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Simultaneous single neuron recording of O2 consumption, [Ca2+]i and mitochondrial membrane potential in glutamate toxicity

Simultaneous single neuron recording of O2 consumption, [Ca2+]i and mitochondrial membrane potential in glutamate toxicity
Simultaneous single neuron recording of O2 consumption, [Ca2+]i and mitochondrial membrane potential in glutamate toxicity
In order to determine the sequence of cellular processes in glutamate toxicity, we simultaneously recorded O(2) consumption, cytosolic Ca(2+) concentration ([Ca(2+)](i)), and mitochondrial membrane potential (mDeltapsi) in single cortical neurons. Oxygen consumption was measured using an amperometric self-referencing platinum electrode adjacent to neurons in which [Ca(2+)](i) and mDeltapsi were monitored with Fluo-4 and TMRE(+), respectively, using a spinning disk laser confocal microscope. Excitotoxic doses of glutamate caused an elevation of [Ca(2+)](i) followed seconds afterwards by an increase in O(2) consumption which reached a maximum level within 1-5 min. A modest increase in mDeltapsi occurred during this time period, and then, shortly before maximal O(2) consumption was reached, the mDeltapsi, as indicated by TMRE(+) fluorescence, dissipated. Maximal O(2) consumption lasted up to 5 min and then declined together with mDeltapsi and ATP levels, while [Ca(2+)](i) further increased. mDeltapsi and [Ca(2+)](i) returned to baseline levels when neurons were treated with an NMDA receptor antagonist shortly after the [Ca(2+)](i) increased. Our unprecedented spatial and time resolution revealed that this sequence of events is identical in all neurons, albeit with considerable variability in magnitude and kinetics of changes in O(2) consumption, [Ca(2+)](i), and mDeltapsi. The data obtained using this new method are consistent with a model where Ca(2+) influx causes ATP depletion, despite maximal mitochondrial respiration, minutes after glutamate receptor activation.
excitotoxicity, glutamate, oxygen consumption
0022-3042
644-655
Gleichmann, Marc
5c37d9d3-144c-40ed-8b30-2a434c0b4bbc
Collis, Leon P.
66f825a3-f4a7-4e85-a1ca-ee16497b8a81
Smith, Peter J.S.
003de469-9420-4f12-8f0e-8e8d76d28d6c
Mattson, Mark P.
6f263544-11d5-46b5-a3b8-2c290cbb923c
Gleichmann, Marc
5c37d9d3-144c-40ed-8b30-2a434c0b4bbc
Collis, Leon P.
66f825a3-f4a7-4e85-a1ca-ee16497b8a81
Smith, Peter J.S.
003de469-9420-4f12-8f0e-8e8d76d28d6c
Mattson, Mark P.
6f263544-11d5-46b5-a3b8-2c290cbb923c

Gleichmann, Marc, Collis, Leon P., Smith, Peter J.S. and Mattson, Mark P. (2009) Simultaneous single neuron recording of O2 consumption, [Ca2+]i and mitochondrial membrane potential in glutamate toxicity. Journal of Neurochemistry, 109 (2), 644-655. (doi:10.1111/j.1471-4159.2009.05997.x). (PMID:19226367)

Record type: Article

Abstract

In order to determine the sequence of cellular processes in glutamate toxicity, we simultaneously recorded O(2) consumption, cytosolic Ca(2+) concentration ([Ca(2+)](i)), and mitochondrial membrane potential (mDeltapsi) in single cortical neurons. Oxygen consumption was measured using an amperometric self-referencing platinum electrode adjacent to neurons in which [Ca(2+)](i) and mDeltapsi were monitored with Fluo-4 and TMRE(+), respectively, using a spinning disk laser confocal microscope. Excitotoxic doses of glutamate caused an elevation of [Ca(2+)](i) followed seconds afterwards by an increase in O(2) consumption which reached a maximum level within 1-5 min. A modest increase in mDeltapsi occurred during this time period, and then, shortly before maximal O(2) consumption was reached, the mDeltapsi, as indicated by TMRE(+) fluorescence, dissipated. Maximal O(2) consumption lasted up to 5 min and then declined together with mDeltapsi and ATP levels, while [Ca(2+)](i) further increased. mDeltapsi and [Ca(2+)](i) returned to baseline levels when neurons were treated with an NMDA receptor antagonist shortly after the [Ca(2+)](i) increased. Our unprecedented spatial and time resolution revealed that this sequence of events is identical in all neurons, albeit with considerable variability in magnitude and kinetics of changes in O(2) consumption, [Ca(2+)](i), and mDeltapsi. The data obtained using this new method are consistent with a model where Ca(2+) influx causes ATP depletion, despite maximal mitochondrial respiration, minutes after glutamate receptor activation.

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Published date: April 2009
Keywords: excitotoxicity, glutamate, oxygen consumption
Organisations: University of Southampton

Identifiers

Local EPrints ID: 190277
URI: http://eprints.soton.ac.uk/id/eprint/190277
ISSN: 0022-3042
PURE UUID: 581c1216-09b2-41ca-81f2-c9f38e0ab3ef
ORCID for Peter J.S. Smith: ORCID iD orcid.org/0000-0003-4400-6853

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Date deposited: 13 Jun 2011 09:02
Last modified: 26 Nov 2019 01:39

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Author: Marc Gleichmann
Author: Leon P. Collis
Author: Mark P. Mattson

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