Download the physiology of a lepidopteran muscle receptor

J. Exp. Biol. (1966), 45, 229-249
With 2 plates and 12 text-figures
Printed in Great Britain
229
THE PHYSIOLOGY OF A LEPIDOPTERAN
MUSCLE RECEPTOR
III. THE STRETCH REFLEX
BY R. DE G. WEEVERS
Zoological Laboratory, University of Cambridge
{Received 23 March 1966)
INTRODUCTION
The proprioceptive equipment of mammals is the most elaborate of any animal
group. Nevertheless, muscle receptors not unlike the characteristically mammalian
muscle spindles are also found among the arthropods, namely in decapod Crustacea
(Alexandrowicz, 1951) and in three orders of insects (Finlayson & Lowenstein, 1958;
Osborne & Finlayson, 1962). Lowenstein & Finlayson (i960) showed that the lepidopteran muscle receptor (MRO) signals both phasic and tonic parameters of imposed
movements, and in a quantitative study of the same sense organ (Weevers, 19666)
it was found that an individual MRO of a caterpillar provides information hardly less
detailed and precise than does a mammalian spindle (Matthews, 1963). The present
study was undertaken in order to discover whether the analogies between the two
systems also extend to the manner in which muscular activity is controlled by stretch
reflexes.
The arthropod reflexes which have received most attention to date are concerned
with 'startle' responses. Thus Pumphrey & Rawdon Smith (1937) described the
excitation of large fibres in the abdominal nerve cord of the cockroach by the action
of air currents on the anal cerci, and Hughes (1953) described similar 'giant' fibres
in the dragonfly nymph. It has since been confirmed that both of these systems are
involved in evasion responses (Roeder, 1948; Fielden, i960). Similarly, Wiersma
(1947, 1949, 1952), among others, has described the physiology of the giant-fibre
system of the crayfish. In each case activation of a sensory pathway, concerned with
detection of environmental changes likely to culminate in noxious situations, stimulates
a rapidly conducting interneurone system with a widespread motor field. Activation
of these interneurones then results in movements appropriate to escape which override other current activities of the animal.
There is less information available about the functioning of those central nervous
mechanisms of a less specialized kind which are involved in reflex adjustment of
posture and locomotor activity. Pringle (1940) described depressor and levator
reflexes in the cockroach leg. He also found that forced flexion of the coxo-trochanteral
joint on one side produced slight but definite depressor inhibition on the other side.
The sense organs involved were presumed to be campaniform sensilla, since pressure
on appropriate regions of the cuticle evoked similar effects, whereas tension on various
tendons did not. He was unable to show any effects on flexor-extensor balance in
15
Exp. Biol. 45, 2
230
R. DE G. WEEVERS
segments other than that stimulated. In contrast, Hughes (1957), who used amputation
and transection of connectives in conjunction with cinematography on the same
animal, had to postulate intersegmental connexions to explain his results. Wilson
(1965) evoked this same stretch reflex using sinusoidal stimulation and found that on
occasion the system could be driven to respond at 30-40 cyc./sec. Thus the duration
of muscle contraction, rather than reflex properties, limits the phasic capabilities of
the legs of cockroaches.
Although the studies of Bush (1962, 1963) were performed on decapod Crustacea
and were concerned with reflex responses to stretch of chordotonal sense organs in the
thoracic appendages, his results are very similar to those which will be described here.
He showed the presence of negative feedback proprioceptive reflexes in a number of
reptantian groups and analysed these more thoroughly than has yet been done for any
invertebrate group. These reflexes are precisely analogous to the myotatic reflexes of
mammals. The peripheral picture is of course considerably complicated in the
Crustacea by the occurrence of neuromuscular inhibition. A large part of Bush's
studies was concerned with muscle antagonism and the respective roles of central and
peripheral inhibition in the control of crustacean locomotion.
Other recently published reflex studies are less directly relevant to the present work
and will only be briefly mentioned- Wilson & Gettrup (1963) described a proprioceptive mechanism controlling wingbeat frequency in grasshoppers; this is a slowly
acting tonic reflex. Fielden (i960) and Mill (1963) demonstrated segmental reflexes in
nymphs of Anax and Aeschna respectively following electrical stimulation of afferent
nerves and Van der Kloot (1963) studied the reflex and other co-ordinating influences
on the spiracular muscle of the pupa of Hyalophora cecropia.
MATERIALS AND METHODS
Last instar larvae of Antheraea peryni were used almost exclusively in the present
investigation. (In one experiment it was necessary to use the pupa). Experiments
performed on larvae required the use of those which had not yet ceased feeding, since
when the caterpillar starts to spin its cocoon it becomes more active, and consistent
stretch reflexes are difficult to obtain. The feeding larva normally spends long periods
motionless except for its jaws and head. The same was found to be true of acute preparations dissected in the manner previously described (Weevers, 1966a). Initially the
sensory discharge from the MRO used to elicit stretch reflexes was monitored continuously. Later it became apparent that the afferent responses to a given stimulus
were so consistent that there was no need for this. It was quite sufficient to check
that the sense organ was functioning properly from time to time. The apparatus used
to stretch the MRO in a controlled manner has already been described (Weevers,
19666). The 'stretcher' mechanogram was displayed as a d.c. signal on the lower
beam of the oscilloscope. The same beam could also be used to display muscle or other
action potentials at the same time.
Muscle activity was recorded intracellularly with fine glass-insulated platinum wire
electrodes of the type described by Ballintijn (1961). The rather obtuse-angled tip
was extremely robust (one electrode lasting for many experiments) and did not usually
penetrate through more than one layer of fibres, even when sufficient pressure was
The physiology of a lepidopteran muscle receptor
231
exerted to bend the thin shaft of the electrode. The Pyrex glass insulation was discontinued about 1 cm. from the tip, leaving a length of about 60 fi of the 12 p diameter
platinum-iridium wire core bare of the staff and springy coating. This gap acted as a
pivoting point when the tip moved laterally. When the electrode was advanced so as to
bow the long shaft, movements of the tip could be tolerated up to 4 mm. laterally
and 1 mm. vertically without displacing the electrode from its intracellular location.
Text-fig. 1. The main muscle groups in abdominal segments 4, 5 and 6 of the caterpillar of the
goatmoth, Cottus ligmperda. Reproduced from Lyonet (1762). The top segment is the most
anterior.
When it was desired to see the shape of an action potential relatively undistorted a
coupling time-constant of one second was used in the preamplifier. Otherwise a 0-002
sec. time-constant was found more convenient, as it eliminated most movement
artifacts cut down hum and showed the mechanogram signal to better advantage.
Electronic apparatus was of the conventional type and has already been described
(Weevers, 1966 a).
Nomenclature
Unlike most other animals utilizing a hydrostatic skeleton, the caterpillar does not
possess antagonistic circular and longitudinal muscle layers. Instead, bundles of
longitudinally and diagonally oriented muscle fibres are interwoven in an extremely
complex fashion; and in addition there is a discontinuous superficial layer of relatively
short muscles (here called integumentary muscles) which are oriented in every direction possible. This adds up to a body musculature more complex than in any other
insect. Lyonet (1762) described the morphology of Cossus Ugniperda caterpillars.
Part of one of his excellent figures is reproduced in Text-fig. 1. Because of its objectivity
and brevity the same nomenclature will be used in the present work. However, to
R. DE G. WEEVERS
232
assist in visualizing the location of muscles being discussed, such descriptive terms as
'dorsal longitudinal muscles' may also be used where this is helpful.
RESULTS
(i) Afferent and efferent conduction pathways
Von Hoist (1934) found that section of a single connective in the abdominal cord
produced flaccid paralysis of the dorsolateral part of the segment immediately posterior
and ipsilateral to the cord hemisection. Whereas stimulation of the integument in this
Record
MRO
Muscle
group E
Record -
Stimulate
Stimulate
"I
Stimulate
I I
Text-fig. 2. (a) Antddromic stimulation of the MRO sensory axon. Electrical stimulation at
each of the arrows produced a 1:1 response at the MRO. (6) The path followed by motor
axons of nerve 2 (see text). Electrical stimulation at each of the points marked by an arrow
produced a 1:1 response in those muscles innervated by nerve 2. In this and in similar subsequent diagrams, anterior ifl to the right. The asterisks show the position of the spiracles.
region could still produce responses in other segments, no muscles could be induced
to contract here by any form of mechanical stimulation. In the absence of more
specific information, von Hoist interpreted this as showing that the paired motor
' centres' for these muscles are situated in the ganglion of the next anterior segment.
Kopec (1919) had observed similar effects but did not comment on them. Since
pathways of this kind would appreciably increase afferent and efferent conduction
times, electrical stimulation of motor and sensory axons was used, together with
histological techniques, to examine this question.
As mentioned in a previous paper (Weevers, 1966c) the sensory axon from the MRO
runs for a short distance separate from all other nerves. Extracellular electrodes could
therefore be used to record action potentials in this single axon when it was stimulated electrically anywhere along its course. Owing to the polarized nature of all but a
very few synapses, this means that one can map the path of the axon into the c.N.s.
Text-fig. 2 a, shows the results of such an experiment. Measurements of the latencies
of responses to stimulation gave, by difference, the conduction time between each of
the arrows. Distances were measured with a micromanipulator having a vernier scale
reading to tenths of a millimeter. The conduction velocity was nearly constant (between
o-8 and 0-9 m./sec.) along the greater part of the three branches of the axon. However,
The physiology of a lepidopteran muscle receptor
233
impulses travelled faster than 2 m./sec. along the anterior intracentral branch within
the ganglion of entry into the C.N.s. No action potentials were recorded in the MRO
sensory axon when stimuli were delivered to the contralateral connectives or to connectives anterior or posterior to the three ganglia shown in Text-fig, za.
Text-fig. 26 shows the results of a similar experiment, where a micro-electrode
was used to record intracellularly from muscles while various points along the possible
paths of the motor axons were stimulated. In this kind of experiment it is more
difficult to achieve the same degree of certainty about the courses of the axons concerned than was possible with the MRO axon; since conduction is orthodromic, any
0-19 cm.
stretch
SO mV.
1 sec
Text-fig. 3. Oscillograph records of action potentials in two muscle groups, showing the
effect of stretching the ipsilateral MRO. (a) was taken from group / and (6) from group G
(see Text-fig, u ) . The time-constant of the reamplifier was set at one second. The second
beam shows the movement of the stretching forceps.
synapse would transmit impulses and one might obtain responses to stimulation of
interneurones. In fact stimulation further afield than the three arrows in Text-fig, zb
did sometimes excite muscles, but the i: i relation between stimulus and response
broke down at frequencies above 2o/sec. Furthermore, careful measurements of
latencies revealed ganglionic delays over and above axon conduction time of 1-2 msec,
in such cases. In contrast, it was not possible to show any ganglionic delay when recording from muscles responding in a 1:1 manner at over 100 stimuli/sec.; such
high-frequency following was seen only with stimulating electrodes located at the
arrows in Text-fig, zb. The conduction velocity along pathways of the latter type was
around 2 m./sec., again being somewhat higher within the ganglion (2-3 m./sec). This
experiment was performed on many different muscles and it became apparent that
only those innervated by nerve 2 behaved as though their motor axons passed along
the ipsilateral connective from the next anterior ganglion. (Nerve 2 is the anterior of the
R. DE G. WEEVEBS
234
two true segmental nerves, nerve i being a branch of the stomatogastric system.) It
was also found, in agreement with von Hoist (1934), that section of a single connective
paralysed about two-thirds of the muscles in the half segment behind the cut. These
were all innervated by nerve 2, and action potentials could not be elicited in such
muscles by any form of sensory stimulation.
Further light was shed on the question of the course of the motor axons of nerve 2
by examination of stained sections of abdominal ganglia. Photomicrographs are
reproduced in PI. 1. The horizontal section in particular shows a tract of relatively
30 r
S 20
a1
•a
a
S
o
a,
I 10
I
Time (sec.)
Text-fig. 4. The frequency of action potentials in muscle group E on the same side of two
adjacent segments, plotted against time. The full line shows the frequency in the fourth
abdominal segment and the dashed line in thefifthsegment. The ipsilateral MRO was stretched
in abdominal segment 4 at the time shown by a heavy line above the abscissa. The time of
releasing is shown by a line below the abscissa. The receptor was stretched from 0-25 to -044 cm.
four times with a pause of 30 sec. between stretches. The curves show the average responses.
large fibres running around the edge of the ganglion from the lateral part of the connective into nerve 2. These showed no sign of branching in any section. These are
among the largest fibres in the cord, as can be seen in the transverse section. The
parasagittal section shows the same bundle of large fibres once again, and in addition
another bundle of (presumably sensory) nerve fibres of which only two are comparable
in size to those in the first bundle. Since recordings showed the majority of efferent
impulses to be larger than afferent ones, the tract of large fibres is almost certainly
motor in function.
(2) Types of reflex resulting from MRO stretch
Text-fig. 3 shows two examples of the clearest and most typical reflex result of
MRO stretch. The records in Text-fig. 3 a were taken from a tonically active muscle
and those in Text-fig. 3 b from a muscle which was inactive at the time of stimulation.
The physiology of a lepidopteran muscle receptor
235
Both muscles were excited, most strongly during the period of stretching, and both
showed some residual excitation after the MRO had been released. When a number of
stretches were given successively, this residual excitation tended to build up.
The shape of the action potential is relatively undistorted in these records as a long
time-constant was used in recording them. These two types represent extreme examples
of the range of different shapes of action potential seen. Thus all muscles were of the
'fast' type with non-facilitating junctional potentials. Active membrane responses
varied in size between the extremes seen in Text-fig. 3 a, b. Precise measurements of
(0)
Contralateral A
Ipsilatera. E
t
1
i i
i
I
. 1 1
i .1 . i
1
Contralateral P
I
I A I ...I. 1 . 1 . 1
1.1
1 .1... °-1SclT>.
Text-fig. 5. Oscillograph records of three different types of stretch reflex in the same »egment. The inset diagram shows the approximate location of the three muscles (see also Textfig. 11). The asterisk shows the position of the spiracle. Movement artifacts were minimized
by using a short recording time-constant. The lower beam signals the movement of the ~j
stretching forceps.
resting potential were not made, but from the change in potential on removing the
electrode from its intracellular location, it was estimated that most action potentials
overshot zero potential by about 3-6 mV.
Records taken from muscles in the segments adjacent to the stretched MRO
revealed that many of these were excited also. In Text-fig. 4 such an intersegmental
reflex is compared with the response of the homologous muscle within the stimulated
segment. The two records were taken simultaneously. The intersegmental reflex was
typically less intense but otherwise similar to the intrasegmental one. As far as this
type of experiment could show, the latencies were little different. Certain contralateral muscle groups were also excited, group A for example, as seen in Text-fig. 5a.
236
R. DE G . WEEVERS
Again the response was little different from the ipsilateral intrasegmental reflex,
though perhaps the contralateral reflex was a little slower to reach peak excitation.
One contralateral muscle, group P, was weakly inhibited by stretching the ipsilateral
MRO, as seen in Text-fig. 56.
Inhibitory reflexes were decidedly unusual. Indeed, none of the longitudinal intersegmental muscles or long diagonal muscles were inhibited by MRO stretch. Unfortunately the integumentary muscles are rather inaccessible and only a few records
were obtained. Some showed excitatory reflexes, but one in particular, group L, was
strongly inhibited by stretch of either the ipsilateral or the contralateral intrasegmental
MRO, as seen in Text-fig. 6.
I
1
r
a
1
Time (sec)
Text-fig. 6. An inhibitory stretch reflex in muscle group L. The location of this muscle is
shown in the inset diagram. The full line in the frequency-time plot shows the response to
stretching the ipsilateral MRO by 0-15 cm. and the dashed line shows the effect of subjecting
the contralateral receptor to a similar stimulus. The full line above and below the abscissa shows
the time of stretching and of releasing the ipsilateral MRO, and the dashed line the time of
stretching and releasing the contralateral MRO. (The average of two such responses.)
(3) Phasic components of the stretch reflex
It was shown previously (Weevers, 19666) that the MRO of the caterpillar provides
the c.N.S. with very detailed information about the rate of extension, and even about
the changes in rate of extension of a segment. It is of interest to examine the way in
which this information is utilized. In fact phasic components can be seen in Textfigs. 3-5, but a low-frequency pulsatile discharge does not yield very good timeresolution; nor is Text-fig. 4 plotted in a way which would show rapid phasic effects.
It was found by chance that embedding the appendages of a caterpillar in dental
cement produced very intense and fairly constant muscular activity. In addition,
motor activity could be increased by isolating a single functional ganglion from its
The physiology of a lepidopteran muscle receptor
237
neighbours. This procedure also rendered the discharge frequency more constant.
The chosen muscle, group E, was also one which normally exhibited a tonic discharge.
(Since group E is innervated by nerve 2, the smallest functional reflex unit was a pair
of adjacent ganglia, owing to the path followed by axons leaving the c.N.s. in this
nerve—see § (1).)
It may be seen from Text-fig. 7 that the reflex has a phasic component which, like
the sensory response (Weevers 19666,), is complex. The sense organ signals displacement, movement and acceleration and all three components appear in the reflex.
The curves of Text-fig. 7 are remarkably close reflexions of the changes in the
afferent discharge from the MRO.
60 r
I
a
I
c
•8 20
50
100
150
Time (percentage of the duration of stretching)
200
Text-fig. 7. The stretch reflex in muscle group E. The ipsilateral MRO in the same segment
was stretched by 0-19 cm. at three different rates. Dotted line 0-008 cm./sec., dashed line
0-18 cm./sec., full line 0-36 cm./sec. The tonic discharge in group E was intensified experimentally (see text). Each curve is the average of two responses.
(4) Summation and facilitation, spatial and temporal
Text-fig. 7 gives a good idea of the responses to stretching a single MRO. This sort
of experiment is necessary for analysis, but the stimulus is a rather artificial one; in an
intact caterpillar, displacements affecting only a single receptor must be unusual.
Text-fig. 8 shows what happened when two receptors were stretched simultaneously.
The ipsilateral and contralateral reflexes reinforced each other to give a response larger
than the sum of the responses to stretching the receptors singly. Similar effects were
seen when receptors on the same side in adjacent segments were stretched simultaneously, though in this case the facilitation was not quite as marked. No clear interactions were observed when receptors further afield were stretched at the same time
as the ipsilateral intrasegmental MRO.
All the reflex data discussed so far were obtained using stretch-stimulation. This is
R. DE G. WEEVERS
238
1
ideal for evoking 'normal responses but is not so well suited for investigating the
synaptic mechanisms responsible for producing such responses. An attempt was
therefore made to clarify the pattern sensitivity and complexity of the pathways
involved in the stretch reflex, using electrical stimulation of the MRO axon. This
technique is a difficult one to apply here because of the short length of single axon
Record
20
1
I
Ipsilateral
receptor
stretched
Both receptors
stretched
simultaneously
10
It
i
i
i
i
l
Contralateral
receptor
stretched
10
Time (sec.)
Text-fig. 8. The stretch reflex in muscle group E. The figure shows the results of stretching
the ipsilateral and contralateral receptors, first separately and then together. Each curve is
the mean of the evoked responses to two stretches with an interval of 2 min. between them.
The full lines above and below the abscissa show the times of stretching and releasing the
MRO singly; the dashed lines show the same thing for simultaneous stretching of the two
MRO in one segment.
available for stimulation; in addition, the steady stretch discharge has to be blocked
by pinching this same axon distally. These difficulties severely limited the data which
could be obtained from available experimental material and also necessitated use of
the pupa, where the length of single axon is greater.
The physiology of a lepidopteran muscle receptor
239
Plate 2 illustrates results which were obtained using this technique. The record
was taken from the dorsal nerve proximal to the spiracle and is therefore a complex
one, but because of the low level of central excitability in the pupa, up to eight units
can be clearly distinguished. In record 1, the smallest of the three units was the axon
innervating the receptor muscle. This exhibited a tonic discharge which was briefly
inhibited following the medium-sized spike passing centripetally along the MRO
axon. The single large spike was in a motor unit showing an excitatory reflex. Other
records yielded a latency for the earliest motor response of 30-33 msec. This seems a
rather long latency, but it should be remembered that synaptic connexions are made
in the next anterior ganglion. In one caterpillar, the conduction time over the same
pathway was about 27 msec. Unfortunately it was not possible to identify in the time
available which pupal muscle was excited when the above-mentioned reflex spike
appeared, so it can only be said that the synaptic delay was probably of the order of
5 msec. The two stimuli seen in record 1 were the third and fourth in a train. There
was no further reflex response after the fourth stimulus.
In record 2 the same units can be seen as in record 1. The large efferent spike
usually did not appear until after the second stimulus, therefore the synaptic effects of
the first MRO impulse persisted for at least 125 msec, and summed with the second
impulse to produce a response. The pathway thus certainly exhibited properties of
temporal summation and ' fatigue' or adaptation. It cannot be said whether temporal
facilitation also occurred. Intracentral recording would be required to decide this.
Record 3 again shows the effects of central adaptation, and two further efferent units
can be identified. In record 4 where the frequency of stimulation was highest the state
of central excitation was sufficient to evoke two spikes from an even larger motor
unit, as well as some smaller ones, and the reflex was beginning to acquire considerable
complexity.
(5) Stretch reflex in a single ganglion
It was mentioned in § (1) that only nerve 2 contained motor axons which originated
in the next anterior ganglion. It was also shown that the MRO axon bifurcated in the
ganglion where it entered the c.N.S. Intersegmental reflexes have been recorded in
both the segment anterior and that posterior to the stretch MRO. The latter observation already implies reflex connexions within the ganglion where the MRO axon
enters. It was of interest to examine whether the MRO axon also made reflex connexions with nerve 3 motoneurones in the ganglion where it entered the c.N.s.
Text-fig. 9 shows an excitatory reflex in a muscle innervated by nerve 3. When
the c.N.s. was intact, excitation was of the typical phasic-tonic variety. On isolating
the relevant ganglion from its neighbours the reflex, though reduced, did not disappear.
In this case some of the characteristic features of the response appear to depend on
intersegmental connexions (possibly non-specific), but there can be no doubt that
synaptic connexions are present within a single ganglion. More cannot be said without
finer surgical techniques.
(6) The spread of the reflex
Recordings of muscle activity were made from as many groups as possible in order
to examine the extent of the reflex effects of stretching a single MRO. In the course
of this investigation it was found that a few muscles, designated by Lyonet (1762) in
R. DE G. WEEVERS
240
his description of the muscular system of the goatmoth caterpillar as separate groups,
did in fact share a common motor axon with another group nearby. The top three
records of Text-fig. 10 show that for each spike in group H there was another in
group G at the same instant, similarly for groups f and g and for 6X and 62. On the
other hand, when recordings were taken simultaneously from pairs of fibres in
' groups' b and c, c and d, and b and d, only two motor axons could be demonstrated,
not three. These two motor units will be called 'bed prox.' and 'bed dist.'
V
/
\
12
: v
i
i
i
i
i
i
i
i
i
i
i
•
•
t
i
i
i /
i /
V
a
I 6
10
Time (sec.)
Text-fig. 9. Stretch reflex in a muscle (ipsilateral group P) innervated by nerve 3 before
(dashed line) and after (full line) isolating the ganglion of the segment tested by section of the
nerve cord anterior and posterior to it. The reflex in the isolated ganglion is the mean of four
tests; the intact C.N.S. was stimulated only twice as the response was clearer.
Tables 1-4 show the results of about twenty experiments on last instar larvae.
Recordings were made from as many muscle groups as possible, before, during and
after stretching one MRO to 1-5 mm. above unstretched length at a rate of 0-2 cm.
per second. This stimulus was repeated four times with a pause of at least 1 minute
between stretches. The mean motor frequencies with the MRO stretched and unstretched were obtained from films of the results of such tests. An inhibitory response
is indicated by a minus sign; all others were excitatory. No attempt was made to
determine the statistical significance of reflex changes in frequency for several reasons:
(1) The parameter of response measured tends to minimize phasic effects.
(2) Several muscles were rather inaccessible and their motor axons were easily
severed unknowingly during dissection.
(3) In many preparations some muscles were difficult to excite at all, so that
The physiology of a lepidopteran muscle receptor
241
although when discharging they did show reflex changes in spike frequency, MRO
stretch often did not initiate a spike train.
For reasons of experimental convenience all results discussed in previous sections
and the data of Tables 1, 3 and 4 were obtained from abdominal segments, mostly the
fifth. Accordingly Table 2 shows a selection of reflex responses to MRO stretch in
f
—(ftrrn
g
bed (distal)
bed (proximal)
bed (distal)
bed (mid,)
bed (proximal)
bed (midj
1 sec.
Text-fig. 10. Simultaneous recordings from pairs of muscles to show which of these share
a common motor axon. The records labelled 'mid!' and 'mid,' were both taken from muscle
fibres near the middle of group bed (see text); ' mid,' was slightly the more proximal.
thoracic segment 3 for comparison with the data of Table 1. The results show that
similar stretch reflexes are present in the thorax, although they may be somewhat
weaker. The reflex effects of stretch in any one preparation were usually very consistent, both in thorax and abdomen.
The tables contain a lot of data, so Text-fig. 11 was drawn in order to show
pictorially the magnitude and location of each reflex. This figure is a diagrammatic
representation of abdominal segments 4, 5 and 6 in a caterpillar dissected in the usual
R. DE G. WEEVERS
242
way. The most intense excitatory reflexes are shown by the most closely spaced lines,
and inhibitory reflexes by dots. Records taken from muscles which are labelled but
not shaded on this diagram showed no change in spike frequency when the MRO was
stretched. Almost all of the other readily accessible muscle groups shown were also
Table 1. Spread of reflex response to MRO stretch. I. Intrasegmental, ipsilateral
MRO unstretchedfrequency of
action potentials
IVi. LLSC1C
group
D
Superficial muscles
±8.D.
o-8
4-S
7'5
A
C
E
bed dist.
ff
bed prox.
a
i
Diagonal muscle
groups: dorsal to
ventral
Mean
F
HG
6
fg
e
4-8
—
8-2
o-o
o-o
o-o
2-7
S-o
13-8
4-3
4-i
i-o
2-3
P
R
L
3 -o
io-6
6-4
o-o
P
y
8
i-3
'
animals
—
no
6
i
2-O
2
4-1
—
—
—
—
136
3-o
—
3'9
2-6
o-8
7-i
7
3
3
3
3
4
o-o
o-o
o-o
6-8
6-4
10-4
6-6
2'2
i-7
2'3
X
Range
—
4-5
—
—
—
—
—
—
2-3
—
—
—
—
o-o
2
Mean
±S.D.
Rang
3-S
8-3
7-8
—
o-o
o-o
o-o
—
i-9
—
—
—
—
—
—
i-3
30
—
5-4
39
—
o-o
o-o
o-o
i-5
3-i
13-8
7-8
o-s
i-8
13-3
In
oiiu&non
of muscle
Longitudinal muscle
groups: dorsal to
ventral
Change in frequency
on stretching MRO
7-2
4
5
4
o-7
i-1
i-4
3-7
2-8
o-8
i-8
—
—
—
—
—
—
2-3
i-7
—
1-9
—
—
—
—
I
2
I
I
I
-i-5
— 1-6
8-7
-S-7
o-o
I
i-7
I
o-o
Pooled data for abdominal segments 6, 5, 4 and 2.
The MRO was stretched in each case by 0-15 cm. at 0-2 cm./sec. from an unstretched length giving
a sensory discharge frequency of 3-7 impulses/sec, (usually between 0-2 and 0-3 cm.). Where the reflex
was inhibitory, the figure under 'change in frequency on stretching' is preceded by a minus sign.
Table 2. Spread of reflex response to MRO stretch.
II. Intrasegmental ipsilateral responses in the thorax (segment 3)
Muscle
group
A
C
E
e
fg
e
y
3-7
o-o
Rise in frequency
on stretching
the MRO
63
5-8
4-2
0-3
07
1-2
07
13
1-7
MRO
unstretched—motor
frequency/sec.
o-o
6-5
60
tested for stretch reflexes by listening for possible changes in spike frequency on a
loudspeaker monitor. As may be seen from this diagram, when compared with the
figure of Lyonet (1762) reproduced in Text-fig. 1, most of the major long muscles
have been tested. Ipsilateral intrasegmental group B could not be examined with
intracellular muscle recordings since this had to be removed to expose the MRO. I
The physiology of a lepidopteran muscle receptor
243
probably does respond reflexly to stretch, however, since extracellular hook electrodes on the nerve leading to this muscle showed the presence of at least three
excited units.
Among the short integumentary muscles, only group P and group X are shown. If
more muscles were drawn the diagram would become rather confusing; and in any
Table 3. Spread of reflex response to MRO stretch.
III. Intrasegmental contralateral
MROiunstretched-motor
Change in frequency
on stretching MRO
frequency/sec.
group
Mean
8.D.
D
A
B
C
E
bed dist.
bed prox
9-S
—
—
I
8-i
—
—
—
i-o
—
—
2
I
4-6
I
o-o
187
—
—
—
—
99
4
2-5
1
o-o
o-o
—
—
—
—
—
3-2
—
of muscle
Longitudinal muscle
groups: dorsal to
ventral
69
o-o
Diagonal muscle
groups: dorsal to
ventral
F
HG
fg
o-o
o-o
3-S
4 0
II-O
4-6
o-o
e
Integumentary muscle I
groups: dorsal to
L
ventral
P
X
7-7
—
—
—
—
—
131
a
i
Range flnimflia
I-O
192
67
5-3
30
1
1
Mean
S.D.
i-o
—
—
9 0
-0-9
1
o-o
2
I-I
o-i
—
—
2
1
0-7
1
O-I
28
—
7.7
—
a
1
3-6
-2-5
4
— I-I
1
— I-O
o-o
Rang
—
-0-5
o-8
—
—
—
—
—
—
—
—
—
—
0-7
—
2-2
—
—
—
—
I-I
o-a
—
—
18
—
i-8
—
Table 4. Spread of reflex responseto. MRO stretch. IV. Adjacent segments
Longitudinal
E
A
Anterior segment
Situation of muscle...
Muscle group...
Ipsilateral
MRO unstretched-motor
frequency per second
Change in motor frequency
on stretching MRO
Contralateral
MRO unstretched-motor
frequency per second
Change in motor frequency
on stretching MRO
Mean
Range
Mean
Range
30
Mean
Range
Mean
Range
7-0
30
—
03
—
i
Diagonal
HG
7-0
62
II-O
3-1
—O-7
39
o-6
—
—
—
—
Superficial
P
X
41
1-9
—
—
—
-o-s
o-i
—
—
—
—
—
8-3
3-7
I-I
—
—
—
—
7-4
—
—
—
—
23
6-a
o-s
-0-5
—
o-o
—
Posterior segment
Ipsilateral
MRO unstretched-motor
frequency per second
Change in motor frequency
on stretching MRO
Contralateral
MRO unstretched-motor
frequency per second
Change in motor frequency
on stretching MRO
Mean
Range
Mean
Range
Mean
Range
Mean
Range
—
64
—
—
4 0
—
0-3
—
—
—
—
—
—
—
—
5-3
3-6
4-5
I-I
2-2
—
—
—
6-i
—
o-i
—
6-8
—
—
—
1-3
— 1-2
I'S
R . DE G . WEEVERS
244
MRO stretch
A. 6
•46/86 muscles
A. 5
Only labelled
muscles tested
Abdominal seg. 4
Text-fig, I I . Diagram of three adjacent abdominal segments of a caterpillar of Antheraea
pernyi. The figure shows the kind (excitatory or inhibitory) and approximate intensity of
reflex effects following stretching of the receptor on the left in the middle of the three segments. Closely spaced lines indicate intense excitation and closely spaced dots intense inhibition. The muscle nomenclature is that of Lyonet (1762)—see Text-fig. 1. The longitudinal
tracheal trunks, the c.N.S. and the paired segmental nerves are included in the diagram to
aid in visualizing the topography of the various muscle groups.
The physiology of a lepidopteran muscle receptor
245
case, owing to their inaccessibility only seven out of twenty such muscle groups have
been tested as yet. Groups P and X are the only muscles in Text-fig. 11 which are
innervated by nerve 3.
MRO
Text-fig. 12. The simplest possible central connexions of a single MRO which could produce a stretch reflex with the observed spread. For simplicity only one muscle innervated by
nerve 2 and one innervated by nerve 3 is shown in each half segment. The ramifications of the
MRO sensory axon are shown by a full line; dashed lines signify motor axons. Asterisks show
the position of the spiracles.
DISCUSSION
Reflex pathways
Present experiments provide strong evidence that motoneurones of nerve 2 in the
abdominal ganglia of silkmoth caterpillars make synaptic contacts only in the ganglion
anterior to the one at which their axons leave the c.N.s. This is in agreement with the
observations of von Hoist (1934) on Agrotis caterpillar. Hughes (1965) described a
similar arrangement for certain cockroach motoneurones. In the caterpillar the MRO
sensory axon divides into at least two branches, probably within the ganglion where it
enters the c.N.s. The anterior of these branches passes up the ipsilateral connective
to the next anterior ganglion where it makes synaptic contact with the motoneurones
of nerve 2, mentioned above. Other sensory axons have a similar anteriorly directed
intracentral branch. In the experiment of Text-fig. 2 a short latency responses following
the stimulus in a one-to-one manner at high frequencies were recorded in branches
of the dorsal nerve which never spontaneously carried centrifugally directed impulses.
Such sensory neurons did not have a posteriorly directed intracentral branch as well
as the anterior branch; only the MRO axon bifurcated in this way.
Another anatomical observation of comparative interest emerged from examination
of thin sections of caterpillar abdominal ganglia. A tract of large nerve fibres was
clearly visible passing from the lateral part of each connective around the anterolateral margin of the ganglion and into the anterior part of nerve 2. In view of the path
16
Exp. Biol. 45, 2
246
R. DE G. WEEVERS
that they follow and in view of their large size these are probably the motor axons
referred to above. If this inference is correct, then motor and sensory axons in the
proximal part of nerve 2 are segregated into anterior and posterior bundles respectively. This is different from the dragonfly larva, where Fielden (1963) showed that the
dorsal and ventral halves of the paraproct nerve roots were respectively motor and
sensory in function.
Direct physiological evidence is needed to prove the existence of horizontally
separated motor and sensory bundles beyond all doubt, but a further observation
provides circumstantial evidence in favour of this idea. In the thorax of the caterpillar a nerve leaves the C.N.S. half way along the 1-2 and the 2-3 connectives. Appropriate stimulation and surgery revealed that this nerve innervated muscles which in
the abdomen would have been innervated by nerve 2 of the ganglion immediately
posterior. The nerve in the corresponding position to the abdominal nerve 2 appeared
in the thorax to contain only sensory fibres. Thus the horizontal separation of motor
and sensory bundles may here be complete. It is not known whether this arrangement has any functional importance, but it could be experimentally convenient (as
the separation of motor and sensory roots has been in the vertebrates). Present
observations likewise shed no light on the functional importance of the anterior
displacement of the synaptic contacts of the motoneurones of nerve 2.
Text-fig. 12 shows the reflex pathways in the caterpillar which have been inferred
from the observations discussed above. This figure also takes account of the spread
of the stretch reflex as shown in Text-fig. 11. The number of synapses drawn is in
every case the minimum which present observations would allow. Thus only the
intersegmental reflex in muscles innervated by nerve 2 of the segment anterior to the
stretched MRO has to be mediated via an interneurone.
The function of the stretch reflex
The paired muscle receptors of the caterpillars are obvious candidates for a role in
co-ordinating peristaltic locomotion. The pattern of muscular activity during crawling
is complex, involving most of the body musculature in a co-ordinated sequence of
contraction and relaxation. In this sequence major groups of muscles within a single
segment act in an antagonistic manner (Barth, 1937). By contrast, the pattern of reflex
changes following stretch of a single MRO (see Text-fig. 11), though widespread, is a
simple one. It consists predominantly of excitation in muscles lying parallel to the
stretched MRO—a classical myotatic reflex. Thus muscles which during peristalsis act
successively or antagonistically are excited by this reflex to contract simultaneously.
Therefore the timing of muscular activity during peristaltic locomotion cannot be
rigidly determined by a reflex involving the MRO (cf. Weevers, 1965). These receptors
function in a more basic role as the sensory elements in a negative feedback reflex
adjusting muscular effort in relation to the load encountered. Intact crawling caterpillars also show such a resistance reflex (von Hoist, 1934; Weevers, 1965).
It is of course possible that the experimental methods used here failed to reveal
more complex proprioceptive responses of fundamental importance because no more
than two MRO could be stretched simultaneously. Certainly, the spatial interactions
between neighbouring receptors exert a profound influence on the intensity of the
reflexes evoked. It can only be said that, in view of von Hoist's (1934) finding that
The physiology of a lepidopteran muscle receptor
247
locomotor waves can pass as many as three denervated segments essentially unchanged,
the pattern of muscular activation is probably largely centrally determined.
Thus the MRO of caterpillars fulfil a role in the reflex control of muscular activity
analogous to the muscle spindles of mammals. It appeared from the work of Eckert
(1961 a, b) on the fast abdominal flexor muscles of crayfish that the very similar muscle
receptors in this group were used differently. But Kennedy, Evoy & Fields (1966)
have shown that the slow abdominal musculature is controlled by a negative feedback
reflex stabilizing bodily position and that the abdominal MRO are the sense organs
involved. Although the receptor elements are different, similar reflexes also stabilize
limb position in decapod Crustacea (Bush, 1963) and in insects (Pringle, 1940; Wilson,
1965). It seems probable that this control mechanism will be found to operate also in
other groups of animals having similar sensory equipment.
SUMMARY
1. When a single MRO of a caterpillar is stretched at least 32 motor units show
clear reflex changes in activity.
2. The great majority of muscles are excited., and the latency of the reflex differs
only slightly from one muscle to another. The response has both tonic and phasic
components which reflect more or less faithfully the magnitudes of the same components in the sensory discharge.
3. Muscles are affected on the contralateral side of the stimulated segment and on
the ipsilateral side of adjacent segments. The reflex fields of neighbouring receptors
therefore overlap; spatial facilitation produces a disproportionate increase in the
overall response when two receptors are stimulated simultaneously.
4. The reflex pathway for muscles innervated by nerve 2 is shown to involve
synaptic connexions in the ganglion of the segment anterior to the stimulated receptor
and responding muscles.
5. The muscles most strongly excited are those which he functionally in parallel
with a stretched sense organ. It is concluded that a major function of the caterpillar
MRO is to mediate a negative feedback reflex tending to stabilize bodily position
independent of load.
I am very grateful to Prof. G. M. Hughes for guidance and encouragement during
this work, and to the D.S.I.R. for financial support in the form of a Research
Studentship.
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"journal of Experimental Biology, Vol. 45, No. 2
R. DE G. WEEVERS
Plate 1
{Facing p. 248)
Plate 2.
Journal of Experimental Biology, Vol. 45, No. 2
4
• >•
'I
I
»
1 t
1
1
> >
1
I
ill
1
1
1
»
1
1
1
1
I,I
,
I
R. DE G. WEEVERS
i
»'
1
1
I
. . .
l i i i i in
0-2 sec.
t
1 1
i.
J
Ji
I .
Axon
pinched
stimulus
Record
t
The physiology of a lepidopteran muscle receptor
249
EXPLANATION OF PLATES
PLATE I
Fig. 1, horizontal, fig. 2, transverse, fig. 3, parasagittal sections of a larval abdominal ganglion.
The tract of motor axons which leaves the C.N.s. via nerve 2 is labelled M. The tract of (probably)
sensory fibres in nerve 2 is labelled S (figs. 1 and 3). The lines in fig. 1 show the planes of the sections
in figs. 2 and 3. The scale of thefiguresis shown on the left. Fixed in 0-2 % osmic in saturated picric;
stained in Heidenhein's iron haematoxylin.
PLATE a
The stretch reflex recorded in nerve 2 of a pupa just proximal to the spiracle. The steady stretch discharge from the MRO was blocked by pinching the sensory axon where it left the cell body. The reflex
was evoked by electrical stimulation of the MRO axon, distal to its junction with the dorsal nerve, at
four different frequencies: (1) i/sec., (3) 8/sec, (3) 23/sec., (4) 36/sec.