Document Detail


Monitoring NAD(P)H autofluorescence to assess mitochondrial metabolic functions in rat hippocampal-entorhinal cortex slices.
MedLine Citation:
PMID:  11431129     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Changes in neuronal energy metabolism, mitochondrial functions and homeostasis of reactive oxygen species are often supposed to induce alterations in neuronal activity in hippocampal slice models. In order to investigate the NAD(P)H autofluorescence signal in brain slice models, methods to monitor NAD(P)H signal in isolated mitochondria as described by Chance et al. [J. Biol. Chem. 254 (1979) 4764] and dissociated neurons as described by Duchen [Biochem. J. 283 (1992) 41] were adapted to recording conditions required for brain slices. Considering different experimental questions, we established an approach to monitor NAD(P)H autofluorescence signals from hippocampal slices of 400 microm thickness under either submerged or interface conditions. Therefore the procedure described here allows the measurement of NAD(P)H autofluorescence under conditions typically required in electrophysiological experiments. Depolarization of plasma membrane caused by electrical stimulation or application of glutamate (100 microM) resulted in a characteristic initial decrease followed by a long-lasting increase in the NAD(P)H autofluorescence signal. H(2)O(2) (100 microM) evoked a strong NAD(P)H signal decrease indicating direct oxidation to the nonfluorescencend NAD(P)(+). In contrast, the increase in NAD(P)H signal that followed a brief inhibition of mitochondrial respiratory chain complex I using rotenone (1 microM) indicated an accumulation of NAD(P)H. However, in presence of rotenone (1 microM) electrically evoked long-lasting NAD(P)H signal overshoot decreased progressively, due to a negative feedback of accumulated NAD(P)H to the citrate cycle. A comparable reduction in NAD(P)H signal increase were observed during low-Mg(2+) induced epileptiform activity, indicating a relative energy failure. In conclusion, the method presented here allows to monitor NAD(P)H autofluorescence signals to gain insight into the coupling of neuronal activity, energy metabolism and mitochondrial function in brain slice models.
Authors:
S Schuchmann; R Kovacs; O Kann; U Heinemann; K Buchheim
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Publication Detail:
Type:  In Vitro; Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Brain research. Brain research protocols     Volume:  7     ISSN:  1385-299X     ISO Abbreviation:  Brain Res. Brain Res. Protoc.     Publication Date:  2001 Jul 
Date Detail:
Created Date:  2001-06-29     Completed Date:  2001-09-13     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  9716650     Medline TA:  Brain Res Brain Res Protoc     Country:  Netherlands    
Other Details:
Languages:  eng     Pagination:  267-76     Citation Subset:  IM    
Affiliation:
Institut für Physiologie, Universitätsklinikum Charité, Humboldt-Universität Berlin, Tucholskystrasse 2, D-10117 Berlin, Germany. sebastian.schuchmann@charite.de
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MeSH Terms
Descriptor/Qualifier:
Animals
Electric Stimulation
Electron Transport / physiology
Energy Metabolism / physiology
Entorhinal Cortex / chemistry,  cytology,  metabolism*
Female
Fluorescence
Hippocampus / chemistry,  cytology,  metabolism*
Indicators and Reagents
Male
Microelectrodes
Mitochondria / chemistry*,  metabolism*
NADP / metabolism*
Potassium / metabolism
Rats
Reactive Oxygen Species / metabolism
Chemical
Reg. No./Substance:
0/Indicators and Reagents; 0/Reactive Oxygen Species; 53-59-8/NADP; 7440-09-7/Potassium

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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