doi:1.13/nture173 Supplementry Text: Wheel-running ctivity hs secondry effects on ehvior Previous studies utilized wheel-running ctivity to ssy the influence the cycles on circdin rhythms 1, 2. Since wheel running ehvior is known to impct circdin photoentrinment, mood, nd lerning 3-7, we ssyed circdin light responses in the cycle in the sence of wheel y mesuring core ody temperture rhythms (Figure S1). All temperture rhythms in mice under the cycle showed period lengths slightly longer thn 24 hours (Figure S1). mice locte the pltform using non-sptil strtegy In ddition to the sptil strtegy mentioned in the mnuscript, non-sptil escpe strtegy relies on circling the pool t the correct distnce from the wll (pltform nnulus) until finding the pltform. To determine if mice plced in the or the cycle used this non-sptil strtegy, we determined the percent of time tht mice spent swimming in the pltform nnulus during the proe tril. We found tht oth the nd the mice spent significntly more time in this re s compred to chnce encounter of the qudrnts (: 4.3±1.1%, n=9, p<.1; : 39.3±2.1%, n=9, p<.1). This indictes tht the mice relied on the non-sptil strtegy to locte the pltform, wheres the mice pired the non-sptil strtegy with cue-sed pproch. Tretment with the ntidepressnt desiprmine restores hippocmpl-dependent lerning In the mnuscript, we show tht chronic tretment with the ntidepressnt fluoxetine ws le to decrese depression relted ehvior nd rescue the lerning deficit oserved in mice housed in the cycle (Figure 3). We sought to investigte whether tretment with n lterntive ntidepressnt could rescue the lerning deficits oserved in mice. Mice received 2 intrperitonel injections (24- nd 1-hour efore testing) of mg/kg desiprmine, tricyclic ntidepressnt 23, prior to the vel Oject Recognition test. We found tht desiprmine dministrtion restored lerning in the cycle exposed mice (Figure S4). Mice housed in the cycle treted with desiprmine showed decresed time spent with the novel oject in greement with previous reports tht desiprmine induces lerning defects in mice plced under norml light environment 24. These results show tht the detrimentl effects of errnt light on lerning could e llevited with ntidepressnt dministrtion. Timing of light dministrtion is crucil for physiology Bright light is used s tretment for sesonl depression in humns 9, 1, wheres, it hs een shown to hve nxiogenic effects in mice 11, 12. Therefore, when determining the effects of light on ehvior, common ssumption is tht light is eneficil to diurnl orgnisms nd versive to nocturnl nimls. However, rts plced in constnt drkness show incresed depression-like ehvior 13, which my e due to lck of eneficil light stimultions in this nocturnl niml. Recent studies in Drosophil indicte tht even in the sme orgnism, preference or voidnce of light chnges during the nimls lifespn 14. Furthermore, light nd drk influence sleep nd lertness in oth nocturnl nd diurnl nimls. Therefore, the direct influence of light on physiology could e disruptive or eneficil depending on the timing of the light input. WWW.NATURE.COM/NATURE 1
Percent time spent wke 1 6 4 19: 7: 19: 7: 19: 7: 19: 7: 19: 7: 19: 7: 19: 7: 19: 7: 19: 7: 19: Time Supplementry Figure 1: The light cycle does not disrupt sleep distriution The verge wkefulness in 3-minute ins for the mice is plotted over 9 dys. Mice were housed in the cycle on 1-4 (solid line long the x-xis) nd in the cycle from -9 (dotted line long the x-xis). There ws disruption in the sleep pttern on Dy, which returned to norml y Dy 6. 2 WWW.NATURE.COM/NATURE
24. Period Length (hours) 24.3 24.1 23.9 23.7 23. c Period Length (hours) d 26 2 24 23 Supplementry Figure 2 Supplementry Figure 2: The light cycle increses the period length of 6 temperture nd generl ctivity rhythms A. Actogrms of core ody temperture rhythms in (grey nd white ckground) nd (yellow ckground) light cycle. B. Mice housed in the light cycle showed n incresed period 24.±.13 (n=) s mesured y core ody temperture, wheres, mice housed in the light cycle showed period of 23.92 ±.7 (n=6). C. Actogrms of generl ctivity rhythms of mice under the light cycle. D. Mice housed in the light cycle showed n incresed period of 24. ±. (n=) s compred to mice housed in the light cycle which showed period of 24.1 ±.1 (n=7). Error r indictes SEM. W W W. N A T U R E. C O M / N A T U R E 3
ZT/CT 1 ZT/CT 7 ZT/CT 13 ZT/CT 19 Rostrl Cudl Rostrl Cudl Opticl Density (.u.) 1. 1. 1.1 1. 1 7 13 19 ZT/CT time Supplementry Figure 3: Rhythmic PERIOD2 expression remins intct in mice housed in the light cycle Figure 3 A. Representtive imges from rostrl to cudl portion of the SCNSupplementry show tht levels of PER2 were rhythmic in mice housed in the nd light cycles peking t ~ZT/CT13. Scle r, µm. Additionlly, locliztion of PER2 positive cells ws similr etween the two groups. Brown punct represent PER2 positive cells. Imges tken t 1x mgnifiction. B. Quntifiction of SCN PER2 expression (n=7- per group t ech time point, ptime<.1). 4 W W W. N A T U R E. C O M / N A T U R E
Amygdl Amygdl Amygdl Amygdl Amygdl Amygdl **** **** cfos + cells per mm2 cfos + cells per mm22 cfos + cells per mm Henul Henul LterlHenul Henul Henul Lterl 2 Suprventriculrnucleus nucleuscnd ndsuprventriculr Suprchismticnucleus nucleus Suprchismtic nucleus ndsuprventriculr Suprchismtic nucleus c c Suprventriculr Nucleus Suprventriculr Nucleus ** 6 4 Suprventri cfos + cells per mm2 cfos + cells per mm22 cfos + cells per mm cfos + cells per mm cfos + cells per mm22 cfos + cells per mm 1 Lterl Hen ** 4 4 1 6 6 Supplementry Figure4 4 Figure Supplementry 1 Supplementry Figure 4: t night induces c-fos expression in the mygdl, henul, nd suprventriculr zone A. (Left) Representtive imges from the mygdl of mouse in drkness nd mouse tht received 1-minute light pulse t pproximtely ZT14. Scle r, µm. (Right) Quntifiction shows incresed c-fos positive cells in the mygdl in response to light pulse (n=3- per group, unpired t-test p<.1). B. (Left) Representtive W W W. Nimges A T U R E. Cfrom O M / NAT U R E the henul of mouse in drkness nd mouse tht received 1-minute light pulse t L Supplemen
tht received 1-minute light pulse t pproximtely ZT14. Scle r, µm. (Right) Quntifiction shows incresed c-fos positive cells in the mygdl in response to light pulse (n=3- per group, unpired t-test p<.1). B. (Left) Representtive imges from the henul of mouse in drkness nd mouse tht received 1-minute light pulse t pproximtely ZT14. Scle r, µm. (Right) Quntifiction shows incresed c-fos positive cells in the lterl henul in response to light pulse (n=3- per group, unpired t-test p=.1). C. (Left) Representtive imges from the suprventriculr nucleus of mouse in drkness nd mouse tht received 1-minute light pulse t pproximtely ZT14. Scle r, µm. (Right) Quntifiction shows incresed c-fos positive cells in the suprventriculr nucleus in response to light pulse (n=3- per group, unpired t-test p=.). Brown punct represent c-fos positive cells. All imges were tken t x mgnifiction. **** indictes p<.1, * indictes p<.. Error r indictes SEM. Totl liquid consumed during sucrose preference test (ml) 1 Body Weight (g) 4 4 3 3 Supplementry Figure : light cycles does not lter totl liquid consumption or ody weight A. Totl mount of liquid consumed during the sucrose preference test did not differ etween mice housed in the nd light cycles (n=,, unpired t-test p=.44). B. Body weight ws similr etween mice housed in the nd cycle (n= per group, unpired t-test p=.6). Error r indictes SEM. 6 WWW.NATURE.COM/NATURE
Open Field Drk Box Elevted Plus Mze Percent time spent in the center Distnce trvelled in the center (Percentge of totl) 1 1 c d Numer of entries to the light zone Percent time spent in the lit zone 1 4 3 1 f g Numer of entries to the open rms Percent time spent in the open rms 1 17. 12. 1 7. 2. e Distnce trvelled in the light zone (m) 4 3 2 1 Supplementry Figure 6: light cycle does not cuse n increse in nxiety-relted ehviors A nd B. Mice housed in the cycle showed no significnt difference in nxiety-relted mesures in the open field s mesured y percent time (A) (n=12 per group, p=.1137) or distnce trveled (B) (n=12 per group, p=.19) in the center of the field. C-E. Mice housed in the nd light cycles showed no significnt differences in nxietyrelted ehvior in the light drk ox s mesured y numer of entries to the light zone (C) (n=12,11, p=.19), percent time in the light zone (D) (n=12,11, p=.), or distnce trvelled in the light zone (E) (n=12,11, p=.9). F nd G. Mice housed in the nd light cycles showed no significnt differences in nxiety-relted ehvior in the elevted plus mze s mesured y numer of entries to the open rms (F) (n=12 per group, p=.6) or percent time spent in the open rms (G) (n=12 per group, p=.3). Error r indictes SEM. WWW.NATURE.COM/NATURE 7
Percent time spent with novel oject 6 4 * * ZT2 ZT14 Supplementry Figure 7: Hippocmpl lerning is oserved oth during dy nd night In the novel oject recognition tsk, mice housed in ZT2 nd ZT14 oth spent more time with the novel oject (ZT2: n=12, p<.1; ZT14: n=11, p=.4). significnt difference ws found etween the two groups (p=.34). Error r indictes SEM. ** Percent time spent with novel oject % 7% % 2% % vehicle * * fluoxetine vehicle fluoxetine Time spent immoile (s) Vehicle Fluoxetine Vehicle Fluoxetine Supplementry Figure : Suchronic fluoxetine tretment does not rescue lerning deficits nor incresed depression relted ehvior induced y the cycle A. Mice housed in the cycle showed significnt preference for the novel oject wheres mice housed in the cycle did not show this preference. This ws lso oserved in mice treted for four-dys with fluoxetine (one smple t-test µ =%, vehicle: n=6, p=.3; fluoxetine: n=7, p=.7, vehicle: n=, p=.; fluoxetine: n=, p=.67). B. Mice housed in the cycle show n incresed mount of time spent immoile in the FST. Amount of time spent immoile is not chnged y fourdy fluoxetine tretment (n=7 per group, two-wy ANOVA p light cycle =.). ** indictes p<.1. Error r indictes SEM. WWW.NATURE.COM/NATURE
9 * Percent time with novel oject 7 3 1 * Vehicle Desiprmine Vehicle Desiprmine Supplementry Figure 9: Administrtion of desiprmine rescues hippocmpl lerning deficits Tretment of mice housed in the cycle with desiprmine prior to the novel oject recognition tsk led to incresed time spent with the novel oject compred to mice treted with vehicle (one smple t-test µ =%. vehicle: n=12, p<.2; desiprmine: n=12, p<.7; vehicle: n=12, p<.24; desiprmine: n=12, p<.4). * indictes p<.. Error r indictes SEM. WWW.NATURE.COM/NATURE 9
Hours Hours Hours Period Length (hours) Period Length (hours) Hours 2. 2.2. 2.24. 24.24. 24.23. pretretment fluoxetine Supplementry Figure 1 fluoxetine pretretment Supplementry Figure 1: Fluoxetine tretment does not lter period length in the 23. 22 Supplementry Figure 1 light cycle A. Actogrms of generl ctivity rhythms of22 mice housed under the light cycle efore nd during fluoxetine tretment. The red rrow indictes the strt of fluoxetine tretment. B. Period length under the light cycle did not chnge fter fluoxetine tretment (n= pired t-test p=.4). Error r indictes SEM. 1 W W W. N A T U R E. C O M / N A T U R E
Time spent immoile (s) WT DTA Corticosterone (ng/ml) WT DTA Supplementry Figure 11: WT nd DTA mice show no sl differences in mood relted ehvior A. Time spent immoile in the FST ws similr etween WT nd DTA littermte mice (n=1 per group, unpired t-test p=.76). B. WT nd DTA mice showed similr sl corticosterone levels t ZT/CT13 (n=1,9, unpired t-test p=.4). Distnce trvelled in the center (Percent of totl distnce) 1 DTA DTA Percent of time spent in the center 1 DTA DTA Supplementry Figure 12: light cycle does not cuse n increse in nxietyrelted ehviors in DTA mice DTA mice housed in the cycle performed similrly to DTA mice housed in the cycle in the open field A. Distnce trvelled in the center ( DTA: n=6 per group, unpired t-test p=.49) B. Percent time spent in the center ( DTA: n=6 per group, unpired t-test p=.7). Error r indictes SEM. WWW.NATURE.COM/NATURE 11
Proposed Model for direct influence of mood nd lerning y light Originl model of light modultion of mood nd lerning Sleep iprgc Circdin Phse Mood Hippocmpl Dependent Lerning Direct effects of light on mood Incresed rhythmic corticosterone levels Circdin disruption model c Sleep deprivtion model Sleep Incresed mni Sleep Deprivtion Decresed Lerning centrl pcemker Circdin phse Incresed depression Supplementry Figure 13: A model for the influence of light on mood nd lerning A. Previous studies suggested tht light regultes mood nd lerning secondrily y first modulting sleep nd circdin rhythms (red inset). In this study (green ox), we revel nother pthwy y which light directly ffects mood through iprgcs without disrupting sleep or cusing circdin rrhythmicity. Furthermore, mood disruptions led to lerning deficits, since we were le to restore lerning in mice mintined in the errnt light environment y dministering ntidepressnt drugs. B. Although, our errnt light cycle lengthened the period of the circdin oscilltor, circdin disruptions, such s those oserved in the clock mutnt mice show mni-like ehviors nd decrese in depression-like ehvior. In contrst, our study found incresed depression-like ehviors under the errnt light conditions. This indictes tht light influences depression independent of circdin rhythm disruptions. C. Interestingly, lthough sleep mounts were not ffected t ll in the cycle, sleep deprived mice show similr deficits to mice mintined under the cycle. 12 WWW.NATURE.COM/NATURE
Supplementry References 1. Mrosovsky, N. & Httr, S. Impired msking responses to light in melnopsinknockout mice. Chronoiol Int, 99-99 (3). 2. Redlin, U. & Mrosovsky, N. Msking of locomotor ctivity in hmsters. J Comp Physiol A 14, 429-37 (1999). 3. Flls, W. A., Fox, J. H. & McAuly, C. M. Voluntry exercise improves oth lerning nd consolidtion of cued conditioned fer in C7 mice. Behv Brin Res 7, 321-31. 4. Grci-Cpdevil, S., Portell-Cortes, I., Torrs-Grci, M., Coll-Andreu, M. & Cost-Miserchs, D. Effects of long-term voluntry exercise on lerning nd memory processes: dependency of the tsk nd level of exercise. Behv Brin Res 2, 2-7 (9).. Greenwood, B. N. et l. Long-term voluntry wheel running is rewrding nd produces plsticity in the mesolimic rewrd pthwy. Behv Brin Res 217, 34-62. 6. Mistlerger, R. E. Effects of dily schedules of forced ctivity on free-running rhythms in the rt. J Biol Rhythms 6, 71- (1991). 7. Solerg, L. C., Horton, T. H. & Turek, F. W. Circdin rhythms nd depression: effects of exercise in n niml model. Am J Physiol 276, R2-61 (1999).. Bldi, E., Lorenzini, C. A. & Corrdo, B. Tsk solving y procedurl strtegies in the Morris wter mze. Physiol Behv 7, 7-93 (3). 9. Rosenthl, N. E. et l. Antidepressnt effects of light in sesonl ffective disorder. Am J Psychitry 142, 3-7 (19). 1. Virk, G., Reeves, G., Rosenthl, N. E., Sher, L. & Postolche, T. T. Short exposure to light tretment improves depression scores in ptients with sesonl ffective disorder: A rief report. Int J Disil Hum Dev, 23-26 (9). 11. Bouwknecht, J. A. et l. Differentil effects of exposure to low-light or high-light open-field on nxiety-relted ehviors: reltionship to c-fos expression in serotonergic nd non-serotonergic neurons in the dorsl rphe nucleus. Brin Res Bull 72, 32-43 (7). 12. Crwley, J. & Goodwin, F. K. Preliminry report of simple niml ehvior model for the nxiolytic effects of enzodizepines. Phrmcol Biochem Behv 13, 7-7 (19). 13. Gonzlez, M. M. & Aston-Jones, G. deprivtion dmges monomine neurons nd produces depressive ehviorl phenotype in rts. Proc Ntl Acd Sci U S A, 49-93 (). 14. Gong, Z. et l. Two pirs of neurons in the centrl rin control Drosophil innte light preference. Science 33, 499-2. WWW.NATURE.COM/NATURE 13