Ultrastructural aspects of the capsular cells in frog muscle spindle.
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Biomedical subjects
Publications and source records attributed to N Fujitsuka.
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The static component of the spindle potential provoked during stretch of isolated muscle spindles of the frog was reversed during the application of depolarizing currents ranging from 0.2 to 5 nA in normal Ringer solution and also in Na+-free Ringer solution. In the same range of current intensities, spontaneous rhythmic hyperpolarizations due to [Ca2+]i-activated GK, an attenuation of membrane impedance, and an anomalous decrease in amplitude of the afferent spikes were observed. All 4 phenomena were abolished by K+ channel blockers (10 mM CsCl, 1-2 mM 4-aminopyridine (4-AP), or 20 mM tetraethylammonium chloride (TEA], Ca2+ channel blockers (5-10 mM CoCl2, MnCl2, 1-2 mM CdCl2 or 0.5 mM verapamil) or 0.1 mM quinine. The amplitude of the static component of the spindle potential was markedly increased at threshold concentration of the K+ channel blockers (5 mM CsCl, 0.1-0.5 mM 4-AP or 5-10 mM TEA), but the component disappeared at that of the Ca2+ channel blockers. The rhythmic hyperpolarizations are associated with the spindle potential, except for its dynamic component, which often triggers a hyperpolarizing deflection. We suggest that both the static component of the spindle potential and rhythmic hyperpolarizations are due to GK(Ca) in the intracapsular axon, either along the terminal or at the branching nodes, or both; and that the receptor potential contributes to, but is not the same as, the spindle potential.
The sensory ending of the frog muscle spindle consists of bulbous swellings interconnected by thin, tube-like axonal branches. This study was made to determine if the bulb or thin tube regions are deformed to the same degree during dynamic stretch, by comparing spindles prepared in the relaxed and stretched states. Isolated muscle spindles were rapidly frozen, either in a completely relaxed state or at the end of dynamic stretch to 130% of the in situ length. Longitudinal sections for ultra-high-voltage electron microscopy revealed that dynamic stretch caused a decrease of 61.7% in bulb diameter, an increase of 88% in bulb length and an increase of 34.6% in bulb volume. The cross-sectional areas of the nonmyelinated terminals in the reticular and compact zones measured in electron micrographs decreased by 79.2% in the bulb region and 66.7% in the tube region during dynamic stretch.
The slope of ventilatory response to hypercapnia at rest was determined in 77 healthy male students by means of the CO2 rebreathing method. It was found that the hypercapnic ventilatory response slope (S) was significantly lower in the lean group with BMI (body mass index) below 19 than that in the normal group, while there were no significant correlation between S and body weight or height. These results indicate that sensitivity of hypercapnia in the lean subjects differed from that of normal and overweight subjects.
The application of 1.5-4 microM dantrolene decreased the threshold and the current sensitivity of the rhythmic hyperpolarizations that occur during depolarization of the sensory nerve terminal in the frog muscle spindle. The higher concentration provoked spontaneous rhythmic changes even without depolarization. Methylxanthines (5 mM caffeine, theophylline or pentylene-tetrazole) increased the threshold and the sensitivity. Electron microscopic observations of the dantrolene-treated spindles revealed numerous electron-dense deposits associated with the cytoplasmic membrane of the sensory terminals and with mitochondrial membranes. The deposits were found to contain K+ and Ca2+ by energy dispersive X-ray microanalysis. Electron-dense deposits containing Ca2+ were usually observed in the inner capsular space and in the mitochondria of the sensory terminals perfused by normal or high Ca2+ Ringer solutions. They were reduced in number following incubation with methylxanthines. The amplitudes of afferent spikes and the spindle potential were increased by methylxanthines in much the same way as by K+ channel blockers, suggesting that GK of the terminal membrane may be reduced by methylxanthines. We suggest that methylxanthines may modulate the terminal responses both as a K+ channel blocker and by enhancing the release of Ca2+ from a storage site, perhaps in the inner capsular space, whereas dantrolene has the opposite effect.
Spindle potential recorded from the sensory nerve terminal of isolated frog muscle spindles disappeared within 20-30 min after the spindle receptor was perfused with Na+-free (Li, Tris or choline) Ringer's solution, whereas the amplitude of spindle potential was not attenuated for periods up to 60 min when the spindles were perfused in a Na+-free Ringer's solution containing both 10 mM TEA and 0.1 mM 4-aminopyridine after being washed with a normal Ringer's solution containing both the K+-channel blockers. It is concluded that the time-dependent decrease in the amplitude of spindle potential during the application of Na+-free solution is not ascribable to a decrease in the inward current carried by Na+, but is due to an increase in an outward current carried by K+.
The presence of a mixed Na+-Ca2+ spike along the sensory terminal of the frog muscle spindle was verified. When the terminal was perfused with Ringer's solution containing 0.1-0.5 mM ruthenium red (RuR), the amplitude and duration of the spike were increased, occurring as a prolonged or a long-lasting depolarization of up to 20-30 s duration following individual afferent spikes evoked spontaneously or antidromically by electrical stimulation. In an isotonic TEA solution, the amplitude and duration of the afferent spikes were increased; however, no prolonged depolarization occurred. Adding 0.2 mM RuR to the TEA solution produced the prolonged and long-lasting depolarization. All responses disappeared in the presence of 3 microM TTX or Na+-free Ringer's solution. An impedance decrease along the terminal was observed during the prolonged or long-lasting depolarization. The prolonged depolarization was blocked by the addition of Ca2+-blockers; the afferent spikes remained. In preparations preincubated with 0.1 mM RuR, increasing CaCl2 in Ringer's solution from 0.2 mM, resulted in shortening of the duration of individual spikes with prolonged depolarization and in increase in the maximum rate of rise (MRR) of the spikes. Preincubation with higher concentrations of RuR produced higher sensitivities in the modifications of the duration and MRR to the change in [Ca2+]O. The responses were retained by adding RuR or RuCl3 to Ca2+-free Ringer's solution containing 0.1-5 mM EGTA, although all responses disappeared in Ca2+-free EGTA Ringer's solution. It is concluded that the RuR-induced prolonged response is produced by an influx of Na+.
The threshold at the terminal node in the capsule of frog muscle spindle, where afferent impulses are initiated, was calculated to be 2 nA on average, from data of intracellularly recorded threshold depolarization of the node against antidromic stimulation and of a mean attenuation ratio of the stimulation current from the stimulation site to the recording site. Using a similar procedure, the absolute value of orthodromically generating current at the node during static stretch of the spindle from the in situ length was calculated to be approximately 0.9 nA. It thus is supposed that at the terminal node the afferent impulses may be triggered by abortive spikes of 1.1 nA or more in amplitude, which are generated along non-myelinated filaments, being superimposed on the generating current.
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Spontaneous cyclic hyperpolarizations along the sensory nerve terminal of frog muscle spindles were observed during the application of 1-9 nA depolarizing currents across an air-gap on which the axon was bridged. An increase in the current intensity increased the amplitude and duration of the cyclic changes. Upon subthreshold depolarization, single or repetitive hyperpolarizations could be elicited after a brief electric pulse or during stretch of the receptors, respectively. The threshold was decreased in higher Ca2+, Sr2+ or Ba2+ solutions. The cyclic changes were reversibly blocked by K+- or Ca2+-blockers and quinine. These results suggest that the changes are due to GK(Ca). The site of origin of the changes was at the branching node in the capsule, as confirmed by the following results: (1) the cyclic changes were abolished upon inactivating the node by UV-irradiation; (2) in normal Ringer's solution, the rate of afferent impulses, which reflects the membrane potential at the encoding site along the non-myelinated filaments, was unmodified by the cyclic changes and was independent of the intensity of the polarizing currents within a certain range; however, it was sensitively dependent on this intensity after treatment with K+-blockers; (3) the amplitude of the impulses reaching the branching node was attenuated during the cyclic changes, but not after GK-blockade.
An increase in the Ca2+ component following individual sodium spike in spontaneous discharges was observed in the isolated sensory terminal of the frog muscle spindle perfused with isotonic sodium solution with a group of anions, of which the size in the aqueous solution ranged from 0.74 to 1.32 times that of hydrated sodium ion. The effects of these anions was counteracted with divalent cations. It is hypothesized that anions similar in size to hydrated sodium ions may form an anion-cation complex at the interaction site of the Na+ carrier, whereby the Na+ may enter the Ca2+ channel. This may be inactivated by the divalent cations.
Blood lactate was determined in 19 untrained subjects after maximal treadmill exercise lasting for about 1 min. It was found that blood lactate increases after exercise, reaching a maximum level 6-9 min after the cessation of exercise, and the average time for the appearance of the peak blood lactate concentration was 7.65 min. Peak blood lactate concentration at 7.65 min (CLA7.65), which was calculated by substituting t (7.65) into the equation for the lactate recovery curve for each subject, agreed well with the observed peak blood lactate concentration (r = 0.98, p less than 0.001). In addition, correlations of r = -0.65, r = -0.78, r = -0.79 were found between CLA7.65 and the running times of 100 m, 200 m, and 400 m sprints, respectively. These results suggest that CLA7.65 may be used as a valid indicator of anaerobic work capacity in man.
A calcium spike in the sensory nerve terminal of the frog muscle spindle could be elicited by electrical pulses, which were given across an air-gap on which the first myelinated segment of the parent axon outside the spindle capsule was bridged, after the sodium spike had been blocked by treatment with tetrodotoxin (TTX). The threshold current was 1.1-2.4 nA higher than that for the sodium spike, suggesting that the calcium channels distribute distally to the site of afferent impulse initiation, or that the density of calcium channels on the encoding site may be less than that along portions distal to the site.
Ventilatory response to CO2 in the Ama (Kachido) was determined by the CO2 rebreathing method in the beginning (March) and during (September) harvest season. It was found that mean slopes of the ventilatory response curve in March and September were 0.76 and 0.73 liters/(min.m2.mmHg), respectively, this difference being insignificant.
Ventilatory response lines to carbon dioxide at rest were determined by the rebreathing method in 10 untrained subjects, 17 sprint swimmers, and 11 long-distance swimmers. It was found that the mean slope of the ventilatory response line of the swimmer was lower than that of untrained group, and the mean slope of the long distance swimmer was lower as compared with the sprint swimmer, though these differences were statistically not significant. The differences in the hypercapnic drive between untrained subjects and swimmers obtained here is discussed in connection with their maximum oxygen uptake.
Lactic acid concentration of venous blood was determined in healthy male subjects after strenuous exercise with and without breath-holding. It was found that in all subjects the peak and total values of lactate during recovery was higher in the breathing run than in the breath-holding run, though the running time was the same.
The ventilatory response to carbon dioxide at rest was determined repeatedly by the rebreathing method; three successive CO2 response lines, with 30-min intervals, were examined in 12 healthy male subjects. Overall mean slopes obtained at 30, 60 and 90 min were 2.95, 2.47 and 2.28 liters/min . mmHg, respectively, the difference being statistically not significant. However, the slopes of four high responders, who were subjects with higher slopes than that of the mean values obtained here, decreased significantly (p less than 0.05) at 60 min as compared with the 30 min test, but this did not occur in the other subjects. Better reproducibility in obtaining the CO2 response slopes in the high responders was observed in the first rather than the 60 or 90 min trails, suggesting incomplete recovery in the humoral agents released during the CO2 test. These results indicated that the CO2 response curve by rebreathing should be determined carefully, especially in the high responder, taking into consideration its individual variability and and interval in consecutive measurements.