Comparison of Identified Leg Motoneuron Structure and Function Between Larval and Adult Manduca Sexta

8/6/2019 Comparison of Identified Leg Motoneuron Structure and Function Between Larval and Adult Manduca Sexta

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O R I G I N A L P A P E R

U. Rose R. B. Levine

Comparison of identied leg motoneuron structureand function between larval and adult Manduca sexta

Accepted: 8 January 2000

Abstract Persistent leg motoneurons of the mothManduca sexta were investigated in larval and adult

animals to compare their dendritic structures, intrinsicelectrical properties and pattern of target innervation.The study focused on two identied motoneurons of theprothoracic leg. Despite the complete remodeling of legmuscles, the motoneurons innervated pretarsal exormuscles in both larval and adult legs. Similarly, al-though the central dendrites regress and regrow, thebranching pattern was similar with the exception of aprominent midline branch that was not present in theadult stage. The intrinsic electrical properties of themotoneurons diered between larval and adult stages.Larval motoneurons had signicantly higher membraneinput resistances and more depolarized resting mem-

brane potentials than did motoneurons in pharate adultsor adults. In all stages, one motoneuron had a lowmaximal ring frequency, whereas the second moto-neuron, which innervated the other half of the muscle,had a high maximum ring frequency. Although the twomotoneurons continued to innervate the same halves ofthe target muscle, their relative eects on muscularcontraction were reversed during metamorphosis alongwith concomitant changes in intrinsic properties. Pre-tarsal exor motoneurons in pharate adults (just prior toemergence) displayed properties similar to those inemerged adults.

Key words Insect

Motoneuron

PropertiesMetamorphosis Behavior

Abbreviations FCO femoral chordotonal organ PrtFlx-ant anterior pretarsal exor motoneuron

PrtFlx-post posterior pretarsal exor motoneuron

PrtFlx pretarsal exor muscle

Introduction

The moth Manduca sexta undergoes extensive reorga-nization during metamorphosis. Larval muscles degen-erate and new adult muscles are generated (Weeks andTruman 1986; Kent et al. 1995; Consoulas et al. 1997).New sensory and central neurons are generated (Bookerand Truman 1987; Witten and Truman 1991), whereasothers die or are respecied to fulll new tasks in the

adult (Truman 1983; Levine and Truman 1985; Weeksand Ernst-Utzschneider 1989; Kent and Levine 1993).Persisting motoneurons undergo dramatic dendritic re-organization during pupal development (Kent and Le-vine 1993), which is under the control of the steroidhormone 20-hydroxyecdysone (Truman and Reiss 1988;Weeks and Ernst-Utzschneider 1989; Weeks et al. 1992;Levine and Weeks 1996). The ability to identify indi-vidual motoneurons in Manduca and follow themthrough metamorphosis allows specic structural andfunctional modications to be linked to changes in be-havior (Streichert and Weeks 1995).

During metamorphosis the short larval thoracic legs,

which participate in crawling and grasping, are replacedby well-articulated adult legs which are used for walking(Kent and Levine 1988; Consoulas et al. 1997). Thelarval crawling motor pattern is dierent from the motorpattern associated with adult walking (Johnston andLevine 1996a, b). Whereas larval crawling is character-ized by simultaneous right and left leg activation withina segment, adult walking involves an alternating gaitsimilar to that characteristic of many insects (Graham1972; Burns 1973; Watson and Ritzmann 1998). In theearly pupal stages, as the leg muscles are being replaced,the thoracic leg motoneurons undergo a substantial re-gression followed by a re-expansion of their dendritic

J Comp Physiol A (2000) 186: 327336 Springer-Verlag 2000

U. Rose (&)1

R. B. LevineDivision of Neurobiology, Room 611, Gould Simpson Building,University of Arizona, Tucson, AZ 85721, USA

Present address:1Department of Neurobiology, University Ulm,Albert-Einstein-Allee 11, 89069 Ulm, Germanye-mail: [email protected]: +49-731-50-22629


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arbor and peripheral axon terminals (Kent and Levine1993; Consoulas et al. 1996).

The present study examined the functional propertiesof identied leg motoneurons in intact preparations. Theacquisition of new behavior and body morphologyduring metamorphosis may require that motoneuronsadapt their membrane properties as well as their eectson target muscles. For example, the rapid movements ofthe adult legs may require persistent motoneurons tochange their integrative properties during pupal devel-opment or may demand alterations in muscle responseto motoneuron activity. A second goal was to comparethe locations of the larval and adult target muscles forpersistent motoneurons. In the one example that hasbeen investigated in depth, larval femoral depressormotoneurons innervated the new femoral depressormuscles in the adult, although dierences in joint artic-ulation caused dierences in the leg movements evoked(Kent and Levine 1988). The generality of this func-tional conservation was examined in the present study.

Material and methods

Animals

Larvae, pupae and adults of the tobacco hornworm, Manducasexta (Lepidoptera; Sphingidae) were obtained from a colony at theUniversity of Arizona. The animals were fed an articial diet (Belland Joachim 1976) and reared under a light/dark regime of 17 hlight and 7 h dark, at 26 C and 60% relative humidity. Staging ofthe animals followed published criteria (Tolbert et al. 1983; Con-soulas et al. 1996, 1997). Briey, the larval stages L0, L1, L2, andL3 represent the rst days of the fth (last) larval instar. Afterpupation, the pupal stages correspond roughly to the days of de-

velopment. For example, P0 represents the day of pupation, P1 andP2 the following 2 days. After the last day of pupal development(pharate adult) the animals undergo eclosion by shedding the pupalcuticle. The day of eclosion was called stage E0.

Preparations

Prior to the experiments animals of all stages were anesthetized bycooling on ice for about 30 min. This was sucient to completelyblock movements for the rst 10 min of the dissection. All exper-iments were carried out in freshly prepared saline (Trimmer andWeeks 1989).

Larval preparation

The head and thoracic segments were removed from the abdominalsegments and were pinned down on a Sylgard-coated (DowCorning) Petri dish with the dorsal side up. The thorax and headwere opened by a midline incision and the body walls were pinneddown laterally. The suboesophageal, pro-and mesothoracic gan-glion chain was left intact but separated from the body by cuttingthe peripheral nerves, leaving only the interganglionic connectivesand one leg nerve (N2a) of the prothoracic segment intact. On thisside, the prothoracic leg was separated from the surrounding bodywall. The remaining head and thoracic segments were removed andthe leg-ganglia preparation was carefully pinned down in the dish.The prothoracic leg was opened by a midline incision on the ventralside without touching nerves or the pretarsal exor muscle. Thelateral sides of the leg were then pinned down with small insectpins. The prothoracic ganglion was xed on a small platform made

of Sylgard with the dorsal side up, and the sheath was carefullyremoved.

Pharate adult and adult preparation

The preparation of pharate adult and adult animals was similar. Aswith the dissection of the larval stage, the prothoracic leg was re-

moved from the body together with the suboesophageal, protho-racic and pterothoracic ganglia (fusion of mesothoracic,metathoracic, and the rst two abdominal ganglia). The cuticle ofthe femur was opened to expose the internal structures. At theentrance of the coxal segment, the main leg trachea was cut andexposed to the air to keep the oxygen supply of the tissue intact.The ganglia were pinned down with the ventral side up and thesheath of the prothoracic ganglion was removed with ne forceps.All preparations were superfused continously with oxygenated sa-line (11.5 ml min)1).

Electrophysiology

Chordotonal organ stimulation

In some preparations, the femoral chordotonal organ (FCO) was

stimulated using a piezo-electric tongue driven by ramp generator(University of Goettingen) amplied by a piezo-electric controller(Thorlabs, MDT 691). The tip of the piezo-electric device was at-tached to the tendon of the chordotonal organ. In pharate adults oradults, the rigid tendon was cut and xed to the tip of a ne clamp.In contrast, the soft and fragile tendon of larvae was stimulated bya hook placed under the tendon. The ramp generator allowed thedeection of the piezo-electric device to be adjusted. The maximaldeection was adapted to match the range of naturally occurringtibial movements.

Extracellular and intracellular recordings

Peripheral nerve activity was recorded with monopolar hookelectrodes made from ne tungsten wires. Hooks were placed under

the nerve of interest and isolated from the bath with petroleum jelly. An Ag/AgCl silver wire was placed in the bath near the re-cording side as an indierent electrode. Muscle potentials wererecorded with glass suction electrodes pulled from borosilicateglass. The tip of the suction electrode was applied to the musclebers of interest and a negative pressure was applied. This tech-nique allowed stable recordings even during muscle contraction. Adisadvantage of this technique was that muscle potentials fromnearby muscles were also recorded. However, these potentialscould be clearly distinguished from the potentials of the recordedmuscle bers by their small size and by visual observation of muscleber contractions.

Intracellular recordings from motoneurons were achieved withthin-wall glass electrodes lled with 3 mol l)1 KCl (resistance 3040 MW). The signal was recorded in bridge mode or, when currentwas injected, in discontinuous current-clamp mode (DCC mode)

with an Axoclamp 2 A amplier. For recordings in DCC mode itwas crucial to keep the resistance and capacitance of the electrodeas low as possible. To achieve sampling frequencies between 4 kHzand 6 kHz, the tip of the electrodes were coated with silicon and thesaline level in the bath was kept as low as possible.

The input resistance (Rin) of each motoneuron was calculatedfrom the slope of the I/V curve in the linear region of negativecurrent. Current pulses from +2 nA to )3.5 nA for 200 ms in stepsof 0.5 nA were injected. All current injections were made using thewaveform tool ofthe Clampexprogram (AxonInstruments). TheI/Vrelation and resulting input resistance were calculated o-line withthe Clampt program (Axon Instruments).

The time constant (sm) of a neuron was determined in DCCmode by averaging the voltage response to 50 current pulses()0.5 nA, 200 ms) followed by an oine calculation applying rst-and second-order exponential equations (Clampt program). The

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longest time-constant of a neuron was considered as sm. Briefertime constants were considered as equalizing time constants (Rall1969; Rall et al. 1992). Although input resistance and time-con-stants were measured from dierent resting membrane potentials inlarval and adult motoneurons, this did not aect the values ob-tained. Input resistance remained constant for larval neurons overthis membrane potential range (see Fig. 5B).

The resting membrane potential of each neuron was determined

by comparing the potential before and after pulling the electroderapidly out of the cell. The action potential threshold of a moto-neuron was determined by injecting steps of positive current from adened membrane potential. In the case of larval motoneurons acontinuous negative current was injected to keep the membranepotential at )60 mV in DCC mode. From this potential, steps ofpositive current from 0.5 nA to 3 nA for 500 ms were injected in0.5-nA increments. Because of their more negative resting mem-brane potentials (ca. 10 mV), motoneurons of pharate adult andadult stages were held at )70 mV prior to injections of the samepositive current steps as in larvae. The number of action potentialsduring each current step was plotted against both the injectedcurrent and the membrane potential. The action potential thresholdof motoneurons were estimated from the mean of the membranepotential reached in response to the current step that rst evokedaction potentials and that reached in response to the previous

current step (i.e., 0.5 nA less). The range of the data were given inaddition to their mean standard error.

Histology

In some recordings the tip of the electrodes were lled with 3%neurobiotin (Vector) in 1 mol l)1 KCl. The neurobiotin was in-

jected into the cell with positive current pulses (400 ms, 2 Hz, 25 nA). Successful stainings were made of six larval and ve pharateadult/adult preparations. The structures of interest were dissectedout, placed in a Petri dish and xed in 4% paraformaldehyde so-lution. Preparations were subsequently washed in 10 mmol l)1

phosphate-buered saline (PBS, pH 7.4) and dehydrated in etha-nol. The preparations were then permeabilized in xylol (5 min) andrehydrated. After several washes in PBS the tissue was incubated in

CY3-conjugated streptavidin (Jackson Research, 1:700 in PBS) for2 h. The staining process was terminated by washing three times inPBS (each step 10 min.). The preparations were nally dehydratedin ethanol and cleared and mounted in methyl salicylate. The la-beling of motoneurons with the lipophilic carbocyanine dye DiI(Molecular Probes) followed the procedure given in Kent and Le-vine (1988) and Prugh et al. (1992). The labeled tissue was viewedwith a Bio-Rad confocal microscope (MRC 600 equipped with aNikon Optiphot-2 microscope and a krypton/argon laser lightsource). Images were scanned as 5-lm optical sections. Sections

were recorded and stored using Confocal Assistant software (BioRad) and subsequently prepared with Corel software (Corel Draw8; Corel Photo-Paint 8).

Statistics

Data are presented as means and their standard errors (SEM). The

two-tailed U-test after Wilcoxon (Sokal and Rohlf 1995) was usedfor comparisons of two samples. Statistical signicance was as-sumed when P 0.05.

Results

Innervation pattern

In larvae, pharate adults and emerged adults, contrac-tions of the pretarsal exor muscle (PrtFlx) resulted inexion movements of the pretarsus. In both larvae andadults the PrtFlx muscle attaches proximally at the cu-

ticle of the femur near the trochanter (Fig. 1). Distally, along tendon extends through the entire tibial and tarsalsegments to the pretarsus (Fig. 1A, Prt). In larvae themuscle consists of two well-separated bundles. Accord-ing to their relative position within the femur, they arereferred to as the posterior and anterior muscle ber

Fig. 1 Drawings of the prothoracic leg of larval (A) and adult (B)Manduca sexta. The larval pretarsal exor muscle (PrtFlx) isdivided into two readily distinguishable muscle ber bundles. Aposterior bundle (PrtFlx-post) extends from the proximal femur tofuse with an anterior muscle ber bundle (PrtFlx-ant). At this pointa tendon originates and runs distally to the pretarsus (Prt). In thepharate adult or adult animal (B) the PrtFlx still resides at a similar

location within the femur, but did not exhibit the well separatedmuscle ber bundles of the larva. However, posterior (PrtFlx-post)and anterior (PrtFlx-ant) parts were still distinguishable on thebasis of their position relative to the tendon (see also Fig. 2D).IN2a intersegmental nerve 1b and 2a; SN sensory nerve; Cx coxa;Ti tibia; Ta tarsus; Prt pretarsus; Tr trochanter; TiExt tibiaextensor muscle; TiFlx tibia exor muscle; PrtFlx pretarsal exormuscle; TaFlx, tarsal exor muscle; UR unguis retractor muscle;AcTiFlx accessory tibia exor muscle; FCO femoral chordotonalorgan. The leg drawings are modied after Consoulas et al. (1996)

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bundles (Fig. 1A, PrtFlx-ant, PrtFlx-post). During pu-pal development the PrtFlx muscle degenerates com-pletely and is replaced by a new muscle (Kent et al.

1995; Consoulas et al. 1997). The adult muscle does notexhibit the well-separated ber bundles that are ob-served in larvae, although posterior and anterior bundlescould be readily distinguished on the basis of their po-sition within the femur and the ber arrangement rela-tive to the tendon (Figs. 1B, 2D). Here, bers attach to atendon that runs almost the entire length of the muscleand separates anterior and posterior bundles.

Retrograde biocytin lls revealed that the PrtFlxmuscle is innervated by three motoneurons which persistduring metamorphosis, as conrmed by persistent la-beling of the adult PrtFlx motoneurons following theintroduction of Di-I into the larval PrtFlx motoneurons

(K. Oanh-Phan, personal communication). We wereable to distinguish among the motoneurons in larvae,pharate adults and adults by their soma position withinthe ganglion and innervation of the pretarsal exormuscle.

In larvae, one motoneuron innervated the posteriorber bundle exclusively, (Fig. 2Aii; Fig. 3Bi, Bii). Thesoma of this motoneuron was located halfway betweenthe lateral perimeter and the midline of the ganglion. Asecond motoneuron, which supplied only the anteriormuscle ber bundle, (Figs. 2Ai, 3Ai, Aii), was situatedon the extreme lateral margin. The third motoneuronhad a similar soma location but caused smooth, graded

Fig. 2AD Neurobiotin-lled motoneurons that innervate the larvaland adult (pharate adult) pretarsal exor muscle. Ai Motoneuron thatinnervates the anterior part of the pretarsal exor muscle exclusively(PrtFlx-ant). Activation of this motoneuron led to a relatively smoothcontraction of the anterior muscle bers only as observed visually.Both motoneurons extend a prominent branch towards the midline.The second, thin, axon in Ai belongs to a motoneuron that had been

impaled briey before PrtFlx-ant. The inset in Ai shows a sketch of thewhole ganglion and the relative position of the motoneuron. (Aii)Dendritic morphology and branching pattern of the motoneuron thatsupplies the posterior part of the pretarsal exor muscle exclusively(PrtFlx-post). The soma is out of the plane of focus. Activation of thismotoneuron caused a twitch-like contraction of the posterior musclebers. B Drawing of the pretarsal exor muscle in the prothoracic legof a larval Manduca sexta. The muscle consists of two separatedmuscle ber bundles, one of which is located posteriorly (PrtFlx-post)and the other anteriorly (PrtFlx-ant) within the femur. Both musclebundles fuse distally where the tendon attaches. C Motoneuronsupplying the pharate adult and adult PrtFlx muscle. The motoneuronshown in Ci innervates the anterior bers only and its activationcaused twitch like contraction. Cii shows the motoneuron thatsupplies the posterior part of the pretarsal exor muscle. Thismotoneuron caused relatively smooth contractions of the muscle. The

dendritic branches of both neurons cover the lateral part of theneuropil but the prominent midline branch seen in the larva wasabsent. Note that the shape of the ganglion changes duringmetamorphosis (compare insets in Ai and Ci). Furthermore, theganglion increased in size and the peripheral nerve 2 (N2, inset in Ci),enters the ganglion anteriorly. D Drawing of a pretarsal exor muscleas it appears in the pharate adult or adult prothoracic leg. The muscleis more compact compared to the larval muscle, but anterior andposterior parts can clearly be distinguished by the ber arrangementand position relative to the tendon

b

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contractions of both anterior and posterior muscle berbundles (not shown).

In pharate adults and emerged adults the relativesoma positions of the motoneurons resembled those inlarvae, so that it remained possible to distinguish amongthe dierent motoneurons. The innervation pattern was

similar at all stages in that both anterior and posteriorber bundles were innervated separately by the twomotoneurons. We therefore refer in all stages to themotoneuron innervating the anterior muscle bers aspretarsal exor-anterior (PrtFlx-ant) and to the moto-neuron innervating the posterior muscle bers as PrtFlx-post. In the larva, PrtFlx-post caused relatively fastcontractions that could be distinguished visually fromthe slower contractions caused in the other half of thepretarsal exor muscle by PrtFlx-ant. By contrast, in thepharate adult or adult, it was PrtFlx-ant that causedthe faster contractions relative to PrtFlx-post.

Intracellular dye injection into motoneurons in lar-

vae, pharate adults and adults revealed densely brancheddendrites in the lateral leg neuropil of the prothoracicganglion. A unique feature of both PrtFlx-ant andPrtFlx-post in larvae was a prominent branch extendingtowards the midline (Fig. 2Ai, Aii, n 6), which was nolonger present in pharate adults or adults (Fig. 2Ci, Cii,n 5). In pharate adults, the third motoneuron had asimilar branching pattern but a smaller soma diameter(n 1). Because PrtFlx-ant and PrtFlx-post had largesomata and could be clearly distinguished at dierentstages, they became the focus of further analysis.

Intracellular dye injection at pupal stage 1 revealedpronounced dendritic regression of PrtFlx motoneurons

(Fig. 4Ai), although PrtFlx-ant and PrtFlx-post were notdistinguished. By pupal stage 7, the dendrites had re-grown substantially, but lacked the midline branch thatcharacterized the larval PrtFlx motoneurons (Fig. 4Aii).

Membrane properties

Membrane properties of individual motoneurons(PrtFlx-ant; PrtFlx-post) were measured in larvae andpharate adults. Adult animals (stage E3, 3 days afteremergence) were also included to examine possible dif-ferences between motoneuron properties before and af-ter adult emergence.

The input resistance of motoneurons was calculatedfrom the slope of the linear region of response to currentinjected into the motoneuron cell body (Fig. 5A, B). Thevalues were signicantly in larvae than in pharate adults.No dierence was found between motoneurons from

pharate adults and emerged adults [Fig. 5B, C; larva:PrtFlx-ant 45.4 3.6 MW, PrtFlx-post 39.3 2.4 MW; pharate adult: PrtFlx-ant 20 1.2 MW,PrtFlx-post 20.2 3.3 MW; adult (E3): PrtFlx-ant 16.1 2.2 MW]. Within a given stage, no signif-icant dierence was found between the PrtFlx-ant andPrtFlx-post motoneurons (Fig. 5C). A pronounced rec-tication, starting at negative currents of 2.5 nA, wasevident in both larval motoneurons but not in pharateadult or in adult motoneurons (Fig. 5A, B).

The time-constants of the motoneurons were deter-mined by averaging the membrane voltage responses to50 0.5-nA current pulses (Fig. 6Ai). The time-constants

Fig. 3A, B Dierential inner-vation of the larval PrtFlx byindividual motoneurons. A, BIntracellular recordings from asingle pretarsal exor moto-neuron (A PrtFlx-ant; B PrtFlx-post) combined with extracel-lular recordings from specic

parts of the pretarsal exormuscle (Ai, Bi posterior bers;Aii, Bii anterior bers). Indi-vidual motoneurons supplydistinct parts of the pretarsalexor muscle. The small poten-tials seen in the muscle record-ings (Ai, Bii) are due to crosstalk from the adjacent musclesbers

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of larval motoneurons were signicantly longer (PrtFlx-ant 23.8 2.6 ms, PrtFlx-post 27.3 1.5 ms)than in pharate adult (PrtFlx-ant 14.7 1.3 ms,

PrtFlx-post 18.2 1.9 ms) or adult (PrtFlx-ant 12.2 1.3 ms). Furthermore, the mean time-constant of the PrtFlx-post within a given stage wasconsistently longer than the time-constant of the PrtFlx-ant (Fig. 6Aii), although the values were not signi-cantly dierent.

The resting membrane potential of the motoneuronsbecame more negative during adult development(Fig. 6B). Larvae had mean resting membrane potentialsof )53.8 2.3 mV (PrtFlx-ant) and )52.4 0.7 mV(PrtFlx-post). The membrane potentials of motoneurons

from pharate adult (PrtFlx-ant )62.2 0.2 mV,PrtFlx-post )62 0 mV) and adult (PrtFlx-ant )64 1.1 mV) were signicantly dierent fromthe larval membrane potentials.

Action potential threshold and maximal action po-tential frequency of individual motoneurons were mea-sured in larvae and pharate adults because theseparameters determine the gradation of motor output inrelation to the input. Multiple steps of positive currentfrom a given membrane potential (larvae )60 mV;pupae and adult )70 mV) were injected and the numberof evoked action potentials were plotted against themembrane potential (Fig. 7Ai, Bi) or directly against the

Fig. 4 Dendritic morphologyof pretarsal exor motoneuronsin pupal stages P1(Ai) and P7(Aii). In pupal stage P1 thedendritic arbors are regressedconsiderably (Ai). Only shortbranches originating from themain neurite are visible. By

later stages (P7) the pretarsalexor motoneurons had re-established many of their den-dritic arbors

Fig. 5AC Input resistance of pretarsal exor motoneurons fromlarva, pharate adult and adult stages. Input resistance was revealed bystep current injection ()3.5 nA to 2 nA) into the motoneuron somataas shown in A. The voltage response was recorded in discontinuouscurrent clamp mode (for details see Material and methods). Inputresistances of larval motoneurons were signicantly higher comparedto pharate adults or adults (C). Note the rectication at negativecurrents larger than )2.5 nA in the larval motoneurons (B). Inpharate adults, the two motoneurons had comparable input resis-tances (B, C). The lines in C indicate signicant dierences betweenlarval and pharate adult stages as determined by a two-tailed U-testafter Wilcoxon, P 0.05. The number of neurons examined is given

in parenthesis

b

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injected current (Fig. 7 Aii, Bii). In larvae, the PrtFlx-ant and PrtFlx-post motoneurons had dierent meanaction potential thresholds although individual valuesvaried considerably (PrtFlx-post: )33.6 4.14, range

)21 to )45 mV, n 5; PrtFlx-ant: )39.0 3.89, range)30 to )46 mV, n 4). However, the mean number ofevoked action potentials was consistently higher inPrtFlx-ant motoneurons (Fig. 7Ai, Aii). Surprisingly,this pattern was reversed in pharate adult stages. Here,the mean number of evoked action potentials in thePrtFlx-post motoneuron exceeded the number of spikesin PrtFlx-ant motoneuron (Fig. 7Bi, Bii). In addition,the PrtFlx-post had a lower threshold compared to thePrtFlx-ant motoneuron as judged from the number ofspike evoked by a given amount of injected current(Fig. 7Bii). A direct comparison of the threshold mem-brane potentials revealed a similar relation (pharate

adult: PrtFlx-post: )47.5 0.29, range )47 to )48 mV,

n 4; PrtFlx-ant: )42.4 3.03, range )37 to )52 mV,n 5; adult: PrtFlx-ant: )40 2.83, range )32 to)44 mV).

To test whether naturally occurring synaptic inputto the motoneurons would reveal similar dierences inthe action potential threshold between PrtFlx-ant andPrtFlx-post, we stimulated the FCO and combined ex-tracellular recordings from the muscle bers of the pre-tarsal exor muscle with intracellular recordings of oneselected PrtFlx motoneuron (Fig. 8). In pharate adult(or adult) the PrtFlx-post motoneuron was reliably ac-tivated by FCO stimulation (Fig. 8Ai, n 22), whereasthe PrtFlx-ant motoneuron reached action potential

threshold only when it was depolarized by intracellularcurrent injection (Fig. 8Aii). In larvae, FCO stimulationwas usually not sucient to activate either motoneuron(n 15).

Discussion

Conservation of target identity

The development of functional and appropriate neuro-muscular connections is dependent on the interactionbetween motoneurons and muscle bers in both verte-

Fig. 6 Membrane time constants (Ai, Aii) and resting membrane

potentials (B) of motoneurons in dierent developmental stages. Themembrane time-constant of PrtFlx-ant and PrtFlx-post was signi-cantly longer in the larva (Ai, Aii) compared to pharate adult or adultstages. The resting membrane potentials of motoneurons shown in Brevealed similar stage-dependent dierences. In larvae, membranepotentials were substantially less hyperpolarized than those ofmotoneurons from pharate adult or adult stages. Lines in Aii and Bindicate signicant dierences between larval and pharate adult stages(two-tailed U-test after Wilcoxon, P 0.05). The number ofmotoneurons examined is given in parenthesis

Fig. 7A, B Activation of pretarsal exor motoneurons in response tointracellular current injections. The number of evoked spikes wasplotted against the resulting membrane potential (Ai, Bi) or directlyagainst the injected current (Aii, Bii). In larvae, the number of evokedspikes was consistently higher in the PrtFlx-ant motoneuron ascompared to the PrtFlx-post motoneuron. In pharate adult or adult(Bi, Bii), this was reversed. Here, the PrtFlx-post motoneuronproduced substantially more spikes at currents above 1 nA. Further-more, the dierence between the PrtFlx-ant- and PrtFlx-postmotoneurons was more pronounced in the pharate adult than in thelarva (Aii, Bii). Note that the injection of current started from dierentholding potentials to reect the naturally occurring resting membranepotentials (larva: )60 mV; pharate adult and adult: )70 mV, seeMaterial and methods)

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brates (Landmesser and Donovan 1984; Vogel and

Landmesser 1987; Grim et al. 1989, Hall and Sanes1993) and invertebrates (Nu esch 1985; Currie and Bate1995; Hegstrom and Truman 1996; Keshishian et al.1996; Bayline et al. 1998; Consoulas and Levine 1997).During leg muscle development in M. sexta, larvalmuscles degenerate completely and are replaced bynewly generated muscles. The degeneration of larvalmuscles is accompanied by regression of motor axonterminals. The innervation of the PrtFlx muscle by thesame motoneurons in larvae and adults may be assuredduring metamorphosis by a sustained interaction be-tween regressed axon terminals and the developingmuscle (Consoulas et. 1996, 1997). The presence ofmotor axons is required for the normal development ofleg muscles during metamorphosis (Luedeman and Le-vine 1996; Consoulas and Levine 1997), and persistentpresynaptic function may be important (Consoulas andLevine 1998). As with the femoral depressor motoneu-ron (Kent and Levine 1993), PrtFlx-ant and PrtFlx-postinnervated muscles with similar locations in the larvaland adult stages.

Dendritic branching

Despite the regression and regrowth of leg motoneurondendrites during metamorphosis (Kent and Levine

1993), the dendritic branching pattern of the pretarsalexor motoneurons in larval and adult stages was sim-ilar. However, the prominent midline branch, which wasonly evident in larval motoneurons, may have a uniquefunction at this stage. For example, a common synapticinput onto the midline branches might be related to thesynchronous activation of right and left leg motoneu-rons during larval locomotion (Johnston and Levine1996b), which is not appropriate for adult walking.Regression of abdominal proleg motoneuron dendritesis clearly related to the loss of a specic synaptic inputand reex behavior during metamorphosis (Streichertand Weeks 1995).

Membrane properties and target function

The intrinsic electrical properties of motoneurons arewell matched to the contractile properties of the musclesthat they innervate (Henneman and Mendel 1981;

Mu ller et al. 1992; Mendell et al. 1994; Rafuse et al.1996; Hughes and Salinas 1999). Motoneuron propertiesare determined by a set of characteristic parameters thatinclude passive and active properties (Rall 1969; Bullock1976; Redman 1976). In vertebrates, fast motoneuronshave lower input resistances, more rapidly conductingaxons and higher rheobase values than intermediate orslow motoneurons (Mendell et al. 1994; Berger et al.1996). In insects, motoneuron responsiveness to injectedcurrent is also correlated with the eect that they exerton the muscle (Meyer and Walcott 1979). It was,therefore, interesting that PrtFlx-ant and PrtFlx-postswitch their relative responses to current injection. This

switch seemed to be correlated with changes in thecontractile properties of the pretarsal exor muscle bersas observed visually. However, further work is necessaryto quantify this observation. If conrmed, this obser-vation, in concert with the electrophysiological changesreported here, suggests that the function of the twopretarsal exor motoneurons reverses during postem-bryonic development perhaps to match alterations in legusage. Interestingly, the soma positions of the slow andfast extensor tibia motoneurons in the locust is reversedin the metathoracic ganglion as compared to the pro- ormesothoracic ganglia which may reect the specializedfunction of the metathoracic leg (Wilson 1979).

Within a given stage, the relative responses of the twopretarsal exor motoneurons to current injection weredierent. In larvae the PrtFlx-post motoneuron had asimilar spiking threshold but a lower maximal ringfrequency than the PrtFlx-ant motoneuron. Althoughreversed, this relation was even more pronounced inmotoneurons of pharate adults or adults. The biggerdierences in spiking threshold and maximal spikingfrequency between PrtFlx-ant and PrtFlx-post moto-neurons from pharate adults and adults may have be-havioral implications. In contrast to the larval legs,which are involved in the relatively simple and slowmovements of crawling or grasping, the adult moth

Fig. 8 Response of PrtFlx motoneurons to stimulation of the FCO inpharate adult. Ramp stimulation (ramp time 0.5 s) of the FCO inpharate adult or adult animals consistently activated the PrtFlx-postmotoneuron only (Ai, middle trace). At resting membrane potentialthe PrtFlx-ant motoneuron receives excitatory input from the FCO.This input was not sucient to reach the spiking threshold (Ai, uppertrace). After depolarizing this motoneuron from )62 mV to )39 mV,the FCO input activated the PrtFlx-ant in phase with the PrtFlx-postmotoneuron (Aii)

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possesses well-articulated legs which must perform avariety of dierent, well co-ordinated movements, in-cluding walking, grooming and grasping. It will be in-teresting to investigate muscle histochemistry in dierentstages to determine whether muscle ber type matchesthe changes seen in the motoneurons (Mu ller et al. 1992;Gu nzel et al. 1993).

Walking behavior is repressed just prior to theemergence of adult silkmoths (Truman 1976). Althoughthis has not been examined in Manduca, we comparedthe electrical properties of PrtFlx-ant motoneuron inpharate adults and emerged adults to determine whetherthis repression was reected in input resistance, ringthreshold or action potential frequency. No dierencesin these parameters were detected, suggesting that be-havioral repression, if present in Manduca, must involvesynaptic inputs to the motoneurons or the action ofneuromodulatory substances that are lacking in thesemi-intact preparation.

The dierence in membrane input resistance and

time-constant between larval and adult motoneuronscould simply re ect the growth of the motoneuronsduring pupal development (cf. Fig. 2Ai, Aii andFig. 2Ci, Cii). Hochner and Spira (1987) reported thatcockroach motoneurons showed decreased input resis-tance during postembryonic growth, which they attrib-uted to an increase in somatic dimensions. Thus, somaticand/or dendritic growth might account for the signi-cant changes in input resistance between identied larvaland adult motoneurons. A similar trend for changes inpassive motoneuron properties and resting membranepotential has been described during vertebrate develop-ment (Viana et al. 1994, Martin-Caraballo and Greer

1999). Changes in the levels of calcium and potassiumcurrents during metamorphosis which were revealed instudies of isolated leg motoneurons (Hayashi and Levine1992; Gru newald and Levine 1998), may also contribute.Whether due to active or passive properties of themotoneurons, changes in the input resistance and time-constant may inuence synaptic integration (Borst andHaag 1996). Further studies must determine how den-dritic remodeling and alterations in intrinsic electricalproperties are related to alterations in synaptic drive andbehavior.

Acknowledgements The authors would like to thank Dr. Christos

Consoulas for helpful discussions. The work was supported byNIH NS24822.

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Comparison of Identified Leg Motoneuron Structure and Function Between Larval and Adult Manduca Sexta