The Journal of Neurophysiology Vol. 84 No. 1 July 2000, pp. 311-324
Copyright ©2000 by the American Physiological Society

Mitochondrial and Intrinsic Optical Signals Imaged During Hypoxia and Spreading Depression in Rat Hippocampal Slices

 ¹Department of Cell Biology,  ²Department of Neurobiology,  ³Department of Physics, and  ⁴Department of Surgery (Neurosurgery), Duke University; and  ⁵Durham Veterans Affairs Medical Center, Durham, North Carolina 27710

Bahar, S., D. Fayuk, G. G. Somjen, P. G. Aitken, and D. A. Turner. Mitochondrial and Intrinsic Optical Signals Imaged During Hypoxia and Spreading Depression in Rat Hippocampal Slices. J. Neurophysiol. 84: 311-324, 2000. 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 (V_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 V_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 V_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 Ca²⁺ was removed from the external medium, resulting in complete synaptic blockade, the mitochondrial response to hypoxia did not significantly differ from control (normal Ca²⁺) 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 Ca²⁺-free medium on the R123 and IOS responses suggests that the protection against hypoxic damage by low Ca²⁺ is not due to the prevention of mitochondrial depolarization.