Instructors 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
Instructors Reference (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 bac(ground 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 instructors discretion
E$am%le &ata
) E%o(ed action potentials
* Recruitment of the lateral giant axons
+ Axonal conduction %elocit"
, Refractor" period
- .idirectionalit" of ner%e impulse
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Instructors Reference
Earthworm Action Potentials
E'ui%ment List( Alternatives
PowerLab )-! 45L6)67# or PowerLab ,8*9! 45L6-97
:hielded -$lead .io Amp &able 45LA*-,17
:hielded Lead ;ires $ Alligator &lips 45LA)9)-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*-= <a&l solution
&or(board
5odeling cla" or similar material
!hree metal pins
Plastic s"ringe
Ruler with millimeter di%isions
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 <er%e .ath at all times @ill the <er%e
.ath with Aust enough Earthworm Ringers solution to fill the small# bottom
chamber If the ner%e contacts the reser%oir of Ringers# a short circuit could
result An alternati%e preparation is to immerse the entire ner%e in mineral oil in
lieu of placing Ringers solution in the chamber
)oise
5ost noise can be reduced or eliminated b" turning off o%erhead fluorescent
lighting and mo%ing the earthworm preparation and .io Amp cables awa" from
&R! monitors &hec( to ma(e sure the chlorided ends of the sil%er wires are in
contact with the worms s(in
5o%ement artifacts are caused b" a worm that is not full" anesthetized 5a(e
sure students full" anesthetize their worms in ethanol and periodicall" appl" the
ethanol8Ringers solution to their worms
!oo much Ringers solution can result in a poor recording Ba%e students blot
excess Ringers from the earthworms with a tissue
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Instructors 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 ma(e 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 wor(s 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" stro(ing the earthworm in a tail$to$head direction
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Instructors Reference
Earthworm Action Potentials
Pre!Laboratory "ui# answers
Bere are the correct answers to the Duiz 'uestions in bold
) <er%e depolarization in%ol%es>
A an increase in membrane potassium permeabilit"
. an increase in membrane sodium %ermeability
& an increase in membrane chloride permeabilit"
* <er%e repolarization in%ol%es>
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 mar(edl" different excitation thresholds
. conduct action potentials more rapidl" than the median giant fiber
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Earthworm Action Potentials
-, behave as a sin+le fiber because of e$tensive cross!connections
between them
)1 Eiant ner%e fibers can conduct action potentials>
A onl" from the rostral end (cerebral ganglion) to the caudal end of the animal
. onl" from the caudal to the rostral end
-, in both directions
)) !he biphasic nature of the action potential recorded extracellularl" is a conse'uence
of>
A the fact that# in an action potential# membrane depolarization is followed b"
repolarization
*, the use of two e$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$on diameter
. is the same for all axons
& would not be decreased in the cold
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Instructors Reference
Earthworm Action Potentials
LabTutor Laboratory Re%ort Answers
<ote> man" of these will be suggested answers# as the students answers will
depend on their collected data
E$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 pea(s of
opposite sign Explain wh" this is
9e recordin+ e$tracellularly and measurin+ the %otential difference between
two recordin+ electrodes, In the absence of an action %otential. there is no
%otential difference between the electrodes, :nce an action %otential arises. a
wave of surface de%olarisation %asses down the nerve, 9hen it %asses under
the first recordin+ electrode. this re+ion of the surface becomes ne+ative
relative to the second electrode, This re+ion ra%idly recovers but the re+ion
under the second electrode become ne+ative for a very short time, This
%ro%a+ation therefore results in a bi%hasic recordin+, *y convention. the
ne+ative recordin+ electrode is %ositioned nearer to the simulatin+ electrodes,
Thus when the surface beneath the first electrode becomes ne+ative. this is
recorded as a %ositive deflection, 9hen the wave reaches the second %ositive
electrode. a ne+ative deflection is recorded,
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Instructors Reference
Earthworm Action Potentials
E$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$ercise <2 &etermination of the refractory %eriod
) Iou will ha%e seen that an action potential generated %er" soon after a
preceding one is not full$size Explain this obser%ation
An action %otential occurs when %ositive ions ()a
=
) are %ermitted to enter the
a$on throu+h s%ecial %rotein channels in the membrane, These channels o%en
briefly when the membrane %otential reaches its threshold, The channels then
must >reset?. and durin+ this time they are closed and inactive. thus. a second
action %otential cannot be %roduced
* @rom "our data# the theoretical maximum fre'uenc" at which ner%e impulses
can be generated b" the median giant fiber was calculated 3o "ou thin( that
the ner%e fiber would be able to generate impulses continuousl" at this rateF
)o, Eventually the ion +radients would dissi%ate as the sodium %um% would be
unable to /ee% u% with the inflow of sodium ions and outflow of %otassium
ions, *ut this %roblem would not be detected for some time,
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Earthworm Action Potentials
+ .riefl" describe the cellular e%ents responsible for the refractor" period 4Bint>
3iscuss the mechanism of repolarization7
An action %otential occurs when %ositive ions ()a
=
) are %ermitted to enter the
a$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$ercise @2 *idirectionality of the nerve im%ulse
) Examine "our data 3oes it show an action potential as beforeF If so#
describe the mechanism behind this result
Results should show an action %otential, Atudents should describe the fact
situation that in this e$%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,
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Instructors Reference
Earthworm Action Potentials
A%%endi$
Earthworm Rin+ers Aolution Reci%e
Table 1. Earthworm Ringers solution recipe to make one liter.
&omponent Amount (g)
<a&l 91
?&l 1)*
&a&l
*
1*
<aB&K
+
1)
3issol%e all components in one liter of distilled water
Add )1ml ethanol to /1ml Earthworm Ringers 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.
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