta psi(p)-sensitive anionic probe DiBAC2(3). This hyperpolarization of delta psi(m) was closely associated with a significant increase in neuronal glucose uptake, NADPH availability, and ATP levels. Statistical analysis of the changes in delta psi(m) or delta psi(p) at a single-cell level revealed two major correlations; those neurons displaying a more pronounced depolarization of delta psi(p) during the initial phase of glutamate excitation entered apoptosis more rapidly, and neurons that displayed a more pronounced hyperpolarization of delta psi(m) after glutamate excitation survived longer. Indeed, those neurons that were tolerant to transient glutamate excitation (18%) showed the most significant increases in delta psi(m). Our results indicate that a hyperpolarization of delta psi(m) is associated with increased glucose uptake, NADPH availability, and survival responses during excitotoxic injury.

Akos A Gerencser - One of the best experts on this subject based on the ideXlab platform.

  • measurement of the absolute magnitude and time courses of mitochondrial membrane potential in primary and clonal pancreatic beta cells
    PLOS ONE, 2016
    Co-Authors: Akos A Gerencser, Shona A Mookerjee, Martin Jastroch, Martin D Brand
    Abstract:

    The aim of this study was to simplify, improve and validate quantitative measurement of the mitochondrial membrane potential (ΔψM) in pancreatic β-cells. This built on our previously introduced calculation of the absolute magnitude of ΔψM in intact cells, using time-lapse imaging of the non-quench mode fluorescence of TetraMethylrhodamine Methyl Ester and a bis-oxonol plasma membrane potential (ΔψP) indicator. ΔψM is a central mediator of glucose-stimulated insulin secretion in pancreatic β-cells. ΔψM is at the crossroads of cellular energy production and demand, therefore precise assay of its magnitude is a valuable tool to study how these processes interplay in insulin secretion. Dispersed islet cell cultures allowed cell type-specific, single-cell observations of cell-to-cell heterogeneity of ΔψM and ΔψP. Glucose addition caused hyperpolarization of ΔψM and depolarization of ΔψP. The hyperpolarization was a monophasic step increase, even in cells where the ΔψP depolarization was biphasic. The biphasic response of ΔψP was associated with a larger hyperpolarization of ΔψM than the monophasic response. Analysis of the relationships between ΔψP and ΔψM revealed that primary dispersed β-cells responded to glucose heterogeneously, driven by variable activation of energy metabolism. Sensitivity analysis of the calibration was consistent with β-cells having substantial cell-to-cell variations in amounts of mitochondria, and this was predicted not to impair the accuracy of determinations of relative changes in ΔψM and ΔψP. Finally, we demonstrate a significant problem with using an alternative ΔψM probe, rhodamine 123. In glucose-stimulated and oligomycin-inhibited β-cells the principles of the rhodamine 123 assay were breached, resulting in misleading conclusions.

  • quantitative measurement of mitochondrial membrane potential in cultured cells calcium induced de and hyperpolarization of neuronal mitochondria
    The Journal of Physiology, 2012
    Co-Authors: Akos A Gerencser, Martin Jastroch, David G Nicholls, Christos Chinopoulos, Matthew J Birket, Cathy Vitelli, Martin D Brand
    Abstract:

    Mitochondrial membrane potential (ΔΨM) is a central intermediate in oxidative energy metabolism. Although ΔΨM is routinely measured qualitatively or semi-quantitatively using fluorescent probes, its quantitative assay in intact cells has been limited mostly to slow, bulk-scale radioisotope distribution methods. Here we derive and verify a biophysical model of fluorescent potentiometric probe compartmentation and dynamics using a bis-oxonol-type indicator of plasma membrane potential (ΔΨP) and the ΔΨM probe TetraMethylrhodamine Methyl Ester (TMRM) using fluorescence imaging and voltage clamp. Using this model we introduce a purely fluorescence-based quantitative assay to measure absolute values of ΔΨM in millivolts as they vary in time in individual cells in monolayer culture. The ΔΨP-dependent distribution of the probes is modelled by Eyring rate theory. Solutions of the model are used to deconvolute ΔΨP and ΔΨM in time from the probe fluorescence intensities, taking into account their slow, ΔΨP-dependent redistribution and Nernstian behaviour. The calibration accounts for matrix:cell volume ratio, high- and low-affinity binding, activity coefficients, background fluorescence and optical dilution, allowing comparisons of potentials in cells or cell types differing in these properties. In cultured rat cortical neurons, ΔΨM is −139 mV at rest, and is regulated between −108 mV and −158 mV by concerted increases in ATP demand and Ca2+-dependent metabolic activation. Sensitivity analysis showed that the standard error of the mean in the absolute calibrated values of resting ΔΨM including all biological and systematic measurement errors introduced by the calibration parameters is less than 11 mV. Between samples treated in different ways, the typical equivalent error is ∼5 mV.

  • measurement of instantaneous velocity vectors of organelle transport mitochondrial transport and bioenergetics in hippocampal neurons
    Biophysical Journal, 2008
    Co-Authors: Akos A Gerencser, David G Nicholls
    Abstract:

    Impaired transport of mitochondria, in dendrites and axons of neurons, and bioenergetic deficit are increasingly recognized to be of pathological importance in neurodegenerative diseases. To study the relationship between transport and bioenergetics, we have developed what to our knowledge is a novel technique to quantify organelle velocity in cultured cells. The aim was to combine measurement of motion and bioenergetic parameters while minimizing photodynamic oxidative artifacts evoked by fluorescence excitation. Velocity determination from sequential fluorescence images is not trivial, and here we describe an application of “optical flow”, the flow of gray values in grayscale images, to this problem. Based on the principles of photon shot noise occurring in low light level fluorescence microscopy, we describe and validate here an optical flow-based, robust method to measure velocity vectors for organelles expressing fluorescent proteins. This method features instantaneous velocity determination from a pair of images by detecting motion of edges, with no assumptions about the separation or shapes of the objects in the image. Optical flow was used in combination with single mitochondrion assay of mitochondrial thiol redox status by mitochondrially targeted redox-sensitive green fluorescent protein and measurement of mitochondrial membrane potential by TetraMethylrhodamine Methyl Ester. Mitochondrial populations of resting cultured hippocampal neurons were analyzed. It was found that mitochondria with more oxidized thiol redox status have lower membrane potentials and are smaller in size. These mitochondria are more motile than the average; however, mitochondrial motility is only slightly dependent on the observed bioenergetic parameters and is correlated the best to the size of the mitochondria.

Martin D Brand - One of the best experts on this subject based on the ideXlab platform.

  • measurement of the absolute magnitude and time courses of mitochondrial membrane potential in primary and clonal pancreatic beta cells
    PLOS ONE, 2016
    Co-Authors: Akos A Gerencser, Shona A Mookerjee, Martin Jastroch, Martin D Brand
    Abstract:

    The aim of this study was to simplify, improve and validate quantitative measurement of the mitochondrial membrane potential (ΔψM) in pancreatic β-cells. This built on our previously introduced calculation of the absolute magnitude of ΔψM in intact cells, using time-lapse imaging of the non-quench mode fluorescence of TetraMethylrhodamine Methyl Ester and a bis-oxonol plasma membrane potential (ΔψP) indicator. ΔψM is a central mediator of glucose-stimulated insulin secretion in pancreatic β-cells. ΔψM is at the crossroads of cellular energy production and demand, therefore precise assay of its magnitude is a valuable tool to study how these processes interplay in insulin secretion. Dispersed islet cell cultures allowed cell type-specific, single-cell observations of cell-to-cell heterogeneity of ΔψM and ΔψP. Glucose addition caused hyperpolarization of ΔψM and depolarization of ΔψP. The hyperpolarization was a monophasic step increase, even in cells where the ΔψP depolarization was biphasic. The biphasic response of ΔψP was associated with a larger hyperpolarization of ΔψM than the monophasic response. Analysis of the relationships between ΔψP and ΔψM revealed that primary dispersed β-cells responded to glucose heterogeneously, driven by variable activation of energy metabolism. Sensitivity analysis of the calibration was consistent with β-cells having substantial cell-to-cell variations in amounts of mitochondria, and this was predicted not to impair the accuracy of determinations of relative changes in ΔψM and ΔψP. Finally, we demonstrate a significant problem with using an alternative ΔψM probe, rhodamine 123. In glucose-stimulated and oligomycin-inhibited β-cells the principles of the rhodamine 123 assay were breached, resulting in misleading conclusions.

  • quantitative measurement of mitochondrial membrane potential in cultured cells calcium induced de and hyperpolarization of neuronal mitochondria
    The Journal of Physiology, 2012
    Co-Authors: Akos A Gerencser, Martin Jastroch, David G Nicholls, Christos Chinopoulos, Matthew J Birket, Cathy Vitelli, Martin D Brand
    Abstract:

    Mitochondrial membrane potential (ΔΨM) is a central intermediate in oxidative energy metabolism. Although ΔΨM is routinely measured qualitatively or semi-quantitatively using fluorescent probes, its quantitative assay in intact cells has been limited mostly to slow, bulk-scale radioisotope distribution methods. Here we derive and verify a biophysical model of fluorescent potentiometric probe compartmentation and dynamics using a bis-oxonol-type indicator of plasma membrane potential (ΔΨP) and the ΔΨM probe TetraMethylrhodamine Methyl Ester (TMRM) using fluorescence imaging and voltage clamp. Using this model we introduce a purely fluorescence-based quantitative assay to measure absolute values of ΔΨM in millivolts as they vary in time in individual cells in monolayer culture. The ΔΨP-dependent distribution of the probes is modelled by Eyring rate theory. Solutions of the model are used to deconvolute ΔΨP and ΔΨM in time from the probe fluorescence intensities, taking into account their slow, ΔΨP-dependent redistribution and Nernstian behaviour. The calibration accounts for matrix:cell volume ratio, high- and low-affinity binding, activity coefficients, background fluorescence and optical dilution, allowing comparisons of potentials in cells or cell types differing in these properties. In cultured rat cortical neurons, ΔΨM is −139 mV at rest, and is regulated between −108 mV and −158 mV by concerted increases in ATP demand and Ca2+-dependent metabolic activation. Sensitivity analysis showed that the standard error of the mean in the absolute calibrated values of resting ΔΨM including all biological and systematic measurement errors introduced by the calibration parameters is less than 11 mV. Between samples treated in different ways, the typical equivalent error is ∼5 mV.

Manus W. Ward - One of the best experts on this subject based on the ideXlab platform.

  • Mitochondrial Membrane Potential and Glutamate Excitotoxicity in Cultured
    2013
    Co-Authors: Cerebellar Granule Cells, Manus W. Ward, Cristina A. Rego, Bruno G. Frenguelli, David G Nicholls
    Abstract:

    The relationship between changes in mitochondrial membrane potential (� � m) and the failure of cytoplasmic Ca 2 � homeostasis, delayed Ca 2 � deregulation (DCD), is investigated for cultured rat cerebellar granule cells exposed to glutamate. To interpret the single-cell fluorescence response of cells loaded with TetraMethylrhodamine Methyl Ester (TMRM � ) or rhodamine-123, we devised and validated a mathematical simulation with well characterized effectors of � � m and plasma membrane potential (� � P). Glutamate usually caused an immediate decrease in � � m of �10 mV, attributable to Ca 2 � accumulation rather than enhanced ATP demand, and these cells continued to generate ATP by oxidative phosphorylation until DCD. Cells for which the mitochondria showed a larger initial depolarization deregulated more rapidly. The mitochondria in a subpopulation of glutamate-exposed cells that failed to extrude Ca 2 � that was released from the matri

  • toxi sim a simulation tool for the analysis of mitochondrial and plasma membrane potentials
    Journal of Neuroscience Methods, 2009
    Co-Authors: Heinrich J Huber, Manus W. Ward, Martin Plchut, Petronela Weisova, Heiko Dussmann, Jakub Wenus, Markus Rehm, Jochen H M Prehn
    Abstract:

    Abstract Changes in the electrochemical gradients across biological membranes are excellent indicators of pathophysiological processes, drug action, or drug toxicity. Our previous studies have utilized the potentiometric probe TetraMethylrhodamine Methyl Ester (TMRM) to characterize changes in mitochondrial function by monitoring alterations in the mitochondrial membrane potential (Δψm) over time during glutamate excitotoxicity. However, fluorescently charged dyes such as TMRM respond to changes in both Δψm and the plasma membrane (Δψp) potentials making whole cell fluorescence data difficult to interpret. Here we have implemented a mathematical model that exploits the Nernstian behaviour of TMRM and uses automated Newton based root-finding fitting (TOXI-SIM) to model changes in TMRM fluorescence from multiple cells simultaneously, providing output on changes in Δψm and Δψp over time. Based on Ca2+ responses, TOXI-SIM allows for an accurate modelling of TMRM traces for different injury paradigms (necrosis, apoptosis, tolerance). TOXI-SIM is provided as a user friendly public web service for trace analysis, with an additional online data base provided for the storage and retrieval of experimental traces ( http://systemsbiology.rcsi.ie/tmrm/index.html ).

  • mitochondrial and plasma membrane potential of cultured cerebellar neurons during glutamate induced necrosis apoptosis and tolerance
    The Journal of Neuroscience, 2007
    Co-Authors: Manus W. Ward, David G Nicholls, Heinrich J Huber, Petronela Weisova, Heiko Dussmann, Jochen H M Prehn
    Abstract:

    A failure of mitochondrial bioenergetics has been shown to be closely associated with the onset of apoptotic and necrotic neuronal injury. Here, we developed an automated computational model that interprets the single-cell fluorescence for TetraMethylrhodamine Methyl Ester (TMRM) as a consequence of changes in either delta psi(m) or delta psi(p), thus allowing for the characterization of responses for populations of single cells and subsequent statistical analysis. Necrotic injury triggered by prolonged glutamate excitation resulted in a rapid monophasic or biphasic loss of delta psi(m) that was closely associated with a loss of delta psi(p) and a rapid decrease in neuronal NADPH and ATP levels. Delayed apoptotic injury, induced by transient glutamate excitation, resulted in a small, reversible decrease in TMRM fluorescence, followed by a sustained hyperpolarization of delta psi(m) as confirmed using the delta psi(p)-sensitive anionic probe DiBAC2(3). This hyperpolarization of delta psi(m) was closely associated with a significant increase in neuronal glucose uptake, NADPH availability, and ATP levels. Statistical analysis of the changes in delta psi(m) or delta psi(p) at a single-cell level revealed two major correlations; those neurons displaying a more pronounced depolarization of delta psi(p) during the initial phase of glutamate excitation entered apoptosis more rapidly, and neurons that displayed a more pronounced hyperpolarization of delta psi(m) after glutamate excitation survived longer. Indeed, those neurons that were tolerant to transient glutamate excitation (18%) showed the most significant increases in delta psi(m). Our results indicate that a hyperpolarization of delta psi(m) is associated with increased glucose uptake, NADPH availability, and survival responses during excitotoxic injury.

  • mitochondrial membrane potential and glutamate excitotoxicity in cultured cerebellar granule cells
    The Journal of Neuroscience, 2000
    Co-Authors: Manus W. Ward, Cristina A. Rego, Bruno G. Frenguelli, David G Nicholls
    Abstract:

    The relationship between changes in mitochondrial membrane potential (Δψm) and the failure of cytoplasmic Ca2+ homeostasis, delayed Ca2+deregulation (DCD), is investigated for cultured rat cerebellar granule cells exposed to glutamate. To interpret the single-cell fluorescence response of cells loaded with TetraMethylrhodamine Methyl Ester (TMRM+) or rhodamine-123, we devised and validated a mathematical simulation with well characterized effectors of Δψm and plasma membrane potential (ΔψP). Glutamate usually caused an immediate decrease in Δψm of <10 mV, attributable to Ca2+ accumulation rather than enhanced ATP demand, and these cells continued to generate ATP by oxidative phosphorylation until DCD. Cells for which the mitochondria showed a larger initial depolarization deregulated more rapidly. The mitochondria in a subpopulation of glutamate-exposed cells that failed to extrude Ca2+ that was released from the matrix after protonophore addition were bioenergetically competent. The onset of DCD during continuous glutamate exposure in the presence or absence of oligomycin was associated with a slowly developing mitochondrial depolarization, but cause and effect could not be established readily. In contrast, the slowly developing mitochondrial depolarization after transient NMDA receptor activation occurs before cytoplasmic free Ca2+([Ca2+]c) has risen to the set point at which mitochondria retain Ca2+. In the presence of oligomycin no increase in [Ca2+]c occurs during this depolarization. We conclude that transient Ca2+loading of mitochondria as a consequence of NMDA receptor activation initiates oxidative damage to both plasma membrane Ca2+ extrusion pathways and the inhibition of mitochondrial respiration. Depending on experimental conditions, one of these factors becomes rate-limiting and precipitates DCD.