Search

Since their discovery in the 1960s, the term paroxysmal depolarization shift (PDS) has been applied to a wide variety of reinforced neuronal discharge patterns. Occurrence of PDS as cellular correlates of electrographic spikes during latent phases of insult-induced rodent epilepsy models and their resemblance to giant depolarizing potentials (GDPs) nourished the idea that PDS may be involved in epileptogenesis. Both GDPs and – in analogy – PDS may lead to progressive changes of neuronal properties by generation of pulsatile intracellular Ca2+ elevations. Herein, a key element is the gating of L-type voltage gated Ca2+ channels (LTCCs, Cav1.x family), which may convey Ca2+ signals to the nucleus. Accordingly, the present study investigates various insult-associated neuronal challenges for their propensities to trigger PDS in a LTCC-dependent manner. Our data demonstrate that diverse disturbances of neuronal function are variably suited to induce PDS-like events, and the contribution of LTCCs is essential to evoke PDS in rat hippocampal neurons that closely resemble GDPs. These PDS appear to be initiated in the dendritic sub-compartment. Their morphology critically depends on the position of recording electrodes and on their rate of occurrence. These results provide novel insight into induction mechanisms, origin, variability, and co-existence of PDS with other discharge patterns and thereby pave the way for future investigations regarding the role of PDS in epileptogenesis.

Biological membranes carry fixed charges at their surfaces. These arise primarily from phospholipid headgroups. In addition, membrane proteins contribute to the surface potential with their charged residues. Membrane lipids are asymmetrically distributed. Because of this asymmetry, the net-negative charge at the inner leaflet exceeds that at the outer leaflet. Changes in surface potential are predicted to give rise to apparent changes in membrane capacitance. Here, we show that it is possible to detect changes in surface potential by an electrophysiological approach; the analysis of cellular currents relies on assuming that the electrical properties of a cell are faithfully described by a three-element circuit (i.e., the minimal equivalent circuit) comprised of two resistors and one capacitor. However, to account for changes in surface potential, it is necessary to add a battery to this circuit connected in series with the capacitor. This extended circuit model predicts that the current response to a square-wave voltage pulse harbors information, which allows for separating the changes in surface potential from a true capacitance change. We interrogated our model by investigating changes in the capacitance induced by ligand binding to the serotonin transporter and to the glycine transporters (GlyT1 and GlyT2). The experimental observations were consistent with the predictions of the extended circuit. We conclude that ligand-induced changes in surface potential (reflecting the binding event) and in true membrane capacitance (reflecting the concomitant conformational change) can be detected in real time even in instances in which they occur simultaneously.

Electrical activity in neurons is highly energy demanding and accompanied by rises in cytosolic Ca(2+). Cytosolic Ca(2+), in turn, secures energy supply by pushing mitochondrial metabolism either through augmented NADH (nicotinamide adenine dinucleotide) transfer into mitochondria via the malate–aspartate shuttle (MAS) or via direct activation of dehydrogenases of the TCA cycle after passing into the matrix through the mitochondrial Ca(2+) uniporter (MCU). Another Ca(2+)-sensitive booster of mitochondrial ATP synthesis is the glycerol-3-phosphate shuttle (G3PS), whose role in neuronal energy supply has remained elusive. Essential components of G3PS are expressed in hippocampal neurons. Single neuron metabolic measurements in primary hippocampal cultures derived from rat pups of either sex reveal only moderate, if any, constitutive activity of G3PS. However, during electrical activity neurons fully rely on G3PS when MAS and MCU are unavailable. Under these conditions, G3PS is required for appropriate action potential firing. Accordingly, G3PS safeguards metabolic flexibility of neurons to cope with energy demands of electrical signaling. SIGNIFICANCE STATEMENT Ca(2+) ions are known to provide a link between the energy-demanding electrical activity and an adequate ATP supply in neurons. To do so, Ca(2+) acts both from outside and inside of the mitochondrial inner membrane. Neuronal function critically depends on this regulation, and its defects are often found in various neurologic disorders. Although interest in neuronal metabolism has increased, many aspects thereof have remained unresolved. In particular, a Ca(2+)-sensitive NADH (nicotinamide adenine dinucleotide) shuttling system, the glycerol-3-phosphate shuttle, has been largely ignored with respect to its function in neurons. Our results demonstrate that this shuttle is functional in hippocampal neurons and safeguards ATP supply and appropriate action potential firing when malate aspartate shuttle and mitochondrial Ca(2+) uniporter are unavailable, thereby ensuring neuronal metabolic flexibility.

Since their discovery in the 1960s, the term paroxysmal depolarization shift (PDS) has been applied to a wide variety of reinforced neuronal discharge patterns. Occurrence of PDS as cellular correlates of electrographic spikes during latent phases of insult-induced rodent epilepsy models and their resemblance to giant depolarizing potentials (GDPs) nourished the idea that PDS may be involved in epileptogenesis. Both GDPs and - in analogy - PDS may lead to progressive changes of neuronal properties by generation of pulsatile intracellular Ca

Acute intermittent porphyria (AIP) is an autosomal dominant inborn error of heme biosynthesis due to a pathogenic mutation in the Hmbs gene, resulting in half-normal activity of hydroxymethylbilane synthase. Factors that induce hepatic heme biosynthesis induce episodic attacks in heterozygous patients. The clinical presentation of acute attacks involves the signature neurovisceral pain and may include psychiatric symptoms. Here we used a knock-in mouse line that is biallelic for the Hmbs c.500G > A (p.R167Q) mutation with ~ 5% of normal hydroxymethylbilane synthase activity to unravel the consequences of severe HMBS deficiency on affective behavior and brain physiology. Hmbs knock-in mice (KI mice) model the rare homozygous dominant form of AIP and were used as tool to elucidate the hitherto unknown pathophysiology of the behavioral manifestations of the disease and its neural underpinnings. Extensive behavioral analyses revealed a selective depression-like phenotype in Hmbs KI mice; transcriptomic and immunohistochemical analyses demonstrated aberrant myelination. The uncovered compromised mitochondrial function in the hippocampus of knock-in mice and its ensuing neurogenic and neuroplastic deficits lead us to propose a mechanistic role for disrupted mitochondrial energy production in the pathogenesis of the behavioral consequences of severe HMBS deficiency and its neuropathological sequelae in the brain.

L-type voltage-gated Ca(2+) channels (LTCCs) are implicated in neurodegenerative processes and cell death. Accordingly, LTCC antagonists have been proposed to be neuroprotective although this view is disputed, because intentional LTCC activation can also have beneficial effects. LTCC-mediated Ca(2+) influx influences mitochondrial function, which plays a crucial role in the regulation of cell viability. Hence, we investigated the effect of modulating LTCC-mediated Ca(2+) influx on mitochondrial function in cultured hippocampal neurons. To activate LTCCs, neuronal activity was stimulated by increasing extracellular K(+) or by application of the GABA(A) receptor antagonist bicuculline. The activity of LTCCs was altered by application of an agonistic (Bay K8644) or an antagonistic (isradipine) dihydropyridine. Our results demonstrated that activation of LTCC-mediated Ca(2+) influx affected mitochondrial function in a bimodal manner. At moderate stimulation strength, ATP synthase activity was enhanced, an effect that involved Ca(2+)-induced Ca(2+)-release from intracellular stores. In contrast, high LTCC-mediated Ca(2+) loads led to a switch in ATP synthase activity to reverse mode operation. This effect, which required nitric oxide, helped to prevent mitochondrial depolarization and sustained increases in mitochondrial Ca(2+). Our findings indicate a complex role of LTCC-mediated Ca(2+) influx in the tuning and maintenance of mitochondrial function. Therefore, the use of LTCC inhibitors to protect neurons from neurodegeneration should be reconsidered carefully.

SummaryObjective An increase of neuronal Cav1.3 L-type calcium channels (LTCCs) has been observed in various animal models of epilepsy. However, LTCC inhibitors failed in clinical trials of epileptic treatment. There is compelling evidence that paroxysmal depolarization shifts (PDSs) involve Ca2+ influx through LTCCs. PDSs represent a hallmark of epileptiform activity. In recent years, a probable epileptogenic role for PDSs has been proposed. However, the implication of the two neuronal LTCC isoforms, Cav1.2 and Cav1.3, in PDSs remained unknown. Moreover, Ca2+-dependent nonspecific cation (CAN) channels have also been suspected to contribute to PDSs. Nevertheless, direct experimental support of an important role of CAN channel activation in PDS formation is still lacking. Methods Primary neuronal networks derived from dissociated hippocampal neurons were generated from mice expressing a dihydropyridine-insensitive Cav1.2 mutant (Cav1.2DHP−/− mice) or from Cav1.3−/− knockout mice. To investigate the role of Cav1.2 and Cav1.3, perforated patch-clamp recordings were made of epileptiform activity, which was elicited using either bicuculline or caffeine. LTCC activity was modulated using the dihydropyridines Bay K 8644 (agonist) and isradipine (antagonist). Results Distinct PDS could be elicited upon LTCC potentiation in Cav1.2DHP−/− neurons but not in Cav1.3−/− neurons. In contrast, when bicuculline led to long-lasting, seizure-like discharge events rather than PDS, these were prolonged in Cav1.3−/− neurons but not in Cav1.2DHP−/− neurons. Because only the Cav1.2 isoform is functionally coupled to CAN channels in primary hippocampal networks, PDS formation does not require CAN channel activity. Significance Our data suggest that the LTCC requirement of PDS relates primarily to Cav1.3 channels rather than to Cav1.2 channels and CAN channels in hippocampal neurons. Hence, Cav1.3 may represent a new therapeutic target for suppression of PDS development. The proposed epileptogenic role of PDSs may allow for a prophylactic rather than the unsuccessful seizure suppressing application of LTCC inhibitors.

Biological membranes carry fixed charges at their surfaces. These arise primarily from phospholipid head groups. In addition, membrane proteins contribute to the surface potential with their charged residues. Membrane lipids are asymmetrically distributed. Because of this asymmetry the net negative charge at the inner leaflet exceeds that at the outer leaflet. Changes in surface potential are predicted to shape the capacitive properties of the membrane (i.e. the ability of the membrane to store electrical charges). Here, we show that it is possible to detect changes in surface potential by an electrophysiological approach: the analysis of cellular currents relies on assuming that the electrical properties of a cell are faithfully described by a three-element circuit - i.e. the minimal equivalent circuit - comprised of two resistors and one capacitor. However, to account for changes in surface potential it is necessary to add a battery to this circuit connected in series with the capacitor. This extended circuit model predicts that the current response to a square-wave voltage pulse harbors information, which allows for separating the changes in surface potential from a true capacitance change. We interrogated our model by investigating changes in capacitance induced by ligand binding to the serotonin transporter (SERT) and to the glycine transporters (GlyT1 and GlyT2). The experimental observations were consistent with the predictions of the extended circuit. We conclude that ligand-induced changes in surface potential (reflecting the binding event) and in true membrane capacitance (reflecting the concomitant conformational change) can be detected in real time even in instances where they occur simultaneously.Statement of SignificanceThe plasma membrane of a cell possesses fixed charges on both surfaces. Surface charges play an important role in many biological processes. However, the mechanisms, which regulate the surface charge densities at the plasma membrane, are poorly understood. This is in part due to lack of experimental approaches that allow for detecting changes in surface charges in real time. Here, we show that it is possible to track alterations in the electric potential at the membrane surface with high temporal resolution by an electrophysiological approach. Importantly, the described method allows for discriminating between a change in surface potential and a change in true membrane capacitance (e.g. a change in membrane area), even if these occur in parallel.

In multi-cellular organisms, cells communicate with each other utilizing chemical messengers. For many of these messenger molecules, the membrane is an insurmountable barrier. Yet, they act by binding to surface proteins often triggering a cascade of reactions inside the cell. Accordingly, studying ligand-receptor interactions at the cellular surface is key to understanding important aspects of membrane biology. However, despite a multitude of approaches to study membrane features, there is a need for developing techniques that can measure ligand binding with high temporal resolution and on a single cellular level. We recently developed a label-free approach to study ligand binding in real time. This methodology capitalizes on changes of the membrane’s surface potential induced by the adsorption of a charged ligand. The resulting apparent alteration of membrane capacitance is measurable by capacitance recordings. Herein, we describe the implementation of the same using recordings obtained from HEK293 cells stably expressing the human serotonin transporter (SERT), which were challenged with the inhibitor cocaine.

Diseases arising from misfolding of SLC6 transporters have been reported over recent years, e.g. folding-deficient mutants of the dopamine transporter and of the glycine transporter-2 cause infantile/juvenile Parkinsonism dystonia and hyperekplexia, respectively. Mutations in the coding sequence of the human creatine transporter-1 (hCRT-1/SLC6A8) gene result in a creatine transporter deficiency syndrome, which varies in its clinical manifestation from epilepsy, mental retardation, autism, development delay and motor dysfunction to gastrointestinal symptoms. Some of the mutations in hCRT-1 occur at residues, which are highly conserved across the SLC6 family. Here, we examined 16 clinically relevant hCRT-1 variants to verify the conjecture that they were misfolded and that this folding defect was amenable to correction. Confocal microscopy imaging revealed that the heterologously expressed YFP-tagged mutant CRTs were trapped in the endoplasmic reticulum (ER), co-localised with the ER-resident chaperone calnexin. In contrast, the wild type hCRT-1 reached the plasma membrane. Preincubation of transiently transfected HEK293 cells with the chemical chaperone 4-phenylbutyrate (4-PBA) restored ER export and surface expression of as well as substrate uptake by several folding-deficient CRT-1 mutants. A representative mutant (hCRT-1-P544L) was expressed in rat primary hippocampal neurons to verify pharmacochaperoning in a target cell: 4-PBA promoted the delivery of hCRT-1-P544L to the neurite extensions. These observations show that several folding-deficient hCRT-1 mutants can be rescued. This proof-of-principle justifies the search for additional pharmacochaperones to restore folding of 4PBA-unresponsive hCRT-1 mutants. Finally, 4-PBA is an approved drug in paediatric use: this provides a rationale for translating the current insights into clinical trials. This article is part of the issue entitled ‘Special Issue on Neurotransmitter Transporters’.

Several shortcomings with currently available pharmacotherapy of epilepsy necessitate the search for new drug targets. Paroxysmal depolarization shifts (PDS) represent the cellular correlates of electrographic (e.g. interictal) spikes. While the ionic basis of PDS is understood in great detail, controversy exists regarding their proposed implication in epilepsy. To address this issue and to consider potential targetability, this mini-review gives an overview of the ionic conductances contributing to PDS and reflects on the hypotheses of their potential pro-epileptic (epileptogenic) and anti-epileptic roles.

OBJECTIVE: An increase of neuronal Ca(v)1.3 L-type calcium channels (LTCCs) has been observed in various animal models of epilepsy. However, LTCC inhibitors failed in clinical trials of epileptic treatment. There is compelling evidence that paroxysmal depolarization shifts (PDS) involve Ca(2+)-influx through LTCCs. PDS represent a hallmark of epileptiform activity. In recent years, a probable epileptogenic role of PDS has been proposed. However, the implication of the two neuronal LTCC isoforms, Ca(v)1.2 and Ca(v)1.3, in PDS remained unknown. Moreover, Ca(2+)-dependent nonspecific cation (CAN) channels have also been suspected to contribute to PDS. Nevertheless, direct experimental support of an important role of CAN channel activation in PDS formation is still lacking. METHODS: Primary neuronal networks derived from dissociated hippocampal neurons were generated from mice expressing either a dihydropyridine-insensitive Ca(v)1.2 mutant (Ca(v)1.2DHP(-/-) mice) or from Ca(v)1.3(-/-) knockout mice. To investigate the role of Ca(v)1.2 and Ca(v)1.3, perforated patch-clamp recordings were made of epileptiform activity which was elicited using either bicuculline or caffeine. LTCC activity was modulated using the dihydropyridines Bay K 8644 (agonist) and isradipine (antagonist). RESULTS: Distinct PDS could be elicited upon LTCC potentiation in Ca(v)1.2DHP(-/-) neurons but not in Ca(v)1.3(-/-) neurons. In contrast, when bicuculline led to long-lasting, seizure-like discharge events rather than PDS, these were prolonged in Ca(v)1.3(-/-) neurons but not in Ca(v)1.2DHP(-/-) neurons. Since only the Ca(v)1.2 isoform is functionally coupled to CAN channels in primary hippocampal networks, PDS formation does not require CAN channel activity. SIGNIFICANCE: Our data suggest that the LTCC requirement of PDS relates primarily to Ca(v)1.3 channels rather than to Ca(v)1.2 channels and CAN channels in hippocampal neurons. Hence, Ca(v)1.3 may represent a new therapeutic target for suppression of PDS development. The proposed epileptogenic role of PDS may allow for a prophylactic rather than the unsuccessful seizure suppressing application of LTCC inhibitors.

Many cellular activities involving cell membrane manifest through changes in membrane capacitance. The accuracy and precision of cell membrane capacitance measurement is limited by the measuring system, which distorts membrane current responses used for analysis of impedance parameters. We developed a practical deconvolution procedure for reconstruction of the time course of membrane current and applied it to current responses recorded at step voltage changes by the whole-cell patch-clamp technique. In contrast to the recorded current responses, the reconstructed current responses could be exactly described by adequate impedance models of the recorded circuit. Deconvolution of membrane currents improved the performance of the square-wave method of high-resolution membrane capacitance recording by providing higher accuracy in a wider range of cell sizes and by eliminating cross-talk errors in parameter estimates. This allowed resolving instabilities in the recording conditions arising from parasitic capacitance and seal resistance variation. Complex tests on hardware models, on simulated data sets, and on living cells confirmed reliability of the deconvolution method for membrane capacitance recording. The aptitude of the method was demonstrated in isolated cardiac myocytes by recording spontaneous vesicular events, by discerning formation of a fusion pore, and by revealing artefacts due to unstable seal resistance.

Monoaminergic signal transmission is terminated by reuptake of monoamines via the closely related transporters for dopamine (DAT), norepinephrine (NET) and serotonin (SERT). SERT is a secondary active transporter that harnesses the chemical energy stored in the gradients of Na+, Cl- and K+ to transport 5-HT uphill against an opposing concentration gradient.The transport cycle of SERT is complex and encompasses several partial-reactions, some of which can directly be monitored utilizing measurements of currents mediated by SERT. However, these measurements are limited to ionic conditions that support currents but fail to provide insights at conditions at which SERT is electrically silent.We therefore performed capacitance measurements during rapid serotonin application and observed that substrate binding to SERT decreases the membrane capacitance. This effect was absent in control cells.Our analysis suggests that the reduction in membrane capacitance occurs as a consequence of the adsorption of 5-HT to a charged residue in the binding site of SERT. This process eliminates negative extracellular surface charges. Surface charge elimination is accompanied by a change in the surface potential, concomitantly affecting the transmembrane potential. The change of the latter then gives rise to a change in the measured membrane capacitance. The substrate-induced decrease in membrane capacitance was still present, when omitting co-substrates. This is consistent with our hypothesis that the reduced capacitance resulted from serotonin binding (adsorption). At sodium-free conditions, the affinity for serotonin was decreased which was reflected by a smaller capacitance reduction. Our findings suggest random order binding reactions of substrate and co-substrates and that serotonin and Na+ bind in a cooperative manner.Financial support was given to WS by the Austrian Funds FWF (P28090-B27).

Intracellular calcium signals are studied by laser-scanning confocal fluorescence microscopy. The required spatio-temporal resolution makes description of calcium signals difficult because of the low signal-to-noise ratio. We designed a new procedure of calcium spike analysis based on their fitting with a model. The accuracy and precision of calcium spike description were tested on synthetic datasets generated either with randomly varied spike parameters and Gaussian noise of constant amplitude, or with constant spike parameters and Gaussian noise of various amplitudes. Statistical analysis was used to evaluate the performance of spike fitting algorithms. The procedure was optimized for reliable estimation of calcium spike parameters and for dismissal of false events. A new algorithm was introduced that corrects the acquisition time of pixels in line-scan images that is in error due to sequential acquisition of individual pixels along the space coordinate. New software was developed in Matlab and provided for general use. It allows interactive dissection of temporal profiles of calcium spikes from x-t images, their fitting with predefined function(s) and acceptance of results on statistical grounds, thus allowing efficient analysis and reliable description of calcium signaling in cardiac myocytes down to the in situ function of ryanodine receptors.

A major challenge of high-resolution cell capacitance measurement methods is to discriminate between the changes of individual impedance parameters of the recording circuit. Square wave methods, in general, are of advantage for their insensitivity to changes in the reversal potential and pipette capacitance but suffer from the deteriorating effect of low-pass filtering in the signal path. We show that the standard correction procedure [1, 2], based on blanking the onset of every current response, corrects this effect only partially, and therefore the calculated impedance parameters are somewhat in error. The filtering also introduces a cell size-/capacitance-dependent crosstalk between the circuit parameters. To overcome this problem, we designed a new method of high-resolution capacitance measurement, based on deconvolution of membrane currents recorded in response to square-wave voltage stimulation. The deconvolution procedure removes distortions introduced by the recording system and allows exact analysis of current responses by the theoretical model of the circuit. The method was tested by extensive simulations, by measurements on equivalent electrical circuits and on living cells (Jurkat T-cells and cardiac myocytes). The method did excel in accuracy and in suppression of cross-talk errors, while keeping all the benefits related to the square wave methods. The method was implemented in the software application MAT-MECAS written in MATLAB.Supported by: APVV-0721-10, VEGA 2/0147/14[1] Lindau, M., Neher E. (1988). Pflugers Arch 411: 137-146.[2] Thompson, R. E., Lindau M., Webb, W. W. (2001). Biophys J 81: 937-948.

Cardiac myocytes are cells with complex membrane structure of unknown dynamics. A good physical measure of the state of the sarcolemma is membrane capacitance. High-resolution capacitance measurements were instrumental in disclosing secretory and endocytotic mechanisms in simpler cells. We used a new method of membrane capacitance recording based on deconvolution of current responses to square wave voltage stimuli (MAT-MECAS: https://sourceforge.net/projects/mat-mecas/) to study membrane dynamics of isolated cardiac myocytes; specifically, the spontaneous and stimulated membrane capacitance changes. We observed spontaneous discrete capacitance events of 1 − 20 fF that may correspond to fusion and fission of single vesicles of 180 − 800 nm in diameter. In addition, large spontaneous stepwise increases of membrane capacitance of 80 − 250 fF were also observed. These large capacitance increases were occasionally preceded by a transient opening of a fusion pore with a conductance of several hundreds of pS that lasted for a few hundreds of milliseconds. Such large stepwise changes of membrane capacitance might be related to the opening of the mouth of transversal tubules. Discrete capacitance increases were also observed after stimulation of myocytes by a train of calcium currents. In these experiments, the discrete capacitance increases of 50 − 160 fF occurred 3 to 20 seconds after stimulation. This might reflect calcium induced fusion of intracellular membrane bodies with the surface membrane. In conditions of permanently raised intracellular concentration of calcium ions, membrane capacitance showed increased activity of step-like capacitance events. These observations support the role of intracellular calcium in regulation of membrane dynamics in cardiac myocytes. Altogether, these findings provide the first evidence of vesicle fusion events related to physiologically relevant membrane activity in cardiac myocytes.Supported by: APVV-0721-10, VEGA 2/0147/14.

The effect of infrared laser stimulation of excitable tissue was explained by the rise of temperature that increases the cell membrane capacitance, which causes cell depolarization and action potential activation (Shapiro et al., Nat. Commun. 3:736, 2012). As in cardiac myocytes the relation of cell capacitance and temperature is not well known, we studied the dependence of membrane capacitance on the bath temperature under steady-state conditions in isolated rat ventricular and atrial myocytes. Individual myocytes were whole-cell patch clamped and the membrane capacitance was recorded by the high-resolution square wave stimulation method (Novak and Zahradnik, Ann. Biomed. Eng. 34:1201, 2006). The bath temperature was increased by means of a temperature controller at a rate of 0.1°C/s from about 23°C to about 33°C. Experiments on 6 atrial and 3 ventricular myocytes provided an average increase of the cell capacitance by 0.30 ± 0.02 %/°C that corresponds to 0.17 ± 0.03 pF/°C for an average myocyte of 56.6 ± 9.9 pF capacitance (mean ± SEM). The increase of capacitance was proportional to the cell capacitance (R=0.81). There were no significant differences between atrial and ventricular myocytes in the temperature sensitivity of membrane capacitance. The temperature increase caused no mechanical activity or changes in the shape of myocytes. These data are in quantitative agreement with the data obtained on HEK cells and bilayers (Shapiro et al., Nat. Commun. 3:736, 2012) and confirm the effect of temperature on cell capacitance in the case of cardiac myocytes, thus provide an independent support for the theory of optical stimulation of excitable tissue.Supported by APVV-0721-10 and by VEGA 2/0203/11 and VEGA 2/0197/11.

Integral calcium release fluxes in mammalian cardiac myocytes increase in amplitude not only due to the increased recruitment of individual release sites but also due to their increased synchronization (1). Here we characterize the voltage dependence of synchronization of individual release sites at high temporal resolution. Calcium spikes and calcium currents elicited in isolated rat ventricular myocytes (2) by 80 ms voltage pulses from −50 to −40 - +50 mV were recorded using Leica TCS AOBS confocal microscope (2 kHz line scan mode) and Axopatch 200B patch-clamp amplifier, respectively. Calcium spikes were identified and analyzed by a new Matlab-based software that highly increased the productivity and reliability of approximation of calcium spikes by the kinetic model of local calcium release (2). Calcium spikes were described by their amplitude, onset latency, time-to-peak, FDHM, time constants of activation and termination, and fractional probability of activation. The synchrony of calcium spikes increased with depolarization and could be fully explained by the voltage dependence of their onset latency that reached a lower limit of about 4 ms at depolarizations between 0 and 40 mV. The rate of calcium spike activation after its onset was independent of the applied voltage or of calcium current amplitude, while the probability of calcium spike activation was related to the voltage dependence of the integral of calcium current. We conclude that both the synchrony of local calcium release activation and recruitment of individual release sites are fully controlled by the activation probability of DHPR calcium channels and by the DHPR-RYR coupling fidelity.Supported by APVV-0721-10, VEGA 2/0203/11, VEGA 2/0917/11.1. Song LS, Sham JS, Stern MD, Lakatta EG, Cheng H. J Physiol. 512:677-91, 1998.2. Zahradnikova A Jr, Polakova E, Zahradnik I, Zahradnikova A. J Physiol. 578:677-91, 2007.