G(o) signaling is required for Drosophila associativelearning

Jacob Ferris1, Hong Ge1, Lingzhi Liu1 & Gregg Roman1,2

Heterotrimeric G(o) is one of the most abundant proteins in the brain, yet relatively little is known of its neural functions in vivo.

Here we demonstrate that G(o) signaling is required for the formation of associative memory. In Drosophila melanogaster,

pertussis toxin (PTX) is a selective inhibitor of G(o) signaling. The postdevelopmental expression of PTX within mushroom body

neurons robustly and reversibly inhibits associative learning. The effect of G(o) inhibition is distributed in both c- and a/b-lobe

mushroom body neurons. However, the expression of PTX in neurons adjacent to the mushroom bodies does not affect memory.

PTX expression also does not interact genetically with a rutabaga adenylyl cyclase loss-of-function mutation. Thus, G(o) defines

a new signaling pathway required in mushroom body neurons for the formation of associative memory.

An associative memory is one that links external stimuli to particularevents, such that the stimuli come to predict the events. In thenegatively reinforced olfactory associative learning assay of Drosophilamelanogaster, flies are presented with an odor (conditional stimuluspaired, CS+) paired with an electric shock (unconditioned stimulus,US). The flies are then presented with a second odor (conditionedstimulus unpaired, CS–). The associative memory is measured as theconditioned avoidance of the CS+ in a T-maze1. The disruption of thecyclic AMP (cAMP) signaling pathway within Drosophila leads toreduced learning scores2–10. The effect of cAMP disruption has beenmapped back to the mushroom body neurons through the targetedexpression of a constitutively active G(s)a and by rescuing the rutabagatype I adenylyl cyclase (rut) phenotype with targeted expression of a rutcDNA7–10. It is thought that the cAMP pathway controls the associationbetween the CS+ and the US within the mushroom body neurons2.

The G(o) heterotrimeric protein is thought to be the most abundantmembrane protein in the vertebrate brain and is activated both bynumerous G protein–coupled receptors (GPCRs) and by amyloidprecursor protein11. Although G(o) can participate in diverse signalingpathways, only a few specific in vivo functions have been ascribed to thismolecule12–14. In Drosophila, G(o)a47A is the only gene encoding thealpha subunit of G(o), and it is expressed throughout the adultbrain14,15. The G(o) protein is much more abundant in the heads ofrutabaga (rut) and dunce learning mutants than in the heads of wild-type flies, suggesting a possible role for G(o) in memory formation16.

The S1 subunit of PTX from Bordetella pertussis catalyzes the transferof an ADP-ribose onto the Ga subunit of the vertebrate G(i/o/t)heterotrimeric G proteins, preventing these proteins from binding toactivated GPCRs (ref. 17). InDrosophila melanogaster, PTX is a selectiveenzymatic inhibitor of G(o) signaling: Drosophila does not have atransducin homolog, and the G(i)a65A protein does not contain the

PTX recognition site, whereas G(o)a does; PTX will ADP-ribosylatea single protein in Drosophila, as seen in western blots and afterisoelectric focusing; and PTX comigrates with G(o)a and is immuno-precipitated by independent G(o)a-specific antibodies15,16,18,19.

RESULTS

PTX inhibits the physiology of memory formation

To determine if G(o) is a mediator of associative memory, we expresseda PtxA transgene within the mushroom body neurons. We selected theP{UASPTX}16 transgenic line because the basal expression of PTX islow in this line and because PTX can be induced by Gal4, albeit in smallamounts (Fig. 1a). G(o)a47A loss-of-function mutant embryos dieduring embryogenesis owing to defects in nervous system and meso-derm development14. In keeping with this result, we found that theinduction of PTX within the developing mesoderm or nervous systemalso resulted in embryonic lethality, indicating that this toxin isfunctional when expressed early in development (SupplementaryTable 1 online).

We examined the role of G(o) in associative memory by inducingPTX expression within the adult mushroom bodies with theP{MBSwitch}12 Gene-Switch driver10. The resulting induction abol-ished the immediate associative memory 3 min after training, which isfrequently taken as a measure of learning (Fig. 1b; P o 0.0001,Bonferroni-Dunn). Although the PTX-uninduced P{MBSwitch}12/P{UASPTX}16 flies also showed reduced learning, their scores werenot significantly lower than the PTX-uninduced P{UASPTX}16/+ con-trol group (P ¼ 0.015; significance with the Bonferroni-Dunn post-hoctest requires Po 0.0033). The induction of PTX within the mushroombody did not alter naıve sensitivities to either odorants or electric shock,indicating that PTX expressed in the mushroom bodies does not affectthe perception of the stimuli (Supplementary Table 2 online).

Received 8 May; accepted 14 June; published online 16 July 2006; doi:10.1038/nn1738

1Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030, USA. 2Present address: Department of Biology andBiochemistry, University of Houston, Houston, Texas 77204, USA Correspondence should be addressed to G.R. (gwroman@UH.edu).

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The severity of the PTX learning phenotype might result from thedeath of the mushroom body neurons. We tested this hypothesis byexamining the integrity of the mushroom bodies after the induction ofPTX and by establishing whether the associative learning phenotypewas reversible. Because Gene-Switch has slow off-rate kinetics20, weused the Gal80ts system with the P247 Gal4 driver9. P247 drivesexpression in B700 a/b- and g-lobe mushroom body neurons21. Weused two independent Gal80ts transgenes to ensure more completeinhibition of Gal4 at 18 1C. After inducing PTX for 12 h at 32 1C, 3-minmemory was almost entirely abolished (Fig. 1c). Using antibodies todownstream of receptor kinase (DRK), which preferentially mark themushroom body22, we found that PTX induction did not alter eitherthe gross structure of the mushroom bodies or the expression of DRK(Fig. 2a). We found similar results using antibodies to cAMP-dependent protein kinase 1 (DCO; data not shown). We also foundthat the effect of PTX was reversible (Fig. 2b): although a 2-h inductionof PTX within mushroom body neurons produced significant inhibi-tion of 3-min memory (P o 0.0001), this effect was completelyreversed after 6 d (P ¼ 0.8274; Fig. 2b). Therefore, the effect of PTXon learning is not due to the death of the mushroom body neurons.

The PTX effect is distributed within mushroom bodies

We next examined whether the effect of PTX on learning was specific tothe mushroom bodies. We induced PTX in the R3 and R4d neurons ofthe ellipsoid body and separately in the dorsally paired medial (DPM)neurons, which innervate the mushroom bodies23 (Fig. 3a,b). Theinduction of PTX with the Gal80ts system in either set of neurons didnot affect performance in the learning assay, suggesting that PTX is cellautonomous. Moreover, PTX induction in the DPM neurons had noeffect on 60-min memory. The inhibition of neurotransmission inDPM neurons by the shibirets transgene completely blocks 60-minmemory but has no effect on 3-min memory23. Thus, PTX and shibirets

have different effects in the DPM neurons, indicating that PTX is not ageneral inhibitor of neurotransmission (Fig. 3b).

We next sought to determine if the requirement for G(o) signaling inolfactory associative learning is dispersed throughout the differentneurons of the mushroom body lobes, or if the requirement is limitedto a subset of these neurons. Several genes have been identified that arepreferentially expressed in the different mushroom body lobes, indicat-ing that these lobes have distinct molecular repertoires22; however,direct tests for lobe function have yet to provide unequivocal and

Figure 2 PTX expression does not kill the

mushroom body neurons. (a) PTX induction did

not affect mushroom body morphology. The wild-type and PTX genotypes were incubated for 12 h

at 32 1C and given a 3-h recovery before

cryosectioning. The prolonged heat treatment

induced PTX expression and eliminated

performance in the negatively reinforced olfactory

associative learning protocol. The a-DRK antibody

stains the a/b and g lobes and the calyces of the

mushroom bodies. Scale bar, 50 mm. (b) The PTX

3-min-memory phenotype is reversible. In this

experiment, PTX was driven by the P247

mushroom body Gal4 line and conditionally

inhibited by two Gal80ts transgenes. A 2-h

induction of PTX was followed by recovery for either 3 h or 6 d. All flies were tested at 7 d of age. Although there was a significant effect of PTX expression

3 h after the 2-h induction (P o 0.0001), there was no apparent effect 6 d later (Bonferroni-Dunn; n ¼ 18 for each group). All values are mean ± s.e.m.

10 ngPTX

hsG4 hsG4PTX PTXhsG4/PTX

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Induced

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Figure 1 PTX expression in adult mushroom bodies eliminates 3-min memory. (a) Flies of the three genotypes were either given a 30-min-long, 37 1C heat

shock, followed by a 3-h recovery period, or they were left at 18 1C for the entire period. Total protein was extracted from the flies, 100 mg was loaded onto

the gel and 10 ng of purified activated S1 subunit was loaded as a positive control. The S1 subunit of pertussis toxin was detected with the 1B7 monoclonal

antibody. The genotypes are as follows: (i) hsG4 ¼ hsGal4 (89-2-1)/+, (ii)PTX/+ ¼ P{UASPTX}16/+, and (iii) hsG4/PTX ¼ hsGal4/P{UASPTX}16. (b) Effect of

inducing PTX expression within mushroom bodies on negatively reinforced associative learning. The induction of PTX by 500 mM RU486 in the presence of the

P{MBswitch} driver inhibited performance in the negatively reinforced olfactory associative learning assay. The PTX-induced flies performed significantly worse

than any of the other control groups (Bonferroni-Dunn, P o 0.0001; n ¼ 16 for each group). (c) The induction of PTX expression in mushroom body neuronsusing the Gal80ts system further demonstrated a requirement for G(o) signaling in olfactory learning. The expression of PTX was induced by a 12-h-long, 32 1C

heat shock, followed by a 3-h recovery period, before training. The PTX-induced flies performed significantly worse than any of the other control groups

(Bonferroni-Dunn, P o 0.0001; n ¼ 12–14 for each group). All values are mean ± s.e.m.

a bG80ts2/+; PTX/P247, G80ts2 Wild type

α/β lobes

Calyces

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differentiated roles for the constituent neurons in associative learn-ing15–17,24,25. The c772 Gal4 line drives expression in B800 neurons ofthe a/b and g lobes21. We found that a 12-h induction with c772 wassufficient to ablate the associative memory, whereas a 2-h inductionwas not (Fig. 3c). There were no differences in the naıve avoidance ofodor or shock between the c772/Gal80ts20; PTX/Gal80ts2 PTX-inducedand PTX-uninduced experimental groups (Supplementary Table 2).There were, however, some differences in naıve odor avoidance betweenthe c772/Gal80ts20; PTX/Gal80ts2 PTX-induced group and the controlgenotypes (Supplementary Table 2), suggesting that PTX induction innon-mushroom-body neurons by c772 may affect odor perception ordiscrimination and that the Gal80ts inhibition may not be complete inthese neurons. The differences in odor avoidance may also participatein the severe c772/PTX phenotype, although it is unlikely to have amajor effect on learning as naıve avoidance scores were not significantlydifferent in the within-genotype control group (P ¼ 0.9759 for 0.1%methylcyclohexanol (MCH), P ¼ 0.4937 for 0.05% MCH, P ¼ 0.9984for 0.1% octanol (OCT), and P ¼ 0.7673 for 0.05% OCT; Fig. 3c andSupplementary Table 2). It is likely that differences in expression levelsbetween c772 and P247 account for the different time courses in theinhibition of learning by PTX between these two lines. The 12-hinduction of PTX in the g-lobe neurons marked by 1471 caused asubstantial, but not complete, loss of 3-min memory (Fig. 3d), as did

the expression of PTX in the a/b-lobe neurons marked by c739(Fig. 3e). Thus, G(o) signaling is required for 3-min memory inboth the g and a/b neurons of the mushroom body as defined by the1471 and c739 drivers, respectively. In contrast, PTX driven by thea/b-lobe driver 17d did not have an observable effect on 3-min memory(Fig. 3f). The mushroom body neurons defined by 17d are most likelythe core neurons of the a/b lobe26, which may be functionally distinctfrom the other neurons of the a/b lobe as they are insensitive to theeffects of PTX in associative memory and have no effect on the rescue ofthe rut learning phenotype8. The fact that associative memory forma-tion was affected by PTX induction in the a/b- and g-lobe neurons, butnot in the putative a/b core neurons, defines a new requirement forG(o) signaling in these lobes for learning and memory and shouldfurther help dissect the memory process in these neurons.

G(o) is independent of rutabaga

We next considered whether the G(o) pathway interacts geneticallywith the rut adenylyl cyclase in associative memory formation. Thepersistent activation of vertebrate G(o) may initially lead to the short-term inhibition of type I adenylyl cyclase, followed by the increasedresponsiveness of this enzyme to G(s)a stimulation, known as hetero-logous sensitization or supersensitization27,28. Thus, PTX may beinterfering with the down regulation of rut activity by G(o), resulting

InducedUninduced

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Gal80ts20/+;PTX/c232,Gal80ts20

Gal80ts20/+;PTX/Gal80ts20

Gal80ts20/+;c232,

Gal80ts20/+

a b c

d e f

Figure 3 The PTX effect on learning maps to the a/b- and g-lobe neurons of the mushroom bodies. In all panels, the white bar indicates a 12-h 32 1C

induction, and the black bar represents the uninduced treatment. (a) c232. The expression of PTX in the ellipsoid body had no effect on 3-min memory. n ¼ 8

for each group. (b) c316. The expression of PTX within the DPM neurons had no significant effect on either 3-min memory (P ¼ 0.8789; first two bars) or 1-h

memory (P ¼ 0.3619; last two bars). White and gray bars, PTX induced; black and dark gray bars, PTX uninduced. n ¼ 13 for each group. (c) c772: a/b and

g lobes. This driver expresses Gal4 in B800 a/b and g lobe neurons35. Gray bars, results after a 2-h 32 1C induction. The 12-h, but not the 2-h, induction of

PTX within these 800 neurons completely ablated 3-min memory (Bonferroni-Dunn, P o 0.0001; n ¼ 13 for each group). (d,e) The induction of PTX withinthe g lobes (1471, d) and the a/b lobes (c739, e) produced a significant but incomplete reduction in 3-min memory (Bonferroni-Dunn, P o 0.0001 for both;

n ¼ 12 for each group). (f) 17d: the presumptive a/b core lobes. The induction of PTX within the a/b-lobe neurons defined by this driver had no apparent effect

on 3-min memory. n ¼ 12 for each group. PTX driven by the 201Y g driver resulted in substantial defects in naıve odor avoidance; hence we could not use this

driver to evaluate G(o)-dependent learning (Supplementary Table 2). All values are mean ± s.e.m.

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in neurons with too much adenylyl cyclase activity. Alternatively, PTXmay inhibit the heterologous sensitization of rut by G(o), leaving theneurons with too little cAMP after G(s) activation. The formerhypothesis predicts that a reduction in rut activity may partiallysuppress the PTX phenotype, whereas the latter suggests that thereduction in rut may act synergistically with PTX. We tested thesepredictions by looking for a genetic interaction between a mildinduction of PTX in the subset of mushroom body neurons definedby P247 and a single copy of rut2080 (Fig. 4a). This rut mutationdemonstrates a semidominant haploinsufficiency, indicating thatlearning is extremely sensitive to the activity levels of this enzyme10.We found that the performance of the rut2080/+; Gal80ts20/+; PTX/P247, Gal80ts2 PTX-induced flies was reduced, but not significantly,as compared to that of the Gal80ts20/+; PTX/P247, Gal80ts2 flies(P ¼ 0.0604; Fig. 4a). This result suggests an additive interactionbetween PTX and the rut2080 heterozygote, but there was evidentlyneither suppression nor a synergistic relationship between PTX and onecopy of rut2080. We further assessed the independence of G(o) functionduring learning from rut in rut homozygotes. The performance of therut2080; Gal80ts20/+; PTX/P247, Gal80ts2 PTX-induced flies was signifi-cantly worse than that of either the PTX-induced flies (P ¼ 0.0075) orthe rut homozygous flies (P ¼ 0.0025; Fig. 4b). Thus, G(o) signalinghas functions in olfactory learning and memory within the mushroombody neurons defined by P247 that are independent of rut.

DISCUSSION

Heterotrimeric G(o) has a considerable role in olfactory associativememory formation. We have shown, through the postdevelopmentalinduction of PTX expression within mushroom bodies, that activationof G(o) is required during the physiological events, which lead to

associative memory formation. The severity ofthe learning phenotype in PTX-induced fliescoupled with the lack of genetic interactionwith rut2080 strongly suggests that the functionof G(o) in associative learning and memory islargely independent of the cAMP pathway.Additional members of this new associativelearning pathway are currently unknown. Onepossibility is that, similar to the role of theG(o) in the vertebrate dorsal root ganglia, theDrosophila G(o) may participate in learningthrough the inhibition of voltage-gated Ca2+

channels (VGCCs; refs. 12,29). These Ca2+

channels are thought to be activated by theodor-induced depolarization of the mush-room body neurons, leading to the release ofsynaptic vesicles and the CS pathway activa-tion of rut (ref. 2). It is plausible that thenegative regulation of the VGCCs may benecessary to restrict the number of activatedsynapses during learning. Nevertheless, it isnow clear that the in vivo functions ofG(o) include the formation of associativememories in Drosophila.

METHODSGenerating the PTX transgene. The S1 subunit of

PTX contains a signal sequence that is cleaved to

activate and export this enzyme. To produce an

active and intracellular S1 subunit, we used poly-

merase chain reaction (PCR) to generate a new

translational start codon at the beginning of the activated form of PTX. A

plasmid containing the S1 subunit coding sequences was acquired from the

American Type Culture Collection. The sequences for the two primers used for

this amplification were as follows: 5¢-AAAACTCGAGAAAACATGGACGA

TCCTCCCGCCACC and 3¢-GATCGGCATGCTGTTCAATTACC.

The PCR product was cloned into the pCRBluntII vector (Invitrogen). The

modified S1 subunit was liberated from this plasmid with XhoI and XbaI and

then cloned into the pPBRETU Drosophila expression vector using the SalI and

XbaI sites30. This construct was then used to generate the PUASPTX.16

transgenic line by microinjection into ry506 embryos.

Fly stocks and genetics. The P{UASPTX}16 insertion was outcrossed into ry506

for seven generations before behavioral testing. This insertion was then

balanced with the isogenized TM3, Sb, hshid chromosome. The

P{MBSwitch}12 line used in these studies contained only the third chromo-

some insertion10. The Gal80ts2 and Gal80ts20 were gifts from S. McGuire (Baylor

College of Medicine, Houston). The 24BGal4; Twist Gal4 drivers were gifts

from M. Semeriva (Universite de la Mediterranee, Campus de Luminy, France).

The c316 Gal4 driver was a gift from S. Waddell (University of Massachusetts

Medical School, Worcester, Massachusetts). The 1471 Gal4 driver was a gift

from T. Preat (CNRS, Gif sur Yvette, France). The c232 Gal4 driver was a gift

from R.J. Davis (Baylor College of Medicine, Houston). The c739, c772, 201Y

and 17d drivers were gifts from R.L. Davis (Baylor College of Medicine,

Houston). The P247 driver was a gift from T. Zars (University of Missouri,

Columbia, Missouri).

The experimental and control genotypes used in all behavioral experiments

were heterozygous for ry506 on their third chromosomes. For example, in the

experiments using P{MBSwitch}, the experimental and control genotypes were

derived from the following crosses: (i) w+; +; P{UASPTX}16, ry506/TM3 X

w1118; +; P{MBSwitch}12, ry+; (ii) w+; +; P{UASPTX}16, ry506/TM3 X w1118; +;

ry+; and (iii) w1118; +; P{MBSwitch}12, ry+ X w+; +; ry506. For the crosses

involving the Gal80ts transgenes, we used the following general crossing strategy

to generate the experimental genotype: w1118; Gal80ts20; P{UASPTX}16,

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P247/+

Figure 4 G(o) function in olfactory learning and memory is independent of rutabaga. All genotypes listed

also contained the Gal80ts2 transgene located on the same chromosome as P247. (a) PTX was induced

in rutabaga2080 heterozygotes for 2 h at 32 1C, followed by a 3-h recovery before training. This induction

significantly reduced learning in the genotypes containing PTX (P o 0.0001). The induction did not

have a significant effect on the nonPTX genotypes (P ¼ 0.396). The rutabaga2080 mutation reduced

the score of the PTX-induced flies, but the effect was not significant (Bonferroni-Dunn; P ¼ 0.0604).

n ¼ 17–22 for each group. (b) PTX was induced in rutabaga2080 homozygous males for 3 h at 32 1C,

followed by a 3-h recovery. The performance of the rut2080; Gal80ts20/+; PTX/P247, Gal80ts2 flies was

worse than that of flies of the rut2080; Gal80ts20/+; P247, Gal80ts2/+ (P ¼ 0.0025) or Gal80ts20/+;

PTX/P247, Gal80ts2 (P ¼ 0.0075) genotypes. These differences are significant with Fisher post-hoc

least squared difference test, but not with the more stringent Bonferroni-Dunn where a probability of

0.0018 is required for significance (0.05/28 comparisons). The absence of 3-min memory in the

rut2080; Gal80ts20/+; PTX/P247, Gal80ts2 PTX-induced group produces a floor effect in this experiment,

leading to a greater potential for type II errors. The PTX induction in Gal80ts20/+; PTX/P247, Gal80ts2

flies led to significantly reduced learning compared to uninduced flies of the same genotype

(P o 0.0001). n ¼ 18 for all groups. All values are mean ± s.e.m.

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ry506/TM3 X w1118; Gal4, ry+. In these latter experiments, the controls were also

heterozygous for ry506.

Gene switch experiments. For behavior testing, Drosophila crosses were raised

at 25 1C and 60% humidity on a 12-h-light, 12-h-dark cycle. Adults were

harvested within 4 d of eclosion and transferred to vials, each containing one

Kimwipe (11.4 cm � 21.3 cm) wetted with 2 ml of either 500 mM RU486 in

2% sucrose, or 2% sucrose alone10. Flies were kept in these vials at 25 1C for 24 h

and then transferred to fresh food vials for another 24 h. Flies were moved to a

testing room at 25 1C and 60–70% humidity under dim red light approximately

30 min before testing. Training was performed using 0.1% MCH and

0.2% OCT in mineral oil as odorants, each presented for 1 min either with or

without an unconditioned stimulus consisting of 12 pulses of electrical shock at

90 V. Flies were then loaded into a T-maze and, after 1 min of rest, allowed

2 min to choose between the two odors1. Performance index (PI) was

determined as follows: (number of flies that chose the CS– – the number of

flies that chose the CS+)/( total number of flies in both tubes). When a

phenotype was found, odor and shock avoidance were tested for all groups

(n ¼ 8). Odorants were presented in the concentrations used for the learning

assay, as well as one-half the concentrations, opposite pure mineral oil, and

untrained flies were exposed to both in the T-maze for 2 min, as above. For

shock avoidance, both arms of the T-maze were replaced with shock tubes, and

a 90-V or a 60-V electric shock was applied to one of the two arms for 2 min.

Gal80ts experiments. Crosses were raised at 18 1C. Adults were harvested

within 4 d of eclosion and transferred to fresh-food vials. For induction of PTX,

flies were moved to a 32 1C environment for a period of 12 h and then moved

to a 25 1C environment for a 3-h recovery period (unless otherwise specified).

PTX-uninduced flies remained at 18 1C. Flies were moved to a testing room no

more than 30 min before testing. The 3-min memory assay was performed as

described above, except that the concentration of OCT was reduced from 0.2%

to 0.1%. For the 60-min memory test, flies were trained as described, then

returned to food vials and loaded into the maze after 60 min.

The reversibility of the PTX learning phenotype was analyzed with three

induction protocols using P247 and two Gal80ts transgenes (Fig. 2b). In the

first protocol, flies of all genotypes were given a 2-h induction treatment at

32 1C, followed by a 3-h recovery at room temperature. The second protocol

also had a 2-h induction at 32 1C, but instead was followed by a 6-d recovery.

The final protocol did not have an induction period. All flies were 7 d old when

scored for associative learning.

The experiment to test the effects of PTX in rut2080 homozygotes was

performed in a new laboratory at the University of Houston with new T-mazes

based on the standard design. In order to achieve a similar induction of PTX,

flies received the induction protocol for 3 h, rather than 2 h, at 32 1C, which

was again followed by a 3-h recovery at 25 1C. To adjust for the greater

sensitivity of the new mazes, odorant concentrations were reduced to 0.05%

MCH and 0.1% OCT.

Note: Supplementary information is available on the Nature Neuroscience website.

ACKNOWLEDGMENTSThe authors wish to thank J. Maynard (Stanford University, Palo Alto,California) for providing the 1B7 monoclonal antibody to PTX, and R. Davis,K. Choi and M. Mancini for sharing space in their laboratories. This work wassupported by NS042185-04 awarded to G.R.

AUTHOR CONTRIBUTIONSThe behavioral experiments were performed by both J.F. and H.G. H.G.performed the immunohistochemistry and L.L. performed the western blotanalysis. G.R. generated the PTX transgene and the tested genotypes.

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Published online at http://www.nature.com/natureneuroscience

Reprints and permissions information is available online at http://npg.nature.com/

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