period

DEVELOPMENTAL BIOLOGY

Embryonic

The per gene product has been demonstrated in a segmental pattern in the midline of the ventral nervous system (CNS). It appears in the optic lobes of the pupal brain, ovaries and testes, where staining is cytoplasmic (Siwicki, 1988 and Saez, 1988).

Spatio-temporal pattern of cells expressing the clock genes period and timeless and the lineages of period expressing neurons in the embryonic CNS of Drosophila

The initial steps towards the generation of cell diversity in the central nervous system of Drosophila take place during early phases of embryonic development when a stereotypic population of neural progenitor cells (neuroblasts and midline precursors) is formed in a precise spatial and temporal pattern, and subsequently expresses a particular sequence of genes. The clarification of the positional, temporal and molecular features of the individual progenitor cells in the nerve cord and brain as well as of their specific types of neuronal and/or glial progeny cells forms an essential basis to understand the mechanisms controlling their development. The present study contributes to this effort by tracing the expression of period and timeless, two genes that encode transcription factors with a key role in the molecular mechanism of the biological clock. Using a combination of genetic markers and immunocytochemistry with antibodies specific for Period and Timeless this study defines the number, location, origin and lineage of period cells in the nerve cord throughout embryogenesis. This study also provides the first description of the expression of timeless in the embryonic central nervous system. A major transformation in the number and types of cells that express period and timeless takes place between embryonic and larval life (Ruiz, 2011).

By using a combination of cell lineage tracing and molecular markers this study uncover the neuroblast or midline precursor origin of the neurons that express per in the nerve cord. In the embryonic brain three populations of cells were found regarding expression of tim and per. One of them comprises 130 cells expressing per, other comprises 160 cells expressing tim and the third is a group of about 20 cells that co-express both genes. Cells expressing either tim, or per, or both exist also in the larval brain, although in much smaller numbers, and part of them initiate expression of per or tim first after some hours of larval life. Considering their lack of expression and their number, the 20 cells that co-express per and tim in the brain at embryonic stage 16 could be the same 20 cells that co-express these genes in the larval brain and function as clock neurons. This is reinforced by their approximate distribution within the protocerebrum and the detection of oscillator features in a similar, probably identical array of per and clock expressing brain neurons at the same stage. Since synaptic activity in central neurons develops in Drosophila during the same phase of development (from late stage 16-17), it is possible that a network of clock neurons becomes functional at this time or shortly thereafter (Ruiz, 2011).

The number of per and tim-expressing cells in the brain increases again during metamorphosis and reaches values in the adult similar to those reported here for the embryo, suggesting that such a large population of cells expressing these clock genes is required twice during life. This is in accordance with the identification, in a careful study of the global profile of gene expression during the life cycle of Drosophila, of a group of genes with expression peaks late in embryogenesis and pupal life (Ruiz, 2011).

Another considerable difference between embryonic and other life stages regards the expression of per and tim in glial cells. Hundreds of glia of different types express per and/or tim in the adult but none or few in the larval brain. This study found that per is not expressed by glial cells in the embryonic CNS, indicating that either per expression is turned on later in some of these cells, or in glial cells born during postembryonic stages. On the contrary, this study found that embryonic glial cells do express tim, although at apparently lower levels than in neurons. The approximate number of cells that was found to co-express repo and tim in the embryo is smaller than the total number of repo-positive cells at this stage, defining two new subpopulations of glial cells. The correlation found in this study between weak anti-Tim fluorescence in Repo-positive cells and strong anti-Tim fluorescence in Repo-negative cells in both the brain and the nerve cord suggests a common regulation of the expression of both genes within CNS cells. In summary, these results indicate that during the few hours from late stage 16 to shortly after larval eclosion there is a significant change in the transcriptional profile of CNS cells, turning off the expression of per and tim in neurons and the expression of tim in glia or eliminating these cells through programmed cell death (Ruiz, 2011).

In the nerve cord the per and tim neurons defined in this stdy are located in close vicinity but are generated by many different NBs. In each NB lineage, only one, or very few cells turn on the expression of either tim or per. Thus, expression of these genes is not inherited by lineage, but needs to be induced in individual postmitotic progeny cells. As suggested by the occurrence of only one positive cell in some of the lineages this is also true for sibling cells derived from individual ganglion mother cells. Differential cell fate among such sibling cells may result form asymmetric division induced by Notch signaling as previously demonstrated (Ruiz, 2011).

A clear, although not complete segregation of per and tim cells was found along the CNS. Within the nerve cord some of the most posterior per cells were located in the engrailed domain (the posterior compartment of the segment) and almost all tim cells were anterior to the engrailed domain. The same relationship was found in the brain, although without the clear segmental pattern of the nerve cord (i.e. lacking correlation with the small engrailed domains of the brain). Instead, the distribution of per and tim cells in the brain can roughly be described as two large fields with abundant overlap, with the majority of the tim cells located in the more anterior field i.e. the same relative position with regards to per cells as observed in the nerve cord (Ruiz, 2011).

The best known function of Per and Tim is as transcriptional regulators acting within molecular feedback loops responsible for the timekeeping mechanism of the biological clock. Within this conceptual frame, the 'traditional' function of Per and Tim in the CNS is restricted to 'oscillator cells' or clock neurons, in which the cycling of gene expression, mRNA and protein, as well as the shuttling of Per and Tim between cytoplasm and nucleus has been documented with several methods. Thus, the embryonic expression of per and tim in cells other than the clock neurons, as the glia, interneurons and motorneurons identified in this study in the nerve cord, suggests that these proteins might have other functions, perhaps in neuronal development (Ruiz, 2011).

Larval and Pupal stages

The circadian timekeeping system of Drosophila functions from the first larval instar (L1) onward. The phase of eclosion and of adult activity rhythms can be set by light pulses delivered to animals entering or progressing through L1. period and timeless are rhythmically expressed in several groups of neurons in the larval CNS both in light/dark cycles and in constant dark conditions. There are a total of six to eight per-expressing cells in the nervous system of L3. These cells are identified as neurons. Among the clock gene-expressing neurons there is a subset of the putative pacemaker neurons, the "lateral neurons" (LNs), that have been analyzed mainly in adult flies. Like the adult LNs, the larval ones are also immunoreactive to a peptide called pigment-dispersing hormone . Neurons not cycling Period protein are also identified and consist of three to five clusters by position. The LNs are the only larval cells that maintain a strong cycling in Per protein from L1 onward, throughout metamorphosis and into adulthood. The phase of Per cycling is similar to that found in the adult, with a peak at 23 hours and a trough at 6 to 12 hours. In these LNs, it is Tim (which also cycles) that is found to be both nuclear and cytoplasmic. These are, therefore, the best candidates for being pacemaker neurons responsible for the larval "time memory" (inferred from previous experiments). Putative dendritic trees of LNs are found to be in close proximity to the terminals of the larval optic nerve Bolwig's nerve, possibly receiving photic input from the larval eyes. LNs also express Pigment dispersing hormone (PDH). In addition to the LNs, a subset of the larval dorsal neurons (DNLs) expresses per and tim. These two groups of neurons, identified as "cycling" neurons expressing per are termed Dorsal neurons-1Larval and Dorsal neurons-2Larval (DNL). Intriguingly, two neurons of this DNL group cycle in PER and TIM immunoreactively, almost in antiphase to the other DNLs and to the LNs. These DNLs also express PDH. Thus, the temporal expression of per and tim are regulated differentially in different neurons. Furthermore, the light sensitivity associated with levels of the TIM protein is different from that in the heads of adult Drosophila (Kaneko, 1997).

The Drosophila clock genes period (per) and timeless (tim) have been studied behaviorally and biochemically, but to date there has been no viable culture system for studying the cell biology of the Drosophila clock. In this study, pupal ring glands were cultured while attached to the central nervous system. Rhythms of period gene expression were observed in the prothoracic gland for 4-7 days. A daily rhythm of Per protein can be entrained by light in culture, even when neural activity is blocked by tetrodotoxin. In cultures maintained for a week in constant darkness, a per-luciferase reporter gene reveals circadian rhythms of bioluminescence. As the first circadian culture system from Drosophila, the prothoracic gland provides unique advantages for investigating the interactions between clock genes and cellular physiology (Emery, 1997).

Circadian control of eclosion: Interaction between a central and peripheral clock

Drosophila displays overt circadian rhythms in rest:activity behavior and eclosion. These rhythms have an endogenous period of approximately 24 hr and can adjust or 'entrain' to environmental inputs such as light. Circadian rhythms depend upon a functioning molecular clock that includes the core clock genes period and timeless. Although a clock in the lateral neurons (LNs) of the brain controls rest:activity rhythms, the cellular basis of eclosion rhythms is less well understood. The LN clock has been shown to be insufficient to drive eclosion rhythms. The prothoracic gland (PG), a tissue required for fly development, contains a functional clock at the time of eclosion. This clock is required for normal eclosion rhythms. However, both the PG clock function and eclosion rhythms require the presence of LNs. In addition, it is demonstrated that pigment-dispersing factor (PDF), a neuropeptide secreted from LNs, is necessary for the PG clock and eclosion rhythms. Unlike other clocks in the fly periphery, the PG is similar to mammalian peripheral oscillators because it depends upon input, including PDF, from central pacemaker cells. This is the first report of a peripheral clock necessary for a circadian event (Myers, 2003).

Lateral neurons (LNs) are considered the central circadian pacemaker. These LNs are required for rest:activity rhythms and are most likely required for controlling the timing of eclosion (adult emergence from the pupal case). Eclosion is considered to be under the control of the circadian system because its timing is gated such that it is restricted to the hours surrounding dawn each day, even for flies that are developmentally ready hours earlier. Because eclosion occurs once in a single fly's lifetime, the multiple events that occur over several days within a population are considered a rhythm. This gating is absent in flies mutant for the clock gene period (per) or timeless (tim) and is also absent in disconnected flies that lack LNs (Myers, 2003).

Neuronal clocks (including those in the LNs) are sufficient to drive rest:activity rhythms, but perhaps not eclosion rhythms. It was of interest to determine whether these clocks would be sufficient for eclosion gating by using fly lines in which the molecular clock functions in neurons only. The gal4-UAS binary system was used to express Tim only in neurons by using the elav^c155-gal4 driver and a UAS-tim transgene in an arrhythmic tim null background. This manipulation does not rescue eclosion rhythms as it does locomotor rhythms in adults (Myers, 2003).

Another circadian mutant line, one that also displays rhythmic rest:activity behavior, was also arrhythmic for eclosion. This fly line, cry^b, is mutant for a circadian photoreceptor, Cryptochrome (Cry). Cry^b affects the sensitivity of the LN molecular clock to pulses of light but does not affect its endogenous rhythm. Emerging evidence now suggests that Cry is a central clock gene in peripheral clocks. Besides demonstrating that the LN clock is insufficient to drive eclosion rhythms, the eclosion data suggest that Cry may also be required within a relevant peripheral clock mechanism (Myers, 2003).

From these data, it is concluded that a LN molecular clock, which can drive rest:activity rhythms, is not sufficient to restore eclosion rhythms. A peripheral clock, then, is necessary to maintain eclosion rhythms, even in the presence of a functioning LN clock (Myers, 2003).

The prothoracic gland (PG), is part of an endocrine structure known as the ring gland. This structure surrounds the heart just anterior to the cardia and is present during all stages of life except adulthood. The PG secretes ecdysteroids, which when converted to the active form of 20-hydroxyecdysone bind to their nuclear hormone receptor (ecdysone receptor, EcR) and affect gene transcription. These alterations in gene expression cause tissue metamorphosis over the course of development. Levels of ecdysteroids peak at the beginning of larval and pupal stages but, during the two days just prior to eclosion, drop to nearly undetectable levels. In Manduca sexta (tobacco hornworm), this drop in ecdysteroid titer is necessary for eclosion to proceed normally (Myers, 2003).

Previous studies suggest that there is some circadian control over PG function. Notably, ecdysone titers cycle in a circadian fashion in Rhodnius prolixus. In Drosophila, Per is present and oscillates in central brain-PG cultures taken from white prepupae. It is not known, however, whether both Per and Tim oscillate in this tissue under free-running conditions (in constant darkness and temperature) immediately preceding eclosion. Presumably, these conditions should be met before one considers the PG a true clock tissue and an appropriate candidate clock tissue involved in the control of eclosion gating (Myers, 2003).

Clock function was assessed in the PG by quantitating Per and Tim levels over the course of the day in intact pupae. Both Per and Tim levels change over the course of the LD cycle, with a significant difference between the peak (late night) and the trough (late day) values. The peak of Per expression is slightly later when compared to Tim. Both expression profiles, though, match those seen in the LNs. Per and Tim still show significant differences in daily expression in constant darkness (DD), although the difference between the peak and trough values is, as in other fly tissues, smaller. Per expression in DD, although significantly different throughout the course of the day, does not match its LD profile. This effect is similar to the delay in Per degradation in DD versus LD seen in head extracts, although the delay is more pronounced within the PG (Myers, 2003).

It is concluded that there is a molecular clock inside cells of the PG at the time when pupae are developmentally ready for imminent eclosion. Because differences between peak and trough levels of Per are smaller, the profiles of daily Tim expression in DD were used to report PG clock function (or clock synchrony within the population) in subsequent experiments (Myers, 2003).

Does circadian output of the PG clock gate eclosion? Because lesions of the PG are lethal, the necessity of the PG clock was established by assaying eclosion rhythms in fly lines in which genetic manipulation had disrupted the clock inside the PG. To disrupt the molecular clock inside the PG, Tim (UAS-tim^2-1) was expressed at all times of day specifically in PG cells by using the Mai⁶⁰-gal4 driver. Expressing Tim in this manner disrupts eclosion rhythms. Peaks are present in the UAS-tim^2-1; Mai⁶⁰-gal4 eclosion profile, but a rhythm and gating are absent. In this line, adult locomotor behavior remains rhythmic (80.3% of adults had a significant circadian period to their rest:activity behavior in constant light. Consistent with a role for the PG in eclosion gating, it was found that there are no significant differences in the daily expression of Tim in the PG of the arrhythmic cry^b flies (Myers, 2003).

Although the clock inside the LNs is not sufficient for eclosion rhythms, the cells still appear to be required for eclosion gating. It is likely that the LNs could control PG clocks, much like the suprachiasmatic nucleus (SCN) of the hypothalamus is believed to drive peripheral clocks in mammals. Anatomical evidence does suggest that the LN axons (containing pigment-dispersing factor, PDF) indirectly innervate the PG (Myers, 2003).

To determine whether LNs are necessary, tests were carried out for the presence of both eclosion rhythms, and a PG clock in a fly line that lacked LN cells. A fly line was used in which LNs were ablated without lesioning many other neurons. This is a more focused disruption than that caused by the disconnected (disco) mutation. LN cells were ablated by driving a cell death gene, head-involution defective (hid), with pdf-gal4. These pdf-gal4 X UAS-hid flies are also arrhythmic for rest:activity behavior as adults. In the PG of pdf-gal4 × UAS-hid and in disco⁰¹ flies, there is no longer any significant difference in Tim expression over the course of the day. Although eclosion gating in the pdf-gal4 X UAS-hid line appears to be present during the first two days in constant darkness, rhythms do not persist. It is speculated that the flies emerging during the first two days of this assay are gated because their exposure to the entraining LD cycle persists until relatively late in development. This may result in limited and short-lived synchrony through unknown mechanisms. Clearly, though, LNs are required to maintain eclosion rhythms (Myers, 2003).

To determine whether a functioning molecular clock inside the LNs is necessary for their influence on the peripheral clock and on eclosion, the effect of disrupting this clock was examined. Tim (UAS-tim^3-1) was expressed in neurons of wild-type flies at all times of day by using an elav^c155-gal4 driver, all in a wild-type background. This perturbation of Tim expression is sufficient to disrupt locomotor rhythms in the adult fly. Although the molecular clock in the LNs is disrupted, there are still daily changes of Tim in the PG, and eclosion remains rhythmic (Myers, 2003).

It is possible that the role of the LNs is to provide, via PDF, a signal to the PG clock. In the LN axons that project to the dorsal brain, PDF expression cycles, with PDF release believed to occur during subjective night. The rhythmicity of PDF release and eclosion correlate well. For instance, in per⁰ and tim⁰ flies, PDF is no longer released in a rhythmic fashion from these dorsal, LN projections, and eclosion is arrhythmic as well. To determine whether PDF is part of the LN output pathway to the PG or involved in eclosion gating, both PG clock function and eclosion were assayed in flies with no functional PDF protein (pdf⁰¹). These flies are arrhythmic for locomotor behavior as well as for eclosion. In the PG, Tim levels were significantly different over the course of the day in the presence of an LD cycle, but neither Per in LD nor Tim in DD showed significant differences in their daily expression profile. These data indicate that the endogenous clock inside the PG cannot function (or entrain) without PDF in the fly. PDF overexpression and anatomical studies suggest that the LNs are the best candidates for a source of PDF relevant to eclosion behavior and the PG clock. PDF, though, is also expressed in a small subset of neurons in the central brain (LN_vs and two to four tritocerebral cells) and in four to six abdominal cells. In addition, there may be other inputs to the PG. For instance, the Tim cycling seen in LD conditions is most likely due to an acute light response, suggesting the presence of photic input to the PG. Supporting this hypothesis are data from R. proxilus, whose PG clock (its presence is inferred from cyclic release of ecdysteroids in culture) is also directly photosensitive (Myers, 2003 and references therein).

Thus, the PG molecular clock is under control of the central clock. This is unlike other Drosophila peripheral clocks, such as those in the antenna, which can operate autonomously. It is also unexpected when one considers data from studies in which PG clock function is directly or indirectly assayed in culture and determined to be independent of the central brain (or independent of any tetrodotoxin-dependent output from the brain, in the case of Drosophila). The PG clock, in fact, is more similar to peripheral clocks in the mammalian circadian system. In addition, it is important to note that edysteroid synthesis (and presumably PG clock function) in the cockroach Periplaneta americana also depends upon input from the central brain. Perhaps, then, the mechanisms controlling peripheral clock function are not the same in different tissues in the same insect or in the same tissue in different insects (Myers, 2003).

From these data, a model for the circadian gating of eclosion emerges. The LNs secrete PDF into the anterior protocerebrum, where it acts on neurons that innervate the PG. Appropriate regulation of PDF levels is critical. Just as the absence of PDF disrupts eclosion, so can excess levels of it in the dorsal brain. However, the mechanisms underlying this PDF overexpression phenotype are unknown (Myers, 2003).

This study introduces a new set of clock cells necessary for the regulation of eclosion rhythms. It is not known, however, whether the LN and PG clocks together are sufficient to control eclosion gating. The current hypothesis holds that, in the ventral nervous system of Drosophila, cells containing Crustacean Cardioactive Peptide (see Cardioacceleratory peptide) are the most likely sites for control of eclosion gating, as indicated by two lines of evidence. The first is that CCAP, in response to eclosion hormone (EH), can activate ecdysis within minutes in Manduca sexta. However, the circadian gate of eclosion cannot be regulated solely by EH or CCAP because flies without either of these sets of neurons still eclose within a circadian gate. The second line of evidence is that some CCAP cells in Drosophila also express LARK, an RNA binding protein that regulates eclosion rhythms. LARK oscillates in a clock-dependent manner inside these cells. Interestingly, PG cells also contain LARK, although there have been no reports of cycling LARK outside of the CCAP cells. The exact mechanism for how and where development and circadian inputs are coordinated to control eclosion gating are still important and open questions (Myers, 2003 and references therein).

Adult

PER protein is found in a wide variety of tissues: in very specific locations in the adult brain, in ventral ganglia, the photorecetor cells of the eyes, reproductive tissue, and the outer layers of epithelial cells in the gut (Saez, 1988 and Siwicki, 1988).

Double-labeling experiments in which neural cells are labelled with anti-ELAV antibody, reveal that many PER positive cells are glial. Thus, cells located at the margins of cortex and neuropil in the optic lobes and the central brain, within the lamina and central brain neuropil, and in the inner chiasm do not express ELAV but do express per (Ewer, 1992).

Genetic analysis reveals that the possible site of the photoperiod response is in a small cluster of per espressing cells in the central brain. These cells, called lateral neurons (LNs) also express the glass gene. The same cells are immunoreactive to pigment dispersing hormone. Release of this hormone is associated with migration of retinal screening pigments during light adaptation in some crustacea (Vosshall, 1995).

Pigment-dispersing hormones (PDH) are a family of octadecapeptides that have been isolated from several crustacean species. An antiserum against the crustacean PDH was used to identify PDH-immunoreactive neurons in the developing nervous systems of wild type Drosophila and the brain mutant disconnected. Particular attention was paid to a group of PDH-immunoreactive neurons at the anterior margin of the medulla, known as the pigment-dispersing factor-containing neurons close to the medulla (PDFMe neurons). This group of neurons seem to be involved in the control of adult circadian rhythmicity. In adults, this group consists of four to six neurons with large somata (large PDFMe neurons) and four neurons with small somata (small PDFMe neurons). Both the small and the large PDFMe neurons are identical to the ventral lateral neurons, a group of neurons containing the Period protein. Both subgroups were usually absent in adults of behaviorally arrhythmic disconnected(disco) mutants. The compound eyes of these mutants are usually disconnected from the optic lobes due to a severe defect in optic lobe development. disco mutants, as a result, have either very tiny rudiments of optic lobes if no connections are made at all (unonnected phenotype) or, if some connections are established (connected phenotype), the optic lobes have an almost normal size but are grossly disorganized. disco mutants are behaviorally arrhythmic, and the lateral neurons are generally absent in adults. In the wild type, PDH immunoreactivity is seen first in the small PDFMe neurons of 4 hour old first-instar larvae. The small PDFMe neurons persist unchanged into adulthood, whereas the large ones seem to develop halfway through metamorphosis. Beside the PDFMe neurons, three other clusters of PDH-immunoreactive neurons stain in the developing nervous systems of Drosophila and are described in detail. Two of them are located in the brain, and the third is located in the abdominal neuromeres of the thoracic nervous system. In the mutant disconnected, the larval and the adult set of PDFMe neurons are absent. The other clusters of PDH-immunoreactive neurons seemed to develop normally. The present results are consistent with the hypothesis that the PDFMe neurons are circadian pacemaker neurons that may control rhythmic processes in larvae, pupae, and adults (Helfrich-Forster, 1997).

Another study shows that the arborizations of a subclass of lateral neuron pigment dispersing cells are always in close proximity to PER-containing glial cells. In the medulla, an optic lobe neuropile, the varicose network of PDH-immunoreactive fibers is covered by a layer of PER-containing glial cells (Helfrich-Förster, 1995).

Transgenic strains of flies were generated carrying a luciferase cDNA fused to the promoter region of the per gene. Because of the rapid turnover of luciferase, the temporal gene expression could be studied in a sensitive, non-invasive assay using whole-fly luminescence of individual living insects. A primary luminescence peak occurs four hours into the night. Luciferase detected a secondary low amplitude luminescence peak in many flies occuring after the onset of light. This secondary peak does not seem to reflect a simple response to lights-on as secondary maxima can be seen in many individuals transferred to constant darkness (Brandes, 1996).

Per and Tim are both expressed in the Malpighian tubules of Drosophila. Per and Tim show a clear rhythm in level in both intact and decapitated flies, indicating that an endogenous rhythm in protein levels is independent of the brain. Per and Tim are present in the nuclei of all secretory cells toward the end of the dark period, but the proteins are not found toward the end of the light period. The cycling of Per and Tim in tubules persists after decapitation; when decapitated flies are exposed to a 12 hour shift in photoperiod, the Per and Tim rhythms in their tubules is also reset by 12 hours, just as in the intact fly. Thus, tubules exhibit free-running cycling of clock proteins, the rhythms of which can be reset by light, in the absence of both brain and eyes. What is the function of the Malpighian tubule clock? Adult flies have behavioral rhythms that may require periods of higher excretory activity (associated with increased metabolic rates). Fluid excretion in fly tubules is driven by transmembrane ion movements. Ion pumps, such as vacuolar ATPase, operate in this tissue, so a circadian clock in the tubules may affect the rates of ion movement across the cell membranes - as in other circadian systems (Giebultowicz, 1997).

Transgenic Drosophila that expressed either luciferase or green fluorescent protein driven from the promoter of the clock gene period was used to monitor the circadian clock in explanted head, thorax, and abdominal tissues. The tissues (including sensory bristles in the leg and wing) show rhythmic bioluminescence; the rhythms can be reset by light. The photoreceptive properties of the explanted tissues indicate that unidentified photoreceptors are likely to contribute to photic signal transduction to the clock. A particularly interesting feature of the GFP expression pattern is its labeling of chemosensory cells. This pattern specifically identifies structures at the base of chemoreceptor bristles in the proboscis, antennae, anterior wing margins, and legs. per-driven bioluminescence in these tissues is rhythmic, showing that there is a functional clock in these cells. Although the evidence for clock control of sensory thresholds in the fly is still circumstantial, the presence of independent clocks indicates a central role for per-dependent clock functions in tissues outside the head (Plautz, 1997a).

To determine the in vivo regulatory pattern of the clock gene period (per), transgenic Drosophila carrying a luciferase cDNA fused to the promoter region of per were examined. Noninvasive, high time-resolution experiments were carried out, allowing high-throughput monitoring of circadian bioluminescence rhythms in individual living adults for several days. This immediately solved several problems (resulting directly from individual asynchrony within a population) that have accompanied previous biochemical experiments in which groups of animals were sacrificed at each time point. Furthermore, numerical analysis methods were developed for automatically determining rhythmicity associated with bioluminescence records from single flies. This has revealed some features of per gene transcription that were previously unappreciated and provides a general strategy for the analysis of rhythmic time series in the study of molecular rhythms (Plautz, 1997b).

Period is involved in a molecular feedback loop in which Per inhibits the transcription of its own mRNA. This feedback causes the Per protein to cycle in a circadian manner, and this cycling in specific regions of the brain (the presumed location of the central pacemaker) is responsible for the rhythmicity of locomotor activity and possibly eclosion. Per has also been detected in several nonneural tissues in the abdomen, but whether Per exhibits free-running and light-sensitive cycles in any of these tissues is not known. A study was performed of the spatial and temporal distribution of a Per-reporter expressed in transgenic flies carrying a per-lacZ construct, which is able to cycle in per-expressing brain cells. This Per-reporter fusion protein cycles in the Malpighian tubules, showing first cytoplasmic accumulation, which is then followed by translocation of the signal into the nucleus. To test whether this rhythm is controlled by the brain, flies were decapitated and assayed for 3 days after decapitation. Expression patterns of the Per-reporter in decapitated flies are nearly identical to those in intact flies reared in normal light-dark cycles, reversed light-dark cycles (phase shifted), and constant darkness. These results suggest that the Malpighian tubules contain a circadian pacemaker that functions independent of the brain (Hege, 1997).

Drosophila mutants for the DCO gene, which encodes the major catalytic subunit of cAMP-dependent protein kinase (PKA), display arrhythmic locomotor activity, strongly suggesting a role for PKA in the circadian timing system. This arrhythmicity might result from a requirement for PKA activity in photic resetting pathways, the timekeeping mechanism itself, or downstream effector pathways controlling overt behavioral rhythms. To address these possibilities, the protein and mRNA products from the period gene were examined in PKA-deficient flies. PER protein and mRNA undergo daily cycles in the heads and bodies of DCO mutants, indistinguishable from those observed in wild-type controls. These results indicate that PKA deficiencies affect the proper functioning of elements downstream of the Drosophila timekeeping mechanism. The requirement for PKA in the manifestation of rhythmic activity was preferentially greater in the absence of environmental cycles. However, PKA does not appear to play a universal role in output functions because the clock-controlled eclosion rhythm is normal in DCO mutants. These results suggest that PKA plays a critical role in the flow of temporal information from circadian pacemaker cells to selective behaviors (Majercak, 1997).

Sequential nuclear accumulation of the clock proteins Period and Timeless in the pacemaker neurons

Antisera against the circadian clock proteins Period (Per) and Timeless (Tim) were used to construct a detailed time course of Per and Tim expression and subcellular localization in a subset of the ventrolateral neurons (vLNs) in the Drosophila accessory medulla (AMe). These neurons, which express pigment-dispersing factor, play a central role in the control of behavioral rhythms. The data reveal several unexpected features of the circadian clock in Drosophila: (1) Tim but not Per is restricted to the cytoplasm of vLNs throughout most of the early night; (2) the timing of Tim and Per nuclear accumulation is substantially different; (3) the two subsets of vLNs, the large and small vLNs, have a similar timing of Per nuclear accumulation but differ by 3-4 hr in the phase of Tim nuclear accumulation. These aspects of Per and Tim expression were not predicted by the current mechanistic model of the circadian clock in Drosophila and are inconsistent with the hypothesis that Per and Tim function as obligate heterodimers. The differing profiles of Tim and Per nuclear accumulation suggest that Per and Tim have distinct functions in the nuclei of vLNs (Shafer, 2002).

One concern of an immunocytochemical study is that differences in staining intensity for two epitopes may reflect a disparity in antibody affinity rather than differences in epitope abundance. Attempts were made to allay this concern by using two to three different Per and Tim antibodies. Similar intensities and patterns of staining are seen for all of the antibodies used in this study, suggesting that differential antibody affinity is not responsible for the differences in staining observed for these two proteins. The best estimate for the relative abundance of Per and Tim has come from the analysis of whole-head extracts; the Tim/Per ratio is ~2:1 at ZT 16 and 1:1 by ZT 23. A variation of this magnitude in the vLNs would be consistent with the relative intensities of staining observed in this study. Given this transient disparity between Tim and Per abundance, the persistence of strong cytoplasmic Tim signals (at times when Per is predominantly nuclear) must not be considered as evidence that these proteins enter the nucleus sequentially. Rather, it is the differing profiles of Tim and Per immunoreactivity in the nuclei of the vLNs on which this conclusion is based (Shafer, 2002).

Several lines of evidence had suggested that Per and Tim are transported into nuclei as a dimeric complex. Biochemical assays in fly-head extracts show that Per and Tim are present as heterodimers throughout much of the night, but the data did not exclude the fact that a fraction of Per and Tim are monomeric or complexed with other proteins. Importantly, it is also uncertain what fraction of the Per/Tim heterodimers are present in vivo and what fraction form in vitro during extract preparation and analysis. Furthermore, these extracts reflect thousands of cells of many different types, including all of the photoreceptors from the compound eye. These contribute the majority of the Per and Tim to the homogenate, effectively swamping out the contribution of the vLNs. There is therefore no reason to expect that the biochemistry of the vLNs will be identical to what has been observed in whole-head extracts. In flies, little or no nuclear Per accumulation is seen without Tim, but this observation is likely attributable to Per instability and consequent low Per levels in mutants that lack Tim. The coexpression of Per and Tim has been shown to be required for the nuclear transport of both proteins in cultured S2 cells. Although these cells have not been shown to express Per and Tim rhythmically, this codependence fits well with the notion that Per and Tim enter the nucleus as a heterodimeric complex. Based on these considerations, it is expected that a time course study would reveal the simultaneous nuclear accumulation of Per and Tim in the vLNs. Instead, the data show that Per appears in nuclei at least 3 hr before Tim, indicating that most, and perhaps all, nuclear Per is not complexed with Tim at these times (Shafer, 2002).

In the large vLNs, Per is already nuclear as well as cytoplasmic when it is first detectable at ZT 16. In the large vLNs, nuclear Per is first detected at times that correlate well with the onset of repression in head extracts. This begins at approximately ZT 15-17 by biochemical criteria. The discrepancy between the timing of Per nuclear accumulation in the small cells and the onset of transcriptional repression in whole-head extracts might reflect differences between the cells and tissues that contribute to the biochemical data (Shafer, 2002).

In contrast to Per, Tim is not nuclear until after ZT 18. This difference with Per is especially prominent in the small cells, where Tim remains cytoplasmic until ZT 22. In both cell types, Tim is degraded in the early morning and is therefore nuclear for only a short time. In other words, Tim becomes nuclear well after transcriptional repression is thought to begin and is degraded well before the next cycle of transcription begins anew (Shafer, 2002).

A central feature of the molecular model of the Drosophila clock is that Per and Tim function as obligate heterodimers in their nuclear transport as well as in the repression of Clock/Cycle-based transcription. However, the different times of nuclear accumulation suggest that Per and Tim are transported independently to nuclei. The lack of high-resolution data for the Doubletime kinase makes it difficult to assess whether the Doubletime nuclear accumulation pattern correlates better with that of Per or of Tim. Importantly, these results do not preclude a role for Tim in the nuclear entry of Per. For example Tim could act catalytically in the cytoplasm to potentiate some required Per phosphorylation event. Moreover, Per and Tim could still enter nuclei as Per/Tim heterodimers, but this would require the subsequent export of Tim to the cytoplasm to explain the apparent absence of nuclear Tim during the middle of the night (Shafer, 2002).

In biochemical assays, Per and Tim are both capable of blocking per and tim transcription in the absence of the other, suggesting that repression does not require the Per/Tim heterodimer. The data indicate that little or no Tim is present in the nuclei of vLNs during times associated with repression in whole-head extracts, suggesting that Per might influence transcription independently of Tim. It has been suggested that Per is still capable of repressing transcription after a mutant form of Tim (TimUL) is degraded by light exposure. It has been concluded that Per can repress transcription in the absence of Tim and it has been suggested that Tim-independent repression by Per normally occurs after dawn. However, there was no reason to challenge the notion that the Per/Tim heterodimer is the agent of nuclear entry and the initiator of repression. The current data indicate that the Tim-independent repression by Per also applies to the onset of repression in the middle of the night. Tim may therefore play no direct role in the feedback repression of Per and Tim transcription (Shafer, 2002).

The persistence of Tim oscillations in the small vLNs but the absence of oscillations in the large vLNs under DD conditions has been noted. This observation has been confirmed, but these data extend this result in several important ways. Per was assayed as well as Tim and these two proteins were followed throughout the first DD cycle. This provides a finer temporal resolution and leads to a surprising conclusion: that the molecular oscillation in the large vLNs appears to arrest during the first subjective day. Based on the patterns of Per and Tim, these cells stop in a state that corresponds to ZT 8-10 under LD conditions (Shafer, 2002).

The individual features of Per and Tim distribution in the large and small vLNs can be added to a growing list of differences between these two classes of PDF-expressing neurons. Cell-specific differences in Per and Tim nuclear accumulation underscore the importance of studying the brain clock in situ and may presage an even more profound heterogeneity among other clock-containing neurons within the brain. This view also reflects the fact that the PDF-positive vLNs represent a minority of clock gene-expressing cells in the central brain. One might therefore expect that the circadian system will vary to an even greater degree among other clock neurons. Such diversity might offer clues as to how different groups of clock neurons interact to create the temporal complexity of behavioral rhythms (Shafer, 2002).

Neural activity and the circadian pacemaker

The ventral lateral neurons (LNvs) of the Drosophila brain that express the period and pigment dispersing factor genes play a major role in the control of circadian activity rhythms. A new P-gal4 enhancer trap line is described that is mostly expressed in the LNvs This P-gal4 line was used to ablate the LNvs by using the pro-apoptosis gene bax, to stop Per protein oscillations by overexpressing per and to block synaptic transmission with the tetanus toxin light chain (TeTxLC). Genetic ablation of these clock cells leads to the loss of robust 24-h activity rhythms and reveals a phase advance in light-dark conditions as well as a weak short-period rhythm in constant darkness. This behavioral phenotype is similar to that described for disconnected mutants, in which the majority of the individuals have a reduced number of dorsally projecting lateral neurons which, however, fail to express Per. In both LNv-ablated and disco mutant flies, Per cycles in the so-called dorsal neurons (DNs) of the superior protocerebrum, suggesting that the weak short-period rhythm could stem from these PDF-negative cells. The overexpression of per in LNs suppresses Per protein oscillations and leads to the disruption of both activity and eclosion rhythms, indicating that Per cycling in these cells is required for both of these rhythmic behaviors. Interestingly, flies overexpressing Per in the LNs do not show any weak short-period rhythms, although Per cycles in at least a fraction of the DNs, suggesting a dominant role of the LNs on the behavioral rhythms. Expression of TeTxLC in the LNvs does not impair activity rhythms, which indicates that the PDF-expressing neurons do not use synaptobrevin-dependent transmission to control these rhythms (Blanchardon, 2001).

Electrical silencing of Drosophila circadian pacemaker neurons through targeted expression of K⁺ channels causes severe deficits in free-running circadian locomotor rhythmicity in complete darkness. Pacemaker electrical silencing also stops the free-running oscillation of Period (Per) and Timeless (Tim) proteins that constitutes the core of the cell-autonomous molecular clock. In contrast, electrical silencing fails to abolish Per and Tim oscillation in light-dark cycles, although it does impair rhythmic behavior. On the basis of these findings, it is proposed that electrical activity is an essential element of the free-running (occuring in complete darkness) molecular clock of pacemaker neurons along with the transcription factors and regulatory enzymes that have been previously identified as required for clock function (Nitabach, 2002).

Rhythmic cycles of clock gene expression and subcellular localization are found in a set of pacemaker neurons that control circadian rhythms of locomotor activity in Drosophila. These pacemaker cells receive light inputs via neuronal signals originating in the eyes and by cell-autonomous expression of Cryptochrome (Cry), a blue-light photoreceptor protein. Either of these signals is sufficient to entrain behavioral rhythms. Some pacemaker cells produce a neuropeptide, Pigment dispersing factor (Pdf), which is likely to function as a circadian output signal. An important area of circadian rhythm research is the relationship between the function of the molecular clock in pacemaker neurons and the central physiological property that distinguishes neurons from other cells -- regulated membrane electrical activity. Synaptic inputs are transduced through transient membrane currents, and downstream outputs are driven by firing action potentials. Activity-dependent free-running circadian rhythms in intracellular Ca²⁺ levels and NMDA-evoked Ca²⁺ currents have been observed in pacemaker neurons of the mammalian suprachiasmatic nucleus (SCN). Clock-dependent circadian rhythms in ion channel mRNA abundance occur in Drosophila heads (Claridge-Chang, 2001; McDonald, 2001). Free-running circadian rhythms in membrane conductance and delayed-rectifier K⁺ channel current have been observed in pacemaker neurons of the molluscan retina. After reversible blockade of action potential firing in cultured SCN neurons by tetrodotoxin treatment, circadian firing rhythms reemerge with unaltered phases, suggesting that neuronal activity is not required for cell-autonomous molecular oscillations in these cells. Nevertheless, it remains a mystery how synaptic inputs to pacemaker neurons entrain the intracellular molecular clock, how the molecular clock controls the electrical activity of pacemakers, and whether electrical activity of pacemaker neurons plays a role in vivo in oscillations of the molecular clock itself (Nitabach, 2002 and references therein).

To begin to address these questions, a reverse genetic approach has been adopted in which either of two distinct K⁺ channels is expressed in the pacemaker neurons of transgenic flies. It has been found that K⁺ channel-mediated electrical silencing of pacemaker neurons leads to severe deficits in circadian locomotor rhythms. This result is not unexpected—if pacemakers cannot communicate with downstream target neurons, there is no way for them to drive circadian behavioral rhythms. It is also found that pacemaker electrical silencing causes rundown and the ultimate stopping of the free-running cell-autonomous intracellular molecular clock (refering to the continued function of the clock in complete darkness), thus indicating an essential role for electrical activity in the cycling of the clock in the absence of environmental cues. However, electrical activity is not required for running of the pacemaker molecular clock when driven by light-dark cycles, indicating a specific function for electrical activity in the function of the free-running clock. On the basis of these results, it is proposed that pacemaker cell electrical activity acts as part of a feedback loop that is necessary for the cycling of the free-running clock (Nitabach, 2002).

Thus, electrical activity is a functional component of the molecular clock of the LN_V pacemaker neurons. Eliminating light-driven inputs to the LN_Vs, while preventing entrainment and causing intercell clock asynchrony, does not prevent the cycling of each LN_V's cell-autonomous molecular clock. Interference with chemical synapse-mediated outputs of Drosophila pacemaker neurons by expression of tetanus toxin does not prevent intracellular molecular clock cycling. In addition, after reversible blockade of action potential firing in cultured mammalian pacemaker neurons, circadian firing rhythms reemerge with unaltered phases. Thus, electrical-activity of the LN_Vs, while involved in entrainment and circadian behavior, would not be considered likely to play a role in intracellular molecular oscillations. It was thus surprising to observe that electrical silencing of the LN_Vs stops their free-running molecular clock. On the basis of this finding, it is proposed that electrical activity is a necessary component of the cell-autonomous feedback loops in the LN_V molecular clock, along with the essential transcription factors and regulatory enzymes that have been previously identified (Nitabach, 2002 and references therein).

In order to address the role of electrical activity of the pacemaker neurons in circadian rhythms, a method for neuronal electrical silencing was employed based upon UAS/GAL4-mediated targeted expression of either of two distinct K⁺ channels. Such manipulations of membrane properties have been shown to be highly effective at shunting synaptic inputs and silencing activity, both in mammalian and Drosophila excitable cells. While it would be desirable to directly measure in vivo the effects of K⁺ channel expression on pacemaker membrane properties, given their location deep within the brain this will require the refinement of existing techniques (Nitabach, 2002).

Two approaches were taken to confirm that effects of K⁺ channel expression in the pacemaker neurons result from electrical silencing. (1) The developmental and behavioral effects of panneuronal expression of dORKdelta-C (see Open rectifier K⁺ channel 1 ), the Kir2.1 (mammalian( inward rectifier, and the 'EKO'-modified voltage-gated K⁺ channel (a genetically modified Shaker K⁺ channel) were compared. Kir2.1 and EKO have each been demonstrated to silence electrical activity in Drosophila neurons and other excitable cells. Panneuronal expression of either dORKdelta-C or Kir2.1 leads to nearly complete lethality, with few larvae hatching from their egg cases and those that do exhibiting extreme sluggishness, as observed with panneuronal expression of multiple copies of EKO. (2) An otherwise-identical nonconducting pore mutant version of dORKdelta (dORKdelta-NC) was generated and this channel was included in all experiments as a negative control. Since dORKdelta-NC-expressing neurons behave in all respects as wild-type, it was concluded that the effects of dORKdelta-C or Kir2.1 are due to an increase in K⁺ conductance at rest -- an alteration in membrane properties known to silence electrical activity both in mammalian and Drosophila neurons (Nitabach, 2002).

One potential difficulty with the use of K⁺ channels for silencing neuronal electrical activity is the possibility that excessive K⁺ efflux may lead to cell death. LN_V pacemaker neurons expressing dORKdelta-C or Kir2.1 were, however, shown to be present in their ordinary number; they appeared healthy, and exhibited their normal dorso-medial projection into the central brain. Projections of dORKdelta-C- or Kir2.1-expressing pacemakers never show the type of beading that is a morphological hallmark of neuronal necrosis and apoptosis. dORKdelta-C- and Kir2.1-expressing LN_V pacemakers continue to synthesize PDF and to transport it down their axonal processes, and dORKdelta-C- and Kir2.1-expressing LN_Vs still exhibit oscillation and nuclear entry of Tim and Per in LD. Thus, pacemaker neurons expressing dORKdelta-C or Kir2.1 are viable and possess a molecular clock capable of oscillating (Nitabach, 2002).

The results establish that electrical activity in the PDF-expressing subset of pacemaker neurons is required for generation of circadian locomotor rhythms. The role of pacemaker electrical activity in free-running circadian molecular oscillations was examined. When dORKdelta-C or Kir2.1 is expressed in the LN_V pacemaker neurons, the LN_V molecular clock runs down and ultimately stops in DD. The rapidity of the rundown correlates with the relative severity of behavioral phenotypes seen in lines C1 and Kir2.1(II). Furthermore, electrical silencing interferes with normal nuclear translocation of Tim in free-running conditions, but not in LD. In contrast, dORKdelta-NC expression has no effect on the normal cycling of LN_V Tim and Per levels in DD, nor on the nuclear translocation of Tim. These results indicate a hitherto unexpected role for pacemaker electrical activity in the cycling of the free-running intracellular molecular clock (Nitabach, 2002).

Blocking pacemaker synaptic output with tetanus toxin, while inducing behavioral arrythmicity, has no effect on molecular oscillation. This suggests that stopping the clock through pacemaker electrical silencing is not mediated by consequent silencing of synaptic outputs. Furthermore, in the complete absence of entraining light-driven inputs, the pacemaker molecular clock continues to oscillate. The results indicate that neuronal electrical activity plays independent roles in pacemaker output signaling and oscillation of the intracellular clock and point to a potentially cell-autonomous function in the free-running LN_V molecular clock (Nitabach, 2002 and references therein).

Pacemaker electrical silencing also causes deficits in circadian behavior in LD -- while wild-type and dORKdelta-NC-expressing flies show an increase in locomotor activity beginning about 2 hr before lights-off, flies expressing Kir2.1 in the LN_Vs increase their activity about 4 hr before lights-off, phenocopying pdf⁰¹ null mutant flies and flies lacking PDF-expressing LN_Vs. This behavioral alteration indicates that electrical silencing still occurs in LD. However, some core elements of the molecular clock still oscillate in abundance and subcellular localization in LD, with high levels of both Tim and Per in the nuclei of Kir2.1-expressing larval LN_V pacemaker neurons at ZT23. While oscillation of Tim levels in LD does not by itself rule out impairment of the clock, the continued oscillation and nuclear translocation of both Tim and Per suggest that the molecular clock continues to function in at least some respects. Continued molecular oscillation in LD also indicates that electrical silencing does not result in gross nonspecific derangement of cellular physiology that prohibits clock function per se. Rather, electrical silencing reveals a specific requirement for electrical activity in the function of the free-running clock and indicates the existence of a light-dependent drive on the molecular clock that can substitute at least partially for electrical activity in LD (Nitabach, 2002).

Cry is a good candidate for a light-dependent drive on the clock that is unimpaired by electrical silencing. Cry has been shown to be involved in cell-autonomously transducing light inputs to the LN_V intracellular clock. It is also possible that the activation of ligand-gated ion channels or G protein-coupled metabotropic receptors via light-dependent synaptic inputs to the LN_Vs might couple directly to the clock without requiring electrical activity. Future experiments will determine whether Cry is required for the light-dependent rescue of Per and Tim cycling from electrical silencing (Nitabach, 2002).

How might membrane electrical activity be coupled to cycling of the molecular clock? Electrical activity in the LN_V pacemaker neurons is required for circadian behavioral rhythms. Electrical activity is also required for cycling of the free-running LN_V intracellular clock, but not for cycling of the light-driven clock. These features of the interaction between neuronal physiology and the molecular clock pose a number of questions for further inquiry (Nitabach, 2002).

What feature of the free-running molecular clock makes it dependent upon electrical activity for continued oscillation? Electrical activity could act as a reinforcing feedback mechanism to keep the clock cycling in the absence of environmental cues. Changes in clock protein levels may be coupled to modulation of membrane electrical activity, which could feed back on clock protein levels. In other words, circadian oscillations in electrical activity could reinforce circadian oscillations in clock protein abundance or function, and vice versa. Light-dark cycles could provide enough of a drive on the clock that reinforcing electrical oscillations are not necessary for clock protein oscillations (Nitabach, 2002).

How does membrane electrical activity affect cycling of clock proteins? A likely candidate for transducing electrical events at the membrane to intracellular processes central to cycling of the clock is calcium entry through voltage-dependent calcium channels. These processes could be mediated by enzymes such as calcium/calmodulin-dependent protein kinases or protein kinase C, or by transcription factors such as CREB, which has already been shown to play a role in Drosophila circadian behavior. Future work is required to determine whether intracellular calcium levels and membrane conductance in the pacemaker neurons oscillate with a circadian rhythm, as has been observed in mammals and snails, and whether such oscillations do indeed feedback on the clock (Nitabach, 2002).

How might clock protein oscillation affect neuronal membrane properties? Ion channels have been demonstrated to be subject to modulation by a diverse set of intracellular signaling pathways. For example, voltage-gated potassium channels are regulated through protein-protein interactions and covalent modification by stably-associated protein tyrosine kinases. Certain inward rectifier potassium channels and voltage-gated calcium channels are modulated by G proteins. It is also possible that clock protein oscillations affect membrane properties by regulating the transcription of ion channel genes or modulatory proteins. Indeed, several Drosophila ion channels, including the Shaker Kv channel, exhibit clock-dependent circadian oscillations. Future work will determine whether and how cycling of the molecular clock causes oscillation of Drosophila pacemaker membrane properties (Nitabach, 2002).

A self-sustaining, light-entrainable circadian oscillator in the Drosophila brain

The circadian clock of Drosophila is able to drive behavioral rhythms for many weeks in continuous darkness (DD). The endogenous rhythm generator is thought to be generated by interlocked molecular feedback loops involving circadian transcriptional and posttranscriptional regulation of several clock genes, including period. However, all attempts to demonstrate sustained rhythms of clock gene expression in DD have failed, making it difficult to link the molecular clock models with the circadian behavioral rhythms. Expression of a novel period-luciferase transgene was restricted to certain clock neurons in the Drosophila brain, permitting the monitoring of reporter gene activity in these cells in real-time. Only a subset of the previously described pacemaker neurons is able to sustain Period protein oscillations after 5 days in constant darkness. In addition, a sustained and autonomous molecular oscillator was identified in a group of clock neurons in the dorsal brain with heretofore unknown function. These 'dorsal neurons' (DNs) can synchronize behavioral rhythms and light input into these cells involves the blue-light photoreceptor cryptochrome. These results suggest that the DNs play a prominent role in controlling locomotor behavior when flies are exposed to natural light-dark cycles. Analysis of similar 'stable mosaic' transgenes should help to reveal the function of the other clock neuronal clusters within the fly brain (Veleri, 2003).

The well characterized LNv's comprise a minority of clock gene-expressing neurons. There is also a more dorsally located group of LNs (LNd), consisting of approximately six cells on each side of the brain, as well as three bilateral groups of clock neurons in the dorsal brain (approximately 15 DN1s, 2 DN2s, and 40 DN3s). All these neurons send projections to the same dorsal brain area to which the s-LNv's project, but not much is known with regard to the function of these cells or whether they contain sustained molecular oscillators. This study finds that although the DN3's are unable to drive behavioral rhythms in DD, Per expression in these dorsal cells—and probably in two other groups of DNs—mediates synchronized locomotion under LD conditions (Veleri, 2003).

This study directs attention toward analyzing the specific functions of the different clock neuron clusters within the fly brain. Although the importance of the s-LNv cells in controlling sustained behavioral rhythms is well established, specific ablation of the 2 LNv groups, or lack of the LNv-specific neuropeptide PDF, does not lead to complete arrhythmicity in free-running conditions. Moreover, expression of a neurotoxin in all (LN plus DN) groups of clock neurons leads to behavioral phenotypes similar to those of flies carrying per⁰¹ and tim⁰¹ loss-of-function mutations; also, disco flies (lacking the LNv and LNd neuronal clusters) still show synchronized behavior under LD cycles. Taken together, these findings point to a contribution of the LNd and DN neuronal clusters in regulating behavioral rhythms (Veleri, 2003).

The neuropeptide pigment-dispersing factor coordinates pacemaker interactions in the Drosophila circadian system

In Drosophila, the neuropeptide pigment-dispersing factor (PDF) is required to maintain behavioral rhythms under constant conditions. To understand how PDF exerts its influence, time-series immunostainings were performed for the Period protein in normal and pdf mutant flies over 9 d of constant conditions. Without pdf, pacemaker neurons that normally express PDF maintained two markers of rhythms: that of Period nuclear translocation and its protein staining intensity. As a group, however, they displayed a gradual dispersion in their phasing of nuclear translocation. A separate group of non-PDF circadian pacemakers also maintained Period nuclear translocation rhythms without pdf but exhibit altered phase and amplitude of Period staining intensity. Therefore, pdf is not required to maintain circadian protein oscillations under constant conditions; however, it is required to coordinate the phase and amplitude of such rhythms among the diverse pacemakers. These observations begin to outline the hierarchy of circadian pacemaker circuitry in the Drosophila brain (Lin, 2004).

Morning and evening peaks of activity rely on different clock neurons of the Drosophila brain

In Drosophila, a 'clock' situated in the brain controls circadian rhythms of locomotor activity. This clock relies on several groups of neurons that express the Period (Per) protein, including the ventral lateral neurons (LN_vs), which express the Pigment-dispersing factor (PDF) neuropeptide, and the PDF-negative dorsal lateral neurons (LN_ds). In normal cycles of day and night, adult flies exhibit morning and evening peaks of activity; however, the contribution of the different clock neurons to the rest-activity pattern remains unknown. Targeted expression of Per was used to restore the clock function of specific subsets of lateral neurons in arrhythmic per⁰ mutant flies. Per expression restricted to the LN_vs only restores the morning activity, whereas expression of PER in both the LN_vs and LN_ds also restores the evening activity. This provides the first neuronal bases for 'morning' and 'evening' oscillators in the Drosophila brain. Furthermore, the LN_vs alone can generate 24 h activity rhythms in constant darkness, indicating that the morning oscillator is sufficient to drive the circadian system (Grima, 2004).

Temperature synchronization of the Drosophila circadian clock

Circadian clocks are synchronized by both light:dark cycles and by temperature fluctuations. Although it has long been known that temperature cycles can robustly entrain Drosophila locomotor rhythms, nothing is known about the molecular mechanisms involved. This study shows that temperature cycles induce synchronized behavioral rhythms and oscillations of the clock proteins Period and Timeless in constant light, a situation that normally leads to molecular and behavioral arrhythmicity. Expression of the Drosophila clock gene period can be entrained by temperature cycles in cultured body parts and isolated brains. The phospholipase C encoded by the norpA gene contributes to thermal entrainment, suggesting that a receptor-coupled transduction cascade signals temperature changes to the circadian clock. The further genetic dissection of temperature-entrainment was initiated, and the novel Drosophila mutation nocte was isolated. nocte mutants are defective in molecular and behavioral entrainment by temperature cycles but synchronizes normally to light:dark cycles. It is concluded that temperature synchronization of the circadian clock is a tissue-autonomous process that is able to override the arrhythmia-inducing effects of constant light. These data suggest that temperature synchronization involves a cell-autonomous signal-transduction cascade from a thermal receptor to the circadian clock. This process includes the function of phospholipase C and the product specified by the novel mutation nocte (Glaser, 2005).

Although it has been known for a long time that temperature can serve as a potent Zeitgeber to entrain circadian rhythms in animals, practically nothing was known about thermal-entrainment mechanisms and, thus, about the genes and molecules involved. This study has revealed that temperature entrainment of clock-protein expression can function at the level of isolated tissues, independent of the antennal thermosensors studied with regard to Drosophila's acute thermal responsiveness described earlier. The situation is therefore similar to that of circadian photoreception in flies: Clock-gene-expressing tissues can be synchronized in the absence of external, image-forming photoreceptors, and this synchronization is probably mediated by the blue-light photoreceptor Cryptochrome. Similarly, these findings suggest the existence of a cell-autonomous thermoreceptor dedicated to temperature entrainment of the circadian clock. Among the candidates for such a receptor are the transient receptor potential vanilloid (TRPV) channels that have been shown to function in thermoreception in mammals and fly larvae (Glaser, 2005).

The nocte mutant was isolated in a novel screen for temperature-entrainment variants; nocte specifically affects synchronization of the circadian clock to this Zeitgeber (namely, temperature cycles). Such mutant flies are drastically impaired in molecular entrainment of Per-LUC reporter-gene rhythms as well as those of native Per and Tim expression. Behavioral rhythms can be entrained by light:dark, but not by temperature cycles, in nocte flies. Moreover, nocte flies do not affect circadian clock function as such because mutant flies are robustly rhythmic in constant darkness. The circadian clock of nocte flies is also properly temperature compensated; their free running period does not change as a function of increasing or decreasing constant ambient temperatures. These findings show that the assumed product of this gene plays a central role in, and is specific for, temperature entrainment (Glaser, 2005).

It has been shown that the norpA gene is involved in the light-entrainment pathway that ends at the brain's clock. The phospholipase C (PLC) encoded by this gene is an essential factor of the canonical photo-transduction cascade within Drosophila's external photoreceptors. Loss-of-function norpA mutations (such as norpA^P24 and norpA^P41, as applied in this study) disrupt this pathway and cause visual blindness -- but they also blind the eyes' contribution to circadian entrainment by light (Glaser, 2005).

In addition, norpA contributes to a temperature-sensitive splicing event at the 3' end of the per gene. Splicing of a per intron is enhanced by relatively cold temperatures, and this enhancement leads to an earlier increase of per mRNA during the daily cycle of per RNA accumulation and decline (Majercak, 1999; Majercak, 2004). Correlated with this early upswing is an advanced behavioral activity peak in cold conditions. Because both phenomena are enhanced by shortened photoperiods and suppressed by long photoperiods, it was suggested that the 3' alternative splicing event serves as a mechanism to adjust the fly's behavior to seasonal changes: More locomotion during the day in the winter, with its short photoperiods, and more behavior in the evening during the summer, to avoid the desiccation effects of midday heat (Glaser, 2005).

But are norpA and the 3'splicing mechanism also important for the more basic features of day-by-day temperature entrainment? The answer, from this analysis, is yes and no: PLC is clearly involved because norpA mutations affect both molecular and behavioral entrainment to temperature cycles. This is not true for the alternative splicing event at per's 3' end: Two types of controls (wild-type and y w) robustly synchronized their behavioral rhythms to temperature cycles in constant light and constitutively expressed roughly equal amounts of the spliced and unspliced versions of per RNA in the same LL and temperature-cycling conditions. Therefore, temporal regulation of the 3' splicing event is not necessary for entrainment to temperature cycles (Glaser, 2005).

The idea is favored that the PLC encoded by norpA has an additional role to its known functions in photo transduction and thermal regulation of splicing. It is possible that a signal-transduction cascade, similar to the visual one operating in the compound eye, is used to transduce the temperature signal to the clock. It is not known whether all clock-gene-expressing tissues also express norpA (although it is notable that norpA is expressed in adult tissues way beyond the external eyes. If not, this could explain why the temperature-entrainment defects in norpA mutants in behavior seem less severe than those of nocte. norpA's function in certain tissues could be replaced by other PLC enzymes encoded by different genes. In this respect, one of the more salient norpA mutant defects that was uncovered, that such flies cannot entrain Per-LUC rhythms to temperature cycles, suggests that the temperature-entrainment pathway involves a receptor-coupled signal-transduction cascade that includes a crucial function for PLC (Glaser, 2005).

Robust temperature-entrained reporter-gene rhythms were observed only in transgenic situations in which two-thirds or the entirety of the Per protein was fused to luciferase. These results suggest that the temperature-entrainment mechanism targets clock proteins (at least Per) and does not rely on transcriptional mechanisms. Interestingly, in Neurospora, temperature entrainment is also mainly regulated at the protein level. This supposition is also supported by the surprisingly robust Per and Tim cyclings observed in fly heads under constant light and temperature cycling conditions. These protein oscillations were severely damped in the temperature-entrainment-defective nocte mutant (Glaser, 2005).

Previous work showed that heat pulses of 37°C can induce stable molecular and behavioral phase shifts when applied during the early night but not during the late night. These heat pulses function at the posttranscriptional level because they result in rapid disappearance of Per and Tim. Nevertheless, responsiveness of the clock to heat pulses seems to be mediated by a different mechanism, when compared with daily entrainment analyzed in the current study, because heat pulses involving elevated temperatures below 37°C (i.e., 31°C and 28°C) did not lead to significant clock-protein degradation, whereas temperature cycles applied in the current study (which were in a physiological range, 17°C to 25°C) did cause fluctuations in protein concentrations (Glaser, 2005).

A surprising finding in this study was that temperature-entrained molecular oscillations of Per-LUC and of endogenous Per and Tim proteins are robust in constant light. It was reported earlier that upon transfer to constant light and temperature, Tim protein is expressed at constitutively low levels -- probably by CRY-mediated light absorption followed by Tim:CRY interaction. Per protein continues to oscillate for about 2.5 days after transfer to LL, after which its expression levels also becomes low and constitutive. In the current experiments, Per and Tim still oscillated after 4 days in LL when temperatures were cycling, and Per-LUC luminescence oscillations continued with robust amplitude for more than 5 days. These results clearly show that temperature cycles override the arrhythmia-inducing effects of constant light and explain why circadian entrainment of behavioral and physiological rhythmicity is observed under these conditions (Glaser, 2005).

Future work will illuminate which molecules mediate temperature entrainment in addition to phospholipase C. Given the drastic and specific effects of the nocte mutation on temperature entrainment, the factor encoded by this gene will almost certainly be revealed to play a central role in this process (Glaser, 2005).

This study has shown that temperature cycles induce molecular rhythms of clock-gene expression, even in the presence of constant light, which normally results in complete molecular and behavioral arrhythmia. Synchronization was observed in isolated peripheral clock tissues and in the brain, demonstrating that the process is tissue autonomous and is responsible for the synchronized locomotor behavior under constant-light and temperature-cycling conditions. The data suggest that the mechanism functions at a posttranscriptional level involving at least the clock protein Per. Phospholipase C is likely to be involved in the signaling mechanism from the thermal receptor to the clock. The novel mutation nocte specifies a factor that is specific for thermal synchronization of the circadian clock in flies, demonstrating that this input pathway can be genetically dissected, as has been similarly shown for the light input into the circadian clock (Glaser, 2005).

A role for blind DN2 clock neurons in temperature entrainment of the Drosophila larval brain

Circadian clocks synchronize to the solar day by sensing the diurnal changes in light and temperature. In adult Drosophila, the brain clock that controls rest-activity rhythms relies on neurons showing Period oscillations. Nine of these neurons are present in each larval brain hemisphere. They can receive light inputs through Cryptochrome (CRY) and the visual system, but temperature input pathways are unknown. This study investigated how the larval clock network responds to light and temperature. Focus was placed on the CRY-negative dorsal neurons (DN2s), in which light-dark (LD) cycles set molecular oscillations almost in antiphase to all other clock neurons. The phasing of the DN2s in LD depends on the pigment-dispersing factor (PDF) neuropeptide in four lateral neurons (LNs), and on the PDF receptor in the DN2s. In the absence of PDF signaling, these cells appear blind, but still synchronize to temperature cycles. Period oscillations in the DN2s were stronger in thermocycles than in LD, but with a very similar phase. Conversely, the oscillations of LNs were weaker in thermocycles than in LD, and were phase-shifted in synchrony with the DN2s, whereas the phase of the three other clock neurons was advanced by a few hours. In the absence of any other functional clock neurons, the PDF-positive LNs were entrained by LD cycles but not by temperature cycles. These results show that the larval clock neurons respond very differently to light and temperature, and strongly suggest that the CRY-negative DN2s play a prominent role in the temperature entrainment of the network (Picot, 2009).

Although the absence of PDF severely affects Drosophila activity rhythms in DD, the exact function of the neuropeptide in the adult clock neuronal network remains unclear. In LD, PDF is required to produce a morning activity peak and to properly phase the evening peak, but not to entrain the brain clock. The behavioral phenotypes of PDF receptor mutants resemble that of the pdf⁰¹ mutant. PDFR is expressed in all clock neurons except the large ventral lateral neurons (l-LNvs), supporting a role of PDF in maintaining phase coherence within the adult clock network in DD. The loss of PER oscillations in the DN2s of pdf⁰¹ larvae demonstrates a clear and novel role of PDF in transmitting not only phase information but also a synchronizing signal without which the receiving neurons are not entrained in LD (Picot, 2009).

The current results show that the CRY-less DN2s are 'blind' neurons that perceive light indirectly. The PDF receptor rescue experiments strongly suggest that PDF acts on its receptor on the larval DN2s themselves, which are located in the vicinity of the LN axon terminals. Furthermore, DN2s possess a wide and dense neuritic network that borders on the axons of the LNs over a large fraction of their length. However, it cannot be ruled out that expression of the receptor in the (PDF-negative) fifth LN is involved in synchronizing the DN2s downstream, through PDF-independent mechanisms (Picot, 2009).

The PDF-negative fifth LN is also a CRY-negative clock neuron, but it cycles in phase with the CRY-positive neurons of the larval brain. The visual input to the PDF-expressing LNs appears sufficient to phase them normally even in cry^b mutants. It could thus be expected to entrain the CRY-less fifth LN in phase with the other larval LNs, as observed, in contrast to the CRY-less DN2s. A direct input from the visual system to the fifth LN is also consistent with its PDF-independent entrainment by LD cycles. Similarly, light entrainment of the larval DN1s in cry^b mutants is consistent with their suggested connection to the visual system. Thus, the CRY-less DN2s would be the only larval clock neurons devoid of such a connection (Picot, 2009).

Adult eclosion rhythms depend on the PDF-expressing LNs and appear to require the PDF-dependent clock that resides in the prothoracic gland. Since the larval DN2s project in the pars intercerebralis, a region of the brain that sends projections to the prothoracic gland, they could play a role in this physiologically important clock function. These results raise the possibility that the damped PER oscillations in the DN2s of the pdf⁰¹ mutants participate to their eclosion phenotype (Picot, 2009).

The DN2s are the only larval clock neurons that are phased identically by light and temperature, but their temperature entrainment appears independent of any LN-derived signal. PER oscillations in the DN2s have a larger amplitude in HC cycles than in LD cycles, also suggesting a prominent role of temperature in their entrainment. Conversely, the molecular oscillations of the PDF-positive LNs have a larger amplitude in LD compared with HC cycles. In the latter, the molecular oscillations of the PDF-expressing LNs seem to follow those in the DN2s, with a large phase change compared with LD conditions. The DN1s and the PDF-negative fifth LN, in contrast, share another phase that is slightly advanced. Interestingly, behavioral and transcriptome data in adult flies indicate that HC cycles result in a general phase advance relative to LD cycles. Cooperative synchronization of the clock by light and temperature likely requires temperature changes to act earlier than light changes since changes in temperature always lag behind changes in solar illumination in nature. The very different relative phasing of the larval clock neurons in HC versus LD cycles suggests different ecological constraints on this life stage, spent mostly burrowed in food, in which light may be a weaker Zeitgeber, and in which the lag between temperature and light changes may be quite different (Picot, 2009).

When a functional clock is absent from the DN2s (and the fifth LN), the larval PDF-expressing LNs are unable to entrain to thermocycles, whereas they autonomously entrain to LD cycles. It remains possible that autonomous temperature entrainment of the larval LNs (but not the DN2s) requires per transcriptional regulation, which the GAL4-UAS system is lacking. But the results demonstrate the existence of a control exerted on the LN clock by CRY-negative clock cells when temperature is the synchronizing cue. Although a role of the fifth LN cannot be ruled out, the absence of autonomous photoperception by the DN2s nicely fits with a role in temperature entrainment. The high cycling amplitude of the DN2s in thermocycles and the locking of the phase of the LNs on that of the DN2s in these conditions strongly support their role in the temperature entrainment of the LNs (Picot, 2009).

Additional studies should investigate whether the DN2s communicate with the LNs via fibers that appear to run along the dorsal projection of the LNs. Alternatively, the dense dendritic-like network of the DN2s could ensure reciprocal exchanges between them and the LNs. A model is thus proposed whereby, in the larval brain, the DN2s and the four PDF-positive LNs form a distinct subnetwork, with the LNs entraining the DN2s in LD, whereas the opposite is true in HC). What becomes of their hierarchy in constant conditions, after entrainment stops? Their relative phases appear to change little at least during the first 2 d after entrainment, whether they have been set in antiphase by LD entrainment, or in phase by HC entrainment. This suggests that, whatever the entraining regimen, the LNs and the DN2s run autonomously in constant conditions. However, it cannot be excluded that one of the two groups still dominates but requires more time after the end of entrainment to shift the phase of the other (Picot, 2009).

The rhythmic behavior of the adult flies that emerge from the temperature-entrained larvae is almost in antiphase compared with the one of flies entrained by light during the larval stage. This strongly suggests that the phase of the adult rhythms is set by the antiphasic oscillations of the larval PDF-positive LNs, consistent with these cells being the only neurons in which molecular cycling persists throughout metamorphosis. It is thus believed that the large phase shift of adult activity can be accounted for simply by the large phase shift of molecular oscillations in the PDF-expressing LNs (Picot, 2009).

It is often assumed that temperature affects the molecular clock directly and identically in all clock cells, as opposed to light, which requires dedicated input pathways. However, in the adult, thermocycles phase the brain clock differently from all peripheral clocks, as judged from whole-tissue oscillations of a luciferase reporter enzyme (Glaser, 2005). Recent data suggest that subsets of clock neurons in the Drosophila adult brain may indeed be dedicated to temperature entrainment. In experiments combining LD and HC entrainment, all DN groups, as well as the less studied lateral posterior neurons (LPNs), seem to preferentially follow thermocycles, whereas the other LNs preferentially follow LD cycles (Miyasako, 2007). Although adult PDF⁺ LNs are able to entrain to thermocycles in the absence of any other functional clock, they do not seem to be required for (and actually slowed down) the temperature entrainment of activity rhythms, whereas the PDF-negative LPNs appear to play a prominent role in such conditions (Picot, 2009).

The current results indicate that a similar specialization toward light or temperature entrainment exists in the larval brain. The DN2s, which appear to be the most temperature-responsive clock neurons, are by themselves completely blind. Conversely, the four PDF-positive LNs, which may be the most light-sensitive clock neurons (with both CRY and the visual system as inputs), appear almost temperature blind, and depend on the DN2s for temperature entrainment. PER-negative DN2s do not allow PER oscillations in the larval LNs, suggesting that entrainment of the latter in HC cycles depends on clock function in the former. The hierarchy of clock neurons thus appears very different during entrainment of the clock network by one or the other Zeitgeber (Picot, 2009).

A subset of dorsal neurons modulates circadian behavior and light responses in Drosophila

A fundamental property of circadian rhythms is their ability to persist under constant conditions. In Drosophila, the ventral Lateral Neurons (LNvs) are the pacemaker neurons driving circadian behavior under constant darkness. Wild-type flies are arrhythmic under constant illumination, but flies defective for the circadian photoreceptor CRY remain rhythmic. Flies overexpressing the pacemaker gene per or the morgue gene, a gene that can protect flies from the disruptive effects of constant light when overexpressed with the tim-GAL4 driver, are also behaviorally rhythmic under constant light. Unexpectedly, the LNvs do not drive these rhythms: they are molecularly arrhythmic, and PDF - the neuropeptide they secrete to synchronize behavioral rhythms under constant darkness - is dispensable for rhythmicity in constant light. Molecular circadian rhythms are found only in a group of Dorsal Neurons: the DN1s. Thus, a subset of Dorsal Neurons shares with the LNvs the ability to function as pacemakers for circadian behavior, and its importance is promoted by light (Murad, 2007).

Recent studies have shown that two groups of cells control circadian behavior. The PDF-positive LNvs are called morning cells (M cells), and the LNds evening cells (E cells), because they control the anticipatory behavior observed before dawn and dusk, respectively. In addition, the LNvs are the cells maintaining circadian behavior in constant darkness and controlling the phase of most circadian neurons of the brain. In their absence, circadian behavior rhythms are lost after a few days in DD. Surprisingly, the current results show that a functional circadian clock in the LNvs is actually not necessary for long-term behavioral rhythms. In flies overexpressing PER, the LNvs are no longer circadianly functional under constant illumination. No oscillation of the circadian protein PDP1 can be detected, and yet these flies remain rhythmic for at least 7 days. Moreover, limiting per overexpression to circadian neurons that do not express PDF is sufficient to obtain circadian behavioral rhythms under constant environmental conditions (Murad, 2007).

It is thought that the neurons maintaining circadian behavior independently of the LNvs are not the E cells. Indeed, when per is overexpressed, no sign of circadian oscillation is seen in the neurons that are thought to control the evening activity: the LNds. In addition, the PDF-negative LNv that might also contribute to the evening activity did not cycle in LL when morgue was overexpressed. Moreover, flies with per overexpression driven by cry-GAL4 were completely arrhythmic under constant light. cry-GAL4 is one of the critical GAL4 drivers used to define the E cells. Importantly, molecular circadian oscillations were detected in only one group of cells when per was overexpressed: the DN1s. Due to the high number of DN3s, it cannot be ruled out that a few cells in the DN3 groups also oscillate. Interestingly, it has been shown that a subset of DN3 neurons can maintain their own circadian oscillations in DD, in the absence of circadianly functional LNvs. However, these DN3 cells were not able to generate rhythmic behavior in DD. While it is possible that light is a necessary cofactor for these self-sustained DN3s to participate in the control of circadian behavior, the hypothesis is favored that it is the DN1s that maintain circadian rhythmicity in LL. This idea is strongly supported by several additional findings. First, the phase of PDP1 molecular oscillations in the DN1s on the third day of LL fits well with the long period of the circadian behavior observed under these conditions in per-overexpressing flies. Second, the behavioral observations made with morgue overexpression also suggest that the critical cells for rhythmicity are not the LNvs, and PER staining in morgue-overexpressing flies gave an independent confirmation that robust circadian molecular oscillations are restricted to the DN1s in LL. Finally, in LNv-rescued cry^b flies, only the DN1s show robust, coherent circadian rhythms in phase with the behavioral rhythms. Remarkably, the DN1s can maintain circadian behavior in LL even when PDF is absent. This indicates that they can work autonomously of LNv output. Interestingly, not all DN1s do oscillate in LL, only about six or seven cells most likely. This shows that the DN1 group is heterogeneous. This is not surprising, since the different groups of circadian neurons were named based on their location in the brain, not on their function or developmental lineage. There is ample evidence for heterogeneity of morphology, gene expression, and behavior within these different groups of cells, including the DN1s (Murad, 2007).

Thus, a subset of DN1s can control and generate circadian behavioral rhythms. They must therefore play an important role in the circadian neuronal circuits. Since ablation of the M cells and E cells results in flies with no morning and evening activity, and no self-sustained rhythms in DD, this could mean that the DN1s are usually functioning downstream of the M and E cells. This is further supported by the fact that in the absence of the neuropeptide PDF—believed to be the critical synchronizing signal secreted by the M cells—the DN1s cannot maintain their circadian rhythms in the long run in DD. The DN1s can thus probably function as a relay connecting the LNvs with the neurosecretory cells of the pars intercerebralis (PI), believed to play an important role in the control of locomotor behavior. A LNvs-DN1-PI pathway has also been suggested based on the anatomical studies of the projections of the small LNvs and the DN1s. The expression of the receptor for PDF in at least a subset of DN1s also supports the existence of a functional connection between them and the LNvs. The implication of this connection is that, in wild-type flies under LL, the LNvs should constantly send a disruptive signal to the DN1s, presumably the nonoscillating secretion of PDF (Murad, 2007).

This leaves us with the following question: if the LNvs and rhythmic PDF secretion are normally required for the DN1s to be rhythmic, why are the DN1s able to free themselves from the disruptive effects of constant light, while at the same time becoming independent of the LNvs? The results show that an important mechanism is the inhibition of the CRY-dependent light input pathway. Indeed, morgue-overexpressing flies are defective in the CRY-dependent behavioral responses to short light pulses, and cry loss-of-function mutations also result in rhythms driven by the DN1s. In the case of per overexpression, it is presumed that the TIM role is reduced, since one of its major functions is to protect PER from proteasomal degradation. TIM is the target of CRY; thus its reduced importance would result in DN1s that are less sensitive to the CRY input pathway. In addition, overexpression of Shaggy, which inhibits CRY signaling, also results in LL rhythms driven by dorsal neurons. However, under natural environmental conditions, inhibition of the CRY input pathway is probably not required for the DN1s to participate in the control of circadian rhythms. Indeed, even in the polar regions of the globe that experience constant light conditions during the summer, the elevation of the sun varies during the day, and this should result in variations of temperature sufficient to synchronize the DN1 circadian clock (Murad, 2007).

The mechanism by which the DN1s avoid becoming arrhythmic in LL as a result of the molecular arrhythmicity of the LNvs, which should result in constant PDF secretion, is not clear yet. It is possible that the presence of light inhibits PDF signaling and thus promotes the role of the DN1s. Light input could come from the eyes, ocelli, or from the DN1s themselves. Alternatively, the DN1s could induce rhythmic PDF secretion. The fact that PDF is not required for LL behavioral rhythms does not exclude this possibility, particularly since the robustness of the rhythms is improved by the presence of PDF (Murad, 2007).

Interestingly, per and morgue overexpression results in a very similar long period phenotype under LL, which could suggest that these two molecules coincidentally affect the period length of the circadian molecular pacemaker in the same way. In DD, however, per overexpression does affect behavioral period length, while morgue does not. The long period phenotype observed in LL actually probably reflects the fact that the CRY input pathway is not completely blocked in the DN1s of per- or morgue-overexpressing flies. Indeed, under very low light intensity, wild-type flies exhibit a long period phenotype as well. In addition, morgue overexpression does not completely block the CRY-dependent responses to short light pulses. Finally and most importantly, LNv-rescued cry^b flies (in which the CRY input pathway is completely nonfunctional in the DN1s) have 24 hr period rhythms. The LNv-rescued cry^b flies show nevertheless a higher degree of arrhythmicity than normal cry^b flies or than flies overexpressing morgue or per. This might be due to the desynchronization observed within the DN3 group of circadian neurons. Indeed, the DN3s do not appear to be desynchronized in per- or morgue-overexpressing flies (Murad, 2007).

A previous report had already shown that LNv-rescued cry^b flies are partially rhythmic, and this was interpreted as evidence for a functional role of CRY directly in the LNvs. The new results show that expression of CRY in the LNvs is probably not very important for the response to constant light. The DN1s are the important cells for this response. Does this mean that CRY is not a photoreceptor in the LNvs? It is thought that CRY actually does function as a photoreceptor in the LNvs as well. CRY is expressed in these cells, and LNv-rescued cry^b flies show very significantly rescued responses to short light pulses. Preliminary experiments with morgue overexpression limited to the LNvs confirm a predominant role of these cells for light-pulse responses. Thus, the CRY input pathway might mediate response to short light pulses by its action in the LNvs and constant light responses by its action in the DN1s (Murad, 2007).

In summary, the work underscores the importance of the DN1s in the control of circadian behavior and responses to light. Earlier genetic studies have indicated that the DN1s modulate the sensitivity of the circadian network to light:dark cycles of very low light intensity. The current results significantly extend this observation by showing the profound impact the DN1s have on the response to constant light and by demonstrating that these cells not only modulate circadian light responses but can also become the driving force controlling circadian locomotor behavior, and this in the absence of environmental cues and functional LNvs. This confers upon them a unique status among non-PDF circadian neurons. One of the striking results is that genetically identical flies rely either on the LNvs or the DN1s for the control of their circadian rhythms, depending on the presence or absence of light. Indeed, the LNvs determine period length in these experiments with per overexpression in DD, but in LL the DN1s set the pace. That the presence or the absence of light can so remarkably shift the dominance from one cell group to the other strongly suggests that the relative contributions of the LNvs and DN1s to the control of circadian rhythms change during the course of the year, particularly at high latitude. The DN1s, which interestingly generate evening activity, would play a more prominent role in the control of circadian behavior during the long days of the summer, while the LNvs would be more important when photoperiods are shorter (Murad, 2007).

Adult circadian behavior in Drosophila requires developmental expression of cycle, but not period

Circadian clocks have evolved as internal time keeping mechanisms that allow anticipation of daily environmental changes and organization of a daily program of physiological and behavioral rhythms. To better examine the mechanisms underlying circadian clocks in animals and to ask whether clock gene expression and function during development affected subsequent daily time keeping in the adult, the genetic tools available in Drosophila were used to conditionally manipulate the function of the Cycle component of the positive regulator Clock/Cycle (Clk/Cyc) or its negative feedback inhibitor Period (Per). Differential manipulation of clock function during development and in adulthood indicated that there is no developmental requirement for either a running clock mechanism or expression of per. However, conditional suppression of Clk/Cyc activity either via per over-expression or cyc depletion during metamorphosis resulted in persistent arrhythmic behavior in the adult. Two distinct mechanisms were identified that may contribute to this developmental function of Clk/Cyc and both involve the ventral lateral clock neurons (LN_vs) that are crucial to circadian control of locomotor behavior: (1) selective depletion of cyc expression in the LN_vs resulted in abnormal peptidergic small-LN_v dorsal projections, and (2) Per expression rhythms in the adult LN_vs appeared to be affected by developmental inhibition of Clk/Cyc activity. Given the conservation of clock genes and circuits among animals, this study provides a rationale for investigating a possible similar developmental role of the homologous mammalian CLOCK/BMAL1 complex (Goda, 2011).

This study created transgenic flies with conditional clock function, in which expression of the essential clock components Cyc and Per was induced or repressed in relevant spatiotemporal patterns. In per⁰¹ [timP>per]^ts flies, which conditionally rescue the per⁰¹ mutation, clock function was conditional and readily reversible. Moreover, adult circadian behavior was restored in flies raised under restrictive conditions. In earlier studies conducted by Ewer widespread transgenic expression of per under control of a heat-shock protein 70 (hsp70) promoter was shown to partially rescue the per⁰¹ mutation resulting in restoration of behavioral rhythms at an abnormally long period length. These long period rhythms could be generated in a conditional manner even when induction was restricted to the adult phase(Ewer, 1999). the present study targeted expression of transgenic per specifically to clock-bearing cells and achieved a more complete conditional rescue of the per⁰¹ phenotype that did not require developmental per expression (Goda, 2011).

Although circadian behavior of per⁰¹ [timP>per]^ts flies at 25°C showed rhythmicity comparable to that observed for wild-type flies, period lengths were at least 2 h longer than those of wild-type flies and molecular rhythms showed a relatively low amplitude. One key difference between the molecular clock circuits in per⁰¹ [timP>per]^ts at the permissive temperature and those of wild-type flies is the constitutively high level of per mRNA expression in the transgenically rescued flies, which could contribute to the increased circadian period length and blunted molecular rhythms in per⁰¹ [timP>per]^ts flies. Wild-type flies exhibit a trough in per transcript levels in the early morning that may facilitate subsequent down-regulation of Per protein levels and optimal induction of CLK/Cyc-regulated. The lack of a trough in per mRNA expression in the conditionally rescued flies could account for a delay in the turnover of Per protein in the morning and, therefore, a lengthened period and blunted CLK/Cyc activity. This hypothesis also explains apparent discrepancies with previous reports, in which increased per gene dosage was associated with a shortened circadian period length and decreased per dosage or expression resulted in longer circadian period lengths. As long as per expression shows strong circadian regulation the timing of Per nuclear entry and Per-mediated transcriptional repression is predicted to be advanced by the introduction of one or two additional copies of the wild-type per gene and delayed by a reduction in per dosage, while neither manipulation is predicted to strongly affect subsequent Per turnover (Goda, 2011).

Adult circadian behavior was also conditional and reversible in [timP>per]^ts flies, which exhibit temperature-dependent over-expression of per. However, developmental over-expression of per during metamorphosis was associated with irreversible behavioral arrhythmia in adults. Likewise, depletion of cyc expression during the metamorphosis in cyc⁰¹ [elav>cyc]^ts flies resulted in disruption of adult circadian locomotor behavior under permissive conditions. Both increased levels of Per and decreased levels of Cyc negatively regulate CLK/Cyc activity. The Clk/Cyc heterodimer functions as the central transcriptional regulator in the Drosophila clock and its activity critically depends on the presence of both Clk and Cyc. Loss of functional cyc expression in the cyc⁰¹ mutant results in both molecular and circadian arrhythmia and constitutively low expression levels for Clk/Cyc-regulated target genes, whereas Per acts as a negative regulator of Clk/Cyc activity by binding and inactivating the Clk/Cyc complex. The arrhythmic locomotor behavior and molecular arrhythmia in the clock neurons observed as a result of per over-expression are, therefore, interpreted to result from constitutive inhibition of Clk/Cyc (Goda, 2011).

Adult behavioral arrhythmia in [timP>per]^ts or cyc⁰¹ [elav>cyc]^ts flies raised under permissive conditions was reversible. However, exposures to restrictive conditions of comparable duration resulted in long-term after-effects only when they occurred during development and, particularly, during the pupal and pharate adult stages. Therefore the effects of circadian arrests during development in [timP>per]^ts or cyc⁰¹ [elav>cyc]^ts flies on adult circadian behavior are attributed to a developmental requirement for Clk/Cyc function beyond its immediate role in maintaining daily time keeping. The requirement for Clk/Cyc activity, but not clock function per se may indicate that one or more transcriptional Clk/Cyc targets play a role in enabling adult circadian locomotor behavior. Such targets would likely be expressed constitutively along with other Clk/Cyc-regulated genes in conditionally arrested per⁰¹ [timP>per]^ts flies, but constitutively down-regulated in circadian arrests due to low Clk/Cyc activity (Goda, 2011).

A central question that remains is what mechanism links developmental Clk/Cyc activity to adult circadian behavior. The experiments indicate that both clock neuron anatomy and the molecular oscillator itself may be involved. Previously published studies of constitutively arrhythmic alleles of the Clk and cyc genes have documented a reduction in PDF expression as well as neuro-anatomical defects in the LN_vs that could be associated with a developmental role for the Clk/Cyc transcription factor. By selectively blocking transgenic rescue of cyc⁰¹ in the PDF-expressing clock neurons this study shows that the reduction of PDF expression and PDF-positive dorsal projections from the s-LN_vs is a cell-type specific phenotype. PDF is known to play an important role in mediating clock-controlled behavior in both LD and DD conditions. The PDF-producing s-LN_vs project towards the dorsal protocerebrum as do DN1, DN2, DN3, and LN_d clock neurons, suggesting that the dorsal s-LN_v projections may play an important part in signaling across the neural clock circuits. In this context, it may be relevant that expression of the PDF Receptor in 'E' cells, a subset of clock neurons including DN1s and LN_ds, has been associated with circadian control of locomotor activity. Moreover, the axonal terminals of the dorsal s-LNv projections undergo clock-controlled rhythms in remodeling that may play a role in circadian signaling. Nevertheless, the observed developmental requirement for Clk/Cyc activity also appears to involve mechanisms other than PDF-mediated signaling for the following reasons. First, developmental over-expression of Per resulted in persistent adult arrhythmia, but did not lead to a loss of PDF-positive dorsal projections from the s-LN_vs. Second, while developmental suppression of Clk/Cyc activity uniformly affected the behavior of adult flies constitutive depletion of Cyc from the PDF-expressing neurons resulted in a variable phenotype in the s-LN_v dorsal projections. Third, the light/dark activity pattern of cyc⁰¹ (elav-Pdf)>cyc flies was strikingly different from that of Pdf⁰¹ flies or flies from which the PDF-expressing cells have been ablated, suggesting that PDF signaling persisted in cyc-depleted LN_vs in spite of the defects in PDF-positive dorsal projections (Goda, 2011).

In principle, neuro-anatomical defects affecting intercellular connectivity rather than cell-autonomous clock function could lead to behavioral phenotypes due to asynchrony among the clock neurons or the loss of output signals. Indeed, apparent separation of molecular and behavioral phenotypes has been reported previously for genetic manipulation of Clk/Cyc function. It may be particularly relevant that rescue of the per⁰¹ phenotype in the PDF-expressing clock neurons restores rhythmic behavior, while rescue of cyc⁰¹ in the same cells restores molecular, but not behavioral rhythms. However, the experimental results also provide support for developmental phenotypes at the level of the adult molecular clock circuits. Immunofluorescence expression analyses indicated that the molecular clock circuits in the adult PDF-expressing clock neurons were affected by developmental over-expression of Per. PDF-expressing LN_vs in adults that were behaviorally arrhythmic due to developmental Per over-expression exhibited adult Per expression with an altered daily profile, but not necessarily at excessively high levels. Future studies may determine the degree to which neuro-anatomical and molecular phenotypes are linked and help determine the effect of intercellular connectivity in the neural clock circuit on the function of molecular circadian rhythms in individual clock neurons (Goda, 2011).

Temperature-dependent resetting of the molecular circadian oscillator in Drosophila

Circadian clocks responsible for daily time keeping in a wide range of organisms synchronize to daily temperature cycles via pathways that remain poorly understood. To address this problem from the perspective of the molecular oscillator, temperature-dependent resetting of four of its core components was monitored in Drosophila: the transcripts and proteins for the clock genes period (per) and timeless (tim). The molecular circadian cycle in adult heads exhibited parallel responses to temperature-mediated resetting at the levels of per transcript, tim transcript and Tim protein. Early phase adjustment specific to per transcript rhythms was explained by clock-independent temperature-driven transcription of per. The cold-induced expression of Drosophila per contrasts with the previously reported heat-induced regulation of mammalian Period 2. An altered and more readily re-entrainable temperature-synchronized circadian oscillator that featured temperature-driven per transcript rhythms and phase-shifted Tim and Per protein rhythms was found for flies of the 'Tim 4' genotype, which lacked daily tim transcript oscillations but maintained post-transcriptional temperature entrainment of tim expression. The accelerated molecular and behavioural temperature entrainment observed for Tim 4 flies indicates that clock-controlled tim expression constrains the rate of temperature cycle-mediated circadian resetting (Goda, 2014).

A group of segmental premotor interneurons regulates the speed of axial locomotion in Drosophila larvae

Animals control the speed of motion to meet behavioral demands. Yet, the underlying neuronal mechanisms remain poorly understood. This study shows that a class of segmentally arrayed local interneurons (period-positive median segmental interneurons, or PMSIs) regulates the speed of peristaltic locomotion in Drosophila larvae. PMSIs formed glutamatergic synapses on motor neurons and, when optogenetically activated, inhibited motor activity, indicating that they are inhibitory premotor interneurons. Calcium imaging showed that PMSIs are rhythmically active during peristalsis with a short time delay in relation to motor neurons. Optogenetic silencing of these neurons elongated the duration of motor bursting and greatly reduced the speed of larval locomotion. These results suggest that PMSIs control the speed of axial locomotion by limiting, via inhibition, the duration of motor outputs in each segment. Similar mechanisms are found in the regulation of mammalian limb locomotion, suggesting that common strategies may be used to control the speed of animal movements in a diversity of species (Kohsaka, 2014).

Age-related changes in the expression of the circadian clock protein PERIOD in Drosophila glial cells

Rhythms in behavioral and other circadian outputs tend to weaken during aging, as evident in progressive disruptions of sleep-wake cycles in aging organisms. However, less is known about the molecular changes in the expression of clock genes and proteins that may lead to the weakening of circadian outputs. Western blot studies have demonstrated that the expression of the core clock protein PERIOD (PER) declines in the heads of aged Drosophila melanogaster flies. This age-related decline in PER does not occur in the central pacemaker neurons but has been demonstrated so far in retinal photoreceptors. Besides photoreceptors, clock proteins are also expressed in fly glia, which play important roles in neuronal homeostasis and are further categorized into subtypes based on morphology and function. While previous studies of mammalian glial cells have demonstrated the presence of functional clocks in astrocytes and microglia, it is not known which glial cell types in Drosophila express clock proteins and how their expression may change in aged individuals. Immunocytochemistry experiments have been conducted to identify which glial subtypes express PER protein suggestive of functional circadian clocks. Glial cell subtypes that showed night-time accumulation and day-time absence in PER consistent with oscillations reported in the pacemaker neurons were selected to compare the level of PER protein between young and old flies. The data demonstrate that some glial subtypes show rhythmic PER expression and the relative PER levels become dampened with advanced age. Identification of glial cell types that display age-related dampening of PER levels may help to understand the cellular changes that contribute to the loss of homeostasis in the aging brain (Long, 2017).

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