the circadian clock provides the interface between an organism and its 24  hr geophysical environment .
as currently accepted , the fly clock is constituted by interlocked negative transcription / translation feedback loops ( ttfls ) . at the core of the system are the activators clock ( clk ) and cycle ( cyc ) that bind to the promoters of the period ( per ) and timeless ( tim ) genes , initiating their transcription .
after translation , the negative autoregulators per and tim become the substrate of several kinases and phosphatases , they dimerize , translocate into the nucleus , and repress the clk / cyc complex .
the second feedback loop is centered around clk and involves the rhythmic expression of par domain protein 1 ( pdp1 ) and vrille ( vri ) ( reviewed in ) .
the blue - light - sensitive protein cryptochrome ( cry ) regulates photo - responsiveness and flies with altered cry function display aberrant circadian light entrainment [ 25 ] and visual behavior .
moreover , immunofluorescence ( if ) and confocal microscopy reveal that -cry immunoreactivity ( ir ) is often found at the level of neuronal projections , suggesting that cry may play additional roles .
null mutants for cry ( cry ) show defects in rhythmic behavior under constant light ( ll ) , so it is possible that cry exerts direct functions in central clock neurons . indeed
clock neurons constitute a network of cells expressing clock genes that are divided into lateral neurons ( six dorsal lateral neurons [ lnds ] , four large ventral lateral neurons [ l - lnvs ] , four small ventral lateral neurons [ s - lnvs ] , a single pdf - null ventral lateral neuron [ pn - lnv ] , and three lateral posterior neurons [ lpns ] ) and dorsal neurons ( 16 dn1s , 2 dn2s , and 40 dn3s ) ( reviewed in ) .
although the classic molecular model portrays the clock as cell autonomous , cellular cross - talk appears to be essential for its function [ 1114 ] .
the circadian neurons can be further grouped or differentiated by the activation of promoters of clock and clock - related genes .
for instance , all clock neurons express tim   whereas the pigment - dispersing factor ( pdf ) promoter is active only in the s- and l - lnvs .
a cry 5.5-kb promoter   is seemingly expressed in all lnvs , in the lnds , and in  two dn1s   although shafer et  al .   reported further expression in two additional dn1s ( called dn1a ) and two dn3s . by combining the expression of these promoters ,
coupled to either gal4 ( to drive transcription of a reporter gene ) or gal80 ( to inhibit gal4 function ) , it is possible to define subsets of clock cells and to manipulate them selectively by expressing genes whose products alter the electrical properties of the neurons or the running of the clock . in our study
we have introduced local alterations in the neuronal network and have investigated the period of locomotor activity under constant darkness and temperature ( dd ) , an  artificial condition where the interactions among neurons is  independent from the light - dark ( ld ) cycle .
we did this by  initially distinguishing among clock cells based on whether they express both the pdf and cry promoters ( pdfcry ) , the  cry promoter only ( pdfcry ) , or neither ( pdfcry ) .
our results suggest a model for the logic that regulates the neuronal network under these conditions .
cry , a c - terminal deletion of cry , renders cry constitutively active , as revealed in a number of molecular ( light - independent binding to per and tim ) and behavioral ( long period of locomotor activity in dd ) phenotypes [ 4 , 5 ] .
based on the discovery that light - activated cry increases neuronal firing , we presumed that cry could activate neurons and enhance their output in dd , which might be reflected in period changes .
we used a tim - gal4 driver and several uas - cry lines and confirmed that overexpression of cry in all clock cells , namely pdfcry  pdfcry  pdfcry , results in 1  hr lengthening of the endogenous period of locomotor activity compared to controls   ( table s1 available online ) .
we noticed that the vast majority of cry flies had simple rhythmicity ( sr ) although a few showed complex rhythms ( cr , more than one periodicity in a single fly ) or arrhythmicity ( ar ) but in proportions no different from controls .
we asked whether all clock cells contribute to the free running period or whether one group of neurons imposes its own rhythmicity to behavior .
we focused on one line ( uas - cry14.6 ) , which will hereafter be referred to as cry. pdfcry > cry ( pdf - gal4 > uas - cry ) and pdfcry  pdfcry > cry ( cry13-gal4 > uas - cry ) flies  both revealed a 1  hr longer period compared to controls .
we obtained a similar value for the pdfcry  pdfcry  pdfcry > cry genotype ( tim - gal4 > uas - cry ) , which argues against the assumption that these three drivers largely differ in their  strength   
then , we expressed cry in smaller groups of neurons by combining the gal80 repressor with gal4 drivers .
we  used the lines cry - gal802e3 m and pdf - gal8096a , each carrying two copies of gal80 , that have been reported to repress uas - dependent expression in the pdfcry  pdfcry and the pdfcry cells , respectively .
to confirm the extent of gal80-mediated inhibition of gal4 activity , we measured anti - gfp ir of pdfcry cells in tim - gal4 > uas - gfp , in tim - gal4  cry - gal802e3 m > uas - gfp , and in tim - gal4  pdf - gal8096a > uas - gfp flies . for both gal80 configurations
, we were able to confirm a good and equivalent level of repression of gal4 activity in pdfcry cells ( figures s1a and s1b ) .
however , in all tim - gal4  cry - gal802e3 m > uas - gfp preparations , we observed a robust anti - gfp signal in three lnds , meaning that gal80 can not efficiently inhibit gal4 in these cells because of lower cry expression ( figures s1a and s1c ) .
gal80-mediated repression of gal4 was inefficient also in four dn1ps and in three or four cry - positive dn3s .
conversely , good repression was achieved in the dn1as , in two dn1ps , and in the pn - lnv ( figure  s1c and data not shown ) .
henceforth we identify cells that strongly or weakly express cry as pdfcry and pdfcry , respectively .
we tested pdfcry  pdfcry > cry ( cry13-gal4  pdf - gal8096a > uas - cry ) and pdfcry  pdfcry  pdfcry > cry ( tim - gal4  pdf - gal8096a > uas - cry ) flies ; for both genotypes the free running period of locomotor activity was not significantly different from controls . however , when we analyzed pdfcry  pdfcry > cry ( tim - gal4  cry - gal802e3 m > uas - cry ) flies , we observed a significant 1.2  hr shortening of the period ( figure  1 ; table s1 ) .
this result can not be explained in terms of residual gal4 expression in the pdfcry cells as we detected similar levels of ir in them when driving gfp in both tim - gal4  pdf - gal8096a and tim - gal4  cry - gal802e3 m flies ( figures s1a and s1b ) .
cry overexpression resulted in a faster rhythm only for the  latter genotype , thereby unveiling the contribution of the pdfcry and pdfcry groups to period , which would otherwise be inhibited by the pdfcry cells ( this is because the two genotypes above differ in period and in the inclusion of the latter group of cells ) .
interestingly , in wild - type flies the dn2 cluster of pdfcry neurons show faster ( 22  hr ) endogenous molecular cycling that is not reflected in the 24  hr behavioral rhythms . under seminatural conditions ,
they show an advance in molecular cycling compared to other clock neurons , also consistent with a faster period [ 20 , 21 ] .
conversely , it has been reported that by reducing the contribution of the pdfcry cells to the network , either by eliminating their main signaling molecule , the neuropeptide pdf   ( figure  s2 ) , or by reducing their numbers   ( table s2 ) , a short activity period is generated .
thus , the overall evidence supports a role for the pdfcry neurons in generating a short - rhythm phenotype when the balance of the network is tilted in their favor , with the dn2s being a likely but perhaps not exclusive component of this function .
the overexpression of cry in pdfcry cells ( pdf - gal4 > uas - cry ) results in a longer period . however , these slower - paced cells are not alone in controlling self - sustained behavior because increasing the impact on the network of the pdfcry and pdfcry neurons ( tim - gal4  cry - gal802e3 m > uas - cry ) results in a shorter period .
the effect is reversed by  adding pdfcry to the ensemble ( tim - gal4  pdf - gal8096a > uas - cry ) , suggesting the latter intervene in a negative control , possibly on the faster pdfcry neurons ( figure  2a ) .
uas - cry ) had no consequence in terms of period ( figure  1 ; table s1 ) .
this suggests a balance , with the pdfcry neurons inhibiting the faster - paced pdfcry cells and in turn the pdfcry neurons inhibiting the slower - paced pdfcry cells ( figure  2a ) .
note that in this context , the words  activation  and  inhibition  do not have a physiological connotation but are used as logical operators .
we wanted to show that the effects observed on period are not specific to cry but are reproducible by other manipulations .
thus , we employed the same gal4/gal80 combinations described above , using effectors that have a known and consistent mode of action independent of the type of neuron .
the overexpression of the depolarization - activated bacterial sodium channel nachbac   using tim - gal4 ( pdfcry  pdfcry  pdfcry  pdfcry ) resulted predominantly in arrhythmicity , confirming the effectiveness of this manipulation ( table s2 ) . limiting nachbac expression to the  pdfcry ( pdf - gal4>uas - nachbac ) neurons generated 50% of flies with complex rhythms , showing a major long - period and a minor short - period component .
moreover , those 30%40% individuals with a single activity rhythm had a period 1.52.5  hr longer than controls ( figure  3a ; table s2 ) .
nachbac overexpression in pdfcry induces longer molecular cycles in these cells and directly increases their output , explaining the origin of the major , slower ( longer period ) activity component [ 22 , 23 ] .
immunocytochemistry experiments have  also indicated that the dn2s show a faster cycle in both controls and nachbac - overexpressing flies .
thus , it is tempting to speculate that fast pdfcry neurons ( we can not exclude that neurons in addition to the dn2s might also be involved ) might be indirectly activated through a positive , pdf - independent connection linking the pdfcry and the pdfcry cells and be responsible for the minor , short activity component detected in this genotype ( figures 2b and s2 ) .
only 13%25% of pdfcry  pdfcry > nachbac ( tim - gal4  cry - gal802e3 m > uas - nachbac ) flies showed single rhythms ( figure  3a ; table s2 ) .
this result supports the hypothesis that the pdfcry cells might inhibit the pdfcry neurons , explaining why they do not sustain behavioral rhythms in this genotype ( figure  2b ) .
subsequently we tested pdfcry  pdfcry  pdfcry > nachbac flies ( tim - gal4  2xpdf - gal80 > uas - nachbac ; figure  3a ; table s2 ) .
now , about 70%80% of individuals had a single activity component with a period 0.51  hr longer than controls .
this implies that the addition of the pdfcry group ( which is  not included in the largely arrhythmic tim - gal4  cry - gal802e3 m > uas - nachbac genotype ) to the ensemble of activated neurons results in the inhibition of the output of the pdfcry cells ( because of the longer activity period ) and of the pdfcry cells ( because the majority of flies are rhythmic ) .
possibly , the latter inhibition is indirect , in agreement with our previous hypothesis ( figure  2b ) .
pdfcry  pdfcry ( cry13-gal4  > uas - nachbac ) neurons caused a decrease in the number of rhythmic individuals and a shortening in the activity period ( now reaching wild - type values for single - rhythm flies ) compared to the pdfcry > nachbac ( pdfgal4 > uas - nachbac ) genotype ( figure  3a ; table s2 ) .
this result is different from the comparison between the same genotypes for cry flies , which were both rhythmic with a period about 1  hr longer than controls ( figure  1 ; table s1 ) .
the different strength of the two activators likely accounts for these discrepancies , which in general result from exaggerated effects on period and on rhythmicity following nachbac overexpression ( figures s3 ) .
we also applied the opposite manipulation , namely the overexpression of the mammalian inward rectifier k channel kir2.1 , a tool for decreasing membrane excitability in  vivo [ 24 , 25 ] .
pdfcry > kir2.1 ( pdf - gal4  > uas - kir2.1 ) resulted in more than a third of the flies being arrhythmic and in the tendency for a shorter period for those showing a single rhythm .
this argues for a disruption in the balance of the network and , in the flies still rhythmic , for a tilt toward the faster pdfcry neurons . extending the misexpression of kir2.1 to include also the pdfcry and the pdfcry cells ( cry13-gal4 
> uas - kir2.1 ) abolished these effects ( figure  3b ; table s2 ) .
finally , we tested the expression of kir2.1 in  pdfcry  pdfcry neurons ( tim - gal4  cry - gal802e3 m > uas - kir2.1 ) , but this genotype was not viable .
we then overexpressed the proapoptotic genes head involution defective ( hid ) and reaper ( rpr )   in pdfcry cells ( pdf - gal4  > uas - hid , uas - rpr ) and observed that 50% of the flies were equally either arrhythmic or showed complex rhythms .
the remaining half cycled with a period of locomotor activity 1  hr shorter than controls .
expression of hid and  rpr in pdfcry  pdfcry cells ( tim - gal4  cry - gal802e3 m > uas - hid , uas - rpr ) resulted in poor viability and 60% of the surviving flies were arrhythmic , further underscoring the importance of these neurons to the network ( figure  3c ; table s2 ) .
the remaining flies showed complex or single rhythms , the latter having periods on average intermediate to their corresponding controls ( table s2 ) .
although we did not verify this experimentally , it is likely that viable and rhythmic flies must have largely escaped the apoptotic response triggered by hid and rpr .
this explanation is in line with the lethality of the tim - gal4  cry - gal802e3 m > uas - kir2.1 genotype and with previously reported variability in the induction of  the apoptotic response in neurons . driving the production of
the dominant - negative clk mutant   in pdfcry  pdfcry cells ( tim - gal4  cry - gal802e3 m > uas - clk ) resulted in virtually complete arrhythmicity .
thus , as with nachbac and hid - rpr overexpression , disrupting ( independently of mechanism ) the pdfcry  pdfcry groups generated a largely arrhythmic profile , which argues against a simple dominant effect of the pdfcry cells on the neuronal network ( figure  3d ; table s2 ) .
the expression of clk in pdfcry cells caused 20%50% arrhythmicity and 10%30% of complex rhythms , depending on the line .
the remaining 40%50% of flies showed a single rhythm of activity with a period 0.51  hr shorter than controls .
this is in line with an increased influence of the pdfcry cells on the network . by expressing clk in pdfcry  pdfcry cells in addition to the pdfcry group ( cry13-gal4  > uas - clk )
, we increased the ratio of single - rhythm flies to 72%94% and reverted to a period closer to  that of the parental control strains , but slightly shorter ( figure  3d ; table s2 ) .
thus , as previously seen with the overexpression of kir2.1 , we could rescue rhythmicity in flies with weakened pdfcry neurons by reducing the inhibitory influence of the pdfcry cells on them and by buffering changes in activation of the pdfcry cells . again
, this shows that the  pdfcry cells do not act alone in determining circadian rhythmicity and its period .
we were unable to obtain viable  pdfcry  pdfcry  pdfcry > clk ( tim - gal4 ,   pdf - gal8096a >
 pdf - gal8096a > uas - clk ) flies were rhythmic with periods indistinguishable from controls ( figure  3d ; table s2 ) .
this result is the same as that observed with cry , again suggesting that the pdfcry and the pdfcry cells inhibit the faster pdfcry and the slower pdfcry cycling groups respectively , so the balance of the network is undisturbed ( figure  2b ) .
cry flies with a long behavioral period exhibit a delay in  the  cycling of clock proteins in pdfcry neurons .
here  we  investigated the cellular changes caused by cry overexpression in pdfcry  pdfcry ( tim - gal4  cry - gal802e3 m > uas - cry ) and in pdfcry  pdfcry  pdfcry ( tim - gal4  pdf - gal8096a > uas - cry ) neurons , resulting in short and normal ( compensatory ) activity rhythms , respectively .
we analyzed the cycling of the clock protein pdp1 in the major classes of clock neurons by if .
figure  4 shows the staining index ( si ) for pdp1  at ct0 , ct6 , ct12 , and ct18 ( circadian time , ct0  = subjective lights on , ct12  = subjective lights off ) during day 2 and 5 in dd .
control flies ( uas - cry ) showed , at day 2 , maximum si at about ct18 for all cell types . at day 5
the si peaked also at ct18 for s - lnvs , lnds , and dn1s , suggesting that these cells cycle with a period of about 24  hr . instead the si maximum was reached earlier ( about ct12 ) by the dn2s , suggesting that these neurons run with a shorter period of 22  hr .
these cells  also showed a pronounced reduction in their cycling amplitude compared to the other clusters .
thus , in control flies , s - lnvs , lnds , and dn1s showed molecular oscillations that were compatible with the corresponding behavioral period attributed to them .
the overexpression of cry in pdfcry  pdfcry cells ( tim - gal4  cry - gal802e3 m > uas - cry ) targeted directly the dn2s , the large majority of dn1s , the dn3s , and half of the lnds .
the dn1s and even more so the dn2s showed a phase advance in pdp1 immune staining at day 2 ; at day 5 both groups reached their maximum at around ct6 .
however , while the dn1s experienced a shortening of their period ( in this genotype compared to the control ) , the dn2s maintained the same faster rhythm seen in wild - type flies .
the lnds were considered as a group because we could not distinguish the three pdfcry from the three pdfcry cells ( we could not discriminate them by anti - cry staining because both the anti - cry and the anti - pdp1 antibodies available to us are made in rabbit ) , which limits our interpretation of these data .
overall , the lnds showed a reduction in cycling amplitude especially at day 5 , perhaps resulting from averaging different cycling profiles in the two groups . the s - lnvs experienced phase advance and shortening of their rhythm such that by day 5 they reached the si maximum at ct6 ( although because of the 6  hr resolution of our data , the actual peak might be a few hours later ) . thus , in this genotype , the s - lnvs , the dn1s , and the dn2s were synchronous and showed endogenous cycling compatible with the short behavioral period . a significant 
time  cell type  anova interaction ( we compared the si of cell types s - lnvs , dn1s , and dn2s , at different time points , at day 2 and 5 ; see legend to figure  4 ) provides support to the view that the advance in the phase of pdp1 expression occurred first in the dn2s , then in the dn1s , and finally in the s - lnvs .
the changes above appeared first at day 2 and were consolidated at day 5 , confirmed with a significant 
we then overexpressed cry in pdfcry  pdfcry  pdfcry cells ( tim - gal4  pdf - gal8096a > uas - cry ) , namely in the dns and in both types of lnds ( the pn - lnv was also affected but it has not been analyzed here ) .
extending the expression of cry to the pdfcry cells caused a broadening in the expression profile of pdp1 in the dns , supporting our hypothesis that the pdfcry modulate the pdfcry cells . especially at day 2 , the levels of pdp1 started to rise earlier ( compared to controls ) than for pdfcry  pdfcry > cry flies , but declined later in all dorsal neurons , suggesting a combination of phase advance and phase delay inputs .
the dn2s maintained their shorter molecular cycling while the dn1s and seemingly the dn3s moved to a 24  hr period .
the lnds , also expressing cry directly , showed the most pronounced phase delay ( supported by a significant 
time  cell type  anova interaction ) and a reduction in cycling amplitude ( although smaller than for the previous genotype ) .
day  time  cell type  anova interaction ( see legend to figure  4 ) .
the profile of pdp1 ir in the s - lnvs ( not expressing cry ) remained unchanged at day 2 and 5 , which , compared to the controls , corresponds to a small delay in phase but no change in period . in this genotype , only the slnvs and the dn1s showed robust endogenous cycling compatible with the behavioral period .
overall , only the s - lnvs and the dn1s consistently showed cellular cycling matching the behavioral periods of the flies in all genotypes .
we suggest that locomotor activity with simple rhythmicity requires synchronization between these two groups of neurons , although the contribution of neurons from other clusters may also be significant . in the pdfcry  pdfcry
> cry ( tim - gal4  cry - gal802e3 m > uas - cry ) flies , the faster rhythm of the dn2s and perhaps of additional unidentified fast pdfcry cells was shared by dn1s and s - lnvs . in pdfcry  pdfcry  pdfcry
> cry ( tim - gal4  pdf - gal8096a > uas - cry ) individuals , the dn2s were still cycling with a faster pace but , as in wild - type flies , the shorter period did not spread to dn1s and s - lnvs , suggesting this is the role of the highly pdf - responsive pdfcry cells ( included in the second but not in the first genotype ) .
the expressions of cry in the dn1ps only   did not result in faster behavioral rhythms , showing that the shorter period originates from a different group of neurons ( table s3 ) ; see figure  2c for a possible scenario .
we employed pdf - geneswitch ( pdf - gs )   to trigger drug - dependent expression of cry in pdfcry neurons either in adults only ( acute ) or through the whole development ( chronic ) .
for acute treatment , flies were fed for 48  hr from the day of eclosion with medium supplemented with either 200  g / ml of the drug ru486 ( induction ) or with an equal volume of vehicle ( 80% ethanol , no induction ) .
note , all flies were raised on this medium and were then examined ( on their respective medium ) for locomotor activity rhythms . for chronic treatment
, flies were exposed to either drug or vehicle during their whole life and adult locomotor activity was examined accordingly . as controls ,
we tested ( heterozygous ) driver and effector flies raised under acute or chronic exposure to drug or on vehicle only ( table s4 ) . under both treatment conditions
, we observed a significant , albeit small , lengthening of the period of locomotor activity for cry-overexpressing flies compared to controls ( figures 5a and 5b ) .
as a further test we directed the expression of the temperature - sensitive cation channel trpa1 in pdfcry and in  pdfcry  pdfcry cells ( figures 5c and 5d ; table  s4 ) .
this channel is inactive at temperatures below 25c in drosophila and originally it was reported as being endogenously expressed in the adult brain in only about a dozen cells , which are not part of the circadian system .
however , recent evidence has shown that trpa1 transcription does occur in at least some cells in each cluster of clock neurons but that the effects of trpa1-null mutations on rhythmic behavior are quite modest .
flies overexpressing trpa1 and ( heterozygous ) parental controls were raised at 18c ( trpa1 is inactive ) and then tested as adults at 18c and 28c ( trpa1 is active ) . compared to controls
, we would expect a longer and a shorter period for pdfcry > trpa1 ( pdf - gal4 > uas - trpa1 ) and pdfcry  pdfcry > trpa1 ( tim - gal4  cry - gal802e3 m > uas - trpa1 ) flies at 28c , respectively . for pdfcry > trpa1 flies ,
the increase in period length with temperature was comparable to controls ( figures 5c and 5d ) .
conversely , we observed a significant decrease in period length for pdfcry  pdfcry > trpa1 flies at higher temperature but not for controls ( figures 5c and 5d ) . in summary ,
the manipulation of the circadian network limited to adults can affect the free - running period of locomotor activity .
however , the modest effects obtained with adult  pdfcry cells suggest that the ontogeny of the clock is particularly sensitive to the status of these neurons .
similar conclusions were reached by depetris - chauvin et  al .   and gorostiza and ceriani .
conversely , here we  show that the pdfcry and the pdfcry cells appear to be less sensitive to developmental effects ( figures 5c and  5d ) .
had shown that overexpression of a constitutive active form of the kinase shaggy ( sgg ) , although able  to shorten the period of locomotor activity when driven in pdfcry cells , was unable to elicit a behavioral effect when directed in pdfcry  pdfcry neurons .
this was in spite of causing a faster molecular oscillation in some cells , for instance the dn2s , and was considered evidence that those cells are not relevant for free - running behavior .
pdfcry > sgg ( tim - gal4  cry - gal802e3 m > uas - sgg ) flies showed a free - running period of locomotor activity no different from controls , in agreement with previous results   ( figure  6a ; table s5 ) .
however , a visual inspection of the activity profiles revealed that these flies , both in ld and dd , showed earlier anticipation of the actual or subjective dark - to - light transition ( figure  6b ) .
perhaps this anticipation of phase reflects a tendency for the shorter rhythm to propagate to the network , which instead is prevented by other neuronal groups .
previous work has suggested that there is a physical and functional interaction between cry and sgg .
there is little or no  cry in pdfcry and pdfcry neurons , which could preclude sgg from exerting any robust effect . according to  our model , the pdfcry repress the pdfcry cells
; thus , increasing the output of the former should reduce the  resistance of the network to the propagation of the faster  rhythm .
we coexpressed cry and sgg and indeed observed a faster free - running period of locomotor activity in pdfcry  pdfcry > sgg , cry ( tim - gal4  cry - gal802e3 m > uas - sgg , uas - cry ) but not in pdfcry  pdfcry > cry ( tim - gal4  cry - gal802e3 m > uas - cry ) flies ( figure  6c ; table s5 ) .
these results reveal that the dn2s are indeed part of the circuit that generates self - sustained  behavior , although they do not exclude that the  establishment of a faster rhythm might require additional pdfcry neurons .
implicitly , they also contradict the view of a defined hierarchical organization of the clock under constant conditions . considering all results reported above
, we favor a model based on a flexible network of circadian neurons , which operates under any environmental condition .
our model complements and extends evidence by others of network organization as opposed to hierarchical dominance among neurons under ld and dd conditions [ 3436 ] .
we have used genetic manipulations limited to discrete groups of neurons to unveil the logic governing self - sustained rhythmic locomotor activity in drosophila .
activity rhythms and observations of molecular cycling in defined cellular groups consistently support a model ( figure  2 ) where endogenous behavioral rhythmicity is based upon synchronization between pdfcry and pdfcry cells .
this requires modulation of the pdfcry cells by the pdfcry neurons , largely but not exclusively via pdf signaling , and multistep feedback adjustment of the latter group of neurons by the former .
the behavioral outcomes of manipulations that alter but do not destroy the equilibrium of the network reveal that the pdfcry cells have a tendency for driving rhythms longer than 24  hr whereas some pdfcry cells promote rhythms shorter than 24  hr .
however , the organization of the network is such that wild - type flies reach a combined 24  hr oscillation .
this requires the pdfcry and the pdfcry cells that tune , through inhibitory interactions , the relative contribution of the other two groups of neurons to the network .
we note that the interposition of the pdfcry and the pdfcry cells adds delay and signal amplification to the feedback regulation that links the pdfcry to the pdfcry neurons and vice versa . in conclusion , we have shown that the endogenous behavioral period reflects the nonadditive interplay of all clock cells and not the dominant action of a single group of neurons ( i.e. , the s - lnvs ) , as previously suggested .
second , our overall results suggest that intercellular coupling is fundamental for synchronizing different cellular oscillators in a coherent clock , and therefore additional elements must complement the known molecular model focused around transcriptional regulators .
finally , we have shown that excitatory and inhibitory interactions are instrumental for changing the strength and the time delay in the coupling among neurons , resulting in different ( collective ) behavioral periods .
this suggests that contrary to current belief , the period of circadian rhythms is not a fixed feature genetically encoded in a group of neurons .
we propose that it is an emerging property of a network of multiple , different circadian oscillators and that it is the wiring of the system , whose logic is genetically encoded , that determines period length .
perhaps not surprisingly , the same principles of negative feedback with amplification and delay , which are central to the ttl model , re - emerge in the constitution of the clock at the intercellular level .
the development of novel tools will be required to investigate how the known molecular mechanisms translate into different cellular features and for the experimental validation of the  logical  connections we postulate in our model .
cry , a c - terminal deletion of cry , renders cry constitutively active , as revealed in a number of molecular ( light - independent binding to per and tim ) and behavioral ( long period of locomotor activity in dd ) phenotypes [ 4 , 5 ] .
based on the discovery that light - activated cry increases neuronal firing , we presumed that cry could activate neurons and enhance their output in dd , which might be reflected in period changes .
we used a tim - gal4 driver and several uas - cry lines and confirmed that overexpression of cry in all clock cells , namely pdfcry  pdfcry  pdfcry , results in 1  hr lengthening of the endogenous period of locomotor activity compared to controls   ( table s1 available online ) .
we noticed that the vast majority of cry flies had simple rhythmicity ( sr ) although a few showed complex rhythms ( cr , more than one periodicity in a single fly ) or arrhythmicity ( ar ) but in proportions no different from controls .
we asked whether all clock cells contribute to the free running period or whether one group of neurons imposes its own rhythmicity to behavior .
we focused on one line ( uas - cry14.6 ) , which will hereafter be referred to as cry. pdfcry > cry ( pdf - gal4 > uas - cry ) and pdfcry  pdfcry > cry ( cry13-gal4 > uas - cry ) flies  both revealed a 1  hr longer period compared to controls .
we obtained a similar value for the pdfcry  pdfcry  pdfcry > cry genotype ( tim - gal4 > uas - cry ) , which argues against the assumption that these three drivers largely differ in their  strength   
then , we expressed cry in smaller groups of neurons by combining the gal80 repressor with gal4 drivers .
we  used the lines cry - gal802e3 m and pdf - gal8096a , each carrying two copies of gal80 , that have been reported to repress uas - dependent expression in the pdfcry  pdfcry and the pdfcry cells , respectively .
to confirm the extent of gal80-mediated inhibition of gal4 activity , we measured anti - gfp ir of pdfcry cells in tim - gal4 > uas - gfp , in tim - gal4  cry - gal802e3 m > uas - gfp , and in tim - gal4  pdf - gal8096a > uas - gfp flies . for both gal80 configurations
, we were able to confirm a good and equivalent level of repression of gal4 activity in pdfcry cells ( figures s1a and s1b ) .
however , in all tim - gal4  cry - gal802e3 m > uas - gfp preparations , we observed a robust anti - gfp signal in three lnds , meaning that gal80 can not efficiently inhibit gal4 in these cells because of lower cry expression ( figures s1a and s1c ) .
gal80-mediated repression of gal4 was inefficient also in four dn1ps and in three or four cry - positive dn3s .
conversely , good repression was achieved in the dn1as , in two dn1ps , and in the pn - lnv ( figure  s1c and data not shown ) .
henceforth we identify cells that strongly or weakly express cry as pdfcry and pdfcry , respectively .
we tested pdfcry  pdfcry > cry ( cry13-gal4  pdf - gal8096a > uas - cry ) and pdfcry  pdfcry  pdfcry > cry ( tim - gal4  pdf - gal8096a > uas - cry ) flies ; for both genotypes the free running period of locomotor activity was not significantly different from controls . however , when we analyzed pdfcry  pdfcry > cry ( tim - gal4  cry - gal802e3 m > uas - cry ) flies , we observed a significant 1.2  hr shortening of the period ( figure  1 ; table s1 ) .
this result can not be explained in terms of residual gal4 expression in the pdfcry cells as we detected similar levels of ir in them when driving gfp in both tim - gal4  pdf - gal8096a and tim - gal4  cry - gal802e3 m flies ( figures s1a and s1b ) .
cry overexpression resulted in a faster rhythm only for the  latter genotype , thereby unveiling the contribution of the pdfcry and pdfcry groups to period , which would otherwise be inhibited by the pdfcry cells ( this is because the two genotypes above differ in period and in the inclusion of the latter group of cells ) .
interestingly , in wild - type flies the dn2 cluster of pdfcry neurons show faster ( 22  hr ) endogenous molecular cycling that is not reflected in the 24  hr behavioral rhythms . under seminatural conditions ,
they show an advance in molecular cycling compared to other clock neurons , also consistent with a faster period [ 20 , 21 ] .
conversely , it has been reported that by reducing the contribution of the pdfcry cells to the network , either by eliminating their main signaling molecule , the neuropeptide pdf   ( figure  s2 ) , or by reducing their numbers   ( table s2 ) , a short activity period is generated .
thus , the overall evidence supports a role for the pdfcry neurons in generating a short - rhythm phenotype when the balance of the network is tilted in their favor , with the dn2s being a likely but perhaps not exclusive component of this function .
the overexpression of cry in pdfcry cells ( pdf - gal4 > uas - cry ) results in a longer period . however , these slower - paced cells are not alone in controlling self - sustained behavior because increasing the impact on the network of the pdfcry and pdfcry neurons ( tim - gal4  cry - gal802e3 m > uas - cry ) results in a shorter period .
the effect is reversed by  adding pdfcry to the ensemble ( tim - gal4  pdf - gal8096a > uas - cry ) , suggesting the latter intervene in a negative control , possibly on the faster pdfcry neurons ( figure  2a ) .
uas - cry ) had no consequence in terms of period ( figure  1 ; table s1 ) .
this suggests a balance , with the pdfcry neurons inhibiting the faster - paced pdfcry cells and in turn the pdfcry neurons inhibiting the slower - paced pdfcry cells ( figure  2a ) .
note that in this context , the words  activation  and  inhibition  do not have a physiological connotation but are used as logical operators .
we wanted to show that the effects observed on period are not specific to cry but are reproducible by other manipulations .
thus , we employed the same gal4/gal80 combinations described above , using effectors that have a known and consistent mode of action independent of the type of neuron .
the overexpression of the depolarization - activated bacterial sodium channel nachbac   using tim - gal4 ( pdfcry  pdfcry  pdfcry  pdfcry ) resulted predominantly in arrhythmicity , confirming the effectiveness of this manipulation ( table s2 ) . limiting nachbac expression to the  pdfcry ( pdf - gal4>uas - nachbac ) neurons generated 50% of flies with complex rhythms , showing a major long - period and a minor short - period component .
moreover , those 30%40% individuals with a single activity rhythm had a period 1.52.5  hr longer than controls ( figure  3a ; table s2 ) .
nachbac overexpression in pdfcry induces longer molecular cycles in these cells and directly increases their output , explaining the origin of the major , slower ( longer period ) activity component [ 22 , 23 ] .
immunocytochemistry experiments have  also indicated that the dn2s show a faster cycle in both controls and nachbac - overexpressing flies .
thus , it is tempting to speculate that fast pdfcry neurons ( we can not exclude that neurons in addition to the dn2s might also be involved ) might be indirectly activated through a positive , pdf - independent connection linking the pdfcry and the pdfcry cells and be responsible for the minor , short activity component detected in this genotype ( figures 2b and s2 ) .
( tim - gal4  cry - gal802e3 m > uas - nachbac ) flies showed single rhythms ( figure  3a ; table s2 ) .
this result supports the hypothesis that the pdfcry cells might inhibit the pdfcry neurons , explaining why they do not sustain behavioral rhythms in this genotype ( figure  2b ) .
subsequently we tested pdfcry  pdfcry  pdfcry > nachbac flies ( tim - gal4  2xpdf - gal80 > uas - nachbac ; figure  3a ; table s2 ) .
now , about 70%80% of individuals had a single activity component with a period 0.51  hr longer than controls .
this implies that the addition of the pdfcry group ( which is  not included in the largely arrhythmic tim - gal4  cry - gal802e3 m > uas - nachbac genotype ) to the ensemble of activated neurons results in the inhibition of the output of the pdfcry cells ( because of the longer activity period ) and of the pdfcry cells ( because the majority of flies are rhythmic ) .
possibly , the latter inhibition is indirect , in agreement with our previous hypothesis ( figure  2b ) .
pdfcry  pdfcry ( cry13-gal4  > uas - nachbac ) neurons caused a decrease in the number of rhythmic individuals and a shortening in the activity period ( now reaching wild - type values for single - rhythm flies ) compared to the pdfcry > nachbac ( pdfgal4 > uas - nachbac ) genotype ( figure  3a ; table s2 ) .
this result is different from the comparison between the same genotypes for cry flies , which were both rhythmic with a period about 1  hr longer than controls ( figure  1 ; table s1 ) .
the different strength of the two activators likely accounts for these discrepancies , which in general result from exaggerated effects on period and on rhythmicity following nachbac overexpression ( figures s3 ) .
we also applied the opposite manipulation , namely the overexpression of the mammalian inward rectifier k channel kir2.1 , a tool for decreasing membrane excitability in  vivo [ 24 , 25 ] .
pdfcry > kir2.1 ( pdf - gal4  > uas - kir2.1 ) resulted in more than a third of the flies being arrhythmic and in the tendency for a shorter period for those showing a single rhythm .
this argues for a disruption in the balance of the network and , in the flies still rhythmic , for a tilt toward the faster pdfcry neurons . extending the misexpression of kir2.1 to include also the pdfcry and the pdfcry cells ( cry13-gal4  > uas - kir2.1 ) abolished these effects ( figure  3b ; table s2 ) . finally , we tested the expression of kir2.1 in  pdfcry  pdfcry neurons ( tim - gal4  cry - gal802e3 m > uas - kir2.1 ) , but this genotype was not viable .
we then overexpressed the proapoptotic genes head involution defective ( hid ) and reaper ( rpr )   in pdfcry cells ( pdf - gal4  > uas - hid , uas - rpr ) and observed that 50% of the flies were equally either arrhythmic or showed complex rhythms .
the remaining half cycled with a period of locomotor activity 1  hr shorter than controls .
expression of hid and  rpr in pdfcry  pdfcry cells ( tim - gal4  cry - gal802e3 m > uas - hid , uas - rpr ) resulted in poor viability and 60% of the surviving flies were arrhythmic , further underscoring the importance of these neurons to the network ( figure  3c ; table s2 ) .
the remaining flies showed complex or single rhythms , the latter having periods on average intermediate to their corresponding controls ( table s2 ) .
although we did not verify this experimentally , it is likely that viable and rhythmic flies must have largely escaped the apoptotic response triggered by hid and rpr .
this explanation is in line with the lethality of the tim - gal4  cry - gal802e3 m > uas - kir2.1 genotype and with previously reported variability in the induction of  the apoptotic response in neurons . driving the production of the dominant - negative clk mutant   in pdfcry
 pdfcry cells ( tim - gal4  cry - gal802e3 m > uas - clk ) resulted in virtually complete arrhythmicity .
thus , as with nachbac and hid - rpr overexpression , disrupting ( independently of mechanism ) the pdfcry  pdfcry groups generated a largely arrhythmic profile , which argues against a simple dominant effect of the pdfcry cells on the neuronal network ( figure  3d ; table s2 ) .
the expression of clk in pdfcry cells caused 20%50% arrhythmicity and 10%30% of complex rhythms , depending on the line .
the remaining 40%50% of flies showed a single rhythm of activity with a period 0.51  hr shorter than controls .
this is in line with an increased influence of the pdfcry cells on the network . by expressing clk in pdfcry  pdfcry cells in addition to the pdfcry group ( cry13-gal4  > uas - clk )
, we increased the ratio of single - rhythm flies to 72%94% and reverted to a period closer to  that of the parental control strains , but slightly shorter ( figure  3d ; table s2 ) .
thus , as previously seen with the overexpression of kir2.1 , we could rescue rhythmicity in flies with weakened pdfcry neurons by reducing the inhibitory influence of the pdfcry cells on them and by buffering changes in activation of the pdfcry cells . again
, this shows that the  pdfcry cells do not act alone in determining circadian rhythmicity and its period .
we were unable to obtain viable  pdfcry  pdfcry  pdfcry > clk ( tim - gal4 ,   pdf - gal8096a >
 pdf - gal8096a > uas - clk ) flies were rhythmic with periods indistinguishable from controls ( figure  3d ; table s2 ) .
this result is the same as that observed with cry , again suggesting that the pdfcry and the pdfcry cells inhibit the faster pdfcry and the slower pdfcry cycling groups respectively , so the balance of the network is undisturbed ( figure  2b ) .
cry flies with a long behavioral period exhibit a delay in  the  cycling of clock proteins in pdfcry neurons . here 
we  investigated the cellular changes caused by cry overexpression in pdfcry  pdfcry ( tim - gal4  cry - gal802e3 m > uas - cry ) and in pdfcry  pdfcry  pdfcry ( tim - gal4  pdf - gal8096a > uas - cry ) neurons , resulting in short and normal ( compensatory ) activity rhythms , respectively .
we analyzed the cycling of the clock protein pdp1 in the major classes of clock neurons by if .
figure  4 shows the staining index ( si ) for pdp1  at ct0 , ct6 , ct12 , and ct18 ( circadian time , ct0  = subjective lights on , ct12  = subjective lights off ) during day 2 and 5 in dd . control flies ( uas - cry ) showed , at day 2 , maximum si at about ct18 for all cell types . at day 5
the si peaked also at ct18 for s - lnvs , lnds , and dn1s , suggesting that these cells cycle with a period of about 24  hr . instead the si maximum was reached earlier ( about ct12 ) by the dn2s , suggesting that these neurons run with a shorter period of 22  hr .
these cells  also showed a pronounced reduction in their cycling amplitude compared to the other clusters .
thus , in control flies , s - lnvs , lnds , and dn1s showed molecular oscillations that were compatible with the corresponding behavioral period attributed to them .
the overexpression of cry in pdfcry  pdfcry cells ( tim - gal4  cry - gal802e3 m > uas - cry ) targeted directly the dn2s , the large majority of dn1s , the dn3s , and half of the lnds .
the dn1s and even more so the dn2s showed a phase advance in pdp1 immune staining at day 2 ; at day 5 both groups reached their maximum at around ct6 .
however , while the dn1s experienced a shortening of their period ( in this genotype compared to the control ) , the dn2s maintained the same faster rhythm seen in wild - type flies .
the lnds were considered as a group because we could not distinguish the three pdfcry from the three pdfcry cells ( we could not discriminate them by anti - cry staining because both the anti - cry and the anti - pdp1 antibodies available to us are made in rabbit ) , which limits our interpretation of these data .
overall , the lnds showed a reduction in cycling amplitude especially at day 5 , perhaps resulting from averaging different cycling profiles in the two groups . the s - lnvs experienced phase advance and shortening of their rhythm
such that by day 5 they reached the si maximum at ct6 ( although because of the 6  hr resolution of our data , the actual peak might be a few hours later ) .
thus , in this genotype , the s - lnvs , the dn1s , and the dn2s were synchronous and showed endogenous cycling compatible with the short behavioral period . a significant 
time  cell type  anova interaction ( we compared the si of cell types s - lnvs , dn1s , and dn2s , at different time points , at day 2 and 5 ; see legend to figure  4 ) provides support to the view that the advance in the phase of pdp1 expression occurred first in the dn2s , then in the dn1s , and finally in the s - lnvs . the changes above appeared first at day 2 and were consolidated at day 5 , confirmed with a significant 
we then overexpressed cry in pdfcry  pdfcry  pdfcry cells ( tim - gal4  pdf - gal8096a > uas - cry ) , namely in the dns and in both types of lnds ( the pn - lnv was also affected but it has not been analyzed here ) .
extending the expression of cry to the pdfcry cells caused a broadening in the expression profile of pdp1 in the dns , supporting our hypothesis that the pdfcry modulate the pdfcry cells . especially at day 2 , the levels of pdp1 started to rise earlier ( compared to controls ) than for pdfcry  pdfcry > cry flies , but declined later in all dorsal neurons , suggesting a combination of phase advance and phase delay inputs .
the dn2s maintained their shorter molecular cycling while the dn1s and seemingly the dn3s moved to a 24  hr period .
the lnds , also expressing cry directly , showed the most pronounced phase delay ( supported by a significant 
time  cell type  anova interaction ) and a reduction in cycling amplitude ( although smaller than for the previous genotype ) .
day  time  cell type  anova interaction ( see legend to figure  4 ) .
the profile of pdp1 ir in the s - lnvs ( not expressing cry ) remained unchanged at day 2 and 5 , which , compared to the controls , corresponds to a small delay in phase but no change in period . in this genotype ,
only the slnvs and the dn1s showed robust endogenous cycling compatible with the behavioral period .
overall , only the s - lnvs and the dn1s consistently showed cellular cycling matching the behavioral periods of the flies in all genotypes .
we suggest that locomotor activity with simple rhythmicity requires synchronization between these two groups of neurons , although the contribution of neurons from other clusters may also be significant . in the pdfcry  pdfcry
> cry ( tim - gal4  cry - gal802e3 m > uas - cry ) flies , the faster rhythm of the dn2s and perhaps of additional unidentified fast pdfcry cells was shared by dn1s and s - lnvs . in pdfcry
 pdfcry  pdfcry > cry ( tim - gal4  pdf - gal8096a > uas - cry ) individuals , the dn2s were still cycling with a faster pace but , as in wild - type flies , the shorter period did not spread to dn1s and s - lnvs , suggesting this is the role of the highly pdf - responsive pdfcry cells ( included in the second but not in the first genotype ) .
the expressions of cry in the dn1ps only   did not result in faster behavioral rhythms , showing that the shorter period originates from a different group of neurons ( table s3 ) ; see figure  2c for a possible scenario .
we employed pdf - geneswitch ( pdf - gs )   to trigger drug - dependent expression of cry in pdfcry neurons either in adults only ( acute ) or through the whole development ( chronic ) . for acute treatment ,
flies were fed for 48  hr from the day of eclosion with medium supplemented with either 200  g / ml of the drug ru486 ( induction ) or with an equal volume of vehicle ( 80% ethanol , no induction ) .
note , all flies were raised on this medium and were then examined ( on their respective medium ) for locomotor activity rhythms . for chronic treatment
, flies were exposed to either drug or vehicle during their whole life and adult locomotor activity was examined accordingly . as controls , we tested ( heterozygous ) driver and effector flies raised under acute or chronic exposure to drug or on vehicle only ( table s4 ) . under both treatment conditions
, we observed a significant , albeit small , lengthening of the period of locomotor activity for cry-overexpressing flies compared to controls ( figures 5a and 5b ) .
as a further test we directed the expression of the temperature - sensitive cation channel trpa1 in pdfcry and in  pdfcry  pdfcry cells ( figures 5c and 5d ; table  s4 ) .
this channel is inactive at temperatures below 25c in drosophila and originally it was reported as being endogenously expressed in the adult brain in only about a dozen cells , which are not part of the circadian system .
however , recent evidence has shown that trpa1 transcription does occur in at least some cells in each cluster of clock neurons but that the effects of trpa1-null mutations on rhythmic behavior are quite modest .
flies overexpressing trpa1 and ( heterozygous ) parental controls were raised at 18c ( trpa1 is inactive ) and then tested as adults at 18c and 28c ( trpa1 is active ) .
compared to controls , we would expect a longer and a shorter period for pdfcry > trpa1 ( pdf - gal4 > uas - trpa1 ) and pdfcry  pdfcry > trpa1 ( tim - gal4  cry - gal802e3 m > uas - trpa1 ) flies at 28c , respectively . for pdfcry > trpa1 flies ,
the increase in period length with temperature was comparable to controls ( figures 5c and 5d ) .
conversely , we observed a significant decrease in period length for pdfcry  pdfcry > trpa1 flies at higher temperature but not for controls ( figures 5c and 5d ) . in summary ,
the manipulation of the circadian network limited to adults can affect the free - running period of locomotor activity .
however , the modest effects obtained with adult  pdfcry cells suggest that the ontogeny of the clock is particularly sensitive to the status of these neurons .
similar conclusions were reached by depetris - chauvin et  al .   and gorostiza and ceriani .
conversely , here we  show that the pdfcry and the pdfcry cells appear to be less sensitive to developmental effects ( figures 5c and  5d ) .
stoleru et  al .   had shown that overexpression of a constitutive active form of the kinase shaggy ( sgg ) , although able  to shorten the period of locomotor activity when driven in pdfcry cells , was unable to elicit a behavioral effect when directed in pdfcry  pdfcry neurons .
this was in spite of causing a faster molecular oscillation in some cells , for instance the dn2s , and was considered evidence that those cells are not relevant for free - running behavior .
pdfcry > sgg ( tim - gal4  cry - gal802e3 m > uas - sgg ) flies showed a free - running period of locomotor activity no different from controls , in agreement with previous results   ( figure  6a ; table s5 ) .
however , a visual inspection of the activity profiles revealed that these flies , both in ld and dd , showed earlier anticipation of the actual or subjective dark - to - light transition ( figure  6b ) .
perhaps this anticipation of phase reflects a tendency for the shorter rhythm to propagate to the network , which instead is prevented by other neuronal groups .
previous work has suggested that there is a physical and functional interaction between cry and sgg .
there is little or no  cry in pdfcry and pdfcry neurons , which could preclude sgg from exerting any robust effect . according to  our model , the pdfcry repress the pdfcry cells
; thus , increasing the output of the former should reduce the  resistance of the network to the propagation of the faster  rhythm .
we coexpressed cry and sgg and indeed observed a faster free - running period of locomotor activity in pdfcry  pdfcry > sgg , cry ( tim - gal4  cry - gal802e3 m > uas - sgg , uas - cry ) but not in pdfcry  pdfcry > cry ( tim - gal4  cry - gal802e3 m > uas - cry ) flies ( figure  6c ; table s5 ) .
these results reveal that the dn2s are indeed part of the circuit that generates self - sustained  behavior , although they do not exclude that the  establishment of a faster rhythm might require additional pdfcry neurons .
implicitly , they also contradict the view of a defined hierarchical organization of the clock under constant conditions . considering all results reported above
, we favor a model based on a flexible network of circadian neurons , which operates under any environmental condition .
our model complements and extends evidence by others of network organization as opposed to hierarchical dominance among neurons under ld and dd conditions [ 3436 ] .
we have used genetic manipulations limited to discrete groups of neurons to unveil the logic governing self - sustained rhythmic locomotor activity in drosophila .
activity rhythms and observations of molecular cycling in defined cellular groups consistently support a model ( figure  2 ) where endogenous behavioral rhythmicity is based upon synchronization between pdfcry and pdfcry cells .
this requires modulation of the pdfcry cells by the pdfcry neurons , largely but not exclusively via pdf signaling , and multistep feedback adjustment of the latter group of neurons by the former .
the behavioral outcomes of manipulations that alter but do not destroy the equilibrium of the network reveal that the pdfcry cells have a tendency for driving rhythms longer than 24  hr whereas some pdfcry cells promote rhythms shorter than 24  hr .
however , the organization of the network is such that wild - type flies reach a combined 24  hr oscillation .
this requires the pdfcry and the pdfcry cells that tune , through inhibitory interactions , the relative contribution of the other two groups of neurons to the network .
we note that the interposition of the pdfcry and the pdfcry cells adds delay and signal amplification to the feedback regulation that links the pdfcry to the pdfcry neurons and vice versa . in conclusion , we have shown that the endogenous behavioral period reflects the nonadditive interplay of all clock cells and not the dominant action of a single group of neurons ( i.e. , the s - lnvs ) , as previously suggested .
second , our overall results suggest that intercellular coupling is fundamental for synchronizing different cellular oscillators in a coherent clock , and therefore additional elements must complement the known molecular model focused around transcriptional regulators .
finally , we have shown that excitatory and inhibitory interactions are instrumental for changing the strength and the time delay in the coupling among neurons , resulting in different ( collective ) behavioral periods .
this suggests that contrary to current belief , the period of circadian rhythms is not a fixed feature genetically encoded in a group of neurons .
we propose that it is an emerging property of a network of multiple , different circadian oscillators and that it is the wiring of the system , whose logic is genetically encoded , that determines period length .
perhaps not surprisingly , the same principles of negative feedback with amplification and delay , which are central to the ttl model , re - emerge in the constitution of the clock at the intercellular level .
the development of novel tools will be required to investigate how the known molecular mechanisms translate into different cellular features and for the experimental validation of the  logical  connections we postulate in our model .
 summarybackgroundin the fruit fly drosophila melanogaster , interlocked negative transcription / translation feedback loops provide the core of the circadian clock that generates rhythmic phenotypes . although the current molecular model portrays the oscillator as cell autonomous , cross - talk among clock neurons is essential for robust cycling behavior . 
 nevertheless , the functional organization of the neuronal network remains obscure.resultshere we show that shortening or lengthening of the  circadian period of locomotor activity can be obtained either by targeting different groups of clock cells with the same genetic manipulation or by challenging the same group of cells with activators and repressors of neuronal excitability.conclusionsbased on these observations we interpret circadian rhythmicity as an emerging property of the circadian network and we propose an initial model for its architectural design . 
Introduction
Results and Discussion
CRY Lengthens or Shortens the Endogenous Period ofLocomotor Activity when Expressed in Different ClockNeurons
Additional Manipulations Consolidate the Model
Expression of CRY Sets off Nonautonomous Effects on Cellular Clocks
Network Perturbations Can Drive Changes in the Period of Locomotor Activity Independently of Development, although Ontogeny Is Not without Effect
Confirming the Model
Conclusions
Author Contributions
Supplemental Information