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Fast imaging in two dimensions resolves extensive sources of Ca²⁺ sparks in frog skeletal muscle

Gustavo Brum, Adom González*, Juliana Rengifo*, Natalia Shirokova* and Eduardo Ríos*

1. Ca²⁺ sparks were monitored by confocal laser-scanning microscopy of fluo-3 at video rates, in fast twitch muscle fibres, stimulated by exposure to caffeine and/or low [Mg²⁺]. Scanning was in two spatial dimensions ('2D') or 2D plus time, at 4 ms per image frame. Sparks were identified in 2D images of normalized fluorescence by an automatic procedure, which also evaluated the event's location and morphometric parameters.

2. Most sparks were circular, but some were elongated, especially in caffeine. Separation of the spark from circular symmetry was quantified by its eccentricity (length/width - 1).

3. In an internal solution with 0·4 mM [Mg²⁺], sparks (989 events in 4 cells) had amplitude 0·73, width 1·94 µm, length 2·12 µm and eccentricity not significantly different from 0. Upon application of 1 mM caffeine, length (of 2578 events in the same cells) increased significantly (by 0·41 µm, or 19 %), width increased by 0·18 µm (9 %) and amplitude decreased slightly. The eccentricity became significantly different from 0, and the sparks' long axis predominantly oriented parallel to the plane of the Z disks.

4. More than 10 % of the events in caffeine had length greater than 4 µm, a relatively flat top, and a sharp termination at both ends of the major axis. Additionally, there was only a weak correlation between eccentricity and amplitude. These properties suggest that the elongated events are produced by simultaneous opening of multiple channels within a junction, rather than anisotropic diffusion of Ca²⁺ or random overlap of round sparks.

5. Elongated events often increased in eccentricity early in their evolution. Then, most remained elongated during their rise and decay, while others spread spatially in the plane of the Z disks. In 1-2 % of the events, the centre of mass migrated in space, over time, at ~0·1 µm ms^-1.

6. These spatio-temporal features require the involvement of multiple release channels, at spatially resolvable locations. Because sources often spread over distances greater than 1 or 2 µm, and arrays of junctional elements (couplons) are at most 1·2 µm long, it must be possible for activation of release to propagate between neighbouring couplons, especially under the influence of caffeine and/or low [Mg²⁺].

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