Giant Neuron Pathway Neurophysiological Activity in Per0 Mutants of Drosophila Melanogaster

In Drosophila melanogaster, the clock gene period (per) has a clearly defined role in the molecular machinery involved in generating free-running circadian rhythms, per mutations also influence rhythms in the Drosophila love song and in the ultradian timescale. The relationship between these two phenomena has so far escaped satisfactory explanation. Here we analyzed the neurophysiological activity of the giant fiber neural pathway in per⁰ flies. Under constant light, and at relatively low stimulation frequencies (1-2 Hz), per⁰¹ flies habituate significantly earlier than they do under 12 h light-dark cycles. The results suggest an involvement of per in phenomena of short-term neural plasticity.     

Restoration of circadian behavioural rhythms in a period null Drosophila mutant (per01) by mammalian period homologues mPer1 and mPer2

BACKGROUND: Recent molecular studies suggest that mammals and Drosophila utilize similar components to generate circadian (approximately equal to 24 h) rhythms. The first identified circadian clock gene, the period (per) gene, is indispensable for behavioural rhythms in Drosophila and is represented in mammals by three orthologues, the relative roles of which are not known. In this study, we investigated the functional conservation of per by introducing the mouse mPer1 and mPer2 genes, driven by the Drosophila timeless (tim) promoter, into Drosophila melanogaster. RESULTS: Behavioural assays showed that both mPer constructs restored rhythms in per(01) flies that are otherwise arrhythmic due to a lack of endogenous per protein (PER). However, the rhythms restored by mPer2 were generally stronger and differed in periodicity from those restored by mPer1. In rhythmic transgenic flies, mPER proteins were expressed in lateral neurones and/or many cells in optic lobe. In addition, cell culture experiments indicated that the Drosophila PER partner, TIM, can form a complex with each of these two mammalian proteins. CONCLUSIONS: This study demonstrates that both mPer1 and mPer2 can function as clock components, and has implications for a functional analysis of the different per genes.     

Genetic dissection of the circadian clock: A timeless story

The period (per) gene is known to play a critical role in the generation and/or maintenance of circadian rhythms in Drosophila. Efforts to identify other molecular components of pacemaking pathways recently led to the isolation of a new clock mutation, timeless (tim). Circadian rhythms are eliminated in tim flies, probably due to the effect tim has on the subcellular localization of period protein (PER). Data indicate that the putative product(s) of the tim locus is essential for timekeeping in Drosophila. In this article I will review the isolation and phenotype of the tim mutation and also discuss its interaction with per.     

DCRY is a Drosophila photoreceptor protein implicated in light entrainment of circadian rhythm

BACKGROUND: Light is the major environmental signal for the entrainment of circadian rhythms. In Drosophila melanogaster, the period(per) and timeless (tim) genes are required for circadian behavioural rhythms and their expression levels undergo circadian fluctuations. Light signals can entrain these rhythms by shifting their phases. However, little is known about the molecular mechanism for the perception and transduction of the light signal. The members of the photolyase/cryptochrome family contain flavin adenine dinucleotide (FAD) as chromophore and are involved in two diverse functions, DNA repair and photoreception of environmental light signals. RESULTS: We report the cloning of a new member of this family, dcry, from Drosophila. Northern blot analysis shows that this gene is expressed in various tissues. The dcry mRNA is expressed in a circadian manner in adult heads, while such rhythmic fluctuation is abolished in the clock-defective per0 and tim0 mutants. The circadian expression is dampened down in constant darkness. The over-expression of the dcry gene alters the light-induced phase delay in the locomotor activity rhythms of flies. CONCLUSION: These results suggest that DCRY is a circadian photoreceptor and that its expression is regulated by circadian clock genes.     

High-resolution analysis of locomotor activity rhythms indisconnected,a visual-system mutant ofDrosophila melanogaster

Free-running locomotor activity and eclosion rhythms ofDrosophila melanogaster, mutant at thedisconnected (disco) locus, are substantially different from the wild-type phenotype. Initial periodogram analysis revealed little or no rhythmicity (Dushayet al., 1989). We have reanalyzed the locomotor activity data using high-resolution signal analysis (maximum-entropy spectral analysis, or MESA). These analyses, corroborated by autocorrelograms, uncovered significant residual circadian rhythmicity and strong ultradian rhythms in most of the animals tested. In this regard thedisco mutants are much like flies expressing mutant alleles of theperiod gene, as well as wild-type flies reared throughout life in constant darkness. We hypothesize that light normally triggers the coupling of multiple ultradian oscillators into a functional circadian clock and that this process is disrupted indisco flies as a result of the neural lesion.This work was supported in part by NIH Grant FM-33205.     

Why a fly? Using Drosophila to understand the genetics of circadian rhythms and sleep

Among simple model systems, Drosophila has specific advantages for neurobehavioral investigations. It has been particularly useful for understanding the molecular basis of circadian rhythms. In addition, the genetics of fruit-fly sleep are beginning to develop. This review summarizes the current state of understanding of circadian rhythms and sleep in the fruit fly for the readers of Sleep. We note where information is available in mammals, for comparison with findings in fruit flies, to provide an evolutionary perspective, and we focus on recent findings and new questions. We propose that sleep-specific neural activity may alter cellular function and thus accomplish the restorative function or functions of sleep. In conclusion, we sound some cautionary notes about some of the complexities of working with this "simple" organism.     

An antibody to the Drosophila period protein labels antigens in the suprachiasmatic nucleus of the rat.

Cell bodies in the rat suprachiasmatic nucleus (SCN) were labeled with an antibody against a small domain of the period (per) protein, the product of a gene in Drosophila that regulates circadian rhythms. In immunoblots of SCN protein extracts, the antibody recognized a band of approximately 115 kD, as well as a heterogeneous antigen ranging from 160 kD to 170 kD. The antibody was found in previous studies to label putative circadian pacemaker neurons in Aplysia and Bulla, as well as the cellular sites of per expression in flies. Taken together, these results suggest that the region of the per protein recognized by this antibody may be widely conserved in neuronal circadian pacemakers.     

Characterization of Andante, a new Drosophila clock mutant, and its interactions with other clock mutants

A new clock mutant, named Andante, has been identified on the X chromosome of Drosophila melanogaster. Andante lengthens the period of the circadian eclosion and locomotor activity rhythms by 1.5-2.0 hours. The phase response curves for the eclosion and activity rhythms, indicating light-induced phase shifts, show a similar degree of lengthening. Andante also lengthens the periods of other clock mutants, including Clock, and alleles of the period locus. Analysis of locomotor activity rhythms reveals that Andante is semi-dominant, and Andante rhythms are highly temperature compensated. The sine oculis mutation, which eliminates the outer visual system, has no effect on the period of Andante. Deficiency mapping indicates that Andante is located in the 1OE1-2 to 1OF1 region of the X chromosome, close to the miniature-dusky locus. Whereas Andante flies have a dusky wing phenotype, dusky flies do not have an Andante clock phenotype.     

Mapping the clock rhythm mutation to the period locus of Drosophila melanogaster by germline transformation.

The Clock (Clk) mutation shortens circadian rhythms of locomotor activity and eclosion from ca. 24 h to 22.5-23 h. Clk was previously mapped, by meiotic recombination, very close to the period(per) locus on the X chromosome. To determine whether Clk is a mutation within the per gene or if the former is separate from the latter, two overlapping genomic fragments were cloned from Clk flies to produce a per-containing 13.2 kb construct, per01 flies (which by themselves are arrhythmic)--when transformed with this construct--expressed short-period rhythms. This indicates that the Clk mutation is contained within this 13.2 kb region and is almost certainly a new "fast-clock" allele of per.     

Cocaethylene synthesis in Drosophila

Cocaethylene is an active cocaine metabolite that targets mammalian neural reward pathways and thus contributes to the reinforcing and addictive properties of ethanol and cocaine. Using gas chromatography-mass spectrometry, we find that fruit flies (Drosophila melanogaster) possess a cellular mechanism through which cocaine can be converted to cocaethylene, presumably via ethanol-sensitive enzymes. These findings illustrate the striking similarity of gene products in humans and flies, which might reflect a homologous role in the metabolic inactivation of cocaine. Further, this conservation of metabolic steps suggests that Drosophila can be used to study cellular, molecular and biochemical processes leading to polydrug abuse and addiction.     

Mosaic analysis in the Drosophila CNS of circadian and courtship-song rhythms affected by a period clock mutation

The period gene in Drosophila melanogaster controls not only daily rhythms associated with adult emergence and behavior, but also a much higher frequency rhythm that accompanies the male's courtship song. This oscillation in the rate of sound production (normal period, ca. one minute) is either sped up (by perS), slowed down, or eliminated in the three classic per mutants. We have conducted a mosaic analysis in which both lovesong and circadian locomotor cycles were examined in a series of flies that were each part perS and part per+. Consistent with previous studies, the focus for per control of the adult's circadian rhythm of locomotion was found to be in the brain. However, several mosaic individuals were found to exhibit a mutant locomotor rhythm but a wild-type song cycle, or vice-versa, enabling us provisionally to map the song-rhythm focus to the thoracic ganglia. That per is expressed only in glial cells in the thoracic nervous system and, in general, mediates slow (hour-by-hour) fluctuations in the levels of its own products are discussed from the standpoint of the current mosaic mapping results and the renewed focus they bring to the gene's influence on an ultradian rhythm.     

Analysis of period circadian expression in the Drosophila head by in situ hybridization

The expression of the period (per) gene of Drosophila melanogaster has been studied by in situ hybridization in the adult's head, where it is required for the fly to exhibit behavioral circadian rhythms. We have used non-radioactive in situ hybridization to obtain a high sensitivity and specificity on head sections, with single cell resolution. Consistent with previous per protein- or per reporter gene-expression, per-expressing cells were detected in the optic lobes and the central brain, as well as in the head sensory organs: eyes, ocelli, maxillary palps and proboscis. In the brain and the eyes, circadian fluctuations of the per mRNA abundance were observed in different per expressing cells.     

Mushroom body influence on locomotor activity and circadian rhythms in Drosophila melanogaster

Whether or not mechanisms underlying circadian locomotor rhythms and learning are related anatomically through the mushroom bodies (MBs) was investigated by monitoring behavioral rhythmicity in flies with MB lesions induced by chemical ablation and by mutations in five different genes. All flies tested were later examined histologically to assess (1) MB neuroanatomy, and (2) the condition of the putative pacemaker cells--the ventral Lateral Neurons (LN(v)s) and their terminals that project to the vicinity of the MB calyces. All groups of flies had normal rhythms except for mushroom body miniature (mbm; only in a wild-type Berlin genetic background) and mushroom body defect (mud). MB ablation had no effect on the gender-specific differences in the rhythmic activity profile that are typical of wild-type flies. However, ablated males had a slightly longer period than untreated males and were more active under constant dark conditions. LN(v)s and their arborization patterns appeared normal in MB-ablated and in most mutant flies. Activity defects of mbm flies were attributed to genetic background rather than to the mutation alone. Misrouted LN(v) projections and approximately 14% arrhythmia of mud flies were uncorrelated and attributed to pleiotropy rather than to specific effects of MB lesions. Our results imply that MBs are not involved in circadian activity rhythms but that they do have an inhibitory effect on activity levels of male flies.     

MUSHROOM BODY INFLUENCE ON LOCOMOTOR ACTIVITY AND CIRCADIAN RHYTHMS IN DROSOPHILA MELANOGASTER

Whether or not mechanisms underlying circadian locomotor rhythms and learning are related anatomically through the mushroom bodies (MBs) was investigated by monitoring behavioral rhythmicity in flies with MB lesions induced by chemical ablation and by mutations in five different genes. All flies tested were later examined histologically to assess (1) MB neuroanatomy, and (2) the condition of the putative pacemaker cells -- the ventral Lateral Neurons (LN v s) and their terminals that project to the vicinity of the MB calyces. All groups of flies had normal rhythms except for mushroom body miniature ( mbm ; only in a wild-type Berlin genetic background) and mushroom body defect ( mud ). MB ablation had no effect on the gender-specific differences in the rhythmic activity profile that are typical of wild-type flies. However, ablated males had a slightly longer period than untreated males and were more active under constant dark conditions. LN v s and their arborization patterns appeared normal in MB-ablated and in most mutant flies. Activity defects of mbm flies were attributed to genetic background rather than to the mutation alone. Misrouted LN v projections and ～14% arrhythmia of mud flies were uncorrelated and attributed to pleiotropy rather than to specific effects of MB lesions. Our results imply that MBs are not involved in circadian activity rhythms but that they do have an inhibitory effect on activity levels of male flies.     

Evening circadian oscillator as the primary determinant of rhythmic motivation for Drosophila courtship behavior

Circadian clocks of Drosophila melanogaster motivate males to court females at a specific time of day. However, clock neurons involved in courtship rhythms in the brain of Drosophila remain totally unknown. The circadian locomotor behavior of Drosophila is controlled by morning (M cells) and evening (E cells) cells in the brain, which regulate morning and evening activities, respectively. Here, we identified the brain clock neurons that are responsible for the circadian rhythms of the close-proximity (CP) behavior that reflects male courtship motivation. Interestingly, the ablation or functional molecular clock disruption of E cells caused arrhythmic CP behavior, but that of M cells resulted in sustained CP rhythms even in constant darkness. In addition, the ablation of some dorsal lateral neurons (LNd) of E cells using neuropeptide-F (NPF)-GAL4 did not impair CP rhythms. These findings suggested that the NPF-negative LNds and DN1s of E cells include cells essential for circadian CP behavior in Drosophila.     

Lateralized rhythms of the central and autonomic nervous systems.

This paper reviews lateralized ultradian rhythms in the nervous system and their unique place in evolutionary development. The rhythmic lateralization of neural activity in paired internal structures and the two sides of the central and autonomic nervous system is discussed as a new view for the temporal and spatial organization of higher vertebrates. These lateralized neural rhythms are integral to the hypothesis of the basic rest-activity cycle. Rhythms of alternating cerebral hemispheric dominance are postulated to be coupled to oscillations of the ergotrophic and trophotrophic states. The nasal cycle is coupled to this cerebral rhythm. This lateralized central and autonomic rhythm is discussed in relationship to ultradian rhythms of neuroendocrine activity, REM and NREM sleep, lateralized rhythms of plasma catecholamines, and other lateralized neural events. The relationship of this phenomenon to stress and adaptation is postulated. The effects of unilateral forced nostril breathing is reviewed as a method to alter cerebral activity, cognition, and other autonomic dependent phenomena.     

Sleep homeostasis in Drosophila melanogaster

STUDY OBJECTIVES: The fruit fly Drosophila melanogaster is emerging as a promising model system for the genetic dissection of sleep. As in mammals, sleep in the fruit fly is a reversible state of reduced responsiveness to the external world and has been defined using an array of behavioral, pharmacologic, molecular, and electrophysiologic criteria. A central feature of mammalian sleep is its homeostatic regulation by the amount of previous wakefulness. Dissecting the mechanisms of homeostatic regulation is likely to provide key insights into the functions of sleep. Thus, it is important to establish to what extent sleep homeostasis is similar between mammals and flies. This study was designed to determine whether in flies, as in mammals, (1) sleep rebound is dependent on prior time awake; (2) sleep deprivation affects the intensity, in addition to the duration, of sleep rebound; (3) sleep loss impairs vigilance and performance; (4) the sleep homeostatic response is conserved among different wild-type lines, and between female and male flies of the same line. DESIGN: Motor activity of individual flies was recorded at 1-minute intervals using the infrared Drosophila Activity Monitoring System during 2 baseline days; during 6, 12, and 24 hours of sleep deprivation; and during 2 days of recovery. Sleep was defined as any period of uninterrupted behavioral immobility lasting > 5 minutes. Sleep continuity was measured by calculating the number of brief awakenings, the number and duration of sleep episodes, and a sleep continuity score. Vigilance before and after sleep deprivation was assessed by measuring the escape response triggered by 2 different aversive stimuli. SETTING: Fly sleep research laboratory at UW-Madison. PARTICIPANTS AND INTERVENTIONS: Adult flies of the Canton-S (CS) strain and 116 other wild-type lines (> or = 16 female and > or = 16 male flies per line). MEASUREMENTS AND RESULTS: In wild-type CS flies, as in mammals, the amount of sleep recovered after sleep deprivation was dependent on prior time awake. Relative to baseline sleep, recovery sleep in CS flies was less fragmented, with longer sleep episodes, and was associated with a higher arousal threshold. Sleep deprivation in CS flies also reduced performance. Sleep duration and continuity increased after 24 hour of sleep deprivation in all the other wild-type lines tested. CONCLUSION: The sleep homeostatic response in fruit flies is a stable phenotype and shares most of, if not all, the major features of mammalian sleep homeostasis, thus supporting the use of Drosophila as a model system for the genetic dissection of sleep mechanisms and functions.     

Salience modulates 20-30 Hz brain activity in Drosophila

Fruit flies selectively orient toward the visual stimuli that are most salient in their environment. We recorded local field potentials (LFPs) from the brains of Drosophila melanogaster as they responded to the presentation of visual stimuli. Coupling of salience effects (odor, heat or novelty) to these stimuli modulated LFPs in the 20-30 Hz range by evoking a transient, selective increase. We demonstrated the association of these responses with behavioral tracking and initiated a genetic approach to investigating neural correlates of perception.