mechanical properties of locust extensor tibiae muscles

Comp. Biochem. Physiol.. I/oL 61A. pp, 85 to 95 0300-9629/78/0801-0085502.00/0 © Pergamon Prex,~ Ltd 1978. Printed in Great Britain

MECHANICAL PROPERTIES OF LOCUST EXTENSOR TIBIAE MUSCLES

M. D. BURNS and P. N. R. USHERWOOD* Department of Zoology, University of Glasgow, Glasgow G12 8QQ, Scotland

(Received 15 November 1977)

Abstract--l. The prothoracic and mesothoracic extensor tibiae muscles of the locust respond to activity in the "slow" extensor tibiae motoneuron (SETi) with very slow contractions and a low fusion frequency, while their phasic contractions are more rapid than those of the metathoracic extensor tibiae muscle.

2. SETi activity can induce a memory or "catch" effect in which a high tension is maintained by a lower frequency than is needed to develop it. "Catch" tension is reduced by phasic contractions of the muscle or by activity in the inhibitory axon.

3. A bundle of tonic fibres isolated from the metathoracic extensor tibiae muscle exhibits co-ordinated rhythmic contractions similar to those recorded from intact muscles.

4. Depolarizations of the tonic fibres coincide with the contractions and are sometimes accompanied by bursts of EPSPs and IPSPs.

5. The tonic fibres are electrically-coupled.

INTRODUCTION

The large size of the extensor tibiae muscle in the jumping leg of the locust has led to its being investigated extensively and much is now known abou t its innervat ion, physiology and pharmacology and mechanical properties. However, studies of this muscle still cont inue to disclose new and interest ing information. For example, it has been shown recently tha t some of its fibres undergo co-ordinated rhy thmic cont rac t ions (Hoyle & O'Shea, 1974; Usherwood, 1974), which are influenced not only by the motor axons which innervate the meta thorac ic extensor t ibiae muscle but also by the ou tpu t from a neurosecretory neurone (Hoyle, 1974; Hoyle & Barker, 1975; Evans & O'Shea, 1977). This paper conta ins further informat ion on the physiology and pharmacology of some of the tonic fibres from this muscle which have been isolated as a small bundle from the rest of the muscle and which main ta in co-ordinated rhythmic activity for long periods in s tandard locust saline.

Unl ike the meta thorac ic extensor t ibiae muscle, the pro thorac ic and mesothoracic homologues have received very little at tention. They differ markedly from the meta thorac ic muscle in gross structure, are considerably smaller and opposed by flexor muscles which are more powerful than themselves. Their roles in locust locomot ion have been examined by Burns & Usherwood (1978), who also describe their anatomy and innervation. In this paper we report on the mechanical responses of the pro- and mesothoracic extensor t ibiae muscles to neural s t imulat ion and show that the ion ic fibres tha t they contain can develop a main ta ined "catch" tension s tronger than that measured by Wilson & Lar imer (1968) in the metathoracic leg. The "catch" can be released by inhibi tory or "fast" axon activity.

* Present address: Department of Zoology, University of Nottingham, Nottingham NG7 2RD, England.

85

MATERIALS AND METHODS

Male and female adult locusts (Schistocerca gregaria) were used.

In situ extensor tibiae nerve-muscle preparations

The insect was fastened down on its back, the femur of the appropriate leg was opened and the extensor tibiae muscle was exposed by removing the flexor tibiae and retractor unguis muscles. The leg was perfused with locust saline (Usherwood & Grundfest, 1965). A strain gauge was connected to either the tibia or the apodeme of the extensor muscle. The motor axons (two excitatory, FETi and SETi, and one inhibitory, CI) to the extensor tibiae muscle were excited with suction electrodes on nerves 3b, 5 and 3c all of which were disconnected from the ganglion. Recordings of electrical activity in extensor muscle fibres were obtained with microelectrodes filled with either 3 M KCI or potassium propionate.

Isolated preparations of tonic extensor tibiae muscle fibres

The metathoracic extensor tibiae muscle of Schistocerca contains two major types of muscle fibres classified by their responsiveness to potassium (Cochrane et al., 1972). The majority are phasic and contract only transiently when the potassium content of the extracellular environment is increased. The rest are tonic and give a sustained contracture with potassium depolarization. Some of the tonic fibres at the proximal end of this muscle are arranged in a discrete bundle which is attached to the centrally-running apodeme of the extensor tibiae muscle by a flange of con- nective tissue (Cochrane et al., 1972).

After removing the flexor tibiae and retractor unguis muscles from the metathoracic leg it was possible to isolate ~hese tonic fibres from the rest of the extensor tibiae muscle by careful dissection. The rhythmic contractions of the isolated tonic fibres were best maintained if the extensor tibiae muscle was perfused with locust saline throughout the operation. However, when a bundle of tonic fibres was first isolated, connected to the transducer and stretched to its natural maximum body length, it sometimes exhi- bited considerable resting tension (0.34).4 g) and it was not until this tension had waned to a low value that spontaneous contractions appeared. During the early stages of these studies, spontaneous contractions of the isolated

86 M. D. BURNS and P. N. R. USHERWOOD

fibres occurred much less regularly than in situ, and sometimes isolated preparations failed to contract spontaneously. Such failure probably resulted from our initial inexperience in handling these delicate preparations since it became apparent as the studies progressed that their rhythmicity was adversely influenced by excessive stretch and also if one or more of the fibres in the bundle were accidently damaged during its isolation. We were much more successful with later preparations, over 90~ giving responses similar to those illustrated in Fig. 3.

Paired Ag/AgCI wire electrodes were used for direct extracellular stimulation of the tonic bundle. Microelec- trodes of 20-30 MD resistance and filled with 3 M potassium propionate were used for either intracellular stimulation or, for recording from, "single,i fibres.

In a few preparations it was possible to isolate the tonic bundle from the rest of the extensor tibiae muscle with the metathoracic trochanter and coxa still attached to the animal. In such in situ preparations mechanical responses to stimulation of the SETi and CI and the effects of such stimulation on the frequency and amplitude of the rhythmic contractions were investigated. It was also possible to examine the influence of centrally-determined activity of the SETi and CI on the electrical and mechanical activity of the tonic fibres.

RESULTS

Prothoracic and mesothoracic extensor tibiae muscles

Like the metathoracic extensor tibiae muscle, the prothoracic and mesothoracic extensor muscles are innervated by four axons (Usherwood, 1962a), three motor axons FETi, SETi, CI (Hoyle & Burrows, 1973) and a fourth small axon (DUMETi) to which a neurosecretory role has been ascribed (Hoyle et al., 1974). The anatomy and innervation of these muscles are described elsewhere (Burns & Usherwood, 1978).

Discrete tension twitches could be measured at the apodeme of the prothoracic and mesothoracic extensor tibiae muscles following single spikes in either the FETi or the SETi. The twitch induced by the FETi produced 4-6 times as much tension as that due to the SETi and this difference meant that only the "fast" twitch was measurable at the distal end of the tibia, the "slow" one being measurable at the apodeme but too small to overcome friction in the femur-tibia joint. The mean amplitude and temporal characteristics of the isometric tension twitches are listed in Table 1. The rise time of the response to an FETi

potential was 3 4 times shorter than the 59 msec of the phasic twitch in the metathoracic extensor tibiae muscle (Cochrane et al., 1972). The relaxation times were more variable but were also shorter than their metathoracic equivalents.

When simultaneous stimuli were delivered to both excitatory axons a summated twitch resulted in which the maximum tension obtainable was considerably less than the arithmetic sum of the individual twitch tensions. The differences between these figures were used to obtain the estimates shown in Table 1 for the proportion of fibres which are dually innervated by the FETi and the SETi (allowing for the effect of the CI which is usually stimulated along with the FETi). Since the ratio of FETi to SETi twitch tensions is lower in the mesothoracic than in the prothoracic muscle, the results suggest that of the two, the mesothoracic extensor muscle contains fewer fibres which receive only FETi endings, unless CI is more effective in reducing the response to FETi in the mesothoracic leg.

Higher frequencies of stimulation give summated tensions with the twitch/tetanus ratios shown in Table 1. The tetanic response to the FETi fatigues fairly rapidly, typically beginning to decay after about 7 sec of 40 Hz stimulation, but it does not show the anti- facilitation typical of cockroach coxal depressor muscles (Becht, 1959; Becht & Dresden, 1956; Usher- wood, 1962b; Iles & Pearson, 1971) and the locust retractor unguis muscle (Usherwood & Machili, 1968). Tetanic tension due to stimu!ation of the SETi developed very slowly. At 100 Hz the half-rise and half-fall times were about 0.3 sec, and fatigue was only visible after stimulation at this frequency for a period exceeding 30 sec.

Measurements of the force developed in response to SETi activity were complicated by three related phenomena. First, the prothoracic and mesothoracic extensor muscles often gradually developed low levels of tension (about 0.02 g) in the absence of any neural excitation. This tension was steady rather than oscil- latory as in the metathoracic muscle (see below), but oscillation did occur in the base tension between SETi potentials when the frequency of stimulation was low (Fig. 2A). Second, the muscles did not completely relax after an SETi induced tetanus and a low level of residual tension remained. The third was a "catch"

Table I. Mechanical characteristics of the prothoracic and mesothoracic extensor tibiae muscles measured at their apodedaes at maximum natural length. Recording conditions were almost isometric.

Tensions are _+1 S.D.

Axon

Max. reduction SETi innervated Peak twitch Twitch rise ½ fall of twitch by fibres with

tension time time Twitch/tet 1 CI spike FETi endings (g) (msec) (msec) ratio (%) ('~ o)

Prothoracic FETi

SETi

Mesothoracic FETi

SETi

0.49 19 21 1/7 0 (_+o.15)

0.07 61 160 ~< 1/17 22 ( + 0.02)

0.43 20 26 1/7 0 (+0.21)

0.10 86 146 1/25 25 (+0.03)

50-55

60-65

Mechanical properties of locust extensor tibiae muscles

A I0 Hz IOOHz

SET,--,, l , I"

'\ IOHz

\ I I Ois

b I O I s

87

C 50Hz

SET ' . . . . . . . . . . . . . . . . . . . . . . . IO Hz

~ q O I s

D

FETi SETi

'°J 5Hz

k k k k k \ x. k k \ k

i I I i i ) i

OIs I !

Fig. 1. Tensions developed by the locust mesothoracic extensor tibiae muscle in response to stimulation of the FETi and SETi axons. Tensions at apodeme with muscle at maximum natural length. (A} "Catch" effect resulting from SETi activity. See text. (B) Effect of four spontaneous FETi potentials on residual tension. (C) Release of "catch" tension by stimulation of FETi. The relaxing effects are additive. (D) Relaxation resulting from simultaneous stimulation of FETi and SETi at about the same

frequency. Beat frequency effects can also be seen.

effect. This appeared when a high frequency burst of stimuli to the SETi axon was inserted into a con- tinuous low frequency train and took the form of a tension plateau following t h e burst which was frequently higher than could be developed with low frequency stimulation alone (Fig. 1A). The muscle relaxed as soon as the low frequency stimulation ceased. The metathoracic muscle shows the same effect but rather more weakly (Wilson & Larimer, 1968). A similar, but stronger effect has been found in crayfish (Blaschko e t al., 1931) and crab leg muscles (E. Rathmayer, personal communication).

Phasic contractions due to FETi activity, as well as producing additional tension, caused a subsequent relaxation in SETi induced tension and released the "catch". Two or three phasic twitches were sufficient to erase residual tension (Fig. IB), but a burst of high

frequency FETi activity was necessary to produce a significant reduction in "catch" tension. The relaxing effects of short bursts were additive (Fig. IC) until a minimum tension was reached which was character- istic of the frequency of SETi stimulation. A low frequency of stimulation to FETi maintained the tension at this minimum level during SETi stimulation (Fig. ID).

In both the prothoracic and mesothoracic extensor tibiae muscles activity in the inhibitory axon produced a considerable reduction in tension due to SETi stimulation but had no effect on FETi responses. A single optimally timed CI potential reduced the twitch resulting from an SETi spike by 20-25~ (Table i). The effect became rapidly smaller with increasing separation between the two impulses, but there is evidence that high frequency trains of

88 M.D. BURNS and P. N. R. USHERWOOD

01 gl llllllUlllllllllllllllllll*llllllllllliaillllllllllllllllllllllllll . . * t l l l l l l b .~ , l i l i l l , I , , , , . . . . . . . . . . . . . . . . . . . . . . . . . . .

¢1 :lOs : .50 HZ

B IOOHz o b

S E ~ - - . . . . . . . . . . . . . . . . . . ~ . . . . t

© 5 s

C I00 Hz SETi .

CI .........................................

o~gI

i I 0 5 s

\

I00 Hz

SET~

i

I 00 Hz CI 10 Hz

,I t0s

Fig. 2. The effects of CI activity on tension induced in the mesothoracic extensor tibiae muscle by SETi. Conditions as in Fig. 1. (A) Stimulation of SETi at 1 Hz. Note oscillations in base tension. Tension variations during CI activity are a beat frequency effect. (B) Tetanic tension with CI activity (curve 1) and without (curve 2). SETi stimulation for curve 1 stopped at (a). and that for curve 2 at (b). (C) Acceleration of relaxation by CI activity. Trace 1 is with CI stimulation, trace 2 is without. The half-time of relaxation is reduced from 240 to 130msec. (D) Release of "catch" tension by CI

activity.

CI potentials produced measurable inhibitory effects lasting up to 100 msec after the end of the train. Higher frequencies in the CI axon had correspond- ingly greater effects (Fig. 2A), the SETi induced twitch being reduced by up to 90% by 100 Hz stimulation of the CI. The inhibitory effect fatigued rapidly at high frequencies.

If the SETi firing frequency rose the effectiveness of inhibition was reduced, but it was still considerable when both axons were stimulated at 100 Hz. Inhibi- tion then slowed down the rise of tension (Fig. 2B), induced a reduction in tetanic tension of about 30~o (a slow effect with a half time of relaxation of about 0.5 sec) and produced a marked acceleration of relaxation after SETi activity had ceased (Fig. 2C). High firing frequencies in CI also released "catch" tension in a manner similar to that of FETi activity (Fig. 2D), but it was less than half as effective in this role, showing that simultaneous activation of CI could not account for all of the releasing action of FETi.

Tonic fibres of metathoracic extensor tibiae muscle

Although the spontaneous rhythmic contractions of the isolated fibre preparations were of similar

duration and frequency to those recorded from intact extensor tibiae muscles (Hoyle & O'Shea, 1974; Ush- erwood, 1974) they had a more variable time course (Fig. 3).

Stimulation of the muscle fibre bundles via paired Ag/AgCI electrodes resulted in twitch contractions, the amplitudes of which were related to the stimulus intensity. During equilibration of a preparation of tonic muscle fibres, before the resting tension had waned to zero, the first in a series of directly-evoked contractions was followed by a transient relaxation of the fibre bundle below the base-line tension. The magnitude o, ~ the fall in tension below the base-line level could be varied by altering either the stimulus intensity or the stimulation frequency. Stimulation of the SETi axon evoked twitch contractions from the tonic fibres (Fig. 4A, B) which had a similar time course to the contractions recorded in response to direct stimulation (Cochrane et al., 1972). The twitch contractions summated at stimulus frequencies less than 1 Hz and at frequencies greater than 5 Hz a smooth tonic contraction was obtained (Fig. 4C, D). Stimulation of CI reduced the amplitude of the responses to SETi. In some preparations the resting ten-


! i lOS

89

) L_ I I I I

2s 5s

t • • • • I OSg

2min 5s

A T

Fig. 3. Spontaneous contractions recorded from bundles of tonic muscle fibres isolated from metathor- aeic extensor tibiae muscles of four different locusts. Note differences in time course and duration

of contractions.

sion of the tonic bundle was transiently lowered during CI stimulation (Fig. 4D) (see also Runion & Ush- erwood, 1970).

Intracellular recordings from a tonic fibre during the appearance of spontaneous contractions were characterized by rhythmic oscillations of the membrane potential, slow depolarizations of the fibres accompanying the contractions. In in situ preparations bursts of EPSPs and IPSPs were frequently recorded from tonic fibres but they were not always correlated with the rhythmic changes in membrane potential referred to above, although they influenced their amplitude, time course and frequency.

The fact that fibres of isolated tonic bundles con- tracted synchronously indicated that they might be electrically coupled. This possibility was tested by injecting hyperpolarizing current into one fibre and recording the effects of this current one to seven fibres away from the current injection site (Fig. 5C-E). The results of this exercise clearly support the idea of some form of electrical coupling between the fibres. Recordings made with two electrodes during the generation of spontaneous contractions by isolated bundles of tonic fibres gave further support since during such contractions the fibres more or less synchronously depolarized and decremental conduction of

electrically-excited potentials occurred between fibres in the bundle. If the depolarizations responsible for the rhythmic contractions of the tonic fibres are myogenic one might reasonably anticipate the presence of a pacemaker site. However, we were unable to find such a site.

In view of the results of earlier studies by Usher- wood & Grundfest (1965), we were rather surprised to find that depolarizing current injections evoked spike-like responses from the fibres of isolated tonic bundles (Fig. 5A, B). To date we have not been able to record electrically excited spike-like activity from these fibres in situ.

Effect of ions and drugs on the spontaneous contractions

The effects of potassium on the rhythmic activity of isolated bundles of tonic fibres were rather variable. Replacement of standard saline which contains 10mM potassium, by saline containing zero potassium resulted, in some preparations, in a reduction in the frequency of the contractions and an increase in their amplitude (Fig. 6A). In other preparations an increase in frequency and a decrease in amplitude was recorded (Fig. 6C). An increase in the potassium

90 M. D, BURNS and P. N. R. USHERWOOD

• SET~

.SE ~-i -SETi + CI

I I IHz SETI IHz SETJ f~i

I I 5HzSET~ t CI

~ 4g

I I Is

I O 2g

15

I I I 5Hz SET~ lOs

t I IHz SETI+Ci

J L I

5Hz SETi + CI I I

lOs

Fig. 4. (A) Twitch contraction of isolated bundle of tonic fibres from locust metathoracic extensor tibiae muscle in response to stimulation of SETi alone. (B) Twitch contraction of the tonic fibres in response to SETi stimulation (left) is compared with the contraction in response to stimulation of the SETi plus CI. (C) Comparison of mechanical responses to SETi alone with response to SETi plus CI for different stimulation frequencies. (D) In this preparation stimulation of SETi plus CI

produced contractions followed by long-lasting relaxations below the base tension.

A C

2 0 m Y

r

E

J~/t / / I I IOO~m

O-TA

~ .4g

t

~ 3g

Fig. 5. (A, B) Electrical responses to injection of hyperpolarizing current pulses (upper traces) recorded from tonic fibres isolated from the metathoracic extensor tibiae muscle. The responses in (A) were obtained from a preparation which did not exhibit spontaneous rhythmic contractions. The regular oscillations of membrane potential recorded from this fibre during the larger current-induced depolarizations indicated the presence of electrically-excitable membrane. The response in (B) which was obtained from a spontaneously contracting preparation also demonstrated the occurrence of electrical excitability. Electrically-excited responses generated in adjacent fibres due to electrical coupling could have accounted for the complex response characteristics. The hyperpolarizations in (C, D) were also obtained from a spontaneously contracting preparation. Current injection into one tonic fibre of this preparation not only hyperpolarized that fibre (C) but also hyperpolarized other fibres of the tonic bundle. The fibre in (D) was located seven fibres distant from the current injection site (see E). Calibra-

tion pulse in (C, D) was 20 mV:5 msec.


I I ~ Zero K + sahne 2 rn~n

91

' ....: .... ! i.

I I I mltn

/!tl(IIf!!I!/ ltfll ll l! I! Itl!l ! tl / [ l / /

I Zero K+- saline I

I- 20rnM K+-sahne I

. . . . .

I lOOm M K+-sahne

Fig. 6. Effect of changes of potassium concentration on the spontaneous contractions of bundles of tonic fibres isolated from the metathoracic extensor tibiae muscle. In (A) standard locust saline containing 10 mM K + was replaced by saline containing 0 mM K÷. Note the reduction in rhythm frequency and increase in contraction amplitude in zero K ÷ saline and the transient contracture following return to standard saline. (B-E) A different preparation showing spontaneous activity in standard

saline (B) and the effects of 0mM K + (C}, 20mM K ÷ (D) and 100mM K + (E).

concentration of the bathing medium above 10mM raised the "resting" tension, i.e. the fibres went into a sustained contracture, and a slight increase in frequency of the spontaneous contractions was observed (Fig. 6D). With high potassium concentrations, i.e. > 100 mM, the spontaneous contractions were abolished (Fig. 6E). The contractures which developed in high potassium saline were often punctuated by quite dramatic falls in tension (Fig. 6E). Recovery from treatment with this saline was relatively slow, presumably due to the influx of KCI during the period of treatment and its subsequent slow efflux (Usherwood, 1969).

Saline containing zero calcium initially increased the frequency and amplitude of the rhythmic contractions but this phase of hyperactivity was soon followed by loss of the contractions (Fig. 7A) except for an occasional large response. When 40 mM magnesium chloride was added to the zero-calcium saline the contractions disappeared almost immediately (Fig. 7A) but when added to standard saline, which contained 2 mM calcium the decline was much less rapid (Fig. 7B).

L-glutamate, the putative transmitter at excitatory synapses on the phasic fibres of the orthopteran extensor tibiae muscle, applied in the bath at

5 × 10 -4 M, abolished the rhythmic contractions and evoked a contracture from the tonic fibres (Fig. 8A). Lower concentrations of this amino acid increased the frequency of the contractions. 7-Aminobutyrate (GABA), the putative transmitter at inhibitory synapses on the extensor tibiae muscle fibres, inhi- bited the contractions but sometimes evoked a contracture from the muscle bundle (Fig. 8D). Contrac- tures in response to GABA are perhaps not surprising since isolation of the tonic fibres involves a lengthy dissection which must inevitably load the fibres with KCI and thereby affect the relationship between the resting potential and the chloride dependent GABA equilibrium potential (Usherwood, 1968, 1969). Picro- toxin, which blocks the action of GABA at the inhibitory synapses, increased the frequency of the contractions, and resulted in a slight increase in the base tension of the tonic bundle (Fig. 8C). It is probable that this drug affects the contractions of the tonic bundle by blocking chloride channels in the extrajunctional membrane of the tonic fibres. Ibotenic acid (10-3M), which activates extrajunctional chloride channels on locust muscle fibres (Lea & Usherwood, 1973a, b), blocked the rhythmic activity. Tetrodotoxin at 10-4M had no effect on the rhythmic activity (Fig. 8B).

92 M. D. BURNS and P. N. R. USHERWOOD

I I I I ~. Zero mM Co 2+ Zero mM Co 2÷ t4OmM Mg 2~

l~ll/l/l///1/lll I o,0 I I ' t

Zero mM Co 2+ 2m~n +40mM Mg 2+

t/tItttIlt~ ~ ~, lllllllllll~1lll111llll Io,~ 1 40 mM Mg 2÷ + 2ram Co 2t4 i2rnln I

Fig. 7. Effect of variation in calcium and magnesium concentrations of locust saline on the spontaneous contractions of isolated bundles of tonic fibres from the metathoracic extensor tibiae muscle. Standard locust saline contains 2 mM Ca -'+ and zero mM Mg 2+. Changes in the concentrations of Ca 2+ and

Mg 2+ were compensated for by changes in the concentration of Na ÷.

', ~ ,//,,,/// /~ , , /, t,!/! , , , , // f

5x 10-4M L-glutamate

B

/ !It I/!!/!tit l!t/! !It!It/i!/It!/ I I I I I0 M-4"Tefrodotoxin tmin

C

l I I I |

I0 M-3Picrotoxin I() -3M Picrotoxin

I I 10-4M GABA

Fig. 8. Effect of various drugs on the spontaneous contractions of bundles of tonic fibres isolated from the metathoracic extensor tibiae muscle. Note slight enhancement of contractions during treatment

with picrotoxin (C).

Mechanical properties of locust extensor tibiae muscles 93

DISCUSSION

The prothoracic and mesothoracic extensor tibiae muscles of the locust show a remarkably wide range of contraction speeds in response to stimulation of the FETi and SETi motor axons. The very rapid response to FETi activity reflects the high speed of contraction necessary in walking since the mechanical advantage of the muscle is higher in these legs than in the metathoracic leg (Burns & Usherwood, 1978) and as a result there must be a greater shortening of the muscles. In contrast, the responses to trains of potentials in the SETi and CI were very slow. Fibres with a tension rise time of nearly 1 sec cannot be useful in the leg movements involved in walking and so they must be specialized for some other pur- poses, one of which seems likely to be the main- tenance of posture. Although the contribution of the extensor tibiae muscles is probably small when the animal is standing on a horizontal surface, it must be considerable when the animal is climbing. During climbing behaviour the SETi axons are in fact con- tinuously active at high frequencies (Burns & Usher- wood, 1978).

The very strong "catch" effect may also be useful in maintaining large postural forces, and may be even stronger under the non-isometric conditions obtain- ing in the intact insect (Wilson & Larimer, 1968). Phasic contractions due to FETi activity provide a remarkably efficient way of releasing the "catch" and prevent its being a hindrance when the animal is walking (Burns & Usherwood, 1978). This relaxation is probably caused by the phasic muscle fibres transiently removing the load from the parallel tonic fibres. Further work involving quick release of the muscle will indicate whether this is so.

Both the slow responses of the prothoracic and mesothoracic extensor tibiae muscles to SETi activity and the very marked "catch" effect are probably due to the existence within these muscles of a large number of tonic fibres with properties similar to those in the metathoracic muscle. This is indicated by the large contracture tension developed by the mesothoracic extensor muscle in high potassium saline (Aidley, 1965). However, the tonic fibres do not seem to be arranged into separate bundles as they are, at least in part, in the metathoracic leg. Diffuse distribution of the tonic fibres would seemingly preclude the de- velopment of synchronized contractions in the absence of some form of nervous co-ordination. Per- haps this explains the absence of rhythmic contractions of the pro- and mesothoracic extensor muscles, although oscillations in base tension were recorded during low frequency SETi stimulation which were not synchronized with the stimuli (Fig. 2A). However, it is possible that the tonic fibres in this leg do contract rhythmically in a co-ordinated fashion in the absence of SETi stimulation but that our recording technique was not sufficiently sensitive to monitor this activity.

It seems likely that the rhythmic contractions of the tonic fibres in the metathoracic extensor tibiae muscle are myogenic. The rhythmic changes in membrane potential, the electrical coupling between the fibres on the proximal tonic bundle, the short-term modulating action of the SETi and CI on the rhyth-

micity, the effects of junctional and/or extrajunctional conductance increases induced by drugs and ions and the longer term modulating effects of neurosecrete(s) (see below) all support this contention. Comparisons with insect visceral and cardiac muscle which show myogenic rhythmicity (Miller, 1975) cannot be avoided. The effects of divalent cations on the rhythmicity might suggest that the contractions are under neural control, possibly either by the spontaneous generation of bursts of action potentials in the endings of SETi or through the activity of "peripheral" neurones (i.e. with cell bodies outwith the CNS) located either on or in the vicinity of the tonic muscle fibres. However, the observed effects of divalent cations could be due to direct action of these ions on the membrane of the tonic fibres. Furthermore, there is no structural evidence for "peripheral" neurons either on or in the vicinity of the tonic fibres in the proximal part of the extensor tibiae muscle. The failure of tetrodotoxin to abolish the rhythmic contractions is further evidence against neural control of the rhythmicity, at least, that is, neural control involving sodium action potentials.

Hoyle & O'Shea (1974) have suggested that conduction and co-ordination between the tonic fibres in the metathoracic extensor tibiae muscle which result in synchronous contractions would require bridges between the fibres. Cochrane et al. (1972) found no evidence for such structures during their ultrastructural studies of the tonic fibres of the locust metathoracic extensor tibiae muscle. However, these fibres are very closely packed with the result that their surface membranes are probably in sufficiently close contact to ensure good electrical communication. If tonic fibres in the extensor muscle other than those found in the proximal bundles are also involved in generating the rhythmic movements of the extensor muscle then how are the contractions of these fibres synchronized with the tonic fibres at the proximal end of the femur? Possibly the answer lies in the neurosecretory neurone (DUMETi) which supplies the metathoracic extensor tibiae muscles of locusts and grasshoppers (Hoyle et al., 1974), although the motorneurones which innervate these muscles may also play a co-ordinating role.

The DUMETi neurone has been shown to be octopaminergic (Hoyle & Barker, 1975; Evans & O'Shea, 1977). Inhibition of the rhythmic contractions of the metathoracic extensor tibiae muscle results from either DUMETi activity or octopamine application to the muscle (Hoyle, 1974; Evans & O'Shea, 1977). However, octopamine is not the only neurosecrete known to influence the rhythmic activity of this muscle. The pentapeptide, proctolin, a myotrophic compound, extracted from cockroaches by Brown (1975) has been recently tested on this muscle by Piek & Mantel (1977). Low concentrations (10-1°M) of proctolin initiate rhythmic contractions in quiescent muscle preparations whereas higher concentrations (10 -8 M) of this peptide evoke a sustained contracture. Proctolin causes a slow contracture of the proc- todeum of Periplaneta americana when applied to this part of the gut at concentrations of 10 - 9 M or greater (Brown, 1975; Brown & Staratt, 1975; Staratt & Brown, 1975). There is no clear evidence for a proc- tolinergic innervation of the locust extensor tibiae

94 M.D. BURNS and P. N. R. USHERWOOD

muscle. Possibly proctolin is released from the endings of FETi, SETi and CI along with the neurotransmitters secreted by these neurons. Dense-core vesicles of the type normally associated with neurosecretion, are found in the endings of locust motorneurones (Rees & Usherwood, 1972). An alternative possibility is that proctolin reaches the tonic fibres via the circulatory system after being released at sites distant from the extensor tibiae muscle. It is, of course, important to determine whether proctolin is, in fact, a locust neurohormone.

Usherwood (1974) proposed that the tonic bundle in the metathoracic extensor tibiae muscle forms an integral part of the blood circulatory system by acting as a valve controlling the flow of haemolymph through the femur and tibiae of the metathoracic leg. The motor nerve supply to the tonic fibres would presumably serve to control phasically the ongoing activity of the valve and thereby the flow of haemolymph through the leg segments whereas longer-term tonic control could be achieved through the activity of neurosecretes such as proctolin and octopamine. Whether the tonic fibres also serve a postural function as proposed by Runion & Usherwood (1970) remains to be established. It seems likely, however, that these tonic fibres are responsible in part, for the "'catch" phenomena described by Wilson & Larimer (1968).

The complex chemical control exerted by the nervous system over the metathoracic tonic muscle fibres provides the neuropharmacologist with exciting ex- perimental material. It seems likely that there are glutamate (excitatory) and GABA (inhibitory) synapses on the tonic fibres in the metathoracic extensor tibiae muscle. Possibly there are in addition synapses between D U M E T i and these fibres. It seems more likely, however, that the site of action of the secretion from this neurone is on the extrajunctional membrane of the tonic muscle fibres, where it mediates an effect via either cyclic A M P or GMP. The immediate target sites of these nucleotides could be protein kinases whose activation results in the phosphorylation of specific membrane proteins (Greengard, 1976) and thereby modification of the electrical properties of this membrane and its capacity to generate potential oscillation. Since Evans & O'Shea (1977) have now demonstrated modulat ion of SETi and CI postsynap- tic potentials by D U M E T i activity, it may be that D U M E T i also has a direct presynaptic or postsynap- tic effect on the endings of these neurones.

SUMMARY

1. Contractions of the prothoracic and mesothoracic extensor tibiae muscles of the locust induced by activity of the "slow" excitatory neurones (SETi) which innervate these muscles have long time courses and low fusion frequencies, but the responses to the "fast" excitatory motoneurones (FETi) are more rapid than those in any of the metathoracic leg muscles. Twitch/tetanus ratios in these muscles are 1:7 for the FETi and 1:20 for the SETi.

2. The response to SETi activity in these muscles shows a memory or "catch" effect stronger than that of the metathoracic muscle, high tensions being maintained by lower firing frequencies than are needed to develop them.

3. The inhibitory input to the extensor tibiae muscles considerably reduces tension responses to the SETi, accelerates relaxation and reduces "catch" tension.

4. FETi induced contractions in these muscles are stronger than the responses to the SETi and will sum with them but, under isometric conditions at least, they cause a subsequent relaxation in the tonic muscle fibres and can reduce "catch" tension.

5. A bundle of tonic fibres has been isolated from the metathoracic extensor tibiae muscle of the locust which exhibits co-ordinated rhythmic contractions similar to those recorded from intact muscles.

6. The contractions are not affected by TTX but they are blocked by either calcium removal from or magnesium addition to standard locust saline.

7. Depolarizations of the tonic fibres coincide with the contractions and are sometimes accompanied by bursts of EPSPs and IPSPs.

Stimulation of the SETi evokes a slow twitch contraction from the tonic fibres, the amplitude of which is diminished by additionally stimulating the inhibitory input.

8. The fibres are electrically-coupled. The rhythmic contractions are probably myogenic.

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