Browsing by Author "Hepp, Sebastian"

Oxygen withdrawal blocks mitochondrial respiration. In rat hippocampal slices, this triggers a massive depolarization of CA1 neurons and a negative shift of the extracellular DC potential, the characteristic sign of hypoxia-induced spreading depression (HSD). To unveil the contribution of mitochondria to the sensing of hypoxia and the ignition of HSD, we modified mitochondrial function. Mitochondrial uncoupling by carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone ( FCCP, 1 mu M) prior to hypoxia hastened the onset and shortened the duration of HSD. Blocking mitochondrial ATP synthesis by oligomycin ( 10 mu g/ml) was without effect. Inhibition of mitochondrial respiration by rotenone (20 mu M), diphenyleneiodonium (25 mu M), or antimycin A (20 mu M) also hastened HSD onset and shortened HSD duration. 3-nitropropionic acid (1 mM) increased HSD duration. Cyanide ( 100 mu M) hastened HSD onset and increased HSD duration. At higher concentrations, cyanide (1 mM), azide (2 mM), and FCCP (10 mu M) triggered SD episodes on their own. Compared with control HSD, the spatial extent of the intrinsic optical signals of cyanide- and azide-induced SDs was more pronounced. Monitoring NADH ( nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide) autofluorescence and mitochondrial membrane potential verified the mitochondrial targeting by the drugs used. Except 1 mM cyanide, no treatment reduced cellular ATP levels severely and no correlation was found between ATP, NADH, or FAD levels and the time to HSD onset. Therefore ATP depletion or a cytosolic reducing shift due to NADH/FADH(2) accumulation cannot serve as a general explanation for the hastening of HSD onset on mitochondrial inhibition. Additional redox couples (glutathione) or events downstream of the mitochondrial depolarization need to be considered.

The cytosolic redox status modulates ion channels and receptors by oxidizing/ reducing their sulfhydryl ( SH) groups. We therefore analyzed to what degree SH modulation affects hippocampal susceptibility to hypoxia. In rat hippocampal slices, severe hypoxia caused a massive depolarization of CA1 neurons and a negative shift of the extracellular DC potential, the characteristic sign of hypoxia- induced spreading depression ( HSD). Oxidizing SH groups by 5,5 '- dithiobis 2- nitrobenzoic acid ( DTNB, 2 mM) postponed HSD by 30%, whereas their reduction by 1,4- dithio- DL- threitol ( DTT, 2 mM) or alkylation by N- ethylmaleimide ( 500 mu M) hastened HSD onset. The DTNB- induced postponement of HSD was not affected by tolbutamide ( 200 mu M), DL- 2- amino- 5-phosphonovaleric acid ( 150 mu M), or 6- cyano- 7- nitroquinoxaline- 2,3-dione ( 25 mu M). It was abolished, however, by Ni2+ ( 2 mM), withdrawal of extracellular Ca2+, charybdotoxin ( 25 nM), and iberiotoxin ( 50 nM). In CA1 neurons DTNB induced a moderate hyperpolarization, blocked spontaneous spike discharges and postponed the massive hypoxic depolarization. DTT induced burst firing, depolarized glial cells, and hastened the onset of the massive hypoxic depolarization. Schaffer- collateral/ CA1 synapses were blocked by DTT but not by DTNB; axonal conduction remained intact. Mitochondria did not markedly respond to DTNB or DTT. While the targets of DTT are less clear, the postponement of HSD by DTNB indicates that sulfhydryl oxidation increases the tolerance of hippocampal tissue slices against hypoxia. We identified as the underlying mechanism the activation of BK channels in a Ca2+- sensitive manner. Accordingly, ionic disregulation and the loss of membrane potential occur later or might even be prevented during short- term insults. Therefore well- directed oxidation of SH groups could mediate neuroprotection.

Previously we reported that sulfhydryl (SH) modulation affects the susceptibility of rat hippocampal slices to severe hypoxia. SH-oxidation by DTNB (5,5'-dithiobis 2-nitrobenzoic acid) or H2O2 postponed the onset of hypoxia-induced spreading depression (HSD), thereby delaying the loss of neuronal function, whereas SH-reduction by DTT (1,4-dithio-DL-threitol) hastened HSD onset. To judge the neuroprotective merit that might arise from a postponement of HSD by oxidants, we have extended our earlier observations by multiparametric recordings and screened for changes in the extracellular K+ accumulation, HSD propagation velocity, and its maximum spread. As parameters for neuronal network function, the failure of synapses during hypoxia and their posthypoxic recovery were analyzed. DTNB (2 mM) or H2O2 (5 mM) postponed HSD but did not attenuate the rise in extracellular K+ concentration ([K+](o)), HSD propagation velocity or its maximum spread. H2O2 slightly postponed the synaptic failure during hypoxia; the posthypoxic recovery of synapses was, however, incomplete. DTNB slowed the synaptic recovery upon reoxygenation. DTT (2 mM) hastened HSD onset, but HSD propagation velocity and tissue invasion were not affected. Upon reoxygenation, however, normalization of [K+](o) was disturbed and synaptic recovery failed. Therefore, SH-reducing conditions at the onset of HSD proved to be devastating for the hippocampal network. In conclusion, the only merit of DTNB or H2O2 treatment is a delayed HSD onset, i.e. some extra time before neuronal function is lost during severe hypoxia. Attenuation of the severe changes during HSD or an improved outcome was not observed. Nevertheless, combination of SH-oxidants with established neuroprotectants might be a potential therapeutic approach. (c) 2008 IBRO. Published by Elsevier Ltd. All rights reserved.