Imaging analysis of clock neurons reveals light buffers the wake-promoting effect of dopamine

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1 Imging nlysis of lok neurons revels light uffers the wke-promoting effet of dopmine Yuhu Shng 1,2, Pul Hynes 2, Niolás Pírez 2, Kyle I Hrrington 3, Fng Guo 1,2, Jordn Pollk 3, Pengyu Hong 3, Leslie C Griffith 2 & Mihel Rossh 1,2 211 Nture Ameri, In. All rights reserved. How nimls mintin proper mounts of sleep yet remin flexile to hnges in environmentl onditions remins unknown. We found tht environmentl light suppressed the wke-promoting effets of dopmine in fly rins. The ten lrge lterl-ventrl neurons (l-lnvs), suset of lok neurons, re wke-promoting nd respond to dopmine, otopmine nd light. Behviorl nd imging nlyses suggested tht dopmine is stronger rousl signl thn otopmine. Notly, light exposure not only suppressed l-lnv responses, ut lso synhronized responses of neighoring l-lnvs. This regultion ourred y distint mehnisms: light-medited suppression of otopmine responses ws regulted y the irdin lok, wheres light regultion of dopmine responses ourred y upregultion of inhiitory dopmine reeptors. Plstiity therefore lters the reltive importne of diverse ues on the sis of the environmentl mix of stimuli. The regultory mehnisms desried here my ontriute to the ontrol of sleep stility while still llowing ehviorl flexiility. Animl sleep is responsive to externl signls suh s light nd soil environment 1 4. Sleep is lso modulted y internl signls suh s the irdin lok nd hnges in sleep pressure. The ltter reflets, for exmple, prolonged periods of wke or sleep. Beuse insomni nd hypersomni often ompny ging nd other helth prolems, it is importnt to understnd how the rin nd its sleep iruitry integrte nd prioritize diverse sleep-relevnt signls, internl s well s externl. Drosophil sleep is modulted y severl internlly generted rousl signls, inluding dopmine, otopmine nd the irdin lok relted neuropeptide pigment-dispersing ftor (PDF) 8. Light is prominent rousl signl for diurnl nimls suh s flies. However, wkefulness does not sle linerly with light intensity, nor does light work in isoltion. For exmple, oth flies nd humns re prone to sleepiness in the middle of the, often ompnied y np or siest 9,1. This indites tht effets of light on the rin nd on sleep iruitry re likely to e omplex nd integrted with other sleep-relevnt signls. Conerning how light ffets sleep iruitry, we nd others hve previously investigted the role of lok neurons in sleep regultion. These studies identified suset of the lok iruit, the ten l-lnvs (five on eh side of the rin), s eing potently wke-promoting 4,8,11. Notly, they only promote wkefulness during the light phse of stndrd lightdrk onditions nd hve no effet when flies re rered in onstnt drkness 4. In ddition, reent study found tht these ells my even medite soil enrihment indued inreses in time sleep need 12. l-lnvs therefore ontriute to sleep regultion s prt of plsti iruit tht is importnt for nimls to dpt to their environment. Its physiologil sis is lrgely unknown, exept tht the l-lnvs inrese their firing rte in response to ute light exposure 13. The ten l-lnvs hve relted neurons nery, the eight smll LNvs (s-lnvs). s-lnvs express neuropeptide, PDF, tht helps the s-lnvs keep time in the drk nd ontriutes to their funtion s mster lok neurons 14. Beuse l-lnvs lso express PDF 14, oth ell groups n e speifilly leled with Pdf-Gl4 driver line. Indeed, rin imging with fluoresene resonne energy trnsfer (FRET)-sed yli AMP reporter driven y Pdf-Gl4 (ref. ) reveled roust responses evoked y otopmine in the l-lnvs, ut not the s-lnvs 16. This is onsistent with the enrihment of mrnas for two otopmine reeptors, MB nd 2, in l-lnvs reltive to their expression in s-lnvs 16. To extend our previous studies, we investigted how light interts with other rousl systems in fly rins. Dopmine is highly potent wke-promoting signl in oth mmmls nd flies. We first found tht 12-h light exposure suppressed dopmine-medited wke promoting effets; tht is, sleep in the drk ws more inhiited thn sleep in the light y dopminergi neuron firing. Given tht the l-lnvs express dopmine reeptors nd re the only known wke-promoting neurons modulted y light in fly rins, we deided to fous on understnding the funtionlity of this iruit node. By omining the split-gfp pproh with funtionl rin imging using FRET-sed yli nuleotide reporter,17 19, we found tht the l-lnvs reeive synpti inputs from dopmine nd otopmine neurons. However, dopmine ppered to e stronger rousl signl thn otopmine in fly rins, t lest for flies rised under sl 12-h:12-h light-drk onditions. By ompring the l-lnv responses evoked y dopmine or otopmine under different light onditions, we found tht light suppressed oth dopmine- nd otopmine-indued AMP responses in the l-lnvs. The dt suggest tht these neurons re n integrtion enter for the externl rousl signl light nd different internl sleep-regulting 1 Howrd Hughes Medil Institute, Ntionl Center for Behviorl Genomis, Brndeis University, Wlthm, Msshusetts, USA. 2 Deprtment of Biology, Brndeis University, Wlthm, Msshusetts, USA. 3 Deprtment of Computer Siene, Brndeis University, Wlthm, Msshusetts, USA. Correspondene should e ddressed to M.R. (rossh@rndeis.edu). Reeived 9 Mrh; epted 17 My; pulished online 19 June 211; doi:1.138/nn.286 nture NEUROSCIENCE VOLUME 14 NUMBER 7 JULY

2 211 Nture Ameri, In. All rights reserved. Figure 1 Light suppressed the wke-promoting effets of dopmine. ( d) Indued firing of dopminergi neurons mrkedly deresed sleep during the drk period in light-drk (LD) or onstnt drkness (DD) onditions followed y sleep reound the following when firing ws returned to norml levels. In onstnt drkness, sleep ws even more severely suppressed, with oth sujetive time nd time sleep eing lmost entirely sent. TH-Gl4 driven expression of TrpA1 trnsiently inresed the tivity of dopminergi neurons when the temperture ws rised from 21 to 27 C t the eginning of the. The ehvior ws monitored for 3 d in either the light-drk ondition or onstnt drkness t 27 C efore returning to 21 C. For simpliity, only 1 d of dt from eh ondition is shown. The dt were olleted from ontrol UAS-TrpA1, ontrol TH-Gl4 (green) nd TH-Gl4; UAS-TrpA1 flies. Error rs represent s.d. ues. We propose tht the opposing effets of environmentl light nd dopmine my llow this simple iruit to uffer expeted flututions in dopmine relese from presynpti prtners. This ility to generte ondition-dependent plsti responses to vrious rousl ues my llow nimls to mintin proper sleep levels while still eing responsive to environmentl hnges. RESULTS Light suppresses the wke-promoting effet of dopmine Given tht oth dopmine nd otopmine hve een shown to promote wkefulness in Drosophil,6,2, we set out to investigte how the externl rousl signl, light, interts with these two internlly generted signls. This ws ddressed y tivting dopminergi or otopminergi neurons in dult rins under different entrinment onditions. We first used TH-Gl4; UAS-TrpA1 fly lines to tivte dopminergi neurons under light-drk or onstnt drkness onditions nd tested the resulting ehviorl effets. The TrpA1 hnnel llows n ute tivtion of dult rin neurons for 2 3 d using shift to wrm temperture 4,21. Unlike the hroni tivtion rried out previously 6, this mnipultion should use few developmentl effets. To this end, we heted flies to 27 C to mildly tivte dopminergi neurons in either light-drk or onstnt drkness onditions. Stimultion of dopminergi neurons using TH-Gl4 mrkedly suppressed totl sleep nd then inresed the mount of sleep on the next during reovery (Fig. 1 d). In ontrst, tivtion of otopminergi neurons in Td2-Gl4; UAS-TrpA1 flies using the sme temperture protool hd no detetle effet on totl sleep in either ondition (Supplementry Fig. 1 nd dt not shown for onstnt drkness onditions). Given tht hroni stimultion of these neurons using sodium hnnel led to mild derese of totl sleep 6, we speulted tht stronger tivtion of otopminergi neurons in dult rins my e neessry to produe signifint totl sleep effets. We then used 3 C to tivte otopmine neurons nd oserved slight derese in the mount of totl sleep s well s time sleep (Supplementry Fig. 1). As tivtion of dopminergi neurons produed stronger effets, we deided to fous on the intertion etween light nd dopmine. Nighttime sleep ws mrkedly suppressed y dopminergi neuron tivtion in light-drk onditions, wheres time ws not ffeted Tle 1 Indued firing of dopminergi neurons mrkedly suppresses sleep Experimentl onditions Light-drk onditions Constnt drkness Control used for sutrtion UAS-TrpA1 TH-Gl4 UAS-TrpA1 TH-Gl4 Chnge in Totl sleep (%).4 ± 2.% 29. ± 2.% 48.9 ± 4.4% ± 4.4% Chnge in Dytime sleep (%) N.S ± 8.% 23. ± 8.% Chnge in Nighttime sleep (%) 29.4 ± 2.% 34 ± 2.% 1.1 ± 4.2% 2 ± 4.2% N.S., not sttistilly signifint (P >.); see Online Methods for lultion of the reltive hnge in sleep time. ± vlues re s.e.m. Sleep per 3 min Sleep per 3 min C (efore tivtion) C (tivtion in DD) UAS-TrpA1 TH-Gl4 TH-Gl4; UAS-TrpA (Fig. 1 nd Tle 1). Moreover, totl sleep in onstnt drkness ws onsistently more sensitive to dopminergi neuron tivtion thn totl sleep in the light-drk ondition (Tle 1). In onstnt drkness, sleep in oth sujetive nd ws suppressed y dopmine tivtion (Fig. 1 nd Tle 1). Moreover, sleep in the sujetive ws more ffeted thn time sleep in the light-drk ondition (Tle 1), suggesting the 12 h of light exposure in light-drk onditions lso suppressed dopmine-medited wke-promoting effets in the time. The wke-promoting l-lnvs respond to dopmine To further investigte the iruitry mehnisms underlying this light effet on dopmine responsiveness, we foused on the l-lnv lok neurons, whih re the only neurons in fly rins known to promote wkefulness in the light phse 4. Dopmine reeptor mrnas (DopR, DopR2 nd D2R) re present in purified l-lnvs. Moreover, ll three mrnas hve quite high l-lnv:s-lnv rtios 16, suggesting tht dopmine reeptors re more undnt in l-lnvs thn in s-lnvs. To ssy for synpses etween dopminergi neurons nd l-lnvs, we first used the split-gfp system to detet possile memrne ontts etween these two lsses of neurons 18,19. The memrnetethered GFP frgment onstrut CD4øspGFP1-1 ws driven y TH- Gl4, whih lels most dopminergi neurons, nd CD4øspGFP11 ws driven y pdf-lexa, whih lels l-lnvs nd s-lnvs. In ll six of the rins tht we imged, reonstituted GFP signls were deteted round the LNv ell ody nd dendriti res, ut not in the opti loe where the xons of the l-lnvs re loted (Fig. 2,). A lk of GFP punt elsewhere in the rin, s well s omplete lk of GFP signl in four ontrol rins indites tht this puntte stining ner the LNv ells odies is, in ft, reonstituted GFP. This method lso deteted memrne ontts etween otopminergi neurons leled y Td2-Gl4 nd the l-lnvs (Fig. 2,d), inditing ontts etween l-lnvs nd oth otopminergi nd dopminergi neurons. Individully leled PPL2 dopminergi neurons hve een shown to projet to the re ontining the LNvs 22,23. To evlute potentil presynpti dopminergi projetions in Sleep per 3 min d Sleep per 3 min C (tivtion in LD) 21 C (reound in DD) the viinity of the LNv dendrites, we stined TH-Gl4 driven UAS mcd8-gfp rins with ntiody to PDF to lel oth dopminergi nd LNv neurons. Dense roriztions of the PPL2 dopminergi neurons were visile ner the LNv dendriti re (dt not shown). As split-gfp nlysis with the Pdf driver does not priori distinguish etween the 89 VOLUME 14 NUMBER 7 JULY 211 nture NEUROSCIENCE

3 211 Nture Ameri, In. All rights reserved. Figure 2 The l-lnvs form memrne ontts with dopminergi nd otopminergi neurons. (,) The memrne-tethered GFP frgment onstrut CD4øspGFP1-1 ws expressed in most dopminergi neurons with TH-Gl4, nd CD4øspGFP11 ws expressed in l- nd s-lnvs with Pdf-LexA. Green, GFP; red, PDF. The fine fiers in the ventrl elongtion in re likely to e the dendrites of the l-lnvs 31. Reonstituted GFP signls were deteted round the LNv ell odies nd dendriti re ut not in the optil loe round the xons of the l-lnvs (n = 6). The digrm indites the orienttion of the rin. D nd M indite the dorsl nd medil sides of the rin, respetively. An imge with higher mgnifition shows the reonstituted GFP signls round the LNv ell ody nd dendriti re (). Note tht the PDF stining in the dendriti res is very wek euse the dendrites do not likely ontin muh of the PDF peptide, resulting in GFP tht does not pper to ololize well with PDF stining in the dendriti res. (,d) The memrne-tethered l-lnvs nd s-lnvs nd my lso lel nonsynpti ontts, we used funtionl imging to ssy synpti inputs from dopminergi neurons to LNvs. Flies rrying Pdf-Gl4 nd UAS-EPAC trnsgenes speifilly express the FRET-sed AMP reporter EPAC in oth sets of PDFpositive ells, l-lnvs nd s-lnvs. We pplied dopmine to utely disseted rins nd determined the effets on AMP levels. We lso YFP hnnel CFP hnnel Rtio nd foreground Figure 3 The l-lnvs responded to dopmine or otopmine pplition y inresing AMP. () Exmple of how FRET imges were proessed using n utomted method s desried in the Online Methods. Briefly, eh video hd two hnnels (YFP nd CFP). The responses of ell to drug were omputed s the men of its CFP/YFP rtios, whih were normlized y signls ptured under the untreted ondition. Cells without sttistilly signifint response differenes over time were merged s group. In this exmple, the l-lnvs, ut not the s-lnvs, inresed AMP in response to th pplition of dopmine. () Dopmine () pplition indued M M Initil segmenttion D D LNv ell odies LNv dendrites l-lnv ventrl elongtion LNv ell odies nd dendrites GFP frgment onstrut CD4øspGFP1-1 ws expressed in most otopminergi neurons with Td2-Gl4. Reonsitituted GFP signls were lso deteted round the LNv ell odies nd dendriti re (n = 1). Sle rs represent 1 µm. n = 44 1 µm Control n = 6 Agonist developed n imge-proessing method to utomtilly ompre the temporl responses of individul l-lnvs or s-lnvs in the sme hemisphere (for detils, see Online Methods nd Supplementry Fig. 2). We oserved strong derese in FRET rtio (yellow fluoresent protein (YFP)/yn fluoresent protein (CFP)) in l-lnvs on th pplition of dopmine, inditing tht the reltive AMP level in Response merge l-lnvs s-lnvs Men of normlized luster intensity overtime (CFP/YFP) s-lnvs l-lnvs d 16 P =.14 n = 1 n = 7 1 µm Control Antgonist Reltive AMP hnges (CFP/YFP%) LD P =.4 DD P =.3 ZT8.1 TTX ZT8.7 TTX ZT9.3 TTX ZT1.2 TTX stronger responses in the l-lnvs thn otopmine (). Left, imging of flies rered in light-drk onditions (LD); right, flies rered in onstnt drkness 1 (DD). () The responses ould e indued y dopmine gonist nd were loked y dopmine ntgonist. The verge fluoresene hnge (re under the reltive AMP hnge urve) ws determined y lulting n verge CFP/YFP rtio inrese from 1 to 44 s. Error r represents s.e.m. Does this pply to error rs in s well? Plese stte expliitly. A dopmine gonist, 1 µm pergolide mesylte, lso indued n inrese of AMP in the l-lnvs with n effet only slightly less thn dopmine lone. The l-lnv dopmine-indued AMP response ws lmost ompletely loked following -min pre-inution with dopmine ntgonist, µm (+)-utlmol hydrohloride. (d) Dopmine-indued responses were ell utonomous; the l-lnv responses to dopmine in oth the presene nd sene of TTX were indistinguishle. The l-lnvs inresed AMP level in response to th pplition of dopmine in light-drk onditions. Responses of individul rin smples from different times of the re shown. The reltive AMP hnges re lulted s the normlized CFP/YFP rtio. Eh urve represents the verge AMP response of ll the visile l-lnvs in one hemisphere. The verge AMP responses from 13 rins re shown. Colored urves, with TTX dded to the utely disseted rins efore th pplition of dopmine. Gry urves, responses reorded without TTX. PDF GFP l-lnv xon l-lnv xon d ZT7.1 ZT7.7 ZT8.2 ZT8. ZT8.7 ZT9.1 ZT9.3 ZT9. ZT9.7 nture NEUROSCIENCE VOLUME 14 NUMBER 7 JULY

4 211 Nture Ameri, In. All rights reserved LD d these ells inresed mrkedly (Fig. 3). We rehed similr onlusion using Pdf-Gl4; UAS-EPAC flies in yw geneti kground, suggesting tht the onnetion etween dopminergi neurons nd l-lnvs is not strin speifi (dt not shown). s-lnvs, in ontrst, showed very wek responses (Fig. 3 nd Supplementry Fig. 3), similr to their wek response to otopmine (Supplementry Fig. 3) 16. Moreover, dopmine (1 µm) indued stronger responses in the l-lnvs thn otopmine (1 µm) in oth light-drk nd onstnt drkness onditions (Fig. 3). Comined with the ehviorl results, this suggests tht dopmine is stronger rousl signl thn otopmine in fly rins. To further test the speifiity of the dopmine-indued responses, we pplied dopmine gonist to disseted rins 24. Pergolide mesylte (1 µm) indued sustntil inrese in AMP in the l-lnvs (Fig. 3). Moreover, pre-inution with ntgonist, (+)-utlmol hydrohloride ( µm) 24, lmost ompletely loked the ility of dopmine to stimulte AMP prodution (Fig. 3). The muh stronger dopmine response of l-lnvs ompred with s-lnvs is onsistent with the reeptor mrna distriution 16. To further test whether the dopmine-indued responses re ellutonomous, we pplied tetrodotoxin (TTX) to the disseted rins efore th pplition of dopmine nd still oserved roust responses. They showed no sttistil differene from the non-ttx responses (Fig. 3d), exept tht the non-ttx groups showed slightly higher vrition. We onlude tht the l-lnvs reeive diret synpti DD e f P = P =.3 DD DD inputs from dopminergi neurons. Tken together with our previous study 16, we onlude tht the l-lnvs, ut not the s-lnvs, re trgets of dopmine nd otopmine neurons. Light suppresses dopmine-medited AMP inreses in the l-lnvs The wke-promoting effets of l-lnvs re plsti, tht is, they re effetive in stndrd 12-h:12-h light-drk onditions, ut not in onstnt drkness 4. To understnd how environmentl hnges ffet the physiology of this iruit node, we rered flies in either light-drk or onstnt drkness nd ssyed the differenes in the l-lnv response to dopmine or to otopmine 21. We first ompred the response mplitude to dopmine etween lightdrk nd onstnt drkness rering. Although we did not oserve differene in the l-lnv response, onstnt drkness rering used sustntil AMP inrese in oth sujetive nd sujetive (Fig. 4 ). The inresed AMP response to dopmine therefore ppered to e light sensitive, ut time insensitive; oth time nd time responses to dopmine were negtively regulted y the 12-h light exposure of light-drk onditions. On the other hnd, otopmine responses were oth light nd time sensitive. l-lnvs from sujetive were more sensitive to otopmine thn those from sujetive. In other words, the 12-h light exposure speifilly suppressed the time response (Fig. 4d f). We lso ompred the response of individul l-lnvs in the sme hemisphere nd oserved heterogeneous responses during the first n = 44 n = 34 n = 39 DD P =.2 n = 22 DD Figure 4 12-h light exposure suppressed the responses of l-lnvs to dopmine or otopmine. ( ) Light exposure suppressed the l-lnv responses to dopmine (). Flies were housed in light-drk onditions () or in onstnt drkness onditions () nd the responses to dopmine during time or sujetive were ompred with tht during time or sujetive. A summry of the reltive hnges of AMP (,) is shown in. The l-lnv responses to dopmine during the /sujetive versus the /sujetive were not signifintly different in either light-drk (LD) or onstnt drkness (DD) onditions. However, omprison etween light-drk nd onstnt drkness onditions showed tht the responses of the l-lnvs to dopmine in onstnt drkness were muh stronger during oth the sujetive nd sujetive thn the responses t the sme irdin times in light-drk onditions. (d f) Dytime light exposure suppressed the time l-lnv responses to otopmine (). Flies were housed in light-drk onditions (d) or onstnt drkness onditions (e) nd the responses to otopmine during time or sujetive were ompred with tht during time or sujetive. Note tht the response mplitude of l-lnvs from sujetive in onstnt drkness ws similr to tht of time in light-drk onditions. A summry of the reltive hnges in AMP (d,e) is shown in f. The responses to otopmine during time, time or sujetive time were similr, wheres the l-lnvs from sujetive were more sensitive to otopmine. P vlues indite signifint differene from ontrol groups (Student s t test). Error rs represent s.e.m. n = 2 n = 26 n = 4 n = VOLUME 14 NUMBER 7 JULY 211 nture NEUROSCIENCE

5 211 Nture Ameri, In. All rights reserved. d per per of onstnt drkness rering. In the most extreme se, the differene etween the responses of ll four l-lnvs ws sttistilly signifint (P <.; Supplementry Fig. 4). We therefore lssified the rin responses into four tegories on the sis of heterogeneity (Supplementry Fig. 4,): rins with homogenous responses, rins with two types of responses, rins with three types of responses nd rins in whih ll four l-lnvs showed different responses. The third nd fourth types were only oserved in smples from onstnt drkness rering, inditing tht the 12-h light exposure lso mde the dopmine- nd otopmine-evoked responses more homogeneous mong neighoring l-lnvs in the sme hemisphere. We speulte tht the synhroniztion mong the neighoring l-lnvs my llow the l-lnvs to produe reltively stle output in light-drk onditions (lso see Disussion). e per per f otopmine responsiveness during onstnt drkness is regulted y two opposing ftors: it is inresed y the irdin lok nd deresed y the light phse of norml light-drk yle. Dytime responses ppered to e more stle, tht is, less ffeted y either light or the lok (see Disussion). To determine whether the phenotype oserved in per 1 mutnts is speifilly used y loss of lok funtion, we tested the otopmineindued responses of the l-lnvs in flies in non-irdin mutnt. Flies without funtionl yellow gene show rhythmi ehvior in onstnt drkness onditions nd should therefore hve norml lok. The l-lnvs from this strin showed - response ptterns to otopmine, similr to ontrol strins (Supplementry Fig. ). Thus, the redued otopmine sensitivity t tht we oserved in the per 1 mutnt is likely result of the sene of the irdin lok. n = 44 n = 2 n = 34 per 1 n = 31 per 1 n = 34 n = 27 per 1 P = P =. n = 26 n = 27 per 1 Figure The irdin lok (PER) speifilly promotes otopmine-indued responses in l-lnvs t. ( ) The l-lnv responses to dopmine were not ffeted y PER. The time () nd time () responses re plotted seprtely. The dopmine-indued responses of the l-lnvs from ontrol rins were ompred with those from per 1 mutnt flies. A summry of the reltive hnges of AMP (,) is shown in. The responses to dopmine were not ffeted y per 1 muttion. (d f) PER positively regulted otopmine-evoked responses y l-lnv t. Flies were housed in light-drk onditions nd the time (d) nd time (f) responses re plotted seprtely. The otopmine-indued responses of the l-lnvs from ontrol rins were ompred with those from per 1 mutnt flies. A summry of the reltive hnges of AMP (d,e) is shown in f. The responses to otopmine during time were not ffeted y per 1 muttion (left), wheres the time responses were mrkedly deresed in the per 1 mutnts (right). Error rs represent s.e.m. Light nd lok regulte otopmine-indued responses As the l-lnvs re lso prt of the lok iruit, we sked whether the irdin lok plys role in regulting their responsiveness to rousl signls. per 1 flies rry null muttion in the ore lok gene period nd therefore lk funtioning irdin lok. Beuse dopmine-indued responses re time insensitive, they my not e regulted y the lok. Consistent with this predition, the dopmine-indued FRET responses in per 1 were omprle to those in ontrol rins (Fig. ). However, l-lnvs from per 1 flies were muh less responsive to otopmine thn ontrols during the, lthough their responses were similr to ontrol flies during the (Fig. d f). In other words nd unlike in wild-type flies, we oserved - differene in per 1 flies (P <.; Fig. f). Thus, the norml time inrese in l-lnv Light upregultes inhiitory dopmine reeptors How then does light suppress the l-lnv AMP responses to dopmine? Dopmine tivtes oth stimultory nd inhiitory reeptors, nd mny mmmlin rin neurons oexpress stimultory D1-like reeptors (D1Rs) nd inhiitory D2-like reeptors (D2Rs) 26. D1Rs modulte neurons y inresing intrellulr AMP, wheres D2Rs ntgonize AMP signling. Thus, D2Rs re importnt for gting ellulr responses to dopmine nd re involved in mny neurologil nd psyhologil disorders 26. Both Drosophil DopR nd DopR2 elong to the D1-like stimultory reeptor sufmily 27, wheres Drosophil D2R is the only known inhiitory reeptor in the fly 28. Moreover, D2R is highly enrihed in l-lnvs ompred with the neighoring s-lnvs 16. nture NEUROSCIENCE VOLUME 14 NUMBER 7 JULY

6 LD LD D2R-RNAi DD DD D2R-RNAi Averged reltive AMP hnge (%) P =.7 n = 44 n = 17 n = 2 n = LD LD D2R-RNAi DD DD D2R-RNAi Figure 6 Light suppresses dopmine responses y upregulting inhiitory dopmine reeptors. (,) D2R negtively regulted dopmine-evoked responses in the l-lnvs. () The l-lnv response to dopmine in light-drk onditions ws mrkedly inresed y knoking down D2R expression in the l-lnvs. The dopmine-indued responses of the l-lnvs from ontrol rins were ompred with those from D2R-RNAi knokdown mutnt flies. () The l-lnv response to dopmine in onstnt drkness onditions ws not ffeted y knoking down D2R expression in the l-lnvs. The dopmine-indued responses of the l-lnvs from ontrol rins were ompred with those from D2R-RNAi knokdown mutnt flies. () Summry of the reltive hnges of AMP shown in nd. The responses in onstnt drkness were omprle with those in D2R knokdown mutnts in light-drk onditions. Error rs represent s.e.m. 211 Nture Ameri, In. All rights reserved. To investigte whether the LNvs use D2R to uffer the effetiveness of dopmine tivtion, we used Pdf-Gl4 to drive the expression of UAS-D2R RNA interferene (RNAi) in the l-lnvs nd s-lnvs. Knokdown of D2R mrkedly inresed the dopmine-indued AMP response in l-lnvs in light-drk onditions (Fig. 6), inditing tht they indeed oexpress inhiitory s well s exittory dopmine reeptors nd tht D2R gtes the dopmine response. In ontrst, the knokdown in flies housed in onstnt drkness onditions showed no effet on AMP inreses (Fig. 6,). Moreover, the D2R knokdown group now showed similr responses in light-drk ompred with onstnt drkness onditions. As ontrol, we imged the s-lnv responses to dopmine in the D2R-RNAi flies nd oserved no detetle effets (Supplementry Fig. 6), onsistent with the ft tht these ells express muh lower levels of D2R thn l-lnvs 16,24. Tken together, these dt suggest tht light-drk onditions led to n upregultion of the D2R inhiitory signling pthwy in the l-lnvs, whih ounter-lnes the tivtion effet of dopmine. DISCUSSION Light uffers the effetiveness of dopmine-medited wke-promoting effets in Drosophil. Dytime sleep is reltively insensitive to dopmine tivtion, wheres time sleep in light-drk onditions is sensitive, ut less so thn time sleep in onstnt drkness onditions. The ten l-lnvs, suset of lok neurons, re the only neurons known to e prt of the light-medited wke-promoting iruits in fly rins. We found tht they re downstrem trgets of dopminergi neurons. They not only formed memrne ontts with dopminergi neurons, ut lso responded to dopmine y inresing AMP levels. This presumly reflets the ft tht l-lnvs express stimultory reeptors for these neurotrnsmitters. The response ws lrgely ell-utonomous, s they still responded to dopmine in the presene of TTX (Fig. 3). We lso found tht the responses re likely to e speifi to dopmine, s they were loked y dopmine ntgonist nd ould e indued y dopmine gonist (Fig. 3). These ells lso reeive diret synpti input from otopminergi neurons (Fig. 2 nd dt not shown for otopmine in the presene of TTX). Dopmine is likely stronger rousl signl thn otopmine in fly rins, t lest for flies rised in stndrd light-drk onditions. An identil stimultion of otopmine neurons in dult rins only mildly suppressed totl sleep, n effet tht ws lso onsiderly smller thn previously reported 6. This previous study used sodium hnnel to onstitutively stimulte otopmine neurons 29. Comined with the ft tht feeding flies with otopmine lso required 2 3 d to suppress sleep nd tht time sleep ws still ffeted even fter otopmine ws removed 7, we suggest tht hroni tivtion of otopminergi neurons my require reonfigurtion of neurl iruits to produe strong ehviorl effets. The s-lnvs re neighors of the l-lnvs nd re key pemker neurons in Drosophil. In ontrst with the l-lnvs, s-lnvs showed very wek responses to dopmine or otopmine in light-drk onditions, likely refleting the ft tht mrnas for these reeptors re muh more undnt in l-lnvs thn in s-lnvs 16. This even inludes the dopmine D2R inhiitory reeptors, whih lso explins why D2R knokdown did not led to detetle AMP inrese in s-lnvs in response to dopmine pplition (Supplementry Fig. 6). Light hs profound effet on niml ehvior. For exmple, extensive light-driven yli gene expression hs een deteted in Drosophil 3. The l-lnvs re lso reported to inrese their firing rte in response to ute light exposure, espeilly during erly morning 13. We found tht the 12-h light exposure of stndrd light-drk housing onditions hs profound effet on l-lnv physiology. Light-drk rering not only mitigtes the stimulting effets of oth dopmine nd otopmine, ut lso synhronizes ell responses. One possile funtion for synhroniztion is tht the l-lnv responses re more stle when synhronized (see elow). Although l-lnvs from light-drk rered flies re less sensitive to oth dopmine nd otopmine thn those from onstnt drkness rered flies, the two signling pthwys re differentilly regulted. Otopmine-medited responses re time sensitive in onstnt drkness, nd otopmine tivtion t is promoted y the lok, ut inhiited y prior light exposure. Mirorry dt indite tht trnsription of the otopmine reeptor 2 peks round zeitgeer time 12 (ZT12), wheres tht for MB peks round ZT6 (ref. 16). Given tht our imging nlysis indited tht mximum time l-lnv responses to otopmine require the lok (Fig. ), it is possile tht the trnsltion or tivities of these reeptors, or the expression of signling moleules downstrem of these reeptors, peks t. In ontrst with otopmine-medited responses, the dopminemedited responses of l-lnvs were time insensitive nd were not ffeted y the per 1 muttion (Figs. 4 nd ). However, light exposure suppressed the l-lnv dopmine responses t ll times, oth time nd time. Given tht downregultion of D2R is suffiient to mimi the responses of flies rered in onstnt drkness nd tht D2R RNAi 894 VOLUME 14 NUMBER 7 JULY 211 nture NEUROSCIENCE

7 211 Nture Ameri, In. All rights reserved. hd no effet in onstnt drkness, light exposure pprently upregultes D2R tivity to dmpen dopmine responsiveness in light-drk onditions. This implies tht there re light-stimulted hnges in either D2R gene expression or regultion, suh s modifition of the D2R reeptor or its downstrem trgets. Light my lso downregulte stimultory D1R signling pthwys in onert with the upregultion of D2R, lthough our results suggest tht expression of D2R n ount for most of the redution in responsiveness. Given tht there re no known inhiitory reeptors for otopmine, the l-lnvs must use different mehnism to effet light-medited modultion of otopmine responsiveness (Supplementry Fig. 7). For exmple, light my downregulte stimultory otopmine reeptors. Nonetheless, ommon theme is tht light inhiits the ility of these two hemils to stimulte the l-lnvs. The ft tht the 12-h light exposure suppressed the ility of dopmine nd otopmine to stimulte l-lnvs suggests tht they do not simply sum different rousl signls. Insted, they re integrted nd perhps sled depending on onditions, suggesting link to ehviorl flexiility. In this senrio, light ppers to e dominnt signl, s its presene during the redues the ility of internl signls to stimulte rousl. However, the l-lnvs use numer of mehnisms, inluding the irdin lok to integrte signls nd produe pproprite responses. The surprisingly wek ehviorl effets of ute stimultion of otopmine neurons rises the possiility tht there re other irumstnes (ge, nutritionl or reprodutive sttus) in whih these inputs eome more importnt. Beuse nimls must mintin proper qulity nd quntity of dily wke nd sleep time, ounter-lning mehnisms suh s those desried here my lso serve to preserve sleep stility in the fly rin. For exmple, the opposing effets of environmentl light nd dopmine my llow the l-lnvs nd perhps other rousl-sleep relevnt neurons to uffer unexpeted flututions in light intensity nd/or dopmine relese from presynpti prtners; tht is, the iruit orgniztion llows the tivity of sleep-relevnt neurons to e mintined in physiologil rnge with reltively stle output. We imgine tht only exeptionl irumstnes would tke preedene over sleep-wke stility; for exmple, y modulting the rtio of stimultory nd inhiitory dopmine reeptors. Our dt suggest tht modultion ould lso our y ltering the synhroniztion of individul ells in group, suh s etween different individul l-lnvs. It will not e surprising if dditionl integrtion mehnisms re lso importnt for the l-lnvs to generte pproprite signls to downstrem iruits, oth to mintin optiml sleep t nd optiml wkefulness during the, tht is, for sleep-wke homeostsis, nd for pproprite responses to emergeny irumstnes. Methods Methods nd ny ssoited referenes re ville in the online version of the pper t Note: Supplementry informtion is ville on the Nture Neurosiene wesite. Aknowledgments We thnk P. Tghert for kindly providing pdfgl4 (X) nd UAS-EPAC flies. We otined UAS-D2R-RNAi lines from the Vienn Drosophil RNAi enter. We re grteful to O. Shfer for tehnil help with AMP imging. We lso thnk E. Dougherty for ssistne in onfol mirosopy, K. Plm nd S. Pestore for dministrtive ssistne nd C. Vesey for omments on the mnusript. The work ws supported in prt y grnts from the US Ntionl Institutes of Helth (PO1 NS to M.R., R1 MH67284 to L.C.G. nd NIH R1 EB742 to P. Hong). AUTHOR CONTRIBUTIONS Y.S. oneived the projet. Y.S., P. Hynes, N.P. nd F.G. performed the experiments. K.I.H., J.P. nd P. Hong developed the lgorithm for the utomted imging nlysis. Y.S., L.C.G. nd M.R. wrote the pper. COMPETING FINANCIAL INTERESTS The uthors delre no ompeting finnil interests. Pulished online t Reprints nd permissions informtion is ville online t reprints/index.html. 1. Gnguly-Fitzgerld, I., Donle, J. & Shw, P.J. Wking experiene ffets sleep need in Drosophil. Siene 313, (26). 2. Ho, K.S. & Sehgl, A. Drosophil melnogster: n inset model for fundmentl studies of sleep. Methods Enzymol. 393, (). 3. Keene, A.C. et l. Clok nd yle limit strvtion-indued sleep loss in Drosophil. Curr. Biol. 2, (21). 4. Shng, Y., Griffith, L.C. & Rossh, M. Light-rousl nd irdin photoreeption iruits interset t the lrge PDF ells of the Drosophil rin. Pro. Ntl. Ad. Si. USA, (28).. Andreti, R., vn Swinderen, B. & Greenspn, R.J. Dopminergi modultion of rousl in Drosophil. Curr. Biol., (). 6. Croker, A. & Sehgl, A. Otopmine regultes sleep in Drosophil through protein kinse A dependent mehnisms. J. Neurosi. 28, (28). 7. Croker, A., Shhidullh, M., Levitn, I.B. & Sehgl, A. Identifition of neurl iruit tht underlies the effets of otopmine on sleep:wke ehvior. Neuron 6, (21). 8. Prisky, K.M. et l. PDF ells re GABA-responsive wke-promoting omponent of the Drosophil sleep iruit. Neuron 6, (28). 9. Hendriks, J.C. et l. Rest in Drosophil is sleep-like stte. Neuron, (2). 1. Shw, P.J., Cirelli, C., Greenspn, R.J. & Tononi, G. Correltes of sleep nd wking in Drosophil melnogster. Siene 287, (2). 11. Shee, V. et l. Lrge ventrl lterl neurons modulte rousl nd sleep in Drosophil. Curr. Biol. 18, 37 4 (28). 12. Donle, J.M., Rmnn, N. & Shw, P.J. Use-dependent plstiity in lok neurons regultes sleep need in Drosophil. Siene 324, 18 (29). 13. Shee, V., Gu, H., Shrm, V.K., O Dowd, D.K. & Holmes, T.C. Cirdin- nd light-dependent regultion of resting memrne potentil nd spontneous tion potentil firing of Drosophil irdin pemker neurons. J. Neurophysiol. 99, (28). 14. Renn, S.C., Prk, J.H., Rossh, M., Hll, J.C. & Tghert, P.H. A pdf neuropeptide gene muttion nd ltion of PDF neurons eh use severe normlities of ehviorl irdin rhythms in Drosophil. Cell 99, (1999).. Shfer, O.T. et l. Widespred reeptivity to neuropeptide PDF throughout the neuronl irdin lok network of Drosophil reveled y rel-time yli AMP imging. Neuron 8, (28). 16. Kul-Eversole, E. et l. Surprising gene expression ptterns within nd etween PDF-ontining irdin neurons in Drosophil. Pro. Ntl. Ad. Si. USA 17, (21). 17. Shkirynov, D. & Levitn, E.S. Prolonged presynpti posttetni yli GMP signling in Drosophil motoneurons. Pro. Ntl. Ad. Si. USA, (28). 18. Feinerg, E.H. et l. GFP reonstitution ross synpti prtners (GRASP) defines ell ontts nd synpses in living nervous systems. Neuron 7, (28). 19. Gordon, M.D. & Sott, K. Motor ontrol in Drosophil tste iruit. Neuron 61, (29). 2. Croker, A. & Sehgl, A. Geneti nlysis of sleep. Genes Dev. 24, (21). 21. Hmd, F.N. et l. An internl therml sensor ontrolling temperture preferene in Drosophil. Nture 44, (28). 22. Ching, A.S. et l. Three-dimensionl reonstrution of rin-wide wiring networks in Drosophil t single-ell resolution. Curr. Biol. 21, 1 11 (211). 23. Mo, Z. & Dvis, R.L. Eight different types of dopminergi neurons innervte the Drosophil mushroom ody neuropil: ntomil nd physiologil heterogeneity. Front. Neurl. Ciruits 3, (29). 24. Sugmori, K.S., Demhyshyn, L.L., MConkey, F., Forte, M.A. & Niznik, H.B. A primordil dopmine D1-like denylyl ylse linked reeptor from Drosophil melnogster displying poor ffinity for enzzepines. FEBS Lett. 362, (19).. Hll, J.C. Systems pprohes to iologil rhythms in Drosophil. Methods Enzymol. 393, (). 26. Boni, A. & Hopf, F.W. The dopmine D2 reeptor: new surprises from n old friend. Neuron 47, (). 27. Hn, K.A., Millr, N.S., Grotewiel, M.S. & Dvis, R.L. MB, novel dopmine reeptor expressed speifilly in Drosophil mushroom odies. Neuron 16, (1996). 28. Hern, M.G. et l. A Drosophil dopmine 2 like reeptor: moleulr hrteriztion nd identifition of multiple lterntively splied vrints. Pro. Ntl. Ad. Si. USA 99, (22). 29. Nith, M.N., Shee, V., Ver, D.A., Blu, J. & Holmes, T.C. Memrne eletril exitility is neessry for the free-running lrvl Drosophil irdin lok. J. Neuroiol. 62, 1 13 (). 3. Wijnen, H., Nef, F., Boothroyd, C., Clridge-Chng, A. & Young, M.W. Control of dily trnsript osilltions in Drosophil y light nd the irdin lok. PLoS Genet. 2, e39 (26). 31. Helfrih-Förster, C. et l. Development nd morphology of the lok-gene-expressing lterl neurons of Drosophil melnogster. J. Comp. Neurol., 47 7 (27). nture NEUROSCIENCE VOLUME 14 NUMBER 7 JULY 211 8

8 211 Nture Ameri, In. All rights reserved. ONLINE METHODS Fly stoks. Flies were rised on stndrd medium with 12-h light:drk yles t 23 C. The pdf-gl4 (X) nd UAS-Ep1-AMP (A nd A) flies were kindly provided y P. Tghert (Wshington University). pdf-gl4/cyo flies were used to express the EPAC sensor in the PDF-expressing l-lnvs nd s-lnvs in fly rins. UAS-D2R-RNAi (II) flies were otined from VDRC. We typilly entrined 1 2 mle flies t C in stndrd light-drk onditions for 3 4 d efore imging. We used fluoresent light t n intensity of 1,6 ± 4 lx. To test the effet of different environmentl onditions on the physiologil responses of the LNvs, we turned off the lights of the inutors t ZT fter 3 d of entrinment nd ontinued housing the flies in the onstnt drkness. The flies were kept in onstnt drkness for less thn 24 h nd were then disseted in the red light to void light exposure. Behviorl nlysis. Individul flies were housed seprtely in 6-mm -mm glss tues (Trikinetis) ontining % grose with 2% surose (wt/vol). We olleted nd entrined 2 -d-old flies under stndrd light-drk onditions, with 12-h light phse nd followed y 12-h drk phse for 3 4 d. To test the effet of het-indued firing y Drosophil TrpA1 hnnels, we entrined flies in stndrd light-drk onditions t 21 C for 3 d nd then rised the temperture of the inutor to 27 C or 3 C t ZT12 for 2 3 d (Fig. 1). For Figure 1, the lights were turned off permnently fter the het tivtion. The temperture ws then returned to 21 C to intivte the TrpA1 hnnel. Both sleep time nd the effet of het on sleep re highly sensitive to genotype. We therefore needed to sutrt the het-indued hnges ourring in the prentl ontrols. We first lulted the het-indued perentge hnge in sleep (SI) for eh genotype, whih is (sleep time t 3 C sleep time t 21 C) / sleep time t 21 C. We then lulted the reltive sleep hnge ( SI), whih is SI = SI experimentl SI ontrol (Tle 1). Split-GFP imging. w-/yw; pdf-lexa, LexAop-GFP11/+; UAS-GFP1-1 / (TH- Gl4 or Td2-Gl4) flies were used to express the GFP11 frgment in the PDFexpressing LNvs nd the GFP1 1 frgment in dopmine or otopmine neurons, respetively. w-/yw; pdf-lexa/lexaop-gfp11; TM6B.T/UAS-GFP1-1 flies were used s ontrols, nd no reonstituted GFP signls were deteted round the LNv ell odies or dendriti res. For immunostining, stndrd fixtion protool ws used. Briefly, the rins were fixed immeditely fter dissetion for 1 h on ie in 4% prformldehyde (vol/vol). Brins were inuted in primry ntiodies for two s t 4 C nd seondry ntiodies for one t 4 C. Sequentil stining ws used to prevent the Alex-488 ntiody to mouse from reting with the rt ntiody to PDF. Brins were sequentilly inuted with four ntiodies, wshing etween eh, in the following order: mouse monolonl ntiody to GFP (Rohe), whih stins GRASP-reonstituted GFP, ut not either GFP frgment lone, Alex 488 ntiody to mouse, rt ntiody to PDF, nd y3 ntiody to rt (Jkson). Brin smples were visulized y Lei TCS SP2 onfol mirosope nd ll imges were tken sequentilly. Brin imging. Live FRET imging ws performed s desried previously with some modifitions 16. Briefly, 3 6-d-old entrined mle flies were disseted in ie-old dult hemolymph-like medium (AHL) 32. We dded 4 µl of 2 C AHL to the imging hmer. An individul rin ws then pled in the hmer. To void rin floting, we tthed smll piee of nylon to the ottom of the hmer with grese. Individul rins were then inserted under the nylon. EPAC expressed in LNvs ws exited with -ms pulses of light using CFP filters. To void light-indued effets, two -mm neutrl density filters (Chrom), 1.3 nd.6, were used to further lok the r lmp light. Fluoresent signls emitted y LNvs were imged every s y n epifluoresent mirosope using 2 ojetive on Zeiss mirosope (Intellegent Imging Innovtions). The imges were olleted with either CFP or YFP filter. The CFP-2432A filter from Semrok nd the Chrom 2 ET CFP/YFP FRET ue with exiter ET436/2x, dihroi T4LP, nd emitter ET3/3m were used. A shutter system ws used to ontrol the rottion of the filter sets. SLIDEBOOK 4.1 softwre (Intelligent Imging Innovtions) ws used for imging nlysis. For suset of the experiments, different setup ws used tht onsisted of n Olympus BX1WI mirosope with CCD mer (Hmmmtsu Or C AG). The quisition system for this setup ws slightly different nd llowed for simultneously reording oth hnnels. The 862v1 JP4 exittion filter (436, Chrom) s well s two-hnnel, simultneous-imging system from Optil Insights with the D48/3m nd D3/4m emission filters were used. Voloity softwre (Perkin Elmer) ws used for quisition nd the CFP nd YFP imges were reorded simultneously. Under these onditions, we determined tht the seline fluoresent signl in LNvs stilized fter imging the neurons for frmes. We were then le to otin relile responses indued y 1 µm foskolin (dt not shown). Otopmine nd dopmine were purhsed from Sigm nd stok solution (1 mm) ws freshly prepred in H 2 O efore the imging 33. Dopmine gonist, pergolide mesylte slt, nd ntgonist, (+)-utlmol hydrohloride, were purhsed from Sigm. A stok solution of pergolide mesylte slt (1 mm) ws prepred in DMSO nd µm (+)-utlmol hydrohloride ws prepred in H 2 O (ref. 24). The stok solutions were stored t 2 C. Pergolide mesylte slt (1 µm) ws used to indue the AMP responses in the LNvs. To lok the dopmine indued responses, rins were pre-inuted with µm (+)-utlmol hydrohloride for min efore pplying 1 µm dopmine. TTX ws purhsed from Sigm nd stok solution (1 µm) ws prepred in H 2 O. The finl onentrtion ws used t 1 µm 34. Brins were pre-inuted in 1 µm TTX for min efore dding dopmine or otopmine. The seline imges were olleted for s efore pplying 1 µm of otopmine or dopmine to the rin. The kground first ws sutrted from the men intensity of CFP nd YFP over l-lnvs or s-lnvs. The kground is the men intensity of nonfluoresent rin region next to the LNvs. The YFP/CFP rtio for eh time point ws lulted nd normlized to the rtio of the first time point, efore drug pplition. The reltive AMP hnges were determined y plotting the normlized CFP/YFP rtio (%) over time. We lso determined the verge fluoresene hnge (re under the reltive AMP hnge urve) y lulting n verge CFP/YFP rtio inrese from 1 to 44 s. Automted dt nlysis nd sttistil nlysis. Eh video hd two hnnels (YFP nd CFP, t resolution of pixels) tht re pre-proessed s the following. First, Gussin kernel (9 9 pixels) ws pplied to redue noise in eh individul imge in eh hnnel. The mirosope stge undergoes some virtion during imge quisition nd it used disseted rins to move slightly. To remove the mehnil movements of the disseted rin during imging, two-step registrtion ws pplied to lign imges in two hnnels. In the first step, the first frmes of oth hnnels were registered ginst eh other. In the seond step, the rest of the frmes in eh hnnel were registered to the first one in the respetive hnnel using mutul informtion sed method (Artyushkov, K.; Automti Imge Registrtion using (Normlized) Mutul Informtion for users of IP toolox, mtlentrl/fileexhnge/414-utomti-imge-registrtion-usingnormlized-mutul-informtion-for-users-of-ip-toolox) After registrtion, the kground signl in eh frme ws modeled s Gussin distriution. A threshold representing 99.9% kground popultion ws seleted to detet the foreground tht ws refined y morphologil imge-proessing opertions 3. To otin roust foreground detetion result, finl foreground msk ws generted to inlude those pixels tht were deteted s foreground in more thn 7% of the time in the whole video. To ount for the noise over time, temporl medin filter (ten frmes) ws pplied to eh pixel in the foreground msk. A referene imge ws generted for eh hnnel y verging the imges reorded in the period efore drug dministrtion (the first ten frmes). This referene imge ws then used to normlize ll imges in the sme hnnel. Both the imge intensity nd the temporl dynmis were used to segment ells into lusters. The initil segmenttion ws omputed using the wtershed trnsform 36 of the grdient of the referene imge from the YFP hnnel. In mny ses, the grdient my hve lrge vritions in ell or more frequently in luster of ells. Thus, ells my e over-segmented. This type of over-segmenttion n e resolved y merging segments with sttistilly indistinguishle differenes in their temporl responses. The response of pixel in n imge frme ws omputed s the rtio CFP/YFP, whih ws normlized y its response in the referene imge. The men nd the s.d. of ll the pixels in segment were lulted. The temporl response of n imge segment ws then omputed s the men of the temporl response of ll pixels in it. The differene etween two different segments ws then ompred to the distriution of differenes etween kground pthes. If none of the initil nture NEUROSCIENCE doi:1.138/nn.286

9 segments were different with P., the segments were merged into single luster. If the s.d. of this luster ws less thn., these segments or ells were onsidered s homogenous group. Segments with differenes in the top.% (P =.) were onsidered to e heterogeneous lss. The null distriution (Supplementry Fig. 2) used for ompring the temporl responses of two segments ws uilt using 1, kground pthes (16 16 pixels) rndomly seleted from ten rins. The differene etween the temporl responses of two segments, = [ 1, 2,, T ] nd = 1, 2,, T, ws lulted s the Euliden distne etween them T D(, ) = ( t t t ) = 1 where t nd t re the responses of segments nd t time t, nd T is the totl numer of frmes in video. The mximum s.d. etween two segments, p nd q, is given s mx [ s ({ pt m } m M = 1,{ qt n } n N = 1)] m, where p 1 t T t is the response of the m th n pixel in segment p in frme t, M is the numer of pixels in segment p, q t is the response of the n th pixel in segment q in frme t, N is the numer of pixels in segment q, nd ( ) = s { pt m } m M,{ qt n } n N 1 M ( pt m N = = 2 x) ( q x) M N m t n 2 + = + n = x = M + N M pt m N + q m t n = 1 n= 1 Finlly, the temporl responses of the remining lusters n e tken to show drug effets. 32. Wng, J.W., Wong, A.M., Flores, J., Vosshll, L.B. & Axel, R. Two-photon lium imging revels n odor-evoked mp of tivity in the fly rin. Cell 112, (23). 33. Cyre, M., Bukinghm, S.D., Ygodin, S. & Sttelle, D.B. Cultured inset mushroom ody neurons express funtionl reeptors for etylholine, GABA, glutmte, otopmine nd dopmine. J. Neurophysiol. 81, 1 14 (1999). 34. Gervsi, N., Thenio, P. & Pret, T. PKA dynmis in Drosophil lerning enter: oinidene detetion y rutg denylyl ylse nd sptil regultion y dune phosphodiesterse. Neuron 6, (21). 3. Soille, P. Morphologil Imge Anlysis: Priniples nd Applitions (Springer- Verlg, Seuus, New Jersey, USA, 23). 36. Meyer, F. Topogrphi distne nd wtershed lines. Signl Proessing 38, 113 (1994). 211 Nture Ameri, In. All rights reserved. doi:1.138/nn.286 nture NEUROSCIENCE