The Journal of Experimental Biology – ACCEPTED AUTHOR MANUSCRIPT 33 32 31 30 29 28 27 26 25 24 23 22 21 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 J ExpBiolAdvanceOnlineArticles.Firstpostedonlineon2July2013asdoi:10.1242/jeb.087809 LISTABBREVIATIONS: OF KEYWORDS RUNING TITLE: CORRESPONDING AUTHOR Medford, MA02155,USA. Ave., 4600,200 Boston Suite University, Tufts Biology Center for andDevelopmental Department Regenerative andTufts Biology, AUTHORS: TITLE: Access themostrecentversionat email: [email protected]: Fax: (617) 627-6121 (617) 627-6161Tel. Medford, MA02155-4243 Ave., 4600,200 Boston Suite University Tufts Center andDevelopmental for Regenerative Tufts Biology Biology Department and Dr. Michael Levin An Automated Training Paradigm Reveals Long-term Memory in Planaria Planaria in Memory Long-term Reveals Paradigm Training Automated An : : flatworms, conditioning, training, learning, planaria, regeneration, behavior Copyright (C)2013. Published byThe Company ofBiologists Ltd Memory Regenerating Planaria in and Its Persistence Through Head Regeneration Head Through Persistence Its and

Tal Shomrat, and Michael Levin Levin Michael and Shomrat, Tal Automated Training Training Apparatus (ATA). Automated http://jeb.biologists.org/lookup/doi/10.1242/jeb.087809 1

The Journal of Experimental Biology – ACCEPTED AUTHOR MANUSCRIPT 66 65 64 63 62 61 60 59 58 57 56 55 54 53 52 51 50 49 48 47 46 45 44 43 42 41 40 39 38 37 36 35 34 tissues, and biological specifically thedynamics ofmemory about brain regeneration, during 2009)?al., Answering about questions thestorage of information in dynamically-remodeling repopulated by ofimplanted stem (Martino etal.,cells 2011; thedescendants Velthoven et van and mental ofancontent patient adult of with decades stored memories when brain his is 1996). tothepersonality Forwhat(Murre 2001;Perry example, etal., andHodges, happens et(Macia al., 2012;Sole et al., 2007), andfor thebiomedicine ofdegenerative brain disease Gomez, 2001), etal., 2009;Sampaio for of thesynthetic bioengineering cybernetic systems for neurobiology basic implications ofthebrain:bodyand engineering (Pfeifer and interface bytime nervous thecentral system. intersection two The ofthese fundamental has processes over stimuli andstored from regeneration, abstracted environmental andtemporal information needed tocreateandmaintain specific anatomical structures during and embryogenesis Biological information multicellular in atorganisms comes in least 2flavors: spatial information, INTRODUCTION humanadults. in disorders for implications thebiomedicineimportant of stem treatments cell-derived brainof degenerative Moreover, ofspecificmemories biologicalsystemlikely tissues. in encoding this is tohave proposeplanariaWe as akey emerging model species for mechanistic oftheinvestigations that will interf shedlightonthefundamental fora foundation objective, high-throughput assays in this molecularly-tractable model system paradigmof memory asavings in after retrieval regenerating anewOurwork head. establishes furtherfor thebraintoregenerate. planaria show evidence exhibit We that trained,decapitated familiarization,environmental andthat memory for this atleast 14 persists days enough –long familiarization protocol. showtrain flatwormsWe anenvironmental exhibit thatworms in manual new thenused attempts previous, this to totrainplanaria. that confounded system We paradigmthatavoided themany andtesting issues acomputerized training brain, we developed regenerating a ofthedynamics in ofmemory investigation establish asystem the To for animal. system may offer opportunity theunique tostudy brain regeneration andmemory thesame in dataClassical suggest that they be may also capable of long-term memory. the Thus, planarian flatworms Planarian are apopular system for themolecular research into mechanisms complex these that enable organisms toregenerate theirentirebody, thebrain. including SUMMARY One ofthemost interesting capabilities of living systems is processing information.

ace betweenand stored memories. patterning body 2

The Journal of Experimental Biology – ACCEPTED AUTHOR MANUSCRIPT 99 98 97 96 95 94 93 92 91 90 89 88 87 86 85 84 83 82 81 80 79 78 77 76 75 74 73 72 71 70 69 68 67 the could survive process ofhead regeneration were a measuring demonstrated by direct 2002;Dasheiff, Inoue etal., 2004; Prados et al., 2013;Stephen, 1963). Acquired memories that well-known photosensitivityand based (Dasheiff protocol onplanarians’ conditioning a classical 1965;Morange, 2006;Sheiman most McConnell, and Tiras, 1996). The common procedure was associative 1966; Corning, learning etal., subsequent (Cherkashin studies 1966; Corning,1967; oftheheadoutside andimprinted on thenew brain during regeneration, toamyriad led of memory stunning 1959). thatsome etal., This suggesting finding, bestored McConnell may remembered theoriginal training 1963;Corning (Best, and 1961;John, 1965; McConnell, oftheand followingamputation head, regenerating theanimals from the tail original sections analysesmolecular-biological regeneration ofadult But oflarge-scale they brains. can learn? andTiras, 1996;TullySheiman 1994), system are etal., planaria tractable auniquely for 1999; 1992;Ray, 2008;Hepper (Alloway, etal., andWaldman, metamorphosis Blackiston 1972; severalidentified organisms insect in which memories survive thedrastic reorganization of information thestored in CNSduring massive remodeling andrepair. studies haveWhile 2006).Alvarado, Thus, planaria are anideal system which in to probe thedynamics of 2011; Loboet al., 2012;Newmark and Sánchez Alvarado, 2002;Salo et al., 2009;Sánchez etal., (Aboobaker, 2011;Gentile after theheadtoberegenerated damage as such structures popular molecular-genetic system for theinvestigation ofthepathways thatallow complex a become have 2011). planaria etal., Recently, Umesono 2004; etal., (Inoue within few days from onlyproportion asmall oftheadult worm: off(or acut damaged) head is rebuilt perfectly (Reddien andSanchez Alvarado, 2004; 2011), etal., andwhole regenerate can worms Wagner andcomplex ofbehaviorsrich as set they theirenvironment. navigate sensory1966). Their are integrated by mechanisms reception worm’s system nervous a the into (Brown Chow, 1975;Brown, fields fields and andmagnetic (Brownelectric 1968), andOgden, 1975;Miyamoto(Mason, andShimozawa, 1985), vibration (Fulgheri andMesseri, 1973), have primitive eyes andother sensory capabilities majority ofneurotransmitters that occur in vertebrate brains (Buttarelli et 2008). al., Planaria (Sarnat brain withthe centralized andNetsky, transmission andshares true synaptic 1985), learn andremember. requires atractable modelsystemwith both– Free-living, planarian flatworms represent the “first” class of organism to have a oforganism a flatworms tohave planarian representFree-living, the“first” class Nearly 55 years could wasbetrainedtoNearly agoit thatplanarians demonstrated 55 learnatask, (neoblasts) populations cell stem Adult their remarkable regenerative abilities underlie a robust CNS andtheability repaira mechanism to 3 including sensitivity chemical to gradients

The Journal of Experimental Biology – ACCEPTED AUTHOR MANUSCRIPT 133 132 131 130 129 128 127 126 125 124 123 122 121 120 119 118 117 116 115 114 113 112 111 110 109 108 107 106 105 104 103 102 101 100 planaria ofregenerative forplatform theuse for thestudy andmemory. oflearning amodern and attempts establishes previous in problems anumber of paradigm thatovercomes (Blackiston etal., 2010;Hicks et al., 2006). aim ofthisThe study tofind was a learning awide paradigms in andtestingand fully ofplanaria range training offeedback objective 2008b).thusal., developed two thatallowWe platforms automated, parallelized, quantitative, et 2008;Oviedo etal., (Nicolas methods manual inherent in theproblems toovercome planaria part As ofour investigations into information by processing dynamically-organizing we fortissues, beguntodevelop elicitingautomated approaches learning have andrecall in 1981). term memorylong 2008; beenquestioned (Abbott and has 2009; etal., Takeda Wong, Travis, 1981).aMorange, difficulties, As 2006;Travis, planarians’ ofthese result even, capacity for and ofquantitative protocols, lack 1970;analysis (Corning andRiccio, Hartry 1964; etal., behavior Manual experiments involvedifficultiessample limited sizes, reproduction precise ofin remained:thedifficultyregeneration has arobust learning ofdeveloping assay planaria. in primary investigations thedynamics molecular-level into ofmemory barrier to duringCNS explanationsofthe results, ofsome the original 2001) et al., now mechanistic (Smalheiser offer and Sweatt, 2009;Levenson Sweatt, 2005) andRNAi 2010;Ginsburg andJablonka, 1996). modern discoveriesWhile such asepi Riccio, 1970;McConnell,whol 1966), andthe some were(Corning others they in cases, in and tobereproduced findings validated failed experiments. difficulties manual inherentin largely thebasic While abandonedduetopractical and imprinted onanewly-regenerating brai approacheshow bestored ofthebrain for can investigating 1967) information outside suggests entire head(Corning, its thathadtoregenerate an animal memory reports ofpersistent The in 2001), regeneration likewise survived ofthehead(Corning, 1966; Ernhart 1959). andSherrick, 1965; odorant Roe,1963)McConnell, cues or(Wisenden toassociate and learning Millard, 1961;and John, Corning,1966; Corning, 1967;Corninget al., 1967; Humphries, 1961; tasks, e.g.,eliciting movement in etal., 1961),Halas other showed studies more formed in thatmemories complex discrimination ratheradaptation thanconsolidation of“real, encoded”and retrieval memory 1962; etal., (Halas generated pieces from etal., headandtail ofpreviously 1959). (McConnell trainedplanarians response ordisplay rate afaster learning ofaconditioned (“savings”) amongworm fragments While induced bylearning While beattributed conditioning could tosensory classical These remarkable discoveriesThese have nothad sufficient impact on thefield andwere aspecific direction inatwo- n –atruly fascinatingpossibility. genetic modification (A 4 e lineofresearch became abandoned(Rilling, choice maze (Best, 1963;Corning rshavsky, 2006;Dayand

The Journal of Experimental Biology – ACCEPTED AUTHOR MANUSCRIPT 167 166 165 164 163 162 161 160 159 158 157 156 155 154 153 152 151 150 149 148 147 146 145 144 143 142 141 140 139 138 137 136 135 134 melted floor, but thedisplaced ofthedish's toadepth of0.2mmbelow thecircles thelevel cuts 0875713A), altered by etching laser (universal laser systems versaLASER VL-300). The laser (Fishermade from polystyrene Scientific, commercially available petri dishes 15x60mm floor surrounded by walls(Fig. electrode 2). Rough-texturedbeen theATA have dishes petri systemofshelf capable video multiple (Marechal 2004;Nolduset al., tracking 2001). etal., animal. any could Therefore, bedonewith toeach protocol oftheoff-the- or(light shock) this feedback with algorithm training no complex instantaneous (Fig. 1B) but oftheanimals tracking we path onaparadigmthatrequires However, settled day, arena. type temperature, of and andexperimental worms,control time including simultaneously ofthe conditions within thesame bias caused by procedures manual and facilitated thetraining andtesting oflarge numbers of 2010; (Blackiston(ATA) etal., Blackiston and Levin, 2012) (Figs.1A,2L,M), which minimized Experiment apparatus METHODS AND MATERIALS memories specific large-scalesurvive regeneration ofneural tissues. amodel as organism ofplanaria for tounderstandhow theuse great opportunities opens entire and simple approach paradigm. This brain regenerationsurvive a“saving” promising in enoughtoallowlong for regenerationwe after andindeed amputation, show thatmemory traces familiarized worms compare to unfamiliarized worms. Most importantly, the memory survives ofBestfindings and Rubenstein, and show astatistically-significant delayshorter feeding for the the support results Our ended. training days for the long-term memory,several checks after protocol, frame. ourshort time Additionally,andRubinstein’s procedure toBest contrast in theprocedure analysis,rigor behavioral arelatively andapplies in toalarge samplein size bias byminimizes caused procedures, manual ofquantitative allowsanunprecedented level andanautomatedbehavior systemetal.,animals) 2010). (Blackiston analysis protocol Our adapting approach, totheuseit with atextured (to provide substrate cues totheclear haptic improve this non-punishi or touse attempts beennolater thatthere appears have 1962;Dufort, 1962),and Best, andit manually-performed contained small experiments sample sizes andlimited controls (Davenport wormsnaïve which neverbeenexposed tothe feeding arena before. As priorin their studies, that planarians wh Best and Rubinstein (Best, 1963; Best and Rubinstein, 1962a; Koopowitz, 1970) showed 1962a;Koopowitz, 1963;Best (Best, andRubinstein, andRubinstein Best The ATA “familiarized”The chamberenvironment aPetri contained dish with rough-textured weFor fully andtesting acustom-made apparatus training used automatedtraining ich hadbeenfedin afamiliar environment will start to eat more quickly than

ng paradigm. Here, we modifiedthis 5 environmental familiarization

The Journal of Experimental Biology – ACCEPTED AUTHOR MANUSCRIPT 201 200 199 198 197 196 195 194 193 192 191 190 189 188 187 186 185 184 183 182 181 180 179 178 177 176 175 174 173 172 171 170 169 168 familiarizedfresh water groupswere period. The adish thedark for in in thecleaning placed with petri dishes in andplaced theATA wormswere out of water taken was The the changed. water). density2ml high water This required every to be changed day. (Bestfission et 1969). al., the Thus, wereworms groups, in held in high density (~12 worms / spontaneous suppressing in foundtobeeffective was theworms also Culturing density athigh (Fig. fragments 4). theheadless worms decapitation allowing testing 10days from after only in tails’ at18°C.held regenerationincubator The also higher rate is 18°C in to10°C, compared at18°C. kept Therefore,was duringtheexperimental were periodtheworms temperature also whenlonger exploration 18°C phase in to10°C). compared (as experiment kept room The andtheirenvironment petri resting plate. Planariansaredish/wells more anddisplay active between andtransfers except duringwater for changes briefperiods theexperimental procedure). were darkness continuous in Worms kept duringtheentire experimental period negative phototaxis to enhanced (McConnell, 1965)(an feature important for thetesting andmaintenance during theHandling experiment liver. withweek beef organic a twice darkness (Morita1984)or andBest, fedonce continuous for size suitable theexperiment (1-1.5 containers cm), were an storedin at10°Cincubator in spontaneously fission. In order spontaneous toprevent fission and allowworms a toreach water naturalspring japonica Springs 2008a). (Oviedo etal., Dugesia experiments, planarian colonies were stored inre Before andis forprocedures, very training tolerance active. high anddissection capabilities, found mediterranea andSchmidtea Dugesia japonica, dorotocephala, species: planarian Dugesia maintenance colonies’ Worm wide). is about0.3mm circle for each cuts laser trough1.5mm and1.2mminner diameterthatthe (the to closer upbeing ends theouter circle cut, diameter ofeach As 2.15mm attheir centers. pattern (Fig.dish. The 2N) is made upofcircles drawn diameter at1.4mmin andspaced polystyrene builds also uparound each circle toaheight of about0.05mm the above floorof the Dugesia japonica Dugesia Every morning, during the training phase, the experimental apparatus was cleaned and cleaned was theexperimental apparatus phase, Every morning, during the training In to addition suppressing fissioning, thekeeping worms in darkness has been reported japonica were thecurrent Dugesia in study used planaria All to be the most suitable for this project. It has remarkable regenerating project.remarkable for It this suitable has tobethemost

6

ctangular plastic containe has tendency ahigh to . After three examining rs, filledwith Poland ,

we we

The Journal of Experimental Biology – ACCEPTED AUTHOR MANUSCRIPT 235 234 233 232 231 230 229 228 227 226 225 224 223 222 221 220 219 218 217 216 215 214 213 212 211 210 209 208 207 206 205 204 203 202 thanwhat of dropsofliver (less they capable with for are onehour,the ATA 1-2 small wereworms fedin among worms. levels thepossibility The starvation eliminate ofdifferential period Feeding duringthetraining before dissection).be digested (to wells eaten foodto allow all small in groups into smaller anddivision before dissection phase forevery regeneration day. experiments wereWorms aPetri-dish in for kept a24-hour rest with water The (12well 2ml worms Fig.2E). filled testing a in plates (Greiner Bio-One: were anddivided outfrom taken theATA into smaller groups andwere 12 in kept multiwell caused by spontaneous fissions were fragments were andtail theworms thechambers, inspected into back Before inserted being andcleaning. forchange thewormswere water outoftheATA phase, during taken thetraining were in group theATA non-textured butin placed (Fig. standard2B). Petri dish Every morning wormswent throughprocedure, thesame simultaneously with thefamiliarized (experimental) waterworms (~12 water) lidswere /2ml andthe closedfor darkness. Unfamiliarized (control) periodwalls (Fig. training 2A). The last 10-11 days. consecutive chambers were The with filled contained aenvironment Petri dish with rough-textured floor surrounded by electrode the ATA animals, wastesting doneonindividual groups). familiarization proceeded in ATA chamber The procedure Training pipettes were usedfor thefamiliarized andunfamiliarized groups. withpipette thetip offtocut make aslightly largerDuringthe opening. training, separate were between cleaned every For testing trial. all worms’ handling, we aplastic used transfer Duringthetesting anddishes) sessionsanimals. theexperimental (ATA-electrodes apparatus density high withwater) (~12 waswormswater of lowenvironment tomaintain levels filled /2ml backplaced into theirexperimental environments. In order tosuppress fission, the experimental were worms procedure the thecleaning Attheendof with 70%. ethanol Kimwipes soaked ATA electrodes,The usedas walls for the withcleaned Kimwipessoaked with 70% andpositionally ethanol randomized between trials. Rough-textured were afterthoroughly andstandardPetri dishes duringthetraining being reused roughwith textured floor aandth Worms were fed throughout the training period, in order to suppress fissioning, order period,in tosuppress were and Worms thetraining fedthroughout Groups were of20-40 anindividual ATA experimental in worms placed chamber (while

part 665102,hydrophobic number surface (no treatment)) tillthe e unfamiliarizedweree groups pl

extracted. After a10 day fa “familiar” environment, were alsocleaned with 7

water in the wells was changed was changed water thewells in aced intostandard Petri dishes. miliarization period, theworms

The Journal of Experimental Biology – ACCEPTED AUTHOR MANUSCRIPT 269 268 267 266 265 264 263 262 261 260 259 258 257 256 255 254 253 252 251 250 249 248 247 246 245 244 243 242 241 240 239 238 237 236 theresults.in not included by offissioning, were caused werewormsinjuries like or thetransfer that pipette, theprocess in issue health ofany worms evidence thatshowed well as as liver), attempted toeatthe never the Anyquadrant, withinliver containing spot. worms criterion 60minutes reach (e.g., thatdidn’t took it thelong worms toreachthe of criterion 3consecutive theminutes in illuminated their natural aversionovercame light (Fig. 2P). anindication As offeeding, we measuredhow 1968) (Fig. no 2L) thus, worm stay would this quadrant in unless forits the desire liver, spot ofliver was illuminated with astrong blueLED light (Azuma etal., 1994;Brown etal., (Inoueworms etal.,2004). generally Sincethe were thelids chamber, andthe was initiated. tracking closed transfer forspot. allWorm chambers averaged<1min. After all the12 worms were the inside starting with theunfamiliarized.we worms The wereworms totheATA inserted chambers with aplastic transfer pipette, alternating in order, would bemore willing to leave the edge andmove to Therefore, theliver was applied away thearena wall from (Fig.that 2J)so familiarized worms with ofwater. 11ml In offood,worms theabsence preferedgeofthedish. tostay onthe (Fig. 2J,K,O), andallowed todryabout 5minutes before placed for being theATA and in filled amountofsmall liver was spreadwith afine paintbrush onsmall area ofthe roughened dishes from used those for the training), surrounded by theATA electrode walls (Fig. 2J,K). A very individual chamber. All chambers contained a rough textured floor (a separate of set dishes familiarized and6unfamiliarized worms were te procedureTesting wereend oftraining before thetest. 1-2 fedagain times with theexperimental environment.association thatwereWorms 11-15 tested days after the before testing.phase In addition, feeding protocolthis is designed to a create positive fissioningduringalongerresting oftheworms suppresses were which 4days later, and tested drop ofliver remaining intact). This procedure “synchronizes” thehunger ofthe level worms were with fedintensively 1-2 ofliver drops every until 20minutes, satiety by (revealed thelast additional ~10ml ofwater. t On familiarization training (days 1,4,7,10). Ju themorningafter in every thirddays of place Fig.2C,D).consuming, Feedingtook To identify feeding, we capitalized upon the planarians’ strong negative phototaxis phototaxis strongnegative we upontheplanarians’ identify To capitalized feeding, ATAThe 12 contains identical chambers (Fig. 1A). During testing trial, each 6

he last morning offamiliarizationhe last training (dayworms the 10), 8 st before feeding, chambers were filledwith re placedinthecham avoid illuminatedareas, sted simultaneously, worm each initsown ward thecenter ofthe dish (Fig. 2P). The bers, oppositetheliver the quadrant with the thewith quadrant the

The Journal of Experimental Biology – ACCEPTED AUTHOR MANUSCRIPT 303 302 301 300 299 298 297 296 295 294 293 292 291 290 289 288 287 286 285 284 283 282 281 280 279 278 277 276 275 274 273 272 271 270 at thebeginning18°C ofthetest until the next session. day, 4days after saving divided into groups of6 familiarized and 6unfam session, thewormswere 3days well/2 savings water;in ml after Fig. 2E). the Attheevening, themultiwall in the back (~12 worms satiety.wereworms plate placed Attheendof session, ofliver drops werephase, in andtheworms thechamber placed allowed toeatuntil were surfaces and therough floor (the fa regenerated worms were in inserted totheATA’s chambers with the surrounding electrode days tonine Seven after ofboth,familiarized groups, decapitation, andunfamiliarized the worms’ homewells, in the saving protocol, Savings Paradigm from decapitation. were atday again 9or 10 fedatdays 7andthen worms decapitation atdays 13after thattested example, days. For than3-4 was test longer therecall to feeding duration between thefirst when the cases in time, asecond were worms fed The recall. were for tested theworms feeding theregenerateddays after decapitation, were wormstofour days after Three fed tosatiety. fragments (Fig. tonine ofeating 4). were Seven headless regenerating capable at18°C, the day.was at18°C. with worms, every waterincubator After As changed theintact 7days of 2ml plates, in of12multiwell ofwater well (Fig. groupsof12worms 2E), in adark one kept in in were fragments 2F,4). ofthepharynx Headless andtheanterior (Figs. side between auricles the the familiarization session. Sothatno brain re Fragments Headless Producing next day. theexperimental period,plates werein in at18°C dark placed atthe thebeginning till ofthetest group ineach was aseparate placed well with of12-well filled 1ml plates, of water(Fig. 2I). As the testingwormswere dividedintotwo groups groups, sequential interferencefrom in moving before, forpossible worms in testing theevening headregenerationincomplete the in case ofthe fragment headless worms. In order toavoid or lesions, by fission, caused pipette, thetransfer forinjuries microscope, generalhealth, At the end of each testing trial, theworms testing At theendofeach were individually, adissection inspected under In tothe contrast headless regularfragments’ protocol, where thefeeding place took in wereWorms after 24hours feeding decapitated which thefinal occurred at theendof

miliarization arena, Fig. 2H). mained, the worms were de 9 of 6familiarized and6unfamiliarizedworms and the worms were fedinthefamiliarization arena. iliarized (Fig. 2I) andplacedbackinthedark at After 30 minutes of exploration capitated atthepoint

The Journal of Experimental Biology – ACCEPTED AUTHOR MANUSCRIPT 336 335 334 333 332 331 330 329 328 327 326 325 324 323 322 321 320 319 318 317 316 315 314 313 312 311 310 309 308 307 306 305 304 differences revealed significant than 8minutes less in wormsreach criteria number of to “unfamiliarized” worms (one P<0.001, tailed U-test, Familiarized worms displayed significantly shorter time to reach criterion compared to the two groups’ motility 1). (Table down before settled phase ontheliver differences were area. Nosignificant found between the during the first minute, a time period while most of the worms were still during their exploration general physical differencescondition between theworms, wetheir movement checked rate inspent the illuminated quadrant near theliver. testing trialsThe lasted 60 minutes. rule outTo we measuredhow for took the it long worms of toreachthecriterion 3 minutes consecutive a strong bluelight illuminated thequadrantwith the floor (the familiar environment). area Asmall of thewas dish coveredwith liver (Fig. 2J,O) and and 6“unfamiliarized” control worms were placed standard, smooth-bottom Petri dish(Fig. 2D). bottom surface (Fig. “unfamiliarized”were The butina 2C). group alsokeptandfedintheATA which thefamiliarized worms were andfedinATA chambers kept inPetri dishes with arough tookenvironment place 4- 15 days after theending ofthe 10days familiarization period, during Methods, described in as andthentracked by test (Fig. retrieval 1). theATA for The familiar rememberWorms afamiliar environment RESULTS behavior. calculated for thefirst minute, when themajori familiarized andunfamiliarized theav worms, any mobility-impairment thatmight beres advance in predicted onthe based workprevious ofBest & Rubinstein (1962a). for check To that reachcriterion in than less 8 wereminutes. Tests onetailed since thedirection was wastodetermine exact test ofthetotalFisher's applied significance statistical number ofworms significance 2001). statistical etal., (Bevins toevaluate Mann-Whitneynonparametric Utest we (Kolmogorov-Smirnov the distributed test), were used normally delay values not Data analysis The ATA’s trackingThe files log were file toexcel converted for dataanalysis. Because the We tested for recall of afamiliarWe environment 4days after thefamiliarization period. Worms were familiarized to theautomated behavior analysis platform (ATA) chambers

ponsible for behavior di 10 During eachtestsession, 6 familiarized worms tywormswere of stillengaged inexploration erage(Pixels/Second) movementrate was individually chambers intheATA with arough liver stain (Fig. 2L). As Fig. 3B&Table 2). Similarly, testingfor the fferences between the indication offeeding,

The Journal of Experimental Biology – ACCEPTED AUTHOR MANUSCRIPT 370 369 368 367 366 365 364 363 362 361 360 359 358 357 356 355 354 353 352 351 350 349 348 347 346 345 344 343 342 341 340 339 338 337 2009; Sánchez Alvarado, 2006; and Reddien, Tanaka 2011;Umesono and Agata, 2009). etal.,Gentile 2011;Newmark andSánchez 2002; Alvarado, Nishimura et 2011; Salo al., etal., 2009; etal., (Aoki neural repair mechanisms behind andpatterning understand themolecular effortsare underway to significant population) CNS, andcomplex cell stem adult by(driven an andof developmental regenerative biology; of because theirextensive capacityregenerative DISCUSSION and brainregeneration.decapitation test, Fig. 3Ac &Table 2). We conclude that some memory of the place familiarization survives familiarized wormsbetween wormsandcontrol Fisher's exact (P=0.013; onetailed, theoriginal revealedwormsfor difference than8minutes less thenumber in of toreachcriterion significant 2&Fig.3B). prior Testing the environment (One to decapitation U-test,P=0.027;Table tailed latency thetesting assay in comparedtoregenerated worms thathad not beenfamiliarized to fragments from original familiarized worms (Fig. 4) displayed significantly shorter feeding had beentrainedonthis task prior todecapitation. thatregeneratedWorms from headless where memorywas by revealed asignificantly faster training ain specific task groupsin that his classical foundin conditioning McConnell protocol), procedures as 1965), (McConnell, thensignificant. for checked theWe phenomenonof (See savings formethods detailed was 2). notstatistically However, (Fig. effect 3B&Table the 10-14decapitation tested days after fragmentsfromshorter latencyunfamiliarized feeding comparedtoheadless worms when regeneration. Headless fragments regenerated from regenerate the (Fig. 4), possibility tocheck allowed memory us brain thatthis survive can withWorms retain regenerated memory also some heads paradigm asavings in conclude that worms canre wormstrained andcontrol (P=0.014; Fisher's exactone tailed, test, Fig.3Ab 2). andTable We worms criterion toreach in thanless 8minutes revealed significant difference between the wormscontrol U-test,P (one tailed < 0.001,Fig.3Aaand2). Table forTesting thenumber of worms displayed significantly shorter time to reach criterion comparedto the unfamiliarized 2). P=0.005,worms (Fisher'sexactbetween test, onetailed, Fig.3Aa&Table thetrained andcontrol Different groups ofwormswere 12-15 days tested following training. The familiarized During the last decade, During thelast decade, haveplanaria an become modelimportant organism the in field memory finding thatthis The 14days for atleast enoughfor –long thebrainto persists member afamiliar environm 11 familiarized worms displayed slightly ent for atleast 14days.

The Journal of Experimental Biology – ACCEPTED AUTHOR MANUSCRIPT 403 402 401 400 399 398 397 396 395 394 393 392 391 390 389 388 387 386 385 384 383 382 381 380 379 378 377 376 375 374 373 372 371 after1962), retrieval allows our 14days protocol atleast from theendof thetraining. frame. (Davenport 1962;Dufort, time Inshort andBest, tomore controls rigorous addition arelatively in size theprocedure toalargesample analysis anddatareporting, andapplies allows procedures, manual objective ofquantitative, level anunprecedented rigorbehavioral in analysis behavior system (Blackiston etal., 2010). This system minimizes bias caused by theirenvironmental familiarizationmodified totheuse andadaptedit with protocol anautomated fed in familiar environment 90 minutes after single, 25 minutes, familiarization session. Here we andRubinstein,(Best 1962a) showed thatworms display ashorter feeding delay, when being been frequently in and used learning memory researchwith Best invertebrates. & Rubinstein recognition (Collett familiarity 2003;Horridge,environmental etal., 2005), environmental not has memory of spatial are invertebratesandants capable and some bees such Although as Environmental familiarity awell-accepted is paradigm for the study memory ofexplicit (Heyser vertebrates 2013;Teyke, in mechanism 1989). andFerris, Chemero, 2012;Heyser and and memory. learning into modern memories, in research explicit-like theplanarian more even central system becomes term memory capabilities. By demonstrating evidenc objective,quantitative, high-throughput protocols for eliciting and characterizing planarian long was a reliable, state-of-the-art tofind that moves approach beyond to controversies past identify ofquantitativeand lack analysis(Corning andRiccio, 1970; 1981).Travis, aim ofthis The work limitations included difficultiesthese precisesample of protocols, sizes, reproduction small in being importantlargelyworkers; tothese by lead questions experiments have recent neglected manual 1966), brain regeneration survive thelimitations ofprevious (McConnell, can memories 2008). extensive Although work and learning onplanarians’ memory suggested that long have memory still is very (Nicolas etal., limited 2008;Nishimura et al., 2010; and Oviedo Levin, forand amodel learning as ofplanaria 2008), etal., theusage 2010;Sacavage Rawls etal., 2011; 2012;Rawls etal., al., and Valdez, 2001;Ramoz et 2008;Raffa 2012;Raffaetal., et al., ofthebrain.processes ofmechanisms of issues on thefundamental memory thephysical andstorage in encoding bears therapiesdisorders neurological ofadult butalso clinical forcell stem implications obvious notonly question has thedynamicsunderstanding ofmemory This during brain regeneration. 2008), this modelLevin, system has thepotential also to offer unique opportunities for However, their rich behavioral dueto repertoir While planaria are now being used for studies of drug addiction andwithdrawal ofdrug(Pagan addiction planaria for studies areused nowbeing While e andability tolearn 1967;Oviedo (C and orning, 12 e for theacquisition of relatively complex,

The Journal of Experimental Biology – ACCEPTED AUTHOR MANUSCRIPT 436 435 434 433 432 431 430 429 428 427 426 425 424 423 422 421 420 419 418 417 416 415 414 413 412 411 410 409 408 407 406 405 404 worms be needto andthe 1901;Pearl, 1903) worm (Bardeen, exceptional decapitated is reflexes in ofthese 1917). wormsWulzen, However,activation decapitated (Pearl, 1903; in and swallowing, the even reflexes ofextending activate thepharynx pharynx, can of thefolded it (Inoue1903)sensations andoptical 2004). etal., (Bestmechanical stimulation and 1962b) Rubinstein, , (Ashchemotactic 1973; etal., Pearl, paradigm,oftactile/ our in as (Pearl, 1903) dependentonsensory , is integration behavior byand initiated thecentral nervous system (Pearl, 1903;Sheiman et al., 2002). The feeding a true complex behavior. Although composed of a series of stereotypic it actions, is coordinated is feeding Planarians’ by thebrain. controlled behavior of“real,retrieval encoded”memoryand effects (Halassensitization etal., et 1961)1962; Halas al., rather than consolidation and environmental familiarity this or protocols), beattributedtopseudoconditioning cannot protocol for worms.parameters theexperimental andcontrol and conditions the experimental and groups,control the indicate importance ofrigor with to respect identical temperature. aresult, As thechanges in latency each in ofthe offeeding are both categories in conditions andmaintenance offeeding through identical andwent time thesame in tested and colony, same were trained category from each the andexperimental from groups control latency between offeeding, thedifferent categories. Even so, any in ofthe experiments, both protocol was similar between thedifferent groups reached. criterion there until is Also, from seen theresults (fig.3B), as thegeneral although feedingthan1minutethird oftheworms less from in thestartof initiate trial andstay testing theproperwhennotmore aheuristic, seems tobeachieved thana As protocol. hunger level worms,we toother andtypeheuristic an additional workers offood, offer this reproducing asvariables manipulation intensity, maintenance temperature, of size the worms, of thespecies Because hunger level is a parameter pivotal this approach in and could beaffected by many assay. this in results theworms in thebest toobserve hunger thecorrect level establish must whenobtained theworms were fed3-4 days beforetested. being Future ofthis procedure users in were our results, procedure, thatthebest 1962a).observed andRubinstein, We Best (Best, 1963; did andRubinstein afunctionperiod,as Best oftheworms’ as starvation variance and significance theresults’ in data)between andfound differences days (unpublished 1-30 this protocol feeding Since measures theworms’ behavior, theretrieval performance in dependentontheirbaseline examined different periods level. is appetite test starvation We Previous Previous havestudies shown thatwhen foodis direct placed in withcontact theopening Importantly, tothemost contrast commonly-used in procedures(classical conditioning 13 , there were stilldifferences in thegeneral

The Journal of Experimental Biology – ACCEPTED AUTHOR MANUSCRIPT 470 469 468 467 466 465 464 463 462 461 460 459 458 457 456 455 454 453 452 451 450 449 448 447 446 445 444 443 442 441 440 439 438 437 to adangerous environment, i.e. one which in theanimal previously received noxious stimulus McConnell, 1965). Evidence suggests thatthis tobehaverefuse at all when inserted thetraininginto apparatuses (Best and 1962b; Rubinstein, to begin can worms while healthy andactive suddenly asteady improvement, after reverse 1966;McConnell,McConnell, 1965). learning curves duringtheWorms’ phase training can thebest seeking learning phenomenon complex we protocol While the observed previously lethargyplanarian's called 1963;Best (Best, 1962b; andRubinstein, Corning,1964; are thedata into analysisbiases introduced by ofanimal subjective behavior. thatnoscoring operator avoids andensures protocol fatigue 2001). etal., The 2004; Noldus done with any of systems oftheoff-the-shelf capable videomultiple tracking (Marechal etal., also be protocol could Therefore, this oranimal. (light shock) toeach feedback instantaneous Our paradigm requires pathtrackingbutno oftheanimals algorithm training complex with 1967). conditioning procedures(Corning,intensity 2013; classical 1967;Pradosetal., in Wells, not required analysis, as manual was required for example in of studies responseconditioned brain-regulatedcomplex, behavior program ofheadregeneration. through theprocess reflex our datashowofatrue or modification. system peripheralnerve thesurvival Thus, theCNS in a duetomodification and not/or latency notjust foundin is difference offeeding pharynx required todemonstrate “retraining” retrieval (Fig.3, 2),Table suggest thatthe intact weeksworms,with afterregenerated training, an worms two tointact tested contrast CNS (Bardeen, 1901;Pearl, 1903;Sheiman etal., 2002). Finally, that our results show thatin and away theedgeofdish, from Fig. 2P,Movie S1), are behaviors that by are controlled the against approach, theirnaturalpreference andacautious (under above making thestronglight thataccepted therecognition offood andmoving directly as toit, ourin with decision case, well- is However, it latency. feeding in tochange systemthe peripheralnervous contribute the movements into liver. cannot We completely rule outthepossibility thatthemodifications in the headmadeafirst contactwith thefood,some just after happened thatextrusion ofthepharynx we observed Moreover, adjacent tothefood. immediately passed fragment when thetail even decapitation), regeneration days after (5-7 fragmentsfooduntil head in withpharynx any notdemonstrate headless did anintact interest 2002), etal., our with(Pearl, 1903;Sheiman observations others’ week) consistent than a and 2002; etal., Sheiman 1917). Wulzen, (Bardeen, 1917)directlystarved (Bardeen, 1901; andtested 1901; afterWulzen, decapitation We never behavior observed We such in our worms ( The procedureThe is ideally suited for automated apparatuswith minimal handling and does 14 phenomenoncouldbe relat time with akindofstereotypic, drilling-like, which japonica, fastedDugesia for less ed tofamiliarization

The Journal of Experimental Biology – ACCEPTED AUTHOR MANUSCRIPT 504 503 502 501 500 499 498 497 496 495 494 493 492 491 490 489 488 487 486 485 484 483 482 481 480 479 478 477 476 475 474 473 472 471 familiarizedoriginal worms, shorter displayed significant feeding latency to compare introduction for theunfamiliarized. Worms fragments thathadregenerated from from headless session was apreviously-enc test. 3hour asingle session,Therefore thefeeding in environment) 4days before theretrieval worms.In our savingsprotocol, regenerated worm etal.,McConnell 1959). Therefore, for we thepresence of checked savings in theregenerated retrained is animals better (faster) relative tothat of to naïve animals (McConnell, 1965; determine whether effect beincreased. can thestrengthofthis workwill explore longer traini unfamiliarized worms (Fig. 3 &Table 2), but this effect was not statistically significant. Future displayed aslightly latencyfeeding shorter average, fragmentsfrom comparedtoregenerated etal., 1967; McConnell 1959). regenerating fromWorms decapitated familiarized worms displaycan after decapitation retrieval andregeneration of anew head (Corning, 1966;Corning, 2004), (Fig. 4). by Encouraged we thelong-term retrieval, whether investigated trainedworms 14daysdays, andin theworms are fully regenerated 2008;Inoue (Agata andUmesono, etal., 2). 14daysleast (Fig.3&Table from theelectrodechemical additional cues also facilitated metal place recognition. products metalelectrolysis such as (Blackiston etal., apossibility 2010), ions there is that off notgive does material was andtheelectrode delivered (Fig.electrode 2). noshock Although chamber walls,which andiridium alternated betweenoxide-coated delrin-plastic titanium ofthe shape oftheflooranddodecagon theroughness experimental worms feel could thewormsphase, around were theedgeonbottomof crawling the Hence, chamber. between wasdifference In mainly thetwo themajority tactile. oftheirexploration environments familiarized (experimental) andthe unfamiliariz the type andamountofwater, food,handling andmaintenance were between identical the activity levelduetothefamiliarization protocol any areworms notduetosimpleofoverall 1). differences Thus, (Table changes behavioral mediterranea tomuch as more sensitive specieslethargy such theapplication allows also andthus withbehavior handling minimal and without reinforcement. negative planarians’This overcomes reported natural protocol 1963). here dataandBest, The involves (Shomrat, unpublished No differences were foundingeneral motility between familiarized and unfamiliarized McConnell’s original results revealed apattern of “savings”, where thelearning curve of can for rememberOur planarians at showhabitats previously-encountered results that (Sanchez Alvarado et al., 2002).(Sanchez etal., Alvarado regenerates a functional head and CNS after 7 head7 Dugesia regenerates andCNSafter japonica afunctional ng phases and furtherng phases optimize different starvation periods to ountered environment for thefamiliarized worms and a first . The training occurred. The incomplete darkness and 15 ed (control) groups. Therefore, thelearned s were fedinthetesting arena (familiarization Schmidtea

The Journal of Experimental Biology – ACCEPTED AUTHOR MANUSCRIPT 537 536 535 534 533 532 531 530 529 528 527 526 525 524 523 522 521 520 519 518 517 516 515 514 513 512 511 510 509 508 507 506 505 theneoblasts, presentin memoryis thatonly rough correlate ofthe Alternatively, a possible is it protocol. trainingsub-optimal or be a results These could duetoinsufficient modification. aretraining after component include thebrainregenerated, the indicating necessity ofCNS not results were thatdid significant experiments foundin hand,no trained worms.On theother memory physicalsubstratum brain. in andits theintact butforofconscious are thenormal relation poorly case onlytopologies this understoodnot for floor”, ofgenetic walls”,physico-chemical material “metal aspects etc.)into or neural network sensory (e.g., (coding ofenvironmental “rough mapping anddecoding) facts unambiguous pastpersisting complete removal of thebrain. Of course, themechanisms thatallow molecularly-tractable oflearned information aunique model provide (metamorphosis), planaria the ability ofmemories tosurvive signi andTiras, 2008;Sheiman (Blackiston etal., 1996) previous work suggested exciting insects in cells. stem While ofthenew back duringtheconstruction adult these recapitulated brainby andareturn in neoblasts somatic ofthe thebrainalterproperties in occurring that experiences possible is it Thus, 2011;Singer, 1952). etal., Singer, 1978;Mondia of theCNS(Geraudieand 2013). communicating cells (Levin, 2012;Mondia etal., 2011; et Oviedo al., 2010; andLevin, Tseng 2012;KennedyDavies, andDehay,2012; Saetzler etal., 2011) andnon-neural but electrically- pathwaysepigenetic ofself-organization thatdeterminepatterns of neural (Arendt, 2005; bothregulated via are learning) andregeneration (plasticity during that brain remodeling andSweatt,Levenson 2005;Zovkic 2013) (Smalheiseret al., andRNAi et 2001). al., It likely is nervous system (Arshavsky, 2006;Day and suchdiscoveries as epigenetic modification thatoccur manyin types, cell notjust the central headremoval. results are However,complete now bysuch plausible mademore modern bymediated pattern central generators) beencoded can andstored tissuesin remaining after Sarnat andNetsky, 1985), is andit howhard toimagine memory (not just traces reflex arcs (Smalheiser with 2001; beenreceived skepticism etal., have results such In past, the 1981). Travis, planarian a has The centralized brainthatguides behavior (Buttarelli etal., 2008; regeneration. exclusively ontothenot located imprinted thebrain, andhadbecome newly-built in brain during unfamiliarized worms (Fig.3&Table 2), suggesting th Our regenerated ofmemory in thepresence tail datareveal fragmentsfrom savings It been long has known thatregeneration both andshapes, is in turn guided by, activity ficant rearrangements ofthe brain andCNS 16 Sweatt,Ginsburg 2010; at memory oftheoriginal environment was and Jablonka, 2009;

The Journal of Experimental Biology – ACCEPTED AUTHOR MANUSCRIPT 571 570 569 568 567 566 565 564 563 562 561 560 559 558 557 556 555 554 553 552 551 550 549 548 547 546 545 544 543 542 541 540 539 538 the possibility theperipheralnervous thatthemodifications in system contribute in tochange thatenvironment makes trained wormsinitiate feeding sooner. cannotWe completely rule out S1, thebehavior notresemble does reflex asimple modification but rather thewhole Movie developedcan oftheliver. in positive tothesmell have As beseen associations could groups andcontrol thetrained was thevicinity, in somewhere andboth texture thatfood toknow odor would beeverywhere thedish in –this means theworms did nothave torely ontherough andtheliver with during theentire procedure, liver fed wormswere Experimental andcontrol is less likely, of because the behavioral complexity ofthelearned task (Fig. 2P & Movie S1). theunfamiliarizedthan in worms, PNS this duetothemodified sensitivity). believe scenario We thenewinto CNS,during the “Saving” short session (which suffices because it is more efficient duringthetrainingto agiven stimuli phase) retrainingthe is new the association brain:“burning” receptor sensitivity andtuned,e.g.,increased/decreased wouldPNS (which been modified have imprinted by any ofthe traces abilities ofthePNS processing otherin organisms. advanced information- program aresearch would theuntapped brain,andthus into suggest an efficient to theperipheral in system thatnormally information complex nervous ofananimal access has and Movie S1). However, if true, this would suggest a remarkable capacity for of integration unlikely by tobeimplemented sensitivity andreflex receptor modifications only (e.g., Fig.2P very thatis portion oftheparadigm, andthecomplexity the behavior need for of theSavings supportedby behavior. also role for Apivotal drives thebrain is the brain indeed the planarian thatworms priorand thefact most totheregrowth nobehavior exhibit likely is ofthebrain,it that 1985)), andNetsky, 2011;Sarnat 2008;Rawls etal., andLevin, 2008;Oviedo etal., (Nicolas ethology complex ofstructure, (in terms biochemistry, and brain andthatofhigher animals the Given thebrainin system, or betweenlearning recall. similarities notinvolving theplanarian andtheregeneratingorgans CNS.However,must possibilities beconsidered. two additional of instructive between interactions the properties andmechanisms such somatic remaining orderbrain in to result in theobserved subsequent recall behavior. Future work investigate must that acquiredimplies information during must on training beimprinted the regenerating (naïve) brain (since the place conditioning association survives decapitation). A straightforward model behavior the (asoccursanimal in savings paradigm). requiring abrief re-exposure tothetrigger order in to consolidate into measurable effects on We that traceofmemorysuggest distributedsome We locations stored in beyond is the Second theSecond is possibility thatthenew regenerated a brain is as Tabula andis notRasa thepossibility is First executed nervous entirely is thatthememory theperipheral by previous memory.Instead, onthismodelthe familiarized worms’ 17

The Journal of Experimental Biology – ACCEPTED AUTHOR MANUSCRIPT 606 605 604 603 602 601 600 599 598 597 596 595 594 593 592 591 590 589 588 587 586 585 584 583 582 581 580 579 578 577 576 575 574 573 572 FUNDING Van Oye two V.McConnell, andJames andmemory oflearning pioneers planaria. in oftherough-textured,Ethan Golden for fabrication work dedicated This toPaul is petri dishes. andthe manuscript, of onearly for Romero, versions Starks comments andPhilip Michael Dany andLaura S.Adams, Vandenberg with for Blackiston, statistics, assistance Douglas forRobert many Cook discussions aboutbehavioral helpful paradigms, Durwood Marshall, Blackiston and with system, andhelp model theplanarian Wendy Douglas Beanefor advice ACKNOWLEDGEMENTS memoryself-organization mechanisms in brain development, and encoding, brain regeneration. advantage hypothesis allows as ofinnovative and,crucial theroleofepigenetic theInvestigation system which in memory be can andbrainthe regeneration studied in same animal. a This is molecularly-tractable areImportantly, theonly planarians andmemory. research oflearning approach opens great opportunities for theuse ofplanaria as a model for modernorganism display significant envi Remarkably, headlessfragments, regenerated from original environment-familiarized worms, memoryMoreover, this long regeneration, enoughtoallow survives full after amputation. are capableofacqui previoussupport findings of Best and Rubenstein (Best and Rubinstein, 1962a) ,thatplanarians Conclusions: anewly-developedin brain(Inoue 2004;Koopowitzal., 1988). et andHolman, between andlearning experiences examine by theinteractions amature PNSmodified specific morebecome or sensitized. less Thus,this system would anovel model provide whichin to have sensory thatcould therough outofmanypossible presence, surface, modes other predictor andthesalient ofits ofliver anassociationbetween thepresence implements learning,(including appropriate modification of PNS, andfacilitation of re-training phases) whole place - hadtotake afterakindthesystem oflearning training, a tochange as stimulus latency.feeding should be However, it notedthat orderin for receptor sensitivity toa This research was research This fundedbyG the thank Punita for Koustubhan generalWe laboratory Junji Morokuma and assistance, Our obtained using results, ahighly-sensitive, objective, quantitative analysis system,

ring arelatively complex, explicit-lik ronmental familiarity inasaving

. Harold and Leila Y.Harold andLeila Charitable Mathers Foundation . 18 e memories ofenvironmental familiarity. paradigm.promising This simpleand particular .

The Journal of Experimental Biology – ACCEPTED AUTHOR MANUSCRIPT 656 655 654 653 652 651 650 649 648 647 646 645 644 643 642 641 640 639 638 637 636 635 634 633 632 631 630 629 628 627 626 625 624 623 622 621 620 619 618 617 616 615 614 613 612 611 610 609 608 607 ofGeneralJournal Physiology ocellus. horizontal Nature magnetic vector. Zoology magnetic rotationcounterclockwise by Physiological dugesia-dorotocephala. theplanarian e1736. through metamorphosis: can amoth remember what itlearned as acaterpillar? Coldgeneral principles. Spring Harbor Protocols molecularly-tractable organisms. model PLoSONE for device automatedsecond-generation behavior andquantitative training analyses of 75, 63-76. Neurobiology differentialand rats: nicotine. of effectsin ofacute chronic Learning andMemory JCompplanaria. Physiol Psychol Science planarian. the brain. ofPhysiologyJournal biochemistry andphysiology. Part A, Physiology theocellusDugesia japonica. in oftheplanarian activity tolight changes feeding behaviour Dugesiaelicits dorotocephala. in Animal Behaviour 113. of synaptic plasticity long-term explain memory consolidation? brain research Progress in electroencephalogram.of theplanarian (Tenebrio-Molitor). Trans Philos planarian. RSoc LondB Sci Biol Biology Cell Trends in tigrina) (Dugesia planaria for preferences. directional REFERENCES Brown, H. M. and Ogden, T. E. T. Ogden, H. and M. Brown, E. T. Ogden, and H. Ito, M., H. Brown, A., Jr. F. Brown, C. Chow, and F. Brown, Blackiston, D.J.,SilvaCasey, and Weiss, E. M.R. Blackiston, D.J.and Levin, M.(2012). training Aversive methods in Xenopus laevis: M. Levin, and Granata, C. C.L., T.,Blackiston, Nicolas, D., Shomrat, A.,Bevins,Armiger, R. Koznarova, J.and T. J. I. Rubinstein, and B. J. Best, I. Rubinstein, and B. J. Best, A. A. Pigon, and B. Goodman, B., J. Best, Best, J.B.(1963). Protopsychology. Maculata. Planaria oftheBrain in Function The Bardeen, C.R.(1901). Azuma,Asai, K.,Okazaki, Y., K.and N. Iwasaki, Ash,F.,O. McClure, Hirsch, W. and J. J. Arshavsky, Y. I. T. Arendt, Aoki,Wake, H.,Sasaki,H.and R., Agata, K. M. T. Alloway, Brain regeneration cells Y. from stem Agata,in pluripotent Umesono,(2008). K.and Aboobaker, A. A. paradigm asimple for cells: stem Planarian (2011). regeneration. Abbott,Wong, S.M.and G. K.

The Journal of generalphysiologyThe Journal 48, 168-176. Science

(2005). Alzheimer's disease as a disorder of dynamic brain self-organization. adisorder ofdynamic as (2005). disease Alzheimer's

164, 565-6. American Zoologist 135, 916-8.

4 21, 304-11. (1972). Retention of Learning through Metamorphosis in Grain Beetle Grain in Beetle Learning through(1972). Metamorphosis of Retention , 175-179. (2006). sevenoftheHebbian sins" "The synapse: the can hypothesis (1966). Effects and after-effects on planarians of reversals ofthe ofreversals onplanarians andafter-effects Effects (1966). 147, 355-78.

51, 237-53.

55, 560-6.

209, 533-5.

12, 471-472. (1968). electrical response The of theplanarian ocellus. Neuroscience (1975). between Differentiation and clockwise (1962b). Maze andassociated learning behavior in (1962a). Environmental fa

(2008). The conditioning andmemory conditioning The (2008). retention of 51, 255-60. Sci Am

363, 2071-8. 19

109, 593-9. 2012. (1968). sensitivity Spectral theplanarian of

5 of studies (1973).a factorwhich Chemical (1969). Fissioning in by planarians: control (1969). Fissioning , 54-62. 208, 54-62. , e14370. Bios (2009). andspectrum Recording analysis 159, 908-14.

(2001). Environmental familiarization 79, 160-170. (1994). Electrical responses and K+ Progress In Neurobiology (2008). Retention ofmemory Retention (2008). miliarity andfeeding in a

21, 796-800. PLoS ONE Comparative (2010). A A (2010). American

80, 99-

3 ,

The Journal of Experimental Biology – ACCEPTED AUTHOR MANUSCRIPT 707 706 705 704 703 702 701 700 699 698 697 696 695 694 693 692 691 690 689 688 687 686 685 684 683 682 681 680 679 678 677 676 675 674 673 672 671 670 669 668 667 666 665 664 663 662 661 660 659 658 657 considerations. DevelopmentalMethodological Psychobiology are equal. created ofExperimentalJournal Psychology. B, Comparative andPhysiological Psychology Reexamined.through Cannibalism byplanaria JComp light. Physiol Psychol theplanarian. in conditioning Biosci andblastema of theadultepidermis newt Zool Exp J regenerate. biology for cell model stem system andnervous regeneration. 1145. discriminationdarkness planaria.in Bollettino - Societa Italiana Biologia Sperimentale Association regeneration planaria in (D. aculatd). In Paper presentedatMidwestern Psychology 13,1319-23.neuroscience 42-48. tigrina)following(Dugesia regeneration. and memory tolearning approaches Chemistry invertebrate research.New oflearning; Press. Plenum York,: planarians. Psychology regenerated in planarians.response Neurobiology in Opinion 1110-1115. Deiatelnosti I.P.Pavlova Nervnoi regenerazei. Imeni opiti s Vysshei i Zhurnal planarii &pharmacologyToxicology :CBP inbehavior planarians: translations to mammals.

34, 633-46. Heyser, C.J.and Ferris, J.S. A. Chemero, J. and Heyser, C. B. Waldman, and G. P. Hepper, P. Keithlee, W. and D. A. Morton, L., Hartry, A. L. Stone, and L. R. James, S., E. Halas, Halas, E.S.,James, R.L.Knutson, and C. S. E. Jablonka, and S. Ginsburg, M. Singer, and J. Geraudie, K. Bartscherer, F. and Gentile,L., Cebria, Fulgheri, Messeri,P. D.and Ernhart, E.N. Sherrick, and C. R.H. Dufort, Day, J.and J.D. Sweatt, Davies, andtop-down epigenome C.W. causation. P. The (2012). (1962).Science On Best,J.B. Behavior. Planarian D.and Davenport, Dasheiff, R.M. Dasheiff,B. D.and D. Riccio, C. and W. Corning, AmericanCorning,W. S.C.and C.,Ratner, Institute Biological Sciences. of W.Corning, C. W.Corning, C. W.Corning, C. E. John, W. and Corning, Collett, T. S.,Graham, P.and Durier, V. I. G. Bogorovskaya, M.and I. A. N.,Sheiman, Cherkashin, Buttarelli, F. R.,Pellicano, Pontieri, C.and F. E.(2008). Neuropharmacology and . St.Louis, Mo. Psychanomic Science 58, 131-139. Behav ProcessesBehav (1962). OnScience Behavior. Planarian (1967). Regeneration (1967). andretention ofacquired information: NASA. (1964). Evidence of right-left discrimination in planarians. ofright-left discrimination in Evidence (1964).

(1966). Retention of a position discrimination after discrimination regenerationof aposition in (1966). Retention 13, 718-25. Journal Journal of comparative andphysiological psychology

5 147, 399-408. Science (1961). Effect of ribonuclease on retention of conditioned onretention ofconditioned ofribonuclease Effect (1961). , 17-18. , (ed. W. Byrne),, (ed. Press. Academic York: W. pp.107-150. New

Science (1978). Nervedependentmacromolecular synthesis thein 89, 232-8. (2010). DNAmethylation andmemory Nature formation. (1973). The use of2different (1973). use reinforcements The in light (2012). Novel object exploration in mice: not all objects notall object mice: exploration in Novel (2012). (2009). Epigenetic learning Epigenetic (2009). non-neural in organisms. (1992). Embryonic olfactory learning in frogs. frogs. Embryonic (1992). in olfactory learning Ecotoxicology & Ecotoxicology Environmental Safety

54, 302-5. (2013). Object thedeveloping exploration in rat: 146, 274-275. (1970). The planarian controversy. In planarian The (1970). (2002). Photonegative response in brown planaria brown in planaria (2002). response Photonegative

(1959). Retention of a maze habit following maze habit (1959). ofa Retention 134, 1363-1365. 20 (2003). Route learning by insects. Comparative biochemistry andphysiology. (2011). The planarian flatworm: an in vivo vivo anin flatworm: (2011). planarian The (1961). Types of responses elicited in responses (1961). of Types (1964). - Planaria Memory Transfer

(1962). An attempt atclassical (1962). Anattempt 138, 400-2.

Dis Model Mech Dis 55, 373-81.

203, 455-60. (1966). Uslovniye reflexi y reflexi Uslovniye (1966). Interface Focus

4 137, 452-6. 53, 196-9. , 12-9. The Journal of The Journal

55, 969-71.

44, 179-97.

Molecular 49, 1141- Quarterly Quarterly (1967). (1967). Current

XVI

2 J , ,

The Journal of Experimental Biology – ACCEPTED AUTHOR MANUSCRIPT 758 757 756 755 754 753 752 751 750 749 748 747 746 745 744 743 742 741 740 739 738 737 736 735 734 733 732 731 730 729 728 727 726 725 724 723 722 721 720 719 718 717 716 715 714 713 712 711 710 709 708 to connectionist models of long-term memory. Reproduction. Asexual macromolecules. as shownXenopus tail, tadpole by ablation. femtosecond-laser S. D. Adams, Dugesia-japonica-japonica. Physiology andPsychology regeneration uponretention of a responseconditioned thein planarian. Journal ofComparative Arbor, Mich. Physiol 23, 460-9. cells. physiology andpathology: theimmune signature driving therapeutic plasticity ofneuralstem measuredmarine invertebrate larvae 3.0.by EthoVision Trends Biotechnolsynthetic biology devices. primer for theworm. reverse-engineering 205-17. gradients. controldiscoveries: of behaviorcell andpattern by formation transmembrane potential Neurosci NatRev formation. the Polyclad Flatworm, Notoplana-Acticola. Planocera-Gilchristi. cortex.primate brain research Progress in system Sci during headregeneration. Zoolog K. Agata, and A. physiology neural,andbehavioral sensory, ofcomparative physiology. A,Neuroethology, Journal 977-90. andtheinvestigation systemoflearning. Neurobiol computer-controlled screening J for drug Physiol Rev Murre, J.M., Graham, K.S.and Hodges, J. R. andMelatonin ofPhotoperiods on Planarian Effects B.(1984). Morita, Best, M.and J. Morange, M. and R. M. Branch, D., R. Orendorff, G., F. Omenetto, M., Levin, P., J. Mondia, A. Miyamoto, Shimozawa, S.and D.P. Kimble, A. L.and Jacobson, J. V., McConnell, V. McConnell, J. McConnell, J. R. P. Mason, M. Schwartz, and L. Bonfanti, S., Pluchino, G., Martino, A. S. M. and Sebire,Marechal, Clare, J.P.,Hellio, C., Macia, J.,Posas, F. and Sole, R. V. M. Levin, and W. S. Beane, D., Lobo, M. Levin, J.D. Levenson, Sweatt, M.and J. M. Holman, and H. Koopowitz, H. Koopowitz, C. Dehay, Kennedy, H.and Alvarado, K.,Umesono,Y., M.,Sanchez Sakai, H.,Okamoto, Inoue, T.,Kumamoto, B. Humphries, Horridge,A. G. byof afamiliar mellifera). Recognition (2005). place thehoneybee(Apis M. Levin, Hicks, C.,Sorocco, D.and 28, 107-36.

BioEssays :newsBioEssays molecular, andreviews in anddevelopmental cellular biology 191, 301-16. (2011). Long-distance are requiredsignals for morphogenesis of theregenerating (2012). Molecular bioelectricity in developmental biology: new tools and recent andrecent tools biology: bioelectricity new (2012). developmental in Molecular Journal ofbiosciencesJournal

(2004). Morphological and (2004). functional Morphological recovery oftheplanarian photosensing 91, 1281-304. Animal Behaviour (1975). Chemo-klino-kinesis planarian (1975).in foodlocation. Chemo-klino-kinesis (2006). What history tells us VI. The transfer of behaviours by ofbehaviours VI. history transfer us The tells (2006). What Annu Rev AnnuRev physiology: invertebrates. in (1966). learning Comparative (1961). Maze learning in planaria. planaria. (1961). in Maze learning (1970). Feeding Behaviour and Role of Brain in Polyclad ofBrain in Flatworm, andRole Feeding Behaviour (1970). Journal ofExperimentalJournal Zoology (1965). A Manual of psychological experimentation on planarians. Ann experimentation onplanarians. psychological of AManual (1965).

Zoological (Tokyo) Science 52, 1-5.

6 , 108-18. (2012). Self-organization andinterareal networks (2012). the in

18, 31-&.

(1988). Neuronal Repair and Recovery of Function in in of Function Recovery NeuronalRepairand (1988). 31, 323-7. PLoS Comput Biol 195, 341-60. American Zoologist

(1985). Chemotaxis in the freshwater planarian planarian thefreshwater in Chemotaxis (1985).

30, 342-9. (2012).computation: the Distributed newwave of 21, 275-83. Brain 21 (2005). Epigenetic mechanisms memory (2005). in Epigenetic (2006). Automated analysis of behavior: A ofbehavior: Automatedanalysis (2006). (2012). planarian regeneration: Modeling a

124, 647-75.

2 Biofouling 231, 273-282. (2001). Semantic dementia: relevance , 389-396. Worm Runner’s Digest

8 , e1002481.

PLoS ONE (2004). Settlement behaviour of behaviour Settlement (2004). 28, 1065-1075.

(2011). Brain regeneration in 20, 211-7. (1959). The The (1959). effects of

6 , e24953. Animal Behaviour 3 , 114–115.

66, 34,

The Journal of Experimental Biology – ACCEPTED AUTHOR MANUSCRIPT 809 808 807 806 805 804 803 802 801 800 799 798 797 796 795 794 793 792 791 790 789 788 787 786 785 784 783 782 781 780 779 778 777 776 775 774 773 772 771 770 769 768 767 766 765 764 763 762 761 760 759 Dependence behavioralRaffa, R.B.(2011). Nicotine pharmacology: from clues planarians. Neuroscience S. M. Rawls, and 430, 143-5. an planarians: of'pharmacologicinstance congruence'. Brain Res butnotamphetamine, cannabinoid (WIN 52212-2), abstinence-induced withdrawal in M. S. Rawls, cognition C. Davidson, A. and M. Young, SystemsIntelligent Body, andEnvironment. Current disease. degenerative Opinion Neurology in animal behaviour: London:J. & A.Churchill. ofdevelopmental journal International biology Girardia theplanarian behavioral tigrina. in ofcocaine aspecific antagonist is parthenolide al. G.D., Wilk, et Tallarida, C.,Bach, Kotturu, D., andbehavior. aging, regulation, andpowerfulversatile system model for molecular ofregeneration, studies adult stem cell acolonymaintaining ofplanarians. polaritycontrol Biol regeneration. duringplanarian Dev M. Levin, and T. Gojobori, RGRaffa andS.Rawls).Co. Landes Austin: In andmechanisms. effects study ofdrug Comput trackingvideo system for automation ofbehavioral experiments. 18-30. by neurons modulated planarian in function cholinergic of brain. Journal planarian Neurochemistry (2011). Regenerationofdopaminergic neurons after 6-hydroxydopamine-induced inlesion RG Co. Landes Austin: Inmodel. and Abuse AModel Planaria: Action for Drug enterstheera genomics.model offunctional Genet NatRev Rawls, S. M., Patil, T., Tallarida, C. S., Baron, S., Kim, M., Song, K., Ward, S. and Ward, S.M.,Patil, M., Song, T., K., S.and S.,Baron, Tallarida, S.,Kim, Rawls, C. A. L.,Ramoz, S.,Bhatt,P.,Reitz, Lodi, Tallarida, Raffa, C.,Tallarida, R.B. B.,R.J., J.M. Valdez, B.and Raffa, R. Raffa, R.B.,Stagliano, G.A., W.,G., J. Powell, Ross, A. Phillips, Ding, G., Z. and Alvarez, K.,Gibson,Prados, J.,Stewart, B.,Howarth, L., J., Hutchinson, C.V., C. G. Gomez, and R. Pfeifer, in memorydysfunction (1996). R. Spectrumof Hodges,Perry, J. and R.J. Pearl, R. C., Tenaglia,M.,Randolph, E., Montgomery, S., O. D.,Deats, R.,Baker, Pagan, (2008b). M. Planarians: a Levin, and Adams, S. D. L., C. Nicolas, J., N. Oviedo, and Establishing M.(2008a). Levin, Adams, D.S.and L., Nicolas, C. Oviedo, N.J., J., Ahn, Hwang, J., M., Gu, Walentek, P.,Kema,I., Oviedo, J., N., Morokuma, regeneration (2008). planarian model a M. as The Levin, context for the and N. Oviedo, A. R. Tegelenbosch, A. and J. Spink, P., L. Noldus, Agata, K. and Y., T. Taniguchi, Kitamura, Nishimura, K., Agata, K. Y. and Kitamura, T., Taniguchi, K., Yoshimoto, T., Inoue, K., Nishimura, theplanarian Abramson, in Analysisofbehavior M.(2008). Levin, and Nicolas, C., C. A. Alvarado, Sánchez and P. Newmark, , 398-414. 33, 398-414. 16, 177-86. 118, 274-9. 208 (2008). The kappa-opioid receptor antagonist nor-BNI inhibits cocaine and cocaine nor-BNI receptor inhibits antagonist (2008). kappa-opioid The (1903). movements and offresh-water The reactions :a study planarians in , 79-84. (2012). Mephedrone ("bath salt") pharmacology: from insights invertebrates.

, 66-83. 5436, 66-83. Creating Brain-Like Intelligence:Creating Brain-Like From Principles toComplex Basic (2010). Long-range neural and gap junction protein-mediated cuesjunction Long-range neural andgap (2010). Cold Cold Spring Harb Protoc (2013). Cue competition effects the Cuecompetition in (2013). Animal planarian. CSH Protoc CSH (2009). Morphological Computation Morphological - Connecting (2009). Brain, (2001). Cocaine withdrawal Planaria. Cocaine (2001). in Planaria: A Model for Drug Action and Abuse Action Model Drug for A Planaria:

, 1217-31. 119, 1217-31.

56, 193-6. 22

2008, pdbprot5053. (2002). Not your aclassic father'splanarian: (2002). Not

9 , 281-5.

, eds. R.Raffaand , eds. S. Rawls), pp.83-94. 339, 188-99. Dugesia japonica Dugesia (2012). Planarians in pharmacology: in Planarians (2012).

2008, pdb.emo101-. 3 , 210-9. (2001). EthoVision: a versatile (2001). aversatile EthoVision:

1193, 51-6. Behav MethodsRes Instrum (2010). Analysis of motor ofmotor (2010). Analysis . Neuroscience Drug andAlcohol Eur JPharmacol , eds. R. R. , eds. 168, The

The Journal of Experimental Biology – ACCEPTED AUTHOR MANUSCRIPT 860 859 858 857 856 855 854 853 852 851 850 849 848 847 846 845 844 843 842 841 840 839 838 837 836 835 834 833 832 831 830 829 828 827 826 825 824 823 822 821 820 819 818 817 816 815 814 813 812 811 810 Developmental Cell science types system. bodyof differentcell during changes in cell thestem by size using in 1727-39. biology: computation. protocell to reproduction from onthe righttrack? McConnell Biol extremity. QRev Biochemistry andPhysiology (Girardia) Dugesia oftheplanarian tigrina. behavior feeding and Y.Praeger. pp.43-76.CT: M.Burmistrov), Westport, Inbeetle. to contributions Russian invertebrate behavior Journalthe humanbrain. Canadian ofSciences Neurological andregeneration. Developmentcells Schmidtea mediterranea 241-5. persons via the tongue. blind Biol regeneration:Planarian achievements andfuture after directions 20years ofresearch. G. Rodriguez-Esteban, and D. M. Molina, M., Iglesias, M., Handberg-Thorsager, 21, 165-74. networks: generatingorder from through disorder Seminars self-organization. cancer in biology duration. M. S. Rawls, 16-24. American Psychologist 51,pg589,1996). engram, (vol andcelebrity abiochemical for learning, quest planarian 1960s regeneration. Letters Neuroscience domestica. PharmacologyBehavioural behavioral produce ofabuse drugs and sensitization cross sensitization planarians. in Planaria

53, 1317-27. Tanaka,Reddien, E.M.and P.W. Agata,Takeda, K.and K. H.,Nishimura, W. S. Stephen, Synthetic (2007). J. Macia, and C. A., Rodriguez-Caso, Munteanu, V., R. Sole, E. Costa, and Smalheiser, H. N.R.,Manev, M. Singer, D. N. Kreshchenko, and V. E. Zubina, M., I. Sheiman, L. K. Tiras, and M. I. Sheiman, G. M. Netsky, H.B.and Sarnat, R. Juste, and A., M. S. P. Robb, A., Newmark, Alvarado, Sanchez A. Alvarado, Sánchez Sampaio, E.,Maris, S. and Bach-y-Rita, P. Abril,Salo, E., Adell, J.F., Cebria, F., Eckelt, T., K.,Fernandez-Taboada, E., A. M. Soto, and C. Sonnenschein, K., Saetzler, Sacavage,Acker, S.,Patel,A.M., H.,Zielinski, G., J.,Phillips, R.B.and Raffa, planarians. Worm in In Roe, K.(1963). oflearning ofthelocus Digest search Runner's Rilling, M. A. Alvarado, Sanchez W. and P. Reddien, Ray, S. S. M.,Patil, T.,Rawls, Yuvasheva, E.andB. Raffa, R.

26, 805-13. Neuroscience LettersNeuroscience

Lugubris (2008). Withdrawal-like in behavior planarians is dependent ondrug exposure Annu Rev Cell Dev Biol Cell Annu Rev (1999). Survival ofolfactory Survival (1999). memory thefly in through metamorphosis Musca (1996). The mystery of the vanished citations: (1996). mystery vanished James McConnell's forgotten The ofthe (1952). The influence of the nerve in regeneration of the amphibian amphibian regeneration in of the of thenerve influence The (1952).

. Worm Runner's Digest 21, 172-85.

27, 169-200. (1963). The influence of varying light intensities onspeed ofmovement intensities ofvarying(1963). influence light The 51, 1039-1039. database as amolecular as database resource for studying platyhelminthes, stem

21, 301-13.

Brain Res 38, 414-418. Trends Neurosci (2006). Planarian regeneration: its end regeneration: Planarian (2006). its is its beginning.

439, 84-8.

259

, 37-40. 129, 5659-65. 20, 725-57.

908, 204-7. (1985). The brain of the planarian as the ancestor of theancestor as brainoftheplanarian The (1985). (1996). Memory(1996). in and planaria andmorphogenesis

(2011). The cellular basis for animal(2011). basis cellular regeneration. The 5 , 40-45.

23 24, 216-8. (2009). maintain a ratio Planarians constant Philos Trans R Soc Lond B Biol Sci Philos Trans LondBBiol RSoc (2001). Brain plasticity: 'visual' acuity of (2001). RNAi and brain function: was was function: andbrain RNAi (2001). , eds. C.I. Abramson Z. P. Shuranova (2004). ofplanarian Fundamentals

(2011). Systems biology Systems (2011). beyond 12, 296-302. (2010). First evidence that evidence First (2010). (2002). Regulation ofthe Regulation (2002). Journal of Evolutionary ofEvolutionary Journal (2002). The Zoological Zoological Int J Dev Int J Cell (2009). (2009).

362, 124, 5 ,

The Journal of Experimental Biology – ACCEPTED AUTHOR MANUSCRIPT 891 890 889 888 887 886 885 884 883 882 881 880 879 878 877 876 875 874 873 872 871 870 869 868 867 866 865 864 863 862 861 of memory andmaintenance. formation Bull 33,67-69. Behaviour toinjured conspecifics assess predation risk and to associate risk with novel cues. C.CorningandS.Ratner), eds. pp.251-254. NewW. York: Plenum. adult cellsplanarianpluripotent stem thatunderlie regeneration. Science processes. Regeneration oftheischemicbrain fuelling by repair cells: engineeredendogenous stem 34,863-9.of neuroscience anevolutionarilybrain--thein japonica primitive Dugesia model. planarian system.nervous normal andmutantDrosophila. ofcontrols ionic pattern formation. PlanarianLearning in Worms. Physiology Anoptichthys-Jordani. Zovkic, I.B.,Guzman-Karlsson, C.and J.D. Sweatt, M. Wulzen, R.(1917). andfeeding reactions Somechemotropic of Planaria maculata. Wisenden, B.D.and Millard, M.C.(2001). flatworms Aquatic chemical cues use from Wells, P.H. W. P. Reddien, and E. I. Wang, E., D. Wagner, J. C. Heijnen, and F. A., van Bel, Kavelaars, T., C. van Velthoven, Agata, K. T. and K.,Inoue, J.,Nishimura, Y., Tasaki, Umesono, Agata, K. Y. and Umesono, L. Kruse, and V. Cambiazo, T., Tully, M. A. Levin, and Tseng, of Construction oftheSocial - Aspects Replication (1981). L. Replicating Travis,G. D. T. Teyke,

62, 761-766.

Brain Res RevBrain Res 164, 655-662. Dev GrowthDev Differ (1989). Learning andRemembering theEnvironment the in Cave FishBlind (1967). Training flatworms in a Van Oye maze. In aVanOye maze. in flatworms Training (1967). Journal ofComparative Physiologya-Sensory Neural andBehavioral

61, 1-13. Social Studies of Science Social Studies Journal ofNeuroscience (2013). Cracking thebioelectric code: (2013).Probing Cracking endogenous Communicative &Integrative Biology

51, 185-95. (2009). Evolution andregenerationEvolution oftheplanarian (2009). central Learn Mem 24 (1994). Memory through metamorphosis in Memory (1994). metamorphosis through

, 61-74. 20, 61-74.

14, 68-74. 11, 11-32. (2011). Clonogenic (2011). areneoblasts Clonogenic (2013). regulation Epigenetic

6

Chemistry ofLearning, Chemistry 332 , 1-8. (2011). Regeneration (2011). The European journal , 811-6. (2009). (2009). Animal Biol Biol

The Journal of Experimental Biology – ACCEPTED AUTHOR MANUSCRIPT 925 924 923 922 921 920 919 918 917 916 915 914 913 912 911 910 909 908 907 906 905 904 903 902 901 900 899 898 897 896 895 894 893 892 well plate. two of6familiarized groups and6unfamiliariz into were divided worms day, before the thetesting Intheevening (I) test. retrieval days before regeneratedwormwere fedintheATA chambers with arough floor (the were (H) 4daysWorms session: plates (G). the12multiwell before test fedin Saving retrieval decapitated atthe point between the and auricles the anterior side ofthe pharynx arrow). (White were worms remained,the thatnobraintissue ensure To afterworm decapitation. before and ofa Illustration (F) was every water changed day. wells’ testing. The until thedark in plates wereworms standardPetri into thedark. in dishes, placed familiarized worms were placedin a dishwith procedure the was Duringthecleaning water changed. were electrodes) and cleaned more thangiven liver they consume. Every could day, arenas(dishes + theexperiment were extensively being worms by day fed (white the morningofthelast liver On arrows). the dropsof 1-2 with small theATA in wormswere fed the 7,10, 1,4, (C&D)In themorning days “unfamiliarized” (control) group wasplaced in st days. “familiarized” (A) The group was in Petri dishes with arough textured bottom,while the Fig. 2. be employed. to feedback each individual animal allows very training and sophisticated to testing paradigms reinforcementnegative was notused these in experiments, butthe ability real-time toprovide providing changes of light or electric inshock response to specific worm positions. Such celleach determine the position ofeach worm andrecord it. The has device provisionsalso for duringtesting trials.behavior data coordinate are processed, allowing anobjective and analysisquantitative animal’s ofeach 2010). worms etal., All (Blackiston of12individual tracking andtraining simultaneous ofblocks 3isolated chambers. Each chambercontained 1 worm apetri in dish, allowing the 1 Fig. LEGENDS FIGURE . The Automated . The Training Apparatus (ATA). Experimental protocol (A) Apicture ofthe 12 fullychannel we automateddevice used. The device contained 4 Resting phase: (E)Resting After 10familiarization days,worms the were in kept 12multiwell for chambers 10consecutive theATA’s in Groups placed phase: ofworms were Training workflow Continuously basic (B) ofthedevice. loop The andindependently, in cameras

25 ed worms andplacedinseparatewells ofa12- arough textured floor and theunfamiliarized andard Petri dishes with smoothbottoms(B). familiar environment),familiar 4

The Journal of Experimental Biology – ACCEPTED AUTHOR MANUSCRIPT 959 958 957 956 955 954 953 952 951 950 949 948 947 946 945 944 943 942 941 940 939 938 937 936 935 934 933 932 931 930 929 928 927 926 Unfamiliarized, (Control) groups. Red line: are form thefamiliarizedpoints points are (Trained),groups, andtheleft from the tested, 10-14 days after decapitating, (Familia Fragments, Regular Protocol which are worms regenerated fragmentsandhave from been tail (time feeding minutes).in The same groupsas A,in including the category ofHeadless exact test). Fisher's p=0.013;one-tailed, (<8 minutes, than 8minutes 11-13been tested, days after with decapitating, theSaving paradigm, reach criterion in less (n=106, red and column) 64.5% ofthe unfamiliarized worms (n=104, black which column), have p=0.014; one-tailed, Fisher's exact test). (c) Saving paradigm: 79.5% of familiarized worms beentestedhave 12-15 days after reach training, criterion in than 8less (<8minutes minutes, wormsred oftheunfam column) (n=70, and67.1% p=0.005; one-tailed,minutes, Fisher's exact test). (b) Intact-14-days: 84.2% offamiliarized beentested whichcolumn), have 4days in after reachcriterion than8minutes training, (<8less familiarized worms (n=225, red column) and48% A. feeding: initiating 3. Fig. a beforewith making turnsharp theliver, and stain initiating toward theliver feeding. in this example, theworm will make more thanone,short, enters to the illuminated quadrant illuminated quadrant (Blue area) containing the liver st thewormsthe start(red around avoiding theedgeofchamber, arrow) are the moving mainly under theilluminatedthe liver quadrant. with the in rightposition thedish toplace enabled This is. where liver thearea marker tolabel reddashed circle). The black stain in the middle is made onthe outer side of thedish by ablack (O) the stain dishoflivercomparison. floor with(inside Enlargement ofthetesting thesmall Enlargement of therough textured bottomof theexperimental environment with worm for theworm’s(N) pharynx. Redarrow camera, from belowseen indicates thetracking by worm as (M) Panel feeding. shows the tobegin animal each measured took it howlong device the quadrant with theliver (openedstain lid ofthe ATA with the light setting during thetest). The (redliver arrow onthe point liver stain) and(L) astrong blue light was illuminating, from above with arough floor (the familiar environment). (J&K) Asmall area ofthedish was covered with familiarized worms and6unfamiliarizedworms we Percentage of worms to reach criterion in less than 8 minutes. (a) than8minutes. Intact-4-days: 60.4% of less in worms of toreachcriterion Percentage Worms in afamiliar in environment displayWorms significantly before phase shorter exploration Testing phase:Testing After theresting period, theretrieval test place. took recall,To 6 test

(P) exploration/foraging during thetest. Typical trail At 26 rized n=164, Unfamiliarized right n=171).The Intact-4-days (Familiarized 6.641±0.47; of theunfamiliarized of worms(n=229, black iliarized worms (n=70, blackcolumn), which re placedindividually ain (dashed red circle). In somecases, as in theATA chambers B. Median delay of delay of Median

The Journal of Experimental Biology – ACCEPTED AUTHOR MANUSCRIPT 973 972 971 970 969 968 967 966 965 964 963 962 961 960 atthepoint and betweendecapitated of theanterior the auricles side the pharynx (red arrows). panels) darknessworm (notwater thesame ofthe andconstant each in 18°C in 4 Fig. # Error bars show SEM. U-test).P=0.027; one-tailed, significance). Blue-line, Saving paradigm (Familiarized 7.166±0.58; Unfamiliarized 8.304±0.55, fragments, Regular Protocol (Familiarized 10.15±0. 5.012±0.49; Unfamilia Unfamiliarized 8. Criterion was minutes 3consecutive theilluminated in quadrant, containing theliver spot. . Decapitation andregeneration. Decapitation Illustration ofwormIllustration our in protocol regeneration sequence ofconditions 12 worms /2ml 341±0.48, P<0.001; one-tailed, U-test). Bl rized 6.991±0.41, P<0.001;one-tailed, U- test). Green line:Headless 27 7; Unfamiliarized 10.325±0.69, No statistical ack line: Intact-14-days (Familiarized . Worms were Worms

The Journal of Experimental Biology – ACCEPTED AUTHOR MANUSCRIPT The Journal of Experimental Biology – ACCEPTED AUTHOR MANUSCRIPT The Journal of Experimental Biology – ACCEPTED AUTHOR MANUSCRIPT The Journal of Experimental Biology – ACCEPTED AUTHOR MANUSCRIPT The Journal of Experimental Biology – ACCEPTED AUTHOR MANUSCRIPT **Saving Protocol: The feeding session before the test was taken place in the in the familiarization arena (ATA*Regular Protocol: The feeding session before thetestwas takenplace thein wormmulti plate wells (Fig. 2G) Legend: F = familiarized; C = controls (unfamiliarized) 2 Table 1 Table after decapitating tested 11-13 days Headless fragments **Savings Protocol after decapitating tested 10-14 days Headless fragments * training days after endof Intact: after end oftraining Intact:

11-13 days after decapitating Headless fragments Intact: Intact: Regular Protocol tested12-15 tested4 days Protocol : Latency Session ofFeedingDuringtheTesting : Motility Session DuringtheTesting tested 12-15 days after end of training 12-15 tested days after endof 4days after tested endoftraining chamber with theelectrode insert and the rough floor (fig. 2H). Reach Criteria 104/115 Unfamiliarized: 106/117 Familiarized: 164/199 Unfamiliarized: 171/201 Familiarized: 70/72 Unfamiliarized: 70/72 Familiarized: 229/238 Unfamiliarized: 225/233 Familiarized : Protocol Protocol tested tested paradigm): (saving / Tested

N

±0.58 8.532 4±0.7 12.93 ±0.49 5.932 ±0.47 8.817 reach criteria F Minutes to

(± s.e.m) Average Latency 12.603 10.339 ±0.55 9.545 ±0.69 ±0.41 7.326 ±0.48 C

±0.58 7.166 10.15 ±0.49 5.012 ±0.47 6.641 ±0.7 ±0.7 reach criteria F Minutes to

(± s.e.m) Latency Median Median Familiarized: Unfamiliarized: 8.102±0.33 8.859±0.27 8.775±0.2 8.818±0.2 7.34±0.24 7.858±0.25 10.325 8.304± ±0.69 ±0.41 6.991 ±0.48 8.341 0.55 C

Average Pixels/Second

significance No statistical P = 0.027 P < 0.001 P < 0.001 Movement Rate Rate Movement (One tailed) (± s.e.m) U-test Statistical Significance

(Onetailed) test (n<8min) Fisher's exact significance No statistical P=0.013 P=0.014 P=0.005