Presynaptic Mitochondrial Calcium Sequestration Influences Transmission at Mammalian Central Synapses

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 ([Ca2+]mito, measured using rhod-2 or rhod-FF), cytoplasmic calcium concentration ([Ca2+]cyto, measured using fura-FF), and the postsynaptic current were monitored during synaptic transmission. Presynaptic [Ca2+]cytorose to 8.5 ± 1.1 μm and decayed rapidly with a time constant of 45 ± 3 msec; presynaptic [Ca2+]mito also rose rapidly to >5 μm but decayed slowly with a half-time of 1.5 ± 0.4 sec. Mitochondrial depolarization with rotenone and carbonyl cyanidep-trifluoromethoxyphenylhydrazone abolished mitochondrial calcium rises and slowed the removal of [Ca2+]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 [Ca2+]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.

[1]  B. Sakmann,et al.  Calcium dynamics associated with a single action potential in a CNS presynaptic terminal. , 1997, Biophysical journal.

[2]  S. Hagiwara,et al.  Differences in Na and Ca Spikes As Examined by Application of Tetrodotoxin, Procaine, and Manganese Ions , 1966, The Journal of general physiology.

[3]  R. Zucker,et al.  Mitochondrial Involvement in Post-Tetanic Potentiation of Synaptic Transmission , 1997, Neuron.

[4]  R. Tuft,et al.  Mitochondrial Ca2+ homeostasis during Ca2+ influx and Ca2+ release in gastric myocytes from Bufo marinus , 2000, The Journal of physiology.

[5]  W. N. Ross,et al.  The spread of Na+ spikes determines the pattern of dendritic Ca2+ entry into hippocampal neurons , 1992, Nature.

[6]  T. Gunter,et al.  Mechanisms by which mitochondria transport calcium. , 1990, The American journal of physiology.

[7]  M. Duchen,et al.  Mitochondria Exert a Negative Feedback on the Propagation of Intracellular Ca2+ Waves in Rat Cortical Astrocytes , 1999, The Journal of cell biology.

[8]  A. González,et al.  Agonist-evoked Mitochondrial Ca2+ Signals in Mouse Pancreatic Acinar Cells* , 2000, The Journal of Biological Chemistry.

[9]  R. Tsien,et al.  Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores. , 1989, The Journal of biological chemistry.

[10]  B. Sakmann,et al.  Pre‐ and postsynaptic whole‐cell recordings in the medial nucleus of the trapezoid body of the rat. , 1995, The Journal of physiology.

[11]  J. García-Sancho,et al.  Chromaffin-cell stimulation triggers fast millimolar mitochondrial Ca2+ transients that modulate secretion , 2000, Nature Cell Biology.

[12]  G. Szabadkai,et al.  Cytoplasmic Ca2+ at low submicromolar concentration stimulates mitochondrial metabolism in rat luteal cells , 2001, Pflügers Archiv.

[13]  G. Spirou,et al.  Specialized Synapse-Associated Structures within the Calyx of Held , 2000, The Journal of Neuroscience.

[14]  J. Russell,et al.  Mitochondria Support Inositol 1,4,5-Trisphosphate-mediated Ca2+ Waves in Cultured Oligodendrocytes* , 1996, The Journal of Biological Chemistry.

[15]  Tullio Pozzan,et al.  Rapid changes of mitochondrial Ca2+ revealed by specifically targeted recombinant aequorin , 1992, Nature.

[16]  I. Forsythe,et al.  Presynaptic Calcium Current Modulation by a Metabotropic Glutamate Receptor , 1996, Science.

[17]  D. Richter,et al.  Oscillations and hypoxic changes of mitochondrial variables in neurons of the brainstem respiratory centre of mice , 2001, The Journal of physiology.

[18]  M. Duchen,et al.  Contributions of mitochondria to animal physiology: from homeostatic sensor to calcium signalling and cell death , 1999, The Journal of physiology.

[19]  G. Matthews,et al.  The Role of Mitochondria in Presynaptic Calcium Handling at a Ribbon Synapse , 2000, Neuron.

[20]  B Sakmann,et al.  Calcium current during a single action potential in a large presynaptic terminal of the rat brainstem , 1998, The Journal of physiology.

[21]  K. Yamagami,et al.  Sequestration of depolarization-induced Ca2+ loads by mitochondria and Ca2+ efflux via mitochondrial Na+/Ca2+ exchanger in bovine adrenal chromaffin cells. , 1999, The Japanese journal of physiology.

[22]  E. Barrett,et al.  Evidence that mitochondria buffer physiological Ca2+ loads in lizard motor nerve terminals , 1998, The Journal of physiology.

[23]  Todd B. Sherer,et al.  Chronic systemic pesticide exposure reproduces features of Parkinson's disease , 2000, Nature Neuroscience.

[24]  E. Stuenkel,et al.  Mitochondria Regulate the Ca2+–Exocytosis Relationship of Bovine Adrenal Chromaffin Cells , 1999, The Journal of Neuroscience.

[25]  E. Barrett,et al.  Stimulation-Evoked Increases in Cytosolic [Ca2+] in Mouse Motor Nerve Terminals Are Limited by Mitochondrial Uptake and Are Temperature-Dependent , 2000, The Journal of Neuroscience.

[26]  D. Bers,et al.  Cytosolic and mitochondrial Ca2+ signals in patch clamped mammalian ventricular myocytes , 1998, The Journal of physiology.

[27]  Margaret Barnes-Davies,et al.  Inactivation of Presynaptic Calcium Current Contributes to Synaptic Depression at a Fast Central Synapse , 1998, Neuron.

[28]  S. J. Smith,et al.  Calcium entry into voltage‐clamped presynaptic terminals of squid. , 1985, The Journal of physiology.

[29]  P. Robinson,et al.  Ca2+ Influx Inhibits Dynamin and Arrests Synaptic Vesicle Endocytosis at the Active Zone , 2000, The Journal of Neuroscience.

[30]  B. Hille,et al.  Mitochondria Shape Hormonally Induced Cytoplasmic Calcium Oscillations and Modulate Exocytosis* , 2000, The Journal of Biological Chemistry.

[31]  Y. Peng,et al.  Caffeine and carbonyl cyanide m-chlorophenylhydrazone increased evoked and spontaneous release of luteinizing hormone-releasing hormone from intact presynaptic terminals , 1999, Neuroscience.

[32]  P. B. Simpson,et al.  Mitochondrial Ca2+ uptake and release influence metabotropic and ionotropic cytosolic Ca2+ responses in rat oligodendrocyte progenitors , 1998, The Journal of physiology.

[33]  R. Regazzi,et al.  Disruption of Rab3–calmodulin interaction, but not other effector interactions, prevents Rab3 inhibition of exocytosis , 1999, The EMBO journal.

[34]  R. Tuft,et al.  Release of Ca2+ from the sarcoplasmic reticulum increases mitochondrial [Ca2+] in rat pulmonary artery smooth muscle cells , 1999, The Journal of physiology.

[35]  W G Regehr,et al.  Calcium Dependence and Recovery Kinetics of Presynaptic Depression at the Climbing Fiber to Purkinje Cell Synapse , 1998, The Journal of Neuroscience.

[36]  M. Duchen,et al.  Mitochondria as Targets for Nitric Oxide-Induced Protection During Simulated Ischemia and Reoxygenation in Isolated Neonatal Cardiomyocytes , 2001, Circulation.

[37]  S. Budd,et al.  Mitochondria and neuronal survival. , 2000, Physiological reviews.

[38]  M. Berridge,et al.  Mitochondrial Ca 2 1 Uptake Depends on the Spatial and Temporal Profile of Cytosolic Ca 2 1 Signals* , 2001 .

[39]  T. Aiuchi,et al.  Effects of probes of membrane potential on metabolism in synaptosomes. , 1985, Biochimica et biophysica acta.

[40]  E. Strehler,et al.  Plasma membrane calcium ATPases as critical regulators of calcium homeostasis during neuronal cell function. , 1999, Frontiers in bioscience : a journal and virtual library.

[41]  Ralf Schneggenburger,et al.  Intracellular calcium dependence of transmitter release rates at a fast central synapse , 2000, Nature.

[42]  T. Peng,et al.  Privileged access to mitochondria of calcium influx through N-methyl-D-aspartate receptors. , 1998, Molecular pharmacology.

[43]  W. Brownell.  Organization of the cat trapezoid body and the discharge characteristics of its fibers , 1975, Brain Research.

[44]  A. Lysakowski,et al.  Dense‐cored vesicles, smooth endoplasmic reticulum, and mitochondria are closely associated with non‐specialized parts of plasma membrane of nerve terminals: Implications for exocytosis and calcium buffering by intraterminal organelles , 1999, The Journal of comparative neurology.

[45]  Alan Fine,et al.  Calcium Stores in Hippocampal Synaptic Boutons Mediate Short-Term Plasticity, Store-Operated Ca2+ Entry, and Spontaneous Transmitter Release , 2001, Neuron.

[46]  I. Forsythe,et al.  Direct patch recording from identified presynaptic terminals mediating glutamatergic EPSCs in the rat CNS, in vitro. , 1994, The Journal of physiology.

[47]  Gary Matthews,et al.  Inhibition of endocytosis by elevated internal calcium in a synaptic terminal , 1994, Nature.

[48]  Charles F Stevens,et al.  Activity-Dependent Modulation of the Rate at which Synaptic Vesicles Become Available to Undergo Exocytosis , 1998, Neuron.

[49]  R. Llinás,et al.  Presynaptic calcium currents in squid giant synapse. , 1981, Biophysical journal.

[50]  G. Rutter,et al.  Subcellular imaging of intramitochondrial Ca2+ with recombinant targeted aequorin: significance for the regulation of pyruvate dehydrogenase activity. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[51]  Leonard K. Kaczmarek,et al.  High-frequency firing helps replenish the readily releasable pool of synaptic vesicles , 1998, Nature.

[52]  M. Berridge,et al.  Mitochondrial Ca2+ Uptake Depends on the Spatial and Temporal Profile of Cytosolic Ca2+ Signals* , 2001, The Journal of Biological Chemistry.

[53]  S. Iwasaki,et al.  Developmental changes in calcium channel types mediating synaptic transmission in rat auditory brainstem , 1998, The Journal of physiology.

[54]  J. Simpson.  THE RELEASE OF NEURAL TRANSMITTER SUBSTANCES , 1969 .

[55]  A. Marty,et al.  Presynaptic calcium stores underlie large-amplitude miniature IPSCs and spontaneous calcium transients , 2000, Nature Neuroscience.

[56]  R. Farrar,et al.  Reduced calcium uptake by rat brain mitochondria and synaptosomes in response to aging , 1985, Brain Research.

[57]  M. Beal.  Energetics in the pathogenesis of neurodegenerative diseases , 2000, Trends in Neurosciences.

[58]  E. Neher,et al.  Presynaptic Depression at a Calyx Synapse: The Small Contribution of Metabotropic Glutamate Receptors , 1997, The Journal of Neuroscience.

[59]  J. Borst,et al.  The Reduced Release Probability of Releasable Vesicles during Recovery from Short-Term Synaptic Depression , 1999, Neuron.

[60]  B Sakmann,et al.  Calcium sensitivity of glutamate release in a calyx-type terminal. , 2000, Science.

[61]  Y. Y. Peng,et al.  Effects of mitochondrion on calcium transients at intact presynaptic terminals depend on frequency of nerve firing. , 1998, Journal of neurophysiology.

[62]  B. Hille,et al.  Mitochondrial Participation in the Intracellular Ca2+ Network , 1997, The Journal of cell biology.

[63]  G. David.  Mitochondrial Clearance of Cytosolic Ca2+ in Stimulated Lizard Motor Nerve Terminals Proceeds without Progressive Elevation of Mitochondrial Matrix [Ca2+] , 1999, The Journal of Neuroscience.

[64]  E. Stuenkel,et al.  Mitochondria regulate the Ca(2+)-exocytosis relationship of bovine adrenal chromaffin cells. , 1999, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[65]  E Neher,et al.  Kinetic studies of Ca2+ binding and Ca2+ clearance in the cytosol of adrenal chromaffin cells. , 1997, Biophysical journal.

[66]  E. Neher,et al.  Calmodulin Mediates Rapid Recruitment of Fast-Releasing Synaptic Vesicles at a Calyx-Type Synapse , 2001, Neuron.

[67]  Suk-Ho Lee,et al.  Kinetics of Ca2+ binding to parvalbumin in bovine chromaffin cells: implications for [Ca2+] transients of neuronal dendrites , 2000, The Journal of physiology.

[68]  E. Neher,et al.  Combining Deconvolution and Noise Analysis for the Estimation of Transmitter Release Rates at the Calyx of Held , 2001, The Journal of Neuroscience.

[69]  K. Gunter,et al.  Transport of calcium by mitochondria , 1994, Journal of bioenergetics and biomembranes.

[70]  B. Hille,et al.  Ca2+ clearance mechanisms in isolated rat adrenal chromaffin cells. , 1996, The Journal of physiology.

[71]  B. Sakmann,et al.  Calcium influx and transmitter release in a fast CNS synapse , 1996, Nature.

[72]  M. Tachibana,et al.  Ca2+ regulation in the presynaptic terminals of goldfish retinal bipolar cells. , 1995, The Journal of physiology.

[73]  T. Rohacs,et al.  Cytoplasmic Ca2+ signalling and reduction of mitochondrial pyridine nucleotides in adrenal glomerulosa cells in response to K+, angiotensin II and vasopressin. , 1997, The Biochemical journal.

[74]  B. Walmsley,et al.  Ultrastructural basis of synaptic transmission between endbulbs of Held and bushy cells in the rat cochlear nucleus , 2002, The Journal of physiology.

[75]  D. Bers,et al.  Oxygen-bridged Dinuclear Ruthenium Amine Complex Specifically Inhibits Ca2+ Uptake into Mitochondria in Vitroand in Situ in Single Cardiac Myocytes* , 1998, The Journal of Biological Chemistry.

[76]  B. Hille,et al.  Dominant Role of Mitochondria in Clearance of Large Ca2+ Loads from Rat Adrenal Chromaffin Cells , 1996, Neuron.

[77]  W. Cascio,et al.  Mitochondrial calcium transients in adult rabbit cardiac myocytes: inhibition by ruthenium red and artifacts caused by lysosomal loading of Ca(2+)-indicating fluorophores. , 2000, Biophysical journal.