LETTER. Aberrant light directly impairs mood and learning through melanopsin-expressing neurons

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1 oi:1.138/nture11673 Aerrnt light iretly impirs moo n lerning through melnopsin-expressing neurons Tr A. LeGtes 1 *, Cr M. Altimus 1 *,HuiWng 2, Hey-Kyoung Lee 2, Sunggu Yng 2, Hiqing Zho 1, Alfreo Kirkwoo 2, E. To Weer 3 & Smer Httr 1,2 The ily solr yle llows orgnisms to synhronize their irin rhythms n sleep wke yles to the orret temporl nihe 1. Chnges in y-length, shift-work, n trnsmeriin trvel le to moo ltertions n ognitive funtion efiits 2. Sleep eprivtion n irin isruption unerlie moo n ognitive isorers ssoite with irregulr light sheules 2. Whether irregulr light sheules iretly ffet moo n ognitive funtions in the ontext of norml sleep n irin rhythms remins unler. Here we show, using n errnt light yle tht neither hnges the mount n rhiteture of sleep nor uses hnges in the irin timing system, tht light iretly regultes moorelte ehviours n ognitive funtions in mie. Animls expose to the errnt light yle mintin ily ortiosterone rhythms, ut the overll levels of ortiosterone re inrese. Despite norml irin n sleep strutures, these nimls show inrese epression-like ehviours n impire hippompl long-term potentition n lerning. Aministrtion of the ntiepressnt rugs fluoxetine or esiprmine restores lerning in mie expose to the errnt light yle, suggesting tht the moo efiit preees the lerning impirments. To etermine the retinl iruits unerlying this impirment of moo n lerning, we exmine the ehviourl onsequenes of this light yle in nimls tht lk intrinsilly photosensitive retinl gnglion ells. In these nimls, the errnt light yle oes not impir moo n lerning, espite the presene of the onventionl retinl gnglion ells n the ility of these nimls to etet light for imge formtion. These finings emonstrte the ility of light to influene ognitive n moo funtions iretly through intrinsilly photosensitive retinl gnglion ells. In mmmls, ll light informtion for imge formtion n regultion of ehviour is etete y the retin n signlle to the relevnt rin trgets through retinl gnglion ells (RGCs). Most RGCs signl light to thlmi rely nulei n then to the visul ortex for imge funtions. A popultion of intrinsilly photosensitive RGCs (iprgcs 3,4 ), whih preominntly signl light informtion for non-imge-forming visul funtions, expresses the photopigment melnopsin n n e istinguishe from most RGCs tht support imge trking n etetion 5. iprgcs projet to severl hypothlmi n preopti res suh s the suprhismti nuleus (SCN), suprventriulr nuleus n ventrolterl preopti re to regulte irin rhythms n sleep. However, they lso projet to limi regions suh s the lterl henul n the meil mygl 4,6, highlighting possile role in the regultion of ognitive funtions. To etermine how errnt light influenes ehviour, we sujete mie to n ultrin yle onsisting of 3.5-h light n 3.5-h rk (). Our previous stuies showe tht this errnt light yle oes not ffet the rhiteture (Supplementry Fig. 1) or the totl sleep levels when ompre to 12 h:12 h light rk () ontrol yle 7. We lso etermine whether the irin timing system ws isrupte in the nimls y mesuring ore oy temperture n generl tivity rhythms. The yle oes not use irin rrhythmiity in either output rhythm (Fig. 1, n Supplementry Fig. 2), lthough the irin perio is lengthene. To etermine whether the yle influenes the moleulr sis of the irin lok, we mesure irin hnges of moleulr lok omponent (PER2) in entrl (SCN) n peripherl (liver) tissues. Mie house in the yle show similr rhythms n loliztion of PER2 expression in the SCN s in littermtes house in the yle (Fig. 1 n Supplementry Fig. 3), initing no isruption of internl rhythmiity of the SCN pemker. Furthermore, Per2 levels in the liver from mie house in the or light yle were intt n showe similr phses (Fig. 1). Together, these t show tht the light yle oes not isrupt sleep or use irin rrhythmiity. Although the irin timing system n sleep remin intt uner the light yle, mie re expose to light pulses t ll irin phses uring the experimentl time ourse owing to the mismth etween the impose light yle n the perio length. Thus, the yle will llow us to etermine the iret influene of errnt light exposure on moo n ognitive funtions. The moel uses light to pper uring the night (tive) phse of the nimls yle. To etermine whih rin regions respon to light presente t night, we mesure expression of the trnsription ftor -Fos in response to n ute light pulse. After exmintion of iprgc trgets tht re prt of or known to influene the limi system, we foun light-inue -Fos expression in the mygl, lterl henul n suprventriulr nuleus (Supplementry Fig. 4). This suggests tht light input prtiulrly when presente t n errnt time of y my influene regions of the rin involve in moo n ognitive funtions. Shorter y-length uring the winter months les to sesonl form of epression known s sesonl ffetive isorer (SAD), n ppropritely time light therpy n llevite the symptoms of SAD 8. We thus investigte whether the yle, whih exposes nimls to light t inpproprite times, uses epression-like ehviours in mie y evluting surose nheoni n ehviourl espir in the fore swim test (FST). Mie house in the yle showe erese surose preferene, initing n inrese in epression-like ehviour (Fig. 1e n Supplementry Fig. 5). This ws further supporte y the FST; mie house in the yle spent signifintly more time immoile thn mie house in the yle (Fig. 1f). An estlishe ssoition with epression is inrese serum ortiosterone levels 9. We mesure serum ortiosterone in nimls house in or yles t four time points ross the y. Although we foun n intt irin rhythm in ortiosterone with similr phse to mie house in the yle, the overll levels of ortiosterone were inrese in mie house in the light yle (Fig. 1g). Inreses in ortiosterone levels s well s nheoni re orrelte with inrese stress n nxiety 1. To ssy nxiety-like ehviour, we 1 Deprtment of Biology, Johns Hopkins University, Bltimore, Mryln 21218, USA. 2 Deprtment of Neurosiene, Johns Hopkins University, Bltimore, Mryln 21218, USA. 3 Deprtment of Biology, Rier University, Lwreneville, New Jersey 8648, USA. *These uthors ontriute eqully to the work. 594 NATURE VOL NOVEMBER 212 Mmilln Pulishers Limite. All rights reserve 212

2 RESEARCH Time (ys) Time (ys) Time (h) Time (h) Time (h) ZT/CT 1 ZT/CT 7 ZT/CT 13 Reltive expression CT/ZT time ZT/CT 19 e Surose preferene (% surose onsume) Figure 1 Aerrnt light inreses epression-like ehviour n ortiosterone levels., Boy temperture rhythms uner the (grey/ white) n (yellow) yles., Generl tivity rhythms uner the (left) n (right) yles., PER2 expression in the SCN ws rhythmi uner the n yles. Sle rs, 2 mm., Liver Per2 expression ws rhythmi (P time 5.72) (n per time point, P light yle ). CT, irin f Time spent immoile (s) * g Cortiosterone (ng ml 1 ) * CT/ZT time time; ZT, zeitgeer time. e, mie showe surose nheoni (n 5 16 () n n 5 15 (), P,.1). f, mie showe inrese immoility in the FST (n 5 22 per group, P 5.2). g, Cortiosterone levels were rhythmi ut inrese in mie (n per time point, P time,.1, P light yle,.1, Bonferroni post-test: P,.5 ZT/CT 1 n P,.1 ZT/CT 13). *P,.5; P,.1; *P,.1; P,.1. Error rs inite s.e.m. use three tests: open fiel test, light rk ox n elevte plus mze. We foun no signifint ifferene etween nimls mintine in the yle n those in the yle (Supplementry Fig. 6). These results show tht the yle oes not glolly influene ehviour ut speifilly eliits epression- n not nxiety-like ehviours. Inrese serum ortiosterone levels n epression hve een losely ssoite with hippompl lerning efiits 11. We onute hippompl-epenent tsk, the Morris wter mze (MWM) (Fig. 2 ). In the MWM, the mie require signifintly more trils to hieve the sme lteny in loting hien pltform ompre with the mie (Fig. 2), espite similr swim spees. All our mesurements were one uring the y in the mie (Supplementry Fig. 7). Previous stuies using the MWM hve shown tht mie use hippompl-epenent sptil strtegy n hippomplinepenent non-sptil strtegy to lote the pltform 12. We sought to etermine whether the efiit in lerning quisition in the mie ws ue to the lk of hippompl-epenent sptil lerning. We therefore onute proe tril, in whih the pltform in the wter mze ws remove from the trget qurnt 13 (Fig. 2). We foun tht mie house in the yle showe signifint preferene for the trget qurnt, wheres mie house in the yle showe no preferene for the trget qurnt (Fig. 2). Using reversl ssy, mie house in the yle require signifintly more trils to lote the pltform in the new qurnt, wheres the mie showe no hnge in the lteny to lote the pltform (Fig. 2). This further supports the epenene of mie on non-sptil espe strtegy, highlighting hippompl- epenent lerning efiit. Sptil lerning efiits re usully ssoite with long-term potentition (LTP) 14 erement in the hippompus. We foun tht mie house in oth the n yles hve similr sl synpti trnsmission t the Shffer ollterls in the hippompus (Fig. 2e, f ). However, mie house in yle showe impire LTP in response to oth one n four pulses of thet urst stimultion (TBS; Fig. 2g, h). We foun no ifferene in low-frequeny stimultion-inue longterm epression (LTD) etween mie house in the n yles (Fig. 2i). Impire LTP with no hnge in LTD hs een previously ssoite with sleep eprivtion 15. The seletive LTP efiits oserve in mie house in the errnt light yle inepenent of sleep eprivtion inites tht lerning impirments ue to inpproprite light exposure n sleep eprivtion my use similr neurl pthwys (see moel in Supplementry Fig. 13). To etermine whether the lerning efiits in the yle exten to tsks involve in hippompl-epenent reognition memory, we onute the novel ojet reognition test 16. Mie house in the yle showe signifint preferene for the novel ojet (Fig. 2j). By ontrst, mie house in the yle showe no preferene for novel ojet from fmilir ojet (Fig. 2j). 22 NOVEMBER 212 VOL 491 NATURE Mmilln Pulishers Limite. All rights reserve

3 RESEARCH LETTER 8 Lighting Mie house in yle yle moel Mie house in yle yle 6 yle Experimentl Alimtion to Nive MWM Reversl MWM moel light yle Proe Dys Time (ys) * 4 8 yle 3 6 yle Q1 Q2 TQ Q3 Q1 Q2 TQ Q yle yle Time (ys) e f g , 1 TBS 1, , , ISI (ms) Fire volley (mv) h i j TBS 15 pplfs yle yle PPF (EPSP2/EPSP1) Pth in qurnt (%) Figure 2 Aerrnt light impirs hippompl lerning, LTP, n reognition memory., Experimentl moel for the MWM., mie showe impire lerning (n 5 21 per group, P intertion 5.41, Bonferroni post-test P,.1 ys 2 n 4)., mie show no signifint preferene for the trget qurnt (TQ) (: n 5 9, P 5.11; : n 5 9, P 5.). Dotte line inites hne (%)., Mie house in the yle showe n inrese lteny in reversl tril, wheres -house mie show similr lteny to the qusition phse (n 5 9 per group, P intertion,.3, Bonferroni post-test: P,.1 ys 15 n 16). e, f, n mie hve EPSP (mv ms 1 ) Lteny to fin the pltform (s) Lteny to fin the pltform (s) Time with novel ojet (%) similr sl synpti relese properties (P intertion 5.76) (n 5 5 per group) (fire volley: P intertion 5.984, slope: P..1). EPSP; exittory postsynpti potentil; ISI, interstimulus intervls; PPF, pire-pulse filittion. g i, mie showe LTP efiits (n 5 5 per group, 1 TBS: P intertion,.1; 4 TBS: P intertion,.1) (g n h) ut no ifferene in LTD (i). pplfs, pire-pulse low-frequeny stimultion. j, mie showe efiit in novel ojet reognition (: n 5 24, P 5.57; : n 5 24, P ). *P,.5; P,.1. Error rs inite s.e.m. To investigte whether ntiepressnts oul resue the lerning efiits oserve in mie, we hronilly ministere fluoxetine to n mie (Fig. 3). Chroni fluoxetine tretment erese epression-like ehviour in mie house in the yle (Fig. 3). Furthermore, this tretment ws le to resue the lerning efiit oserve in the novel ojet reognition test (Fig. 3). In support of this ehviourl resue of hippompl funtion, hroni fluoxetine tretment lso resue the LTP efiit inue y the yle (Fig. 3). Suhroni fluoxetine tretment i not resue the inrese epression or lerning efet oserve in mie house in the yle (Supplementry Fig. 8), onsistent with pulishe reports 17. We lso use esiprmine, triyli ntiepressnt, n foun tht esiprmine restore lerning (Supplementry Fig. 9 n Supplementry Informtion). These results show tht the etrimentl ehviourl hnges inue y errnt light exposure n e llevite with ntiepressnt ministrtion. To etermine the mehnism y whih fluoxetine restores lerning, we mesure the irin perio in mie trete with fluoxetine. Although fluoxetine n phse shift the irin osilltor 18, hroni ministrtion of fluoxetine i not hnge the length of the irin perio in mie house in the yle (Supplementry Fig. 1). We then exmine the ortiosterone rhythms in fluoxetine-trete mie n showe tht ortiosterone rhythms persist with lower overll levels ompre with untrete mie (Fig. 3e). These t inite tht fluoxetine tretment oes not lter irin rhythms ut inste lowers the level of ortiosterone, whih oul le to lower epression-like ehviour n etter lerning. Stuies hve suggeste tht iprgcs, in ition to ffeting reflexive, irrine-epenent non-imge forming visul funtions, might iretly influene higher ognitive funtions n rin proessing of emotions To etermine iretly whether iprgcs meite the effets of the yle on moo n lerning, we teste mie lking iprgcs (Opn4 DTA/DTA, herein referre to s DTA mie). Although most iprgcs re lte, these mie still retin more thn 95% of RGCs n re ple of imge formtion 5. First, we ompre wil 596 NATURE VOL NOVEMBER 212 Mmilln Pulishers Limite. All rights reserve 212

4 RESEARCH 2 Time spent immoile (s) Dys Vehile n = 2 n = 2 * Vehile vehile vehile fluoxetine fluoxetine Vehile (n = 1) 18 mg kg 1 y 1 fluoxetine (n = 1) Vehile (n = 1) 18 mg kg 1 y 1 fluoxetine (n = 1) Time with novel ojet (%) Cortiosterone (ng ml 1 ) e 2 * * * type to DTA littermtes n showe no ifferenes in seline for the FST n ortiosterone levels (Supplementry Fig. 11). We then ple DTA mie uner n yles n foun no ifferene in nxiety-like ehviours etween the two groups (Supplementry Fig. 12). By ontrst, the inrese epression-like ehviour, lerning efiits n hippompl LTP erement oserve in the yle in wil-type nimls were not oserve in DTA mie expose to this yle (Fig. 4). These results inite tht the negtive influene of the errnt light yles on ehviour requires iprgcs. Thus, suonsious light etetion in humns vi iprgcs my e responsile for the epression n lerning efiits oserve uner isruptive light environments. Mnipultion of the light environment n le to isruptions in irin rhythms n sleep n lso use moo n lerning efets in mie n humns 2,23,24. These stuies hve le to moel in whih light ffets ognition exlusively through the moultion of sleep n irin pthwys (Supplementry Fig. 13). Here, we provie further moel in whih light iretly influenes moo leing to lerning efiits in the ontext of n intt irin timing system n norml sleep istriution n rhiteture (Supplementry Fig. 13). Further support tht the effets of the yle re not ue to irin isruptions originte from ehviourl omprisons to nimls lking funtionl irin osilltor (for exmple, irin mutnt- or SCN lesione- nimls), in whih reue epression-like ehviour is oserve,26 (Supplementry Fig. 13). Tken together, 15 1 Vehile 5 NOR FST CT time Vehile * fluoxetine Figure 3 Chroni ntiepressnt ministrtion resues lerning., Timeline for fluoxetine ministrtion n testing. NOR, novel ojet reognition., Chroni fluoxetine reue immoility time in the FST (n 5 1 per group, P intertion 5.92, Bonferoni post-test: P,.1 versus vehile)., mie trete with fluoxetine showe restore novel ojet preferene ( vehile : n 5 9, P 5.3; fluoxetine : n 5 9, P 5.48; vehile : n 5 1, P 5.77; fluoxetine : n 5 1, P 5.4)., Chroni fluoxetine resue the -inue LTP efiit (n 5 5 ( vehile), n 5 5 ( fluoxetine), n 5 5 ( vehile), n n 5 4 ( fluoxetine); P intertion,.1). e, Chroni fluoxetine erese ortiosterone levels in mie (n per time point). ortiosterone levels re plotte for omprison. (P intertion 5.53, Bonferroni post-test: P,.1 CT 13 n P,.1 CT 19.) *P,.5; *P,.1; P,.1. Error rs inite s.e.m. Time with novel ojet (%) 1 5 DTA * * DTA 4 TBS these t provie eviene tht the errnt light effets on moo re iret. Furthermore, the epression-like ehviour use y the errnt light yle n serve s n lterntive experimentl moel for epression reserh in roents, inepenent of geneti mnipultions or versive tretments suh s repetitive restrint or eletri shoks. METHODS SUMMARY Ault (4 8 months) mle mie (B6/129 F 1 hyri; Jkson Lortory) were use for ll experiments involving wil-type mie ple uner ifferent light environments. Ault littermte DTA mie ple uner ifferent light environments were of B6/129 kgroun (6 8 months) n were rise in our lortory. DTA mie oul not e use until they were t lest 6 months ol euse elimintion of the melnopsin ell popultion in these mie is not omplete until this time 5. All mie were iniviully house in stnr niml fility ges with ess to foo n wter liitum. All mie were initilly entrine to 12 h:12 h light rk yle (), fter whih the lights for one group were swithe to 3.5 h:3.5 h light rk yle () for 2 weeks. The light intensity uring the light portion ws,8 lx, hosen to use no irin rrhythmiity. All experiments were one in orne with the regultions set forth y Johns Hopkins University n Rier University Animl re n use ommittee. Behviourl nlysis. Mie were mintine in the or light yle for 2 weeks efore testing unless otherwise inite. Boy temperture n generl tivity were mesure to evlute irin rhythmiity. Surose preferene n the FST were use to ssess epression-like ehviour. The MWM n novel ojet reognition test were use to ssess hippompl-epenent lerning. Cellulr n moleulr nlysis. To exmine the irin timing system, immunohistohemistry n qrt PCR were use to quntify PER2 expression in the SCN n liver, respetively. Serum ortiosterone levels were quntifie y ELISA. Eletrophysiologil reorings from the orsl hippompus were performe to evlute hippompl funtion inluing LTP n LTD. Full Methos n ny ssoite referenes re ville in the online version of the pper. Reeive 4 April 211; epte 11 Otoer 212. Pulishe online 14 Novemer Reppert, S. M. & Wever, D. R. Coorintion of irin timing in mmmls. Nture 418, (22). 2. Foster, R. G. & Wulff, K. The rhythm of rest n exess. Nture Rev. Neurosi. 6, (). Time spent immoile (s) DTA DTA DTA DTA Figure 4 iprgcs meite impirment of moo n lerning y errnt light., Mie lking iprgcs showe signifint preferene for the novel ojet when house in oth the n light yles (n 5 12 per group, P,.1)., DTA mie house in the yle performe similrly to those house in the yle in the FST (n 5 6 () n n 5 5 (), unpire t-test: P 5.93)., DTA mie h similr LTP to DTA mie in response to four-pulse TBS. *P,.1. Error rs inite s.e.m. 22 NOVEMBER 212 VOL 491 NATURE Mmilln Pulishers Limite. All rights reserve

5 RESEARCH LETTER 3. Berson, D. M., Dunn, F. A. & Tko, M. Phototrnsution y retinl gnglion ells tht set the irin lok. Siene 295, (22). 4. Httr, S., Lio, H. W., Tko, M., Berson, D. M. & Yu, K. W. Melnopsin-ontining retinl gnglion ells: rhiteture, projetions, n intrinsi photosensitivity. Siene 295, (22). 5. Güler, A. D. et l. Melnopsin ells re the prinipl onuits for ro one input to non-imge-forming vision. Nture 453, (28). 6. Httr, S. et l. Centrl projetions of melnopsin-expressing retinl gnglion ells in the mouse. J. Comp. Neurol. 497, (26). 7. Altimus, C. M. et l. Ros-ones n melnopsin etet light n rk to moulte sleep inepenent of imge formtion. Pro. Ntl A. Si. USA 15, (28). 8. Lm, R. W. & Levitn, R. D. Pthophysiology of sesonl ffetive isorer: review. J. Psyhitry Neurosi., (2). 9. Nestler, E. J. et l. Neuroiology of epression. Neuron 34, 13 (22). 1. MEwen, B. S. Protetive n mging effets of stress meitors. N. Engl. J. Me. 338, (1998). 11. Cryn, J. F. & Holmes, A. The sent of mouse: vnes in moelling humn epression n nxiety. Nture Rev. Drug Disov. 4, 7 79 (). 12. Bli, E., Lorenzini, C. A. & Corro, B. Tsk solving y proeurl strtegies in the Morris wter mze. Physiol. Behv. 78, (23). 13. Vorhees, C. V. & Willims, M. T. Morris wter mze: proeures for ssessingsptil n relte forms of lerning n memory. Nture Protools 1, (26). 14. Bliss, T. V. & Collingrige, G. L. A synpti moel of memory: long-term potentition in the hippompus. Nture 361, (1993). 15. MDermott, C. M. et l. Sleep eprivtion uses ehviorl, synpti, n memrne exitility ltertions in hippompl neurons. J. Neurosi. 23, (23). 16. Honey, R. C., Wtt, A. & Goo, M. Hippompl lesions isrupt n ssoitive mismth proess. J. Neurosi. 18, (1998). 17. Dulw, S. C., Holik, K. A., Gunersen, B. & Hen, R. Effets of hroni fluoxetine in niml moels of nxiety n epression. Neuropsyhophrmology 29, (24). 18. Sprouse, J., Brselton, J. & Reynols, L. moultes the irin iologil lok vi phse vnes of suprhismti nuleus neuronl firing. Biol. Psyhitry 6, (26). 19. Lokley, S. W. et l. Short-wvelength sensitivity for the iret effets of light on lertness, vigilne, n the wking eletroenephlogrm in humns. Sleep 29, (26). 2. Vnewlle, G. et l. Spetrl qulity of light moultes emotionl rin responses in humns. Pro. Ntl A. Si. USA 17, (21). 21. İyiliki, O., Ayin, E. & Cneyli, R. Blue ut not re light stimultion in the rk hs ntiepressnt effet in ehviorl espir. Behv. Brin Res. 23, (29). 22. Wrthen, D. M., Wiltgen, B. J. & Provenio, I. Light enhnes lerne fer. Pro. Ntl A. Si. USA 18, (211). 23. Fonken, L. K. et l. Influene of light t night on murine nxiety- n epressive-like responses. Behv. Brin Res., (29). 24. M, W. P. et l. Exposure to hroni onstnt light impirs sptil memory n influenes long-term epression in rts. Neurosi. Res. 59, (27).. Royl, K. et l. Mni-like ehvior inue y isruption of CLOCK. Pro. Ntl A. Si. USA 14, (27). 26. Ttroğlu, O., Aksoy, A., Yilmz, A. & Cneyli, R. Effet of lesioning the suprhismti nulei on ehviorl espir in rts. Brin Res. 11, (24). Supplementry Informtion is ville in the online version of the pper. Aknowlegements Wewoullike tothnk T. Goul, G. Blln A. Sw for their expert vie on the ehviourl tests. We woul like to thnk R. Kuruvill for her ritil reing n vie on this mnusript. We woul lso like to thnk the mouse tri-lortory for suggestions n vie. This work ws supporte y the Dvi n Luile Pkr Fountion grnt to S.H. Author Contriutions T.A.L., C.M.A., H.Z., E.T.W. n S.H. esigne experiments. T.A.L. n C.M.A. rrie out experiments. H.W., H.-K.L., S.Y. n A.K. esigne n performe eletrophysiologil experiments. T.A.L., C.M.A., H.Z., E.T.W. n S.H. wrote the pper. Author Informtion Reprints n permissions informtion is ville t The uthors elre no ompeting finnil interests. Reers re welome to omment on the online version of the pper. Corresponene n requests for mterils shoul e resse to S.H. (shttr@jhu.eu). 598 NATURE VOL NOVEMBER 212 Mmilln Pulishers Limite. All rights reserve 212

6 RESEARCH METHODS Animls n housing. Ault (4 8 months) mle mie (B6/129 F 1 hyri; Jkson Lortory) were use for ll experiments involving wil-type mie ple uner ifferent light environments. Ault littermte DTA mie ple uner ifferent light environment were of B6/129 kgroun (6 8 months) n were rise in our lortory. DTA mie oul not e use until they were t lest 6 months ol euse elimintion of the melnopsin ell popultion in these mie is not omplete until this time 5. All mie were iniviully house in stnr niml fility ges with ess to foo n wter liitum. All mie were initilly entrine to 12 h:12 h light rk yle (), fter whih the lights for one group were swithe to 3.5 h:3.5 h light rk () for 2 weeks. The light intensity uring the light portion ws,8 lx, hosen to hve no irin rrhythmiity. All experiments were one in orne with the regultions set forth y Johns Hopkins University n Rier University Animl re n use ommittee. Boy temperture mesurements. Boy temperture mesurements were me using G2 E-mitter telemetri proes from Mini Mitter (Respironis). The telemetri proe ws implnte into the peritoneum n suture to the insie of the ominl wll. Mie were given 1 week to reover from surgery efore ny reoring. Reorings were otine using Vitlview softwre (Respironis), t rte of 3 mesurements per hour. Throughout, experiment mie were mintine in their home ge without perturtion n the light yles were juste to test response to oth the n light yles. Cirin perio ws etermine y fitting regression line to the onsets of tivity over 7-y perio using Clokl (Atimetris). Perio lengths of mie house in the n light yles were ompre using n unpire Stuent s t-test. Generl tivity mesurements. Generl tivity ws mesure using infrre motion etetors from Mini Mitter (Respironis). Mie were house iniviully, n the motion etetor ws mounte to the top of the ge so tht generl tivity oul e monitore throughout the n light yle. Dt were ollete in 1-min ins using Vitlview softwre (Respironis). Cirin perio length ws etermine y fitting regression line to the onsets of tivity over 7-y perio using Clokl (Atimetris). Perio lengths of mie house in the n light yles were ompre using n unpire Stuent s t-test. Moleulr rhythm mesurements. Mie were house in either the (n 5 16) or the (n 5 16) light yle for 2 weeks, uring whih generl tivity ws monitore in mie to etermine their irin phse. Mie were smple ross four time points. Mie uner the yle were smple t ZT 1, 7, 13 n 19, n mie uner the yle were smple t CT 1, 7, 13 n 19, whih ws etermine using their generl tivity rhythm. Eh mouse ws remove from its ge, n til loo ws quikly smple. The mouse ws then nesthetize with 1 ml of vertin (2 mg ml 21 ). One nesthetize, smll smple of liver ws isolte. The mouse ws then perfuse trnsrilly with.9% sline followe y 4% prformlehye. The rin ws remove n postfixe overnight in 4% prformlehye followe y ryoprotetion n emeing in OCT ompoun (Skur Finetek). Rel-time PCR. Liver smples were homogenize n RNA extrte using the RNesy mini kit (Qigen). The Retrosript kit (Amion) ws use to reverse trnsrie poly(a) RNAs. Rel-time quntittive PCR ws performe with iq SYBR Green Supermix n the icyler iq rel-time PCR etetion system (Bio- R). Eh smple ws nlyse in triplite retions of 5 ml. Primers for Per2 were forwr: 59-GCCTTCAGACTCATGATGACAGA-39 n reverse: 59-TTT GTGTGCGTCAGCTTTGG-39. Primers for 18S rrna (internl ontrol) were forwr: 59-CGCCGCTAGAGGTGAAATTC-39 n reverse: 59-TTGGCAA ATGCTTTCGCTC-39. Dt were nlyse using the DDC t metho, normlizing eh smple to the internl ontrol, n reltive messenger RNA ws etermine s the perentge of the mximum vlue oserve in the experiment. Dt were nlyse y two-wy nlysis of vrine (ANOVA) followe y Bonferroni post-ho test to exmine ifferenes over time s well s potentil light yle effets on Per2 expression. PER2 immunohistohemistry. Brins were setione (4 mm) y ryostt through the rostrl ul extent of the SCN, n were store free floting in.1 M phosphte uffer. Free-floting setions were inute in loking uffer (.1 M phosphte uffer, 3% triton,.5% ovine serum lumin n 1% got serum) for 2 h. Setions were then inute in rit nti-per2 (Alph Dignosti Interntionl; 1:4, in loking uffer) overnight n visulize with Vetstin horserish peroxise kit (Vetor Ls) using 3,39-iminoenziine (DAB; Sigm). Setions were mounte on mirosope slies, ehyrte n overslippe with Permount. Setions were imge t 31 mgnifition with Zeiss Axio Imger M1 mirosope. Optil ensity ws mesure using ImgeJ. Dt were nlyse y two-wy ANOVA followe y Bonferroni post-ho test to exmine ifferenes over time s well s potentil light yle effets on PER2 expression. Cortiosterone mesurement. Serum ws isolte from ollete til loo n ssye for ortiosterone y ELISA (Assypro). Dt were nlyse y two-wy ANOVA followe y Bonferroni post-ho test to exmine ifferenes over time s well s light yle effets on ortiosterone levels. For fluoxetine experiments, ortiosterone levels were nlyse y two-wy ANOVA followe y Bonferroni post-ho to exmine ifferenes over time s well s the effet of fluoxetine. For the experiment ompring wil-type n DTA mie, serum ws smple from wil-type n DTA mie t ZT 13/CT 13 se on generl tivity rhythms (s esrie ove). The smples were proesse s esrie ove. Cortiosterone levels of wil-type n DTA mie were nlyse y unpire Stuent s t-test. Light-inue -Fos expression. Mie were house uner 12 h:12 h light rk yle n were expose to 1-min light pulse t ZT 14 (2 h fter light offset), fter whih they were ple k in the rk for further 8 min. Control mie remine in the rk until nesthetiztion. Ninety minutes fter the strt of light presenttion, mie were eeply nesthetize with 1 ml of vertin (2 mg ml 21 ). One nesthetize, the mie were perfuse trnsrilly with.9% sline followe y 4% prformlehye. Brins were remove, postfixe overnight in 4% prformlehye, n then trnsferre to.1 M phosphte uffer. Brins were setione (4 mm) through the rostro ul extent of the SCN using virotome (Worl Preision Instruments). Setions were store free floting in.1 M phosphte uffer. Every other setion ws stine immunohistohemilly for -Fos. Setions were inute in loking uffer (.1 M phosphte uffer, 3% Triton X-1 n.5% ovine serum lumin) for 2 h. Setions were inute in rit nti--fos (Cliohem A-5; 1:2,) overnight t 4 uc n then visulize with got nti-rit Vetstin horserish peroxise kit (Vetor Ls) using DAB (Sigm) s hromgen. Setions were mounte on mirosope slies, ehyrte n overslippe with Permount. Slies were viewe n imge on Zeiss Axio Imger.M1 mirosope t 35 mgnifition. Photoshop n ImgeJ were use to ount -Fos-positive ells n mesure the re of region ounte from. The numer of -Fos positive ells ws normlize to the re of the region, n these vlues were ompre etween mie tht reeive light pulse n rk ontrols. These t were nlyse using n unpire Stuent s t-test. Surose nheoni. Mie were house in the presene of two wter ottles 1 y efore testing to limte them to the ottles. Surose preferene ws ssesse over 2 ys. Eh y, one ottle ontining 1% surose n one ottle ontining wter were introue t the eginning of the tive phse. The position of these ottles ws swithe 6 h lter, n the ottles were remove t the en of the tive phse. Bottles were weighe t the eginning n en of the tive perio to mesure mount onsume. Surose preferene ws lulte y iviing the mount of surose onsume y the totl mount onsume (wter n surose). The perentge of surose onsume y mie in the n yles ws ompre y Stuent s t-test. FST. Mie were iniviully ple in n inesple ontiner of wter ( uc) for 6 min. Behviour ws monitore y vieo mers positione in front of the pprtus n sore y vieo trking system (Fore Swim Test, Bioserve). Time spent immoile for the lst 4 min of the test ws lulte. Inrese time spent immoile is initive of inrese epression-relte ehviour. The mount of time spent immoile uring the lst 4 min ws nlyse y Stuent s t-test. For fluoxetine experiments, the mount of time spent immoile uring the lst 4 min were nlyse y two-wy ANOVA followe y Bonferroni post-ho to ompre light yle n rug tretment. Open fiel. Mie were iniviully ple in the entre of lrge, rightly lit ren (5 lx, m) n llowe to explore for 5 min. Behviour ws monitore from ove y vieo mer onnete to omputerize vieo trking system (Anymze, Stoelting). The pprtus ws lene thoroughly etween eh tril. The perentge istne trvelle n time in the entre of the ren were mesure. These mesures were ompre etween mie house in the n yles using n unpire Stuent s t-test. Light rk ox. The light rk ox onsiste of two omprtments equivlent in size (2 3 2 m); one re is rightly lit (6 lx) n the other is imly lit (,1lx). A smll opening joins the two omprtments, so the mie oul freely move etween the two res. Mie were rk pte for 1 h efore testing. At the eginning of the test, rk-pte mie were ple in the lit omprtment fing wy from the opening n llowe to freely explore for 5 min. Behviour ws monitore from ove y vieo mer onnete to omputerize vieo trking system (Anymze). The pprtus ws lene thoroughly etween eh tril. The numer of trnsitions etween the two omprtments s well s the time spent n istne trvelle in the lit room were mesure s initions of nxiety-relte ehviour. These mesures were ompre etween mie house in the n yles using n unpire Stuent s t-test. Elevte plus mze. The pprtus onsiste of two open rms (42 3 6m) opposite to one nother n two rms enlose y wlls ( m) opposite 212 Mmilln Pulishers Limite. All rights reserve

7 RESEARCH LETTER of one nother forming ross. The rms were seprte y entrl pltform (6 3 6 m). The mze ws elevte (33 m) suh tht the open rms onvey openness, unfmilirity n elevtion. The light intensity in the open rms ws,6 lx, wheres the light intensity in the lose rms ws,2 lx. Mie were ple in the entre of the elevte plus mze fing one of the open rms. Behviour ws monitore from ove y vieo mer onnete to omputerize vieo trking system (Anymze). The pprtus ws lene thoroughly etween eh tril. The time spent n the istne trvelle in the open rms were mesure s initions of nxiety-relte ehviour. These mesures were ompre etween mie house in the n yles using n unpire Stuent s t-test. MWM. The wter mze onsiste of irulr pool (15 m in imeter) with room temperture wter (26 28 uc). The wter ws me opque with the ition of non-toxi white tempur pint to hie the espe pltform. The pltform ws me from PVC piping with top (1 minimeter) pintewhite n sumerge in the pool suh tht 1 m of wter overe the pltform hiing it from sight. For visul trils, flg me from 5-ml onil tue overe with oloure tpe ws ple on the pltform. During the quisition, proe n reversl trils, four ues were tthe to the sie of the pool equiistnt from one nother, n the entire pool ws surroune y plin urtin to lok ny other visul ues. Performne ws sore using vieo trking system (Anymze) with mer mounte ove the pool. The light intensity t the wter surfe ws pproximtely 5 lx. Mie were teste in four stges: visul, quisition, proe n reversl (see Fig. 2). Before sptil trining, mie were trine to espe the pool using the visul flg lote on top of the pltform to fmilirize them with the test. This ws performe four times with n inter-tril intervl of (3 min), n the pltform ws move etween eh tril. We lso use this visul trining to sreen for n remove nimls tht show floting ehviour in the wter mze. Animls tht flote in two or more trils uring trining were not teste in the sptil tsk to prevent onfouns from floting ehviour. During the quisition phse, mie were trine (one tril per y for 12 ys) to fin the hien pltform using the four visul istl ues surrouning the pool. The mouse ws rnomly ple in ifferent re of the pool t the strt of eh tril with the pltform mintine in the sme qurnt (trget qurnt). The pltform ws remove on y 13 in the proe tril. The swimming in eh qurnt n speifilly the preferene for the trget qurnt ws mesure to evlute sptil memory using omputerize vieo trking system (Anymze). Reversl trining egn on y 14 when the pltform ws move to the qurnt opposite the originl trget qurnt. Mie were trine s esrie for the quisition phse. Lteny to lote the pltform uring the quisition n reversl phses ws nlyse y two-wy ANOVA followe y Bonferroni post-ho test to exmine hnges in lteny throughout the ourse of the experiment s well s the effet of light yle exposure. Proe tril ws nlyse y lulting the perentge time spent in the trget qurnt n performing one-smple t-test to etermine whether this ws signifintly ove %. Novel ojet reognition. Novel ojet reognition ws ompose of three stges: limtion to the novel ojet ren, fmilir ojet exposure n finlly novel ojet exposure. Mie were first remove from their home ge, limte to the empty testing ren (light intensity of 5 lx) for 1 min, n susequently returne to their home ge 24 h efore inluing two ojets in the ren. The y fter limtion, mie were returne to this ren with two ientil ojets tht they oul freely explore for 1 min, fter whih they were returne to their home ge for 1 h. At the en of the 1-h perio, mie were ple k into the ren with one of the ojets hnge to novel ojet, n were llowe to explore oth the fmilir n novel ojets for 5 min. Behviour ws monitore from ove y vieo mer onnete to omputerize vieo trking system (Anymze), n the perentge of time spent with eh ojet ws lulte. Wil-type mie spen more time with the novel ojet, however, mie with reognition memory efiits will not e le to istinguish the novel from the stle ojet. Ojets h een previously teste to ensure tht nimls showe no initil preferene for prtiulr ojet. The ientity of the ojets (fmilir versus novel) ws ounterlne. The ojets n ren were thoroughly lene etween eh tril to remove oour ues. Ojet reognition ws nlyse y lulting the perentge time spent with the novel ojet n performing one smple t-test to etermine whether this ws signifintly ove 5%. Slie eletrophysiology. Coronl (.4 mm) hippompl slies were prepre s esrie 27 in ie-ol issetion uffer (2.6 mm KCl, 1.23 mm NH 2 PO 4,26mM NHCO 3, mm surose, 1 mm extrose, 3 mm MgCl 2 n 1 mm CCl 2, ule with 5% CO 2,95%O 2 ). Reorings were one in similr uffer ut with the surose reple y NCl n the temperture rise to 3 uc. Synpti responses were evoke t.33 Hz stimulting the Shffer ollterls with.2-ms pulses (onentri ipolr eletroes, FHC), n reore extrellulrly in CA1 strtum ritum. LTP ws inue y TBS, onsisting of one or four thet epohs elivere t.1 Hz. Eh epoh, in turn, onsiste of 1 trins of four pulses (t 1 Hz) elivere t 5 Hz. LTD ws inue y low frequeny stimultion (1 Hz, 15 min). These protools were elivere fter 2 min of stle seline trnsmission. All hippompl slie eletrophysiologil reorings were performe n nlyse y n experimenter lin to the tretment of the nimls. Two-wy ANOVA ws use to nlyse fire volley ifferenes etween the n tretments. The slopes for the liner fit of the fire-volley slope reltionship were ompre y t-test. ministrtion. Mie were house in either the or yle for two weeks (n 5 2 per group) with foo n wter liitum. For hroni tretment, this ws followe y 3-week tretment of 18 mg kg 21 y 21 of fluoxetine (Sigm). For suhroni tretment, this ws followe y 4-y tretment of 18 mg kg 21 y 21. ws ministere in the rinking wter n ontrol mie reeive tp wter. Dosge ws lulte se on the verge mount of wter onsume per y n mouse weight. onsumption ws lso mesure uring tretment to etermine the mount onsume per mouse. Perio length mesurement with fluoxetine tretment. Mie were house uner infrre motion etetors s esrie ove. Mie were house in the light yle for 2 weeks fter whih fluoxetine (18 mg kg 21 y 21 ) ws ministere in the rinking wter for 3 weeks. Cirin perio ws etermine y fitting regression line to the onsets of tivity over 7-y perio. Perio lengths efore n fter 3 weeks of fluoxetine tretment were ompre y pire t-test. Desiprmine ministrtion. Desiprmine (Sigm) ws issolve in sterile wter with 5% Tween-2. Eh mouse reeive 16 mg kg 21 esiprmine intrperitonelly 24- n 1-h efore testing in the novel ojet reognition prigm. The sme volume of vehile (wter with 5% Tween-2) ws ministere intrperitonelly to ontrol mie. Sttistil nlysis. All sttistil nlysis ws performe using GrphP Prism. Speifi tests use to nlyse t re esrie their respetive setion of the methos. 27. Lee, H. K., Min, S. S., Gllgher, M. & Kirkwoo, A. NMDA reeptor-inepenent long-term epression orreltes with suessful ging in rts. Nture Neurosi. 8, (). 212 Mmilln Pulishers Limite. All rights reserve