Instructor’s Reference Earthworm Action Potentials In this set of experiments students will obtain extracellular recordings of action potentials from an anesthetized earthworm ( Lumbricus spp ) !he" will examine the threshold potential# the all$or$none response# the refractor" period# and the conduction %elocit" of the ner%e Written by staff of ADInstruments.
Material Provided for the Earthworm Action Potentials LabTutor Laboratory Instructor’s Reference (this ( this document )
&ontains information for teachers about troubleshooting# anal"zing data# and altering the student protocols for this experiment !his document also includes the answers to the pre lab 'uiz# and suggested answers for the lab report page of Lab!utor experiment Laboratory andout
&ontains the rele%ant bacground material and a summar" of the experiments Ideall" this should be pro%ided to the students prior to the class session Pre!lab "ui#
!his ma" be used at the instructor’s discretion E$am%le &ata E%oe E%oed d actio action n poten potentia tials ls * Recruitm Recruitment ent of the the late lateral ral giant giant axons axons + Axon Axonal al condu conducti ction on %eloc %elocit" it" , Refr Refrac acto tor" r" per perio iod d - .idirecti .idirectional onalit" it" of ner%e ner%e impulse impulse
Page of /
0*112 A3Instruments
Instructor’s Reference Earthworm Action Potentials E'ui%ment List Alternatives • • • • • • • • • • • • •
PowerLab -! 45L667# or PowerLab ,8*9! 45L6-97 :hielded -$lead .io Amp &able 45LA*-,17 :hielded Lead ;ires $ Alligator &lips 45LA9-7 :timulator &able $ .<& to Alligator &lips 45LA*-17 :il%er wire plus bleach Earthworms (st least 91 mm long) Petri dish or similar -= Ethanol in 1*-=
ardware substitution !he following PowerLab units are compatible with Lab!utor and will function for these experiments> ,8*-!# ,8*1!# ,:! !he PowerLab ,8*-# ,8*1# ,:P and ,8+1 will also function for this experiment with external front$ends &onnection of single and multiple front$ends is described in the hardware documentation erve %re%aration !he ner%e must not be allowed to desiccate# but it should be gentl" blotted on a tissue before use ?eep the co%er on the
5o%ement artifacts are caused b" a worm that is not full" anesthetized 5ae sure students full" anesthetize their worms in ethanol and periodicall" appl" the ethanol8Ringer’s solution to their worms !oo much Ringer’s solution can result in a poor recording Ba%e students blot excess Ringer’s from the earthworms with a tissue Page * of /
0*112 A3Instruments
Instructor’s Reference Earthworm Action Potentials *iolo+ical issues Unable to separate the response between the median and lateral giant axons
:ome earthworms ha%e nearl" identical threshold %oltages for the median and lateral ner%es In this case# it will not be possible to mae two distinct recordings !his is normal %ariation# and not a malfunctioning of the e'uipment Recording from the dorsal side versus the ventral side of the earthworm
!his experiment wors best when the recording is done from the dorsal surface of the earthworm !he dorsal surface of the earthworm will feel smoothC the %entral side is characterized b" the presence of bristles# called seti that can be felt b" stroing the earthworm in a tail$to$head direction
Page + of /
0*112 A3Instruments
Instructor’s Reference Earthworm Action Potentials Pre!Laboratory "ui# answers Bere are the correct answers to the Duiz 'uestions in bold A an increase in membrane potassium permeabilit" . an increase in membrane sodium %ermeability & an increase in membrane chloride permeabilit" * A, an increase in membrane %otassium %ermeability . an increase in membrane sodium permeabilit" & an increase in membrane calcium permeabilit" + 3uring an action potential> A cell sodium concentration is changed appreciabl" . cell potassium concentration is changed appreciabl" -, there are no measurable chan+es in any cell ion concentrations , !he relati%e refractor" period> A occurs before the absolute refractor" period *, re%resents a time durin+ which a stimulus of +reater than normal intensity may elicit a new action %otential & is a time when the %oltage$sensiti%e sodium channels can not be reacti%ated under an" conditions - An earthworm giant axon> A contains man" indi%idual ner%e fibers . consists of a single ner%e fiber -, is com%osed of individual cells. one in each se+ment. lin/ed throu+h +a% 0unctions to its nei+hbors in ad0acent se+ments 9 !he earthworm %entral ner%e cord A contains onl" giant axons . contains onl" smaller ner%e fibers -, contains %redominantly smaller nerve fibers but with three +iant a$ons 2 !he fibers first excited as the stimulus %oltage is increased are the> A man" small fibers *, median +iant fiber & lateral giant fibers 6 !he ad%antage of a giant ner%e fiber is that it> A, conducts action %otentials faster than a smaller fiber . is less easil" excited & re'uires no sensor" input to trigger an action potential / !he two lateral giant ner%e fibers> A ha%e maredl" different excitation thresholds . conduct action potentials more rapidl" than the median giant fiber
Page , of /
0*112 A3Instruments
Instructor’s Reference Earthworm Action Potentials -,
behave as a sin+le fiber because of e$tensive cross!connections between them
1 iant ner%e fibers can conduct action potentials> A onl" from the rostral end (cerebral ganglion) to the caudal end of the animal . onl" from the caudal to the rostral end -, in both directions !he biphasic nature of the action potential recorded extracellularl" is a conse'uence of> A the fact that# in an action potential# membrane depolarization is followed b" repolarization *, the use of two e$tracellular electrodes s%aced some distance a%art to record the %otential & the different conduction %elocities of different axons * &onduction %elocit" measured in the %entral cord when both median and lateral giant axons are stimulated> A, varies de%endin+ u%on a$on diameter . is the same for all axons & would not be decreased in the cold
Page - of /
0*112 A3Instruments
Instructor’s Reference Earthworm Action Potentials LabTutor Laboratory Re%ort Answers man" of these will be suggested answers# as the students answers will depend on their collected data
E$ercise 12 Evo/ed action %otentials3 threshold and all!or! nothin+ res%onse ;hat is the relationship between stimulus strength and response amplitude in a single axonF The action %otential am%litude should be similar across all recordin+s, This is /nown as the all!or!none res%onse,
* An intracellularl" recorded ner%e action potential approximates 61 mG ;h" is "our recorded action potential so much smallerF Most of the loss of am%litude is due to internal current flow in the worm4s body, The am%litude also de%ends on the recordin+ conditions2 how easily current can flow outside the worm, If the re+ion between the recordin+ electrodes is very wet with saline. the %ea/ deflection may be as small as 56 78, The recorded res%onse may thus be less than a thousandth of the am%litude of the action %otential itself,
+ !he t"pical shape of the action potential is biphasic with two peas of opposite sign Explain wh" this is 9e recordin+ e$tracellularly and measurin+ the %otential difference between two recordin+ electrodes, In the absence of an action %otential. there is no %otential difference between the electrodes, :nce an action %otential arises. a wave of surface de%olarisation %asses down the nerve, 9hen it %asses under the first recordin+ electrode. this re+ion of the surface becomes ne+ative relative to the second electrode, This re+ion ra%idly recovers but the re+ion under the second electrode become ne+ative for a very short time, This %ro%a+ation therefore results in a bi%hasic recordin+, *y convention. the ne+ative recordin+ electrode is %ositioned nearer to the simulatin+ electrodes, Thus when the surface beneath the first electrode becomes ne+ative. this is recorded as a %ositive deflection, 9hen the wave reaches the second %ositive electrode. a ne+ative deflection is recorded,
Page 9 of /
0*112 A3Instruments
Instructor’s Reference Earthworm Action Potentials E$ercise 52 Recruitment of the lateral +iant a$ons !here was an increase in the number of fibers responding as the stimulus strength was increased ;hat is this t"pe of beha%ior calledF Is this of ph"siological significance to the earthwormF The %henomenon is called recruitment, It has no %articular %hysiolo+ical si+nificance to the earthworm as the median and lateral +iant fibers sub serve different functions,
E$ercise ;2 &etermination of nerve conduction velocity It is usuall" assumed that an action potential starts immediatel" at the cathode If this were true# both methods for calculating conduction %elocit" would pro%ide the same answer ;hen a strong stimulus intensit" is used# the action potential ma" begin some distance awa" from the cathode Hnder these conditions# the difference method would be more accurate 3id "ou obser%e an" important difference between the conduction %elocit" %aluesF Answer de%ends on students’ results,
E$ercise <2 &etermination of the refractory %eriod ou will ha%e seen that an action potential generated %er" soon after a preceding one is not full$size Explain this obser%ation An action %otential occurs when %ositive ions (a =) are %ermitted to enter the a$on throu+h s%ecial %rotein channels in the membrane, These channels o%en briefly when the membrane %otential reaches its threshold, The channels then must >reset?. and durin+ this time they are closed and inactive. thus. a second action %otential cannot be %roduced
* @rom "our data# the theoretical maximum fre'uenc" at which ner%e impulses can be generated b" the median giant fiber was calculated 3o "ou thin that the ner%e fiber would be able to generate impulses continuousl" at this rateF o, Eventually the ion +radients would dissi%ate as the sodium %um% would be unable to /ee% u% with the inflow of sodium ions and outflow of %otassium ions, *ut this %roblem would not be detected for some time,
Page 2 of /
0*112 A3Instruments
Instructor’s Reference Earthworm Action Potentials + .riefl" describe the cellular e%ents responsible for the refractor" period 4Bint> 3iscuss the mechanism of repolarization7 An action %otential occurs when %ositive ions (a =) are %ermitted to enter the a$on throu+h s%ecial %rotein channels in the membrane, These channels o%en briefly when the membrane %otential reaches its threshold, The channels then must >reset?. and durin+ this time they are closed and inactive, Althou+h the reset time is very fast. it is detectable as the refractory %eriod, A second stimulus that occurs durin+ this time will not cause an action %otential,
E$ercise @2 *idirectionality of the nerve im%ulse Examine "our data 3oes it show an action potential as beforeF If so# describe the mechanism behind this result Results should show an action %otential, tudents should describe the fact situation that in this e$%eriment the action %otential is bein+ elicited at some %oint alon+ the a$ons len+th rather than at the a$on hilloc/ as would ha%%en %hysiolo+ically, As a result the action %otential %ro%a+ates in both directions,
* In most# if not all# ner%e fibers there is a preferred natural direction of conduction (orthodromicJ) ;hat determines thisF Action %otentials can travel alon+ an a$on in either direction, owever. in natural systems. the direction of %ro%a+ation is functionally unidirectional. as action %otentials arise near the cell body and then travel down the a$on, The median nerve fiber in earthworms is usually oriented to conduct action %otentials in an anterio!%osterior direction, The lateral +iant neurons are oriented to conduct action %otentials %osterio!anterior,
Page 6 of /
0*112 A3Instruments
Instructor’s Reference Earthworm Action Potentials A%%endi$ Earthworm Rin+er’s olution Reci%e Table 1. Earthworm Ringer’s solution recipe to make one liter. &omponent
Amount (g) 91 1* 1* 1
3issol%e all components in one liter of distilled water Add 1ml ethanol to /1ml Earthworm Ringer’s solution for the final solution (1= %8%)
-hloridin+ silver wire To chloride silver wire; Obtain a small Petri dish. Place a small amount of household bleach into the Petri dish. Place one end of the silver wire into the bleach until it becomes blackened. Rinse the wires with distilled water before using them. • • •
•
Page / of /
0*112 A3Instruments
