Lab6BiomechanicsofFrogSkeletalMuscle

I.

iWorx,Wayne,sp2014

Purpose
Thisexerciseisdesignedtodemonstratesomemechanicalandphysiological
propertiesofskeletalmuscleusingthegastrocnemiusmuscleofafrog.

II.

PerformanceObjectives
Attheendofthisexercisethestudentshouldbeableto:
1.
2.
3.
4.
5.
6.
7.
8.
9.

III.

Defineminimal(threshold),subminimal,maximal,andsupramaximalstimulus.
Explainwhatismeantbyagradedresponse.
Drawadiagramofthesetupusedinthislabexercise.
Calculatetheworkthatisdoneinagivenmuscle.
Describethefunctionofaforcedisplacementtransducer.
Describewhathappenstomusclecontractionwhentheloadisincreased.
Definetwitch,treppe,tetanusandincompletetetanus.
Explainthedifferencebetweenasingleanddoublepithofthefrog.
PostalldatatoGoogleDocs.

Introduction
Thegastrocnemiusmusclecontainsmanymusclefibers.Eachfiberhasitsown
thresholdandrespondsallornonewhenstimulated.Itisknownthatallofthefibersina
muscledonothavethesamethresholdandthatastimulusappliedtoamuscledoesnot
necessarilyexciteallthefiberinit.Thethresholdisthatintensity(voltage)ofstimulus
whichbringsresponse.Astheintensity(voltage)ofthestimulusisincreasedabovethe
threshold,moreandmorefibersarestimulatedandtheresponsebecomesgreaterand
greater.Eventually,however,stimulusintensity(voltage)isreachedbeyondwhichthe
responseisconstant.Thisstimulus,calledthemaximalstimulusmarksthepointwhere
allofthefibersinthemusclearestimulatedandrespondingallornone.Stimuliabove
thismaximalstimulusarecalledsupramaximalstimuli.Stimulibelowthethresholdthat
donotinitiateamechanicalresponsearecalledsubthresholdstimuli.
Thefrogmuscleisusedinthislaboratoryexerciseinplaceofmammalianmuscle
becauseofitstolerancetotemperaturechangeandhandling.Theresultsaresimilarto
whatwouldbeseeninmorecarefullycontrolledmammalianexperiments.

III.

PreparationoftheFrog

Afterthefroghasbeendoublypithedyouarereadytoremoveamusclefortesting.Oneofthe
largestandeasiestmusclestoobtainisthegastrocnemiusofthelowerleg.
Placethefrogonacleandissectingpan.Besurethatneitherthepannorthedissecting
instrumentshavebeencontaminatedwithpreservativessuchasformaldehyde.Youwillbe
providedwithpansandtoolsreservedspecificallyforthedissectionoffreshmaterials.
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Itisnotnecessarytotiedownthefrogwhenusingthefemurclamppreparation.Asthemuscle
isdissectedassignoneofyourlabpartnersthetaskoffrequentlyfloodingthesurgicalareawith
frogringersusingasmallbeakeranddisposablepipet.Themusclemustnotdryoutwhile
dissectingoritwillbeuseless.
Withforceps,lifttheskinfromonethighandcuttheskincompletelyaroundthelegusing
scissors.Pullthecutendbackandpeeltheskinofftheleg.Minimizethetouchingofthe
muscletissuewithcontaminateddissectingequipment.KeeptheareamoistwithfrogRingers.
Withabluntprobeorthebluntendoftheforcepsseparatethebodyofthegastrocnemiusfrom
theunderlyingbone.ThedistalendofthemuscleisattachedtotheAchillestendon,awhiteto
yellowstrandoffibroustissue.LoosenaportionoftheAchillestendonfromthebonethen
insertapieceofthreadabout10incheslongaroundthetendonandtieitsecurely.Cutthe
tendondistaltothisknot.Theproximalendofthegastrocnemiusisattachedtothefemur.Do
notremoveitfromthebone.Carefullyremoveallothermusclesfromtheupperlegbutleave
thegastrocnemiusattached.Thencutthelowerlegbone(tibia)atorslightlydistaltotheknee
jointwithstrongscissorsorbonecutters.Cuttheproximalendofthefemurleavingatleastone
halfinchofboneattachedtothemuscle.Youshouldnowhaveapreparationresemblingthatin
thefigure.Withtheproximalendofthegastrocnemiusattachedtothefemurandthedistalend
attachedtoaportionofthread.Thisisthemusclebonepreparationyouwilluseforyour
musclephysiologyexperiments.ConnectthefemurtothefemurclampasshowninVI.Steps
IV,VandVIcanbedoneconcurrently.
IV.

iWorxFrogSkeletalMusclePhysiologySetup.Ifthesystemisalreadysetup,skip
toV.Standardizethemovementofthetransducer
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.

MakesurecomputerandiWorxinterface(blackbox)areunplugged)
AttachthecableoftheDisplacementTransducertothebottomchannel3input
plug
Insertthestimulatorcableplugintochannel3oftheiWorxbox
(whichisattachedtothestimulatorsectionoftheiWorxinterface)
Plugthecomputerpowercordintothepoweroutlet.
PlugtheiWorxpowercordintotheoutlet
TurnontheiWorxinterface
Turnonthecomputer
Afterthecomputerhasbootedup,clickontheLabscribeicon
On the Main window, pull down the Settings menu and
select the Skeletal Muscle-Summation-Tetanus-LS2 settings file.
After a short time, LabScribe will appear on the computer screen as configured
by the Skeletal Muscle-Summation-Tetanus-LS2 settings.

Yourequipmentisnowreadyfortheexperiment
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V.

Standardizethemovementofthetransducer(StarthereifLabscribeisready)
1.

Whenthetransducerrodispulledbythecontractingmuscle,theamplitudeofthe
contractionwillbegivenasvoltsintheupperrightofthewindowasV2
Recordtheabsolutevalueandignorenegativesign.

2.

Toconvertthisreadingtoactualmillimetersofmusclecontraction

V1.

a.

Clickrecord.

b.

Pullthetransducerrodashighasitwillgoandthereleaseit.

c.

Clickstop.

d.

Measuretheactualdistancethattherodmoved(thisisusuallyabout
3034mm).Seeabovepictures.

f.

Clickautoscaleanddoubleorsinglemountainiconsasneeded.

g.

Clickthe2cursoriconandmeasuretheheightofthedeflectionon
channel3byplacingthefirstbluelineonthepeakofthe
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deflectionand
deflectionhas

thesecondbluelineonthebaseline(valley)afterthe
returnedtoits originalposition.
h.

ReadandrecordtheVoltsreadingaboveandtotherightofthechannel
(ignorenegativesigns).Thisvoltagereadingisequivalenttothe
mmofmovementmeasuredbytheruler

j.

Dividethemmofmovementbythevoltagetogetaconversion
factor.Thiswillallowyoutodeterminethedistancethemusclehas
contracted,whichisnecessarytocalculatetheworkdonebythemuscle.

k.

Wheneveryoumeasureamplitudeofacontractioninvoltageyoucan
nowconvertittomillimetersofcontractionbymultiplyingthis
yourconversionfactordeterminedabove.

actual

valueby

VI. Setupthefrogmusclepreparationasfollows:
1.

Compressthefemurinthefemurclamp

2.

Adjusttheclampssothatthethreadisverticalandthedisplacementtransducer
rodrestsjustontheupperstopwithnoslackinthethread;makesurethe
vertical

3.

Positionthestimulatorelectrodessothattheylieagainstthemuscleabout
midwaybetweenthekneeandthetendon;thetwoelectrodesshouldnot
another,butbothmustbeincontactwiththemuscle

thread

touchone
4.

Placetwonickels(10g)intheweightpan

5.

Occasionallyaddfrogringerstokeepthemusclepreparationmoist.

iWorx,Wayne,sp2014

VII. ExercisesinFrogMusclePhysiology
A.

Determiningthethresholdstimulus.

1.

Setupthestimulator(select<preferences>inEditmenu)orusethestimulator
panelandsetthefollowing:
PulseWidth
Delay
Amplitude
Frequency
#ofPulses

=
=
=
=
=

10ms
50ms
0Volts
0.5Hz
1

Thestimulatorcontrolpanelisshownbelow.Don'tforgettoclick"apply"aftereach
change.

Ifyoudontseethestimulatorcontrolpanel,clickthestimulatoriconshowncircledingreenand
thestimulatorcontrolpanelwillbeshown.

2.

Type0vinthemarksbox,clickRecordtobeginrecordingandpressenter.The
stimulusandarecordofthemusclesresponse(ifany)willbedisplayedin
channel#3
andchannel#4,respectively.
3.

Quicklyclickstop.

4.

Clickthe<2cursor>iconsothattwoblueverticallinesappearovertherecording
window.

5.

Adjustthedisplaytimeifneededandmovethemousetoplacethecursorontheleftline,
clickthemouseanddragittothepeakofthefirstresponsewave(ifthereisone).Drag
thesecondcursor,tothebaselineaftertheresponse.
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6.

Thevalueinthechannelthreetitlearea,aboveandtotherightofthechannelthree
windowisthevaluefortheamplitudeofcontractioninvoltsatthestimulusvoltage
used.Recordthisvalueinthetableonyourdatasheet.

7.

Convertthisamplitudevoltagetomillimetersofcontractionbymultiplyingitbythe
conversionfactordeterminedinpartVandrecordthisvalueinthetableinyourdata
sheet.

8.

Openthestimulatordialogboxandchangethestimulusamplitudeto0.25v.Dontforget
toclickapplyaftereachchangeinthestimulatordialogbox.

9.

Repeatsteps2through7,thistimetyping.25vinthemarksboxbeforestarting.

10.

Repeattheaboveprocedureincreasingthevoltagein0.25voltincrementsuntilyouare
stimulatingthemusclewith5volts.

11.

Onceyouhavecompletedyourtableyoushouldbeabletocompletetheobservations
below:

12. Thethresholdstimulusistheminimumstimulusneededtogetanykindofaresponse
fromthefrogmuscle.

13. Recordthethresholdstimulusinthedatasection
14.

C.

Useyourdatatomaketherequiredgraphsasindicatedinthedatasection.

TimingtheMuscleTwitch

1.

Setthestimulusvoltageatthevalueyoudeterminedwasthemaximalstimulus(usually
34volts)

2.

Typetwitchinthemarksarea;clickRecordandquicklypressentertomarkthe
record andproduceasingletwitch

3.

Clickstop.

4.

Ifnecessary,rightclickafterrecordingtoinverttrace.

5.

Ifnecessaryadjustthedisplaytimeasneededbyclickingthe<halfdisplaytime>
iconor<doubledisplaytime>oneormoretimestospreadthewaveformoutto
facilitateyouranalysis.

iWorx,Wayne,sp2014

6.

Scrolltothebeginningofthesectionofdatayouwanttoinvestigate;clickAutoScaleif
necessary.

7.

Clickthe2cursoriconsothattwoblueverticallinesappearovertherecordingwindow
andmeasure:tinthediagrambelowLequalslatentperiod,Cequalscontractionphase,R
equalsrelaxationphaseandAistheamplitudeofcontraction.

8.

Theamplitudeofthetwitchandconvertittommasbefore.

9.

Thedurationofthelatentperiod:Placeonecursorlineatthestimulus
mark(inwindow4)andtheotheronwindow3justbeforeacontraction
andreadT2T1inupperrightcornerofscreen.Thisistheduration
thelatentperiodinseconds.

begins
of

10.

D.

Usethedoublecursorstomeasurethedurationoftheperiodofcontractioninthe
sameway

11.

Usethedoublecursorstoalsomeasurethedurationoftheperiodofrelaxation.
Convertthesevaluestomillisecondsandrecordthemonyourdatasheet.

12.

Printacopyofthistracingforyourgroup.Pastethegraphinthedatasectionof
thislabexercise.

DeterminingtheEffectofLoadonSkeletalMuscle

iWorx,Wayne,sp2014

Withinlimits,increasesintheload(i.e.,passivetension)placedonamusclebeforeitcontracts
resultsincorrespondingincreasesinthestrengthofthecontractions(i.e.,activetension).There
ishowever,amaximumtensionthatamusclecanexert,andbeyondthatlimit,increasesinload
(passivetension)resultinweakerandweakercontractions(activetension).
1.

Setthestimulusvoltagetoproducemaximalstimulusasdeterminedpreviously(usually
between3to5volts)

2.

ResetstimulatorsettingstothoseusedinpartConthepreviouspage

3.

With2nickelsintheweightbaskettype10gonthekeyboard

4.

ClickRecordandpressEntertomarktherecordandproduceasingletwitch.

5.

ClickStop.

6.

Addtwomorenickels(10g)totheweightbasketforatotalof20g

7.

Repeattherunasabove(#3&4)

8.

Continueincreasingtheweightintheweightbasketin10g(twonickel)incrementsuntil
thereisnodiscernablecontractionofthemuscle

DataAnalysis:Effectofloadonmuscle
9.

Adjustthedisplaytimeandautoscaleasneeded

10.

ScrolltothebeginningofthissectionofdataorusetheMarksicontofindeachofthe
aboveruns

11.

13.

Clickthe2cursoriconsothattwoblueverticallinesappearovertherecording
windowandforeachtwitchateachweight,measuretheamplitudeofthetwitchbothasa
voltagemeasurementandafterconvertingittomillimetersusingyourconversionfactor
andrecordonyourdatasheet
Calculatetheworkdoneforeachweightthatproducedameasurablecontractionofthe
muscleas:
workdone(gmm)=weightofload(g)Xamplitudeofcontraction(mm)

13.

E.

Recordthisvalueinthetableonyourdatasheet

ObservingGradedMuscleResponsetoIncreasedStimulusFrequency
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1.

Openthestimulatordialogboxandchangethenumberofpulsesto250,makesurethe
stimulusisstillsettothemaximalstimulusvoltageasdeterminedearlier,usetwo(2)
nickelsintheweightbasket.

2.

Type0.5Hzinthemarksbox;clickRecordandpressEnteronthekeyboardtomark
yourrecord.

3.

Clickstop

4.

Increasethefrequencyto1,5,10andthen20Hzandrepeatsteps2and3.Noticethat
thefirstfewcontractionsincreaseinintensityeventhoughthestimulusisthesame.
Noticealsothatatacertainfrequencythemuscledoesnothavesufficienttimetofully
relaxsothattheresponsedoesnotreturntobaseline,thisissummation.

5.

Recordthefrequencyatwhichsummationfirstappearsonyourdatasheet

6.

Printagroupcopyofthetracingonyourscreenatthisfrequency

7.

Asyoucontinuetoincreasethefrequencyofstimulation,noticethatatsomefrequency
thereisnorelaxationatallbetweeneachstimulus,thisistetanus.

G:

MuscleFatigue

Musclefiberscannotcontinuallylift.Afterashorttime,themusclewillloseitsabilityto
shortenandwillultimatelyfail.Duetotheaccumulationofwasteproductsandthedepletionof
storedenergymaterials,amuscleissaidtohavelostitscontractilityandbecomefatigued.

1.

Setthestimulatorvoltagetothemaximalstimulusvoltageasdetermined
previously.

2.

Setthenumberofpulsesto0.Thiswillprovideaninfinitenumberofpulses.

3.

Clickrecordandobservethewaveformproduced.Toseetheeffectofcontinuous
stimulationthedoublemountainiconmayhavetobeclickedseveraltimestoput
muchmoredataonthescreen.

4.

Afterseveralminutesorwhentheamplitudedropssignificantly,stopthe
recording.

5.

Compressthedatabyusingthedoubleorsinglemountainicons.

6.

Pastetherecordingwhereindicatedinthedata.

iWorx,Wayne,sp2014

InducingTreppe,ortheStaircasePhenomenon(optionaliftimepermits)
Aninterestingphenomenoncansometimesbeseenduringtheearlystagesofaseriesoftwitches.
Uponapplicationofasinglestimulusofadequateintensity,themusclecontracts.With
successivestimuli,itcontractsalittlemorestronglybysmallincrements,makingthispartofthe
myographicrecordappearlikeastaircase.Forthisreason,theeffecthasbeencalledthe
"StaircasePhenomenon"or"Treppe".Itisbelievedthattheslightincreaseintemperatureofthe
musclefibersduringcontractionincreasestheircontractilityandthatthismaybetheexplanation
fortheincreasedstrengthofcontractions.
1

Dissectandsetupthegastrocnemiusfromtheotherleg.

2.

Usingamaximalstimulusasdeterminedpreviously,stimulatethemusclewith5
stimulisetatafrequencyof1.

3.

IfyouhadtimetodoTreppepasteyourrecordingwhereindicatedinthedata
section.

VIII. Completingyourreport.Yourlabreportshouldhave:
1.

Alldatasheetscompleted

2.

Agraphofgradedresponseandmaximalstimulus

3.

Agraphofloadversusworkdone

4.

Aprintoutofamuscletwitchwithallphasesofatwitchcontractionlabeled.

5.

Aprintoutshowingsummation.

6.

Aprintoutshowingtetanus.

CleanupandDisposal
1.
2.
3.
4.
5.

Wrapfrogandmusclepreparationinpapertowelsorplasticbagprovidedanddiscardin
trashcan.
Cleanalldissectingequipmentwithsoapandwater,blotdrywithpapertowelsandreturn
totray
Disconnectthefemurclampfromyourstation,rinsewithDIwater,andreplaceonthe
ringstandatyourstation
WetapapertowelwithDIwaterandwipedownthetransducer,stimulatorprobesand
clamps,blotdrywithapapertowel
TurnofftheiWorxstation.
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iWorx,Wayne,sp2014

6.

Remembertosprayyourworkareawithdisinfectantbeforeleavingthelabroom.

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DataSheet

iWorx,Wayne,sp2014

Exercisesin
FrogSkeletalMusclePhysiology
Conversionfactortoconvertvoltagetommofdisplacement:___________
Recordthevaluesinthetablebelow:
stimulus
voltage
contraction
(volts)
contraction
(mm)
stimulus
voltage
contraction
(volts)
contraction
(mm)

DeterminingtheThresholdStimulus
Atwhatstimulusvoltagedidameasurablecontractionfirstoccur?_____________
Doallmotorunitshavethesamestimulusthreshold?Explain
___________________________________________________________________________
___________________________________________________________________________
___________________________________________________________________________
ObservingGradedMuscleResponsetoIncreasedStimulusIntensity

1.

Usingthedatafromtheprevioussection,makeagraphplacingstimulusvoltageonthe
xaxisandtheamplitudeofcontractioninmillimetersontheyaxis.Pastethe
graph below.

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ObservingGradedMuscleResponsetoIncreasedStimulusIntensity

2.

Notetherangeofstimulusvoltagesinwhichyouproducedagradedresponsebythe
muscle.Whatisoccurringinthemuscleatthispoint?
___________________________________________________________________
___________________________________________________________________
____________________________________________________________________
____________________________________________________________________

3.

Theamountofcontractionshouldpeakandremainrelativelyconstantatsomestimulus
voltage;thisisyourmaximalcontractionandmaximalstimulusvoltage

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Whenthecurvelevelsoffyouhavereachedmaximalstimulusvoltage.Estimatethe
maximalstimulusvoltagefromyourgraphandrecorditonyourdatasheet.Whycant
themusclecontractanymoresinceyouarestillincreasingthestimulusafterthispoint?
___________________________________________________________________
___________________________________________________________________
____________________________________________________________________
____________________________________________________________________

Whatwasthestimulusvoltageatmaximalcontraction?____________

Whatwashappeningtothemusclecellswithinthemuscleasthestimulusvoltagewasincreased
uptomaximalstimulusintermsofrecruitmentofmotorunits?

____________________________________________________________________
____________________________________________________________________

TimingtheMuscleTwitch
Stimulusvoltageused:___________

Amplitudeofcontraction(mm):___________

Durationsof:
LatentPeriod(ms):

_____________

PeriodofContraction(ms): _____________
PeriodofRelaxation(ms):

_____________

Labelthetracingofatwitchwitheachphaseandthedurationofeachphaseandpastewhere
indicated.
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MuscleTwitch

Howdothesevaluescomparewiththetimeintervalsgiveninlectureortext?Explain.
____________________________________________________________________
____________________________________________________________________

InducingTreppe,ortheStaircasePhenomenon
Optional.Ifthiswasattemptedbyyourgroup,includethedataandprintoutwhereindicated.
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InducingTreppe

ObservingGradedMuscleResponsetoIncreasedStimulusFrequency
1.Whatwasthefrequencythatproducedsummation:

___________?

2.Whatwasthefrequencythatproducedtetanus:

___________?

InducingMuscleFatigue
1.

Didmusclefatigueoccur?Howwasthisdetermined?
____________________________________________________________________
____________________________________________________________________

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2.

Explainwhatishappeninginthemusclecellsduringfatigue.
____________________________________________________________________
____________________________________________________________________

DeterminingtheEffectofLoadonSkeletalMuscle

Load
(g)

Amplitudeof
Contraction
(volts)

Amplitudeof
Contra
ction
(mm)

WorkDone
(g/mm)

10g
20g
30g
40g
50g
60g
70g
80g
Didtheamplitudeofthecontractionincreaseordecreasewithincreasingload?Explain.
____________________________________________________________________
____________________________________________________________________
____________________________________________________________________
____________________________________________________________________
Didtheworkdoneincreaseordecreasewithincreasingload?Explain.

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____________________________________________________________________
____________________________________________________________________

Explainyourresultsintermsofenergyrequired,andtheinterplaybetweenisotonicand
isometriccontractions.

____________________________________________________________________
____________________________________________________________________
____________________________________________________________________
____________________________________________________________________
Makeacombinedgraph.Foronedatasetplottheloadonthexaxisandtheamplitudeof
contractioninmmontheyaxis.Fortheseconddatasetplotloadonthexaxisandworkdone
ontheyaxis.Pastethegraphwhereindicated.

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CombinedGraph

DontforgettopostyourdatatoGoogleDocs!

Printoutsforsummationandtetanusshouldfollowthispage!!

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