mushroom bodies suppress locomotor activity in drosophila...

Mushroom Bodies Suppress LocomotorActivity in Drosophila melanogasterJean-Rene Martin,1 Roman Ernst, and Martin HeisenbergLehrstuhl fur GenetikBiozentrumD-97074 Wurzburg, Germany

Abstract

Locomotor activity of single, freelywalking flies in small tubes is analyzed inthe time domain of several hours. To assessthe influence of the mushroom bodies onwalking activity, three independentnoninvasive methods interfering withmushroom body function are applied:chemical ablation of the mushroom bodyprecursor cells; a mutant affecting Kenyoncell differentiation (mushroom bodyminiature1); and the targeted expression ofthe catalytic subunit of tetanus toxin insubsets of Kenyon cells. All groups of flieswith mushroom body defects show anelevated level of total walking activity. Thisincrease is attributable to the slower andless complete attenuation of activity duringthe experiment. Walking activity in normaland mushroom body-deficient flies isclustered in active phases (bouts) and restperiods (pauses). Neither the initiation northe internal structure, but solely thetermination of bouts seems to be affected bythe mushroom body defects. How thisfinding relates to the well-documented roleof the mushroom bodies in olfactorylearning and memory remains to beunderstood.

Introduction

Attempts at structure/function mapping in theinsect brain have implicated the mushroom bodies(MBs) in a variety of complex behavioral functions.Most conclusively, the MBs have been shown to berequired for olfactory learning and memory (Men-

zel et al. 1974; Heisenberg et al. 1985; de Belle andHeisenberg 1994; Connolly et al. 1996). They werealso invoked as centers for courtship behavior indifferent insects. Electrical stimulation in brain ar-eas close to or in the MBs elicited courtship behav-ior in freely moving crickets and grasshoppers(Huber 1960; Otto 1971; Wahdepuhl and Huber1979; Wahdepuhl 1983). In Drosophila melano-gaster gynandromorphs, Hall (1979) showed thatthe MBs (or tissue in their vicinity) had to be malefor flies to display male courtship behavior. Morerecently, with the new P[GAL4] enhancer-traptechnique (Brand and Perrimon 1993) a feminizingtransgene (transformer1) was expressed specifi-cally in parts of the MBs (besides other tissues).This intervention caused the males to court bothsexes indiscriminately (Ferveur et al. 1995; O’Dellet al. 1995).

Much less is known about MB function in mo-tor control. Unilateral MB ablation caused aberrantcocoon spinning in Cecropia larvae (van der Klootand Williams 1953), and MB lesions in adult malecrickets led to an elevated general motor activity(Huber 1955). Locusts and bees with MB lesionsalso showed a general increase of behavioral activ-ity. In addition, many of the animals displayed ri-valing motor patterns (Howse 1974). Increased lo-comotor activity was noted for mushroom bodyminiature1 (mbm1) mutant flies (Heisenberg et al.1985). Recently, in search of an implication of MBsin the circadian clock, J.S. de Belle, J. Wulf, andC.H. Forster (pers. comm.) also found flies withchemically ablated MBs to be more active thantheir respective controls. All these results suggestthat MBs might play a role in the generation, regu-lation, or coordination of motor patterns.

Locomotor activity in Drosophila has been re-corded by many methods and in different contexts.To mention just a few, in an early study, spontane-ous walking activity in an open field apparatus wasused to select for active/inactive Drosophilastrains (Connolly 1966, 1967) and later for single1Corresponding author.

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gene mutants with aberrant locomotor activity(Meehan and Wilson 1987; Burnet et al. 1988;O’Dell and Burnet 1988). In an object fixation task(Buridan’s paradigm) developed by Gotz (1980),single flies with clipped wings are allowed to walkfreely on a platform between two opposing inac-cessible landmarks (vertical black stripes). Differ-ent parameters, such as walking speed, straight-ness of walk, walking activity, and time course ofactivity are extracted from a video record (Gotz1989; Strauss et al. 1992; Strauss and Heisenberg1993). Locomotor activity of flies has also beenextensively analyzed with respect to circadianrhythms. Walking activity in these recordings ismonitored in the range of hours to days and isevaluated for circadian and ultradian oscillations(Konopka and Benzer 1971; for review, see Hall1995). Foraging is another example of locomotion.In this case, the distance away from a feeding sitea larva or adult fly reaches in a certain amount oftime is the parameter of interest (Sokolowski 1980;de Belle and Sokolowski 1987; Pereira and So-kolowski 1993). All these tests consider walkingeither in the time domain of minutes or, as in thecase of the biological clock, days. Here, we presentan analysis of the pattern of walking activity in thetime window of several hours and investigate theinfluence of the MBs on this aspect of behavior.

At present, only Drosophila provides severalindependent noninvasive techniques of blockingthe MB pathway. We use three approaches: chemi-cal ablation of the MB precursor cells; a single genemutant affecting MB development; and targetedgene expression in the MBs. This latter techniquealready mentioned above, takes advantage of en-hancer-trap lines expressing the yeast transcriptionfactor GAL4 (Brand and Perrimon 1993) in groupsof Kenyon cells. In our case, GAL4 drives expres-sion of a transgene for the catalytic subunit of teta-nus toxin (Cnt-E; Sweeney et al. 1995), which isknown to block synaptic transmission in the re-spective neurons by cleaving synaptobrevin, adocking factor for synaptic vesicles (Niemann et al.1994). We show that blocking the MB pathwayinvariably causes an increase in walking activity.Spontaneously walking, MB-deficient flies have dif-ficulty stopping.

Materials and Methods

FLIES

Stocks (D. melanogaster) were maintained at

25°C on a standard cornmeal–molasses medium ina 16-hr light–8-hr dark cycle at 60% humidity. Wild-type strains Canton-S and Berlin (WT-Berlin) andthe mutant mbm1 were used. This mutant was iso-lated by mass histology (Heisenberg and Bohl1979) following ethylmethane sulfonate mutagen-esis and has been back-crossed to WT-Berlin sev-eral times. The P[GAL4] enhancer-trap lines gener-ated as described by Brand and Perrimon (1993)were kindly provided by K. Kaiser (P[GAL4]201Y;Yang et al. 1995) and by T. Raabe (P[GAL4]H24and P[GAL4]17D; H. Pfister and T. Raabe, unpubl.).We used the gene for tetanus toxin light chaininserted on the second chromosome in a Canton-Sgenetic background as a UASGAL4 reporter (calledCnt-E). The Cnt-E stock was kindly provided by C.O’Kane (Sweeney et al. 1995). The three enhancer-trap lines are homozygous viable. Because GAL4-directed tetanus toxin expression was tested inflies heterozygous for both the P[GAL4] andP[UASGAL4–tetanus toxin] constructs, the corre-sponding heterozygous P[GAL4]/Canton-S flies weretested as controls. For hydroxyurea (HU) ablation ofMB neuroblasts we followed the protocol of de Belleand Heisenberg (1994). The HU ablation was per-formed on wild-type Canton-S flies; experimental andcontrol animals were treated strictly in parallel.

MEASURING WALKING ACTIVITY

The apparatus was a transparent rectangularchamber (40 × 3 × 3 mm3) with an infrared lightgate situated in the middle. A moist filter paperpermitted the fly to drink water. Single female fliesat the age of 3 days were tested. Recordings lastedfor 4.5 hr and were conducted in complete dark-ness. The light gate was sampled at a rate of 1 Hz.If the fly had passed the light gate once or severaltimes since the last sampling event the computerrecorded a count (stored as a value of 1); if the flyhad not passed the light gate during that one sec-ond interval the computer stored a value of 0 (nocount). Experiments were performed at 25°C and50%–60% humidity always starting at 6:00 pm. Thedata represent the mean of three recordings per-formed on three successive days. Because therewere no significant differences between record-ings on successive days, data were pooled. In ad-dition, all experiments were performed threetimes at three different periods of the year 1997(May, September, and November). In all threeexperiments, MB-disturbed flies showed elevatedlocomotor activity compared to their controls.

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The data presented are from the November record-ing.

PROCESSING DATA

Data analysis was programmed in C++ (Micro-soft Visual C++). For statistical analysis Statistica(StatSoft, Inc.) was used. Total activity was ap-proximated as the total number of counts for eachfly during the recorded time. For the time course ofactivity, counts were summed for successive 10-min periods. Time intervals between consecutivecounts (intercount intervals; ICIs) were calculatedas the sum of the 0 events between counts. Toreveal the clustering (bout structure) in the timetraces, we calculated for each fly a minimumpause, which is the shortest ICI separating bouts.To obtain the minimum pause we plotted the cu-mulative ICI frequencies (on a logarithmic scale)for increasing ICI duration (log-survivorship curve;Machlis 1977; Slater and Lester 1982; Sibly et al.1990). This curve provided an unambiguous crite-rion for the minimum pause for each fly. To obtainthe number and duration of bouts as well as theduration of the pauses between bouts, the onsetand end of each bout were determined in the timetraces using the individual minimum pauses as acriterion (for a more detailed description of thisanalysis, see J.-R. Martin, R. Ernst, and M. Heisen-berg, in prep.). As is commonly found for events intime series, the parameters extracted were not nor-mally distributed. Therefore, we subjected the datain Figure 5 (B–D) to a log transformation to ap-proximate a normal distribution and then calculatethe means and standard errors of the mean, whichwere weighted to the number of observations foreach fly, as a conservative statistical test (Sachs1992). For a more reliable representation of thebout structure, all bouts consisting of a singlecount were removed together with the subsequentpause. Also the first bout of each fly was omitted.The results are not qualitatively affected by thisprocessing step.

IMMUNOHISTOCHEMISTRY

Immunohistochemistry methods of Buchner etal. (1988) were used. Briefly, Drosophila adultheads were fixed for 4 hr in buffered 4% paraform-aldehyde and washed overnight in 25% sucrose so-lution. Frontal sections were cut on a cryostat mi-crotome at 10 µm thickness and incubated over-

night at 4°C with a monoclonal anti-tetanus toxinantibody (1:10,000). Antibody and unpublished in-formation were kindly provided by J. Thierer andH. Niemann. Biotin–streptavidin coupled to per-oxidase (ABC Kit; Vectastain) with diaminobenzi-dine as the chromogen was used to visualize theprimary antibody.

Results

METHODS OF MB PATHWAY INTERVENTION

Three kinds of intervention were used to gen-erate flies with defective MBs (MB-disturbed flies).One was the use of mutants. We chose the strainmbm1 in the WT-Berlin genetic background. Thedegree of MB neuropil reduction in females of thisstock has been quantitatively determined by deBelle and Heisenberg (1996). The mean volume ofthe calyx was about 10% of that in the WT-Berlincontrol. After that study the structural phenotypein the stock started to revert toward wild type.Therefore, the stock was crossed back to WT-Ber-lin and was reisolated from a single F2 paired mat-ing. In the present line, the mutant phenotype is aspronounced as published previously. Approxi-mately 80% of the animals have no or minute MBson both sides. In the remaining 20%, the sizeranges from small to normal. [Histology was per-formed on mbm1 females of the same vials fromwhich experimental animals were taken. Mean ca-lyx volume over all mbm1 female flies was about10% of that in WT-Berlin (data not shown).]

In the second kind of intervention, MB forma-tion during development was suppressed by feed-ing HU to first instar larvae (Prokop and Technau1994). This treatment leads to a nearly completeloss of MB Kenyon cells in the adult. All HU flieswere subjected to paraffin histology after the be-havioral experiment. Of the 34 flies tested, onlyone had small MBs on both sides. Its performancein the behavioral experiment did not fall outsidethe range of behavioral phenotypes of the remain-ing animals and was included in the mean data.

In the third intervention we applied theP[GAL4] enhancer-trap technique (Brand and Per-rimon 1993). Three lines expressing the transcrip-tion factor GAL4 in subsets of Kenyon cells wereused to up-regulate in these cells the expression ofthe transgene for a tetanus toxin light chain (Cnt-E;Sweeney et al. 1995). One necessary criterion forselecting the lines was that flies had to survive toadulthood in combination with the Cnt-E trans-

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gene. In many MB-specific P[GAL4] lines, this wasnot the case. Even in one of the lines used here(P[GAL4]201Y), introduction of the Cnt-E-trans-gene led to low viability.

The expression patterns of the three lines aredepicted in Figure 1. They differ in the intensity oflabeling in the MBs, in the types of Kenyon cellsexpressing the transgene, and in the staining pat-terns outside the MBs. The line P[GAL4]201Y, al-ready extensively described and used in other stud-ies (O’Dell et al. 1995; Yang et al. 1995; Connolly

et al. 1996), exhibits staining preferentially in theg-lobes whereas the a- and b-lobes are only weaklystained. This pattern is also apparent in cross-sec-tions of the pedunculus. The strong peripheralstaining derives from fibers of the g-lobe (g-fibers)and the small dark core belongs to fibers that causethe diffuse staining in the a- and b-lobe (a/b-fi-bers). Interestingly, the a8-lobe (Ito et al. 1997)which is formed by Kenyon cells of the g-lobe sys-tem is also only weakly stained, suggesting a fur-ther subdivision of Kenyon cell types.

In the P[GAL4]17D line (Pfister 1997) expres-sion of Cnt-E is localized in Kenyon cells of the a-and b- but not the g-lobes (a/b-fibers; no g-fibers).In the pedunculus, the staining is confined to thecentral core (Fig. 1). Caudally, this bundle splitsinto four tracts (not shown), revealing the clonalorigin of the respective Kenyon cells as describedby Ito et al. (1997). In the MBs, the lineP[GAL4]H24 (Pfister 1997) shows an expressionpattern restricted only to the g-lobes. In peduncu-lus cross-sections, faint staining is seen at the pe-riphery, corresponding to the weak staining in theg-fibers. Again, no staining of the a8-lobe is ob-served. In addition, line P[GAL4]H24 exhibits stain-ing in some other parts of the brain. In the centralcomplex (CC) the transgene is expressed in agroup of ring-neurons of the ellipsoid-body (EB)(Hanesch et al. 1989). About 40 cell bodies locatedrostro latero ventrally to the EB can be counted oneither side of the brain. They send their fibers to-ward the EB where they arborize in the peripheryof the ring. In addition, patchy staining is observedin the antennal lobes. The expression patterns ofthe three lines outside the brain and throughoutthe life cycle have not been studied in detail.

LOCOMOTOR ACTIVITY

Flies continuously walk in the tubes from oneend to the other and back for extended periods oftime. Their activity is recorded only at the momentwhen they pass the light gate in the middle of thetube’s long axis. During the interval between con-secutive passages at the light gate they normallywalk to the end of the tube, walk around there asif trying to escape, and finally return. Turningaround before the end of the tube is rare (e.g., Fig.3F, below). Periods of walking are interrupted byperiods of rest for varying lengths of time. The totalnumber of light gate crossings (counts) of each fly,therefore, are taken as its total walking activity(Fig. 2).

Figure 1: Tetanus toxin light chain staining in adultbrains of the three groups of P[GAL4]/Cnt-E flies used inthis study. The tetanus toxin expression is revealed byanti-tetanus toxin antibody. The expression pattern cor-relates well with that of other reporter genes (e.g., b-galactosidase; data not shown). Cryostat frontal sections(10 µm) at the level of the a- and b/g-lobes (left) andpeduncule cross-sections at the level of the CC (right).Line P[GAL4]201Y exhibits an extensive staining in theg-lobe, whereas only faint staining is observed in the a-and b-lobes. In the peduncule, most of the staining isrestricted to an outer ring (g-fibers). The small dot in thecenter may correspond to the immunopositive fibers ofthe a- and b-lobes (a/b-fibers). In line P[GAL4]17Dstaining is found in the a- and b-lobes but not the g-lobes. In the peduncule, staining is restricted to the cen-tral core, absence of staining in outer ring correlates withabsence of g-lobe staining. Line P[GAL4]H24 shows, inthe MBs, a staining only in the g-lobe. The faint stainingrestricted to the periphery of the peduncule correspondsto the g-lobe staining. The difference in the size of thestained patterns between the three P[GAL4] lines is at-tributable to a difference of head sizes.

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The five groups of MB-disturbed flies (mbm1,HU, and the three types of P[GAL4]/Cnt-E flies)present a common effect: Their total walking ac-tivity is significantly increased in comparison to therespective controls. In P[GAL4]201Y/Cnt-E flies ac-tivity is nearly doubled. Note that different controlsare used for all five groups. In Figure 2 (left), weshow the activity of Canton-S to allow a compari-son with the P[GAL4] flies, which are all on a Can-ton-S genetic background. The activity of WT-Ber-lin, which is the appropriate control for mbm1 isslightly lower than that of Canton-S. In the case ofthe HU flies (Canton-S), controls and experimentalanimals were processed strictly in parallel. OnlyHU was omitted for the controls. For the P[GAL4]/Cnt-E flies, the controls are heterozygous for theP[GAL4] transposon, as are the P[GAL4]/Cnt-Eflies. Surprisingly, all three P[GAL4] controls have asignificantly lower activity than Canton-S. Appar-ently, the transposon causes a decrease of locomo-tor activity. Because the three lines have the trans-poson inserted at different locations in the genomethe effect is likely attributable to the P[GAL4] trans-poson itself rather than to position effects.

TIME COURSE OF WALKING ACTIVITY

To explore further the increase in total activ-

ity, its time course was plotted. During the 4.5 hrexperiment activity was determined separately foreach successive 10-min period. Figure 3 shows thedecay of walking activity as a function of time foreach group of flies. Control flies (e.g., Canton-S andHU controls; Fig. 3A–B) exhibit two componentsin the time course: an early phase lasting 60–120min in which the decay of activity is fast and a latephase in which it is slow or reaches a plateau. Thesteep part of the curve represents the fly’s fadingresponse to the new environment or the previoushandling, whereas the plateau is attributable to thefly’s spontaneous activity (Connolly 1967; Meehanand Wilson 1987). All five types of MB-disturbedflies show a smaller decrease of activity with timethan the corresponding controls.

Some differences can be noted in the timecourses of activity in the five kinds of flies. Bothmutant mbm1 and HU-flies (Fig. 3A,B), in whichMBs are almost completely removed, start their ac-tivity at about the same level as the control flies butshow very little attenuation of activity during the firsthour. In P[GAL4]201Y/Cnt-E flies (Fig. 3C), the early,reactive component seems to be largely missing.P[GAL4]H24/Cnt-E and P[GAL4]17D/Cnt-E flies (Fig.3D,E) driving the tetanus toxin in small (but differ-ent) subsets of Kenyon cells show a general upwardshift of the curve over the whole time course.

Figure 2: Total walking activity during 4.5 hr for each group of MB-disturbed flies compared to their respective controls.(CS) Canton-S; (WTB) WT-Berlin; (Con) Control. Total activity is represented as the mean ±S.E.M. over n (number in eachcolumn) flies. For each group, a one-way ANOVA of total activity revealed significant difference (P value below eachgroup column). The P value below the WTB-mbm1 column is for the WT-Berlin and mbm1 comparison only (CS notincluded).



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As an example, histograms of time intervalsbetween successive counts (ICIs) for wild-type Ber-lin and mbm1 flies are plotted in Figure 3F. Be-cause the total activity of each group varies, fre-quencies are expressed as relative values to permita better comparison. As pointed out above, thedecay of the frequency curve toward small inter-vals may be attributable to the special design of the

apparatus. The fly needs a certain time to reach theend of the tube and to come back to the light gate.For intervals above 13 sec, the curve shows thatshort ICIs are frequent and long ones rare.

WALKING ACTIVITY IS CLUSTERED IN BOUTS

Visual examination of the time traces sug-

Figure 3: (A–E) Time course of walking activity for each group of MB-disturbed flies compared to their respectivecontrols. The curves represent the means ±S.E.M. of activity for successive 10-min periods. See legend in each graph. Samedata as in Fig. 2. In F as an example, histograms (plotted as a continuous line) of the relative mean frequency [%; ±S.E.M.(dotted curve)] of ICIs over increasing ICI duration between 1 and 60 sec are shown for mbm1 and its controls.

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gested that the activity was not uniformly or ran-domly spread but was clustered in bouts of activity,as are many other behaviors (e.g., Slater 1974; Ma-chlis 1977). In behavioral analysis, a bout is char-acterized by the repeated and temporally clusteredoccurrences of a behavior. Short intervals betweenthe events occur within bouts; long intervals sepa-rate two successive bouts. As will be described indetail elsewhere (J.-R. Martin, R. Ernst, and M. Hei-senberg, in prep.), we determined whether walk-ing activity under our experimental conditions, in-deed, had a bout structure. For this purpose wecalculated for the ICIs of each fly a so-called log-survivorship curve, which is the cumulative fre-quency of ICIs (on a logarithmic scale) as a func-tion of increasing ICI duration (on a linear scale).The linear part of this function indicates the de-creasing probability for ICIs of increasing durationwithin bouts. At the critical minimum pause defin-ing bouts, the probability abruptly increases abovethe value extrapolated from the linear part of thecurve. In fact, for each single fly this break in thelog-survivorship curve is obvious and allows us todetermine an individual criterion for the minimumpause. Figure 4 shows the histogram of the meanof this minimum pause for each group of flies. Dif-ferences between experimental and control

groups are generally small, with the exception ofP[GAL4]H24.

The determination of the minimum pause foreach fly enabled us to describe walking activity interms of bout structure. The time traces were char-acterized by four parameters, which were partiallydependent on each other: the number of bouts, thenumber of counts per bout, the duration of bouts,and, finally, the duration of interbout pauses. InFigure 5A the histogram of the mean number ofbouts for each group of flies is depicted. As shownin Figures 2 and 3 all MB-disturbed flies have anincrease in total walking activity. This increasemust be reflected somewhere in the pattern of ac-tivity, either in the number of bouts, or in the num-ber of counts/bout, for example, in the intrinsicbout structure. In mbm1, P[GAL4]H24/Cnt-E andP[GAL4]17D/Cnt-E flies, the number of bouts isnot significantly affected compared to the appro-priate controls. HU flies show a significant de-crease, while P[GAL4]201Y/Cnt-E flies show a sig-nificant increase in the number of bouts comparedto their respective controls (Fig. 5A).

In all groups of MB-disturbed flies there is atendency toward an increase in the mean of thenumber of counts/bout (Fig. 5B). This increase ishighly significant for mbm1 and still significant

Figure 4: Minimal pause, determining the bout structure, for each group of MB-disturbed flies compared to theirrespective controls. Minimal pause, in seconds, is represented as the mean ±S.E.M. For each group, a one-way ANOVA wasperformed (P value below each group column). The P value below the WTB-mbm1 column is for the WT-Berlin and mbm1

comparison only.



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(P < 0.05) for HU-flies (see Materials and Methodsfor the different estimates of confidence limits).Because a time series recording is a continuousprocess, the increase in the number of counts/bout has to change the bout structure by oneor both of two possible ways: Either the dura-tion of the bouts is increased while the frequencyof counts is unchanged, or the frequency of countsis increased without an increase in the durationof bouts. Figure 5C shows that bout durationclosely parallels counts/bout. It seems, there-fore, that the increase in total activity is repre-sented by an increase in bout duration, leavingthe frequency of counts unchanged (data notshown).

In all five groups, the duration of the inactiveperiod between two successive bouts (pause)is inversely affected compared to the durationof the bouts (Fig. 5D) although, by the con-servative criterion, the decrease is significantonly for P[GAL4]H24/Cnt-E flies. This inverserelationship between the duration of bouts andthe duration of pauses implies that the periodbetween consecutive starts of bouts is fixed andthat the pause is the dependent variable (i.e.,period − bout duration = pause). The resultsuggests that only bout duration, but neither pe-riod nor pause, is the primary target of MB func-tion.

The defects in locomotor activity, both in theoverall level and in the time structure are not anunspecific side effect of any brain damage. Expres-sion of the tetanus toxin in other parts of the brainby various P[GAL4] enhancer-trap lines not affect-ing MBs did not generate such defects. Mutationsaffecting different substructures of the CC, forexample, no-bridge1 (nob1), ellipsoid-body-open(ebo1, ebo2, ebo3, and ebo4, central-body-defect1

(cbd1) did not cause an increase in locomotor ac-tivity (but showed different locomotion pheno-types; J.-R. Martin, R. Ernst, and M. Heisenberg, inprep.).

Discussion

Recording locomotion of individual Dro-sophila flies for several hours, we have shown herethat MBs act as inhibitors of walking activity. Thethree different and independent methods used toablate or disturb the MBs, all lead to a substantialincrease in total locomotor activity. Surprisingly,the degree of this increase seems not to be cor-related with the number of Kenyon cells affectedby the intervention. In P[GAL4]H24/Cnt-E andP[GAL4]17D/Cnt-E only small fractions of Kenyoncells express the tetanus toxin while the increasein activity over the controls is as large as in mbm1

or HU-flies in which 90% or more of the Kenyoncell fibers are missing. This non-linear relationshipmight indicate that the effect on overall activitydepends on the MB as an integral structure and thata loss of a small number of Kenyon cells alreadyabolishes total MB function. Alternatively, traces oftoxin might invade and block the nonstained fi-bers. However, the interpretation is complicatedby the fact that the P[GAL4] transposons by them-selves seem to have a suppressive effect on walk-ing activity. In comparison to Canton-S, theP[GAL4]201Y/Cnt-E and P[GAL4]H24/Cnt-E flieshave only a small and P[GAL4]17D/Cnt-E flies noincrease of total walking activity.

During the first few hours in the small tubewalking activity of Drosophila gradually dimin-ishes. As pointed out by Connolly (1967), the de-clining component of the activity can be inter-preted as a response to the previous handling and/or the novel situation of the tube. The increasedactivity in MB-disturbed flies is, at least in part,attributable to a slower and less complete attenu-ation of activity. In all five experimental groups,not only the overall attenuation but also the initial(negative) slope of the time course is less steepthan in the respective controls. These two param-eters, amplitude and initial slope of attenuation,correlate better than overall activity with the as-

Figure 5: (A) Mean number of bouts ±S.E.M. for each group of MB-disturbed flies compared to their respective controls.Original data are the same as in Fig. 2. For each group, a one-way ANOVA was performed (P value below each groupcolumn). For HU- and P[GAL4]201Y flies, a significant difference is seen compared to their controls. (B–D) Threeparameters of the bout structure: counts per bout; duration of bouts; and duration of pauses. For statistical analysis, the datawere converted to log scale and the means and standard errors of the means, weighted to the number of observation, werecalculated. For each group, a one-way ANOVA was performed (P value below each group column). The additional Pvalues inside the boxes indicate the statistical significance of the differences with the pooled number of bouts as samplesizes (n). For presentation, the data are reconverted to normal values (note that the S.E.M. is not symmetrically spread onboth side of the mean). Data are the same as in Fig. 2. The P values below the WTB-mbm1 column are for the WT-Berlinand mbm1 comparison only (CS not included).



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sumed degree of impairment afflicted to the MBs.As would be expected from the number of Kenyoncells expressing the toxin, P[GAL4]H24/Cnt-E andP[GAL4]17D/Cnt-E flies are the least affected inthese two parameters of the reactive component.

Our analysis of the data shows that the activityis distributed neither randomly nor regularly withrespect to time. Rather, time is divided into periodsof rest and walking (bouts). The pattern generatorfor this bout structure is not yet known. We sug-gest that the MBs are part of, or have a specificinfluence on, the pattern generator. Although ourdata set is still too small for drawing definitive con-clusions, it provides several interesting observa-tions. The main difference between MB-disturbedflies and controls lies in the duration of the bouts.The number of bouts seems not to be directly af-fected as shown for mbm1 and two of theP[GAL4]/Cnt-E flies in Figure 5A. A significant in-crease is observed only for P[GAL4]201Y/Cnt-E.These flies are also exceptional because they showno increase in bout duration. Their activity patternseems to be influenced by additional defects in thebrain. The reduced number of bouts in HU-flies canbe explained by the extremely large mean boutduration and variance in these flies.

In the present interpretation, the pattern gen-erator consists of three components, bout initia-tion, bout termination, and the regulation of theinternal structure of the bout. If the number ofbouts is not directly affected by the MBs as we havejust argued, their initiation must also be MB inde-pendent. As a consequence, an increase in boutduration entails shortening of the following pause(Fig. 5, cf. C and D). Also, the MBs seem not toinfluence the internal bout structure. Counts perbout closely parallel duration of bouts (Fig. 5, cf. Band C) and in four of the five groups, the minimumpause is little changed (Fig. 4). Therefore, the MBsseem to contribute specifically to bout termina-tion. This does not necessarily imply that the MBsare an integral part of the pattern generator. Forinstance, they might exert their influence by regu-lating arousal, which in turn would influence boutduration.

Our results corroborate and extend the con-clusions drawn by Huber and co-workers (Huber1955, 1960; for review, see Huber 1963, 1965;Otto 1971; Wahdepuhl 1983) from their ablationand stimulation experiments in crickets and grass-hoppers. In their experiments, the animals withMB lesions had an elevated locomotor activity, andsome of them sang until completely exhausted.

These authors also found that injury to the CC in-hibited behavioral activity, and they proposed asimple model in which the MBs suppress and theCC up-regulates (initiates) it. More recently in Dro-sophila, the behavioral analysis of structural brainmutants in combination with genetic mosaics anddeoxyglucose activity staining has confirmed theCC as a pre-motor control center for walking andflight (Strauss et al. 1992; Strauss and Heisenberg1993; Ilius et al. 1994; Bausenwein et al. 1994).Whether the pattern generator for the bout struc-ture of walking activity described here is specifi-cally affected by the CC, and if so in which way,will have to be investigated.

Recently it has been reported that rats withhippocampal lesions walk more than control ratsand these rats also exhibit a perturbation in thepattern of walking activity. Although the hippo-campal formation has projections to the striatum,which is known to modulate locomotion, themechanism by which this lesion-induced changeoccurs remains enigmatic (Whishaw et al. 1995,1997). It will be interesting to find out whethersimilar behavioral modifications are found in micewith genetic lesions in the hippocampus (Mayfordet al. 1996; Wilson and Tonegawa 1997).

Although Kenyon cells have many characteris-tic properties in common and may well be consid-ered an isomorphic set of functionally equivalentneurons, it has long been known that they com-prise different morphological types (for review,see Schurmann 1987). Recently, in Drosophila, theP[GAL4] enhancer-trap technique has permitted aconvenient way to visualize and manipulate suchfiber types (Yang et al. 1995; Ito et al. 1997). In theP[GAL4]H24/Cnt-E flies only a small subset ofKenyon cell fibers of the g-lobe (g-fibers), while inthe P[GAL4]17D/Cnt-E flies only a/b-fibers and nog-fibers express the tetanus toxin gene (Fig. 1).One might expect to find behavioral impairmentsto be defective in only one and not the other ofthese two groups of flies. However, our data pro-vide no evidence that the suppressive effect of theMBs on walking might be associated with only oneand not the other type of Kenyon cells. It is pos-sible that locomotion is similarly affected by thedifferent MB subsystems. Further investigationswith additional P[GAL4] enhancer-trap lines andwith different behavioral impairments will beneeded. Moreover, we did not investigate whetherthe walking performance of individual mbm1 orHU flies might be correlated with their structuralimpairments. No such correlation had been ob-

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served for olfactory learning with mbm1 (Heisen-berg et al. 1985).

The only other MB function demonstrated asunambiguously as the one discussed here is theimpairment of olfactory learning and memory.How are these two behavioral effects related?What do they have in common? Obviously, the an-swer to these questions requires further behavioralexperiments. Connolly et al. (1996) have shownthat Gs signaling in Kenyon cells is necessary forodor learning by expressing a constitutively activeGsa protein subunit in Kenyon cells. Preliminaryexperiments with the same effector gene and thethree P[GAL4] lines of the present study indicatethat Gs signaling is also important for the suppres-sion of walking activity (J.-R. Martin, R. Ernst, andM. Heisenberg, in prep.). In addition to more re-fined methods of intervention, other kinds of be-havioral paradigms need to be exploited.

AcknowledgmentsWe are indebted to K. Kaiser and T. Raabe for

generously providing us the GAL4 enhancer trap-lines and S.Sweeney and C. O’Kane for the UAS-tetanus toxin line(Cnt-E). We are also indebted to J. Thierer and H. Niemannfor the tetanus toxin antibody and to K. Oechsner and H.Kaderschabek for excellent technical support. We areespecially grateful to Markus Reif for helpful discussion onstatistical analysis of the data, and T. Zars for critical readingof the manuscript. J.-R.M. was supported by the PasteurInstitute, Paris, France, and M.H. by grants of the DeutscheForschungsgemeinschaft (He 986) and Fonds der ChemischenIndustrie.

The publication costs of this article were defrayed inpart by payment of page charges. This article must thereforebe hereby marked ‘‘advertisement’’ in accordance with 18USC section 1734 solely to indicate this fact.

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Received January 26. 1998; accepted in revised form April17, 1998.



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