these rhythms have a molecular basis and depend on self - sustaining transcriptional / post - translational feedback loops ( ttfls ) ( darlington et  al . , 1998 ,
in drosophila , clk and cyc drive circadian oscillations by promoting rhythmic transcription of several key genes , including per , tim , and cwo , which repress clk - cyc - mediated transcription ( allada and chung , 2010 ) . in addition to transcriptional control , post - transcriptional and post - translational regulatory processes play essential roles in circadian timekeeping ( kadener et  al . , 2009 , kim et  al . , 2002 , lerner et  al . , 2015 ,
, 2004 , so and rosbash , 1997 , yu et  al . , 2009 ) .
the complexity of the circadian system extends beyond the single - cell level . in drosophila ,
they are clustered in six major subgroups : small and large ventral - lateral neurons ( s - lnvs , l - lnvs , and the fifth s - lnv ) , dorsal - lateral neurons ( lnds ) , and three subgroups of dorsal neurons ( dns13 ) . the neuropeptide pigment dispersing factor ( pdf )
, the main neuromodulator of the circadian neuronal network , is expressed in the lnvs .
pdf is essential for normal circadian activity patterns in light : dark cycles ( ld ) and for persistent circadian rhythms in constant darkness ( dd ) ( hyun et  al . , 2005 , lear et  al . , 2005 , mertens et  al . , 2005 , renn et  al . , 1999 ) .
, 2005 , i m and taghert , 2010 , shafer et  al . , 2008 ) .
, 2004 , welsh et  al . , 2004 ) , as well as in neurospora and cyanobacteria ( brunner and kldi , 2008 , kitayama et  al . ,
2008 ) . in this context , the main function of the circadian neuronal network is readjusting individual circadian oscillators , hence facilitating resonance or coherence in the network ( abraham et  al .
, 2010 , busza et  al . , 2007 , depetris - chauvin et  al . , 2011 ,
. however , several studies provided evidence for a role of neuronal connectivity in the timekeeping process per se , in flies ( peng et  al . , 2003 , weiss et  al . , 2014 )
( nitabach et  al . , 2002 , nitabach et  al . , 2005 but also see depetris - chauvin et  al . , 2011 ) and mammals ( bernard et  al . , 2007 , takahashi et  al . , 2010 ) . nevertheless , in drosophila
, the extent to which the molecular and neuronal circadian networks are intertwined is still not well understood .
pdf has a central role in the timekeeping process , as it coordinates phase and amplitude of molecular oscillations of downstream neurons ( collins et  al . , 2014 , liang et  al . , 2016 , lin et  al
peng et  al . , 2003 , seluzicki et  al . , 2014 ,
moreover , pdf signaling impacts the ttfl , by promoting the stabilization of the proteins tim and per ( li et  al . , 2014 , seluzicki et  al . , 2014 ) .
however , the effect of this regulation on clk - driven transcription is unclear , and pdf might be merely an output of the dominant pacemaker cells ( depetris - chauvin et  al . , 2011 ,
fernndez et  al . , 2007 , nitabach et  al . , 2005 , shafer and yao , 2014 ) . on the other hand
, clk has a key role in development of the pdf - expressing neurons ( allada et  al . , 2003 ,
) , but nothing is known about the mechanism employed for clk regulation over pdf expression .
thus , in this context , the interaction between the neuronal network and the molecular oscillator of individual neurons is far from being established . in mammalian systems
, those issues have been addressed using fluorescent reporters ( kuhlman et  al . , 2003 , nagoshi et  al . , 2004 ,
however , in drosophila luciferase reporters are more commonly used ( roberts et  al . , 2015 , sehadova et  al . , 2009 ,
, we developed and utilized fluorescent transcriptional reporters for tim and pdf in an ex  vivo brain culture setup , which allows us to perturb and monitor circadian transcription with spatiotemporal precision . using this approach
interestingly , we found that neuronal activity also modulates pdf transcription , likely utilizing a similar pathway as clk , involving the transcription factors drosophila hormone receptor - like 38 ( dhr38 ) and stripe ( sr ) . in sum
, our results suggest the existence of a tight inter - cellular feedback loop , involving the transcription factor clk and the neuropeptide pdf , that tightly wraps together the neuronal network and circadian molecular oscillators .
to follow clk - cyc driven transcription in  vivo , we generated a  circadian fluorescent transcriptional reporter .
it contains drosophila codon - optimized td - tomato fluorophore downstream to 6.4 kb of the timeless control region .
we fused the td - tomato to a pest motif and a nuclear localization signal ( nls ) to produce a short - lived , nuclear - localized signal ( figures 1a and s1a ) .
as expected , the reporter is strongly induced by addition of clk in a system lacking this transcription factor ( drosophila s2 cells ; figure  s1b ) .
five of the lines displayed strong oscillations in tomato levels across the day ( figure  s1c ) .
we choose one line ( # 7 ) with moderate fluorescent intensity , low background , and high specificity for circadian neurons for further experiments ( figure  s2a ) .
immunostaining with anti - tim and anti - pdf antibodies shows that the reporter is specifically co - expressed with tim in all circadian neuronal subgroups , including the lnvs ( figures 1b and s2b ) .
the line displays mrna and protein oscillations in fly head extracts with similar phase and amplitude to endogenous tim gene products ( figures 1c , 1d , s2c , and s2d ) , likely due to the long maturation time of the tdtomato fluorophore ( 1  hr at 37c and probably longer at 25c ) .
the reporter also recapitulates timeless expression temporally , as we detected synchronized tomato oscillations that peak at zt19 across the circadian neuronal network both in light : dark ( ld ) as well as in free running conditions ( figures 1e , s2e , and s2f ) . to determine whether the timtomato transgene can be used to report acute changes in clk activity in  vivo
the uas - clkgr transgene directs the expression of a fusion between clk and the glucocorticoid receptor ligand - binding domain .
this fusion protein acts as a dominant negative of clk ( weiss et  al . ,
2014 ) , but addition of the artificial glucocorticoid analog dexamethasone ( dex ) activates the fusion provoking a quick and large increases in clk - dependent transcription ( kadener et  al . , 2007 , mcdonald and rosbash , 2001 , weiss et  al . , 2014 ) .
we drove expression of clkgr with the timgal4 driver ( timclkgr - timtomato flies ) and observed a strong upregulation in signal from the reporter , within the circadian cells , in brain of flies incubated with dex ( figure  s2 g ) .
our timtomato reporter also displayed a strong increase in tomato in flies carrying a clksv40 transgene ( figure  s2h ) .
circadian rhythms are monitored from cultured adult drosophila brains using luciferase reporters ( roberts et  al . , 2015 ,
2002 ) . to further establish the use of the brain culture system for long - term studies of the circadian network , we dissected fly brains and cultured them in ld for 5  days .
we visualized vri and pdf levels by post - culture immunostaining and found that vri oscillations persisted in all circadian neuronal groups with similar phase , peaking at zt15 ( figures 1f and s2i ) .
we utilized this brain culture in combination with our reporter to follow clk - driven transcription immediately after induction of clk activity in all circadian cells ( using timclkgr - timtomato flies ) .
as expected , addition of dex strongly upregulates the reporter in all neuronal groups ( movies s1 , s2 , and s3 ) .
the response starts within less than 24  hr , reaching a plateau after approximately 4860  hr ( figure  s3 ) .
our results demonstrate that dynamic changes in clk - mediated transcription can be manipulated and simultaneously monitored ex  vivo using our reporter across the circadian neuronal network .
we followed by using our reporter in the brain culture system with the clkgr transgene to determine the relationship between clk - driven transcription in the lnvs and the rest of the circadian neuronal network .
we generated flies expressing the uas - clkgr transgene under the control of the pdfgal4 driver together with the reporter ( pdfclkr - timtomato flies ) .
the number of timtomato positive cells was used as a proxy for signal intensity . as expected , expression of the dominant - negative protein clkgr in
the lnvs resulted in a low number of cells expressing detectable levels of the reporter at zt19 ( figure  2a ; vehicle ) .
we observed that a large proportion of the dns and lnds still displayed strong tomato signal .
addition of dex dramatically increased the number of cells expressing the timtomato reporter in the lnvs .
interestingly , the induction of clk transcriptional activity in the lnvs suppressed reporter signal in the dns and lnds ( figure  2a ; dex ) .
the frequency plot of the number of cells expressing tomato illustrates this compensatory response ( figure  s4a ) .
the activation of clkgr in the lnvs appeared to consistently inhibit tim transcription in the dns and in some lnds . to determine the kinetic of this response , we monitored , by live imaging , tomato signal from brains of pdfclkgr - timtomato flies immediately after addition of dex to the culture ( movie s4 ; figures 2a , bottom right , and s4b ) .
we observed a quick repression in tomato signal in the dn1s , shortly after addition of dex .
a quick response in this cellular group is consistent with the physical interaction between s - lnvs and dn1s ( seluzicki et  al . , 2014 ) .
tomato signal in the l - lnvs remained high throughout this time course . to determine whether this response depends on pdf signaling
, we performed the same culture assay utilizing pdfclkgr - timtomato flies that also carry a mutation in the pdf receptor ( pdfr ; hyun et  al . , 2005 ) .
indeed , the presence of the pdfr mutation eliminated the difference in tomato signal between vehicle - treated and dex - treated brains in all neuronal subgroups ( figure  2b ) , demonstrating that this response depends on pdf signaling . surprisingly , in pdfr;pdfclkgr - timtomato flies , we did not detect any difference in tomato signal between vehicle - treated and dex - treated brains even in the s - lnvs and l - lnvs ( figure  2b , bottom ) .
this strongly suggests that pdf signaling is required for the cell - autonomous increase in clkgr - driven transcription induced by addition of dex . in addition , we observed that introduction of the pdf receptor mutation lead to a 4-fold increase in tomato signal in the s - lnvs of cultured brains ( figure  s4c ) .
importantly , the increase does not depend on the expression of clkgr ( figure  2c ) , showing that pdf signaling regulates clk - mediated transcription , at least in cultured brains .
these results indicate that pdf signaling suppresses clk - transcriptional activity in the main pacemaker neurons .
indeed , at the same culturing conditions described above , treatment of pdfclkgr brains with dex resulted in a significant elevation in pdf levels as indicated by quantification of pdf signal intensity in the lnv cell bodies and the number of pdf positive cells ( figure  s5a , top right and bottom right ) . to assess whether clk regulates pdf at the transcriptional level , we generated transgenic flies carrying a pdftomato transcriptional reporter , which we built using the same fluorescent protein under the control of a 2.4-kb genomic region from the pdf gene ( figure  s5c ; park et  al . , 2000 ) .
the reporter is highly specific , thus making identification of pdf - expressing cells straightforward ( figure  s5c , right ) .
we generated pdfclkgr flies that carry this reporter ( pdfclkgr - pdftomato ) . despite the above - mentioned positive effect on pdf levels , activation of clkgr by addition of dex decreased pdf transcription specifically in the s - lnvs , as indicated by the intensity of tomato signal ( figure  3a ) .
this result suggests that clk activity in the lnvs inhibits pdf transcription . to confirm this post - developmental effect of clk on pdf transcription in  vivo
, we knocked down clk in the lnvs of pdftomato brains using the pdfgal4 driver ( pdfgal4-pdftomato - clk ) . in agreement with the result described above , downregulation of clk in developed pdf - expressing cells resulted in more than 3-fold upregulation in tomato intensity in the s - lnvs and with a mild increase in pdf levels ( figures 3b and s5b ) .
overall , we conclude that clk suppresses pdf transcription post - development . to determine the mechanism by which clk regulates pdf expression , we identified putative regulators of pdf transcription using a yeast one - hybrid ( y1h ) screen .
for doing so , we generated five partially overlapping fragments from the 2.5-kb pdf promoter ( figure  s6 ) and utilized a previously described library of 650 drosophila transcription factors ( hens et  al . , 2011 ) .
we then focused on candidates that are strongly enriched in the s - lnvs and known to be activated by the mef2 and clk transcription factors ( abruzzi et  al .
mef2 has been shown to mediate between clk and the plasticity of the terminals in the pdf - expressing cells .
we assessed the effect of these factors on pdf transcription by expressing rnai transgenes in the pdf - expressing cells of flies which also carry the pdftomato reporter ( pdfgal4-pdftomato - rnai ) .
interestingly , downregulation of dhr38 and sr led to a strong upregulation of pdf transcription ( figures 4a and 4b ) , suggesting that both transcription factors inhibit pdf transcription . consistently with this result , downregulation of sr lead to an increase in pdf levels ( figures 4b and s7c ) .
however , downregulation of dhr38 either in the pdf or tim - expressing cells reduced pdf levels , suggesting that this transcription factor regulates pdf expression at different levels ( figures 4a , s7a , and s7b ) .
interestingly , dhr38 and sr were also identified as activity regulated genes in drosophila ( fujita et  al . , 2013 ;
x. chen and m. rosbash , personal communication ) suggesting neuronal firing could regulate pdf levels .
thus , we determined whether pdf transcription is regulated by neuronal firing by using the heat - activated cation channel dtrpa1 ( rosenzweig et  al . , 2005 ) in the pdf - expressing cells of flies that also carry the pdftomato reporter ( pdfgal4-pdftomato - trp ) .
dhr38 protein levels have been shown to reach a maximal expression within 2  hr of stimulation of the dtrpa1 channel ( fujita et  al . , 2013 ) .
thus , we determined the changes in tomato expression and pdf immunoreactivity in the lnvs of flies that were entrained at 25c and stimulated at 33c for 2  hr ( figure  4c ) .
we observed that both experimental and control groups respond to the heat stimulation by downregulating tomato and pdf levels ( figure  4c ) , plausibly due to the partial overlap in expression of the endogenous dtrpa1 channel  rna and protein  with the pdf - expressing cells and the s - lnvs specifically ( das et  al .
nevertheless , the decrease in tomato and pdf signal intensity , relative to the basal levels at 25c , was significantly stronger in flies overexpressing the channel in the s - lnvs ( figure  4c ) , and the expression of the trp channel strongly contributed to downregulate pdf expression in the s - lnvs at 33c ( figure  s7d ) . we conclude that neuronal activity of the lnvs suppresses pdf expression in the s - lnv .
this predicts that pdf should regulate pdf transcription , at least indirectly . to test this
we found that both pdf transcription and pdf immunostaining were strongly downregulated in pdfr flies ( figure  5a , left and right , accordingly ) , suggesting that pdf signaling is necessary for continuous pdf expression . to determine whether continuous activation of pdf signaling will increase pdf transcription , we utilized the tethered - pdf ( t - pdf ) technology ( choi et  al .
, 2009 , choi et  al . , 2012 ) . indeed , expression of t - pdf transgene in the pdf - expressing cells leads to a significant increase in pdf transcription , compared to controls , as assessed by the pdftomato reporter ( figure  5b ) .
together , the results presented in this section demonstrate that pdf signaling positively modulate pdf transcription .
here , we utilized an ex  vivo brain culture system combined with recently developed fluorescent transcriptional reporters to analyze interactions between the neuronal network and the molecular oscillator in the drosophila circadian system .
we found that activation of clk in the lnvs inhibits clk activity in the other circadian groups .
we showed that clk and pdf regulate each other and that neural activity regulates pdf transcription , probably through a common pathway involving the direct clk targets hr38 and sr .
we also found that pdf signaling is required for pdf  transcription , adding additional complexity to this cycle .
together , this study identified a tight connection between the core molecular circadian pacemaker , neuronal activity , and pdf .
we present an important technical innovation : the use of fluorescent reporters in dissected brains in culture to study circadian regulation .
our reporter system can be used to evaluate pdf and clk - dependent transcription at single - cell resolution .
, we were able to visualize and follow dynamics in clk activity across the circadian network .
our vri staining demonstrated that long - term ex  vivo brain culture supports coherent molecular oscillations in all neuronal groups .
as reported ( ayaz et  al . , 2008 ) , we also observed some degree of abnormality in pdf staining of these cultures .
our data revealed that pdf signaling negatively regulates clk activity in the s - lnvs ( figures 2c and s4c ) .
the timtomato reporter is the best tool available to specifically evaluate clk - driven transcription in the s - lnvs .
as , tim and per levels are under strong post - transcriptional control , which can also be regulated by pdf signaling ( li et  al . , 2014 ,
the latter is analogous to the one described for vasoactive intestinal polypeptide ( vip ) and per1 in the mammalian suprachiasmatic nucleus ( scn ) ( maywood et  al . , 2006 ) .
the effect of pdf signaling on clk - driven transcription in the s - lnvs can likely be extended to the other circadian cells ( figures 2a and 2b ) . however , further studies are needed to determine the mechanism of this compensatory effect .
this is mainly due to the opposite effects that clk - driven transcription has on pdf transcription and overall pdf levels .
the lack of ability to activate the clkgr transgene ( figure  2b ) could be due to several reasons , including : ( 1 ) the tim promoter is already active up to its maximal capacity ; ( 2 ) clkgr can not be activated by the addition of dex in the pdfr mutants ; and ( 3 )  a combination of both options .
it is also possible that pdf signaling somehow interferes with dex induction of clkgr , although we did not find experimental support for this possibility .
since we show that in the absence of pdf signaling there is a 4-fold increase in signal originated from the timtomato reporter in s - lnvs ( figure  s4c ) and since similar increase was observed independently of clkgr expression ( figure  2c ) , we can conclude that in the absence of pdf signaling endogenous clk activity is higher .
we speculate that the observed increase in endogenous clk activity overrides the ability to activate clkgr in the pdfr flies .
previous studies support the notion that clk positively regulate the development of the pdf cells ( allada et  al . , 2003 , lerner et  al . , 2015 , park et  al . , 2000 , zhao et  al . ,
, we show that specific induction of clk activity in the adult main pacemaker cells , as well as silencing clk post - development of the pdf cells using the pdfgal4 driver , result in repression of pdf transcription ( figure  6 , model ) .
this suggests opposite effects of clk on pdf transcription either pre and post - development of the lnvs or within and outside the lnvs . as pdf signaling
positively regulates pdf expression , the continuous expression of pdf by the lnvs is a consequence of a balance between clk and pdf activities .
neuronal electrical activity might have a crucial role in this balance , as it can suppresses pdf transcription in the main pacemaker cells through hr38 and sr .
the effect of clk on pdf seems to also involve those factors that are direct clk and mef2 transcriptional targets ( abruzzi et  al .
, 2011 , sivachenko et  al . , 2013 ) and are in the lnvs ( kula - eversole et  al . , 2010 ) and represses pdf transcription ( figure  6 , model : red inhibitory arrow ) .
although clk , hr38 , sr , and neuronal activity downregulate pdf transcription , their effects on pdf neuropeptide levels are intriguing .
knockdown of hr38 ( figure  4a ) and activation of clkgr ( figures 3a and s5a ) yielded opposite effects on the pdftomato reporter and pdf levels ( up and down or down and up , respectively ) .
this suggests that hr38 is involved in the regulation of pdf by clk but also that there might be a more complex post - translational effects on pdf .
however , neural stimulation downregulates both pdf transcription and protein levels , reinforcing the idea of additional regulatory factors and possibly post - translational control over pdf expression / processing / secretion . hence
, our model predicts that both neuronal firing and clk activity are indirectly regulating pdf transcription , not only through promoting activities of targets genes such as dhr38 and sr , but also through the post - translational control over pdf ( sivachenko et  al . , 2013 ) since , as we show here , pdf feeds back to regulate pdf transcription ( model , green dotted arrow ) .
this positive auto - regulation introduces complexity to the clk - pdf feedback loop , and it is in agreement with a previously proposed model for pdf auto - receptor signaling in the lnvs ( choi et  al . , 2012 ) .
this intriguing feature of the loop should be addressed in future studies in order to extend our understanding of how post - translational mechanisms for pdf regulation affect its own transcription .
the regulation of pdf signaling on pdf transcription can be also predicted from the strong and mutual regulation between clk and pdf ; however , we favor a more direct effect of pdf signaling on pdf transcription ( model , green dotted arrow ) .
interestingly , the transcription factor ttk is expressed in all tim positive circadian neurons except the lnvs ( nagoshi et  al . , 2010 ) , suggesting it might be an important endogenous regulator of pdf spatial expression .
pdf signaling was shown to be required for normal pattern of oscillations in clk - driven transcription under constant darkness ( peng et  al . , 2003 ) .
based on the upregulation of timtomato in pdfr mutant , we postulate that in the pacemaker cells pdf feedback is required for normal pattern of oscillations in clk - driven transcription under light : dark cycles ( figure  6 , model : inhibitory red arrow ) .
since zt19 is the peak of tomato expression in the s - lnvs ( figure  1e , white circle ) , upregulation at this zt can not be a consequence of a phase shift ; thus , it must reflect an increase in the amplitude of tomato oscillation .
this demonstrate that in the absence of pdf signaling , the lnvs lose control of their own circadian molecular clockwork ( collins et  al .
, 2014 , peng et  al . , 2003 ) , likely because it is overridden by signals from the lnds and/or the dns ( weiss et  al . , 2014 , yao and shafer , 2014 ) . in sum ,
our findings challenge the notion that pdf is a merely output of the circadian system .
indeed , we demonstrate the existence of an uncharacterized and essential clk - pdf - clk regulatory loop in the lnvs that integrates pdf signaling into the single - cell molecular oscillator . the question of whether pdf feeds back to the same cells that secrete it within the subgroup of s - lnvs or whether its role as a communication agent is also applied within this subgroup remains to be answered in order to determine the final impact of this loop on the concept of cellular autonomy in the fly brain .
to generate the timtomato reporter construct , a drosophila codon optimized tdtomato - nlsx3-pest coding sequence was fused with a sv40 3 utr in a pcasper4 vector downstream to a 6.4-kb dna fragment containing the promoter and 5 utr stretching into to the second exon atg of the timeless gene . to generate the pdftomato reporter
, we utilized a similar approach but using a previously a 2.45-kb fragment containing the genomic region upstream to the pdf gene transcription start site inserted in a pattb based vector . to generate dna baits of the pdf promoter for drosophila transcription factor screen ( y1h )
, we used a carrier vector containing the 2.45-kb promoter of the pdf gene as a template to generate five overlapping pcr fragments approximately 600  bp long ( see the supplemental experimental procedures ) .
the reporter transgenic fly lines were generated by bestgene using the p - element - mediated germline transformation for tim reporter and phic31 integrase method into the pattp2 site for pdf reporter .
timgal4 , pdfgal4,pdfr , uas - clkgr ; uas - dcr2 , hr38 , sr 
 , clk 
 , uas - t - pdf , uas - trpa1 , and clksv40 fly lines were previously described ( choi et  al .
, 2007 , hyun et  al . , 2005 , kaneko and hall , 2000 , lerner et  al .
, 2015 , perkins et  al . , 2015 , renn et  al . , 1999 ,
flies were dissected ( leaving trachea and imaginal discs ) in 1   pbs on a clean surface at room temperature .
each brain was immediately placed in a separate well of a 96-well plate containing 150  l drosophila shield and sang m3 insect medium ( 10% fbs , insulin [ 10  g / ml ] , and a 1:100 dilution of antibiotic solution ) . for activation of clkgr in culture ,
the medium was prepared with a supplement of 10  mg / ml dexamethasone ( d4902 ; sigma ) , kept in 100% ethanol , and diluted 1:500 ( dex ) or with a 1:500 dilution of 100% etoh only ( vehicle ) .
brains were taken out from the well , cleaned in ice - cold 1   pbs , and then fixed with 4% paraformaldehyde ( pfa ) for 20  min at room temperature .
brains were then washed with ice - cold 1   pbs before downstream application ( visualization or immunolabeling ) ( see the supplemental experimental procedures ) .
brains were then placed on a 12-m cell - culture insert ( millipore ) inside a 35-mm culturing dish containing 800  l of culture medium as previously described ( ayaz et  al . , 2008 ) and entrained for 24  hr at 25c in ld .
medium was supplemented with dex or vehicle before time - lapse imaging was performed ( one frame/30 or 40  min ) .
nikon eclipse ti confocal microscope was used to perform time - lapse imaging ( olympus 20 super plan flour , 0.5 numerical aperture [ na ] , long distance 8,200  mm , air lens ) and visualization ( olympus 40 plan fluor 1.3 na , 240  mm ,  60 plan apo 0.9 na , 150  mm , oil lenses ) of fluorescence . for relative intensity quantification ,
both the sum and the average signal intensity of an auto or manually detected rois were calculated .
signal was summed across all confocal planes surrounding the neuronal cell bodies belong to a certain sub - neuronal group in the brain .
the average signal of the whole population of samples was then calculated , and statistical significance was determined . for cell - number - based quantification , microscope settings were set as permissive for weak signal ( high excitation energy and low detection threshold ) .
the number of cells in a certain neuronal group was determined based on the existence of a positive ( weak or strong ) tomato or pdf signal .
tomato signal in the s - lnvs of reporter brains was quantified from brains over - stained for pdf and by evaluation of anatomical location .
s2 cells were maintained using standard procedures and transfected using 1.5  l trans - it insect transfection reagent ( mirus ) .
the timtomato and timyfp ( lerner et  al . , 2015 ) were transfected in equal molarity ( 400 and 200  ng , respectively ) with 100  ng pactin - clkv5 or pmt - clksv40 .
12  hr post - transfection , cu was added to a final  concentration of 1  mm .
the yeast one - hybrid screens were conducted as described in hens et  al .
 summarycircadian clocks generate 24-hr rhythms in physiology and behavior . despite numerous studies , 
 it is still uncertain how circadian rhythms emerge from their molecular and neural constituents . here 
 , we demonstrate a tight connection between the molecular and neuronal circadian networks . using fluorescent transcriptional reporters in a drosophila ex  vivo brain culture system 
 , we identified a reciprocal negative regulation between the master circadian regulator clk and expression of pdf , the main circadian neuropeptide . 
 we show that pdf feedback is required for maintaining normal oscillation pattern in clk - driven transcription . 
 interestingly , we found that clk and neuronal firing suppresses pdf transcription , likely through a common pathway involving the transcription factors dhr38 and sr , establishing a direct link between electric activity and the circadian system . in sum 
 , our work provides evidence for the existence of an uncharacterized clk - pdf feedback loop that tightly wraps together the molecular oscillator with the circadian neuronal network in drosophila . 
Introduction
Results
Discussion
Experimental Procedures
Author Contributions