Pheromone responsiveness threshold depends ontemporal integration by antennal lobeprojection neuronsMasashi Tabuchia,b,1,2, Takeshi Sakuraib,1, Hidefumi Mitsunob, Shigehiro Namikib,3, Ryo Minegishia,b,4,Takahiro Shiotsukic, Keiro Uchinoc, Hideki Sezutsuc, Toshiki Tamurac, Stephan Shuichi Hauptb,5, Kei Nakatanid,and Ryohei Kanzakib,6

aDepartment of Advanced Interdisciplinary Studies, Graduate School of Engineering, and bResearch Center for Advanced Science and Technology, Universityof Tokyo, Meguro-ku, Tokyo 153-8904, Japan; cNational Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8634, Japan; and dGraduate School of Lifeand Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan

Edited by John G. Hildebrand, University of Arizona, Tucson, AZ, and approved August 9, 2013 (received for review July 22, 2013)

The olfactory system of male moths has an extreme sensitivity withthe capability to detect and recognize conspecific pheromonesdispersed and greatly diluted in the air. Just 170 molecules of thesilkmoth (Bombyx mori) sex pheromone bombykol are sufficient toinduce sexual behavior in the male. However, it is still unclear howthe sensitivity of olfactory receptor neurons (ORNs) is relayedthrough the brain to generate high behavioral responsiveness. Here,we show that ORN activity that is subthreshold in terms of behaviorcan be amplified to suprathreshold levels by temporal integration inantennal lobe projection neurons (PNs) if occurring within a specifictime window. To control ORN inputs with high temporal resolution,channelrhodopsin-2 was genetically introduced into bombykol-re-sponsive ORNs. Temporal integration in PNs was only observedfor weak inputs, but not for strong inputs. Pharmacological dissec-tion revealed that GABAergic mechanisms inhibit temporal integra-tion of strong inputs, showing that GABA signaling regulates PNresponses in a stimulus-dependent fashion. Our results show thatboosting of the PNs’ responses by temporal integration of olfactoryinformation occurs specifically near the behavioral threshold, effec-tively defining the lower bound for behavioral responsiveness.

optogenetics | pheromone orientation behavior | transgenic silkmoth |olfaction

Olfaction is a key element in many aspects of animal behav-ior, such as foraging, oviposition, and mate recognition. In

many moth species, a special class of odorants called sex pher-omones plays a critical role for identification of and orientationto potential mates. Because sex pheromones emitted by femalesare greatly diluted and dispersed in the air, sophisticated olfac-tory systems to detect minute amounts of sex pheromones andprocessing systems to translate subtle peripheral sensory respon-ses into appropriate behavioral responses have evolved in malemoths. Theoretical calculations have shown that the detectionof 170 molecules of the sex pheromone bombykol [(E,Z)-10,12-hexadecadienol] can trigger sexual behavioral responses in malesof the silkmoth Bombyx mori (1). The physical and chemicalmechanisms in the antennae, the sensilla, and the olfactory re-ceptor neurons (ORNs) responsible for this remarkably highsensitivity, which can detect even a single molecule, are wellunderstood (2, 3). However, it is unclear how a small number ofspikes from a small number of ORNs is processed centrally toallow for high behavioral responsiveness.In moths, pheromone molecules are detected by specialized

antennal ORNs that express particular pheromone receptorgenes (4–10). The axons of ORNs convey pheromone informationto the first olfactory center, the antennal lobe (AL; an analog ofthe vertebrate olfactory bulb). The AL is composed of a number ofglomeruli where ORNs establish connections with two typesof neurons: projection neurons (PNs), which relay olfactory in-formation to higher brain regions, and local interneurons (LNs),

which are involved in processing olfactory information withinthe deutocerebrum (11). In particular, male ALs have a specializedpheromone-processing unit called the macroglomerular complex(MGC), which comprises several glomeruli (12). In the silkmoth,the toroid glomerulus in the MGC is known to exclusively processbombykol information (13).Previous reports have shown that the sensitivity of phero-

mone-responsive ORNs is ∼1,000-fold lower than that of PNstuned to the same pheromone compound (14, 15), suggestingthat the AL network amplifies ORN inputs. One possible sourceof amplification is the high convergence ratio between ORNsand PNs (16). Considering the small size of most antennae,spatial integration would be likely to occur; however, the factthat weak stimuli activate a few ORNs at best calls for additionalmechanisms to explain the high behavioral sensitivity.

Significance

The olfactory system of male moths exhibits the ability to de-tect minute amounts of sex pheromones. How this extremesensitivity is achieved remains unclear. Using optogenetictechniques to activate a pheromone-responsive olfactory re-ceptor neuron, our results reveal that weak olfactory inputs,but not strong inputs, are temporally integrated in second-orderprojection neurons to promote behavioral responsiveness.Furthermore, temporal integration of strong olfactory inputs isinhibited by GABAergic mechanisms, indicating that GABAsignaling suppresses the amplification of strong stimuli. Thetimescale of this temporal integration corresponds well to thetemporal dynamics of odor signals in the natural environment,suggesting that the olfactory systems of male moths use thismechanism to detect weak pheromone signals in the air.

Author contributions: M.T., T. Sakurai, K.N., and R.K. designed research; M.T., H.M., S.N.,R.M., K.U., and S.S.H. performed research; T. Shiotsuki, H.S., and T.T. contributed new re-agents/analytic tools; M.T. and T. Sakurai analyzed data; andM.T., T. Sakurai, S.S.H., and R.K.wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Freely available online through the PNAS open access option.1M.T. and T. Sakurai contributed equally to this work.2Present address: Department of Neurology, Johns Hopkins University, Baltimore, MD 21287.3Present address: HHMI Janelia Farm Research Campus, Ashburn, VA 20147.4Present address: Department of Mechanical and Control Engineering, Tokyo Instituteof Technology, 2-12-1 Ookayama, Meguro-ku, Tokyo 152-8552, Japan.

5Present address: Department of Biological Cybernetics, Bielefeld University, 33615 Bielefeld,Germany.

6To whom correspondence should be addressed. E-mail: kanzaki@rcast.u-tokyo.ac.jp.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1313707110/-/DCSupplemental.

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One candidate explanation is temporal integration, which isa fundamental mechanism for contributing to the amplificationof synaptic inputs (17–19). In the olfactory system, the temporalintegration properties of AL neurons and their relevance for theinitiation of behavior are unknown. Temporal integration in theolfactory system has been challenging to investigate in detailbecause of the difficulty of accurately controlling olfactorystimuli within a given time period. Under natural conditions,odor molecules are distributed in odor plumes with intermittentlocal dynamics of odor exposure lasting >100 ms, followed byodor-free air lasting several hundreds of milliseconds (20–24).However, another study showed that “bursts” of odor withina plume structure can be as little 10–20 ms (25, 26). Therefore,the stimulation of pheromone-responsive ORNs must be con-trolled in the millisecond range.To examine the role of temporal integration in the generation

of low thresholds for pheromone-induced behavioral responses,we generated GAL4/UAS transgenic silkmoths expressing chan-nelrhodopsin-2 (ChR2), a light-gated ion channel from green al-gae, in bombykol-responsive ORNs. The transgenic silkmothsshowed light-activated pheromone orientation behavior, and wesucceeded in controlling the activity of bombykol-responsiveORNs with single-spike resolution. Using paired-pulse photo-stimulation at various interstimulus intervals (ISIs), we testedwhether temporal integration in the AL network could be thebasis of the high behavioral sensitivity.

ResultsReplacing Pheromone Stimuli with Light. We generated a GAL4/UAS transgenic silkmoth line expressing ChR2 under the controlof a putative promoter sequence of Bombyx mori olfactoryreceptor-1 (BmOR1) (27), the gene encoding the olfactory re-ceptor for the major sex pheromone (bombykol) in the silkmoth.RT-PCR with ChR2 gene-specific primers showed that ChR2 wasexpressed only in male antennae bearing both the BmOR1-GAL4 and UAS-ChR2 transgenes (Fig. 1A). Two-color fluores-cence whole-mount in situ hybridization of male antennaerevealed that ChR2 was expressed in almost all (96.8%) BmOR1-expressing cells in transgenic silkmoths containing BmOR1-GAL4 and UAS-ChR2 (Fig. S1 and Table S1). In the antennae ofBmOR1-GAL4/UAS-ChR2 male moths, fluorescence signals ofmCherry fused with ChR2 were detected in ORNs whosedendrites innervated pheromone-responsive sensilla (Fig. 1B).Axons of ChR2-expressing neurons terminated exclusively in thetoroid (Fig. 1C), which is the bombykol-processing glomerulus inthe silkmoth MGC (13). These results show that ChR2 expres-sion was restricted to bombykol-responsive ORNs.Upon blue-light stimulation, ChR2-expressing male moths

exhibited wing-flapping behavior, which always accompaniespheromone orientation behavior in silkmoths (Fig. 1D andMovie S1). The initiation of the behavioral response was de-pendent on the intensity and duration of illumination (Fig. 1 Eand F). No behavioral response was observed in ChR2-express-ing moths without all-trans retinal (ATR) injection or devoid ofantennae, in response to yellow-light stimulation, in parentalcontrol moths bearing either the GAL4 or UAS transgene aloneor in normal moths (Fig. 1D, Figs. S2 and S3, and Movie S2).The analysis of the body orientation direction after single-

pulsed photostimulation demonstrated that the moths displayedzigzag turning typical of pheromone orientation behavior (Fig.1G) (28). Based on different behavioral parameters, there wereno significant differences between stimulation with bombykoland with blue light (Fig. 1G and Table S2). Therefore, blue-lightstimulation in the ChR2-expressing male silkmoths can be usedto mimic olfactory stimulation with pheromones.To confirm that ChR2-assisted photoactivation can control the

activity of bombykol-responsive ORNs, we recorded the elec-trophysiological responses to blue-light stimulation in male long

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Fig. 1. Light stimulation triggers pheromone orientation behavior in ChR2-expressing transgenic silkmoths. (A) RT-PCR analysis of the ChR2 gene. RT-PCR was conducted with total RNA from the antennae of male moths car-rying BmOR1-GAL4 and UAS-ChR2, UAS-ChR2 alone, or BmOR1-GAL4 alone.The minus sign (−) indicates negative controls without reverse transcriptase.Actin was used as a positive control. (B) The expression of ChR2 in the an-tennae of male moths bearing both BmOR1-GAL4 and UAS-ChR2 was visu-alized by fluorescence of mCherry fused with ChR2. White arrows indicatethe dendrites of ORNs that innervate olfactory sensilla. No mCherry signalswere detected in the proximal axon. (Scale bar: 50 μm.) (C) Terminal axonalarborization of ChR2-expressing neurons visualized by fluorescence ofmCherry fused with ChR2 (magenta) in a representative confocal section.The label was confined to the toroid (dotted line) of the MGC. The overallneuropil structure was imaged by autofluorescence (green). T, toroid; D,dorsal, M; medial. (Scale bar: 100 μm.) (D) Blue-light stimulation inducedpheromone orientation behavior only in the ATR-injected BmOR1-GAL4/UAS-ChR2 male moths (n = 20). A single 100-ms pulse of 1.19 mW/mm2 bluelight was used as a stimulus. (E) Light intensity-dependent increase in thepercentage of ChR2-expressing male moths responding to blue-light stimu-lation (n = 20). A series of blue-light intensities (100-ms duration; 0.04–1.19mW/mm2) was applied. (F) Illumination-duration-dependent increase in thepercentage of ChR2-expressing male moths (n = 35) responding to blue-lightstimulation (1.69 mW/mm2). (G) Dynamics of the cumulative body-orienta-tion angle during tethered walking in a representative ChR2-expressingmale moth in response to photostimulation (Upper) or bombykol stimula-tion (Lower). Unilateral stimuli of 200-ms duration with 1.69-mW/mm2 bluelight (Upper) or 100 ng of bombykol (Lower) triggered similar zigzag be-havioral patterns. Stimulus onset (at t = 0) is indicated by arrows; an angle of0° indicates the initial forward bearing of the moth.

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sensilla trichodea that contained a bombykol-responsive ORN.All of the tested long sensilla trichodea in transgenic mothsresponded to both bombykol and blue-light stimulation with thesame spike amplitude, indicating that light-evoked spikes werederived from the bombykol-responsive ORNs; long sensilla tri-chodea in parental control moths showed no responses to bluelight (Fig. 2A and Fig. S4). The responses of bombykol-re-sponsive ORNs to light stimulation were dependent on the in-tensity and duration of the light stimulus (Fig. 2 B and C).In the brain, ChR2-assisted photoactivation of bombykol-

responsive ORNs triggered the same typical neural activity pat-terns as bombykol stimulation in toroid PNs and neck motorneurons, whose activity is indicative of steering during phero-mone orientation (29). We thus confirmed that the response ofbombykol-responsive ORNs to ChR2-assisted photostimulationmimics the bombykol-induced response in the brain (Fig. S5).

Temporal Integration Lowers the Behavioral Threshold. To de-termine whether intermittent ORN inputs could be effectivelyintegrated to induce behavior, we observed behavioral responsesto 3-ms paired-pulse photostimulation separated by various ISIs.Only 17–28% of moths showed a behavioral response at ISIsfrom 100 to 180 ms, but behavioral responsiveness was elevatedmore than twofold at ISIs of <80 ms (Fig. 3). Thus, ISIs of <80ms in paired-pulse photostimulation led to temporal integrationand enhanced behavioral responsiveness. Where in the braindoes this behaviorally relevant temporal integration occur? Onepotential candidate is the AL, and we therefore proceeded toanalyze temporal integration properties of AL PNs.

Temporal Integration in PN Responses at Behaviorally RelevantTimescales. We recorded PN responses to paired-pulse photo-stimulation separated by various ISIs using the same paradigm asin the behavioral experiments (Fig. 4). At longer ISIs (100–180ms), PN responses to the stimuli were clearly temporally segre-gated and similar in amplitude. This segregation represents thecondition in which responses to successive stimuli do not interact(Fig. 4C). In contrast, PN spike responses tended to increase atan ISI of 80 ms and were significantly elevated at ISIs of ≤60 ms,compared with single responses at long ISIs (Fig. 4C). Theseresults show that ORN inputs are temporally integrated in PNson a behaviorally relevant timescale.Temporal integration may impede the capability to resolve

odor dynamics within plumes at high stimulus intensities. Thus,we tested whether temporal integration occurs specifically whenthe stimulus amplitude is near the behavioral threshold (3-msduration; Fig. 1F), but not at stimulus levels that induce maxi-mum behavioral responsiveness (10-ms duration; Fig. 1F). We

defined ORN inputs induced by 3- and 10-ms stimulus durationsas “weak” and “strong” ORN inputs, respectively, based on thebehavioral responsiveness they elicited. In contrast to the resultsfor weak ORN inputs, no significant difference in spike outputwas observed between long and short ISIs with strong ORNinputs (Fig. 4). These results suggest that supralinear integrationof weak, but not strong, ORN inputs occurred in PNs.We confirmed that ChR2-expressing ORNs showed sublinear

integration of individual responses to paired-pulse photostimulationat all ISIs and stimulus durations used (Fig. S6) and that no adap-tation or facilitation occurred in the ORNs. Latency scatter due toconduction times in the antennal nerve was also negligible (Fig. S7).

Origins of Temporal Integration in PNs. AL PN firing patterns aregenerated by ORN and AL LN inputs, both of which may beinvolved in controlling temporal integration. The LNs are pre-dominantly GABAergic and play an important role in modifyingPN temporal dynamics (30–32). To examine the involvement ofGABAergic mechanisms in PN temporal integration, we applied

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Fig. 2. Light-evoked ORN responses in BmOR1-GAL4/UAS-ChR2 transgenic moths. (A) Representative recordings of bombykol-responsive ORNs to blue-lightstimulation (1.69 mW/mm2) for 100-ms duration, as indicated by the bars under the recording traces. The genotypes are given on each trace. (B) Intensity-dependent increase in spike counts induced by blue-light stimulation for 100-ms duration in bombykol-responsive ORNs expressing ChR2. Data are shown asmeans ± SEM (n = 18). (C) Duration-dependent increase in spike counts induced by blue-light stimulation at 1.69 mW/mm2 in bombykol-responsive ORNsexpressing ChR2. Data are shown as means ± SEM (n = 48).

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Fig. 3. Temporal integration in the initiation of pheromone orientationbehavior induced by paired-pulse photostimulation. Shown are percentagesof moths responding behaviorally to paired-pulse photostimulation (3-msduration and 1.69 mW/mm2) at various ISIs. Data groups labeled with dif-ferent letters differ significantly (P < 0.05; Fisher’s exact test). The percent-age of moths displaying a behavioral response was significantly increasedwith ISIs of <80 ms (n = 36).

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two different GABA receptor blockers, picrotoxin as a GABAAreceptor blocker and 3-N-[1-(3,4-dichlorophenyl)ethylamino]-2-hydroxypropyl cyclohexylmethyl phosphinic acid as a GABABreceptor blocker (32), and conducted the same paired-pulsephotostimulation during PN recordings (Fig. 5). Application ofthese blockers caused increases in the spontaneous firing rate,light-evoked spike count, and peak instantaneous spike fre-quency of PNs (Fig. S8), suggesting a disinhibitory effect by

GABA receptor blockade. In response to weak ORN inputs, PNtemporal integration properties in the presence of these blockerswere similar to those in saline controls; a paired-pulse stimulusevoked a greater spike output than that attributable to linearsummation for ISIs of 20–80 ms (Fig. 5C). This result impliesthat temporal integration may be an intrinsic property of thePNs. In response to strong ORN inputs, PN temporal integration,which was not evident at ISIs of <80 ms in saline controls, be-came significant in the presence of the GABA receptor blockers(Fig. 5C). Previous work has suggested that GABAergic signalingcan regulate temporal precision of PN response to ORN stim-ulation (31). We thus examined this issue, using our optogeneticORN stimulation. As shown in Fig. S8, the interquartile range ofPN spike latency is increased in the presence of GABA receptorblockers, suggesting that temporal precision of PN responses isdependent on GABA signaling. Thus, our data suggest thattemporal integration is abolished by GABAergic mechanisms inthe presence of strong ORN inputs to favor increased temporalresolution for plume tracking and that GABA signaling is im-portant for temporal precision of PN responses.

DiscussionThe extreme sensitivity of male moths to female pheromonesarises from pushing olfactory systems nearly to their physical andchemical limits. We show that information from ORNs is furtherprocessed and that temporal integration in AL PNs contributesto enhanced behavioral sensitivity at low stimulus intensities,whereas GABAergic mechanisms inhibit temporal integration athigh stimulus intensities to increase temporal resolution. Takingadvantage of the high temporal resolution of ChR2-assistedphotoactivation, we demonstrated that ChR2 makes it possibleto control ORN activity on a millisecond timescale.In response to weak ORN inputs, PN responses showed

supralinear integration properties at ISIs of <80 ms in paired-pulse photostimulation (Fig. 4). This supralinear integrationlikely occurs when the first afferent synaptic potential is tem-porally integrated with a subsequent one (17), indicating that thetime window within which ORN inputs can effectively interact inPNs is in the range of 40 to 80 ms. The timescale of integration inPNs appears to be relatively long compared with the temporalcharacteristics of cortical neurons in the somatosensory and vi-sual systems of mammals (33, 34). This long integration timeallows nonsimultaneous synaptic inputs to effectively generatespike outputs, and thus PNs act as temporal integrators to am-plify synaptic inputs from ORNs.In the present study, temporal integration was not evident in

response to strong ORN inputs (Fig. 4), but supralinear in-tegration could be obtained by applying GABA receptor block-ers (Fig. 5). Previous studies have reported that GABAergic LNshave synaptic connectivity with both ORNs and PNs (35–37) andthat GABA release from LNs inhibits synaptic transmission fromORNs to PNs (31, 38). Indeed, pharmacological blockade ofGABA receptors is likely to have a disinhibitory effect on PNresponses (31, 32, 39), correlated with the strength of ORNinputs (39–41). Consistent with these observations, our resultsshowed that inhibition by GABAergic LNs contributed toshaping PN responses only in the presence of strong, but notweak, ORN inputs. The details of this GABAergic mechanismremain to be elucidated.Although the application of GABA receptor blockers induced

temporal integration of strong ORN inputs at ISIs of 40–100 ms,supralinear output was not observed at the shortest ISI of 20 mseven in the presence of blockers (Fig. 5C). This result raises thepossibility that paired-pulse stimulation induces short-term syn-aptic depression, as shown in the AL of Drosophila (42).The integration time window for PN responses in the present

study was identical to that during which behavioral responsescould be enhanced by paired-pulse photostimulation. This

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finding suggests that temporal integration of successive spikes byPNs is a basic mechanism contributing to the high behavioralsensitivity of moths to pheromone stimuli. It has been shown thatwithin a plume, the concentration of odor is highly dynamic, withbursts of odor lasting as little as 10–20 ms (25, 26). Temporalintegration is limited to a time window of up to 80 ms, whichapproximately corresponds to the duration of multiple bursts. Incontrast, the durations of the odor plumes themselves are sig-nificantly longer, and previous work has examined the behavioral

and electrophysiological responses of insect olfactory systems tothese stimuli (43–46). Thus, the insect olfactory system is able tointegrate ORN inputs between multiple bursts within single odorplumes for enhanced sensitivity as well as to retain temporalinformation of plume dynamics occurring at longer timescales(23–26). Our use of optogenetic techniques has allowed us todescribe the cellular and behavioral responses at timescalesrelevant to “intraplume” odor dynamics, which provides insightsinto the signal processing properties of the olfactory system.In addition to temporal structure of stimuli, the properties of

the PNs appear to be sensitive to odor concentration, because athigh stimulus intensities, temporal integration is inhibited byGABAergic signaling, thus making this system responsive toa wide range of odor concentrations. These features may bebehaviorally advantageous for male silkmoth pheromone-plumetracking, particularly at close range of a female. Together, ourresults demonstrate that temporal integration properties of PNsare matched to the temporal properties of odor signals and playan important role in enhancing behavioral sensitivity.

Materials and MethodsFor details, see SI Materials and Methods.

Construction of the pBacUAS-ChR2 Vector. The DNA fragment encodingmammalian codon-optimized ChR2 fused withmCherry was amplified by PCRusing pLenti-CaMKIIa-hChR2-mCherry-WPRE (47) as a template and theprimers 5′-CTCCTAGGATGGACTATGGCGGCGCTTTG-3′ and 5′-CTCCTAGGT-TACTTGTACAGCTCGTCCATG-3′. The recognition sequence for BlnI wasadded to the 5′ ends of both primers. PCR was performed with PrimeSTARHS DNA polymerase (R010A; Takara Bio) under the following thermal pro-gram: 98 °C for 2 min and 30 cycles of 98 °C for 10 s, 64 °C for 15 s, and 72 °Cfor 2 min, followed by a final extension at 72 °C for 5 min. The amplifiedDNA was digested with BlnI and inserted into the BlnI site of the pBacMCS[UAS-A3-kynurenine 3-monooxygenase (KMO)] vector (48). The resultantvector was named pBacUAS-ChR2.

Generation of Transgenic Moths. The pBacUAS-ChR2 vector was purified byusing a HiSpeed Midi Plasmid Purification kit (Qiagen), dissolved ata concentration of 0.2 mg/mL in 0.5 mM phosphate buffer (pH 7.0) con-taining 5 mM KCl, and mixed with an equal volume of 0.2 mg/mL helperDNA encoding piggyBac transposase. This mixture was microinjected intoeggs of the w1-pnd strain that were collected 3–6 h after oviposition, asdescribed (49). The injected eggs were reared to adults and crossed toobtain G1 progeny. The screening of positive clones was performed im-mediately after the hatch by detecting the body pigmentation inducedby Bombyx KMO expression under the control of the cytoplasmic actingene (A3) promoter, as described (48). G1 adults were crossed withindividuals of the BmOR1-GAL4 line (27), and G2 adults were used asa GAL4/UAS strain.

Injection of Exogenous ATR. ATR injection was performed to provide a chro-mophore as a cofactor for ChR2. ATR is essential for ChR2 activation, andinsects do not intrinsically possess ATR. Therefore, we injected exogenous ATRinto the abdomen of silkmoths. For the experiments, we used moths 2–5 hafter ATR injection because this time window coincided with high ATRconcentration in the moth antennae as confirmed by HPLC (Fig. S2).

Light Stimulation. A high-power blue LED with peak wavelength of 465 nm(LBW5AP-JYKY-35-Z; Osram Opto Semiconductors) generated the photo-stimulation in all experiments.

ORN Recordings. The moth was immobilized on a plastic plate, and the an-tennae were stabilized by dental wax. Spikes were recorded by inserting anelectrolytically sharpened tungsten wire electrode into the base of a sensil-lum trichodeum. As a reference electrode, a platinum plate was inserted inthe neck of the moth. Photostimulation was generated by the blue LEDsystem described above.

PN Recordings. Two methods were used to record PN responses, intracellularrecordings (Fig. S4) and cell-attached recordings (Figs. 4 and 5).

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Fig. 5. GABAergic mechanisms in the AL network inhibit PN temporal in-tegration of strong ORN inputs. (A) A representative PN response underblockade of GABAA and GABAB receptors. With 3- and 10-ms duration,paired-pulsed stimulation induced a greater spike count at 40-ms ISI than at120-ms ISI. Arrows indicate the timing of stimulus presentation. The plussymbol (+) indicates spike timing. (B) PSTHs of PN spike responses underblockade of GABAA and GABAB receptors. Spikes were counted in 20-ms binsand averaged (n = 6). (C) Effect of GABA receptor blockade. The firing ratewas calculated by counting spikes during 500 ms from the onset of the firstlight pulse (n = 6). *P < 0.05 (Wilcoxon signed rank test). Data are shown asmeans ± SEM.

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ACKNOWLEDGMENTS. We thank Dr. Karl Deisseroth (Stanford University)for providing the pLenti-CaMKIIa-hChR2-mCherry-WPRE plasmid; Dr. ShigeruMatsuyama (University of Tsukuba) for purification of bombykol; Kenji Higo(Tokyo Institute of Technology) for technical advice about optical components;

and Tomoki Kazawa (University of Tokyo) and Dr. Mark Wu (Johns HopkinsUniversity) for comments on the manuscript. This work was supported bythe Japan Society for the Promotion of Science (JSPS) (M.T., S.S.H., and R.M.)and JSPS Scientific Research (B) Grant 21370029 (to R.K.).

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