Free-running Locomotor Activity Rhythms Research Articles

ВЛИЯНИЕ МАГНИТНЫХ ФЛУКТУАЦИЙ НА ЦИРКАДНУЮ СТРУКТУРУ ДВИГАТЕЛЬНОЙ АКТИВНОСТИ У DANIO RERIO (ПРЕДВАРИТЕЛЬНЫЕ ДАННЫЕ)

The locomotor activity of zebrafish (Danio rerio) has a pronounced, well-studied circadian rhythm. Under constant illumination, the period of free-running locomotor activity in this species usually becomes less than 24 hours. To evaluate the entraining capabilities of slow magnetic variations, zebrafish locomotor activity was evaluated at constant illumination and fluctuating magnetic field with a period of 26.8 hours. Lomb-Scargle periodogram revealed significant free-running rhythms of locomotor activity and related behavioral endpoints with a period close to 27 hours. Obtained results reveal the potential of slow magnetic fluctuations for entrainment of the circadian rhythms in zebrafish. The putative mechanisms responsible for the entrainment are discussed, including the possible role of cryptochromes.

Effect of periodic social interaction and odour presentation of same and opposite-sex conspecifics on free-running mice

ABSTRACT Social interaction, a non-photic cue, plays a role in the synchronization of circadian rhythm under constant environmental conditions. However, little is known about the effect of sex-specific interaction and sex-specific odor on the free-running circadian clock of mice. Hence, in the present study, we investigated the effect of social interaction and odor presentation of same and opposite-sex conspecifics on the free-running rhythm of mice. In this investigation, stranger and dummy mice (saturated with either male- or female-associated odor) were separately exposed to the same and opposite-sex conspecifics of male and female runners and receivers, respectively for 30 min at CT15 for 10–12 d. The results showed that the daily exposure of runners with same and opposite-sex strangers entrained the free-running locomotor activity rhythm in a gender-specific manner. However, daily exposure of same and opposite-sex conspecific odors masked the circadian rhythm of receiver animals by increasing the activity. Further, social interaction and odor presentation caused lengthened and shortened period length, respectively. These results suggest that periodic social interactions can produce entrainment and conspecific odor can produce only masking on free-running circadian locomotor activity rhythm in mice.

Entrainment of circadian rhythms of locomotor activity by ambient temperature cycles in the dromedary camel

In the dromedary camel, a well-adapted desert mammal, daily ambient temperature (Ta)-cycles have been shown to synchronize the central circadian clock. Such entrainment has been demonstrated by examining two circadian outputs, body temperature and melatonin rhythms. Locomotor activity (LA), another circadian output not yet investigated in the camel, may provide further information on such specific entrainment. To verify if daily LA is an endogenous rhythm and whether the desert Ta-cycle can entrain it, six dromedaries were first kept under total darkness and constant-Ta. Results showed that the LA rhythm free runs with a period of 24.8–24.9 h. After having verified that the light–dark cycle synchronizes LA, camels were subjected to a Ta-cycle with warmer temperatures during subjective days and cooler temperatures during subjective nights. Results showed that the free-running LA rhythm was entrained by the Ta-cycle with a period of exactly 24.0 h, while a 12 h Ta-cycle phase advance induced an inversion of the LA rhythm and advanced the acrophase by 9 h. Similarly, activity onset and offset were significantly advanced. All together, these results demonstrate that the Ta-cycle is a strong zeitgeber, able to entrain the camel LA rhythm, hence corroborating previous results concerning the Ta non-photic synchronization of the circadian master clock.

Selection for reproduction under short photoperiods changes diapause-associated traits and induces widespread genomic divergence.

The incidence of reproductive diapause is a critical aspect of life history in overwintering insects from temperate regions. Much has been learned about the timing, physiology and genetics of diapause in a range of insects, but how the multiple changes involved in this and other photoperiodically regulated traits are inter-related is not well understood. We performed quasinatural selection on reproduction under short photoperiods in a northern fly species, Drosophila montana, to trace the effects of photoperiodic selection on traits regulated by the photoperiodic timer and/or by a circadian clock system. Selection changed several traits associated with reproductive diapause, including the critical day length for diapause (CDL), the frequency of diapausing females under photoperiods that deviate from daily 24 h cycles and cold tolerance, towards the phenotypes typical of lower latitudes. However, selection had no effect on the period of free-running locomotor activity rhythm regulated by the circadian clock in fly brain. At a genomic level, selection induced extensive divergence from the control line in 16 gene clusters involved in signal transduction, membrane properties, immunologlobulins and development. These changes resembled those detected between latitudinally divergent D. montana populations in the wild and involved SNP divergence associated with several genes linked with diapause induction. Overall, our study shows that photoperiodic selection for reproduction under short photoperiods affects diapause-associated traits without disrupting the central clock network generating circadian rhythms in fly locomotor activity.

The Circadian Clock of the Ant Camponotus floridanus Is Localized in Dorsal and Lateral Neurons of the Brain.

The circadian clock of social insects has become a focal point of interest for research, as social insects show complex forms of timed behavior and organization within their colonies. These behaviors include brood care, nest maintenance, foraging, swarming, defense, and many other tasks, of which several require social synchronization and accurate timing. Ants of the genus Camponotus have been shown to display a variety of daily timed behaviors such as the emergence of males from the nest, foraging, and relocation of brood. Nevertheless, circadian rhythms of isolated individuals have been studied in few ant species, and the circadian clock network in the brain that governs such behaviors remains completely uncharacterized. Here we show that isolated minor workers of Camponotus floridanus exhibit temperature overcompensated free-running locomotor activity rhythms under constant darkness. Under light-dark cycles, most animals are active during day and night, with a slight preference for the night. On the neurobiological level, we show that distinct cell groups in the lateral and dorsal brain of minor workers of C. floridanus are immunostained with an antibody against the clock protein Period (PER) and a lateral group additionally with an antibody against the neuropeptide pigment-dispersing factor (PDF). PER abundance oscillates in a daily manner, and PDF-positive neurites invade most parts of the brain, suggesting that the PER/PDF-positive neurons are bona fide clock neurons that transfer rhythmic signals into the relevant brain areas controlling rhythmic behavior.

On the Role of Histamine Receptors in the Regulation of Circadian Rhythms.

Several lines of evidence suggest a regulatory role of histamine in circadian rhythms, but little is known about signaling pathways that would be involved in such a putative role. The aim of this study was to examine whether histamine mediates its effects on the circadian system through Hrh1 or Hrh3 receptors. We assessed both diurnal and free-running locomotor activity rhythms of Hrh1 -/- and Hrh3 -/- mice. We also determined the expression of Per1, Per2 and Bmal1 genes in the suprachiasmatic nuclei, several areas of the cerebral cortex and striatum under symmetric 24 h light-dark cycle at zeitgeber times 14 and 6 by using radioactive in situ hybridization. We found no differences between Hrh1 -/- and wild type mice in the length, amplitude and mesor of diurnal and free-running activity rhythms as well as in expression of Per1, Per2 and Bmal1 genes in any of the examined brain structures. The amplitude of free-running activity rhythm of the Hrh3 -/- mice was significantly flattened, whereas the expression of the clock genes in Hrh3 -/- mice was similar to the wild type animals in all of the assessed brain structures. Therefore, the knockout of Hrh1 receptor had no effects on the circadian rhythm of spontaneous locomotion, and a knockout of Hrh3 receptor caused a substantial reduction of free-running activity rhythm amplitude, but none of these knockout models affected the expression patterns of the core clock genes in any of the studied brain structures.

Possible treatment of circadian sleep disorders using daily ketotifen administration

Classic H1 histamine receptor (H1R) antagonists are currently used as over-the-counter sleep aids. However, their overdose administrations rather produce wakefulness. Our previous rat model studies suggest that ketotifen, an early-phase, second-generation H1R antagonist may be a more desirable sleep aid. Here, we further examined daily injections of ketotifen (3 mg/kg, intraperitoneally, for 5 days) 3 h before light onset time in rats and analyzed the effect of ketotifen on the successive free-running locomotor activity rhythms under constant dim red light. We found significant phase-advancement by the daily ketotifen injections, suggesting possible treatment of circadian sleep disorders using daily ketotifen administration.

Now you see me, now you don’t: The locomotory activity rhythm of the Asian garden dormouse (Eliomys melanurus) from Saudi Arabia

The locomotory activity rhythms of Asian garden dormice, Eliomys melanurus from Saudi Arabia were investigated under controlled laboratory conditions. Animals were maintained under different lighting conditions for a period of two weeks each. Dormice exhibited predominantly nocturnal activity during the light/dark (LD) (mean % nocturnal activity: 95.5±0.9%) and dark/light (DL) (mean % nocturnal activity: 96.8±0.7%) light cycles. All animals expressed free-running rhythms of locomotor activity when subjected to constant darkness (mean τ: 24h06±0h09). Upon inversion of the LD cycle to DL, activity re-entrained to the new light cycle within 3 days. When the dark component of the day was lengthened to 8L:16D the active time increased significantly from 11h43±0h05 to 13h43±0h22. In contrast, when the dark component was shortened from 12L:12D to16L: 8D, the active time decreased significantly to 7h58±0h04. No difference was apparent in the locomotor activity at 20 and 30°C however, dormice became very inactive at 10°C.Overall, locomotor activity patterns of the Asian garden dormouse largely resemble those of other species of dormice. However, Asian garden dormice spent long periods of time inactive both during light and darkness, when exposed to an ambient temperature of 10°C, which may be related to the dramatic and sudden change in temperature. This period of inactivity may reflect a bout of torpor or the entering into a state of hibernation.

Accurate and precise circadian locomotor activity rhythms in male and female Indian pygmy field mice, Mus terricolor

We report some basic circadian characteristics in Mus terricolor, the nocturnal pygmy field mouse. Both male and female M. terricolor entrain well to a 12:12 light:dark schedule. Activity is completely nocturnal in distribution in LD. ρ and α in LD differ significantly between males and females. Free-running circadian locomotor activity rhythm in DD was 23.69 h (males) and 23.46 h (females). In LD, ψ was −0.20 h and −0.15 h in males and females, respectively. Rhythms were accurate (6.02 in males/7.30 in females [h−1]) and precise (4.83 in males /4.74 in females [h−1]). Fifteen-minute perturbations with ∼70 lux white light in DD led to predictable phase advances and delays. Mus terricolor took ∼7 days to re-entrain to a 6 h advance in the ambient light schedule, with rapid resynchronization to a 6 h phase delay. These results indicate that M. terricolor is a good model animal for in-depth research in circadian biology.

Involvement of circadian oscillation(s) in the photoperiodic time measurement and the induction of reproductive diapause in a northern Drosophila species

An ability to predict forthcoming changes in environmental conditions and get prepared for them in advance is crucial for the survival and reproduction of organisms living in a seasonally changing environment. We have studied the possible involvement of circadian oscillator(s) in the photoperiodic timer controlling seasonal responses by tracing Drosophila montana females’ diapause induction in constant darkness and in a classical Nanda–Hamner experiment. Nearly all females developed ovaries in continuous darkness, which shows the direct development to be their default developmental pathway in the absence of photoperiods. In Nanda–Hamner experiment the females’ diapause incidence was close to zero in light:dark cycle 12:4 (photoperiod 16h) and increased to nearly 100% in 12:8 and 12:12 (photoperiods 20 and 24h). In longer photoperiods (28–72h) the females’ diapause percentages decreased gradually along with an increase in the length of the dark period, showing no peaks of high diapause incidence in the multiples of 24h. These findings suggest that the photoperiodic timer of D. montana is based on heavily damping circadian oscillator(s) or that it lacks strong oscillators. Damping of the photoperiodic timer under prolonged nights and constant darkness fits well with our earlier finding that these flies lose their locomotor activity rhythm in constant darkness, and suggests that the mechanisms underlying females’ photoperiodic diapause response and their free-running locomotor activity rhythm may be partly based on same oscillators.

Modulation by adiponectin of circadian clock rhythmicity in model mice for metabolic syndrome

To assess the effect of adiponectin on the circadian rhythm disturbances associated with metabolic syndrome, we generated a KK/Ta mouse line expressing the human adiponectin transgene in the liver. Locomotor activity of control C57BL/6 mice was highest during the beginning of the dark period and low during the light period. Under constant darkness, the length of locomotor activity rhythm of control mice was slightly shorter than 24 h. In KK/Ta mice the peak of locomotor activity was blunted and significant activity was observed during the light period. Furthermore, KK/Ta mice showed shorter average period length of free-running locomotor activity rhythm when compared with control mice. However, the transgenic expression of adiponectin in the liver significantly altered the circadian rhythm of locomotor activity and the length of free-running rhythm of KK/Ta mice towards those of C57BL/6 mice. In the liver and skeletal muscles from control mice, mRNA levels of Arntl and Cry1 were increased during the dark period, whereas those of Dbp, Cry2, Per1 and Per2 were elevated during the light period. KK/Ta mice exhibited phase advances in circadian rhythms of Arntl, Dbp, Cry2 and Per2 in both tissues. The phase shifts of the circadian clock gene expression in the liver were attenuated in adiponectin-transgenic mice. These results suggest that adiponectin is a peripheral regulator of the circadian clocks in the brain and peripheral organs, and may be a novel target for the treatment of obesity-associated disorders of circadian rhythms.

Larval ethanol exposure alters free-running circadian rhythm and per Locus transcription in adult D. melanogaster period mutants

Alcohol consumption causes disruptions in a variety of daily rhythms, including the circadian free-running rhythm. A previous study conducted in our laboratories has shown that larval ethanol exposure alters the free-running period in adult Canton-S Drosophila melanogaster. Few studies, however, have explored the effect of alcohol exposure on organisms exhibiting circadian periods radically different than (normal) 24-h. We reared Canton-S, period long, and period short Drosophila melanogaster larvae on 10%-ethanol supplemented food, and assessed their adult free-running locomotor activity and period transcript at ZT 12. We demonstrate that in Canton-S larval ethanol exposure shortens the adult free-running locomotor activity but does not significantly alter period mRNA levels at ZT 12. Period long mutants exposed to larval ethanol had significantly shortened adult free-running locomotor activity rhythms and decreased period mRNA levels, while period short mutants lengthened their free-running rhythm and showed increased period mRNA levels at ZT 12 after being exposed to larval ethanol. These results indicate that the effects of ethanol on the circadian clock might depend upon the baseline circadian period of the organism or that period mutant gene expression is sensitive to developmental ethanol treatment.

Flies in the North

The circadian clock plays an important role in adaptation in time and space by synchronizing changes in physiological, developmental, and behavioral traits of organisms with daily and seasonal changes in their environment. We have studied some features of the circadian activity and clock organization in a northern Drosophila species, Drosophila montana, at both the phenotypic and the neuronal levels. In the first part of the study, we monitored the entrained and free-running locomotor activity rhythms of females in different light-dark and temperature regimes. These studies showed that D. montana flies completely lack the morning activity component typical to more southern Drosophila species in an entrained environment and that they are able to maintain their free-running locomotor activity rhythm better in constant light than in constant darkness. In the second part of the study, we traced the expression of the PDF neuropeptide and the CRY protein in the neurons of the brain in D. montana adults and found differences in the number and location of PDF- and CRY-expressing neurons compared with those described in Drosophila melanogaster. These differences could account, at least in part, for the lack of morning activity and the reduced circadian rhythmicity of D. montana flies in constant darkness, both of which are likely to be adaptive features during the long and dark winters occurring in nature.

Lights out, let’s move about: locomotory activity patterns of Wagner’s gerbil from the desert of Saudi Arabia

We investigated the circadian activity rhythm in the little-studied Wagner’s gerbil (Gerbillus dasyurus) from the Raydah Protected Area, southwestern Saudi Arabia. In order to assess whether these animals possess an endogenous rhythm of locomotor activity that entrains to the light: dark cycle, they were subjected to three distinct light cycles: an LD cycle (12 h light/12 h dark), a DD cycle (constant darkness) and a DL light cycle (an inverse of the LD light cycle). All eight individuals studied exhibited entrainment of their activity to the light cycles. Under LD, the total percentage of activity during the dark phase was 93.7 ± 1.8%. Activity was distributed throughout the night (mean peak activity 22:46 ± 0:14). All eight animals expressed distinct endogenous free-running rhythms of locomotor activity (mean τ = 23:55 ± 0:36). During constant darkness, animals still displayed more activity during the subjective night (75.6 ± 0.4%). Under the DL light cycle, the total percentage of activity was 92.7 ± 1.8% during the dark phase. In conclusion, the daily locomotor activity rhythm of Wagner’s gerbil is strongly entrained by the light:dark cycle with the most activity concentrated during the night and consequently this desert-dwelling mammal may thus be considered truly nocturnal.

L-5-hydroxytryptophan resets the circadian locomotor activity rhythm of the nocturnal Indian pygmy field mouse, Mus terricolor

We report that L-5-hydroxytryptophan (5-HTP), a serotonin precursor, resets the overt circadian rhythm in the Indian pygmy field mouse, Mus terricolor, in a phase- and dose-dependent manner. We used wheel running to assess phase shifts in the free-running locomotor activity rhythm. Following entrainment to a 12:12 h light-dark cycle, 5-HTP (100 mg/kg in saline) was intraperitoneally administered in complete darkness at circadian time (CT)s 0, 3, 6, 9, 12, 15, 18, and 21, and the ensuing phase shifts in the locomotor activity rhythm were calculated. The results show that 5-HTP differentially shifts the phase of the rhythm, causing phase advances from CT 0 to CT 12 and phase delays from CT 12 to CT 21. Maximum advance phase shift was at CT 6 (1.18 ± 0.37 h) and maximum delay was at CT 18 (-2.36 ± 0.56 h). No extended dead zone is apparent. Vehicle (saline) at any CT did not evoke a significant phase shift. Investigations with different doses (10, 50, 100, and 200 mg/kg) of 5-HTP revealed that the phase resetting effect is dose-dependent. The shape of the phase-response curve (PRC) has a strong similarity to PRCs obtained using some serotonergic agents. There was no significant increase in wheel-running activity after 5-HTP injection, ruling out behavioral arousal-dependent shifts. This suggests that this phase resetting does not completely depend on feedback of the overt rhythmic behavior on the circadian clock. A mechanistic explanation of these shifts is currently lacking.

Lights Out, Let's Move About: Locomotory Activity Patterns of Wagner's Gerbil from the Desert of Saudi Arabia

We investigated the circadian activity rhythm in the little-studied Wagner’s gerbil (Gerbillus dasyurus) from the Raydah Protected Area, southwestern Saudi Arabia. In order to assess whether these animals possess an endogenous rhythm of locomotor activity that entrains to the light:dark cycle, they were subjected to three distinct light cycles: an LD cycle (12 h light/12 h dark), a DD cycle (constant darkness) and a DL light cycle (an inverse of the LD light cycle). All eight individuals studied exhibited entrainment of their activity to the light cycles. Under LD, the total percentage of activity during the dark phase was 93.7 ± 1.8%. Activity was distributed throughout the night (mean peak activity 22:46 ± 0:14). All eight animals expressed distinct endogenous free-running rhythms of locomotor activity (mean i = 23:55 ± 0:36). During constant darkness, animals still displayed more activity during the subjective night (75.6 ± 0.4%). Under the DL light cycle, the total percentage of activity was 92.7 ± 1.8% during the dark phase. In conclusion, the daily locomotor activity rhythm of Wagner’s gerbil is strongly entrained by the light:dark cycle with the most activity concentrated during the night and consequently this desert-dwelling mammal may thus be considered truly nocturnal.

Circadian rhythms of locomotor activity in the reddish‐grey musk shrew (Eulipotyphla: Soricidae) from South Africa

The circadian rhythm of locomotor activity in a southern African shrew, the reddish-grey musk shrew Crocidura cyanea was investigated. Thirteen individuals were subjected to three successive light cycles, each cycle lasting approximately 2 weeks: an LD cycle (12 h light/12 h dark), a DD cycle (constant darkness) and a DL cycle (an inverse of the LD cycle). All of the animals exhibited entrainment of their activity to the LD and DL lighting regimes. Locomotor activity of C. cyanea occurred predominantly during the dark phases of the LD cycle and the DL cycle. Under LD, the mean active phase (α) of C. cyanea was 10.8±0.3 h and the total percentage of activity was 78.9% during the dark phase. When subjected to constant darkness, the mean active phase increased to 13.2±01.8 h and all animals expressed free-running rhythms of locomotor activity (mean±1 sd=23.0±0.55 h; range=22.4–23.7 h). On the reverse LD cycle, the mean active phase was lowest (09.3±0.16 h) and the total percentage of activity was 71.5% during the dark phase of the cycle. The reddish-grey musk shrew possesses a strong circadian rhythm of locomotor activity that predominantly occurs during the dark phase and consequently the musk shrew may essentially be considered a nocturnal mammal.

Knowing when to stop: Rhythms of locomotor activity in the high-shore limpet, Cellana grata Gould

The high shore limpet, Cellana grata, forages whilst awash, moving upshore with the rising tide and retreating downshore on the ebbing tide to become inactive in refuges. Spraying inactive, emersed individuals with seawater at low tide invokes a locomotory response, with limpets moving up the shore. Controlled laboratory experiments under continuous white or red light (to simulate light or dark periods respectively) and continuous emersion, immersion or seawater spray showed that C. grata possesses a free-running endogenous rhythm of locomotor activity. This rhythm was maintained over 30 days in continuous seawater spray and white light. Maximum entropy spectral analysis (MESA) revealed two major components to this rhythm, at 7.2 h and 12.4 h. The 12.4 h component is of a circatidal nature and appears to initiate activity, allowing individuals to anticipate immersion by the incoming tide, although this clock can be over-ridden by strong wave splash or spraying vigorously with seawater. The 7.2 h period, however, was the most significant component and is suggested to act as a stopwatch enabling the limpet to assess the duration of each foraging excursion in order to prevent being stranded at the wrong height on the shore. The environmental stimulus for both components of the endogenous rhythm in C. grata appears to be the time of first exposure to wave wash from the incoming tide. C. grata, therefore, has behavioural rhythms entrained to initiate and also terminate activity, which play a role in the limpet maintaining a fixed vertical level on the shore when inactive.

Double-stranded RNA-mediated suppression of Period2 expression in the suprachiasmatic nucleus disrupts circadian locomotor activity in rats

Circadian behavioral rhythms in mammals are controlled by a central clock located in the suprachiasmatic nucleus (SCN). PER2, the protein product of the clock gene, Period 2 (Per2), is expressed rhythmically in the SCN [Beaule C, Houle LM, Amir S (2003) Expression profiles of PER2 immunoreactivity within the shell and core regions of the rat suprachiasmatic nucleus: Lack of effect of photic entrainment and disruption by constant light. J Mol Neurosci 21:133-148] and has been implicated in the control of circadian behavioral rhythms based on the evidence that genetic mutations in Per2 abolish free running locomotor activity rhythms in mice [Zheng B, Larkin DW, Albrecht U, Sun ZS, Sage M, Eichele G, Lee CC, Bradley A (1999) The mPer2 gene encodes a functional component of the mammalian circadian clock. Nature 400:169-173; Bae K, Jin X, Maywood ES, Hastings MH, Reppert SM, Weaver DR (2001) Differential functions of mPer1, mPer2, and mPer3 in the SCN circadian clock. Neuron 30:525-536]. Such mutations eradicate PER2 expression in the SCN and disrupt the SCN molecular clockwork, however, they also affect PER2 in the rest of the brain and body leaving open the possibility that the changes in behavioral rhythms might be influenced, at least in part, by disruptions in PER2 functioning outside the SCN. We used RNA interference-mediated transient knockdown of Per2 to study the effect of selective suppression of PER2 expression in the SCN, per se, on behavioral circadian rhythms. We found that transient suppression of PER2 in the SCN disrupted free running locomotor activity rhythms for up to 10 days in rats. Infusions of control dsRNA into the SCN or infusions of dsRNA to Per2 immediately dorsal to the SCN had no effect. These results constitute evidence for a direct link between PER2 expression in the SCN and the expression of behavioral circadian rhythms in mammals.

Electrical Silencing of PDF Neurons Advances the Phase of non-PDF Clock Neurons inDrosophila

Drosophila clock neurons exhibit self-sustaining cellular oscillations that rely in part on rhythmic transcriptional feedback loops. We have previously determined that electrical silencing of the pigment dispersing factor (PDF)-expressing lateral-ventral (LN(V)) pacemaker subset of fly clock neurons via expression of an inward-rectifier K(+) channel (Kir2.1) severely disrupts free-running rhythms of locomotor activity-most flies are arrhythmic and those that are not exhibit weak short-period rhythms-and abolishes LN(V) molecular oscillation in constant darkness. PDF is known to be an important LN(V) output signal. Here we examine the effects of electrical silencing of the LN(V) pacemakers on molecular rhythms in other, nonsilenced, subsets of clock neurons. In contrast to previously described cell-autonomous abolition of free-running molecular rhythms, we find that electrical silencing of the LN(V) pacemakers via Kir2.1 expression does not impair molecular rhythms in LN(D), DN1, and DN2 subsets of clock neurons. However, free-running molecular rhythms in these non-LN(V) clock neurons occur with advanced phase. Electrical silencing of LN(V)s phenocopies PDF null mutation (pdf (01) ) at both behavioral and molecular levels except for the complete abolition of free-running cellular oscillation in the LN(V)s themselves. LN(V) electrically silenced or pdf 01 flies exhibit weak free-running behavioral rhythms with short period, and the molecular oscillation in non-LN(V) neurons phase advances in constant darkness. That LN( V) electrical silencing leads to the same behavioral and non-LN( V) molecular phenotypes as pdf 01 suggests that persistence of LN(V) molecular oscillation in pdf 01 flies has no functional effect, either on behavioral rhythms or on non-LN(V) molecular rhythms. We thus conclude that functionally relevant signals from LN(V)s to non-LN(V) clock neurons and other downstream targets rely both on PDF signaling and LN(V) electrical activity, and that LN( V)s do not ordinarily send functionally relevant signals via PDF-independent mechanisms.