INTRINSIC RHYTHM AND BASIC TONUS IN INSECT SKELETAL...

Jf. exp. Biol. (1978), 73, 173-203 173With 21 figures

Printed in Great Britain

INTRINSIC RHYTHM AND BASIC TONUS ININSECT SKELETAL MUSCLE

BY GRAHAM HOYLE

Department of Biology, University of Oregon, Eugene, OR. 97403, U.S.A.

{Received i^July 1977)

SUMMARY

The jumping muscle of orthopterous insects contains fibres that possess anintrinsic rhythm (IR) of slow contraction. The contributing fibres aregenerally synchronized, but as many as three or four pacemakers are present.The frequency, amplitude and duration of IR contractions fluctuateerratically over a 24 h period. Metathoracic DUM neurone bursts suppressIR for a few minutes. Other, unidentified dorsal neurones enhance itsamplitude. In addition to IR, the extensor tibiae shows intrinsic basic tonus(BT). BT is relaxed for several s by low-frequency burst output from un-identified metathoracic dorsal neurones. DUM neurone bursts may enhanceextensor BT, relax it, or leave it unaffected.

The effects on IR of various regimes of activity in the slow extensortibiae (SETi) and the common inhibitor (CI) axons were examined.

CI affects IR when stimulated at frequencies above 2 Hz. It causes ampli-tude depression and reduced duration of individual IR contractions as wellas increased frequency. At 30 Hz and above, CI completely suppresses IR.An enhanced IR contraction starts within a few milliseconds of the termina-tion of a CI train.

At low frequencies (below 10 Hz) SETi causes increased frequency anddecreased amplitude of IR, with a depressed IR contraction followingcessation of the SETi burst. At frequencies above 15 Hz the SETi-evokedcontraction dominates tension development, though IR summates with itduring the rising phase. In quiescent preparations not showing IR, SETistimulation at 10 Hz often started up IR.

Single SETi or FETi impulses can initiate an IR contraction, and causealtered phasing, with up to a quintupling of frequency.

After a critical period has elapsed following the onset of an IR contraction,a single impulse in any one of the three axons will terminate it abruptly. Theearly termination is followed by a reduced interval which is proportional tothe reduced IR contraction time. The rhythm of accumulated readiness togo into an IR contraction is independent of the pacemaker rhythm thatinitiates the contraction.

INTRODUCTION

The large, anterior, dorsal, unpaired median (DUM) neurones of orthopterousinsects (Plotnikova, 1969) were first studied physiologically by Kerkut, Pitman &Walker (1969), who found their cell bodies to have overshooting action potentials.Crossman et al. (1971) filled some of them with dye in the cockroach and like

174 G. HOYLE

Plotnikova (1969), found tentative evidence for their sending an axon into a leg nerve.This possibility was fully substantiated by Hoyle et al. (1974) for the locust Schisto-cerca gregaria DUM neurone, DUMETi, whose axons were followed into its finalterminals and found to make neurosecretory-type endings in the jumping muscle.

In testing the possible action of DUMETi on muscle properties and neuromusculartransmission I was unable to find any effect of the axon on transmission, but theextensor tibiae would sometimes very slowly extend spontaneously in a rhythmicalmanner. Such movements had been noticed in Dr P. N. R. Usherwood's laboratory,who had demonstrated them to the Scottish Electrophysiological Society. In a note tothe Physiological Society, Anderson et al. (1970) state: 'tonic fibres... undergospontaneous rhythmic contractions in vivo and in vitro. These contractions are possiblymyogenic and could conceivably be synchronized by electrical coupling between thefibres.'

The movement is of variable amplitude and time-course, both in different prepara-tions or different legs of the same animal and for the same leg at different times,although it can be constant over several hours. When at its most pronounced, thetibia extends through an arc of 300. How was it possible to have missed if for so long ?The answer is that it is actively suppressed when the animal is disturbed and handled,and the suppression is usually long-lasting. Also, it is seldom evident in Locustamigratoria preparations, on which the early neuromuscular research was carried out,unless the leg nerve has been cut for some hours. In all insects that show it, a few daysof denervation, followed by excision of the leg, are sufficient to reveal that it is anintrinsic feature, at least of the extensor tibiae muscle. It will be referred to hereafteras the intrinsic rhythm (IR). An example, recorded as isometric force by a transducerattached to the extensor tibiae of an isolated Schistocerca gregaria femur, is shown inFig. 2. Another reason for the late discovery of IR is that bathing the muscle in insectRinger's solutions tends to suppress it. It has now been found that a hidden, orRinger-suppressed IR can be recovered by adding acetylcholine (io~8 M) and eserineto the saline (G. Hoyle & E. Florey, in preparation).

In the intact animal the IR movements are sufficiently strong to initiate resistancereflexes (Hoyle & O'Shea, 1974). During the extension phase the antagonistic flexortibiae (FITi) muscle is excited and the slow extensor (SETi) is inhibited. Duringrelaxation the flexion movement reflexly causes SETi to be excited. The net result isthat there is very little actual movement in the intact animal, which is another reasonwhy IR failed to be noticed. The animal is, in effect, continually performing a set ofisometric exercises.

The locust IR has acquired a special interest since it was found that it is suppressedby DUMETi (Hoyle, 1974). The suppression is mimicked by some biogenic amines,the most effective of which is octopamine, which works at a concentration as low as2 x io~10 M (Hoyle, 1975).

These observations also bring into focus the neglected question of the generationof tonus and its control by central nervous systems. Before about 1930 there had beenmany theories about the basis of tonus in skeletal muscle. A favourite hypothesis hadbeen that of Langelaan (1915, 1922), known as the dual theory of tonus, whichclaimed that there are two separate aspects to tonus: a basic, or 'plastic' element thatis modulated by the sympathetic nervous system, and a 'contractile' element

-••"t

Rhythm and tonus in insect muscle 175

if

Fig. 1. Drawing of basic preparation used to study the intrinsic rhythm (IR) and basic tonus(BT) of locust or grasshopper extensor tibia muscles, and their modulation by neural stimu-lation or drug action. Muscle fibres responsible for IR are located exclusively in the proximalregion of the muscle. Electrodes were used to stimulate identified motor neurones, as follows:(a) fast extensor tibiae (FETi); (6) common inhibitor (CI); (c) slow extensor tibiae (SETi);(d) dorsal unpaired median neurone innervating extensor tibiae (DUMETi), or other dorsallylocated neurone.

dependent upon ordinary motor innervation. The ideas were reviewed by Stanley Cobbin 1925 (Cobb, 1925), who was impressed only by the elegant, eventually to becomeclassical, work of Liddell & Sherrington (1924, 1925) on the myotatic reflex. He con-cluded 'that tonus is a beautifully graded series of proprioceptive reflexes.' And thatwas that; even invertebrate myo-neural physiologists have never chosen to challengethis conclusion except in the case of the 'catch' mechanism of some molluscanobliquely striated muscles.

In arthropods, studies of the membrane potential-tension relationships of individualmuscle fibres revealed some potential anomalies. Atwood, Hoyle & Smyth (1965)found some fibres in the crab claw closer muscle whose resting membrane potentialwas essentially at the excitation-contraction coupling threshold (Ec). In the levator

176 G. HOYLE

of the eyestalk of the crab Podophthalmus, Hoyle (1968) found several muscle fibreswith resting potentials below Ec. They are in a constant state of partial contractionthat is their normal state, not a pathological one, which serves the useful function ofkeeping the eyestalks raised. This is so in the absence of excitatory nerve input. Whenonly the inhibitory axon supplying the muscle was stimulated, the muscle relaxed. Theslow excitatory axon acts reflexly to supplement this intrinsic tonus and move therelevant eyestalk, in conjunction with the inhibitor. This system is clearly a dual tonuscontrol mechanism.

The present work, though principally a study of IR, supports the prematurelydiscarded notion of dual mechanisms of tonus in striated muscles. As in the crusta-ceans, not all muscle fibres of a muscle contribute: only specialized slow fibres areinvolved.

MATERIALS AND METHODS

The species studied were the locusts Schistocerca gregaria Forskal = 5. americana(Dirsch, 1974), Locusta migratoria and Schistocerca vaga = S. nitens nitens (Dirsch,1974), and the grasshoppers Romdlea microptera and Brachystola magna. The principalpreparation used (Fig. 1) consisted of a dorsal dissection in which the wings wereremoved and the thorax was bisected longitudinally followed by removal of the heartand gut. The wing bases were clamped firmly and the body and legs, but not theabdomen, firmly bedded in soft dental wax. A hard-wax-coated platform was micro-manipulated under the metathoracic ganglion. The tibiae were cut off just below theknee joint, and the latter opened up. The extensor tibiae apodemes were cut close totheir distal insertions and seized by fine forceps tips attached directly to micro-manipulated RCA 5734 mechano-electronic transducer tubes, providing isometricforce measurement. The apodeme position corresponded to a femero-tibial angle ofapproximately 400. Some recordings were also obtained of tension, or movement ofthe tibia, from whole animals and preparations in which the whole leg was left intact.In these, force was registered by tying the tibia to the force transducer; movement wasregistered by a photocell, with a light flag attached to the tibia to interrupt a lightbeam.

The intrinsic rhythm is sometimes altered, or even abolished, by perfusing the legwith saline, so the present experiments were all carried out with the muscle bathed inits own haemolymph. A petroleum jelly cup was fashioned around the metathoracicganglion so that it could be bathed independently in locust saline without the latteraffecting the leg muscle.

Ganglion cells were excited either directly, by an intracellular electrode, or in-directly via small insulated wires or a suction electrode applied to the ganglion surface.The slow extensor tibiae axon (SETi) was excited via small hook electrodes placedunder nerve N3b, the common inhibitor (CI) by similar electrodes placed under N3C,and the fast extensor (FETi) by electrodes touching N5.

RESULTS

Occurrence of the intrinsic rhythm

IR was found in most preparations whilst the muscles were still bathed in haemo-lymph, and the patterns observed were substantially similar in all genera and species.


LMUUUUUUJUU

JdLJUUUUUuLUUUUUul

uuJJFig. 2. Intrinsic rhythm (IR) of metathoracic extensor tibiae of .S1. gregaria recorded as iso-

metric force changes from the isolated leg. Calibration: vertical, 0-5 g; horizontal, 1 min.

It is not possible to say, even from a long-term recording, which species a given recordcame from. Some preparations did not show IR within the first 2 h of setting up, butthen it began to appear. In these, the muscle was in a state of strong tonic contractionafter being set up, and the indication that a rhythm would appear was an abruptrelaxation having a time-course similar to that of an IR relaxation. It might then beminutes before a contraction followed, and likewise before the next relaxation butgradually the rhythm speeded up to 3 typical one. IR persisted, essentially unchanged,when recorded from an isolated leg or femur (Fig. 2).

Hoy/e & Fforey (in preparation) studying similar Locusta preparations found thatthe majority did not show either a strong tonus or IR. However, they found that IRcould quickly be induced by adding acetylcholine (io~8g/ml threshold) and eserineto ordinary locust saline and perfusing the leg with it. Other tricks that revealed IRwere isolation from other locusts for 2 weeks, and denervation.

i78 G. HOYLE

B /I

D A A A A

Fig. 3. IR recorded from left (L) and right (R) extensor tibiae at the same time from differentspecies, sexes and stages of saltatory orthopterans. A, S. gregaria, (i) adult $; (ii) adult <$\ (iii)fifth instar nymphal 6"- B, S. vaga $. C, Romalea microptera $. D, Locusta migratoria $.Calibration: vertical bar, o-i g; time, 1 per s.

Rhythm and tonus in insect muscle

B

179

Fig. 4. IR recorded at diflFerent times during a 24 h period with 12 h artificial light, 12 h dark,from left and right legs of S. gregaria 6". A-E and I-K during daylight. F-H at night. Bar =1 min. Average interval between records was about 2 h.

The unit IR contractionEach IR contraction consists of three components: (1) a slow rising phase, when

tension is increasing; (2) a peak, which is sometimes sharp but usually extended intoa plateau; and (3) relaxation, which is relatively fast compared with the rise time. Apause, or interval, follows each contraction. The three principal variables encounteredwere: the amplitude of the excursion, the total duration of the contraction and theinterval between successive contractions.

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The basic IR for a given muscle can be determined only in the isolated leg. Notonly is IR affected by direct neural output from the ganglion, it is also affected byneurohumoral material released into the blood. Ideally, the isolated limb should beperfused by a simple standard saline solution. Unfortunately, all salines so far testedhave reduced or stopped the rhythm completely in some specimens. An improvementin IR stability in saline occurred in Locusta, which is particularly sensitive to saline,by using lowered K+ (2 ITIM/I, Hoyle & Florey, in preparation), but no improvementwas noticed in either Schistocerca or Romalea. In the isolated femur, the extensortibiae showed a strong IR that persisted with little change in amplitude, duration andfrequency for several hours. The tension developed was maximally about 0-2 g. Forthe example shown in Fig. 3, the mean duration of an IR contraction was 4-8 s, themean frequency 5-6 per min. During a period of 3 h the same preparation showed a4 s minimum and a y s maximum duration, and 4 per min minimum and 6 per minmaximum frequencies. During the whole second hour, however, the variance in theseparameters was below 15%.

Variations in IR

In the intact animal, the amplitude of extensor tibiae IR contractions fluctuated,in all species, in an unpredictable manner. The longest single IR lasted for almost4 h, and the shortest one, 1 s. The frequency varied from 1 per 4 h to 8 per min. Thetime to peak contraction varied from o-8 to 40 s for the same muscle at different times.The corresponding times from peak contraction to 70% relaxation were 0-8-1-4 s.Examples from different species are illustrated in Fig. 3, taken, in each case, about 1 hafter setting up the preparation. However, the detailed patterns for a particularspecies are meaningless in view of the extensive variations at different times, as therecords shown in Fig. 4 clearly demonstrate. These records were samples of a con-tinuous record from one S. gregaria at various times during a 24 h period. During thistime there were many changes in amplitude, frequency and duration of IR contrac-tions, and also in the presence or absence and size when present, of relaxation under-shoot.

It will be shown that frequency, amplitude and undershoot are under central nervouscontrol, probably by neurohumoral activity. It is not known what factors determinethe durations of IR contractions, which change slowly, in a manner which suggestsneurohumoral action, over a remarkably wide range.

In addition to slow, smooth transitions, many occurred abruptly (Fig. 5). In lightof experience, to be described below, of the effects of ordinary motor nerve activityon IR, it seems that abrupt changes in IR may be caused by effects of either SETi,CI or both axons.

Site of IR and its electrophysiological concomitants

It was anticipated that the source of IR would be an oscillatory depolarization, butsince during earlier studies on locust muscles only stable membrane potentials wererecorded in widespread sampling, it was likely that only a relatively small number offibres are involved. There could have been multiple sites, or there might be a singleregion involved. Very long, flexible glass capillary microelectrodes filled with 2 M-K-acetate, first tested for their ability to bend without giving artifacts, were used. A few

Rhythm and tonus in insect muscle 18:

Attenuation ofIR characteristic

of DUMETiaction

Uu

Acceleration of IR-acharacteristic accompaniment

of weak SETi discharge(which antagonizes DUMETi action)

Leg extends, then flexes topartially extended position

"ILeg extends then flexes.Weak suppress

of weak DUM neurone discharge

B • w u u u w u i ^ ^Small twitches [arising out of

R * | Normal IR

IRLeg extendsthen flexes Vslowly

Period of interference with IR characteristic of low-frequency Cl discharge

Fig. 5. Three different types of change in IR patterns in the absence of extrinsic stimulation.These are due to interactions between the normal motor innervation and IR. S. gregaria <J.A, Marked attennuation of left IR occurring at the same time as slow extension in the right legonly. Later, a prolonged extension occurred in the left leg, accompanied by increased IRfrequency at reduced amplitude, whilst IR frequency fell in the right leg. These events can beexplained (see Hoyle, 1978) on the basis of a DUMETi discharge at the same time as a briefSETi burst to the right leg, followed by a prolonged SETi burst to the left leg. B, The rightleg was showing a large, quick extra contraction (arrow — compare with second, normal con-traction). Fast extension of right leg only (caused by FETi) was accompanied by the attenuationof IR of left leg without change in its frequency. This type of action is caused only by dis-charge of the common inhibitor. Increase in frequency of IR of right leg that follows extensionis associated with prolonged, but declining, SETi discharge. C, Marked attenuation of IR,with fluctuating amplitude and slight increase in frequency are attributable to CI firing, withprogressively increasing frequency, between arrows. Time marks: 1 s, A, C; 2 s, B.

muscle fibres showing depolarization waves at the same time as IR (Fig. 6) werelocated in a large bundle of fibres located on the outside of the apodeme between 6and 3 mm distant from the proximal border of the femur. Fibres examined elsewherein the muscle showed no depolarization waves. The active fibres were in the sameregion found by Hoyle & Florey (in preparation) to be the one where drugs applied insmall drops most readily affected IR. There is an obvious shortening of fibres in thisregion during IR in the isolated or denervated leg. This bundle was isolated from thewhole of the distal part of the muscle, and also the more proximal fan-shaped region,and continued to develop IR tension equal to at least 80% of that developed by thewhole leg. Hence, fibres in this region are largely, or entirely, responsible for IR. Thedepolarizations shown by these fibres generally matched rather closely the shape ofIR contractions. They ranged from the barely detectable, to ones having 24 mV peakamplitude. It is presumed that the larger waves were recorded from pacemaker fibres

182 G. HOYLE

r I

SmV

Fig. 6. A-D, Membrane potential changes in presumed pacemaker muscle fibres generatingIR: R. microptera. In each experiment the whole extensor tibiae tension is recorded on theupper trace. Intracellular (K acetate) electrode recording is on the lower traces. Calibrationsignal of 5 mV in A. Unidentified DUM neurones were stimulated via an external suctionelectrode at times indicated by bars. Note the close parallel between the electrical potential andthe total tension. Calibrations: vertical bar = o-i g, 7-0 mV. Time marks: 1 s, A-D; bar =10 s in E.

and the smaller from one weakly electrically coupled to them. As IR amplitude andduration fluctuated naturally, so did those of the depolarization wave. That the wavewas not an artifact was demonstrated when strong SETi contractions and FETitwitches were interposed in the records.

DUMETi and other DUM neurones were stimulated whilst recording intracellu-larly from an IR 'pacemaker' muscle fibre. The amplitude of the IR contractions wasreduced following the neural stimulation and it was found that the depolarizing waveswere correspondingly reduced (Fig. 6).

The onset of depolarization in presumed pacemaker fibres preceded the onset oftension development in the whole bundle by a few milliseconds, but occurred insome fibres showing a small wave after the onset of tension. The onset of relaxationwas not well correlated with tension decline, and in some fibres the onset of relaxationoccurred before repolarization. But this anomaly could be explained on the basis ofdifferences between contraction times of individual fibres. It will be shown below thatthe rhythmtcity can be markedly different in different regions of the same muscle.


Flex

Fig. 7. Evidence that IR can occur in the flexor tibiae of S. gregaria. A, In this experimentmovement was being recorded from left and right tibiae using a pair of photo cells. The rightleg only, showed rhythmic flexions (downwards movements from baseline) alternating, at aslightly different period with the extensor IR. B, This record was obtained during an experi-ment in which a force transducer was attached to the tibia of an intact preparation. Rhythmicaldownwards deflexions indicated that a flexor IR was present. The movement initiated excita-tion of the slow extensor, which in turn triggered an early extensor IR contraction but this, inturn, activated flexor excitation.

When the slow axon discharged, as in Fig. 6 A, following the second ganglionstimulation, and also Fig. 6C, the SETi contractions were accompanied only by avery small depolarization. FETi fired during the experiment shown in Fig. 6E, asshown by the twitch at the arrow, and there was no e.j.p. Neighbouring fibres generallyhad relatively large, facilitating SETi e.j.p.s and also large CI i.j.p.s, but some alsoresponded to FETi with large e.j.p.s and overshooting spikes. There is no branch ofDUMETi to the region.

How many of the muscles show IR ?

The following muscles were examined for signs of IR: flexor tibiae, tarsal levator,tarsal depressor, retractor unguis, anterior coxal rotator, coxal adductors, coxallevators, trochanteral levators and dorsal longitudinal flight. Only in the flexors wasevidence obtained for the existence of an IR. During isometric recordings made byattaching the force transducer to the tip of the tibia, two tension rhythms, withdifferent amplitudes and durations occurred. One of them was an enhanced tension,attributable to the extensor. The other was of tension reductions and could only havebeen produced by the flexor (Fig. 7 A). On another occasion (Fig. 7B) there wasstrong interaction from reflexes, but the dominant rhythm was a flexion. However,rhythmic contractions were not obtained from any preparation during direct isometricrecording from the flexors, so intrinsic rhythmicity is either rarely present in flexorsor is easily suppressed there.

Aberrant IRs

In a few preparations complex polyrhythms occurred. This condition was seen toarise out of a regular rhythm in three preparations, and in two others it graduallychanged into a smooth IR. Intracellular recordings made from pacemaker musclefibres at these times showed normal-looking IR depolarizations. It seems probable,then, that a highly irregular baseline occurs when IRs of individual fibres of fibreclusters cease to be synchronized, and vice versa.

Examples of polyrhythm, in which two or more units repeating at different fre-quencies could be recognized, are shown in Fig. 8. Figure 8 A shows one in which alarge IR and a smaller one in the same muscle were very similar in shape and frequency.They summated together in simple algebraic fashion, showing clearly that they weregenerated by two independent pacemakers affecting two different sets of muscle fibres.

G. HOYLE

Fig. 8. Evidence for multiple IR pacemaker regions, that are not always synchronized. A,Two pacemaker regions with slightly dissimilar periods. The contractions summate when inphase. B, Three independent pacemakers in left extensor, normal IR in right extensor: i?.microptera. The three units had slightly different periods: unit no. 3 was the fastest. This hadsmall, late oscillations superimposed. There was also a strong basic tonus that was lost duringthe recovery period after each contraction of the larger unit 1 (indicated by R = relaxation ofBT). Time marks: 1 per s. C-D, Examples of preparations that showed periodic fluctuation inamplitude. Calibration: vertical bar = o-ig; time marks: 1 per s.

Another example, shown in Fig. 8B, is more complex: there were at least fourcomponents. One occurred at the same frequency as the stronger unit and wassynchronized with it (1), leading to a step in the rising phase (arrows). Another (2)was small, and of short duration whilst a third (3) was small but of long duration. Thelatter had a ripple on its plateau phase. The muscle bundle had a strong basic tonus,and following the unit 1 IR contraction there was a large relaxation undershoot (R).But when the onset of unit 3 overlapped the unit 1 relaxation the undershoot wasmissing. The sub-rhythms commonly summed simply: more complex interactionsometimes occurred, and all sometimes merged into a single normal IR. Thus thereare a few IR pacemakers in the jumping muscle or perhaps every muscle fibre involved


has its own intrinsic rhythmicity, but somehow the fibres are normally synchronized.The question of how synchrony is brought about is of considerable interest in itself,and will be the object of future studies.

A further type of aberration could not so readily be explained. It consisted of aperiodic fluctuation in amplitude, that was at times highly regular (Fig. 8C, D). Itwas as if there was a basic period causing equal contractions in about half the fibres,with progressive recruitment of other fibres on the second, third and fourth repetitionsonly.

When IR contractions had a long plateau phase, this tended to be interrupted byshallow quick relaxations, suggesting that some contributing units had a higherfrequency than the dominant one.

Independence of IR on left and right sides

It is to be expected that intrinsic rhythms of the two sides be completely indepen-dent, but in the intact or nearly-intact insect the possibility exists of neural co-ordination. There were indeed times when the left and right legs had perfectlysynchronized IRs, of similar amplitudes and widths, but synchrony did not last forlong, and it was concluded that in all cases it resulted from co-incidence. At timesthere was a marked disparity on the two sides, in regard to all parameters. There was,however, a strong tendency for the same types of overall fluctuation, frequency oramplitude shift, to occur in IRs of the two sides at about the same times. This waspresumably because many central nervous influences affect both sides at the sametime, as has to be the case for actions caused by unpaired neurones. Nevertheless, themagnitudes of the effects were seldom exactly matched on the two sides. On nooccasion was an IR observed to be synchronized to respiratory movements.

Basic tonus (BT) and neurones that affect it

In some preparations there was an undershoot of the tension baseline during therelaxation phase of each IR contraction, with slow return to the starting tension. In theintact animal, over a long period, the undershoot would come and go (see Fig. 4). Theundershoot could occur only because there is a basic weak background tonus in themuscle fibres in which IR occurs. The undershoot was greatest when the backgroundtonus was moderately large. When this relaxed, the tension undershoot disappeared.The background tonus appeared to be developed independently of IR, but when it wasvery large, as was occasionally the case, IR was reduced until, in some cases, it vanishedaltogether, to re-appear after the tonus had subsided somewhat. It became obviousduring experiments on intact animals that basic tonus is under some kind of centralnervous control. Octopamine, or stimulation of DUM neurones, not only inhibits IRbut also affects the basic tonus, the common reaction being relaxation (see Fig. 1 inHoyle, 1975), but sometimes contraction occurs (Fig. 9).

Whilst exploring the effects of a small electrode applied to the dorsal surface of theganglion, a site was consistently found that had a dramatic relaxing action on basictonus (BT) of the extensor tibiae (Fig. 10). This effect, unfortunately, has not beenseen whilst stimulating any individual neurone intracellularly. The effective site wasin region C4 (Burrows & Hoyle, 1973) and it affected BT on the stimulated side only.At the same time the amplitude of IR was reduced, but only if the frequency and total

186 G. HOYLE

2-5xlO-9Moctop.

2 - 5 X I 0 -

2-5x10octop.

Fig. 9. Opposite actions of octopamine on basic tonus of left and right legs of the same animal:Romalea, central nervous system intact. At the first arrow for each leg, and the second arrowalso for the left leg, a c o i ml drop of 2̂ 5 x 10-9 M DL-octopamine was added as shown in Fig. 1.Atthesecondarrow, for the right leg, and the third for the left leg, a o-1 ml drop of 2-5 x IO"8MDL-octopamine was added. The left leg had little BT, and what there was relaxed (R) followingoctopamine application. The right leg had a very strong BT, as evidenced by the marked under-shoots in the IR tension records. Paradoxically, the addition of octopamine led to an increase (C)in BT. Time marks: 1 per s.

D

Fig. 10. Relaxation of basic tonus during localized stimulation applied to unidentified neuronein left dorsal quadrant D4. Frequencies of stimulation: A, B, C, I per a s ; D, 1 per s; E,5 per s. The rate, extent and duration of relaxation are functions of the number as well as thefrequency, of stimuli. The action greatly outlasts the stimulation, suggesting that a neuro-secretory action is occurring. Note that the amplitude, but not the frequency of IR was re-duced, briefly, following a burst of stimulation at the higher frequencies. The IR inhibitingaction had a different time course to the relaxing action. IR amplitude was enhanced duringthe time BT was relaxed. Arrows indicate abrupt return of BT. These experiments are im-portant because they give some indication of the amount of basic tonus and the extent to whichit is under central nervous control. Calibrations: 01 g, 10 s.


Fig. 11. Experiments similar to those illustrated in Fig. 9, showing the complexity of therelation between stimulation frequency and duration on BT and IR. Frequencies: A, 1 Hz;B, C, 2 Hz. Calibration: o'i g, 10 s.

number of stimuli were above critical levels. There was a delayed, rebound increasedin amplitude and frequency of IR if a sufficiently high frequency was used (Fig. 10E).

A pair of shocks at a separation of 4 s was sufficient to produce a detectable fall inBT, that lasted for about 70 s. Five shocks at the same frequency produced a largerdecrease in BT, of 70 mg, that lasted for 180 s. Ten shocks at the same frequencyrelaxed BT by 120 mg for 210 s. At this frequency the amplitude of IR contractionswas enhanced (as they started from a lower BT level) and there was only a very smallslowing of IR frequency. At a shorter interval (2 s) a single IR was attenuated, and at ayet shorter interval (0-5 s) IR was completely suppressed for three cycles, attenuatedfor a further three, but followed by a period of enhancement (Fig. 10E). A 10 sstimulation at 2/s led to an abrupt relaxation of BT that lasted 396 s.

The rate of relaxation of BT, as well as its maximum extent, was also a functionof the frequency of the stimulation. The duration of the period of relaxation of BTwas a function of both the frequency and the number of shocks (Figs. 10, 11). As isalso true for neurones enhancing ER, it is especially desirable to locate the specificneurones responsible for the extraordinary graded relaxation of BT.

Is there a relation between BT and IR ?

BT is clearly present in the muscle fibres responsible for IR. Is it only present inthese fibres ? At the present time this question cannot be answered and furtherresearch will be needed to resolve it, but it seems unlikely, in principle. DUMneurone activity generally relaxes BT, but there have been exceptions. The same wastrue in the experiments in which octopamine caused relaxation of BT in one leg butenhancement in the other. Since the undershoot was not changed, the increase in BTwas probably in muscle fibres other than those responsible for IR.

Nevertheless, as was mentioned earlier, IR is small when BT is high and may notbe visible at all until BT relaxes. Occasionally, individual IR contractions were seento lengthen, erratically, until an individual IR failed to relax (Fig. 12). This state wasindistinguishable from one in which there was relatively strong BT.

i88 G. HOYLE

Fig. 12. Continuous record of IR in Brachystola in which the IR contractions abruptly becameprolonged. Note that at the anticipated normal relaxation time relaxation started, but then ledinto a long series of minute contraction waves followed by smooth contraction. The last IR ledinto steady tonus that lasted indefinitely.

Neurones affecting IR amplitude

No individual neurones were identified that caused IR amplitude to increase. Butduring localized stimulation of the dorsal surface of the ganglion, a site was found inquadrant B 5 of the map in Burrows & Hoyle (1973) that produced a marked, long-lasting enhancement of the amplitude of IR contractions (Fig. 12). The amplitudeincrease was not accompanied by a change in IR frequency, for low frequencies ofstimulation, but at frequencies above about 10 Hz the frequency was increased at thesame time as the amplitude was enhanced.

The amplitudes of both sides of the animal were affected at the same time, within1 s of the onset of stimulation when this was commenced at about the time an IRcontraction was anticipated. A frequency of about 10 Hz for 2-3 s was required toproduce the effect. The results and stimulation requirements are compatible withtheir being associated with a DUM neurone. However, all DUM neurones that havebeen penetrated and directly stimulated with a microelectrode have produced inhibi-tion, not enhancement, of IR, probably via octopamine release. It is possible that oneof the smaller DUM neurones was responsible. However, only one of them travels inthe extensor nerve, namely DUMETi, which is a strong inhibitor.

The enhancement occurred at exactly the same time on both sides provided theeffective dorsal site was stimulated at a time when IRs of the two sides were expectedto occur together (Fig. 13B). The delay in action cannot have been more than about0-5 s. This suggests that the sites of release of the neurohumoral agent responsible areclose to the extensor muscles.

The decay of the enhancement was a function of the duration of stimulation.Maximum enhancement following a 20 s stimulation at 10 Hz was 8-25 x , and this


Fig. 13. Amplitude enhancement of IR caused by stimulation of an unidentified dorsal un-paired neurone of the metathoracic ganglion of S. gregaria. Movement records were made withphotocells and flags attached to each tibia, that interrupted a light beam. Approximately linearoutput in relation to femero-tibial angle. The solid bar indicates the period of stimulation ofthe dorsal neurone, at 10 Hz. A, The complex activity in A suggests that SETi may have firedat a low frequency for about 25 s after about 10 s of stimulation, terminating when a FETitwitch occurred. Then there must have been inhibition, or a flexor burst, to abbreviate thefirst IR contraction after the twitch. Time marks: 1 per s. B, Nearly synchronous enhance-ment of the left and right IR contractions. There was a slight brief acceleration of IR of theleft side only. Time marks: 1 per s.

decayed to one-half (4-1 x ) 35 s after cessation of stimulation. Following a 5 s stimu-lation at 10 Hz, the maximum amplitude enhancement was 6-4 x , and this decayedto one-half in 15 s, i.e. by the next contraction. Return to normal took 4 min in theformer experiment and 2 min in the latter. Thus the amount of enhancement, and itsdecay rate, are a function of the period of stimulation.

Interactions of motor axons with IRWhenever the whole animal was being studied, there was the possibility of spon-

taneous and reflex neural outputs occurring in SETi or CI that might interact with,and affect IR. Experiments were undertaken to investigate the effects of variousfrequencies, durations and timing of CI and SETi on IR.

Common inhibitor (CI)

CI does not share innervation of the muscle fibres on which DUMETi endingsoccur (Hoyle, 1978). If the latter act only on the fibres they innervate, a close directinteraction between the two neurones would not be expected. However, DUMETimakes neurosecretory-type terminals (Hoyle et al. 1974), so its action may be on moredistant targets. No influence of DUMETi on CI action was observed, but CI wasfound to have strong effects on IR. When CI was stimulated at the same time as anindividual IR contraction, it reduced its amplitude and duration. But unlike a DUMneurone, its action consisted only of that on the immediate contraction, there was no

7 EXB 73

190 G. HOYLE

B

(iv)

(v)

Fig. 14. Interactions of CI with IR. A, Threshold frequency for effects of CI on IR in averagepreparation of £. gregaria. Slight attenuation occurs at 1 Hz, quite marked attenuation at5 Hz. Larger effects occur at the same frequencies in R. microptera. B, Effects of attenuating IRby CI action on frequency, amplitude and IR subsequent to a period of inhibition, in fivepreparations. CI stimulation was at 30 Hz, applied immediately after the onset of an IR con-traction, and terminated as soon as it started it to cause attenuation, (i) In this preparation anIR contraction was suppressed by a brief CI burst, but subsequent to this first suppressionmuch longer CI bursts were required, (ii, iii, iv) A preparation in which brief CI bursts con-sistently terminated an IR contraction though there was a tendency of IR to break through,(v) A preparation in which there was an opposite action to that occurring in A, namely afacilitation, or a progressive enhancement of the ability of CI to block an IR contraction. In alater test on the same preparation facilitation did not occur, but there was no diminution as inA. Note that in every case CI activity led to a faster IR, especially in the third test of B. Timemarks: 1 per s.

persistent effect reducing later contractions. When stimulated between bursts, atexactly the same repetition rate as the IR frequency, there was no long-term actioneither. Such bursts sometimes caused a small relaxation, sometimes a small contrac-tion, or were without direct mechanical effect. Stimulation of CI for periods longerthan a single IR cycle consistently altered IR, even at frequencies below 10 Hz, byreducing both the amplitude and duration of individual IR contractions (Fig. 14A).The reductions were slight and they declined with time under continued CI


stimulation. This was true for CI frequencies below about 25 Hz, above which IRsuppression was total.

Interposition of a strong CI burst during the course of an IR contraction quicklyand fully suppressed it. However, if a second similar or even longer CI burst wasapplied during subsequent IR contractions, it was generally much less effective thanthe first (Fig. i4B(i)), although preparations varied considerably in this respect anda few showed the opposite effect (Fig. i4B(v)), first series). Several examples areshown in Fig. 14 in order to illustrate the extent to which inhibitability of IR by CIvaried from moment to moment (Fig. i4B(i), second and third series). When an IRcontraction was not completely inhibited a series of small contractions occurred atirregular intervals (Fig. i4B(ii), second series).

At frequencies below 15 Hz, amplitude and also duration, were at first reduced, butthe attenuation waned rapidly. At higher frequencies all the effects were furtherenhanced until, at 25-30 Hz complete suppression occurred. Following inhibition ofIR by CI, the first contraction is larger than normal and longer in duration. Its onsetimmediately follows cessation of CI stimulation. There was never a pause, such aswould occur sometimes following only partial suppression of IR.

The slow extensor

Single impulses in the slow extensor axon (SETi) cause a minute twitch in mostlocust/grasshopper preparations. This is generally quite small, even compared withan IR contraction, and is often not visible on IR records, but when SETi was excitedat a low rate it nevertheless greatly influenced IR. The first SETi impulse to arrivetended to initiate a contraction (Fig. 15), even if the next IR contraction to be expectedin the normal sequence was not due to start for several seconds. This triggering actionwill be described in detail later. It results in synchronizing of IR with SETi impulsesat rates of 1 per 10 s to 1 per 5 s. At somewhat higher, but still low, frequencies ascaling process occurs, such as one IR contraction for every second, third or fourthSETi impulse, depending on the SETi stimulation rate (Fig. 15 A). Also, there is atendency for SETi impulses to become progressively less effective with time, intriggering IR.

The amplitude of individual IR contractions was decreased slightly at the sametime as IR frequency was increased by SETi action. Also the duration, as well as theamplitude, of an individual IR contraction was reduced. The extent of this reductionwas increased the greater the SETi frequency, but some preparations were affectedto a much greater extent than others. In general, if the IR frequency was low, and theduration of an individual IR contraction was long, the latter was but little changed bySETi. But if IR frequency was higher and duration of individual contraction short thelatter was greatly shortened. Examples of these different effects are shown in Fig. 16in which D(i) should be compared with Fig. 15E. It was in many ways surprising tofind that SETi, which itself causes depolarizing junctional potentials and tension,reduces the amplitude of IR.

A few preparations did not show IR, as noted also by Hoyle & Florey (in prepara-tion) who found that addition of eserine and io~6ACh to saline bathing inactiveLocusta preparations initiated IR. In the present experiments, it was found that insuch preparations IR could be initiated by stimulating SETi at a moderate frequency

7-2

G. HOYLE

(iii)

JUJUULJULUJUUULJU_ H o s i i i i i i i i

I I I I I I I I I I I I I I I I I I I I ! I I

B (i)

C (i)

Fig. 15. Phase-shifting of IR by SETi. Preparations A, C, D, E, F did not show a twitch con-traction in response to a SETi impulse. B gave small twitches. A (i, ii) Increased IR frequency,with reduced amplitude and duration of individual contractions, during SETi stimulation ati s. Note compensatory long intervals after cessation of stimulation. A (iii) IR became syn-chronized with SETi at -j^- s for a while, after which only every second, third, fourth or fifthimpulse triggered an IR contraction. Note long delay before next contraction after cessation ofstimulation. B (i) Initiation of IR in a preparation in which it appeared to be absent, bystimulation of SETi at 10 Hz. IR ceased as soon as SETi stimulation was stopped. B (ii) Sameas Bi, but with stimulation of CI interposed, at 50 Hz. The inhibition was complete, and alsosuppressed IR. C (i) IR became synchronized with SETi stimulation at -£% s. C (ii) Increase inIR frequency, during SETi stimulation at i s. C (iii) Same by SETi stimulation at 5/s.C (iv) Same by SETi stimulation at 10 Hz, that leads to slow tension development. D (i) IRacceleration by SETi frequency of 5 Hz. D (ii) Break up of IR into irregular contractions at10 Hz. E, Preparation in which IR was markedly accelerated by SETi stimulation at 10 Hz,but without break-up. F, Preparation that showed IR break-up during SETi stimulation at5 Hz. Second stimulation was at 10 Hz. The first IR contraction following cessation of a SETiburst was depressed in C (iv) and F.

(Fig. 16B). IR thus evoked continued, however, only as long as SETi was excited andit was quelled by combining CI with SETi action (Fig. 15 B(ii)). The effects ofstimulation of SETi at frequencies of 5-15 Hz were different in different preparationsand generally very interesting. In some preparations stimulation of SETi at 10 Hz ledonly to a slight acceleration of IR (Fig. i5D(i)). In others, it produced an erratic


SETi - 5 H z

(iii) JUUUL

B

5 Hz

8 Hz

CI 10 Hz 20 Hz 30 Hz

SETi 8 Hz

JUUl

CI 20 Hz CI 30 Hz

— SETilO Hz40 Hz 20 Hz

Fig. 16. A, Interactions of SETi with IR in S. gregaria. (i) SETi, stimulated at 5 Hz, thoughnot causing a contraction, weakly attenuated IR and markedly increased its frequency. Theshapes of individual IR contractions became erratic. The first IR contraction following SETistimulation was depressed (dep.) in height, (ii) SETi, stimulated 6 Hz, which in this prepara-tion just led to contraction, attenuated IR, increased its frequency and made individualcontractions erratic, (iii) SETi, stimulated at 8 Hz had more pronounced actions on IR.B, Comparison of SETi effects with CI effects. CI, stimulated at 10 Hz, led to partial attenua-tion of IR, combined with a marked increase in IR frequency. There was progressive attenua-tion of IR. At 20 Hz there was strong attenuation and greater increase in frequency. At 30 Hzthere was complete inhibition of IR. The first IR contraction following a period of strong in-hibition at 20 and 30 Hz but not at 10 Hz occurred immediately after the inhibitory stimulationwas stopped and it was enhanced (en.) in both amplitude and duration. C, Combined effectsof SETi and CI on IR in R. microptera. (i) SETi only, at 8 Hz. (ii) SETi at 8 Hz, combinedwith CI at 2 Hz, as indicated, and CI alone. At these frequencies CI almost completelyinhibited IR, but the addition of a background of SETi at 8 Hz counteracted CI inhibition,(iii) With SETi at 10 Hz and CI at 20 Hz a smooth, slow contraction occurred, but IR wascompletely suppressed. With SETi at 10 Hz and CI at 30 Hz, the slow contraction was sup-pressed, but IR, although greatly attenuated, was evident, (iv) With SETi at 10 Hz, CI at 40 Hzcompletely inhibited both slow contraction and IR. Note the step in SETi tension rise at thetime when an IR contraction would be expected. Time marks: 1 per s.

194 G. HOYLE

contraction (Fig. 15 D (ii)) reminiscent of those sometimes initiated by causing repeatedsuppression of IR by CI bursts (Fig. i4B(iv, v)). In others there was a strong suppres-sion of IR (Fig. 15 F). The explanation of these various effects seems to be that theyare due to a combination of two actions. One is that a SETi impulse tends to initiatean IR prematurely during an inter-IR contraction period. Secondly, when an IRcontraction is actually under way, a SETi impulse tends to cause it to terminate pre-maturely. Both effects will be described separately in a later section. Variations in therelative strengths of these two independent actions, between preparations, can accountfor the differences seen. The balance between the tendency of a SETi impulse toinitiate an IR contraction prematurely and the tendency to cause its early terminationonce under way, also explains the effects of increasing frequency of stimulation ofSETi on IR. As SETi frequency is increased, the frequency of IR rises but theduration of individual IR contractions falls (Fig. 15 C).

Comparison of SETi and CI effects

In some respects, the actions of SETi and CI on IR are similar: both lead toattenuation and acceleration. But during long-continued low-frequency CI stimula-tion the suppressed IR breaks through and eventually resumes nearly normal ampli-tude, duration and frequency. By contrast, the reduction in amplitude, shortening ofduration and increase in frequency of IR that occur during SETi stimulation continueindefinitely.

The major difference is that following a period of CI inhibition of IR there is animmediate rebound; an IR contraction starts as soon as CI stimulation ceases. It willbe recalled that both the amplitude and the duration of this rebound IR contractionare greater than normal (Fig. 16B). By contrast, following cessation of SETi action,there is no immediate rebound IR. Furthermore, the first IR contraction followingSETi action tends to be of less than average amplitude (Fig. 16A, dep.).

Effects of combined SETi and CI action on IR

Since the principal actions of CI and SETi, stimulated at low frequencies, on IRare similar, namely increased IR frequency but decreased IR duration, it might beexpected that they would reinforce each other. On the other hand, the membranepotential changes that each effects are basically opposed to each other. The principaldifference in their actions is that CI produces more powerful attenuation than doesSETi.

When CI and SETi were stimulated together at low frequencies the dominantaction was slow contraction, with suppression of IR. It required a CI:SETi ratio of3:1 or greater for suppression of the SETi contraction (Fig. 15 C(iii)). At this ratio IRwas still apparent, whereas with CI alone at this frequency it was completely sup-pressed. Thus SETi slightly antagonizes the inhibitory effect of CI on IR.

Combinations of DUMETi, SETi and CI

Little interaction was observed when DUM neurones were stimulated at the sametime as SETi and CI. Stimulation of SETi after first causing a strong burst in a DUMneurone that inhibited the extensor tibiae IR led to contractions that resembled IRrather than normal slow ones. SETi counteracts the combined suppressing actions of

Rhythm and tonus in insect muscle J95

SETi; 6 Hz

DUMETiSETi-

Cl-10 Hz~20Hz

Fig. 17. Partial antagonism of DUM neurone action by SETi, S. gregaria. A, DUMETiexcited by break from hyperpolarizing pulse; SETi at 6 Hz during the inhibited phase im-mediately initiated attenuated IR at a high frequency. B, SETi excited at 10 Hz, causing a slowcontraction that was partially inhibited by stimulating CI at 20 Hz. IR was accelerated, butgreatly attenuated. During the course of the combined SETi/CI stimulation, DUMETi wasdepolarized. There was only slight additional suppression of IR, and normal IR resumed soonafter cessation of stimulation.

both CI and DUMETi and restores IR if the frequency is not too high. In theexperiment shown in Fig. 17A, a DUM neurone burst was elicited, that completelysuppressed IR. During the inhibition, a brief SETi train at 6 Hz was applied. Thisimmediately initiated IR, at a faster than normal rate. Thus SETi is easily able toovercome DUM neurone inhibition and restore IR.

In another experiment SETi was stimulated at 10 Hz, initiating a contraction thatwas then inhibited by stimulation of CI at the same time, at 20 Hz (Fig. 17B). A weakslow contraction slowly built up, with superimposed, very weak, IR contractions atincreased frequency. During this period, a burst was elicited from a DUM neuronethat inhibited IR. No additional attenuation of the abortive IR was caused. NormalIR was restored earlier than it would normally have been following such a burst. Thelonger period of SETi stimulation opposed the long-term action of DUMETi.

Natural modulation of IR

Whenever the whole animal is being studied, or in preparations in which the nerveshave not been severed, there is a possibility of SETi, CI or any of the DUM neuronesaffecting IR. Examples of such action were shown in Fig. 5 and can now be interpretedin light of the effects obtained on IR whilst stimulating these axons.

In Fig. 5 A, the interpretation of the abrupt changes in IR are that excitation of theslow extensor tibiae (SETi) to the right leg only occurred, followed soon after by aprolonged SETi burst to the left leg. At the same time as the right SETi was excitedthe dorsal unpaired median neurone that innervates both extensor tibiae, DUMETi,fired. The DUMETi action was responsible for the abrupt attenuation of the left IR

196 G. HOYLE

I I I I I I I I

Fig. 18. Early triggering and early termination of IR by FETi (A, B, D) or SETi (C) actionR. microptera. SETi gave an unusually large twitch in this preparation. A, FETi impulses at1 per 10 s. No attenuation of IR. B, Same as A, but at higher rate of 1 per 7 s. Contractionswere not triggered after seventh stimulus, but note attenuation. C, SETi stimulated at 1 per10 s. D, Effects of FETi at various times after the onset of IR contractions. When FETioccurred less than the critical time of 1 -4 s after the onset, the time-course was unaltered. Butwhen given more than 1 -4 s after the onset of an IR contraction, the latter was terminated. Theinterval between an attenuated IR contraction and the next ensuing one was decreased inproportion to the extent of attenuation. Time bar = 10 s.

contraction and for the long interval before the next, small right IR contractionfollowing the right SETi burst. How, though, can the acceleration in frequency of theleft IR be explained, when we know DUMETi had released material that decreasesIR frequency. The answer lies in the difference between the two SETi discharges,combined with the fact that SETi also interacts with IR. The left SETi dischargecontinued at a low frequency after the initial burst, as evidenced by the continuedtension plateau. When a SETi neurone fires, the frequency of IR in that leg is increasedand the amplitude is diminished (Hoyle, 1978): these actions evidently override thefrequency-diminishing action of the DUMETi secretion.

In the middle set of IRs shown in Fig. 5 B, a right SETi burst was matched by asimultaneous burst in the common inhibitor. This reduced the left IR, but continuing,low-frequency right SETi firing after the burst caused acceleration of the right IR.Finally, in Fig. 5C is shown common events: variable attennuation of IR amplitude,with slight increase in frequency. These are both attributable to the presence of anaccelerating prolonged discharge in the common inhibitor (CI). The erratic nature ofthe changes in IR reflects a fluctuating frequency in CI.

Initiation and termination of IR contractions by FETi, SETi and CI

Following a FETi twitch the muscle does not relax fully, but instead goes into apremature IR contraction. Provided this was not caused within the first 2 s aftertermination of a preceding IR contraction it was similar in size and duration to thepreceding one; having only been initiated early. The FETi impulse would trigger anIR contraction of normal amplitude and duration as early as i-8s following the


FETi

SETi 20.Hz..

Fig. 19. Critical period for early termination of freely occurring IR contraction. FETi failed tofire at second stimulation. A, Single FETi shock at various intervals after onset of contraction.The critical time in this instance is 1 -55 s. B, Same preparation as A, but with SETi stimulatedin a burst at 20 Hz for 1 s (first and third), 02 s (second) and 04 s (fourth). A shorter burstfailed to cause termination of IR contraction even though given after the critical time (second).Second burst overlapped critical time. Time i/s.

termination of a preceding one, whatever the natural IR frequency. IR could routinelybe driven synchronously at frequencies up to 5 x normal by repeated FETi triggering(Fig. 19) and sometimes even higher, though with a slight reduction in amplitude andduration.

If a FETi impulse occurred during the middle of an IR contraction, relaxation tothe baseline occurs, i.e. the IR contraction is aborted. A critical time must follow itsonset, before which it is not so terminated. This time was greater the lower thefrequency of IR, or the longer the duration of individual IR contractions, which tendto have a long duration when their frequency is low. A linear relationship was foundbetween the IR rest interval and the critical time for early termination by a FETiimpulse. After the frequency of IR has been increased by FETi triggering for severalcycles, upon cessation the normal interval is resumed right away.

Single SETi impulses also caused triggering and early termination in some prepara-tions (see Figs. 16, 18). Others did not, but in these a SETi burst was effective if it was

198

MlHJc i iiiiiiiiiiiiiiiii mi mini

71

G. HOYLE

IIIIIIIIIIIIIIIII

y^-~\ D ^ ^

Ci 1 1 L

\_-——ci nun

. n.CI J J J

SETiCI

Fig. 20. Early termination of an IR contraction by a single CI impulse. A, Effect of low fre-quency (1 per 2 s) of stimulation of CI on IR. Individual contractions are shortened: inter-preted as indicating that next CI impulse after critical period induces early termination. B,First four CI impulses in a train at 2 Hz each produced a slight relaxation of IR contraction.Fifth impulse caused termination. C, Single CI impulse terminates IR contraction whenarriving 2-5 ms after onset (critical period, 2-3 s), only slight relaxation when arriving after 1 -9 s.D, Effect of single early CI impulse is to reduce IR contraction height by about 12 %; threeCI impulses reduce it by 18 %. E, Comparison of termination by CI with that achieved by asingle SETi impulse. F, Similar to C, but at critical time and just before it. Calibrations:0-07 g vertical; bar = 10 s, A; 2 s, B-F.

sufficiently long and of sufficiently high frequency (Fig. 19 B). The ones that didrespond to a single SETi impulse gave unusually large SETi e.j.p.s and twitches.Single CI impulses never initiated early IR contractions, but it will be recalled thatrepeated CI impulses increase IR frequency, which could mean that there is at least aslight tendency for CI junctional potentials to trigger them. Nevertheless, single CIimpulses were effective in initiating early termination (Fig. 20 A). The critical timesfor initiating early termination were slightly longer for SETi and CI than for FETi inthe same preparations.

The quantitative relationship between the mean periods of IR contractions andduration of an IR contraction for naturally different durations are shown in Fig. 21B.The relationship between the duration of IR contraction (in one preparation) and thetime before the next IR contraction started is shown in Fig. 21C. The duration wascut short by exciting FETi at various periods after the onset of a natural (i.e. nottriggered) IR contraction. Both of these graphs are linear, with similar slopes, andboth hint at an underlying relaxation oscillator-type of mechanism that is controlledby slow chemical accumulation. If an IR contraction is cut short the next one is boundto occur earlier, by an amount that is strictly linearly related to the abbreviatedduration. The early onsets are not quite sufficient to keep the interval betweentermination constant and normal: there is slight overcompensation, so that theinterval becomes less than normal following curtailment. In the normal unstimulatedpreparation, the duration of individual IR contractions slowly alters. There is a smalldecrease in frequency as the duration of contraction increases (Fig. 21B, lower line).


B

P-d

30

28

26

24

22

20

18

161 A 1 1 1 1 1 1 1 1 1

p'-d'

30

28

26

, 24P

22

20

18

16

0 10 s

14 l i i l l0

d'10 s

Fig. ai. Relationship of IR frequency to duration. A, Diagram of IR to show parameters.p = period of IR, d — duration of individual IR contraction, CT = critical period - minimumtime after onset of IR contraction at which an interposed FETi impulse arriving at verticalarrow leads to its early termination; d' = duration of IR contraction aborted by neural excita-tion after critical period. Broken lines indicate declining phases after abortion at differenttimes and next IR contraction, p' = reduced period associated with aborted contraction; B,Relationship of mean period of IR in R. microptera (solid circles) as well as p-d (solid triangles)to duration of contraction. C, Relation of interval between an aborted IR contraction and thenext ensuing one (p') to the duration of the aborted one (d') in one preparation of R. microptera.Also plotted is p'-d'.

DISCUSSION

The present results show that in some insect skeletal muscles there are threeindependent mechanisms determining tonus. Each of the three types of tonusmechanism will be given a descriptive title. Only one category of tonus mechanism,from among several once thought to exist, survived 'critical' appraisals in the mid-19208. The favoured one is low-frequency tetanus in one or a few motor units inner-vated by ordinary excitatory motor neurones. This will be termed tetanus tonus andit is one of the three forms present in locust and grasshopper jumping muscles.Arthropods in general have specialized slow motor neurones, often innervatingspecialized slow muscle fibres, that serve a similar function, possibly more efficiently,

200 G. HOYLE

by way of graded, frequency-dependent synaptic depolarizations at distributed,multiple terminals on single muscle fibres. The other two tonus mechanisms areintrinsic to muscle fibres: basic tonus (BT) (Langelaan, 1922) and the intrinsic rhythm(IR). The former is the steady tension that is developed by some muscle fibres in theabsence of motor nerve synaptic excitation. The latter is the slow myogenic rhythmdescribed in this paper. There is some interaction between BT and IR, and alsobetween IR and tetanus tonus. This is to be expected since all three act via the samemuscle fibres and all act by way of membrane potential shifts.

During studies on the action of octopamine, which is considered to be released byDUM neurones (Hoyle, 1975), it was found that BT is relaxed in many preparations,but enhanced in a few and unaffected in others. Neither octopamine action nor DUMneurone action exactly mimic the actions of the specific BT relaxing neural elementsencountered (see p. 185).

The first example of BT to be discovered in an arthropod muscle was the pupalmoth spiracular muscle (Beckel & Schneiderman, 1957). Here it is sufficiently greatthat the spiracular valves are closed in the absence of neural input; C02 is the naturalrelaxing agent. Similar phenomena also exist in adult locust spiracular muscles(Hoyle, 1961). BT was later found in a crustacean eye-stalk muscle (Hoyle, 1968)where it can be relaxed, very rapidly, by the peripheral inhibitory axon. There was noevidence, from the present work, that the locust peripheral inhibitory axon can relaxBT. In fact, stimulation of CI led to the development of tension in most preparations.It might have been concluded that CI does not innervate muscle fibres responsiblefor BT, but since CI often gives depolarizing i.p.s.p.s this could not be ascertainedwithout further tests.

The frequency of stimulation of the key neurone required to achieve a markedreduction in BT was only 1 per 2 s and even a pair of stimuli produced a detectableeffect. The low frequency and small number of stimuli that were effective in relaxingBT together with a long duration of the action, strongly suggest that secretion of aneurohumoral type of material is responsible for the relaxation.

IR is reduced when the BT level increases and is lost completely when it is high.Conversely, as BT falls IR amplitude is enhanced. It must be presumed that there arefunctions for IR and BT in the jumping muscle. Although the strength of these issmall, not more than 0-3 g, it should be borne in mind that a whole mature locustweighs barely 2-0 g. BT in the muscle is probably seldom sufficiently large to put thelocust into a raised posture, but it does prevent complete slackness when SETi issilent which is often the case when the locust is at rest.

IR is masked in the intact animal by the resistance reflexes it evokes, though move-ments caused by it are still recordable from the whole animal (Hoyle & O'Shea,1974). The peak tension developed in a large IR contraction is about equalled by asteady discharge in SETi at 10 Hz.

The existence of BT, and perhaps also IR, provides a previously unsuspectedpossible reason for the existence of common peripheral inhibitory neurones. Becauseboth can be expected to interfere with the generation of behaviour, they need to besuppressed at times. However, CI, although it does reduce BT, does not suppress itcompletely, perhaps because fast muscle fibres are not innervated by it. The as yetunidentified dorsal neurones that suppress BT are very much more effective.


The results described have shown that there is a subtle interaction between themechanism generating IR and effects associated with each of the ordinary motoraxons. There are three ways in which the interactions might occur. They could becaused indirectly by the independent mechanical actions of the axons. This seemsunlikely, because SETi and CI exert miniscule effects on tension individually, or atthe low frequencies that affect IR. A second way in which the mediation might occuris by a direct action of the SETi and CI transmitter substances on the muscle fibresactive in IR. This could be mediated either by conventional close synaptic action orafter diffusion through haemolymph. If the muscle fibres in which IR originates areinnervated by CI and SETi then DUMETi must exert its action on the pacemakerafter diffusing, or being carried, in the haemolymph, since DUMETi terminalsapparently only accompany FETi (Hoyle, 1978).

There is no obvious function for IR. Usherwood (personal communication) hassuggested that it may aid the flow of haemolymph. Since it must interfere with normalreflex and centrally programmed behaviour, it is desirable to be able to turn it offwhen normal locomotor activity is called for, and that is something DUM neurones do.

The minimum latency for attenuation by DUMETi is about 100 ms (Hoyle, 1974),so the IR initiation site must be fairly close to the terminals. However, the musclefibres on which these terminals occur are of fast type and they are not innervated byeither the common inhibitor or the slow extensor. Since the latter affect IR with evenshorter latencies, of about 50 ms, either IR is initiated in slow muscle fibres receivingCI and SETi innervation, or else all the relevant transmitter substances affect IRafter diffusing a short distance to the generator sites.

The three types of terminals release different substances. CI is associated with aprobable release of GABA and causes increased chloride conductance, and eitherhyperpolarizing, or weakly depolarizing i.j.p.s, whilst SETi causes only depolarizinge.j.p.s, possibly by release of L-glutamate (Usherwood & Cull-Candy, 1975). It isparticularly surprising that SETi impulses attenuate, rather than simply sum with,IR. So the interaction between the motorneurone effects and IR are unlikely to besimple indirect consequences of membrane potential shifts but more probablyrepresent actions on the ion conductance changes of the pacemaker.

The effects of two of the three axons in initiating an anticipated IR contractionprematurely, or of all three in terminating it early, are subtle. FETi is the morepowerful in both respects: a single FETi impulse will trigger an IR contraction aslittle as 2 s after termination of a preceding one when the anticipated natural intervalis from 7 s to more than 30 s. An IR having a natural frequency of i/min will followfor 1 h at a rate as high as 6/min when triggered by FETi impulses although theamplitude will diminish slightly. Single SETi impulses would trigger an early IRcontraction in preparations with large SETi e.j.p.s, whereas a burst was required inthose with small e.j.p.s. Neither a single, nor a burst of CI impulses triggers an IRcontraction but during a low-frequency train of CIs the IR frequency always increasedslightly, which may represent a similar, though weak, effect.

The critical time for termination of an IR contraction is directly proportional to itsfrequency. IR contractions are themselves briefer at higher frequencies though thereis no simple relationship between IR frequency and duration. The critical time forSETi was slightly longer than for FETi, and that for CI about the same as that for

202 G. HOYLE

SETi. Since a single CI impulse can terminate an IR contraction early, the actioncannot be mechanical. The only aspect the three impulses always have in common isthat they initiate some outward current flow. That may be sufficient to start repolariza-tion of the pacemaker muscle fibres after the critical period. FETi and SETi firstinitiate inward current flow, and that may be what initiates the early IR contractions.The capability to generate an IR depolarization wave clearly resets faster than, and isindependent of, the pacemaker to initiate it. Duration of IR contraction, frequency,critical time for attenuation and amplitude are all interrelated, although the first threecan vary independently of the others to a considerable extent. The problems associatedwith these phenomena are familiar ones to students of cardiac pacemaking. This newpreparation for studying such matters may therefore have a wider potential interestthan its relevance to insect muscle physiology and behaviour.

This research was supported by National Science Foundation Researach Grant No.BNS 75-00463.

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GABA onto insect central neurones. Comp. Biochem. Physiol., 31, 611-633.LANGELAAN, J. W. (1915). On muscle tonus. Brain 38, 235-380.LANGELAAN, J. W. (1922). On muscle tonus. De Boer's experiments on the frog. Brain 45, 434-453.LIDDELL, E. G. T. & SHERRINGTON, C. S. (1924). Reflexes in response to stretch (myotatic reflexes).

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Note added in proof. Whilst this paper was in press T. Piek & P. Mantel (1977)published an article on the extensor tibiae of Locusta tnigratoria in which they notedthe normal absence of IR in locust saline. However, they found that addition of io~9

mole per litre proctolin to the saline evoked IR. They also noted (their Fig. 3) twotypes of relaxation of tonus. One (ist asterisk) was equivalent to termination of a verylong-lasting IR (see Fig. 12 of the present paper); after this relaxation IR was un-covered. The other (2nd asterisk) was clearly a relaxation of BT. Both were inducedby proctolin.

PIEK, T. & MANTEL, P. (1977). Myogenic contractions in locust muscle induced by proctolin and bywasp, Philanthus triangulum, venom. J. insect Physiol., 33, 321-325.

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