J. Phy8iol. (1969), 205, pp.

499-513 499
With 2 plate8 and 6 text-figure8
Printed in Great Britain

THE INFLUENCE
OF STIMULUS PARAMETERS ON CONTRACTIONS OF ISOLATED
FROG MUSCLE FIBRES
BY R. RtDEL* AND S. R. TAYLORt
From the Department of Physiology, University College London,
Gower Street, W.C..1
(Received 16 July 1969)
SUMMARY
1. Reversible changes in twitch and tetanus contractions of isolated
frog muscle fibres were produced by varying the duration and strength of a
transversely applied d.c. stimulus.
2. When the stimulus strength was at least 1 1 times threshold the peak
force of the contraction elicited was reduced by stimulus durations longer
than 1 msec at 20 0C, and by durations longer than 3 msec at 2-5 0C.
3. As the strength of a prolonged stimulus was progressively increased to
3 times rheobase or more, peak twitch and tetanus force declined to
40-70 % of the amplitude with short (0*2-0-5 msec) stimuli. The time to
peak of the twitch was slightly decreased and the first phase of relaxation
was accelerated.
4. Cine-micrography of fibres which were permitted to shorten under a
light load revealed that the side of the fibre adjacent to the anode was
unactivated under conditions which also reduced peak force output.
5. With stimulus durations longer than 20 msec, peak force, time to
peak, and relaxation time increased as the stimulus strength increased
from 2-5 to 3 times rheobase. This might have been produced by a catho-
dal contracture following the action potential.
6. The results are discussed in relation to previous studies of activation
in striated muscle.
INTRODUCTION
In the initial stages of investigating the activation of single muscle fibres
during a twitch, we attempted to determine, as a matter of course, the
optimal strength and duration for our transverse stimulating current. The
results were, in general, like those previously reported for whole muscle
* Supported by the Deutsche Forschungsgemeinschaft.
t Postdoctoral Rehabilitation Research Fellow of the U.S. Department of Health,
Education and Welfare.
500 R. RUDEL AND S. R. TAYLOR
(Close & Hoh, 1968) in showing a reduction of twitch force with certain
suprathreshold combinations of strength and duration. But direct obser-
vations of contracting single fibres have given us additional information
to use in interpreting their behaviour. These findings have enabled us to
reassess the evidence that Kelly, Fry & Fry (1964) used to question the
definition of a twitch response.
A preliminary account of this work has already been reported (Riidel
& Taylor, 1969).
METHODS
Dissection
Twitch fibres were isolated from the dorsal head of the semitendinosus muscle of
the frog Rana temporaria. The animals were kept at 2-4° C for at least 2 weeks before
use. To start the dissection a large bundle of fibres from the side where the nerve
enters the muscle was removed with a pair of fine scissors, and subsequently a single
fibre was separated from one of the small remaining bundles using knives made from
pieces of stainless-steel razor blade.
The dissection was carried out at room temperature while the muscle was immersed
in Ringer solution that contained tubocurarine (10-5 g/ml.). The fibre was usually
stored overnight at 2-4° C in Ringer solution of the following composition (mM):
NaCl 115; KCI 2*5; CaCl2 1-8; Na,HPO4 2-15; NaH2PO4 0-85 (pH 7.2).
Mounting
The fibre was transferred from the dissection dish into a glass experimental trough
by means of a Perspex scoop. This trough contained curare-free Ringer solution
whose temperature could be controlled by running either room temperature tap
water or an antifreeze-ice mixture through an attached heat exchanger. The fibre
was mounted horizontally at slack length (i.e. the length assumed by the unrestrained
fibre). One tendon of the fibre was hooked to an extension attached to the anode of an
RCA 5734 mechano-electronic force transducer. The other tendon was hooked to a
lever attached to the jewelled bearing of a watch movement. Tension was applied to
the lever via a thread tied to the end of a watch mainspring (Huxley & Peachey,
1961). The compliance of the lever-spring arrangement was about 0 5 mm/mg. A
movable stop could be positioned in front of the lever to convert contractions with
shortening to isometric contractions. Both tendon attachments were mounted on
screw-driven slides with vernier scales so that the position of each end of the fibre
was independently adjustable.
Microscopy
The screw-driven slides and the experimental trough were mounted on the stage
of an ordinary light microscope. The fibre was illuminated by a low-voltage tungsten
lamp, with a green filter to improve contrast. The numerical aperture (n.a.) of the
illuminating cone was set to 0-3, which made a reasonable compromise between the
desires for high contrast and a narrow depth of field. The fibre was observed through
a Zeiss D* water immersion objective (focal length 4-4 mmn, n.a. 0.75) in combination
with two different eyepieces. A x 25 eyepiece with a calibrated graticule was used
while determining the cross-sectional area of the fibre by measuring both its width
and its vertical thickness (Blinks, 1965, p. 48) and while setting the striation spacing
to the desired value. The average sarcomere length was determined by counting
striations in several different 50 #t lengths near the middle of the fibre. A x 3 eyepiece
 STIMULUS PARAMETERS AND CONTRACTION 501
was used while taking cine photographs (64 frames/see, on Ilford Pan F or Kodak
High Contrast 16 mm negative film) of the central optical section between 0-5 and
1-0 mm from the end of the fibre attached to the force transducer. This was within
the region where the striation spacing does not substantially differ from that in the
middle part of the fibre (Huxley & Peachey, 1961). At this point the fibre rested
lightly on a piece of 2 mm diameter glass rod laid across the bottom of the trough.
For analysis, positive enlargements with an over-all magnification of x 640 were
made from the cine film.
Stimulation
The fibre was stimulated transversely by a pair of bright platinum plate electrodes
(20 mm long, 4 mm high, and 10 mm apart) across which we could apply up to 20 V
with a coinstant voltage generator. The electric field in the area where the fibre was
situated was found to be homogeneous. For a square pulse applied to the electrodes,
the voltage across the bath dropped by about 1-5 V during the first 1 msec owing to
electrode polarization, and then stayed constant. The field strengths given are the
values applied to the electrodes.
At the beginning of an experiment we determined rheobase and recorded several
twitches and tetani with short (0.2-0.5 msec) stimuli until the responses were repro.
ducible. Throughout this investigation twitches were elicited at least 1 min apart
and not before 10 min after a tetanus.

RESULTS
Isometric contractions
The strength-duration relationship for threshold stimulation of our iso-
lated fibres was in general like that found by Ramsey & Street (1941). In
our experiments the rheobasic current density for transverse stimula-
tion ranged from 20 to 50 mA/cm2 depending on the fibre diameter and the
chronaxie was 0-5 msec at 50 C and 0-2 msec at 200 C.
Dependence of the twitch on stimulus duration. The reduction of the peak
force of an isometric twitch on prolongation of a stimulus of 2-2 times
rheobase is illustrated in Text-fig. 1. Here, a stimulus of 2 msec duration
at 20° C decreased the peak force to 65 % of the value elicited by a 0-5
msec pulse (Text-fig. 1 A, B). Further prolongation of the stimulus to
10 msec slightly reinforced this effect as shown in Text-fig. 1 C. The de-
crease was completely reversible, for a 0-5 msec pulse given at the end of
this series yielded the same response as in the beginning (not illustrated).
At low temperature the stimulus had to be longer than at room tempera-
ture in order to produce this decrease in peak force. The records in the
lower row of Text-fig. 1 were obtained with stimuli of the same strength
as before, after the fibre had been cooled down to 50 C, and the general
effects of cooling on the twitch can be seen, viz. an increase in amplitude
and duration of the force (Hill, 1951). At this temperature, prolongation
of the stimulus from 0- 5 to 2 msec did not affect the twitch (Text-fig. 1 D,
E). However, further prolongation reduced the amplitude of the force to
65 % at 5 msec, and even more at 10 msec (Text-fig. 1 F). Also in this case,
502 R. RUDEL AND S. R. TAYLOR
a control pulse of 2 msec at the end of the series established the reversibility
of the effect.
The reduction of force was accompanied by changes in the time course
of the contraction which are more easily seen at the lower temperature.
The time from the beginning of the stimulus to the peak of the contraction
was slightly decreased. Also, the shoulder which quite often appears on
the falling phase of the twitch (Text-fig. 1 D, E) disappeared when the
contraction was induced by a prolonged stimulus (Text-fig. 1F). That is
200 C
05 2 10
A B C
100 msec
3 kg/cm2 1
200 msec

05 2 10
D E F
Text-fig. 1. The change in the isometric twitch of an isolated fibre (29. i. 69)
in response to single stimuli of increasing duration. Oscilloscope records: Top
row at 200 C, time calibration 100 msec; bottom row at 50 C, time calibra-
tion 200 msec. The duration in msec of each stimulus is indicated by the
number ascribed to the stimulus signal. Resting striation spacing was set
to 1-96 ,u at 200 C, and 2-18 ,u at 50 C. The stimulus strength was 2-2 times
rheobase, throughout. At each temperature the responses were elicited
approximately 1 min apart.

to say, the earliest phase of relaxation was speeded up but the total twitch
duration was usually unchanged. This is graphically illustrated in Text-fig.
2 which was compiled from the results of a similar experiment carried out
with another fibre at 20 C. The stimulus strength was 341 times rheobase
throughout, the stimulus duration was the variable plotted on the abscissa
(logarithmic scale). The graphs show that for a stimulus longer than 2 msec
up to a duration of 20 msec the peak amplitude of force dropped, accom-
panied by a slight decrease in the time to the peak of the contraction as
well as a decrease in the relaxation time from the peak to 75 % of the force
In like manner, when a curarized whole muscle is stimulated with transverse
pulses, prolonging the stimulus from 0-2 to 10 msec decreases the twitch amplitude
(Kelly et al. 1964; Sandow & Isaacson, 1966; Close & Hoh, 1968). But when curare
is absent the force of the contraction elicited is greatly increased as the stimulus
duration is prolonged (Kelly et al. 1964). We have confirmed this latter observation
using whole extensor longus digiti IV muscles from our frogs, but found that the
same was not true with isolated semitendinosus fibres. It should be noted that most
 STIMULUS PARAMETERS AND CONTRACTION 503
of our experiments were carried out in curare-free solution after a fibre had been
isolated in the presence of curare, but we also investigated several fibres which had
been isolated without ever being curarized as well as some which were curarized
during the experiment. In all cases the results were unvarying and unequivocal,
viz. prolonging the stimulus decreased the peak amplitude of the contraction.
4 X 1 l l l l

~
b4 ~ ~ ~ ~ ~ ~ ~ ~ ~ 61
0~~~~~~~~~~~~~~~~~~~~~~~~~~~

C 1

100 msec

° L2t0 Oxv I
N
V

0.-_wSI
/cm
I 150
0
_ ~ ~ ~ ~~
' ,7 /v AA2
/^
i E
100X
_ A_

0.1 0-2 0.5 1 2 5 10 20 S0 100

Stimulus duration (msec)
Text-fig. 2. A summary of the changes in isometric contractions of an
isolated fibre (19. ii. 69) in response to single stimuli of increasing dura-
tion. Open circles: variation of peak tension; the ascribed numbers indicate
the order in which the responses were elicited. Filled triangles: time to peak
of these contractions. Open triangles: relaxation time from 100 to 75 % of
peak. The superimposed oscilloscope records in the inset are responses no.
11, 12 and 14. The temperature was 20 C, the stimulus strength 3-1 times
rheobase, and the resting striation spacing 2-08 ,.
amplitude. The contractions elicited by stimuli longer than 20 msec will
be dealt with in a later section.
Dependence of the twitch on stimulu8 strength. A threshold stimulus what-
ever its duration within the range tested (0-1-100 msec) elicited a twitch
with the same peak force. When the stimulus strength was at least 1-1
times threshold, the dependence of the twitch on the stimulus strength was
different for short and long stimuli. For stimuli less than 2 msec at 200 C
and less than 4 msec at 2 to 50 C the twitch height was not dependent on
the stimulus strength. This is illustrated in Text-fig. 3 for twitches elicited
504 R. RttDEL AND S. R. TA YLOR
by stimuli of 1 msec duration at 30 C. No change in amplitude was recorded
when the stimulus was increased from threshold to about twice threshold.
For longer stimuli there was a dependence of peak force on the stimulus
strength as shown in Text-fig. 3 for 10 msec stimuli. Here, threshold was
lower, and with threshold stimulation the force amplitude was as large as
for the preceding 1 msec stimulus. With increasing field strength, how-
ever, the twitch amplitude decreased, resulting in only 40 % of the maxi-
mum value at 2-5 times threshold.
12

01
03 1 msec 05

E~~~~ 9 6N 1684l
0.
C4

1l l
in 7

° _10 1 2 1 3

5 7 9 11 13 15
Stimulus strength (V/cm)
Text-fig. 3. The dependence of the twitch tension of an isolated fibre
(26. ii. 69) on stimulus strength. Open circles: responses to 1 msec stimuli;
filled circles: responses to 10 msec stimuli. The ascribed numbers indicate
the order in which the responses were elicited. The temperatuire was 30 C,
and the resting striation spacing 2-18 /s.
Dependence of the tetanus on stimulus strength and duration. A similar
reduction of the maximum force of a tetanic contraction was observed
when the duration of the single pulses in the stimulus train was prolonged.
This reduction occurred only if the prolonged stimuli were above threshold,
and the previous force amplitude was restored on returning to short stim-
uli. An example of the effect is shown in Text-fig. 4, where an isolated
fibre, kept at 5° C, was stimulated for 1 sec at 20 Hz with pulses of 3-6
times rheobase strength and of either (A) 0-5 msec or (B) 10 msec duration.
The almost fused contraction (A) reached a plateau value of 245 mg
with the train of short pulses while with long pulses (B) maximum force
was only 190 mg, dropping slightly during the course of stimulation.
 STIMULUS PARAMETERS AND CONTRACTION 505
Contractions with shortening
The mechanism that was involved in the reduction of force on stimulus
prolongation was made evident when the fibre was allowed to shorten under
the same stimulating conditions in which the isometric contraction was
attenuated. During such shortening under a very light load the fibre
developed a wavy pattern all along the side adjacent to the anode. Plates
1 and 2 show selected frames from a cine film taken of the same fibre
from which the records of Text-fig. 1 were obtained.
so C
20 Hz

1 kg/cm2

sec

0-5 msec 10 msec

per stimulus per stimulus
Text-fig. 4. The change produced in the isometric tetanus of an isolated
fibre (29. iv. 69) by increasing the duration of each stimulus in a train, at
20 Hz frequency (50 C), lasting for 1 sec. The stimuli are shown on the
lower oscilloscope trace and were 3*6 times rheobase. (A) stimulus duration
0 5 msec; (B) stimulus duration 10 msec. Response A was elicited 1 min
after B. Resting striation spacing was 2-00 ,u.

Twitch. In P1. 1 A the fibre was at rest with a striation spacing set to
2-06 ,t, bathing temperature was about 200 C. The moment of peak short-
ening after a 0-5 msec stimulus is shown in P1. 1 B. The pattern of shorten-
ing was apparently uniform throughout the fibre. If, however, a 10 msec
stimulus was applied, the shortening was homogeneous only during the
first quarter of the twitch. Then (P1. 1 C), a sequence of broad dark and
light transverse bands began to develop mainly along the half-cylinder of
the fibre exposed to the anode. With continued shortening this pattern
became more distinguished, as is clearly seen in P1. 1 D which was taken
at the peak of shortening. Lowering the temperature to 5° C slowed the
time course of the shortening and then these broad bands were evident
from about 35 msec until 160 to 190 msec after the start of a 10 msec
stimulus. During this period the apparent sarcomere length was shorter
than 19 /t.
Tetanus. This pattern of obviously asymmetrical shortening was much
more pronounced when the fibres were stimulated with trains of prolonged
506 R. RtUDEL AND S. R. TA YLOR
stimuli. At the plateau of a tetanus striation spacings of less than 1-6 #
were reached. During such shortening the broad dark and light bands
became more distinct and the distance between them shrank. The sarco-
lemma on the side of the fibre exposed to the anode became scalloped and
it was evident that the myofibrils lying in the part of the fibre showing the
bands were not straight but folded in waves of 40-45 ,u length and up to
10 ,ct amplitude. Plate 2 shows two pictures of the same fibre as in P1. 1
during the plateau of contractions induced by 10 msec pulses of 50 Hz
frequency at a temperature of 20° C. The only difference in the experi-
mental conditions between the contractions in P1. 2A and B was the
reversal of polarity of the stimulating electrodes. The wavy pattern in
each case extended through more than the half-cylinder of the fibre lying
next to the respective anode.
It should be noted that a similar pattern of wavy fibrils can be observed
on stimulation of a fibre which has been made inexcitable either by remov-
ing sodium ions from the bathing medium or by local injury. Further-
more, we sometimes observed wavy fibrils even without any stimulation in
slightly damaged fibres. We were, therefore, careful to establish that our
results were not influenced by damage possibly caused by these long,
intense pulse trains. For example, the fibre depicted in P1. 1 and 2 was
able to produce the unusually large tetanic tension of about 5 kg./cm2
at the end of the series described.
Extreme prolongation of the stimulu8
When the stimulus duration was greatly prolonged, i.e. beyond about
20 msec, in the warm or in the cold the contraction amplitude was no
longer decreased by stimulus strengths of 2 to 3 times rheobase, but rose
again towards or exceeded the value obtained with a short stimulus. For
example, in the experiment illustrated in Text-fig. 2 peak force increased
when the stimulus duration was prolonged to about 30 msec. The con-
traction amplitude in this case happened to almost equal that of a fused
tetanus elicited by a train of chronaxie stimuli. For longer pulses, up to
more than 100 msec, peak force was approximately the same as for a
chronaxie stimulus. In other experiments the peak force increased more
gradually between 20 and 30 msec, and, furthermore, could be increased
or decreased in a graded manner by increasing or decreasing the stimulus
intensity.
A detailed investigation of these effects was complicated by the occa-
sional appearance of obviously supernumerary responses. Text-figure 5,
for example, displays the results of an experiment at 50 C in which we
observed the effects of varying the stimulus strength on contractions
elicited by a 50 msec pulse. Contraction 1 was at 1 1 times rheobase and
 STIMULUS PARAMETERS AND CONTRACTION 507
yielded about 20 % less force than a previous response to a chronaxie
stimulus. On increasing the stimulus strength the twitch amplitude de-
creased further (responses 2 and 3). In contraction 3, however, 80 msec
after the cessation of the stimulus, the force rose again to an amplitude al-
most twice that of response 1. A subsequent stimulus of the same strength
did not elicit this secondary contraction (response 4), but on slightly
further increasing the stimulus strength another response with two steps
occurred (response 5). This time the secondary contraction started earlier,

I kg/cM2

I~~~~
3
2
~~100 msec 4
Vc

Text-fig. 5. The changes in isometric contractions of an isolated fibre

(17. iv. 69) in response to 50 msec stimuli of increasing field strength. The
ascribed numbers alongside the tracings indicate the order in which the
stimuli were given and the responses elicited. Stimulus no. 1 was 1 1 times
rheobasic strength. Stimuli nos. 3 and 4 were identical to each other. The
temperature was 50 C and the resting striation spacing 2-08 /-Z.

about 20 msec after the end of the stimulus, causing an inflexion in the
rising phase of the response. These types of contraction, which seemed to
be multiple responses, appeared in only some of the fibres studied, and
became less frequent or disappeared entirely with repeated trials. When
the stimulus strength in the experiment of Text-fig. 5 was further increased
to 3 times rheobase a contraction (response 6) resulted in which force
reached almost the same amplitude as in response 1, but without distinct
indication of summation of the effects of more than one excitation. This
latter type of contraction (i.e. response 6) was observed with all the fibres
examined, was reproducible, and is the only type plotted for the long
pulse durations (30-100 msec) in Text-fig. 2.
As already mentioned, the time to peak of contraction decreased slightly
on prolongation of the stimulus from 2 to 20 msec, but for contractions
508 R. RUDEL AND S. R. TAYLOR
elicited by longer pulses the time to peak was increase I (see closed tri-
angles and inset in Text-fig. 2). This prolongation of the time to peak
began at about 2-5 times rheobase and increased with increasing strength
of a long stimulus. In Text-fig. 5, for example, the time to peak of response
6 took 108 msec as compared to 80 msec in response 4.
Relaxation was often slowed in contractions elicited by very long
stimuli (open triangles and inset Text-fig. 2), particularly at high stimulus
strengths. In the experiment of Text-fig. 5, for example, response 6 was
elicited by a stimulus of 3 times rheobasic intensity, and the time from
peak to half-relaxation took 116 msec, compared to 42 msec in response 1,
and to 50 msec for a subsequent response to a chronaxie stimulus. On
return to a weaker or shorter stimulus in general, contraction would again
assume the normal time course. The force amplitude, however, was occa-
sionally up to twice as large as it originally was for a chronaxie stimulus.
This suggests that an extremely long pulse of several times rheobasic
intensity produced after-effects similar to those produced by short trains
of stimuli (Close & Hoh, 1968).

DISCUSSION
We have described the relations between certain changes in the con-
traction of an isolated muscle fibre and the duration and strength of a
transversely applied d.c. stimulus. This is summarized in Text-fig. 6.
Many other workers of course have appreciated the importance of
determining the optimum conditions for studying muscle contraction, but
we have explored a different dimension of this problem by correlating
some of the findings from isometric studies with direct observations of the
behaviour of the myofibrils while shortening.
Brown & Sichel (1940) and Close & Hoh (1968) have previously observed
that the peak amplitude of twitch contractions of curarized whole muscle
is decreased by moderate prolongations of the stimulus. We have now
established that this is true for twitches and tetani of individual fibres as
well. Thus, we refute the inference of Kelly & Fry (1965) that isolated
single fibres should behave like uncurarized whole muscle and respond
with increasing amplitudes of twitch tension to increasing stimulus pulse
duration between 0-2 and 10 msec at room temperature. Isolated fibres do
not behave like uncurarized whole muscles because the latter are probably
excited both directly by the stimulus and indirectly via the nerve stump
remaining after dissection. Katz (1936), for example, showed that a muscle
responds repetitively during the application of a prolonged stimulus to its
nerve. In our single muscle fibres, on the other hand, the nerve stump
was so short that the ending was probably depolarized and incapable of
 STIMULUS PARAMETERS AND CONTRACTION 509
conducting action potentials. The fact that our experiments with and
without curare gave the same results supports this supposition.
When a fibre was permitted to shorten under the same stimulating
conditions which produced a decrease in force, the myofibrils in the region
of the fibre adjacent to the anode were thrown into waves. Wavy fibrils in
intact muscle fibres have previously been described by Huxley & Gordon

3~~~~~~~~~~~~
20 C
0
5)

-C

E
4-i ~A

0
01 1 10 100
Stimulus duration (msec)
Text-fig. 6. A summary of the relations between contraction of an isolated
single fibre and the strength and duration of transversely applied d.c. stimu-
lus in the cold. The stimulus duration in msec is plotted in a logarithmic
scale on the abscissa; the stimulus strength in units of rheobase is plotted on
the ordinate. In area (A) the stimuli are subthreshold and there is no
response. In area (B) all the responses are like those with a 05 msec (i.e.
chronaxie) stimulus (0). There is no change in peak tension, time to peak
or relaxation time. In area (C) peak tension is reduced, time to peak is
slightly decreased, and the early phase of relaxation is accelerated. In area
(D) peak tension and time to peak are increased, relaxation is slowed, and
occasionally there are multiple responses. At room temperature, the whole
of area (B) is shifted to the left with chronaxie at 02 msec.

(1962) who suggested that the waves were produced when inactive myo-
fibrils were bent upon themselves by actively contracting myofibrils. By
the same criterion, Gonzales-Serratos observed passively shortened myo-
fibrils during depolarization contractures (1965), and in addition was able
to produce wavy fibrils by longitudinally compressing resting muscle fibres
embedded in gelatin (1966). We conclude that the wavy fibrils observed
510 R. RUDEL AND S. R. TAYLOR
under the present circumstances also delineate an unactivated part of the
shortening muscle fibre.
The stimulus duration at which attenuation of contraction first appeared
roughly corresponded to the duration of the action potential spike i.e.
about 4 msec in the cold and 2 msec in the warm (unpublished observa-
tions). It is to be expected that when the stimulus strength is several times
greater than threshold and persists for the period in which ordinarily an
action potential would depolarize the entire membrane, activation should
occur only in a fraction of the fibre on the cathodal side since only in that
part would the transmembrane potential difference exceed the level of the
mechanical threshold. This is probably why unactivated myofibrils are
usually visible across more than half the fibre width. It is also obvious that
only a fraction should be activated when one stimulates a fibre that cannot
produce an action potential, and graded, twitch-like responses involving
only the cathodal part have been studied in several types of muscle fibre
(Brown & Sichel, 1936; Sichel & Prosser, 1940; Sugi & Kosaka, 1964).
Similar considerations might account for the findings with barnacle
muscle by Hoyle & Abbott (1967).
The mechanisms involved in increasing peak force when the stimulus is
very long (20-100 msec), seem less certain. The obviously multiple res-
ponses (i.e. 3 and 5 in Text-fig. 5) might have been caused by a delayed
anode-break excitation or spontaneous firing and the resulting contraction
probably involved the whole fibre.
But the reproducible contractions (e.g. 6 in Text-fig. 5) were probably
not produced by anode-break excitation since the time to peak does not
increase proportionately as the stimulus duration is prolonged beyond
30 msec (Text-fig. 2). Another difference from the multiple responses is
that this type of contraction evidently involves only the cathodal side of
the fibre. Sugi & Kosaka (1964) and Sugi & Ochi (1967) found that apply-
ing an intense stimulus to a fibre causes what is ordinarily a local contrac-
tion to spread to the side of the fibre opposite the point of stimulation.
However, we have not seen any indication of this, and a prolonged pulse
of high intensity always created a pattern of activation similar to that
shown in P1. 2. When we increased the field strength up to the maximum
which our stimulator could produce, i.e. about 150 to 200 mV across the
fibre diameter, wavy fibrils extended across more than 80 % of the optical
section without interruption.
It is well known that a directly stimulated muscle fibre will respond
repetitively to a long duration pulse of slightly greater than rheobasic
strength (Ramsey & Street, 1941; Jenerick, 1956; Mashima & Washio,
1968). At higher strength, however, repetitive firing is inhibited after one
or two action potentials and a steady depolarization is produced for the
 STIMULUS PARAMETERS AND CONTRACTION 511
duration of the pulse (Mashima & Washio, 1968). Thus, the reproducible
responses to very long pulses may possibly be produced in this latter
manner, the initial spike being followed by a maintained depolarization
along the cathodal side of the fibre.
The shoulder on the falling phase of the twitch has been observed and
studied by several workers (Funke, 1874; Hartree & Hill, 1921; Gilson,
Schoepfle & Walker, 1947; Aubert, 1956; Close & Hoh, 1968) but its essen-
tial qualities are still open to conjecture. Likewise, our results do not
provide sufficient evidence to allow more than a guess about its origin.
The excitation process, which begins at the muscle fibre surface, evidently
reaches the myofibrils by a route along the transverse tubular system
(Huxley & Taylor, 1958). But after the sarcolemma has been removed
from a muscle fibre, graded or 'all-or-none' activation can also be pro-
duced, apparently by depolarization of some element of the internal mem-
brane system which is electrically continuous longitudinally over many
sarcomeres (Costantin & Podolsky, 1967; also see Birks & Davey, 1969).
Our experiments have shown that a prolonged stimulus inhibits the spread
of activation throughout a muscle fibre (Pls. 1 and 2), and under these
same conditions the shoulder disappears (Text-figs. 1 and 2). But if the
effects of a prolonged stimulus were merely to decrease the number of a
homogeneous population of myofibrils acting in parallel, one would expect
no remarkable change in the time course of the response. Hartree & Hill
(1921) suggested that the shoulder may represent a superimposed con-
traction initiated by separate elements within the muscle fibre and, in
light of the foregoing, one may suppose that these separate elements
could be different parts of the internal membrane system.
The general slowing of relaxation which occurs especially after a long,
intense stimulus (Text-fig. 5) may perhaps be related to a direct effect of
electrical stimulation on release or re-uptake of calcium by the sarco-
plasmic reticulum (SR), as is suggested by the findings with electrical
stimulation of SR fragments (Lee, Ladinsky, Choi & Kasuya, 1966). The
voltage gradient in the interior of our fibres must have been much smaller
than the applied gradient because of the relatively high membrane resis-
tance. But, as Costantin & Podolsky (1967) have pointed out, the results
of Lee et al. (1966) with SR fragments were obtained also with a very
small gradient.
We are very grateful to Professor A. F. Huxley for providing the prolonged
stimulus of the environment of his laboratory, for valuable advice and encourage-
ment, and are indebted to him and Dr B. R. Jewell for reading and criticizing the
manuscript. We are also obliged to Miss G. Shaw for dependable and expert technical
assistance.
512 R. RUDEL AND S. R. TA YLOR

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The Journal of Physiology, Vol. 205, No. 2 Plate 1
A - B +

C- + u +

R. RUDEL AND S. R. TAYLOR (Facing p. 512)

The Joutrnal of Physiology, Vol. 205, No. 2 Plate 2

+ - -+

_rr E~EI

tl3|
fil

_w
LA ~ ~ kI_il

IA B

R. RiCDEL AND S. R. TAYLOR

 STIMULUS PARAMETERS AND CONTRACTION 513
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EXPLANATION OF PLATES
PLATE 1
Selected frames from cine records showing shortening of an isolated fibre under a
light load. Same fibre as in Text-fig. 1. Temperature 200 C (A), before stimulation,
striation spacing 2-06 #u; (B), at the peak of shortening induced by a 0*5 msec
stimulus; (C), about 20 msec after the beginning of a 10 msec stimulus; (D) at the
peak of shortening induced by a 10 msec stimulus. The anode was at the right, and
the field strength was the same as in Text-fig. 1. Calibration mark represents 50 /Z.
Exposure time 5 msec.
PLATE 2
Selected frames from cine records taken at the plateau of shortening induced by
trains of 10 msec pulses at a frequency of 50 Hz. Same fibre as in P1. 1. (A), anode at
the left; (B), anode at the right. The temperature, field strength, and resting stria-
tion spacing were the same as in P1. 1. Calibration mark represents 50 ,t. Exposure
time 5 msec.