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, 22 (14), 5840-7

Presynaptic Mitochondrial Calcium Sequestration Influences Transmission at Mammalian Central Synapses

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Presynaptic Mitochondrial Calcium Sequestration Influences Transmission at Mammalian Central Synapses

Brian Billups et al. J Neurosci.

Abstract

Beyond their role in generating ATP, mitochondria have a high capacity to sequester calcium. The interdependence of these functions and limited access to presynaptic compartments makes it difficult to assess the role of sequestration in synaptic transmission. We addressed this important question using the calyx of Held as a model glutamatergic synapse by combining patch-clamp with a novel mitochondrial imaging method. Presynaptic calcium current, mitochondrial calcium concentration ([Ca(2+)](mito), measured using rhod-2 or rhod-FF), cytoplasmic calcium concentration ([Ca(2+)](cyto), measured using fura-FF), and the postsynaptic current were monitored during synaptic transmission. Presynaptic [Ca(2+)](cyto) rose to 8.5 +/- 1.1 microM and decayed rapidly with a time constant of 45 +/- 3 msec; presynaptic [Ca(2+)](mito) also rose rapidly to >5 microM but decayed slowly with a half-time of 1.5 +/- 0.4 sec. Mitochondrial depolarization with rotenone and carbonyl cyanide p-trifluoromethoxyphenylhydrazone abolished mitochondrial calcium rises and slowed the removal of [Ca(2+)](cyto) by 239 +/- 22%. Using simultaneous presynaptic and postsynaptic patch clamp, combined with presynaptic mitochondrial and cytoplasmic imaging, we investigated the influence of mitochondrial calcium sequestration on transmitter release. Depletion of ATP to maintain mitochondrial membrane potential was blocked with oligomycin, and ATP was provided in the patch pipette. Mitochondrial depolarization raised [Ca(2+)](cyto) and reduced transmitter release after short EPSC trains (100 msec, 200 Hz); this effect was reversed by raising mobile calcium buffering with EGTA. Our results suggest a new role for presynaptic mitochondria in maintaining transmission by accelerating recovery from synaptic depression after periods of moderate activity. Without detectable thapsigargin-sensitive presynaptic calcium stores, we conclude that mitochondria are the major organelle regulating presynaptic calcium at central glutamatergic terminals.

Figures

Fig. 1.
Fig. 1.
Rapid time course of presynaptic [Ca2+] transients in the calyx of Held.A, DIC image of a calyx of Held (arrow) surrounding a postsynaptic MNTB neuron (asterisk). Scale bar, 15 μm. Patch pipette is to the right.B, Fluorescence image from the same calyx filled with fura-FF via diffusion from the patch pipette. C, Calcium currents (bottom trace) recorded in the calyx of Held in response to four voltage steps (−80 to 0 mV depolarization, 2 msec duration at 100 Hz; top trace). D,[Ca2+]cyto time course during stimulation recorded simultaneously with the calcium current from the same terminal as in C using fura-FF imaging at a frame frequency of 100 Hz (stimulation at arrow as shown in C).
Fig. 2.
Fig. 2.
Slow time course of presynaptic mitochondrial [Ca2+] transients. A, DIC image of the presynaptic terminal. Scale bar, 15 μm. B,Projected stack of fura-FF images focused through the entire terminal.C, Projected stack of rhod-2 images showing that fluorescence was concentrated in the terminal but was absent from the pipette and axon regions. D,[Ca2+]mito rose rapidly on stimulation (as in Fig. 1C, black trace) but decayed slowly. Four groups of four stimuli (the same protocol as in Fig. 1Crepeated 4 times 150 msec apart, gray trace) had a similar time course and amplitude, indicating saturation of the dye. Mitochondrial depolarization (perfusion of 25 μm rotenone and 1 μm FCCP in the presence of 5 μg/ml oligomycin) completely blocked calcium accumulation by the mitochondria after one group of four stimuli (bottom black trace).E, Rhod-FF fluorescence signals were not saturated by four groups of four stimuli (gray trace) compared with one group of stimuli (black trace). Thetraces in D and E are shown normalized to the change in fluorescence after one group of stimuli. F, Summary data show that mitochondrial calcium uptake was inhibited by mitochondrial depolarization and 10 μm TPP+ but not 1 μmthapsigargin. Mitochondrial calcium sequestration saturated rhod-2 (Kd, 570 nm) but not rhod-FF (Kd, 19 μm).Asterisks indicate statistical significance (p < 0.05).
Fig. 3.
Fig. 3.
Mitochondrial calcium sequestration buffers [Ca2+]cyto. A, The [Ca2+]cyto transient (using the same stimulation protocol as Fig. 1) was significantly slowed by mitochondrial depolarization, but the peak rise was not significantly altered (97 ± 6% of control; n = 5;p = 0.66). B, The τfast [Ca2+]cyto decay was slowed by mitochondrial depolarization and TPP+but not by thapsigargin (129 ± 9% of control;n = 3; p = 0.08). C, D, Trains of presynaptic depolarization (−80 to 0 mV, 1 msec stimulation repeated 20 times at 200 Hz) produced rises in [Ca2+]cyto that were also significantly slowed by mitochondrial depolarization or 1 μm Ru360. E, F, [Ca2+]mito recorded with rhod-FF increased during stimulation (at arrow as inC). This increase was blocked by mitochondrial depolarization or Ru360. Asterisks indicate statistical significance (p < 0.05).
Fig. 4.
Fig. 4.
Presynaptic mitochondrial calcium sequestration influences synaptic transmission. A, The postsynaptic MNTB neuron and presynaptic calyx were simultaneously voltage clamped. Scale bar, 10 μm. B, Trains of stimuli (as in Fig. 3C) produced a markedly depressing EPSC train. At 500 msec after the end of the train, the EPSC had recovered to 81% of control amplitude. C, D,Presynaptic calcium currents recorded in response to the train were unaffected by mitochondrial depolarization. E,Postsynaptic currents normalized to the magnitude of the first EPSC. The absolute magnitude of the EPSC was −4.6 ± 0.4 nA before mitochondrial depolarization and −4.3 ± 0.4 nA after. This 6 ± 2% reduction was a time-dependent run-down and was not statistically significant (n = 3;p = 0.07). Mitochondrial depolarization induced by rotenone and FCCP in the presence of oligomycin had no effect on the initial train amplitude and time course, but it significantly reduced the relative magnitude of the test EPSC. F, Identical results were observed after mitochondrial depolarization or by blocking the calcium uniporter with intracellular Ru360 (1 μm).G, Inclusion of 1 mm EGTA in the presynaptic patch pipette accelerated the decay and reduced the effect of mitochondrial depolarization on the presynaptic calcium transient.H, Presynaptic EGTA at 1 mm abolished the effects of mitochondrial depolarization on the rate of recovery from synaptic depression. Asterisks indicate statistical significance (p < 0.05).

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