Ca²⁺- and Metabolism-Related Changes of Mitochondrial Potential in Voltage-Clamped CA1 Pyramidal Neurons In Situ

Abstract

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.

Protective role of neuronal KATP channels in brain hypoxia

“…K ATP channels in brain hypoxia also not affected by depleting endoplasmic reticulum Ca 2+ stores with cyclopiazonic acid (Fig.·3C). On the contrary, the Cai response to anoxia or cyanide is both mimicked and occluded by the protonophore FCCP (Fig.·3C), which dissipates the mitochondrial transmembrane H + gradient (Schuchmann et al, 2000). These results suggest that the moderate anoxic rise of Cai is caused by mitochondrial Ca 2+ release .…”

Mitochondrial Calcium Ion and Membrane Potential Transients Follow the Pattern of Epileptiform Discharges in Hippocampal Slice Cultures

“…2؉ ion uptake and extrusion in changes of ⌬⌿m It was shown that changes in ⌬⌿m evoked by a prolonged (15 s) depolarization step above the threshold of Ϫ40 mV were dependent on Ca 2ϩ ion influx through voltage-gated calcium channels in pyramidal cells (Schuchmann et al, 2000). Therefore, we hypothesized that the loss of ⌬⌿m during SLE was dependent on an increase in [Ca 2ϩ ] i .…”

“…To give a relative estimation of the change in ⌬⌿m, we compared SLEassociated cytosolic Rh-123 elevation with the effect of protonophore application at the end of an SLE sequence. The protonophores FCCP and CCCP were shown to completely dissipate ⌬⌿m and to increase the intensity of cytosolic Rh-123 fluorescence (Duchen, 1992;Schuchmann et al, 2000;Kovács et al, 2001). Application of a protonophore resulted in an overall increase in the cytosolic Rh-123 fluorescence by 132 Ϯ 9% (n ϭ 3 cells), whereby fluorescence of single mitochondria disappeared (Fig.…”

“…⌬⌿m was monitored using a voltage-sensitive mitochondrial dye, rhodamine-123 (Rh-123) (Schuchmann et al, 2000), whereas measurements of [Ca 2ϩ ] m were performed using the mitochondria-specific Ca 2ϩ ionsensitive dyes rhod-2 and rhod-ff (Kovács et al, 2001;Billups and Forsythe, 2002).…”

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

“…As a result of the decreased H + conductance in response to these ATP-synthase inhibitors, mitochondria become hyperpolarized (as indicated by the monitoring of ?? m by e.g., Rh123) Schuchmann et al, 2000).…”