Current iology 19, 1654 1659, Octoer 13, 2009 ª2009 Elsevier Ltd ll rights reserved DOI 116/j.cu.2009.08.017 Socil Fcilittion of Long-Lsting Memory Retrievl in Drosophil Report Mrie-nge Chud, 1,2,5 Guillume Isel, 1,5 Lure Kiser, 2,3,4 nd Thoms Pret 1, * 1 Genes nd Dynmics of Memory Systems, Neuroiology Unit, Centre Ntionl de l Recherche Scientifique, École Supérieure de Physique et de Chimie Industrielles, 10 rue Vuquelin, 75005 Pris, Frnce 2 Développement, Evolution, et Plsticité du Système Nerveux, Centre Ntionl de l Recherche Scientifique, 1 venue de l Terrsse, 91198 Gif-sur-Yvette Cedex, Frnce 3 Institut Ntionl de l Recherche gronomique Centre de Versilles-Grignon, UMR 1272, Physiologie de l Insecte Signlistion et Communiction, Route de St Cyr, 78026 Versilles Cedex, Frnce Summry Recent studies demonstrte tht socil interctions cn hve profound influence on Drosophil melnogster ehvior [1 8] nd cuticulr pheromone ptterns [8 10]. Olfctory memory performnce hs mostly een investigted in groups, nd previous studies hve reported tht grouped flies do not interct with ech other nd ehve in the sme wy s individul flies during short-term memory retrievl [11 13]. However, the influence of socil effects on the two known forms of Drosophil long-lsting ssocitive memory, nesthesi-resistnt memory (RM) nd longterm memory (LTM), hs never een reported. We show here tht RM is displyed y individul flies ut is socilly fcilitted; flies trined for RM interct within group to improve their conditioned performnce. In contrst, testing shows LTM improvement in individul flies rther thn in group. We show tht the socil fcilittion of RM during ing is independent of the socil context of trining nd does not involve nonspecific ggregtion. Furthermore, we demonstrte tht socil interctions fcilitte RM retrievl. We lso show tht socil interctions necessry for this fcilittion re specificlly generted y trined flies: when single flies trined for RM re mixed with groups of nive flies, they disply poor retrievl, wheres mixing with groups trined either for RM or LTM enhnces performnce. Results nd Discussion RM Is Fcilitted in Group Tests Independently of the Lerning Context We use n olfctory conditioning prdigm during which first odor ssocited with electric shocks is followed y second *Correspondence: thoms.pret@espci.fr 4 Present ddress: Institut de Recherche pour l Développement, UR 072, Lortoire Evolution, Génomes, et Spécition, UPR 9034, Centre Ntionl de l Recherche Scientifique, 1 venue de l Terrsse, 91198 Gif-sur-Yvette Cedex, Frnce 5 These uthors contriuted eqully to the work. odor presented lone [11, 14]. Memory performnce is normlly mesured on groups of flies in T-mze, in which flies re llowed to choose etween oth odors for given time [11]. Two forms of consolidted long-lsting memory cn e otined y repetition of lerning trils: long-term memory (LTM) is formed fter spced conditioning, wheres mssed conditioning leds only to the formtion of nesthesi-resistnt memory (RM) [15, 16]. LTM is ffected y prtil protein-synthesis inhiition nd thus depends on de novo protein synthesis, wheres RM is insensitive to the sme level of inhiition [15]. Two models hve een proposed to explin the reltionship etween LTM nd RM. In the first, oth forms of memory coexist fter spced conditioning [15, 17]. In the second model, RM nd LTM re exclusive, nd only LTM is expressed fter spced conditioning [16]. For convenience, the memory formed fter mssed conditioning will e referred to s RM, nd the memory formed fter spced conditioning will e referred to s LTM, lthough the existence of the two models will e discussed when pproprite. We studied the influence of group interctions on RM y compring individul nd group performnce during the test. Flies were mssed trined in groups of 30 40 flies nd tested either individully or in groups 24 hr or 48 hr fter trining. RM scores of groups outstrip individul scores (Figure 1; two-wy NOV test, significnt socility fctor, p = 5.9 10 25 ) when flies were tested t oth 24 hr nd 48 hr (nonsignificnt time fctor: p = 0.089. Nonsignificnt time 3 socility interction: p = 0.86). Fly interctions in group therefore hve positive effect on RM performnce. Strikingly, t 48 hr individul RM scores were not different from zero, wheres group scores remined high (Figure 1; t test, p > ). In this first set of experiments, flies were mssed trined in group, nd so it is plusile tht the higher memory score displyed y flies tested in groups ws due to similr socil setting during trining nd testing. We therefore serched for potentil context effects occurring during mssed conditioning y compring individul nd group trining. The group enhncement of RM performnce occurred similrly fter individul nd group trining, indicting tht it is independent of the lerning context nd rther involves socil interctions during the test (Figure 1; two-wy NOV, test context fctor, p = 05; lerning context fctor: p = 0.25; test 3 lerning interction, p = 0.73). Moreover, the socil effect on RM ws not sex specific nd did not rely on sexul interctions (Figure S1). We then serched for similr socil effect on LTM performnce. No positive group effect ws induced fter spced conditioning; performnce ws higher in individul flies thn in groups (Figure 1C; two-wy NOV test, significnt socility fctor, p = 0.029; nonsignificnt time fctor, p = 0.079; nonsignificnt time 3 socility interction, p = 0.24). ecuse the time fctor ws mrginl, we nlyzed 24 hr nd 48 hr testing times sepertely, nd it ppered tht the difference etween scores of individul nd group flies ws mrginl t 24 hr nd significnt t 48 hr (t test, 24 hr: p = 0.052. 48 hr: p = 0.043). Thus, socil interctions incresed memory retrievl fter mssed ut not fter spced conditioning, supporting the view tht
Socil Fcilittion of Memory Retrievl 1655 C 0.70 Group trining 5x spced: 24 hr 5x mssed: 48 hr Individul trining 5x spced: 48 hr Figure 1. RM Is Socilly Fcilitted in Group Tests Independently of the Lerning Context () Twenty-four hour nd 48 hr olfctory memory induced y the mssed conditioning protocol (RM) of groups of flies, tested s individuls (n = 120) or in groups (n = 10 15). RM scores of groups outstrip the level of individul scores (p = 5.9 10 25 ). () Twenty-four hour RM scores, mesured in individul flies (n = 120 individuls) or in groups of flies (n = 10). Flies were either trined in groups (left) or trined individully (right). The group enhncement of RM performnce occurred similrly fter individul or group trining (p = 05). (C) Twenty-four hour nd 48 hr olfctory memory induced y the spced conditioning protocol (LTM) of groups of flies, tested s individuls (n = 100 120) or in groups (n = 10 18). Performnce fter spced conditioning ws higher in individul flies thn in groups (p = 0.029). In ll pnels, dt correspond to the men 6 SEM of the memory score. LTM ut not RM is present fter spced conditioning [16]. lterntively, if the memory otined fter spced conditioning is composed of oth LTM nd RM, s is often proposed [15, 17], it could men tht the ehviorl expression of LTM is predominnt over tht of RM ecuse it is retrieved more efficiently. fter mking these initil oservtions, we scrutinized the ehviorl mechnisms underlying the socil improvement of RM. The Positive Group Effect on RM Is Due to Socil Fcilittion of RM Retrievl, Not to ggregtion The socil effect tht tkes plce during RM retrievl could e explined y three min hypotheses. In the first, the socil phenomenon would not e directly relted to memory, nd during the test, flies trined with mssed conditioning would simply tend to ggregte in the tue where flies re most undnt. In the second hypothesis, the socil phenomenon would involve communiction out odor qulity linked to conditioning: during the test, flies trined with mssed conditioning would send signl(s) telling their neighors tht they should either void the conditioned odor or orient to the unconditioned odor (the perception of this signl could e restricted or not to flies trined with mssed conditioning). In the third hypothesis, socil interctions would fcilitte memory retrievl. To discriminte etween these hypotheses, we tested the performnce of single flies conditioned with the mssed protocol to void one odor nd mixed them with groups conditioned with the sme protocol to void the opposite odor. If socil interctions were linked to nonspecific ggregtion or odor-specific communiction, we would expect decresed score for individul mssed-trined flies when they re mixed with group trined for the opposite odor (s compred to mssed-trined flies tested lone). However, if socil interctions fcilitte RM retrievl, these single flies should hve high scores, similr to those of their mixed groups (ut specific for their respective odor). Interestingly, individul mssed-trined flies tested mong group of mssed-trined flies conditioned to the opposite odor reclled their own conditioned odor etter thn when they were tested individully; they showed score superior to mssed-trined flies tested individully nd similr to tht of mssed-trined flies tested in group (Figure 2; one-wy NOV test, p = 3). The fct tht group interctions increse the RM performnce of single flies even if the group is trined for the opposite odor indictes tht socil interctions ct on RM retrievl nd do not involve ggregtion. This conclusion ws strengthened y the oservtion tht the performnce of groups of mssed-trined flies ws not ffected during the test y the presence of groups trined for the opposite odor with either the spced (Figure 2) or the mssed (Figure 2C) protocol. Only Flies with RM re Influenced y Socil Interctions during Memory Retrievl If groups of flies trined with the mssed protocol fcilitte RM retrievl, they should hve no influence on flies tht hve no RM. To chllenge this hypothesis, we used two groups of flies, nive wild-type flies nd mssed-trined rdish (rsh) mutnts tht re deficient for RM [15]. When mixed with group of mssed-trined flies, nive flies displyed score not different from zero (Figure 3; t test, p = 0.55), showing tht the group effect produced y mssed-trined flies requires the presence of memory. Similrly, rsh flies trined for RM nd mixed with group of wild-type flies trined for the sme odor displyed low score similr to tht of control rsh flies (Figure 3; t test, p = 0.55). The performnce of rsh
Current iology Vol 19 No 19 1656 C ed in mssedtrined group (opposite odor) 5x spced: 24 hr group mixed group group (30 flies) group (60 flies) mixed groups, trined to void sme odor mixed groups, trined to void opposite odors Figure 2. The Positive Group Effect on RM Is Due to Socil Fcilittion of Memory Retrievl () Oserved 24 hr RM scores mesured in individul flies (n = 192 individuls), in individul flies tested mong groups of mssed-trined flies conditioned to void the opposite odor (n = 192 individuls), nd in groups of flies (n = 32). Dt represent the men 6 SEM of the memory score. One-wy NOV followed y Tukey sttistic: lowercse letters indicte significnt differences t p < 0.05. Groups of mssed-trined flies in which individul flies were introduced hd scores of 0.43 6 0.02, similr to those of control groups (t test with Dunn-Sidk correction, p =, n = 96). () Oserved 24 hr RM nd LTM scores when oth groups were mixed t testing (n = 13 mixed groups) re compred with scores of nonmixed groups of mssed-trined (n = 16 groups) nd spced-trined (n = 11 groups) flies. Scores of mssed- nd spced-trined flies were not influenced y the mixing (t test; p = 0.34 for RM nd p = 0.61 for LTM). (C) Oserved 24 hr RM scores when oth groups of mssed-trined flies were conditioned to void opposite odors nd mixed t testing (n = 16) re compred with scores of mixed groups of mssed-trined flies conditioned to void the sme odor (n = 16) nd with scores of groups of 30 nd groups of 60 mssed-trined flies (n = 12). Scores of mssed-trined flies were not influenced y mixing with flies trined for the opposite odor (one-wy NOV test, p = 0.76). For ll pnels, dt represent the men 6 SEM of the memory score. flies ws therefore not enhnced y socil interctions with trined wild-type flies. Only flies tht disply RM re positively influenced y mssed-trined flies during the memory test. These results re in greement with the view tht socil interctions enhnce RM performnce y fcilitting memory retrievl. The Socil Fcilittion of RM Retrievl Requires Interctions with Trined Flies Which types of interctions re t the origin of the socil fcilittion of RM retrievl? In prticulr, re these interctions specific to flies trined with the mssed conditioning protocol, or does the simple fct of eing in group fcilitte RM retrievl? We first investigted this issue y testing single mssed-trined flies mixed during the test with groups of nive flies. Groups of nive flies did not fcilitte RM retrievl; individul mssed-trined flies mixed with nive groups showed poor performnce, similr to tht of mssed-trined flies tested individully, nd oth showed significntly lower scores thn mssed-trined flies tested in groups (Figure 4; one-wy NOV test, p = 3). In second step, we mixed single mssed-trined flies with groups of mssed-trined flies or of spced-trined flies conditioned for the sme odor. Interestingly single mssedtrined flies mixed with either mssed- or spced-trined flies showed score significntly higher thn tht of mssedtrined flies tested individully, nd similr to the score of mssed-trined flies tested in groups (Figures 4 nd 4C; one-wy NOV test, respectively p = 0.022 nd p = 8 for the mixing in mssed-trined group nd in spcedtrined group). These results show tht the simple fct of eing in group is not enough to produce socil fcilittion of RM retrievl nd tht specific interctions with trined flies re necessry. The socil interctions t the origin of this enhnced memory retrievl re not specific to mssed-trined flies; they re lso oserved with spced-trined groups. This could e due to the fct tht RM is present fter oth mssed nd spced conditioning [15, 17]. lterntively, only LTM might e present fter spced conditioning [16], ut groups of oth mssed- nd spced-trined flies might shre common feture tht is perceived y mssed-trined flies in the test sitution. For exmple, in the presence of the conditioned odor, oth trined groups might produce n lrm molecule tht fcilittes RM retrievl y enhncing ttention or motivtion. LTM-trined flies my perceive the lrm ut experience no visile effect on their performnce, possily ecuse their memory retrievl is lredy very efficient. y investigting memory performnce in different socil situtions of trining nd testing, we hve shown tht the memory performnce of group does not lwys reflect individul performnce. Previous studies did not report differences in short-term memory performnce etween flies tested individully nd those tested in groups, in either versive or ppetitive olfctory memory [12, 13]. We hve demonstrted tht long-lsting versive memory performnces displyed y groups cn outstrip performnces of individuls: RM performnce tested individully is lower thn performnce tested in groups. This effect is strong; the conditioned response of odor voidnce ws no longer expressed in individuls t 48 hr ut ws still clerly detected in groups. Drosophil is known to e ggregtive [1, 2]. One could therefore imgine tht mssed-trined flies hve wek individul memory nd ggregte during the test, either pssively
Socil Fcilittion of Memory Retrievl 1657 NS NS control group mixed group wild-type rsh - nïve Figure 3. Nive Wild-Type Flies nd rdish (rsh) Mutnts with Deficient RM re Not Influenced y Flies Trined with Mssed Conditioning () Oserved 24 hr memory scores of mssed-trined flies nd nive flies when oth groups were mixed t testing (n = 12 mixed groups). Nive flies displyed score not different from zero (p = 0.55). () Oserved 24 hr RM scores for wild-type nd rsh flies when oth groups were mixed t testing (n = 13 mixed groups) re compred to RM scores of wild-type groups nd rsh groups lone (n = 13). The performnce of rsh flies ws not enhnced y socil interctions with mssed-trined wild-type flies (p = 0.55). In ll pnels, dt represent the men 6 SEM of the memory score. NS indictes nonsignificnt difference. or y communicting signls specific to the conditioned or unconditioned odor. On the contrry, our results indicte tht the socil effect during the test is due to fcilittion of memory retrievl. The poor RM performnce of individuls does not reflect poor lerning or memory loss ut rther reflects memory-retrievl deficit tht occurs in solitry flies nd tht cn e compensted y socil interctions within group. lthough the moleculr nd cellulr mechnisms t ply in this process remin to e determined, n ttrctive hypothesis is tht, during memory retrievl, trined flies produce stress signls tht lrm their mtes nd enhnce their mtes ttention or motivtion to respond. Stressed flies hve een shown to relese repellnt odornts tht include CO 2 [18], nd it will e importnt to test whether this prticulr signl plys role in mediting socil fcilittion of RM. ny puttive lrm molecule secreted y trined flies during the test (i.e., in the presence of the conditioned odor) would represent signl out the existence of dngerous environment without specifying the nture of the dnger. In tht context, it will e interesting to investigte whether the socil effect is only oserved fter versive conditioning nd not fter ppetitive conditioning. Socil informtion shring, used y wide rnge of species, is n dvntgeous solution in which nimls dpt their ehvior to the environment y extrcting knowledge from neighors [19 24]. This phenomenon is not restricted to vertertes or colonil insects; for instnce, the wood cricket uses socil informtion to dpt its predtor-voidnce ehvior [21]. The ese with which influences of the socil environment on ehvior cn e quntified, nd the vriety of powerful genetic tools ville, mke Drosophil n idel model for future studies of socility, lerning, nd memory [24]. Experimentl Procedures Conditioning Procedure Drosophil melnogster wild-type strin Cnton-Specil (CS) nd rdish (rsh) mutnt flies were rised t 18 C nd 60% humidity in 12:12 hr light: drk cycle. Flies were trined with clssicl olfctory versive conditioning protocols s descried [14] except tht for repeted (mssed or spced) conditioning, five cycles were used insted of ten. These experimentl conditions promote stronger RM performnce (Figure S2) thn tht previously reported [14, 15]. Conditioning ws performed on smples of 30 40 flies ged etween 2 nd 3 dys. In the cse of individully trined Drosophil, flies were gthered into groups of 35 flies directly fter trining. Memory Test Memory tests were performed t 25 C nd 80% reltive humidity under dim red light. For group memory tests, flies were tested in the T-mze pprtus for 3 min. men group memory score nd its stndrd error were then clculted from ten to 18 groups. memory score represents the normlized proility of correct choice. For individul memory tests, single flies were collected without nesthesi from groups just efore the test nd introduced lone into the T-mze [11] to choose etween octnol or methylcyclohexnol odor over 3 min period. t the end of the test period, the presence of the fly in one odor comprtment or the other ws recorded. To compre the memory scores of individuls nd groups, we first pooled 12 consecutive individul results (six flies conditioned with ech odor) to clculte memory score similr in essence to tht of group score [12, 14]. men individul memory score nd its stndrd error were then clculted from eight to 16 pools. In the cse of tests with mixed groups, we needed to differentite etween two popultions of flies. To do this, we mrked flies of one of the two groups y clipping the tips of their wings under rief CO 2 nesthesi 24 hr efore trining. We checked tht flies with clipped wings displyed norml scores fter mssed nd spced conditioning. Mixed groups ech consisted of 30 flies either trined for RM nd LTM with opposite odors (Figure 2) or oth trined for RM with opposite odors (Figure 2C); groups were mixed just efore testing. In the experiment with mixed groups of nive or rsh flies nd CS mssed-trined flies (Figure 3), pproximtely 12 nive or rsh flies were mixed with the group of out 30 mssed-trined flies just fter conditioning. Control groups consisted of 30 rsh or CS mssed-trined flies. In experiments testing single mssed-trined flies mixed in groups of out 30 flies (nive, mssed-trined, or spced-trined flies) (Figure 4), we mrked single flies nd introduced them into groups just efore testing. We clculted scores of these single flies s for single flies tested lone. Sttisticl nlyses Men scores were compred vi two-tiled t tests in the cse of two groups (Figures 2, 2, 3, nd 4; see lso Figure S2) or one-wy NOV in the cse of severl groups. This ws followed y the post-hoc Tukey-Krmer
Current iology Vol 19 No 19 1658 ed in group of nïve flies ed in mssed-trined group test if results were significnt (Figures 2 nd 4). Men scores of Figures 1 nd 2CndFigure S1 were nlyzed y two-wy NOV including clcultion of the interction etween oth considered fctors (GLM procedure of the softwre R ); susequently, the post-hoc Tukey-Krmer test ws used for ssessing significnce in the sme fctor mong more thn two groups (Figure S1). The individul 48 hr RM score of Figure 1 nd the scores of nive flies (Figures 3 nd 4) were tested for significnce with t test for single men ginst zero [25]. For ll sttisticl nlyses, we used the significnce level lph = 0.05, except when we used dt in two comprisons, where the significnce level lph = 0.025 ws used, fter ppliction of the Dunn-Sidk correction [25]. Supplementl Dt Supplementl Dt include two figures nd cn e found with this rticle online t http://www.cell.com/current-iology/supplementl/s0960-9822(09)01589-9. cknowledgments We thnk Ydin Dudi nd memers of our lortory for helpful discussions. We thnk ntoine rnc for help with R. We thnk Vlérie Goguel nd Niki Scplehorn for their vlule comments on the mnuscript. This work ws supported y the gence Ntionle pour l Recherche, the Fondtion ettencourt-schueller, nd the Fondtion pour l Recherche Médicle (to T.P.). M.-.C. ws supported y grnt from the Université Pris 13, Ecole Doctorle Vivnt et Sociétés (Ministry of Reserch). G.I. ws supported y the Fondtion pour l Recherche Médicle. Received: June 18, 2008 Revised: ugust 1, 2009 ccepted: ugust 3, 2009 Pulished online: Septemer 24, 2009 C ed in spced-trined group Figure 4. The Socil Fcilittion of RM Retrievl Requires Interctions with Trined Flies () Oserved 24 hr RM scores, mesured in individul flies (n = 96 individuls), in individul flies tested mong groups of nive flies (n = 96 individuls), or in groups of flies (n = 16). Groups of nive flies did not fcilitte RM retrievl. We verified tht groups of nive flies hd score not different from zero when mssed-trined fly ws introduced (score = 24 6 0.020; t test, p = 0.85, n = 64). () Oserved 24 hr RM scores, mesured in individul flies (n = 120 individuls), in individul flies tested mong groups of mssed-trined flies conditioned to void the sme odor (n = 120 individuls), nd in groups of flies (n = 20). Groups of mssed-trined flies fcilitted RM retrievl. Groups of mssed-trined flies in which single mssed-trined flies were introduced hd norml scores (RM score = 0.37 6 0.02, not different from the control group score; t test with Dunn-Sidk correction, p = 0.21, n = 60). References 1. Lefrnc,., Jeune,., Thoms-Orillrd, M., nd Dnchin, E. (2001). Non-independence of individuls in popultion of Drosophil melnogster: Effects of sptil distriution nd dispersl. C. R. cd. Sci. III 324, 219 227. 2. Tinette, S., Zhng, L., nd Roichon,. (2004). Coopertion etween Drosophil flies in serching ehvior. Genes rin ehv. 3, 39 50. 3. Gnguly-Fitzgerld, I., Donle, J., nd Shw, P.J. (2006). Wking experience ffects sleep need in Drosophil. Science 313, 1775 1781. 4. Yurkovic,., Wng, O., su,.c., nd Krvitz, E.. (2006). Lerning nd memory ssocited with ggression in Drosophil melnogster. Proc. Ntl. cd. Sci. US 103, 17519 17524. 5. Levine, J.D., Funes, P., Dowse, H.., nd Hll, J.C. (2002). Resetting the circdin clock y socil experience in Drosophil melnogster. Science 298, 2010 2012. 6. Fujii, S., Krishnn, P., Hrdin, P., nd mrein, H. (2007). Nocturnl mle sex drive in Drosophil. Curr. iol. 17, 244 251. 7. Mery, F., Vrel, S..M., Dnchin, E., lnchet, S., Prejo, D., Coolen, I., nd Wgner, R.H. (2009). Pulic versus personl informtion for mte copying in n inverterte. Curr. iol. 19, 730 734. 8. Krupp, J.J., Kent, C., illeter, J.C., znchi, R., So,.K.C., Schonfeld, J.., Smith,.P., Lucs, C., nd Levine, J.D. (2008). Socil experience modifies pheromone expression nd mting ehviour in mle Drosophil melnogster. Curr. iol. 18, 1373 1383. 9. Kent, C., znchi, R., Smith,., Formos,., nd Levine, J.D. (2008). Socil context influences chemicl communiction in D. melnogster mles. Curr. iol. 18, 1384 1389. (C) Oserved 24 hr RM scores, mesured in individul flies (n = 144 individuls), in individul flies tested when mixed with groups of spcedtrined flies (n = 144 individuls), or in groups of flies (n = 28). Groups of spced-trined flies fcilitted RM retrievl. We checked tht groups of spced-trined flies in which single mssed-trined flies were introduced hd scores tht would normlly e expected fter spced conditioning (LTM score = 0.39 6 0.02, n = 84). Dt represent the men 6 SEM of the memory score. One-wy NOV followed y Tukey sttistic: lowercse letters indicte significnt differences t p < 0.05.
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