most animals possess circadian clocks that measure the time of day and allow the organism to adapt to daily environmental changes . the clock generates circadian rhythms of approximately 24  hours in many biological processes , such as behavior , metabolism , and physiology , enabling animals to anticipate and adapt to environmental changes . the oscillatory mechanism of the clock is self - sustained but must be synchronized to external time cues in order to allow adaption to changing environmental conditions . in most cases , light is the most critical entrainment cue and must be integrated into circadian clock circuits in the brain . the brain of most animals contains a central clock . in the fruit fly drosophila melanogaster , approximately 150 neurons in the brain have been identified as clock neurons based on the cyclic expression of the genes and proteins that play central roles in the circadian clock . each cluster is named according to its location and the size of individual neurons , as shown in figure  1 . it remains unclear whether all clusters of clock neurons are important for controlling behavioral rhythms ; however , several studies have suggested that they are not all of equal importance . the left hemisphere shows the distribution of the cryptochrome ( cry)-positive ( blue ) and cry - negative ( white ) clock neurons . the right hemisphere shows the compound eye and the hofbauer - buchner ( hb ) eyelet . the small and large lateral neuron ( s - lnv and l - lnv ) clusters express the pigment - dispersing factor ( pdf ) protein and are regarded as the clock neurons controlling the morning ( m ) activity peak . the lateral dorsal neurons ( lnd ) and the hb eyelet is directly connected to the s - lnv neurons through projections to the surrounding area . in contrast , the connections between the compound eyes and the clock neurons have not yet been elucidated . 
  the circadian clock network in the drosophila brain . the left hemisphere shows the distribution of the cryptochrome ( cry)-positive ( blue ) and cry - negative ( white ) clock neurons . the right hemisphere shows the compound eye and the hofbauer - buchner ( hb ) eyelet . the small and large lateral neuron ( s - lnv and l - lnv ) clusters express the pigment - dispersing factor ( pdf ) protein and are regarded as the clock neurons controlling the morning ( m ) activity peak . the lateral dorsal neurons ( lnd ) and the hb eyelet is directly connected to the s - lnv neurons through projections to the surrounding area . in contrast , the connections between the compound eyes and the clock neurons have not yet been elucidated . the best - studied clock neurons are the large and small lateral ventral neurons ( l - lnvs and s - lnvs ) . these neurons express pigment - dispersing factor ( pdf ) , a neuropeptide that acts as a circadian neuromodulator . pdf plays an important role in the circadian network as an intercellular messenger , synchronizing daily rhythms between pdf neurons and other clock neurons . pdf - null flies display weak circadian activity rhythms with a shortened period of approximately 22  hours when kept in constant darkness ( dd ) and a phase - advanced evening activity peak in the presence of a 12-hour:12-hour light - dark cycle ( ld ) . interestingly , flies lacking the l - lnv and s - lnv neuron clusters show identical behavioral phenotypes , suggesting that pdf is the principal output of these neurons . thus , pdf - expressing clock neurons as well as pdf itself strongly influence activity rhythms . the gal4/uas system is a genetic tool commonly used for tissue - specific manipulation of gene expression in drosophila . several useful gal4 lines have been available since the year 2000 that target subsets of clock neurons , allowing us to study their function . pioneering studies using the gal4/uas system were performed by grima et  al . and stoleru et  al . in 2004 . these studies investigated different subsets of clock neurons by restoring clock function to specific neuron clusters in the clock - impaired period ( per ) mutant or by ablating clusters expressing the apoptosis - inducing head involution defective gene . the flies without functional pdf neurons lost anticipatory morning ( m ) activity , whereas the flies without functional lateral dorsal ( lnd ) and dorsal neuron ( dn ) clusters lost anticipatory evening ( e ) activity under ld conditions . thus , both studies concluded that the pdf - positive clock neurons are important for morning activity , and lnd and dn clusters are important for evening activity . in 2006 , the 5 s - lnv was identified as a non - pdf - expressing clock neuron that was located in close proximity to the l - lnv cluster and was classified as an e clock neuron . the structure of the cry protein is very similar to that of the bacterial 64 photolyase . the cry protein was first identified in arabidopsis thaliana , suggesting that it is highly conserved . whereas wild - type flies are able to synchronize their circadian rhythms to a new ld condition within one day , cry mutant flies require approximately 7  d to adapt . the circadian clock responds to a light pulse during the night by advancing or delaying activity rhythms , depending on the time at which the light - pulse is given . these responses can be described by a phase - response curve . compared to wild - type flies , cry mutants cry is expressed in many clock neurons , including m and e neurons . upon light exposure , cry binds to the timeless ( tim ) protein , an essential clock component , and leads to its ubiquitination by the jetlag protein and subsequent degradation . the light - induced degradation of tim destabilizes the period ( per ) protein , another core clock component and a binding partner of tim , and thus pauses the circadian oscillation of per and tim levels . these interactions between the per , tim , and cry proteins provide a simple and clear - cut explanation of how the drosophila clock is reset by light . however , not all clock neurons express cry , and levels of cry expression differ among clock neurons . furthermore , not all clock neurons show similar responses to light - pulses given during the night . ( 2010 ) demonstrated that a light pulse early in the night induces degradation of tim in e neurons , but not in m neurons , suggesting that the phase resetting of the clock by a light pulse is not the same in all clock neurons and depends on the time of day . more interestingly , cry - dependent light input to m neurons can cause cry - independent tim degradation in e neurons . thus , m neurons can direct the light response of e neurons through neuronal communication . a bright light pulse lasting for several minutes during the night is sufficient to induce tim degradation in all clock neurons . in response to a light pulse of reduced intensity and increased duration , the 5 s - lnv neuron shows the most efficient tim degradation compared to other clock neurons , suggesting that individual clock neurons differ in cry - dependent light sensitivity . these studies used light pulses to examine light responses , revealing fine temporal properties of the clock in response to a short light - pulse . however , responses to light pulses differ from entrainment to ld conditions that consist of distinct periods of day and night . when cry is re - expressed in e neurons using the gal4/uas system in the cry mutant background , these flies show responses similar to those of wild - type flies to an 8-hour phase delay of the ld cycle , re - entraining nearly within one day . in contrast , cry mutants require nearly a week to completely synchronize to the phase - delayed ld cycle . interestingly , e neurons in cry mutants show rapid re - entrainment of molecular cycling of the clock protein par domain protein ( pdp1 ) . however , re - entrainment of clock protein cycling in other clock neurons occurs slowly , similar to re - entrainment of behavioral rhythms . thus , e neurons are the first to reset in response to light input from the visual system , followed by other clock neurons . light - activated cry also influences the neural activity of l - lnv neurons by increasing action potential firing . this effect is independent of the cry - tim interaction and opsin - based photoreception . the role of these cry - induced increases in neuronal activity is thus far unknown , but a possible involvement in light entrainment should be considered . drosophila have 3 different external photoreceptors : compound eyes , ocelli , and hofbauer - buchner eyelets ( hb eyelets ) . the compound eyes are the largest photoreceptive structure and are thought to be the most important to light entrainment . studies using eye mutants have demonstrated that the compound eyes play roles in measuring day length and detecting moonlight . flies display 2 distinct activity peaks at approximately dawn and dusk in 12  hour:12  hour ld cycles , termed the m and e peaks , respectively . these 2 activity peaks respond to changing photoperiod and to dim light during the night . under long - day conditions or during nights with moonlight , the m and e peaks move away from one another , thus creating a larger phase angle between the peaks . drosophila mutants lacking compound eyes can not adapt their m and e activity peaks to long - day conditions . and do not respond to moonlight . these findings suggest that the light input from the visual system affects the m and e neurons in opposite manners , namely phase - advances the m oscillator and phase - delays the e oscillator . moonlight also has direct effects on activity , known as masking effects , in that it increases nighttime activity levels . these masking effects are mediated by the compound eyes but are not influenced by cry . the compound eyes consist of approximately 800 ommatidia , each of which contains 8 photoreceptor cells ( r18 ) expressing different rhodopsins ( rh16 ) . mutant flies lacking rh1 and rh6 , which are expressed in r16 and r8 cells , respectively , do not display masking effects on activity behavior in response to moonlight . twilight , the gradual change in light intensity at dawn and dusk , also affects activity rhythms , though it is often neglected in laboratory experiments . simulation of twilight conditions causes shifts of the m and e activity peaks into the dim light zones . the effects of twilight on behavior exceed those of moonlight , and activity rhythms during twilight simulation more strongly resemble activity rhythms under natural conditions . the effects of twilight on behavior are also mediated by the compound eyes , particularly by the 2 inner photoreceptor cells r7 and r8 . thus , different light - sensing mechanisms have different roles in modulating and entraining activity rhythms . comparisons between mutants that lack all external photoreceptors or lack only the compound eyes showed significant contribution of the compound eyes to entrainment , compared to the minor contribution of the ocelli . the same is true for the hb eyelets , which are remnants of the larval photoreceptors , the bolwigs organs , and express the rhodopsin 5 and 6 ( rh5 and rh6 ) genes . in both larval and adult brains , the projections of bolwigs organs and hb eyelets directly contact the pdf - positive lnvs . the larval bolwigs organs use acetylcholine as a neurotransmitter , whereas the adult hb eyelets express both acetylcholine and histamine . application of cholinergic agonists increases ca and cyclic amp ( camp ) levels in both dissociated larval lnvs and in adult lnv clusters in intact brains . bolwigs organs , together with cry , are essential for light entrainment of the larval clock neurons . however , it remains unclear how significantly hb eyelets contribute to light entrainment of the adult clock . two studies have investigated the roles of the rh5 and rh6 proteins that are expressed in the hb eyelets and in photoreceptor cell r8 of the compound eyes . flies with triple - mutation of rh5 , rh6 , and cry display slower light re - entrainment than cry single mutants in following a 6-hour phase shift of the ld cycle , suggesting that the hb eyelets contribute to light entrainment . flies with double - mutation of cry and the hdc gene encoding histidine decarboxylase are unable to synchronize to an ld cycle . both receptors are expressed in interneurons located between the lamina and the medulla and may also be present in other brain regions . however , it is not yet known how histamine receptor - positive cells transmit signals to clock neurons . a recent study demonstrated that cry interacts with the phototransduction complex through the inactivation no afterpotential d protein in the compound eyes . cry mutant flies show reduced nocturnal light sensitivity in electroretinograms and a weaker optomotor response compared to wild - type flies . however , cry expressed in the eyes may have a minor contribution to light entrainment , since flies expressing cry only in the eyes do not show significant improvement in entrainment compared to cry mutants . the structure of the cry protein is very similar to that of the bacterial 64 photolyase . the cry protein was first identified in arabidopsis thaliana , suggesting that it is highly conserved . cry mutant flies display failures in light entrainment . whereas wild - type flies are able to synchronize their circadian rhythms to a new ld condition within one day , cry mutant flies require approximately 7  d to adapt . the circadian clock responds to a light pulse during the night by advancing or delaying activity rhythms , depending on the time at which the light - pulse is given . these responses can be described by a phase - response curve . compared to wild - type flies , cry mutants cry is expressed in many clock neurons , including m and e neurons . upon light exposure , cry binds to the timeless ( tim ) protein , an essential clock component , and leads to its ubiquitination by the jetlag protein and subsequent degradation . the light - induced degradation of tim destabilizes the period ( per ) protein , another core clock component and a binding partner of tim , and thus pauses the circadian oscillation of per and tim levels . these interactions between the per , tim , and cry proteins provide a simple and clear - cut explanation of how the drosophila clock is reset by light . however , not all clock neurons express cry , and levels of cry expression differ among clock neurons . furthermore , not all clock neurons show similar responses to light - pulses given during the night . for example , tang et  al . ( 2010 ) demonstrated that a light pulse early in the night induces degradation of tim in e neurons , but not in m neurons , suggesting that the phase resetting of the clock by a light pulse is not the same in all clock neurons and depends on the time of day . more interestingly , cry - dependent light input to m neurons can cause cry - independent tim degradation in e neurons . thus , m neurons can direct the light response of e neurons through neuronal communication . a bright light pulse lasting for several minutes during the night is sufficient to induce tim degradation in all clock neurons . in response to a light pulse of reduced intensity and increased duration , the 5 s - lnv neuron shows the most efficient tim degradation compared to other clock neurons , suggesting that individual clock neurons differ in cry - dependent light sensitivity . these studies used light pulses to examine light responses , revealing fine temporal properties of the clock in response to a short light - pulse . however , responses to light pulses differ from entrainment to ld conditions that consist of distinct periods of day and night . when cry is re - expressed in e neurons using the gal4/uas system in the cry mutant background , these flies show responses similar to those of wild - type flies to an 8-hour phase delay of the ld cycle , re - entraining nearly within one day . in contrast , cry mutants require nearly a week to completely synchronize to the phase - delayed ld cycle . interestingly , e neurons in cry mutants show rapid re - entrainment of molecular cycling of the clock protein par domain protein ( pdp1 ) . however , re - entrainment of clock protein cycling in other clock neurons occurs slowly , similar to re - entrainment of behavioral rhythms . thus , e neurons are the first to reset in response to light input from the visual system , followed by other clock neurons . light - activated cry also influences the neural activity of l - lnv neurons by increasing action potential firing . this effect is independent of the cry - tim interaction and opsin - based photoreception . the role of these cry - induced increases in neuronal activity is thus far unknown , but a possible involvement in light entrainment should be considered . drosophila have 3 different external photoreceptors : compound eyes , ocelli , and hofbauer - buchner eyelets ( hb eyelets ) . the compound eyes are the largest photoreceptive structure and are thought to be the most important to light entrainment . studies using eye mutants have demonstrated that the compound eyes play roles in measuring day length and detecting moonlight . flies display 2 distinct activity peaks at approximately dawn and dusk in 12  hour:12  hour ld cycles , termed the m and e peaks , respectively . these 2 activity peaks respond to changing photoperiod and to dim light during the night . under long - day conditions or during nights with moonlight , the m and e peaks move away from one another , thus creating a larger phase angle between the peaks . drosophila mutants lacking compound eyes can not adapt their m and e activity peaks to long - day conditions . and do not respond to moonlight . these findings suggest that the light input from the visual system affects the m and e neurons in opposite manners , namely phase - advances the m oscillator and phase - delays the e oscillator . moonlight also has direct effects on activity , known as masking effects , in that it increases nighttime activity levels . these masking effects are mediated by the compound eyes but are not influenced by cry . the compound eyes consist of approximately 800 ommatidia , each of which contains 8 photoreceptor cells ( r18 ) expressing different rhodopsins ( rh16 ) . mutant flies lacking rh1 and rh6 , which are expressed in r16 and r8 cells , respectively , do not display masking effects on activity behavior in response to moonlight . twilight , the gradual change in light intensity at dawn and dusk , also affects activity rhythms , though it is often neglected in laboratory experiments . simulation of twilight conditions causes shifts of the m and e activity peaks into the dim light zones . the effects of twilight on behavior exceed those of moonlight , and activity rhythms during twilight simulation more strongly resemble activity rhythms under natural conditions . the effects of twilight on behavior are also mediated by the compound eyes , particularly by the 2 inner photoreceptor cells r7 and r8 . thus , different light - sensing mechanisms have different roles in modulating and entraining activity rhythms . comparisons between mutants that lack all external photoreceptors or lack only the compound eyes showed significant contribution of the compound eyes to entrainment , compared to the minor contribution of the ocelli . the same is true for the hb eyelets , which are remnants of the larval photoreceptors , the bolwigs organs , and express the rhodopsin 5 and 6 ( rh5 and rh6 ) genes . in both larval and adult brains , the projections of bolwigs organs and the larval bolwigs organs use acetylcholine as a neurotransmitter , whereas the adult hb eyelets express both acetylcholine and histamine . application of cholinergic agonists increases ca and cyclic amp ( camp ) levels in both dissociated larval lnvs and in adult lnv clusters in intact brains . bolwigs organs , together with cry , are essential for light entrainment of the larval clock neurons . however , it remains unclear how significantly hb eyelets contribute to light entrainment of the adult clock . two studies have investigated the roles of the rh5 and rh6 proteins that are expressed in the hb eyelets and in photoreceptor cell r8 of the compound eyes . flies with triple - mutation of rh5 , rh6 , and cry display slower light re - entrainment than cry single mutants in following a 6-hour phase shift of the ld cycle , suggesting that the hb eyelets contribute to light entrainment . flies with double - mutation of cry and the hdc gene encoding histidine decarboxylase are unable to synchronize to an ld cycle . both receptors are expressed in interneurons located between the lamina and the medulla and may also be present in other brain regions however , it is not yet known how histamine receptor - positive cells transmit signals to clock neurons . a recent study demonstrated that cry interacts with the phototransduction complex through the inactivation no afterpotential d protein in the compound eyes . cry mutant flies show reduced nocturnal light sensitivity in electroretinograms and a weaker optomotor response compared to wild - type flies . however , cry expressed in the eyes may have a minor contribution to light entrainment , since flies expressing cry only in the eyes do not show significant improvement in entrainment compared to cry mutants . pdf is a circadian neuropeptide specifically expressed in the l - lnv and s - lnv clusters and serves as an intercellular communication signal between clock neurons . the pdf receptor ( pdfr ) is expressed in many clock neurons including the s - lnvs . pdf mutants or flies lacking pdf - expressing neurons show a phase - advanced e activity peak under ld conditions . they are also incapable of shifting m and e peaks under long - day conditions . furthermore , ablation of the l - lnv cluster attenuates the response to a light - pulse late in the night . flies with double - mutation of pdf and cry show an intriguing phenotype , completely lacking the e peak under ld conditions . this phenotype suggests a model in which cry and the visual system entrain the m and e neurons by either direct or indirect mechanisms ; furthermore , pdf - expressing neurons transduce light input from the visual system to e neurons , such that e neurons in pdf - cry double mutants are unable to receive light input . i m and taghert ( 2011 ) additionally demonstrates that the m peak in the double mutants is a masking effect but is not driven by the circadian clock , indicating that pdf and cry are essential for light entrainment . thermogenetics is a suitable genetic tool for the manipulation of clock neuron activity because the effect of temperature on the clock is relatively moderate compared to that of light , which is used for optogenetics . trpa1 is a temperature - sensitive cation channel that induces neuronal depolarization in response to increasing temperature . interestingly , it appears that phase - response curves in response to temperature pulses applied to flies overexpressing trpa1 in pdf neurons mimic phase - response curves in response to light pulses . thus , light inputs , likely from the visual system , may normally excite pdf neurons , consequently resetting downstream neurons such as the e neurons and leading to behavioral phase shifts . this pathway may be mediated by specific adenylate cyclases , camp , and the protein kinase a signaling cascade that promotes tim degradation in e neurons . the neuronal circuits linking the visual system to the clock neurons have not yet been described , with the exception of the direct connection between hb eyelets and pdf neurons described above . it has not yet been demonstrated that light inputs from all visual organs converge on the pdf neurons , not on the e neurons , which would support the hypothesis that pdf neurons provide the only pathway for light inputs to the clock that are not mediated by cry . this has already been shown for the larval brain , which has simpler circadian circuits involving only 18 clock neurons and only 2 light - input pathways : bolwigs organs and the cry protein . previous studies have revealed the astonishing complexity of light entrainment in the circadian system of the fly , which has otherwise been considered a rather simple organism . the compound eyes convey light signals to the pdf neurons via histaminergic signaling through interneurons , leading to the resetting of the molecular clock in the pdf neurons . bolwigs organs and hb eyelets use both acetylcholine and histamine to signal to pdf neurons ; this pathway seems to consist of a direct connection between the visual organs and clock neurons . both pathways first reset pdf - expressing neurons , which in turn reset the pdf - negative clock neurons . cry is expressed in the clock neurons and directly interacts with tim to reset the molecular clock . although more detailed studies are required for an exhaustive understanding of the complete mechanism , including unknown light - input pathways , the relentless efforts of many researchers in our field have been steadily revealing new details on how the drosophila light - input systems entrain the neuronal clock network ( fig .  2 ) . cry is expressed in many clock neurons , which transmit light information to cry - negative clock neurons . histamine , dopamine , and serotonin have been suggested as neurotransmitters that convey light inputs from the compound eyes . the hb eyelets use acetylcholine and histamine as neurotransmitters and directly target the pdf neurons . pdf plays a role in intercellular communication between the pdf neurons and other clock neurons that express the pdf receptor . cry is expressed in many clock neurons , which transmit light information to cry - negative clock neurons . histamine , dopamine , and serotonin have been suggested as neurotransmitters that convey light inputs from the compound eyes . the hb eyelets use acetylcholine and histamine as neurotransmitters and directly target the pdf neurons . pdf plays a role in intercellular communication between the pdf neurons and other clock neurons that express the pdf receptor . crycryptochromecyclic ampcampddconstant darknessdndorsal neuroneeveninghb eyeletshofbauer - buchner eyeletsldlight - darklnlateral neuronmmorningpdfpigment - dispersing factorpdp1par domain protein 1perperiodtimtimeless hofbauer - buchner eyelets pigment - dispersing factor this work was supported by the jsps ( kakenhi 23870021 , 25840121 , and 15h05600 ) and the german research foundation ( dfg ; fo207/103 and sfb1047 , inst 93/7841 ) .
abstractlight is the most important environmental cue to entrain the circadian clock in most animals . in the fruit fly drosophila melanogaster , 
 the light entrainment mechanisms of the clock have been well - studied . 
 the drosophila brain contains approximately 150 neurons that rhythmically express circadian clock genes . 
 these neurons are called  clock neurons  and control behavioral activity rhythms . 
 many clock neurons express the cryptochrome ( cry ) protein , which is sensitive to uv and blue light , and thus enables clock neurons deep in the brain to directly perceive light . 
 in addition to the cry protein , external photoreceptors in the drosophila eyes play an important role in circadian light - input pathways . 
 recent studies have provided new insights into the mechanisms that integrate these light inputs into the circadian network of the brain . in this review 
 , we will summarize the current knowledge on the light entrainment pathways in the drosophila circadian clock .