Short- and Long-Term Modification of Reflex Function During Learning and Metamorphosis in Manduca

8 Pages·1996·3.3 MB·English

by  J C Weeks

Reference:Biol. Bull 191:62-69.(August. 1996)
Short- and Long-Term Modification of Reflex Function
During Learning and Metamorphosis in Manduca
JANIS C. WEEKS AND EMMA R. WOOD
InstituteofNeuroscience, UniversityofOregon, Eugene, Oregon 97403-1254
Howevermuch onemaydisparagethestolidabdominal approximately 6 wk. Manduca hasproven tobeideal for
crawlingbehaviorofcaterpillars, thefactremainsthatthey investigatingthe neural mechanismsofbehavior, aswell
possessprobably the most versatile abdomens in the ani- as how hormones the ecdysteroids and juvenile hor-
malkingdom. . . . Theabdomenistothecaterpillarwhat mone (JH) reorganize the nervous system during
athre.mss.aa.mrcecatlteiormpaielplteahrressyoanmr.uesstucbhespcerhfiezcotphcarteenripcilbleaarsstsa.nAdtpootneentainadl Tmheitsamroervpiehwosfioscu(sreesvioenweadnbeyurWaeleckisrcuaintdthLaetvimneed,ia1t9e92s).a
moths. Theymustbepreparedtomeetallthechallengesthat simpledefensivewithdrawal reflex ofthe larval prolegof
lifepresents to a slow-moving, earthboundgrazer while be- Manduca, and describes how reflex function is modified
ginningtheconstructionofthefragilelurredbodyandgossa- in two contexts: during the dismantling ofthe circuit at
merwingsthat willofferthemthefreedomojtheair. pupation and during nonassociative learning in the lar-
Dethier(1980) val stage. The signals for these two forms of plasticity
differ, as do the associated electrophysiological changes
Introduction that underliethe behavioralchanges. Even in thissimple
reflex circuit, a variety ofneural mechanismscontribute
In his book. The World of the Tent-Makers, from
to behavioral modifications that occur on time scales
which the above quotations are taken, V. G. Dethier from seconds to days and permit the insect to interact
cbhlreonndicolfelsyrtihceprloespei,dopphtielroasnophliyf,e acnycdleastinuteanobasbesrovratbiionng adaptively with the world around it.
ofanimal behavior. Asdetailed in thisbook, the insect's
life is marked by countless challenges and opportunities The Proleg Withdrawal Reflex
towhichthenervoussystem must respond inanadaptive
manner. Although the basic neural circuits underlying Manduca larvae have 8 pairs of legs: 3 pairs of seg-
many behaviors are preassembled in the egg, these be- mented legs on the thorax and 5 pairs ofprolegs on the
haviors must be modified to accommodate the exigen- abdomen (see Fig. 2). Our studies have focused on the
ciesoftherealworld. Furthermore,duringmetamorpho- prolegslocatedon abdominal segmentsthreethroughsix
sis, the neural circuits for larval behaviors must be re- (A3-A6). Returning to Dethier'squotation above, ifthe
placed with those for pupal and adult behaviors. This caterpillar's abdomen is like a person's arms, then the
reviewdescribesourinvestigations into howthe nervous prolegs are the hands on those arms: they are used to
system strikesa balance between constancy and flexibil- reach forandgrasp objects, tocrawl, andtosensedistur-
ity in generatingbehavioral output. bances. The tip ofeach proleg bears an array ofmecha-
Our experimental animal is the tobacco hornworm. nosensory hairs, the planta hairs (PHs; Weeks and Ja-
Manduca sexta. which transforms from egg to moth in cobs, 1987). Stimulation ofthe PHsevokesipsi- orbilat-
eral retractionoftheprolegsin thestimulatedsegment
Received30November 1995;accepted 19March I99h. the proleg withdrawal reflex (PWR; Fig. 1A). PThWeRmajor
This paper was originally presented at a symposium titled FinJinx featuresofthe neuralcircuit fortheipsilateral have
Food:NeuroethologicalAspectsofForaging. Thesymposium washeld been identified (Fig. 1B): thesensory neuronsthat inner-
attheUniversityofMassachusetts.Amherst.from6to8October1995. vate PHsexcite prolegretractor motoneurons via mono-
62
LEARNING AND METAMORPHOSIS IN MANDl'CA 63
planta retractor muscle (APRM). Each proleg has one
PPRM and one APRM. One PPR innervates each
PPRM, whereas each APRM is innervated by a pair of
similar APRs (Weeks and Truman, 1984; Sandstrom
and Weeks, 1996). These motoneuronsand musclesrep-
PWR
resentthe output pathway ofthe reflex circuit.
PWR
Long-Term Modification ofthe Circuit
During Metamorphosis
DismantlingoftheP\\'R circuit
Over the final few days oflarval life, the prolegs col-
lapse and stiffen, most oftheir retractor muscles degen-
PWR
erate(seebelow),and the can no longerbeelicited.
Loss ofthe behavior results in part from the weakening
ofcentralexcitatory pathwaysin thecircuit, asindicated
B by a developmental decrease in the ability ofelectrical
stimulationoftheprolegsensorynervetoevokereflexive
firing in proleg motoneurons(Jacobs and Weeks, 1990).
The decreased excitation could involve changes in sen-
sory neurons, interneurons, motoneurons, or all three,
but a previous finding that the dendritesofPPR and the
APRs regressdramatically overthe same period that the
PROLEGRETRACTORMUSCLE PWR
disappears (Fig. 2) suggested that they might be a
key locus of change. As reviewed elsewhere (Weeks el
a/., 1996), dendritic regression istriggered by a prepupal
SENSORYNEURON
peak ofecdysteroids in the hemolymph.
PLANTAHAIR Because dendrites are the sites of synaptic input, we
hypothesized that the regression of motoneuron den-
Figure 1. PWRbehaviorandneuralcircuit. (A)The PWR. Apair drites would weaken their inputs from sensory neurons
ofprolegsisshown in ventral viewwiththeventral midlinemarkedby and interneurons. To test this idea, we stimulated the
a dashed line. Tactile stimulation ofPHs(filled arrow)evokes proleg proleg sensory nerve while recording the amplitude of
withdrawal, as shown in the successive drawings (open arrows). (B)
PWRcircuit. The prolegtip(left), a prolegretractormuscle(middle), thecompoundexcitatorypostsynapticpotential(cEPSP)
and the ganglion ofthe same abdominal segment (right) are shown evoked in PPR and the APRs. The cEPSP reflects both
schematically.Neuronsareindicatedbyfilledcircles.EachPHisinner- the mono- and polysynaptic components ofthe PWR.
vatedbyasinglesensoryneuronwithacellbodyintheprolegepidermis During the larval-pupal transformation, cEPSP ampli-
ailpinsndielraagtinecraa(lxAoCPnhP)RthsaaytnnapdprsoAejPsec,Rtasnmtdootptoohnleeyugsayrnnoganlpsitoi(nc.MpNPa)tHhvwsiaeanymssootnrhyorsnoyeunugahrpotininsct.eerxcncheiout--e tmuodteodneecurreoansedinbpyutgrreeastiesrtatnhcaen(5J0a%codbesspaintdeaWneeiknsc,rea1s9e90i;n
rons(INs).Thediagramshowstheneuralcircuitfortheleftprolegonly. Streichertand Weeks, 1995). Tobetterdefinethecontri-
bution ofdifferent neurons to this decrease, we took ad-
vantage ofa unique characteristic ofManduca: the abil-
synaptic, cholinergic connections and by polysynaptic ity to manipulate the levels ofecdysteroids and JH to
pathways involving unidentified interneurons (Weeks generate heterochronic mosaic neural circuits composed
and Jacobs, 1987; Peterson and Weeks, 1988; Trimmer ofneuronsindifferentdevelopmental states(e.g., Levine
and Weeks, 1989, 1991;Jacobs and Weeks, 1990;Sand- et a/.. 1989). It isthen possible to determine whetherde-
strom and Weeks, 1991; Streichert and Weeks, 1995). velopmental changesin particularneuronsare necessary
The interneuronal pathway includes excitatory and in- or sufficient for the developmental change in circuit
hibitory interneurons, but the excitatory components function. For this experiment, we generated hetero-
predominate. All ofthenecessaryneuralcomponentsfor chronic mosaic insects in which the PH sensory neurons
thePWRarelocatedintheganglion ofthesamesegment ononeprolegwereretained in theirlarval formwhilethe
as the stimulated proleg. Studies of plasticity in PWR centralcomponentsofthePWRcircuitunderwentpupal
function have focused on two identified motoneurons, development. Notably, motoneuron regression in mo-
PPR and APR (Fig. 2), which innervate the principal saic hemisegments was the same as in unmanipulated
planta retractor muscle (PPRM) and the accessory controls. In themosaichemisegments,cEPSPamplitude
64 J. C. WEEKS AND E R. WOOD
3456
34
5
t r f r
PROLEGS
PHARATE
LARVA DAY PO DAY P2 ADULT ADULT
PPR DIES
(A3-A6) 100pm (A3-A6)
DIES
(A5.A6)
APR
SURVIVES DIES
(A3.A4) (A3.A4)
(A3-A6) (A3-A6)
(A3.A4)
Figurt2. Lifehistoryofprolegmotoneurons.(Top) Drawingsshowalarva,pupa,andadultMaiulitca
withabdominalsegments3,4.5,and6indicated.(Bottom(Cameralucidadrawingsofcobalt-stainedPPRs
andAPRsareshownonday L3 (final instarlarvabeforetheonsetofmetamorphosis),day PO(firstdayof
pupallife),dayP2(thirddayofpupallife),pharateadult(thefinaldayofpupallife),andintheadultmoth
severaldaysafteremergence.Thesegmentallocationsoftheneuronsareindicatedinparentheses. PPRsin
allsegmentsregressbyday POanddiebyday P2.APRsinallsegmentsregressbyday PO.butonlythosein
segmentsA5andA6diebydayP2.TheAPRsthatsurviveinsegmentsA3andA4showdendriticregrowth
duringthepupalstageanddieafteradultemergence. Datafrom WeeksandErnst-Utzschneider(1989).
decreased to the same extent as in normal pupae, sug- EPSP amplitude was correlated with the position ofthe
gesting that metamorphic changes in PH sensory neu- PH on the proleg (Fig. 3A), but the key finding was the
ronsare not necessaryand metamorphicchangesin cen- EPSP amplitude decreased significantly in all regions of
tral neurons (motoneurons and interneurons) are suffi- the PH array during the larval-pupal transformation
cient for the weakening of excitatory pathways in the (Fig. 3B). Analysis ofEPSP shape indices indicated that
PWR circuit (Jacobs and Weeks, 1990; Streichert and the decreased EPSP amplitude in pupae resulted from a
Weeks, 1995). decrease in synapticcurrent, consistent with a reduction
One limitation of these experiments was that the in the number ofsynaptic contacts between the sensory
cEPSP isa mixture ofmono- and polysynaptic inputsto neurons and motoneurons as the motoneurons regress
themotoneuronsand hencedoesnotreveal howindivid- (Streichert and Weeks, 1995).
ual synapses change during metamorphosis. To test In summary, these experiments revealed a specific
more explicitly the hypothesis that dendritic regression mechanism that contributes to the dismantling of an
disconnects synaptic inputs to the motoneurons, we outmoded neural circuitattheentryintoanewlifestage:
measured the amplitude of monosynaptic EPSPs pro- the hormonally driven regression of motoneuron den-
duced in APR by individual PH sensory neurons in lar- drites "sheds" sensory synapsesthat constitute the input
vae and heterochronic mosaic pupae. At both stages. pathway for the reflex. In a broadercontext, steroid hor-
LEARNING AND METAMORPHOSIS IN MANDl'CA 65
B
DAY L3 DAY PO, MOSAIC
ANT MID POST ANT MID POST 1.00
O o O<
0.00 ANT MIDPOST ANT MIDPOST
L3 PO, MOSAIC
Figure3. Developmentalchangesin synapticconnectionsbetween PH sensory neuronsandAPR. In
(A), the drawings at top are maps ofsockets (circles)corresponding to individual PHs in the PH array.
Dashed lineindicatesthedistal borderofthe PH array, anddotted linesindicateanterior(ANT), middle
(MID),andposterior(POST)regions.Filledcirclesindicatethesocketsstimulated(at I Hz)toproducethe
EPSPs shown below. Traces show signal-averaged responses recorded in an APR while stimulating the
indicatedsensoryneuronsondayL3(left)and,inanotherpreparation,ondayPOinamosaichemisegment
(right). Arrowsindicatestimulusartifacts.(B)ComparisonofEPSPsondaysL3andPO.Themeanampli-
tude ( SEM) ofEPSPsevoked in APRs by PH sensory neurons located in the three regionsofthe PH
arrayareshownonday L3andindayPOmosaics.ThenumberofPH sensory neuronstestedisindicated
above each bar. On both days ofdevelopment, EPSPs produced by anterior and posterior PH sensory
neuronsweresignificantlysmallerthanthosefrom middlesensoryneurons(*indicatesP<0.001). Inday
POmosaics, mean EPSPamplitudewassignificantlysmallerthanonday L3 inallthreeregionsofthePH
array(tindicatesP<0.001). DatafromStreichertandWeeks(1995).
mones cause simultaneous changes in neuronal struc- (Streichert and Weeks, 1994; reviewed in Levine and
ture and behavior in many animals (e.g., Forger and Weeks, 1996). TheAPRsthatsurviveinsegmentsA3and
Breedlove, 1987; Woolley and McEwen, 1993; Smith et A4 grow new dendritic arbors, are respecified for new
a/., 1995), but it is typically difficult to test whether the functions, and die after adult emergence (Fig. 2). In seg-
relationshipiscausal ormerelycorrelational. Atpresent, ment A4, the APRs' larval target muscle, APRM, degen-
the PWR ofManduca is the best-documented example eratesandtheAPRsappeartoinnervateanewabdominal
inwhichacausal relationshiphasbeen demonstrated us- extensor muscle (Weeks and Ernst-Utzschneider, 1989).
ing electrophysiological methods. Progress is also being In segmentA3, thelarval APRMspersist in reduced form
made in a numberofothersystems, both vertebrate and duringthe pupal stage. Afterthe prolegsdegenerate, these
invertebrate(reviewed in Weeksand Levine, 1995). APRMsinserton theventralabdominalwallatalocation
that is covered externally by the wings ofthe developing
adult moth (see Fig. 2). At pupation, the APRs and
FatesofleftovercomponentsofthePtt'R circuit APRMs in segment A3 begin to be driven in a rhythmic
inpupae motor pattern that circulates hemolymph within the lu-
men ofthedevelopingwing(Sandstrom, 1993; Lubischer
What happens to members of the PWR circuit after et ui. 1995 and unpub. data). Thisisa remarkableexam-
theirlarval function isobsolete?The PH sensory neurons ple of functional respecification: returning to Dethier's
die (Jacobs and Weeks, 1990; Streichert and Weeks. analogy, the neuromuscularsystem ofthe hands(prolegs)
1995),andthefatesoftheinterneuronsareunknown.The has been respecified to function like a heart! We plan to
proleg motoneurons exhibit two pupal fates: death or re- investigate the ontogeny of synaptic inputs that drive
specification. All ofthe PPRs, the APRs in segments A5 APRs in the new circulatory motor pattern that, intrigu-
and A6, and their target muscles die in response to the ingly, begins to be expressed when the APRs' dendrites
prepupal peak ofecdysteroids (Fig. 2; reviewed in Weeks are maximally regressed. Studiesofhow the APRsare in-
et u/.. 1996). Recent experiments utilizingcell culture in- corporated into this newcircuitwillcomplementourpre-
dicate that the ecdysteroids act directly on proleg moto- vious work examining how the APRs are phased out of
PWR
neurons to activate an intrinsic program of cell death the circuit attheend oflarval life.
66 J. C. WEEKS AND E. R. WOOD
Short-Term Modification ofthe PVVRCircuit ing dishabituation. The PWR exhibits several ofthe de-
During Learning fining characteristics of habituation (Thompson and
PWR Spencer, 1966). including spontaneous recovery from
Prior to the dismantling ofthe circuit at pupa-
toiroant,oMrya-nrdeaurcead Msapenndduacbaoustpe2nwdkthienirthlearlvaarlvallifsetiangep.laLsatbi-c aehfanfbdeicttrueoahftaibioinntteufraostltilioomnwuilfnuogslltiohnwetienrcgveasldsiaostnhiaonbrietosufpaotsnitsoienmudl(eaWctirieeolmn,eannatdn,
cups containing artificial diet. This habitat does not re-
Weeks, 1992, 1996).
semble their natural environment: the stemsand foliage
ofsolanaceousplants. Whenprovidedwithplants, larvae PWAlRthaorueghprealslenotfitnhethneeugraanlgleiloenmeonfttsheresquaimreedsefgormetnhte
colfintghewiirtthitmheeisrtpartoiloenagrsya,neditthheorrafceiecdilenggsoarnd"rsepsetinndg.m"osItn (mFeing.tsIaBr),e lwiekewdiisdensuoftfikcineontwtowhmeetdhieartethheasbeitnueautriaoln aelned-
tshtirsatpeosatnudrea,rteheunPdeHfsletcytpeidc:allsytipmruoljiectthaptaradlelfellecttotthheesPuHbs- dPiWshRabituationofthereflex. Toaddressthisquestion, the
include movements ofthe foliage produced by the wind the expwearsimteesntteadlafsteegrmseunrgticfarlolymitshoelarteisntgotfhethgeanCgNliSo.n Ionf
or the activity of other insects. In a cinematographic PWR
theseneurally isolated bodysegments, habituation
analysisofMatu/uca larvae, Reinecke el til. (1980) noted was robust but dishabituation was negligible (Wiel and
that feedingcan be interrupted byexternal disturbances,
Weeks, 1996; Wiel, 1995). Thus, habituationcan bepro-
potentiallyaffectinggrowth rate.Therefore,itmaybead- duced within a single body segment, whereas dishabitu-
vantageous for larvae to disregard routine stimuli that
ation involves intersegmental processes.
are nonthreatening. This issue has not been addressed in
a natural settingbut, under more controlled conditions,
we have now shown that PWR exhibits thetwo simplest Neuralcorrelates ot'PWR habituation
formsofnonassociative learning: habituation and disha- We
next sought to identify the locusorloci within the
bituation. Habituation is a decrease in the amplitude of PWR
circuitthat areresponsible forresponsedecrement
a response to a repeated stimulus, whereas dishabitua-
during habituation. Peripheral mechanismssuch as sen-
tion is an increase in a decremented (habituated) re-
sory adaptation or muscle fatigue could contribute, as
sponse after presentation ofa novel or noxiousstimulus could central changes within the PWR circuit. A semi-
(Thompson and Spencer. 1966). These forms of plas-
tltiieacalivltiyynssgiegtrnhvieefistcyoasnftti,eltmsertsieomnuustliit.mivuBenetdloaonwnoevweels,ednaesnsocdrrytibhseetribemebuhlyaipvowihtoirelanel- aacinhbntaddanocWgtmeeieesnlkaweslce,trrge1oa9pin8hng9yvls)oiilowovnaleosdag.iuncsTdaeoldatepttloraietcmpehisaentrdaawtthepierottonhhleeecgrpoonctsseisipnistbt(rliaTenlrgcniooemnuftmrreaainrl-
and electrophysiological experiments that investigate
PWR bution ofsensory adaptation, a train ofelectrical pulses
these short-term modifications of function in that mimickedthe normal pattern ofPH sensory neuron
Manduca.
activity during a 500-ms deflection was applied to a PH
socket to activate its sensory neuron. The PH sensory
Habituation anddishabituation ofPll'R behavior
neuron was activated in an identical pattern on each
Habituation and dishabituation of the PWR were stimulus trial. To eliminate the possible contribution of
tested in isolated larval abdomens, which show fewer muscle fatigue, the response to each stimulus was mea-
spontaneous movements and greater stereotypy of the sured asthe number ofaction potentials (spikes)evoked
PWR
than do intact insects (Weeks and Jacobs, 1987). in the ipsilateral proleg motor nerve. In response to the
Thestimulus, abriefdeflection ofasingle PH,wasdeliv- same stimulation protocol used in isolated abdomens
ered byan automatedapparatus(Fig. 4A). The response, (20trials, 60sinterval), themotoneuronsexhibitedasig-
measured as the evoked force ofproleg withdrawal, was nificant response decrement, representing a central neu-
measured by an isometric force transducer attached to ral correlate of habituation (Wood et a/.. 1994, and in
the proleg tip. Dishabituating stimuli were delivered by preparation: Wiel. 1995). Moreover, followinga 30-min
pinchingthe bodywall dorsal totheexperimental proleg rest after trial 20, the response recovered spontaneously
with a pairofforceps. to prehabituation levels, indicating that the response
Figure 4B shows a representative example ofhabitua- decrement resulted from repeated stimulation rather
tion and dishabituation of the PWR. The PH was de- than nonspecific factors. Thus, changes in the central
flected for 500 ms once every 60 s for 25 trials. The neural circuit forthe PWR contribute to habituation.
evokedforceofprolegwithdrawaldecreasedsignificantly Where in the circuit do these changes occur? The
between trials 1 and 20. reflecting habituation of the monosynaptic excitatory connections from PH sensory
PWR. and pinching the body wall between trials 20 and neurons to proleg motoneurons (Figs. IB, 3A) were
21 caused a significant recovery on trial 21. demonstrat- likely sites ofplasticity for two reasons. First, homosyn-
LEARNING AND METAMORPHOSIS IN MANDL'CA 67
20 --
expt(n= 10)
VACUUM E O oont(n= 10)
8
"8 io-
o
FORCE
<D
1 5H
B.
CO
10 15 2021 25 10 15 20
Trial f Trial
Figure4. Habituation anddishabituationot the PWR.(A) Behavioral preparation. An isolatedabdo-
menwasgluedtoacurvedplatformandasinglePHwasdeflectedbyattachmenttoasmallstyrofoamball
thatwassucked upapipettebycontrollednegativepressure(VACUUM).Theforceofprolegwithdrawal
wasmeasured(ingrams)b\ anisometricforcetransducer(FORCE)attachedtotheprolegtip.(B)Habitu-
ationanddishabituationinisolatedabdomens.APHwasdeflectedfor500msat60-sintervalsfor25trials,
andtheevoked forceofprolegwithdrawal wascalculated asthedifference between thepeak forceduring
the PH deflection minusbaseline forcebeforethedeflection. Thirtysecondsaftertrial 20. thebody wall
dorsal totheexperimental prolegwaspinched with forceps(arrow). Significant habituation occurred be-
tweentrials 1 and20(*indicatesP<0.01),andsignificantdishabituationoccurredbetween trials20and
21 (\indicatesP<0.01);11= 15abdomens.(C)DecreaseinactivityevokedinPPRbyrepeatedPHsensory
neuron stimulation. The preparation consisted ofan isolated proleg tip and the ganglion ofthe same
segment. An intracellular recordingwas made from the ipsilateral PPR. A train ofelectrical pulses that
mimickedthe normal patternofsensory neuron activityduringa 500-ms PH deflectionwasapplied toa
PHsensoryneuron.Experimentalpreparations(expt)received20trialsata60-sISI.whereascontrol(cont)
preparationsreceived2trialsata 19-min ISI. Theresponsetoeachstimuluswasmeasuredasthenumber
ofspikesevokedoverbaseline in PPR. duringthe 1000msfollowingstimulusonset. PPR'sresponsede-
creased significantly between trials 1 and 20 in experimental (n = 10; * indicates P < 0.01) but not in
control ( = 10; P> 0.05) groups. Values in (B)and (C)are mean SEM. Data from Wiel and Weeks
(1992, 1996),Wielela/.,(1995,andinpreparation)andWiel(1995).
aptic depression ofsynaptic transmission from sensory activity-dependent plasticity facilitation, depression,
neurons to postsynaptic neurons is a primary mecha- and post-tetanic potentiation which come into play
nism ofhabituation in several other systems, including under different patterns of sensory neuron activity
the gill- and siphon-withdrawal reflexes ofAplysia and (Weeks and Jacobs, 1987; Trimmer and Weeks, 1991).
the tail flip escape reflex ofcrayfish (reviewed by Krasne These phenomena appear to be homosynaptic and
andGlanzman, 1995). Second, previousexperimentsin- caused by alterations in neurotransmitter release from
volving PPR demonstrated that these connections ex- sensory neuron terminals. Two formsofplasticity at the
hibit considerable short-term plasticity. The EPSPs are synapses the activation of presynaptic mAChRs and
cholinergic and mediated by nicotinic acetylcholine activity-dependent homosynaptic depression decrease
receptors (nAChRs) on PPR (Weeks and Jacobs, EPSP amplitude and were thus possible candidates for
PWR
1987; Trimmer and Weeks, 1989, 1993). There are also mediating habituation.
two classes of muscarinic cholinergic actions: activa- Before examining EPSP amplitudes, we first con-
tion of presynaptic muscarinic acetylcholine receptors firmed that PPR's evoked activity decreased during ha-
(mAChRs) reduces EPSP amplitude (Trimmer and bituation training. Thiswassuggestedbythefindingthat
Weeks. 1989), whereas postsynaptic activation of evokedactivity in the prolegmotornervedecreased dur-
mAChRs on PPR activates an inward cationic current ing habituation training (see above) but, in these whole
that lowers PPR's spike threshold (Trimmer and Weeks nerve recordings, PPR's spikes could not be distin-
1989, 1993; Trimmer, 1994). In addition to the musca- guished from those ofother motoneurons. Accordingly,
riniceffects, EPSPamplitudeisaffectedbythreetypesof we repeated this experiment while recording intracellu-
68 J. C. WEEKS AND E. R. WOOD
larly from PPR and measuring its response directly. As proleg motoneuron dendrites and the resultant decrease
shown in Fig. 4C, the number ofspikes evoked in PPR in sensory input provides a mechanism to explain the
decreased significantly over20trialsdeliveredat 60-sin- progressiveweakeningand lossofthe reflex at pupation.
tervals. Furthermore, in control preparations that re- Some ofthe neuromuscularcomponents are "recycled"
ceivedjust two stimuli, oneat the time oftrial 1 and the as members ofthe pupal circulatory system. In larvae,
second at the time of trial 20, the number of spikes the reflex is modified by experience and, despite the
evoked in PPR did not change significantly (Fig. 4C). known plasticity in synaptic transmission from PH sen-
Thus, response decrement resulted from repeated pre- sory neurons to proleg motoneurons, the properties of
sentation ofthestimulus. these synapses appear to remain stable during habitua-
Finally, usingthe sameexperimental andcontrol pro- tion. Thus, the specific neural mechanismsthat underlie
tocols, we measured the amplitude ofEPSPs evoked in habituation ofthe PWR elude usat present. On a larger
PPR by the stimulated PH sensory neuron before and scale, the PWR is only one of many behaviors that in-
after training. PPR's membrane potential, spike thresh- volvethe prolegs, and the relationshipbetween elements
PWR
old and input resistance, and the magnitude ofpaired- ofthe circuit and the circuits that produce these
pulse facilitation ofthe EPSP were also measured. Con- other behaviors remains unexplored. The caterpillarab-
trary to our expectation, the amplitude of the unitary domen and its "hands" should continue to provide in-
EPSPevoked in PPR by the PH sensory neuron used for sights into how the nervous system balances constancy
trainingdidnotchangedetectablyineitherexperimental with change when generatingbehavior.
or control preparations (Wiel ct at.. 1995, and in prepa-
ration; Wiel, 1995). Therefore, it appears unlikely that Acknowledgments
eitherofthetwo known decrementingformsofplasticity
contributeto PWR habituation. Oftheotherparameters Research funding was provided by NIH (R01
measured. PPR's spike threshold became significantly NS23208, K04 NS01473 and T32 GM07257), an NSF
more hyperpolarized and input resistance significantly Presidential Young Investigator award (J.C.W.), and
increased in both the experimental and control groups grants from the Whitehall Foundation, M.J. Murdock
(Wiel ct a/., 1995 and in preparation; Wiel, 1995). Be- Trust, Oregon Medical Research Foundation and the
cause these values changed in both groups, they cannot U.S. Department ofEducation. We thank Dr. Devon E.
account for habituation; furthermore, both of the Wiel for providing unpublished data, and Dr. Wiel and
changes would tend to make PPR more excitable and Dr. William M. Roberts for comments on the manu-
thereforelesslikelytoexhibit a responsedecrement. script.
PWR
These data suggest that habituation is mediated
neither by changes in synaptic transmission from PH LiteratureCited
sensory neurons to motoneurons nor by changes in the
intrinsic electrical properties of the motoneurons. The Dethier, V.G. 1980. The ll'arltl<>/1lie Tent-Makers. Universityof
former finding was surprising, given the range ofplastic MassachusettsPress.Amherst. MA.
mechanisms known to be in place at these synapses (see Fischer,T. M.,andT.J.Carew. 1993. Activity-dependentpotentia-
tionotrecurrentinhibition:amechanismlordynamicgaincontrol
above). The functional role ofplasticity attheseconnec- in the siphon withdrawal reflex ofAplysia. J Nt-iin>\ci 13: 1302-
tions therefore remains elusive. These findings indicate 1314.
thatwemustlookelsewherein thecircuitforthechanges Forger, N. G., and S. M. Breedlove. 1987. Seasonal variation in
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