Mitochondrial Calcium Uptake Modulates Synaptic Vesicle Endocytosis in Central Nerve Terminals

Presynaptic calcium influx triggers synaptic vesicle (SV) exocytosis and modulates subsequent SV endocytosis. A number of calcium clearance mechanisms are present in central nerve terminals that regulate intracellular free calcium levels both during and after stimulation. During action potential stimulation, mitochondria rapidly accumulate presynaptic calcium via the mitochondrial calcium uniporter (MCU). The role of mitochondrial calcium uptake in modulating SV recycling has been debated extensively, but a definitive conclusion has not been achieved. To directly address this question, we manipulated the expression of the MCU channel subunit in primary cultures of neurons expressing a genetically encoded reporter of SV turnover. Knockdown of MCU resulted in ablation of activity-dependent mitochondrial calcium uptake but had no effect on the rate or extent of SV exocytosis. In contrast, the rate of SV endocytosis was increased in the absence of mitochondrial calcium uptake and slowed when MCU was overexpressed. MCU knockdown did not perturb activity-dependent increases in presynaptic free calcium, suggesting that SV endocytosis may be controlled by calcium accumulation and efflux from mitochondria in their immediate vicinity.     

The Effect of Acetylcholine Release on Choline Fluxes in Isolated Synaptic Terminals

Abstract: As in intact tissues, choline influx into synaptosomes is enhanced after a period of depolarization induced release of acetylcholine. The activation of uptake is dependent on the presence of Ca²⁺ and inhibited by high Mg²⁺ concentrations in the medium during depolarization. Choline transport in erythrocytes was not activated by prior treatment with potassium. The permeability constant of the synaptosome membrane to choline was found to be 2.7 × 10^?8 cm·s^?1 and to acetylcholine 1.8 ′ 10^?8 cm·s^?1. Choline influx has been studied after pre-loading synaptosomes with choline. Different radiolabels were used to measure efflux of preloaded choline and influx simultaneously. Isotopic dilution in flux studies was estimated and corrected for. Influx was stimulated by high internal concentrations of choline, and efflux similarly stimulated by high outside concentrations of choline. The maximal influx and efflux at saturating opposite concentrations of choline were equal with a value of about 500 pmol·min^?1 per mg synaptosomal protein. A reciprocating carrier would explain the equality of the maximal influx and efflux. Acetylcholine competes with choline for binding to the carrier but is itself hardly transported. Increased acetylcholine concentrations were shown to inhibit both choline influx and efflux from the trans position. Raising intrasynaptosomal acetylcholine concentrations by pre-loading abolished the stimulation of influx by prior depolarization. It is proposed that high concentrations of acetylcholine immobilize the carrier on the inside of the synaptic membrane. The stimulation of choline influx consequent upon depolarization is caused by release of ACh which results in relief of this immobilisation. The enhanced supply of choline achieved by this mechanism is likely to be important in maintaining stores of the acetylcholine in vivo.     

Effects of Calcium on Sugar Transport in Azotobacter vinelandii

A fast and environmentally safe procedure was used to study sugar uptake by Azotobacter vinelandii. Transport experiments were performed in a 24-well plate and aerated by rapid oscillatory vibration. Samples were washed by centrifugation and dissolved in biodegradable scintillation cocktail for counting. At cell concentrations up to 6 × 10⁸ cells per ml, the uptake of sucrose was a function of time and was proportional to the cell concentration. This modified uptake assay was used to test the effect of cations on sugar uptake in A. vinelandii. Results showed that Ca²⁺ at 1 to 2 mM stimulated sucrose uptake by decreasing the apparent K_m of sucrose transport. Higher Ca²⁺ concentrations inhibited sucrose uptake in this organism.     

Effects of Chronic Ethanol Treatment on the Expression of Calcium Transport Carriers and NMDA/Glutamate Receptor Proteins in Brain Synaptic Membranes

Abstract: Acute exposure to ethanol inhibits both the NMDA receptors and the Na/Ca-exchange carriers in neuronal membranes. This alters intraneuronal signaling pathways activated by Ca²⁺. Neurons exposed chronically to ethanol exhibit enhanced density and activity of NMDA receptors and increased maximal activity of the exchangers. In the present study, the expression of brain synaptic membrane proteins with ligand binding sites characteristic of NMDA receptors and of exchange carriers were determined after chronic ethanol administration (15 days) to rats. Such treatment caused an increase in the expression of the NMDAR1 receptor subunit, 15% above the levels in the pair-fed controls, as well as of three subunits of a complex that has properties characteristic of NMDA receptors, the glutamate, carboxypiperazinylphosphonate, and glycine binding proteins. Increases for the three binding proteins were 49, 50, and 62%, respectively. The expression of the 120-kDa exchanger proteins was increased by 14% and that of a 36-kDa exchanger-associated protein by 33%. Both the binding proteins and the exchangers returned to basal levels within 36–72 h after withdrawal from ethanol. No changes were detected in synaptic membrane Ca²⁺, Mg²⁺-ATPases. The enhanced expression of receptor and exchanger-associated proteins may explain the increases in the density and activity of NMDA receptors and exchange carriers after chronic ethanol treatment.     

The Mechanics of Calcium Transport

With the recent atomic models for the sarcoplasmic reticulum Ca²⁺-ATPase in the Ca²⁺-bound state, the Ca²⁺-free, thapsigargin-inhibited state, and the Ca²⁺-free, vanadate-inhibited state, we are that much closer to understanding and animating the Ca²⁺-transport cycle. These snapshots of the Ca²⁺-transport cycle reveal an impressive breadth and complexity of conformational change. The cytoplasmic domains undergo rigid-body movements that couple the energy of ATP to the transport of Ca²⁺ across the membrane. Large-scale rearrangements in the transmembrane domain suggest that the Ca²⁺-binding sites may alternately cease to exist and reform during the transport cycle. Of the three cytoplasmic domains, the actuator (A) domain undergoes the largest movement, namely a 110° rotation normal to the membrane. This domain is linked to transmembrane segments M1–M3, which undergo large rearrangements in the membrane domain. Together, these movements are a main event in Ca²⁺ transport, yet their significance is poorly understood. Nonetheless, inhibition or modulation of Ca²⁺-ATPase activity appears to target these conformational changes. Thapsigargin is a high-affinity inhibitor that binds to the M3 helix near Phe²⁵⁶, and phospholamban is a modulator of Ca²⁺-ATPase activity that has been cross-linked to M2 and M4. The purpose of this review is to postulate roles for the A domain and M1–M3 in Ca²⁺ transport and inhibition.     

Effects of the Calcium Channel Blocker Otilonium Bromide on Seizure Activity in Rats With Pentylenetetrazole-Induced Convulsions

Neurochemical Research - Millions of people suffer from drug-resistant epilepsy. New therapeutic approaches for removing this life-affecting disease are required. The activation of T-type calcium...     

The Structural Specificity of Choline Transport into Cholinergic Nerve Terminals

Abstract: The accumulation of choline, homocholine, and 4-hydroxybutyl-trimethylammonium by rat brain synaptosomes was measured; the choline uptake mechanism transported homocholine but not hydroxybutyltrimethylammonium, which, in addition, did not block choline accumulation. In cats'superior cervical ganglia, preganglionic nerve stimulation increased the accumulation of homocholine, but not that of hydroxybutyltrimethylammonium. It is concluded that the substrate specificity of the choline transport mechanism is such that increasing the N-O atom distance by one methylene group retains affinity, but increasing this distance by two methylene groups does not.     

Distribution of Motor Cortical Neuron Synaptic Terminals on Monkey Parvocellular Red Nucleus Neurons

We determined the location of 54 horseradish peroxidase (HRP)-labeled motor cortical neuron synaptic terminals on 17 parvocellular neurons in the monkey red nucleus. Synaptic terminals and their postsynaptic elements were identified and reconstructed, using light- and electron-microscopic techniques, from serial thick and thin sections. Terminals were found on proximal and distal dendrites of small and medium-sized parvocellular neurons, where they formed excitatory synapses. Some were 180 μm from cell somata. Approximately half of the labeled terminals, aside from those located at dendritic origins, were situated strategically at or near dendritic branch points. Since monkey parvocellular neurons show little activity during movement, the obvious next question is this: How and in what way does motor cortex influence these cells?     

The Integration of Mitochondrial Calcium Transport and Storage

The extraordinary capacity of isolated mitochondria to accumulate Ca(2+) has been established for more than 40 years. The distinct kinetics of the independent uptake and efflux pathways accounts for the dual functionality of the transport process to either modulate matrix free Ca(2+) concentrations or to act as temporary stores of large amounts of Ca(2+) in the presence of phosphate. One puzzle has been the nature of the matrix calcium phosphate complex, since matrix free Ca(2+) seems to be buffered in the region of 1-5 microM in the presence of phosphate while millimolar Ca(2+) remains soluble in in vitro media. The key seems to be the elevated matrix pH and the third-power relationship of the PO(4)(3-) concentration with pH. Taking this into account we may now finally have a model that explains the major features of physiological mitochondrial Ca(2+) transport.     

Quantal Characteristics of Synaptic GABA Release under Conditions of Stimulation of Single Synaptic Terminals of Cultured Cortical Cells of the Rat

We studied evoked inhibitory postsynaptic currents (eIPSC) using local electrical stimulation of single presynaptic terminals of cultured rat neocortical neurons. According to pharmacological and kinetic properties, these currents were qualified as GABA_A-activated. Using autocorrelation analysis of distributions of the eIPSC amplitudes, which were in all cases polymodal, we examined quantal characteristics of the above eIPSC. These results were compared with the values of quantal parameters (N, p, Q, and m) of the current families obtained using approximation by binomial distribution. Amplitude histograms of spontaneous miniature IPSC recorded under conditions of the minimum quantal release of the neurotransmitter were normal (close to Gaussian) with the mode within a 10 pA range, which is very close to analogous parameters calculated using autocorrelation and binomial techniques.     

The Vesicle Protein SAM-4 Regulates the Processivity of Synaptic Vesicle Transport

Axonal transport of synaptic vesicles (SVs) is a KIF1A/UNC-104 mediated process critical for synapse development and maintenance yet little is known of how SV transport is regulated. Using C. elegans as an in vivo model, we identified SAM-4 as a novel conserved vesicular component regulating SV transport. Processivity, but not velocity, of SV transport was reduced in sam-4 mutants. sam-4 displayed strong genetic interactions with mutations in the cargo binding but not the motor domain of unc-104. Gain-of-function mutations in the unc-104 motor domain, identified in this study, suppress the sam-4 defects by increasing processivity of the SV transport. Genetic analyses suggest that SAM-4, SYD-2/liprin-α and the KIF1A/UNC-104 motor function in the same pathway to regulate SV transport. Our data support a model in which the SV protein SAM-4 regulates the processivity of SV transport.     

The Accumulation and Transport of Calcium in Barley Roots

The accumulation and transport of Ca by various zones of 6-day old barley roots (Hordeum vulgare L.) were examined with special reference to their relationship to the salt status of the root. The initial salt content had a profound effect on Ca transport and a lesser effect on Ca accumulation. High-salt roots transported Ca in much larger amounts than did low salt roots. In low salt roots the apical zone was more active in transporting Ca to the conducting tissue than were the mid or basal zones However, in high salt roots all zones were about equally active in transporting Ca. The metabolic inhibitor, DNP, had little effect on accumulation but inhibited transport very effectively. The effect of DNP was more pronounced on transport from the apical zone than from the other root zones. Calcium applied anywhere along the root length moved only basally and its polarized longitudinal movement was maintained irrespective of the salt status of the root. The movement of Ca was characterized by a rapid release of preabsorbed Ca and a ready exchange of apoplastic Ca. The hypothesis is presented that cellular Ca is in a relatively mobile state. Its entry into the symplasm is the rate limiting step in longitudinal transport and its overall movement is metabolically controlled.     

力竭游泳对大鼠心肌线粒体钙运输的影响

 力竭游泳对大鼠心肌线粒体钙运输的影响丁树哲,王文信,连克杰,许豪文（华东师范大学体育系运动生化实验室,上海２０００６２）线粒体钙运输在细胞功能调节方面有重要作用．线粒体通过摄取与释放钙,对其跨膜质子、不依赖底物及产物抑制的ＡＴＰ合成、磷酸化偶联等均有...     

The Anticonvulsant and Neuroprotective Effects of Oxysophocarpine on Pilocarpine-Induced Convulsions in Adult Male Mice

Epilepsy is one of the prevalent and major neurological disorders, and approximately one-third of the individuals with epilepsy experience seizures that do not respond well to available medications. We investigated whether oxysophocarpine (OSC) had anticonvulsant and neuroprotective property in the pilocarpine (PILO)-treated mice. Thirty minutes prior to the PILO injection, the mice were administrated with OSC (20, 40, and 80 mg/kg) once. Seizures and electroencephalography (EEG) were observed, and then the mice were killed for Nissl and Fluoro-jade B (FJB) staining. The oxidative stress was measured at 24 h after convulsion. Western blot analysis was used to examine the expressions of the Bax, Bcl-2, and Caspase-3. In this study, we found that pretreatment with OSC (40, 80 mg/kg) significantly delayed the onset of the first convulsion and status epilepticus (SE) and reduced the incidence of SE and mortality. Analysis of EEG recordings revealed that OSC (40, 80 mg/kg) significantly reduced epileptiform discharges. Furthermore, Nissl and FJB staining showed that OSC (40, 80 mg/kg) attenuated the neuronal cell loss and degeneration in hippocampus. In addition, OSC (40, 80 mg/kg) attenuated the changes in the levels of Malondialdehyde (MDA) and strengthened glutathione peroxidase and catalase activity in the hippocampus. Western blot analysis showed that OSC (40, 80 mg/kg) significantly decreased the expressions of Bax, Caspase-3 and increased the expression of Bcl-2. Collectively, the findings of this study indicated that OSC exerted anticonvulsant and neuroprotective effects on PILO-treated mice. The beneficial effects should encourage further studies to investigate OSC as an adjuvant in epilepsy, both to prevent seizures and to protect neurons in brain.     

Characterization of Nucleotide Transport into Rat Brain Synaptic Vesicles

ATP transport to synaptic vesicles from rat brain has been studied using the fluorescent substrate analogue 1,N6-ethenoadenosine 5'-triphosphate (epsilon-ATP). The increase in intravesicular concentration was time dependent for the first 30 min, epsilon-ATP being the most abundant nucleotide. The complexity of the saturation curve indicates the existence of kinetic and allosteric cooperativity in the nucleotide transport, which exhibits various affinity states with K0.5 values of 0.39 +/- 0.06 and 3.8 +/- 0.1 mM with epsilon-ATP as substrate. The Vmax values obtained were 13.5 +/- 1.4 pmol x min(-1) x mg of protein(-1) for the first curve and 28.3 +/- 1.6 pmol x min(-1) x mg of protein(-1) considering both components. This kinetic behavior can be explained on the basis of a mnemonic model. The nonhydrolyzable adenine nucleotide analogues adenosine 5'-O-3-(thiotriphosphate), adenosine 5'-O-2-(thiodiphosphate), and adenosine 5'-(beta,gamma-imino)triphosphate and the diadenosine polyphosphates P1,P3-di(adenosine)triphosphate, P1,P4-di(adenosine)tetraphosphate, and P1,P5-di(adenosine)pentaphosphate inhibited the nucleotide transport. The mitochondrial ATP/ADP exchange inhibitor atractyloside, N-ethylmaleimide, and polysulfonic aromatic compounds such as Evans blue and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid also inhibit epsilon-ATP vesicular transport.     

Effects of Maintenance Electroshock on the Oxidative Damage Parameters in the Rat Brain

Although several advances have occurred over the past 20 years concerning refining the use and administration of electroconvulsive therapy to minimize side effects of this treatment, little progress has been made in understanding the mechanisms underlying its therapeutic or adverse effects. This work was performed in order to determine the level of oxidative damage at different times after the maintenance electroconvulsive shock (ECS). Male Wistar rats (250–300 g) received a protocol mimicking therapeutic of maintenance or simulated ECS (Sham) and were subsequently sacrificed immediately after, 48 h and 7 days after the last maintenance electroconvulsive shock. We measured oxidative damage parameters (thiobarbituric acid reactive species for lipid peroxidation and protein carbonyls for protein damage, respectively) in hippocampus, cortex, cerebellum and striatum. We demonstrated no alteration in the lipid peroxidation and protein damage in the four structures studied immediately after, 48 h and 7 days after a last maintenance electroconvulsive shock. Our findings, for the first time, demonstrated that after ECS maintenance we did protocol minimal oxidative damage in the brain regions, predominating absence of damage on the findings.     

Calcium Dependence of Synaptic Vesicle Recycling Before and After Synaptogenesis

Abstract: Using an immunocytochemical assay to monitor synaptic vesicle exocytosis/endocytosis independently of neurotransmitter release, we have investigated some aspects of vesicle recycling in hippocampal neurons at different developmental stages. A calcium- and depolarization-dependent exocytotic/endocytotic recycling of synaptic vesicles was found to take place in neurons already before the formation of synaptic contacts. The analysis of synaptic vesicle recycling at different calcium concentrations revealed the presence of two release components: the first one activated by low calcium concentrations and sustaining vesicle recycling before synaptogenesis, and a second one activated by high calcium concentrations, which is specifically turned on after the establishment of synaptic contacts. These data suggest that formation of synapses correlates with the activation of a putative low-affinity calcium sensor, which allows synaptic vesicle exocytosis to be triggered and turned off over extremely short time scales, in response to large increases in the level of intracellular calcium.     

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.     

Surprising Effects of Synaptic Excitation

Typically, excitatory synaptic coupling is thought of as an influence that accelerates and propagates firing in neuronal networks. This paper reviews recent results explaining how, contrary to these expectations, the presence of excitatory synaptic coupling can drastically slow oscillations in a network and how localized, sustained activity can arise in a network with purely excitatory coupling, without sustained inputs. These two effects stem from interactions of excitatory coupling with two different forms of intrinsic neuronal dynamics, and both serve to highlight the fact that the influence of synaptic coupling in a network depends strongly on the intrinsic properties of cells in the network.This work was partially supported by the National Science Foundation, under award DMS-0414023