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

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

Ca²⁺ sparks in atrial myocytes.

<p>A, Series of confocal 2-D images of a spontaneous Ca²⁺ spark originating from the SS j-SR, recorded at 0.96 ms intervals (top). Bottom: reconstructed line scan images (x-t and y-t) from the 2-D series. <b>B</b>, analogous to panel A: spontaneous Ca²⁺ spark recorded from a CT nj-SR Ca²⁺ release unit. <b>C</b>, movement of the x-y -coordinates of maximal fluorescence during a SS Ca²⁺ spark. Left: the spatial coordinates of peak fluorescence intensity were recorded along the dashed lines in x- and y-dimension. Right: x-t and y-t plots of the location of maximal fluorescence, indicating a preferential movement of the point source of Ca generating the spark in centripetal direction.</p

Ca²⁺ release signals in ventricular myocytes.

<p>A, series of confocal 2-D images of the early phase of a Ca²⁺ transient evoked by electrical field stimulation in a ventricular myocytes. White circles mark the first appearance of individual j-SR Ca²⁺ release sites. t = 0 indicates the image frame where the first activated CRU was detected. <b>B</b>, cumulative recruitment of individual j-SR CRUs. CRUs were detected in a region of interest encompassing the j-SR and measuring 60 µm×7.5 µm. Detected CRUs were normalized to number of CRUs/100 µm². <b>C</b>, evolution of [Ca²⁺]_i at individual SS and CT j-SR release sites. The traces were recorded from a 1 pixel sized regions (0.09 µm²). <b>D</b>, series of confocal 2-D images of the early phase of a Ca²⁺ transient evoked by electrical field stimulation in an atrial myocyte and recorded from a focal plane positioned in the SS j-SR space at the bottom of the cell. <b>E</b>, normalized Ca²⁺ transients recorded from ventricular j-SR (black), CT nj-SR atrial (blue) and SS j-SR atrial (red) release sites.</p

Subcellular Ca²⁺ fluxes during ECC identified by fast Ca²⁺ imaging.

<p>A, global Ca²⁺ transient (ΔF/F₀) elicited by electrical field stimulation of a ventricular myocyte (panel <b>a</b>). Marker ‘1’ indicates the application of the electrical stimulus. Markers ‘2’ and ‘3’ were positioned according to the analysis in panel b. Panel <b>b</b>: first derivative of ΔF/F₀ (s⁻¹) from panel a, representing Ca²⁺ flux. Markers ‘2’ and ‘3’ indicate abrupt changes in Ca²⁺ flux rate, and marker ‘4’ indicates maximal Ca²⁺ flux. Panel <b>c</b>: spatially averaged Ca²⁺ flux under control conditions (Ctl) and during inhibition of SR function with thapsigargin (TG; 1 µM) and caffeine (Caff; 10 mM). <b>B</b>, subcellular Ca²⁺ transients (ΔF/F₀) elicited by electrical field stimulation and recorded from subsarcolemmal (SS) j-SR and central (CT) nj-SR regions of an atrial myocyte (panel <b>a</b>). Panel <b>b</b>: first derivative of SS and CT Ca²⁺ signals representing subcellular Ca²⁺ flux rates. Markers ‘1’ to ‘4’ as in panel A. Panel <b>c</b>: subcellular (SS, CT) Ca²⁺ flux rates in control and in the presence of thapsigargin+caffeine. 2-D images were recorded 1719 Hz time resolution.</p

Ultra-fast Ca²⁺ spark recordings.

<p>A, x-t line scan image recorded at 40,000 lines/s. <b>B</b>, Top: one pixel-wide (0.3 µm) ΔF/F₀ profile recorded from the center of the spark. For noise reduction data were averaged to 10,000 lines/s. The red trace represents a 5-point moving average. Bottom: d(ΔF/F₀)/dt (s⁻¹), first derivative of the ΔF/F₀ signal of the rising phase of the spark. Vertical dashed lines mark maxima of the d(ΔF/F₀)/dt signal identifying maxima of Ca²⁺ release flux. The discrete peaks of the d(ΔF/F₀)/dt signal were used to identify step-like increases of the ΔF/F₀ signal (marked by horizontal solid black lines). Grey bars indicate discrete d(ΔF/F₀)/dt levels. A discrete d(ΔF/F₀)/dt level was defined when at least two d(ΔF/F₀)/dt peaks of the same amplitude were observed.</p

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

AP-induced Ca²⁺ transients in atrial myocytes.

<p>A, series of confocal 2-D images of the early phase of a Ca²⁺ transient evoked by electrical field stimulation. White ovals mark the first appearance of individual Ca²⁺ release sites of the j-SR in the SS region. t = 0 indicates the image frame where the first activated CRU was detected. <b>B</b>, cumulative recruitment of individual j-SR CRUs. CRUs were detected in a SS region of 75 µm×2 µm and normalized to number of CRUs/100 µm². <b>C</b>, Sequential 2-D images of an individual SS j-SR release site, marked by the white box in panel A, recorded at 0.58 ms time intervals. <b>D</b>, Evolution of [Ca²⁺]_i at an individual SS CRU and an adjacent non-release site at a distance of ∼1 µm. The traces were recorded from a 1 pixel sized regions (0.09 µm²). <b>E</b>, comparison of SS (j-SR) and CT (nj-SR; recorded at a distance of ∼4 µm from the cell membrane) Ca²⁺ transients.</p