Frog Twitch Muscle Research Articles

Analysis of Calcium (Ca) Transients during Excitation-Contraction Coupling in Frog Twitch Muscle Fibers

Singly-dissected fibers were micro-injected with furaptra, a rapidly-responding Ca indicator. Resting fluorescence (FR) and action-potential evoked changes (ΔF) were measured at 16 oC; ΔF/FR was scaled to units of ΔfCaD, the change in fraction of furaptra in the Ca-bound form. Measured ΔfCaD was compared with ΔfCaD simulated with a kinetic model of the underlying myoplasmic Ca movements. During the period 30-200 ms after an action potential, simulated ΔfCaD decayed toward baseline more slowly than measured ΔfCaD. If ΔfCaD was simulated with a modified model that incorporated competition between Mg and Ca for occupancy of the regulatory sites on troponin (assumed dissociation constant of Mg's reaction with the regulatory sites, 2.2 mM; assumed myoplasmic free [Mg], 1 mM), good agreement with measured ΔfCaD was observed. The results support the conclusion that Mg, at physiological levels, competes with Ca for occupancy of the regulatory sites, as indicated in some experiments from fragmented preparations, including tension-pCa measurements in skinned fibers and biochemical studies of isolated troponin molecules. ΔfCaD in frog was also compared with our previous measurements and simulations of ΔfCaD in mouse fibers (reviewed in J. Gen. Physiol., 2012). In frog, the SR Ca release flux elicited by an action potential appears to be the sum of two components. The time course of the first component is similar to that of the entire flux waveform in both fast-and slow-twitch mouse fibers, while that of the second is several-fold slower; the fractional release amounts are ∼0.85 (first component) and ∼0.15 (second component). An anatomical basis for two release components in frog is the presence of both junctional and para-junctional Ca release channels, whereas the mouse fibers have only junctional channels (Felder and Franzini-Armstrong, 2002). Supported by NIH (GM 086167)

Calcium buffering properties of sarcoplasmic reticulum and calcium-induced Ca2+ release during the quasi-steady level of release in twitch fibers from frog skeletal muscle

Experiments were performed to characterize the properties of the intrinsic Ca2+ buffers in the sarcoplasmic reticulum (SR) of cut fibers from frog twitch muscle. The concentrations of total and free calcium ions within the SR ([CaT]SR and [Ca2+]SR) were measured, respectively, with the EGTA/phenol red method and tetramethylmurexide (a low affinity Ca2+ indicator). Results indicate SR Ca2+ buffering was consistent with a single cooperative-binding component or a combination of a cooperative-binding component and a linear binding component accounting for 20% or less of the bound Ca2+. Under the assumption of a single cooperative-binding component, the most likely resting values of [Ca2+]SR and [CaT]SR are 0.67 and 17.1 mM, respectively, and the dissociation constant, Hill coefficient, and concentration of the Ca-binding sites are 0.78 mM, 3.0, and 44 mM, respectively. This information can be used to calculate a variable proportional to the Ca2+ permeability of the SR, namely d[CaT]SR/dt ÷ [Ca2+]SR (denoted release permeability), in experiments in which only [CaT]SR or [Ca2+]SR is measured. In response to a voltage-clamp step to −20 mV at 15°C, the release permeability reaches an early peak followed by a rapid decline to a quasi-steady level that lasts ∼50 ms, followed by a slower decline during which the release permeability decreases by at least threefold. During the quasi-steady level of release, the release amplitude is 3.3-fold greater than expected from voltage activation alone, a result consistent with the recruitment by Ca-induced Ca2+ release of 2.3 SR Ca2+ release channels neighboring each channel activated by its associated voltage sensor. Release permeability at −60 mV increases as [CaT]SR decreases from its resting physiological level to ∼0.1 of this level. This result argues against a release termination mechanism proposed in mammalian muscle fibers in which a luminal sensor of [Ca2+]SR inhibits release when [CaT]SR declines to a low level.

Effects of phorbol esters on excitation-contraction coupling and protein kinase C activity of frog twitch muscle fibers.

By recording the calcium transients evoked by voltage-clamp depolarizing pulse with arsenazo III as a calcium indicator, it has been shown that 1 micromol/l phorbol 12,13-dibutyrate (PDBu), a protein kinase C (PKC) agonist, causes a transient potentiation and then a depression of the calcium transients of twitch muscle fibers in frogs. PDBu also produces an initial translocation and activation of PKC, which is followed by a down-regulation. To find out whether the effect of PDBu on the calcium transients depends on PKC, a correlated study of the effect of phorbol esters on calcium transients and PKC activity was performed. The calcium transients and PKC activity were similarly affected by PDBu in ordinary and cold-accommodated frogs, but the effects occurred more quickly in the latter. However, they still changed in parallel as in ordinary frogs. 1 or 10 micromol/l, 4-alpha-phorbol, a PKC-inactive analogue of phorbol ester, caused a partial depression of the calcium transients in cold-accommodated frogs, while PKC activity was not affected. Moreover, the transient potentiation of the calcium transients induced by 1 micromol/l PDBu could be antagonized by the PKC inhibitors 10 micromol/l chelerythrine chloride or 10 micromol/l polymyxin B (PMB). All these results suggest that: (1) the transient potentiation of calcium transients induced by PDBu is caused by activation of PKC; (2) phorbol ester can depress the calcium transients by a mechanism that is independent of PKC.

The effects of chloride ions in excitation-contraction coupling and sarcoplasmic reticulum calcium release in twitch muscle fibre.

Using the sucrose vaseline gap technique, experiments were carried out on isolated frog twitch muscle fibre to investigate the role of chloride ions in excitation-contraction coupling. In current clamp conditions, replacement of chloride ions by impermeant anions led to an increase of the amplitude of the early after potential and of the amplitude of the twitch. Addition of a chloride channel blocker, anthracene-9-carboxylic acid gave similar results. In voltage clamp conditions, replacement of chloride ions by impermeant anions induced a decrease of the outward current and an increase of both the amplitude of the contraction and of the resting tension. Addition of anthracene-9-carboxylic acid gave similar results except that resting tension was not modified. Replacement of chloride ions by impermeant anions resulted in a shift of the tension-voltage relationship toward negative potentials and in an increase of the amplitude of the contraction at all potentials. Outward currents were also reduced at all potentials but no shift of the current-voltage relationship was observed. Similar results were obtained upon addition of anthracene-9-carboxylic acid. Rapid filtration experiments were performed on isolated sarcoplasmic reticulum vesicles to study the role of chloride ions in Ca2+ release. Under conditions where KCl was present in the intra- and extravesicular media, removal of chloride ions from the release solution produced a 2-fold increase in the rate of Ca(2+)-induced Ca2+ release. Together, these results suggest that, besides their involvement in the action potential time course, chloride ions could exert a negative control on the sarcoplasmic reticulum Ca2+ release.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of activation of protein kinase C on excitation-contraction coupling in frog twitch muscle fibres.

Intracellular Ca2+ transients were recorded from frog twitch muscle fibres in response to voltage-clamp depolarizing pulses, using arsenazo III as an intracellular Ca2+ indicator. The effect of the activation of protein kinase C (PKC) on the Ca2+ transients was studied. With 1 microM phorbol 12,13-dibutyrate (PDBu), a PKC activator, the peak of the Ca2+ transients increased to about 120% of control during the first 0.5 h, and then decreased gradually to a plateau of 44% of control within the following 2 h. This effect of PDBu could be alleviated significantly by PKC inhibitors, 10 microM polymyxin B (PMB) or 30 microM 1-(5-isoquinolinylsulphonyl)-2-methyl-piperazine (H-7). Moreover, PDBu caused an upward shift of the strength/duration curve. In Li(+)-loaded muscle fibres the Ca2+ transients could not fully recover after 80 mM K+ exposure for 15 min, while the post-K+ Ca2- transients could be completely restored in the fibres not loaded with Li+. In the presence of 10 microM PMB or 30 microM H-7, a full restoration of the post-K+ Ca2+ transients was seen in Li(+)-loaded fibres. PMB supplemented after high-K+ exposure also could result in a complete recovery of the post-K+ Ca2+ transients in Li(+)-loaded fibres. The role of PKC in modulating excitation-contraction coupling in frog twitch muscle fibres is clearly indicated, but the mechanism(s) and physiological significance remain to be established.

Ratio of Dihydropyridine to Ryanodine Receptors in Mammalian and Frog Twitch Muscles in Relation to the Mechanical Hypothesis of Excitation-Contraction Coupling

An indirect argument in favour of the mechanical hypothesis of excitation-contraction (EC) coupling [Schneider & Chandler (1973) Nature242, 244-246] is the fixed stoichiometry between the voltage-driven dihydropyridine receptor (DHP-R) on the transverse tubule (TT) and the ryanodine-sensitive Ca2+ channel (RyR) of sarcoplasmic reticulum (SR) terminal cisternae (TC), based mainly on the structural arrangement described by Block et al. at junctional triads of toadfish swimbladder muscle [Block, B., et al. (1988) J. Cell Biol.107, 2587-2600]. We calculated the overall DHP-R/RyR ratios for a wide variety of rabbit twitch muscles, as compared to fast- and slow-twitch muscles of the rat, and to frog sartorius, following quantification of high-affinity binding sites for tritiated PN200-110 and ryanodine. To this purpose, we carried out our binding measurements on both skeletal muscle membranes detached from myofibrils by extensive extraction with low-ionic strength medium,and on the myofibrillar residue containing a proportion of binding sites. Our results expressed per gram of muscle allow the following main conclusions: i) The overall DHP-R/RyR ratio is approximately 2 in both fast-twitch and slow-twitch muscle of the rabbit, implying that most, if not all, Ca2+-channels at junctional triads are stoichiometrically associated with the voltage sensor; in agreement with Block′s model; ii) There is a major inconsistency in these values, rather than in the absolute values of RyR sites, on side by side comparison of fast-twitch and of slow-twitch muscles of the rabbit and rat, in that the DHP-R/RyR ratio is invariably lower for the rat, i.e.,only about 1.2. Since the absolute values of DHP-R sites, according to our calculations, agree well with those obtained for the same muscles by Lamb & Walsh [(1987) J. Physiol. 393, 595-617], it seems improbable that they were underestimated. On the assumption that there is a fixed stoichiometry between DHP-R and RyR at the junctional contact area between TT and TC, the relatively large excess of RyR sites in the rat, expressed per g. muscle, should be attributed to the presence of a proportion of Ca2+-channels that are located outside this area, i.e.,are extrajunctional [Dulhunty, A., et al. (1992) Proc. R. Soc. Lond. (B) 247, 69-75]. We also find similarities in DHP-R/RyR ratios between twitch muscles of the rat and the frog; iii) Our findings lend themselves to speculate that the presence of extrajunctional Ca2+-channels, which are natural candidates for Ca2+-dependent modulation of depolarization-induced Ca2+-release, is a species-specific feature, independent of the class of Vertebrates to which animals belong and of the particular triadic pattern of the muscle.

Inward barium current and excitation-contraction coupling in frog twitch muscle fibres

The role of barium ions in excitation-contraction coupling was studied in single isolated frog semitendinosus fibres. Simultaneous recordings of membrane currents and contraction under voltage-clamp conditions in a sucrose-vaseline gap device show that barium ions have a reversible inhibiting effect on contraction. This inhibiting action was correlated to the entry of barium ions via the DHP-sensitive tubular calcium channel. Cytological observations and X-ray microanalysis performed on the fibres used in the electrophysiological experiments indicate that barium ions do not accumulate in the junctional sarcoplasmic reticulum; they can freely diffuse in the intermyofibrillar space and they accumulate in mitochondria. Calcium release experiments performed on isolated sarcoplasmic reticulum vesicles show that barium ions are not able to induce calcium release from calcium-loaded vesicles, they behave as calcium release inhibitors. These results are discussed in relation with the possible role of the slow Ca current in excitation-contraction coupling.

Depression of calcium transients after exposure to high K+ solution in Li(+)-loaded frog twitch muscle fibres and its reversal by exogenous myo-inositol.

Using Arsenazo III as a myoplasmic calcium indicator, we have studied the calcium transients evoked by voltage-clamp depolarizing pulses in frog twitch muscle fibres which had been temporarily depolarized by 80 mmol/L K+ in the absence or presence of myoplasmic Li+. After the high K+ exposure, for either a short (15 min) or long (1 h) time, the post-K+ calcium transients could gradually be restored to the level of the pre-K+ ones, if the fibres were not loaded with Li+. In contrast, the post-K+ calcium transients of Li(+)-loaded fibres could not fully recover, and were depressed in a Li+ concentration-dependent manner. The mean amplitude of the post-K+ responses recorded more than 3.5 h after 15 min high K+ exposure was reduced to 56% of pre-K+ control in the fibres which had been loaded with Li+ in 20 mmol/L Li+ Ringer's solution. This depression could be prevented or partially reversed by exogenous myo-inositol. More depression could be induced by 1 h high K+ exposure, but the presence of exogenous myo-inositol could not clearly prevent the post-K+ calcium transients from reduction. Assuming that high K+ exposure caused a depletion of myo-inositol and probably other changes in the metabolism of inositol phospholipids in Li(+)-loaded fibres, we conclude that some metabolites of phosphoinositides may play modulation roles in excitation-contraction coupling in frog twitch muscle fibres.

Design and control of the head retractor muscle in a turtle, Pseudemys (Trachemys) scripta: II. Efferent innervation.

The head retractor muscle (RCCQ) of Pseudemys scripta is a useful model in which to study the mechanisms animals use to vary the force and timing of movement. Single fibers in this muscle differ significantly in attachments, length, diameter, taper characteristics, and histochemical properties, suggesting that they may be energetically and architecturally specialized for different roles in head movement. In the present paper, we report the peripheral and central efferent innervation of these diverse muscle cells, and we ask how the design of the neural apparatus is matched to the properties of its target muscle fibers. Three out of four bellies in RCCQ are supplied by multiple segmental nerves. The territories of these nerves are separated rostrocaudally within the muscle belly; thus, long muscle fibers cross the territories of two or more segmental nerves. Motor terminals in RCCQ resemble those on frog twitch muscles. Their sizes (length, bouton number) are correlated with the diameters of their target muscle fibers. Each muscle fiber bears 2-14 terminals evenly spaced (approx. 5 mm apart) along its length. Thus, single muscle fibers in RCCQ are multiterminally, and long fibers are multisegmentally innervated. Control experiments indicate that the axons in each segmental nerve arise from different motor neuron populations. Thus, short, in-series fibers are supplied by different motor neurons, and individual long fibers in RCCQ are polyneuronally innervated. These data help explain how long muscle fibers with relatively slow conduction speeds can generate rapid head movements, and they raise questions about the central mechanisms that coordinate the recruitment of RCCQ motor neurons.

Reappraisal of the role of sodium ions in excitation-contraction coupling in frog twitch muscle.

Tetanic and twitch tension were recorded on isolated frog twitch fibres under experimental conditions modifying the influx of sodium ions. In current clamp conditions replacing Li+ for Na+ did not modify the electrical activity but drastically decreased the plateau of tetanic tension. In voltage clamp conditions replacing Li+ for Na+ did not modify the inward currents but induced a marked decrease of the plateau of the tetanic tension for depolarizations between the activation threshold and the reversal potential of sodium current. Under veratridine treatment, during tetanic depolarization, a slow inward sodium (or lithium) current developed. This induced a parallel increase of the tetanic tension which was much more pronounced in sodium than in lithium containing solution. The twitch tension obtained during short depolarization was increased by greater than 100% during veratridine treatment with a sizeable decrease (40%) of the delay between the end of depolarization and the beginning of tension. All these results could be reproduced in calcium-free solution. Our data confirm that the entry of sodium ions (and to a lesser extent of lithium ions) is able to modulate the release of calcium from the sarcoplasmic reticulum (SR). We discuss these results in terms of a model where sodium ions entering the compartment between the tubular membrane and the SR junctional membrane carry counter charges through the SR K+ channels and help to maintain the SR Ca2+ release. This could occur in particular during a physiological tetanic contraction where the junctional compartment is probably filled with Na+ ions and depleted of K+ ions.

Intracellular diffusion in the presence of mobile buffers. Application to proton movement in muscle

Junge and McLaughlin (1987) derived an expression for the apparent diffusion constant of protons in the presence of both mobile and immobile buffers. Their derivation applies only to cases in which the values of pH are considerably greater than the largest pK of the individual buffers, a condition that is not expected to hold in skeletal muscle or many other cell types. Here we show that, if the pH gradients are small, the same expression for the apparent diffusion constant of protons can be derived without such constraints on the values of the pK's. The derivation is general and can be used to estimate the apparent diffusion constant of any substance that diffuses in the presence of both mobile and immobile buffers. The apparent diffusion constant of protons is estimated to be 1-2 x 10(-6) cm2/s at 18 degrees C inside intact frog twitch muscle fibers. It may be smaller inside cut fibers, owing to a reduction in the concentration of mobile myoplasmic buffers, so that in this preparation a pH gradient, if established within a sarcomere following action potential stimulation, could last 10 ms or longer after stimulation ceased.

Dependence of contractile responses by some calcium antagonists on external calcium in the skeletal muscle.

Dependence of contractile potentiation by calcium antagonists on external calcium was investigated on the frog's twitch muscle. Low concentrations (10(-6) to 10(-5) M) of nicardipine and verapamil enhanced the peak tension of both twitch and electrically induced contracture in the presence of calcium. In the calcium-free media the drugs suppressed the contractile tension. Caffeine contracture was inhibited by the calcium antagonists at 20 degrees C. This inhibition was caused by an early onset of relaxation, which was not observed at 7 degrees C. The results suggest that some interaction between calcium ions and drug molecules at the voltage sensor on the transverse tubular membrane, which regulates E-C coupling but is not directly related to the functional calcium channel, may play an important role for the phenomena. The inhibitory action of calcium antagonists on the caffeine contracture in the presence of calcium is probably independent of the potentiating effect seen in the depolarization-induced contractions.

Effect of prolonged in vitro lithium treatment on calcium transients in frog twitch muscle fibres and its reversal by exogenous myo-inositol

Using arsenazo III as an intracellular indicator to monitor the calcium transients elicited by voltage-clamp depolarizing pulse, the effect of prolonged in vitro lithium treatment on excitation—contraction coupling in frog twitch muscle fibres was investigated. Incubation in 10 mM Li + Ringer's solution for 2 days caused a 46% increase in the amplitude of the calcium transients, while treatment with 30 mM Li + for 2 days produced a depression of 44%. Shortening the bathing time to 1 day, the decrease of calcium transients caused by 30 mM Li + was reversed to a small increase. For the 2-day incubation, both the increase in the amplitude with 10 mM and the decrease with 30 mM Li + were abolished by the presence of 1 mM myo-inositol in the bathing medium. These results imply that the turnover of inositol phospholipids is involved in regulating excitation—contraction coupling in skeletal muscle fibres.

Effect of barium ions on excitation-contraction coupling of frog twitch muscle fibres.

The mechanism for the Ba2(+)-induced potentiation of the twitch tension in frog skeletal muscle was investigated. No significant difference in the peak amplitude of the calcium transients evoked by voltage-clamp depolarizing pulse was found between the fibres bathed in Ca2+ (control) and Ba2+ Ringer's solution, whereas the calcium transients evoked by the action potentials from the fibres in Ba2+ Ringer's solution were increased by about 64%, compared with control. In comparison with control, the action potentials of the fibres in Ba2+ Ringer's solution had a similar overshoot but a significantly longer time course. Our results suggest that excitation-contraction coupling in frog twitch muscle fibres is not altered by replacing Ca2+ with Ba2+. The Ba2(+)-induced potentiation of contraction may be accounted for by broadening of the action potentials.

Changes in Na channel properties of frog and rat skeletal muscles induced by the AaH II toxin from the scorpion Androctonus australis

The effects of the mammal toxin II isolated from the venom of the scorpion Androctonus australis Hector (AaH II) were studied under current and voltage clamp conditions in frog (semitendinosus) and rat (fast e.d.l. and slow soleus) skeletal twitch muscle fibres. In both species, AaH II induced a dose-dependent prolongation of the action potential (AP) leading at saturating concentration to APs with long plateaus of about 1.5 s in frog and 5 s in rat e.d.l. and soleus fibres. The concentrations to induce 50% of the maximal effect (K0.5) were 9.1 x 10(-9) M in the frog and 1.4 x 10(-9) M in the rat. AaH II increased the time constants of inactivation of the peak Na current and induced a maintained Na current that was greater in rat e.d.l. and soleus (31.6% of peak current amplitude at -30 mV; K0.5 = 0.8 x 10(-9) M) than in frog (16.5%; K0.5 = 15.5 x 10(-9) M) muscles. Peak and maintained Na currents were TTX-sensitive and had identical threshold and reversal potentials. The half-maximum maintained permeability occurred at a potential 20 mV more positive than the peak permeability. Recovery from inactivation and steady-state inactivation of the inactivating Na current remained unchanged. The maintained current deactivated with normal fast kinetics. The action of the toxin reversed poorly on washout but could be largely removed by conditioning depolarizations more positive than the reversal potential of the Na current. Our results suggest that, in vertebrate skeletal muscle fibres, AaH II affects all the Na channels and are consistent with the hypothesis that the maintained current originates from a reopening of previously inactivated Na channels.

Effects of hypertonic solutions on calcium transients in frog twitch muscle fibres.

1. The effects of hypertonic solutions on excitation-contraction (e.-c.) coupling in frog skeletal muscle fibres were investigated using Arsenazo III to monitor intracellular calcium transients in voltage-clamped fibres. 2. In solutions made hypertonic with sucrose or sodium chloride, the size of the Arsenazo signal evoked by a 5 ms depolarization to 0 mV was little altered by increases in tonicity up to about twice normal, but declined in higher tonicities, and was almost completely suppressed at 4 times normal tonicity. 3. The latency to onset of the Arsenazo signal was increased in hypertonic solutions (2.3 and 3.1 times normal tonicity), but the decay time constant of the signal was little changed with tonicities up to 2.3 times normal. 4. The rheobase potential for a just-detectable Arsenazo signal was shifted about 4 mV more negative by increases in tonicity up to 2.3 times normal, but further increases reversed the direction of the shift, and in 3.95 times normal tonicity the rheobase was 10 mV more positive than in normal Ringer solution. 5. With short (less than 10 ms) pulse durations the depolarization needed to elicit a threshold Arsenazo signal increased steeply with increasing tonicity. Changes in the strength-duration curve could be accounted for by an increase in the time constant for build-up of a hypothetical coupler in the e.-c. coupling process. 6. Solutions of about twice normal tonicity are commonly used to suppress muscle contraction. Since the size of the Arsenazo signal was only slightly reduced by this tonicity, the main effect is presumably on the contractile proteins.

Histochemical study of calcium on T-tubule membranes and in the sarcoplasmic reticulum, in frog twitch muscle fibres at rest and during activity.

The trigger calcium hypothesis of signal transmission between T-tubules and terminal cisternae (TC) of the sarcoplasmic reticulum (SR) in twitch muscle fibres implies the presence of calcium along T-tubule membranes at rest and its release upon excitation. To test this hypothesis, calcium was immobilised using a fixing and precipitating solution of glutaraldehyde in phosphate buffer at pH 8.0 and the calcium was substituted for by lead. Simultaneous tension recordings revealed the occurrence of contractions or a burst of twitches upon perfusion with the fixative. Procaine or tetrodotoxin (TTX) was used to inhibit this activity. In fibres without fixative-induced activity, precipitates were observed along T-tubules and in adjoining parts of TC. In activated fibres, tubular and TC precipitates were absent. These results are consistent with the trigger calcium hypothesis. In fibres activated by depolarisation, calcium returned to TC after passing successively through different parts of the SR.

Minimal latency of calcium release in frog twitch muscle fibres.

Intracellular release of calcium in frog skeletal muscle fibres was monitored by the use of arsenazo III, in response to voltage clamped depolarizing pulses. A latency of a few milliseconds was evident between the onset of depolarization and the first detectable rise in the arsenazo-calcium signal, and this decreased logarithmically as the depolarization was increased. The minimal latency with strong depolarization (to +20 to +100 mV) was about 2 ms at 5 degrees C. This delay appears to be sufficiently long to be compatible with a chemically mediated coupling mechanism between depolarization and calcium release from the sarcoplasmic reticulum.

Differences in synaptic efficacy at neuromuscular junctions in frog twitch muscles.

A comparison was made of neuromuscular junctions in cutaneous pectoris and cutaneous dorsi muscles of Rana pipiens in order to study mechanisms controlling synaptic efficacy. Other than a small difference in junctional size, the two muscles were structurally and functionally very similar. Despite these similarities, cutaneous pectoris junctions had substantially higher synaptic safety margins. With intracellular recording, it was apparent that the difference in safety margin was due to a large difference in transmitter release. In low-Ca2+ solutions, levels of evoked and spontaneous release were 4 times higher in the cutaneous pectoris. When corrected for differences in nerve terminal size at identified junctions, there remained a 3-fold difference in evoked release and a 6-fold difference in spontaneous release per unit terminal length. Differences in normal Ringer solution were 1.8- and 2.5-fold for evoked and spontaneous release, respectively. There was no simple relationship between synaptic efficacy and the total amount of nerve terminal supported by each motoneurone in different frog muscles. We concluded that there can be large differences in synaptic efficacy without correlated structural differences visible with the light microscope.

New optical spike in frog twitch muscle fibers injected with a potential probe dye NK2935.

Absorbance responses posterior to action potentials were recorded from frog twitch fibers loaded by an impermeant potential probe dye NK2935 that was chemically identical with WW781. Extracellularly loaded NK2935-related responses (EOS) increased in absorbance around 640nm wavelengths and were like action potential in waveform but were slightly preceded by action potential. Model analysis showed that EOS consisted of two components, namely 39% non-delay component reflected with sarcolemma action potential and 61% first-order delay component reflected with transverse tubule (T) action potential (delay-constant tau 1 = 0.48 ms). The T-disrupted fibers showed only the non-delayed response. Intracellularly injected NK2935-related responses (IOS) were also composed of a single spike of the same direction swing as in the EOS case described by the term "ipsidirection," but were clearly preceded by EOS and followed by Ca transient, birefringence, transparency and latency relaxation responses. The T-disrupted fibers showed a small "sidedness" response characterized by both opposite direction swing, namely "contradirection" and non-delay against EOS. Model analysis showed that IOS consisted of two main components, namely second-order delay component with ipsidirection (tau 2 = 0.8 ms) and the further high-order delay component with contradirection. Based on spatio-temporal heterogeneity of NK2935 binding inside fibers and potential-absorbance relation in sarcolemma, the former origin was thought to be in junctional sarcoplasmic reticulum (jSR) responding to T excitation with a transient "positive polarization" estimated roughly 20 mV. Similarly, the latter was possibly in free SR where a slow "negative polarization" estimated -0.9 mV might appear associated with Ca release.