title: Mitochondrial and intrinsic optical signals imaged during hypoxia and spreading depression in rat hippocampal slices.
abstract: During hypoxia in the CA1 region of the rat hippocampus, spreading-depression-like depolarization (hypoxic spreading depression or HSD) is accompanied by both a negative shift of the extracellular DC potential (DeltaV(o)), and a sharp decrease in light transmittance (intrinsic optical signal or IOS). To investigate alterations in mitochondrial function during HSD and normoxic spreading depression (SD), we simultaneously imaged mitochondrial depolarization, using rhodamine-123 (R123) fluorescence, and IOS while monitoring extracellular voltage. Three major phases of the R123 signal were observed during hypoxia: a gradual, diffuse fluorescence increase, a sharp increase in fluorescence coincident with the HSD-related DeltaV(o), primarily in the CA1 region, and a plateau-like phase if reoxygenation is delayed after HSD onset, persisting until reoxygenation occurs. Two phases occurred following re-oxygenation: an abrupt and then slow decrease in fluorescence to near baseline and a slow secondary increase to slightly above baseline and a late recovery. Parallel phases of the IOS response during hypoxia were also observed though delayed compared with the R123 responses: an initial increase, a large decrease coincident with the HSD-related DeltaV(o), and a trough following HSD. After reoxygenation, there occurred a delayed increase in transmittance and then a slow decrease, returning to near baseline. When Ca(2+) was removed from the external medium, resulting in complete synaptic blockade, the mitochondrial response to hypoxia did not significantly differ from control (normal Ca(2+)) conditions. In slices maintained in low-chloride (2.4 mM) medium, a dramatic reversal in the direction of the IOS signal associated with HSD occurred, and the R123 signal during HSD was severely attenuated. Normoxic SD induced by micro-injection of KCl was also associated with a decrease in light transmittance and a sharp increase in R123 fluorescence but both responses were less pronounced than during HSD. Our results show two mitochondrial responses to hypoxia: an initial depolarization that appears to be caused by depressed electron transport due to lack of oxygen and a later, sudden, sharp depolarization linked to HSD. The depression of the second, sharp depolarization and the inversion of the IOS in low-chloride media suggest a role of Cl(-)-dependent mitochondrial swelling. Lack of effect of Ca(2+)-free medium on the R123 and IOS responses suggests that the protection against hypoxic damage by low Ca(2+) is not due to the prevention of mitochondrial depolarization.
authors.affiliation: Department of Physics, Duke University, Durham, North Carolina 27710, USA.
authors.forename: S
authors.initials: S
authors.lastname: Bahar
authors.forename: D
authors.initials: D
authors.lastname: Fayuk
authors.forename: G G
authors.initials: GG
authors.lastname: Somjen
authors.forename: P G
authors.initials: PG
authors.lastname: Aitken
authors.forename: D A
authors.initials: DA
authors.lastname: Turner
mesh_terms: D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002712:Chlorides / Q000494:pharmacology; D013181:Cortical Spreading Depression / Q000502:physiology*; D019706:Excitatory Postsynaptic Potentials / Q000187:drug effects / Q000502:physiology; D005456:Fluorescent Dyes; D006624:Hippocampus / Q000503:physiopathology*; D002534:Hypoxia, Brain / Q000503:physiopathology*; D066298:In Vitro Techniques; D008297:Male; D008928:Mitochondria / Q000502:physiology*; D055095:Optics and Photonics; D011189:Potassium Chloride / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D020112:Rhodamine 123
chemical_list: D002712:Chlorides; D005456:Fluorescent Dyes; D020112:Rhodamine 123; D011189:Potassium Chloride; D002118:Calcium