The spatio-temporal properties of calcium transients in hippocampal pyramidal neurons in vitro.

The spatio-temporal properties of calcium signals were studied in cultured pyramidal neurons of the hippocampus using two-dimensional fluorescence microscopy and ratiometric dye Fura-2. Depolarization-induced Ca2+ transients revealed an asynchronous delayed increase in free Ca2+ concentration. We found that the level of free resting calcium in the cell nucleus is significantly lower compared to the soma, sub-membrane, and dendritic tree regions. Calcium release from the endoplasmic reticulum under the action of several stimuli (field stimulation, high K+ levels, and caffeine) occurs in all areas studied. Under depolarization, calcium signals developed faster in the dendrites than in other areas, while their amplitude was significantly lower since larger and slower responses inside the soma. The peak value of the calcium response to the application of 10 mM caffeine, ryanodine receptors (RyRs) agonist, does not differ in the sub-membrane zone, central region, and nucleus but significantly decreases in the dendrites. In the presence of caffeine, the delay of Ca2+ signals between various areas under depolarization significantly declined. Thirty percentage of the peak amplitude of Ca2+ transients at prolonged electric field stimulation corresponded to calcium release from the ER store by RyRs, while short-term stimulation did not depend on them. 20 µM dantrolene, RyRs inhibitor, significantly reduces Ca2+ transient under high K+ levels depolarization of the neuron. RyRs-mediated enhancement of the Ca2+ signal is more pronounced in the central part and nucleus compared to the sub-membrane or dendrites regions of the neuron. In summary, using the ratiometric imaging allowed us to obtain additional information about the involvement of RyRs in the intracellular dynamics of Ca2+ signals induced by depolarization or electrical stimulation train, with an underlying change in Ca2+ concentration in various regions of interest in hippocampal pyramidal neurons.

Error correction due to background subtraction in ratiometric calcium measurements with CCD camera.

BACKGROUND: Ca2+ plays an important role in many physiological processes and an accurate study of these signals is important. In modern fluorescence microscopy, a charge-coupled device (CCD) camera is widely deployed for calcium imaging. The ratiometric method is used for the fluorescence dye Fura-2 and Grynkiewitz's formula (Grynkiewicz et al., 1985) is commonly used to convert fluorescence to free Ca2+ concentration ([Ca2+]). But the need to subtract the background signal can lead to a big error in ratiometric calcium measurements. When the error due to background subtraction occurs, the fluorescence ratio of 340 nm divided by 380 nm lights may be twice as large as the actual value. Under conditions when the excitation intensity is not adjusted to ensure the same throughput of the objective lens for ultraviolet dye illumination, the indicator does not gradually bleach out for channels with a wavelength of 340 nm and 380 nm light, which lead to an additional error in determining the concentration of Ca2+. NEW METHOD: Here we present a new approach for calculating [Ca2+] from the ratiometric fluorescence of Fura-2 dye imaged by a CCD camera. It is designed to optimize [Ca2+] measurements with photobleaching correction without background subtraction error. A mathematical method is also provided for removing the existing underestimated value of fluorescence at an excitation wavelength of 340 nm and compensating for the bleaching rate for both channels with wavelengths of 340 nm and 380 nm using a power function. RESULTS: In cultured neurons, the calculations of the free Ca2+ concentration during Ca2+ transients estimated by the old and new methods, determine it to the same extent. This comparison was made under conditions without errors through background subtraction. If there is this error, the old method calculates [Ca2+] with a much higher, rather than the actual value. CONCLUSIONS: We present a modified Grynkiewitz's formula for calculation [Ca2+] for ratiometric dye, such as Fura-2 imaged by a CCD camera, with photobleaching correction without background subtraction error.

Ca(2+) release events in cardiac myocytes up close: insights from fast confocal imaging.

The spatio-temporal properties of Ca(2+) transients during excitation-contraction coupling and elementary Ca(2+) release events (Ca(2+) sparks) were studied in atrial and ventricular myocytes with ultra-fast confocal microscopy using a Zeiss LSM 5 LIVE system that allows sampling rates of up to 60 kHz. Ca(2+) sparks which originated from subsarcolemmal junctional sarcoplasmic reticulum (j-SR) release sites in atrial myocytes were anisotropic and elongated in the longitudinal direction of the cell. Ca(2+) sparks in atrial cells originating from non-junctional SR and in ventricular myocytes were symmetrical. Ca(2+) spark recording in line scan mode at 40,000 lines/s uncovered step-like increases of [Ca(2+)]i. 2-D imaging of Ca(2+) transients revealed an asynchronous activation of release sites and allowed the sequential recording of Ca(2+) entry through surface membrane Ca(2+) channels and subsequent activation of Ca(2+)-induced Ca(2+) release. With a latency of 2.5 ms after application of an electrical stimulus, Ca(2+) entry could be detected that was followed by SR Ca(2+) release after an additional 3 ms delay. Maximum Ca(2+) release was observed 4 ms after the beginning of release. The timing of Ca(2+) entry and release was confirmed by simultaneous [Ca(2+)]i and membrane current measurements using the whole cell voltage-clamp technique. In atrial cells activation of discrete individual release sites of the j-SR led to spatially restricted Ca(2+) release events that fused into a peripheral ring of elevated [Ca(2+)]i that subsequently propagated in a wave-like fashion towards the center of the cell. In ventricular myocytes asynchronous Ca(2+) release signals from discrete sites with no preferential subcellular location preceded the whole-cell Ca(2+) transient. In summary, ultra-fast confocal imaging allows investigation of Ca(2+) signals with a time resolution similar to patch clamp technique, however in a less invasive fashion.

Using two dyes with the same fluorophore to monitor cellular calcium concentration in an extended range.

We extend the sensitivity of quantitative concentration imaging to an approximately 1000-fold range of concentrations by a method that uses two fluorescent dyes with the same fluorophore, having different affinity for the monitored species. While the formulation and illustration refer to a monitor of calcium concentration, the method is applicable to any species that binds to multiple indicators with the same spectral properties. The use of a common fluorophore has the virtue of leaving vast regions of the electromagnetic spectrum available for other applications. We provide the exact analytic expression relating measured fluorescence to [Ca(2+)] at equilibrium and an approximate analytic expression that does not require the equilibrium assumption. The sensitivity of the method is calculated numerically for two useful dye pairs. As illustrative application of the enhanced measurement, we use fluo-4 and fluo-4FF to image the calcium wave produced by a cardiac myocyte in response to a small artificial calcium spark.

Refractoriness of sarcoplasmic reticulum Ca2+ release determines Ca2+ alternans in atrial myocytes.

Cardiac alternans is a recognized risk factor for cardiac arrhythmia and sudden cardiac death. At the cellular level, Ca(2+) alternans appears as cytosolic Ca(2+) transients of alternating amplitude at regular beating frequency. Cardiac alternans is a multifactorial process but has been linked to disturbances in intracellular Ca(2+) regulation. In atrial myocytes, we tested the role of voltage-gated Ca(2+) current, sarcoplasmic reticulum (SR) Ca(2+) load, and restitution properties of SR Ca(2+) release for the occurrence of pacing-induced Ca(2+) alternans. Voltage-clamp experiments revealed that peak Ca(2+) current was not affected during alternans, and alternans of end-diastolic SR Ca(2+) load, evaluated by application of caffeine or measured directly with an intra-SR fluorescent Ca(2+) indicator (fluo-5N), were not a requirement for cytosolic Ca(2+) alternans. Restitution properties and kinetics of refractoriness of Ca(2+) release after activation during alternans were evaluated by four different approaches: measurements of 1) the delay (latency) of occurrence of spontaneous global Ca(2+) releases and 2) Ca(2+) spark frequency, both during rest after a large and small alternans Ca(2+) transient; 3) the magnitude of premature action potential-induced Ca(2+) transients after a large and small beat; and 4) the efficacy of a photolytically induced Ca(2+) signal (Ca(2+) uncaging from DM-nitrophen) to trigger additional Ca(2+) release during alternans. The results showed that the latency of global spontaneous Ca(2+) release was prolonged and Ca(2+) spark frequency was decreased after the large Ca(2+) transient during alternans. Furthermore, the restitution curve of the Ca(2+) transient elicited by premature action potentials or by photolysis-induced Ca(2+) release from the SR lagged behind after a large-amplitude transient during alternans compared with the small-amplitude transient. The data demonstrate that beat-to-beat alternation of the time-dependent restitution properties and refractory kinetics of the SR Ca(2+) release mechanism represents a key mechanism underlying cardiac alternans.

Properties of Ca2+ sparks revealed by four-dimensional confocal imaging of cardiac muscle.

Parameters (amplitude, width, kinetics) of Ca(2+) sparks imaged confocally are affected by errors when the spark source is not in focus. To identify sparks that were in focus, we used fast scanning (LSM 5 LIVE; Carl Zeiss) combined with fast piezoelectric focusing to acquire x-y images in three planes at 1-µm separation (x-y-z-t mode). In 3,000 x-y scans in each of 34 membrane-permeabilized cat atrial cardiomyocytes, 6,906 sparks were detected. 767 sparks were in focus. They had greater amplitude, but their spatial width and rise time were similar compared with all sparks recorded. Their distribution of amplitudes had a mode at ΔF/F(0) = 0.7. The Ca(2+) release current underlying in-focus sparks was 11 pA, requiring 20 to 30 open channels, a number at the high end of earlier estimates. Spark frequency was greater than in earlier imaging studies of permeabilized ventricular cells, suggesting a greater susceptibility to excitation, which could have functional relevance for atrial cells. Ca(2+) release flux peaked earlier than the time of peak fluorescence and then decayed, consistent with significant sarcoplasmic reticulum (SR) depletion. The evolution of fluorescence and release flux were strikingly similar for in-focus sparks of different rise time (T). Spark termination involves both depletion of Ca(2+) in the SR and channel closure, which may be synchronized by depletion. The observation of similar flux in sparks of different T requires either that channel closure and other termination processes be independent of the determinants of flux (including [Ca(2+)](SR)) or that different channel clusters respond to [Ca(2+)](SR) with different sensitivity.

Synthetic localized calcium transients directly probe signalling mechanisms in skeletal muscle.

The contribution of Ca2+-induced Ca2+ release (CICR) to trigger muscle contraction is controversial. It was studied on isolated muscle fibres using synthetic localized increases in Ca2+ concentration, SLICs, generated by two-photon photorelease from nitrodibenzofuran (NDBF)-EGTA just outside the permeabilized plasma membrane. SLICs provided a way to increase cytosolic [Ca2+] rapidly and reversibly, up to 8 µM, levels similar to those reached during physiological activity. They improve over previous paradigms in rate of rise, locality and reproducibility. Use of NDBF-EGTA allowed for the separate modification of resting [Ca2+], trigger [Ca2+] and resting [Mg2+]. In frog muscle, SLICs elicited propagated responses that had the characteristics of CICR. The threshold [Ca2+] for triggering a response was 0.5 µM or less. As this value is much lower than concentrations prevailing near channels during normal activity, the result supports participation of CICR in the physiological control of contraction in amphibian muscle. As SLICs were applied outside cells, the primary stimulus was Ca2+, rather than the radiation or subproducts of photorelease. Therefore the responses qualify as 'classic' CICR. By contrast, mouse muscle fibres did not respond unless channel-opening drugs were present at substantial concentrations, an observation contrary to the physiological involvement of CICR in mammalian excitation­contraction coupling. In mouse muscle, the propagating wave had a substantially lower release flux, which together with a much higher threshold justified the absence of response when drugs were not present. The differences in flux and threshold may be ascribed to the absence of ryanodine receptor 3 (RyR3) isoforms in adult mammalian muscle.

A novel method for spatially complex diffraction-limited photoactivation and photobleaching in living cells.

Photoactivated probes have gained interest as experimental tools to study intracellular signalling pathways all the way to the molecular level. However technical limitations of the means to activate such compounds have put constraints on their use in spatially highly restricted subcellular areas. The Mosaic digital illumination system uses a high-speed array of individually addressable, tiltable micromirrors to direct continuous-wave laser light onto a specimen with diffraction-limited precision. The system, integrated into a Nikon A1R confocal microscope, was used to uncage Ca²âº or IP3 and conduct photobleaching experiments from multiple geometrically complex subcellular regions while simultaneously measuring [Ca²âº]i with high-speed confocal imaging.

Reciprocal amplification of ROS and Ca(2+) signals in stressed mdx dystrophic skeletal muscle fibers.

Muscular dystrophies are among the most severe inherited muscle diseases. The genetic defect is a mutation in the gene for dystrophin, a cytoskeletal protein which protects muscle cells from mechanical damage. Mechanical stress, applied as osmotic shock, elicits an abnormal surge of Ca(2+) spark-like events in skeletal muscle fibers from dystrophin deficient (mdx) mice. Previous studies suggested a link between changes in the intracellular redox environment and appearance of Ca(2+) sparks in normal mammalian skeletal muscle. Here, we tested whether the exaggerated Ca(2+) responses in mdx fibers are related to oxidative stress. Localized intracellular and mitochondrial Ca(2+) transients, as well as ROS production, were assessed with confocal microscopy. The rate of basal cellular but not mitochondrial ROS generation was significantly higher in mdx cells. This difference was abolished by pre-incubation of mdx fibers with an inhibitor of NAD(P)H oxidase. In addition, immunoblotting showed a significantly stronger expression of NAD(P)H oxidase in mdx muscle, suggesting a major contribution of this enzyme to oxidative stress in mdx fibers. Osmotic shock produced an abnormal and persistent Ca(2+) spark activity, which was suppressed by ROS-reducing agents and by inhibitors of NAD(P)H oxidase. These Ca(2+) signals resulted in mitochondrial Ca(2+) accumulation in mdx fibers and an additional boost in cellular and mitochondrial ROS production. Taken together, our results indicate that the excessive ROS production and the simultaneous activation of abnormal Ca(2+) signals amplify each other, finally culminating in a vicious cycle of damaging events, which may contribute to the abnormal stress sensitivity in dystrophic skeletal muscle.

Reactive oxygen species contribute to Ca2+ signals produced by osmotic stress in mouse skeletal muscle fibres.

Ca(2+) sparks, localized elevations in cytosolic [Ca(2+)], are rarely detected in intact adult mammalian skeletal muscle under physiological conditions. However, they have been observed in permeabilized cells and in intact fibres subjected to stresses, such as osmotic shock and strenuous exercise. Our previous studies indicated that an excess in cellular reactive oxygen species (ROS) generation over the ROS scavenging capabilities could be one of the up-stream causes of Ca(2+) spark appearance in permeabilized muscle fibres. Here we tested whether the cytosolic ROS balance is compromised in intact skeletal muscle fibres that underwent osmotic shock and whether this misbalance contributes to unmasking Ca(2+) sparks. Spontaneous Ca(2+) sparks and the rate of ROS generation were assessed with single photon confocal microscopy and fluorescent indicators fluo-4, CM-H(2)DCFDA and MitoSOX Red. Osmotic shock produced spontaneous Ca(2+) sparks and a concomitant significant increase in ROS production. Preincubation of muscle cells with ROS scavengers (e.g. MnTBAP, Mn-cpx 3, TIRON) nearly eliminated Ca(2+) sparks. In addition, inhibitors of NAD(P)H oxidase (DPI and apocynin) significantly reduced ROS production and suppressed the appearance of Ca(2+) sparks. Taken together, the data suggest that ROS contribute to the abnormal Ca(2+) spark activity in mammalian skeletal muscle subjected to osmotic stress and also indicate that NAD(P)H oxidase is a possible source of ROS. We propose that ROS-dependent Ca(2+) sparks are an important component of adaptive/maladaptive muscle responses under various pathological conditions such as eccentric stretch, osmotic changes during ischaemia and reperfusion, and some muscle diseases.

Transfer and tunneling of Ca2+ from sarcoplasmic reticulum to mitochondria in skeletal muscle.

The role of mitochondrial Ca2+ transport in regulating intracellular Ca2+ signaling and mitochondrial enzymes involved in energy metabolism is widely recognized in many tissues. However, the ability of skeletal muscle mitochondria to sequester Ca2+ released from the sarcoplasmic reticulum (SR) during the muscle contraction-relaxation cycle is still disputed. To assess the functional cross-talk of Ca2+ between SR and mitochondria, we examined the mutual relationship connecting cytosolic and mitochondrial Ca2+ dynamics in permeabilized skeletal muscle fibers. Cytosolic and mitochondrial Ca2+ transients were recorded with digital photometry and confocal microscopy using fura-2 and mag-rhod-2, respectively. In the presence of 0.5 mM slow Ca2+ buffer (EGTA (ethylene glycolbis(2-aminoethylether)-N,N,N',N'-tetraacetic acid)), application of caffeine induced a synchronized increase in both cytosolic and mitochondrial [Ca2+]. 5 mM fast Ca2+ buffer (BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid)) nearly eliminated caffeine-induced increases in [Ca2+]c but only partially decreased the amplitude of mitochondrial Ca2+ transients. Confocal imaging revealed that in EGTA, almost all mitochondria picked up Ca2+ released from the SR by caffeine, whereas only about 70% of mitochondria did so in BAPTA. Taken together, these results indicated that a subpopulation of mitochondria is in close functional and presumably structural proximity to the SR, giving rise to subcellular microdomains in which Ca2+ has preferential access to the juxtaposed organelles.

Mitochondrial redox state and Ca2+ sparks in permeabilized mammalian skeletal muscle.

Intact skeletal muscle fibres from adult mammals exhibit neither spontaneous nor stimulated Ca(2+) sparks. Mechanical or chemical skinning procedures have been reported to unmask sparks. The present study investigates the mechanisms that determine the development of Ca(2+) spark activity in permeabilized fibres dissected from muscles with different metabolic capacity. Spontaneous Ca(2+) sparks were detected with fluo-3 and single photon confocal microscopy; mitochondrial redox potential was evaluated from mitochondrial NADH signals recorded with two-photon confocal microscopy, and Ca(2+) load of the sarcoplasmic reticulum (SR) was estimated from the amplitude of caffeine-induced Ca(2+) transients recorded with fura-2 and digital photometry. In three fibre types studied, there was a time lag between permeabilization and spark development. Under all experimental conditions, the delay was the longest in slow-twitch oxidative fibres, intermediate in fast-twitch glycolytic-oxidative fibres, and the shortest in fast-twitch glycolytic cells. The temporal evolution of Ca(2+) spark frequencies was bell-shaped, and the maximal spark frequency was reached slowly in mitochondria-rich oxidative cells but quickly in mitochondria-poor glycolytic fibres. The development of spontaneous Ca(2+) sparks did not correlate with the SR Ca(2+) content of the fibre, but did correlate with the redox potential of their mitochondria. Treatment of fibres with scavengers of reactive oxygen species (ROS), such as superoxide dismutase (SOD) and catalase, dramatically and reversibly reduced the spark frequency and also delayed their appearance. In contrast, incubation of fibres with 50 microm H(2)O(2) sped up the development of Ca(2+) sparks and increased their frequency. These results indicate that the appearance of Ca(2+) sparks in permeabilized skeletal muscle cells depends on the fibre's oxidative strength and that misbalance between mitochondrial ROS production and the fibre's ability to fight oxidative stress is likely to be responsible for unmasking Ca(2+) sparks in skinned preparations. They also suggest that under physiological and pathophysiological conditions the appearance of Ca(2+) sparks may be, at least in part, limited by the fine-tuned equilibrium between mitochondrial ROS production and cellular ROS scavenging mechanisms.

Action of hypoxia on different types of calcium channels in hippocampal neurons.

Whole-cell patch clamp and polarographic oxygen partial pressure (pO2) measurements were used to establish the sensitivity of high-voltage-activated (HVA) Ca2+ channel subtypes of CA1 hippocampal neurons of rats to hypoxic conditions. Decrease of pO2 to 15-30 mm Hg induced a potentiation of HVA Ca2+ currents by 94%. Using selective blockers of N- and L-types of calcium channels, we found that inhibition of L-type channels decreased the effect by 54%, whereas N-type blocker attenuated the effect by 30%. Taking into account the ratio of currents mediated by these channel subtypes in CA1 hippocampal neurons, we concluded that both types of HVA Ca2+ channels are sensitive to hypoxia, however, L-type was about 3.5 times more sensitive to oxygen reduction.