Ca(2+)- and metabolism-related changes of mitochondrial potential in voltage-clamped CA1 pyramidal neurons in situ.
In hippocampal slices from rats, dialysis with rhodamine-123 (Rh-123) and/or fura-2 via the patch electrode allowed monitoring of mitochondrial potential (DeltaPsi) changes and intracellular Ca(2+) ([Ca(2+)](i)) of CA1 pyramidal neurons. Plasmalemmal depolarization to 0 mV caused a mean [Ca(2+)](i) rise of 300 nM and increased Rh-123 fluorescence signal (RFS) by </=50% of control. The evoked RFS, indicating depolarization of DeltaPsi, and the [Ca(2+)](i) transient were abolished by Ca(2+)-free superfusate or exposure of Ni(2+)/Cd(2+). Simultaneous measurements of RFS and [Ca(2+)](i) showed that the kinetics of both the Ca(2+) rise and recovery were considerably faster than those of the DeltaPsi depolarization. The plasmalemmal Ca(2+)/H(+) pump blocker eosin-B potentiated the peak of the depolarization-induced RFS and delayed recovery of both the RFS and [Ca(2+)](i) transient. Thus the DeltaPsi depolarization due to plasmalemmal depolarization is related to mitochondrial Ca(2+) sequestration secondary to Ca(2+) influx through voltage-gated Ca(2+) channels. CN(-) elevated [Ca(2+)](i) by <50 nM but increased RFS by 221% as a result of extensive depolarization of DeltaPsi. Oligomycin decreased RFS by 52% without affecting [Ca(2+)](i). In the presence of oligomycin, CN(-) and p-trifluoromethoxy-phenylhydrazone (FCCP) elevated [Ca(2+)](i) by <50 nM and increased RFS by 285 and 290%, respectively. Accordingly, the metabolism-related DeltaPsi changes are independent of [Ca(2+)](i). Imaging techniques revealed that evoked [Ca(2+)](i) rises are distributed uniformly over the soma and primary dendrites, whereas corresponding changes in RFS occur more localized in subregions within the soma. The results show that microfluorometric measurement of the relation between mitochondrial function and intracellular Ca(2+) is feasible in whole cell recorded mammalian neurons in situ.
Schuchmann
S
S
Institut für Physiologie, Humboldt-Universität Berlin, Universitätsklinikum Charité, D-10117 Berlin, Germany.
Lückermann
M
M
Kulik
A
A
Heinemann
U
U
Ballanyi
K
K
D000818:Animals; D002118:Calcium / Q000502:physiology*; D002138:Calibration; D002462:Cell Membrane / Q000502:physiology; D003486:Cyanides; D003956:Dialysis; D004558:Electric Stimulation; D004594:Electrophysiology; D004801:Eosine Yellowish-(YS); D005260:Female; D005456:Fluorescent Dyes; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D008297:Male; D008564:Membrane Potentials / Q000502:physiology; D008839:Microelectrodes; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism* / Q000648:ultrastructure; D009415:Nerve Net / Q000166:cytology / Q000502:physiology; D018408:Patch-Clamp Techniques; D017966:Pyramidal Cells / Q000378:metabolism / Q000502:physiology* / Q000648:ultrastructure; D051381:Rats; D017208:Rats, Wistar; D012238:Rhodium
D003486:Cyanides; D005456:Fluorescent Dyes; D012238:Rhodium; D002118:Calcium; D004801:Eosine Yellowish-(YS)