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• W2014452352 abstract "Synaptic mechanisms of plasticity are calcium-dependent processes that are affected by dysfunction of mitochondrial calcium buffering. Recently, we observed that mice deficient in mitochondrial voltage-dependent anion channels, the outer component of the mitochondrial permeability transition pore, have impairments in learning and hippocampal synaptic plasticity, suggesting that the mitochondrial permeability transition pore is involved in hippocampal synaptic plasticity. In this study, we examined the effect on synaptic transmission and plasticity of blocking the permeability transition pore with low doses of cyclosporin A and found a deficit in synaptic plasticity and an increase in base-line synaptic transmission. Calcium imaging of presynaptic terminals revealed a transient increase in the resting calcium concentration immediately upon incubation with cyclosporin A that correlated with the changes in synaptic transmission and plasticity. The effect of cyclosporin A on presynaptic calcium was abolished when mitochondria were depolarized prior to cyclosporin A exposure, and the effects of cyclosporin A and mitochondrial depolarization on presynaptic resting calcium were similar, suggesting a mitochondrial locus of action of cyclosporin A. To further characterize the calcium dynamics of the mitochondrial permeability transition pore, we used anin vitro assay of calcium handling by isolated brain mitochondria. Cyclosporin A-exposed mitochondria buffered calcium more rapidly and subsequently triggered a more rapid mitochondrial depolarization. Similarly, mitochondria lacking the voltage-dependent anion channel 1 isoform depolarized more readily than littermate controls. The data suggest a role for the mitochondrial permeability transition pore and voltage-dependent anion channels in mitochondrial synaptic calcium buffering and in hippocampal synaptic plasticity. Synaptic mechanisms of plasticity are calcium-dependent processes that are affected by dysfunction of mitochondrial calcium buffering. Recently, we observed that mice deficient in mitochondrial voltage-dependent anion channels, the outer component of the mitochondrial permeability transition pore, have impairments in learning and hippocampal synaptic plasticity, suggesting that the mitochondrial permeability transition pore is involved in hippocampal synaptic plasticity. In this study, we examined the effect on synaptic transmission and plasticity of blocking the permeability transition pore with low doses of cyclosporin A and found a deficit in synaptic plasticity and an increase in base-line synaptic transmission. Calcium imaging of presynaptic terminals revealed a transient increase in the resting calcium concentration immediately upon incubation with cyclosporin A that correlated with the changes in synaptic transmission and plasticity. The effect of cyclosporin A on presynaptic calcium was abolished when mitochondria were depolarized prior to cyclosporin A exposure, and the effects of cyclosporin A and mitochondrial depolarization on presynaptic resting calcium were similar, suggesting a mitochondrial locus of action of cyclosporin A. To further characterize the calcium dynamics of the mitochondrial permeability transition pore, we used anin vitro assay of calcium handling by isolated brain mitochondria. Cyclosporin A-exposed mitochondria buffered calcium more rapidly and subsequently triggered a more rapid mitochondrial depolarization. Similarly, mitochondria lacking the voltage-dependent anion channel 1 isoform depolarized more readily than littermate controls. The data suggest a role for the mitochondrial permeability transition pore and voltage-dependent anion channels in mitochondrial synaptic calcium buffering and in hippocampal synaptic plasticity. mitochondrial permeability transition pore voltage-dependent anion channel long term potentiation paired pulse facilitation cyclosporin A tetraphenylphosphate field excitatory postsynaptic potential theta burst stimulation carbonyl cyanide 3-chlorophenylhydrazone The mitochondrial permeability transition pore (MPT)1 is a complex believed to be composed of the voltage-dependent anion channel (VDAC) in the mitochondrial outer membrane, the adenine nucleotide transporter in the inner membrane, and cyclophilin-D in the matrix and is found in mitochondria of all eukaryotic cells. Although the majority of research on the function of the MPT and its components has focused on apoptosis (1Kroemer G. Biochem. Soc. Symp. 1999; 66: 1-15Crossref PubMed Scopus (182) Google Scholar, 2Shimizu S. Matsuoka Y. Shinohara Y. Yoneda Y. Tsujimoto Y. J. Cell Biol. 2001; 152: 237-250Crossref PubMed Scopus (332) Google Scholar, 3Bauer M.K. Schubert A. Rocks O. Grimm S. J. Cell Biol. 1999; 147: 1493-1502Crossref PubMed Scopus (239) Google Scholar), the MPT has recently been shown to play a role in learning and synaptic plasticity in mice (4Weeber E.J. Levy M. Sampson M.J. Anflous K. Armstrong D.L. Brown S.E. Paylor R.E. Sweatt J.D. Craigen W.J. J. Biol. Chem. 2002; 277: 18891-18897Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar) as well as in other physiological cellular functions (5Pfeiffer D.R. Gunter T.E. Eliseev R. Broekemeier K.M. Gunter K.K. IUBMB Life. 2001; 52: 205-212Crossref PubMed Scopus (55) Google Scholar). In both pathological and physiological functions, the induction of the MPT is mediated by mitochondrial calcium influx above a certain threshold, and once opened, the MPT conducts small substrates and ions out of the mitochondrial matrix (6Bernardi P. Physiol. Rev. 1999; 79: 1127-1155Crossref PubMed Scopus (1343) Google Scholar). It has therefore been proposed that following physiologic calcium influx into the mitochondrial matrix, formation of the MPT might be a physiological mechanism of mitochondrial calcium release (5Pfeiffer D.R. Gunter T.E. Eliseev R. Broekemeier K.M. Gunter K.K. IUBMB Life. 2001; 52: 205-212Crossref PubMed Scopus (55) Google Scholar). We are interested in determining the role of mitochondrial calcium regulation in synaptic function. The most extensively characterized synapses in the mammalian central nervous system are the Schaffer collateral synapses between CA3 pyramidal axons and their postsynaptic targets on the dendrites of CA1 pyramidal neurons. As mitochondria are typically found in CA3 presynaptic terminals as well as CA1 dendrites, we hypothesized that CA3-CA1 hippocampal long term potentiation (LTP) and paired pulse facilitation (PPF) would be sensitive to alterations in mitochondrial calcium regulation by the MPT because both depend on calcium-dependent processes within synaptic terminals. In this study, we examined the effect of blocking the MPT on LTP and PPF using low doses of cyclosporin A (CsA). In addition, we monitored the changes in calcium dynamics caused by CsA by imaging presynaptic terminals loaded with the fluorescent calcium indicator Fura-2. We found that blocking the MPT caused a transient increase in the resting calcium in presynaptic terminals, correlating with changes in synaptic transmission and plasticity. The increase caused by CsA was abolished by depolarizing mitochondria with tetraphenylphosphate (TPP+) prior to CsA exposure indicating a common target,i.e. mitochondria. In addition, the characteristics of the effects of TPP+ and CsA on resting calcium were similar, suggesting the possibility of a common mechanism of action. To more precisely determine the effect of blocking the MPT on mitochondrial calcium handling, we employed an in vitroassay to study calcium handling in isolated brain mitochondria and found that CsA induces earlier and more rapid mitochondrial depolarization and subsequent calcium release, which may explain the transient increase of calcium in presynaptic terminals. Similarly, VDAC1-deficient mitochondria depolarized and subsequently released their mitochondrial calcium stores more readily than wild-type littermate controls, which may explain their similar electrophysiological deficits (4Weeber E.J. Levy M. Sampson M.J. Anflous K. Armstrong D.L. Brown S.E. Paylor R.E. Sweatt J.D. Craigen W.J. J. Biol. Chem. 2002; 277: 18891-18897Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). Based on these results we propose a model to explain how the MPT serves to help prevent mitochondrial calcium overload and subsequent depolarization. Overall, our studies are consistent with an important role for synaptic mitochondria in calcium regulation and specifically suggest a role for the MPT in neuronal calcium regulation. Hippocampal slices (400 μm) were prepared as previously described (7Roberson E.D. Sweatt J.D. J. Biol. Chem. 1996; 271: 30436-30441Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Hippocampal slices were bathed (1 ml/min) with artificial cerebrospinal fluid (125 mm NaCl, 2.5 mm KCl, 1.24 mmNaH2PO4, 25 mm NaHCO3, 10 mmd-glucose, 2 mmCaCl2, 1 mm MgCl2, and 10 μm cyclosporin A as indicated) in an interface chamber maintained at 30 °C bubbled with 95% O2, 5% CO2. The Schaffer collateral axons were stimulated with a bipolar electrode, and the field excitatory postsynaptic potential (fEPSP) was recorded in the stratum radiatum of area CA1. Responses were monitored every 20–30 s for 20 min to ensure a stable base line. Measurements are shown as the average slopes of the fEPSP from four to six individual traces and are normalized to 10 min of base-line recordings. Base-line stimulus intensities were adjusted to produce an fEPSP at 50% of the maximal response. 100 Hz LTP was induced by stimulating twice at high frequency (100 Hz) at base-line intensity for 1 s each, 20 s apart. Theta burst stimulation (TBS) consisted of three trains of stimuli delivered at 20-s intervals, each train composed of 10 stimulus bursts delivered at 5 Hz, with each burst consisting of four pulses at 100 Hz. PPF was induced by two single stimuli at base-line intensity, 30 milliseconds apart. CsA (Alexis Pharmaceuticals) was dissolved in 99% ethanol (final ethanol concentration, 0.1%) and added to artificial cerebrospinal fluid while stirring for 10 min to increase solubilization. Thorough rinsing of all plastic tubes and parts exposed to CsA with ethanol and water prevented contamination of control slices. The hippocampal slices were prepared as described above, submersed in artificial cerebral spinal fluid, and maintained with 95% O2, 5% CO2bubbling at 30 °C. As described in detail in Wu and Saggau (8Wu L.G. Saggau P. J. Neurosci. 1994; 14: 645-654Crossref PubMed Google Scholar), Fura-2 AM (Molecular Probes) dissolved in 80:20 Me2SO:pluronic acid was pressure-injected in the stratum radiatum of area CA1 where the AM-ester dye is taken up by CA3 axons, the AM-ester was cleaved, and the cell-impermeable Fura-2 was transported to the presynaptic terminals. An area in stratum radiatum of CA1 about 1 mm away from the injection site was illuminated at a single excitation wavelength, 340 or 380 nm as indicated. Fluorescence was collected with a 50× objective, filtered by a long pass filter (490 nm), and converted into an electrical signal by a single photodiode. The resting presynaptic calcium was determined by ratiometric measurements of fluorescence excited at both 340 and 380 nm in the absence of CA3 stimulation. For PPF measurements, two traces recorded 1 min apart were averaged to improve the signal-to-noise ratio. Hippocampal slices were prepared and incubated in an interface chamber as described above. High doses of CsA (250 μm), low doses of CsA (10 μm), or no CsA in 0.5% Tween 20, 0.5% ethanol was added to the artificial cerebrospinal fluid for 30 min. The slices were removed from the interface chamber and homogenized in buffer containing 20 mm Tris, 1 mm EGTA, 1 mm EDTA, 1 mm Na4P2O7, protease inhibitor mixture (Sigma), 1 mm p-nitrophenyl phosphate, 0.25 mm Na3VO4, pH 7.5, at 4 °C. The homogenates were run on a 12% SDS-PAGE, transferred to a nylon membrane (Millipore), and probed with phosphospecific antibodies to phosphorylated synapsin I (αSer603 and αSer62/67 phosphospecific antibodies were generous gifts from the lab of Paul Greengard; αSer553 was purchased from Santa Cruz Biotechnology) and total synapsin (α-synapsin was purchased from Cell Signaling Technology). The blots were developed using the ECL system (Amersham Biosciences). Each experiment was repeated at least three times. Mouse hippocampal slices were prepared and maintained as previously described for electrophysiological experiments. The slices were exposed to 50 mm KCl for 10, 2, or 0 min before perchlorate extraction. A luciferase-based assay from Molecular Probes was used to quantify the ATP concentration and normalized to protein concentration, as determined by BCA protein assay (Pierce). Mouse brain mitochondria were isolated by Percoll centrifugation as outlined in Ref. 9Sims N.R. J. Neurochem. 1990; 55: 698-707Crossref PubMed Scopus (343) Google Scholar. One mouse forebrain was minced on ice and homogenized in a small glass homogenizer with 3 ml of a 12% Percoll solution in isolation buffer composed of 0.32 m sucrose, 1 mm K-EGTA, 10 mm Tris, pH 7.1. The Percoll homogenate was carefully added on top of 3.5 ml of 26% Percoll, which was layered over 3.5 ml of 40% Percoll in a tube and centrifuged at 30,700 × g for 5 min at 4 °C. The cloudy layer at the interface of the lower two Percoll solutions was aspirated, washed 1:4 (v/v) with isolation buffer, and centrifuged at 16,700 ×g for 10 min at 4 °C. The pellet was resuspended in 1% bovine serum albumin (in isolation buffer) and spun at 8,000 ×g for 12 min at 4 °C. The pellet was then washed in 900 μl of bovine serum albumin-free isolation buffer, centrifuged at 8,000 × g for 12 min at 4 °C and finally resuspended in 750 μl of bovine serum albumin-free isolation buffer. Isolated mitochondria were tested for respiration using an oxygraph (biological oxygen monitor, YSI model 5300) and a Clark-type electrode (oxygen probe, YSI model 5331) as described in Ref. 10Trounce I.A. Kim Y.L. Jun A.S. Wallace D.C. Methods Enzymol. 1996; 264: 484-509Crossref PubMed Google Scholar. Only mitochondria with a respiratory ratio of >2 on succinate were used for the in vitro assays. In addition, only mitochondria that possessed an intact outer membrane, as tested indirectly by the cytochrome c assay (11Miro O. Casademont J. Grau J.M. Jarreta D. Urbano-Marquez A. Cardellach F. Br. J. Rheumatol. 1998; 37: 1047-1053Crossref PubMed Google Scholar), were used. Isolated mitochondria stored on ice can be reliably used for experiments for up to 3 h. 300 μl of buffer (125 mm mannitol, 75 mm sucrose, 10 mm KCl, 10 mm Tris-HCl, 5 mmKH2PO4, 5 mm glutamic acid, 5 mm pyruvic acid, 5 mm succinic acid, 5 mm malic acid; pH adjusted with KOH to 7.3; osmolarity = 300 mosmol) was prewarmed in a stirring measurement chamber for 1 min at 32 °C. To the warming buffer, ADP was added to a final concentration of 100 μm and CsA (dissolved in 99% EtOH), or an equivalent volume of EtOH for control was added to a final concentration of 1 μm as indicated. Mitochondria were added to a final protein concentration of 60–70 μg/ml (∼50 μl of mitochondrial preparation) and incubated for 2 min to warm up to 32 °C. Extramitochondrial calcium was monitored using a calcium-selective electrode (World Precision Instruments) and recorded every 20 s. A CaCl2 bolus was added to reach the desired calcium concentration as indicated. CsA and other drugs were added either 2 min before the calcium bolus or after calcium was maximally buffered, defined as 40 s of zero net flux. Ruthenium red, an inhibitor of the mitochondrial calcium uniporter, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), a mitochondrial proton uncoupler, and TPP+ were purchased from Sigma. In these experiments, we observed that there is a range of calcium release rates that varies between mitochondrial preparations and depends partly on the initial calcium bolus, the mitochondrial concentration, the background strain of mouse, and the degree of mitochondrial depolarization. The traces we show represent the average release rates for one set of experiments, where each set was run at least three times with different mitochondrial preparations. Approximately half of all CA3 presynaptic terminals contain at least one mitochondrion (26Ventura R. Harris K.M. J. Neurosci. 1999; 19: 6897-6906Crossref PubMed Google Scholar), and mitochondria are also typically found in dendrites of CA1 pyramidal neurons (Fig.1). We investigated the role of these mitochondria in synaptic transmission and plasticity. As previously reported in Weeber et al.(4Weeber E.J. Levy M. Sampson M.J. Anflous K. Armstrong D.L. Brown S.E. Paylor R.E. Sweatt J.D. Craigen W.J. J. Biol. Chem. 2002; 277: 18891-18897Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar), we found that 10 μm CsA significantly attenuates hippocampal 100 Hz LTP at Schaffer collateral synapses in area CA1 of the mouse hippocampus (Fig.2A). We sought to investigate the basis of this effect by assessing acute changes caused by CsA. Immediately upon the addition of CsA, synaptic transmission began to increase and reach a plateau at ∼25% above base line (Fig.2B, also evident in the base lines of panels Aand C). Interestingly, although CsA attenuated LTP induced by a 100 Hz stimulating protocol (Fig. 2A), it did not appear to attenuate LTP induced by TBS (Fig. 2C), even when the increase in base-line synaptic transmission is subtracted from the theta burst-induced potentiation (139% potentiation in controlsversus 128% in CsA-exposed slices, p = 0.11). 100 Hz stimulation (Fig. 2A) and TBS (Fig.2C) are believed to differ in their mechanism of LTP induction because of differences in the characteristics of calcium influx into synaptic terminals (12Cavus I. Teyler T. J. Neurophysiol. 1996; 76: 3038-3047Crossref PubMed Scopus (164) Google Scholar), which may affect different biochemical pathways. For example, theta burst LTP involves the MAPK pathway in the mouse hippocampus, whereas 100 Hz LTP does not (27Selcher J.C. Weeber E.J. Christian J. Nekrasova T. Landreth G.E. Sweatt J.D. Learn Mem. 2003; 10: 26-39Crossref PubMed Scopus (139) Google Scholar). Thus our studies suggest that the effect of CsA on synaptic plasticity affects biochemical pathways that are important for 100 Hz LTP but not theta burst LTP; however, it is important to note that the CsA-elicited increase in base-line synaptic transmission complicates interpretation of our TBS-induced LTP result. One question that arises from our CsA experiments is whether the locus of action is mitochondrial as opposed to other potential sites of action. TPP+ has been shown to attenuate post-tetanic potentiation at the crayfish neuromuscular junction by causing presynaptic mitochondrial depolarization (22Tang Y. Zucker R.S. Neuron. 1997; 18: 483-491Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar). In the mouse hippocampal area CA1, exposure to TPP+ 20 min prior to a 100 Hz LTP simulation paradigm caused a marked reduction in LTP induction, not unlike CsA exposure (Fig. 2D). Although the mechanisms of action of TPP+ and CsA are different, the similarity of the effect of TPP+ and CsA on early LTP insinuates a common final effect on mitochondrial calcium handling. Unlike CsA, however, sustained mitochondrial depolarization by TPP+ eventually causes swelling and alterations in metabolic activity (see Fig. 5), resulting in a loss of synaptic activity (Fig. 2D). Overall, however, our data indicating that TPP+ application blocks LTP induction in a fashion reminiscent of CsA is consistent with our interpretation that CsA acts at a mitochondrial locus. We also assessed the effects of MPT inhibition on shorter term forms of synaptic plasticity, in particular PPF. We particularly followed PPF because it is generally held to involve presynaptic calcium handling. The effect of CsA exposure on PPF, becoming evident ∼20 min after the addition of CsA, was a reduction in the second fEPSP compared with the first fEPSP such that PPF was attenuated (Fig.3). These findings are consistent with our previous observations of the effect of CsA on hippocampal synaptic plasticity (4Weeber E.J. Levy M. Sampson M.J. Anflous K. Armstrong D.L. Brown S.E. Paylor R.E. Sweatt J.D. Craigen W.J. J. Biol. Chem. 2002; 277: 18891-18897Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). In summary, our electrophysiologic studies indicate that CsA has both an immediate effect to increase synaptic transmission, followed by a delayed attenuation of two forms of synaptic plasticity, PPF, and 100 Hz LTP. We hypothesized that the effect of CsA in synaptic terminals is in part caused by inhibition of the MPT and subsequent alterations in mitochondrial calcium handling. One specific concern with the use of CsA to block the MPT is the possibility of inhibition of the protein phosphatase, calcineurin. Typically, the concentrations of CsA used to block calcineurin are at least 10–25-fold greater than used in this study (13Lu Y.F. Tomizawa K. Moriwaki A. Hayashi Y. Tokuda M. Itano T. Hatase O. Matsui H. Brain Res. 1996; 729: 142-146Crossref PubMed Scopus (54) Google Scholar, 14Mulkey R.M. Endo S. Shenolikar S. Malenka R.C. Nature. 1994; 369: 486-488Crossref PubMed Scopus (905) Google Scholar). In addition, several considerations suggest that calcineurin is not inhibited in our studies. Hippocampal slices from mice lacking calcineurin exhibit enhanced PPF (15Malleret G. Haditsch U. Genoux D. Jones M.W. Bliss T.V. Vanhoose A.M. Weitlauf C. Kandel E.R. Winder D.G. Mansuy I.M. Cell. 2001; 104: 675-686Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar), and FK506, another calcineurin inhibitor, does not affect PPF (16Lu Y.F. Hayashi Y. Moriwaki A. Tomizawa K. Matsui H. Neurosci. Lett. 1996; 205: 103-106Crossref PubMed Scopus (33) Google Scholar). FK506 also reduces base-line synaptic transmission (16Lu Y.F. Hayashi Y. Moriwaki A. Tomizawa K. Matsui H. Neurosci. Lett. 1996; 205: 103-106Crossref PubMed Scopus (33) Google Scholar). None of these effects of calcineurin inhibition are observable in our experiments with low doses of CsA. Nevertheless, to confirm that low doses (10 μm) of CsA do not inhibit calcineurin in the hippocampal slice to the same extent as high doses (250 μm) of CsA do, we used phosphospecific antibodies to detect the phosphorylation state of a calcineurin substrate, synapsin I. Hippocampal slices incubated under the same conditions as for electrophysiology, in the presence or absence of high or low doses of CsA, were harvested for Western blotting with phosphospecific antibodies to synapsin I phosphorylated at Ser603, Ser553, and Ser62/67, all of which have been shown to be sensitive to calcineurin (17Jovanovic J.N. Sihra T.S. Nairn A.C. Hemmings Jr., H.C. Greengard P. Czernik A.J. J. Neurosci. 2001; 21: 7944-7953Crossref PubMed Google Scholar). Incubation in 250 μm CsA resulted in dramatically enhanced phosphorylation of all three synapsin I phosphorylation sites, which is likely due to inhibition of dephosphorylation by calcineurin (Fig. 4). Equally dramatic is the lack of phosphorylation in slices exposed to 10 μm or no CsA (Fig. 4). Taken together with our electrophysiological observations, these findings indicate that the low doses of CsA used in this study do not cause calcineurin inhibition and suggest that low doses of CsA are acting via a different mechanism. Among the known targets of CsA, mitochondrial cyclophilin-D has one of the lowest Kivalues in vitro (18Galat A. Eur. J. Biochem. 1993; 216: 689-707Crossref PubMed Scopus (316) Google Scholar) and has been shown to mediate the functional inhibition of the MPT by CsA in isolated mitochondria (19Crompton M. Virji S. Doyle V. Johnson N. Ward J.M. Biochem. Soc. Symp. 1999; 66: 167-179Crossref PubMed Scopus (186) Google Scholar). In addition, our prior studies of hippocampal slices from VDAC-deficient mice showed a similar electrophysiological phenotype to CsA-exposed wild-type slices. Thus, it is likely that the effect of CsA on hippocampal electrophysiology results from inhibition of the MPT. Inhibition of the MPT by CsA may perturb the mitochondrial proton gradient important in ATP production (20Brustovetsky N. Dubinsky J.M. J. Neurosci. 2000; 20: 8229-8237Crossref PubMed Google Scholar). To address the possibility that CsA may alter hippocampal ATP concentrations, we measured total ATP concentration in hippocampal slices in the presence and absence of 10 μm CsA and found no significant change in ATP concentration caused by 10 μm CsA (Fig.5). To induce synaptic calcium influx as might occur during synaptic activity, hippocampal slices were exposed to 50 mm KCl for 2–10 min and then harvested for ATP quantification. Interestingly, ATP levels dropped to the same extent after KCl depolarization in both CsA-exposed slices and controls and stayed constant throughout KCl exposure (Fig. 5A). Thus, CsA did not affect ATP concentration before or after KCl depolarization, which suggests that changes in synaptic function caused by CsA are not due to altered ATP availability. Sustained exposure to 100 μm TPP+ caused a significant decline in levels of ATP in the hippocampal slice after 40 min of incubation (Fig. 5B). The decline in ATP correlated with the degradation of electrophysiological signals in hippocampal slices continuously exposed to 100 μm TPP+(Fig. 2D). In contrast, hippocampal slices exposed to 10 μm CsA did not show altered ATP levels and accordingly maintained electrophysiological integrity (Fig. 5B). The effect of CsA on synaptic function suggested a two-phase mechanism: an immediate increase in base-line synaptic transmission, followed ∼20 min later by an attenuation in synaptic plasticity (Fig. 2). We hypothesized that the changes in synaptic function might be due to secondary changes in mitochondrial calcium handling. To determine the effect of CsA on calcium handling, we monitored cytosolic calcium within presynaptic terminals exposed to CsA in acute hippocampal slices using an adaptation of our previously published protocol (8Wu L.G. Saggau P. J. Neurosci. 1994; 14: 645-654Crossref PubMed Google Scholar). Briefly, the calcium indicator Fura-2 was applied to the axons of the Schaffer collateral pathway and transported to presynaptic terminals in CA1. This allowed us to measure relative changes in fluorescence caused by calcium binding to Fura-2 (Fig.6A). Using this approach, we were able to monitor the real time effects of CsA on presynaptic calcium. Immediately upon exposure to 10 μm CsA, resting presynaptic calcium was rapidly increased, followed by a more gradual return to base line by 30 min (Fig. 6B). Application of the vehicle, ethanol, had no effect (Fig. 6C). Thus, the increase in base-line synaptic transmission that we observed in Fig. 2B correlates with a transient increase in resting calcium over the same time period. Interestingly, the return of resting calcium to base-line levels occurs at about the same time that the PPF deficit began (Fig. 3). To confirm the target of CsA on mitochondrial calcium stores, we performed the same experiment using TPP+ to depolarize mitochondria. By depolarizing mitochondria, we effectively remove the ability of mitochondria to take up calcium. As expected, exposure to 100 μm TPP+ immediately caused mitochondrial depolarization and consequent calcium release (Fig. 6D) caused by loss of the mitochondrial proton gradient. When CsA was added to the TPP+ bath 30 min later, there was no effect (Fig.6D), suggesting that the target of CsA and TPP+are the same, i.e. mitochondrial calcium stores. When the TPP+ was washed out for 10 min, mitochondria recovered, and CsA regained its ability to cause an increase in resting calcium (Fig. 6E). CsA did not measurably affect stimulus-evoked calcium transients using the paired pulse protocol (Fig. 6F), suggesting that rather than participating in the dynamic handling of calcium during paired pulse stimulation, the MPT is more involved in regulating resting presynaptic calcium concentration. Thus, we can now correlate changes in resting presynaptic calcium handling with alterations in synaptic function in hippocampal slices exposed to CsA. Surprisingly, the effect of TPP+ on resting presynaptic calcium remarkably resembled the effect of CsA (Fig. 6E), again suggesting a common mechanism. Because TPP+ is known to depolarize mitochondria as part of its mechanism of causing calcium release, we used an in vitro assay of calcium handling in isolated mitochondria to determine whether CsA might be affecting mitochondrial calcium handling as well. Intact and functional isolated brain mitochondria rapidly buffered extramitochondrial calcium via the mitochondrial calcium uniporter, which could be blocked by ruthenium red (Fig.7A). Calcium influx is driven by a respiration-dependent electrochemical gradient across the mitochondrial inner membrane. CCCP, which dissipates the mitochondrial electrochemical gradient, also blocked calcium influx and, in addition, induced mitochondrial depolarization and subsequent calcium release when added at the point of maximal buffering (Fig.7A). Overwhelmed by the amount of calcium used in thisin vitro assay, control mitochondria eventually depolarized and subsequently released their stores of calcium. These observations are consistent with previously published studies on mitochondrial calcium handling (21Nicholls D.G. Budd S.L. Physiol. Rev. 2000; 80: 315-360Crossref PubMed Scopus (1052) Google Scholar) and indicate that our isolated mitochondrial preparation contains intact and functional mitochondria capable of sequestering calcium. Exposure of mitochondria to CsA prior to a 5–10 μmcalcium bolus resulted in an increased rate of calcium influx (Fig.7B) and an earlier and more rapid mitochondrial depolarization and subsequent calcium release (Fig. 7C). CsA exposure to at least three different mitochondrial preparations consistently resulted in an increased influx rate and more rapid calcium release (1.6–2.0-fold faster influx and release in each preparation) and consequently greater extramitochondrial calcium. Thus, the transient increase in cytosolic calcium observed in presynaptic terminals exposed to CsA (Fig. 6B) may have been due to triggering of mitochondrial depolarization and subsequent calcium release. Mitochondrial depolarization by CsA is not a direct effect, as with TPP+ and CCCP, because CsA added after maximal calcium buffering did not induce mitochondrial depolarization and subsequent calcium release (Fig. 7D). For CsA to depolarize mitochondria, it had to be present before calcium influx. One interesting possibility is that the increased rate of calcium influx caused by CsA triggered an earlier and more rapid mitochondrial depolarization and subsequent calcium release. We interpreted the effect of CsA on mitochondrial calcium handling as being due to inhibition of the MPT. To confirm this interpretation, we evaluated the effects of genetic deletion of an outer pore component of the MPT, VDAC1, using the in vitromethod above. Similar to CsA-exposed wild-type mitochondria, VDAC1-deficient mitochondria depolarized and released calcium more rapidly compared with littermate controls (Fig. 7E), which ultimately resulted in greater extramitochondrial calcium. This common feature of mitochondrial depolarization and increased extramitochondrial calcium may explain the electrophysiological similarity of CsA exposed wild-type hippocampal slices and VDAC-deficient hippocampal slices in synaptic plasticity (4Weeber E.J. Levy M. Sampson M.J. Anflous K. Armstrong D.L. Brown S.E. Paylor R.E. Sweatt J.D. Craigen W.J. J. Biol. Chem. 2002; 277: 18891-18897Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). Our data along with other recent reports suggest that mitochondrial calcium buffering is an important regulator of synaptic function (22Tang Y. Zucker R.S. Neuron. 1997; 18: 483-491Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar, 23Billups B. Forsythe I.D. J. Neurosci. 2002; 22: 5840-5847Crossref PubMed Google Scholar). Extracellular field recordings in hippocampal slices exposed to low doses of CsA, which functionally inhibits the mitochondrial permeability transition pore, showed significant deficits in PPF and 100 Hz LTP and an increase in base-line synaptic transmission. Calcium imaging of presynaptic terminals loaded with Fura-2 demonstrated that CsA induces an increase in resting calcium that may account for the acute change in synaptic transmission. Although the changes in presynaptic calcium and base-line synaptic transmission were acute effects of CsA, the deficits in short term synaptic plasticity began as the resting presynaptic calcium returned to base line, ∼20 min after CsA exposure. This delayed outcome of the synaptic plasticity deficit might be due to a secondary effect of the transient calcium increase or to prolonged mitochondrial depolarization. The former possibility is supported by previous studies showing that elevated presynaptic calcium can activate calcium-calmodulin kinases that phosphorylate the vesicle-associated protein synapsins; synapsin phosphorylation may result in impaired presynaptic plasticity (17Jovanovic J.N. Sihra T.S. Nairn A.C. Hemmings Jr., H.C. Greengard P. Czernik A.J. J. Neurosci. 2001; 21: 7944-7953Crossref PubMed Google Scholar). However, as indicated in Fig. 4, 10 μm CsA exposure does not result in phosphorylation of synapsin I at any of the sites tested, suggesting that synapsin phosphorylation is not likely mediating the changes in synaptic plasticity caused by CsA. Nevertheless, there are numerous other calcium-sensitive pathways within synaptic terminals that may impinge on mechanisms important in synaptic plasticity. Alternatively, the transient increase in calcium may represent a more generalized mitochondrial malfunction resulting from prolonged depolarization. In support of this idea are recent studies showing that respiratory chain uncouplers affecting mitochondrial calcium handling disrupt synaptic plasticity in the calyx of Held (23Billups B. Forsythe I.D. J. Neurosci. 2002; 22: 5840-5847Crossref PubMed Google Scholar) and the crayfish neuromuscular junction (22Tang Y. Zucker R.S. Neuron. 1997; 18: 483-491Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar). The location of mitochondria in presynaptic terminals does not lend itself well to buffering calcium influx from the extracellular space during single synaptic depolarizations (24Brocard J.B. Tassetto M. Reynolds I.J. J. Physiol. 2001; 531: 793-805Crossref PubMed Scopus (71) Google Scholar) because this type of calcium influx may cause calcium elevations restricted to a smaller submembranous space. Rather, mitochondria appear to be more important in regulating the overall resting calcium level. To understand the role of the MPT and VDACs in regulating the resting calcium level, we employed an in vitro assay of mitochondrial calcium buffering. Studies of calcium buffering in isolated mitochondria are useful for understanding the roles of channels, exchangers, and pores in calcium influx and/or efflux, but their behavior in vivomight differ from their function in vitro. For example, although a calcium bolus of low micromolar concentration is necessary to observe the effect of CsA in isolated mitochondria, resting calcium levels in presynaptic terminals do not likely reach these levels. In addition, isolated mitochondria include those from both presynaptic and postsynaptic compartments as well as glia. For these reasons, this assay only serves as a model for the role of the MPT in calcium regulation, which might help to explain the function of mitochondrial buffering in the intact presynaptic terminal in synaptic transmission and plasticity. It appears that in vitro mitochondrial calcium handling can be functionally divided into two stages, the uptake phase and the release phase. The uptake phase is dominated by calcium uptake by the uniporter. Blocking the MPT with CsA resulted in an increased net uptake during this phase. There are two workable hypotheses to explain this effect of CsA. First, if CsA hyperpolarizes the mitochondrial membrane, the drive for calcium influx would be greater and would thus cause increased calcium influx. Alternatively, one could suppose that the MPT conducts calcium release and, if blocked by CsA, would lead to a greater net influx. Either mechanism is plausible to explain the results of this assay but more importantly, the upshot is the same, that is, excessive calcium influx caused by blockage of the MPT results in calcium overload and subsequent mitochondrial depolarization. The result of mitochondrial depolarization is rapid calcium release from isolated mitochondria, as observed with CCCP. Consistent with this mechanism, adding CsA at the point of maximal calcium buffering did not have any effect (Fig. 5), indicating that the effect of CsA is dependent on calcium influx specifically. It is not yet known how calcium exits the mitochondria during the release phase, but based on our studies, it is not likely to be via the MPT or the Na+/Ca2+ exchanger (data not shown) because inhibitors of these pathways did not affect calcium release during this phase. The trigger for calcium release appears to be mitochondrial depolarization by cation overload. Although there is a correlation between increasing the rate of calcium uptake by adding CsA and inducing earlier calcium release, mitochondria can buffer anywhere from 5 to 75 μm total calcium before releasing it. Thus, it is not the total calcium uptake, but the rate of calcium uptake, that appears to play a role in triggering calcium release in ourin vitro preparation. The increase in resting calcium stimulated by CsA strongly resembles the increase stimulated by TPP+, suggesting at least a common locus of action and possibility a common mechanism of action in the presynaptic terminal. TPP+, a lipophilic cation, works by carrying its positive charge across mitochondrial membranes into the negatively charged matrix and depolarizing it by virtue of overloading the matrix with positive charges. CsA is believed to work by binding to cyclophilin-D, the matrix component of the MPT, and functionally blocking the pore. The in vitro assay results suggest that blockage of the MPT leads to calcium (or other cation) overload and subsequent mitochondrial depolarization, complete with rapid calcium release. Thus, we can now speculate why CsA and TPP+ have the same effect on resting presynaptic calcium. They both cause mitochondrial depolarization leading to calcium release; TPP+ does it by virtue of loading positive charges into the matrix, whereas CsA does it by overloading the matrix with calcium or other cations such as potassium, sodium, and/or protons. Mitochondria lacking VDAC1 were also more likely to depolarize and subsequently release their calcium stores. VDAC1-deficient mitochondria may be more vulnerable to calcium overload, resulting in more rapid calcium release as observed in in vitro assays of VDAC1-deficient isolated mitochondria. Although previously known mitochondrial calcium-specific transporters and channels have only been observed on the mitochondrial inner membrane, the mechanism of calcium transport across the mitochondrial outer membrane is not known. VDACs are the only known channels that may be able to conduct calcium into the intermitochondrial space (25Gincel D. Zaid H. Shoshan-Barmatz V. Biochem. J. 2001; 358: 147-155Crossref PubMed Scopus (319) Google Scholar) and therefore may play an important function in calcium influx into the mitochondrial matrix. Although calcium flux into the mitochondrial matrix has hitherto been attributed solely to the calcium uniporter of the mitochondrial inner membrane, we think it is interesting that VDAC-1-deficient mitochondria were impaired in mitochondrial calcium uptake in this assay (Fig. 7F). This finding is consistent with the growing notion that the mitochondrial outer membrane is not simply a molecular sieve but rather a regulated barrier that is involved in calcium flux into the intermitochondrial space, the source of calcium for the uniporter. Thus, the role of VDACs in calcium flux may not only be as a component of the MPT but also as calcium regulators across the mitochondrial outer membrane. Putting all the clues together, our study defines the roles of VDACs and the MPT in synaptic function. Based on the in vitroassay results, the MPT and VDACs serve to regulate the release of cations from mitochondria to prevent overload and subsequent depolarization. VDAC deficiency is detrimental for the proper function of the MPT because the absence of VDAC1 resulted in an increased likelihood of mitochondrial depolarization and subsequent calcium release as well. The overall result of mitochondrial depolarization in presynaptic terminals, induced by TPP+, CsA, or the absence of VDACs, is dysregulation of resting calcium, which may impinge on signal transduction pathways important in synaptic transmission and plasticity." @default.
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• W2014452352 title "Mitochondrial Regulation of Synaptic Plasticity in the Hippocampus" @default.
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