Mitochondrial Inhibition Prior to Oxygen-Withdrawal Facilitates the Occurrence of Hypoxia-Induced Spreading Depression in Rat Hippocampal Slices

Abstract

Gerich, Florian J., Sebastian Hepp, Irmelin Probst, and Michael Mü ller. Mitochondrial inhibition prior to oxygen-withdrawal facilitates the occurrence of hypoxia-induced spreading depression in rat hippocampal slices. J Neurophysiol 96: 492-504, 2006. First published April 12, 2006 doi:10.1152/jn.01015.2005. 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 M) prior to hypoxia hastened the onset and shortened the duration of HSD. Blocking mitochondrial ATP synthesis by oligomycin (10 g/ml) was without effect. Inhibition of mitochondrial respiration by rotenone (20 M), diphenyleneiodonium (25 M), or antimycin A (20 M) also hastened HSD onset and shortened HSD duration. 3-nitropropionic acid (1 mM) increased HSD duration. Cyanide (100 M) hastened HSD onset and increased HSD duration. At higher concentrations, cyanide (1 mM), azide (2 mM), and FCCP (10 M) triggered SD episodes on their own. Compared with control HSD, the spatial extent of the intrinsic optical signals of cyanide-and azideinduced 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.

Optical and pharmacological tools to investigate the role of mitochondria during oxidative stress and neurodegeneration

“…Choosing the appropriate filter sets, we have been able to measure NADH and FAD levels simultaneously from the same location in acute hippocampal slices (Figs. 4B,5C; see also (Gerich et al, 2006;Hepp et al, 2005)). …”

“…Interestingly, the administration of DPI induced a decrease in NADH autofluorescence and an increase in flavin adenine dinucleotide (FAD) levels suggesting that mitochondrial respiration was stimulated. This effect, however, was probably caused independently of the inhibition of complex I by DPI (Gerich et al, 2006). In brain preparations, rotenone has been one of the most widely used inhibitors to study the role of the electron transport chain in the production of ROS.…”

“…The application of CN − (2 mM) to CA1 neurons causes hyperpolarization in cells with less negative resting membrane potential, and transient depolarization in cells with more negative resting membrane potential, indicating that CN − activates K + channels. Applied to interface hippocampal slices, 1 mM CN − (as well as 2 mM azide) trigger spontaneous spreading depression episodes despite the presence of O 2 (Gerich et al, 2006). Activation of K ATP channels by CN − was found in dorsal vagal neurons, where K ATP channels were activated independent of ATP depletionobviously as a direct consequence of impaired mitochondrial metabolism or mitochondrial depolarization (Müller et al, 2002).…”

“…Such ratio-like approaches might allow correction for cell swelling or changes in the intrinsic optical properties of the tissue under investigation, and we have used them to verify and compare the targeting of mitochondrial metabolism by various mitochondrial inhibitors in rat hippocampal slices (Gerich et al, 2006). All of the above methods involve one-photon excitation at near-UV wavelengths, which inevitably leads to pronounced light scattering, photobleaching and particularly photodamage of tissue samples.…”

NADH hyperoxidation correlates with enhanced susceptibility of aged rats to hypoxia

“…For example, a previous study demonstrated that the exogenous application of the uncoupler carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) in rat hippocampal slices caused a reversible decrease in NADH levels (i.e. oxidation) due to the occurrence of mitochondrial depolarization (as shown by an increase in R123) and the activation of mitochondrial respiration (Gerich et al, 2006).…”