J Comp Physiol A (1997) 180: 597±610
Ó Springer-Verlag 1997
ORIGINAL PAPER
William C. Lemon á Richard B. Levine
Segmentally distributed metamorphic changes in neural circuits controlling abdominal bending in the hawkmoth Manduca sexta
Accepted: 10 January 1997
Abstract During the metamorphosis of Manduca sexta the larval nervous system is reorganized to allow the generation of behaviors that are speci®c to the pupal and adult stages. In some instances, metamorphic changes in neurons that persist from the larval stage are segmentspeci®c and lead to expression of segment-speci®c behavior in later stages. At the larval-pupal transition, the larval abdominal bending behavior, which is distributed throughout the abdomen, changes to the pupal gin trap behavior which is restricted to three abdominal segments. This study suggests that the neural circuit that underlies larval bending undergoes segment speci®c modi®cations to produce the segmentally restricted gin trap behavior. We show, however, that non-gin trap segments go through a developmental change similar to that seen in gin trap segments. Pupal-speci®c motor patterns are produced by stimulation of sensory neurons in abdominal segments that do not have gin traps and cannot produce the gin trap behavior. In particular, sensory stimulation in non-gin trap pupal segments evokes a motor response that is faster than the larval response and that displays the triphasic contralateralipsilateral-contralateral activity pattern that is typical of the pupal gin trap behavior. Despite the alteration of re¯ex activity in all segments, developmental changes in sensory neuron morphology are restricted to those segments that form gin traps. In non-gin trap segments, persistent sensory neurons do not expand their terminal arbors, as do sensory neurons in gin trap segments, yet are capable of eliciting gin trap-like motor responses.
W. C. Lemon1 (&) á R.B. Levine Arizona Research Laboratories, Division of Neurobiology, University of Arizona, Tucson, AZ 85721, USA Present address: Department of Environmental Science, Policy & Management, Division of Insect Biology, 201 Wellman Hall, University of California, Berkeley, CA 94720, USA, Tel.:+1-510/643-1227, Fax: +1-510/642-7428, e-mail:
[email protected] 1
Key words Insect á Larva á Metamorphosis á Nervous system á Pupa Abbreviations A1 ®rst abdominal segment á A2 second abdominal segment á A3 third abdominal segment, etc. á DNA anterior branch of the dorsal nerve á DNL lateral branch of the dorsal nerve á DNP posterior branch of the dorsal nerve á ISM intersegmental muscle á JH juvenile hormone
Introduction During development in segmental organisms such as leeches and insects, a segmentally reiterated population of precursor cells produces a largely similar population of neurons that may become specialized through segment-speci®c cell death and dierentiation (Bate et al. 1981; Doe and Goodman 1985; Shankland and Martindale 1989; Stewart et al. 1991; Weeks and Ernst-Utzschneider 1989; Weeks et al. 1992; reviews: Levine and Macagno 1990; Shankland 1991). There are a variety of mechanisms that lead to segment-speci®c dierentiation of neurons, including intrinsic dierences in segment founder cells (Martindale and Shankland 1990), expression of homeotic genes (Aisemberg et al. 1993), interactions with other neurons (Blair et al. 1990) and interactions with peripheral organs (Baptista et al. 1990; French et al. 1992). Thus, re¯ex or pattern-generating circuits may be segmentally reiterated to produce similar types of behavior in multiple segments or complex whole-animal behavior (Lockery and Kristan 1990; Shaw and Kristan 1995; Wittenberg and Kristan 1992) or become specialized to yield segment-speci®c circuits and behavior (Pearson et al. 1985). Of interest in this context are the neural circuits underlying the bending re¯ex of the hawkmoth Manduca sexta. Observations of abdominal bending movements in larvae and pupae indicate that there are stage-speci®c dierences in the segmental organization of the behav-
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ior. In larvae, tactile stimulation of mechanosensory hairs on the abdomen evokes slow lateral bending movements often involving the entire abdomen. In pupae, however, the evoked bending response is rapid, restricted to three abdominal segments and often involves movement of a single segment. Although the larval behavior involves many abdominal segments the underlying neural circuits undergo hormonally dependent changes during metamorphosis to produce the segmentally restricted ``gin trap'' re¯ex in the pupa (Bate 1973a, b, c; Levine and Truman 1983, 1985; Levine et al. 1985; Waldrop and Levine 1989, 1992). It is not known, however, whether the metamorphic changes responsible for the change in behavior are restricted to those segments that participate in the gin trap re¯ex. During the larval re¯ex, motor neurons that innervate the ipsilateral intersegmental muscle (ISM) motor neurons in several segments are excited and respond with a long, low-frequency barrage of action potentials. Contralateral motor neurons are inhibited or weakly excited (Waldrop and Levine 1989). In the pupa, stimulation of the sensory neurons that persist from the larval stage and later innervate gin trap sensory hairs, causes a triphasic ipsilateral/contralateral motor neuron ®ring pattern that leads to rapid closing and re-opening of the pupal gin trap. Ipsilateral ISM motor neurons innervating muscles of the next anterior segment are activated to produce a brief, high-frequency burst of action potentials followed by pronounced inhibition. Contralateral motor neurons are activated brie¯y during a period of co-activity, then inhibited during ipsilateral activity, then ®re again after the ipsilateral activity (Waldrop and Levine 1989). The neural circuit underlying the pupal gin trap re¯ex develops during the ®nal days of larval life from neurons that were present and functional in the larva (Levine and Truman 1983, 1985; Levine et al. 1985; Waldrop and Levine 1989, 1992). The re¯ex involves sensory neurons that project to the next anterior segmental ganglion, interganglionic and local interneurons, and ISM motor neurons (Levine et al. 1985; Waldrop and Levine 1989, 1992). During the ®nal days of larval life, many of the larval mechanosensory neurons die, but a subset in the region of the developing gin trap persist and expand their terminal arborizations within the central nervous system under steroid hormone control (Levine et al. 1985, 1986; Levine 1989). The change in sensory neuron arborization is associated with a change in the behavioral response to their stimulation. Gin traps are only present in pupal segments A5, A6, and A7, whereas the larval abdominal segments are more uniform. In segments that do not develop gin traps several things may happen at the larval-pupal transition. First, the neural circuits underlying larval abdominal bending could persist into the pupal stage where stimulation of the non-gin trap sensory neurons could produce a larval-like motor response. Indeed, stimulation of pupal gin trap sensory neurons that have been treated with juvenile hormone so that they retain their larval
morphology produces a larval-like motor response (Levine et al. 1986, 1989). Second, the neural circuits responsible for producing the larval behavior could degenerate in non-gin trap segments. Segment speci®c degeneration of neuronal elements has been shown in other neural circuits in Manduca (Jacobs and Weeks 1990; Weeks and Ernst-Utzschneider 1989). Third, the neural circuits in non-gin trap segments could be modi®ed in a unique way to produce a new motor response. Finally, circuit modi®cations that occur in gin trap segments could also occur in the homologous non-gin trap circuits. The purpose of this study was to determine whether the change from the multisegmental larval response to the segmentally restricted pupal response re¯ected segmental dierences in the central nervous system. By examining the evoked motor activity in each abdominal segment in pupae and the corresponding segments in larvae we revealed that metamorphic changes in motor patterns occur in every abdominal segment irrespective of the development of gin traps. Each abdominal segment is capable of producing a pupal speci®c motor pattern, but expression of the gin trap re¯ex is restricted to the three articulated segments.
Materials and methods Animals and behavioral tests Larvae and pupae of Manduca sexta were tested to determine which segments were involved in the stage speci®c abdominal re¯exes. Animals were obtained from a laboratory colony maintained at the University of Arizona. Larvae were fed a de®ned diet (Bell and Joachim 1976) and reared under long day photoperiod (17:7 L:D). Behavioral tests were performed on intact larvae and pupae. To test the bending re¯ex in larvae (third day of the ®fth instar; stage L2), the animal was gently held by its thorax so that the abdomen was pendent. The amount of pressure needed to hold an animal in this manner did not elicit the larva to bend or struggle. Furthermore, holding the animal in this way prevented the abdominal prolegs from contacting a substrate, which enhanced the evoked response to tactile stimulation of the lateral body wall surface. To test the bending re¯ex in pupae, i.e., gin trap re¯ex (Bate 1973a, b, c; Levine and Truman 1983, 1985; Levine et al. 1985; Waldrop and Levine 1989, 1992) animals (®rst day of the pupal stage; stage P0) were placed ventral side up on a ¯at surface. A bending re¯ex was evoked by gently stroking speci®c sensory hairs with a ®ne-tipped paint brush. An eort was made to avoid touching more than one hair at a time and to avoid contact with the surrounding body wall cuticle. Electrophysiology: isolated nerve cords After being cold anesthetized with ice, animals were dissected along the dorsal midline. All dissections and physiological recordings were carried out under saline [mmol á l)1: 140 NaCl, 5 KCl, 4 CaCl2, 28 glucose, 5 HEPES, pH 7.4; Trimmer and Weeks (1989)]. In larvae and pupae, the anterior branch of the dorsal nerve (DNA), and the posterior branch of the dorsal nerve (DNP), on each side of each abdominal ganglion were dissected free from the surrounding tissue. Then the abdominal nerve cord from the second thoracic ganglion to the terminal ganglion was removed from the body and placed in a sylgard-lined dish and submerged in saline. The nerve cord was pinned dorsal side up and one or more ganglia were
599 desheathed with ®ne forceps after 1 min of exposure to a 3% solution of collagenase-dispase (Boehringer-Mannheim). Suction electrodes were used to record motor activity from the posterior branch of the right and left dorsal nerves (DNP) of one or more segments (Fig. 1). In the larval and pupal stages the DNP contains the axons of ®ve motor neurons whose somata are located in the ganglion one segment anterior to the nerve and the ISMs that they innervate (Levine and Truman 1985). These muscles cause the closure of the gin trap in the segment posterior to the DNP. In addition, this nerve contains the axons of two external muscle motor neurons, a large midline neuromodulatory cell with a bifurcating axon, and several mechanosensory axons (Levine and Truman 1985). In the larva there are also two additional ISM motor neurons with axons in the DNP (Levine and Truman 1985). To con®rm that the extracellular motor activity recorded from the DNP represented the temporal properties of the activity of individual motor neurons, intracellular recordings were obtained from ISM motor neuron somata. There are several bilateral pairs of ISM motor neuron somata in the larva that are located along the dorsal midline of the ganglion. All but one pair of these motor neurons persist into the pupal stage. These motor neurons innervate ventral ISMs in both larvae and pupae (Levine and Truman 1985). Motor neuron somata in one or more ganglia were impaled with glass microelectrodes ®lled with 2 mol á l)1 potassium acetate (resistance 50±70 MX). Intracellular and extracellular recordings were ampli®ed with Getting 5A microelectrode ampli®ers and AM Systems dierential ampli®ers, respectively, and stored on video tape using a eight-channel PCM recorder (Vetter Instruments). Suction electrodes were used to electrically stimulate sensory neuron axons in the anterior branch of the right and left dorsal
nerves (DNA) of various segments. In most cases, the sensory nerve was stimulated in the segment posterior to the motor nerves being recorded (Fig. 1). The sensory nerve roots were stimulated with a pulse train (400-ms trains of 0.1-ms pulses at 100 Hz) that was similar to the response of sensory neurons to stimulation of the mechanosensory hairs within the gin traps (Bate 1973a). The stimulus intensity was just above threshold for evoking a motor response in the next anterior segment. Unlike the graded responses produced in response to varying stimulus intensities in some wholebody behaviors (Kristan et al. 1982; Wittenberg and Kristan 1992; Shaw and Kristan 1995), abdominal re¯exes in Manduca pupae appear to be largely an all-or-none event. Higher intensity stimuli evoked motor responses that were indistinguishable from activity evoked at threshold levels and lower intensity stimuli failed to evoke motor responses (Waldrop and Levine 1989). In larvae, responses to stimuli with intensities above threshold were graded but remained distinct from the pupal responses (Waldrop and Levine 1989). In the pupal stage, the DNA contains approximately 20 sensory axons whose somata and dendrites are associated with hairs in the ipsilateral gin trap in the same segment. These sensory neurons are also present in the larva and their axons project into the central nervous system through the same nerves (Levine et al. 1986, 1989; Levine 1989). This nerve also contains a small number of mechanosensory neurons that do not participate in the pupal gin trap response (Bate 1973a). A previous study showed that stimulation of the entire A5 DNA in pupae reliably evoked a response that resembled the response elicited by stimulation of the sub-branch of the A5 DNA, which innervates only the hairs in the gin trap region, or tactile stimulation of the gin trap sensillae (Waldrop and Levine 1989). In the present study, tactile stimulation of the appropriate mechanosensory hairs in larvae and pupae evoked motor responses that closely resembled the motor responses produced by electrical stimulation of the entire DNA. For example, the extracellular responses to tactile stimulation shown in Fig. 3 is similar to the pupal extracellular responses to electrical stimulation shown in Fig. 2. Electrophysiology: semi-intact preparations
Fig. 1 Schematic diagram of the isolated nerve preparation used in these experiments. Three adjacent abdominal ganglia are shown. A suction electrode for stimulation was placed on the right or left sensory nerve (DNA). These nerves carry the axons from the sensory neurons innervating the mechanosensory hairs on the anterior lateral body wall of larvae and pupae. The sensory neuron axons terminate in the next anterior ganglion. Recording electrodes were placed in the next anterior segment on the left and right motor nerves (DNP) that contain the axons of the motor neurons that innervate the ventrolateral intersegmental muscles. Microelectrodes were used to impale the somata of these motor neurons that are located one segment anterior to the muscles they innervate. Diagrams of a motor neuron with a shaded dendritic ®eld and a sensory neuron are shown on the right side
To record from semi-intact pupal preparations, pupae were cold anesthetized and a piece of cuticle was removed on the dorsal surface from A2 to A6 leaving all gin traps intact. The dorsal fat body and the gut were removed. The pupa was secured with pins through A4 into a sylgard-lined dish and the body cavity was ®lled with saline. Suction electrodes were introduced through the window in the dorsal cuticle and placed en passant on the intact left and right DNP of one or more segments. Because input from the large, dorso-lateral abdominal stretch receptor, whose axon is carried in the lateral branch of the dorsal nerve (DNL), can signi®cantly aect ISM motor neuron activity (Lemon and Levine 1993; Tamarkin and Levine 1996), the DNL was cut in all of the semi-intact preparations and was not stimulated in the isolated nerve cords. In the semi-intact preparations, the gin trap sensory hairs were stimulated with the tip of a ®ne brush and motor neuron activity was ampli®ed and recorded as described above. Records of motor activity were transferred from video tape to a computer for analysis using Datapac (Run Technologies). The duration of activity and the latency to activity onset in the extracellular records were calculated. These two variables were used to discriminate between larval and pupal motor responses. Upon repeated stimulation, animals show signs of habituation and sensitization that contribute to variation in the behavior (W.C. Lemon, unpubl. obs.). To decrease the variability of the responses that were analyzed and to maintain independence of samples for statistical analysis, consistent rules were used to determine which of the many motor responses evoked from each preparation was selected for analysis. Preparations that reliably showed activity in three adjacent segments, one of which was the segment of interest, were selected for analysis. Preparations that did not reliably produce responses in three segments were judged to be unhealthy and were not included in the analysis. Because the second stimulus to a given
600 segment was more reliable than the ®rst stimulus at evoking a motor response, only the activity evoked during the second stimulus event was analyzed. Responses subsequent to the second stimulation may have been further subject to habituation or sensitization and, therefore, were not used. Responses to subsequent stimulation of adjacent segments were also not used for the same reason. Thus, each motor response used to construct the average motor patterns and to compare burst durations and latencies was obtained from a separate preparation. This conservative approach failed to include the variety of responses that the individual preparations were capable of producing (Waldrop and Levine 1989). Quanti®ed motor responses were compared with analysis of variance using Statview for the Macintosh. Histology At pupation sensory neurons innervating the gin trap in A5 show an expansion in their terminal arborizations in the CNS (Levine et al. 1985, 1986; Levine 1989). The metamorphic fate of the larval sensory neurons in A4, a segment that does not develop a gin trap, was determined by ®lling of single sensory hair with cobalt in larvae and pupae. A pool formed from petroleum jelly was placed around a sensory hair near the anterior margin of the abdominal segment and ®lled with 2% CoCl2. A hair within the pool was plucked with ®ne forceps, thus damaging and exposing the sensory neuron dendrites, and the pool was sealed with petroleum jelly. The intact animal was kept for four days at 4 °C. Then, the abdominal nerve cord was removed from the animal, ®xed and silver intensi®ed (Davis 1982). Camera lucida drawings of central projections of ®lled axons in whole mounted ganglia were measured by tracing the two-dimensional projections on a digitizing tablet using Sigma Scan software (Jandel Scienti®c). The number of branches per sensory neuron and the total length of all branches per neuron were calculated and compared using ANOVA in conjunction with ScheeÂ's post hoc comparisons.
Results Comparison of the bending re¯ex evoked in dierent abdominal segments: behavioral observations In larvae, gentle tactile stimulation of mechanosensory hairs in any abdominal segment of a larva produced a bending response that involved the entire abdomen. In the typical response, abdominal movement began in the most posterior segments and proceeded anteriorly until every abdominal segment was bent toward the stimulus. A single stimulus evoked a response that lasted from 5 to 15 s. The behaviors evoked by gentle tactile stimulation of dierent abdominal segments did not appear dierent from each other. In pupae, gentle tactile stimulation of the mechanosensory hairs within the gin traps produced a rapid movement in one or more of the three articulated segments (A5±A7) that caused the abdomen to bend toward the stimulus (Lemon and Levine, (1997). In 36% of the cases (24 of 66), the response comprised rapid movements that closed and re-opened the stimulated gin trap without any obvious movement in the other gin trap segments. In 53% of the cases (35 of 66), stimulation of the hairs in one gin trap evoked rapid movements in all three articulated segments, which acted to close each gin
trap ipsilateral to the stimulus as the abdomen bent toward the stimulus. Frequently, this behavior was followed by an opening of all three gin traps and a straightening of the abdomen. In the remaining 11% of the cases (7 of 66), stimulation evoked motor activity that closed only two of the gin traps. The pupal abdomen is not articulated and cannot bend anterior to A5, but tactile stimulation of speci®c mechanosensory hairs (see below) in A4 could produce closure of one or more of the gin traps in the articulated abdominal segments. The response to A4 stimulation was less reliable in that 82% (36 of 44) of the stimuli did not evoke movement. Furthermore, the stimulus intensity needed to evoke activity via A4 was greater than the intensity required to evoke activity via a single gin trap segment. When stimulation of A4 evoked a response, the rapid bending toward the stimulus always involved all three articulated segments, which was sometimes followed by opening of one or more gin traps and straightening of the abdomen. Only rarely (7%, 3 of 44) did stimulation of A4 cause only the ipsilateral gin trap in A5 to close. No overt movement was detected in gin trap segments in pupae when the cuticle in A1±A3 was gently stimulated. These pupal segments are mostly covered by the developing adult wings and lack mechanosensory hairs on their lateral surfaces. However, deformation of the pupal cuticle overlying the wings could evoke abdominal rotation or behavioral responses that were similar to the bending response evoked by stimulation of A4. Comparison of the bending re¯ex evoked in dierent abdominal segments: motor activity in isolated nerve cords In the isolated larval nerve cord, electrical stimulation of the sensory nerve in A5 (a presumptive gin trap segment) evoked motor activity in the DNP both ipsilateral and contralateral to the site of stimulation. The evoked activity consisted of a long lasting, low frequency barrage of action potentials in the DNP of A4 on the ipsilateral side and weaker excitation, weak inhibition, or no apparent activity, on the contralateral side (Fig. 2A). The evoked activity in the isolated nerve cord resembled the motor responses recorded in semi-intact larval preparations that resulted in a slow lateral ¯exion of all the abdominal segments (Levine et al. 1985; Waldrop and Levine 1989). We de®ne this long duration motor response evoked by stimulation of sensory neurons in a presumptive gin trap segment as the ``larval abdominal bending'' motor pattern. In the larval isolated nerve cord, electrical stimulation of the sensory nerve in segment A4 (not a presumptive gin trap segment) evoked a motor response in the DNP of A3 that resembled the larval abdominal bending motor pattern in A4 evoked by stimulation of segment A5 (Fig. 2B). The typical response to stimulation of A4 was a long lasting burst of action potentials on the
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Fig. 2A±D Responses of motor neurons innervating ISMs in A4 and A3 to electrical stimulation of the sensory nerve in A5 (A, C) and A4 (B, D), respectively. The DNA was stimulated at 100 Hz for 400 ms (bar at bottom). Asterisks indicate large stimulus artifacts in the recordings. Intracellular and extracellular responses from motor neurons innervating muscles ipsilateral to the stimulus (Ipsi) and muscles contralateral to the stimulus (Contra) one segment anterior of the site of stimulation are shown for a ®fth instar larva (A, B) and a ®rst day pupa (C, D). The time scales of the larval and pupal records are the same: A stimulation of larval segment A5 produces a long duration response that lacks alternating bursts; B stimulation of larval segment A4 also produces a long duration response that lacks alternating bursts; C stimulation of pupal segment A5, which has a gin trap, produces a short duration response with alternating bursts of activity; D stimulation of pupal segment A4, which does not have a gin trap, also produces a short duration response with alternating bursts of activity. The intracellular records show that the activity of individual motor neurons is faithfully represented by the extracellular records. Intracellular calibration bar 10 mV
ipsilateral side and a weaker burst, weak inhibition, or no activity, on the contralateral side. Because stimulation in either a presumptive gin trap segment (A5) or a non-presumptive gin trap segment (A4) in isolated larval nerve cords evoked similar motor patterns and similar behaviors, we also de®ne the response evoked by stimulation of sensory neurons in A4 as the ``larval abdominal bending'' motor pattern. In the isolated pupal nerve cord, electrical stimulation of the sensory nerve that innervates the gin trap hairs in
A5 evoked a brief motor response in A4, both ipsilateral and contralateral to the site of stimulation (Fig. 2C). The evoked response was characterized by a brief burst of motor activity on the side contralateral to the stimulation followed by contralateral inhibition and a longer burst of activity on the ipsilateral side. This was followed by a prolonged burst of activity on the contralateral side. The evoked activity in isolated pupal nerve cords is similar to the motor responses recorded in semi-intact pupal preparations, which appear to be correlated with closing and opening of the stimulated gin trap (Bate 1973b; Waldrop and Levine 1989; Lemon and Levine 1997). We de®ne this contralateral-ipsilateral-contralateral motor response evoked by stimulation of sensory neurons in a gin trap segment as the ``gin trap'' motor pattern. During the larval-pupal metamorphic transition, abdominal segments A5±A7 become morphologically distinct from the more anterior abdominal segments A1± A4. Externally, A5±A7 develop articulations at the anterior border of each segment with specialized gin trap structures, which are not present in A1±A4. Abdominal segments A1±A4 are beneath the developing adult wings and cannot move. Although A4 does not develop a gin trap, electrical stimulation of A4 sensory axons in pupae always (15 of 15) produced a motor response that was similar to the gin trap motor pattern evoked by A5
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stimulation. The response in the next anterior segment evoked by A4 stimulation was short in duration and showed the temporal patterning characteristic of the gin trap motor pattern (Fig. 2D). These results reveal that although the sensory neurons whose axons are carried in the A4 DNA do not innervate gin trap hairs in pupae, they are still capable of eliciting a gin trap-like motor response in motor neurons of the next anterior segment. This suggests that elements of the neural circuit that underlies abdominal bending motor patterns in larvae were modi®ed in A4 at the larval-pupal transition even though this segment did not develop a gin trap. We de®ne the characteristically pupal contralateral-ipsilateral-contralateral motor response to stimulation of segments that do not contain gin traps as a ``gin trap-like'' motor pattern. The typical response (27 of 27) to stimulation of segments A4 or A5 in pupae was characterized by a single ipsilateral burst of motor activity interposed between two contralateral bursts of activity. This was in contrast to the primarily ipsilateral response to stimulation of segments A4 or A5 in larvae. The distinct coordination patterns were used to distinguish between larval and pupal motor patterns. Comparison of the bending re¯ex evoked in dierent abdominal segments: motor activity in semi-intact preparations To con®rm that tactile stimulation of sensory hairs evokes motor responses that are similar to activity evoked by electrical stimulation, extracellular recordings of motor neuron activity were obtained from semi-intact pupae. The semi-intact preparation retained the lateral body wall and intact DNA nerves while input from the other major branch of the dorsal nerve (DNL), which includes input from the dorso-lateral abdominal stretch receptor, was removed. Sensory input from the abdominal stretch receptor is believed to play a role in the production of re¯ex bending in intact animals (Lemon and Levine 1992). Stimuli delivered with a ®ne-tipped brush to either the anterior-lateral body wall of A4 or the gin trap sensillae in A5 in semi-intact pupal preparations (n 3), evoked a contralateral-ipsilateral-contralateral motor response that resembled the temporal patterns evoked by electrical stimulation of the entire A4 or A5 DNA, respectively (Fig. 3). In parallel with our behavioral data in intact pupae, gentle tactile stimulation of A4 in semi-intact preparations evoked motor responses in only two of six stimulations as compared to six of six for A5 stimulation. In contrast, electrical stimulation of the A4 DNA reliably evoked a gin traplike motor response. Electrical stimulation of the DNA may simulate intense tactile stimulation of sensory hairs and recruit more of the sensory neurons than normally would be activated by gentle tactile stimulation of sensory hairs. Furthermore, synchronous stimulation of all the DNA sensory neurons may evoke a more reliable
Fig. 3A, B Extracellular records of responses of motor neurons innervating ISMs in A3 (A) and A4 (B) in a semi-intact preparation to tactile stimulation of the sensory hairs in the next posterior segment (A4 and A5, respectively). The stimulus used to evoke these responses (the tip of a ®ne brush) normally evokes a rapid closure and opening of one or more gin traps in an intact animal. Note the single ipsilateral burst of motor activity interposed between two bursts of contralateral activity. Comparison of responses to tactile stimulation and responses to electrical stimulation suggests that electrical stimulation evokes motor responses similar in duration and coordination to those evoked by tactile stimulation
motor response than would be produced by the asynchronous recruitment of sensory neurons during natural sensory hair stimulation. Comparison of the bending re¯ex evoked by stimulation of dierent abdominal segments In isolated larval nerve cords, stimulation of any abdominal DNA branch induced a motor response in the next anterior abdominal segment. The evoked activity in each segment was characterized by a long duration burst of activity in ipsilateral motor neurons of the next anterior segment, and more variable levels of contralateral motor activity (Fig. 4). In contrast to the posterior abdominal segments, the evoked motor response in the more anterior abdominal segments (A1±A3) began with a variable delay after the stimulus and had more pronounced contralateral activity. Typically, A1 produced responses with the longest delays and the most pronounced contralateral activity. In some responses the contralateral and ipsilateral activity were of similar intensities and durations (e.g., A1 in Fig. 4). In isolated pupal nerve cords, stimulation of the DNA in any abdominal segment also produced a response in the next anterior segment. The response in each segment showed the gin trap motor pattern: two brief bursts of contralateral activity with an interposed single burst of ipsilateral activity (Fig. 5). In Fig. 5, the intracellular
603 Fig. 4 Typical responses of larval ISM motor neurons to stimulation of the sensory nerve in the next posterior segment. All these records were obtained from a single ®fth instar larva. Each record shows extracellular contralateral (Contra DNP) and ipsilateral (Ipsi DNP) recordings and an intracellular ipsilateral (Ipsi ISM MN) recording of activity in motor neurons innervating muscles in one segment. The sensory nerve in the next posterior segment was stimulated at 100 Hz for 400 ms (bar and corresponding stimulus artifact). The responses in each segment are prolonged and do not show alternating bursts of activity. In segments A1±A3 the motor response is preceded by a variable delay and there is greater activity in the contralateral motor neurons than there is in segments A4±A7. Intracellular calibration bar 10 mV
records from the contralateral motor neurons clearly show the two periods of contralateral activity. In contrast to larval responses (Fig. 4), the pupal responses were more variable within abdominal segments. In many responses, the contralateral-ipsilateral alternation was not strictly maintained and even showed periods of contralateral-ipsilateral co-activity (e.g., A1 and A3 in Fig. 5). In some responses, one of the bursts was absent, but the duration of the remaining bursts of motor activity remained characteristically brief (e.g., A7 in Fig. 5) compared to larval responses. The average motor patterns produced by each of the abdominal segments of larvae and pupae showed distinct stage speci®c dierences in latency to activity onset
and duration (Fig. 6). A comparison of the larval and pupal motor patterns revealed dierences in burst durations between developmental stages and similarities among abdominal segments. In every abdominal segment of larval isolated nerve cords the ipsilateral and contralateral motor neurons produced long bursts of activity (Fig. 6C). There were no signi®cant dierences detected in ipsilateral burst duration among segments (F 2:11, P 0:0738) or in contralateral burst duration among segments (F 1:73, P 0:154) in isolated larval nerve cords, although ipsilateral bursts were longer than contralateral bursts (F 20:0, P 0:0001). In pupal isolated nerve cords (Fig. 6D), the duration of the ®rst contralateral burst did not dier signi®cantly among the
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Fig. 5 Typical responses of pupal ISM motor neurons to stimulation of the sensory nerve in the next posterior segment. All these records were obtained from a single ®rst day pupa. Each record shows extracellular ipsilateral (Ipsi DNP) and contralateral (Contra DNP) recordings and an intracellular contralateral (Contra ISM MN) recording of activity in motor neurons innervating muscles in one segment. The stimulation paradigm is identical to that in Fig. 4. The responses in each segment have a much shorter duration than those of larvae and show alternating bursts of activity. There are typically two bursts of activity in the contralateral side that alternate with one burst of activity on the ipsilateral side. The intracellular records highlight these double bursts. Intracellular calibration bar 10 mV
abdominal segments (F 1:67, P 0:143). However, both the ipsilateral burst duration (F 10:1, P 0:0001) and the duration of the second contralateral burst (F 5:57, P 0:0001) diered signi®cantly among the abdominal segments in pupae. There were distinct stage speci®c features of the motor patterns produced in each abdominal segment (Fig. 6). Comparisons between stages revealed that larval ipsilateral burst durations in any given segment were always signi®cantly longer than pupal ipsilateral burst durations (F 210:9, P 0:0001), the ®rst pupal contralateral burst duration (F 27:01, P 0:0001) and the second pupal contralateral burst (F 61:54, P 0:0001). The contralateral larval bursts were also longer than pupal ipsilateral bursts (F 47:8, P 0:0001), the ®rst pupal contralateral bursts (F 101:0, P 0:0001) and the second pupal contralateral burst (F 61:5, P 0:0001). There were also dierences in timing of activity between larvae and pupae. With the exception of A7, contralateral and ipsilateral activity in larval isolated nerve cords overlapped substantially and often showed synchronous onsets of activity. In contrast, the onsets of ipsilateral and contralateral activity in pupal nerve cords were asynchronous and, with the exception of A7, the two sides often alternated with each other. Although the onsets of ipsilateral activity and the second contralateral burst of activity were also asynchronous, they showed substantially overlapping periods of activity. The activity produced by A7 in response to stimulation of the DNA in A8 diered from other segments in both the larval and pupal stages. In larvae, stimulation evoked only an ipsilateral response in A7. In pupae, A7 activity showed near synchronous onsets of the ®rst contralateral burst and the ipsilateral burst of activity and a pronounced advance in the onset of the second contralateral burst of activity. Although the response in A7 showed three bursts of activity, it lacked the alternating pattern between ipsilateral activity and the ®rst burst of contralateral activity seen in other segments. However, in all the pupal abdominal segments including A7, the second contralateral burst never occurred simultaneously with, or before, the ipsilateral activity. In summary, although there are dierences among segments in larvae and pupae, there are stage speci®c similarities in the responses. Thus, metamorphic reorganization of bending re¯ex circuits occurred throughout the abdomen and was not restricted to articulated segments that developed gin traps. Multisegmental responses to stimulation of a single segment As described above (also see Levine et al. 1985) stimulation of the mechanosensory hairs in a single segment in intact larvae produced a bending behavior that involved
605
Fig. 6A±D Quanti®cation of motor patterns: A, B the quanti®ed responses from many animals were used to create average motor patterns produced by each segment. The quanti®ed responses were also used to compare coordination of motor activity during larval and pupal responses. To maintain sample independence and to avoid sensitization, one response from each nerve cord was quanti®ed and included in the statistical analyses (see Materials and methods); C average motor patterns from many isolated larval nerve cords. The left end of each thick bar represents the mean onset of motor activity and the associated thin bar represents the standard error of the mean. The length of each thick bar represents the mean duration of motor activity and the thin bar on the right represents the standard error of the mean. The thin bar at the lower left of each average motor response represents the stimulus (400 ms, 100 Hz) applied to the next posterior segment. Note the long mean duration of the motor activity elicited from each segment and the substantial amount of overlap between ipsilateral (Ipsi) and contralateral (Contra) activity. The data shown are from 47 dierent nerve cords that reliably produced responses to stimulation of three adjacent segments; D average motor patterns from 63 isolated pupal nerve cords. Note that the mean durations of the pupal responses are much shorter than those seen in larvae and there is less overlap between ipsilateral and contralateral bursts of activity. One consistent feature of pupal responses is the double burst of contralateral activity that is not seen in larvae
the entire abdomen. In isolated nerve cords, stimulation of any single abdominal segment always elicited motor responses in many segments (eight of eight preparations), both anterior and posterior to the stimulated segment (Fig. 7A). For example, stimulation of the A5 DNA produced motor responses in the ipsilateral DNP in each segment. Furthermore, the majority of the preparations (®ve of eight) showed a posterior to anterior delay in the onset of motor activity such that the onset of A7 activity coincided with the stimulus while the onset of activity in more anterior segments (e.g., A2) was delayed (Fig. 7A). Intact larvae also show a posterior to anterior progression of abdominal movements during bending behavior. In the example shown in Fig. 7A, the delay in onset of activity in A2 with respect to the onset of activity in A7 was approximately 650 ms. Although the nerve cord is quite ¯exible and can be stretched at least 50% farther, on average, the distance from A2 to A7 in third-day larvae is approximately
606
Fig. 7 A Stimulation of one larval sensory nerve evokes motor responses in many segments. Extracellular motor neuron responses were simultaneously recorded from each right DNP in segments A2±A7 in a ®fth instar larva. The ipsilateral sensory nerve in segment A5 was stimulated at 100 Hz for 400 ms (bar). Each segment produced a long duration response similar to the ipsilateral responses seen in Figs. 2 and 4. B Stimulation of one pupal sensory nerve evokes motor responses in many segments. The recording paradigm was identical to that in Fig. 7A, but the stimulus was applied to the contralateral sensory nerve to highlight the contralateral double bursts. Each segment produced two short duration bursts of activity similar to the contralateral responses seen in Figs. 2 and 5
50 mm. If the delay between segments were due solely to action potential conduction time, the conduction velocity in this example would have to be a very slow 77 mm á s)1. This suggests that the delay in motor recruitment between segments was not due simply to conduction time. In intact pupae, evoked bending movements are restricted to the three articulated abdominal segments, A5, A6 and A7 (Bate 1973b; Waldrop and Levine 1989). However, in the isolated pupal nerve cord, stimulation of a single pupal segment also produced motor responses in many abdominal segments both anterior and posterior to the stimulated segment (12 of 12 preparations; Fig. 7B). In contrast to larvae, there was no pronounced posterior to anterior increase in delay to the onset of motor activity in pupae (Fig. 7B). The coordination of multisegmental responses in the pupa is discussed in detail elsewhere (Lemon and Levine, 1997). Identi®cation of sensory neurons in non-gin trap segments Cobalt back®lls of the sensory neurons were used to identify the locations of sensory hairs that are innervated by neurons with their axons in the DNA. This information was compared to the behavioral response to tactile stimulation of speci®c sensory hairs in intact animals. In pupal segments containing gin traps (A5±7), the sensory hairs that were innervated by the DNA and
were capable of evoking a gin trap re¯ex were restricted to the gin traps and the immediately surrounding cuticle [Fig. 8a, see also Bate (1973a)]. Other hairs on the body wall did not evoke a behavioral response when stimulated individually. By contrast, the sensory hairs innervated by the A4 DNA that were capable of producing a gin trap-like behavior when stimulated were restricted to an area anterior and ventral of the spiracle. This area was slightly more ventral than would be expected if sensory neuron locations were strictly homologous among segments. Unlike the large sensory hairs in the gin traps, the sensory hairs that were innervated by sensory neurons in the DNA of A4 were morphologically indistinguishable from short, thin hairs elsewhere on the body surface that did not elicit gin trap-like re¯exes. Each of these hairs was innervated by a single sensory neuron with a peripheral morphology similar to those in gin trap segments (Fig. 8b). Although tactile stimulation of the pupal cuticle in segments A1±A3 did not produce obvious movement, electrical stimulation of the pupal sensory nerve in these segments produced the characteristic pupal motor response. In segments A1±A3 much of the lateral body wall of the pupa is covered by the developing adult wings. To determine whether sensory structures were present in these segments the sensory nerves were ®lled toward the periphery with cobalt. Cobalt ®lls of the DNA in these segments revealed sensory neurons with dendrites beneath the cuticle, but which did not innervate any cuticular hairs (Fig. 8C). Some of these sensory dendrites may respond to deformation of the cuticle, because gin trap-like behavior and abdominal rotation can be evoked by deforming the wings. Anterograde cobalt ®lls of sensory neurons innervating mechanosensory hairs on the wall of A4 and A5 in larvae and pupae showed segment-speci®c changes in the sensory terminals in the CNS. Sensory neurons innervating hairs within the gin trap in A5 had signi®cantly higher total length and number of branches of the terminal arborizations in A4 than did the sensory neurons in larvae [Fig. 8D±E, Table 1; see also Levine (1989); Levine et al. (1985,1986)]. Sensory neurons in-
607
Fig. 8 A Location of sensory hairs that are able to elicit gin trap-like re¯exes in segments that have gin traps and a segment that does not have a gin trap (shaded regions). Cobalt ®lls of the entire DNA toward the periphery and behavioral assays indicate that the sensory neurons that evoke the pupal behavior and have axons in the DNA in segment A4 innervate mechanosensory hairs that are located more ventrally than the sensory hairs innervated by the DNA in segments that contain gin traps. B In segment A4 sensory neurons in the DNA innervate mechanosensory hairs on the anterior lateral body wall. The neurons were ®lled by exposing the cut distal end of the entire DNA to cobalt. Each neuron sends a dendrite into a single hair and an axon, which cannot be seen in this picture, into the central nervous system. The small clear circles surrounding the ends of the dendrites are the cuticular sockets at the base of the hairs. Scale bar 50 lm. C In A3, sensory neurons can be seen under the developing wing of the pupa. This sensory neuron was ®lled by exposing the DNA in segment A3 to cobalt. Although there are no mechanosensory hairs under the wing, the sensory neurons have dendrites that terminate in the epidermis beneath the pupal cuticle. Scale bar 50 lm. D±G Camera lucida drawings of the terminal arborizations of cobalt ®lled sensory neurons in larvae and pupae. Sensory neurons that have their terminals in the ganglion in A4 innervate mechanosensory hairs on the lateral anterior margin of A5, which does develop a gin trap. These gin trap neurons signi®cantly expand their terminal arbors at pupation (D±E, see Table 1). Sensory neurons that have their terminals in the ganglion in A3 innervate mechanosensory hairs on the lateral anterior margin of A4, which does not develop a gin trap. These neurons do not expand their terminal arbors at the larval-pupal transition (F±G). Scale bar 100 lm
nervating hairs in A4 did not have signi®cantly dierent terminal arborizations in A3 in larvae and pupae (Fig. 8F±G, Table 1).
Discussion During development in segmental organisms such as Manduca sexta, segmentally reiterated populations of neurons become specialized by segment speci®c cell death and dierentiation. For example, the larvae of Manduca have abdominal prolegs in A3±A6 that show a withdrawal re¯ex in response to stimulation of mechanosensory hairs on the prolegs (Weeks and Jacobs 1987; Trimmer and Weeks 1991). At the larval-pupal transition, the external proleg structures and the muscles are lost in all segments; however, there are segmentspeci®c fates of the motor neurons responsible for the proleg withdrawal (Weeks and Ernst-Utzschneider 1989), with the motor neurons in some segments persisting to participate in dierent behaviors at later stages (Lubischer et al. 1995). The abdominal bending behavior is not lost but becomes modi®ed at the larval-pupal
608 Table 1 Total length and number of branches in terminal arborizations of sensory neurons that innervate hairs in gin trap segments (A5) and sensory neurons that innervate hairs in non-gin trap segments (A4) in larvae and pupae
Larvae A4 Larvae A5 Pupae A4 Pupae A5
Length (lm, mean SE)
No. of branches (mean SE)
n
518 537 461 850
26 27 22 54
4 4 7 8
15 48 57 53a
F 13:12 P 0:0001
2.7 6.0 3.1 6.3a
F 9:46 P 0:0005
a Indicates that the value is signi®cantly dierent than neurons in all other groups (post hoc Schee F-test, P < 0.05)
transition (Bate 1973a, b, c; Levine and Truman 1983, 1985; Levine et al. 1985; Waldrop and Levine 1989, 1992). Every abdominal segment in the larva produced the motor pattern responsible for larval bending. Stimulation of the sensory nerve in any larval segment evoked stereotyped larval motor activity in the next anterior segment and in more distant segments. Somewhat surprisingly, we also found that, although there are gin traps in only A5±A7, every abdominal segment in the pupa produced a pupal-speci®c motor pattern similar to the motor pattern responsible for the gin trap re¯ex. Stimulation of the sensory nerve in any pupal abdominal segment, including those that did not have gin traps, evoked a gin trap-like motor pattern in the next anterior segment and in more distant segments. Thus, the neural circuits underlying bending in every abdominal segment undergo modi®cations at the larval-pupal transition. Metamorphic changes in the larval bending circuits apparently occur throughout the abdominal nervous system and produce neural circuits capable of producing new pupal motor patterns in every abdominal segment. Although the neural circuits in each abdominal segment are modi®ed at the larval-pupal transition, the motor patterns produced by the various segments within a stage are not identical. Both larval and pupal motor responses show segmental variation. Stimulation of larval segments A2±A4 produces a response that is preceded by a variable delay which is not seen when other segments are stimulated and prolonged synchronous bilateral motor activity. The function of the synchronous bilateral motor activity in the anterior segments and the variable delay among segments in larvae is not clear. One possibility is that synchronous bilateral activity produces stiening and that these segments stien as well as bend. Some pupal responses also show overlap between ipsilateral and contralateral bursts. The resulting co-activity may also produce stiening of a segment in addition to bending (Lemon and Levine 1997). The larval pattern of coordination is modi®ed in every abdominal segment at that larval-pupal transition producing new pupal motor patterns. The
pupal motor patterns also are not identical in every segment, although there are consistencies in the way burst durations and patterns are coordinated. One obvious segmental dierence in the pupal responses was that the responses were less reliable in non-gin trap segments. Thus, the dierences in coordination and reliability seen between larval and pupal motor patterns suggest that similar, but not identical, developmental changes were occurring throughout the abdomen. In response to tactile stimulation of sensory hairs in the larva, motor responses were produced throughout the abdomen. This response was consistent with the behavioral response to sensory hair stimulation, which involves the entire abdomen (Levine et al. 1985; Waldrop and Levine 1989). Pupal motor responses were also produced throughout the abdomen, although only A5±A7 are capable of bending. The restriction of the behavior to these three segments appears to be a consequence of the changes in the biomechanical properties of the cuticle. The larvae of Manduca possesses a ¯exible hydrostatic exoskeleton that can bend and stretch along the length of the abdomen. The pupal cuticle, however, tans and becomes rigid within minutes of pupal ecdysis. The only ¯exible articulations in the entire exoskeleton lie at the abdominal segment boundaries that also contain the gin traps. Thus, motor activity evoked by sensory hair stimulation occurs in every pupal abdominal segment, but biomechanical constraints restrict the movement to the three articulated gin trap segments. The movement is further restricted to the segment anterior to the stimulated gin trap by the phasing of the pupal motor responses (Lemon and Levine 1997). In isolated nerve cords, motor responses could be elicited in every abdominal segment, but in intact pupae, gentle tactile stimulation of the cuticle in A2±A3 did not evoke any bending behavior. The dendrites of the sensory neurons in the DNA in A2±A3 end blindly on the abdominal wall underneath the developing adult wings, and only deformation of the cuticle could evoke strong re¯ex behavior. Similarly, electrical stimulation of the A4 DNA in pupal nerve cords evoked a triphasic gin trap-like motor response much more reliably than did tactile stimulation of A4 sensillae in intact pupae. In semi-intact pupae, however, the motor responses to tactile stimulation of A4 sensillae resembled those evoked by electrical stimulation of the A4 DNA, although they were evoked less reliably. Thus, electrical stimulation probably represented a powerful stimulus, but evoked the same motor response as that caused by natural stimulation. Cobalt ®lls of single sensory hairs into the central nervous system showed that pupal sensory neurons innervating hairs in gin traps develop larger terminal arborizations than sensory neurons innervating homologous hairs in segments that do not have gin traps. However, stimulation of both types of sensory neurons produced a motor response that was characteristically dierent from that in larvae. This suggests the sensory neurons have segment speci®c fates, and that the de-
609
velopmental changes in the motor response to stimulation of these sensory neurons are not entirely dependent on changes in the size of the terminal arborizations. Previous studies have concluded that changes in sensory neuron morphology are necessary to produce the gin trap behavior because JH analog treated gin trap sensory neurons in larval-pupal mosaics retained larvallike terminal arborizations and evoked a larval-like motor response (Levine et al. 1986, 1989). This expansion is not sucient, however, for the production of the gin trap re¯ex because in the converse situation, ecdysteroid treated sensory neurons expanded their arbors within the otherwise larval CNS, yet did not evoke the pupal re¯ex (Levine 1989; Levine et al. 1989). The signi®cance of the expanded arborizations of sensory neurons in gin trap segments may lie in their unique ability to evoke segmentally restricted gin trap closure (Lemon and Levine 1997) and in their ability to evoke the behavior more reliably with gentle tactile stimulation than sensory neurons in non-gin trap segments. Thus, although there is some degree of specialization in the gin trap segments, developmental changes in the central nervous system that are responsible for producing unique pupal re¯exes are not segmentally restricted. The signi®cance of the reiteration of this re¯ex circuit in non-gin trap segments is unclear, but may be related to the production of multisegmental bending and rotational movements that are common in Manduca pupae that are disturbed in their burrows. Acknowledgements We thank Drs. Rebecca Johnston, Andrea Novicki and Brian Waldrop and two anonymous reviewers for their comments on earlier versions of the manuscript. Thanks to Kimiko Della Croce for measuring the neuronal arborizations. This work was supported by grants from the NSF (BNS 11174) and NIH (NS24822) to RBL. WCL was supported by a postdoctoral fellowship (NS 09008) and a training grant (NS 07309) from NIH.
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