NADH in the pyramidal cell layer of hippocampal regions CA1 and CA3 upon selective inhibition and uncoupling of oxidative phosphorylation.
N2-laser-induced fluorescence in combination with the time and spectral resolution of fluorescent NADH molecules allows on-line measurement of relative NADH concentration with high spatial resolution (diameter of optical fibre 200 microns, lambda(exc) = 337 nm, lambda(det) = 460 nm). Energy metabolism was impaired in submerged rat hippocampal slices using the inhibitors amytal, 3-nitropropionate (3-np), sodium cyanide (1 mM each) and the uncoupling agent 2,4-DNP (200 microM). A microprocessor-controlled repeated positioning of the optical fibre in CA1 and CA3 pyramidal cell layers, and CA1 stratum radiatum (CA1SR). Time-dependently, NADH fluorescence increased reversibly upon perfusion with amytal and cyanide. It was unchanged by perfusion with 3-np for 40 min and rapidly decreased upon perfusion with 2,4-DNP. The CA1/CA3 ratio of NADH fluorescence mildly decreased to 0.92 +/- 0.04 (mean +/- S.D.) at 10 min (P < 0.05) and 0.89 +/- 0.05 at 20 min (P < 0.01) upon perfusion with amytal. The CA1/CA3 ratio increased to 1.56 +/- 0.28 at 10 min (P < 0.01) and 1.29 +/- 0.35 at 20 min (P < 0.05) upon application of 2,4-DNP. Fluorescence in CA1SR was similar to fluorescence in CA1 upon perfusion with 2,4-DNP and similar to CA3 upon perfusion with amytal. We conclude that NADH fluorescence can be measured with high regional selectivity and specificity in hippocampal slices. Selective inhibition of mitochondrial complex I and uncoupling of energy metabolism differentially impair NADH concentration in different hippocampal areas.
Riepe
M W
MW
Department of Neurology, Humboldt University (Charité), Berlin, Germany. riepe@rz.charite.hu-berlin.de
Schmalzigaug
K
K
Fink
F
F
Oexle
K
K
Ludolph
A C
AC
D019297:2,4-Dinitrophenol / Q000494:pharmacology; D000654:Amobarbital / Q000494:pharmacology; D000818:Animals; D005470:Fluorometry; D006624:Hippocampus / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D066298:In Vitro Techniques; D008297:Male; D009243:NAD / Q000378:metabolism*; D009574:Nitro Compounds; D010085:Oxidative Phosphorylation; D011422:Propionates / Q000494:pharmacology; D017966:Pyramidal Cells / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D012966:Sodium Cyanide / Q000494:pharmacology; D014475:Uncoupling Agents / Q000494:pharmacology
D009574:Nitro Compounds; D011422:Propionates; D014475:Uncoupling Agents; D009243:NAD; D000654:Amobarbital; D012966:Sodium Cyanide; D019297:2,4-Dinitrophenol; C015392:3-nitropropionic acid