Plasticity of local GABAergic interneurons drivesolfactory habituationSudeshna Dasa,1, Madhumala K. Sadanandappaa,1, Adrian Dervanb, Aoife Larkinb, John Anthony Leeb,Indulekha P. Sudhakarana, Rashi Priyaa, Raheleh Heidarib, Eimear E. Holohanb, Angel Pimentelc, Avni Gandhid,Kei Itoe, Subhabrata Sanyalf, Jing W. Wangg, Veronica Rodriguesa,d,2, and Mani Ramaswamia,b,c,d,3

aNational Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore 560065, India; bSchool of Genetics and Microbiology, School ofNatural Sciences, and Trinity College Institute of Neuroscience, Trinity College, Dublin 2, Ireland; cDepartment of Molecular and Cellular Biology, University ofArizona, Tucson, AZ 85721; dDepartment of Biological Sciences, Tata Institute of Fundamental Research Mumbai 400005, India; eInstitute of Molecular andCellular Biosciences, University of Tokyo, Tokyo 1130032, Japan; fDepartment of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322;gNeurobiology Section, Division of Biological Sciences, University of California at San Diego, La Jolla, CA 92093

Edited* by Jeffrey C. Hall, University of Maine, Orono, ME, and approved June 30, 2011 (received for review April 24, 2011)

Despite its ubiquity and significance, behavioral habituation ispoorly understood in terms of the underlying neural circuit mech-anisms. Here, we present evidence that habituation arises frompotentiation of inhibitory transmission within a circuit motifcommonly repeated in the nervous system. In Drosophila, priorodorant exposure results in a selective reduction of response to thisodorant. Both short-term (STH) and long-term (LTH) forms of olfac-tory habituation require function of the rutabaga-encoded adeny-late cyclase inmultiglomerular local interneurons (LNs) thatmediateGABAergic inhibition in the antennal lobe; LTH additionally requiresfunction of the cAMP response element-binding protein (CREB2)transcription factor in LNs. The odorant selectivity of STH and LTHis mirrored by requirement for NMDA receptors and GABAA recep-tors in odorant-selective, glomerulus-specific projection neurons(PNs). The need for the vesicular glutamate transporter in LNs indi-cates that a subset of these GABAergic neurons also releases gluta-mate. LTH is associated with a reduction of odorant-evoked calciumfluxes in PNs aswell as growth of the respective odorant-responsiveglomeruli. These cellular changes use similar mechanisms to thoserequired for behavioral habituation. Taken togetherwith the obser-vation that enhancement of GABAergic transmission is sufficient toattenuate olfactory behavior, these data indicate that habituationarises fromglomerulus-selective potentiation of inhibitory synapsesin the antennal lobe. We suggest that similar circuit mechanismsmay operate in other species and sensory systems.

learning | memory | homeostasis | feedback

Habituation is a specific form of implicit learning in whichrepeated exposure to an unreinforced stimulus results in

a decreased behavioral response (1–3). By filtering out suchconstant sensory input, habituation enhances an animal’s abilityto focus its cognitive resources on novel or salient features ofthe environment. Thus, habituation serves as a building block fornormal cognition (2, 4). Although it has been studied in manydifferent contexts, causal connections between mechanisms, neu-ronal changes, and behavioral habituation have not been clearlyidentified (2). Given our current understanding, it remains un-clear whether similar or distinct mechanisms underlie differentforms of habituation; also unclear is how these mechanismscompare with those mechanisms used in consolidation or ex-tinction of associative memory (1, 5).The olfactory system provides an experimentally accessible

circuit in which to analyze mechanisms that underlie differenttimescales of behavioral habituation (4, 6, 7). Particularly useful isthe adult Drosophila olfactory system, which has organizationsimilar to that of mammals (8, 9). Here, olfactory sensory neurons(OSNs) expressing a single type of functional odorant receptormolecules (ORs) send axons to the antennal lobe and synapseonto (i) glomerulus-specific projection neurons (PNs) that projectto the mushroom body and lateral horn, (ii) multiglomerularlocal interneurons (LNs) that mediate both intraglomerular and

transglomerular inhibition (10–12), and (iii) other PN and LNtypes in early stages of characterization (13–17). The antennallobe is also innervated by neuromodulatory cells, which couldregulate both olfactory responses and olfactory plasticity (18–22).Different odor-induced behaviors show robust habituation in

Drosophila (23). Larval short-term olfactory habituation (STH),which is dependent on the rutabaga (rut) -encoded type I adeny-late cyclase and central N-Methyl-D-aspartate (NMDA) recep-tors, shows significant selectivity for the exposed odorant (24–28).In adults, transient habituation of the odor-evoked, reflexivejump response requires rut as well as other cAMP signalingcomponents (29, 30). In addition, a long-term olfactory habitua-tion (LTH) of the avoidance response to benzaldehyde, a chem-ical potentially sensed by nonolfactory mechanisms, requires rutand persists for several days (31–33). A more recent study char-acterized LTH of the CO2 response, better understood in terms ofthe relevant, Gr21a-expressing sensory neurons and their targetprojection neurons (VPN) in the V glomerulus (34, 35), which isassociated with a reduced olfactory startle response, selectivegrowth of the CO2-responsive V glomerulus, decreased CO2-evoked calcium influx in VPN terminals in the lateral horn, and anincreased CO2-induced calcium influx into processes of a subclassof LNs within the V glomerulus (35). However, mechanisms thatunderlie these phenomena as well as whether they cause habitu-ation remain unknown.To understand the mechanisms of odorant-selective STH and

LTH in terms of (i) the necessary genes, (ii) the cell types in whichthey function, and (iii) consequent neuronal plasticity necessaryfor habituation, we standardized behavioral assays for STH andLTH to selected odorants and combined them with genetic, be-havioral, anatomical, and live imaging methods feasible in Dro-sophila. Our results indicate that olfactory habituation arisesbecause of plasticity in the antennal lobe. In addition, they suggesta simple and theoretically generalizable model for habituation inother systems.

Author contributions: S.D., M.K.S., A.D., A.L., I.P.S., R.P., R.H., E.E.H., A.P., A.G., S.S., V.R.,and M.R. designed research; S.D., M.K.S., A.D., A.L., J.A.L., I.P.S., R.P., R.H., E.E.H., A.P.,A.G., S.S., and V.R. performed research; K.I., J.W.W., and V.R. contributed new reagents/analytic tools; S.D., M.K.S., A.D., A.L., J.A.L., I.P.S., R.P., R.H., E.E.H., A.P., A.G., S.S., V.R.,and M.R. analyzed data; and M.R. wrote the paper.

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.

See Commentary on page 14711.1S.D. and M.K.S. contributed equally to this work.2Deceased November 10, 2010.3To whom correspondence should be addressed. E-mail: mani.ramaswami@tcd.ie.

See Author Summary on page 14721.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1106411108/-/DCSupplemental.

E646–E654 | PNAS | September 6, 2011 | vol. 108 | no. 36 www.pnas.org/cgi/doi/10.1073/pnas.1106411108

Dow

nloa

ded

by g

uest

on

Aug

ust 2

8, 2

021

ResultsSTH and LTH in Drosophila. After a 30-min exposure either to 15%CO2 or 5% ethyl butyrate (EB), Drosophila show a diminishedolfactory avoidance response as measured in a Y-maze test (Fig.1 A and B and raw data in Tables S1). This STH is selective forthe odorant used during training and recovers with a half-life ofabout 20 min (Fig. 1 B and C).Because CO2 and EB are sensed by nonoverlapping odorant

receptors expressed in distinct sets of OSNs expressing Gr21a andOr83b, respectively, odor-selective habituation could, in princi-ple, arise from adaptation of odorant-receptor signaling pathwaysin distinct groups of OSNs. However, several observations suggestthat STH can be induced without receptor activation and thus,cannot be explained by adaptation of the odorant-receptor sig-naling pathway.Thirty minutes of direct depolarization of CO2-sensing, Gr21a-

positive OSNs expressing the heat-activated Transient ReceptorPotential ion channel A1 (TRPA1) channel (36, 37) results ina reduced response to CO2 but not to EB (Fig. 1D Left and Figs.S1 and S2). Similar 30-min activation of OSNs expressing Or83bresults in selectively reduced avoidance of EB but not of CO2

(Fig. 1D Right and Figs. S1 and S2). Thus, as reported for Dro-sophila larvae (28), odorant-selective STH (i) does not requireodorant receptor activation and (ii) can arise from a processdownstream of action potential firing in OSNs.

After a more prolonged exposure period, where Drosophilaadults are maintained in the presence of an odorant (e.g., 5%CO2) for 4 d, flies tested in a Y-maze (Fig. 1A) show a diminishedavoidance response (Fig. 1E) that lasts for several days (Fig. 1F)(31, 35). LTH to CO2 causes minimal change in response to EB(Fig. 1E Left). Similar LTH can be observed to EB after 4-d ex-posure to 20%EB. Like LTH to CO2, LTH to EB is selective, andthe decreased response to EB occurs with little effect on CO2

sensitivity (Fig. 1E Right). LTH to CO2 and EB are both rever-sible. When habituated animals are restored to normal rearingenvironment, normal olfactory sensitivity returns within a fewdays (Fig. 1F). As shown for STH, a “phantom” LTH to CO2 orEB can be induced by prolonged (4-d) depolarization of appro-priate OSNs. Thus, LTH can also occur without odorant receptoractivation through a process downstream of action potential firingin OSNs (Fig. 1G).A biochemical similarity in the neuronal signaling cascades that

underlie STH and LTH is indicated by the observation that bothSTH and LTH are greatly diminished in rutabaga (rut2080)mutants (Fig. 2 and Fig. S3A) deficient in an adenylate cyclaseactivated by the coincidence of increased cytosolic calcium andG protein-coupled receptor (GPCR) activation (38–41). Meas-urements of basal odor-evoked behavior in naïve flies show thatthe observed defects in habituation cannot be ascribed to alteredodorant sensitivity in rutmutants (Fig. S3B and Tables S1 and S3).

Fig. 1. Properties of STH and LTH in Drosophila. (A) Y-maze used to measure aversive odorant response of adult flies. (B) White bars, response to odorants(5% CO2 or 10

−3 dilution EB) before exposure; black bars, after 30 min exposure to either 15% CO2 (Left) or 5% EB (Right). (C) Recovery profile of STH to CO2

and EB. White bars indicate the percent response of naïve flies, and the black bars indicate the same for the 0- and 30-min recovered flies after odor exposure.***, comparisons between preexposed and exposed animals at t = 0; +++, comparisons between exposed animals after 0 and 30 min of recovery. One-wayrepeated measure ANOVA shows significant difference between naive and CO2 (F = 28.237, P < 0.001) and naive and EB treatments (F = 15.564, P < 0.001).Posthoc testing showed a significant difference between naïve and 0-min recovered flies (q = 10.507, P < 0.001; Student-Newman-Keuls (SNK) test) and 0- and30-min recovered flies (q = 6.634, P < 0.001; SNK) for CO2 treatment. The same test for EB treatment showed significant difference between naïve and 0-minrecovered flies (q = 7.813, P < 0.001) and 0- and 30-min recovered flies (q = 4.993, P = 0.003). (D) TrpA1-mediated direct activation (29 °C) of OSN subsets for 30min reduces behavioral responses mediated by these OSNs. White bars, control responses at 22 °C. (E) Four-day exposure to 5% CO2 (Left) or 20% EB (Right)causes odorant selective LTH. (F) Recovery profile of LTH to CO2 and EB. White bars indicate the percent response of naïve flies, and the black bars indicate thesame for the 0- and 6-d recovered flies after odor exposure. ***, comparisons between mock-exposed and 4-d odor-exposed animals 1 d after exposure; +++,comparisons between exposed animals after 1 and 6 d of recovery. One-way repeated measure ANOVA shows significant difference between mock and CO2

(F = 239.598, P < 0.001) and mock and EB treatments (F = 82.124, P < 0.001). Posthoc testing showed a significant difference between naïve and 0-d recoveredflies (q = 30.958, P < 0.001) and 0- and 6-d recovered flies (q = 15.422, P < 0.001) for CO2 treatment. The same test for EB treatment showed significantdifference between naïve and 0-d recovered flies (q = 16.689, P < 0.001) and 0- and 6-d recovered flies (q = 14.467, P < 0.001). (G) Forced activation of TRPA1-expressing OSNs for 4 d causes odorant-selective LTH. At least eight sets of flies (n) were used for each measurement (exact n and P values are shown in TableS1). Control data showing WT behavior in various Gal4 and Upstream Activation Sequence (UAS) transgene stocks examined in isolation are shown in Fig. S2.Bars show the mean ± SEM. ++P < 0.01; ***P < 0.001. The student’s t test was used for all statistical comparisons except for Fig. 1 C and F.

Das et al. PNAS | September 6, 2011 | vol. 108 | no. 36 | E647

GEN

ETICS

PNASPL

US

SEECO

MMEN

TARY

Dow

nloa

ded

by g

uest

on

Aug

ust 2

8, 2

021

cAMP Signaling Is Required in GABAergic LNs for Habituation. Theoften unique, anatomical location where rut is required for a givenlearning task defines cell types in which neuronal plasticity is re-quired for the specific form of memory (42–44). To identifyneurons whose intrinsic properties change during olfactory ha-bituation, we looked to identify the cell type(s) in which rut isrequired (Fig. 2, Figs. S2 and S3, and Tables S1 and S3).Tissue-restricted expression of a WT rut+ transgene in either

the LN1 class of antennal lobe local interneurons (45) (Fig. S2F and G) or GABA-expressing glutamic acid decarboxylase(GAD1) -positive neurons was sufficient to rescue rut2080 defectsin STH or LTH (Fig. 2A–C and Fig. S3C andD). Arguing againsta developmental requirement in these cells, experiments using theGal80ts conditional expression system show that rut+ expression inadult LN1 neurons is sufficient to rescue the habituation defectsin rut2080 (Fig. 2 A–C and Figs. S2 F and G and S3 C and D).In contrast, rut+ expression in OSNs, PNs, or MB247-positivemushroom-body neurons had no effect on the rut2080 mutantphenotype (Fig. 2 A–C and Fig. S3 C and D). This finding indi-cates that rutabaga function is necessary for both STH and LTHresides principally in adult inhibitory local interneurons in theantennal lobe.The conclusion that rut is necessary in LN1 neurons is also

supported by the observation that LN1 orGAD1 promoter-drivenexpression of either of two previously validated transgenic RNAiconstructs against rut (43) also blocked habituation to EB (Fig.2D and Fig. S3F). Together, these observations indicate that rut-dependent changes in the intrinsic properties of inhibitory localinterneurons are necessary for behavioral habituation.

LTH Is Accompanied by a rut-Dependent Reduction in PN Responses toOdorant. The observed requirement for rut in LNs suggests thatbehavioral habituation arises from rut-dependent strengthening

of GABAergic LN1 synapses in the antennal lobe. This observa-tion predicts first, that odor-evoked responses in PN dendrites willbe reduced after habituation and second, that the reduction in PNresponse will be dependent on rut function. We tested thesepredictions by measuring the effects of 4-d EB exposure on EB-evoked responses in dorso-medial (DM)2 and DM5 glomeruli,which respond to EB under the conditions used here (48).EB-evoked responses of PN dendrites in DM2 and DM5 were

significantly reduced after habituation. We imaged odor-evokedcalcium fluxes inGH146-Gal4, UASGCaMP3/+ flies (also termedGH146>GCaMP3/+), which carry one copy each of GH146-Gal4and UAS-GCaMP transgenes heterozygous to a WT + chromo-some. Two-second pulses of 0.5% EB induced strong calci-um fluxes measured as

ÐΔF/F, the fractional increase in GCaMP

fluorescence induced by odor stimulation (Fig. 3 A and B andTable S4). However, 4-d exposure to EB caused a significant re-duction in EB-evoked GCaMP responses compared with paraffin-exposed controls (DM2 P = 0.004, DM5 P < 0.001). In contrast,4-d exposure to 5% CO2, previously shown to reduce CO2-evokedresponses in the V projection neuron (35), did not reduce the EBresponse (Fig. 3B). Furthermore, EB exposure had no effect onthe 3-octanol (3-Oct) response in the dorsal-central (DC)2 glo-merulus (Fig. 3B Right). DC2 has previously been shown to re-spond to 3-Oct (49) and in our experiments, was unresponsive to0.5% EB. These data indicate that LTH is accompanied by a se-lective reduction of PN responses to the habituated odor.If the observed reduction in PN response causes behavioral

habituation, then it should not occur in nonhabituating mutants.Consistent with this premise, 4-d EB exposure had no significanteffect on EB-evoked PN responses of nonhabituating, rut2080/y;GH146 > GCaMP3/UAS rut+ flies (Fig. 3B). This observationtightens the relationship between the observed physiologicalplasticity and behavioral LTH.

Fig. 2. The rut-encoded adenylate cyclase is required in GABAergic/LN1 neurons for habituation. (A) Schematic showing the major classes of olfactory neuronsand Gal4 lines that mark them. GAD1 predominantly marks GABAergic neurons and 95% of LN1-expressing interneurons (45–47). The LN1 promoter is highlyrestricted to a subgroup of antennal lobe interneurons, is distinct from a different set of LN2-positive inhibitory interneuron class, and shows minimal overlapwith kra-Gal4–marked excitatory LNs (45) (Fig. S2 F andG). (B and C) Histograms show the efficiency of STH (B) and LTH (C) to EB of rut2080 flies expressing aWTrut+ transgene (44) in different classes of olfactory neurons. White bars indicate olfactory response behavior of mock-treated siblings; dark bars indicate re-sponse after the relevant period of odorant exposure. (D) RNAi-mediated knockdown of rut using a validated transgenic RNAi construct (VDRC5569) (43) in LN1or GAD1 neurons blocks STH and LTH to EB. Similar results obtained with an independent rut RNAi line, VDRC101759, are not shown. Additional Gal4 and UAStransgene alone controls are shown in Fig. S2. Bars show the mean ± SEM determined through the Student t test [n > 8 (Tables S1 and S3); ***P < 0.001].

E648 | www.pnas.org/cgi/doi/10.1073/pnas.1106411108 Das et al.

Dow

nloa

ded

by g

uest

on

Aug

ust 2

8, 2

021

GABAergic Transmission from LNs Is Necessary and Its Enhancement IsSufficient for Habituation. If the reduced PN response arises fromenhanced GABAergic transmission onto PNs, then synapticoutput from LN1 neurons should be necessary for the reducedolfactory aversion in habituated animals, which was previouslyobserved for larval STH (28). To test this prediction for adultSTH and LTH, we expressed a dominant, conditional dynamin(Shits1) in local interneurons and used a rapid temperature shift,from 22 °C (permissive) to 32 °C (restrictive) to conditionallyreduce transmitter release from LNs during any specified timeinterval (50). At permissive temperatures (22 °C), LN1 > Shits1

Drosophila showed normal STH to EB and CO2. However, un-like control flies that show robust habituation at 32 °C (Fig. S4Aand Tables S2 and S3), LN1 > Shits1 flies behaved like naiveunhabituated animals when tested at temperatures (32 °C) fordynamin function (Fig. 4A and Fig. S4B). Therefore, synapticoutput from LN1 neurons is necessary for display of STH (Fig.4A). We also found that transmitter release from LN1 neuronswas necessary during the exposure period (Fig. 4A), an obser-vation that we consider later in this paper.Transmitter release from LN1 interneurons is also required for

the expression of LTH to CO2 and EB. Thus, although bothcontrol and LN1 > Shits1 adults exposed to EB or CO2 at per-missive temperatures for 4 d show habituated behavior whentested at room temperature, LN1 > Shits1 flies alone behave likenaive unhabituated animals when tested at temperatures non-permissive for Shits1 function (Fig. 4B and Fig. S4C).To test if GABAergic transmission from LNs to PNs mediates

the expression of habituated behavior, we asked whether habitu-ation defects were also evident when the function of GABAreceptors was knocked down in odorant-responsive PNs throughthe transgenic RNAi line (UAS-Rdli) previously shown to knock-down ionotropic GABAA (Rdl) receptors (51) (Fig. S4F andTables S1 and S3). Knockdown of GABAA receptors in definedsubsets of PNs selectively blocked habituation to their respectiveodorants without affecting the basal olfactory response (Fig. 4 CandD and Fig. S4D andE). Thus,Rdl RNAi expression inGH146-positive neurons that communicate the odor of EB (48) blockedSTH and LTH to EB without affecting habituation to CO2. Incontrast, Rdli RNAi expression in the CO2-responsive projectionneuron (VPN) blocks STHandLTH toCO2without affecting STHor LTH to EB (Fig. 4 C and D). Taken together with the obser-

vation that synaptic output from predominantly GABAergic LN1neurons is required for display of habituation, these data point toa model in which PN inhibition by LN1 neurons is necessaryfor the decrease in olfactory avoidance behavior observed afterhabituation.To determine whether increased transmission from LN1 neu-

rons could be potentially sufficient to attenuate olfactory behav-ior, we expressed the heat-activated, cation-permeable TRPA1channel in LN1 cells and compared olfactory response indices forthese LN1 > TRPA1 flies at 22 °C, when TRPA1 channels wouldbe silent, with responses measured just after the flies were shiftedto 29 °C, when LN1 neurons show increased activity (Fig. S1).Activation of LN1 neurons was sufficient to substantially reduceaversion of naïve LN1 > TRPA1 flies to CO2 or EB (Fig. 4E).Thus, transmitter release from LN1 neurons is necessary (Fig. 4 Aand B), and the potentiation of LN1 activity is sufficient (Fig. 4E)for the attenuated behavioral response observed in habituation.

PN-Specific NMDA Receptors May Explain Odorant Selectivity inHabituation. Forced activation of LN1 neurons results in a non-selective reduction in the behavioral response to both CO2 andEB (Fig. 4E). This finding is easily explained by the fact that mostLN1 local interneurons are multiglomerular and would be ex-pected to inhibit most, if not all, PNs (45). However, the obser-vation that behavioral habituation is odorant-selective posits theneed for a mechanism to ensure glomerulus-selective potentia-tion ofLN1 synapses in vivo (Fig. 5A). Howmay synapse-selectivepotentiation of GABAergic outputs be controlled?Previous studies have shown that presynaptic LTP of GABAergic

synapses usually arises from a heterosynaptic mechanism thatrequires retrograde signaling, which is often mediated throughpostsynaptic NMDA receptor activation (52, 53). Although glu-tamate is not the main excitatory transmitter in insect brains, it islikely that NMDA receptors, which typically mediate synapseplasticity rather than synaptic depolarization (54), serve evolu-tionarily conserved functions in synaptic plasticity (55). We,therefore, asked whether postsynaptic NMDA-type glutamatereceptors on PNs could contribute to such glomerular selectivityof LN potentiation.The Drosophila NMDA receptor, a complex of NR1 and NR2

subunits, is expressed in many neurons of the adult antennal lobe(55). Levels of NR1 are substantially reduced by panneural ex-

Fig. 3. LTH to EB is accompanied by a rut-dependent decrease in the EB-evoked response of PNs. (A) Representative images of odor-evoked responses in DM2,DM5, and DC2 imaged in brains ofGH146Gal4, UASGCaMP3/ + flies. Pseudocolored images show responses (ΔF) during a 2-s puff of 5 × 10−3 EB (DM2 and DM5)or 3 × 10−2 dilution of 3-Oct (DC2). (Bottom) Representative traces of mean fluorescence change (ΔF/F) in the DM2 and DM5 (EB-evoked) and DC2 (3-Oct–evoked) in 4-d paraffin oil-exposed (solid lines) or 4-d EB-exposed (dashed lines)flies. (B) Quantitative analysis (expressed as percent ofmock) of odor-evoked PNresponses in the indicated glomeruli and genotypes. White bars indicate responses in mock-exposed flies, black bars indicate responses in 20% EB-exposed flies,and gray bars indicate responses in 5%CO2-exposed animals. The experiment uses rut2080; UAS-rut+flies in place of rut2080, because (i) like rut2080, these animalsdo not habituate (Fig. 2) and (ii) they show naïve GCaMP responses indistinguishable from WT controls. In the DM2 glomerulus, two-way ANOVA yieldeda significant difference between EB/mock treatments [F(1,82) = 11.227, P< 0.001]. Posthoc testing showed a significant difference betweenmock and EB-exposedWT flies (q = 4.167, P = 0.004; SNK) and no difference for rut2080; UAS-rut+ flies (q = 2.280, P = 0.111; SNK). For WT, n = 6 and n = 4 (mock and EB-exposed,respectively). For rut, n = 12 and n = 8 (mock and EB-exposed, respectively). In the DM5 glomerulus, two-way ANOVA yielded a significant difference betweenEB/mock treatments [F(1,79) = 6.469, P = 0.013)]. Posthoc testing showed a significant difference between mock and EB-exposed flies in WT flies (q = 5.554, P <0.001; SNK) and no difference for rut2080; UAS-rut+ flies (q = 0.804, P = 0.572; SNK). In EB-exposed flies, WT differed from the rut2080; UAS-rut+ genotype (q =5.002, P < 0.001). For WT, n = 6 and n = 7 (mock and EB-exposed, respectively). For rut, n = 8 and n = 8 (mock and EB-exposed, respectively). Bars represent meanarea under the curve (first 5.5 s of responses) + SEM (**P < 0.01, ***P < 0.001).

Das et al. PNAS | September 6, 2011 | vol. 108 | no. 36 | E649

GEN

ETICS

PNASPL

US

SEECO

MMEN

TARY

Dow

nloa

ded

by g

uest

on

Aug

ust 2

8, 2

021

pression of an RNAi transgene, dsNR1 (Fig. S5A). Expression ofdsNR1 in CO2-responsive VPN-positive PNs blocked STH andLTH to CO2 without affecting habituation to EB (Fig. 5 B andC).In contrast, NMDAR knockdown in EB-responsive GH146-expressing PNs efficiently blocked STH and LTH to EB withoutaffecting habituation to CO2 (Fig. 5 B and C and Fig. S5C). Thus,the NMDA receptor is required in a PN-specific manner forodorant selective habituation.In mammals and Drosophila, NMDA receptors respond to the

coincidence of postsynaptic depolarization and glutamate release(54, 55). PN depolarization is driven by OSN input. To deter-mine the source of glutamate in the Drosophila antennal lobe, weexamined the distribution of the vesicular glutamate transporter(DVGLUT) in vivo. DVGLUT is expressed widely in the an-tennal lobe, including a significant subset of LN1 local inter-neurons (Fig. S5B). RNAi-based knockdown of either GAD1 orDVGLUT in LN1 blocks STH and LTH, indicating that LN1terminals are an important source of both GABA and glutamaterequired for habituation (Fig. 5D). Disruption of STH and LTH

after knockdown of DVGLUT in GAD1-expressing, GABAergiccells (47) further suggests that habituation requires glutamatecorelease from GABAergic neurons, a phenomenon previouslyobserved in mammalian nervous systems and recently linked toinhibitory synapse plasticity (56–58) (Fig. 5 E and F). In contrast,mushroom-body expression of the VGLUT RNAi construct inMB247-expressing neurons had no effect on STH (Fig. S5D).The postulated need for glutamate release from inhibitory local

interneurons in the lobe can also explain why STH requires shifunction in LN1 cells during initial odorant exposure (Fig. 4A).Together, the data can explain glomerulus-selective potentia-

tion of LN–PN synapses by postulating that (i) odor stimulationcauses glutamate release onto PNs in multiple glomeruli, (ii)glomerular specificity arises because glutamate activates NMDAreceptors only on PNs that show coincident, odorant-induceddepolarization, and (iii) NMDAR signaling in selected PNs thenmediates, possibly through retrograde signaling (52, 53), the po-tentiation of GABAergic transmission onto these PNs.Additional observations (Fig. 2 B and C and Fig. S3F and Fig.

S5E) showing that the key signaling components (rut, NMDAR,and VGLUT) are required in adult-stage neurons provide addi-tional data consistent with this model.

Glomerulus-Selective Structural Plasticity Requires the Same Mecha-nisms Required for Odorant-Selective LTH. Previous work has shownthat 4-d exposure to CO2 or EB causes selective increase in thevolume of the V and DM2 glomeruli, respectively (35). Ourexperiments confirmed these observations (Fig. S6) and addi-tionally showed that 4-d EB exposure, which causes EB-selectiveLTH (Fig. 1), also causes particularly robust growth of the EB-responsive DM5 glomerulus that mediates the behavioral re-sponse to EB (48) (Fig. S6 A and B). We tested whethermechanisms required for odorant-selective habituation were alsorequired for glomerulus-selective structural plasticity.rut2080 mutants that do not show LTH to either CO2 or EB also

do not show associated increases in glomerular volume. Re-markably, expression of aWT rut+ transgene inLN1 interneuronsorGAD1-expressing neurons not only restores normal LTH to rutmutants but also restores respective glomerulus-selective growth(Fig. 6A and Fig. S6C). Thus, rut is required in LN1/GAD1 cellsfor both behavioral LTH and LTH-associated structural change.Similar to their role in behavioral habituation,NMDAreceptors

are required in odorant-responsive PNs for glomerulus-selectivestructural plasticity. Consistent with synapse-specific requirementfor NMDARs, knockdown of NR1 in VPN neurons blocked notonly LTH to CO2 but also the CO2-induced increase in V glo-merular volume. However, these flies (which showed normal LTHto EB) showed normal EB-evoked growth of the DM5 glomerulus(Fig. 6B and Fig. S6D). In contrast, knockdown of NR1 inGH146neurons blocked LTH to EB as well as the associated increase inDM5 glomerular volume without affecting LTH to CO2 or asso-ciated growth of the V glomerulus. Thus, postsynaptic NMDARsin PNs contribute not only to odorant-selective habituation butalso to glomerulus-selective structural plasticity.Knockdown of DVGLUT in GAD1 neurons again showed

effects analogous to observations in behavioral habituation.Consistent with the need for glutamate secretion from multi-glomerular LNs, knockdown of DVGLUT in GAD1-expressingneurons blocked both CO2- and EB-induced structural plasticity(Fig. S6E).It is interesting that the GABAA receptor (Rdl), which is es-

sential for expression of habituated behavior (Fig. 4 C and D), isnot necessary for LTH-associated structural plasticity (Fig. S6F).This finding is probably because the stable expression of GABAAreceptors is necessary for expressing the physiological conse-quence of plasticity in the antennal lobe, namely for the in-creased GABAergic transmission onto PNs. However, they arenot required for the changes per se.

Fig. 4. GABAergic transmission from LN1 interneurons is necessary andpotentially sufficient for the expression of habituation memory. (A and B)UAS-Shits1/+; LN1-Gal4/+ and UAS-Shits1/+ flies tested for STH (A) or LTH (B) toEB at ∼23 °C (room temperature) or 32 °C either during odor exposure (blueschematic) or testing (red schematic). (C andD) Knockdown of the Rdl GABAA

receptor (through UAS-RdlRNAi; abbreviated as Rdli) in VPN or GH146 neu-rons selectively blocks STH/LTH to odorants that activate these respective PNs.(E) LN1Gal4/UAS-TRPA1 show reduced responses to EB and CO2 when testedat 29 °C (dark bars) compared with 22 °C (white bars). Bars indicate mean ±SEM. ***P < 0.001 (Student t test); n of between 4 and 10 sets of flies con-tribute to each data point (exact n and P values in Tables S1–S3).

E650 | www.pnas.org/cgi/doi/10.1073/pnas.1106411108 Das et al.

Dow

nloa

ded

by g

uest

on

Aug

ust 2

8, 2

021

CREB Is Required for LTH but Not STH. In several neurobiologicalpreparations, structural plasticity and transcriptional gene ex-pression mediated in part by the cAMP response element-bindingprotein (CREB) transcription factor is required for consolidatingshort-term memory into a more stably stored, long-term memory.We therefore asked if STH and LTH could be distinguishedbased on their need for CREB function. We found that a 1-hinduction of CREB2b, an inhibitory isoform of CREB, througha heat-inducible hsCREB2b transgene blocked LTH without af-fecting STH (Fig. 7A and Tables S1–S3). Similar effects couldalso be seen when CREB2b was transiently induced in the LN1cells using the Tub-Gal80ts system (Fig. 7B and Tables S1 and S3).CREB2b induction not only blocked LTH but also associatedstructural plasticity (Fig. S7). Thus, CREB-dependent transcrip-tion in the LN1 class of local interneuron mediates specific formsof plasticity required for long-term but not short-term habituation(Fig. 7B). Taken together, these data point to a simple circuitmodel for habituation, which is outlined in Fig. 8.

DiscussionCircuit Mechanism of Olfactory Habituation. A key observation isthat rut function is uniquely required in adult-stage GABAergiclocal interneurons for STH and LTH (Fig. 2). This observationcontrasts with the rut requirement in mushroom-body neuronsfor olfactory aversive memory (42, 44). The demonstration offundamentally different neural mechanisms used in olfactoryhabituation and olfactory-associative memory elegantly refutesa proposal of the Rescorla–Wagner model that habituation (andextinction) may be no more than associations made with anunconditioned stimulus of zero intensity (5).The requirement for rut in inhibitory LNs also indicates that

intrinsic properties of multiglomerular LNs change during ha-bituation. However, logic, as well as anatomical and functionalimaging data, indicate that glomerulus-selective plasticity mustbe necessary if LN changes produce odorant-selective habitua-tion (Figs. 3 and 6). A potentially simple mechanism for glo-merulus-specific potentiation of LN terminals is suggested by thespecific requirement for postsynaptic NMDAR in odorant-re-sponsive glomeruli (Figs. 5 and 8).

The observation that LTH and STH show similar dependenceon rut, NMDAR, VGLUT, GABAA receptors, and transmitterrelease from LN1 cells indicates a substantially shared circuitmechanism for the two timescales of habituation. The data pointto a model in which transient facilitation of GABAergic synap-ses underlies STH; long-lasting potentiation of these synapsesthrough CREB and synaptic growth-dependent processes under-lies LTH (Fig. 8). This finding differs in three ways from synapticfacilitation that underlies Aplysia sensitization (40). First, it refersto inhibitory synapses, with potentiation that may involve a spe-cific heterosynaptic mechanism similar to that used for inhibitoryLong Term Potentiation (iLTP) in the rodent ventral tegmentum(52, 53). Second, by presenting evidence for necessary glutamatecorelease from GABAergic neurons, it proposes the involvementof a relatively recently discovered synaptic mechanism for plas-ticity (56). Third, it posits an in vivo mechanism to enable glo-merulus-specific plasticity of LN terminals.It is pleasing that, in all instances tested, physiological and

structural plasticity induced by 4-d odorant exposure requires thesame mechanisms required for behavioral LTH (Figs. 3–6 andFigs. S6 and S7). When taken together, these different lines ofexperimental evidence come close to establishing a causal con-nection between behavioral habituation and accompanying syn-aptic plasticity in the antennal lobe.It is important to acknowledge that, although our experiments

show that plasticity of LN–PN synapses contributes substantiallyto the process of behavioral habituation, it remains possible thatplasticity of other synapses, such as of recently identified excit-atory inputs made onto inhibitory LNs (16, 17, 59), also accom-pany and contribute to olfactory habituation (60).

Potentially General Mechanism for Habituation? The conserved or-ganization of olfactory systems suggests that mechanisms of ol-factory STH and LTH could be conserved across species (8, 9).Although this prediction remains poorly tested, early observa-tions indicate that a form of pheromonal habituation in rodents,termed the Bruce effect, may arise from enhanced inhibitoryfeedback onto mitral cells in the vomeronasal organ (61–63).

Fig. 5. NMDA receptors are required in PNs and VGLUT in LNs for odorant-selective habituation. (A) Diagram showing a single multiglomerular LN thatforms synapses with two different PNs. (B and C) Effects of PN-specific NMDAR knockdown on STH and LTH to CO2 and EB. VPN > dsNR1 selectively blocks CO2

habitiuation; GH146 > dsNR1 selectively blocks EB. (D–F) Behavioral effect of GAD1 or DVGLUT knockdown in LNs and GAD1 expressing neurons. Expressionof an RNAi against GAD1 (VDRC32344) or DVGLUT (VDRC104324) in LN1- (D) or GAD1-expressing neurons (E and F) blocks STH and LTH to EB and CO2. Barsindicate mean ± SEM calculated using the Student t test. ***P < 0.001; n > 7 (Tables S1–S3 show specific n values, and Fig. S2 has additional Gal4 and UAStransgene alone controls).

Das et al. PNAS | September 6, 2011 | vol. 108 | no. 36 | E651

GEN

ETICS

PNASPL

US

SEECO

MMEN

TARY

Dow

nloa

ded

by g

uest

on

Aug

ust 2

8, 2

021

Less obviously, two features of the circuit mechanism that wedescribe suggest that it is scalable and generalizable. First, se-lective strengthening of inhibitory transmission onto active glo-meruli can be used to selectively dampen either uniglomerular(CO2) or multiglomerular (EB) responses; thus, the mechanismis scalable. Second, the antennal lobe/olfactory bulb uses a circuitmotif commonly repeated throughout the brain, in which an

excitatory principal cell activates not only a downstream neuronbut also local inhibitory interneurons, which among other things,limit principal cell excitation (64–66).It is possible that, in nonolfactory regions of the brains, a sus-

tained pattern of principal neuron activity induced by a prolonged,unreinforced stimulus could similarly result in the specific po-tentiation of local inhibition onto these principal neurons. Sub-sequently, the pattern of principal cell activity induced by a secondexposure to a now familiar stimulus would be selectively gatedsuch that it would create only weak activation of downstreamneurons. In this manner, the circuit model that we propose forolfactory habituation could be theoretically generalized. We ex-pect more studies to test the biological validity of this observation.

Experimental ProceduresDrosophila Stocks. Unless otherwise stated, all flies were raised at 25 °Con standard cornmeal agar medium. The stocks were obtained from stockcenters or through the generosity of Drosophila colleagues as listed in SIExperimental Procedures.

Meauring Olfactory Responses and Habituation. Olfactory avoidance wasmeasured using an upright Y-Maze apparatus (Fig. 1). Odorant was drawnthrough one arm of the maze, and control air was drawn through the otherarm. Flies starved overnight were allowed into the entry tube, and theirpreference for the armwith the odorant (O) vs. the control (C) armwith air wasquantified as a response index [RI; the difference in the number of flies in theodorant and control arms as a fraction of the total flies RI = (O − C)/(O + C)]. Atleast eight batches were assayed for each data point, RI values were normal-ized to the appropriate control (e.g., response of naïve flies for STH, paraffin-exposedflies for LTH to EB, and air exposed for LTH to CO2), and the data wereplotted as percent control response. Except where otherwise indicated, com-parisons were carried out by the unpaired Student t test. All batches werecoded, and the experimenter was blind to the genotype being tested.STH. Two-day-old flies (unless otherwise stated) starved overnight werepretested to 5% CO2 or 10−3 dilution EB to measure the naïve RI. RIs wereagain determined after the same set of flies was exposed to either 15% CO2

or 5% EB for 30 min.LTH. The protocol was adapted from refs. 31 and 35. Flies aged between0 and 12 h were collected; 5% CO2 habituation involved 4-d exposure to CO2

(air control). EB habituation involved 4-d incubation with a perforated tubewith odorant 1:5 dilution of EB in light liquid paraffin oil (paraffin control).For TrpA1-mediated “phantom” habituation, control flies were maintainedand tested at room temperature (22 °C); experimental flies were tested atroom temperature after exposure to 29 °C for 30 min or 4 d.

Fig. 7. Brief induction of an inhibitory isoform of CREB2 (CREB2b) in LNsblocks LTH. (A) Effect of a single 37°C heat pulse (1 h) applied to 0- to 10-h-old hs-dCREB2b adults on STH and LTH. (B) Effect of a single 29°C heat pulse(8 h) applied to 0- to 6-h-old LN1Gal4; TubGal80ts > UAS dCREB2b adults onLTH. Heat pulses applied to control WT flies had no effect on STH or LTH,and the heat pulse to LN1Gal4; TubGal80ts > UAS dCREB2b had no effect onSTH. Bars indicate mean ± SEM computed using the Student t test. ***P <0.001. White bars, mock-exposed; black bars, odor-exposed (Tables S1 and S3show exact n and P values). CREB2b induction also blocked LTH-associatedstructural plasticity (Fig. S7).

Fig. 6. LTH is associated with glomerular-specific volume changes. (A) Effectof CO2 or EB exposure on glomerular plasticity in rut2080; UAS-rut+ mutantflies expressing rut+ in LN1- or GAD1-expressing cells. (B) PN-specific NMDARknockdown causes glomerulus-specific block of structural plasticity inducedby CO2 (Left) or EB (Right). Additional control data and glomeruli are shownin Fig. S6 A–D. Bars indicate mean ± SEM computed using the Student t test.n varies between 8 and 16 for each data point. ***P < 0.001; **P ≤ 0.01.White bars, mock-exposed; black bars, odor-exposed.

Fig. 8. A circuit model for the establishment of olfactory habituation. Asimplified diagram of OSN–LN–PN connections (Fig. 2A) in a single glomeruluswith labels coded black for events involved in naive olfactory behavior (cho-linergic transmission from OSNs to LNs and PNs as well as GABAergic trans-mission from LNs to OSNs and PNs), green for shared signaling pathwaysinitiated during the onset of STH and LTH habituation, red for hypothesizedchanges that have occurred after STH and LTH (increased GABAergic trans-mission from LNs to PNs suggested here to be presynaptic), and blue for sig-naling (CREB activation) and synaptic changes that occur specifically with LTH.

E652 | www.pnas.org/cgi/doi/10.1073/pnas.1106411108 Das et al.

Dow

nloa

ded

by g

uest

on

Aug

ust 2

8, 2

021

Brain Dissection, Immunohistochemistry, and Volume Measurements. Brainswere dissected and stained using standard conditions (67). For measurementsof glomerular volume, 3D confocal stacks (FV1000; Olympus) were analyzedusing Amira 5.2.0. Comparisons were made using the unpaired Student ttest. Except for the rut rescue analysis where male flies were used, 10–15female flies from each genotype, and at least 10 glomeruli were analyzed todetermine the mean and SEM. Investigators performing the measurementswere blind with respect to the genotype and experimental treatment.

Imaging Calcium Dynamics in Vivo. The procedure was as previously describedwith minor modifications (12) (SI Experimental Procedures). Analysis ofchanges in fluorescence was performed (with user blinded to genotype)using a custom Matlab script. WT and rutabaga flies (mock or EB-exposed)were compared using a two-way ANOVA. A Student Newman–Keuls posthoctest was used where appropriate. A Student t test was used when onlycomparing two groups of data.

ACKNOWLEDGMENTS. In acknowledgement of her formidable biologicalinsight, demanding, yet inspirational mentorship and generous friendshipthat guided this work, MR, SD, MKS, IPS, RP, AG and SS dedicate this paperto the memory of our co-author, Veronica Rodrigues. We thank AlbertoFerrus for introducing us to long-term habituation. We acknowledge A. Fiala,E. Buchner, M. Heisenberg, R. Davis, G. Norman, V. Jayaraman, L. Looger, L. Liu,A.-S. Chiang, G. Miesenbock, L. Vosshall, and S. Sachse for fly stocks andreagents,L. Vosshall for sharing data on CO2 habituation before publication,C. Root and S. Kim for advice on antennal nerve/odor stimulation and imaging,J. Truman for drawing our attention to the Bruce effect, and F. Chee andV. Kumar for early help with brain imaging. We thank K. VijayRaghavan,B. Keverne, S. O’Mara, T. Weinert, R. Parker, J. Joseph, J. P. Labrador,P. Paranjpe, and A. Chiang for useful discussions and/or comments on the man-uscript. I.P.S. was supported by a Council for Scientific and Industrial Research(Government of India) postgraduate fellowship. This work, seededwith supportfrom the Fogarty International Research Collaboration Award and MinorityScientist Supplement Awards from the National Institute on Drug Abuse/Na-tional Institutes of Health, was funded by grants from Department of Biotech-nology (DBT), India (to V.R.) and Science Foundation Ireland (to M.R.).

1. Thompson RF, Spencer WA (1966) Habituation: A model phenomenon for the studyof neuronal substrates of behavior. Psychol Rev 73:16–43.

2. Glanzman DL (2009) Habituation in Aplysia: The Cheshire cat of neurobiology.Neurobiol Learn Mem 92:147–154.

3. Rankin CH, et al. (2009) Habituation revisited: An updated and revised description ofthe behavioral characteristics of habituation. Neurobiol Learn Mem 92:135–138.

4. Wilson DA, Linster C (2008) Neurobiology of a simple memory. J Neurophysiol 100:2–7.

5. Rescorla RA, Wagner AR A theory of Pavlovian conditioning: Variations in theeffectiveness of reinforcement and nonreinforcement. Classical Conditioning II, edProkasy AHBWF New York (Appleton-Century-Crofts), pp 64–99.

6. McNamara AM, Magidson PD, Linster C, Wilson DA, Cleland TA (2008) Distinct neuralmechanisms mediate olfactory memory formation at different timescales. Learn Mem15:117–125.

7. Dalton P, Wysocki CJ (1996) The nature and duration of adaptation following long-term odor exposure. Percept Psychophys 58:781–792.

8. Su CY, Menuz K, Carlson JR (2009) Olfactory perception: Receptors, cells, and circuits.Cell 139:45–59.

9. Vosshall LB, Stocker RF (2007) Molecular architecture of smell and taste in Drosophila.Annu Rev Neurosci 30:505–533.

10. Olsen SR, Wilson RI (2008) Lateral presynaptic inhibition mediates gain control in anolfactory circuit. Nature 452:956–960.

11. Wilson RI, Laurent G (2005) Role of GABAergic inhibition in shaping odor-evokedspatiotemporal patterns in the Drosophila antennal lobe. J Neurosci 25:9069–9079.

12. Root CM, et al. (2008) A presynaptic gain control mechanism fine-tunes olfactorybehavior. Neuron 59:311–321.

13. Shang Y, Claridge-Chang A, Sjulson L, Pypaert M, Miesenböck G (2007) Excitatorylocal circuits and their implications for olfactory processing in the fly antennal lobe.Cell 128:601–612.

14. Lai SL, Awasaki T, Ito K, Lee T (2008) Clonal analysis of Drosophila antennal lobeneurons: Diverse neuronal architectures in the lateral neuroblast lineage.Development 135:2883–2893.

15. Chou YH, et al. (2010) Diversity and wiring variability of olfactory local interneuronsin the Drosophila antennal lobe. Nat Neurosci 13:439–449.

16. Huang J, ZhangW, QiaoW, Hu A, Wang Z (2010) Functional connectivity and selectiveodor responses of excitatory local interneurons in Drosophila antennal lobe. Neuron67:1021–1033.

17. Yaksi E, Wilson RI (2010) Electrical coupling between olfactory glomeruli. Neuron 67:1034–1047.

18. Dacks AM, Green D, Root CM, Highorn AJ, Wang JW (2009) Serotonin modulatesolfactory processing in the antennal lobe of Drosophila. J Neurogenet 23:366–377.

19. Fiala A (2007) Olfaction and olfactory learning in Drosophila: Recent progress. CurrOpin Neurobiol 17:720–726.

20. Krashes MJ, et al. (2009) A neural circuit mechanism integrating motivational statewith memory expression in Drosophila. Cell 139:416–427.

21. Nassel DR, Winther AM (2010) Drosophila neuropeptides in regulation of physiologyand behavior. Prog Neurobiol 92:42–104.

22. Root CM, Ko KI, Jafari A, Wang JW (2011) Presynaptic facilitation by neuropeptidesignaling mediates odor-driven food search. Cell 145:133–144.

23. Engel JE, Wu CF (2009) Neurogenetic approaches to habituation and dishabituationin Drosophila. Neurobiol Learn Mem 92:166–175.

24. Wuttke MS, Tompkins L (2000) Olfactory adaptation in Drosophila larvae. JNeurogenet 14:43–62.

25. Cobb M, Domain I (2000) Olfactory coding in a simple system: Adaptation inDrosophila larvae. Proc Biol Sci 267:2119–2125.

26. Boyle J, Cobb M (2005) Olfactory coding in Drosophila larvae investigated by cross-adaptation. J Exp Biol 208:3483–3491.

27. Colomb J, et al. (2007) Complex behavioural changes after odour exposure inDrosophila larvae. Anim Behav 73:587–594.

28. Larkin A, et al. (2010) Central synaptic mechanisms underlie short-term olfactoryhabituation in Drosophila larvae. Learn Mem 17:645–653.

29. Asztalos Z, Arora N, Tully T (2007) Olfactory jump reflex habituation in Drosophilaand effects of classical conditioning mutations. J Neurogenet 21:1–18.

30. Störtkuhl KF, Hovemann BT, Carlson JR (1999) Olfactory adaptation depends on theTrp Ca2+ channel in Drosophila. J Neurosci 19:4839–4846.

31. Devaud JM, Acebes A, Ferrús A (2001) Odor exposure causes central adaptation andmorphological changes in selected olfactory glomeruli in Drosophila. J Neurosci 21:6274–6282.

32. Devaud JM, Acebes A, Ramaswami M, Ferrús A (2003) Structural and functionalchanges in the olfactory pathway of adult Drosophila take place at a critical age. JNeurobiol 56:13–23.

33. Keene AC, et al. (2004) Diverse odor-conditioned memories require uniquely timeddorsal paired medial neuron output. Neuron 44:521–533.

34. Suh GS, et al. (2007) Light activation of an innate olfactory avoidance response inDrosophila. Curr Biol 17:905–908.

35. Sachse S, et al. (2007) Activity-dependent plasticity in an olfactory circuit. Neuron 56:838–850.

36. Rosenzweig M, et al. (2005) The Drosophila ortholog of vertebrate TRPA1 regulatesthermotaxis. Genes Dev 19:419–424.

37. Pulver SR, Pashkovski SL, Hornstein NJ, Garrity PA, Griffith LC (2009) Temporaldynamics of neuronal activation by Channelrhodopsin-2 and TRPA1 determinebehavioral output in Drosophila larvae. J Neurophysiol 101:3075–3088.

38. Bourne HR, Nicoll R (1993) Molecular machines integrate coincident synaptic signals.Cell 72(Suppl):65–75.

39. Levin LR, et al. (1992) The Drosophila learning and memory gene rutabaga encodesa Ca2+/Calmodulin-responsive adenylyl cyclase. Cell 68:479–489.

40. Bailey CH, Kandel ER, Si K (2004) The persistence of long-term memory: A molecularapproach to self-sustaining changes in learning-induced synaptic growth. Neuron 44:49–57.

41. Gervasi N, Tchénio P, Preat T (2010) PKA dynamics in a Drosophila learning center:Coincidence detection by rutabaga adenylyl cyclase and spatial regulation by duncephosphodiesterase. Neuron 65:516–529.

42. Blum AL, Li W, Cressy M, Dubnau J (2009) Short- and long-term memory in Drosophilarequire cAMP signaling in distinct neuron types. Curr Biol 19:1341–1350.

43. Pan Y, et al. (2009) Differential roles of the fan-shaped body and the ellipsoid body inDrosophila visual pattern memory. Learn Mem 16:289–295.

44. Zars T, Fischer M, Schulz R, Heisenberg M (2000) Localization of a short-term memoryin Drosophila. Science 288:672–675.

45. Okada R, Awasaki T, Ito K (2009) Gamma-aminobutyric acid (GABA)-mediated neuralconnections in the Drosophila antennal lobe. J Comp Neurol 514:74–91.

46. Tanaka NK, Ito K, Stopfer M (2009) Odor-evoked neural oscillations in Drosophila aremediated by widely branching interneurons. J Neurosci 29:8595–8603.

47. Ng M, et al. (2002) Transmission of olfactory information between three populationsof neurons in the antennal lobe of the fly. Neuron 36:463–474.

48. Semmelhack JL, Wang JW (2009) Select Drosophila glomeruli mediate innateolfactory attraction and aversion. Nature 459:218–223.

49. Wang JW, Wong AM, Flores J, Vosshall LB, Axel R (2003) Two-photon calcium imagingreveals an odor-evoked map of activity in the fly brain. Cell 112:271–282.

50. Kitamoto T (2002) Targeted expression of temperature-sensitive dynamin to studyneural mechanisms of complex behavior in Drosophila. J Neurogenet 16:205–228.

51. Liu X, BuchananME, Han KA, Davis RL (2009) The GABAA receptor RDL suppresses theconditioned stimulus pathway for olfactory learning. J Neurosci 29:1573–1579.

52. Nugent FS, Penick EC, Kauer JA (2007) Opioids block long-term potentiation ofinhibitory synapses. Nature 446:1086–1090.

53. Castillo PE, Chiu CQ, Carroll RC (2011) Long-term plasticity at inhibitory synapses. CurrOpin Neurobiol 21:328–338.

54. Collingridge GL (2003) The induction of N-methyl-D-aspartate receptor-dependentlong-term potentiation. Philos Trans R Soc Lond B Biol Sci 358:635–641.

55. Xia S, et al. (2005) NMDA receptors mediate olfactory learning and memory inDrosophila. Curr Biol 15:603–615.

56. El Mestikawy S, Wallén-Mackenzie A, Fortin GM, Descarries L, Trudeau LE (2011) Fromglutamate co-release to vesicular synergy: Vesicular glutamate transporters. Nat RevNeurosci 12:204–216.

57. Noh J, Seal RP, Garver JA, Edwards RH, Kandler K (2010) Glutamate co-release atGABA/glycinergic synapses is crucial for the refinement of an inhibitory map. NatNeurosci 13:232–238.

Das et al. PNAS | September 6, 2011 | vol. 108 | no. 36 | E653

GEN

ETICS

PNASPL

US

SEECO

MMEN

TARY

Dow

nloa

ded

by g

uest

on

Aug

ust 2

8, 2

021

58. Szabadits E, et al. (2011) NMDA receptors in hippocampal GABAergic synapses andtheir role in nitric oxide signaling. J Neurosci 31:5893–5904.

59. Hu A, ZhangW,Wang Z (2010) Functional feedback frommushroombodies to antennallobes in the Drosophila olfactory pathway. Proc Natl Acad Sci USA 107:10262–10267.

60. Maffei A, Nataraj K, Nelson SB, Turrigiano GG (2006) Potentiation of corticalinhibition by visual deprivation. Nature 443:81–84.

61. Brennan P, Kaba H, Keverne EB (1990) Olfactory recognition: A simple memorysystem. Science 250:1223–1226.

62. Wilson DA, Sullivan RM, Leon M (1985) Odor familiarity alters mitral cell response inthe olfactory bulb of neonatal rats. Brain Res 354:314–317.

63. Gao Y, Strowbridge BW (2009) Long-term plasticity of excitatory inputs to granulecells in the rat olfactory bulb. Nat Neurosci 12:731–733.

64. Kullmann DM, Lamsa KP (2007) Long-term synaptic plasticity in hippocampalinterneurons. Nat Rev Neurosci 8:687–699.

65. Buzsaki G (2006) Rhythms of the Brain (Oxford University Press, New York).66. Pouille F, Scanziani M (2004) Routing of spike series by dynamic circuits in the

hippocampus. Nature 429:717–723.67. Chiang A, Priya R, Ramaswami M, Vijayraghavan K, Rodrigues V (2009) Neuronal

activity and Wnt signaling act through Gsk3-beta to regulate axonal integrity inmature Drosophila olfactory sensory neurons. Development 136:1273–1282.

E654 | www.pnas.org/cgi/doi/10.1073/pnas.1106411108 Das et al.

Dow

nloa

ded

by g

uest

on

Aug

ust 2

8, 2

021