classes ofnon-neuronalelectrogenesis followedbyamoredetailed and Passano,1968).Thefollowing isabriefdescriptionoffour sheets foundintheHydrozoa(Mackie,1965;Mackie,1976;Mackie Mackie, 1997;Leysetal.,1999)andthetwo-dimensionalepithelial © 2015.PublishedbyTheCompanyofBiologistsLtd|JournalExperimentalBiology(2015)218,537-550doi:10.1242/jeb.111955 *Author ([email protected]) for correspondence UK. Physiology Bristol,BristolBS81TD, andPharmacology,School of University of parenchyma ofglassspongessuchas cells, thesematricesincludetheloosethree-dimensional them intoadifferent cellmatrix. Besidesnetworksofneuronal-like selection co-optedmuchthesamesetofionchannelsbutinserted in anumberofdifferent evolutionary lineages.Ineachcase,natural and othersingle-celledspecies.Infact,propagatingimpulses arise propagation inmulticellularlifeformshavetheiroriginsbacteria It isevidentthatmanyoftheionchannelvariantsinvolvedinsignal Ionic currents,Sponges KEY WORDS:,Ctenophores,Electrogenesis,Ionchannel, digitale between slowandfastswimminginthehydrozoanjellyfish and swimming,betweententaclecontractionswimming Integrative interactionsdiscussedhereincludethosebetweenfeeding coordinated movementsdependoninteractionsbetweenthem. controlled byseparateneuronalsystems,integratedresponsesand performance. Althoughdifferent aspectsofcnidarianbehaviourare theme istheroleofinactivatingpotassiumchannelsinregulating specified bytheirselectivityandvoltagedependence.A recurring by cellularanatomy;activecomponentsincludeionchannels components includethedecrementalspreadofcurrentdetermined processes makeuptheseanalogueapplications.Passive generated pacemakeractivity. A smallnumberofmembrane-based ‘apps’ tointegrateincomingsensoryinformationwithinternally excitement’ andutilizestheequivalentofasetbasicelectrical Neuronal electrogenesis,goesbeyondsimplyrelaying‘statesof and passiveneuronalpropertiesinteracttogiveintegratedbehaviour. subject ofextensivestudy. Theaimhereistoshowhowtheiractive focused ontheCnidaria,whosenervoussystemshavebeen data relatingtoctenophorenervesmeansthatourattentionis in ctenophoresandcnidariansbutthedearthofelectrophysiological impulses. Neuronalelectrogenesismayhaveevolvedindependently Beroe ovata,allofwhicharecapablegeneratingelectrical of hydrozoanjellyfishandtheeggcellmembranesctenophore parenchyma ofglasssponges,thetwo-dimensionalepithelialsheets metazoan cellsasdisparatetheloosethree-dimensional in avarietyofanimalandplantlineages.Consideredhereare Electrogenic communicationappearstohaveevolvedindependently Robert W. Meech* neuronal integration Electrogenesis inthelowerMetazoaandimplicationsfor REVIEW Introduction: electrogenesis in early Metazoa inearly electrogenesis Introduction: ABSTRACT . Rhabdocalyptus (Leys and 10 little effect buttheactionpotentialswerereversiblyblockedby sodium chlorideinthebathingmediumwithcholinehad 12°C). Ifa Electrical eventspropagatewithin 2015). Mackie, 2007),foramoregeneralaccount,seeMeech(Meech, electrogenesis intheProtozoa,seeMeechandMackie(Meech exhibited bytheCnidaria.Foranaccountthatincludesaspectsof account ofintegrativeaspectsneuronalelectrogenesisas spread throughouttheepitheliumataspeedof24–31 potentials areelicitedbyelectricalormechanicalstimulation and the scypho-andcubo-medusae(Mackieetal.,1984].Action gap junctions[whichareabsentinotherformsofjellyfishsuch as Passano etal.,1967)ismadepossiblebythepresenceofepithelial as Impulse propagationintheexumbrellaofhydrozoanjellyfish such into thecellsofflagellatedchamberthatstopstheirpumping. (Naitoh andEckert,1969),itseemslikelythatistheinfluxofCa al., 1999;LeysandMeech,2006).Byanalogywith (R.W.M. andG.O.Mackie, unpublishedresults).Pipettes were formed asealthatwasabout fourtimesthepipetteresistance (about 10 μmtipdiameter)tothesurfaceof theepitheliumwhereit technique on the ‘loosepatchclamp’ technique(Stühmer et al.,1983).Usingthis currents generatedbyadepolarizing commandarebeststudiedwith behaves likeasensorysurface. of harm’s way(Mackie,1965).Thewholeoftheexumbrella water isdirectedforwardsandthecolonypropelledbackwards out exumbrella causescontractioninitsbell-likenectophores;a jet of propagate ataspeedof0.17–0.3 and 1997). InRhabdocalyptus,actionpotentialslastforatleast5s recorded inresponsetoelectricalstimulation(LeysandMackie, grafted attachmentsthat‘all-or-nothing’ actionpotentialshavebeen clumps ofcellspreparedfromtheirowntissue.Itisthese patch clampexperiments,buttheydorecognize,andaccept,grafted have failed.Thecellsalsoresistforminghigh-resistancesealsfor all attemptstopenetrateglassspongecellswithmicroelectrodes 1983). Thesearenottheeasiestanimalstorecordfromandthusfar immediate cessationofpumping(Lawnetal.,1981;Mackie the inflowingwater, areassociatedwithaprotectiveresponse–an more naturally, alocalmechanicaldisturbancesuchassedimentin chambers topumpseawater. Stimuli suchasacurrentpulseor, Rhabdocalyptus allow thefreeflowofioniccurrent.Likeothersponges trabecular reticulumisasyncytiumwithcytoplasmicbridgesthat Electrogenesis intheglasssponge Electrogenesis inanexcitabletwo-dimensionalsheet The delicatenatureoftheepithelial tissuemeansthattheionic Sarsia mmol l and − 1 Nanomia Co Nanomia, weappliedarelativelylarge micropipette Nanomia 2+ as iftheinwardcurrentiscarriedbyCa is afilterfeederthatusesdelicateflagellated colony meetsanobstruction,impulseinthe (Mackie, 1964;MackieandPassano,1968;

Rhabdocalyptus dawsoni cm Rhabdocalyptus s − 1 . Substituting50%ofthe Paramecium because its

2+ cm (Leys et s − 1 537 (at 2+

The Journal of Experimental Biology potentials asin would expectamaintainedstimulationtogenerateseriesofaction different. Thecurrentin two specieswestudied( Interestingly, wefoundthatthekineticsofcurrentineach transient outwardpotassiumcurrentlikethatin electrode voltageclamp R.W.M., unpublishedresults).Whendepolarized underatwo- japonica free saline,justasintheexumbrellarepitheliumof potentials ineitherNa 538 Analysis ofthegenomes of possible torecordactionpotentialsinoocytesfromdifferent remaining inwardcurrentwilldepolarizethecell. stimulus theoutwardcurrentgraduallyinactivatessothatany an actionpotentialunlessthestimulusispersistent.With amaintained current. Thefunctionofthiscurrentistopreventtheoocytegenerating dominated byarapidlyactivatingandinactivatingoutwardpotassium currents usingatwo-electrodevoltageclampandfoundthattheywere polyspermy. Hagiwaraetal.(Hagiwaraal.,1981)studiedthe oocytes theactionpotentialispartofmechanismthatprevents potential orevenaseriesofactionpotentials.Inmanyfertilized maintained depolarizingcurrenteventuallygeneratesanaction reniformis, the‘SeaPansy’ (Hagiwaraetal.,1981).Injectionofa 1 Fig. was foundtoexhibittwoinwardcurrents:onecarriedbyNa (Armstrong andBezanilla,1974).Innerve-freeregions, (see Stühmeretal.,1983)anddigitalprocessing(P4technique) loose sealweresubtractedbyacombinationofananaloguecircuit between thepipettetipandsurroundingmediumthrough negative pipettepressureofabout5 180–280 filled withseawaterandsopipetteresistanceswereintherange REVIEW neuron-like structures (Hernandez-Nicaise,1973; Hernandez-Nicaise, (see Moroz,2015).Electronmicroscopy showssynapse-likeand ctenophore neuronsevolvedindependently ofothernervoussystems Pleurobrachia bachei Electrogenesis inctenophoremuscle cells Electrogenesis inoocytes activated around0 and wascarriedbyCa Using techniquessimilartothoseofHagiwaraetal.,itwas Beroe shows recordsfromanoocyteofthecnidarian (Schwab andJosephson,1982). K seFg 2)(A.Bilbaut,M.-L.Hernandez-Nicaiseand (see Fig. Ω . Sealswereencouragedtoformusingamaintained Renilla

mV, theotherappearedatpotentialsclosertorest (Moroz etal.,2014)supportsthe possibilitythat 200 ms + -free orCa oye Fg 1A). oocytes (Fig. Beroe 2+ B. mitrata . Thus,onemightexpecttoseeaction B. ovata Mnemiopsis leidyi oocytes exhibitedarapidlyactivating 2+

mmHg. Leakagecurrentflowing -free salinebutnotinNa and inactivated soquicklythatone 10 20 mV –7 A B. mitrata)wasmarkedly (Ryan etal., 2013)and 9.5 14 19 23 V 0 5 I Nanomia Euphysa Renilla Renilla + /Ca 2+ + - . , observed tosummateandgiveriseovershootingactionpotentials. communication). Flurriesofexcitatorydepolarizations were the enzymetreatment(M.-L.Hernandez-Nicaise,personal observed, inEMsections,tohaveretainedtheirsynapsesdespite and R.W.M., unpublishedresults).Some fragmentshavebeen isolated fragmentofmuscle(A.Bilbaut,M.-L.Hernandez-Nicaise appear tobeexcitatorysynapticeventsrecordedinanenzymically showswhat 3 in viewoftheabsenceotherpublishedwork.Fig. primary focusofourwork,anincidentalobservationisinterest hydroskeleton. to maintainthetensionacrossmesogleastiffen itintoa with theirseparatefunctions,whichinthecaseofradialfibresis component ineach(Bilbautetal.,1988a).Thesepropertiescorrelate that providesthesignalforcontraction,formsanimportant their electrophysiologydiffers markedly, theinwardcalciumcurrent impulses. Two-electrode voltage-clamprecordsshowthatalthough depolarizing command,whereasradialfibresproducemultiple generate single,long-lastingactionpotentialsinresponsetoa distinguished: radial,longitudinalandintestinal.Longitudinalfibres 1984). InBeroe ovate 1988a; Bilbautetal.,1988b;Anderson,1984;Steinand (Hernandez-Nicaise etal.,1980)orafterisolation(Bilbaut to beaccessibleintracellularmicropipetteseither muscles, however, althoughhighlystretchsensitive,arelarge enough but alsobecausetheyareburiedwithinastiff mesoglea.Ctenophore This islargely becauseofthesizesupposedneuronalelements, marked absenceofelectrophysiologicalrecordings(Tamm, 2014). we cansaylittleabouthowctenophorenervesfunctionbecauseofthe of twodistinctlyseparatenervenets(Jageretal.,2011). Unfortunately, 1991) whileimmunofluorescencestudiesdemonstratetheexistence structural domains. (such asselectivity, gatingetc.)arealsoseparatedintospecific molecule toalarge extentastand-aloneunit,butchannelfunctions process ofelectrogenesis.Not onlyiseachindividualchannel classes ofion-selectivechannel thatcontributetothecomplex independently ofothercomponents’. Suchisthecasewithmany module is‘acomponentof asystemthatoperateslargely Schlosser andWagner (SchlosserandWagner, 2004).Forthem,a significance ofmodularityinevolutionhasbeenreviewed by the modularnatureofion-selectivechannels.Thedefinition and blocks forimpulsegenerationmightseemsurprisingwereitnot for The frequencywithwhichnaturalselectionassembledthebuilding The elementsofelectrogenesis 50 ms Although astudyoftheBeroe 10 The JournalofExperimentalBiology(2015)doi:10.1242/jeb.111955 –7 A (Hagiwara etal.,1981). shown byopencircles, continuous traces, start ofeachcurrenttrace.Holdingpotentialfor Depolarizing commandvoltageinmVshownatthe commands undervoltageclamp(shownatright). transient K depolarization aredelayedbyarapidlyactivating potentials (shownatleft)elicitedduringaprolonged Fig.

.ElectrogenesisinaRenilla 1. , threemainclassesoffibremaybe + current recordedduringdepolarizing − nervous systemwasnotthe 76 mV; holdingpotentialfordata − 30 mV. FromHagiwaraetal. oocyte. Action in situ

The Journal of Experimental Biology 100 nA REVIEW expressed it.To beafullymobileexploratory unit,ananimalmust relaying ‘statesofexcitement’ asSherrington (Sherrington,1906) backwards. Neuronalelectrogenesis, however, goesbeyondsimply bumps intoabarrierandthe colony immediatelypropelsitself spreads andthewholestructure stopspumpingwater; purely reactive;aglasssponge takesinsediment,themessage Electrogenesis, inthedifferent formswehaveexaminedthus far, is neural integration. system, inparticularthecontributionofitselectricalproperties to systems. Here,Idiscusstheperformanceofhydrozoannervous predicted bythectenophoregenomewiththatofothernervous Moroz (Moroz,2015)hascomparedthetransmittermake-up understanding ofthefactorsthatdeterminetheirformandfunction. comparisons betweendifferent neuralsystemsshouldincreaseour independent evolutioninctenophoresandcnidarians.Thatbeing so, electrogenesis fromion-channelmodulesisconsistentwith its fully operationalwatch. completed subassembliescouldbequicklyputtogethertomakea interrupted, helostonlyasmallproportionofhisworkandthe watch tobemadeofsubassemblies.Thatway, werehetobe pieces. Thesuccessfulwatchmakerwastheonewhodesignedhis When theywereinterrupted,theirpartlyassembledwatchesfellto highly soughtafterwasthattheywereconstantlybeinginterrupted. two highlyregardedwatchmakers.Theproblemwiththeirbeingso highlighted bySimon(Simon,1962)intheformofaparableabout Implications for integration neuronal The apparenteasewithwhichnaturalselectiongenerates The advantageofamodularsystemconstructionwas B A 0m 80mV 50mV 80 mV 50 mV 400 ms 120 mV Nanomia later). reducing tentacledragexhibited by (Donaldson etal.,1980;Mackie, 1964)andthedifferent ways of swimming’, whichin Variations inthegroundplanincludecircuitry for‘escape the initiationofswimminguntil afterthetentacleshavecontracted. by tentacleretractionasifacommon mechanismensuresadelayin is anothercommonfeature.Inmanyspecies,swimmingpreceded 1955b), suggestingthataninhibitoryinputtothepacemakersystem hydromedusae stopswimmingwhiletheyarefeeding(Horridge, pacemaker system(SatterlieandSpencer, 1983).Many rhythm byadistinctsetofelectricallycoupledneurons – the However, onecommonfeatureistheproduction ofaregularswim been achallengetodefine‘groundplan’ fortheirneuralcircuitry. as ‘pointing’ (Romanes,1877)]. 1940)] andescape,mouthmanipulationduringfeeding[described tentacle length,defensiveresponses[called‘crumpling’ (Hyman, identifying fourmainareasofinterest:swimming,control of necessarily restrictedtoashortsectionattheend.Ifollowothersin hydrozoan jellyfish;linkswiththescypho-andcubo-medusaeare channels inmechanismsofintegrationandcoordination expense ofanother. responses; coordinationrequirestheoperationofonefunctionat requires thatall-or-nothing signalsbetranslatedintograded coordinated andexecutedinanefficient manner. Neuralintegration and integratetheinformationobtainedsothatresponsescanbe take stockofitsenvironment;itmustsamplemultiplemodalities Hydromedusae aresuchadiversegroupofanimalsthatithas To beginthissectionofthereview, Ifocusontheroleofion 20 ms The JournalofExperimentalBiology(2015)doi:10.1242/jeb.111955 50 nA Aglantha unpublished results. Bilbaut, M.-L.Hernandez-NicaiseandR.W.M., temperature inartificialseawater. DatafromA. ( Holding potential, on therighthandarefrom Fig. Traces ontheleftarefrom commands undervoltageclamp(toptraces). currents (bottomtraces)elicitedbyvoltage current pulseissub-threshold.(B)Ionic trace). Two superimposedtraces;theshorter response toadepolarizingcurrentpulse(top (A) Actionpotentialrecorded(bottomtrace)in B. mitrata).Measurementsatroom

.ElectrogenesisinaBeroe 2. and Chelophyes Nanomia − 60 .ovata);− mV (B. and replace crumpling Beroe mitrata Beroe ovata Muggiaea egg. 70 ; those . mV (see 539

The Journal of Experimental Biology Nicaise andR.W.M., unpublishedresults. membrane potential.Holdingpotential− commands. Notethattheclampwasimperfectatpositivepotentialsbecausemicropipettecouldnotsupplylargecurrents necessarytocontrolthe appearance ofadoublespike.(D)Voltage-clamp recordsfromsamemusclefragmentshowingthetypicalresponseofalongitudinal muscletodepolarizing (see Bilbautetal.,1988a).InC,fallingphaseoftheEPSP (anditsshort-circuitingeffect) appearstoendjustbeforethepeak ofthe actionpotentialgivingthe (B,C) Seriesofspontaneousdepolarizingevents,possiblysynapticinorigin,recordedfromafragmentlongitudinalmuscleproduced byenzymicdigestion Note themultiplenuclei(arrowheads)andthinsarcolemmalevaginations(arrows).FromHernandez-Nicaiseetal.(Hernandez-Nicaise etal.,1980). 540 muscle responsewithdistancefrom thepreysite( forgradationofmanubrial manubrium (m)ofanunfedanimalat rest.(C)Lateralflexion(pointing)toasitewherefoodisheldbythetentacles. (D)Mechanism copepod). Tentacularlips. (B)Peduncle (p)and contractionandflexionbringsthepreytomarginwhilemanubrium pointsacrosstoitwithflared Fig. contrast. Scalebar,extensively; theendingsanchoruponepithelium,whichishiddenbypharyngealcircularmuscles.Nomarskiinterference 20 Fig. REVIEW become increasingly separated. See text for more details. A–C from Mackie et al. (Mackie et al., 2003); D adapted and redrawn from Mackie(Mackie, 1984). become increasingly separated.Seetextformoredetails. A–CfromMackieetal.(Mackie etal.,2003);Dadaptedandredrawn from at eachoftherecording sitesshown.ThesecondFimpulse travelsmoreslowlythanthe first andsummationbecomeslesseffective asthe actionpotentials requirement forintegrationandcoordinationdependsoninteraction process ofnaturalselection(Simon,1962).Despitethis,the but thereaftertheyarelikelytobefavouredduringthetrialanderror cost ofmakingnerveconnections(seeLaughlinandSejnowski,2003) nerve circuitmodulesmayhavearisenasawayofminimizingthe nodes thataresparselyconnectedtoinotherclusters’.Such a networktobemodularifitcontains‘highlyconnectedclustersof a separateneuronalsystem.Cluneetal.(Cluneal.,2013)consider is somewhatmodularinnaturethateachundertheinfluenceof The nervouscontrolofthesedifferent aspectsofjellyfishbehaviour

.Food captureinAglanthadigitale.(A)Ciliarybeatingdriveswaterpast theextendedtentacles(arrows)bringingthemintocontactwithprey(a 4. Electrogenesisinenzymicallyisolated smoothmusclefrom 3. A A B m p 50 mV; experiments conductedinartificialseawateratroomtemperature.DatafromA.Bilbaut,M.-L.Hernandez- C * CD B ). Eachsetoftraces(a–c)represents apairofFimpulses(top)andthegradedmuscleresponses(bottom) 60 mV 20 ms Beroe ovata conduction system’ (MackieandMeech,1995a;Mackie movements ofthetentaclesarecoordinatedby‘tentacle (Romanes, 1877;Horridge,1955a).In and bringthemtothemargin hasbeendescribedpreviously The wayinwhichthetentaclescapturesmallplanktonicorganisms and betweenslowfastswimmingin feeding andswimming,betweententaclecontractionswimming between modules.Here,Ifocusonthreesuchinteractions: Interaction betweenfeedingandswimming D a . (A)Pharyngealendofalivingradialmusclecell.Thefibrebranches * 50 nA 100 mV The JournalofExperimentalBiology(2015)doi:10.1242/jeb.111955 b 10 ms a bc c Aglantha –50 mV . , thegraded μ m.

The Journal of Experimental Biology REVIEW Meech, 1995a;Mackie andMeech,1995b,Mackie Meech,2000). tentacle giantaxon (TG)andtheslowtentaclesystem (TS)(seeMackieand tentacle nervesincludethose thatfeedintotheFsystem(TF), et al.,2004);(7)therelaysystem(R) (MackieandMeech,1995a);(8) 1995a; MackieandMeech,1995b); (6)thenitricoxidepathway(NO)(Moroz carrier (C)systemandthemotorgiant axon(MG)(MackieandMeech, vibration receptorsatthebellmargin mediatesescapeswimmingviathe impulses spreadinalldirections;(5) theringgiantaxon(RG)excitedby pathway arecoupledbygapjunctions;conductionisunpolarizedsothat inhibits swimming(MackieandSingla,1997);thecellsinthisepithelial Meech, 2000);(4)theexumbrellarconductionsystem(EX)whichalso (Mackie etal.,2012);(3)theswimpacemakersystem(P)and endodermal system(E)thatmediateslipflaringandswimminginhibition pointing movementsseenduringfeeding(Mackieetal.,2003);(2)the 1995a). Thepathwaysinclude:(1)theflexionsystem(F)thatmediates the piggyback effect isdescribed inMackieandMeech(MackieMeech, A minussignindicatesinhibitoryinput;allothers areexcitatory. The system (RI)withthemotorgiantaxons(MG)andbetweenepithelial cells. incomplete membranepartitionsatthejunctionsofrootletinterneurone wall ofthepeduncleandmanubriumareshown.Gapjunctionsshown as manipulation. Threeoftheeightlongitudinalmusclebands(blue)lyingin the pathways involvedinswimming,thecontroloftentaclelengthandmouth Fig. of smallaxonswithFMRFamide-likeimmunoreactivity. These muscles) inthemanubriumwheretheyareassociatedwithbundles underside ofthebell.Theydo,however, persist(aslongitudinal Aglantha circular, striatedswimmuscles.Therearenolocalcontractionsin contraction arelocatedintheectodermwheretheyoverlie 1955b). Theradial,smoothmusclesresponsibleforthelocal towards themanubriumduringtransferprocess(Horridge, Aequorea, alocalcontractionofthemargin servestobringthefood 4.Inlessprolatespeciessuchas ingestion asshowninFig. bends towardthefood(points)andengulfsitwithitslipspriorto 1995b). Oncethepreyisatmargin, themanubrium(mouth) NO RG

.Nervecircuitsin 5. RI C R F P Piggyback effect Bidirectional input Excitatory synapticinput Inhibitory input for thegoodreasonthatradialmusclesareabsentfrom Manubrium FN STG TS NO TF MG Peduncle Sensory cell Anoutlineoftheprincipal Aglantha digitale. Tentacle FE EX arise inthemanubrium duringingestionareconducted inanerve Neoturris breviconis which similaritiesofgroundplan canaidtheexperimentalist.In with threedifferent hydrozoanspecies;anexampleofthe wayin to themouth. trapped preyandinhibitionserves tofacilitatethetransferoffood strong watercurrentsgeneratedduringswimmingtendtodislodge period ofinhibitiondependedonthestimulusduration.Perhaps the to thelipsorfollowingelectricalstimulationofmanubrium; the 2003). Swimmingwasinhibitedwhenfoodpresenteddirectly (Horridge, 1955b)andAglantha different systems(Spencerand Arkett,1984). a schematicdiagramtoshowtherelationshipsbetween these 6Bis input fromothersourcessuchasthe‘B’ and‘O’ systems.Fig. baseline oscillations,buttheprecisepatternofactivitydepends on neurons showwhatappeartobeslowendogenouslygenerated motor neuronsystem.Inmanyhydrozoanspecies,swim recorded actionpotentialsaresynchronousthroughouttheswim Electrical coupling(viagapjunctions)meansthatintracellularly subumbrellar musculatureviachemicalsynapses(Spencer, 1975). nerve ringatthebaseofbell,aredirectlylinkedto 1983). These‘swimmotorneurons’,whicharelocatedintheinner 6A)(AndersonandMackie,1977;SatterlieSpencer, (see Fig. been identifiedandstudiedinmostdetail neurons thatgeneraterhythmicswimminginhydromedusaehave make synapticcontactwiththepacemakersystem.The ‘E’ system(see Fig.5).Atthemargin theEsystemisbelievedto manubrium, theyalsogofromthemanubriumtomargin viathe Mackie, 1984). velocities) hasbeendemonstratedinthestemof to this(butinvolvingtwonerveswithdifferent conduction other musclebandsfurtheraway. Anintegrativemechanismsimilar closest tothefoodsitewillbeenhancedrelativecontractionat responses inthemanubrium,contractionofmuscleband 4Billustrates,ifsuccessiveimpulsesproducesummating Fig. further theimpulsestravelmoreseparatedtheywillbe.As slowly thanthefirst(Mackieetal.,2003).Thismeansthat the secondoftwoimpulsesevoked250 seems torequiremultiplefiringofthesmallaxonbundleandthat A possibleexplanationstemsfromtheobservationthatpointing excitation mayfailtobeconveyedtheassociatedmusclebands. way oftheFsysteminnervering.Itappears,however, thatthis excited, butimpulseswillspreadtoothersmallaxonbundlesby muscle bandinthemanubrium. to thatsiteresultsfromimpulsestravellingdirectlytheassociated point onthemargin neartheoriginofasmallaxonbundle,pointing 5).Wecylinder, supposethatwhenfoodiscapturedata F, inFig. by circularpathwayswithinthenervering(shownasahorizontal position betweenradialcanalsasifthepathwayswerelinked when thestimulatingelectrodewasmovedtoanintermediate 15 stimulation site.Suchpointingwasfoundtobeaccuratewithin small axonbundlejoinsthemargin, themanubrium‘pointed’ tothe ring atthebaseofoneradialcanals,i.e.apointwhere al., 2003). the belltowardsmargin alongsideeachradialcanal(Mackieet 5;theyrundown axons areshownaspartofthe‘F’ systeminFig. Analysis oftheinhibitorypathway concerneddependedonwork Many hydromedusaestopswimmingwhiletheyarefeeding During feeding,nerveimpulsesnotonlytraveltowardsthe The musclebandsclosesttothefoodsitewillbefirst When deg, 70%ofthetime(Mackieetal.,2003).Itwasjustasaccurate Aglantha The JournalofExperimentalBiology(2015)doi:10.1242/jeb.111955 preparations werestimulatedontheouternerve the trainsofactionpotentials(E impulses)that is noexception(Mackieetal.,

ms aparttravels73%more Polyorchis penicillatus Nanomia (see 541

The Journal of Experimental Biology more detailsseePrzysiezniakandSpencer(PrzysiezniakSpencer, 1992).(H)Availability of (Przysiezniak andSpencer, 1994).(F)Availability of 542 medusae, suchas has yettobedemonstratedanatomically(Mackieetal.,2003). where thepacemakerneuronsarelocatedbutdirectsynapticcontact this Esystemrunaroundthemargin adjacenttotheinnernervering, and ringcanals(MackieMeech,2008).Neuritesbelonging to plexus locatedintheendodermalwallsofstomachandradial Fig. REVIEW peak current.Formoredetails,seePrzysiezniakandSpencer(Przysiezniak andSpencer, 1994). ( (to +50 current after subtracting the non-inactivating component. The curves are Boltzmann functions fitted to the average peak current. Holdingpotential, current aftersubtractingthenon-inactivatingcomponent.ThecurvesareBoltzmannfunctionsfittedtoaveragepeakcurrent. steps) ontheavailabilityofinactivatingcurrent.At preceded byaconditioningcommandlasting1 2.2–9.2 Mackie etal.(Mackieal.,2012).(E)InactivatingK synaptic potential(lowertrace)coincidentwithanexternallyrecorded‘B’ systemimpulse;fourexcitatoryeventswerealigned system pulse.(D)Excitatory by aspontaneousdepolarizingexcitatorypostsynapticpotential(EPSP;arrow)thatiscoincidentwithanexternallyrecordedB canal.TheIPSPinhibitory potential(bottom)inresponsetoanexternallyrecordedEevent(top)afterelectricalstimulusthenearbyradial wasterminated 1984). (C)Intracellularlyrecorded All threenetworksconsistofaringelectricallycouplednervecells.AdaptedfromSpencerandArkett(SpencerArkett, swim motorneuronstothe‘O’ indicatedbyfilledtriangles. system(green)intheouternervering;otherconnectionsmaybepresent.Excitatorysynapsesare and fromunicellularreceptors(orange).Excitatoryinputstothe‘B’ isanexcitatorypathwayfromthe systemareshownarisingintheocelli(purple).Also inner nervering,theswimmotorneurons(red).They, inturn,receiveanexcitatorysynapticinputfromneuronsofthe‘B’ (yellow)intheouternervering system bypacemakersinthe representation ofthering-shapedneuronalnetworksshowingknownsynapticcontacts.Themuscleepithelium(blue)isdirectlyexcited (G) Availability of averaged ±s.e.; tomaximalcurrentand command. Fast(opencircles)andslow(filledcomponentsobtainedbyfittingexponentialcurvestorawdata.Datanormalized i.6C).Ina potential (IPSP) inanearbyswimmotor neuron (Fig. the peripherywasassociated withaninhibitorypostsynaptic 2012). Followingstimulation, the appearanceofEimpulseat intracellularly whilestimulating theEsystemaxons(Mackieetal., species andithasproved possible torecordfromthem In Aglantha N =8). Data normalized to the maximal current after subtracting the non-inactivating component. The solid curve is a Boltzmann function fittedtotheaverage =8). Datanormalizedtothemaximalcurrentaftersubtractingnon-inactivating component.ThesolidcurveisaBoltzmannfunction There appearstobetheequivalentofanEsysteminother Polyorchis

.ExcitatorysynapticinputtoPolyorchispenicillatus 6. mV) precededbya1

ms fromthebeginningoftestpulse.Average valuesfor i.5)andPolyorchis (shown inFig. N the swimmotorneuronsare larger thaninother I Ca =14). ThecurvesarefittedBoltzmannfunctions.Holdingpotential; at different conditioningpotentials.Eachtestcommand(to+10 Sarsia B A

s conditioningcommand.Peakcurrentsmeasuredat12 (Hernandez-Nicaise andPassano,1967),

s. Thesuperimposedcurrenttracesshowtheeffect oftheconditioninglevel(range (Mackie etal.,2012). + I Na currents recordedundervoltageclampinresponsetotestcommands+50 − 20 at different conditioningpotentials.Eachtestcommand(to+10 mV theinactivatingcomponent( mV µV mV C µV E D swim motorneurons.(A)Polyorchis 100 –55 –45 –60 –50 0 50 0 0 0 I Ca –50 –20 –90 mV are shownbyfilledcircles( Time (s) Time (s)

1 2

1 2 100 ms The propertiesofasimilarcurrent (herecalled molluscan neurons(Hagiwaraet al.,1961)andcommonlycalled the range availability ofthiscurrentdepends onpreconditioningpotentialsin Przysiezniak andSpencer(Przysiezniak andSpencer, 1994).The 6E) swim motorneurons(seeFig. which isarapidlyactivatingtransient K number of‘background’ currents(ConnorandStevens,1971),oneof rhythmically firingcells,theinter-pulse intervalisdetermined bya apparent whenweconsiderthepacemakermechanism. In potential wasabout–69 about 700 typical example,thehyperpolarizationreachedamaximum after 1 nA

mV) precededbya1 The significanceofthisunusuallyprolongedIPSP becomes

− ms afterthecommandonset.Average valuesareshownbyfilledcircles 80 mV. Formoredetails,seeGrigorievetal.(Grigorieval.,1996). I Kfast − ) isabsent,leaving ms andstayedthereforabout500 20 to− The JournalofExperimentalBiology(2015)doi:10.1242/jeb.111955 I H G F Kfast n =6); forI (photograph providedbyClaudiaMills).(B)Partial at different conditioningpotentials. Eachtestcommand K+ current availability Ca2+ current availability Na+ current availability –0.2 –0.2 90 0.2 0.4 0.6 0.8 1.0 1.2 0.2 0.4 0.6 0.8 1.0 0.2 0.4 0.6 0.8 1.0 1.2

s conditioningcommand.Peakcurrentsmeasured –100 –100 –80 0 0 0 Conditioning potential(mV) –60 –50 i.6H);themoredepolarized the mV (seeFig. Ba

open circles( –80 –60–40 mV. I Kslow

–40 –60 –40

mV) precededbya2 . FromPrzysiezniakandSpencer

–20 02040

–30 –20 n andaveraged.DEarefrom =7). Datanormalizedtomaximal

–20 have beenexaminedby +

− current firstidentifiedin mV. Eachtestcommand

–10 90 to−

0 I Kfast 20

ms. Itsreversal s conditioning mV; 10 ) inPolyorchis − 80 mV mV. For I KA .

The Journal of Experimental Biology In this servestoremovethedrag that theywouldotherwisegenerate. tentacles extended,shortenthem justbeforeswimming.Presumably, ‘sink-fishing’ andspendmuchoftheirtime driftingwiththeir Mackie, 1963);Leuckartiaraoctona REVIEW Polyorchis IPSP willincreasetheavailabilityof from inactivationiscompletewithin1 membrane, themoreinactivatedchannelsbecome.Asrecovery through ajunctionalconductance Fig. reality. FromBennett(Bennett,1966).(D)Theanisometricnatureofthespreadcurrentin ifsuchashuntisphysical transformation). Leakageofcurrentisthereforeequivalenttoamembraneshuntinthevicinityjunction.Itnotknown (bottom;Wye-delta resistance originatingmid-waythroughthejunction(top)ortoresistancesinparallelwithpre-andpost-junctional Bennett andZukin(BennettZukin,2004).(C)Equivalentcircuitsfora‘leaky’ isrepresentedeitherbya junction.Thecurrentpathfromthejunction isintroduced.From of thetimeneededtochargeanddischargecapacitance;assumingajust-measurabledetectionlimitsignificantsynapticdelay capacitance actinginparallel.FromBennettandZukin(BennettZukin,2004).(B)Aattenuated andslowedbecause rapidlyrisingpre-synapticimpulseis 1 progressively attenuatedandslowedatfrequenciesabove about for theswimsystemin 2004). Spencer(Spencer, 1981)hasmeasured thefrequencycut-off 7A,B)(Bennett,1966;BennettandZukin, low-pass filter(seeFig. of electricalcoupling,whichmakestheentiresystembehave like a excitatory post-synapticpotentials,EPSPs–seebelow)isa result depolarizing effect ofanybackground inwardcurrent. increase inmembraneconductance,whichwillalsooppose the to delaythenextpacemakerimpulse.Thiseffect isinadditiontothe Interaction betweententaclecontraction andswimming circular direction has fewer leaky gap junctions as might be expected from the elongated form of the individual cells. From Kerfoot etal.(Kerfootal.,1985). circular directionhasfewerleakygapjunctionsasmightbeexpectedfromtheelongatedformofindividualcells.FromKerfoot Measurements inacirculardirection(opencircles)andradial(closedcircles).Thedifference thatthecurrentpathin incurrentspreadsuggests (seeJacketal.,1975). steady statechangeinmembranepotentialdeclineswithdistancefromapointsourceasmodifiedBesselfunction(solidline) produced bycurrentinjectedatdifferent totwodimensionsandthe distancesfromtherecordingsite.Inathinsheetofcoupled cellscurrentflowisrestricted

Hz. The slowtimecourseoftheswimmotorneuronIPSPs(and Proboscidactyla flavicirrata

.Low-passfilteringinelectricallycoupled neurons.(A)Equivalentcircuit.A processfromthepresynapticneuronconnectswithpost-synapticcell 7. and otherhydromedusae[ V B A Polyorchis pre Measured g

j Voltage . Currentthroughthejunctionalconductanceflowstogroundpost-synapticmembraneand delay , synchronous tentacle shortening I

(Russell, 1953)]thatengagein s (Przysiezniak,1993),atypical Kfast Detection Eperetmus typus g j and findsthatsignalsare level and asaconsequence,tend Time C Presynaptic impulse Postsynaptic potential g post V (Mackie and post

mV/5 nA D C 0.4 0.8 the swimsystem pacemakerbysomehidden mechanism.Itisas 1973). Nevertheless,asingle B systemspikeappearstoadvance frequency tocausethetentacles toresponddirectly(Passano, 1977; SpencerandArkett,1984; Mackieetal.,2012). to half-decay, 0.5 and slowlydepolarizingEPSPs (Fig. maintained tentacleshorteningaswellgeneratingsynchronized throughout. Bsystempulsefrequenciesof1 to haveanendogenouscomponentandittooissynchronous potential spikinginBneurons,likethattheswimsystem,appears the epithelialcelllayer(SatterlieandSpencer, 1983).Action neurons thatliealongthemid-lineofouternerveringjustbelow Spencer, 1975).Theyoriginatefromasystem ofelectricallycoupled Passano etal.,1967;OhtsuandYoshida, 1973;Mackie,1975; variously called‘marginal’ or‘pre-tentacle’ pulses(Passano, 1965; the nervering(seeFig. system, whichprovidesaninputtotheswimsystemfromallaround the delaycanlastforaslong3 siphonophore occurs lessthan1 V V pre pre In In g g pre pre Polyorchis, tentaclelengthisunderthecontrolof‘B’ Sarsia, activityintheBsystemis oftenattoolowa Aglantha The JournalofExperimentalBiology(2015)doi:10.1242/jeb.111955 ‘Leaky’ junction Distance (mm) Muggiaea, withitsmoreextensivearrayoftentacles, Equivalent 0.5 1.0 myoepithelium. Meanmembranepotentialdisplacement

s beforeaswimburst(Spencer, 1975)butinthe s) intheswimsystem(Anderson andMackie, g j

6B). TheseBsystemimpulseshavebeen

s (R.W.M., unpublishedresults). g g post post V V 6D; meanduration,1.3 post post

s − 1 , ormore,cause

s; time 543

The Journal of Experimental Biology membrane voltagethatreducestheavailabilityof Fig. and Spencer, 1992;Grigorievetal.,1996).Astheshadedregionin a secondoutwardlydirected K addition tothecurrentsreported byInoueetal.(Inoueal.,2005), myoepithelium usingtheloose patchclamptechnique,wefoundin contraction wasofuniform strength. Whenweexaminedits were fullycontracted,then,whenitdidfinallyswim, each inactivation intherange 1994) whereastheinwardcalciumandsodiumcurrentsshowlittle results). Unlike action potentialduration(R.W.M. andG.O.Mackie,unpublished repolarization, thiscurrentwould ensureagreateruniformityin inactivate summate andtakethemembranetowardsthreshold,buttheyalso therefore thattheEPSP inputhastwoeffects: EPSPsnotonly little effect onthechannelscarryinginwardcurrent.Itappears Ca studies, have evolveddifferent ways tomanagetheirtentaclenets.Inrecent that leadstotheincreasedstrengthofcontraction. potential becomesprogressivelylonger. ItistheenhancedCa fast inactivatingK This meansthatwitheachsucceedingimpulse,lessand K 544 current becomesavailableforrepolarization.TheinwardNa In inactivated after1 is half-inactivatedwiththemembraneat 750 ms (seeFig.6D),longenoughtopartiallyinactivate 1984; Mackieetal.,2012)reachabroadflatmaximumafterabout action potentialthreshold. summating EPSPsintheswimsystemeventuallyexceed apart, thedelayisabout750 depends ontheinter-pulse interval.IftheBsystempulsesare1 will initiateaswimsystemimpulseaftercharacteristicdelaythat membrane tothreshold.A pairofBsystemimpulses,however, if asingleEPSP resetsthepacemakerwithoutraising REVIEW course. system arelinkedbytwoprocesses,eachfollowingadifferent time Passano’s (Passano,1973)conclusion thattheBsystemandswim available potassiumcurrent.Thisexplanationwouldaccountfor Tentacle draginsiphonophores and soCa potential, recoveronlyslowlyfrominactivation( channels responsibleforrepolarizingthemembraneduringanaction cells byInoueetal.(Inoueal.,2005)showsthatthepotassium 1981). A voltage-clampanalysisof enzymicallyisolatedmyoepithelial during aseriesofswims,showgradualincreaseinduration(Bone, can beachievedwiththesameexpenditureofenergy. configuration ismorestreamlined,agreatervelocityofmovement and tentaclesoftheanimalhavehadtimetocontractoverall the swimvelocitylow;laterinsequence,whenstem and sowhenthedragishigh,energy canbeconservedbykeeping energy expendedistheproductofdragandvelocity(Vogel, 1994) the tentaclenetasitisdrawnintoamorestreamlinedshape.The Weaker swimsduringtheearlystagesofsequencereconfigure strength duringaswimsequence(Bone,1981;Chainetal.,1981). elaborate tentacle‘net’ totrapitsprey, swims aregraduatedin The twosiphonophores,Chelophyes EPSPs in Action potentialsrecordedfromthemyoepitheliumof Chelophyes appendiculata 2+ 6F–H shows,swimsystemEPSPsactwithintherangeof currents recoverintimeforthenextimpulse(i.e.within200 Muggiaea 2+ I Kfast Polyorchis entry isenhancedastheplateauphaseofaction and resetthepacemakerbyreducinglevelof Chelophyes, therefore,tentacle contractionin

+ s at− was foundtodelayswimminguntilitstentacles current (PrzysiezniakandSpencer, 1994). 30 swim motorneurons(SpencerandArkett, − mV (Fig. 50 to− , anothersiphonophorethatdeploysan

ms. Presumably, inthiscase, 60 + 6H; PrzysiezniakandSpencer, current. Bycontributingto mV (Fig. − and 52 mV andisalmostfully Muggiaea τ 6F,G; Przysiezniak 1. sat =13.2 I Kfast while having Chelophyes appear to − I Kfast 70 2+ + mV). entry , the I

ms) Kfast and

s + neurons. a specializedmodule,whichcomprises aseparatesetofpacemaker of whicharerepetitivelyactive. Rhythmicswimmingisinitiatedby a subsidiarysystemof‘lateral neurons’ (Weber etal.,1982),none into asetofeightmultinucleate giantaxons(seeMackie,1989)plus the availabilityofrepolarizingK Mackie, 1993b).Consequently, duringaburstofslowswimspikes, this currentrecoversfrominactivationonlyslowly(Meechand low amplitudeCa later ones(seeMeechandMackie,1993a).Slowswimsdependon adopted byChelophyes;itsearlyswimsaresomewhatweakerthan before swimminggetsstarted, Thus, duringslowswimming,insteadofshorteningitstentacles swimming stopsthetentaclesstarttoextendagain’ (Mackie,1980). extended, butthentheyshortenandcurlinward.…Justbefore phase, perhapsforthreeorfourcontractions,thetentaclesarestill bursts ofactivityvariablelength.‘Duringtheearlypartthis (Fig. transmitted totheringgiantandsobacktentacles. way ‘valve’,sothatactivityinitiatedinthegiantmotoraxonsisnot thereby ensuringthatthetentaclescontractfirst.Italsoactsasaone- system isthatitdelaysthemessagegettingtobellmusculature, motor giantaxonsinthesubumbrella.Oneeffect ofthecarrier and tentaclegiantaxonsalsobetweenthering Fig. of smallneuronsintheouternervering(the‘carriersystem’ in destroyed, thetentaclegiantscanbeexcitedbystimulatingasystem to minimizeanysynapticdelay. However, iftheringgiantis 1980) suggestthattentacleandringgiantsarecoupledelectrically (Donaldson etal.,1980).RobertsandMackie(RobertsMackie, (the tentaclegiantinFig. rapidly shortenintotightcoilsasaresultofactivityinlarge axon both formsofswimming.Duringanescapeswim,thetentacles (see Fig. escaping frompredators,andslowswimming,whengatheringfood Aglantha it isinthesiphonophoresitsmanagementsurprisinglycomplex. Although thetentaclenetin considerable inbuiltdelay. Muggiaea In system whichisunderthecontrolofrelay(seeFig. stronger (Fig. contractile responseinthemyoepitheliumbecomesprogressively the slowswimspikesincreaseinduration,andastheydoso, by arapidlyinactivatingoutwardK (Mackie andMeech,1985).Theseneuronalspikesarerepolarized Tentacle dragin Interaction between slowInteraction andfast swimming in swims. tentacle contractionmaybeinterposedwithinthesequenceof slow 1995b). Ifitdoesso,firesthetentaclegiantsothatastronger EPSPs maycausetheringgiantaxontospike(MackieandMeech, pacemaker, relayandcarriersystemsfire together, theirsummating giant). Therelaysystemalsoexcitesthecarriersystem.When the tonic contractions(ratherthanthetwitchesproducedbytentacle during slowswimmingthetentaclesgothroughaseriesofgraded The relaysystemis,inturn,excitedbythepacemakerso that Slow swimming,performedby The lengthofthetentaclesisalsoregulatedviaaslowtentacle Aglantha digitale 5). Thiscarriersystemactsasanintermediarybetweenthering 9A), isusedtogainheightinthewatercolumn.Itoccurs Aglantha 8 andFig.9A).Thetrailingtentacleswouldcompromise is unusualinthatitexhibitsbothfastswimming,when precedes swimactivation,whichoccursonlyaftera The JournalofExperimentalBiology(2015)doi:10.1242/jeb.111955 9D). Aglantha digitale swims eitheras a meansofraisingitsposition in 2+ the swimmotorneuronshave become fused ‘spikes’ inthe motorgiantaxons(Fig.

5) thatrunswithinthetentacleitself Aglantha digitale Aglantha + current decreases.Thismeansthat + Aglantha current, andasin uses astrategyakintothat is notasextensive when ‘sink-fishing’ Aglantha Chelophyes,

9B)

5).

The Journal of Experimental Biology (Vogel, 1994),whichmayaccount forthefactthat means thateachescapeswim consumes alarge amountof energy the muscleepitheliumandvelocity ofthewaterleavingbell advance ofthebellsothatasignificant reversethrustisavoided. Fig. substantial thrusttojettheanimal forwardby5–10bodylengths.As 1980). Itisthehighvelocityofejectedwaterthatproduces the water thatfillsthebellisforced(seeFig. the bellmargin closingdown toformanozzlethroughwhichthe The endresultisthattheentiremyoepitheliumexcitedat once, REVIEW threshold fortheNa in thesubumbrella.Thesynapticdepolarizationexceeds the synaptic eventsineachofthemotorgiantaxonsthattravelradially giant axon.Actionpotentialsintheringgeneratesharplyrising al., 1988)sensevibrationsandtriggeractionpotentialsinthering among single-belledjellyfish. ‘escape swimming’).Thisdualformofswimmingappearsunique the watercolumn(‘slowswimming’)orforprotection(high-speed motor giantensurethatexcitationspreadsrapidly(about39 the myoepitheliumandlateralneuronselectricallycoupledto the Neuromuscular synapsesallalongtheaxoncarryexcitation to across themusclesheetbetweengiantmotoraxons(seeFig. propagates thelengthofeachaxontravelingatabout4 B A The squarerelationshipbetween thekineticenergy supplied by During anescapeswim,‘haircells’ atthebellmargin (Arkettet 8C shows,duringtherefilling processthemargin widens in 0 + 76 32023393 293 210 93 60 27 action potential(Fig.

8A) (Donaldsonetal.,

9B), whichthen

Aglantha Change in diameter 0.4 0.8 1.2 0.4 0.8 1.2 0 0 0 300600900 C

cm

m

uses 9C).

s s − − 1 1 ) . Na The motoraxonshavetwothresholds,oneforthehigh-threshold channels (MackieandMeech,1985;MeechMackie,1993a). 1981; Pinatoand Midtgaard,2005). from dendriticfieldsinthevertebrate brain(LlinásandYarom, (Fig. contraction areelicitedbytwodifferent formsofpropagatingsignal (Singla, 1978;Kerfootetal.,1985).Thedifferent patternsof ‘giant’ motoraxonsthatsynapsedirectlywiththemuscleepithelium in responsetostimulation. expected tooperateasasingleunitproducinguniformcontractions are electricallycoupled(Kerfootetal.,1985)andassuchwould be a physiologicalproblembecausethecellsofmuscleepithelium ejected stayslowandrelativelylittleenergy isconsumed.Thisposes remaining openwithawideaperturesothatthevelocityofwater contraction takesplaceintheupperhalf,baseofbell the bellduringaslowswim(seeFig. an energy-saving slowswimmode(Mackie,1980).Examinationof fast jetpropulsionrelativelyrarely. Whencollectingfood,itenters slower low-amplitude‘spike’ basedonCa amplitude slowswimCa the Ca kingdom, theselow-amplitude Ca escape swimming.Althoughapparently uniqueintheinvertebrate Time (ms) Surprisingly, thetwoformsofswimarisefromsingleset + -dependent actionpotential,and oneforthelow-threshold,low- 9B); afastovershootingNa 2+ spike topropagateindependently, belowthethresholdfor The JournalofExperimentalBiology(2015)doi:10.1242/jeb.111955 2+ 0.0038 ( of eachframe.(B)Video framesat0.1 Time (ms)afterstimulusisshownatthebottom vibration receptorsatthebaseoftentacles. elicited byaglassprobeusedtodisturb digitale. (A)Video framesofanescapeswim Fig. A of theswim.Mid-wayupbellvaluesfor damping coefficient, change inanun-dampedsystem, the valuesduringslowswimare0.18( 1750 0.0038 ineachcase; from R.W.M., unpublishedresults. captured at1/300 intervals ofasingleslowswim.Frameswere x performance ofadampedoscillator: drawn accordingtoanequationforthe including thoseinB.Linesthroughthedata measured fromindividualvideoframes that atthemidpoint(opencircles).Data the bell(closedcircles)issignificantlylessthan (bottom) thechangeindiameteratbaseof including thoseinA.Duringaslowswim measured fromindividualvideoframes mid-bell diameter(opencircles).Data (closed circles)recoversmorerapidlythanthe its length;duringrecoverythebasediameter (top) thejellyfishbellcontractsuniformlyalong diameter overtime.Duringanescapeswim in belldiameter, spike. Thedifference inthresholdallows = 0 A are 0.675(fastswim),1.0(slowswim); 0

.Two modesofswimmingin 8. exp

ms (slowswim).Atthebaseofbell, + b – 2+ -dependent actionpotentialanda bt ), 1250(τ (sin 2 spikes resemblethoserecorded

8B) revealsthatmostofthe π A

t 0 s. (C)Changesinbell / ). Temperature, 10°C.Data τ is themaximumdiameter 2+ ), wherex t τ is timeand is480 influx throughT-type

ms (fastswim), is thereduction τ b istheperiod is the Aglantha

s b A 545 0 is ),

The Journal of Experimental Biology suggests aconstantdelayof1.2 adjacent myoepithelium.Preparationpartiallyimmobilizedin65 recording siteinthemotorgiantaxonabout5.5 between synapticinputfrompacemakersystemandCa Stimulation sitefixed;stimulusjustsupra-threshold.Thelinethroughthepointsgivesaconductionvelocityof34 post-synapticpotential. and thesiteofstimulationlateralneuron;ordinate:delay(ms)betweenstimulusappearancedepolarizing siteinthemyoepithelium neuron monitoredintracellularlyatsitesintheadjacentmyoepithelium.Abscissa:distance(mm)betweenintracellularrecording potential recordedintracellularlyinaparticularlylargelateralnerve(10 546 seawater; temperature10°C.(DatafromG.O.MackieandR.W.M., unpublished.)(D)Sequenceofspontaneouslow-thresholdCa recording sitesR1,R2inthemotorgiantaxonare2 tothemotoraxon.Intracellular are distributedroundthebellrunningbesidearadialcanal;onlytwoshownhere.Lateralneuronselectricallycoupled cylinder. motorgiantaxons Synapsesareshownbetweeninnernerveringandmotorgiantaxons,axonsmyoepithelium.Eight bell; theinnernervering,containingavarietyofdifferent Fig. systemsincludingpacemakerneurones andtheringgiantaxon(see the animalupwatercolumnuntilsequencerestarts.FromMackie(Mackie,1980).(B)(Top):body wallatthebaseof Diagramofasectionthe ofslowswims(right)takes asymmetrical swimsbringitintoanuprightposition(bottom);thetentaclesbecomeprogressivelycontractedasextendedseries Fig. REVIEW diameter) have similar membranes, the attenuation factor should be about 30 (equivalent to the ratio of input resistances i.e. diameter diameter) havesimilarmembranes,theattenuationfactorshouldbeabout30(equivalenttoratioofinputresistancesi.e. (30 μmdiameter)andneuron(3 appears tobeanattenuatedversionoftheactionpotentialinlateralneuron.Ifjunctionalresistanceislowandaxon traces; inone,thestimuluswassubthresholdandartefactisseenalone.Thesuprathresholdsignal(amplitude,3.8±0.12 (C) Propertiesoflateralneurons.(Top): Intracellular signalrecordedfrommotorgiantaxonfollowing1 escape swim;theresponseofmyoepitheliumatR3appearsafterashortsynapticdelay(0.7±0.1 at R1andR2followingabriefdepolarizingcurrentpulse( swimming, eachlow-amplitudeCa intracellularly inthegiantmotor axon(seeFig. Mackie, 1995). EPSP (arrow)decreases exponentially asitspreadsalongthemotorgiantaxon.Restingpotential: circuits theCa the pacemakercellsynapses, the highconductanceEPSP short- the nerveringatbaseof bell (MeechandMackie,1995).Near from aslowlyrisingsynapticpotential setoff bypacemaker cellsin with achangeinthewaveform oftheCa excitation passesfromthebasetoapexofbelliscorrelated during slowswimming.Theincreasedstrengthofcontraction as as wehaveseen,thestrengthofcontractionisdistinctlyregionalized contraction dependingonthekindofimpulseinmotoraxon but, − 62 The musclesheetnotonlyproducesdifferent strengthsof

mV (right);myoepitheliumrestingpotential .Electrophysiologyofswimmingin 9. D A 50 mV 30 mV 2+ 0 spike, butthiseffect fadesasthesynapticpotential

1 Time (s)

2 ms; takentobethetimeinitiateactionpotentialplussynapticdelay. Preparationsimmobilizedin82 2+

3 spike inthemotoraxondevelops Aglantha digitale.(A)

− mm fromthenervering;bottomtraceshowscorrespondingpostsynaptic electricaleventsrecordedfrom 4 77 mV. FromMackieandMeech(MackieMeech,1985)Kerfootetal.(Kerfootal.,1985).

mm apart;R3isinthemyoepithelium,80 40 mV B 2+ 2+ E 50 mV

9E). Duringslow * spike electrogenesisatdifferent pointsalongthemotorgiantaxon.Theamplitudeofpacemaker ). (Bottomright):AnovershootingNa spike recorded *

mmol . m30m 6.6mm 3.0mm 0.6 mm 0m 2ms 40 ms R2 R1 l − μm diameter).(Bottom):Graphofconductionvelocityactionpotentialalonglateral 1 Mg 2+ is shownsinkingpassivelywithitstentaclesextendedreadytotrapprey(left); seawater. FromMeechandMackie(Meech andMackie,1993a).(E)Interaction R1 R2 regenerative Ca during fastandslow swims(MeechandMackie, 2006)(R.W.M. and the motoraxons,havetwoseparate thresholdsthatcomeintoplay loose patchclamptechniquehas shownthatthemusclecells,like events canpropagateindependently throughthesheet.Useof Ca spreads electrotonicallyalongtheaxon.Asaconsequence, full 4 regenerative Ca in thestrengthoflocalcontraction. the amplitudeofexcitatorypotentialinmuscleandanincrease

mm (inafull-grownanimal,2 R3 The cellsofthemuscleepithelium appearcapableofgenerating 2+ spike onlydevelopsatdistancesfromthebasegreaterthanabout μm fromR2.(Bottomleft):A propagatingCa The JournalofExperimentalBiology(2015)doi:10.1242/jeb.111955 + Inner nervering Synapse Radial canal Myoepithelial cell Lateral neuron Motor giantaxon -dependent actionpotentialisrecordedatR2duringafictive − 2+ 2+ 66

ms s.d.;n

ms stimulustonearbylateralnerve;threesuperimposed spike intheaxoniscorrelatedwithanincrease events eventhoughthereisno evidence thatsuch mV. AdaptedfromMeechandMackie(Meech 40 ms R2 R3 =6). Axonrestingpotential

cm s −

cm inlength).Thepresenceofafully 1 (mean, 39 C Delay (ms) mV mV –66 –62 –80 –38 5 0 1 2 3 4 5 6 7 8 0 1.252.503.755.00 0 1.252.503.755.00 0 2+

cm 5), isrepresentedbyasingle Distance (mm) spikes (toptrace)at

0.5 mV; mean±s.d.; N 3/2 Time (ms) s − ). (Centre):Action 1 ; N

1.0 − 2+ 72 =3). Extrapolation spike isrecorded

1.5

mV (left); mmol

2.0 l –1 Mg =4) 2+ μm

The Journal of Experimental Biology myoepithelium duringslowswimming. the lateralneuronsplayanypartinspreadingexcitation myoepithelium ispresentedinFig. isolated. Anexplanationfortheanisometricnatureof bell sothatthebasalandapicalregionsareelectricallysemi- motor axontowardsthenext,itspreadsonlyashortdistanceup although currentspreadsalongdistancearoundthebellfromone flow towardsthebellapex(Fig. steady statecurrentflowinacirculardirectionand177 anisometric withthetwo-dimensionalspaceconstantof770 over distance(seeJacketal.,1975).In source, suchasasynapse,wouldbeexpectedtodecreaserapidly dimensional sheetofconductingtissue.Potentialsfromapoint transition isthatthethinmuscleepitheliumbehaveslikeatwo- the apex(49.2±1.5 synaptic eventis21.8±2.0 spike. Towards thebaseofbell,averageamplitude amplitude ofthesynapticdepolarizationarisingfromslowswim myoepithelium duringaslowswimappearstodependuponthe channels associatedwithweakercontractions generated bytheslowswimspikeactivateonlylow-threshold to generateastrongcontraction.ThesmalleramplitudePSPs REVIEW two-dimensional networkoflarge neuronscovering thesubumbrella (Anderson andSchwab,1981; AndersonandSchwab,1983)isa neurons totheswimmusculature andfromonerhopaliumtoanother in hydrozoanjellyfish. contraction isincontrasttothealmost synchronizedcontractionseen (Horridge, 1959;Passano, 1965). Thisslowlypropagated the fastestpacemakerdeterminingoverallswimfrequency of contractionoriginatingfromtherhopalia(Romanes,1877) with inter-swim interval(Horridge,1959).Swimmingconsists ofawave longer periodsofcontinuousactivityalbeitwithahighlyvariable by sink-fishingHydrozoa,arerarely, ifever, seen.Instead,thereare In theScyphozoa,intermittentburstsofswimmingsuchasexhibited inherent rhythmintheirsensoryintegratingcenter. and cubozoans(Mackieetal.,1984)whetherornotthere isan include theapparentabsenceofelectricalcouplinginscyphozoans rhopalia. Otherdifferences between thethreeclassesofjellyfish the swimpacemakersaregatheredtogetheringanglion-like around thebellmargin. IntheScyphozoaandCubozoa,however, synchronized byelectricallycoupledpacemakerneuronsdistributed refills withwater. Inthehydrozoa, swimcontractionsare mesoglea duringcontractionofthebellislargely regainedasit Jellyfish swimrhythmicallyinsuchawaythatenergy storedbythe range approximately 10 lasting inwardcurrentevenwithdepolarizingcommandsaslow close totheholdingpotentialof G. O.Mackie,unpublishedresults).A seriesofvoltagecommands Scyphomedusae Comparison withScyphozoa and Cubozoa higher thresholdchannels;theassociatedCa PSP inthemyoepithelium,whichislarge enoughtoactivatethe escape swimactionpotentialinthemotorgiantaxongeneratesafast muscle spikethresholdsseenduringslowandfastswimming.The dependence ofthesetwocomponentscanaccountforthedifferent peak valueat+20 The basetoapexvariationinthestrengthofcontraction The motornervenetthattransmits excitationfrompacemaker mV. Thislow-thresholdcurrenthasapeakamplitudeat − 40 to+20 − 40 mV showasecondinwardcurrentthatreaches

mV. Currentsrecordedinresponsetostepsthe mV. Theactivationcurveshowingthevoltage

mV). Anexplanationfortheabruptnessof mV, lessthanhalfthatmeasuredtowards

7D) (Kerfootetal.,1985).Thus,

− 7C,D. Thereisnoevidencethat 80 mV haverevealedalong- Aglantha 2+ influx islarge enough , theepitheliumis μm forradial μm for delay beingreducedto0.5 paired diffuse nervenetstimuliwereevenmoreeffective, withthe are spacedoutbyafastoutwardpotassiumcurrent(K account foreachofthem:i.e.toassumethatpacemakerimpulses behave sosimilarlythatitistemptingtousethesamemodel activation occurred2–3 was activatedimmediatelyafterthepacemakertolessthan1 preceding swim.Itrangedfrom4 interval betweentheimpulseindiffuse nervenetandthe (Passano, 1965)foundthattheprecisetimingdependedon so longasitcomesmorethan1–2 significantly reducedbyasinglestimulustothediffuse nervenet, taken forthemaintaineddepolarizingEPSP itselftoinactivateK inactivation. Nowthedelayrepresents(slightlylonger)time swim intervalinanundisturbedanimallasts4–10 state (Romanes,1877;Passano,1965).In net. Thedurationofthisdelaycanbeusedtomonitorthepacemaker excitation andtheappearanceofswimimpulsesinmotornerve hydrozoan Bsystem,thereisasignificantdelaybetweenits and caninitiateswimimpulses(Passano,1965).Aswiththe Romanes, 1885),triggersslowmanubrialreflexes(Passano,1982) (Horridge, 1956)setsoff tentaclecontraction(Romanes,1877; the EPSP amplitudedeclineswithrepeatedstimuli. facilitation betweenmembersofthisnervenetisabsentandinfact release chemicaltransmitter(Anderson,1985).Interneuronal synapses witheithersideofthesynapticjunctionbeingableto communication betweenneuronsdependsuponbi-directional surface, andabletotransmitactivityinanydirection.In including obstacle avoidance,courtshipand mating(Lewisand Cubomedusae arecapableofa widerangeofcomplexbehaviour successful behaviouralresponse. generates alternateelectrophysiological applicationstoachievea this isanotherexampleofthe wayinwhichnaturalselection cell andevokefurthertransmitterreleasethere,soon.Perhaps post-synaptic cell;thiswould,inturn,depolarizethepre-synaptic depolarizations wouldbesufficient toevoketransmitterreleaseina network withalowertransmissionthreshold,low-amplitude unusually highthresholdfortransmission(+20 produce brief(about20 (Anderson, 1985),thebidirectionalsynapsesofmotornerve net generated bychemicalsynapses?AccordingtoAnderson Scyphozoa (Mackieetal.,1984).Couldaprolongedinput be known electricalcouplingthere,whichisthoughttobeabsent in the swim motorneuronsiswellestablished,buttheyareattributed to the the motorresponse.In necessary toaccountforthestrikingdelaybetweenstimulusand system onpacemakerneuronsin so thattheswimimpulsecanbefired. suppose thattheK threshold. Atlongerintervals,afterthepacemakerimpulse,wemay than normal,anydelayrepresentingtherisetimeofEPSP to depolarizing inputfromthediffuse nervenetwillbemoreeffective current, butitislikelythatsomeinactivationwillremain.Ifsoa refractory period.ThismayallowtherecoveryofmuchK a swimthepacemakerwillbeunresponsivebecauseof normally recoverfrominactivationratherslowly. Immediatelyafter to delaytheriseofmembranethreshold.Suchcurrents Cubomedusae The diffuse nervenetin Activity inasecondnervenet,calledthe‘diffuse nervenet’ A similarmodelwasproposed(above) fortheeffect oftheB The JournalofExperimentalBiology(2015)doi:10.1242/jeb.111955 A Polyorchis current willhavefullyrecoveredfrom

s later. AswiththehydrozoanBsystem,

ms) EPSPsonlybecausetheyhavean

s orless. Cassiopea , thepresenceoflongEPSPsin

s ormoreifthediffuse nervenet

s afteraswimimpulse.Passano Polyorchis. Inbothcases,itis and theBsystemin Cassiopea, thenormal mV ormore).Ina

s, butthiscanbe A ) thattends Cyanea, Sarsia 547

s if A A

The Journal of Experimental Biology characteristics sequentially. Thispropertymay beinvolvedina axons exhibittrainsofaction potentialsthatchangetheir or-nothing’ actionpotentialsareidentical andthatmanyinvertebrate integrated behaviour. Alongthewaywehavenotedthatnotall‘all- which activeandpassiveproperties ofneuronsinteracttoproduce released. strength ofmusclecontractionortheamountneurotransmitter enters thecellduringelectricalactivity. Thiswilldeterminethe 548 inactivating K nerve andmusclemembranes.A keyactivefactoristheroleof include thevoltage-dependentcharacteristicsofionchannels in current asdeterminedbycellularanatomywhileactivefactors integrative role.Passivefactorsincludethedecrementalspread of active factorscombinetotailoreachneuronalstructure to its local potentialsandsynapticpotentials.Ineachcase,passive and included: all-or-nothing actionpotentials,pacemaker potentials, responsiveness thatcouldberecordedintracellularly. These properties ofneuronsbyitemizingthedifferent formsof Horridge (BullockandHorridge,1965)categorizedtheintegrative place whenthebellwasrefilling.Fifty-nineyearslater, Bullockand during jellyfishswimmingbypreventingcontractionfromtaking Bethe andnotedthattherefractoryperiodensuredcoordination (Sherrington, 1906)referredtotheworkofRomanes,Nageland In ‘TheIntegrativeActionoftheNervousSystem’ Sherrington repolarization dependsonarapidlyinactivatingK muscle cells(seeInoueetal.,2005),itislikelythatimpulse we knowoftheionicbasiselectrogenicityinothercnidarian being associatedwithintervalsof0.5 on theprecedinginter-pulse intervalwithmaximumfacilitation polyinnervated musclecell.Thepotentialamplitudedepends amplitudes arisefromvariationsinthesynapticinputto time course.Satterlie(Satterlie,1979)suggeststhatthedifferent coupled electricallyandthesynapticeventshavearelativelyshort myoepthelial cells(Satterlie,2011) northenervenetneuronsare the strongerforceofcontraction(Satterlie,1979).Neither generating gradedmusclepotentials:thelarger themusclepotential, suppress theiractivity. Theyalso spreadtothesubumbrellamuscle, Nolen, 2001). independent couplingproducesthebestoutcome(Satterlieand the animalistrackingprey. Modellingtrialssuggestthatsemi- possibility ofasymmetricswimmingsuchasmaybecalledforwhen between pacemakerstobeabsolute,however, therewouldbeno upward anddownwardadjustmentsinfrequency. Were thelinkage system hastheadvantagethatasynapticinputcanproduceboth because eachcycleisdrivenbythefirsttoreachthreshold.Sucha somewhat fasterthanthenaturalrhythmofindividualpacemakers enabling directionalswimming.Thenormalswimfrequencyis four pacemakingrhopaliaisthatthisprovidesanidealmeansof Nolen, 2001)havesuggestedthatthereasoncubomedusaeonly 2009) makingsteeringpossible.SatterlieandNolen(Satterlie influence theoutputofassociatedpacemaker(GarmandMori, ocelli. Whenstimulated,thedifferent typesofeyeareableto rhopalium hastwolarge eyesequippedwithlensesandtwopairsof Long, 2005).Inadditiontobeingthesiteofpacemakeractivity, each REVIEW Conclusion potential amplitudewouldbelarger. less repolarizingcurrentwouldbeavailableandsothemuscle shorter intervals,recoveryfrominactivationwouldbeincomplete: requires severalsecondstorecoverfullyfrominactivation.With In thepresentreview, Ihavefocusedonexamplesoftheway in Impulses thatoriginateintherhopaliaactonotherto + channels indeterminingtheamountofCa

s (Satterlie,1979).Fromwhat + current that 2+ that interaction betweensystems.We notethatin with feedingandthecontroloftentaclelengththisrelieson afforded bypacemakerneurons,butswimmingmustbecoordinated food. Efficient locomotioninjellyfishisbasedonthetiming which themanubriuminAglantha variety ofcoordinatedactionssuchastheratherprecisewayin supported bygrantsfromTheRoyalSociety andTheWellcome Trust. Research CouncilofCanada(Grantno. OGP000142701).Earlierworkwas Research fundingwasprovidedbythe NaturalSciencesandEngineering The authordeclaresnocompetingorfinancialinterests. meeting, whichwassupportedbytheNationalScienceFoundation(NSF). support. Thisworkwaspresentedatthe‘EvolutionofFirstNervousSystems II’ research andIamtrulygratefultomyBristolcolleaguesfortheirfriendship University ofBristol,UKhasformanyyearsprovidedasecurebasemy have beenimpossiblewithouttheenthusiasticencouragementofSallyLeys. The Columbia, Canada,withsupportfromtheDirector(AndrewSpencer)andwould sponge permission toincludeunpublisheddatahere.Micropipettestudiesontheglass Hernandez-Nicaise andAndreBilbautIthankthemforhappytimes for and attheUniversityofNice.ItwascarriedoutincollaborationwithMarie-Luz research wascarriedoutattheStationZoologique,Villefranche-sur-Mer, France thank ClaudiaMillsforherfriendship,unfailinginterestandhelp.Thectenophore thank theDirectorandstaff fortheirskilledhelpandwarmhospitality. Iparticularly Cnidaria wasperformedattheFridayHarborLaboratories,Washington, USA andI encouragement, adviceandinspirationthathehasfreelygivenme.Work on George Mackie,myfriendandcollaborator. Ithankhimforallthesupport, This manuscripthasbenefittedgreatlyfromthemanydiscussionsIhavehadwith nesn P. A.V. Anderson, References Funding Competing interests Acknowledgements crumpling areconfinedtothemanubrium.However, to accommodateescapeswimmingandtheradialmusclesrequiredfor (Hyman, 1940),isnotadefensiveoptionbecausethebellhasevolved Aglantha muscles andtheavailabilityofelectricalcoupling.Forexample,in as thepropertiesofananimal’s skeleton, thearrangementofits neuronal modulesconcernedareconstrainedbyholisticfactorssuch and errorduringthecourseofevolution,butchangesindifferent These successfulbehaviouralstrategieshavearisenasaresultoftrial when swimming,controllingtheirtentacles,escapingorfeeding. axon. input andslowswimelectrogenicityatthebaseofmotorgiant anisotropic spreadofcurrentandtheinteractionbetweensynaptic contractions duringslowswimmingasaconsequenceofthe impulse inthemotorgiantaxon.Italsoproducesregionalized different strengthsofcontractiondependingonthekindactive electrically coupledmyoepithelium.Themusclesheetproduces neurons inAglantha interactions betweenactiveandpassivepropertiesofthegiantmotor over voltage-gatedcomponents.Finally, wehaveexaminedhowthe the durationofallsynapticinputs,therebyincreasingtheirinfluence properties oftheelectricallycoupledpacemakersystemenhances differently expressedneuronalmodules. holistic constraintsthatapplytothemolecularcomponentsof applicable toall;speciesdifferences providethemeanstoexplore them forexperimentalpreparationsthatarelikelytoprovidedata between jellyfishspecieshavegivenusthechanceto‘cherrypick’ that takesplacewhenitfeedsisanexampleofthis.Similarities operates inamore‘classical’ fashionandtheinhibitionofswimming the ctenophore Different speciesofCnidariaexhibitawiderangemechanisms Rhabdocalyptus ‘crumpling’ behaviour, whichiscommoninmanyjellyfish The JournalofExperimentalBiology(2015)doi:10.1242/jeb.111955 Mnemiopsis (1984). Theelectrophysiology ofsinglesmoothmusclecells from were carriedoutatBamfieldMarineStation,British combine togenerategradedresponsesinthe . J.Comp.Physiol.B is directedtowardsasourceof 154 , 257-268. Polyorchis Aglantha the passive also

The Journal of Experimental Biology onr .A n tvn,C.F. Stevens, and J.A. Connor, P. A.V. Anderson, and Q. Bone, B.M., Chain, G.A. Horridge, and T. H. Bullock, ln,J,Mue,J-.adLpo,H. Lipson, and J.-B. Mouret, J., Clune, aiaa . uao .adSio N. Saito, and K. Kusano, S., Hagiwara, Donaldson, enne-ias,M-. ake .O n ec,R.W. Meech, and G.O. Mackie, M.-L., Hernandez-Nicaise, M.-L. Hernandez-Nicaise, L.M. Passano, and M.-L. Hernandez-Nicaise, M. Yoshii, and S. Yoshida, S., Hagiwara, A.N. Spencer, and J. Przysiezniak, J.D., Spafford, N.G., Grigoriev, S. Mori, and A. Garm, ibu,A,HradzNcie .L,Leh .A n ec,R.W. Meech, and C.A. Leech, M.-L., Hernandez-Nicaise, A., Bilbaut, oe Q. Bone, G.O. Mackie, and P. A.V. Anderson, nesn .A .adSha,W. E. Schwab, and P. A.V. Anderson, W. E. Schwab, and P. A.V. Anderson, REVIEW enne-ias,M.-L. Hernandez-Nicaise, orde G.A. Horridge, orde G.A. Horridge, ae . hoiR,Ai . aru . uincE,Mne M. 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