Journal of Physiology (1996), 494.1, pp.279-285

The pattern of stimulation influences the amount of oscillatorywork done by frog muscle

E. Don Stevens

Department of Zoology, University of Guelph, Guelph, Ontario N1G 2W1, Canada

1. A doublet is the interjection of an additional action potential at the beginning of a regularmotoneuron discharge pattern. The present experiments were designed to estimate themagnitude of the effect of doublets on the capacity of isolated frog sartorius muscle to dowork during oscillatory length changes.

2. For the work loop method, the muscle was subjected to sinusoidal length changes at 4 Hz.Work was calculated from the loop formed when force was plotted against length. Workdone was positive when the muscle was shortening and was negative when the muscle waslengthening; net work was the difference.

3. Adding pulses at the start of the stimulus pulse train increased isometric force and markedlyincreased net work per cycle. Adding one pulse increased the net work by about 52 %, butincreased isometric force by only 24 %.

4. Eighty per cent of the maximum net work was achieved by adding only three pulses at thestart of a low-frequency pulse train, i.e. 80% of the work achieved with seventeen pulsescould be achieved with only six pulses.

5. The maximum net work per stimulus pulse was achieved with a stimulus train that consistedof five pulses with two being additional initial pulses at 5 ms interpulse intervals, i.e. atriplet.

6. The results of the present study imply that doublet stimulation patterns may be importantto reduce the fatigue that occurs during artificial neuromuscular stimulation of skeletalmuscle.

In vivo changes in the level of work output by muscles are

achieved by changes in motoneuron firing patterns. With an

increase in the intensity of exercise, any of the followingcould contribute to the increase in work done by a particularmuscle: (1) recruitment of previously quiet motoneurons;(2) increase in the discharge frequency of active moto-neurons; (3) earlier recruitment of active motoneuronsduring one limb cycle; and (4) doublets (Milano, Grelot,Bianchi & Iscoe, 1992).

A doublet is the interjection of one action potential at thebeginning of a regular discharge pattern (see Fig. 1 for a

simulated doublet stimulus pattern). Usually they are

restricted to the first or second interpulse interval. If, forexample, a particular motoneuron has a maximum dischargefrequency of 40 Hz, then the normal interpulse interval is25 ms. When a doublet occurs, then the first interpulseinterval is markedly decreased, to -5 or 10 ms, and theremaining interpulse intervals in the stimulus train are

25 ms. The result of the doublet is a rapid burst of twoaction potentials, followed by a discharge at a regularfrequency (see Fig. 1). Triplets and quadruplets also occur.

Doublets have been recorded from motoneurons in a numberof preparations. They have been reported for populations ofmotoneurons in normal rats (Hennig & Lomo, 1985, 1987).Zajac & Young (1980b) reported that motor units fireddoublets 100% of the time at slow walking speeds and firedfewer doublets at faster walking speeds. In man, doubletshave been reported in motor units involved in locomotion(Denslow, 1948; Norris & Gasteiger, 1955; GurfinkelMirskii, Tarko & Surguladze, 1972; Desmedt & Godaux,1977, 1978; Kudina & Alexeeva, 1992), wrist movement(Bawa & Calancie, 1983), and respiration (Adams, Datta &Guz, 1989). A doublet should cause additional calciumrelease (Blinks, Riidel & Taylor, 1978) and additional forcedevelopment; then force could be sustained by the lowerfrequency that occurs after the initial rapid phase. Thisincrease in force could result in a marked increase in thework done per movement cycle even though the maximumforce achieved would not be altered appreciably. It has beenargued that doublets are of adaptive significance becausethey should reduce the metabolic cost of doing work(Bigland-Ritchie, Thomas, Rice, Howarth & Woods, 1992).Theoretically, maximum work will be achieved if a muscle is

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E. D. Stevens

Table 1. Number of stimulus pulses, duration of stimulus train and intervals between stimuluspulses within each stimulus train for the stimulus patterns used for experiment A

Pattern Number Train 1st 2nd 3rd 4th 5thof pulses duration interval

(ms) (ms)31-base 3 66 32 32Doublet 4 80 5 42 33Triplet 5 80 5 5 36 33Quadruplet 6 80 5 5 5 32 32Continuous 17 80 5 5 5 5 5 ...

Parameters for data shown in Figs 1 and 2. Pulse width was always 0 4 ms. Numbers have beenrounded to the nearest millisecond.

fully and instantaneously activated at the onset ofshortening (Luiker & Stevens, 1994). Thus, a doublet ortriplet should contribute to rapid achievement of maximumforce and should increase work done per movement cycle.The goal of the present experiments was to estimate themagnitude of the effect of doublets on the capacity ofisolated muscle to do work during oscillatory length changesusing the work loop method (Josephson, 1985). A furthergoal was to compare the work done per stimulus usinguniform stimulus patterns with work done with stimuluspatterns containing doublets, triplets and quadruplets. Itwas hypothesized that doublets will influence work morethan they will influence isometric force because the rate offorce development is important in work and doubletsmarkedly influence the rate of force development.

METHODSFrogs (Rana pipiens) were captured locally and maintained at20°C. They were fed mealworms daily. All procedures wereapproved by the University of Guelph Animal Care Committee.Frogs were stunned, then double pithed and the sartorius musclewas dissected out. A bundle of fibres from the centre of the musclewas used. The bundle was split to reduce the thickness of the sheetof fibres. Care was taken to ensure the cuts were made parallel tothe fibres so that none were damaged. The tendon end of the musclewas attached to the movable arm of an ergometer (CambridgeTechnology) and the other end was tied to a force transducer.Average muscle length was 36-1 + 0-8 mm (mean + S.E.M.),sarcomere spacing estimated by laser diffraction was2-27 + 0-02 um and muscle bundle wet weight was 25 + 2-8 mg.The muscle was superfused with physiological saline solutioncontaining (mM): NaCl, 86; KCl, 1; CaCl2, 1P8; MgSO4, 1; KH2PG4,1-9; NaHCO3, 10. Extracelluar pH was 7-74 and was controlledwith a Wostoff pump that maintained the ratio of C02: 02 to 1: 99.All measurements were made at 20°C. All saline solutionscontained substrate as pyruvate (10 mM) plus antibiotics(streptomycin sulphate, 28 U ml-' and penicillin-G, 63 U ml-'),and were made fresh daily.

The muscle bundle was stimulated with platinum plate electrodes.Stimulus voltage (about 6-5 V) was adjusted to 1P2 times thatrequired to produce maximal isometric twitch force. Muscle restinglength (L.) was adjusted to produce maximal isometric twitch force.

To measure work and power, a sinusoidal signal was used to cyclemuscle length about Lo. The amplitude of the length change cycle

(i.e. strain or excursion amplitude expressed as a fraction of L.) was0 07 (Lo ± 0 035). The phase (i.e. the timing of the start of thestimulus train relative to the imposed length change cycle) was15%; muscle shortening occurs from 25 to 75% (both given as aproportion of the length change cycle).

The cycle frequency (i.e. the frequency of the imposed lengthchange) was 4 Hz. Stimulus train duration was about one-third ofthe total cycle duration. These conditions yield very high poweroutput for this preparation at the test temperature.

Work is the product of force and displacement and was calculatedby integration of the force-displacement curve yielding an area inV x V, which was multiplied by calibration factors to yield work inJoules per cycle, and divided by muscle mass to standardize thework per cycle in Joules per kilogram. Positive work is the workdone by the muscle during shortening and negative work is thework done on the muscle to lengthen it. Net work is the differencebetween positive and negative work. Work measurements weremade at 8 min intervals. For each measurement the muscle wascycled for four complete length changes and stimulated for the lastthree cycles. Work was calculated from the average of the secondand third cycles, but the three differed by less than 2%. Isometricforce measurements were made using the same protocol except thatmuscle length was held constant.

The stimulus trains were created and stored as arrays so that thestimulus pattern could be controlled precisely. Stimulus pulse widthwas 0 4 ms. A 200 Hz stimulus train gave a fused tetanus. Twotypes of stimulus patterns were used: (1) base frequency only and(2) base frequency plus additional initial pulses. The base frequencypattern was the same stimulus pattern as commonly is used instudies on isolated muscle, i.e. a constant stimulus frequency. Fortrains with additional initial stimuli, individual pulses were addedafter the first pulse of a typical pulse train at 5 ms interpulseintervals. This pattern consisted of an initial rapid burst followedby a slow sustained pattern. The stimulus train durations, numberof pulses per train, and intervals between pulses are given inTables 1 and 2 for the different stimulus patterns used.

In experiment A, a base pattern of 31 Hz was used and initialpulses (1, 2 or 3) were added after the first pulse at 5 ms interpulseintervals (i.e. at 200 Hz) (see Table 1). Eight preparations wereused.

In experiment B, the base pattern was varied from 15 Hz to 200Hz and with or without added initial pulses. The pattern of initialpulses always consisted of adding three pulses at 5 ms interpulseintervals (see Table 2). Eight preparations were used.

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J Physiol. 494.1 Doublets increase work done in frog muscle

Table 2. Number of stimulus pulses, duration of stimulus train and intervals between stimuluspulses within each stimulus train for the stimulus patterns used for experiment B

Pattern Number Train 1st 2nd 3rd 4th 5th 6th 7thof pulses duration

(ms)2 673 665 668 7610 7513 785 806 808 8010 8012 8014 8017 80

interval(ms)67321611865555555

3216 1611 118 86 65 55 55 55 55 55 55 5

1611 11 11 118 8 8 ...

6 6 6 ...

6532 3316 1611865

16 1611 118 86 65 5

Parameters for data shown in Figs 3 and 4. Pulse width was always 0 4 ms. Numbers have beenrounded to the nearest millisecond.

RESULTSAs expected, isometric force and net work per cycleincreased with an increase in the number of stimuli in thepulse train (Fig. 1). Maximum work was achieved with an

80 ms stimulus train at 200 Hz.

In experiment A, one, two, or three additional initial pulseswere added to a base stimulus frequency of 31 Hz. The pulseswere added after the first pulse at the beginning of the pulsetrain at an interpulse interval of 5 ms (Table 1). Addingpulses at the start of the pulse train increased isometric forceand markedly increased net work per cycle. Adding one pulse

increased the net work by about 52 %, but increasedisometric force by only 24 %. Eighty per cent of themaximum net work was achieved by adding only threepulses at the start of the train, i.e. 80% of the work achievedwith seventeen pulses was produced by only six pulses. Thepulse train at 31 Hz with one extra initial pulse yielded more

net work per cycle than a train with four pulses given at200 Hz only (shown by the square symbols in Fig. 1). Network per stimulus pulse and force per stimulus pulse were

affected differently (insets in Fig. 1). Maximum net work per

stimulus pulse was achieved with a stimulus train that

0

z

Figure 1. The effect of adding initial stimulus pulseson net work per cycle and on isometric force in frogsartoriusd, doublet; t, triplet; q, quadruplet. Cycle frequency was4 Hz; strain, 0 07; stimulus phase, 15% (as a proportionof the length change cycle). The pattern of stimuli in thetrain is shown beside each point. The base stimulus ratewas 31 Hz; the additional initial stimulus rate was200 Hz. Train duration and interpulse intervals for eachstimulus pattern are given in Table 1. The insets show network per cycle and isometric force divided by the totalnumber of pulses in the stimulus train. The square

symbols show the response to the initial part of thestimulus train with 4 pulses at 200 Hz only.Means+s.E.M., n= 8.

5

0

40

q

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0 5 10 15

30 FN

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2 4 6 8 10 12 14 16 18

Total number of pulses

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15-base31 -base62-base92-base123-base154-base15-plus31 -plus62-plus92-plus123-plus154-plusContinuous

15

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E. D. Stevens

consisted of five pulses with two being additional initialpulses at 5 ms interpulse intervals. Force per stimulus pulsedecreased with additional initial pulses. The effect of addingpulses on the net work per cycle and on isometric force atlonger sarcomere lengths was virtually identical to that at L.(data not shown).

Figure 2 shows exemplary isometric force traces and workloops at a base stimulus frequency of 31 Hz. Each additionalinitial pulse caused a further increase in the initial rapiddevelopment of force. Although the force trace was far froma fused tetanus at this low base frequency, the stimuluspattern was sufficient to maintain some force throughoutthe shortening phase and thus contributed significantly tothe amount of work done per cycle. The small loop at thebottom of the work loops is a passive work loop with nostimulus; this loop is clockwise so the net work is negative.The train durations of the stimulus patterns used in thisexample were identical: 802 ms. Thus there were smalldifferences in the base frequency when initial pulses wereadded.

In experiment B, only the effect of adding three additionalinitial pulses was tested and the base frequency was varied.The effect of adding three initial pulses was greater at lowerbase frequencies and decreased as base stimulus frequencyapproached fusion frequency (Fig. 3). Adding the initialpulses increased net work per cycle much more than it

17

o 50 101050 5

E

FStIrse

0 50 100 150 200 250 2Time (ms) 1

StC

17 ~~~~~~~~~tI6 ~~~~~~~~~~cil

0

0

increased isometric force. Adding three initial pulses to abase frequency of 15 Hz resulted in a fourfold increase inthe net work per cycle. Adding to a base frequency of 31 Hzdoubled the net work per cycle. The insets in Fig. 3 showthat the maximum work per stimulus pulse occurred at abase frequency of 31 Hz with the added initial pulses (i.e. atrain of six pulses, 3 pulses at 31 Hz plus 3 additional initialpulses) whereas maximum isometric force per stimulus pulseoccurred at a base frequency of 31 Hz with no added initialpulses (i.e. a train of three pulses). The effect of addingpulses on the net work per cycle and on isometric force wasvirtually identical at longer sarcomere lengths (data notshown).

Figure 4 shows work loops without, and with, threeadditional initial pulses at 5 ms interpulse intervals. At thelower base frequency of 15 or 31 Hz there was a markedincrease in initial force development, and at 31 Hz fairlyhigh force was maintained throughout the shortening partof the cycle.

DISCUSSIONOther workers have studied the effect of non-uniformpatterns of stimulation on force developed by isolatedmuscle but none have studied work done by muscle. Wilsonand co-workers modulated the frequency of stimulation in asinusoidal fashion using invertebrate limb muscles (Wilson

Pigure 2. Exemplary force traces and work loops at a basetimulus frequency of 31 Hzn each case the top trace is for 17 stimulus pulses at 200 Hz, theecond is for 6 pulses (3 at 31 Hz plus 3 initial pulses at anaterpulse interval of 5 ms), the third is for 5 pulses (3 at 31 plusinitial; a triplet), and the bottom is for 4 pulses (3 at 31 plusinitial; a doublet). Cycle frequency, 4 Hz; phase oftimulation, 15%; strain, 0 07; train duration, 80 2 ms (duty cycle,)l32; the fraction of the imposed length change cycle duringvhich the muscle was stimulated). The small loop at the bottom ofhe work loops is a passive work loop with no stimulus; it islockwise and is net negative work.

41-4386 40-0

Muscle length (mm)

J Physiol. 494.1282

J Physiol.494.1 Doublets increase work done in frog muscle

15 F

_e0)

03

zFigure 3. The effect of adding three initial pulses at5 ms interpulse intervals to different base stimulusfrequencies on net work per cycle and isometric forcein frog sartorius muscleIn each panel the circle symbols are for the base frequencyalone, the square symbols are for the base frequency plusthe 3 initial pulses. Train duration and interpulseintervals for each stimulus pattern are given in Table 2.The open triangle is the work or force produced by theinitial 4 pulses only. The insets show net work per cycleand isometric force divided by the total number of pulsesin the stimulus train. Means + S.E.M., n = 8.

1 0 -

5

0

40

30 I

CY)Eze00LL

20 -

10 I-

o '0

& Larimer, 1968; Wilson, Smith & Demptser, 1970). Theyshowed that force could be maintained with a low level ofexcitation after an initial period of high stimulationfrequency. They also showed that the phenomenon was dueto events within the muscle itself and not a property of theneuromuscular junction. Partridge (1966) obtained similarresults using mammalian skeletal muscle. The results of thepresent experiments are concordant with previous

0 100 200

20 40 60 80 100 120 140Base frequency (Hz)

160 180 200

experiments, and in addition show that the effect is muchmore marked when measuring work done rather thanmeasuring isometric force.

The effect of non-uniform stimulation patterns also hasbeen studied in situ by Zajac & Young (1980a), who showedthat the optimal stimulus pattern always began with one ortwo short interpulse intervals (<10Ims) followed by a lowerstimulus frequency. They defined optimal as maximal

40-0Muscle length (mm)

B

41-4 38-6 40-0 41-4Muscle length (mm)

Figure 4. Exemplary work loops at base stimulus frequencies of 15, 31, 62, 92 and 200 HzA, loops produced by base stimulus pattern with no additional initial pulses. B, work loops produced bybase stimulus patterns with 3 additional initial pulses at 5 ms interpulse intervals. Cycle frequency, 4 Hz;phase of stimulation, 15%; strain, 0 07; train duration, 80-2 ms (duty cycle, 0 32). The small loop at thebottom of the work loops is a passive work loop with no stimulus.

283

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E. D. Stevens

isometric tension-time area per stimulus pulse. Burke,Rudomin & Zajac (1970) showed that doublet stimulation ofsingle motoneurons in cats caused a long lasting elevation ofisometric force and pointed out that it is in some wayssimilar to the 'catch property' reported for some invertebratemuscles. Stein & Parmiggiani (1979) attempted to estimatethe stimulus pattern that would produce the maximalisometric force impulse (i.e. force-time integral) for astimulus train delivered to cat soleus muscle. The mosteffective pattern was a short interpulse interval, a doublet,then a single long interval, followed by a series atintermediate intervals. For the cat soleus these interpulseinterval times were < 15, 130-160 and 100-120 ms (or, asfrequencies; 67, 6f3-7f7 and 8 3-10 Hz). Binder-Macleod &Barrish (1992) used doublets, triplets and quadruplets in astudy of the force-frequency curve of rat soleus muscle insitu. They measured isometric force and calculated the forceimpulse and average force for a stimulus train lasting 2 s orseventy pulses, whichever was shorter. A doublet increasedthe average force by about 20 %, increased the force impulseby about 13% and increased peak isometric force by about18%. The results of the present study for peak isometricforce with isolated frog muscle are similar (24 %).

Doublets have been recorded from motoneurons in a numberof preparations. They have been reported for populations ofmotoneurons in normal rats (Hennig & Lomo, 1985, 1987).They occur in the phrenic motoneurons during fictivecoughing and vomiting (Milano et al. 1992) and in hindlimbmotoneurons in decerebrate cats (Zajac & Young, 1980b).Zajac & Young reported that motor units fired doublets100% of the time at slow walking speeds and fired fewerdoublets at faster walking speeds. They also showed thatsimilar stimulation patterns in situ produce maximumisometric force impulse (Zajac & Young, 1980a). Whenrespiratory drive is increased in anaesthetized cats, manymotoneurons to the intercostal muscles start dischargingwith doublets (Kirkwood, Munson & Westgaard, 1988). Inman, doublets have been reported in motor units involved inlocomotion (Denslow, 1948; Norris & Gasteiger, 1955;Gurfinkel et al. 1972; Desmedt & Godaux, 1977, 1978;Kudina & Alexeeva, 1992), wrist movement (Bawa &Calancie, 1983), and respiration (Adams et al. 1989). Theexperiments on wrist movement showed that doubletsoccurred at the onset of slow recruitment and also at theonset of ballistic movements in the majority of single motorunits studied. They suggested that the two arise from twodistinct processes in the spinal cord. They reported thatduring long slow periods of force development, the outputfrom motoneurons initially consisted of a train of repetitivedoublets that changed to a train of pulses at constantinterpulse intervals. Repetitive doublets also were observedin motoneurons innervating a number of other limb muscles.Triplets and quadruplets have also been reported in vivo,and triplets have been observed in the motoneurons ofhumans (S. J. Garland, personal communication). Bawa &Calancie (1983, their Fig. 5) and Desmedt (1981, his Fig. 5)show triplets in their recordings from single motor units.

It has been argued that doublets are of adaptive significancebecause they should reduce the metabolic cost of doing work(Bigland-Ritchie et al. 1992). In active muscle, energy isconsumed by several processes, the dominant ones beingmyosin ATPase and calcium pumping. Estimates of the costof calcium pumping are high and vary from 20 to 50%(Homsher & Keen, 1978). Presumably, the initial doubletcould produce high calcium concentrations for activation andthe subsequent low stimulation frequency could effectsufficient release of calcium to balance its uptake. Continuoushigh-frequency stimulation would result in more calciumrelease (Blinks et al. 1978), more calcium pumping andwould cost more. The results of the present study show thatwork values near maximal can be achieved with fewerpulses by introducing a few stimuli at a high frequency atthe beginning of the stimulus train. If the base stimulusfrequency is low then a doublet can increase the amount ofnet work done per cycle by 50 %. The slight decrease in theamount of work done by using a lower base stimulationfrequency is more than offset by the expected decrease incost of doing the work. That is, it should be more efficient touse a doublet to achieve the same amount of work (Milano etal. 1992). Milano et al. suggested that doublets 'may berelevant to the problem of diaphragmatic fatigue; they maypermit development of a given transdiaphragmatic pressureat a lower mean discharge frequency, with a consequentreduction in metabolic cost.' At present, this idea is untestedin diaphragm muscle. Bevan, Laouris, Reinking & Stuart(1992) compared regular stimulus trains (16 pulses at 26 Hz)with those containing triplets (16 pulses, triplet then 23 Hz)in a fatigue protocol in motoneurons innervating the tibialisposterior muscle of the anaesthetized cat. Initial peak forcewas 2A4 times greater and the force-time integral was1P5 times greater with the triplet stimulus pattern in fastfatigue motor units. Throughout the 6 min fatigue trial,peak force and the force-time integral were always greaterfor the triplet stimulus pattern compared with the regularpattern. The efficacy of the triplet stimulus patternincreased as the fatigue trial proceeded. They concludedthat initial short interpulse intervals were valuable formaintaining force production during fatigue. Otherexperiments have shown that if doublets are used tomaintain force for repeated isometric contractions, thenthere is less fatigue (Binder-Macleod & Barker, 1991). Withthe human elbow moving at 65 deg s-', doublets occur at theonset of the movement, at peak velocity and at movementtermination (S. J. Garland, personal communication). Thesedoublets increase in prevalence with fatigue.

There are two questions, as yet unanswered, that arise fromthe present studies and the other studies on non-uniformstimulation patterns. First, if it is more efficient to usedoublets, why are they not used in every circumstance?Second, in the present study it has been found thatmaximum work per pulse was achieved by adding twoadditional pulses, i.e. with a triplet rather than a doublet.Thus, one would expect that workers studying the in vivooccurrence of non-uniform stimulation patterns should

284 J Physiol.494.1

J Physiol. 494.1 Doublets increase work done in frog muscle 285

observe triplets more often. Perhaps there is a costassociated with producing doublets and triplets that has yetto be explained.

The results of the present experiments may have somerelevance to pathological conditions. Rapid muscle fatigue isa problem associated with using artificial neuromuscularstimulation to control skeletal muscle in persons withdamaged or diseased motor control systems. The presentexperiments show that using non-uniform stimulationpatterns may reduce the rapid fatigue seen in these patients.That is, similar amounts of work done can be achieved withmany fewer stimulus pulses using doublets or triplets. Theeffect should be much more marked using workingcontractions such as those used during normal locomotionthan would be seen in isometric contractions.

In summary, the present results confirm the findings ofprevious studies that showed the importance of stimulationpattern in determining force output. They extend thosefindings to show that doublets cause a much larger increasein net work per cycle than in isometric force.

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AcknowledgementsThis work was supported by a Natural Sciences and EngineeringResearch Council of Canada operating grant. I thank Liz Niimi fortechnical assistance. Drs John Brooke and Jane Garland provideduseful comments on a draft of the paper.

Author's email addressE. D. Stevens: dstevens@uoguelph.ca

Received 9 August 1995; accepted 21 February 1996.