Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such behavior is spontaneous locomotor activity. Here we describe our protocol that utilizes Drosophila population monitors and a tracking system that allows continuous monitoring of the spontaneous locomotor activity of flies for several days at a time. This method is simple, reliable, and objective and can be used to examine the effects of aging, sex, changes in caloric content of food, addition of drugs, or genetic manipulations that mimic human diseases.     

Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila

Most life forms exhibit daily rhythms in cellular, physiological and behavioral phenomena that are driven by endogenous circadian (≡24 hr) pacemakers or clocks. Malfunctions in the human circadian system are associated with numerous diseases or disorders. Much progress towards our understanding of the mechanisms underlying circadian rhythms has emerged from genetic screens whereby an easily measured behavioral rhythm is used as a read-out of clock function. Studies using Drosophila have made seminal contributions to our understanding of the cellular and biochemical bases underlying circadian rhythms. The standard circadian behavioral read-out measured in Drosophila is locomotor activity. In general, the monitoring system involves specially designed devices that can measure the locomotor movement of Drosophila. These devices are housed in environmentally controlled incubators located in a darkroom and are based on using the interruption of a beam of infrared light to record the locomotor activity of individual flies contained inside small tubes. When measured over many days, Drosophila exhibit daily cycles of activity and inactivity, a behavioral rhythm that is governed by the animal''s endogenous circadian system. The overall procedure has been simplified with the advent of commercially available locomotor activity monitoring devices and the development of software programs for data analysis. We use the system from Trikinetics Inc., which is the procedure described here and is currently the most popular system used worldwide. More recently, the same monitoring devices have been used to study sleep behavior in Drosophila. Because the daily wake-sleep cycles of many flies can be measured simultaneously and only 1 to 2 weeks worth of continuous locomotor activity data is usually sufficient, this system is ideal for large-scale screens to identify Drosophila manifesting altered circadian or sleep properties.     

Quantification of Locomotion and the Effect of Food Deprivation on Locomotor Activity in Drosophila

A new method to quantify locomotor behavior in Drosophila is presented, and compared with previous methods. It is based upon a radar wave, reflected by moving flies. A problem associated with the new apparatus is that its output is dependent on fly size. However, for the case the weight of the experimental flies has been determined, a correction is proposed. The method has been used by studying the effect of starvation upon locomotion in Drosophila melanogaster. It was found that starved flies are much more active than well fed flies. The importance of this effect under several conditions is discussed.     

The Dual-Oscillator System of Drosophila melanogaster Under Natural-Like Temperature Cycles

Dual-oscillator systems that control morning and evening activities can be found in a wide range of animals. The two coupled oscillators track dawn and dusk and flexibly adapt their phase relationship to seasonal changes. This is also true for the fruit fly Drosophila melanogaster that serves as model organism to understand the molecular and anatomical bases of the dual-oscillator system. In the present study, the authors investigated which temperature parameters are crucial for timing morning and evening activity peaks by applying natural-like temperature cycles with different daylengths. The authors found that the morning peak synchronizes to the temperature increase in the morning and the evening peak to the temperature decrease in the afternoon. The two peaks did not occur at fixed absolute temperatures, but responded flexibly to daylength and overall temperature level. Especially, the phase of the evening peak clearly depended on the absolute temperature level: it was delayed at high temperatures, whereas the phase of the M peak was less influenced. This suggests that the two oscillators have different temperature sensitivities. The bimodal activity rhythm was absent in the circadian clock mutants Clk^Jrk and cyc⁰¹ and reduced in per⁰¹ and tim⁰¹ mutants. Whereas the activity of Clk^Jrk mutants just followed the temperature cycles, that of per⁰¹ and tim⁰¹ mutants did not, suggesting that these mutants are not completely clockless. This study revealed new characteristics of the dual-oscillator system in Drosophila that were not detected under different photoperiods. (Author correspondence: yoshii@cc.okayama-u.ac.jp)     

Social Interactions and the Circadian Rhythm in Locomotor Activity in the Cockroach Leucophaea maderae

The role of social interactions in entrainment has not been extensively studied in the invertebrates. Leucophaea maderae is a gregarious species of cockroach that exhibits extensive social interactions. Social interactions associated with copulation between the sexes have been shown to be regulated by the circadian system. We show here that social interactions between males are also under circadian control. We examined the question of whether or not these rhythmic social contacts could function as zeitgebers capable of regulating circadian phase and period. Animals initially in phase that were housed as groups or pairs of single sex or mixed sex in constant darkness for 2–7 weeks were found to drift out of phase. Their behavior was not significantly different from individual animals maintained in isolation. Further, animals that were initially out of phase by 12 h housed as groups or pairs were not significantly different in phase from animals that were isolated. The results show the circadian clocks of cockroaches are remarkably insensitive to the extensive social interactions that occur between individuals.     

Cyclic Presence and Absence of Conspecifics Alters Circadian Clock Phase But Does Not Entrain the Locomotor Activity Rhythm of the Fruit Fly Drosophila melanogaster

Circadian clocks use a wide range of environmental cues, including cycles of light, temperature, food, and social interactions, to fine-tune rhythms in behavior and physiology. Although social cues have been shown to influence circadian clocks of a variety of organisms including the fruit fly Drosophila melanogaster, their mechanism of action is still unclear. Here, the authors report the results of their study aimed at investigating if daily cycles of presence and absence (PA) of conspecific male visitors are able to entrain the circadian locomotor activity rhythm of male hosts living under constant darkness (DD). The results suggest that PA cycles may not be able to entrain circadian locomotor activity rhythms of Drosophila. The outcome does not change when male hosts are presented with female visitors, suggesting that PA cycles of either sex may not be effective in bringing about stable entrainment of circadian clocks in D. melanogaster. However, in hosts whose clock phase has already been set by light/dark (LD) cycles, daily PA cycles of visitors can cause measurable change in the phase of subsequent free-running rhythms, provided that their circadian clocks are labile. Thus, the findings of this study suggest that D. melanogaster males may not be using cyclic social cues as their primary zeitgeber (time cue) for entrainment of circadian clocks, although social cues are capable of altering the phase of their circadian rhythms. (Author correspondence: vsharma@jncasr.ac.in, vksharmas@gmail.com)     

Novel masking effects of light are revealed in Drosophila by skeleton photoperiods

Here, we show that in a skeleton photoperiod where all midday light is removed from a standard laboratory 12:12 LD photoperiod, a large diurnal peak of activity is revealed that is continuous with the E peak seen in constant dark (DD). We further show that the circadian clock gene tim regulates light-dependent masking of daytime activity, but the clock gene per does not. Finally, relative to wild-type flies, mutants for both of these clock genes showed increased nighttime activity in the skeleton photoperiod but not in the standard photoperiod. This result suggests that nighttime activity is suppressed by the intact circadian clock, and in its absence, by exposure to a standard photoperiod. These results support and extend the literature addressing the complex interactions between masking and clock-controlled components of overt circadian rhythms.     

Multi-Oscillatory Control of Eclosion and Oviposition Rhythms in Drosophila melanogaster: Evidence from Limits of Entrainment Studies

The eclosion and oviposition rhythms of flies from a population of Drosophila melanogaster maintained under constant conditions of the laboratory were assayed under constant light (LL), constant darkness (DD), and light/dark (LD) cycles of 10:10 h (T20), 12:12 h (T24), and 14:14 h (T28). The mean (±95% confidence interval; CI) free-running period (τ) of the oviposition rhythm was 26.34 ± 1.04 h and 24.50 ± 1.77 h in DD and LL, respectively. The eclosion rhythm showed a τ of 23.33 ± 0.63 h (mean ± 95% CI) in DD, and eclosion was not rhythmic in LL. The τ of the oviposition rhythm in DD was significantly greater than that of the eclosion rhythm. The eclosion rhythm of all 10 replicate vials entrained to the three periodic light regimes, T20, T24, and T28, whereas the oviposition rhythm of only about 24 and 41% of the individuals entrained to T20 and T24 regimes, respectively, while about 74% of the individuals assayed in T28 regimes showed entrainment. Our results thus clearly indicate that the τ and the limits of entrainment of eclosion rhythm are different from those of the oviposition rhythm, and hence this reinforces the view that separate oscillators may regulate these two rhythms in D. melanogaster.     

Multi-Oscillatory Control of Eclosion and Oviposition Rhythms in Drosophila melanogaster: Evidence from Limits of Entrainment Studies

The eclosion and oviposition rhythms of flies from a population of Drosophila melanogaster maintained under constant conditions of the laboratory were assayed under constant light (LL), constant darkness (DD), and light/dark (LD) cycles of 10:10 h (T20), 12:12 h (T24), and 14:14 h (T28). The mean (±95% confidence interval; CI) free-running period (τ) of the oviposition rhythm was 26.34 ± 1.04 h and 24.50 ± 1.77 h in DD and LL, respectively. The eclosion rhythm showed a τ of 23.33 ± 0.63 h (mean ± 95% CI) in DD, and eclosion was not rhythmic in LL. The τ of the oviposition rhythm in DD was significantly greater than that of the eclosion rhythm. The eclosion rhythm of all 10 replicate vials entrained to the three periodic light regimes, T20, T24, and T28, whereas the oviposition rhythm of only about 24 and 41% of the individuals entrained to T20 and T24 regimes, respectively, while about 74% of the individuals assayed in T28 regimes showed entrainment. Our results thus clearly indicate that the τ and the limits of entrainment of eclosion rhythm are different from those of the oviposition rhythm, and hence this reinforces the view that separate oscillators may regulate these two rhythms in D. melanogaster.     

Locomotor activity is regulated by D2-like receptors in Drosophila: an anatomic and functional analysis

In mammals, dopamine 2-like receptors are expressed in distinct pathways within the central nervous system, as well as in peripheral tissues. Selected neuronal D2-like receptors play a critical role in modulating locomotor activity and, as such, represent an important therapeutic target (e.g. in Parkinson's disease). Previous studies have established that proteins required for dopamine (DA) neurotransmission are highly conserved between mammals and the fruit fly Drosophila melanogaster. These include a fly dopamine 2-like receptor (DD2R; Hearn et al. PNAS 2002 99(22):14554) that has structural and pharmacologic similarity to the human D2-like (D2R). In the current study, we define the spatial expression pattern of DD2R, and functionally characterize flies with reduced DD2 receptor levels. We show that DD2R is expressed in the larval and adult nervous systems, in cell groups that include the Ap-let cohort of peptidergic neurons, as well as in peripheral tissues including the gut and Malpighian tubules. To examine DD2R function in vivo, we generated RNA-interference (RNAi) flies with reduced DD2R expression. Behavioral analysis revealed that these flies show significantly decreased locomotor activity, similar to the phenotype observed in mammals with reduced D2R expression. The fly RNAi phenotype can be rescued by administration of the DD2R synthetic agonist bromocriptine, indicating specificity for the RNAi effect. These results suggest Drosophila as a useful system for future studies aimed at identifying modifiers of dopaminergic signaling/locomotor function.     

Varying the Length of Dim Nocturnal Illumination Differentially Affects the Pacemaker Controlling the Locomotor Activity Rhythm of Drosophila Jambulina

Photic entrainment of animals in the field is basically attributed to their exposure to the dimly lit nights flanked by the dawn and dusk twilight transitions. This implicates the functional significance of the dimly lit nights as that of the twilight transitions. Recently, the authors have demonstrated that the dimly lit night at 0.0006 lux altered the attributes of the circadian rhythm of locomotor activity of Drosophila jambulina. The present study examined whether the durations of such dimly lit nights affect the entrainment and free-running rhythmicity of D. jambulina. Flies were subjected for 10 days to two types of 24-h lighting regimes in which the photophase (L) was at 10 lux for all flies but the scotophase, which varied in duration from 9 to 15?h, was either at 0 lux (D phase) for control flies or 0.0006 lux (the artificial starlight or S phase) for experimental flies. Thereafter, they were transferred to constant darkness (DD) to compare the after-effects of the dimly lit nights on the period (τ) of free-running rhythm in DD with that of the completely dark nights. Control flies were entrained by all LD cycles, but the experimental flies were entrained only by five LS cycles in which the duration of the S phases ranged from 10 to 14?h. The two LS cycles with very short (9?h) and long (15?h) S phases rendered the flies completely arrhythmic. Control flies started activity shortly before lights-on and continued well after lights-off. The experimental flies, however, commenced activity several hours prior to lights-on but ended activity abruptly at lights-off as the result of a negative masking effect of nocturnal illumination. Length of the midday rest was considerably shorter in the control than in the experimental flies in each lighting regime. The active phase in the control flies was predictably shortened; nonetheless, it was invariable in the experimental flies as the nights lengthened. Transfer from lighting regimes to DD initiated robust free-running rhythmicity in all flies including the arrhythmic ones subjected to LS cycles with 9 and 15?h of scotophases. The τ was profoundly affected by the nocturnal irradiance of the prior entraining lighting regime, as it was always shorter in the experimental than in the control flies. Thus, these results indisputably demonstrate the changes in fundamental properties of the circadian pacemaker of D. jambulina were solely attributed to the extremely dim nocturnal irradiance. This strain of D. jambulina is entrained essentially by the dimly lit natural nights, since it is never exposed to the prevailing photic cues such as the twilight transitions or bright photoperiod, owing to the dense vegetation of its habitat. (Author correspondence: drdsjoshi08@gmail.com)     

Period-Dependent Oscillatory Free-Run in the Locomotor Activity Rhythm of the Field Mouse Mus booduga Under Skeleton Photoperiodic Regimes

The entrainment behaviour of the circadian rhythm of locomotor activity in the field mouse Mus booduga was studied in order to evaluate the role of the animals' free-running period (τ) and the duration of skeleton photoperiods in determining entrainment of animals with τ values beyond and close to the “limits of entrainment”. We predicted that animals with τ lesser than the lower “limit of entrainment” would entrain only to short skeleton photoperiods (≤ 6 h) and not to longer skeleton photoperiods. Experimental animals (n = 25) were entrained to light/dark (LD) 12:12 h schedule, and then subjected to various skeleton photoperiods in which the duration of one of the two intervals of darkness was successively reduced while holding the zeitgeber period (T) constant. Some animals (n = 9) entrained to long as well as short photoperiods, whereas others (n = 5) entrained only to extremely short skeleton photoperiods of 6 h or less. The mean τ of the animals entraining to all photoperiods (23.78 ± 0.22 h) was significantly greater than that of the animals that entrained only to very short skeleton photoperiods (22.43 ± 0.41 h) (t _{df 12} = 5.3, p < 0.001). We also selected a few animals (n = 11) with average τ value of 23.13 ± 0.38 h and studied them under several skeleton photoperiods. To our surprise the animals which were subjected to restricted dark intervals invariably underwent “phase-jump” assuming the longer dark interval as “subjective night”. We suggest that the observed variation in entrainment behaviour might be due to the variation seen among individual animals in τ and the shape of their PRC. These results support the view that the duration of the skeleton photoperiod and the τ of an individual animal interact to determine its entrainment, and underscore the relevance of inter-individual variation in circadian organisation to studies of circadian rhythms.     

Entrainment of Eclosion Rhythm in Drosophila melanogaster Populations Reared for More Than 700 Generations in Constant Light Environment

In this paper, we report the results of our extensive study on eclosion rhythm of four independent populations of Drosophila melanogaster that were reared in constant light (LL) environment of the laboratory for more than 700 generations. The eclosion rhythm of these flies was assayed under LL, constant darkness (DD) and three periodic light‐dark (LD) cycles (T20, T24, and T28). The percentage of vials from each population that exhibited circadian rhythm of eclosion in DD and in LL (intensity of approximately 100 lux) was about 90% and 18%, respectively. The mean free‐running period (τ) of eclosion rhythm in DD was 22.85 ± 0.87 h (mean ± SD). Eclosion rhythm of these flies entrained to all the three periodic LD cycles, and the phase relationship (ψ) of the peak of eclosion with respect to “lights‐on” of the LD cycle was significantly different in the three periodic light regimes (T20, T24, and T28). The results thus clearly demonstrate that these flies have preserved the ability to exhibit circadian rhythm of eclosion and the ability to entrain to a wide range of periodic LD cycles even after being in an aperiodic environment for several hundred generations. This suggests that circadian clocks may have intrinsic adaptive value accrued perhaps from coordinating internal metabolic cycles in constant conditions, and that the entrainment mechanisms of circadian clocks are possibly an integral part of the clockwork.     

Genetic Variants Affecting Phenoloxidase Activity in Drosophila melanogaster

In Drosophila melanogaster, two new variants affecting the activity of phenoloxidase were found in natural populations at Gomel in Belorussia and at Krasnodar in Russia. Prophenoloxidases, A ₁ and A ₃ , in these variants had the same mobilities on native electrophoresis as the wild type. However, enzymatic activities in their activated states were much lower than in the wild type, whereas the existence of prophenoloxidase proteins was demonstrated. Egg-to-adult and relative viabilities in the variants did not decrease at temperatures between 18 and 29°C. Genetic analyses indicated that the genes showing the phenotype of variants are new alleles of Mox and Dox-3 on the second chromosome.     

Possible Mechanism of Interaction of GABAergic-Adenosinergic Systems in the Regulation of Theophylline-Induced Locomotor Activity Under Its Nontolerant and Tolerant Conditions

The measurement of locomotor activity (LA) of theophylline (Th) nontolerant (10 mg/kg, p.o.) rats using agonist and antagonist of different neurotransmitters either in single or in their different combinations suggest that an inhibition of central GABAergic activity as well as adenosinergic and serotonergic activities through the stimulation of dopaminergic activity followed by an inhibition of cholinergic system may stimulate LA in Th nontolerant condition. Further, it is suggested that the development of tolerance to Th restored the LA to control value may be due to an activation of adenosinergic system which possibly withdrew the inhibition occurred in central cholinergic, GABAergic and serotonergic activities followed by the modulation of dopaminergic system.     

果蝇昼夜节律的分子机制研究进展

果蝇由于遗传易操作性而成为一个研究昼夜节律分子机制的理想模式生物 . 到目前为止,通过遗传学和生物化学方法已经鉴定到 10 多个时钟基因 (clock genes) 和许多时钟相关基因,包括时钟输入基因和钟控基因 . 这些时钟基因以及它们的相应产物组成两个互相依赖的转录 / 翻译反馈环路,从而调节行为和生理的昼夜节律 . 果蝇这种核心钟的工作原理同样见于哺乳动物 .     

Reduced Enzyme Activity Following Hsp70 Overexpression in Drosophila melanogaster

Acclimation to environmental change can impose costs to organisms. One potential cost is the change in cell metabolism that follows a physiological response, e.g., high expression of heat shock proteins may alter specific activity of important enzymes. We examined the significance of this cost in a pair of Drosophila melanogaster lines transformed with additional copies of a gene that encodes the heat shock protein, Hsp70. Heat shock induces Hsp70 expression in all lines, but lines with extra copies produce much more Hsp70 than do excision control strains. The consequence of this supranormal Hsp70 expression is to reduce specific activity of both enzymes analyzed, adult alcohol dehydrogenase (ADH), which is heat sensitive, and lactate dehydrogenase, which is not. Strain differences were most pronounced under those conditions where Hsp70 expression was maximized, and not where the heat stress denatured proteins. That result supported the idea that Hsp70 expression is constrained evolutionarily by its tendency to bind nascent peptides when overabundant within the cell.     

鱼藤酮对果蝇运动行为及果蝇头部多巴胺合成相关酶基因表达的影响

为了探讨鱼藤酮对黑腹果蝇Drosophila melanogaster运动行为的影响与其头部多巴胺水平之间的关系,我们测定了鱼藤酮对黒腹果蝇成虫运动行为、头部多巴胺水平及酪氨酸羟化酶和多巴脱羧酶基因表达的影响。结果表明：与取食未加入药剂饲料的果蝇相比雌成虫用0.2～0.8 mmol/L、雄成虫用0.1～0.8 mmol/L浓度药液配制的饲料连续饲养6 d后运动能力显著下降,在0.8 mmol/L浓度下雌、雄成虫的运动能力分别仅为对照的55.6%和49.1%。取食用0.8 mmol/L浓度药液配制饲料6,12和21 d的果蝇雌、雄成虫头部多巴胺水平均显著下降,雌成虫头部多巴胺水平分别为对照雌成虫的83.2%,72.3%和59.8%;雄成虫头部多巴胺水平分别为对照雄成虫的79.3%,66.8%和53.2%。用0.8 mmol/L浓度鱼藤酮处理6,12和21d,雌成虫头部酪氨酸羟化酶基因（pale）的表达水平分别为对照的76.3%,51.4%和37.3%,多巴脱羧酶基因（Ddc）的表达水平分别为对照的87.1%,78.2%和63.5%, 均显著下降。结果提示,鱼藤酮可干扰果蝇成虫头部酪氨酸羟化酶和多巴脱羧酶基因的表达,导致果蝇头部多巴胺水平下降,进而影响了果蝇的运动行为。     

Evidence for genomic regulation of the telomeric activity in Drosophila melanogaster

The structural integrity of TART elements has been used as reporter of instability at chromosomal ends in numerous Drosophila stocks and over time in an unstable stock. The results show that telomeric activity is a regulated process that may differ between the stocks as well as over time within a stock. This revised version was published online in July 2006 with corrections to the Cover Date.     

Investigation of Natural Genetic Variation in the Circadian System of Drosophila melanogaster. II. Biometrical Analyses of Locomotor Activity Rhythms Recorded in Constant Darkness

The locomotor activity rhythms of 24 isochromosomal strains of Drosophila melanogaster were recorded in constant conditions, using an experimental design suitable for the serial screening with so many strains using limited equipment. The data obtained were subjected to biometrical genetic analysis. The results show that four characteristics of the rhythms (period, phase, definition, and waveform) display genetically based variation in expression and that each appears to be affected by a range of genes.