lab

MASSACHUSETTS INSTITUTE OF TECHNOLOGYDepartment of Electrical Engineering and Computer Science

Department of Mechanical EngineeringHarvard-MIT Division of Health Sciences and Technology

Quantitative Physiology: Cells and Tissues2.791J/2.794J/6.021J/6.521J/BEH370J/HST541J

Fall, 1999

The Compound Action Potentialof the Frog Sciatic Nerve

1 Introduction

1.1 Overview

This laboratoryprojectis intendedto provideanopportunityto learnabout(1) designinganexper-iment, (2) acquiring,processing,andinterpretingexperimentaldata,and(3) communicatingtheresultsto others.

You andyour laboratorypartnerwill dissecta sciaticnerve from a frog andmount it in anexperimentalchamber, so that you canstimulatethe nerve andmeasureits electricalresponses.Your work is divided into two parts. First you will make some“Basic Observations” that areoutlinedin this laboratorymanual. Then,you andyour partnerwill carry out a projectthat youdesign.With carefulexperimentaldesignandsomethought,youwill beableto measureimportantpropertiesof electricalresponsesof neurons.

Youmayfeeluneasy:how areyousupposedto do(muchlessdesign)anerveexperimentbeforeyou’velearnedanythingaboutnerves?Relax:this laboratorywasdesignedto becompletedbeforewelearnabouttheoriesfor whatshould happen.Thepointof thelaboratoryexerciseis to determinewhatdoes happen.Thus,it will bemoreimportantto gatherconvincing evidencethatyour resultsarereliable(i.e., would you get thesameresultsif you repeatedyour experiment?)thanto arguethatyour resultsareconsistentwith sometheory. This order, in which experimentalobservationsprecedethedevelopmentof theoreticalideas,is typical in thedevelopmentof scientificknowledge.Doing well in the laboratorywill only requirerelatively simple ideasaboutall-or-noneactionpotentials,thresholds,propagationvelocities,and refractoryproperties. Adequatebackgroundmaterialis containedin pages1 to 20 of Volume2 of thecoursetext.

1.2 Structure of the Sciatic Nerve

The sciaticnerve (a macroscopicstructure)is the main nerve trunk from the spinalcord to theleg. It consistsof a bundleof nerve fibers(microscopicstructures),eachof which is theaxonof aneuron,whosecell bodyis in (or near)thespinalcord. We will usetheterms“fiber” and“axon”interchangeably. Axonsarelong,cylindrical processesthatprojectfrom thecell bodyof a neuronandthatactasa conduitfor neuralmessagescalledactionpotentials.Someaxonsconductactionpotentialstowardthebrain: they arecalledafferentfibers. Others,calledefferentfibers,conductactionpotentialsaway from thebrain.Thesciaticnervecontainsbothefferentandafferentfibers.

Axons within the sciaticnerve differ in importantstructuralandfunctionalways(Figure1).Myelinatedaxonsare surroundedby myelin, which looks in cross-sectionlike tightly-packed,concentricringsaroundtheperimeterof theaxon.Otheraxonslack myelin. Axonsthat innervateinternalorgansandglandstendto besmallerin diameter(

�������������m) thanthosethat innervate

skeletalmuscles( � � � � m). Smallerdiameteraxonstendto conductactionpotentialsmoreslowlythan larger diameteraxons(Figure2). The thresholds,refractoryperiods,and the durationsofactionpotentialsdiffer acrosstypesof axons.Interpretationof thepropertiesof compoundactionpotentialinvolvesthinkingof thesciaticnerveasaheterogenouspopulationof nervefibers.

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Figure1: Crosssectionof thesciaticnerveof arabbit(Young,1951). Eachof theseoutlinesis the darkly-stainedlayer ofmyelin that surroundsa singlemyelinatednerve fiber. Fiberdiametersvary. Unmyelinatedfibers,whicharemuchsmallerthan myelinatedfibers, are not visible with the histologicalmethodusedto preparethetissuefor thispicture.

Diameter (µm)

0�10

2�

0

3�

0

4�

0

0�

5�

10 15 20

Con

duct

ion

velo

city

(m

/s) bullfrog

Figure2: Conductionvelocity versusfiber diameterfor individ-ual myelinatednerve fibersin thebullfrog sciaticnerve (Tasaki,1953,adaptedfrom Figure65). The slopeof the line indicatesthat thebestfitting constantof proportionalityis 2 m/sper � m.Thefiber diameterwasmeasuredneartheinsertionof thenerveinto thegastrocnemiusmuscle.Temperaturewas ����� C.

1.3 Compound Action Potentials

As actionpotentialspropagatealonganaxon,they produceelectricpotentialsthatcanberecordedfrom the surfaceof the nerve. Whena nerve bundle is stimulated,many axonsproduceactionpotentialssynchronously. Theresultingelectricresponsesrecordedfrom thesurfaceof thenervearecalledcompound action potentials to distinguishthemfrom theaction potentials generatedbyindividualaxons.

Compoundactionpotentialscanbe measuredasshown in Figure3. Stimuluselectrodesareappliedto oneendof thenerve; recordingelectrodesarelocatedalongthenerve. If thenerve isstimulatedwith acurrentpulseof sufficientamplitude,theactionpotentialsproducedin thefiberspropagatetoward the recordingelectrodes.The aggregateeffect of the many actionpotentialsisanextracellularwaveof negativepotential,moving alongthesurfaceof thenerve. If therecordingelectrodesare widely spaced(Figure 3, left panel), the wave of negative potentialproducesanegativepulsein recordedvoltage ������� asit passesthe recordingelectrode.At a later time, thenegative wave of extracellularpotentialpassesthe

�recordingelectrode,whereit contributesa

positivepulseto therecordedvoltage������� . If theelectrodesaremorecloselyspaced(centerpanel),thenegative andpositive partsof therecordedvoltage ������� merge,andtheresultingwaveformiscalleda diphasic compound action potential. The propagationcanbe blocked by a numberofmethodsincludingmechanicalmethods(pressureappliedto thenerve or crushingthenerve withforceps),electricalmethods(passinga blocking level of currentthroughthe nerve), or chemicalmethods(applyingpotassiumchloride,local anesthetics,cocaine,or tetrodotoxinto thenerve). If

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Nerve

v! (t)

i"(t)

t#

i"(t)

+$ −

v! (t)t#

wave of negative potential reaches "+" electrode

wave of negative potential reaches "−" electrode

Nerve

v! (t)

i"(t)

t#

i"(t)

+$ v! (t)+$−

v! (t)t#

diphasic waveform

Widely spaced recording electrodes Closely spaced recording electrodes Closely spaced with neural block

Nerve

i"(t)

t#

i"(t)

−

v! (t)t#

monophasic waveform

Figure 3: Threeschemesfor measuringcompoundaction potentials. In eachscheme,a current %�&('*) isappliedto two stimuluselectrodes,andavoltageresponse+�&('*) is measuredacrosstwo recordingelectrodes(upperpanels).Theresponseto a pulseof currentconsistsof two, temporallyseparatedcomponents,if therecordingelectrodesarewidely spaced(left panels).A diphasicwaveformresultsfor morecloselyspacedrecordingelectrodes(centerpanels). A monophasicwaveform resultsif a portion of the nerve betweentherecordingelectrodes(shown asa darkband)is alteredto block transmissionof actionpotentials(rightpanels).

thecompoundactionpotentialis blockedbetweenthetwo recordingelectrodes(right panel)sothatit doesnot reachthe

�recordingelectrode,a monophasic compound action potential is recorded.

The monophasiccompoundactionpotentialconsistsof oneor morepeaksof (negative) po-tential that laston theorderof a millisecond.1 Themaximumamplitudevarieswith therecordingconditionsbut rarelyexceedsa few milli volts. Multiple peaksmight representrepeatedactionpo-tentialsproducedby thesamefibersor alternatively, sub-populationsof fibersthatdiffer in theirpropagationvelocitiesandthusproducecomponentswith differentdelays.

Theshapeof thecompoundactionpotentialdependsonthepopulationof excitedfiberswithinthenerve. If all of thefibersin thenerve have similar diameters,thenthepropagationvelocitiesfor actionpotentialswill be similar. The resultingmonophasiccompoundactionpotentialwillbe brief. If the nerve containsa heterogeneousmix of fiberswith differentdiameters(as in thebullfrog sciaticnerve), thenthemonophasiccompoundactionpotentialwill becomebroaderasitpropagates.If thedistancebetweenthestimulatingandrecordingelectrodesis sufficiently large,themonophasiccompoundactionpotentialmayconsistof severalpeaks— eachcorrespondingtoa differentsubpopulationof fibersthatconductsactionpotentialsat a differentvelocity. Studyingtheelectricalpropertiesof thedifferentpeakshasled to insightsaboutthe thresholds,refractoryperiods,and velocitiesof propagationof action potentialsin different subpopulationsof fibers(Patton,1960;Ochs,1965;Ruchetal., 1965).

1Theconventionof plottingnegativemonophasicactionpotentialsasupwarddeflections(which is notusedin thislaboratorymanual)is commonlyusedin physiologicalpublications.

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1.4 Protocol is Important

Thewritten recordof your experimentis theoriginal recordof your work andthebasisfor every-thing thatyou conclude.Suchrecordsareoftencalledprotocol, andareusuallykept in a specialnotebookwith sewn binding (not a loose-leafnotebook),whosepageswerenumberedwhenthenotebookwasprinted. Recordsarewritten in this protocol book in ink. Theprotocolbook is thepermanentrecordof yourexperiment.

Protocolshouldbe sufficiently detailedso that (1) the experimentcould be repeatedat somelatertime,(2) resultsfrom differentexperimentscanbecompared,and(3) sothatyourprocedurescan be reconstructedat a later time without relying on your memory. The first pagefor eachexperimentshouldgive the dateand time, as well as a brief descriptionof the purposeof theexperiment.All relevantproceduresandobservationsshouldbeenteredin theprotocolbookandthetimeshouldbeindicatedregularly.

Nothingshouldeverbeerased.If anerroris detectedin someprocedureor somereading,thatshouldbenotedin thebook.Theoriginalobservationshouldundernocircumstancesbeobliteratedso that it cannotbe read. Perhapsyou will subsequentlyfind that the original observation wascorrectandthat the correctionwasin error. The generalrule is to write down everythingthat isdonethat may later becomerelevant. Scientistsarerarely sorry that they wrote too muchin theprotocolbook— only thatthey wrotetoo little.

You do not have to purchasea specialprotocolbook for this laboratorysession.However,wedoaskthatyou takeprotocolduringyourexperiment,asasteptowardlearningabouteffectivepracticesin experimentalwork. Youwill beaskedto attachyourprotocolto your laboratoryreport,andwewill assesstheprotocolaspartof thegradingprocedure.

1.5 Use of Animals in Research

In this laboratoryyou will dissecta sciaticnerve from a bullfrog. As with all experimentswithanimals,the appropriatenessof this experimentandthe proceduresthat areusedwerereviewedandapprovedby theMIT Committeeon Animal Care. In general,themethodsmeettheguidingprinciplesof theAmericanPhysiologicalSociety:

Animal experimentsareto beundertakenonly with thepurposeof advancingknowl-edge. Considerationshouldbe given to the appropriatenessof experimentalproce-dures,speciesof animalsused,andnumberof animalsrequired.

Only animalsthat are lawfully acquiredshall be usedin this laboratory, and theirretentionanduseshall be in every casein compliancewith federal,state,andlocallawsandregulations,andin accordancewith theNIH guide.

Animals in the laboratorymust receive every considerationfor their comfort; theymustbeproperlyhoused,fed,andtheir surroundingskeptin sanitarycondition.

Appropriateanestheticsmustbeusedto eliminatesensibilityto painduringall surgicalprocedures.

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2 Methods

This sectiondescribestheexperimentalmethods.Additional information,includingphotographsof thesurgicalprocedures,is availableathttp://umech.mit.edu/6.021J/index.html.A video-tapeddemonstrationof thesurgerywill beshown duringthefirst 20 minutesof your lab-oratorysession.Arrive on time soyou canstarttheproceduresimmediatelyafterviewing thethetape.

This is a physiologyexperimentandexperimentalanimalscancarrydisease.You maynot eator drink in thelaboratory, andyoumustwearrubbergloveswhenyouhandletheanimals.

2.1 Anesthesia

A bullfrog (Rana catesbeiana) is anesthetizedby immersingit for about20 minutesin tapwaterthat containsMS222 (Ethyl 3-aminobenzoate,methanesulfonicacid), an anestheticcommonlyusedfor aquaticanimals. The concentrationof anestheticis 0.1 % by weight. The frog shouldbecomelimp.

2.2 Solutions

Throughoutthedissectionandthecourseof theexperiment,thefrog sciaticnerve shouldbekeptmoist in Ringer’s solution (Table 1). Ringer’s solution approximatesthe ionic compositionofthe extracellularfluids of the frog, and a sciatic nerve bathedin Ringer’s solution can remainelectricallyresponsive for hours.KRinger’s andSucrosesolutionsarealsoavailablefor studentsinterestedin investigatingtheeffectsof changesin ion concentrationon electricalresponses.

Concentration Ringer’s KRinger’s Sucrose(mmol/L)NaCl 85 1.8 0KCl 1.8 85 0Na, HPO- 2.75 2.75 0CaCl, 1.7 1.7 0NaHCO. 25 25 0Dextrose 4 4 0Sucrose 0 0 241

Table 1: Compositionsof bathingsolutions.Thepredominateionsin Ringer’sareNa/ andCl 0 , which is typical for normalextracellularfluids. Thepredominateionsin KRinger’s areK / andCl 0 , which is typical for intracellularfluids. The sucrosesolution containsneitherNaClnorKCl, but sucroseis addedsothatthissolutionhasthesameosmolarityastheothers.The pH of eachsolutionhasbeenadjustedto7.4.

2.3 Dissection of the Sciatic Nerve

General precautions.

1 Usethebluntestinstrumentthatwill accomplishthejob. Finerinstrumentsmaycausedam-age,e.g.,ruptureof bloodvessels.1 Cut parallelto thenerve. It is elasticbut is easilycut if approachedperpendicularly.

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1 Never closethe jaws of the scissorswithout visual confirmationthat the nerve will not becut.1 Keepthenervewetwith Ringer’ssolution(notwith blood)atall timesduringthedissection.1 Avoid picking up thenerve with forceps:handlingshouldalwaysbedoneby gently liftingthenerve on a blunt instrumentor by anattachedthread.Stretchingcandestroy theactionpotentialgeneratingmechanismin thenervefibers.

Detailed description. Thesciaticnerve runsdown theleg betweenthelargethighmuscles(Fig-ure4), i.e., betweenthesemimembranosusandboth thevastusexternusandinternusmuscles.Itbranchesseveraltimesandtheperonealbranchis founddeepbetweenthetwo largecalf muscles,i.e., theperoneusandgastrocnemiusmuscles.

G2

astocnemius muscle

Peroneus muscle

Vastus internus muscle

Vastus externus muscle

S3

emimembranosus muscle

Figure4: Dorsal (back)view of frog musculature(CarolinaBiological SupplyCompany, 1965,adaptedfrom Review Sheet55).

1. Thefrog is thesourceof your experimentalmaterial.Weighthefrog andmeasureits lengthfrom snoutto vent. Recordtheseas part of your recordof assemblingan experimentalsystem.

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2. Decapitatethefrog with scissors.With apithingneedle,destroy (i.e.,moshup) thebrainandthespinalcord. Thefrog’s muscleswill twitch duringthesemanipulations.Pithinggreatlyreducessubsequentmusclecontractions,thussimplifying thedissection.

3. Placethe frog on its back in the dissectingtray. Graspthe thin wall of skin andmusclethat overlie the visceraof the lower abdomenwith forcepsandpull it up andaway fromthe viscera. Using dissectingscissors,make a horizontalincision in the thin wall, andcutthroughthewall — first to theleft andup theleft sideof theabdomen,thento theright andup theright sideof theabdomen.Lift the thin wall of skin andmuscleup andcut it off toexposetheviscera.

4. Slidethevisceraup to uncover thespinalcolumnandemerging sciaticnerve. Thenerve iswhite andcylindrical andshouldbedistinguishedfrom tendonsandthesurfaceconnectivetissue(fascia)of muscles,both of which arewhite but areusuallyflatter andshinier thannerves. Cut off the portion of the frog above the spinalcord entranceof the sciaticnerve,includingtheviscera.

5. Grabthespinalcolumnwith apairof forcepsandwith anotherpairof forcepsgraba fold ofskinon thefrog andpull down to removetheskin from thelegs. If thisprovesdifficult, grabthemuscleson eachsideof thespinalcolumnwith a pair of forcepsandwith a third pair offorcepsgraba fold of skinon thefrog andpull down to remove theskin.

6. Freethesciaticnervefromsurroundingtissueby cuttingthesurroundingtissueparallelto thenervebeingcarefulnot to cut thenerve. After theportionof thesciaticnervein theabdomenhasbeenfreedof othertissue,slidesmallcurvedforcepsunderneaththesciaticnervenearitsinsertioninto thespinalcord,attachsurgical threadto forcepsandpull through.Tie off oneof thesciaticnervesat thebackbonewhereit leavesthespinalcord,leaving about2 inchesof threadattachedfor manipulatingthenerve. Cut thenervebetweentheknot of threadandthespinalcord.

7. Freethe portion of the nerve in the abdomenfrom all surroundingtissuefrom the part at-tachedto the threaddown to thehip joint. Try to minimize bleedingwhenyou dissectthenerve. Often thesmallerbloodvesselscanbecrushedwith a hemostatandthensafelycutwithout causingmuchbleeding. If you inadvertentlycut a large vessel,clampit shutwitha hemostatandwashaway thebloodwith frog Ringer’s solution.Be sure to keep the nervewet at all times.

8. Whenthe nerve is free,poke a hole throughthe frog’s backby spreadingthe musculatureapartandpull the threadgently throughfrom the ventral(abdominal)to the dorsal(back)side.Do not tugon thenerve.

9. Dissectingthe nerve at the hip is tricky. Switch to the dorsalside(flip the frog over) andexposethenerve in thethigh whereit lies deepbetweenthelargethigh muscles.To do this,pull apartthetwo largegroupsof thigh musclesandpin themdown sothattheir innersidesareexposed.Cut throughthe overlying membranesanduncover the nerve andthe nearbybloodvessel.Dissectthenerve freenearthehip joint. Oncethenerve is visualizedon bothsidesof thehip joint dissectthroughthe hip joint. Take your time hereandcut paralleltothe nerve anddetachthe tissuefrom the nerve. Alwaysmake certainthat the nerve is notbetweenthebladesof thescissorswhenyou cut. You will noticethat thesciaticnerve hasmany branchesthroughoutits course.Thesemaybecutatany time,althoughfor bestresults

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cut themwhentheentirenerve is freeof surroundingtissues.Cuttinga branchof thenervemaycausemusclecontractions.

10. Midway down the thigh the sciaticnerve branchesin two. You want the branchthat goesabove the kneeanddown to the foot (the peronealnerve). Separateandpin the musclesof thecalf apart. Follow thenerve from the thigh to theknee. Thedissectionof thenervethroughthekneeis similar to its dissectionthroughthehip. Beforefreeingthenerve at theknee,dissectthenerve in thecalf. Whenyou aresureyou canvisualizethenerve, cut thetendonwhich coversthenerve at theknee. Hemostatandcut any small bloodvesselsthatcrossover thenerve.

11. Freethenervedown thelengthof thecalf to theankle.Tie off thenerveat theankle,leavingabout2 inchesof threadfor mounting. Cut the nerve just beyond whereit is tied, andcutawayany remainingbranches.

12. Measurethelengthof thenervewithoutstretchingit.

2.4 Mounting the Nerve in the Chamber

Make surethe stimulusis off by disconnectingthe stimulusandrecordingelectrodes.Keepingthe nerve moist, lay it over the silver electrodesin the nerve chamberwith the large endof thenerve over the closely spacedstimulatingelectrodes(Figure 5). Insert the threadsthroughthe

R1

R2

R3

R4

R5

R6

R7

R8

R9

R10

R11

R12

S1

S2

S3 Stimulating electrodes

Plexiglass cover

Recording electrodes

Figure5: Thenerve chamber. Thereare12recordingelectrodesspaced1 cm apart.Thereare3 stimulatingelectrodesspaced0.5cm apart.

holesin theendsof thechamberandextend(do not stretch)thenerve to its original length.Thensecureit in placewith tapeattachedto thethreadandchamber. A smalldropof Ringer’s solutionshouldbelaid at eachelectrode-nerve crossingto ensureelectricalconnection.Cover thebottomof thechamberwith Ringer’s solution.Be carefulnot to short-circuittheelectrodes;do not allowthe Ringer’s solution to reachthe level of the electrodesin the chamber. Squirt a few dropsofRinger’s solutionon therim of thechamberandcoverwith a Plexiglassslabto provideanairtightseal.This keepstheatmospherein thechambersaturatedwith waterandpreventsthenerve from

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drying out. The nerve will be surroundedby moist air which actsasan electric insulator. Thethreeclosely-spacedelectrodesarenormally usedfor electricallystimulatingthe nerve, andtheremainingelectrodesarenormallyusedfor recording.

2.5 Stimulation and Recording

A block diagramof the experimentalapparatusis shown in Figure6. The signalgeneratorpro-ducestwo outputs:a triggerpulsethatusedto synchronizetheoscilloscopeandthecomputer, andstimuluspulsesthatpassthroughthestimulusisolatorto producepulsesof currentto stimulatethenerve. The voltageresponsefrom thesurfaceof thesciaticnerve is amplifiedby thedifferentialamplifierandfed to boththeoscilloscopeandthecomputer.

S3

timulus Isolator

S3

ignal G2

enerator

Differential A4

mplifierC5

omputer

Two channel O6

scilloscope

+

−

+

−

+$−

Nerve

Trigger

Figure6: Block diagramexperimentalarrangement.

2.5.1 Stimulus Generation

Thesignalgenerator(GrassmodelS88)producesa periodicstimulus. Eachperiodconsistsof apair of pulseswhoseparametersareindependentlycontrolled(Figure7). Theoutputof thesignalgeneratordrivesa stimulusisolationunit (Axon Instrumentsmodel Isolator-10) that convertsaninputvoltage,which is referencedto ground,to anoutputcurrentthatis isolatedfrom ground.Forthis laboratorythestimulusisolatoris presetto the“1 mA” settingwhichgivesa transconductanceof 100

A/V.

Theoutputof thestimulusisolatordrivesthestimulatingelectrodesof thenervechamber(Fig-ures5 and6). Thecenterelectrodeis thecathode.Thetwo outsideelectrodesshouldbeconnectedtogetherto serve asanodes.This stimulationconfigurationreducesthethresholdcurrentrequiredto stimulatethenerveandreducesthespreadof thestimuluscurrentalongthenerve.

A technicalproblemwith electricstimulationis thataportionof thestimuluscurrentproducesavoltageinput to therecordingamplifierwhich is calleda stimulus artifact whichcanobscurethenerve response.Thecircuit topologyshown in Figure6 is intendedto reducethestimulusartifact.However, the sizeof the stimulusartifact is alsoaffectedby the geometricalarrangementof thestimulusandrecordingelectrodes.The lengthsof unshieldedwires shouldgenerallybe kept assmallaspossible.

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S3

1 duration

S3

2 duration

S3

1 v7 olts S

32

v7 olts

S3

1 delay

S3

2 delay

Train period

Repeat

Trigger Trigger

Figure7: Thestimulusconsistsof a periodictrain of pulseswhoserepetitionrateis controlledby changingtheTrainRateknobof thesignalgenerator. Eachperiodconsistsof two pulses(S1andS2)whosedelays(S1delayandS2delay)from thetriggerpulse,amplitudes(S1volts andS2volts), anddurations(S1durationandS2duration)caneachbecontrolled.

2.5.2 Response Measurement

Responsesof thesciaticnervepreparationarefragile(voltagesaresmall,impedancesarehigh)andareeasilycorruptedbyelectricinterference(especiallyfrom thecomputer).Therefore,anamplifieris usedto amplify andbuffer the neuralresponses.The amplifier is a differentialamplifier withtwo high-impedanceinputs: a non-inverting input (redwire) andan inverting input (greenwire).Theamplifierhasanominalgainof 100(themeasuredgains,whichmayvaryby asmuchas12%from the nominalvalue,areindicatedon eachamplifier),a frequency responsethat is flat in thefrequency range

�8�9��:�;�<�kHz, andawidebandnoisefloor of 0.7mV rms.Theamplifiersaturates

at outputvoltagesof =?> V.The oscilloscopehastwo input channels.Onechannelis usedto monitor the stimulusand

the other is usedto monitor the response.The oscilloscopealso hasa trigger input, which isconnectedto thesynchronizationoutputof thesignalgenerator. Thistriggeringarrangementallowssynchronizationof thescopesweepwith thestimuluspulses.

As part of your setupprocedure(i.e. beforeyou attemptto stimulatethe nerve), checkouttheoperationof theGrassS88signalgenerator. Connectthe “Monitor Output” of theS88to theoscilloscope.Setup a stimulusprotocolconsistingof a train of two pulseswith a train periodof200ms. Setthedelayof thefirst pulseto beminimal andits durationto 100

s. Setthedelayof

the secondpulseto 2.5 ms andits durationto 200

s. Measurethe amplitudeandtiming of thestimuluswith the oscilloscope.Make surethat thescope’s horizontalsweepvernieris setin thecalibratedposition.

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2.6 Data Acquisition and Processing

The laboratorycomputer(an IBM compatible)is interfacedto an analog-to-digitalconversionsystemthat hastwo analoginputs: channelA andchannelB. The analog-to-digitalconversionsystemis programmedto monitor channelB for the arrival of a synchronizationpulsefrom thestimulusgenerator. Whena synchronizationpulseis detected,periodicsamplingof channelA,which is connectedto theoutputof thepreamplifier, beginsimmediately. Thesamplinginterval is20

sandthesampleshave16bit resolutionwithin therange

���<�to

�<�volts. Samplingcontinues

for 15 msto giveasequenceof 750samples.

2.6.1 Activating the Software

Themainprogramfor acquiringandprocessingdatais calledfrog. This programstoresresultsandtemporaryfiles in the currentworking directory. To avoid confusingyour datawith that ofanothergroup,eachlaboratorygroupshouldstoreresultsin a separatedirectory. Pleaseusethefollowing namingconvention.If youarein laboratorygroupB2, thentypetheDOScommands

C:> mkdir \frog\b2C:> cd \frog\b2

to make a new directorynamedc:\frog\b2 and to make that directory the currentworkingdirectory. Thentype

C:\FROG\B2> frog

to activatethemainprogram.Thefrog programprovidesfacilities to acquiredata,to save andplot results,andto obtain

hardcopy. All of thesefacilitiesaredirectlyaccessiblefrom asinglemaincontrolscreenillustratedin Figure8. Thecontrol screenincludesa graphicarea(top half) whereresultscanbeplotted,atext area(bottomhalf) wherenotesaboutresultscanbe recorded,anda collectionof buttonstoactivatespecificfacilitiesdescribedin thefollowing sections.

2.6.2 Acquisition of Data

Threebuttonsprovide dataacquisitionfacilities. PressingGet Wave causesthe computerto(1) pauseuntil it receives a synchronizationpulsefrom the stimulusgenerator, (2) convert thenext 15msof outputfrom thepreamplifierto adigital representation,and(3) displaytheresultingwaveformin thegraphicarea.PressingContinuous causesthecomputerto performGet Waveoperationsrepeatedly. This modeof operationsimulatestheoperationof anoscilloscopeandtheresultingdisplayshouldlook quitesimilar to theoscilloscopedisplay. WhenContinuous hasbeenpressed,otherdataacquisitionbuttonsareerased,andonly onefunctioncanbeactivated–Stop Continuous. PressingtheAvg 10 buttoncausesthecomputerto collect10consecutiveresponsesandto displaytheaverage.Newly acquireddataareplottedasawhitetracein thegraphicportionof thescreen.

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0 1 2 3 4 5Time (msec)

–5

5

Ele

ctro

de V

olta

ge (

mv)

Stored Waveforms

Next Page

A0: 31-Aug-94 6:58 -- amplitude = 27 uA; duration = 0.1 msA1: 31-Aug-94 6:59 -- amplitude = 28 uA; duration = 0.1 msA2: 31-Aug-94 7:03 -- amplitude = 29 uA; duration = 0.1 msA3:A4:A5:A6: 31-Aug-94 7:12 -- positive electrode = #3, negative electrode = #4 --- before crushing nerveA7: 31-Aug-94 7:10 -- positive electrode = #3, negative electrode = #4 --- after crushing nerveA8:A9:

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__

A7 –3.1

__

A6 –4.3

__

__

__

t = 1.520

Notes

Continuous

+ β

Get Wave Avg 10 G = 100 Print Plot

Print Notes

– ––

+++

– ––

+++

– + – +

Figure8: Appearanceof monitorscreenaftertheacquisitionof datavia thefrog program.Thewaveformsshown arefrom a bullfrog sciaticnerve in responseto stimuluspulsespresentedwith a repetitionrateof10/s,anamplitudeof 28 � A, anda durationof 100 � s. Onewaveform(dashedline) wasrecordedbefore— andthe other (solid line) wasrecordedafter — the nerve wascrushedat a locationbetweenthe tworecordingelectrodes.

2.6.3 Saving Waveforms

Waveformscanbestoredon thecomputer’s disk for lateranalysis.Storageidentifiersconsistofa singleletter(A-Z) followedby a singledigit (0-9). Whendatahave beenacquiredandhave notyet beenstored,theSave Wave buttonappearson thescreen.Pressingthis buttonpromptsfor asave identifierasfollows

Save as: __ Type save ID or <esc> to abort.

Type a letter anda numberor pressthe<esc> key to exit the prompt. Notice that this promptandall othersin thefrog programarecaptive in thesensethatthey precludeall otheroperationswhile they areonthescreen.Youcandismissthispromptwith avalid responseor<esc> to abort.

2.6.4 Storing Notes with Waveforms

One line of notescan be associatedwith waveformsto facilitate on-line waveform identifica-tion and manipulation. Notesare displayedin the lower part of the screen,10 at a time. The

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Next Page andPrev Page buttonsselectthesetof tenwaveformswhosenotesaredisplayed.Whena waveformis saved, thepageof notesassociatedwith thatwaveformis automatically

selectedandthedateandtime-of-dayareautomaticallyenteredonthenoteline for thatwaveform.You mayaddtext to thenoteor modify existing text by pressingthebuttonto theleft of thenote.Thisbuttonactivatesaneditingmodeprompt.Presstext keysto addtext,<Backspace> to deletetext, or strike theReturn key to exit theprompt.

Notescanbemodifiedatany time. Simplyselecttheappropriatepage(usingtheNext PageandPrev Page buttons)andclick on thebuttonto theleft of theappropriatenote.

Relation between notes and protocol records. Notesarenot substitutesfor appropriateentriesin your protocolbook! Notesonly provide informationaboutonemeasurement.It is generallyimpossibleto understandthecontext of onemeasurementin isolation. Thecontext, working hy-potheses,testsetup,andtentativeconclusionsshouldall berecordedin yourprotocolrecordsalongwith a recordof waveformIDs.

2.6.5 Plotting Waveforms

Any newly acquiredwaveformthathasnotyetbeensavedto thediskplusasmany assevenstoredwaveformscanbesimultaneouslyplottedin thegraphicarea.Buttonsprovide facilitiesto choosewaveforms,adjustscales,andobtainnumericalplot valuesusinga mouse-controlledcursor, asdescribedin thefollowing sections.

Scales. Voltagesdisplayedon thescreenarescaledby a constantG. To make thedisplayedvolt-agescorrespondto thoseat the amplifier’s input, usethe G= button to setG to the gain of theamplifier, which is nominally100(moreexactvaluesareindicatedoneachamplifier).

The maximumandminimum valuesof the scaleson both the abscissaandordinatecanbechangedusingthe nearby+, ++, -, and-- buttons. Pressing+ or ++ increasesthe numberbya smallor largeamountrespectively. Pressing- or -- decreasesthenumberby a smallor largeamountrespectively.

Selecting waveforms. Thecolor-keyedbuttonsto theright of theplot canbeusedto selectstoredwaveformsfor display. Pressingoneof thosebuttonsinitiatesapromptingsequence.Entera letterandadigit to selecta storedwaveformor strike thespacebarto erasetheentryor strike<esc> toabortthepromptingsequence.

Using the cursor. To obtainnumericalvaluesfrom the plottedwaveforms,move the cursortothegraphicalareaof thedisplayandpressthemousebutton. A vertical line will appear, andthepositionof that line canbechangedby moving themouseleft or right. The time selectedby thevertical line is indicatedjust underthe lastof thecolor-keyedbuttonsto theright of theplot (seeFigure8). Valuesof theplottedwaveformsarealsoindicatedasnumberstypedto theright of thecorrespondingcolor-keyedbutton.

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2.6.6 Hardcopy

Hardcopiesof thegraphicalportionof thedisplaycanbeobtainedby pressingthePrint Plotbutton. This button producesan encapsulatedPostScriptfile in the currentdirectory and theninitiatesa sequenceof messageson thecampuscomputernetwork thatwill ultimatelyproduceasheetof paperon thelaserprinterlocatedin thelaboratory.

Hardcopiesof thenotescanbeobtainedby pressingthePrint Notes button. This buttonproducesa text file namedNOTES.TXT in thecurrentdirectoryandsendsthatfile off to thelaserprinter.

TheencapsulatedPostscriptfiles andNOTES.TXT arenot deletedwhentheprinting is com-plete. Thesefiles provide opportunitiesto print additionalcopiesof theplotsor to electronicallypastethe plots into your laboratoryreport. The file for the first plot that you make is namedPLOT001.EPS, thesecondis namedPLOT002.EPS, andso forth. Typesettingandelectroniccut-and-pasteareNOT required:thesefacilitiesaremadeavailableonly for thosewhowish to usethem.

2.6.7 Simple Waveform Arithmetic

Waveformscanbescaled(i.e. multiplied by a constant)andaddedto otherwaveformsusingthe A@ button. Pressingthis button addsthe waveform that is plottedwith a red tracewith a

scaledreplicaof thewaveformplottedwith abluetrace.Thescalefactor @ is solicitedby apromptthat is invoked eachtime the A@ button is pressed.The resultingwaveform is displayedusingawhite traceandcanbesavedto thediskby pressingtheSave Wave button.

2.6.8 File Formats

This sectionprovides information abouthow the frog programsaveswaveformson the disk.This informationis notnecessaryto usethefrog programor to obtainhardcopies.However, thisinformationcould be usedto convey dataobtainedfrom the frog programto otherwaveformprocessingor plotting programs.The useof otherprogramsis not required: the information isprovidedonly for thebenefitof studentswhowish to makeuseof otherprograms.

Whenwaveformsaresavedto thedisk, two filesareproduced— abinaryfile andanasciifile.The file namesarederived from the waveform identifier. The binary file associatedwith A0 iscalledA0.BIN andtheasciifile is calledA0.ASC. Thebinaryfile containstwo fields.Thefirst is100bytesin lengthandcontainsthenotesassociatedwith thewaveform.Thefirst byteof thisfieldidentifiesthelengthof thenotestext andsubsequentbytescontaintheasciicodesfor thetext. Thesecondfield in thebinaryfile contains750sixteen-bitintegersrepresentingthewaveform.A valueof

�B� �> CD> represents�?�<�

V anda valueof � �> CD> represents �<� V. Theasciifile contains751linesof text. Thefirst line containsnotesassociatedwith thewaveform.Subsequentlinescontaindecimalnumbersrepresentingvoltagesin mV.

The numbersin both the .BIN and.ASC files representvoltagesmeasuredat the input tothe analog-to-digitalconverterof the computer. Neitherof theserepresentationsaccountfor thepreamplifiergainfactorG.

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2.6.9 Exiting the Program

Thefrog programcanbeterminatedatany timeby pressingtheExit key. Datasessionscanberesumedsimply by restartingtheprogram.No dataarelost by exiting andrestarting.

2.6.10 Network Services

The laboratorycomputersare connectedto the campuscomputernetwork. Useof the campuscomputernetwork is NOT required. This information is includedonly to help thoseinterestedin transferringtheir resultsto anothercomputer(for furthersignalprocessingor for figureprepa-ration). The laboratorycomputerssupportremotecomputerlogin usingtelnet. To activatetelnet type

C:\FROG\B2> telnet comp-name

wherecomp-name representsthenameof thecomputer(e.g.athena.dialup). Thelaborato-ry computerssupportfile transfersto othercomputersusingftp. To activateftp type

C:\FROG\B2> ftp comp-name

wherecomp-name representsthenameof thecomputer(e.g.athena.dialup).

2.6.11 Floppy Disks

The laboratorycomputerssupport1.4 Mbyte floppy disks,which canbe usedto save your filesfor future use(e.g., to make figuresfor your laboratoryreport)or to transferyour files to othermachines.Useof thefloppy disksis not required.

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3 Experiments

3.1 Basic Observations

Thepurposeof theseobservationsis give you someexperiencewith basicfeaturesof compoundactionpotentials,beforeyou begin your project. Try to completethesethreeobservationsin lessthan1/2 hour.

1. Determinethresholdcurrent(about10minutes).

Threshold is definedas “a point at which a physiologicaleffect begins to be produced.”Determinethe threshold current for the compoundactionpotentialfor pulseswith 100

s

duration,repeatingat a rateof 10/s.Usethetwo mostdistalrecordingelectrodes.

Your methodshouldbe well defined,thoughit may have somearbitraryconstraints.Startwith a weakstimulusandgraduallyincreasethe level. You shouldobserve a CAP about2 msafterthestimuluswith a currentof a few tensof

A. Noticethat theverticalaxisgain

of your oscilloscopecaninfluenceyour ability to detecta response.Is thecompoundactionpotentialan“all-or-none”response?

Cautions. Avoid largecurrents,which candamagethenerve. Make surethat thestimulusisolator is set for “1 mA” and rememberthat this meansthat 100

A of currentwill be

deliveredto thenerve for everyvolt from theGrassS88.

2. Identify responsecomponents(10 minutes).

Observe that the recordedwaveform consistsof an early componentthat occurswithin afractionof a millisecondafterthestimulusanda later, diphasiccomponentthatoccursaftera delayof a few milliseconds.Which of theseis thecompoundactionpotential?Explain.Whatis theothercomponent?

3. Createamonophasicresponse(10 minutes).

Recordacompoundactionpotentialfrom themostdistalpairof recordingelectrodes.Next,crusha smallportionof thenerve,mid-waybetweentherecordingelectrodesusingforcepsor ahemostat.Recordthenew responseandcompareit to thepre-crushwaveform.Describetheeffectof crushingon thecompoundactionpotential.

Notice. This responserecordcanserve asa standardagainstwhich you cantestresponseslaterin thesessionto assessthestabilityof yourpreparation.

3.2 Your Project

Your project is the main educationalexperienceof this laboratory. You andyour partnershouldplan your project well in advanceof going to the laboratory. You neednot study materialoncompoundactionpotentials(or cellularmechanisms)beyondreadingpages1-20 in Volume2 ofthe coursetext. The goal is to designandconductan experimentaltestof an hypothesisrelatedto compound-action-potential(CAP) behavior. If your knowledgeof theoriesfor neuroelectricphenomenais weak,that’sfine; you only needto think throughyour testof your hypothesis.

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The following exampleprojectsareconcernedwith stimulus-responserelations,spatialvari-ationsin the response,or effectsof external agents. You may chooseoneof theseor you canconstructan entirely new one. The descriptionsgiven hereare incomplete. Your plan shouldbe well-defined.Keepit simpleso that you cancompletethe work, includingsomepreliminaryplotting of resultsandthinking aboutinterpretationwithin a one-hourlab session(dissectingthenerveanddoingthebasicobservationswill takeat least2 hours).Yourexperimentalmanipulationsshouldavoid actionsthatmayalterthenerve’s responsivenessovera long term.

3.2.1 Projects Exploring Stimulus-Response Relations

CAP responsesaregenerallynot linearly relatedto thestimulus.A projectmayfocusona featureof theCAPandits dependenceon thestimulus.Fourexamplesfollow.

1 Iso-response contours

Motivation: Non-linearsystemsarehardto characterize.Onepossibility is to look for a setof inputsthatare“equivalent” in thatthey produceidenticalresponses.

Hypothesis:A setof rectangular-pulsestimuli (height E andduration F ) canbe found forwhich theCAP responseis identical.

Methods: For eachof 10 pulsedurations,determinethe pulseheight requiredto producea particularresponse.The function EG�HFI� thatdescribesthestimulusparametersyielding afixedresponseis a “iso-responsecontour”calleda “strength-duration”curve.

1 Characterizing post-response excitability

Motivation:After anervehasrespondedto astimulus,its responsepropertiesaretemporarilychanged.Themostprominentchangeis thatfor afew millisecondsafteraresponsethenerveis lessresponsive,or “refractory.”

Hypothesis:The refractorinessof a frog sciaticnerve canbe describedby an iso-responsecurve (suchasFigure1.13on page13 of Volume2 of thecoursetext) for thesecondof twostimuli.

Method: Stimulatewith a pair of shortpulses. Vary the interval J betweenthe first andsecond. Determinehow the heightof the secondpulse EK, must be chosenso that EK,L�MJN�describesaniso-responsecontourfor theresponseto thesecondpulse.

1 Does refractoriness disappear for small CAP’s?

Motivation:A simplestochasticdescriptionfor apopulationof nervefibersmightbethatallareidenticalbut eachhasa thresholdvaluethatis describedby aprobabilitydistribution. Inresponseto a shortstimuluspulseall neuronswill respondwhosethresholdsareexceededby thestimulus.With thismodel,if astimulusis sosmallthatonly asmallfraction(say1%)of thefibersrespond,then99%will notberefractoryfor asecondstimulusdeliveredshortlyafterward.

Hypothesis:If stimuluslevel is low enough,theresponseto asecondpulsewill beessentiallyidenticalto thatof anidenticalfirst pulseindependent of theinterpulsepulsedelay.

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Method:Straightforward.

1 Response components.

Motivation: Peripheralnervesaremadeup of nerve fibersof differentdiameters(Figures1and2). Plotsof numbersof fibersin particulardiameterrangeshave peaksindicatingdis-tinct sizeclasses.Thesecategoriesmight have differentresponseproperties,e.g. differentthresholds,propagationvelocities,refractoryperiods.

Hypothesis:Smallernerve fibershave higher thresholdsand lower propagationvelocitiesthanlargerfibers.

Method: Investigatethe fine structureof the monophasiccompoundaction potential. Isit unimodal,or are theremultiple peaks?Two peaksmay representpopulationsof fiberswith differentdiameters.Determinewhethereachof thepeakshasthesameor a differentthreshold.

1 Where is the CAP initiated?

Motivation:Theelectricstimulusis deliveredto aregionof thenervedefinedby thestimuluselectrodelocations. Doesa propagatedactionpotentialbegin at oneor the otherstimuluselectrode?

Hypothesis:Theanodeis thesitewheretheCAP is initiated.

Methods:Measurethetime of occurenceof somefeatureof theresponsefor differentcon-figurationsof the stimuluselectrodes.For example, interchangethe anodeand cathode.Alternatively, connecttheoutputof thestimulusisolatorto electrodesthatareusuallyusedfor recording(i.e.,R1-R12).

3.2.2 Projects Exploring Spatial Variations in Response1 CAP amplitude and nerve diameter

Motivation: If eachnervefibergeneratesasimilar response,thena largenerveconsistingofmany nerve fibersshouldgeneratea largerCAP thana smallernerve which containsfewerfibers.

Hypothesis:CAPamplitudewill decreasemonotonicallywith decreasingnervediameter.

Methods:Try to demonstratethat thehypothesizedrelationholdsfor severalstimuluscon-figurations,e.g.stimulatingat boththesmallandlargeendsof thenerve.

1 CAPs represent propagating waves.

Motivation: Neuralsignalsgeneratedat oneendof the nerve producea CAP at the otherend. A commonview is that a stereotypedpulsatilewaveformpropagatesalongthe nervefibersfrom oneendto theother.

Hypothesis:All featuresof theCAP’sspatialdependencealongthenervecanbeinterpretedasasumof waveformsgeneratedby apoolof similarneuronsin whichpropagationvelocityincreaseswith fiberdiameter.

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Methods:Observe waveformof CAP at differentrecordinglocationsandcomparefeaturesof the response(e.g. time of peak,heightof peak,width of peak)to predictionsbasedonyourhypothesis.

1 Superposition of responses from branches.

Motivation:Carefuldissectionof thenervebelow thefrog’s kneecanyield two branchesofroughly equalsize. Thesebranchescanthenspanover commonrecordingelectrodesandtherebyextendthelengthof many fibersin therecordingchamber.

Hypothesis:Theresponserecordedfrom theregion wheretwo branchesarein contactwiththerecordingelectrodesis thesumof theresponsesrecordedwith eachbranchalone.

Methods: Recordwith branch“a” alone,branch“b” alone,andboth “a” and “b” on theelectrodes.Try to maintainthequality of contactof electrodesto nerve branches.Controls(repeatsaftermanipulationof branches)will beimportant.

3.2.3 Projects Exploring External Agents

Numerouspossibilitiesexist for applyingdisturbancesthat may affect the neuralresponse.Cat-egoriesof agentsincludechemical,electric,mechanical,andthermal. Chemicalpossibilitiesin-cludeions thatareimportantin actionpotentialgeneration(Na/ andK / ), andagentsthataffectneuralresponsiveness(e.g. novacaine,caffeine,alcohol).Electricdisturbancesincludeactive ap-plicationof (say)directcurrentto a sectionof thenerve, or passive alterationof theconductivityof thenerve’senvironment.In everycasethemethodsaresimilar. We presentoneexample.

1 Conductivity around the nerve and propagation velocity.

Motivation: If actionpotentialspropagatethroughtheflow of currentsfrom oneregion of anerve fiber to anadjacentregion, thendecreasedresistanceto currentflow shouldspeedupthepropagation.

Hypothesis:Increasingtheconductancebetweentwo locationson thenerve on theoutsideof thenervewill increasethepropagationvelocity in thatregion.

Methods:Stimulateat oneend,recordat theother. Placeanexternalresistorbetweentwoelectrodesnearthecenter. Describethechangein responseandinferredpropagationvelocity.

3.3 Practical Considerations in Choosing a Topic

Projectscan involve any of the topicsdiscussedin Section3.2 or a topic of your own creation.If you createa novel project,you mustobtainany suppliesor equipmentthat is not part of thestandardlaboratorysetupdescribedin Methods.For example,studyingtheeffectsof caffeineonthecompoundactionpotentialis a goodproject. However, we do not stockcaffeine: you wouldhave to obtainthecaffeine. Also, studyingthe effectsof the shapeof theelectricstimulus(e.g.,rectangularversustriangularwaveforms)couldbe interesting.However, our electricalstimulator(GrassS88) generatesonly rectangularpulses. You would have to obtain a suitablewaveformgenerator.

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Whenyou chooseyour topic, rememberthatyour experimentshouldtake approximatelyonehourto complete.Think throughhow many measurementsyou will needto makeandhow long itwill take to make eachmeasurement.For example,it couldtake a long time — possiblyhours—to reversetheeffectsof a drug. Reversaltime oftendependson thesizeof thedose.Therefore,itis generallyprudentto investigateeffectsof smalldosesfirst.

Formulateaspecificandtestablehypothesis,andcenteryourprojecton thathypothesis.Avoidvaguehypotheses,suchas: “I would like to understandhow temperatureaffects the compoundactionpotential.” Insteadchoosea morenarrrowly focusedhypothesis,suchas: “Decreasingthetemperatureof a nerve will decreasethe conductionvelocity of the compoundactionpotential.”This more-focusedhypothesisis testable:it canbe trueor false. If you form a clearhypothesis,you will beableto plana logical setof measurementsto testthehypothesis,andyou will beableto cometo aclearconclusionwhenyou write your report.

Whenyou do your experiment,you maygetunexpectedresults.For example,you mayhaveplannedto measuretemperature-inducedchangesin velocity of the compoundactionpotential,but you mayalsofind largechangesin amplitude.Theremayevenbea temperaturebelow whichthereis no compoundactionpotentialat all. You shouldexplore unexpectedresultsand try tounderstandtheir bases.Your aim shouldbe not simply to rejector acceptyour hypothesis,butto develop insight into the phenomena.For example,perhapscompoundaction potentialsfailto occurat low temperaturebecausethe thresholdcurrentis larger at low temperatures.If so, itmight bebetterto determinethethresholdcurrentfor eachnew temperatureandthenmeasuretheconductionvelocitywhenthestimuluscurrentis equalto (for example)twicethethresholdcurrent.

Keepin mind that this is anexperimental project. Your goal is to characterizewhat happens,not why it is happening.Theoreticalideasaboutneuralmechanismswill be the basisfor yoursecondproject(theHodgkin-Huxley project).

3.4 Control Observations

Control observationsare importantpartsof the designof any experiment,particularlywhenin-vestigatingliving systems,which arelabile. Thepurposeof a controlobservationis to determinewhetherthevariablethat is directly manipulatedby theexperimenteris theonethat controls thechangein response.

Suppose,for example,thatyou wish to determinetheeffect of crushingthenerve on thecom-poundactionpotential.You could (1) measuretheresponsebetweentwo electrodes,sayR7 andR8, (2) crushthenerve at a point betweenthosetwo electrodes,and(3) repeatthemeasurement.Now supposethattheresponsesaredifferent.How doyouknow thatthedifferencewascausedbythecrushing?Perhapsin theprocessof crushing,you alsomovedthenerve to a differentpartoftheelectrodethatis corrodedsothatit makesapoorelectricconnectionto thenerve! Perhapsyoustretchedthenerve sothat it wasdamagedat a sitethat is remotefrom thecrushingsite! Perhapsyou tilted theexperimentalchamberjust enoughsothatsalinein thebottomof thechambernowshortsoutoneof theelectrodes!

Control observationsare intendedto assessthe extent to which factorsthat are not part oftheexperimentaldesignarecontributing to responsepatterns.A goodcontrolobservationfor thenerve crushingexamplewould be to measurenot only the responsebetweenR7 andR8 but also

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the responsebetweenR6 andR7. The intentionalmanipulationis the crushingbetweenR7 andR8, which shouldhave no effect on the responsebetweenR6 andR7. Therefore,if crushingthenerve alsochangesthe responsemeasuredbetweenR6 andR7, thenthe manipulationdid morethanjust affect thenerveat thesiteof thecrush.

Onevariablethat is not underthe control of the experimenteris the passageof time. Eachobservation that you make will happenat a differenttime, andit is importantto know theextentto which differencesin responsessimply reflect the passageof time. To assessthis effect, onecandesigna control in which a particularmeasurementis periodically repeatedthroughouttheexperiment.For example,onecouldmeasurethediphasicresponsebetweenR4andR5to a10

A

pulseof currentwith a durationof 100

s at the beginning of the experimentandat 10 minuteintervalsthroughouttheexperiment.If theresponsesdiffer, thenthepassageof timeis animportantfactorthatmustbeconsideredwhenthedataareinterpreted.

4 Proposals, Reports, and Logistics

4.1 Scheduling a Laboratory Session

Studentsshouldscheduleatimeslot for their laboratorysessionby submittinganelectronicsched-ule form, availableon our homepage(http://umech.mit.edu/6.021J/index.html).At that time, you mayalsorequesta partner. Thelaboratoryaccomodates8 studentsin eachses-sion. We will work out the laboratoryscheduleon the afternoonof September17. You havethe bestchanceto get your mostdesiredtime slot by submittingyour scheduleform BEFORESEPTEMBER 17 AT NOON. After initial laboratoryassignmentshave beenmade,you will bepermittedto changeyour laboratorysessiononly if you requestthe changemorethan24 hoursbeforeyourassignedslotandonly if thereis anopenslot in thescheduleatyourdesiredtime. Thelaboratoryschedulewill bepostedon theWWW.

4.2 Proposal

After your laboratorysessionhasbeenscheduled,you shouldmeetwith your partnerto planyourprojectandto write a proposal.The proposalshouldcontaina brief statementof thehypothesisyou proposeto testandthe methodthat you will useto test it. Includea list of the experimentsyou will performandthe measurementsyou plan to make in eachexperiment. Indicatehow themeasurementswill be usedto helpyou testyour hypothesis.Theproposalshouldfit on a singlesheetof paper. A sampleproposalis shown in Figure9.

The proposalshouldbe submittedbefore5 pm on Monday, September20, 1999. Electronicsubmissionvia email2 to [email protected] is encouraged.Thestaff will review theproposalsandmake suggestions.Acceptableproposalswill beAPPROVED; flawedproposalswill beRE-JECTED. Proposalsthatarenotapprovedmayberevisedandresubmitted.All proposalsmustbeapprovedbefore5 pmonFriday, September24,1999.

2Pleasesubmitsimpleasciitext; pleasedonot submitvendor-specificformats.

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Proposal: Effects of Temperature on Compound ActionPotentials of the Frog Sciatic Nerve

Partner Names (E-mail): PartnerOne([email protected])andPartnerTwo ([email protected])

Hypothesis: Decreasingtemperaturewill decreasethevelocity of thecompoundactionpotentialof thefrog sciaticnerve.

Background: The speedof many chemicalreactionstendsto increaseas temperatureincreas-es. Therefore,it seemsplausiblethat the speedof neuralconductionmight similarly dependontemperature.

Procedure: Wewill obtaina100mL sampleof Ringer’ssolutiononthedaybeforeour laboratorysession,andplacethesolutionin a refrigeratorto chill it to approximately4� C. After performingthe basicobservationsdescribedin the laboratorymanual,we will rinse the nerve with chilledRinger’ssolutionandplace10mL of chilledRinger’ssolutionin thebottomof thenervechamber.We will placea small thermocouplein contactwith the distal end of the nerve to monitor it’ stemperature.Wewill thenrecorddiphasiccompoundactionpotentialsat threepairsof electrodes:R1 andR2; R3 andR4; andR5 andR6. Thesemeasurementswill berepeatedoncea minutefor10minutes.Wewill recordthetemperatureat thetime thateachmeasurementis made.Thespeedof thecompoundactionpotentialwill bemeasuredby dividing thedistancebetweenthefirst andlastpairsof electrodes(4 cm) by thedifferencebetweenthetimesof thefirst negativepeakof thecompoundactionpotentialmeasuredon thefirst andlastelectrodepairs.Measurementsusingthecenterpair of electrodeswill be usedto checkconsistency: if the speedmeasuredfrom the firstpair to middlepair differssignificantlyfrom thatmeasuredfrom thefirst pair to lastpair, we willinvestigatethesourceof thedifference.

We obtaineda thermocoupleby specialarrangementsthatwe madewith theTA. We have alsotestedthebasicmethodby measuringthetemperatureof a pieceof threadthatwasmountedin anexperimentalchamberasthoughit werea nerve. We found that the temperatureincreasedfromabout15� C to about25� C over a time courseof about10 minutes.We thereforeexpectthatwehave time to repeatthis experimentthreetimesto checktherepeatabilityof our results.

Figure9: Sampleproposal.

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4.3 Project Report

Theprojectreportshouldbeconcise.Do notrepeatmaterialthatis easilyreferenced.For example,thereis noneedto reproduceany figurefrom thislaboratorymanualor from thecoursetext: simplyrefer to it. Technicalwriting is necessarilydirectedat someparticularintendedaudience.Writetheprojectreportasthoughit wereto bepublishedin a journal that is readprimarily by studentswho have taken this subject.Thus,you canassumesomeworking knowledgeaboutthesubject.Theprojectreportshouldcontainthefollowing sections.

Cover Page. Onthecoverpageincludethetitle of thelaboratorysession,theauthors’names,yourlaboratorysubsection,thedatesof thelaboratorysession(s),andthenameof yourpartner(ifnotaco-author).

Abstract. The abstractis a oneparagraph( O 100 words)summaryof the report including thequestioninvestigated,the methodsused,and the principal resultsand conclusions.Yourintendedaudienceshouldbe ableto understandthe abstractwithout having to readany ofthereport.Thissectionshouldbewritten last.

Introduction. The introductionis a brief section(about1 page)designedto motivatethereader.Includebackgroundinformationontheproblem,hypothesesto betested,significanceof thework, etc. You maygive citationsto materialin this laboratorymanualor elsewhere. Theintroductionshouldbedirectlyrelevantto yourreport;broaddiscussionsof neurophysiologyor brainfunctionarenotneeded.

Methods. Briefly describeany specialmethodsthat you usedthat arenot in this laboratoryde-scription.Givedetailsof calculationsusedto obtaintheresults.Do not repeatmaterialthatis in this description;just referto it.

Results. Describethemeasurements(whetheror not they fit with expectations)in theresultssec-tion. Generally, resultscanbecommunicatedmoreefficiently andaccuratelywith picturesandgraphsthanwith wordsalone.Describethoseaspectsthatareimportantto your inter-pretation,but omit interpretationsfrom thissection.A collectionof printedgraphswithoutawrittendescriptionof their relevanceis unacceptableasaresultssection.Studentsfrequentlyerron thesideof includinga largenumberof graphsandlittle descriptionof their relevance.You needonly includeresultsthatarerelevantto your conclusions.Most of ushave troubleleaving thingsout. Ask yourselfwhy a particularresult is a necessarypart of your story;omit thosethataren’t necessary.

Discussion. In the discussionsection,assessthe resultsfor dependabilityandaccuracy, andin-terpretresultsin the light of otherknowledge. Thediscussionsectioncanincluderelevantspeculationsandideasfor improving theexperimentto testthehypothesesmorerigorously.

Appendix. The appendixshouldincludea copy of theprotocoltaken during the laboratoryses-sion.

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4.4 Grade

Thegradefor theexperimentalprojectwill bebasedontheproposalandontheprojectreportusingthefollowing considerations:

Proposal (10%). Theproposalshouldreflecta carefullyconstructedplan. Thinking aboutwhatyou planto do beforeenteringthe laboratoryis crucial to collectingmeaningfuldatawhenyouarethere.Lateproposalswill receivenocredit.

First draft & Critique (10%). Yourfirst draftandyourcritiqueof acolleague’sfirst draftwill begradedprimarily for completeness.

Protocol (10%). Theprotocolshouldprovide enoughinformationsothata readercanfollow thecourseof theexperiment.Indeciphereablescribbleswill receivenocredit.

Report structure (10%). Thesectionsof thereportshouldbecoherent.

Clarity/Conciseness (20%). A goodreport is easyto read. The contentof eachparagraphandeachgraphshouldbeclear. Everythingincludedin thereportshouldbe therefor a reason.Pointswill bedeductedfor extraneousmaterial.Reportsshouldbelessthan10 pageslong,unlesstherearegoodreasonsfor additionalpages.

Conceptual correctness (20%). Are thereclearerrors?Are theresultsconfused?Are theresults(whichfollow directlyfrom themeasurements)confusedwith theinterpretations(whichrelyon informationotherthanwhatwasobserved)?

Insightfulness (20%). Insightfulnesscanbedemonstratedby (1)proposinganexperimentalmethodthatcanresolvesomescientificissue,(2) carryingoutexperimentsand/oranalysesthatleadto clearconclusions,(3) preparinga report that demonstratesa clearunderstandingof thestrengthsandweaknessesof your resultsandanalyses.Simply performingoneof the s-tandardexperimentsandshowing unmotivatedmeasurementswill receive 0 points. Cleverdesignof anexperimentor imaginativeanalysisof theresultswill receive20points.Demon-stratingaclearunderstandingof yourexperiment,youranalyses,andwhatcanbeconcludedis sufficient for 10points.

DUE DATES ARE FIRM, AND THERE IS A SEVERE LATENESS PENALTY. The gradefor a latereportwill bemultipliedby a latenessfactor

PRQ �����S 0UTWV - ��� > S 0UTWVYX ,where � is the numberof hourslate. The latenessfactor is plottedin Figure10. Notice that themaximumgradefor a reportthatis morethanONE DAY LATE is lessthan50%.

References

CarolinaBiologicalSupplyCompany (1965).Frogmusculature.

Ochs,S. (1965).Elements of Neurophysiology. JohnWiley & Sons,Inc.,New York, NY.

25

1 10 100

TZ

ime t past deadline (hours)

o[ ne day0�

.0

0�

.5

1.0

Late

ness

fact

or L

Figure10: Latenessfactor.

Patton,H. D. (1960). Specialpropertiesof nerve trunksandtracts. In Ruch,T. andFulton,J.F.,editors,Medical Physiology and Biophysics, pages66–95.W. B. SaundersCompany, Philadel-phia,PA.

Ruch,T. C., Patton,H. D., Woodbury, J. W., andTowe, A. L. (1965). Neurophysiology. W. B.SaundersCompany, Philadelphia,PA.

Tasaki,I. (1953).Nervous Transmission. C. C. Thomas,Springfield,IL.

Young,J.Z. (1951).Doubt and Certainty in Science. Oxford UniversityPress,London.

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