What can we teach Drosophila? What can they teach us?

TRENDS in Genetics Vol.17 No.12 December 2001

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719Review

Scott Waddell*

Dept of Neurobiology,University ofMassachusetts MedicalSchool, Worcester, MA01655, USA.*e-mail: [email protected]

William G. Quinn

Dept of Brain andCognitive Sciences,Department of Biology,Center for Learning &Memory, MassachusettsInstitute of Technology,Cambridge, MA 02139,USA.

Drosophila can be trained to run from an odor thatthey previously experienced with an electric shock1,2

(Fig. 1). This simple learning requires the fly tointegrate and associate two cues, the odor (theconditioned stimulus) and the electric shock(unconditioned stimulus). Fruit flies can also learn to run towards an odor that they experienced with afood reward3, and to associate visual and spatialinformation with a heat punishment4,5.

As with other questions in biology, learning andmemory is amenable to the rapid forward geneticsavailable in Drosophila. This amounts to mutatingflies (with chemicals or transposable elements) andtesting their progeny for those with a learning ormemory defect. These techniques are detailed inBox 1. For olfactory learning mutants, the ability tosmell, sense the electric shock and run are routinelytested to confirm a specific learning deficit.

The development of a reliable learning assay1 ledto chemical mutagenesis of flies and screens forstrains deficient in learning or memory. To date, onlyolfactory learning paradigms have been used toscreen for learning-defective mutant flies. The firstgenetic screen yielded four informative mutants;dunce (dnc)6, rutabaga (rut)7, amnesiac (amn)8 andradish (rsh)9, and the jury is still out on turnip (tur)10.Homozygote tur–/– flies have behavioral defects that indirectly affect learning scores11. However,heterozygote tur/+ flies are healthy but have amemory defect10,11.

Detailed behavioral analysis of dnc, rut, amnand rsh mutants revealed several temporally andgenetically distinct memory phases. Similar tohumans, flies remember better after multipletraining trials than they do after single sessions12.Training with interspersed rest intervals (spacedtraining) induces long-term memory (LTM) that can persist for days. Fly LTM is reduced by half in

flies that are fed the protein-synthesis inhibitorcycloheximide before and after training or in flies thatare anesthetized (by cold shock) after training. Theremaining half is both unaffected by cycloheximideand is resistant to anesthesia. Apparently, these twocomponents (susceptible to cycloheximide/anesthesiaand resistant) comprise all of LTM (Ref. 12). Thersh mutation specifically abolishes the anesthesia-resistant component of LTM (ARM) (Refs 9,12),providing our only clue to ARM. rsh mutant flies thatare fed cycloheximide have no memory at 24 hours12.

The cAMP cascade and the mushroom bodies

A goal for researchers of learning and memory is torelate behavioral kinetics – of learning acquisitionand memory decay – to the chemical kinetics ofrelevant second-messenger enzymes andtranscription factors. Drosophila mutants haveimplicated a handful of gene products as central tolearning and memory.

The dnc, rut and amn genes have been cloned andcharacterized. The protein products are all thought toparticipate in the same biochemical pathway (cyclicadenosine 3,5′-monophosphate; cAMP). The dnc geneencodes a cAMP-specific phosphodiesterase13,14. Therut gene encodes a Ca2+/calmodulin-dependent, type Iadenylyl cyclase7,15. So, the RUT enzyme makescAMP, and the DNC enzyme degrades it. These genesare also preferentially expressed in the same brainregion – the mushroom bodies (MBs; Fig. 2)16,17. MBscan be removed from the adult fly brain by pulse-feeding hydroxyurea to larvae18, or by mutations19.Such studies indicate that the MBs are not needed forolfactory perception18,19, or for simple forms of visual,tactile and motor learning20, but they are required forolfactory learning and memory18,19.

Both the dnc and rut gene products are highlyexpressed in the intrinsic cells of the MBs – theKenyon cells (KCs; Fig. 2)16,17. Disrupting normalcAMP signaling in the MBs by expressing aconstitutively activate G-protein abolishes olfactorylearning21. RUT adenylyl cyclase (AC) is thought to be activated by concurrent Ca2+entry (from electricalactivity) and stimulation through G-proteins,possibly by a receptor-coupled system22. Thismechanism suggests the RUT AC (acting in the MBs)as the coincidence detector underlying theconvergence of pathways from the odor and theelectric shock. In fact restoration of rut geneexpression exclusively to the MBs is sufficient to

A number of single gene mutations dramatically reduce the ability of fruit flies

to learn or to remember. Cloning of the affected genes implicated the adenylyl

cyclase second-messenger system as key in learning and memory. The

expression patterns of these genes, in combination with other data, indicates

that brain structures called mushroom bodies are crucial for olfactory learning.

However, the mushroom bodies are not dedicated solely to olfactory

processing; they also mediate higher cognitive functions in the fly, such as

visual context generalization. Molecular genetic manipulations, coupled with

behavioral studies of the fly, will identify rudimentary neural circuits that

underly multisensory learning and perhaps also the circuits that mediate

more-complex brain functions, such as attention.

What can we teach Drosophila?

What can they teach us?

Scott Waddell and William G. Quinn

restore normal capability for olfactory learning to rutmutant flies23. However, if RUT is the detector ofcoincident odor and shock signals, rut mutant fliesshould be completely unable to learn23. But, rutmutant flies show nearly half of wild-type learningability2,23, suggesting that other, rut-independent,biochemical mechanisms must account for theremaining memory.

The amn gene encodes an apparentpre-proneuropetide neurotransmitter24,25. AMN ismost abundant in two brain cells termed dorsalpaired medial (DPM) neurons, that seem to bemodulatory neurons, and that project to all the lobesof the MBs (Fig. 2). Expressing the amn gene in DPMcells restores normal olfactory memory to amnmutant flies. Blocking synaptic transmission from theDPM neurones with a dominant–negative shibiretransgene (Box 1) blocks one-hour memory, but leaves immediate learning intact26. Thus, the AMNneuropeptide, released onto the MB lobes, couldtrigger a prolonged activation of the cAMP cascade,which is required for the consolidation of initialmemory into more permanent memory. This makessense because neuropeptides stimulate intracellularsignalling cascades for longer than classicaltransmitters such as glutamate. In support of thisidea, amn mutant flies are defective in mid-termmemory (MTM)27 and LTM (preliminary data cited in Ref. 12). The amn-expressing DPM cells project tothe five lobes of the MBs but not to the MB calyces26,consistent with the notion that the AMN peptide(s)stimulate RUT cyclase in the MB lobes and modulateMB output (Fig. 3).

Cyclic AMP-dependent protein kinase (PKA) is the primary downstream target of cAMP induction.Expression of the PKA catalytic and regulatorysubunits is elevated in the MBs (Refs 28,29), incomparison with other brain regions, consistent witha central role of the MBs in learning. Disrupting PKAglobally with inducible inhibitory transgenes acutelyreduces olfactory learning30. Furthermore, fliesmutated in the genes for the catalytic or regulatorysubunits of PKA are deficient in learning29,31,32. Theduration of PKA activation is believed to determinewhether short-, medium-32 or long-term memory isformed. Recent experiments with honeybees haveconvincingly demonstrated that repetitive, spacedtraining causes persistent PKA activation thatengenders LTM (Ref. 33).

CREB and LTM

A major target for PKA phosphorylation is thetranscription factor cAMP-response-element-bindingprotein (CREB). Studies of flies with inducible CREB transgenes show that CREB is crucial forprotein-synthesis-dependent LTM formation34,35.Experiments on the Drosophila larval neuromuscularjunction (NMJ) suggest that CREB-dependenttranscription is crucial for new gene expression thatincreases synaptic efficacy36. However, whereasupregulation of the cAMP pathway (by the dncmutation) leads to both a structural and functionalincrease in synaptic properties, CREB affects onlyfunctional plasticity. This finding suggests thatcAMP-regulated transcription factors other thanCREB might control a different set of genes thatmediate structural changes at the NMJ. Theseremain to be identified.

More mutants

A behavioral screen for transposon-inducedmutations that affect olfactory learning has identifiedthree additional genes: latheo (lat)37, nalyot (nal)38

and linotte (lio)39,40. The lat gene encodes a componentof the origin recognition complex (ORC) involved inDNA replication41. Adult flies with the hypomorphic(reduced function) latP1 allele have a 20% reduction inMB volume. Surprisingly, LAT protein is also presentin the presynaptic termini of motor neurons, and latmutants have defects in both basal transmission andsynaptic plasticity at the NMJ42. It is possible thatLAT has a developmental role as well as a separaterole in adult behavioral plasticity (see below).

The nal mutation is a hypomorphic allele of anessential gene encoding the myb-related ADF1transcription factor38. The nalP1 allele has a mildeffect on learning and a pronounced effect on LTM.The nalP1 mutation also causes a modest reduction inthe number of synaptic boutons at the larval NMJ,whereas increased nal expression appears to cause amodest increase in the number of boutons. The ADF1transcription factor might be involved in regulatingthe structural aspect of synaptic plasticity.


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Fig. 1. Odor-aversion learning in Drosophila, using electric-shock reinforcement2. During training,flies experience an odor in conjunction with electric shock punishment. During subsequent testing, the flies preferentially avoid the shock-associated odor. A group of ~100 flies is trained in achamber (a), with an inner surface covered with an electrifiable printed-circuit grid1. Odors aredelivered into the chamber by an air current. The flies are exposed to one odor (e.g. 3-octanol; OCT)while the walls of the chamber are electrified (CS+). They then experience another odor(e.g. 4-methylcyclohexanol; MCH) without shock (CS−). The flies are then tested for learning ormemory performance by transporting them to a choice point between converging air currentssuffused with the two odors (b). After two minutes the flies that have run into each odor arm aretrapped, anesthetized and counted. A score is calculated as (number of flies avoiding CS+ minusnumber of flies avoiding CS–) divided by the total number of flies. A single learning index is theaverage from two groups of flies trained to avoid each of the two odors. Here ‘learning’ denotes ameasure of performance, within two minutes after training, whereas ‘memory’ is a measure of thesame performance at longer times after training.

The identity of the gene affected in the linottemutant is controversial. The lio gene either encodes anovel protein39 or it is an allele of the receptor tyrosinekinase derailed40. Regardless, lio mutants havestructural brain defects that extend to the MBs andcentral complex (CC)43,44. Whether these defects give rise to the diminished learning performance,however, is undetermined. The brain defect has been

rescued with the tyrosine kinase43, and the learningdefect has been rescued with the novel gene39.

A transposon-induced mutant screen looking forgenes that are highly expressed in the MBs hasidentified three new genes involved in olfactorylearning: leonardo (leo)45, volado (vol)46 and fasciclinII(fasII)47. The leo gene encodes a 14-3-3 cell signalingprotein48. LEO plausibly affects learning by



721Review

Box 1. Techniques used in fly memory genetics

Feeding flies chemicals

Chemical mutagens and metabolic blockers (such ascycloheximidea, hydroxyureab and cAMP pathway effectorsc,d)can normally be fed to flies in food or in a sucrose solution.

Temperature-sensitive mutants

In some cases, temperature-sensitive mutant alleles of genes haveallowed a temporal dissection of the role of a particular gene inlearninge. The flies are raised at the permissive temperature tonegate developmental effects. They are then subjected to therestrictive temperature during training, testing or in the interveninginterval to ascertain when normal gene function is critical. Anadvance based on this principle is shibirets1(see below)f.

Heat-inducible transgenes

The heat shock (hsp70) promoter allows acute induction of geneexpression. Transgenic flies can be generated that carry any geneone wishes under the control of the heat shock promoterg. Byheat-pulsing the flies, the gene can be induced before, during, orafter training. However, this gene expression is global, it occurs inall tissues.

Transposon mutagenesis

Transposable P-elements have been engineered so that theirtransposition is controllable by crossing in an active transposasegene into a fly harboring these elements. Transposition is almostrandom, and the resulting P-insertion often disrupts theexpression of the neighboring gene. In this way, P-elements canbe used as a mutagenh. One great advantage of the P-mutagenesisapproach is the rapid clonability of the flanking gene.

GAL4-enhancer trapping and region-specific gene expression

This method, currently unique to Drosophila, allows exquisiteregion-specific gene expressioni. A P-element carrying the GAL4gene preceded by a minimal promoter (which is not sufficient todrive its expression) is transposed around the fly genome. If theelement inserts near a gene, GAL4 expression is often driven by enhancer elements with similar region specificity to theneighboring gene. GAL4 is a yeast transcription factor thatnormally has no function in flies. Any gene can be expressed withthe same region specificity as the GAL4 factor by inserting itdownstream of a GAL4 responsive promoter (UAS[GAL4]) in thesame fly line. Several hundred lines that express GAL4 in veryprecise regions of the fly brain have been characterizedj,k. Theexpression pattern of a GAL4 fly line can be analyzed by crossingthe flies to flies carrying a visible reporter gene such asβ-galactosidase or green-fluorescent protein driven by aUAS[GAL4] promoter. Once the region specificity of expression is

known, any gene controlled by a UAS[GAL4] promoter can beexpressed with that specificity by crossing the enhancer-trapGAL4 line with flies carrying a the UAS[GAL4] transgene.

UAS[GAL4]

–shibirets1

This method combines the tissue specificity of GAL4 enhancer-trap insertions with temporally regulated inactivation ofneurotransmission. The shibire gene encodes a dynamin – aprotein involved in recycling of synaptic vesiclesl,m. The mutantshi ts1 allele encodes an abberant dynamin protein thatdominantly disrupts synaptic transmission at temperaturesabove 29°C – quickly and reversiblyn. Expression of the shi ts1

transgene can be confined to a limited subset of neurons using aGAL4 enhancer-trap insertionf. Therefore, synaptic transmissionfrom the particular neurons that express shi ts1 can be blocked byshifting the flies to elevated temperaturef. This method permitsunprecedented spatial and temporal control of cell function.

References

a Tully, T. et al. (1994) Genetic dissection of consolidated memory inDrosophila. Cell 79, 35–47

b de Belle, J.S. and Heisenberg, M. (1994) Associative odor learning inDrosophila is abolished by chemical ablation of mushroom bodies. Science263, 692–695

c Moore, M.S. et al. (1998) Ethanol intoxication in Drosophila: genetic andpharmacological evidence for regulation by the cAMP signaling pathway.Cell 93, 997–1007

d Asztalos, Z. et al. (1991) On the pharmacological phenocopying of memorymutations in Drosophila: alkylxanthines accelerate memory decay. Behav.Genet. 21, 495–511

e Li, W. et al. (1996). Effects of a conditional Drosophila PKA mutant onolfactory learning and memory. Learn. Mem. 2, 320–333

f Kitamoto, T. (2001). Conditional modification of behavior in Drosophila bytargeted expression of a temperature sensitive shibire allele in definedneurons. J. Neurobiol. 47, 81–92

g Drain, P. et al. (1991) cAMP-dependent protein kinase and the disruption oflearning in transgenic flies. Neuron 6, 71–82

h Cooley, L. et al. (1988) Insertional mutagenesis of the Drosophila genomewith single P elements. Science 239, 1121–1128

i Brand, A.H. and Perrimon, N. (1993) Targeted gene expression as a means ofaltering cell fates and generating dominant phenotypes. Development118, 401–415

j Connolly, J.B. et al. (1996). Associative learning disrupted by impaired Gssignaling in Drosophila mushroom bodies. Science 274, 2104–2107

k Yang, M.Y. et al. (1995) Subdivision of the Drosophila mushroom bodies byenhancer-trap expression patterns. Neuron 15, 45–54

l van der Bliek, A.M. and Meyerowitz, E.M. (1991) Dynamin-like proteinencoded by the Drosophila shibire gene associated with vesicular traffic.Nature 351, 411–414

m Chen, M.S. et al. (1991) Multiple forms of dynamin are encoded by shibire, aDrosophila gene involved in endocytosis. Nature 351, 583–586

n Poodry, C.A. et al. (1973) Developmental properties of Shibire: a pleiotropicmutation affecting larval and adult locomotion and development. Dev. Biol.32, 373–386

modulation of synaptic excitability49. LEO interactswith the presynaptic Ca2+-dependent potassiumchannel Slowpoke (dSlo) the via Slowpoke-bindingprotein Slob50. Through this interaction, LEO appearsto regulate the voltage sensitivity of the dSlo channel.

The vol gene encodes an α-integrin subunit46.Analysis using a globally inducible vol transgene in a viable vol– mutant background unexpectedlyimplicated acute changes in cell adhesion(presumably in the MBs) in short-term memory.VOL is strongly concentrated in a subpopulation ofsynaptic boutons in the central nervous system (CNS)neuropil and to a variable subset of synaptic boutonsat NMJs51. Synaptic arbors at the NMJs of volmutants are structurally enlarged and have abberanttransmission properties and plasticity.

Mutant fasII flies have an olfactory learningdeficit47. An acute role for fasII in adult fly learningwas shown by temporally controlling fasII expression with the heat shock promoter. FasII is a homolog of mammalian neural cell adhesionmolecule (N-CAM) and Aplysia ApCAM52. At thelarval NMJ, fasII downregulation is necessary foractivity-dependent synaptic growth and for theincrease in synaptic sprouting and efficacy generatedby elevated cAMP52,53.

Developmental versus acute role of a gene in learning

To really understand the roles of these mutants andgene products in learning and memory, we need toseparate an acute learning effect of a mutation from

a developmental effect. In reality, both probablycontribute to the learning defect in most cases.

Some learning genes have clear developmentalroles – null mutants die prematurely. Somehypomorphic mutant alleles of these genes produceviable flies with a learning deficiency. Mutant larvaecarrying either viable or lethal alleles of learninggenes have been examined for morphology, basalsynaptic function and plasticity of their NMJs. dncand rut mutations have been studied extensively andcause pronounced alterations in synaptic morphologyand plasticity at the larval NMJ (see Refs 54,55 andreferences therein). vol mutants have structural andfunctional abberations51. lat and leo mutants show noobvious changes in morphology but have pronouncedeffects on transmission and plasticity42,49. Bycontrast, nal and fasII mutations affect synapticstructure but not function38,47. The amn gene is notessential for development – flies in which the gene isdeleted are viable25. However, amn mutation alsoaffects NMJ synaptic morphology56. The finding thatall of the mutants are altered in some aspect at thelarval NMJ suggests that comparable changes atcentral synapses could in part account for the adultlearning defect.

A few studies have analyzed the adult brains ofmutants; some uncovered changes in brain anatomy.The brains of nal, vol and fasII adult flies areapparently normal38,46,47, whereas adult flies with the hypomorphic latP1 allele have a 20% reduction inMB volume41, and the MBs of lio mutant flies haveaberrant projections43. Whether these anatomicaldefects are directly responsible for the observedlearning defect is unclear.

It is sometimes possible to infer an acute role for agene in neural signaling by restoring normal learningto adult mutant flies after transgene induction inadult flies; for example, the vol mutant, where thelearning defect was fully restored by transientexpression of the gene (with the hsp70 heat-shockpromoter) in adult flies46. Likewise, acute transgeneinduction has demonstrated an adult role for DNC(Ref. 57), PKA (Ref. 30), CREB (Refs 34,35), NF1 (afactor that might participate in the activation of RUTAC)58 and FasII (Ref. 47) in learning and memory.One study claims that the mechanism of action of theamn mutant might be developmental59. Temporal andglobal transgene induction in adulthood (with thehsp70 promoter) did not restore olfactory memory,whereas expression of the amn gene under the controlof its own promoter – throughout development and inadulthood – did restore memory. However, the authorsdid not report whether global transgene induction only in development restored memory. Therefore, it is possible that amn has both a developmental andacute role, or that it requires to be driven accurately in a few cells to restore memory in adults. Expressinga neurotransmitter throughout the brain (with noregard for anatomical specificity) is perhaps not theoptimum way to assess its normal role in memory.



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AL

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Fig. 2. The principal Drosophila brain structures involved in olfactorylearning. A frontal view of the Drosophila midbrain. Olfactoryinformation from the antennae and palpus enters the brain through theantennal lobes (AL). Neurones in the antennoglomerular tract (AGT,only shown on the left side) relay this information to the mushroombodies (MB). Kenyon cells (KCs) receive information to their dendritesthat reside in the MB calyces (CA). There are ~2500 KC axons per MBside. These neurons project in parallel from the calyx down thepedunculus (ped) to the MB lobes. There the axons branch into either,the α–β or α′–β′ lobe sets or they project unbranched into the γ lobe.Two dorsal paired medial (DPM) cell bodies are shown. The right-handDPM is shown innervating the MB lobes. CC, central complex.

The role of the MBs in different learning tasks

Learning from olfactory cues is easily studied infruit flies. However, the flies also learn to respond to visual4, postural60 and spatial cues5. Kenyonspeculated, more than one hundred years ago, thatinsect MBs represent centers of multiple sensoryintegration (smell, sight, taste and touch)61. Weknow that the MBs are critical for short-termolfactory memory18,19. By contrast, the MBs areabsolutely not required for the fly to learn simplevisual, tactile or motor tasks20. However, visualdeprivation reduces MB calycal volume62, whichsuggests that visual information reaches the MBs.

This anatomical finding supports a stunning result:increasing the complexity of a visual learning taskrecruits the MBs63.

Flies can associate visual images (such as anupright or inverted T-shape) with heat punishment in the flight simulator (Fig. 4)4,63. After training they will selectively avoid the visual image that was associated with heat exposure. Forming theassociation between the visual image and the heatdoes not require the MBs (Ref. 63); flies withmalformed or missing MBs, as a result of chemicalablation or the mushroom body miniature (mbm)mutation, are able to learn this visual task.

The visual task can be made more difficult bychanging the illumination conditions of the apparatus(color of light, or flashing and constant light) betweentraining and testing63. Wild-type flies can cope withthis; they differentiate the context (the lightingconditions) from the relevant predictive cues (visuallandmarks), and they learn to respond to the cues. By contrast, flies without MBs learn only when theillumination conditions are kept constant63.Apparently, the MBs are required to ‘contextgeneralize’– to subtract the unreliable ‘background’from the pertinent cues. These experiments are aremarkable new demonstration of a higher-orderinformation processing function for the fly MBs. Theyimply that Drosophila MBs have a much greater rolethan just processing olfactory information.

What behaviors do MBs control?

The experiments described above demonstrate thatthe MBs are centers of multisensory informationprocessing in the fly brain. It is possible that synapticcommunication through the MBs might govern a‘reasoned’ response to the various stimuli to the MBs.Recent experiments with olfactory stimuli supportthis possibility.

The MBs were known to be required for olfactorylearning18,19. Recent experiments from two groupsindicate that MB synaptic output is required to recallolfactory memory, but it is apparently not required toeither acquire or store the olfactory information64,65.These experiments took advantage of the spatial andtemporal control of cell inactivation that is possiblewith the dominant temperature-sensitive shits1

transgene (detailed in Box 1). By expressing shits1

in the MBs, chemical synaptic transmission from the MBs could be switched off by simply changingtemperature. Switching the MBs off during trainingor in the period in between training and testing did not affect memory. However, blockingneurotransmission from the MBs during testingabolished memory. This tells us that memories arestably encoded in or before the MBs and are read outthrough the MBs (Fig. 3).

We do not know exactly where in the brain theMBs signal to. However, one defined function of theMBs is to modulate, or control motor responses.Damaging MBs with mbm, with a tetanus-toxin



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Fig. 3. How the cAMP cascade might mediate learning and memory in Drosophila. A mushroom-body (MB) neuron receives olfactory input, via interneurons in the antennoglomerular tract (AGT) thatsynapse in the MB calyx. MBs also receive electric-shock input through unknown neurons.Presynaptic termini of the MB neuron, residing in the MB lobes, are innervated by modulatoryneurons like the dorsal paired medial (DPM) neurons that might release AMN neuropeptide(s).Activation of the RUT adenylyl cyclase leads to elevation of cAMP levels in the relevant MB neurons.Longer-term stimulation of the cascade by AMN might lengthen the association and help consolidatethe memory. Depending on the conditions of training and the duration of cAMP elevation, theexperience results in short-lived modification of synaptic connectivity (short-term memory; STM) orin longer lasting functional and structural changes (long-term memory; LTM) in that neuron.Persistent or repeated activation of cAMP-dependent protein kinase (PKA) appears to bring aboutenduring synaptic changes via CREB-dependent gene activation. Recall of olfactory memory requiressynaptic transmission from MB neurons. DCO, PKA catalytic subunit; PKA-R1, PKA regulatorysubunit; dnc PDE, cAMP phosphodiesterase encoded by the dunce gene; Gs, stimulatory G protein;RUT, type I adenylyl cyclase; NF1, neurofibromin, rsh, radish gene product; rut, rutabaga geneproduct; VOL, volado gene product; FasII, fascicilinII gene product.

transgene expressed from a MB-specific promoter, or with chemical ablation, increases spontaneouswalking activity66. The CC is a pre-motor controlcenter for walking and flight67, and therefore wemight expect that some MB output will projecttowards the CC. However, such a direct connectionremains to be identified.

Sleep, memory and circadian clocks

Work in mammals suggests that sleep is important inmemory consolidation68. Flies also have prolongedperiods of inactivity that have some of thedistinguishing characteristics of mammaliansleep69,70. At these times, the flies become immobileand unresponsive to sensory stimuli. Furthermore, ifflies are deprived of rest, they are more likely to restin the next stretch of time.

The Drosophila circadian clock is tightly linked to the rest–activity cycle. In fact, walking behavior is the favored assay for analysis of circadianrhythm71. The small ventrolateral neurons (s-LNvs)of the adult brain, which are critical for normalcircadian locomotor-activity rhythm, project to near the MB calyces72,73 Therefore, the MBs might be an intermediate brain structure that

modulates locomotor rhythm. This can be testedwith MB-less flies.

A fly homolog of the CREB transcription factor,dCREB2, is a possible molecular link betweenmemory formation and circadian rhythms of activity.A dCREB2-mutant fly has a shortened circadianrhythm74. Furthermore, in normal flies, dCREB2activity cycles with a 24-hour period, with a peakoccurring just after darkness74. It is possible that restdrives CREB activity levels and thereby promotesmemory consolidation. The requirement of fly rest inmemory formation will no doubt be investigated withsleep-deprived flies.

Attention!

The demonstration of context generalization in thefly63 strongly suggests that flies will be useful to studybrain functions more complex than associativelearning. In fact, another set of observations in theflight simulator suggests that flies, like largeranimals, have defined attentional states75.

In free flight, in the absence of heat reinforcement,wild-type flies attend to the visual images painted onthe surrounding panorama75. In a six-minute flightthey spend the vast majority of the time flyingtowards the contrasting images. By contrast, mutantdnc and amn flies seem inattentive to visual cues;they fly in random directions. The learning mutantshave apparently normal vision, but they could have avisual attention deficit.

The possibility that flies have attention states,and that attention might be altered in learningmutants, has intriguing implications for learning



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Fig. 4. Visual learning in the flight simulator. A fly is suspended in anopaque arena with visual images painted on the surface; T-shapes inthis case. Flight orientation can be conditioned by punishing the fly withheat when it flies towards a given image. Learning is measured as thefly’s preferential avoidance of the image after the heat is withdrawn.The fly does not actually move anywhere. It is tethered in space and itssideways torque is translated into counter rotation of the panorama.The arena is evenly illuminated and the color of the lighting (context)can be changed with a filter. Figure kindly provided by Reinhard Wolf.

studies. Inability to attend to the correct cues couldhave catastrophic effects on associative learningperformance. Impaired attention in learning mutant flies might be analogous to ‘attention deficit disordered’ schoolchildren looking around at their friends and out the window rather thanconcentrating on the blackboard. It will beinteresting to determine the regions of the fly brainthat mediate these behaviors.

Conclusion and future prospects

A partial model for fruit-fly learning was firstproposed in Ref. 16 and is updated in Fig. 3. Short-term memory depends on receptor-coupled adenylylcyclase activation in MBs and is affected by Gsα, dnc,rut, PKA, vol and fasII. Medium-term memoryrequires the amn neuropeptide and PKA. Long-termmemory is split into two independent forms –anesthesia-resistant memory (dependent on the rshgene) and protein-synthesis-dependent long-termmemory (dependent on CREB).

Remarkably, all the altered genes identified inlearning mutants have expression patterns that pointto the MBs as centers of olfactory and multimodallearning76. Even the amn gene, which is not expressedin the MBs, fits this pattern – the neurons where itsproduct is concentrated ramify over all the MBlobes26. Hence, the MBs currently fascinate flyreseachers much as the hippocampal formationfascinates mammalian memory researchers.

The current evidence points to the MB synapses as the site where the cAMP-dependent, memory-relevant modification occurs. Recall of immediate,30-minute and 3-hour olfactory memory requiressynaptic transmission through these neurons64,65.This synaptic modification potentially represents amemory trace23. It will be interesting to see whether

recall of protein-synthesis-dependent LTM alsorequires synaptic transmission from MB neurons.Because CREB is expressed ubiquitously throughoutthe brain77, long-term olfactory memory mechanismscould be more widely distributed.

At present, our knowledge of MB anatomy is fairly superficial. All the 2500 Kenyon cells of eachMB project in parallel down the pedunculus, and then they branch into five lobes28. We know theseprocesses branch into three direction categories (α–β, α′–β′ or unbranched into γ; Fig. 2)28, and weknow that even within these branch categories thereare identifiable neuronal subsets78. However, we donot know why they are organized as they are, or howthis elaborate structure relates to informationprocessing and memory storage by the fly. Onestudy23 suggests that the γ lobe might be mostimportant for olfactory learning, and another64

suggests that the α and β lobes are most important for recall of olfactory memory.

It will be important to understand the temporaland spatial patterns of neural activity in the brain. Byexpressing a transgene of the Ca2+-indicator proteinaqueorin in the MBs, Rosay et al.79 found that MBshave an intrinsic intracellular Ca2+oscillation, withan average period of five minutes in normal flies. Theamplitude of these Ca2+oscillations is dramaticallygreater in amn mutants. When combined with morerestricted GAL4 drivers, such a technique could allowreal understanding of MB functional organization.Voltage80 and cAMP-sensitive81 reporter proteins arelikely to be co-opted to this method as they appear.

It is clear that flies are not automata. Their tinybrains are capable of much more than hard-wiredreactions. The molecular, cellular and anatomicalorganization of brain function in flies should provide abasis for understanding cognition in higher animals.



725Review

Acknowledgements

We thank Mark Alkemaand Justin Blau for theircritique of earlier versionsof this manuscript and theanonymous reviewers fortheir constructivecriticism. We also thankJ. Douglas Armstrong andJosh Dubnau fordiscussion of their workprior to publication. Workin our lab is supported bya grant from the NIH toW.G.Q.

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