Development of spontaneous and evoked behaviors in the medicinal leech

Development of Spontaneous and EvokedBehaviors in the Medicinal Leech

SHIRLEY A. REYNOLDS, KATHLEEN A. FRENCH,* ANDREAS BAADER,

AND WILLIAM B. KRISTAN, JR.

Department of Biology , University of California at San Diego,La Jolla, California 92093–0357

ABSTRACTThe ontogeny of behavior in an organism must reflect developmental events in the

nervous system, and it thus provides a noninvasive measure of neuronal development. Thisapproach may be particularly fruitful in the medicinal leech because the neuronal basis ofseveral behaviors has been characterized in adult leeches, providing a rich backgroundagainst which behavioral development can be interpreted. We have investigated the order inwhich behaviors arise during the period of embryonic development and have determined thetime at which each behavior is first expressed. Some behaviors, such as lateral ridgeformation, germinal plate bending, spiral twisting, and sidewinding, were produced spontane-ously by embryos. Others, such as shortening, circumferential indentation, local bending, andelongation, occurred only when they were elicited by weak mechanical stimulation. Suchstimulation rarely evoked a behavioral response in young embryos (at 45% of the timerequired for complete embryonic development, 45% ED), but by 80% ED embryos responded tonearly 100% of the stimuli presented. In embryos older than 50% ED, the behavior mostfrequently evoked by stimulation of the anterior end, the posterior end, or the rear sucker wasshortening. Stimulation of the midbody usually evoked behavior other than shortening,illustrating that the body was behaviorally compartmentalized, at least in part. Somebehaviors observed during embryogenesis are never seen in adult leeches. For example, inresponse to stimulation of the midbody, young embryos produced a behavior that we havecalled ‘‘circumferential indentation,’’ whereas older embryos produced local bending, aresponse previously described for adults. The switch from circumferential indentation to localbending may signal the formation of new synaptic connections. J. Comp. Neurol. 402:168–180,1998. r 1998 Wiley-Liss, Inc.

Indexing terms: nervous system; embryogenesis;ontogeny, neuroethology; Hirudo

The ability of the central nervous system to produceadaptive behaviors depends absolutely on the precisionwith which neurons communicate with one another, andthe developmental mechanisms that produce such preciseconnections have been investigated intensively. Impres-sive progress has been made in elucidating the processesand molecules that allow axons and dendrites of develop-ing neurons to navigate appropriately and to recognizeappropriate synaptic targets, in both vertebrate and inver-tebrate species (Kuwada, 1985; Kuwada et al., 1990;Goodman and Shatz, 1993; Harris and Holt, 1995; MacFar-lane et al., 1995; Chien and Harris, 1996). However,anatomical and electrophysiological investigations cannotcompletely reflect the ability of the developing nervoussystem to generate behavior because they are often limitedto considering only a few neurons at a time. The mostdirect and unequivocal assay for the presence of an arrayof precise and appropriate connections is to observe an

embryo’s behavior as it develops, although this task hasbeen difficult or even impossible in many species.

In species for which this approach has succeeded, obser-vations of behavior as it develops have provided valuableinsight into the function of developing nervous systems. Insome species, such as frogs (Stehouwer and Farel, 1985;Roberts and Tunstall, 1994; Perrins and Roberts,1995a,b,c), crickets (Bentley and Hoy, 1970), Manduca(Weeks and Levine, 1995; Weeks et al., 1997), and themollusk Aplysia (Carew, 1989) a developing individualpasses through intermediate stages in which it must fend

Grant sponsor: NIMH; Grant number: MH43396; Grant sponsor: NSF;Grant number: IBN11432.

*Correspondence to: Kathleen A. French, Dept. of Biology, 0357, U.C.S.D.,9500 Gilman Drive, La Jolla, CA 92093–0357. E-mail: [email protected]

Received 28 October 1997; Revised 23 July 1998; Accepted 28 July 1998

THE JOURNAL OF COMPARATIVE NEUROLOGY 402:168–180 (1998)

r 1998 WILEY-LISS, INC.

for itself in the environment, so behavior during thesestages is both readily observable and useful for evaluatingwhich neurons have established functional connections.

For other species, such as chick, methods have long beenavailable for experimental manipulations and long-termobservation of development (Hamburger and Balaban,1963; Hamburger et al., 1965), even though the embryosare not naturally exposed to the external environment.Chicks actively hatch out of their shells, so the survival ofa chick depends directly on its ability to perform this vitalbehavior, and the study of the neuronal basis of hatchingbehavior has added to our understanding of how motoroutput becomes organized (Bekoff, 1992; Johnston andBekoff, 1996).

Most observations of a developing nervous system haveapproached it either from the sensory side (Allendoerferand Shatz, 1994; Knudsen and Brainard, 1995; Shatz,1996; Antonini and Stryker, 1996; Hensch and Stryker,1996) or from the motor side (Bekoff, 1992; Perrins andRoberts, 1995a,b,c). It has been more difficult to study thedevelopment of complete behavioral circuitry, includinginterneurons. Simpler nervous systems potentially offerthe opportunity to expand the approach to the completecircuits. For example, in Manduca sexta it has beenpossible to study the development of neurons that mediatelarval (Novicki and Weeks, 1995, 1996; Tamarkin andLevine, 1996; Wood et al., 1997) and pupal (Lemon andLevine, 1997a,b) behaviors, including interneurons, andextending this type of study should add to our understand-ing how circuits are constructed.

Behavior does not have to be expressed in an organism’snormal embryonic repertoire to provide useful informationabout the development of the nervous and muscularsystems. Embryos of the medicinal leech, Hirudo medicina-lis, normally spend their entire period of embryogenesisinside an eggcase, called a ‘‘cocoon,’’ protected from theouter world (Stent et al., 1992), yet they will surviveoutside the eggcase if given appropriate conditions, andthey produce recognizable behaviors before developmenthas proceeded halfway. Several of these behaviors arerecognizable either from the adult repertoire or as compo-nents of more complex adult behaviors, and the richneuroethological background available for leech behaviorfacilitates the interpretation of behavioral ontogeny inneuronal terms.

The underlying neuronal activity that produces severalbehaviors in Hirudo has been described in more or lessdetail. These behaviors include local bending (Lockery andKristan, 1990; Kristan et al., 1995), shortening (Witten-berg and Kristan, 1992; Shaw and Kristan, 1995), swim-ming (Brodfuehrer et al., 1995), crawling (Baader andKristan, 1995), and the generation of the heartbeat (Cala-brese et al., 1995). In addition, the early development ofthe leech (Fernandez and Stent, 1982; Stent et al., 1992),the formation its nervous system (Stent et al., 1992), andthe differentiation of several identified neurons (Frenchand Kristan, 1994; Wolszon, 1995; Wolszon et al., 1995;Shankland, 1995; Jellies and Johansen, 1995) has beendescribed in great detail. This combination of well-described neuronal circuitry and detailed understandingof embryogenesis makes the medicinal leech an idealsystem in which to investigate how individual neuronsacquire their adult phenotype and connect to one anotherproperly to form behavioral circuits. This study character-izes the onset of several behaviors to establish when each

of the behavioral circuits is first likely to be fully func-tional.

The development of Hirudo ordinarily takes approxi-mately 28 days at room temperature, but the timing varieswith temperature (Reynolds et al., 1998). Newly hatchedjuvenile leeches look and behave very much like smallerversions of adults, although they lack mature reproductivestructures and behaviors. We removed embryos from theircocoon prematurely to answer several questions about thedevelopment of leech behavior: What spontaneous behav-iors are exhibited by embryos, and when do they develop?How do responses to mechanical stimulation develop, anddo these responses change over the course of embryogen-esis? Do developing leeches exhibit any behaviors that arenot seen in adults? Do behaviors appear in an order that isconsistent from embryo to embryo? Is there a rostrocaudalgradient of behavioral development that parallels theearly rostrocaudal gradient of segmental development?This paper describes the onset of several simple behaviors.The locomotory behaviors swimming and crawling arecomplex (Reynolds and Kristan, 1989; Reynolds et al.,1992) and will be addressed in separate papers.

MATERIALS AND METHODS

Experimental animals

The progenitor leeches for this study were obtained fromLeeches, USA (Westbury, NY). Embryos were allowed todevelop in their cocoon at room temperature (about 22°C)for 10 days following egg deposition and were then re-moved from the cocoon and placed in individually num-bered Petri dishes and held at 20°C throughout theremaining days of observation. Data from the first 16hours after the embryos were placed at 20°C were dis-carded.

We observed 30 leech embryos from five different co-coons; the time at which the cocoons were opened varied.As a result, the embryos had to be referred to a singlestaging system and the timing of events normalized to thedevelopment of embryos that were held at 20°C through-out embryogenesis. The details of normalization are de-scribed in Reynolds et al. (1998).

The stage of each embryo is expressed as the percentageof embryonic developmental time, or ‘‘% ED’’ (see theaccompanying paper, Reynolds et al., 1998, for details).Stages were estimated to the nearest 0.5% ED, the finestresolution achievable with this staging scheme.

Methods of observation

Behavioral observations were made through a Wild M7Adissection microscope (63–303) every 8 hours for 17 days(the time between 45% embryonic developmental time, or45% ED, and 104% ED). The time at which the bilaterallypaired ventrolateral stripes appeared was taken as 100%ED (Reynolds et al., 1998), because the time of emergencefrom the cocoon, which is analogous to hatching or toeclosion, is quite variable and typically takes place a fewdays later than the appearance of the ventrolateral stripes.

Each time an embryo was observed, its spontaneousbehavior was noted before it was stimulated. All behaviorswere subjectively assessed as weak or strong when theywere present. A behavior was determined to be ‘‘weak’’ if itsamplitude was smaller than usual, if it was less vigorousthan usual, or if it occurred in a smaller than usualfraction of the embryo’s body. In all cases, the behavior was

ONTOGENY OF LEECH BEHAVIOR 169

evaluated compared to its characteristics in embryos thatwere sufficiently advanced that the behavior was elicitedeasily. Mechanical stimulation consisted of gentle prod-ding with a smooth loop at the end of a fine (0.001’’) wireprobe, which produced a calibrated force of 0.10–0.15 mN.During each observation session, we stimulated eachanimal three times in each of four regions of the body: theanterior end, the middle one-third of the body, the poste-rior of the body directly anterior to the rear sucker, and onthe rear sucker. The order of stimulation was randomized,with the exception that no region was stimulated morethan twice in succession. The interstimulus interval was15 seconds, or occasionally longer when the animal wasstill moving 15 seconds after the stimulus was delivered.Two additional embryos from each cocoon served as con-trols for any effects of practice. These embryos were testedfor their response to mechanical stimulation, but only 2–3times over the course of the study, rather than every 8hours.

Embryos sometimes produced a combination of twoseparately identifiable behaviors in response to a singlestimulation. In such an instance, each of the two behaviorswas recorded as that animal’s response to the stimulus. Asa result, when all of the responses to a particular type ofstimulation were summed, they sometimes added up tomore than 100%.

To check for a possible rostrocaudal gradient of develop-ment in the midbody region, we subdivided the midsectionroughly into the anterior one-third, the middle one-third,and the posterior one-third. We then varied the site ofstimulation among these areas, recording the location ofthe stimulus and the response evoked. We expected that atleast some behaviors would appear first in the anteriorregion and then spread caudally over time.

Leeches have been shown to respond behaviorally tolight, so to minimize any effects of illumination, mostobservations were made under red light, to which leechesare insensitive (Kretz et al., 1976).

The age of onset listed for each behavior is the time atwhich the behavior first appeared, averaged over allanimals, with the exception of sidewinding, which was

extremely rare. For this behavior, the ages of onset and oflast appearance represent the average times for thoseanimals in which sidewinding was observed at least once.

RESULTS

Spontaneous behaviors

Even before behavioral responses could be evoked bymechanical stimulation, leech embryos produced behaviorthat we judged to be spontaneous. It should, however, benoted that these behaviors could have been elicited bystimuli that we overlooked or thought were innocuous. Forexample, early in our studies we observed an increase inspontaneous wriggling and writhing movements in em-bryos that had advanced beyond 55% ED. However, thisbehavior began at about the time the eyes developed, so itseemed possible that the increase in movement could be aresponse to illumination by bright white light. Indeed, thebehavior no longer occurred when we started doing obser-vations with red light instead. Operationally, we consid-ered movements to be spontaneous if they occurred whenthe embryo was illuminated by red light and in theabsence of any apparent mechanical stimulation.

Lateral ridge formation. In this behavior, longitudi-nally oriented ridges of tissue appeared on the surface ofthe germinal plate at the approximate location of theboundary between ventral and dorsal territories of theembryo (Fig. 1). Ridges could occur anywhere along theanterior-posterior extent of the longitudinal axis and oftenspanned just a few segments, but sometimes a ridgeextended almost the entire length of the germinal plate.Ridges could form on both sides of the embryo simulta-neously; they never formed on the midline. The averagestage at the onset of this behavior was 41.5% ED. Althoughlateral ridge formation could be elicited by mechanicalstimulation, these ridges also appeared frequently in theabsence of obvious mechanical stimulation. We hypoth-esize that lateral ridges form when the dorsoventral (orflattener) muscles (Stuart, 1970) contract. In adult leeches,these muscles cause the cigar-shaped body to flatten, and

Fig. 1. Beginning at 41.5% embryonic development (ED) embryosproduced one of the earliest observable embryonic behaviors, theproduction of lateral ridges. A: Embryo (at 45% ED) at rest. B: Anembryo at 45% ED that is producing two lateral ridges, one on each

side of the midline. Arrows labeled 1 indicate the anterior andposterior ends of one ridge; arrow marked 2 indicates the ridge on theother side. Black arrows indicate the edge of the germinal plate.Anterior is to the left. Background grid unit 5 1 mm.

170 S.A. REYNOLDS ET AL.

they generate sharp edges along the lateral boundaries ofthe body. Lateral ridges are first formed at about the samestage as the earliest reported activity in flattener muscles(J. Jellies, personal communication). Lateral ridge forma-tion as an identifiable and separate behavior tapered offand disappeared around 60% ED. At this stage, theembryos were nearly cylindrical, in contrast to earlierstages when the germinal plate is nearly planar, andcontraction of the dorsoventral muscles in embryos olderthan 60% ED flattened the lateral edge of the embryonicbody, similar to the effect of flattener muscle contraction inadult leeches.

Germinal plate bending. At about 52% ED, the germi-nal plate began to produce movements separate frommovements generated by peristalsis of the larval sac. Thefirst movements were sideways bending of the germinalplate in the same plane as the plate (Fig. 2). Initiallyembryos bent sideways only at the anterior end, with therest of the germinal plate remaining stationary (Fig. 2A);lateral bending was not associated with lateral ridgeformation. This behavior began at the time when thegerminal plate became slightly rounded, rather than flat,and when dorsal fusion was just beginning at the anteriorend. As time progressed, more and more of the germinalplate participated in bending. By 56% ED, the middle ofthe germinal plate could bend (Fig. 2B), and by 60.5% EDthe embryo could bend along the whole length of its body(Fig. 2C). Bending of the germinal plate was also elicitedby mechanical stimulation, and in that case it was catego-rized as avoidance, although the movements sometimesappeared similar to those of spontaneous bending.

Spiral twisting. During the first half of embryogen-esis, the embryos typically rest ventral side up, whereasadult leeches almost always orient with the dorsal side up.For a limited period during the transition between thesetwo orientations, embryos engaged in a behavior that wehave called spiral twisting. During this behavior theanterior part of the embryo’s body usually was raised upabove the substrate and twisted, so the anterior part of theembryo was held dorsal side up, while the posterior halfremained lying ventral side up (Fig. 3B). Spiral twistingoccurred intermittently between 61% ED and 77.5% ED.The behavior was observed in 61% of all observationsduring that time period, defining the observation of oneembryo at one time point as one observation, and was rareoutside that period.

Dorsoventral flexions. At about the same time whenthe embryos engaged in spiral twisting, they also sporadi-cally engaged in a rhythmic behavior in which most of thebody bent either dorsally or ventrally. These contractionscaused curvature in a larger fraction of the body than didthe waves of peristalsis that travel along the larval sac orthe waves of sequential contractions that will move alongthe body when the embryos begin to swim or to crawl. Thisbehavior was recognized as a separate entity later in thecourse of this work than were the other behaviors, so it wasrecorded for only a subset of the embryos. However, atsome of the time points between 61% ED and 75% ED,every embryo in the group was performing this behavior.

Sidewinding. In this relatively rare embryonic behav-ior, the leech executed an intersegmentally coordinatedcontraction that passed as a wave from the anterior to theposterior of the body in a manner reminiscent of swim-ming, except that sidewinding consisted of lateral undula-tions (Fig. 3C), rather than the dorsoventral undulations

typical of adult swimming (Kristan et al., 1974). Some-times the anterior end of the leech was raised off thesubstrate as it moved to the side at the beginning of a cycle.Sidewinding was seen in 34.6% of the observations, obser-vation being defined as in Spiral twisting above, between67% ED and 77.5% ED and rarely before or after thatperiod. Twenty-three of the thirty embryos observed wereseen performing this behavior at least once.

Responses to mechanical stimulation

Leech embryos respond to weak mechanical stimulation,delivered as described in Materials and Methods, with avariety of responses. The nature of the response elicited byany particular stimulus depended on the stage of theembryo and the location of the stimulus. Several of theseresponses are described in Table 1, where they are ar-ranged according to the time at which they were firstobserved. In general, responses evoked in young embryostended to be simple, local muscle contractions. As develop-ment progressed, the response to a particular stimulusbecame more complicated and involved more of the em-bryo’s body. This pattern suggests that the local circuitrywithin each segment is completed first, and that laterdevelopment in the nervous system connects the indi-vidual segments with one another and with the two brains.As a result, the effects of a locally delivered stimulusspread over increasingly large segments of the body, andthe behavior evoked becomes more complex in pattern.

Embryos began responding to mechanical stimulation at42–46% ED, although at this time their responses wereinfrequent and very weak. In early test sessions (42–45%

Fig. 2. Bending of the germinal plate began at the anterior of theembryo; as development proceeded, a larger fraction of the germinalplate was able to bend. A: Diagram illustrating an embryo at 52%embryonic development (ED) bending, but only at the head. B: Anembryo at 56% ED was able to bend along about half the length of thegerminal plate. C: An embryo at 60% ED could curl along the entirelength of the germinal plate. A and B show a ventral view of theembryo, the position in which embryos of these stages are typicallyobserved. Dots along midline represent the ventral ganglia. C shows adorsal view, the typical position of this developmentally advancedembryo. Stippled regions represent the larval sac tissue. Anterior is upin all sketches.


ED), the most frequently encountered response to mechani-cal stimulation was no response in any body region (Fig. 4);only about 4% of our mechanical stimuli (described inMaterials and Methods) elicited a response. Over the next30% ED, responsiveness increased, so by 75% ED nearly

90% of all stimuli evoked a response. As embryos devel-oped, they responded to a larger percentage of stimuli andthe responses became stronger. The details of this generalpattern are discussed below. Two embryos from eachcocoon served as controls for any possible effect of repeatedstimulation on the development of behavioral responses;these embryos were stimulated only once every 7 to 12days. On each such occasion, the responses of these controlembryos were similar to the responses of their siblingsthat experienced stimulation trials three times each day.We conclude that the increase in responsiveness that weobserved in our test embryos was not due to the experimen-tal manipulations, nor was there any indication thatstimulation modified the types of behaviors evoked.

Regional differences in behavioral responses. Dur-ing development, embryos became increasingly responsiveto mechanical stimulation, and the nature of the responsevaried depending on which region of the body was stimu-lated (Table 2). In the following description, a response isclassified as major if it occurred in response to at least 15%of all stimuli delivered during the study period and inaddition if it was elicited by more than 50% of the stimulidelivered during at least one time point. A response isclassified as rare if it occurred in response to less than 5%of the stimuli delivered over the entire study period. Theremaining responses are classified as minor. Note that asingle behavior can be classified in different categories,depending on which region of the embryo was stimulated.For example, shortening was a major response to stimula-tion of the anterior end, the posterior end, and the tail ofembryos, but it was a minor response to stimulation of themiddle region.

Responses to anterior stimulation. Between 42 and47% ED, few active responses were elicited, and themajority of these were either lateral ridge formation (LR)or a nonspecific increase (I) in ongoing movement (Fig. 5A).Initially shortening (Sh) accounted for only a small percent-age of the responses, but starting at 47.5% ED, thispercentage increased greatly. By 63% ED, and continuingthrough the rest of embryogenesis, most stimuli deliveredto the anterior end evoked a shortening response, eitheralone or in combination with other responses (Table 2).

Avoidance was the most common minor response tostimulation at the anterior, occurring 6.4% percent of thetime. Seventy percent of these avoidance responses were

Fig. 3. Three behaviors seen only in embryos. A: Circumferentialindentation, induced by weak mechanical stimulation to the midbody,consists of muscle contraction that extends around a single segment ofthe germinal plate. Stimulation to the anterior or posterior ends of theanimal only rarely produced this response. Arrowheads indicate theindentation on both sides of the embryo. Ventral view; anterior istoward the upper left. B: Spiral twisting. Embryo spirals its anteriorend upward, lifting it off the substrate. Dorsal view (at anterior,labeled with an asterisk) changing to a ventrolateral view (at theposterior, indicated by an arrowhead). C: Sidewinding. The embryoundulates side-to-side, thereby propelling itself across the substrate.Occasionally the head (labeled with an asterisk) was raised duringthis action. Dorsal view (note the line of dorsal fusion); anterior istoward upper left. A was originally a halftone photograph; B and Coriginated from a standard videotape of ongoing behavior. A wasscanned with a flatbed scanner and transfered into Photoshop soft-ware for Macintosh (Adobe). For B and C, a single frame was acquiredusing the frame-grabbing function of NIH Image. Contrast of theimages was enhanced by shifting the balance among shades of grayusing Photoshop, and the resulting images were treated with a medianfilter function. Labels were added using Photoshop. Background gridunit 5 1 mm.


paired with shortening, making shortening and avoidancethe most common paired response to a single stimulus atthe anterior. In a small number of cases and beginning at78% ED (when the front sucker was first able to attach tothe substrate), shortening was combined with the releaseof the front sucker (FSR). In contrast, anterior stimulationof embryos older than 78% ED almost never caused releaseof the rear sucker (RSR). Many times when an embryoolder than 78% ED had both suckers attached, the embryoresponded to stimulation at the anterior end with a suddentensing of the body that clearly produced tension on bothsuckers as the body shortened, but the suckers were notreleased. This response was scored as shortening eventhough the distance between the attached front and rearsuckers did not change.

Circumferential indentation, local bending, and elonga-tion were rare responses to stimulation at the anteriorend, although they were major or minor responses in theother body regions.

Responses to stimulation at the midbody. Stimula-tion at the midbody elicited responses that were quitedifferent from stimulation of the anterior (Fig. 5B). As foranterior stimulation, responsiveness to stimulation in themidbody region gradually increased over time, but theresponses to stimulation in this region differed markedlyfrom the responses to anterior stimulation. For example,few stimuli to the midbody region elicited shortening.Instead, the major responses were circumferential indenta-tion (Fig. 3A) and local bending (Table 2). Between 46.5%ED and 57% ED, circumferential indentation was thetypical response to stimulation. After 66% ED, the percent-age of circumferential indentation responses decreased,and the number of local bending responses increased, sothat by the end of embryogenesis circumferential indenta-tion had practically disappeared (Fig. 6). Indeed, circumfer-ential indentation is never seen in the adult leech; insteadadults respond to moderately strong touch that is confinedto one segment by producing a local bend.

Mechanical stimulation elicited combined responses moreoften in the midbody than it did at the anterior end. The

TABLE 1. Behaviors Elicited in Leech Embryosby Mechanical Stimulation

Behavior Description Stage at onset

No response No change in the embryo’songoing behavior following astimulus.1

—

Lateral ridgeformation

Described under ‘‘SpontaneousBehaviors’’ in text (see Fig.1B). Note that the time ofonset is different for sponta-neous and evoked lateralridges

45.0% ED4

Shortening Contraction of the longitu-dinal musculature in a fewsegments or along entirebody, causing shortening ofa few segments at the site ofstimulation, or a shorteningof the whole body.

51.0% ED

General increase inactivity

An increase in either ampli-tude or duration (or both) inthe level of ongoing bodymovements.

52.0% ED

Circumferentialindentation

An indentation around anentire stimulated segmentin response to a touch (Fig.3A).

52.5% ED

Rear suckerpuckers

Contraction of an unattachedrear sucker to make the ven-tral surface of the suckermore puckered. This was arare response.

55-70% ED

Avoidance A large part of the body bendsaway from a stimulus.

56.5% ED

Local bend A localized longitudinal short-ening on one side of the body(the side touched), withsimultaneous relaxation onthe opposite side, producinga localized bending of thebody away from the site of astimulus (Described foradult leeches by Kristan,1982).

59.0% ED

Elongation Lengthening of a few seg-ments at the site of stimula-tion, or a lengthening of thewhole body.

63.5% ED

Sucker release Release of the front or rearsucker following stimula-tion. This response usuallyoccurred in combinationwith a shortening response.2

81.0% ED for rear sucker;88.5% ED for front sucker

Crawling A coordinated movement alonga substrate, using bothsuckers and alternatingelongations and contrac-tions. (Crawling by adultleeches is described inStern-Tomlinson, et al.,1986).3

81.5% ED

Swimming Repeated cycles of antiphasicwaves of dorsal and ventralcontraction of the longitu-dinal musculature, causingwaves of dorsoventral undu-lation to pass through thebody producing a forwardpropulsion of the leech.(Adult leech swimming isdescribed in Kristan et al.,1974.)

.84% ED

Sucker attachment Setting down and adhering ofthe front or rear sucker ontothe substrate. Usuallyoccurred when embryocrawled in response tostimulation.

.85% ED

1Seen throughout developmental time, but with decreasing frequency as embryogenesisproceeded.2Only 75% of the embryos tested ever responded by releasing their front sucker,primarily because leeches at rest rarely have their front suckers attached to thesubstrate. Embryos are able to release their suckers earlier than the times indicatedhere, but as part of a behavior, typically in crawling, rather than as a direct response tostimulation.3For a behavior to have counted as crawling, the embryo must have completed at leastone complete crawl cycle with each behavioral component completed and organized inthe proper order. Embryos in this study never completed more than one crawl cycle inresponse to weak mechanical stimulation, and even one complete cycle was rare.Embryos typically crawled immediately after shortening.4ED, embryonic development.

Fig. 4. Overall responsiveness of embryos to weak mechanicalstimulation. Mean response of 24 embryos that were stimulated threetimes in each of four body regions (head, middle one-third of body,posterior end of body, rear sucker). Lines show the percentage ofstimuli that evoked any behavioral response in each body regionplotted against developmental time.


two behaviors that were elicited simultaneously mostoften were circumferential indentation and local bending.Shortening was a minor response in this region of the body,and avoidance, a minor response to anterior stimulation,was a rare response at the midbody, as was a generalincrease in ongoing movement and lateral ridge formation.Rear sucker release was extremely rare, and the frontsucker was never released in response to midbody stimula-tion.

Responses to stimulation of the posterior end. Theprofile of responses to stimulation at the posterior end wasmore similar to responses at the anterior end than it wasto responses in the midbody (Fig. 5C). Shortening was themajor response to stimulation in this region, although itwas elicited less frequently by posterior stimulation thanby anterior stimulation. Elongation was also a majorbehavior elicited by posterior stimulation, but only late inembryogenesis: it was a rare response prior to 77% ED, butwas elicited by 35% of all posterior stimuli after 77% EDand by 47% of all posterior stimuli between 95% and 100%ED. Elongation often occurred along with the seeminglyincompatible behavior of shortening. In fact, these twobehaviors were produced sequentially, with rapid shorten-ing followed by a slower elongation. Typically, the entirebody would elongate, even if shortening was localized tothe posterior end. A rapid rebound or relaxation fromshortening back to the original length was not scored aselongation.

Although in some ways responses to posterior and toanterior stimulation resembled one another, posteriorstimulation evoked more behavioral combinations thandid stimulation of the anterior end. The behavioral combi-nations usually included shortening (Fig. 7). One behaviorthat occurred spontaneously, but that was never evoked byposterior stimulation either singly or in combination, wasfront sucker release. Rear sucker release was observedonce the rear sucker was able to stick down (after 78% ED).After that time, rear sucker release became a minorbehavior (occurring in response to 5.1% of posterior stimuli)that was evoked almost exclusively in conjunction withshortening. A small percentage of posterior stimuli elicitedcircumferential indentation and local bending, with atemporal pattern similar to that seen at the midbody.

Responses to stimulation of the rear sucker. Theresponses to stimulation of the rear sucker (Fig. 5D)strongly resembled responses to stimulation of the poste-rior and anterior ends of the embryo. For instance, shorten-ing was the most common response to rear sucker stimula-tion, and it had a developmental onset similar to thepattern at the anterior and posterior ends (Fig. 8). In olderembryos that had gained control of their suckers, shorten-ing was sometimes followed by release of the rear suckerfrom the substrate. The incidence of rear sucker release inresponse to stimulation of the rear sucker, while still nothigh, was more than double the occurrence when theposterior part of the body was stimulated. One complica-tion was that it was impossible to determine whethersuckers actively released or were instead pulled off thesubstrate as an embryo shortened. Stimulation of the rearsucker almost never elicited circumferential indentationor a local bend.

Charting behavioral development. Most behaviors,whether spontaneous or evoked by mechanical stimula-tion, first appeared at approximately the same stage ofdevelopment in all embryos from all cocoons tested. Thisreliability allows behavioral development to be plotted onthe same time line that describes the development ofmorphological features (Fig. 9; see Reynolds et al., 1998).For some exclusively embryonic behaviors, such as lateralridge formation, the age at which the behavior ceased hasbeen included in the time line. For behaviors that developslowly, such as germinal plate bending, different stages ofthe behavior (anterior end, middle, whole) are indicated.Table 3 lists these and other behavioral developments inchronological order of appearance.

We saw no convincing evidence of a rostrocaudal gradi-ent in the development of evoked behaviors, despite theclear morphological rostrocaudal gradient visible earlierin the developing nervous system (Fernandez and Stent,1982). Such a gradient might help to explain the slight lagin responsiveness of the rear sucker compared to that ofthe anterior end during the period of 42% ED to 53% ED(Fig. 4), but it cannot be the whole explanation becauseduring this same time the midbody was often more respon-sive than was the anterior end.

TABLE 2. Summary of Regional Differences in Response to Mechanical Stimulation1

Stimuli delivered to:

Anterior Mid body Posterior Rear sucker

Percent of stimuli that evokedno response

26.8 26.3 28.3 32.4

Percent of stimuli that evokedsingle behaviors

67.1 61.0 56.2 53.0

Percent of stimuli that evokedcombined behaviors

6.1 12.7 15.5 14.6

Major behaviors (% of total) Shortening (67.6) L. bend (49.8), circ. in. (21.6) Shortening (55.3), elongation(15.6)

Shortening (61.3)

Minor behaviors (% of total) Avoidance (6.4) Shortening (6.1), L. bend andcirc. in. (5.1)

Shortening and elongation (6.9),avoidance (5.5)

Elong. (7.5), rear sucker release(5.6) shorten. and rear suckerrelease (5.0)

Most common combined behav-iors (% of total)

Shorten. and avoidance (4.5) L. Bend and circ. in. (5.1) Shortening & elongation (6.9) Shortening and rear suckerrelease (5.0)

Front or rear sucker released (%of total)

Front 0.9/rear 0.1 Front 0.00/rear 0.04 Front 0.00/rear 2.2 Front 0.1/rear 5.6

Number of single behaviors andcombinations

19 (8 single, 11 combo) 25 (7 single, 18 combo) 36 (10 single, 26 combo) 34 (12 single, 22 combo)

Percent of stimuli that evoked arare behavior

4.9 8.4 11.0 9.0

1Numbers indicate the percentage of all stimuli that evoked each indicated response during the period from 45% embryonic development (ED) to 100% ED. See text for definition ofmajor, minor, and rare. Behaviors are abbreviated as follows: Circ. in., circumferential indentation; L. bend, local bend; LR, lateral ridge formation; The percent listed for a singlebehavior is the total percentage of stimuli which evoked that behavior, whether singly or in combination with other behaviors.


We used two behaviors, circumferential indentationand local bending, to look for a rostrocaudal gradientin the onset of behaviors. The midbody was dividedapproximately into thirds, and the location of stimu-lation was varied among the three regions, noting thelocation of each stimulus along with the response elicited(see Materials and Methods). When an embryo re-sponded to the three trials comprising one set of stimuli

with at least one circumferential indentation and onelocal bend, we compared the location of the stimuli tosee if the animal consistently responded with cir-cumferential indentations at one site and local bendingat another site. If a rostrocaudal gradient exists,with the anterior part of the embryo being more de-velopmentally advanced, anterior stimulation would beexpected to produce local bends at the same time when

Fig. 5. Behavioral response profiles in four body regions. A: Re-sponses to stimulation of the head. B: Responses to stimulation of themiddle third of body. C: Responses to stimulation of the posterior endof body. D: Responses to stimulation of the rear sucker. Responses toweak mechanical stimulation varied both with stimulus location andwith developmental time (in this study from 45% to 100% embryonicdevelopment, ED). Each filling pattern represents a different behavior(see key above); the area occupied by each pattern depicts thepercentage of stimuli that evoked the response at each observation.

NR, no response; I, general increase in activity ; LR, lateral ridgeformation; A, avoidance; CI, circumferential indentation; LB, localbend; CI & LB, circumferential indentation and local bend combined;Sh, shortening; E, elongation; Sh & E, shortening and elongationcombined; Sh & A, shortening and avoidance combined; Sh & RSR,shortening and rear sucker release combined; ‘‘Other’’ includes allother evoked behaviors, all of which were rare as defined in the textand most of which occurred in response to stimulation of the rearsucker.


posterior stimulation evoked only circumferential in-dentations. We observed no rostral-caudal gradientin the switch from circumferential indentation tolocal bending when responses evoked in anterior andin posterior segments of the midbody region were com-pared.

DISCUSSION

We observed the spontaneous behavior of embryonicleeches from the time at which the germinal plate is firstcapable of movement until just before the time at whichembryos would normally emerge from the cocoon with anearly mature behavioral repertoire. In addition, we pro-vided weak mechanical stimulation to determine the timecourse for the onset of evoked behavior as well. Controlembryos that were stimulated only infrequently confirmed

that repeated mechanical stimulation in no way modifiedthe pattern of behavioral ontogeny.

Some spontaneous behaviors, including front and rearsucker release, shortening, and elongation, appeared to becomponents of crawling. A more detailed account of theonset of crawling will be presented in a subsequent report.Other spontaneously occurring behaviors were exclusivelyembryonic behaviors, i.e., they were no longer seen by theend of embryogenesis. Embryonic behaviors included lat-eral ridge formation, spiral twisting, circumferential inden-tation, and sidewinding.

The disappearance of most embryonic behavior may beexplained by concurrent developmental events in theembryo. The formation of lateral ridges disappears asembryos change from the flat germinal plate into the cigarshape that accompanies dorsal fusion (Reynolds et al.,1998). Contraction of the dorsoventral (flattener) muscles

Figure 5 (Continued.)


is likely to produce both lateral ridges in early embryosand flattening in later embryos and adults. One end ofthese muscles inserts lateral to the dorsal midline, and theother end inserts lateral to the ventral midline, explaining

why these ridges that form in the early embryo never crossthe ventral midline. A single lateral ridge often extendsthrough several segments, suggesting that the motorneurons driving these muscles may fire simultaneously inseveral adjoining segments. Such coordinated activityimplies either that the neurons are electrically coupledintersegmentally at this stage, as has been seen for othermotor neurons (Wolszon et al., 1995), or that an interneu-ronal network activates them in concert. This ipsilateralintersegmental coupling appears to be stronger and/or todevelop earlier than coupling across the midline, as lateralridges frequently form on only one side of the germinalplate at a time. In an adult leech, the dorsoventral musclescontract only when an animal swims (Weeks, 1982).

Spiral twisting, another exclusively embryonic behavior,may indicate the onset of a righting response (Gee, 1913).During the early part of development, leech embryosalmost always lie ventral side up; only later do they adoptthe adult-like orientation with the dorsal side up. Leecheshave no known gravity-sensing detectors, such as stato-cysts, but they typically maintain their dorsal surfaceupward in response to both tactile and photic stimuli (Gee,1913; Sawyer, 1986). It is possible that spiral twistingbegins with the development either of the (as yet unidenti-fied) sensory apparatus underlying this response or of theneuronal circuitry needed to produce it. Once the embryois able to remain with the dorsal side up, the behaviordrops out.

Circumferential indentation is an exclusively embryonicevoked behavior that seems to be related to local bending.The same mechanical stimulus that evokes a circumferen-tial indentation in a young embryo usually evokes a localbend in a late embryo or in an adult. In addition, circumfer-ential indentation wanes as local bending becomes moreprominent (Fig. 6). This pattern suggests that circumferen-tial indentation may be an early stage in the developmentof local bending, which occurs because the circuitry under-lying local bending is incomplete in an early embryo. Wepredict that particular synaptic connections are missing inearly embryos, and that when these connections become

Fig. 6. Weak mechanical stimulation applied to the middle of thebody typically evoked either circumferential indentation or localbending, depending on the stage of the embryo. Circumferentialindentation arose earlier in development and practically disappearedby the end of embryogenesis, when the majority of midbody stimulielicited local bending. Sometimes a single response elicited bothcircumferential indentation and local bending (see Fig. 5B). Thesketches depict local bending (right) and circumferential indentation(left). Anterior is up. Stippled region shows the larval sac. Arrows withexpanded tails represent the point of stimulation. Arrows with straighttails indicate data corresponding to diagram.

Fig. 7. Shortening was elicited either alone or in combination withother behaviors. Of all the shortening responses elicited by stimula-tion of the posterior end of the animal, in about one-quarter of thecases shortening was combined with other behaviors. The mostcommon behaviors elicited in conjunction with shortening were elonga-tion (shortening and elongation combined, Sh & E), avoidance (short-ening and avoidance combined, Sh & A), and rear sucker release(shortening and rear sucker release combined, Sh & RSR). Sh & Otherincludes shortening combined with circumferential indentation, localbending, a general increase in activity, lateral ridge formation, orcrawling.

Fig. 8. In embryos at 65% embryonic development (ED) or older,shortening was elicited by the majority of stimuli applied to the head,the posterior end, or the rear sucker. Inset diagram illustrates thebehavior: stimulation of the head provoked shortening toward theposterior, whereas stimulation of the posterior or rear sucker causedshortening toward the anterior. Stimulation of the middle one-third ofthe body usually elicited behaviors other than shortening, for example,local bending. Arrows with expanded tails indicate the point ofstimulation.


active, the response to touch shifts to the local bend.Experiments to test whether these two behaviors shareunderlying neuronal circuitry are underway (Eisenhartand Kristan, 1996).

Sidewinding is a rare and exclusively embryonic behav-ior that remains mysterious. Sidewinding must requirecomplex intersegmental coordination to produce the undu-lations that propel the embryo, yet we know of no adultcorrelate of this behavior nor of any purpose for it duringembryogenesis.

Regional differences in embryonic behavior

We found no evidence for a rostrocaudal gradient ofbehavioral development. Although the anterior end of theembryo was initially more responsive to mechanical stimulithan was the rear sucker, the midbody region was ofteneven more responsive than the anterior end at the sametime. In looking closely at responses to stimulation ofdifferent locations within the midbody region, we found noevidence that the switch from circumferential indentation

to local bending as the dominant response to a mechanicalstimulus took place any earlier at the anterior than at theposterior of this region. One exception may be lateralmovement of the germinal plate; early embryos were ableat first to bend only at the anterior end, then with timethey were able to bend progressively further down thegerminal plate until they could curl along the whole lengthof the body. It is possible that neuronal connections drivingthe longitudinal muscles that bend the germinal platedevelop first in anterior segments, but it is also possiblethat the neuronal capacity for whole-body bending is inplace early and remains unexpressed because of physicalconstraints imposed by the large yolk sac and the rela-tively small size of the embryo early in development.

Mechanical stimulation of different body regions of leechembryos evoked different responses. The most commonresponse to stimulating the anterior end or the rear suckerwas shortening. Shortening was also the most commonresponse to stimulation at the posterior, although elonga-tion became quite common toward the end of embryogen-esis. In response to stimulation at the midbody, there wasover time a gradual switch from circumferential indenta-tion to local bending.

As in embryos, adult leeches can produce a variety ofdifferent behavioral responses to mechanosensory activa-tion, and the constellation of responses produced variesdepending on the site of stimulation (Kristan et al., 1982).The common consequence of all major evoked responses(shortening, local bending, even elongation) is to pull thestimulated region away from the point of stimulation.Shortening removes the leech from the point of contactwhen either end of adults or late embryos is touched (Shawand Kristan, 1995), and shortening is coordinated with therelease of the front or back suckers when they becomefunctional in embryos. However, if a leech were to shortenwhen touched in the midbody region, the behavior would

Fig. 9. Time line for development of behavior in Hirudo embryos,with references to morphological staging markers. Time is given as apercentage of total embryonic developmental time (% ED). A verticalline indicates the average time at which each behavior or feature wasfirst observed. Behaviors are shown above the line and morphologicalfeatures are shown below the line. The timing and duration ofbehaviors that are produced only by embryos are indicated by closedbars above the time line. The ends of the bar representing dorsoven-tral flexions are dashed lines to indicate that these behaviors werecarefully observed in only a small group of embryos, producing

uncertainty about the precise times at which the behavior arises anddisappears. RS, rear sucker; GP, germinal plate; SP, slight puckers ofyolk sac; Dors. Fus., dorsal fusion. Single asterisk indicates a behaviorelicited by mechanical stimulation. Double asterisks indicate behaviorwas produced during spontaneous locomotion in early embryos, butoccurred in response to stimulation only later in development; thetime shown here is the mean time at which the behavior occurred inresponse to a single weak stimulus. Behaviors are described more fullyin the text, and morphological features are described more fully inReynolds et al. (1998).

TABLE 3. Mean Time Each Behavior Was First Observed and Last TimeExclusively Embryonic Behaviors Were Observed

Age (% ED) Developments

41.5–60% ED Lateral ridges form52% ED Germinal plate bends at head53.5% ED Shortening (evoked)55% ED Increase in wriggling (evoked), circumferential indentation

(evoked)56% ED Germinal plate bends at midbody58.5% ED Avoidance (evoked)60.5% ED Germinal plate bends along whole body60–77.5% ED Local bending (evoked)61–77.5% ED Spiral twisting63.5% ED Elongation (evoked)67–77.5% ED Sidewinding79.5% ED Rear sucker release (evoked)85% ED Front sucker release (evoked)


most likely just expose a different part of the body to thesource of stimulation, rather than to move the leech awayfrom the irritation. Local bending produces withdrawalmore effectively in this region of the body (Lockery andKristan, 1990), and it is the most likely behavior inresponse to touch directed at the midbody region. Theelongation response that is seen when older embryos arestimulated at the posterior ends generally results in aflattening or lowering of the stimulated area, although itmay instead be the prelude to crawling or swimming if thestimulus persists. Hence, the sequential combinations ofbehaviors seen in response to stimulating different bodyregions may foreshadow the emergence of more complexbehavioral acts.

Comparison with other leech species

In Hirudo, the dorsal and ventral pressure-sensitivecells (PD and PV, respectively) provide sensory input for atleast some of the behaviors described in this study (Kris-tan et al., 1982). The PD and PV cells of Hirudo arehomologous to those of the glossiphoniid leech Haemente-ria ghilianii (Kramer and Kuwada, 1983). In the PD and PVcells of Haementeria, the primary peripheral axon growsout into its target region of the germinal plate at stage10(0/5) in the Haementeria staging system. A day or 2 later(stages 10[1/5] to 10[2/5]), spontaneous postsynaptic poten-tials can be recorded in the P neurons (Kuwada andKramer, 1983). At this time all ganglia of the ventral nervecord are completely formed, and dorsal fusion has not yetoccurred. This stage corresponds roughly to the same stage(about 50–60% ED) at which we see the development ofsimple behavioral responses to mechanical stimuli inHirudo.

The behavioral development of Hirudo medicinalis re-sembles that of Haementeria ghilianii in some features. InHaementeria, spontaneous peristaltic movements flow inlongitudinal waves along the embryo before any evocablebehaviors arise. The first evocable behavior to develop isshortening, which arises approximately halfway throughembryogenesis. In both Haementeria and Hirudo, shorten-ing initially occurs only in response to mechanical stimula-tion of the anterior end of the animal, and at that time thebehavior is limited to the anterior portion of the body;later, stimulation evokes shortening of the entire body(Stent et al., 1992). Locomotory behaviors develop late inembryogenesis in both species.

Knowing the stage at which behaviors arise duringembryogenesis will facilitate further investigation into theneuronal basis of behaviors, adding the leech to thegrowing number of species in which the ontogeny ofneuronal circuits can be explored. The neuronal circuitrythat underlies many behaviors in adult leeches, localbending (Lockery and Kristan, 1990; Kristan et al., 1995),shortening (Wittenberg and Kristan, 1992; Shaw andKristan, 1995), swimming (Kristan et al., 1974; Brodfue-hrer et al., 1995), and crawling (Baader and Kristan,1995), is understood in more or less detail. A description ofthe ontogeny of behavior provides an indication of stageswhen it is appropriate to investigate changes taking placewithin each identified neuron and in the connectionsbetween neurons forming the circuits that ultimatelyproduce behaviors.

For example, the time during which the apparentlygradual transition from circumferential indentation tolocal bending takes place might be especially interesting,

and close examination of the two behaviors provides someindication of where to begin the study. Circumferentialindentation and local bending look very similar on thestimulated side of the embryo. The difference between thetwo behaviors is apparent on the contralateral side of thestimulated segment: the contralateral side indents duringcircumferential indentation, and it relaxes and bendsduring a local bend. In addition, local bending can spreadto adjacent ganglia (Wittenberg and Kristan, 1992),whereas circumferential indentation appears to take placein only one segment. The transition from one behavior intothe other may well be a time when intraganglionic connec-tions are restructured and novel interganglionic connec-tions are formed to bring about a change in behavioralresponse. Future experiments will explore this process.

ACKNOWLEDGMENTS

We thank all members of our lab for spirited discussions.NIMH (MH43396) and NSF (IBN11432) to W.B.K.

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Development of spontaneous and evoked behaviors in the medicinal leech