vision affects mushroom bodies and central complex in...

Vision Affects Mushroom Bodies and Central Complex in Drosophila melanogaster Mart in Bar th l a n d Mar t in H e i s e n b e r g Theodor-Boveriqnstitut fiir Biowissenschaften Lehrstuhl for Genetik D-97074 Wt~rzburg, Germany

Abstract

The bra in of Drosoph i la is s tructurally altered by sensory stimuli that the flies receive dur ing their adult life. Size and fiber n u m b e r of the m u s h r o o m bodies, central complex, and optic lobes are influenced by social, spatial, or olfactory cues. Recently, the optic lobes have been shown to depend on the light regime that flies experience. Structural plasticity in the bra in is thought to be a correlate of funct ional adaptat ions and long-term memory . We therefore extend our investigation of volume changes to the calyces of the m u s h r o o m bodies and the central complex. We show that rear ing flies in constant light for 4 days increases the volume of both structures by up to 15% compared to rear ing t hem in total darkness. Much of this difference develops dur ing the first day. The effect of light is not h o r m o n a l l y mediated, as monocu la r ly deprived flies develop a smal ler ipsilateral calyx. Mutant analysis suggests that light generates its effects th rough k n o w n visual pathways. In contrast to the optic lobes, in the calyx and central complex structural changes can be l inked to cAMP signaling, as in the mutants dunce z and a m n e s i a c 1 no volume differences are observed. Surprisingly, the mutan t ru tabaga ~ shows a p r o m i n e n t l ight-dependent volume increase in the calyx and central complex, dissociating structural f rom behavioral plasticity. In complete darkness wild-type flies grow larger calyces under crowded condit ions in their no rma l culture vials than if kept in small groups on fresh food. This st imulat ing effect of crowding is not

1Corresponding author.

observed in any of the cAMP mutants, including ru tabaga 1.

Introduct ion

Animals and man adapt their nervous systems to the changing outside world. The developmental process cannot fine-tune the system by phyloge- netic information alone and, hence, provides the substrate for adjustments by experience. Experi- ence-dependent developmental plasticity in the brain has long been studied in higher vertebrates (Wiesel and Hubel 1963; Bennett et al. 1964; Floeter and Greenough 1979; Tieman and Hirsch 1982), later also in insects (Technau 1984; Bailing et al. 1987; Kral and Meinertzhagen 1989; Fahr- bach and Robinson 1995; Heisenberg et al. 1995; Gronenberg et al. 1996) and is considered to be a structural correlate of long-term memory.

For Drosophila melanogaster we recently demonstrated that rearing in darkness decreases the volume of the optic lobes, especially the lamina and the lobula plate, by up to 30% (Barth et al. 1997b). A dark period as short as 6 hr during the subjective day at the onset of imaginal life has a significant effect, and the optic lobes remain sensitive to dark periods during at least the first 4 days.

Here we investigate whether dark rearing affects two structures in the central brain, the central complex, and the mushroom bodies. The mushroom bodies in particular have received considerable attention because of their role in olfactory learning and memory (Heisenberg et al. 1985; de- Belle and Heisenberg 1994), and structural plasticity (Technau 1984; Balling et al. 1987; Withers et al. 1993; Durst et al. 1994; Fahrbach and Robinson 1995; Heisenberg et al. 1995; Gronenberg et al. 1996). For example, in bees significant volume changes in the calyces are associated with behavioral development and/or foraging experience of the workers (Withers et al. 1993, 1995; Fahrbach

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Barth and Heisenberg

and Robinson 1995). Furthermore, visual stimulation at the time of the first reconnaissance flight may lead to an increase in the collar region, a sub- component of the calyx that receives mainly visual input (Durst et al. 1994; Withers et al. 1995).

In D. melanogaster, flies reared under enriched conditions have more Kenyon cell fibers in the peduncle of the mushroom bodies (Technau 1984; Bailing et al. 1987) and larger calyces (Hei- senberg et al. 1995) than do their deprived siblings. In pairs of flies the volume of the mushroom body calyx depends on the sex of the partner (Heisen- berg et al. 1995), but it has not yet been shown whether, like in the honeybee, visual experience has an effect on the size of the mushroom bodies. This is of particular interest because in contrast to the honeybee, in Drosophila direct fiber connections from the optic lobes to the mushroom bodies have, so far, not been found. Moreover, in no species have the mushroom bodies been shown to be involved in any kind of visual behavior, as yet.

In the present study we investigate (1) whether rearing in different light regimes affects the volume of the calyces and the central complex, and (2) how these structures grow in constant light and constant darkness during the first 2 days of adulthood. In addition, (3), the availability of a large collection of mutants with known behavioral or molecular defects enables us to study some of the underlying mechanisms for such effects. Tak- ing earlier results (Barth et al. 1997b) and the present study together, we (4) present evidence that at least three biochemically distinct molecular mechanisms underlie the volume changes in the brain.

Materials and Methods

ANIMALS AND REARING CONDITIONS

D. melanogaster of the wild-type stock Canton S (WT CS) and the following mutants were used: no receptor potential A P24 (norpAV24), histidine decarboxylase (hdcJk91~ dunce (dncl), rutabaga (rutl), amnesiac (amnl). According to our standard protocol, they were allowed to lay eggs over- night on a petri dish containing 5% sucrose and 3% agar to which some live brewer 's yeast had been added. The next morning, batches o f - 2 0 0 eggs were transferred to each of several 200-ml vials containing 40 ml of standard medium (corn meal, molasses, no fresh yeast) and a filter paper. Cul-

tures were kept in an incubator at 25~ and 60% relative humidity on a 16:8 light/dark cycle. On day 9 flies eclosed and were collected in the morning either (1) within 1 hr of the experiment (see Fig. 2, below) or (2) within 3 hr for all other experiments.

Flies were anesthetized on ice, sexed, and as- signed to an experimental group: either constant light (LL) or constant darkness (DD). In our standard experiment, flies were kept in groups of 10- 20 animals of the same sex; except for the first experiment, only males were used. DD flies were transferred to lightproof boxes that permitted air to flow through and were maintained together with the LL flies in a room with 25~ and 40%-60% humidity; lighting was provided by full-spectrum fluorescent lights (40 W at -40 cm distance, color 25, Universal-Weiss, Osram, Germany) that flick- ered at 20 kHz. Flies were reared under these conditions for between 1 hr and 4 days. If not stated otherwise, 4 day-old flies were used. Each experimental group consisted of 10-20 flies; except for the monocular deprivation study, only one calyx per fly was measured (n = number of flies).

In one experiment (Fig. 5, below), flies were kept for 4 days after eclosion in the vials were they had developed and were placed either in DD or LL conditions. They had contact with at least 200 other flies of the same and the opposite sex and, because of the age of the culture, were supplied with severely altered food. With this treatment we hoped to create an environment that was dramati- cally different from the standard rearing conditions described above, where only up to 20 males live in a fresh vial.

In experiments with monocularly deprived flies, one eye (either left or right) was painted with an opaque, black, water-soluble paint (Deka-Lack no. 318, black) within 1 hr after eclosion. This layer absorbs -99.9% of the light (Barth et al. 1997b) and destroys the optics of the compound eye. In all experiments except for the developmental study, fly collection began in the morning between 9:30 and 11:00.

MASS HISTOLOGY

Collected flies were processed for mass histology. Paraffin sections were inspected by fluores- cence microscopy, and volumes of the neuropil regions were evaluated by planimetric measure- ment of their autofluorescent profiles (Heisenberg

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VISION AFFECTS CENTRAL BRAIN IN DROSOPHILA

et al. 1995). For the analysis of the central complex, three neuropi l regions, noduli, ell ipsoid body, and fan-shaped body, were measured together; the protocerebral bridge was omitted.

Flies that unde rwen t different t reatments or mutant and wild-type flies, were arranged in ran- dom order in collars (Heisenberg and B6hl 1979) to evaluate brain sizes wi thout knowing the rearing condi t ions or genotype of the respect ive flies. We carefully fol lowed our standard protocol in any respect to keep larval rearing condi t ions and the histological t rea tment as constant as possible, but still found considerable variation b e t w e e n different exper iments in the volume of the two structures. Therefore, our standard 4-day visual deprivat ion expe r imen t wi th WT CS flies was part of the de- sign of each exper imen t to verify that our manipu- lations and techniques remained effective. Addi- tionally, each expe r imen t was at least repeated

once. To test the status of our mutant lines, the three

learning mutants (dnc*, rut*, and amn*) were tested again in a n e w learning paradigm (Wust- m a n n et al. 1996), w h e r e they failed to show sig- nif icant learning coefficients.

Results

VISUAL EXPERIENCE AFFECTS THE VOLUME OF THE CALYCES

To investigate w h e t h e r visual exper ience during adul thood ult imately affects the volume of the calyces of the m u s h r o o m bodies, we monocular ly bl indfolded newly eclosed flies by paint ing over one eye. The animals were then kept for the nex t 4 days in constant light (LL). To control for possible effects of the paint itself, a second set of flies was kept in total darkness (DD).

In both females and males we found a significantly larger vo lume of the calyx on the side of the open eye than on that of the covered one (t = 2.6, P < 0.05; paired t-test; males and females combined; Fig. 1). During the course of this study, we conduc ted - 1 0 exper iments , each wi th - 1 0 - 1 5 animals. In total, the vo lume of the calyx on the side of the open eye was -7% larger than on the opposi te side. In the control animals, in w h i c h bo th sides did not receive any visual input, no such differences could be found (t = 0.1, P > 0.9; paired t-test, males and females combined; Fig. 1). Thus,

40 I[ - - occluded eye 6" I E= 3o - r -

0 O0 LL O0 LL

WT CS Females INT CS Males

Figure 1: Effects of monocular deprivation and rearing in darkness on the volume of the calyces in female (left) and male (right) WT CS flies. Underneath the open eye in the light (open bars) the volume was significantly larger than underneath the occluded eye in the light (striped bars). Keeping flies in complete darkness, however, had an even more volume-reducing effect (black bars) (females: DDD = 16, nLu = 13; males: nDD = 23, riLL = 23).

the differences b e t w e e n the two sides observed in the light must be ascribed to the differential visual input the flies received during the first 4 days of their adult life. Because there were no obvious sex differences in the magni tude of the effect, we used only male flies in all subsequent exper iments .

The flies kept in total darkness (DD), originally considered to be the control group, unexpec ted ly exhib i ted even more robust effects of visual deprivation during the course of the exper iments (see Barth et al. 1997b). Whereas the difference between the calyces on the side of the occluded eye and on the opposi te side was only as m u c h as 8%, the differences b e t w e e n the calyces in LL and DD flies was as m u c h as 15% (for females, F - 3.6, P < 0.05, ANOVA; for males, F - 3 . 5 , P < 0.05, ANOVA). This d iscrepancy can ei ther be ascribed to the fact that some light (at least 0.1%) is still reaching the occluded eye, or by the assumption that some of the st imulation ( " expe r i ence" ) provided by the open eye reaches the contralateral calyx.

Taken together, these results indicate that the calyces were changed in volume as a funct ion of the visual input they received during adulthood. W h e n animals raised in LL or DD condit ions were compared, the effects were more p rominen t as opposed to compar ing the depr ived and undepr ived side in the same flies. Therefore, we omit ted paint- ing the eyes but kept the flies under LL or DD condi t ions instead.

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EFFECTS OF LIGHT ON THE DEVELOPMENT OF THE CALYCES AND CENTRAL COMPLEX IN ADULT FLIES

So far w e have shown that visual deprivation affects the vo lume of the calyces in the adult fly. Do these changes derive from an increase in volume in the light as in the optic lobes (Barth et al. 1997b) or f rom a vo lume loss in darkness? Is light necessary for the adult calyx to develop and grow, or does it start large at eclosion and diminish with- out light?

To dist inguish b e t w e e n these possibilities we reared flies under LL or DD condit ions and sacri- f iced them for histology at different t imes after eclosion. In this expe r imen t we also measured the volume of the central complex. Irrespective of the light regime, the calyces increased in volume during the first 6 hr after eclosion Thereafter, they increased in size for another 6 hr in LL flies (F = 5.6, P < 0.001, ANOVA), whereas in DD flies the calyx volume leveled off and remained about constant for the rest of the 48-hr measur ing per iod (F - 3.1, P < 0.05, ANOVA). Thus, as early as 9 hr posteclosion, a difference b e t w e e n LL and DD flies was apparent , and after 12 hr in both groups of flies the calyx had reached its final size. The volume difference b e t w e e n LL and DD flies varied somewha t for the four samples b e t w e e n 9 and 48 hr, but taking all data together, LL flies had a significantly h igher calyx volume than DD flies did (data f rom 9-48 hr posteclosion pooled: t = 2.9, P < 0.005, t-test; Fig.2A).

A different t ime course could be observed for the deve lopment of the central complex. The light- i ndependen t increase in vo lume ex tended over the entire 48-hr per iod (for LL flies, F = 4.1, P < 0.005, ANOVA; for DD flies, F = 4.8, P < 0.0005, ANOVA; Fig. 2B), but as in the case of the calyx, the increase was faster in the light. The difference b e t w e e n the two rearing condit ions appeared even earlier than for the calyx. It was already significant at 6 hr after eclosion (t = 2.6, P < 0.05, t-test) and stayed largely constant for the rest of the 2 days.

Taken together, both the calyces and the central complex increased in size during the 2 days of the exper iment . Interestingly, the calyx grew only for the first 12 hr of the exper iment , whereas the central complex kept growing during the entire exper imenta l period. Both structures clearly showed l ight-dependent and l ight- independent growth. In the calyx the l ight- independent growth was conf ined to the first 6 hr of imaginal life, in the

A

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Age after eclos~on (hours)

E o

ii

111 .... t0 ~i/~'•

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3 6 9 12 24 48

Age after eclos,on (hours)

Figure 2: Development of the calyces and the central complex during the first 48 hr after emergence. (A) The calyces increase in volume during the first 6 hr in both groups; thereafter LL flies keep increasing fr iLL = 17 (1 hr), 10 (3 hr), 9 (6 hr), 19 (9 hr), 8 (12 hr), 8 (24 hr), 9 (48 hr); RDD = 19 (] hr), 10 (3 hr), 9 (6 hr), 19 (9 hr), 9 (12 hr), 8 (24 hr), 11 (48 hr)]. (B) In contrast, the central complex increases during the entire 48-hr period and the first significant difference between LL (�9 and DD (0) flies can be observed at 6 hr posteclosion (same animals as in A).

central complex no l ight- independent growth was apparent in the first 12 hr but it was significant b e t w e e n 12 and 48 hr. Light-dependent growth occured in the calyx b e t w e e n 6 and 12 hr, and slightly earlier in the central complex , b e t w e e n 3 and 9 hr after eclosion.

DOES LIGHT EXERT ITS EFFECTS THROUGH THE VISUAL SYSTEM?

Because paint ing one c o m p o u n d eye had a differential effect on the two calyces and, as dis- cussed previously (Barth et al. 1997b), also on the two optic lobes, we assumed that the effect was

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media ted by the c o m p o u n d eyes. To test this sug- gestion we studied the effect of the light regime on the vo lume of the calyx and the central complex in two bl ind mutants. One of them, n o r p A e24, is de- fective in a phosphol ipase C (PLC), an enzyme catalyzing an essential step of the phototransduc- t ion cascade. This gene is preferential ly expressed in the c o m p o u n d eyes. No volume differences between LL and DD flies were detected for the calyces (t = 1.0, P > 0.1, t-test; Fig. 3A) and the central c o m p l e x (t = 0.4, P > 0.5, t-test; Fig. 3B).

In the second mutant, b d c jk910 histamine, the

main neurot ransmit ter of pho torecep to r cells in flies (Hardie 1987) is not synthesized. Melzig et al. (1996) have shown that in h d c jk910 synaptic trans-

mission b e t w e e n the retinula cells of the com- p o u n d eye and the large monopola r cells of the lamina is blocked. In an earlier study (Barth et al. 1997b) w e had found that the volume changes in

A

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B

WT CS hdc jkez~ norpA

~'E 125[

~ lO0

~ 75 ,

-~ 50 o i -~ 25 I j e-

~ 0 NT CS hcl~ k910 norloA

Figure 3: Effects of rearing in DD or LL regimes for WT CS flies and the mutants hdc 2k91~ and norpA m4. (A) The two mutants lack volume changes in the calyces, but the WT CS flies show a significant difference between LL (open bars) and DD (solid bars) flies (WT CS: nDD = 1 7, nLL = 11; hdcJkg~~ nDD = 11, nLL= 12; norpAP24: nDD = 15, nLL= 17). (B) In the central complex, however, the hdc 2k91~ mutant shows the difference between DD and LL flies as does the WT CS, but not the norpA z24 mutant (same flies as in A).

the lamina of this mutant strain were still present, w h i c h was in l ine wi th the observat ion that these changes were contr ibuted primari ly by the presyn- aptic pho torecep to r endings. In line wi th a block in synaptic t ransmission in the lamina, no light- dependen t volume increase was found in the lobula plate of h d c Jk91~ flies, suggesting that this structural plasticity was related to visual experience. In this study we did not find a significant difference b e t w e e n the calyces of LL and DD flies (t = 0.8, P > O. 1; WT CS control, t - 3.7, P < 0.005, t-tests; Fig. 3A). Surprisingly, however , the volume of the central c o m p l e x was still inf luenced by light in the mutant ( t - 2.6, P < 0.05; WT CS control, t - 1.6, P > O. 1, t-tests; Fig. 3B). This may indicate that h d c jk910 is not comple te ly bl ind and that the

residual level of synaptic t ransmission in the lamina is sufficient for the central complex but not the calyces to p roduce the l ight-dependent extra growth during early adulthood. We did not detect any his tamine in bdc jk910 m u t a n t flies using

immunohis tochemis t ry and an anti-histamine anti- body, but this t echnique is not sensitive to low levels of the antigen. The mutant might be a strong h y p o m o r p h instead of a null, h is tamine might be provided by other metabol ic pathways, or it might be taken up from the food (Melzig et al.

1996). In any case, the main photot ransduct ion cas-

cade in D r o s o p h i l a seems to be necessary for both the plasticity in the calyces and the central complex. On the other hand, only the l ight-dependent growth of the calyces depends on a proper ly func- t ioning his taminergic pathway.

INVOLVEMENT OF THE cAMP CASCADE

Meanwhile, it is wel l established that the cAMP signaling cascade is involved in learning and m e m o r y (for review, see Davis 1996). In an earlier study of structural plasticity in the D r o s o p h i l a

brain, it has also been implicated in the regulation of fiber n u m b e r in the pedunc le of the m u s h r o o m body (Bailing et al. 1987). We therefore investi- gated three of the wel l -known learning mutants: d n c 1 and r u t 1 (Davis 1996) wi th defects in the cAMP signalling cascade, and a m n ~ (Feany and Quinn 1995), w h i c h is thought to be involved in this pa thway as well.

As repor ted earlier (Barth et al. 1997b), none of the three mutat ions interfered wi th the light- dependen t volume changes in the optic lobes, in

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l ine wi th the low Dnc-like immunoreact iv i ty in these structures (Nighorn et al. 1991). We were therefore curious to see w h e t h e r they would affect the vo lume changes in the central brain. The mutants d n c ~ and a m n ~ did not show l ight-dependent g rowth regulation of the calyces or the central c o m p l e x (for the calyces: d n c ~, t - 0.5, P > 0.5; a m n ~, t = 0.4, P > 0.5; t-tests; for the central complex: d n c 1, t = 0.7, P > 0.5; a m n 1, t - 0.6, P > 0.5, t-tests). Surprisingly, however , in the third mutant, r u f f , the calyx and the central complex were larger in LL than in DD flies (calyx: t - 4 . 0 , P < 0.001; WT CS control, t = 3.7, P < 0.005; central complex: t = 3.0, P < 0.05; WT CS control, t = 2.3, P < 0.05, t-tests; Fig. 4). This effect was robust and of the same magni tude as in the WT CS controls.

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B 1 5

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Figure 4: Effects of rearing under DD and LL conditions in WT CS (DDD = 12, nL, : 10) and in three learning and memory mutants [dnc I (nDD = 12, n u = 10), a m n I (DDD = 10, riLL -- 10); ruff (DDD -- 1 1, riLL = 10)]. (A) In the WT CS and rut I flies, the calyces exhibit significant volume differences between DD (solid bars) and LL (open bars) flies, whereas the two other mutants do not. (B) For the central complex, the same pattern Can be observed (same flies as in A).

REGULATION OF CALYX SIZE BY SOCIAL CUES IS ABOLISHED IN r u t l

In earlier studies of exper ience-dependent structural plasticity in the D r o s o p h i l a brain (Technau 1984; Bailing et al. 1987; Heisenberg et al. 1995), flies raised singly in small food vials (deprived envi ronment) were compared to flies living in large bisexual groups in flight cages (enr iched environment) . Therefore, the stimuli responsible for the effects were difficult to assess. They could be social, spatial, or olfactory cues. This difficulty was one of the reasons to exper imen t wi th light in this study. Technau (1984) had shown that even in the dark the flight cage was an enr iched environment, but he did not measure w h e t h e r light alone had an effect. In the study of Balling et al. (1987) both mutants d n c 1 and ru t 1 were shown to lack the effects of the depr ived and enr iched environments on the n u m b e r of Kenyon cell fibers. Be- cause these complex envi ronmenta l situations also affect the size of the calyx (Heisenberg et al. 1995) we wan ted to know w h e t h e r the calyx in r u t 1 mutant flies still r esponded to the types of exper ience provided by the enr iched vs. depr ived environments. We therefore reared wild-type and mutant d n c 1, r u t 1, and a m n I flies ei ther in small groups of

-15 males under LL and DD condit ions as in the above exper iments , or in large bisexual groups of >200 flies, again under the two condit ions (LL/ DD). In the dark, wild-type flies in the large bisexual groups grew larger calyces than the flies in the small male groups (t = 2.1, P < 0.05, t-test). This exper ience-dependen t effect in DD flies was missing in all of three mutants, including r u t 1

(P > 0.5 for all three mutants; Fig. 5). Under constant light (LL), no difference in calyx size b e t w e e n flies f rom the small and large groups could be detected ei ther in wi ld type or mutants (data not shown).

D i s c u s s i o n

Our interpretat ion of the present results on central brain structures and the earlier one on the optic lobes (Barth et al. 1997b) is summarized in Figure 6. Visual stimuli enter the system via the photoreceptors of the c o m p o u n d eyes and from there exert the i r effects on subsequent neuropi l regions of. the brain. Effects are largest in the lamina. For flies raised in constant light the lamina volume is up to 30% larger than for flies raised in

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30 R"

20

>o

x lO

m (.3

~ "deprived" ~ "enriched"

NT CS dnc amn rut

Figure 5: WT CS DD flies that are kept under enriched conditions increase the volume of the calyces compared to flies kept under deprived conditions (ndeprived = 19, ner~r~che d = 24). In all three learning and memory mutants this experience-dependent volume increase is missing

(dncl--ndeprived = 12, nenriched = 14; amnl--ndeprived = 10, nenriched = 11 ; rutl--ndeprived = 11, nenriched = 13).

constant darkness. In flies raised in a LD cycle the lamina volume is intermediate. In the lamina the photorecep tor terminals are responsible for >80% of the volume increase, and the monopola r cells only contr ibute a minor fraction. Volume differences in the medul la are considerably smaller than in the lamina and may be contributed, to a large extent, by the R7/8 pho torecep to r endings. No size differences b e t w e e n LL and DD flies were observed in the lobula, but in both the medul la and lobula, size changes have been observed wi th other types of exper ience .

The mutant norpA P24, w h i c h is supposed to be comple te ly bl ind but still can sense temperature, humidity, odors, and CO 2, serves as an important control. Its brain develops the same in constant light and in total darkness, indicat ing that in the wi ld type it is vision and not some undetec ted sec- ondary stimuli, such as l ight-dependent growth of microorganisms or consis tency of the food, that ult imately cause the size differences. A proper ly funct ioning photo t ransduct ion cascade seems to be indispensable. The only reservation to this con- clusion comes from the fact that the norpA gene has other funct ions besides that in vision. How- ever, none of the other sensory modali t ies seem to be complete ly b locked (Riesgo-Escovar et al.

1995). As may be expected, a block in synaptic trans-

mission be tween the pho torecep tor cells and the large lamina monopola r cells as in hdc jk91o does

not abolish the volume difference b e t w e e n LL and DD flies in the lamina. We take this effect to be entirely presynapt ic and a s sume that no differences for the monopolar cells b e t w e e n LL and DD

flies would be found. In line wi th this notion, no significant differences b e t w e e n LL and DD flies in the lobula plate (Barth et al. 1997b) and the calyces of b d c jkglO m u t a n t flies are observed.

It is quite unexpec ted , therefore, to find a l ight-dependent size difference in the central com- p lex of b d c jk910 flies. Considering that in the mutant norpA e24 no such effect is observed we pro- pose that in contrast to these animals, bdc jkglO

flies are not comple te ly bl ind and that the remain- ing visual exper iences are sufficient for the light- dependen t deve lopment of the central complex but not of the other structures tested. This may be a threshold effect. If, for instance, the volume difference in the central c o m p l e x would reflect the r ichness of the pract iced behavioral repertoire, a marginal level of visual information might be suffi- c ient to activate it. On the other hand, the size of the lobula plate and calyx might be related more directly to the salience of sensory stimulation. Al- ternatively, the central complex might receive visual informat ion by a hi therto u n k n o w n pa thway not dependen t on his tamine as neurotransmitter .

The most striking result of the present investigation is the direct effect of visual exper ience on the calyces of the m u s h r o o m bodies. Our finding that each eye has a stronger inf luence on the ipsilateral calyx than on the contralateral one seems to indicate that this inf luence is exer ted by neural circuitry rather than hormonal control. In Dro- sophila, direct connect ions from the optic lobes to the m u s h r o o m bodies have not been reported yet, and evidence for the process ing of visual informa- t ion in these structures has been missing so far. This is different for other insect species such as honeybees , w h e r e fiber connect ions are k n o w n to project f rom the optic lobes to the calyces (Mobbs 1982) and visual input to the calyces has been demonstrated (Fahrbach and Robinson 1995). More- over, visual exper ience has been suggested to play a role in volume changes in the calyces that are observed about at the t ime of the first orientat ion flight of the workers (Durst et al. 1994).

Our results f rom the three cAMP-related mutant lines point to at least three b iochemical ly dis- t inct processes under lying the volume changes:

1. For the observed l ight-dependent regulation of neuropi l size in the lamina and lobula plate cAMP signaling does not have an important role (Barth et , al. 1997b). It is tempt ing to speculate that the under lying funct ional changes should be i ndependen t of learning and memory. In line

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Barth and He isenberg

nther Sensory

Stimuli

tperience"

V i s u a l ~ - ~ '

Optic Lobe Central Brain

Figure 6: Schematic drawing of the Drosophila brain summarizing the structural changes in the optic lobe and the central brain as a consequence of visual stimulation (1-4) and other sensory stimuli or experience (5). Mutants that block structural plasticity in these neuropils are (I) norpAP24; (2) norpA P24 and hdcJk91~ (3) norpA Pc4, dnc 1, amn 1, but not rut I and hdcJk91~ (4) norpA P24, hdc jk91~ dnc 1, amn 1, but not rut1; (5) dnc 1, amn 1, and rud (norpA e24 and hdc jkgz~ were not tested). (CE) Compound eye; (La) lamina; (dM) distal medulla; (pM) proximal medulla; (Lo) Lobula; (Lp) Lobula plate; (MB) mushroom body; (Cal) calyx; (P) peduncle; (CC) central complex; (FB) fan-shaped body; (EB) ellipsoid body; (N) noduli.

with this assumption, we have recently found that wild-type LL and DD flies show robust differences in optomotor behavior (M. Barth, un- publ.). It is also possible that some kind of cAMP-independent visual learning underlies these changes.

2. In the calyx and central complex, however, cAMP seems to be important, as evidenced by the interference of the d n c I and a m n 1 mutations with the light-dependent extra growth. The observation that a mutation in the adenylate cyclase (rut/) does not abolish this regulation might indicate that the regulation requires only low levels of cAMP or alternatively, that a different adenylate cyclase is involved (Levin et al. 1992). This finding distinguishes the increases in volume observed here from the plasticity in the number of Kenyon cell fibers of the peduncle (Balling et al. 1987). Interestingly, the adenylate cyclase of the r u t gene is found at high concentrations in the peduncle but not in the calyx itself (Han et al. 1992). A similar dis- tribution has been reported recently for a dopamine (D2) receptor proposed to stimulate the r u t cyclase (Han et al. 1996; Davis 1996).

3. The third type of structural plasticity is the increase in the size of the calyx because of crowding (Fig. 5). All three mutations including r u t /

interfere with this regulation. In this experiment the different experiences of flies in small all-male groups and in large bisexual groups of >200 animals may resemble those in the deprived and enriched environments used by Bail- ing et al. (1987). In this study in which Kenyon cell fibers in the peduncle were counted, the effect was abolished by mutations in both genes, d n c and rut. Therefore, the 8% size increase in the calyx because of crowding observed in our experiment (Fig. 5) may also reflect, at least in part, an increase in the number of Kenyon cells.

Thus, in mushroom bodies two different cellular mechanisms of structural plasticity seem to be present: one based on volume changes; the other on the outgrowth and retraction of Kenyon cell fibers (Balling et al. 1987). The case of r u t further suggests that the two structural processes may have, to some degree, different underlying molecular mechanisms.

The distinction of the three mechanisms of structural plasticity by biochemical criteria is further supported by the growth dynamics of the optic lobe (Barth et al. 1997b) and the central brain (Fig. 2). Whereas the lamina and the lobula plate increase in size only when light is present as a

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stimulus, the mushroom bodies and the central complex grow in the absence of right during the first hours after eclosion. This indicates that their development depends in part on other stimuli than light and suggests that the molecular and cellular machinery underlying these changes may be different as well.

Do the structural changes have functional consequences? The present approach does not answer this question and for each individual structure this question may be open. The basic principle underlying the phenomena under study seems to be that some neural structures, if used, increase in size until some new equilibrium is reached. For instance, the retinula cell endings in the lamina increase rapidly in response to light for a limited time early in life (Barth et al. 1997b). Their conical shape in the light (as opposed to the more cylin- drical one in darkness) may just reflect the time- averaged local current density in the membrane, which should decline from distal to proximal in the lamina and might not have any functional consequences (Hill et al. 1994).

On the other hand, this assumption is not very plausible. For instance, the right regime also changes the number of synaptic contacts between retinula cells and monopolar lamina neurons in large flies (Kral and Meinertzhagen 1989), and as mentioned above, in Drosophila, other riving conditions affect the number of Kenyon cell fibers in the mushroom bodies (Technau 1984). From what is known about nervous systems, it would be the exception rather than the rule if structural changes had no functional consequences. Moreover, for several years data are now accumulating that dem- onstrate massive behavioral consequences of the very rearing conditions that cause the structural changes (Hirsch et al. 1990). For instance, flies kept for 4 days in one of several light regimes (LL, DD, LD) prefer mates that were raised under the same conditions (Barth et al. 1997a; see also Hirsch and Tompkins 1994; Hirsch et al. 1995). A similar mate preference has been observed for flies that were either raised solitary or in groups of 50 animals (Ellis and Kessler 1975; see Heisenberg et al. 1995 for the corresponding structural effects in the brain).

The cAMP-sensitive volume differences in the calyx and central complex invite speculations that they are at least in part related to learning and memory. First, a structural correlate for the ability to learn and remember is suggested by the failure of all three learning and memory mutants to in-

crease calyx volumes under enriched conditions (Fig. 5). Second, Guo et al. (1996) showed recently that learning ability in a visual discrimination task that is impaired in central complex mutants (Weidtmann 1993) increases during the first 2 days of adult life. It may be more than a mere coinci- dence that during this whole period the size of the central complex increases as well (Fig. 2). In contrast, the light-dependent extra growth of the calyx and central complex is confined to a strikingly short period early in life. Either the capacity for this type of growth is quickly exhausted or the flies' visual experiences in the food vials have little novelty value after that initial period.

We have little doubt that the structural changes in the central brain observed here are closely related to behavioral adaptations. As shown previously (Barth et al. 1997b), 6 hr of darkness during the first day is enough time to still manifest in the volume of the lamina after 4 more days in constant light. Behavioral exposures may have de- tectable structural consequences in the brain as well. For instance, spaced training in a defined learning task can be extended over a period of several hours (Tully et al. 1994). In addition, the structural changes might be better characterized in specially selected enhancer trap lines (deBelle 1995; Yang et al. 1995). Such experiments would therefore ultimately serve the goal of understand- ing the problem of memory formation and its structural basis.

Acknowledgments This research was supported by grants from the German

Science Foundation (DFG) and the Graduiertenkolleg Arthropodenverhalten to M.B. and grant He986 of the DFG to M.H. We thank M. Reif for critically reading the manuscript and for many hours of fruitful discussions.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.

References Bailing, A., G.M. Technau, and M. Heisenberg. 1987. Are the structural changes in the adult Drosophila mushroom bodies memory traces? Studies on biochemical learning mutants. J. Neurogenet. 4" 65-73.

Barth, M., H.V.B. Hirsch, and M. Heisenberg. 1997a. Rearing in different light regimes affect courtship behaviour in Drosophila melanogaster. Anim. Behav. 53" 25-38.

Barth, M., H.V.B. Hirsch, I.A. Meinertzhagen, and M.

& 227






Heisenberg. 1997b. Experience-dependent developmental plasticity in the optic lobe of Drosophila melanogaster. J. Neurosci. 17" 1493-1504.

Bennett, E.L., M.C. Diamond, D. Krech, and M.R. Rosenzweig. 1964. Chemical and anatomical plasticity of brain. Science 146" 610-619.

Davis, R.L. 1996. Physiology and biochemistry of Drosophila learning mutants. Physiol. Rev. 76:299-317.

deBelle, J.S. 1995. Drosophila mushroom body subdomains: Innate or learned representations of odor preference and sexual orientation? Neuron 15: 245-247.

deBelle, J.S. and M. Heisenberg. 1994. Associative odor learning in Drosophila abolished by chemical ablation of mushroom bodies. Science 263: 692-695.

Durst, C., S. Eichm~iller, and R. Menzel. 1994. Development and experience lead to increased volume of subcompartments of the honeybee mushroom body. Behav. Neural Biol. 62: 259-263.

Ellis, L.B. and S. Kessler. 1975. Differential post-eclosion housing experiences and reproduction in Drosophila. Anim. Behav. 23" 949-952.

Fahrbach, S.E. and G.E. Robinson. 1995. Behavioral development in the honey bee: Toward the study of learning under natural conditions. Learn. & Mem. 2: 199-224.

Feany, M.B. and W.G. Quinn. 1995. A neuropeptide gene defined by the Drosophila memory mutant amnesiac. Science 268: 869-873.

Floeter, M.K. and W.T. Greenough. 1979. Cerebellar plasticity: Modification of Purkinje cell structure by differential rearing in monkeys. Science 206: 227-229.

Gronenberg, W., S. Heeren, and B. HOlldobler. 1996. Age-dependent and task-related morphological changes in the brain and the mushroom bodies of the ant Camponotus floridanus. J. Exp. Biol. 199:2011-2019.

Guo, A., L. Li, X. Shou-zhen, F. Chun-hua, R. Wolf, and M. Heisenberg. 1996. Conditioned visual flight orientation in Drosophila: Dependence on age, practice, and diet. Learn. & Mem. 3: 49-59.

Han, P.L., L.R. Levin, R.R. Reed, and R.L. Davis. 1992. Preferential expression of the Drosophila rutabaga gene in mushroom bodies, neural centers for learning in insects. Neuron 9:619-627.

Han, K.A., N.S. Millar, M.S. Grotewiel, and R.L. Davis. 1996. DAMB, a novel dopamine receptor expressed specifically in Drosophila mushroom bodies. Neuron 16" 1-20.

Hardie, R.C. 1987. Is histamine a neurotransmitter in insect photoreceptors? J. Comp. Physiol. A 161 : 201-213.

Heisenberg, M. and K. B6hl. 1979. Isolation of anatomical brain mutants of Drosophila by histological means. Z. Naturforsch. 34c: 143-147.

Heisenberg, M., A. Borst, S. Wagner, and D. Byers. 1985. Drosophila mushroom body mutants are deficient in olfactory learning. J. Neurogenet. 2" 1-30.

Heisenberg, M., M. Heusipp, and T. Wanke. 1995. Structural plasticity in the Drosophila brain. J. Neurosci. 15" 1951-1960.

Hill, A.A.V., D.H. Edwards, and R.K. Murphey. 1994. The effect of neuronal growth on synaptic integration. J. Computational Neurosci. 1" 239-254.

Hirsch, H.V.B. and L. Tompkins. 1994. The flexible fly: Experience-dependent development of complex behaviors of Drosophila melanogaster. J. Exp. Biol. 195" 1-18.

Hirsch, H.V.B., D. Potter, D. Zawierucha, T. Choudhri, A. Glasser, R.K. Murphey, and D. Byers. 1990. Rearing in darkness changes visually-guided choice behavior in Drosophila. Visual Neurosci. 5" 281-289.

Hirsch, H.V.B., M. Barth, S. Luo, H. Sambaziotis, M. Huber, D. Possidente, H. Ghirardella, and L. Tompkins. 1995. Early visual experience affects mate choice in Drosophila melanogaster. Anim. Behav. 50- 1211-1217.

Kral, K. and I.A. Meinertzhagen. 1989. Anatomical plasticity of synapses in the lamina of the optic lobe of the fly. Phil. Trans. R. Soc. Lond. B Biol. Sci. 323" 155-183.

Levin, L.R., P.-L. Han, P.M. Hwang, P.G. Feinstein, R.L. Davis, and R.R. Reed. 1992. The Drosophila learning and memory gene rutabaga encodes a Ca2+/ calmodulin-responsive adenyly cyclase. Cell 68: 479-489.

Melzig, J., S. Buchner, F. Wiebel, R. Wolf, M. Burg, W.L. Pak, and E. Buchner. 1996. Genetic depletion of histamine from the nervous system of Drosophila melanogaster eliminates specific visual and mechanosensory behaviour. J. Comp. Physiol. A 179" 763-773.

Mobbs, P.G. 1982. The brain of the honey bee Apis mellifera. The connections and spatial organization of the mushroom bodies. Phil. Trans. R. Soc. Lond. B Biol. Sci. 298: 309-354.

Nighorn, A., M.J. Healy, and R.L. Davis. 1991. The cyclic AMP phosphodiesterase encoded by the Drosophila dunce gene is concentrated in the mushroom body neuropil. Neuron 6: 455-467.

Riesgo-Escovar, J., D. Raha, and J.R. Carlson. 1995. Requirement for a phospholipase C in odor response: Overlap between olfaction and vision in Drosophila. Proc. Natl. Acad. Sci. 92: 2864-2868.

Technau, G. 1984. Fiber number in the mushroom bodies of

& 228






adult Drosophila melanogaster depends on age, sex and experience. J. Neurogenet. 1 �9 113-126.

Tieman, S.B. and H.V.B. Hirsch. 1982. Exposure to lines of only one orientation modifies dendritic morphology of cells in the visual cortex of the cat. J. Comp. Neurol. 211 : 353-362.

Tully, T., T. Preat, S.C. Boynton, and M. Del Vecchio. 1994. Genetic dissection of consolidated memory in Drosophila. Cell 79: 35-47.

Weidtmann, N. 1993. "Visuelle Flugsteuerung und Verhaltensplastizit~t bei Zentralkomplex-Mutanten von Drosophila melanogaster]." pH.D. thesis. W~rzburg University, W~rzburg, Germany.

Wiesel, T.N. and D.H. Hubel. 1963. Effects of visual deprivation on morphology and physiology of cells in the cat's lateral geniculate body. J. Neurophysiol. 26" 978-993.

Withers, G.S., S.E. Fahrbach, and G.E. Robinson. 1993. Selective neuroanatomical plasticity and division of labour in the honeybee. Nature 364: 238-240.

�9 1995. Effects of experience and juvenile hormone on the organization of mushroom bodies of honeybees. J. Neurobiol. 26:130-144.

Wustmann, G., K. Rein, R. Wolf, and M. Heisenberg. 1996. A new paradigm for operant conditioning of Drosophila melanogaster. J. Comp. Physiol. A Behav. Physiol. 179: 429-436.

Yang, M.Y., J.D. Armstrong, I. Vilinsky, N.J. Strausfeld, and K. Kaiser. 1995. Subdivision of the Drosophila mushroom bodies by enhancer-trap expression patterns. Neuron 15: 45-54.

Received March 5, 1997; accepted in revised form June 25, 1997.

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10.1101/lm.4.2.219Access the most recent version at doi: 4:1997, Learn. Mem.

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