J. Exp. Biol. (196a), 39, 71-88
With 1 plate and 5 text-figures
Printed in Great Britain
71
PERIPHERAL REFLEX INHIBITION IN THE CLAW
OF THE CRAB, CARCIN U8 MAENAS (L.)
BY B. M. H. BUSH
Department of Zoology, University of Cambridge
{Received 7 March 1961)
INTRODUCTION
Peripheral inhibition in the pereiopods of decapod Crustacea was demonstrated
physiologically by Biedermann (1887), Hoffmann (1914), Wiersma (1933), and Pantin
(1936). It has since been subjected to extensive analysis by stimulating single motor
and inhibitor axons (e.g. Wiersma & Ellis, 1942), and to biophysico-chemical studies
on isolated neuromuscular preparations (e.g. Fatt & Katz, 1953; Boistel & Fatt, 1958;
Hoyle & Wiersma, 1958). However, there is as yet little experimental evidence on the
functional significance of peripheral inhibition to the living animal.
The classical view, based upon the histological ' double-fibre' innervation of each
muscle, originated from Biedermann's observation that stimulation of the claw nerve
in the crayfish caused either contraction of the claw-opener muscle with inhibition of
the closer, or, at higher intensities, contraction of the closer and inhibition of the
opener. Each muscle was supposed to receive a motor and an inhibitor axon, the
latter always responding together with the motor axon of the antagonist muscle.
Hoffmann (1914) and others compared this theory of peripheral inhibition of antagonistic muscles with the vertebrate system of reciprocal central inhibition of antagonists
(Sherrington, 1913). He showed in Astacus that, after sectioning the nerve containing
the motor axon of the claw opener, any tonus in this muscle could be diminished by
abdominal stimulation, evidently through peripheral reflex inhibition. Furthermore,
the 'adaptation' in reflex opening, which occurred in the intact claw in response to
repeated or continuous abdominal stimulation, was abolished when the nerve containing the opener inhibitor was cut. From this he concluded that an additional
function of the inhibitor axons was to safeguard the muscles against excessive excitation. Recently, Eckert (1959) reported that passive (imposed) opening of the claw in
Astacus evoked a strong reflex discharge in the opener inhibitor, whereas passive
closing elicited a discharge predominantly in the opener motor axon. On this basis
he too advocates the theory of peripheral inhibition of antagonists.
However, from the limb innervation patterns of various Decapoda (Wiersma,
1941; Wiersma & Ripley, 1952), it is clear that only the one or two 'specific inhibitor'
neurones of each limb could inhibit individual muscles, for the remaining ' common
inhibitor' innervates two or three pairs of antagonists (see Text-fig. 1 in Bush, 1962).
These authors suggested that, while the specific inhibitors probably serve primarily
to permit independent action of the motor-coupled ' opener' and ' stretcher' muscles
in each limb, the common inhibitors do not play a part in the normal locomotory
movements. Instead they may be used only during the moulting process, for instance,
72
B. M. H. BUSH
to inhibit contractions resulting from sensory bombardment of the central nervous
system, or (Wiersma, 1958 a) from direct mechanical stimulation of the peripheral
branches of the motor fibres. Pantin (1936) suggested that peripheral inhibition
might serve to bring to an end contractions evoked by ' trigger' responses of the kind
described by Blaschko, Cattell & Kahn (1931), in which a single shock interjected
into a low-frequency motor train elicited a sharp rise in tension which then remained
at the new level. Wiersma & Adams (1950) found that this type of response occurs in
most 'fast' crustacean nerve-muscle systems and in some opener and stretcher ones,
but is absent or small in 'slow' systems. Katz (1949) suggested that peripheral
inhibition might serve to bring about graded relaxation by a balance of motor and
inhibitor impulses.
Further knowledge of reflex responses involving inhibitor neurones is important
in considering the true role of peripheral inhibition in Crustacea. For example, are
the specific peripheral inhibitor neurones normally used in reflex inhibition of the
muscles which they innervate? To what extent is central inhibition of the appropriate
motoneurones involved ? A clearly defined reflex in many chelate decapods is that of
closing of the claw in response to touching its inner edge. In the present paper this
response will be seen to be accompanied by simultaneous reflex inhibition of the opener
muscle. From mechanical and electrical recording of the opener muscle response,
and from the corresponding activity in the opener motor and inhibitor axons, it will
be shown that this inhibition is mainly of peripheral rather than central origin. Further
analysis of the experimental records indicates a correlation between the degree of
fluctuation, or the 'phasicity', of the recorded tension and the amount of peripheral
inhibitory activity in evidence.
METHODS
The shore crab, Carcinus maenas (L.), proved the most suitable animal available
for these experiments. As in all Brachyura the pereiopod opener muscles receive
specific inhibitor innervation, and in this species reflex opening of the claw could be
relatively easily evoked. A few comparative experiments were performed on the crayfish, Astacus paUipes Lereboullet.
The animal was strapped on to a Perspex plate with its dorsal surface uppermost
and held down by means of wire loops over the limbs. Its walking legs were extended
laterally, and its chelipeds partially flexed so that their dactyli pointed anteriorly.
The plate was secured in a dish of filtered sea water, or van Harreveld's (1936)
crayfish saline for Astacus. Reflex opening of the crab's claw could then be elicited,
and usually maintained for several seconds by light stroking of, or pressure, on,
the lateral carpo-propodite joint membrane using a round-tipped metal probe. In
Astacus opening was evoked by stroking the dorsal surface of the propus. Reflex
closing, or inhibition of this opening, was then elicited by similar tactile stimulation to
the inside toothed edge of either the 'pollex' (the immovable ramus of the propus)
or the dactylus of the open claw. The beginning and end of each probe stimulus
was marked on the oscillograph by the amplified discharge of small capacitance on
make and break of contact between the probe and the moist integument.
Mechanical interference by the closer muscle in the response of the opener was
eliminated by exposing and transecting the closer motor axons just proximal to their
Reflex inhibition in crabs
73
flxst dichotomy (Text-fig. 1) 2 or more days before an experiment. To prevent autotomy of the experimental limb its coxo-basal levator tendons were severed before
operating. Although the closer tendon (apodeme) was left intact, the damping effect
of the closer muscle on the opener response was small, particularly since the latter
was recorded nearly isometrically. By thus reducing dactylus movement to a low level,
proprioceptive feedback from the joint receptor attached to the closer apodeme
(Burke, 1954) was also minimized.
Cf joint
Propus"
Text-fig. 1. Diagram of the bundles of efferent axons innervating the closer (ce) and opener
(oe) muscles of the left cheliped; the stretcher (se) and bender (be) axon bundles are also indicated. X, point at which the closer axons were transected; R, approximate electrode position
used in recording the opener axon responses.
The isometric lever used in most experiments was a 10x0-5 cm. flat metal spring
clamped at a variable distance from its moving end to permit stiffness adjustment. The
displacement of this end by the muscle was recorded with an RCA 5734 moving-anode
transducer, whose output was led to one beam of the oscilloscope. In some later
experiments a simple torsion-wire lever was used, the torque being recorded by
means of a phototransistor and lens system. Either lever was connected to the preparation through a 15 x 0-025 c m - length of copper wire, which was hooked into the dorsal
integument of the dactylus near the opener articulation, in such a way that its pull
was in line with that of the muscle. When the wire was tautened the dactylus rested
about one-third open. The total movement thus permitted was ca, 1 mm., or 5-8%
of the resting length of the muscle, which is small compared to its total possible length
change in situ of ca. 40 % of this length.
The extracellular electrical response of the opener muscle was recorded by means of a
single micromanipulated platinum wire electrode, the earthed saline bath providing an
'indifferent' electrode. In crabs it was sufficient to bring the end of the electrode into
contact with the moist intact integument over the opener muscle, but in Astacus it was
necessary to make contact with the muscle surface itself through a hole in the shell.
To record the reflex discharges in the opener axons, these were exposed and separated from the dorsal nerve trunk of the propus in the region of their earliest branches to
khe muscle (Text-fig, 1). The isolated axon bundle could then be raised in the hooked end
74
B. M. H. BUSH
of a platinum electrode and cut distally to simplify recording. When the simultaneous1
response of the opener muscle was required, however, the exposed but intact axon
bundle was raised just above the fluid level by slipping a small glass rod transversely
beneath it. The tip of a straight electrode was then brought into contact with the raised
portion of the axons, the surface tension of the fluid interface helping to maintain this
contact. Later an electrode was developed whereby it was possible to record the axon
responses beneath the fluid surface. This consisted of an Araldite-insulated platinum
wire running down the centre of a no. 22 hypodermic syringe needle. The axons made
contact with both sheath and centre-electrode while lying in a fine transverse groove
near the end of the needle.
Condenser-coupled Grass P4 preamplifiers were used to record the electrical
response of the opener muscle and axons, the mechanical response being led directly
into the second channel of the Cossor 1049 (mk. Ill) oscilloscope. Before exposing
the closer or opener axons for sectioning or recording, the mounted animal in its
saline bath was cooled in a refrigerator at o° to — 50 C. for 2-4 hr., this serving to
' anaesthetize' it and to reduce the rate of blood flow, thereby facilitating preparation.
RESULTS
A. Reflex inhibition of claw opening
In all preparations reflex opening of the claw with denervated closer muscle could
be reflexly inhibited by stroking the inside of the claw. Isotonic recording of the dactylus movement, used in some early experiments, sometimes failed to show this inhibition, perhaps due partly to the slight residual stiffness of the closer muscle, or to
proprioceptive feedback. Isometric recording, however, revealed reflex inhibition in
response to the large majority of' inhibitory stimuli'. The rate and degree of mechanical
inhibition varied considerably, particularly with the intensity of the inhibitory
stimuli, and inversely with the strength of the 'excitatory stimulation'. With repeated
inhibitory stimuli of similar intensities this rate generally declined, probably owing
mainly to central adaptation. The rate of inhibition was evidently also influenced by
the ' excitability' of the preparation and by other less well-defined factors.
Reflex inhibition of claw opening having been thus demonstrated, the question
arose whether this is brought about by central suppression of the opener motoneurone
response, or by peripheral inhibition through the specific opener inhibitor. Technically the simplest approach to this problem was to record the electrical response of
the opener muscle during such reflex inhibition. More direct evidence was subsequently obtained by recording the concomitant activity of the opener axons themselves.
The electrical response of the muscle
Extracellularly recorded oscillograms of the muscle-action potentials accompanying
the isometric response of the claw-opener muscle during excitatory and inhibitory
stimulation illustrates the mechanism of the observed reflex excitation and inhibition
(PI. 1, records A-G). During purely excitatory stimulation a broad correspondence
is evident between muscle tension and neuromuscular 'facilitation' as represented by
the heights of the muscle potentials. This correspondence appears both in the overall
development and mean level of the tension, and in the small individual fluctuations
Reflex inhibition in crabs
75
superimposed upon the mean tension. The mean tension increases as the mean height
of muscle potentials increases, that is, with the level of neuromuscular facilitation.
However, the tension is not proportional to the absolute magnitude of the facilitated
muscle potentials, owing partly to prolonged influence of peripheral inhibition
resulting from the inhibitory stimuli. In the small tension fluctuations the rate of
tension rise varies directly with the rate of muscle potential growth, which in turn
varies with motor frequency. Each declining phase in these fluctuations is commonly
preceded by a few attenuated muscle potentials indicating peripheral inhibition.
Each inhibitory stimulus usually elicited a sharp decline in tension, 30-200 msec,
after the start of the stimulus. This mechanical inhibition was accompanied by various
degrees of attenuation of the muscle potentials, the degree varying with the prevailing
frequency of muscle potentials (i.e. of motor impulses), and also no doubt with the
frequency of inhibitor impulses. Such attenuation indicates peripheral inhibition,
namely, 'supplemented' (or a) inhibition, in which an inhibitor impulse coincides
with a motor impulse at the neuromuscular junction or precedes it by a short interval
(ca. 0-10 msec.) (Wiersma & Heifer, 1941). Sometimes, however, little attenuation
was evident during mechanical inhibition (PI. 1, record B). These responses illustrate
the presence of ' simple' (or /?) inhibition.
Central inhibition of the opener motor discharge plays at most only a minor role
in bringing about the recorded mechanical inhibition during inhibitory stimuli. In
the first place, in many of the inhibitory responses there was no significant reduction
in motor frequency (PI. 1, records B, D). Secondly, where such a reduction was
apparent, as in about 30-40% of the recorded inhibitory responses (A, C), this might
have been due to complete suppression of the 'missing* muscle potentials by a
(i.e. peripheral) inhibition. Even if this were not the case, however, it is improbable
that any centrally imposed decrease in motor frequency of the order involved could
alone have brought about the observed rapid decline in tension. For the claw-opener
muscle in Carcinus is evidently capable of maintaining considerable tensions, once
achieved, on a relatively low frequency of motor impulses. Any tension decline
resulting from a pronounced decrease in motor frequency, even when this involves a
few particularly long intervals between impulses (of the order of 100-150 msec),
is always slow in the absence of peripheral inhibition (ends of records B, G, PI. 1).
In similar experiments on Astacus, reflex inhibition of claw opening was also effected
primarily by means of peripheral inhibition. Instances of both a and jS inhibition
in the opener muscle are common in the oscillograms, though the proportion and mean
reduction in height of attenuated muscle potentials is smaller than in Carcinus. Some
central inhibition of the motor discharge is also apparent in several of the inhibitory
responses, and probably contributed a little towards the tension decline. However,
even in these cases the relaxation was clearly initiated by peripheral inhibition.
Responses of the opener axons
When recording from the opener axon bundle at the proximal end of the muscle
during excitatory and inhibitory stimulation, two types of spike potential, differing
in height and sometimes also in form, were generally distinguishable. Inhibitory
stimulation alone normally elicited spikes of only one of the two types, often the larger,
excitatory stimulation evoked both the larger and the smaller spikes, though
76
B. M. H. BUSH
usually the latter predominated. Thus the two types of spike clearly represented impulses in the specific opener inhibitor axon and the single opener motor axon, respectively. This identification of the impulses was confirmed in two experiments by
separating the two larger axons in the opener efferent bundle and recording their
responses separately. The two axons were then stimulated individually showing which
was motor and which inhibitor.
During excitatory stimulation inhibitory stimuli caused a pronounced increase
in inhibitor frequency, and sometimes also a slight though variable decrease in motor
frequency. It was not uncommon, however, for the motor frequency to increase somewhat on inhibitory stimulation. Clearly the primary consistent reflex response to
inhibitory stimuli was a significant increase in inhibitor frequency. Any change in
motor frequency was evidently a secondary and much more variable effect.
In none of the experiments in which opener axon responses were recorded has
any convincing indication of activity in the opener branch of the common inhibitor
been observed. Owing to its small diameter (10-15 p, as against 40-50/x of the opener
motor and specific inhibitor axons), this branch was seldom seen clearly in the opener
axon bundle during the experiments, at ca. 20 x magnification. However, it was
undoubtedly normally present, as shown by the use of methylene-blue staining in
several of the experimental and other claws. It is, furthermore, unlikely that its
response was always too small to appear above the noise level of the recording
system. It seems, therefore, that in the responses studied in these experiments the
common inhibitor was not active.
Axon responses and muscle potentials
Simultaneous recording of the activity in the opener axons and the electrical
response of the muscle confirmed the above identification of the nerve impulses, and
illustrated the facilitation of muscle potentials and the a and /? inhibition previously
observed (PL 1, records H-L). Thus each motor impulse elicits a muscle potential,
following the recorded impulse with a delay of ca. 15 msec. At high motor frequencies
these muscle potentials grow markedly, to a maximum facilitated height of the initial
depolarizing phase. The ' diphasic' potentials which occasionally appear at high motor
frequencies may perhaps represent regenerative spikes in some or many muscle fibres
(K). This might indicate a greater degree of facilitation that is manifest in the depolarizing phase alone. Evidence on this point is, however, lacking.
As previous workers have observed, an inhibitor impulse alone causes no extracellularly measurable electrical effect. However, when in these records an inhibitor
impulse occurs between 15-20 msec, before and 2-3 msec, after a motor impulse, the
ensuing muscle potential undergoes attenuation. This becomes maximal when the
inhibitor impulse precedes the motor by 1-5 msec. (These values are quite similar to
those in Cancer; Wiersma & Heifer, 1941.) The overall amount of attenuation, therefore, and hence the proportion of a to /? inhibition, will increase with the inhibitor
frequency, a result which can be seen in these records (cf. H, J; PL 1). Furthermore,
attenuation of at least some muscle potentials must necessarily occur at inhibitor
frequencies as low as 50/sec, when the inhibitor and motor impulses occur at
random with respect to each other, as they appear to do. With inhibitor frequencies
of 150/sec. or more all muscle potentials will be considerably attenuated, giving almost!
Reflex inhibition in crabs
77
fbmplete a. inhibition. These estimates are largely borne out in the oscillograms of the
present experiments. Furthermore, these and subsequent experimental records show
that opener inhibitor (and motor) frequencies of this order are not uncommon. Higher
frequencies, up to ca. 200/sec. for two, three or four impulses in succession, do
occasionally occur. Thus in this species phases of complete a inhibition, as were
apparent in some of the previous experiments, are evidently a physiological possibility
(7, J; cf. A).
Occasionally in these experiments a strong ' outburst' of motor impulses occurred,
sometimes even during or immediately following an inhibitory stimulus. The resulting
muscle potentials manifested marked facilitation (PI. 1, records H, K, L). In preparations in which the muscle was still responding mechanically, such motor outbursts
were accompanied by vigorous claw opening. Often the inhibitor discharge frequency remained low or dropped further, suggesting central inhibition of the inhibitor neurone (H). Sometimes, however, the motor burst was quickly suppressed
by an equally strong inhibitor discharge (L). The motor burst itself appeared to be of
central origin, though it was probably somehow 'triggered' from without. Similar
motor outbursts of a central nature, often accompanied by silent periods in the inhibitor discharge suggestive of central inhibition of this neurone, have occasionally
been observed in the records of axon responses alone. The phenomenon will be discussed later in regard to central inhibition.
Axon responses and tension inhibition
Considerable difficulty was experienced, for undefined reasons, in obtaining simultaneous opener axon and isometric responses. Only two such experiments were
successful, and of these only one yielded satisfactory permanent records (PI. 1,
records M-O). Nevertheless, these records showed quite unequivocally that the reflex
inhibition of claw opening under consideration was brought about primarily by a greatly
enhanced discharge of the specific opener inhibitor neurone. That is, the reflex inhibition was essentially peripheral in nature. There was in fact no evidence in these
two experiments of any central inhibition of the reflexly elicited discharge of the opener
motoneurone.
A sharp increase in inhibitor discharge frequency occurred 50-100 msec, after the
start of each inhibitory stimulus, the interval presumably representing mainly central
latency. The inhibitory response comprised an initial 'burst' of 6 or 7 inhibitor impulses at high frequency (140-170/sec. in five inhibitory stimuli and 200/sec. in a
sixth), followed by 70-90 inhibitor impulses per second (120 in the sixth), continuing
throughout the inhibitory stimulus and sometimes for periods of up to 2 sec. after it.
Clearly these inhibitor frequencies were high enough to cause considerable muscle
potential attenuation, and even maximal attenuation during the initial burst. Prior
to each inhibitory stimulus the inhibitor frequency ranged from 15 to 30 impulses
per second. The ratio of motor to inhibitor impulse frequencies during the six inhibitory responses considered above was about 0-5 in the initial inhibitor burst, and
0-45-0-62 in the ensuing train, as compared with 1-3-2-6 during the 1 sec. preceding
the initial burst. The continuation of the relatively high-frequency inhibitor discharge
after the inhibitory stimulus explains the long 'after-inhibition' in the mechanical
response observed in some previous experiments (e.g. PI. 1, record C).
78
B. M. H. BUSH
The onset of tension decline followed the beginning of the inital high-frequenc^
inhibitor burst by a rather variable interval (peripheral latency) of 30-220 msec.
This latency appeared to depend upon the ratio of inhibitor/motor frequencies just
preceding the sharp increase in inhibitor discharge (Text-fig. 2). The point at which
the curve obtained cuts the vertical axis {ca. 270 msec.) presumably represents the
minimum time required for inhibitory facilitation to develop from zero to the effective
threshold level with the motor frequency that prevailed. The asymptote of the curve
(at ca. 30 msec.) probably represents largely the inhibitory coupling delay. It may
also be significant that the values of the ratio inhibitor/motor frequency around which
the curve appears to flatten off (o-6-o-7) approximate to the probable Rc value for this
muscle. (This value has not been determined for Carcinus's claw-opener muscle, but
280 \
E
~200
0
| 160
\ ^
c
0 120
5-
S 80
3
r—
0
0
1
1
0-1
02
1
1
1
1
1
0-3 0-4
0-5 06
07
Inhibitor/motor frequency
*
1
1
08
09
1-0
Text-fig. 2. Latency of mechanical inhibition as a function of inhibitor/motor frequency during
the 100 msec, preceding the effective high-frequency inhibitor discharge.
it probably lies between 0-5 and 0-7, as in other crabs: Wiersma & Ellis, 1942). In
any event, the general relationship represented by Text-fig. 2 illustrates an important effect of peripheral inhibition upon the opener muscle, to be considered later.
In contrast to the wide variation, by a factor of ca. 9, in the latency of tension inhibition, the average and maximum rate of decline of tension during the inhibitory
stimuli varied by a factor of only 1-4. No correlation was found between rate of
tension decline and inhibitor frequency, or ratio of inhibitor to motor frequencies,
although these also varied by a factor of ca. 1 -4. The reason for this is probably that
the ratio inhibitor/motor frequency (mean 1-7) was well above the Rc value for this
muscle. It is likely, therefore, that the higher rates of tension decline during the
inhibitory stimuli in these experiments were about maximal.
B. The effect of peripheral inhibition on the opener muscle
Tension decline without peripheral inhibition
The preceding section (A) dealt primarily with tension decline due to relatively
strong peripheral inhibitory activity, that is 'active' tension decline. It is of interest
to compare these records with records of 'passive' tension decline on cessation of
excitatory stimulation, and in the absence of the strong peripheral inhibition which
Reflex inhibition in crabs
79
ffesults from inhibitory stimulation. It should be recalled here that the continuous
motor and inhibitor activity seen in the oscillograms from this investigation was in
response to continuing'excitatory stimulation' (to the outer carpo-propodite articular
membrane of the cheliped). But the motoneurone and to some extent also the
inhibitor generally continued to discharge for several seconds following cessation of
this stimulation, the discharge frequency gradually diminishing, sometimes rather
erratically, to zero. Thereafter, and before onset of excitatory (or inhibitory) stimulation, when the preparation was at rest, both opener axons usually remained completely
'silent'.
As already seen, active decline of tension, once initiated, was commonly fairly
rapid and continuous throughout the strong inhibition resulting from the inhibitory
stimulation (Text-fig. 3J). In contrast, passive decline in these experiments was
always considerably more gradual, and was often also erratic and discontinuous
(Text-fig. 3 a-c). The more gradual decline was of course a result of continuing opener
motor discharge, coupled with a low or negligible continued discharge of the inhibitor.
In records where this decline after cessation of stimulation was fairly steady, there
1 sec
Text-fig. 3. (a—c) Passive tension decline (thick tension line), compared with (d) active decline during
inhibitory stimulation (bar.) Arrows indicate cessation of excitatory stimulation.
was commonly little or no muscle-potential attenuation, indicating low inhibitory
activity (c). Presumably, therefore, the form of the decline in these records was fairly
closely indicative of the diminishing level of excitatory facilitation.
When the decline was erratic, however, with transient rises in tension, this was
evidently a result of interaction of peripheral inhibition with the remaining excitatory
facilitation, and a consequent alternating dominance of excitation and inhibition
at the low frequencies involved. In addition, fluctuation of the tension appears in
places to have been aided by an almost rhythmic irregularity in the motor discharge
(a). Probable reasons for this rhythmicity and fluctuation will be discussed later.
The transient rise in tension, or at least the arrest of its decline, commonly following
only one or two muscle potentials in these records, suggests an increased 'sensitivity'
of the contractile process to motor impulses. In fact this effect probably represented
a restoration by these motor impulses of the coupling, broken by inhibition (cf. Hoyle
& Wiersma, 1958), between the still-facilitated excitation and the contraction.
Tension development
Large and rapid rises in tension in this investigation were generally brought about
fjy brief, relatively high-frequency bursts of motor impulses—or rapidly facilitating
80
B. M. H. BUSH
muscle potentials (Text-fig. 4; PL 1, records E, G, O). Each burst commonly com"
prised three to six impulses, though occasionally two in quick succession were effective.
It can be shown graphically that in general the rate of rise of tension is directly related
to the frequency of the effective motor impulses (or muscle potentials). Moreover,
while similar rates of rise are produced by similar mean frequencies in the effective
motor bursts, the actual sequence or 'pattern' of impulses in the burst is relatively
unimportant in determining the rate (Text-fig. 4). That is, while this muscle responds
to different motor frequencies in a typically crustacean manner, its neuromuscular
junctions are evidently not significantly 'pattern sensitive'. The same has been found
true of several other opener and stretcher nerve-muscle systems, and also of 'slow'
systems (Wiersma & Adams, 1950).
Text-fig. 4. Tension rises resulting from various groupings of muscle potentials
(i.e. of motor impulses).
As it is to be expected the linear relationship between rate of tension rise and motor
frequency does not obtain when inhibitor impulses occur during the motor burst.
In addition, however, when a relatively high inhibitor frequency occurs during the
100-200 msec, immediately preceding the effective motor burst, the rate of rise of
tension is diminished. This is most clearly seen in the tension and axon response
records, when an arbitrary period of 100 msec, preceding the first motor impulse of
the burst eliciting the tension rise is considered. Those tension rises whose effective
motor bursts are preceded by an inhibitor/motor frequency ratio less than 2/3 are
approximately linearly related to the motor frequency, while those with a ratio greater
than 2/3 fall below this line (Text-fig 5). It is possible that the value 2/3 for this ratio
is equal to, or perhaps greater than, the Rc value for this nerve-muscle system, that is,
the value required to produce complete mechanical inhibition when the motor and
inhibitor axons are stimulated simultaneously. A similar result was evident in the
muscle potential records, where pronounced attenuation just before an effective
motor burst indicated a relatively high preceding inhibitor/motor frequency ratio.
During each tension rise in records M, N, O (PI. 1), a burst of inhibitor impulses
occurred, their frequency somewhat below that of the motor impulses eliciting the
rise. These bursts began shortly after the increase in tension started, and continued,
often at diminishing frequency, until after it ended. Apparently the tension rises were
brought to an end by these inhibitor bursts. Evidence of similar inhibitory bursts L"
Reflex inhibition in crabs
81
The muscle potential records is commonly seen in the form of brief intervals of greatly
attenuated potentials towards the ends of the rising phases (PI. i, records B, F, G).
The significance of this inhibitor activity during tension rises will be discussed later.
A further effect of peripheral inhibition is illustrated in records D-F (PI. i).
Normally a high-frequency group (i.e. a burst) of several motor impulses is required
to cause a substantial rise in tension. Nevertheless, under certain conditions two or
three widely spaced motor impulses, or even only one, may cause a significant tension
rise, albeit transient. As observed in the preceding section the muscle manifests an
increased 'sensitivity' to motor impulses, which is probably attributable to interaction
100 r
Jj 80
60
s
"o
V
£ 20
40
80
120
160
Motor frequency (Impulses/sec.)
200
Text-fig. 5. Rate of rise in tension as a function of frequency of the motor impulses evoking the rise.
Ratio of inhibitor/motor impulses during the 100 msec, period preceding each motor burst;
(O) < ! ; ( • ) >f. Horizontal lines: possible error in measurement of motor frequency.
between excitatory facilitation and inhibition. In the absence of inhibition the tension
would probably have remained at the higher level of its fluctuations. Intermittent
groups of inhibitor impulses partially' decouple' the motor facilitation, thereby depressing the tension temporarily, until the next motor impulses arrive. Thus, in effect,
peripheral inhibitory activity appears to increase the sensitivity of the muscle to motor
impulses.
Tension maintenance and fluctuation
While some of the oscillograph records from the experiments of this study show
remarkably steady tensions (PI. 1, record G), others manifest considerable flucutation
in the tension level (PI. 1, records A, D). Some preparations displayed predominantly steady tensions, others predominantly fluctuating. In the former, the slight
stiffness of the intact but denervated closer muscle might have had a damping effect
on the recorded relaxation of the opener. Less isometric recording would have
enhanced such an effect. Nevertheless, since both the residual stiffness of the closer
muscle and the degree of movement permitted by the 'isometric' recording were
6
Exp. Biol. 39, 1
82
B. M. H. BUSH
small, and similar in different preparations, it is unlikely that these two factors coula
alone have been responsible for the considerable differences in steadiness of the
recorded opener tension. Moreover, both steady and fluctuating records were sometimes obtained from a single preparation (B), and intermediate degrees of fluctuation
were not uncommon (F). Other factors were therefore probably involved. The
question now arose whether any variables in the experimental records could be correlated with tension.
Table i. Frequencies of opener muscle potentials accompanying steady, maintained
and non-maintained, and fluctuating tensions
Average*
Moderate tensions (10) ("Steady, but not
-( quite maintained
(30-70 % of maximum)
(.Maintained
High tensions (5)
Maintained
( > 70 % maximum)
Control: moderate
Not maintained
and high (22)
(fluctuating)
Instantaneous!
Mean
Range
Mean
Range
12-6
8-23
IO'2
7-i S
21-5
24-8
14-29
17-8
21-27
2O-2
3I-S
20-48
13-23
18-20
—
• Average frequency (per sec.) for 250—750 msec, periods.
t Reciprocal of longest interval between muscle potentials accompanied by steady tension.
( ) no. of 250—750 msec, periods considered.
Analysis of these records shows that steady tensions were commonly associated with
relatively low motor, or muscle-potential frequencies, of the order of 15-20/sec.
(PL 1, records B, G; cf. Table 1). In these records a steady tension level was occasionally held for up to 100 msec, following a muscle potential. Evidently this muscle is
capable of a high degree of summation, such that tetanic contractions can occur with
motor frequencies as low as 10-15 impulses/sec. In other records, however, low
motor frequencies are accompanied by fluctuating tension (D). Moreover, high motor
frequencies, however irregular, do not necessarily lead to tension fluctuation (G).
Clearly a further factor is involved in tension fluctuation. This is peripheral inhibition.
The proportion of attenuated muscle potentials is in general greater in records
displaying predominantly fluctuating tension than in comparable records with mainly
steady tensions but with similar mean tension levels and muscle potential frequencies
(cf. records D and G, PL 1). That is, there appears to be a positive correlation between
degree of tension fluctuation and amount of peripheral inhibitory activity in evidence.
Closer inspection of the records shows that the falling phases of the tension fluctuations
are generally preceded by brief periods of considerable attenuation of muscle potentials
indicating high-inhibitor/motor-frequency ratios, these no doubt being responsible
for the declines in tension. Thus the tension fluctuations may have been due entirely
to the higher inhibitor frequencies during these periods. These in turn may have been
due to greater grouping of inhibitor impulses and/or higher overall inhibitor frequencies in the fluctuating tension records than in the steady tension recordings.
However, while the tension fluctuations certainly are often associated with pronounced grouping of inhibitor impulses (record B, PL 1), they also appear sometimes
when grouping of inhibitor impulses is not clearly evident (Z)). Moreover, tension
fluctuation does not always occur when inhibitor impulses are well grouped. The
Reflex inhibition in crabs
83
brief periods of several attenuated muscle potentials which often accompany or follow
tension rises are not always immediately followed by tension decline (G). Furthermore, impulse-frequency determinations in the axon response records indicated that
the less steady tension plateaux are associated not with higher 'instantaneous'
values of the ratio inhibitor/motor frequency, but with higher absolute values of the
overall inhibitor (and motor) frequency. These rather tentative observations suggest
that there may be a second, indirect and less obvious influence of peripheral inhibition
on the stability of the recorded tension. This will be discussed later.
DISCUSSION
(A) It has been found that, in the experimental situation studied here, practically
all reflex inhibition of reflex opener activity takes place peripherally, through the
specific opener inhibitor axon, and not centrally by inhibition of the motoneurone.
Control experiments to record the opener axon responses to the same stimuli in
preparations with the closer muscle and its innervation intact showed that similar
peripheral reflex inhibition does indeed accompany active reflex closing. There is,
therefore, no doubt that it occurs in the normal animal despite the somewhat different proprioceptive feedback involved. Furthermore, there is no conclusive evidence
from this investigation that the opener motoneurone can be inhibited centrally.
The pereiopods of all DecapodaReptantia except Palinura each possess a 'specific'
opener inhibitor neurone (Wiersma & Ripley, 1952; see Text-fig. 1, Bush, 1962).
Thus there appears to be no a priori reason why in these animals all reflex—or other—
inhibition of' opening', in claws and in walking legs, should not be peripheral in nature.
In fact the common opener-stretcher motor innervation confers on peripheral inhibition a definite advantage over central inhibition, except perhaps where simultaneous
inhibition of both these muscles is required. The same argument applies, of course,
to the 'stretcher' muscles of all Reptantia, since they also receive specific inhibitor
innervation. However, the other pereiopod muscles can be individually inhibited only
by central inhibition of their motor neurones, since their only inhibitor innervation is a
branch of the 'common inhibitor', which supplies all these muscles (Wiersma &
Ripley, 1952).
In contrast to the opener-stretcher motoneurone, the opener inhibitor does appear
to be subject to central inhibition. For instance, on those occasions when a
'spontaneous' burst discharge occurred in the motoneurone, this was often accompanied by a pronounced slowing down or brief cessation, for 100-300 msec, in the
moderately high-frequency inhibitor discharge. Similar inhibitor 'silent' periods
were not infrequently observed during the brief motor bursts eliciting large and rapid
tension rises. On the other hand, the motor discharge never exhibited silent periods of
this kind, again indicating absence of central inhibition.
Another instance of central inhibition of the claw-opener inhibitor in Carcimts
was observed in an experiment in which the responses of the opener inhibitor were
recorded during tetanic stimulation of either circum-oesophageal commissure. Such
stimulation elicited a discharge of 10-20 impulses per second in the opener motor
axon. Any existing discharge in the opener inhibitor, for instance, in response to
stroking the inside of the claw, or to holding it fully open (Bush, 1962), was quickly
suppressed when commissure stimulation commenced, but resumed again on cessation
6-2
84
B. M. H. BUSH
thereof. Conversely, during commissure stimulation, claw stroking or passive opening
elicited at most but a few inhibitor impulses, in contrast to the usually strong reflex
response of the inhibitor to these stimuli.
Several instances are apparent in the oscillograms from the present study in which
a strong discharge of the opener inhibitor followed closely upon a similarly strong,
spontaneous discharge of the motoneurone. These 'following' responses in the inhibitor occurred even when there was no mechanical response, so that they could not
have been a result of any proprioceptive feedback. Rather did the inhibitor seem to
exhibit a central 'tendency' to discharge together with the motoneurone. Such an
inbuilt tendency would safeguard the opener muscle against excitation whenever the
stretcher was activated, but would necessitate central inhibition of the opener inhibitor to permit opener excitation. Central inhibition of this inhibitor has already
been seen to occur. A simple mechanism for automatic following of motor by inhibitor discharge might lie in excitatory synapses, with appropriate facilitatory properties, of motor axon branches on the inhibitor neurone. However, the existence
and nature of any such motor-inhibitor coupling remains speculative, and must await
experimental evidence.
Both from theoretical considerations and from the preceding experimental observations it is clear that central inhibition must play an important part in the reflex
activity of Crustacea. Other instances of central reflex inhibition in Crustacea have
been observed in ' Sherrington's interneurone' in the oesophageal commissure
(Wiersma, 19586), and in the abdominal nerve cord of the crayfish (Hughes &
& Wiersma, i960). Single motor fibres to the abdominal swimmerets in the crayfish
have been found to be inhibited by stimulation of certain small axon-bundles in the
commissure, as well as being excited by others. Sherrington's fibre in the crayfish
is excited by coxo-basal extension in the homolateral walking legs and flexion of the
heterolateral ones, and is inhibited by the opposite movements. Central reflex
inhibition has also been observed in insects, as for instance in the mutually antagonistic leg depressor and levator reflexes in the cockroach (Pringle, 1940). Reflex
excitation of the extensor trochanteris is accompanied by simultaneous inhibition
of motor activity in the 'antagonistic' extensor tibiae, and vice versa. In the absence
of peripheral inhibition in insects, of course, reflex inhibition must necessarily be
central. It is now evident that most reflex inhibition in Crustacea must also be
central, the exceptions being those muscles provided with specific inhibitors. More
evidence of peripheral reflex inhibition in these muscles will be presented in a subsequent paper (Bush, 1962).
(B) The oscillograms from this investigation exhibit a wide range of stability of
the recorded tension. This variation was not attributable simply to differences between
preparations in the degree of mechanical damping of the opener response, or in the
'isometricity' of recording. The more stable tensions were often accompanied by
low motor frequencies, but fluctuating tension was commonly associated with relatively high inhibitor frequencies. It is possible that the variation in tension stability may
have resulted from differences in inhibitor activity. These differences may have arisen
from several possible sources.
The intensity of the 'excitatory stimulation' employed, being difficult to control,
varied considerably, giving rise to roughly corresponding variations in the response
Reflex inhibition in crabs
85
frequencies of both motoneurone and inhibitor. This variation was similar in
different preparations, however. Strong 'inhibitory stimuli' also elicited similar
inhibitor frequencies in different preparations. Thus, while variation in both excitatory and inhibitory stimulation intensities resulted in varying response frequencies
in each preparation, this variation was not significantly responsible for differences
in motor or inhibitor frequencies between preparations. However, differences in
sensory sensitivity, or in the number of afferents stimulated, may have been partially
involved. In addition, the central' excitability' of the two efferent neurones, particularly the inhibitor, appeared to differ somewhat in different preparations. For example,
the preparation represented in PI. 1 by record G never exhibited as strong or prolonged
a discharge of the inhibitor as was recorded in C, from another preparation, although
the two animals displayed similar responses to similar inhibitory stimuli. There
were thus strong indications of differences in inhibitor activity independent of the
stability of the opener-muscle tension.
Nevertheless, some of the peripheral inhibition recorded in these oscillograms clearly
was correlated with the recorded fluctuations in tension. It has been observed that
tension rises were almost invariably accompanied or immediately followed by relatively high-frequency inhibitor impulses. In the light of the succeeding paper (Bush,
1962), it is probable that these inhibitor discharges were evoked by proprioceptive
feedback elicited by the slight opening movement of the dactylus which occurred
with each rise in tension—the mechanical recording not being completely isometric.
Thin of course assumes that active opening elicits a proprioceptive reflex response
similar to that elicited by passive opening; though likely, this has not yet been demonstrated experimentally. It is now evident that the more the tension fluctuates in
the present experiments, the more inhibitor activity of proprioceptive origin there
will be.
Although in these oscillograms tension rise was almost always accompanied by
proprioceptive reflex discharge of the inhibitor, this did not always result in tension
decline, and hence eventually in fluctuation of the tension. Furthermore, there is
no clear evidence that this feedback inhibition was stronger in just those cases where
tension decline did follow it. Nor is it likely that the strength of the feedback differed
greatly in different preparations, particularly since neither the isometricity of recording nor the angle at which the carpo-propodite joint was bent varied significantly.
(The position of the propus affects the strength of the proprioceptive reflex response
to dactylus movement; Bush, 1962.) Evidently, therefore, neither the tension fluctuation themselves, nor the differences between preparations in the stability of
the tension, were due simply to the inhibition resulting from this proprioceptive
feedback.
Certain observations in the last section of the results suggested that peripheral
inhibition may have had a second, indirect influence on the stability of the recorded
tension in these experiments, in addition to its more obvious action of suppressing
the tension directly. In section (A) of the results it was seen that the delay with which
mechanical inhibition followed a sudden high-frequency inhibitor discharge depended
upon the ratio of inhibitor/motor frequency immediately preceding the strong
inhibition causing the tension decay. As this ratio increased the delay decreased,
reaching a minimum with a ratio of approximately 7/10 (see Text-fig. 2). That is, the
86
B. M. H. BUSH
greater the ' background' inhibitor activity the shorter this delay. This effect of pen
pheral inhibition may underlie the second indirect influence on tension stability
referred to above. For greater background inhibition would then increase the probability of tension decline occurring as a direct consequence of a brief burst of inhibitor
impulses. In different terms the effect might be regarded as one of increasing the
' sensitivity' of the muscle to inhibitor impulses. In addition, it has already been noted
that continuous peripheral inhibitory activity appears to increase the sensitivity of
the muscle to motor impulses, in such a way that one or two motor impulses become
capable of eliciting a tension rise which, in the absence of inhibition, could be evoked
only by a burst of several motor impulses.
Both these effects are probably attributable to the existence of a balance between
sustained motor and inhibitory facilitation. Under these conditions the resultant
tension level may be lower than it would be for the same motor frequency in the
absence of the inhibitor activity. In any event these two effects of peripheral inhibition would enhance any tendency for the tension to fluctuate, as a direct consequence
of irregularities in the motor or inhibitor discharge frequencies. Any such tension
fluctuations would, in turn, lead to greater and more varying activity of the inhibitor
—and motor—neurones, owing to the proprioceptive feedback elicited by the concomitant movements of the dactylus. Thus an 'oscillating' system may well arise
under these conditions, unless other opposing influences were brought to bear upon
it. Whether or not such oscillation and tension fluctuation occurred might depend
largely upon the presence of a level of inhibitory facilitation high enough to ensure
that tension decay follows the onset of high-frequency inhibition with little delay.
Immediately after a brief phase of strong inhibition the tension would then rise again
to the level representing the prevailing balance between excitation and inhibition.
The rate and amplitude of such tension rise could be enhanced by grouping of motor
impulses. A further observed effect of the sustained facilitation of inhibition referred
to above was that caused by moderately high inhibition preceding a motor impulse
burst eliciting tension rise, namely, partial suppression of the rate of this rise. While
not influencing the frequency of tension fluctuations this effect might reduce the
amplitude of the fluctuations, thus antagonizing the effect of motor impulse grouping.
These theoretical considerations correspond reasonably well with the experimental observations from the muscle-potential and axon-response recordings and
associated tension variations. They suggest that the claw-opener muscle of Carcinus,
and perhaps also other crustacean muscles which behave rather 'tonically' in the
absence of peripheral inhibition, may tend to behave more ' phasically' in the presence
of a background discharge of the inhibitors innervating them. This could prove to be
of considerable functional importance. Further critical experiments are required to
test this hypothesis.
SUMMARY
i. Reflex opening of the claw of the crab, Carcinus mamas (or of the crayfish,
Astacus pallipes), elicited by mechanical stimulation at the carpo-propodite joint, can
be reflexly inhibited by stroking the inside of the claw. By electrical and mechanical
recording from the opener muscle (the apodeme or motor axons of the antagonistic
closer muscle being cut), and by electrical recording from the motor and inhibitor
Reflex inhibition in crabs
87
Rons to this muscle, it is shown that this reflex inhibition is effected mainly peripherally, through the inhibitor axon specific to the opener muscle. Central inhibition of
the motor axon plays at the most a minor part.
2. The externally recorded action potentials of the opener muscle show strong
peripheral inhibition of both the a ('supplemented') and j3 ('simple') forms (i.e.
with and without attenuation of muscle potentials, respectively) during the reflexinhibitory stimuli, as well as sporadic attenuation at other times. Attenuation of a
muscle potential results, in Carcinus claw opener, when an inhibitor impulse precedes
the motor impulse by less than 15-20 msec, and is maximal when this interval is less
than approximately 5 msec. Thus a inhibition predominates over /? inhibition at the
higher frequencies of discharge.
3. The frequency of impulses in the opener motor axon commonly ranges from 20
to 80/sec. with brief bursts of 80-200/sec. during reflex contraction of the opener
muscle, and does not fall significantly during reflex-inhibitory stimulation. The opener
inhibitor, which often manifests an irregular low-frequency 'background' discharge
during reflex opening, responds to concurrent reflex-inhibitory stimulation with a
sharp increase in frequency to 100-200 impulses/sec, initially and falling to 50-100/sec.
with continued reflex inhibition. Reflex-inhibitory stimulation alone elicits only inhibitor impulses.
4. Following cessation of stimulation the tension declines 'passively'. Owing to
continuing motor activity, however, this decline is often slower than during active
inhibition; it is often also erratic due to the occurrence of inhibitor impulses.
5. When a relatively high ratio of inhibitor/motor impulse frequencies precedes a
burst of motor impulses eliciting a rise in tension, the rate of this tension rise is lower
than that excepted from the frequency of motor impulses evoking the rise.
6. When there is a relatively high-frequency 'background' discharge in the opener
inhibitor the tension in the muscle shows rapid fluctuations, whereas with lower
inhibitor frequencies fairly steady tensions are sustained. This is in part because the
peripheral latency of mechanical inhibition varies inversely with the ratio of inhibitor/
motor frequencies preceding the inhibition.
It is suggested that a background of peripheral inhibition, by making possible
quicker relaxation, may be an important factor in rapid movement.
I am grateful to Prof. J. W. S. Pringle, F.R.S., for his advice and encouragement
during the course of this work, and to Prof. C. A. G. Wiersma for critically reading
the typescript. My thanks are also due to Prof. D. W. Ewer, who introduced me to
this problem. The work was carried out during the tenure of a Union Scholarship
(Union of South Africa).
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3USH, B. M. H. (1962). Proprioceptive reflexes in the legs of Carcinus maenas (L.). J- Exp. Biol. 39,
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EXPLANATION OF PLATE
Recordt A-G. Oscillograms of isometric and extracellular reflex responses of the claw-opener muscle
to continuous 'excitatory stimulation' and interposed 'inhibitory stimuli'.
Records H-L. Opener axon (lower) and extracellular (upper) responses to excitatory and inhibitory
stimulation, separately (H, I) and concurrently (J-L). Smaller impulses, motor; larger, inhibitor
(marked with a dot below each impulse: amplitude change in K was an artifact due to fluid movement).
Records M-O. Opener axon and isometric responses to excitatory stimulation (M) terminated at arrow,
(N) initiated at arrow, and (0) with three interposed inhibitory stimuli. Smaller impulses, motor;
larger, inhibitor; the variation in height of the impulses in M was due to fluid movements.
These records are from five different preparations: (1) A-B, (2) C-F, (3) G, (4) H-L, (5) M-O.
Arrows: upward, start; downward, end of excitatory stimulation (when present within the portions
shown). Horizontal bars: inhibitory stimuli. The lowest level of the tension trace in each record represents zero tension, the highest is almost maximal for the preparation (except in F, N). Calibration:
ca. 10—20 g. tension; ca. 1—2 mV. amplitude of muscle potentials; and cd. 5 mV. axon impulses. Time:
(A) 2 msec.; {B-G, M-O) 1 sec.; (H-L) 0-4 sec.; oscillations in tension trace: 50 eye./sec.
Journal of Experimental Biology, 39, No. 1
t"'
Plate 1
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B. M. H. BUSH
. 88)