Proc. Ntl. Acd. Sci. USA Vol. 73, No. 5, pp. 1684-1688, My 1976 Genetics dunce, mutnt of Drosophil deficient in lerning (conditioned behvior/olfctory discrimintion/behvior genetics) YADIN DUDAI*, YUH-NUNG JAN*, DUNCAN BYERS*, WILLIAM G. QUINNt, AND SEYMOUR BENZER* * Division of Biology, Cliforni Institute of Technology, Psden, Clif. 91125; nd t Deprtment of Biology, Princeton University, Princeton, New Jersey 854 Contributed by Seymour Benzer, Februry 13, 1976 ABSTRACT Norml Drosophil lern to void n odornt ssocited with electric shock. An X-linked mutnt, dunce, hs been isolted tht fils to disply this lerning, in spite of being ble to sense the odornt nd electric shock nd showing essentilly norml behvior in other respects. The mechnism of lerning presumbly involves ltered interctions mong neurons, bsed on moleculr events. Assuming tht the biochemicl pprtus responsible for plsticity is specified by the genes, one might isolte mutnts deficient in lerning. This could led to dissection of the lerning process. In Drosophil, the method of mosic nlysis (1) could permit one to pinpoint the criticl ntomicl foci which, when ltered by muttion, cuse lerning deficiency. Quinn, Hrris, nd Benzer (2) demonstrted lerning in Drosophil using n olfctory discrimintion prdigm. Pseudoconditioning, hbitution, nd odor preference were excluded s explntions. We report here the isoltion nd chrcteriztion of mutnt tht ppers to hve norml sensory nd motor mechnisms, yet is unble to lern in this prdigm. MATERIALS AND METHODS Flies nd Mutgenesis. Norml D. melnogster of the Cnton-Specil (C-S) wild-type strin were mintined on cornmel medium (3). Mutgenesis by ethylmethnesulfonte ws ccording to Lewis nd Bcher (4). Treted mles were mted to virgin, ttched-x femles, so tht ech F1 progeny mle crried treted X-chromosome received from his fther. Ech individul F1 mle ws mted to ttched-x femles, producing stock in which the mles crried identicl, potentilly mutnt, X-chromosomes. To screen for muttions ffecting lerning bility, we tested ech such popultion of mles. The Lerning Test. The prdigm ws similr to tht of Quinn et l. (2). The pprtus (Fig. l) consisted of two sliding Plexigls holders ccepting plstic test tubes (Flcon no. 217, 17 X 1 mm). Tube 1 ws "rest tube" with bout 15 holes t the fr end, mde with hot needle. Tubes 2 through 5 contined copper grids etched on flexible epoxy bcking (Fig. lb), rolled up to fit inside the tubes with connecting tbs folded out. Odornts were spred over the grids in.2 ml of solution in bsolute ethnol, llowing 5 min for evportion. The shock voltge ws 9 V c (6 Hz). Between experiments, grids were clened in ethnol nd the pprtus ws wshed with sop nd wter. Before testing, mles were seprted from femles in nitrogen tmosphere, which immobilizes flies but does not ffect subsequent lerning (5). For trining, bout 4 flies were plced in the strt tube nd the pprtus ws lid horizontlly, with tubes 1 through 5 pointing t horizontl 15-W dylight fluorescent lmp (Westinghouse F15T8/D). After the flies hd explored the rest tube for bout 1 min, the pprtus ws held verticlly nd tpped shrply on rubber mt to bring the flies to the bottom of the strt tube. The holders were slid to set the strt tube in register with tube 2 nd the pprtus ws replced on the tble. The flies, strongly phototctic, rn towrd the light nd were shocked in the presence of odornt A. After 3 sec, the flies were shken bck into the strt tube, nd the slide shifted so tht the flies could run into the rest tube gin for 3 sec. Next ws similr run for 3 sec into tube 3, contining odornt B but no shock, followed by 3 sec rest. The complete sequence ws repeted twice, thus totling three trining trils with ech odornt. To test for lerning, we then shifted the flies to tube 4, contining odornt A on fresh grid (but with no voltge pplied). After 15 sec, the number of flies in the strt tube ws counted. After 3 sec rest, the flies were tested ginst tube 5, contining odornt B (fresh grid, no voltge), nd the number of flies in the strt tube t 15 sec ws recorded. Selective voidnce of the shock-ssocited odornt indicted lerning. To control for odor bis, we performed reciprocl experiment with fresh popultion of flies, in the sme pprtus, with the voltge trnsferred from tube 2 to tube 3. For ech hlf-experiment, the frction of flies voiding the shock-ssocited odornt minus the frction of flies voiding the control odornt ws determined. The lerning index A ws defined s the verge of the two vlues. A = 1 represents perfect lerning; A = indictes no lerning. Behviorl Tests. The bility of flies to sense the odornts on the grids ws mesured in the choice-chmber pprtus of Fig. 3. A new group of flies ws used for ech test. Tubes 1 nd 2 contined grids, one coted with.2 ml of n odornt in ethnol, the other being n ethnol blnk. As in the lerning experiments, the ethnol ws llowed to evporte beforehnd. Ech grid ws used for two runs. About 6 flies were introduced into tube nd shken into the sliding comprtment. The pprtus ws then lid horizontlly with tubes 1 nd 2 prllel to fluorescent lmp (t 6 cm distnce) to equlize the light intensity in both rms. The sliding comprtment ws then shifted into register with tubes 1 nd 2. After 3 sec, the slide ws shifted wy nd the number of flies trpped in ech tube (nd the smll number remining in the chmber) ws counted. Phototxis nd geotxis were mesured by countercurrent distribution (6). Flying bility ws mesured s described by Benzer (7). Spontneous locomotor ctivity ws mesured by the totl distnce wlked s function of time in long glss tube of 8 mm inside dimeter (8). All lerning nd other behviorl experiments were crried out t 2-23O. Chemicls. 3-Octnol, 4-methylcyclohexnol, nd gerniol were from K&K Lbortories (Plinview, N.Y.). Benzldehyde, myl cette, nd menthol were from Mllinckrodt Chemicl Works (St. Louis, Mo.). Cproic cid ws from Ntionl Biochemicls Corp. (Clevelnd, Ohio), nd steric cid nd quinine sulfte were from Mtheson, Colemn nd Bell (Norwood, Ohio). 1684
Genetics: Dudi et l. fluorescent lmp rest tube 3 4 5 7flies II strt tube b FIG. 1. Apprtus used for trining nd testing flies. () Two plstic blocks holding tubes slide on dovetil joint, so tht the strt tube cn be shifted into register with tubes 1 through 5. Tube 1, the rest tube, is perforted for ventiltion. Tubes 2 through 5 contin grids with odornts A or B. Tubes 2 nd 3 re used for trining. The lightning bolt indictes voltge on the grid. Tubes 4 nd 5 re for testing. (b) Printed-circuit grid for shocking flies. The brs of the grid re.5 or 1 mm wide nd re seprted by.5 mm. The grid is rolled up nd inserted into tube, which is plugged into the pprtus. Conductive tbs for pplying voltge re bent round the tube rim to the outside. RESULTS Isoltion of dunce. Norml flies, during trining, void the shock-odornt combintion, but show much smller voidnce of the control odornt (Fig. 2). On testing, they still preferentilly void the odornt tht hd been ssocited with shock, yielding lerning index A of.31 (SEM i.2, n = 18). The following criteri were chosen for lerning-deficient mutnt: (i) during testing, the flies fil to void selectively the shock-ssocited odornt; (ii) the bnorml performnce cnnot be scribed to sensory or motor defects. Approximtely 5 mutgenized X-chromosome lines were tested, using 3-octnol nd 4-methylcyclohexnol s odornts A nd B. About 2 of these lines met the first criterion; of these, only one lso stisfied the second. The strin ws mde homozygous, nd nmed dunce * (dnc). Behvior of dunce in the Lerning Prdigm. Fig. 2 shows results of lerning tests of the mutnt strin compred with norml flies. During the three trining trils, the mutnt flies show norml phototxis nd re deterred by the chrged grid. When subsequently tested, however, they do not selectively void the shock-ssocited odornt. To determine whether the deficiency in performnce of dunce is specific to the odornts used in screening, vrious other odornts were tested, including lcohols, cids, ldehydes, nd esters. The results re shown in Tble 1. All the combintions listed re effective lerning cues for norml flies, but not for dunce. Differentreinforcements were tried. Norml flies showed lerning for voltges rnging from 2 to 14 V; dunce showed very little. Quinine sulfte powder, dusted onto grid, cn substitute for shock s negtive reinforcement for norml flies (2) (A =.21, Tble 1). The lerning bility of dunce is deficient with this reinforcement (A =.5, Tble 1). Term of derision pplied to followers of John Duns Scotus, theologin, d. 138, regrded by 16th century humnists s n enemy of lerning. Tble 1. Proc. Ntl. Acd. Sci. USA 73 (1976) 1685 Lerning performnce of norml nd dunce flies Lerning index (A) Odornts used Norml flies dunce.5% 4-Methylcyclohexnol.5% 3-octnol.31 ±.2 (18).4 ±.2 (17).25% 3-Octnol blnk.28 ±.6 (4) -.2 ±.3 (4).5% 3-Octnol blnk.48 ±.2 (4).1 ±.1 (4) 1.% 4-Methylcyclohexnol blnk.28 ±.3 (3).8 ±.1 (3).25% Menthol blnk.24 ±.3 (4).5 ±.4 (3).5% Gerniol blnk.24 ±.6 (4).5 ±.2 (4).5% 4-Methylcyclohexnol 1.% steric cid.29 ±.5 (3).1 ±.2 (3).25% Cproic cid blnk.29 ±.5 (3).4 ±.7 (3).5% Benzldehyde blnk.28 ±.4 (3).6 ±.4 (3).1% Benzldehyde.5% myl cette.3 ±.3 (4).3 ±.8 (4).5% 4-Methylcyclohexnol.5% 3-octnol, with quinine s negtive reinforcement.21 ±.4 (3).5 ±.4 (3) Ethnol ws used s blnk. Vlues re men 4 SEM with the numbers of experiments in prentheses. Ability of the Mutnt to Sense the Odornts. There is tendency of flies to void some of the odornts used. In the lerning prdigm, this is lrgely overcome by the strong phototctic drive. The choice-chmber pprtus of Fig. 3 ws designed to use this voidnce s test of the bility of flies to sense odornts. The frction of flies entering the odornt tube is plotted in Fig. 4 for vrious substnces. It ppers tht dunce cn sense the odornts. In order to find out whether the poor performnce of dunce could be due to phototctic drive so strong s to override ny lerned voidnce, flies trined in the stndrd prdigm were tested in the choice-chmber pprtus, using the control odornt in one tube nd the shock-ssocited odornt in the other, the light intensity being equl in both tubes. Norml flies selectively void the shock-ssocited odornt; dunce flies gin show poor lerning. In this experiment, the lerning index is defined s the frction of flies present in the control odornt tube minus the frction in the shock-ssocited odornt tube t 3 sec. The vlues obtined were.33 (SEM i.3, n 7) for norml flies nd.13 (SEM ±.5, n = 7) for dunce. Other Tests on dunce. The externl morphology of dunce flies is norml. The vibility of dunce eggs, lrve, nd pupe is norml, nd the dults hve norml life spn. The mutnt hs essentilly norml phototxis, geotxis, flight, locomotor ctivity, nd sexul courtship. In crowded culture bottles, dunce flies do seem to become somewht weker nd more sluggish thn norml. The electroretinogrm (9) is norml. Synptic
1686 Genetics: Dudi et l. Proc. Ntl. Acd. Sci. USA 73 (1976) -o - U1). E (n -(11 C - C).5K.4.61-.3-.2 F.1k norml dunce shock odornt shock odornt -- 1 2 3 Te st 1 2 3 Test Trining trils Trining trils FIG. 2. Behvior of norml nd dunce flies during trining nd testing. Frction of flies in strt tube fter 15 sec is shown for the three trining trils nd for the test. Ech point represents the men : SEM for 11 experiments;.5% 3-octnol nd.5% 4-methylcyclohexnol served s odornts. The lerning index, A, is the frction of flies voiding the shock-ssocited odornt during the test minus the frction voiding the control odornt during the test. ro) control odornt control odornt loc rest tube rest tube trnsmission t the neuromusculr junction in the lrv, including fcilittion (1), is norml. To test for possible difference in the electricl conductivity of the dult cuticle which could ffect sensitivity to shock, we mde mesurements of the current pssing through single flies stepping cross the grid lines, using n pplied dc voltge in series with n oscilloscope s detector. Both norml nd dunce gve similr deflections, corresponding to leg-to-leg resistnce of the order of 19 ohms. The effect of prolonged shock on the flies ws ssessed by mesuring their subsequent phototxis. Flies were forced into tube contining grid nd shocked with 9 V c for 6 sec. Fifteen seconds lter, they were tested for phototxis by countercurrent distribution. Both norml nd dunce flies showed similrly reduced phototxis, the probbility of response per tril being decresed from.9 to.6. After bout 1 min, the phototxis of both strins recovered to norml. fluorescent lmp 1 fluorescent lmp b FIG. 3. Choice-chmber pprtus for testing the bility of flies to sense odornts. () The flies re shken from the strt tube into the sliding comprtment. (b) The comprtment is shifted into register with tubes contining odors A nd B. After 3 sec, the comprtment is shifted out of register nd the numbers of flies trpped in ech tube nd in the centrl comprtment re counted. Genetics. The X-linked dunce muttion is incompletely recessive. Heterozygous dnc/ + femles hve A of.23 (SEM I.2, n = 5) in comprison to.31 (SEM L.2, n = 18) for +/+ flies. Hemizygous mles nd homozygous femles re eqully deficient in lerning; A =.1 (SEM.2, n = 4) for mles nd.5 (SEM 4.6, n = 4) for femles. To mp the muttion, dunce mles were mted to femles homozygous for the following mrkers: yellow (y), chocolte (cho), crossvwinless (cv), vermilion (v), forked (f), nd norml llele of yellow (y+) locted ner the.centromere (11). The heterozygous F1 femles were then crossed to norml mles, yielding mles whose X-chromosomes hd n opportunity to undergo recombintion. The F2 mles included vrious recombinnts for the morphologicl mrkers nd dnc. These mles could not be tested for the presence of dnc becuse some of the mrker genes ffect the ctivity of flies, interfering with lerning performnce. To overcome this difficulty, recombinnt mles were individully mted to dnc/dnc femles to produce femle progeny heterozygous for the recessive morphologicl mrkers, which do not ffect lerning bility when heterozygous. These flies were either homozygous or heterozygous for dnc, depending upon whether the dnc gene ws present in the F2 mle. Testing these femles revels whether dnc ws present in the F2 mle, since dnc/dnc femles lern much more poorly thn dnc/+. The results re shown in Tble 2 nd indicte tht the dnc gene is between y nd cho. DISCUSSION During trining, norml flies perceive the light, respond to it phototcticlly, sense the odornt nd the electric shock, nd integrte the sensory inputs. An ssocition occurs between shock nd odornt so tht, in lter testing, the odornt cts s cue for voidnce. Poor performnce in this prdigm could result from defects in ny of these steps. Wht could the defect in dunce be? (i) Although the mutnt hs essentilly norml phototxis, cn sense the odornts, nd is deterred by the electric shock,
Genetics: Dudi et l..61 Proc. Ntl. Acd. Sci. USA 73 (1976) 1687 (V.5! 4--- 3^ ) c -o -3i C_).12 C513OH + L- T = norml C: dunce oc,~ XCO/ odornt used (vs. blnk) FIG. 4. Response of norml nd dunce flies to vrious odornts in the choice-chmber pprtus of Fig. 3. Two-tenths milliliter of the specified odornt in ethnol ws spred on one grid. The other grid ws blnk with.2 ml of ethnol lone. Before use, the grids were dried for 5 min to evporte the ethnol. Ech column represents the verge (+ SEM) of 1 experiments, with 5-7 flies per experiment. A smll number of flies remin in the centrl comprtment, so blnk vs. blnk is slightly less thn 5%. there my exist some subtle sensory or motor process which is defective in dunce tht becomes importnt under the conditions of the prdigm. (ii) The defect could be in certin pthwys connecting sensory input nd motor output. The fct tht dunce is ble to void the odornts under conditions where lerning is not required shows tht some sensory-motor connections re intct. Nevertheless, others might be needed during lerning. Using other sensory modlities, the mutnt flies might lern. Tble 2. Clss Mpping of the dunce muttion Lerning index (A) Prentl chromosomes dunce.5 ±.3 ychocuvfy+.32 ±.3 Recombinnt chromosomes cv v fy+.1 ±.3 vfy+.11 ±.2 f Y+.9 ±.2 {.7 ±.1 (n = 6).26 ±.3 (n = 4)J y cho.2 ±.2 y cho cv.25 ±.4 y cho cv v.2 ±.1 y cho cvuf.28 ±.3 Lerning performnce of the prentl types nd ech recombinnt clss. Ech chromosome ws tested over dunce in heterozygous femles to eliminte effects of mrkers. For recombinnt types, ech. A is the verge for 9-11 independently rising recombinnts. For the prentl types, the A represents the verge of 9-11 determintions. The recombinnt clsses y+ nd cho cv v f y+ re indistinguishble from one of the prentl types with the mrkers used. The y recombinnts could be clerly divided into two groups, ech with A typicl of one of the prentl types. The results plce the presumptive loction of dnc between y nd cho; i.e., between mp positions. nd 5.4 t the left tip of the X-chromosome. (iii) The dunce muttion my interfere with ccessory neurl processes necessry for lerning. For exmple, defects in rousl nd motivtion my interfere with informtion storge nd retrievl (12), even though the mchinery for lerning is intct. (iv) The dunce muttion my disrupt moleculr mechnism underlying neurl plsticity. Severl moleculr mechnisms hve been implicted in lerning. Some involve neurotrnsmitter metbolism (12) or protein synthesis (13). Biochemicl mesurements on dunce nd norml flies might revel possible defects in such mechnisms. Phrmcologicl tretments, if successful in reversing the mutnt phenotype, could indicte the nture of the lesion. Preliminry experiments hve not yet yielded ny such clues. It is possible tht the defect is confined to very smll ntomicl region. If so, mosic nlysis should focus ttention on the crucil region. Isoltion of dditionl mutnts might revel vrious steps in the process of lerning. We thnk Ms. Brbr Butler for expert technicl ssistnce. This work ws supported by grnt from the Ntionl Science Foundtion. Y.D. is fellow of the Europen Moleculr Biology Orgniztion. Y.-N.J. is supported by the Scottish Rite Schizophreni Reserch Progrm. D.B. is fellow of the Gordon Ross Medicl Foundtion. 1. Hott, Y. & Benzer, S. (1972) "Mpping of behvior in Drosophil mosics," Nture 24,527-535. 2. Quinn, W. G., Hrris, W. A. & Benzer, S. (1974) "Conditioned behvior in Drosophil melnogster," Proc. Ntl. Acd. Sci. USA 71, 78-712. 3. Lewis, E. B. (196) "A new stndrd food medium," Drosophil Informtion Service 34, 117-118. 4. Lewis, E. B. & Bcher, F. (1968) "Method of feeding ethyl methne sulfonte (EMS) to Drosophil mles," Drosophil Informtion Service 43, 193. 5. Byers, D. & Quinn, W. G. (1974) "Experiments on lerning in Drosophil: effects of nesthesi nd screening for lerning mutnts," Cliforni Institute of Technology Biology Annul Report, p. 15. 6. Benzer, S. (1967) "Behviorl mutnts of Drosophil isolted by countercurrent distribution," Proc. Ntl. Acd. Sci. USA 58, 1112-1119.
1688 Genetics: Dudi et l. 7. Benzer, S. (1973) "Genetic dissection of behvior," Sci. Am. 229, 24-37. 8. Ghysen, A. & Benzer, S. (1974) "Studies on sluggish mutnts of Drosophil," Cliforni Institute of Technology Biology Annul Report, p. 17-18. 9. Hott, Y. & Benzer, S. (1969) "Abnorml electroretinogrms in visul mutnts of Drosophil," Nture 222, 354-356. 1. Jn, Y. N. & Jn, L. (1975) "Neurophysiology of the Drosophil lrvl nerve-muscle preprtion," Cliforni Institute of Technology Biology Annul Report, pp. 7-71. Proc. Ntl. Acd. Sci. USA 73 (1976) 11. Lindsley, D. L. & Grell, E. H. (1968) "Genetic vritions of Drosophil melnogster," Crnegie Inst. Wshington *Publ. no. 627. 12. Kety, S. S. (197) "The biogenic mines in the centrl nervous system: their possible roles in rousl, emotion, nd lerning," in The Neurosciences, Second Study Progrm, ed. Schmitt, F.. (The Rockefeller University Press, New York), pp. 324-36. 13. Glssmn, E. (1969) "The biochemistry of lerning: n evlution of the role of RNA nd protein," Annu. Rev. Biochem. 38, 65-646.