Proc. Natl. Acad. Sci. USAVol. 93, pp. 9875-9880, September 1996Neurobiology

Expression of Drosophila mushroom body mutations inalternative genetic backgrounds: A case study of the mushroombody miniature gene (mbm)

(genetic modifiers/brain anatomy/odor learning/olfaction/walking)

J. STEVEN DE BELLE* AND MARTIN HEISENBERGt*Max-Planck-Institut fur Biologische Kybernetik, Spemannstrasse 38, D-72076 Tubingen, Germany; and tTheodor-Boveri-Institut fur Biowissenschaften, Lehrstuhlfur Genetik, Universitat Wurzburg, Am Hubland, D-97074 Wurzburg, Germany

Communicated by M. Lindauer, Universitat Wurzburg, Wurzburg, Germany, April 9, 1996 (received for review December 12, 1995)

ABSTRACT Mutations in 12 genes regulating Drosophilamelanogaster mushroom body (MB) development were eachstudied in two genetic backgrounds. In all cases, brain struc-ture was qualitatively or quantitatively different after replace-ment of the "original" genetic background with that of theCanton Special wild-type strain. The mushroom body minia-ture gene (mbm) was investigated in detail. mbm supports themaintenance of MB Kenyon cell fibers in third instar larvaeand their regrowth during metamorphosis. Adult mbm' mu-tant females are lacking many or most Kenyon cell fibers andare impaired in MB-mediated associative odor learning. Weshow here that structural defects in mbm' are apparent onlyin combination with an X-linked, dosage-dependent modifier(or modifiers). In the Canton Special genetic background, thembm' anatomical phenotype is suppressed, and MBs-developto a normal size. However, the olfactory learning phenotype isnot fully restored, suggesting that submicroscopic defectspersist in the MBs. Mutant mbm' flies with full-sized MBshave normal retention but show a specific acquisition deficitthat cannot be attributed to reductions in odor avoidance,shock reactivity, or locomotor behavior. We propose thatpolymorphic gene interactions (in addition to ontogeneticfactors) determine MB size and, concomitantly, the ability torecognize and learn odors.

The corpora pedunculata or mushroom bodies (MBs) areprominent neuropil structures in the insect central brain. Theyconsist mainly of a bulbous calyx and tightly packaged arraysof thin parallel fibers of the so-called Kenyon cells (KCs)(1-6). In bees and flies, a wealth of evidence attests to MBinvolvement in associative odor conditioning (for reviews, seerefs. 6-10). For instance, chemical ablation of the MBs inDrosophila abolishes conditioned odor avoidance while leavingother aspects of behavior, including various forms of learning,intact (11, 12). Comparable behavioral patterns have also beendocumented for genetic variants with MB structural defects(13, 14). Moreover, the gene products of dunce (dnc), rutabaga(rut), and the catalytic subunit (DCO) of protein kinase A,which participate in the cAMP signaling cascade (15-18) andaffect olfactory conditioning in flies (19-22), are all prefer-entially expressed in the MBs (23-25).Not only are the MBs associated with behavioral plasticity,

but they also undergo conspicuous structural changes duringthe lifetime of a fly. Most KCs shed and regenerate theirprojections at the onset of metamorphosis, probably to replacelarval synaptic connections with adult circuitry (26). At eclo-sion, fiber number depends on larval rearing conditions andcan still be modified during adult life through experience (27,28). This structural plasticity is not observed in the short-termlearning mutants dnc' and rut' (29).

Here we describe yet another source of MB structural vari-ability, one that is based upon interactions among polymorphicgenes. Mutations in 12 genes are known to affect MB grossanatomy (2, 8, 10, 13, 14). When placed into a genetic backgroundderived from the Canton Special (CS) wild-type strain, all ofthemexhibit alternative brain anatomy phenotypes. We have investi-gated this phenomenon in detail for the mushroom body minia-ture gene (mbm). Mutant mbm flies have a sexually dimorphicanatomical defect specific to the MBs and a correlated odorlearning deficit (8, 13, 14). MBs in mbm1 females developnormally until the beginning of the third larval instar, whereuponKC projections start to degenerate. By the end of larval devel-opment, MB neuropil is usually no longer discernible, althoughKC perikarya persist. Unlike in wild-type flies, regeneration ofKC projections at the onset of metamorphosis is not observed (8,13). We show here that the mbm' developmental defect isrestored in alternative genetic backgrounds, whereas a substantialodor conditioning impairment persists.

MATERIALS AND METHODSFlies and Genetic Background. We used wild-type CS

(maintained in Wurzburg since 1978) as the standard controlstrain in all anatomical and behavioral analyses. mbm' (2-0.0,21B8-C6) (formerly mbmN337; Fig. 1 D and E) was identifiedin a mass histology screen of ethylmethane sulfonate-treatedcn bw sp chromosome-2 lines (provided by C. Nusslein-Volhard, Max-Planck-Institut fur Entwicklungsbiologie, Tu-bingen, Germany) (13). It was initially separated from thegenetic markers by outcrossing with the wild-type Berlin strain.For more than a decade, the fidelity of the mutant anatomicalphenotype has been maintained by periodical artificial selec-tion for high penetrance and expressivity in the progeny ofmultiple single-pair matings (13). Other MB structural mu-tants used are listed in Table 1.To control for potential polygenic variability in brain anat-

omy and behavior (discussed in ref. 36), we constructed twonew mbm' strains by replacing the original genetic backgroundwith that of CS (Fig. 1). Chromosome balancer strains usedwere In(1)FM7a, y3ld SC8 wa vOfB (FM7a), al Bl/In(2LR)O, CydplvIpr cn2 (al Bl/CyO), and Sb H329/In(3LR)TM6b, Hu e Tb(Sb H/TM6b) (32). We used al dp bpr cpx sp/CyO for chromo-some-2 genetic markers (32). Chromosome4 CS alleles were"passively" exchanged in both crossing schemes. Other mutationsinfluencing MB gross anatomy were outcrossed similarly.XCS/YCS; mbm1; 3CS; 4cs (mbm1; CS; Fig. 1C) was made by

substitution of whole mbm1 second chromosomes into CSusing FM7a, al Bl/CyO, and Sb H/TM6b balancer strains.XCS/YCS; mbm1(CS); 3cS; 4cs [mbm1(CS); Fig. 1B] was con-

structed using the external marker aristaless (al, 2-0.4, 21C1-2,- 30 kb proximal to mbm1; T. Twardzik, unpublished data). alwasseparated from aldp bprcpxsp and placed in the CS background

Abbreviations: MB, mushroom body; KC, Kenyon cell; CS, CantonSpecial; MCH, 4-methylcyclohexanol; BAL, benzaldehyde.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

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FIG. 1. Brains and genetic constitution of CS (A), mbm1(CS) (B),mbm';CS (C), mbm' d(D), and mbm1 9(E) adult flies. Frontalparaffin sections ("40 ,um from the caudal margin of the brain)photographed under a fluorescence microscope (30). MB calyces(arrowheads) predict accurately the condition of peduncles and lobes(not shown). All observed anatomical defects appear to be restrictedto the MBs at this level of analysis. Chromosome pairs (1-4) of CS andmbm1 origins are shown as filled and shaded bars, respectively. Thedistal chromosome-2L position of mbm is indicated. al+ was used toensure that the mbm1 mutation was not lost through recombinationduring the construction of mbm1(CS). (Bar = 50 ,um.)

by repeated genetic recombination [al(CS)]. Xs/Ylc; alBl/CyO;3cs; 4CS (alBl/CyO;CS) was constructed from alBl/CyO and CSby chromosome substitution using FM7a and Sb H/TM6b. Weplaced mbm' in the CS background by repeated recombinationwith al(CS) and selection of unmarked heterozygous mbm1al+/mbm+ al progeny. mbm' al+(CS) chromosomes were thenisolated using al Bl/CyO;CS.- Alleles of CS origin constitute>95% of the genetic background in mbm1(CS).

Flies were maintained at equal density in 180-ml plasticbottles containing 40 ml of standard Drosophila medium(cornmeal, agar, molasses, yeast, and nipagin) at 250, 60% RHwith a 16L:8D light regime.

Histology and Brain Volume. Newly constructed mbm1;CS andmbm1(CS) flies were placed in mass histology "collars," fixed inCarnoy's solution, and embedded in paraffin (30). Heads were cutin 7-,um serial frontal sections and inspected under a fluorescencemicroscope. Flies sampled from behavioral experiments (seebelow) were examined similarly and photographed (Fig. 1). Allgenotypes were represented equally in each preparation. Brainneuropil substructure volumes were derived from planimetricmeasurements of these sectioned flies (11, 28).

Classical Conditioning. Associative odor learning and sensoryacuity controls were tested in the conditioning paradigm of Tullyand Quinn (19) as described by de Belle and Heisenberg (11).Groups of -100 2- to 6-day-old flies were trained to avoid either4-methylcyclohexanol (MCH) or benzaldehyde (BAL) pairedtemporally with 120 V dc shock pulses and tested for learning.Naive flies were used in odor and shock avoidance controlexperiments (11, 36). To assess memory, flies were stored onfresh medium for either 30 min or 3 h between training andtesting (21). A performance index represents the average nor-malized percent avoidance of the shock-paired odor (learning andmemory) or the individual stimuli (sensory acuity) (36).Locomotor Activity. Aspects of walking behavior were mea-

sured in Buridan's Paradigm (31, 37), as described by Strauss etal. (38). Single 2- to 6-day-old flies with wings clipped (at theanterior cross-vein) were tracked and recorded for 15 min usinga video scanning device. Data were analyzed with regard to totalpath length, mean walking speed, and landmark orientation.

Fast phototaxis (39) was also used as a measure of locomo-tor activity, as described by Strauss et al. (38). Scores for eachgenotype were calculated from groups of -80 flies as thepercent of all possible transitions toward light in a counter-current device during 5 consecutive 5-s intervals.

RESULTSInfluence of Genetic Background on Expression of MB

Mutations. Many brain structure mutants require occasionalartificial selection to sustain their anatomical defects (2).Several independent lines are established, and the one with themost severe or "typical" defect is propagated. Generally, theselines tend to revert toward wild-type over a period rangingfrom two or three generations to several years. However, insome instances, lines drift toward more severe or even newstructural phenotypes. By systematically placing alleles of allgenes known to influence MB gross anatomy into the CScontrol genetic background, we can now provide more thananecdotal accounts of these phenomena.Table 1 summarizes the changes in brain structure observed

for 15 alleles of 11 genes affecting MB gross anatomy in adultflies. In all cases, brain structure phenotypes changed uponoutcrossing. For ellipsoid body open (ebo), only the mostsevere alleles affected MB anatomy. Structural defects of somemutants became either more [e.g., mushroom body defect(mud)] or less [e.g., mushroom bodies deranged (mbd)] ex-treme. For most mutants, genetic background influencedchanges in certain regions of the central brain and not inothers. Of these, some had defects that became either more[e.g., small mushroom bodies (smu)] or less [e.g., vacuolarpedunculi (vap)] MB-specific. We observed only one example

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Table 1. Expression of mutant MB structural genes in alternative genetic backgrounds*

Original genetic backgroundt

central brain deranged (ceb, 1-23, 7F1-8A5) [singed (sn, 1-21.0, 7D1-2)]cebl (ceb849) (8, 31): CA greatly enlarged & misshapen. KC axons

form extra "lateral CA." PE & lobes absent or thin. EB openventrally, flattened, or divided. FB split in most flies.

ceb2 (ceb892) (8, 31): Similar to cebl but MB defects less severe.PE & lobes present but poorly developed. EB defects in allflies. FB defects in most flies. 9-sterile.

calyx bulging (cxb, 2-73) [curved (c, 2-75.5, 52D3-9)]cxbI (cxbN7l) (8, 13, 32): CA size & shape variable. PE

generally present but lobes absent. EB open ventrally.NO misshapen. 9-sterile.

ellipsoid body open (ebo, 1-3.8, 3D4)§ [white (w, 1-1.5, 3C2)]ebo3 (ebo678) (31, 33): CA & PE misshapen. Lobes absent. EBopen ventrally, flattened, & occasionally divided. FB split inmany flies.

ebo4 (ebo'041) (31, 33): MB defects similar to ebo3. EB openventrally, flattened, & often divided. FB split in many flies.

fused mushroom bodies (fum, 2-centromere)fumI (fumN799) (32): fry-lobes fused across the sagittal

mid-plane. Penetrance incomplete.mushroom bodies deranged (mbd, 1-57, 17B) [forked (f, 1-56.7,

15F1-3) to carnation (car, 1-62.5, 18D1-2)]mbdl (mbdKS65) (2, 8, 13, 14, 26, 32): CA greatly enlarged &

misshapen. KC axons wound into "turbans." PE & lobesabsent in most flies. MB defects highly penetrant &strongly expressed. EB open ventrally or flattened. NOmisshapen.

mushroom body miniature B (mbmB, 2-31) [basket (bsk, 2-33)]mbmBI (mbmBN806) (8, 13, 32): CA 1/4 to 1/2 size. PE &

lobes thin. EB open ventrally. NO often misshapen. 9-sterile.mushroom body miniature C (mbmC, 2-35) [bsk (2-33)]mbmCI (mbmCN28) (8, 13, 32): CA -1/4 size. PE & lobes

thin or absent. AL reduced. EB open ventrally. NO oftenmisshapen.

mushroom bodies reduced (mbr, 1-51) [garnet (g, 1-44.4, 12B6-7)to f (1-56.7, 15F1-3)]mbrl (mbr6O8): CA -1/4 size. PE & lobes thin. EB open

ventrally. NO usually misshapen. Semilethal & d-sterile.mushroom body defect (mud, 1-50, 12E9-11) [g (1-44.4, 12B6-7)

to f (1-56.7, 15F1-3)]mudl (mudl(S63) (2, 26, 32, 34): CA enlarged (to 10 timesnormal size) & misshapen. PE & lobes absent. ALenlarged. CC & other structures often misshapen.Semilethal & 9-sterile.

mud2 (mudUB686) (32, 34, 35): Similar to mud' but defectsmore severe. Semilethal & 9-sterile.

mud3 (32, 34): Similar to mud' but defects more severe.Semilethal & 9-sterile.

mud4 (32, 34): Similar to mud'. PE & lobes occasionallyobserved. Semilethal & 9-sterile.

small mushroom bodies (smu, 1-11, 4F12) [crossveinless (cv,1-13.7, 5B)]smuI (smu1707): CA -1/4 size. PE & lobes thin. EB open

ventrally. NO usually misshapen, 9-sterile.vacuolar pedunculi (vap, 1-54.2) [f (1-56.7 15F1-3)]

vap1 (vapKS67) (2, 32): Large vacuoles restricted to the PEs.EB open ventrally & NO misshapen in many flies.

CS genetic backgroundi

cebl(CS): CA larger & more misshapen than in cebl. PE &lobes absent. CC more disturbed.

ceb2(CS): MB & CC phenotypes more severe than in ceb2.Homozygous viable.

cxb1;CS: CA & PE less mutant than in cxbl. Lobes usuallyabsent. EB & NO defects less severe. 9-sterile.

cxb1(CS): Severe MB & CC phenotypes reverted towardwild type after a few generations. CA often slightlymisshapen. CC defects similar to cxb1;CS. Homozygousviable.

ebo3(CS): MB defects initially less severe than in ebo3 &gradually drifted toward wild type. CC defects stable &similar to ebo3.

ebo4(CS): MB defects similar to ebo3(CS). CC defectsstable & similar to ebo4.

fum';CS: MB defect & penetrance similar to fum1. EBopen ventrally or flattened in some flies.

mbd';CS (14): MB defect expression & penetrance weakerthan in mbdl. CC defects persist in most flies.

mbdl(CS) (14): Severe MB phenotype reverted towardwild type after a few generations. CC defects persist inmost flies.

mbmB1;CS & mbmB1(CS): MB defects similar to mbmB1.CC defects less penetrant. 9-sterile.

mbmCI;CS: Similar to mbmC1. CC defects less penetrant.mbmC1(CS): Wild type. Mutant allele may have been

lost.

mbr1(CS): MB & CC defects similar to mbrl. OL (lobula)often disorganized. Semilethal & d-sterile.

mudl(CS) (34): Defects more severe than in mud'.Semilethal & 9-sterile.

Outcrossing unsuccessful.

mud3(CS) (34): Defects more severe than in mud3.Semilethal & 9-sterile.

mud4(CS) (34): Defects more severe than in mud4. PE &lobes absent. Semilethal & 9-sterile.

smul(CS): MB defects similar to smul. CC & other brainstructures normal. 9-sterile.

vapl;CS & vapl(CS): PE-specific vacuoles smaller than invap1. Brain more dissociated. CC defects similar to vap1.

*Genetic symbols and map positions are enclosed in parentheses. Genetic markers used for outcrossing are enclosed in square brackets (31). Brainswere examined in 7-gm serial frontal paraffin sections under a fluorescence microscope (30). CA, calyx; PE, peduncle; CC, central complex; FB,fan-shaped body; EB, ellipsoid body; NO, noduli; AL, antennal lobe; OL, optic lobe.

tPrevious allele designations are enclosed in parentheses. X-linked and chromosome-2 mutations were generated in Berlin and cn bw sp, respectively(13, 31). Genetic backgrounds were further derived from Berlin and a variety of genetically marked strains.

*Outcrossing procedures were similar to those described for mbm'. ";CS" and "(CS)" indicate outcrossing by chromosome substitution andrecombination, respectively.§MBs appeared wild type in ebol (eboKS234) (31), ebol(CS), ebo2 (eboKs263) (13, 31, 33), and ebo2 (CS).

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of optic lobe involvement in this structural variability [mush-room bodies reduced (mbr)]. There were several cases in whichbrain anatomy reverted to near wild-type condition afteroutcrossing to CS. One of these was the mbm gene, on whichwe will focus for the rest of this report.

Brain Anatomy of mbm. The originally isolated mbm1 mu-

tant exhibits a striking sexual dimorphism. In females, MBs arenearly (if not completely) absent, whereas those of males are

reduced to about 3/4 of the CS control (Figs. 1 E andD and 2A).One or more dosage-dependent X-linked mbm modifiers havebeen implicated in this sex-specific expression pattern [desig-nated as suppressor of mbm, su(mbm); see ref. 8 and below].In contrast, MBs of both mbm1;CS and mbm'(CS) were notsexually dimorphic (data not shown) and were not significantlydifferent in volume from CS (Figs. 1 C and B and 2A).

All mbm1 anatomically mutant phenotypes were restricted tothe MBs at the level of the light microscope. The antennal lobes,which are derived in part from neuroblasts that are geneticallyand developmentally related to those giving rise to KCs (34, 41),showed no significant volume differences in any of the strains(Fig. 2B). The antennal glomerular tract also did not appear tobe affected, except that reduced calyces had proportionally fewerantennal glomerular tract inputs in structurally mutant mbm flies(data not shown). In contrast to a previous report (13), we did notobserve optic lobe defects in the mbm' flies examined here.We were concerned that mbm' alleles might have been lost

through genetic recombination during mbm1;CS andmbm1(CS) construction. Heterozygous female progeny ofmbm';CS and mbm'(CS) females crossed with mbm' maleshad MBs reduced to -1/2 the normal size, whereas males from

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FIG. 2. Brain substructure volumes derived from planimetric mea-surements of 7-,um serial sections of flies sampled from those testedin behavior experiments (see Fig. 3). Bars represent mean ± SE ofmean brain hemisphere values, n = 20 [10 d and 10 Y for CS,mbm'(CS), and mbm1;CS] or 10 (for mbm1 d and 9) flies per bar. (A)Calyx volumes were significantly different [ANOVA (40), F[4,751 =

116.07, P < 0.0001]. A Student-Numan-Keuls test (40) revealed thefollowing relationship: [CS = mbml(CS) = mbm';CS] > mbm1 d >mbm1 $ (P s 0.05). (B) Antennal lobe volumes. A two-way ANOVAtesting the effects of genotype, sex, and interaction was not significant(F[7,72] = 1.80, P = 0.10).

these crosses appeared wild-type (data not shown). Thissuggests that, on average, genetic modifiers have an additiveeffect on the mbm MB gross anatomy phenotype. In particular,X chromosomes derived from CS suppressed mbm defects[they are su(mbm)], whereas those from the original mbm'strain did not [and are su(mbm)+]. Homozygous su(mbm)+;mbm+ flies had wild-type MBs (data not shown).

Classical Conditioning of mbm. To separate and assess theeffects of mbm and modifiers on learning ability, we comparedoriginal and outcrossed mbm1 strains using an olfactory condi-tioning paradigm (11, 19). Learning scores for mbm1 femaleswere 36% of the CS control, whereas those for males were 58%(Fig. 3A). Despite full restoration of calyx volume in mbm1;CSand mbm1(CS) (Figs. 1 C and B and 2A), associative odorlearning remained impaired (to the same degree in both genders,data not shown) and was not significantly higher than in males ofthe original mbm1 strain (Fig. 3A).

Previous studies indicate that Drosophila MBs are not requiredfor simple odor detection and that mbm flies have no obviousolfactory deficits (11, 13, 14). We have confirmed these findingsusing odor avoidance controls in a context relevant to ourconditioning paradigm. The mbm1 mutation in all genetic back-grounds had no significant effect on the ability of naive flies toavoid undiluted MCH (Fig. 3B) and BAL (Fig. 3C). Avoidanceof diluted odorants was also not impaired in mbm'(CS) flies.

Only fully outcrossed mbm1(CS) flies responded normally to120 V as well as 20 V shock (Fig. 3D). Shock avoidance of bothmbm' and mbm';CS was significantly reduced. Several conclu-sions can be drawn from these findings. First, the mbm gene isprobably not responsible for reduced shock avoidance. Second,genetic elements responsible for low shock reactivity must resideon chromosome-2 since mbm1;CS flies that have retained onlythis chromosome from the original stock (see Fig. 1C) inheritedthe defect. Third, learning impairments cannot be attributed to areduced salience of the reinforcer because shock avoidancedifferences between mbm1;CS and mbm1(CS) were not reflectedin their learning scores (Fig. 3, compare D and A). Further,mbm1;CS and both genders of the original mbm1 strain had anequivalent reduction in shock avoidance, which does not explainthe more severe learning impairment of mbm1 females (Fig. 3,compareD andA). The central registering ofshock probably doesnot involve the MBs since shock avoidance is normal in MB-lessflies generated by chemical ablation (11).Memory retention curves for CS and mbm1(CS) were "paral-

lel" [the ANOVA genotype X time interaction component wasnot significant (F[2,30] = 1.85, P = 0.18)], indicating that mbm1affects associative odor learning but not memory (Fig. 3E).Performance indexes averaged over all retention intervals formbm1(CS) were 69% of the CS control. This "acquisition"phenotype is similar to those of the olfactory conditioningmutants latheo and linotte (36, 42).Locomotor Activity ofmbm. Odor and shock avoidance control

experiments assert that the learning impairments in mbm1(CS)were not due to a general debilitation. To provide additionalsupport for this conclusion, we compared walking and orientationofmbm1(CS) and CS flies in Buridan's paradigm (31, 37, 38) andin fast phototaxis (39) (Table 2). mbm1(CS) was not significantlydifferent from CS in total track length, mean walking speed, andlandmark orientation. Although fast phototaxis differences weresignificant, the relatively high score of mbm1(CS) (81 ± 1compared with 85 ± 1 for CS) indicates that poor learning doesnot reflect a locomotor activity deficit.

DISCUSSIONThe observation that mutant gene expressivity and penetranceare genetic background-dependent is not new. Chromosome lossinduced by the Fs(3)Horka mutation ranges in frequency from2% to over 20% in alternative genetic environments (43). eyeless(ey) expression is partially compensated by selection pressureagainst the deleterious mutant phenotype and gradual accumu-

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FIG. 3. Classical conditioning and sensory acuity. Bars or symbolsrepresent mean SE of performance indexes (n = 6 per bar or

symbol). All but two performance index samples were distributednormally. Statistical analyses of arcsine-transformed data (40) (notshown) and of the raw data (below) gave the same results. (A)Learning was significantly different (ANOVA, F[4,2s] = 20.91, P <0.0001). An Student-Numan-Keuls test revealed the following rela-tionship: CS > [mbmr(CS) = mbm1;CS = mbm' d] > mbm1 9] (P c0.05). (B and C) Odor avoidance. (B) Responses of naive flies to pureand 10-2 dilutions of MCH versus air. A two-way ANOVA testing theeffects of genotype, sex, and interaction on avoidance of undilutedMCH was not significant (F[7,40] = 2.08, P = 0.07). For CS andmbm1(CS), a two-wayANOVA detected a significant effect of dilution

lation of modifiers (44). Expression of the naturally polymorphicforaging gene, influencing both larval and adult feeding behavior,is modified by a variety of additional genetic elements (45, 46).The striking result with the structural mutants of the centralbrain, and MBs in particular, is that many of them have vastlydifferent phenotypes after introducing a new wild-type geneticbackground. Genes such as central brain deranged (ceb) andmudare analogous to the ey example (above) in that outcrossingappears to have removed accumulated "suppressor" modifiers,resulting in more severe phenotypes. By contrast, outcrossing ofgenes such as calyx bulging (cxb), mbd, and mbm seems to havere-introduced "suppressor" modifiers (previously artificially se-lected against to maintain structurally mutant strains), resulting inless severe or even wild-type phenotypes. Thus, some brain struc-ture mutations would likely not be detected by histological screensmade in certain genetic backgrounds. In all cases except mbm, theMBs and the central complex are both involved, implying commongenetic or epigenetic interactions in the development of thesestructures. In general, outcrossing generates a higher degree ofstructural variability in the MBs than in the central complex.A Polygenic Basis for the mbm Phenotype. For mbm1, a

comparison of original (derived mainly from cn bw sp and Berlinstrains) and CS genetic backgrounds reveals an interesting pat-tern of genetic interaction. mbm expression is modified by anX-linked suppressor [su(mbm)]. CSX chromosomes suppress themutant brain structure phenotype in mbm1 flies and 1/2 of thelearning deficit of females. Moreover, we find X-linked mbmsuppression in the background of an additional weaker mbmallele (8, 32), in C(l)DX, y w f (32), and in In(1)EN, In(1)dl-49,y vfcarY/O (13) but not in Berlin, Oregon R.,Df(2L)al/CyO (32),and several other laboratory strains (data not shown). It isintriguing that a mutation as severe as mbm' seems to be fullycompensated with respect to brain structure in a su(mbm)background. Flies of the reciprocal genotype [su(mbm)+/su(mbm)+; mbm+/mbm+] appear anatomically wild-type as well.Thus, only specific allelic combinations of these two genes giverise to an MB phenotype. The difference between the two is thatmutant mbm alleles are fully recessive, whereas su(mbm) + isdosage-dependent.MB Functional Specificity. The present account extends pre-

vious studies (13) in that, regardless of genetic background, mbm'is an associative odor learning mutant. Abnormal development ofmbm' in the original genetic background is highly MB-specific.We therefore favor the view that a microscopically invisible MBdefect remains in mbm1(CS). Unlike dnc, mt, and amnesiac (19,21, 22, 47), memory decay within 3 h of training is normal inmbm'(CS), indicating that MBs are important for conditionedacquisition but not later phases of memory retention.

Behaviorally, flies are not severely impaired by either geneticor chemical insults to the MBs. Walking, landmark orientation,circadian activity rhythms (48), male courtship (49), and flightbehavior (12) are all superficially normal in mbm mutants and in

only (F[1,20o = 21.65, P = 0.0002). Effects of genotype and interactionwere not significant. (C) Responses of naive flies to pure and 10-2dilutions of BAL versus air. A two-way ANOVA testing the effects ofgenotype, sex and interactions on avoidance of undiluted BAL was notsignificant (F[?7,40] = 1.86, P = 0.10). For CS and mbm1(CS), a two-wayANOVA detected a significant effect of dilution only (F[1,201 = 42.15,P < 0.0001). Effects of genotype and interaction were not significant.(D) Shock avoidance. Responses of naive flies to 120 and 20 V dcshock. Avoidance of 120 V shock was significantly different (ANOVA,F[4,251 = 5.17, P = 0.0035). An Student-Numan-Keuls test revealed thefollowing relationship: [CS = mbm'(CS)] > [mbm1;CS = mbm' d =mbm1 9] (P ' 0.05). For CS and mbml(CS), a two-way ANOVAtesting the effects of genotype, voltage, and interaction was notsignificant (F[3,201 = 0.54, P = 0.66). (E) Memory retention ofmbm1(CS) was lower than the CS control at all time intervals. Atwo-way ANOVA detected significant effects of genotype (F[1,30] =48.22, P < 0.0001) and time (F[2,301 = 33.95, P < 0.0001) but notgenotype X time interaction (F[2,301 = 1.85, P = 0.18).

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9880 Neurobiology: de Belle and Heisenberg

Table 2. Walking behavior of D. melanogaster adults

Experiment CS mbm(CS) Test statistic P

Buridan's Paradigm*Track length 6.40 ± 0.59 m 7.38 ± 0.35 m t[181 = 1.43 0.18Mean speed 2.13 ± 0.02 cm/s 2.13 ± 0.01 cm/s t[181-= 0 1.00Orientationt -9.44 ± 9.430 -10.66 ± 8.830 t[90] 0.09 0.93

F[45,45]= 1.14 0.66Fast Phototaxist

Transitions 85.3 ± 1.0% 80.9 ± 1.2% t[18] = 2.91 0.0094

*Values are mean + SE of 2- to 6-day-old female flies (n = 10). Fly position was sampled every 0.2 s for 15 min (4.5 X 103measurements per fly) (31, 38). All comparisons were not significant.

tAngles of landmark orientation (in 50 groupings) were distributed normally. Hypotheses concerning the mean and variancewere addressed using the t test and the variance ratio test, respectively (40).tValues are mean ± SE scores for =80 2- to 6-day-old flies (n = 10). Distributions were normal and differences weresignificant.

MB-ablated flies. Although MB-visual system connections are

known in other insects (1, 3) and may exist in Drosophila (50),MBs are not required in associative visual pattern and colordiscrimination learning (12). Behavioral data presented herecorroborate the current concept that Drosophila MBs have a

specific role in mediating odor learning (9-11, 13, 14).Our observations accentuate the importance of controlling

genetic background in behavioral and neurogenetic studies. Inparticular, they advocate the use of several "defined" geneticbackgrounds instead of only one. This bears heavily on con-clusions about single genes "determining" aspects of complexbehavior such as learning (ref. 51 and references therein).Gene expression is context-dependent and meaningful de-scription of it must account for other hereditary (and nonhe-reditary) factors (45). Compensation for this added effort maybe the realization of novel functional interactions.

We thank A. Dinkelacker, T. Wanke, and J. Wulf for technicalassistance; R. Wolf for the planimetric measurement computer pro-gram; R. Strauss for assistance with Buridan's paradigm; T. Twardzikfor permission to discuss unpublished results; and K. A. Osborne andR. Strauss for reading preliminary versions of the manuscript. Finan-cial support was provided by the Max-Planck-Gesellschaft (J.S.d.B.),the Deutsche Forschungsgemeinschaft (Grant HE986 to M.H.), andthe Human Frontier Science Program (H.E.).

1. Strausfeld, N. J. (1976) Atlas of an Insect Brain (Springer, Berlin).2. Heisenberg, M. (1980) in Development and Neurobiology ofDrosoph-

ila, eds. Siddiqi, O., Babu, P., Hall, L. M. & Hall, J. C. (Plenum, NewYork), pp. 373-390.

3. Mobbs, P. G. (1982) Philos. Trans. R. Soc. London B 298, 309-354.4. Schurmann, F.-W. (1987) in Arthropod Brain: Its Evolution, Develop-

ment, Structure, and Functions, ed. Gupta, A. P. (Wiley, New York),pp. 231-264.

5. Yang, M. Y., Armstrong, J. D., Vilinsky, I., Strausfeld, N. J. & Kaiser,K. (1995) Neuron 15, 45-53.

6. Menzel, R., Durst, C., Erber, J., Eichmuller, S., Hammer, M.,Hildebrandt, H., Mauelschagen, J., Muller, U., Rosenboom, H.,Rybak, J., Schafer, S. & Scheidler, A. (1994) in Neural Basis ofBehavioral Adaptations, eds. Schildberger, K. & Elsner, N. (Fischer,Stuttgart), pp. 81-102.

7. Hammer, M. & Menzel, R. (1995) J. Neurosci. 15, 1617-1630.8. Heisenberg, M. (1989) in Fundamentals ofMemory Formation: Neu-

ronal Plasticity and Brain Function, ed. Rahmann, H. (Fischer, Stutt-gart), pp. 3-45.

9. Davis, R. L. (1993) Neuron 11, 1-14.10. Heisenberg, M. (1994) in Neural Basis ofBehavioralAdaptations, eds.

Schildberger, K. & Elsner, N. (Fischer, Stuttgart), pp. 61-79.11. de Belle, J. S. & Heisenberg, M. (1994) Science 263, 692-695.12. Wittig, T., Dill, M. & Heisenberg, M. (1995) Proceedings of the 23rd

Gottingen Neurobiology Conference, eds. Elsner, N. & Menzel, R.(Thieme, Stuttgart), p. 4.

13. Heisenberg, M., Borst, A., Wagner, S. & Byers, D. (1985) J. Neuro-genet. 2, 1-30.

14. de Belle, J. S. & Heisenberg, M. (1995) in Proceedings of the 23rdGottingen Neurobiology Conference, eds. Elsner, N. & Menzel, R.(Thieme, Stuttgart), p. 11.

15. Dudai, Y. (1988) Annu. Rev. Neurosci. 11, 537-568.16. Aszodi, A., Muller, U., Friedrich, P. & Spatz, H.-C. (1991) Proc. Natl.

Acad. Sci. USA 88, 5832-5836.17. Qui, Y., Chan, C. N., Malone, T., Richter, L., Beckendorf, S. K. &

Davis, R. L. (1991) J. Mol. Bio. 222, 553-565.18. Levin, L. R., Han, P.-L., Hwang, P. M., Feinstein, P. G. & Davis, R. L.

(1992) Cell 68, 479-489.19. Tully, T. & Quinn, W. G. (1985) J. Comp. Physiol. A 157, 263-277.20. Drain, P., Folkers, E. & Quinn, W. G. (1991) Neuron 6, 71-82.21. Tully, T. & Gold, D. (1993) J. Neurogenet. 9, 55-71.22. Tully, T., Pr6at, T., Boynton, S. C. & Del Vecchio, M. (1994) Cell 79,

35-47.23. Nighorn, A., Healy, M. J. & Davis, R. L. (1991) Neuron 6, 455-467.24. Han, P.-L., Levin, L. R., Reed, R. R. & Davis, R. L. (1992) Neuron 9,

619-627.25. Skoulakis, E. M., Kalderon, D. & Davis, R. L. (1993) Neuron 11,

197-208.26. Technau, G. & Heisenberg, H. (1982) Nature (London) 295, 405-407.27. Technau, G. M. (1984) J. Neurogenet. 1, 113-126.28. Heisenberg, M., Heusipp, M. & Wanke, C. (1995) J. Neurosci. 15,

1951-1960.29. Balling, A., Technau, G. M. & Heisenberg, M. (1987) J. Neurogenet.

4, 65-73.30. Heisenberg, M. & Bohl, K. (1979) Z. Naturforsch. C 34, 143-147.31. Strauss, R. & Heisenberg, M. (1993) J. Neurosci. 13, 1852-1861.32. Lindsley, D. L. & Zimm, G. G. (1992) The Genome of Drosophila

melanogaster (Academic, San Diego).33. Ilius, M., Wolf, R. & Heisenberg, M. (1994)J. Neurogenet. 9, 189-206.34. Prokop, A. & Technau, G. M. (1994) Dev. Biol. 161, 321-337.35. Fischbach, K. F., Boschert, U., Barleben, F., Hoube, B. & Rau, T.

(1987) J. Neurogenet. 4, 126-128.36. Boynton, S. & Tully, T. (1992) Genetics 131, 655-672.37. G6tz, K. G. (1980) in Development and Neurobiology of Drosophila,

eds. Siddiqi, O., Babu, P., Hall, L. M. & Hall, J. C. (Plenum, NewYork), pp. 391-407.

38. Strauss, R., Hanesch, U., Kinkelin, M., Wolf, R. & Heisenberg, M.(1992) J. Neurogenet. 8, 125-155.

39. Benzer, S. (1967) Proc. Natl. Acad. Sci. USA 58, 1112-1119.40. Zar, J. H. (1984) Biostatistical Analysis (Prentice-Hall, Englewood

Cliffs, NJ), 2nd Ed.41. Ito, K. & Hotta, Y. (1992) Dev. Biol. 149, 134-148.42. Dura, J.-M., Preat, T. & Tully, T. (1993) J. Neurogenet. 9, 1-14.43. Szabad, J., Mdth6, E. & Puro, J. (1995) Genetics 139, 1585-1599.44. Okagi, M. (1966) Genetica 37, 391-402.45. de Belle, J. S. & Sokolowski, M. B. (1987) Heredity 59, 73-83.46. Pereira, H. S., MacDonald, D. E., Hilliker, A. J. & Sokolowski, M. B.

(1995) Genetics 141, 263-270.47. Tully, T., Boynton, S., Brandes, C., Dura, J.-M., Mihalek, R., Preat,

T. & Villella, A. (1990) Cold Spring Harbor Symp. Quant. Biol. 55,203-211.

48. Wulf, J., de Belle, J. S. & Helfrich-Forster, C. (1996) Proceedings ofthe 24th Gottingen Neurobiology Conference, eds. Elsner, N. & Schnit-zler, H.-U. (Thieme, Stuttgart), in press.

49. O'Dell, K. & de Belle, J. S. (1995) J. Neurogenet. 10, 40.50. Technau, G. M. (1983) Ph.D. thesis (Universitat Wurzburg, Wurz-

burg, Germany).51. Gerlai, R. (1996) Trends Neurosci. 19, 177-181.

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