II
ON THE EXCITATION OF CRUSTACEAN MUSCLE. I
BY C. F. A. PANTIN, M.A.
(From the Laboratory of Experimental Zoology, Cambridge,
and the Marine Biological Laboratory, Plymouth.)
(Received 15th April, 1933.)
(With Six Text-figures.)
INTRODUCTION.
the nerves of Crustacea have certain properties peculiar to themselves,
the mechanism of nervous action seems to be essentially the same as in the Vertebrata (Levin, 1927; Furusawa, 1928; Hill, 1929). But the combined functional unit
of nerve and muscle behaves so differently in the two groups that even the applicability of the all-or-none principle to nervous conduction in the Crustacea has been
called in question (Hoffmann, 19146; Jordan, 1928). Richet published his work on
the crayfish in 1879, and since that time many have studied the neuromuscular
system of the higher Crustacea by different methods. A remarkable variety of
phenomena have been recorded, but no successful attempt has yet been made which
relates all these to each other. In their variety, the responses of the isolated crustacean limb resemble those of a reflex arc rather than a peripheral system as found
in the vertebrates. Summation, tonus and after-discharge, inhibition and reciprocal
action of antagonistic muscles have all been recorded. The muscle itself may respond
to various stimuli by more than one type of contraction, which may be either long
and slow or rapid and twitch-like. The contrast between this and the properties of
vertebrate skeletal muscle is great.
The problems which require to be attacked may be summarised as follows.
(a) Summation. Richet himself showed that the claw muscles of Astacus
possess enormous powers of summation. In the Crustacea, a single stimulus applied
to the nerve never appears to evoke more than a small response, and as many
as a dozen stimuli may be given before excitation is successfully carried to the
muscle. This failure of a single stimulus is due neither to the nerve nor to the muscle
of themselves. Single stimuli cause good responses in the muscle when applied
directly; and a single stimulus applied to the nerve certainly propagates an impulse,
though this fails to excite the muscle (Lucas, 1917). Lucas showed that summation
was not a local effect at the electrodes. By a process of exclusion some have suggested that the region responsible for summation is the junction between nerve and
muscle. How summation is effected is not yet known. From experiments on the
conduction of pairs of stimuli Lucas suggested that the effect was due to the second
stimulus of a pair falling within the supernormal phase following the first, and thereALTHOUGH
12
C. F. A. PANTIN
fore being better conducted. But, as Blashko, Cattell and Kahn (1931) point out,
this explanation is inadequate, because a large succession of stimuli is often required
before summation reaches a level at which a response takes place. The nature of
summation requires further investigation.
(b) Tonus. Though a single stimulus of normal intensity fails to produce a
response, prolonged tonic contractions may follow a very strong stimulus. Such
contractions also follow amputation of a crustacean limb and may endure several
minutes. By observation of the action currents in the nerve Barnes (1932) indicates
that all such effects are due to the ability of crustacean nerve to give rise to a persistent discharge of stimuli when damaged, as by amputation or strong stimulation.
Barnes concludes that the marked tonus phenomena of the isolated crustacean limb
are due to such causes and are not the result of inherent" tonus " of the muscle itself.
This is of great importance because such tonic contractions are so evident that much
work has been done attempting to elucidate the neuromuscular mechanism partly
in terms of natural tonus in the muscles (von Uexkiill, 1913, 1915). But in view of
the pronounced powers of summation of the limb, responses to stimuli applied
while the nerve is automatically discharging impulses at an unknown frequency
may be very complex. To study such a mechanism it seems necessary as far as
possible to avoid conditions in which tone is present.
(c) Inhibition. The most striking feature of the crustacean limb is the high
development of peripheral inhibition. In his investigations on Astacus claw
Biedermann (1887) showed that whereas a weak tetanic stimulus applied to the
nerve caused contraction of abductor muscle of the dactylopodite, it caused simultaneously inhibition of the adductor muscle. On the other hand, a strong stimulus
causes inhibition of the abductor and excitation of the adductor. There is thus an
apparent reciprocal inhibition, the direction of action of which can be controlled
merely by altering the strength of the stimulus. It is remarkable that artificial
stimulation of a cut nerve can produce such co-ordinated effects, and no satisfactory explanation of this has yet been given. As Knowlton and Campbell (1929)
point out, the abductor-excitor and adductor-inhibitor fibres may have a lower
threshold than their antagonists, and may therefore be excited by stimuli subliminal
to the latter. But above a certain strength of stimulus the adductor becomes excited, and the abductor inhibited. We are thus led to the strange conclusion that
in the adductor excitation can dominate inhibition, whereas inhibition dominates
excitation in the abductor. This presents a most interesting problem.
Biedermann showed the existence of the double innervation of crustacean
muscle. His work was extended by Mangold (1905) to a great variety of arthropods.
Biedermann suggested that of the two nerve fibres supplying the muscle cells one
was excitatory and the other inhibitory. The correctness of this view was confirmed by Hoffmann (1914). Apart from this, the neuromuscular arrangement
possesses a most important feature. The whole abductor muscle is supplied by
branches of two single axons alone, the one inhibitor and the other excitor. Even
in the large adductor there are only three or four motor fibres which by repeated
branching serve the whole muscle. Moreover, a single axon may supply the muscles
On the Excitation of Crustacean Muscle
13
of more than one joint. The mechanism of graded contraction here presents a
problem which differs entirely from that in vertebrates.
In view of the complex response of the muscles, it is natural to look for peripheral nerve networks and ganglia. Hoffmann and the earlier workers find no
evidence of these. They find no anastomosis of one axon with another nor with the
numerous sensory fibres running in the nerve. Von Uexkiill and his colleagues have
described ganglion cells and nerve nets, but the evidence does not seem satisfactory.
It is also difficult to reconcile his interpretation of excitation with our knowledge
of nervous action, and the weight of physiological evidence is definitely in favour of
Biedermann and Hoffmann.
Of the mechanism of inhibition in Crustacea we have as yet little knowledge.
Frohlich (1908) pointed out that failure of response took place when the frequency
of stimulation was raised, and suggested that this was due to Wedensky inhibition.
But Levin and Furusawa have found that strong and rapid stimuli depolarise the
nerve which then ceases to conduct; and it is necessary to separate true inhibition
from effects such as these.
(d) Quick and slow contraction. The evidence for more than one contractile
mechanism in the muscle rests chiefly on the work of Keith Lucas (1907, 1917).
In general, a weak stimulus causes a slow contraction, whereas a stronger one causes
a twitch. Two sources of evidence should be distinguished: first, two kinds of contraction may be elicited in response to a battery of single stimuli; secondly, Lucaa
obtained two kinds of contraction in response to single stimuli; and these responses
possessed different excitation times. Before the responses to a battery of stimuli
can be taken as evidence of two contractile mechanisms, the possibility that the rate
of response is controlled by summation needs investigation. On the other hand, the
existence of two types of response to single stimuli at least seems to rest on unequivocal evidence. But the conditions here differ greatly from those which govern
responses to a succession of stimuli. The contractions, when obtained at all, involve
only a small number of muscle fibres. They are only evoked for a somewhat short
period following amputation. Moreover, the evidence here is drawn from work
on the highly specialised adductor muscles of the chela of the closely related
lobster and crayfish: nor is the double mechanism invariably encountered even in
this muscle itself (Lucas, 1907, 1917).
In the following experiments the muscles employed have been chiefly those
which control the dactylopodite of the walking legs in Carcinus maenas. These do
not suffer the specialisation of function found in the chela muscles. Most of the
recorded variation of response in crustacean muscle depends upon changes in the
intensity of the stimulus. It is therefore necessary first to determine the relation of
the response to the intensity of some stimulus of constant form. In this paper the
response of the muscle to alternating current of constant frequency and variable
intensity is analysed.
14
C. F. A. P A N T I N
MATERIALS AND METHODS.
In the leg of the shore-crab the dactylopodite hinges on two processes on either
side of the penultimate joint. There is a flexor and an extensor muscle attached over
the inner surface of this joint. Each is inserted on to an apodeme. That of the
flexor is attached to the dactylopodite below the hinge, and that of the extensor above
the hinge. The attachment of the extensor was usually cut, leaving a simple flexor
preparation. Contractions of the muscle were recorded isometrically, so that their
height is proportional to the tension developed. The leg was cut off at the point of
natural autotomy. It was found that perfusing the leg enormously prolonged its
viability: therefore the limb was usually perfused through a rubber tube slipped over
the cut end of the leg. The perfusion fluid was as follows :
NaCl
KC1
CaClg
MgClj
NaHCO,
o-6 M
o-6 M
0-4 M
0-4 M
ioo
2-5
3-5
7-0
to />H 7
parts
parts
parts
parts
This mixture falls within the limits of concentration for Carcinus maenas blood
(Bethe, 1929). A mixture with 3-5 parts MgClj often gave even better results. These
media were very satisfactory, and preparations survived over 8 hours' continuous
experimentation and only very slowly deteriorated. Experiments were conducted
at room temperature in winter and spring. During the whole course of the work
temperatures ranged between 12 and 17° C , and did not vary more than 20 during
any one experiment.
Dissection of the nerve tends to render the limb inexcitable, so that experiments
were done as far as possible on the whole limb. To avoid local depolarisation, it was
found desirable to make the region of stimulation diffuse. Platinum or silver electrodes were inserted into the limb or into the perfusion tube. Sometimes an induction coil was used, but usually the stimulus was provided by a small transformer
working off the mains (220 volts, 90 cycle), the current of which was almost sinusoidal. The transformer yielded 12 volts, and was subject to small variations. These
were corrected by a resistance in series with the transformer. The transformer was
connected through this resistance to a potentiometer, from which was obtained the
desired intensity of stimulus. When possible, stimuli were given regularly each
minute and were of constant duration (2-4 sec). When first perfused, the limb
frequently exhibited tonic contractions. These passed off within a few minutes.
Perfusion was continued 10 or 20 min. before experiments were begun.
RESPONSES TO ALTERNATING CURRENT STIMULATION.
Biedermann and others have shown that on increasing the strength of the
stimulus, the abductor muscle of Astacus claw passes from contraction to inhibition.
A similar increase in stimulus causes the adductor muscle of the claw to pass from
inhibition to contraction. Independently of this, it has been found that increase in
strength of stimulus may cause contraction of the adductor to pass from a slow type
On the Excitation of Crustacean Muscle
if
to a twitch. The following experiments show that none of these effects is peculiar
to a particular muscle; all can be obtained from the same muscle, and there is a
definite relation between them. When the nerve of the leg of Cardnus is stimulated
by alternating current for 4 sec. every minute, a sequence of responses is obtained
from the flexor muscle of the dactylopodite, according to the strength of stimulus.
There is a well-defined threshold above which the response rapidly reaches a
maximum. The contraction is slow, and the tension is still increasing slightly at
the end of 4 sec. stimulation. Fig. 1 A illustrates this. The signal marks the duration of the 4-sec. stimuli, after each of which the drum was stopped. From the
threshold at 0-9 volt the contraction rises to a maximum at about 1-5 volts. In this
preparation these values were above the average. Further increase in strength of
"o-75 0-9 1-05
I-2
K30
Maximum (i)
0-75 volt
1-5
1-65
1-8
1-95
2-1
2-25 2-4 2-55
2-1
Inhibition
1-20 volts
Block above tracing = 1 second.
B
3-0
3-3
3-6
3-9 volts
Maximum (2)
2-25 volts
Fig. 1. For explanation of figures see text,
stimulus leads not to increase in height of contraction, but to a progressive failure
of response. But on increasing the stimulus still more, tetanic contractions reappear,
so that there is a second maximum. This contraction, however, differs significantly
from that of the lower voltage. It is very much more rapid and twitch-like, and its
latent period is much shorter. Fig. i B illustrates the two types of contraction on a
fast drum. The tetanus generated in the first maximum shows a smooth development of tension; the summit of a second maximum tetanus is sometimes irregular.
The first maximum of contraction and the failure of response with the higher
intensity of stimulus are quite comparable with the excitation followed by inhibition
previously recorded in the abductor (= extensor) muscles of the crayfish claw. But
in that case there is no reappearance of contraction with still greater stimulation.
On the other hand, the Cardnus flexor muscle differs from the adductor (= flexor)
i6
C. F. A. PANTIN
muscle of the crayfish claw, because the latter shows no contraction with stimuli of
low intensity, but only inhibition of any tonus which may be already present in the
claw. However, in both leg and claw the flexor muscle passes from inhibition to
contraction if the stimulus is sufficiently increased. The flexor of the dactylopodite
of Carcinus leg therefore shows the features peculiar to both muscles of the crayfish
chela. In Carcinus leg, inhibition overmasters contraction over a certain range of
stimulation intensities only. Further, the contractions above and below the inhibitory region correspond to quick and slow contractions respectively. The relative
speed of these is of the same order as those noted by Keith Lucas in Homarus claw.
The failure of response first appears at the beginning of contraction, and with
increasing stimulation intensity its effect is extended. It finally disappears, with
strong stimuli, at the end of the contraction (Fig. i A). This series of responses was
obtained with the electrodes placed in a variety of positions in the limb. Increasing
the strength of stimulus above 4 volts often leads to a decrease in the second
maximum contractions. But whereas the responses recorded above are easily
repeatable, these strong stimuli temporarily damage the tissue. If the intensity of
stimulus is raised so that the second maximum contractions begin to fail, it is found
that the contractions are temporarily lessened at all voltages.
Sometimes the region of failure is so great that the second maximum of contraction does not appear, or is reduced, even for very strong stimuli. This often
develops in preparations subjected to very long experimental treatment. The behaviour is now the same as that recorded for the abductor of the crayfish claw.
Failure of response between the two regions of contraction was often less complete
than in Fig. 1, and was completely absent in about 2 per cent, of the crabs.
Both quick and slow contractions are evoked through the nerve, and are not
due to direct stimulation of the muscle by irradiation of current from the distant
site of stimulation. Cutting the nerve where it enters the muscle abolishes all
response to the stimulation of the nerve.
INHIBITION OF THE FIRST MAXIMUM.
We have supposed that the failure of contraction for stimuli of medium intensity
is due to the stimulation of inhibitory fibres whose threshold is above that of the
excitatory fibres responsible for the first maximum of contraction. Fig. 2 A shows
that this is the case. The nerve to the flexor muscle was stimulated by two pairs of
electrodes. One pair (A) was placed in the proximal part of the leg; a second pair
(B) was arranged to stimulate the nerve before it entered the muscle. The usual
sequence of responses was obtained from either pair of electrodes, though at slightly
different intensities of stimulus.
Threshold
First contraction maximum
Region of failure
Secend contraction maximum
A
B
volts
volts
0-4
o-6-i-o
1-3-1-8
24-30
0-25
0-5-0-9
1-0—1-5
24-30
On the Excitation of Crustacean Muscle
17
In Fig. 2 A the signal below the muscle tracing indicates the duration of stimuli
through the electrodes B, the signal below this that through the electrodes A.
Underneath is shown a i-min. time interval. The stimulus through B was set in the
I 0-6 volt
Imaximuanfl)
I t-5 volts
(inhibition
Block — 1 minute interval.
Flew
Extensor
Stimuli of 4 seconds duration once a minute. Inhibition in the flexor is incomplete.
From left to right stimuli increase from o-6 to 2-7 volts by increments of 0-15 volt.
B maximum (1)
B maximum (a)
A inhibition
A inhibition
Block below signal = 1 minute interval.
Fig. 2.
first maximum of contraction at o-6 volt; that through A in the region of failure of
response at 1-5 volts. In the record a short stimulus is first given through A, causing
no response. A short stimulus is then sent through B, giving a response. Then a
prolonged stimulus is sent through B, giving a tetanus, during which two short
JBB-Xli
i
18
C. F. A. PANTIN
stimuli are sent through A. These immediately inhibit the tetanus as long as they
last. The record concludes with a short stimulus through A, producing a scarcely
perceptible response, followed by a longer stimulus through B giving an uninterrupted tetanus.
The inhibitory apparatus can be fatigued. Prolonged stimulation at the inhibitory intensity is usually followed by the gradual development of a partial contraction. In this condition the power of inhibiting a first maximum tetanus
becomes reduced.
RECIPROCAL INHIBITION.
The behaviour of the extensor muscle resembles that of the flexor. Like it, the
extensor shows a first maximum of slow contraction, followed by inhibition with
stimuli of greater strength. A further increase in stimulus usually produces some
evidence of a second maximum, of more rapid contraction. But in the extensor,
inhibition is far more complete than in the flexor, and the second maximum is often
absent. In the experiment illustrated in Fig. 2 B, the tendons of the two muscles
were recorded simultaneously. The stimuli were of 4 sec. duration, and were applied
once a minute. The threshold of the first maximum and the region of inhibition
occurred at stimulation intensities which were usually considerably different for the
two muscles, as in Fig. 2 B. The contractions of the extensor were evoked at lower
intensities than in the flexor. On raising the stimulus, the extensor became inhibited, while the flexor contracted. Such a detailed reciprocal correspondence
between the two muscles, as in Fig. 2 B, does not always occur, though it usually
does so in those limbs which are in the best physiological condition. At times the
first maximum and region of inhibition may occur over the same range of stimulation
in both muscles. But in view of the fact that both muscles may show two maxima
of contraction, with a region of inhibition between them, and that these do not
usually occur at the same stimulation intensity in the two muscles, it is perhaps
scarcely surprising that apparent reciprocal inhibition may be found.
Perfusion sometimes alters the relative thresholds of contraction and inhibition
in the two muscles, especially when it is first commenced. So that the reciprocal
correspondence of these various phases in the two muscles at any stimulation
intensity might temporarily be changed to a synergic action.
RELATION OF INHIBITION TO THE SECOND MAXIMUM.
Since the second maximum of contraction takes place at stimulation intensities
above that at which inhibition appears, it seems that its relation to inhibition must
differ from that of the first maximum contraction. The second maximum of contraction must take place in spite of the simultaneous stimulation of inhibitory
fibres. If the preparation is stimulated to give a tetanic contraction by a stimulus
sufficiently intense to evoke the second maximum of response, it is found that the
superimposition on another part of the nerve of a stimulus of an intensity corresponding to inhibition fails to arrest the contraction. This is illustrated in Fig. 2 C
(flexor muscle). Electrodes were placed in the limb close to the penultimate joint.
On the Excitation of Crustacean Muscle
19
A tetanus was evoked by stimulating in the region of the second maximum. The
signals in the lower line show the superimposition of stimuli by electrodes in the
upper part of the limb. These stimuli were set at an intensity corresponding to inhibitory failure. It will be seen that they fail to inhibit the contraction. As a control
the record is followed by a similar experiment in which the tetanus is evoked by
stimulation in the first maximum at the electrodes nearer the muscle. Well-marked
inhibition can be obtained from the upper electrodes. Failure of inhibition can also
be shown when a second-maximum contraction is evoked by stimulation of the
upper part of the limb and the "inhibitory" stimulus is applied over the muscle.
These experiments show that a response evoked in the "second maximum " of contraction is incapable of inhibition by superimposed stimuli.
Successive prolonged stimulations in the second maximum often tend to reduce
greatly the height of the response. When this occurs it is found that stimulation at
intensities corresponding to the second maximum can produce increasingly marked
inhibition when superimposed on a first maximum tetanus. It is as though the
range of inhibition were extending over the range of the second maximum. If,
when the preparation is in this condition, a tetanus is set up in the second maximum,
stimulation of the nerve in another part of the limb at an intensity corresponding to
inhibition may actually cause a small increase in the height of the tetanus. We have
already seen that the inhibitory fibres are capable of fatigue. Superimposed inhibition during a second maximum contraction may thus fatigue these fibres and
thereby allow an increased height of contraction.
It appears that although a superimposed stimulus is incapable of inhibiting the
second maximum response, stimulation at this intensity does involve inhibitory
fibres and that these have a material influence on the second maximum contraction.
The abruptly terminated form often found in the second contraction (Fig. 2 C)
and its mode of development (Fig. 1 A) suggest that the contraction occurs in spite
of the inhibitory action, but that the height of the contraction may be limited owing
to the simultaneous stimulation of the inhibitory fibres. Fatigue of this contraction
permits the action of inhibitory fibres to decrease the effective height which the
second contraction reaches.
These experimental results may therefore be summarised as follows. Increasing
intensity of stimulation produces first a contraction. This is a slow contraction and
is easily inhibited by suitable stimuli. Further increase in intensity results in inhibition of this contraction in which both excitor and inhibitor fibres are being
simultaneously stimulated. Still further increase in intensity results in a rapid
twitch-like contraction. This is incapable of inhibition by superimposed stimuli,
and the contraction takes place in spite of the inhibitory mechanism. But the inhibitory mechanism is not without influence on these contractions. It apparently
limits the tension developed. Fatigue of the contraction increases the power of
inhibitory fibres to limit the contraction; fatigue of the inhibitory mechanism
increases slightly the power of the excitor mechanism to evoke contraction. Not
only can both quick and slow contractions be elicited from the muscle, but there is
an important difference in their relation to inhibition.
20
C. F. A. PANTIN
THE RELATION OF THE QUICK AND SLOW CONTRACTIONS: FATIGUE.
The great difference in the speed of the quick and slow contractions, the different
thresholds, and their very different relation to inhibition seems to suggest that these
involve separate contractile mechanisms. But the following experiments strongly
suggest that this is not the case. In the first place, if we are dealing with two
separate systems which suffer excitation independently and at different stimulation
intensities, it should be possible to fatigue one system by prolonged stimulation
without appreciably affecting the other. Attempts to demonstrate this all failed. If
A
B
A. Signal
=•
i
minute
intervals.
Block
below
signal
•=
duration
of
continuous
stimulation (8 minutes)
at o-75 volt (maximum (i)). At 5th, 6th, 7th and 8th signal, stimuli of 4 seconds duration are
superimposed via a second pair of electrode* on nerve. These four stimuli are marked on the
block, and were of strength fz, 1-5, 2-1 and 3-0 volts respectively. Note absence of response.
From loth to 14th signal, 4-second stimuli at 0-75 volt given (maximum (1)). Note rapid recovery
after prolonged tetanus.
B. As in A, but stimuli superimposed via a second pair of electrodes over muscle. Block below
signal = duration of continuous stimulation
(16 minute*) at 0-75 volt (maximum (1)). Superimposed
stimuli marked on block; strengths, i#a, i-8, 2-4, 3-0, 3-7, 4-5, 5-3, 6-o, o-o, 13-1 volts respectively.
Note contractions of muscle to direct stimulation. Atfirstand last signals of whole series, 4-second
stimuli of 0-75 volt given. Note rapid recovery after fatigue.
Fig. 3. Effect of superimpo»ing stimuli via electrodes. A on the nerve and B on the muscle,
during continuous stimulation of the nerve.
the nerve is continuously stimulated by alternating current, a full tetanus is only
maintained for about 1 min. Fatigue then rapidly sets in (Fig. 3 A). If at one site
the limb is stimulated to fatigue in either the first or second contraction maximum,
stimuli superimposed at any other point in nerve fail to produce a response. This is
true even when the fatigue is generated in the first maximum and the superimposed
stimuli are of sufficiently great strength to correspond to the second maximum.
The fatigue is not that of the muscle itself, for this will still respond to direct
stimulation. In Fig. 3 B a fatigue was produced by stimulating the nerve in the first
maximum. Four-second stimuli were then superimposed by electrodes placed
directly over the muscle itself. Good contractions are obtained when the stimulation
On the Excitation of Crustacean Muscle
21
intensity is sufficiently increased. The intensity required to evoke a response by
direct stimulation of the muscle is far greater than that required for stimulation of
the nerve. Further, continuous direct stimulation of the muscle produces a prolonged tetanus which only falls to 50 per cent, of its greatest height in about 8 min.
Recovery from prolonged direct stimulation is very slow, whereas recovery from
fatigue following stimulation of the nerve even for as long as 20 min. is very rapid
(Fig-3)The fatigue of the nerve is probably due to depolarisation. But these experiments indicate that whatever its cause it is not possible to fatigue the conducting
fibres responsible for the slow contraction without fatiguing those of the twitch,
and vice versa. This suggests that the conducting mechanism in both cases is the
same; a conclusion in accordance with the observation of Blashko, Cattell and Kahn
that both quick and slow contractions appear to have the same threshold.
RATE OF CONTRACTION.
When inhibition is marked the quick and slow contractions appear quite distinct
from each other. But when inhibition is incomplete the initial rate of contraction
0-62 volt
"I
0
1-0 sec.
Contraction rate with increasing intensity of stimulus
Fig. 4-
can be observed throughout the whole range of stimulation intensity. In Fig. 4 are
represented the responses of a flexor muscle to continuous alternating current
stimuli of increasing intensity. The origin corresponds to the onset of the stimulus.
At first sight there appears to be a regular gradation of response. But closer examination shows that the rates of contraction over the major part of the response do
to some extent fall into two distinct groups. As Blashko, Cattell and Kahn found,
increase in stimulus above the threshold first raises the height of the contraction
rather than its rate. Nevertheless, measurement of the slope of the contraction
curves does indicate some gradation between the quick and slow contractions. The
contraction rate above the threshold first increases rapidly, then very slowly in the
region of the first maximum, then rapidly over the region of inhibition, and finally
approaches a more or less constant value for the second maximum in which the rate
MM,
C.
F.
A.
PANTIN
of contraction is about ten times as fast as that of the slow contraction. The contraction rate for the fully developed twitch appears to be identical with that for
strong direct stimulation of the muscle. The latent period is, however, very much
longer.
The contraction rate of the slow contraction may be considerably influenced by
previous stimulation. A previous stimulus increases the rate at which contraction
takes place in response to a subsequent stimulus of the same intensity.
These experiments indicate that there is a real tendency for the rates of contraction of the muscle to fall into two groups, quick and slow, under varying intensities of a stimulus of constant form. But there is some gradation between these,
Redevelopment of tension after quick releases during:
A (direct stimulation
of muscle)
B (2nd maximum
contraction)
C (ist maximum
contraction)
Redevelopment of tension after quick releases during:
D
E
F
G
Stages of development of slow contraction
H
Fig. 5. Time intervals in seconds.
and the rate of the slow contraction is not constant but can be influenced by previous
stimulation.
Despite this, it can be shown that both quick and slow contractions, once they
are developed, involve the same contractile mechanism. Hill (1926) has shown that
the development of tension in a stimulated muscle depends upon its viscouselastic properties. A sudden release of the muscle during a tetanus is followed by a
redevelopment of tension of the same form as that which takes place when the
stimulation commences. This is true for the flexor muscle of Carcinus leg when
directly stimulated to give a maximal tetanus (Fig. 5 A). The redevelopment of
tension following a sudden release approximates to the initial contraction in form.
The same thing is true of the muscle when stimulated to give a maximal response
On the Excitation of Crustacean Muscle
23
by the nerve when the stimulus is of sufficient strength to evoke the second maximum. In Fig. 5 B this is shown, and the similarity of behaviour to that of the directly
stimulated muscle is evident. (The muscles used in Fig. 5 possessed a very welldeveloped second maximum of contraction.) But when the slow contraction is
allowed to develop a tetanus by stimulation in the first maximum, a sudden release
of the muscle is followed by a redevelopment of tension not of the slow type, but of
a type precisely resembling that found in tetani produced by the quick contraction
or by direct stimulation of the muscle (Fig. 5 C). Hence the slow development of
tension in the slow contraction is a phenomenon associated with the initial development of tension when stimulation commences only, and is not apparently due to the
excitation of a different type of contractile machine.
The conditions which govern this slow initial development of tension gradually
pass away as the slow contraction develops. Fig. 5 (D-H) shows the effect of quick
releases on the development of tension during the actual development of the slow
contraction. It will be seen that the tension is not redeveloped at its full rate until
the tetanus is fully developed; and that the greater the degree which the contraction
has reached before the release is made, the more rapid is the redevelopment of
tension. It seems that both quick and slow contractions when induced by a battery
of stimuli involve the same conducting elements and the same contractile elements.
LATENT PERIOD.
While increase in intensity of the stimulus affects the rate of contraction, it also
exerts a profound effect upon the latent period. Fig. 6 illustrates the relation of
latent period to increase in intensity of stimulus. Continuous alternating current
stimuli were applied in the usual manner and the latent period measured from the
beginning of the stimulus. The previous treatment of the preparation affects the
latent period, so that the latter varies somewhat for the same stimulation intensity
at different stages of an experiment.
Two things are evident. First, that the latent period is extraordinarily long,
especially for those weaker stimuli which evoke the slow contraction of the first
maximum of response. The latent period of the muscle itself directly stimulated is
about 7-ioc. This is of the same order as that of frog's skeletal muscle. The latent
period of the contraction when the nerve is stimulated in the leg ranges from about
50 to 300CT, an interval of a magnitude wholly different from that found in frog's
skeletal muscle. The long and variable latent period was noted by Richet (1879)
and Piotrowski (1893), who suggested that the effect was due to delay between
nerve and muscle. The latent periods described by these workers are of the same
order as those found in Carcinus leg.
The latent period shortens as the intensity of stimulus is increased. This shortening is very rapid in that region of low intensity of stimulus which precisely corresponds with that required to evoke the slow contraction. As the second contraction
maximum is approached the contraction becomes more and more twitch-like, the
latent period gradually becomes constant at about 500-, giving the curve a more or
24
C. F. A. PANTIN
less hyperbolic character. It is not until the latent period becomes almost constant
that "twitch" develops its full rapidity. Fig. 6 shows this definite relation between
the latent period and the response of the muscle to stimulation of the nerve. The
slow contraction is characteristic of responses with a prolonged latent period.
Inhibition is effective where the latent period is shortening towards its asymptotic
value, while the quick contraction develops when the latent period has reached this
value, of about 50c.
This relation indicates that the quick and slow contractions may be responses of
the same contractile mechanism to different conditions of excitation of the nerve.
In view of the great length of the latent period it suggests that the slow contraction
as produced in these experiments is due to slow summation of stimuli at the junction
between the nerve and the muscle. The latent period of the muscle itself is only
VolU
Maximum (2)
(quick contraction)
2-0-
Region of
effective inhibition
Maximum (1)
(alow contraction)
1-0-
1
100
Threshold
3A0
300 tr
200
oftDOMfe dlrrrt
Fig. 6.
about 7-100-. The velocity of conduction in crustacean nerve is about 6-10 metres
per second (v. Buddenbrock, 1928), so that an interval of about 4 or 50- may be due
to this cause. Instrumental mechanical delay certainly cannot be responsible for
latent periods of the order of 2OOcr and which vary continuously as in Fig. 7. The
remaining part of the latent period must be due to delay between nerve and muscle.
This long latent period of the slow contraction is not merely the result of the
setting in motion of a slow train of events which will reach accomplishment even
if the stimulus is cut off before contraction commences. A stimulus not continued
throughout the latent period is unable to produce a response in the slower contraction.
The long period of delay indicates that in the slow contraction many stimuli are
applied to the nerve before a response is observed, and that these first stimuli are
ineffective but predispose the muscle to later ones. If this is so it seems that the
quick and slow contractions depend like so many other peculiar properties of
crustacean muscle upon its marked powers of summation.
On the Excitation of Crustacean Muscle
25
DISCUSSION.
Full discussion will be left to a succeeding paper. We have seen that increasing
stimulation by alternating current of the dactylopodite muscles of Carcinus leg
results in a slow contraction, inhibition, and finally a quick contraction. This is not
due to a special method of stimulation, because the same effects can be obtained with
induction shocks. Not only do the contractions differ in rate, but differ also in their
relation to inhibition. Nevertheless, the effects of fatigue and of quick releases of
tension suggest that the same conducting and contractile elements are involved in
both contractions. It is possible to explain these things in terms of summation. The
long latent period of the slow contraction and its rapid decrease with increasing
intensity of stimulation suggest that this contraction only results from the summation
of many stimuli. But if we suppose that the passage from the slow to the quick
contraction depends primarily on summation, we imply that increase in stimulation
intensity without change in frequency increases the number of impulses sent down
the nerve. In the older work in which induction shocks were used, this may be so,
because raising the intensity may cause the make-shocks to be effective as well as
the break-shocks. With an alternating current stimulus it is possible that at low
intensities not every variation during the current cycle excites the nerve, and that
therefore increase in intensity will increase the frequency of excitation. It should
be remembered that with alternating current stimulation the current required for
the rapid contraction is 3-4 times as great as that required for the slow. Blashko,
Cattell and Kahn observed a passage from a slow to a quick contraction with induction shocks of increasing intensity in which the make-shocks were short-circuited.
As we shall see in a later paper, this is only found under special conditions in which
the current intensity is greatly increased.
The interpretation of both types of response in terms of facilitation acting upon
a single type of contractile unit meets with certain apparent difficulties. Blashko,
Cattell and Kahn showed that if during a slow tetanus due to a series of shocks an
extra single shock is interjected a rapid contraction may follow due to this extra
shock. This, however, seems fully explicable in terms of the shortened time interval
between the interjected shock and the preceding and following shocks of the series.
It may merely be the result of the more rapid facilitation at the higher frequency.
Keith Lucas figured responses to short batteries of stimuli in which apparently
two distinct forms of contraction, quick and slow, were evoked in succession, a rapid
twitch being followed by a slow contraction which commenced before the twitch
had passed away (and vice versa). But considering the time-scale of Keith Lucas
responses and their form, a strong parallel seems to exist between them and effects
which can be produced by a succession of stimuli sufficiently strong to excite not
only the activity of the muscle but also its inhibitory fibres. In these a rapid contraction often is followed by an apparent slow one, due to the secondary incidence
of inhibition on the tetanus already induced.
On the view outlined above, the slow contraction in response to a battery of
stimuli is the result of a statistically increasing number of muscle fibres being called
26
C. F. A. PANTIN
into action as each successive stimulus passes down the nerve. The rate of contraction would thus be a measure of the statistical rate of facilitation at the individual
junctions between the nerve terminations and the individual muscle fibres. Very
frequent stimuli on the other hand cause very rapid facilitation with a consequent
twitch-like response in which almost all the muscle fibres are simultaneously called
into play.
Inhibition is effective on the slow contraction, but not upon the quick. Therefore, if summation alone is responsible for the difference between them, inhibition
must bear a relation to the rate of summation. It must only be effective when the
frequency of excitation is low. This might help us to understand the peculiar feature
of the crustacean claw, that in a pair of antagonistic muscles excitation may dominate
inhibition in the one whereas the reverse occurs in the other. This can be tested by
a study of the relation of the response to frequency of stimulation, and of the influence of inhibition upon it. It will be considered in the succeeding paper of this
series.
SUMMARY.
1. A brief account is given of the present position of the problem of neuromuscular action in the Crustacea.
2. A method is described by which the leg of Carcinus maenas may be perfused
and stimulated. By this method the muscle remains in good condition for some
8 hours.
3. By stimulating the nerve in Carcinus leg with alternating currents of increasing intensity a aeries of varied responses is obtained. Above the threshold a
contraction is developed of a comparatively slow type. With increase of intensity of
the stimulus the response fails, owing to the excitation of inhibitory nerves. But at
still greater intensities contraction reappears. This contraction, however, is very
rapid. Tetani developed from the slow contraction are easily inhibited. Tetani
developed from the rapid contraction cannot be inhibited by superimposed stimuli.
4. The relation of the quick and slow contractions is considered. It is not
possible to fatigue one without fatiguing the other. Experiments show that on
suddenly releasing the tension of the muscle during a tetanus, the tension always
redevelops in a manner similar to the development of tension in the quick contraction, even though the tetanus be developed initially by the slow contraction. The
same contractile mechanism is involved in both cases.
5. The latent period of contraction on stimulation of the nerve is very long, and
ranges from 30CXT at the threshold. That for direct stimulation of the muscle is
7-1 ocr. Above the threshold the latent period shortens rapidly with increasing
stimulus. Over this region the contractions are of the slow type. The latent period
becomes asymptotic to 50c as the intensity is increased. At this value the contractions are of the quick type. Inhibition is effective where the latent period begins
to approach its asymptotic value.
On the Excitation of Crustacean Muscle
27
6. It is suggested that all the varied phenomena observed are related to the
power of summation of crustacean muscle; that the slow contraction in response to
a battery of stimuli is not due to a different contractile mechanism from the quick
one, but that it is a summation effect by which a statistically increasing number of
muscle fibres are brought into action as successive impulses pass down the nerve.
These experiments were commenced at the Marine Biological Laboratory,
Plymouth, and I wish to express my thanks to the Director, Dr E. J. Allen, for the
facilities I enjoyed whilst working there. The apparatus employed in this work was
provided in part by a grant from the Government Grants Committee of the Royal
Society, to which I wish to express my thanks.
REFERENCES.
BARNES, T. C. (1932). Amer. Journ. Phyriol. 99, 321.
BETHE, A. (1929). Pflugert Arch. 221, 344.
BIEDERMANN, W. (1887). Sitxungtberichte d. Akad. Wien, 95, 7 and 96, 8.
BLASHKO, H. ( CATTELL, M C K . and KAHN, J. L. (1931). Joum. Pkytiol. 73, 25.
BUDDENBROCK, W . VON (1928). Grundritt der Vergleichenden Phytiologie.
Berlin.
FROHUCH, F. W. (1908). Zeit.f. AUg. Phytiol. 7, 393.
FURUSAWA, K. (1929). Joum. Phytiol. 67, 325.
HILL, A. V. (1926). Proc. Roy. Soc. B, 100, 108.
(1929). Proc. Roy. Soc. B, 105, 153.
HOFFMANN, P. (1914a). Zeit.f. Biol. 63, 411.
(19146). Zeit.f. Biol. 64, 247.
JORDAN, H. J. (1928). Arch. Nierland. Phytiol. 13, 570.
KNOWLTON, F. P. and CAMPBELL, C. J. (1929). Amer.Journ. Phytiol. 91, 19.
LEVIN, A. (1927). Joum. Phytiol. 63, 113.
LUCAS, K. (1907). Joum. Phytiol. 35, 310.
(1917). Journ. Phytiol. 51, 1.
MANCOLD, E. (1905). Zeit.f. AUg. Phytiol. 6, 135.
PIOTROWSKI, G. (1893). Journ. Phytiol. 14, 163.
RICHET, C. (1879). Arch, de Phytiol. Norm, et Path. 6, 262 and 522.
UEXKOLL, J. VON and GROSS, F. (1913). Zeit.f. Biol. 60, 334.
UEXKOLL, J. VON and TIRALA, L. G. (1915). Zeit.f. Biol. 65, 25.