Caenorhabditis elegans selects distinct crawling andswimming gaits via dopamine and serotoninAndrés Vidal-Gadeaa, Stephen Toppera, Layla Younga, Ashley Crispa, Leah Kressina, Erin Elbela, Thomas Maplesa,Martin Braunerb, Karen Erbguthb, Abram Axelrodc, Alexander Gottschalkb, Dionicio Siegelc,and Jonathan T. Pierce-Shimomuraa,1

aSection of Neurobiology, Waggoner Center for Alcohol and Addiction Research, and cDepartment of Chemistry and Biochemistry, University of Texasat Austin, Austin, TX 78712; and bInstitute of Biochemistry and Frankfurt Institute for Molecular Life Sciences, Johann Wolfgang Goethe Universität,60438 Frankfurt am Main, Germany

Edited by Paul W Sternberg, California Institute of Technology, Pasadena, CA, and approved September 7, 2011 (received for review May 31, 2011)

Many animals, including humans, select alternate forms of motion(gaits) to move efficiently in different environments. However, it isunclear whether primitive animals, such as nematodes, also usethis strategy. We used a multifaceted approach to study how thenematode Caenorhabditis elegans freely moves into and out ofwater. We demonstrate that C. elegans uses biogenic amines toswitch between distinct crawling and swimming gaits. Dopamineis necessary and sufficient to initiate and maintain crawling afterswimming. Serotonin is necessary and sufficient to transition fromcrawling to swimming and to inhibit a set of crawl-specific behav-iors. Further study of locomotory switching in C. elegans and itsdependence on biogenic amines may provide insight into how gaittransitions are performed in other animals.

locomotion | optogenetics | caged amines | ablation | magneticmanipulation

Animals from widely diverse taxa (e.g., from annelids tochordates) move efficiently in different environments by

selecting alternate forms of motion, sometimes referred to asgaits (1, 2). Gait transitions are often facilitated by dopamineand serotonin (3–10). One needs only look at individuals withdisrupted aminergic systems (e.g., Parkinson’s disease) to graspthe importance that bioamines have for motor transitions inparticular and for survival in general (11). Despite recent prog-ress, limited knowledge exists regarding when different motorgaits first evolved and what neural strategies are used to switchbetween them (2).The nematode Caenorhabditis elegans lives in the soil and

decaying vegetation, which offer land and water microniches(12). Although most research on C. elegans locomotion has fo-cused on crawling, worms are nevertheless apt swimmers, easilyorienting to chemical cues while immersed (13). Local variationin water availability likely necessitates C. elegans to routinelyenter and exit aquatic environments.C. elegans is a genetically tractable animal whose 302 neurons

and ≈8,000 known synapses make it a promising model systemfor the study of transitions between motor patterns. On an agarsubstrate and in water, C. elegans moves forward with dorso-ventral bending. At present, two opposing views exist regardingthe nature of locomotory patterns in C. elegans. First, crawlingand swimming may be at opposite extremes of a single gait and,as such, represent the output of a single neural circuit (14–18).Second, crawling and swimming may represent different gaitsproduced by functionally distinct neural circuits (3, 13). We useda multifaceted approach spanning in-depth behavioral assays,neuron ablations, optogenetics, and photolysis of caged aminesto determine whether swimming and crawling are the product ofone or two functionally distinct neuronal circuits and next in-vestigated the roles played by dopamine and serotonin as wormsnaturally transition between water and land.

ResultsSwimming and Crawling Have Distinct Kinematics. To study howC. elegans transitions between these environments, we used

a video analysis system to quantify body curvature of individualWT worms as they crawled into and out of puddles on an agarsurface (Fig. 1 A–C and Fig. S1). By dividing the length of theworm into 12 equal segments and measuring the angles betweenadjacent segments, our software extracted the changes in bodycurvature over time as the animals performed different behaviorsin different environments (Fig. S1). Worms crawled on agar bypropagating dorsoventral bends from head to tail. These trav-eling bends had an average amplitude of 135° and occurred ata frequency of 0.5 Hz (Fig. 1 D, G, and H). While crawling,worms often engaged in a foraging behavior (consisting of 10-Hzhead oscillations) and occasional pumping of their feeding organ(the pharynx). After submersion, worms ceased crawling for 6 sbefore the onset of the first swimming cycle. After this delay(hereafter referred to as “swim onset”), worms swam continu-ously for extended periods (>45 min). Swimming was accom-plished by dorsoventral bends that averaged 45° in amplitude andoccurred at a frequency of 1.7 Hz (Fig. 1 E, G, and H). Foragingand pumping ceased during swimming. Worms swam with theircharacteristic alternation of dorsal and ventral “C”-shaped pos-tures until the puddle was absorbed by the agar. Once the heightof the puddle dropped below the thickness of the worm, theycrawled away, dragging residual water (Fig. 1C). After emer-gence, crawling resumed with kinematics resembling those be-fore submersion (including foraging and pumping; Fig. 1 D–I,Fig. S2, and Movie S1). As shorthand, we henceforth refer tobehavior on an agar substrate exposed to air as behavior in the“land” condition and behavior in a puddle of water as behaviorin the “water” condition.

Worms in Viscous Liquids Alternate Between Swim- and Crawl-LikeGaits. The distinct kinematics and subbehaviors observed duringcrawling and swimming suggested that they may be generated byfunctionally distinct patterns of neural activity (gaits). Alterna-tively, the differences in crawl and swim kinematics may be en-tirely explained by the different mechanical constraints imposedby wet and dry environments (14–18). Behavioral evidence forgait-like forms of motion is obtained by examining locomotionover a range of speeds (2). For instance, on a treadmill, humansmove with distinct kinematics at low (walking) and fast (running)speeds. Importantly, humans cannot display a walk–run hybridand instead switch between bouts of fast walking and slow run-ning at intermediate speeds (≈1.9 m/s) indicating two funda-mental gaits (2).

Author contributions: A.V.-G., S.T., and J.T.P.-S. designed research; A.V.-G., S.T., L.Y., A.C.,L.K., E.E., T.M., and J.T.P.-S. performed research; A.V.-G., S.T., A.C., M.B., K.E., A.A., A.G.,D.S., and J.T.P.-S. contributed new reagents/analytic tools; A.V.-G., S.T., L.Y., andJ.T.P.-S. analyzed data; and A.V.-G. and J.T.P.-S. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Freely available online through the PNAS open access option.1To whom correspondence should be addressed. E-mail: jonps@mail.utexas.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1108673108/-/DCSupplemental.

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We performed gait analysis using viscous methylcellulosesolutions to constrain speed. As previously found (16, 17), wormsin low viscosity (0.024 Pa s) swam, slowing their swimming asviscosity was increased (10 and 50 Pa s; Fig. 2A). Unexpectedly,worms in higher viscosities repeatedly switched between distinctbouts of swim- and crawl-like motion (Fig. 2A and Movie S2). Asin crawling (Fig. S2), the slower form of locomotion was morevariable in cycle durations and coincided with foraging andpumping. All-points histograms of cycle durations for animalsmoving in three different viscosities confirmed that motion atlow viscosity could be explained by one state, whereas motion athigher viscosities could only be explained by two states. This isevidenced by unimodal and bimodal Gaussian distribution fitsfor the low- and higher-viscosity conditions, respectively (n = 30;Fig. 2 B–D). Interestingly, a bimodal distribution was also foundfor the sensory cilia mutant che-3 at low viscosity [or in water

(13)], suggesting that sensory input is required to maintainswimming and/or prevent switching to crawl-like motion (Fig.S3). Thus, although swimming frequency decreases with in-creasing viscosity, C. elegans shows distinct crawling and swim-ming gaits when its motion is constrained by viscous solutions.

Localized Mechanical Stimulation Causes Swim-to-Crawl Transition.To test whether C. elegans selects crawling in response to me-chanical pressure (such as experienced when contacting thesubstrate), we performed two additional experiments. For ourfirst experiment, we slowly compressed worms in water betweentwo glass slides. Worms displayed normal swimming until com-pressed to less than the thickness of their bodies (75–90 μm),whereupon they switched between bouts of swimming andcrawling (Fig. 2 E–G). Further compression induced constantcrawling accompanied by foraging and pumping. For our second

Fig. 1. Crawling and swimming have dis-tinct kinematics. A crawling worm entersa puddle (A) and starts swimming (B) untilthe puddle is absorbed and crawlingresumes (C). (D–F) Curvature matrices rep-resent changes in body shape over time.Body position is represented on the y axis(with head at the bottom and tail at thetop). Time is plotted on the x axis, and bodycurvature is represented by a trichromaticscale whereby blue = 120° dorsal, red = 120°ventral, and white = no curvature. For ex-ample, changes in neck curvature vs. timecan be extracted from the second (from thebottom) row in the matrix and used tocompare the slow and deep bends used incrawling with the fast and shallow bendsobserved during swimming (D, Lower). Weperformed this analysis as each individualworm crawled initially (D), swam in a pud-dle (E), and eventually began crawlingagain after emergence (F). Crawling andswimming bends are distinguishable byfrequency (G) and angular excursion (H).We used velocity (obtained by tracking thecentroid of animals) to report changes in the efficiency of locomotion (I). Here and henceforth, all bars report means with SEM, n > 12, blue backgroundindicates water environment, F indicates foraging. *P < 0.05, and **P < 0.001.

Fig. 2. Transition between crawling andswimming gaits is influenced by pressure.As viscosity was increased (by alteringmethylcellulose concentrations), wormsalternated between bouts of swim- andcrawl-like cycle durations (A). All-pointscycle-duration histograms show one vs.two types of motion in lower (B) andhigher viscosities, respectively (C and D).Swimmingworms inwater were reversiblyinduced to crawl by compression betweenglass slides (E), showing similar kinematicsas those on “land” (agar) during stimula-tion (F and G). Swim-to-crawl transitionswere also induced by feeding worms ironand pulling them to the substrate with anelectromagnet (H). (I) Worms with ironparticles in specific body regions (yellowbars) transitioned to crawling when stim-ulated, whereas worms with iron in otherparts of their body (red, green) remainedswimming even while pinned down to thesubstrate. Worms pinned down by theanterior portion of their bodies continuedswimming but engaged in foraging whilebeing pulled (blue).

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experiment, we fed worms iron particles and placed them inwater above an electromagnet. Worms with iron distributedthroughout the gut exhibited normal swimming while the magnetwas OFF (Fig. 2 H and I). Turning the magnet ON reversiblypulled these worms to the substrate, causing them to crawl andforage (Fig. 2H, yellow in Fig. 2I; Movie S3). Similar to a pre-vious report on semiimmobilized preparations (19), worms withiron in more restricted portions of their body continued to swimwhile stuck in place (blue, green, and red in Fig. 2I). We con-clude that contacting the substrate with specific areas of the bodyis sufficient to induce transition from swimming to crawling.

Dopaminergic Signaling Is Necessary for Swim-to-Crawl Transition.Our magnet experiment revealed that pressure on areas of known(mechanosensitive) dopaminergic innervation could inducecrawling (20). Genetic ablation of all eight dopaminergic neurons[achieved in the Pdat-1::ICE strain (21)] resulted in severe defectsin freely transitioning from swimming to crawling. Specifically,Pdat-1::ICE worms crawled as well as initiated and maintainedswimming normally; however, once puddles receded, they becameimmobile for up to 30 min (Fig. 3 A–E and Movie S4). Only a fewindividuals succeeded initiating, but failed to maintain, crawlingafter emergence. Throughout this time, however, Pdat-1::ICEworms could crawl away when prodded, demonstrating that do-pamine neurons are required for the swim–crawl transition ratherthan general crawl performance. This impairment was mirroredby the tyrosine hydroxylase mutant, cat-2, deficient in dopamine

synthesis and by the trp-4mutant, which lacks a mechanoreceptorexpressed in dopaminergic neurons (22, 23) (Fig. 3 D and E).Although laser ablation of two of the three classes of dopami-nergic neurons in C. elegans (ADE and PDE) recapitulated thedefect reported above, ablation of the third class (the four CEPs)had no effect on the swim-to-crawl transition (Fig. 3 F and G).Downstream, we found that deletion of D1-like (but not D2-like)dopamine receptors (dop-1 or dop-4 vs. dop-2 and/or dop-3, re-spectively; Fig. S4) likewise perturbed the swim-to-crawl transi-tion (Fig. 3 H and I).

Dopamine Is Sufficient to Induce Swim-to-Crawl Transition in Water.To test whether dopamine was sufficient to induce the swim-to-crawl transition, we exposed swimming worms to exogenousdopamine. This produced crawl-like bouts (with foraging andpumping) in swimming WT worms (but not in mutants lackingD1-like receptors; Fig. 4A). By expressing the photoactivatablecation-channel Channelrhodopsin-2 (ChR2) in all dopaminergicneurons (under the control of Pdat-1), we could trigger the swim-to-crawl transition in swimming worms (Fig. 4 B and D andMovie S5). Pdat-1::ChR2 worms continued to crawl and forage inwater for the duration of light stimulation. Lack of D1-likereceptors was sufficient to partially (dop-1) or completely (dop-4)prevent this effect (Fig. 4B).Dopamine has been previously implicated in reducing the

crawling rate of worms entering a lawn of bacteria in the so-called basal-slowing response whereby crawling velocity decrea-ses and the worm frequently moves backward to pivot in placewhile eating (24). Consistent with this result, we found thatphoto-activation of dopamine neurons in animals crawling onunseeded (blank) agar plates induced an immediate transition toa slower form of crawling with the same kinematics and in-creased reversal frequency observed in basal-slowing (Fig. S5).We conclude that dopamine can induce discrete, context-de-pendent changes in locomotory patterns: from swimming tocrawling for worms in water, and from crawling to basal-slowingbehavior on land.Because dopamine released in the worm can act humorally in

addition to synaptically, the complete wiring diagram of thenervous system of C. elegans cannot be easily used to determinewhich neurons are altered by dopamine to affect the observedtransition (25). We therefore attempted to narrow down the sitesof dopamine action by synthesizing and injecting light-sensitive,caged dopamine into the posterior and anterior regions of theworm (26). We found that worms thus treated swam normally inall cases but that only photo-uncaging of dopamine in their an-terior section caused the worms to recapitulate the behaviors seenwith stimulation of the dopaminergic neurons by ChR2 (Fig. 4C).Together these results demonstrate that dopamine signaling is

necessary and sufficient for worms to switch from swimming tocrawling gaits. We propose that dopamine released by the ADEand PDE neurons may activate D1-like dopamine receptors lo-cated in the anterior half of the animal to trigger the transitionfrom swimming to crawling gaits.

Serotonin Signaling Is Necessary for Crawl-to-Swim Transition.A testof available aminergic mutants for crawl-to-swim impairmentsrevealed that the tryptophan hydroxylase mutant tph-1, which isunable to synthesize serotonin, crawled normally before and af-ter submersion but was severely impaired in swim initiation (Fig.5 A–C). Delayed swimming onsets were also observed for sero-tonin receptor mutants (Fig. 5D). C. elegans has six classes ofserotonergic neurons (VC-4, -5, RIH, AIM, ADF, NSM, andHSN). Ablation of VC-4, -5, AIM, or NSM neurons delayedswim onset, implicating serotonin signaling in swim initiation(Fig. 5E).Serotonin was also necessary to maintain normal swimming

and to prevent crawling in water (Movie S6). Paralleling theeffects of dopamine exposure in WT (and untreated che-3mutants in low viscosity), tph-1 mutants displayed crawl-likebouts while submersed (Fig. S6 A and B). Ablation of the VC-4, -5,

Fig. 3. Dopamine is necessary for swim-to-crawl transition. Animals lackingdopaminergic neurons (Pdat-1::ICE ) can crawl (A), enter water, and swimnormally (B), but become immobile upon emergence (C). Dopamine pro-duction and dopamine mechanoreceptor mutants are likewise impaired, asillustrated by their crawling frequencies (D) and linear velocities (E). Abla-tion of ADE and PDE dopamine neurons disrupts swim-to-crawl transition (Fand G). D1-like dopamine receptors (dop-1 and dop-4) are required forswim-to-crawl transition (H and I).

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RIH, or AIM neurons recapitulated the tph-1 phenotype (Fig. 5D and E). Although most ablated animals showed no change inswimming amplitudes (Fig. S7), some showed significantincreases in swimming velocities (Fig. S6D) resulting from boutsof high-amplitude tail bends (Fig. S8). Among the seven de-scribed serotonin receptors in C. elegans, velocity was most im-paired for mutants missing LGC-40 or both SER-1 and SER-7(Fig. S6F). These serotonin-receptor mutants (and those lackingSER-5) exhibited poor swim coordination and showed crawl-likebouts accompanied by foraging and pumping (Fig. 5 F and G).

Serotonin Is Sufficient to Induce Crawl-to-Swim Transition on Landand to Delay Crawl Onset. Exogenous serotonin was sufficient todelay crawl onset when puddles receded. It prolonged swim-likemovement in shallow puddles, in a manner reminiscent of fishflailing on land, until all water had completely disappeared (Fig.6A and Movie S7).Consistent with a role in blocking crawl-initiation and main-

taining swimming, exogenous serotonin delayed onset of photo-induced crawling of Pdat-1::ChR2 worms in water (Fig. 6B).Conversely, photoactivation of ChR2 in serotonergic neurons(via Ptph-1::ChR2 worms) drastically hastened swim onset (Fig.6C) and dramatically increased frequency of bend propagationon land (Fig. 6 D and E and Movie S8). These findings suggestedthat the balance between serotonin and dopamine could bias gaittransitions in C. elegans. We investigated this possibility andfound supporting evidence in the dopamine synthesis mutant(cat-2) in which lower than normal dopamine levels could ex-plain a faster than normal swim onset (Fig. 6D). Moreover, av-erage swim onsets for mutants with impairments in production ofboth amines were similar to WT (bas-1, 4.47 ± 1.89 s; cat-4,5.98 ± 1.65 s; bas-1;cat-4 double mutant, 5.46 ± 2.36 s). Takentogether, these data lead us to conclude that serotonin signaling

is necessary and sufficient for worms to switch from crawling toswimming gaits. Furthermore, it seems likely that the balancebetween serotonin and dopamine influences gait transitions inC. elegans.

DiscussionSwimming and Crawling as Distinct Motor Gaits. Consistent withprevious reports, we found that worms modulated their swim-ming gait as liquid viscosity was increased (15–17). However, thebimodal Gaussian distributions seen for their cycle durations athighest viscosities, and the associated foraging and pumping seenduring only the slowest form, strongly suggest that these animalsare alternating between two distinct gaits. Foraging and pha-ryngeal pumping are two (of a number of) behaviors presentwhen worms crawl but absent when worms swim. This impliesa discrete change in global behaviors occurring when wormstransition between environments.Previous studies have suggested that all of the kinematic dif-

ferences between crawling and swimming are the product ofchanges in the physical environment and not due to discretechanges in neural activity (14–18). The transition from swimmingto crawling seen for worms in our magnet experiment suggeststhat crawling can be induced in water by mechanical stimulationof specific areas of the worm’s body. This is further supported bythe observation that worms with iron in alternate areas of theirbodies continued to swim when pulled (and pinned) against thesubstrate by a magnetic field (Fig. 2I). Additional evidencesupporting swimming and crawling as distinct gaits came fromthe time required by unrestrained WT worms to begin swimmingafter entering water (6 s). During this time (swim onset), wormsneither crawled nor swam. Furthermore, the existence of mutantswith behavioral defects specific to transitions between theseenvironments (as seen in strains deficient in dopamine and se-

Fig. 4. Dopamine is sufficient for swim-to-crawl transition. (A) Exogenous dopamine caused WT (but not dop-1) worms to exhibit crawling bouts in water. (B)Photoactivation of ChR2 in dopamine neurons reversibly induced crawl-like behavior in WT but less so for D1-like receptor mutants. (C) Photo-uncaging ofdopamine injected into the anterior (solid bars) but not posterior half of animals reversibly induced crawl-like behavior in WT but not in a dop-1 background.Representative neck curvature traces shown below. (Scale bar for A–C on left, 50° by 1 s.) (D) Time course of neck curvature before, during, and afterphotoactivation (indicated by blue bar) of ChR2 in dopamine neurons of a WT worm in water.

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rotonin signaling; Fig. 7 A and C) can only be understood if theworms were transitioning between different behaviors. Together,these data indicate that crawling and swimming must be theoutputs of (at least) partially distinguishable neural networks.Recent studies of locomotion in C. elegans have made exten-

sive use of modeling techniques to quantitatively describe thephysical interactions between environment and this tiny organ-ism (15, 17, 27–30). Indeed, any thorough understanding of lo-comotion must include knowledge of the physical properties ofboth the animal and its environment. These strategies, coupledwith approaches focusing on the evolution, anatomy, physiology,and natural history make C. elegans a promising system for un-derstanding the production and control of adaptive locomo-tory gaits.

Dopamine Mediates the Transition from Swimming to Crawling. Onland, dopamine has been shown to modulate C. elegans crawlingbehavior by decreasing velocity when worms enter a patch offood in the so-called basal-slowing behavior (24). We found thatlight activation of dopaminergic neurons in worms crawling onland (in the Pdat-1::ChR2 strain) recapitulated this finding (Fig.4). In water, activation of dopamine neurons via D1-like sig-naling induced an immediate switch from swimming to a versionof how the worm crawls off food (Fig. 4 B and D). Althoughactivation of dopamine neurons decreases velocity in bothcontexts, loss of dopamine signaling produces opposite results.Namely, worms with reduced dopamine signaling continue crawl-ing quickly as if food is absent upon entering a food patch (24),whereas the same dopamine deficient worms cease all movementupon transitioning from water to land (Fig. 3 C and E). More-over, the basal-slowing response relies on D2-like dopaminesignaling rather than the D1-like signaling pathway we observedto be required for the swim-to-crawl transition (24). Dopamine,

therefore, seems to affect locomotion in a context-dependentway, slowing crawling on land through D2-like pathway and in-ducing crawling as animals exit water through a D1-like pathway.Our results also provide an explanation for the swimming

behavior of mutant worms that lack the dopamine reuptaketransporter DAT-1 in previous studies. Worms lacking dat-1become paralyzed after extended swimming (10 min) duringwhich internal dopamine presumably accumulates to high con-centration (SWIP, swim-induced paralysis) (31). Our findings areconsistent with the SWIP phenotype because dopamine releaseis proposed to be down-regulated during swimming. As dopa-mine levels rise without reuptake, the dat-1 mutant may switchfrom swimming to crawling before eventual paralysis. This isconsistent with our finding that worms submerged in water switchfrom swimming to crawl-like behavior and foraging after lightactivation of dopaminergic neurons (in the Pdat-1::ChR2 strain;Fig. 4 B and D).

Serotonin Mediates the Transition from Crawling to Swimming. Likedopamine, the role of serotonin on behavior modulation alsoseems to be context-dependent. Worms deprived of food areproposed to release higher levels of serotonin when the animalcrawls back onto a food patch (24). The extra serotonin dra-matically decreases speed, which presumably ensures that thestarved worm eats to satiation (24). By contrast, we found thatphoto-activation of serotonergic neurons increased bend fre-quency and speed to swim-like levels. We also found that ex-posure to serotonin hastened swim onset and prolonged swim-ming in worms emerging from puddles. Conversely, lack ofserotonin resulted in dramatic delay in swim onset and in de-ficient swimming behavior. Therefore, although it remains to beseen whether other neuromodulators play a role in the transitionfrom crawl to swim [analogous to the role of octopamine for gait

Fig. 6. Serotonin is sufficient for crawl-to-swim transition. Exogenous andendogenous serotonin delayed onset of crawling after puddle absorption forWT worms on land (A), and onset of photostimulated crawling for Pdat-1::ChR2 worms in water (B). Altering the dopamine-serotonin balance towardserotonin either genetically via cat-2 or by photostimulation of serotonergicneurons caused faster than WT swim onset. The opposite effect was accom-plished by photostimulation of dopaminergic neurons (C). Conversely, pho-tostimulation of serotonergic neurons in crawling worms produced swim-likefrequencies (D). (E) Time course of neck curvature of Ptph-1::ChR2 worm onland before, during, and after photoactivation of serotonergic neurons.

Fig. 5. Serotonin is necessary for crawl-to-swim transition. tph-1 mutantsdeficient in serotonin synthesis crawl normally (A) but are impaired in swiminitiation and performance (B); after emergence, crawling resumes normally(C). Swim onset was delayed in serotonergic pathway mutants (D) and inworms with their AIM, NSM, or VC-4 and -5 serotonergic neuron classesablated (E). Serotonergic pathway mutants showed defective swimming (F),including bouts of crawl-like behaviors (G).

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switching in the leech (3)], serotonin is clearly important in theinduction and maintenance of swimming in C. elegans.

Dopamine and Serotonin in Behavioral Transitions. Dopamine andserotonin are emerging as universal switches turning behaviorsON or OFF. More than 30 y ago, serotonin was found to beinvolved in initiating and maintaining swimming and fictive swimrhythms in the nerve cord of the leech (32, 33). More recently,dopamine has been shown to switch a fictive swim rhythm toa fictive crawling rhythm in leech (3). The role of serotonin inrapid locomotory rhythms and dopamine in slow locomotoryrhythms is surprisingly conserved across animals as evolutionarilydiverse as leech (3, 9), sea-slugs (4, 7), lamprey (5, 8), and mouse(6). Even in humans, loss of dopamine neurons in Parkinson’sdisease leads to debilitating problems in initiating and switchingbetween motor programs (analogous to the immobility of do-pamine-deficient worms emerging from water) (11). The pro-found importance that selecting the correct motor pattern has onsurvival may explain how dopamine and serotonin have retainedtheir roles as behavioral switches during 1.1 billion years of di-vergent evolution (34). Further study of crawl–swim switching inC. elegans has the potential to uncover the fundamental neuralmechanisms underlying how dopamine enables the natural tran-sitions between motor patterns, as well as how motor switchingbecomes dysfunctional in Parkinson’s disease.

Materials and MethodsC. elegans were grown on nematode growth media (NGM) agar platesseeded with OP50 bacteria at 20 °C as previously described (35). Informationon strains, viscosity, pressure and magnetic assays, statistical analysis, laserablation of neurons, behavioral pharmacology, and optogenetics is in SIMaterials and Methods.

Behavioral Assays. Each assay was conducted on 10–30, never-starved, youngadult worms. Worms were cleaned of bacteria by allowing them to crawl onan empty plate before each experiment. After transfer to assay plate, wormswere allowed a 2-min acclimation period. Movie recordings were made at 30

frames/s, 344 pixels/mm using a Flea2 camera (Point Grey Research) andStreamPix software (NorPix). Data were analyzed blind to experimentaltreatment and genotype.Single worms. The behavior of a wormwas video recorded for 2min on a blankNGM agar plate (Movie S1) before a 3-μL drop of NGM buffer was placed inits path, allowing the worm to crawl into the puddle. After ≈5 min thepuddle was absorbed by the agar enough that the worm could escape andresume crawling. Filming continued for 5 min after puddle emergence.Animal midlines (13 points) of single worms were derived as previously de-scribed using a custom image analysis algorithm available upon request (13)(ImagePro; Media Cybernetics). The series of 11 angles formed by the mid-line was represented in a color-coded “curvature column” (Fig. S1). A timeseries of curvature columns formed a “curvature matrix” (e.g., Fig. 1B) inwhich blue and red stripes represent the waves of dorsal and ventral cur-vature, respectively, passing along the body. Curvature matrices were di-vided into neck-bend cycles according to curvature of the second mostanterior row in the curvature matrix using IgorPro (Wave Metrics). Thequality of each digitized worm midline was manually checked, super-imposing it on the original video frame, and corrected if necessary for everyvideo frame used in this study (≈1,000,000 frames).Groups of worms. The behavior of 15 worms confined within a square copperframe (1.2 cm per side) on a blank NGM agar plate was recorded before,during, and after a 150-μL drop of NGM buffer was placed in the frame andwicked away with a Kimwipe tissue. Worm centroids were detected andtracked using ImagePro.

ACKNOWLEDGMENTS. We thank Kurt Svoboda, Dorothy Paul, HansHofmann, Lauren O’Connell, and Serge Faumont for critical reading of man-uscript drafts; and Villu Maricq, Robert Horvitz, Richard Komuniecki, andShohei Mitani for worm strains. Additional strains were kindly provided bythe Caenorhabditis Genetics Center, which is funded by the National Insti-tutes of Health (NIH) National Center for Research Resources; the C. elegansGene Knockout Project at the OklahomaMedical Research Foundation, which ispart of the International C. elegans Gene Knockout Consortium; and the Na-tional BioResource Project in Japan. This work was supported by the Universityof Texas at Austin, Alcohol Beverage Medical Research Foundation, NIHNational Institute of Neurological Disorders and Stroke Grant R01NS075541(to J.T.P.-S.), Deutsche Forschungsgemeinschaft Grant SFB807-TP11, andCluster of Excellence Frankfurt (to A.G.).

1. Bradford EH (1897) Examination of human gait. J Boston Soc Med Sci 2:20–21.2. Alexander RM (2003) Principles of Animal Locomotion (Princeton Univ Press,

Princeton).3. Mesce KA, Pierce-Shimomura JT (2010) Shared strategies for behavioral switching:

Understanding how locomotor patterns are turned on and off. Front Beh Neurosci 4:1–5.

4. Katz PS, Frost WN (1995) Intrinsic neuromodulation in the Tritonia swim CPG: Sero-tonin mediates both neuromodulation and neurotransmission by the dorsal swiminterneurons. J Neurophysiol 74:2281–2294.

5. Wallén P, et al. (1989) Effects of 5-hydroxytryptamine on the afterhyperpolarization,spike frequency regulation, and oscillatory membrane properties in lamprey spinalcord neurons. J Neurophysiol 61:759–768.

6. Dunbar MJ, Tran MA, Whelan PJ (2010) Endogenous extracellular serotonin modu-lates the spinal locomotor network of the neonatal mouse. J Physiol 588:139–156.

7. Woodward OM, Willows AOD (2006) Dopamine modulation of Ca(2+) dependent Cl(-)current regulates ciliary beat frequency controlling locomotion in Tritonia diomedea.J Exp Biol 209:2749–2764.

8. McPherson DR, Kemnitz CP (1994) Modulation of lamprey fictive swimming andmotoneuron physiology by dopamine, and its immunocytochemical localization in thespinal cord. Neurosci Lett 166:23–26.

9. Esch T, Mesce KA, Kristan WB (2002) Evidence for sequential decision making in themedicinal leech. J Neurosci 22:11045–11054.

10. El Manira A, Grillner S (2008) Switching gears in the spinal cord. Nat Neurosci 11:1367–1368.

11. Voon VP, et al. (2009) Chronic dopaminergic stimulation in Parkinson’s disease: Fromdyskinesias to impulse control disorders. Lancet Neurol 8:1140–1149.

12. Félix M-A, Braendle C (2010) The natural history of Caenorhabditis elegans. Curr Biol20:R965–R969.

13. Pierce-Shimomura JT, et al. (2008) Genetic analysis of crawling and swimming loco-motory patterns in C. elegans. Proc Natl Acad Sci USA 105:20982–20987.

14. Gray J, Lissmann HW (1964) The locomotion of nematodes. J Exp Biol 41:135–154.15. Berri S, Boyle JH, Tassieri M, Hope IA, Cohen N (2009) Forward locomotion of the

nematode C. elegans is achieved through modulation of a single gait. HFSP J 3:186–193.

16. Korta J, Clark DA, Gabel CV, Mahadevan L, Samuel ADT (2007) Mechanosensation andmechanical load modulate the locomotory gait of swimming C. elegans. J Exp Biol210:2383–2389.

17. Fang-Yen C, et al. (2010) Biomechanical analysis of gait adaptation in the nematodeCaenorhabditis elegans. Proc Natl Acad Sci USA 107:20323–20328.

18. Boyle JH, Berri S, Tassieri M, Hope IA, Cohen N (2011) Gait modulation in C. elegans:It’s not a choice, it’s a reflex! Front Beh Neur 5:1–3.

19. Faumont S, Miller AC, Lockery SR (2005) Chemosensory behavior of semi-restrainedCaenorhabditis elegans. J Neurobiol 65:171–178.

20. White JG, Southgate E, Thomson JN, Brenner S (1986) The structure of the nervoussystem of the nematode Caenorhabditis elegans. Philos Trans R Soc Lond 314:1–340.

21. Hills T, Brockie PJ, Maricq AV (2004) Dopamine and glutamate control area-restrictedsearch behavior in Caenorhabditis elegans. J Neurosci 24:1217–1225.

22. Li W, Feng Z, Sternberg PW, Xu XZS (2006) A C. elegans stretch receptor neuron re-vealed by a mechanosensitive TRP channel homologue. Nature 440:684–687.

23. Kang L, Gao J, Schafer WR, Xie Z, Xu XZS (2010) C. elegans TRP family protein TRP-4is a pore-forming subunit of a native mechanotransduction channel. Neuron 67:381–391.

24. Sawin ER, Ranganathan R, Horvitz HR (2000) C. elegans locomotory rate is modulatedby the environment through a dopaminergic pathway and by experience througha serotonergic pathway. Neuron 26:619–631.

25. Chase DL, Pepper JS, Koelle MR (2004) Mechanism of extrasynaptic dopamine sig-naling in Caenorhabditis elegans. Nat Neurosci 7:1096–1103.

26. Lee TH, Gee KR, Ellinwood EH, Seidler FJ (1996) Combining ‘caged-dopamine’ pho-tolysis with fast-scan cyclic voltammetry to assess dopamine clearance and releaseautoinhibition in vitro. J Neurosci Methods 67:221–231.

27. Karbowski J, et al. (2006) Conservation rules, their breakdown, and optimality inCaenorhabditis sinusoidal locomotion. J Theor Biol 242:652–669.

28. Sznitman J, Purohit PK, Krajacic P, Lamitina T, Arratia PE (2010) Material properties ofCaenorhabditis elegans swimming at low Reynolds number. Biophys J 98:617–626.

29. Park S-J, Goodman MB, Pruitt BL (2007) Analysis of nematode mechanics by piezor-esistive displacement clamp. Proc Natl Acad Sci USA 104:17376–17381.

30. Petzold BC, et al. (2011) Caenorhabditis elegans body mechanics are regulated bybody wall muscle tone. Biophys J 100:1977–1985.

31. McDonald PW, et al. (2007) Vigorous motor activity in Caenorhabditis elegans re-quires efficient clearance of dopamine mediated by synaptic localization of the do-pamine transporter DAT-1. J Neurosci 27:14216–14227.

32. Willard AL (1981) Effects of serotonin on the generation of the motor program forswimming by the medicinal leech. J Neurosci 1:936–944.

33. Nusbaum MP, Kristan WB, Jr. (1986) Swim initiation in the leech by serotonin-con-taining interneurones, cells 21 and 61. J Exp Biol 122:277–302.

34. Blair JE, Shah P, Hedges SB (2005) Evolutionary sequence analysis of complete eu-karyote genomes. BMC Bioinformatics 6:53.

35. Brenner S (1974) The genetics of Caenorhabditis elegans. Genetics 77:71–94.

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