Side bridge geometry after quick-freezing of stimulated and unstimulated frog skeletal muscle fibers

1983 ◽  
Vol 41 ◽  
pp. 464-465
Author(s):  
J.R. Sommer ◽  
R. Nassar ◽  
S. Walker
Keyword(s):  
Skeletal Muscle ◽  
Time Course ◽  
Muscle Fibers ◽  
Freeze Fracture ◽  
Freeze Substitution ◽  
Frog Skeletal Muscle ◽  
Specimen Holder ◽  
Skeletal Muscle Fibers ◽  
Quick Freezing ◽  
The Impact

Quick-freezing allows the structural analysis of timed perturbations of morphology. We are presenting preliminary results concerning the feasibility of studying directly the side bridge geometry of actin-myosin interactions within the time course of a twitch in single intact frog skeletal muscle fibers, both by freeze-substitution and freeze-fracture after quick-freezing, and following various time intervals between stimulation and impact of the fibers on a liquid He-cooled copper block.Materials and Methods. The quick-freezing device was a "Slammer"(Polaron) for which the electronics had been redesigned; they are capable, in combination with a Grass S48 stimulator, of any stimulation interval between 0 and 1 sec prior to freezing, including tetanus. The actual elapsed time between stimulation and freezing is recorded with a digital clock. Single intact tendonto- tendon frog skeletal muscle fibers (semitendinosus of r. temporaria) or toe muscle bundles (r.pipiens) were isolated by sharp dissection and placed between coextensive Pt stimulation wires on blackened 2% agarose, the height of which on the specimen holder was adjusted appropriately with respect to a spacer ring both, to calibrate the impact time and to prevent smashing of the fibers.

The SR of skeletal muscle: Its structure after quick-freezing and freeze-etching, following field stimulation of single intact muscle fibers

1984 ◽  
Vol 42 ◽  
pp. 300-301
Author(s):  
J.R. Sommer ◽  
R. Nassar ◽  
N.R. Wallace
Keyword(s):  
Skeletal Muscle ◽  
Striated Muscle ◽  
Muscle Fibers ◽  
Freeze Fracture ◽  
Electron Gun ◽  
Skeletal Muscle Fibers ◽  
Quick Freezing ◽  
Intact Muscle ◽  
Similar Images ◽  
Freeze Etching

It is known that the P faces of freeze-fractured SR of fixed and cryoprotected striated muscle fibers are studded with particles, whereas the E faces remain smooth, except for two staggered rows of pits in the junctional SR (JSR) which face transverse tubules (junctional pits). Freeze-fracture after quick-freezing of native skeletal muscle provides similar images (1). We have used freeze-etching to look at the SR's structure in single intact skeletal muscle fibers (r.temporaria) without stimulation, following varied post-stimulation intervals, and in tetanus. Single intact skeletal muscle fibers were isolated and quick-frozen as previously reported (2). After quick-freezing, the fibers were transferred to a Balzers 301 device and etched for 3 minutes at -100°C, followed by unidirectional Pt evaporation with an electron gun and carbon coating.

Quantitative x-ray elemental imaging in stimulated single intact skeletal muscle fibers: The fate of calcium in the presence of ryanodine

1988 ◽  
Vol 46 ◽  
pp. 90-91
Author(s):  
J. Sommer ◽  
P. Ingram ◽  
A. LeFurgey ◽  
R. Nassar ◽  
T. High
Keyword(s):  
Skeletal Muscle ◽  
Electrical Stimulation ◽  
Time Course ◽  
Imaging System ◽  
Quantitative Imaging ◽  
Muscle Fibers ◽  
Frog Skeletal Muscle ◽  
X Ray ◽  
Skeletal Muscle Fibers ◽  
Freeze Dried

We are involved in a continuing series of experiments aimed at a complete description,in terms of morphology and quantitative topochemistry, of the time course of spatial distributions of physiologically important elements during excitation-contraction coupling (ECC) at different time intervals (fractions of msec) following electrical stimulation of single, intact frog skeletal muscle fibers. In this present study wg report such distributions for Ca after 1,2 and 3 min of electrical stimulation in the presence of 2x10-4 M ryanodine, an alkaloid that, in time, causes irreversible muscle contractures.Single, intact frog skeletal muscle fibers were quick-frozen, cryosectioned, freeze-substituted and in one case freeze-fractured. The freeze-dried cryosections were subjected to electron probe X-ray microanalysis (EPXMA) in a JEOL 1200EX analytical electron microscope equipped with a Tracor Northern X-ray detector and a fully quantitative imaging system. Both, 64/64 pixel images (ambient temp.), and small raster probes (cold stage,-115 °C) for better statistics, were obtained, each from the same section.

Last second twitch-response test of single intact skeletal muscle fibers prior to quick-freezing after various stimulation intervals

1983 ◽  
Vol 41 ◽  
pp. 526-527
Author(s):  
J.R. Sommer ◽  
R. Nassar ◽  
I. Taylor
Keyword(s):  
Skeletal Muscle ◽  
Time Course ◽  
Morphological Changes ◽  
Muscle Fibers ◽  
Visual Observation ◽  
Time Interval ◽  
Time Intervals ◽  
Twitch Response ◽  
Skeletal Muscle Fibers ◽  
Quick Freezing

Conventional chemical fixation of muscle fibers is not suited to disclose morphological changes occurring with a time course of only a few msec, e.g.during excitation-contraction-coupling. We have taken advantage of a quick-freeze method to be able to study single intact frog skeletal muscle fibers (r.temporaria) after various time intervals following electrical stimulation. The electronics were designed to permit any time interval from 0 to more than 1 sec between stimulation and impact of the specimen on a liquid He-cooled copper block. It is important to monitor the twitch-response to stimulation. Given the geometry of the freezing device ("Slammer", Polaron), and the fact that the device does not operate vibration-free, it is quite difficult to design and built an effective monitor able to record the actual twitch-response following the stimulation of an isolated single muscle fiber during its descent prior to freezing. We have built a simple device that allows visual observation of the twitch-response within a few seconds prior to the definitive stimulation during the specimen drop.

How to sample the entire length of a single muscle fiber quick-frozen after electrical point stimulation for high resolution EM

1992 ◽  
Vol 50 (1) ◽  
pp. 776-777
Author(s):  
Joachim R. Sommer ◽  
Teresa High ◽  
Betty Scherer ◽  
Isaiah Taylor ◽  
Rashid Nassar
Keyword(s):  
Skeletal Muscle ◽  
High Resolution ◽  
Action Potential ◽  
Muscle Fiber ◽  
Freeze Fracture ◽  
Freeze Substitution ◽  
Electrical Field Stimulation ◽  
Skeletal Muscle Fiber ◽  
Freeze Dried ◽  
Quick Freezing

We have developed a model that allows the quick-freezing at known time intervals following electrical field stimulation of a single, intact frog skeletal muscle fiber isolated by sharp dissection. The preparation is used for studying high resolution morphology by freeze-substitution and freeze-fracture and for electron probe x-ray microanlysis of sudden calcium displacement from intracellular stores in freeze-dried cryosections, all in the same fiber. We now show the feasibility and instrumentation of new methodology for stimulating a single, intact skeletal muscle fiber at a point resulting in the propagation of an action potential, followed by quick-freezing with sub-millisecond temporal resolution after electrical stimulation, followed by multiple sampling of the frozen muscle fiber for freeze-substitution, freeze-fracture (not shown) and cryosectionmg. This model, at once serving as its own control and obviating consideration of variances between different fibers, frogs etc., is useful to investigate structural and topochemical alterations occurring in the wake of an action potential.

Inaction of saxitoxin-oximes on the sodium channel of frog skeletal muscle fibers

Toxicon ◽  
1987 ◽  
Vol 25 (2) ◽  
pp. 159-165 ◽  
Author(s):  
S.L. Hu ◽  
C.Y. Kao ◽  
F.E. Koehn ◽  
H.K. Schnoes
Keyword(s):  
Skeletal Muscle ◽  
Sodium Channel ◽  
Muscle Fibers ◽  
Frog Skeletal Muscle ◽  
Skeletal Muscle Fibers

scholarly journals Anatomical distribution of voltage-dependent membrane capacitance in frog skeletal muscle fibers.

1989 ◽  
Vol 93 (3) ◽  
pp. 565-584 ◽  
Author(s):  
C L Huang ◽  
L D Peachey
Keyword(s):  
Skeletal Muscle ◽  
Muscle Fibers ◽  
Fiber Surface ◽  
Membrane Capacitance ◽  
Hypertonic Solution ◽  
Charge Movement ◽  
Frog Skeletal Muscle ◽  
Transverse Tubule ◽  
Skeletal Muscle Fibers ◽  
Voltage Dependent

Components of nonlinear capacitance, or charge movement, were localized in the membranes of frog skeletal muscle fibers by studying the effect of 'detubulation' resulting from sudden withdrawal of glycerol from a glycerol-hypertonic solution in which the muscles had been immersed. Linear capacitance was evaluated from the integral of the transient current elicited by imposed voltage clamp steps near the holding potential using bathing solutions that minimized tubular voltage attenuation. The dependence of linear membrane capacitance on fiber diameter in intact fibers was consistent with surface and tubular capacitances and a term attributable to the capacitance of the fiber end. A reduction in this dependence in detubulated fibers suggested that sudden glycerol withdrawal isolated between 75 and 100% of the transverse tubules from the fiber surface. Glycerol withdrawal in two stages did not cause appreciable detubulation. Such glycerol-treated but not detubulated fibers were used as controls. Detubulation reduced delayed (q gamma) charging currents to an extent not explicable simply in terms of tubular conduction delays. Nonlinear membrane capacitance measured at different voltages was expressed normalized to accessible linear fiber membrane capacitance. In control fibers it was strongly voltage dependent. Both the magnitude and steepness of the function were markedly reduced by adding tetracaine, which removed a component in agreement with earlier reports for q gamma charge. In contrast, detubulated fibers had nonlinear capacitances resembling those of q beta charge, and were not affected by adding tetracaine. These findings are discussed in terms of a preferential localization of tetracaine-sensitive (q gamma) charge in transverse tubule membrane, in contrast to a more even distribution of the tetracaine-resistant (q beta) charge in both transverse tubule and surface membranes. These results suggest that q beta and q gamma are due to different molecules and that the movement of q gamma in the transverse tubule membrane is the voltage-sensing step in excitation-contraction coupling.

scholarly journals Ultraslow contractile inactivation in frog skeletal muscle fibers.

1990 ◽  
Vol 96 (1) ◽  
pp. 47-56 ◽  
Author(s):  
C Caputo ◽  
P Bolaños
Keyword(s):  
Skeletal Muscle ◽  
Time Course ◽  
Muscle Fibers ◽  
Membrane Depolarization ◽  
Frog Muscle ◽  
Slow Time ◽  
Inactivation Curve ◽  
Lanthanum Ions ◽  
Skeletal Muscle Fibers ◽  
Spontaneous Relaxation

After a contracture response, skeletal muscle fibers enter into a state of contractile refractoriness or inactivation. Contractile inactivation starts soon after membrane depolarization, and causes spontaneous relaxation from the contracture response. Here we demonstrate that contractile inactivation continues to develop for tens of seconds if the membrane remains in a depolarized state. We have studied this phenomenon using short (1.5 mm) frog muscle fibers dissected from the Lumbricalis brevis muscles of the frog, with a two-microelectrode voltage-clamp technique. After a contracture caused by membrane depolarization to 0 mV, from a holding potential of -100 mV, a second contracture can be developed only if the membrane is repolarized beyond a determined potential value for a certain period of time. We have used a repriming protocol of 1 or 2 s at -100 mV. After this repriming period a fiber, if depolarized again to 0 mV, may develop a second contracture, whose magnitude and time course will depend on the duration of the period during which the fiber was maintained at 0 mV before the repriming process. With this procedure it is possible to demonstrate that the inactivation process builds up with a very slow time course, with a half time of approximately 35 s and completion in greater than 100 s. After prolonged depolarizations (greater than 100 s), the repriming time course is slower and the inactivation curve (obtained by plotting the extent of repriming against the repriming membrane potential) is shifted toward more negative potentials by greater than 30 mV when compared with similar curves obtained after shorter depolarizing periods (10-30 s). These results indicate that important changes occur in the physical state of the molecular moiety that is responsible for the inactivation phenomenon. The shift of the inactivation curve can be partially reversed by a low concentration (50 microM) of lanthanum ions. In the presence of 0.5 mM caffeine, larger responses can be obtained even after prolonged depolarization periods, indicating that the fibers maintain their capacity to liberate calcium.

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