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1.  A single pair of neurons links sleep to memory consolidation in Drosophila melanogaster 
eLife  null;4:e03868.
Sleep promotes memory consolidation in humans and many other species, but the physiological and anatomical relationships between sleep and memory remain unclear. Here, we show the dorsal paired medial (DPM) neurons, which are required for memory consolidation in Drosophila, are sleep-promoting inhibitory neurons. DPMs increase sleep via release of GABA onto wake-promoting mushroom body (MB) α'/β' neurons. Functional imaging demonstrates that DPM activation evokes robust increases in chloride in MB neurons, but is unable to cause detectable increases in calcium or cAMP. Downregulation of α'/β' GABAA and GABABR3 receptors results in sleep loss, suggesting these receptors are the sleep-relevant targets of DPM-mediated inhibition. Regulation of sleep by neurons necessary for consolidation suggests that these brain processes may be functionally interrelated via their shared anatomy. These findings have important implications for the mechanistic relationship between sleep and memory consolidation, arguing for a significant role of inhibitory neurotransmission in regulating these processes.
DOI: http://dx.doi.org/10.7554/eLife.03868.001
eLife digest
Sleep affects memory: if you do not sleep well after a learning task, chances are you will not be able to recall whatever you tried to learn earlier. This is seen in almost all animals ranging from the fruit fly Drosophila, to mice and humans. However, the precise details of how memory and sleep are connected remain unclear.
Drosophila is an excellent model for teasing out the connections between memory and sleep. This is because its brain has a simple and well-studied memory region that contains a pair of nerve cells called the dorsal paired medial neurons. These neurons enable memories to be stored for the long term. Here, Haynes et al. asked whether these neurons can also affect sleep, and if so, how.
The experiments show that the dorsal paired medial neurons promote sleep in fruit flies. The neurons release a signaling molecule called GABA, which is detected by a type of neighboring ‘mushroom body’ neuron that usually promotes wakefulness. This leads to increases in the levels of chloride ions in the mushroom body neurons, but no change in the levels of calcium ions and a molecule called cAMP, which indicates that GABA inhibits these cells. Flies that have lower levels of two receptor proteins that detect GABA sleep less than normal flies.
Haynes et al.'s findings suggest that dorsal paired medial neurons deactivate their neighbors to promote sleep in fruit flies. This result was unexpected because current models of memory formation propose that dorsal paired medial neurons can activate the mushroom body neurons. Understanding how inhibiting mushroom body neurons influences memory will require researchers to reassess these models.
DOI: http://dx.doi.org/10.7554/eLife.03868.002
doi:10.7554/eLife.03868
PMCID: PMC4305081  PMID: 25564731
learning and memory; circuits; sleep; D. melanogaster
2.  A big picture of a small brain 
eLife  null;3:e05580.
A detailed map of the neurons that carry information away from the mushroom bodies in the brains of fruit flies has improved our understanding of the ways in which experiences can modify behaviour.
doi:10.7554/eLife.05580
PMCID: PMC4275571  PMID: 25537193
mushroom body; olfactory learning; associative memory; behavioral valence; sleep; D. melanogaster
3.  Up all night on a redeye flight 
eLife  2014;3:e02087.
A protein called RYE has a central role in the regulation of sleep.
doi:10.7554/eLife.02087
PMCID: PMC3912630  PMID: 24497548
Drosophila; sleep; acetylcholine signaling; cycling; sleepless/Lynx-1; homeostasis; D. melanogaster
4.  Neuron-specific protein interactions of Drosophila CASK-β are revealed by mass spectrometry 
Modular scaffolding proteins are designed to have multiple interactors. CASK, a member of the membrane-associated guanylate kinase (MAGUK) superfamily, has been shown to have roles in many tissues, including neurons and epithelia. It is likely that the set of proteins it interacts with is different in each of these diverse tissues. In this study we asked if within the Drosophila central nervous system, there were neuron-specific sets of CASK-interacting proteins. A YFP-tagged CASK-β transgene was expressed in genetically defined subsets of neurons in the Drosophila brain known to be important for CASK function, and proteins present in an anti-GFP immunoprecipitation were identified by mass spectrometry. Each subset of neurons had a distinct set of interacting proteins, suggesting that CASK participates in multiple protein networks and that these networks may be different in different neuronal circuits. One common set of proteins was associated with mitochondria, and we show here that endogenous CASK-β co-purifies with mitochondria. We also determined CASK-β posttranslational modifications for one cell type, supporting the idea that this technique can be used to assess cell- and circuit-specific protein modifications as well as protein interaction networks.
doi:10.3389/fnmol.2014.00058
PMCID: PMC4075472  PMID: 25071438
Drosophila; GAL4/UAS; mass spectrometry; MAGUK; CASK; immunoprecipitation
5.  Regulation of dopamine release by CASK-β modulates locomotor initiation in Drosophila melanogaster 
CASK is an evolutionarily conserved scaffolding protein that has roles in many cell types. In Drosophila, loss of the entire CASK gene or just the CASK-β transcript causes a complex set of adult locomotor defects. In this study, we show that the motor initiation component of this phenotype is due to loss of CASK-β in dopaminergic neurons and can be specifically rescued by expression of CASK-β within this subset of neurons. Functional imaging demonstrates that mutation of CASK-β disrupts coupling of neuronal activity to vesicle fusion. Consistent with this, locomotor initiation can be rescued by artificially driving activity in dopaminergic neurons. The molecular mechanism underlying this role of CASK-β in dopaminergic neurons involves interaction with Hsc70-4, a molecular chaperone previously shown to regulate calcium-dependent vesicle fusion. These data suggest that there is a novel CASK-β-dependent regulatory complex in dopaminergic neurons that serves to link activity and neurotransmitter release.
doi:10.3389/fnbeh.2014.00394
PMCID: PMC4235261  PMID: 25477794
Drosophila melanogaster; neurotransmitter release; CASK; dopamine; locomotion; Hsc70-4
6.  Short neuropeptide F is a sleep-promoting inhibitory modulator 
Neuron  2013;80(1):171-183.
SUMMARY
To advance the understanding of sleep regulation, we screened for sleep-promoting cells and identified neurons expressing neuropeptide Y-like short neuropeptide F (sNPF). Sleep-induction by sNPF meets all relevant criteria. Rebound sleep following sleep deprivation is reduced by activation of sNPF neurons and flies even experience negative sleep rebound upon cessation of sNPF neuronal stimulation, indicating that sNPF provides an important signal to the sleep homeostat. Only a subset of sNPF-expressing neurons, which includes the small ventrolateral clock neurons, is sleep-promoting. Their release of sNPF increases sleep consolidation in part by suppressing the activity of wake-promoting large ventrolateral clock neurons, and suppression of neuronal firing may be the general response to sNPF receptor activation. sNPF acutely increases sleep without altering feeding behavior, which it affects only on a much longer time scale. The profound effect of sNPF on sleep indicates that it is an important sleep-promoting molecule.
doi:10.1016/j.neuron.2013.07.029
PMCID: PMC3792499  PMID: 24094110
7.  Neuromodulatory control of sleep in Drosophila melanogaster: Integration of competing and complementary behaviors 
Current opinion in neurobiology  2013;23(5):819-823.
The transition between wake and sleep states is characterized by rapid and generalized changes in both sensory and motor processing. Sleep is antagonistic to the expression of important behaviors, like feeding, reproduction and learning whose relative importance to an individual will depend on its circumstances at that moment. An understanding of how the decision to sleep is affected by these other drives and how this process is coordinated across the entire brain remains elusive. Neuromodulation is an important regulatory feature of many behavioral circuits and the reconfiguring of these circuits by modulators can have both long-term and short-term consequences. Drosophila melanogaster has become an important model system for understanding the molecular and genetic bases of behaviors and in recent years neuromodulatory systems have been shown to play a major role in regulation of sleep and other behaviors in this organism. The fly, with its increasingly well-defined behavioral circuitry and powerful genetic tools, is a system poised to provide new insight into the complex issue of how neuromodulation can coordinate situation-specific behavioral needs with the brain’s arousal state.
doi:10.1016/j.conb.2013.05.003
PMCID: PMC3783581  PMID: 23743247
8.  Larval Population Density Alters Adult Sleep in Wild-Type Drosophila melanogaster but Not in Amnesiac Mutant Flies 
Brain Sciences  2014;4(3):453-470.
Sleep has many important biological functions, but how sleep is regulated remains poorly understood. In humans, social isolation and other stressors early in life can disrupt adult sleep. In fruit flies housed at different population densities during early adulthood, social enrichment was shown to increase subsequent sleep, but it is unknown if population density during early development can also influence adult sleep. To answer this question, we maintained Drosophila larvae at a range of population densities throughout larval development, kept them isolated during early adulthood, and then tested their sleep patterns. Our findings reveal that flies that had been isolated as larvae had more fragmented sleep than those that had been raised at higher population densities. This effect was more prominent in females than in males. Larval population density did not affect sleep in female flies that were mutant for amnesiac, which has been shown to be required for normal memory consolidation, adult sleep regulation, and brain development. In contrast, larval population density effects on sleep persisted in female flies lacking the olfactory receptor or83b, suggesting that olfactory signals are not required for the effects of larval population density on adult sleep. These findings show that population density during early development can alter sleep behavior in adulthood, suggesting that genetic and/or structural changes are induced by this developmental manipulation that persist through metamorphosis.
doi:10.3390/brainsci4030453
PMCID: PMC4194033  PMID: 25116571
Drosophila; sleep; development; social isolation and enrichment; population density; amnesiac; or83b
9.  Autonomous Circuitry for Substrate Exploration in Freely Moving Drosophila Larvae 
Current biology : CB  2012;22(20):1861-1870.
Summary
Background
Many organisms, from bacteria to human hunter-gatherers, use specialized random walk strategies to explore their environment. Such behaviors are an efficient stratagem for sampling the environment and usually consist of an alternation between straight runs and turns that redirect these runs. Drosophila larvae execute an exploratory routine of this kind that consists of sequences of straight crawls, pauses, turns, and redirected crawls. Central pattern generating networks underlying rhythmic movements are distributed along the anteroposterior axis of the nervous system. The way in which the operation of these networks is incorporated into extended behavioral routines such as substrate exploration has not yet been explored. In particular, the part played by the brain in dictating the sequence of movements required is unknown.
Results
We report the use of a genetic method to block synaptic activity acutely in the brain and subesophageal ganglia (SOG) of larvae during active exploratory behavior. We show that the brain and SOG are not required for the normal performance of an exploratory routine. Alternation between crawls and turns is an intrinsic property of the abdominal and/or thoracic networks. The brain modifies this autonomous routine during goal-directed movements such as those of chemotaxis. Nonetheless, light avoidance behavior can be mediated in the absence of brain activity solely by the sensorimotor system of the abdomen and thorax.
Conclusions
The sequence of movements for substrate exploration is an autonomous capacity of the thoracic and abdominal nervous system. The brain modulates this exploratory routine in response to environmental cues.
doi:10.1016/j.cub.2012.07.048
PMCID: PMC4082562  PMID: 22940472
10.  A gustatory receptor paralog controls rapid warmth avoidance in Drosophila 
Nature  2013;500(7464):580-584.
Behavioral responses to temperature are critical for survival, and animals from insects to humans show strong preferences for specific temperatures1, 2. Preferred temperature selection promotes avoidance of adverse thermal environments in the short-term and maintenance of optimal body temperatures over the long-term1, 2, but its molecular and cellular basis is largely unknown. Recent studies have yielded conflicting views of thermal preference in Drosophila, attributing importance to either internal3 or peripheral4 warmth sensors. Here we reconcile these views by demonstrating that thermal preference is not a singular response, but involves multiple systems relevant in different contexts. We previously found that the Transient Receptor Potential (TRP) channel TRPA1 acts internally to control the slowly developing preference response of flies exposed to a shallow thermal gradient3. Here we find that the rapid response of flies exposed to a steep warmth gradient does not require TRPA1; rather, the Gustatory receptor (Gr) Gr28b(D) drives this behavior via peripheral thermosensors. Grs are a large gene family widely studied in insect gustation and olfaction and implicated in host-seeking by insect disease vectors5–7, but not previously implicated in thermosensation. At the molecular level, Gr28b(D) misexpression confers thermosensitivity upon diverse cell types, suggesting it is a warmth sensor. These data reveal a new type of thermosensory molecule and uncover a functional distinction between peripheral and internal warmth sensors in this tiny ectotherm reminiscent of thermoregulatory systems in larger, endothermic animals2. The use of multiple, distinct molecules to respond to a given temperature, as observed here, may facilitate independent tuning of an animal’s distinct thermosensory responses.
doi:10.1038/nature12390
PMCID: PMC3758369  PMID: 23925112
Gr28b; thermosensation; TRPA1; TRP; thermosensor; thermoreceptor
11.  Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions 
The Drosophila larval neuromuscular preparation has proven to be a useful tool for studying synaptic physiology1,2,3. Currently, the only means available to evoke excitatory junctional potentials (EJPs) in this preparation involves the use of suction electrodes. In both research and teaching labs, students often have difficulty maneuvering and manipulating this type of stimulating electrode. In the present work, we show how to remotely stimulate synaptic potentials at the larval NMJ without the use of suction electrodes. By expressing channelrhodopsin2 (ChR2) 4,5,6 in Drosophila motor neurons using the GAL4-UAS system 7, and making minor changes to a basic electrophysiology rig, we were able to reliably evoke EJPs with pulses of blue light. This technique could be of particular use in neurophysiology teaching labs where student rig practice time and resources are limited.
doi:10.3791/1133
PMCID: PMC2762902  PMID: 19289998
12.  Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions 
The Drosophila larval neuromuscular preparation has proven to be a useful tool for studying synaptic physiology1,2,3. Currently, the only means available to evoke excitatory junctional potentials (EJPs) in this preparation involves the use of suction electrodes. In both research and teaching labs, students often have difficulty maneuvering and manipulating this type of stimulating electrode. In the present work, we show how to remotely stimulate synaptic potentials at the larval NMJ without the use of suction electrodes. By expressing channelrhodopsin2 (ChR2) 4,5,6 in Drosophila motor neurons using the GAL4-UAS system 7, and making minor changes to a basic electrophysiology rig, we were able to reliably evoke EJPs with pulses of blue light. This technique could be of particular use in neurophysiology teaching labs where student rig practice time and resources are limited.
doi:10.3791/1133
PMCID: PMC2762902  PMID: 19289998
13.  Alternative splicing of the eag potassium channel gene in Drosophila generates a novel signal transduction scaffolding protein 
The Drosophila eag gene has been shown to regulate neuronal excitability (Wu et al., 1983), olfaction (Dubin et al., 1998), associative learning (Griffith et al., 1994) and larval locomotion (Wang et al., 2002a). Not all of the roles of this gene in these processes can be explained by its function as a voltage-gated potassium channel (e.g. Zhong and Wu, 1991). In this study, we show that the eag gene is spliced in a PKA- and PKC-regulated manner to produce a protein lacking channel domains. This protein, in the context of activated PKA, can engage cellular signaling pathways that alter cell structure. Nuclear localization is necessary for C-terminal-mediated effects, which also require MAPK. The requirement for PKA/PKC activation in the synthesis and function of this novel protein suggests that it may couple membrane events to nuclear signaling to regulate neuronal function on long time scales.
doi:10.1016/j.mcn.2008.11.005
PMCID: PMC2646804  PMID: 19130887
14.  Multimodal Sensory Integration of Courtship Stimulating Cues in Drosophila melanogaster 
Mechanisms for identifying appropriate mating partners are required for any species to survive. In many types of animals, males employ multiple sensory modalities to initially search for females and to subsequently determine if they are fit and/or receptive. In this paper we will detail the multiple types of sensory information that are used to initiate and drive courtship in Drosophila melanogaster and discuss the importance of context in the interpretation of chemosensory cues. We find that food-related olfactory cues increase the salience of the aversive pheromone cis-vaccenyl acetate.
doi:10.1111/j.1749-6632.2009.04367.x
PMCID: PMC2795581  PMID: 19686165
Drosophila; olfaction; gustation; hearing; cis-vaccenyl acetate; pheromone
15.  NEUROSCIENCE 
Nature  2008;451(7174):24-25.
In many species, males have developed strategies to safeguard their genetic material from dilution by that of competing males. Fruitflies achieve this by altering the behaviour of their partners.
doi:10.1038/451024a
PMCID: PMC2742166  PMID: 18172487
16.  PDF cells are a GABA-responsive wake-promoting component of the Drosophila sleep circuit 
Neuron  2008;60(4):672-682.
The daily sleep cycle in humans and other mammals is driven by a complex circuit within which GABAergic sleep-promoting neurons oppose arousal systems. The latter includes the circadian system, aminergic/cholinergic systems as well as neurons secreting the peptide orexin/hypocretin, which contribute to sharp behavioral transitions (Lu and Greco, 2006). Drosophila sleep has recently been shown also to be controlled by GABAergic inputs, which act on unknown cells expressing the Rdl GABAA receptor (Agosto et al., 2008). We identify here the relevant Rdl-containing cells as a subset of the well-studied Drosophila circadian clock neurons, the PDF-expressing small and large ventral lateral neurons (LNvs). LNv activity regulates the total amount of sleep as well as the rate of sleep onset, and both large and small LNvs are part of the sleep circuit. Flies mutant for either the pdf gene or its receptor are hypersomnolent, and PDF acts on the LNvs themselves to control sleep. These features of the Drosophila sleep circuit, GABAergic control of sleep onset and maintenance as well as peptidergic control of arousal, support the idea that features of sleep circuit architecture as well as the mechanisms governing the behavioral transitions between sleep and wake are conserved between mammals and insects.
doi:10.1016/j.neuron.2008.10.042
PMCID: PMC2734413  PMID: 19038223
17.  Generalization of courtship learning in Drosophila is mediated by cis-vaccenyl acetate 
Current biology : CB  2007;17(7):599-605.
Summary
Reproductive behavior in Drosophila has both stereotyped and plastic components that are driven by age- and sex-specific chemical cues. Males who unsuccessfully court virgin females subsequently avoid females that are of the same age as the trainer. In contrast, males trained with mature mated females associate volatile appetitive and aversive pheromonal cues and learn to suppress courtship of all females. Here we show that the volatile aversive pheromone that leads to generalized learning with mated females is (Z)-11-octadecenyl acetate (cis-vaccenyl acetate, cVA). cVA is a major component of the male cuticular hydrocarbon profile, but it is not found on virgin females. During copulation, cVA is transferred to the female in ejaculate along with sperm and peptides that decrease her sexual receptivity. When males sense cVA (either synthetic or from mated female or male extracts) in the context of female pheromone, they develop a generalized suppression of courtship. The effects of cVA on initial courtship of virgin females can be blocked by expression of tetanus toxin in Or65a, but not Or67d neurons, demonstrating that the aversive effects of this pheromone are mediated by a specific class of olfactory neuron. These findings suggest that transfer of cVA to females during mating may be part of the male’s strategy to suppress reproduction by competing males.
doi:10.1016/j.cub.2007.01.053
PMCID: PMC1913718  PMID: 17363250
Learning and memory; olfaction; Drosophila; pheromones; cis-vaccenyl acetate
18.  Identifying behavioral circuits in Drosophila melanogaster: Moving targets in a flying insect 
Current opinion in neurobiology  2012;22(4):609-614.
Drosophila melanogaster has historically been the premier model system for understanding the molecular and genetic bases of complex behaviors. In the last decade technical advances, in the form of new genetic tools and electrophysiological and optical methods, have allowed investigators to begin to dissect the neuronal circuits that generate behavior in the adult. The blossoming of circuit analysis in this organism has also reinforced our appreciation of the inadequacy of wiring diagrams for specifying complex behavior. Neuromodulation and neuronal plasticity act to reconfigure circuits on both short and long time scales. These processes act on the connectome, providing context by integrating external and internal cues that are relevant for behavioral choices. New approaches in the fly are providing insight into these basic principles of circuit function.
doi:10.1016/j.conb.2012.01.002
PMCID: PMC3340460  PMID: 22285110
19.  DlgS97/SAP97, a Neuronal Isoform of Discs Large, Regulates Ethanol Tolerance 
PLoS ONE  2012;7(11):e48967.
From a genetic screen for Drosophila melanogaster mutants with altered ethanol tolerance, we identified intolerant (intol), a novel allele of discs large 1 (dlg1). Dlg1 encodes Discs Large 1, a MAGUK (Membrane Associated Guanylate Kinase) family member that is the highly conserved homolog of mammalian PSD-95 and SAP97. The intol mutation disrupted specifically the expression of DlgS97, a SAP97 homolog, and one of two major protein isoforms encoded by dlg1 via alternative splicing. Expression of the major isoform, DlgA, a PSD-95 homolog, appeared unaffected. Ethanol tolerance in the intol mutant could be partially restored by transgenic expression of DlgS97, but not DlgA, in specific neurons of the fly’s brain. Based on co-immunoprecipitation, DlgS97 forms a complex with N-methyl-D-aspartate (NMDA) receptors, a known target of ethanol. Consistent with these observations, flies expressing reduced levels of the essential NMDA receptor subunit dNR1 also showed reduced ethanol tolerance, as did mutants in the gene calcium/calmodulin-dependent protein kinase (caki), encoding the fly homolog of mammalian CASK, a known binding partner of DlgS97. Lastly, mice in which SAP97, the mammalian homolog of DlgS97, was conditionally deleted in adults failed to develop rapid tolerance to ethanol’s sedative/hypnotic effects. We propose that DlgS97/SAP97 plays an important and conserved role in the development of tolerance to ethanol via NMDA receptor-mediated synaptic plasticity.
doi:10.1371/journal.pone.0048967
PMCID: PMC3492131  PMID: 23145041
20.  Song Choice Is Modulated by Female Movement in Drosophila Males 
PLoS ONE  2012;7(9):e46025.
Mate selection is critical to ensuring the survival of a species. In the fruit fly, Drosophila melanogaster, genetic and anatomical studies have focused on mate recognition and courtship initiation for decades. This model system has proven to be highly amenable for the study of neural systems controlling the decision making process. However, much less is known about how courtship quality is regulated in a temporally dynamic manner in males and how a female assesses male performance as she makes her decision of whether to accept copulation. Here, we report that the courting male dynamically adjusts the relative proportions of the song components, pulse song or sine song, by assessing female locomotion. Male flies deficient for olfaction failed to perform the locomotion-dependent song modulation, indicating that olfactory cues provide essential information regarding proximity to the target female. Olfactory mutant males also showed lower copulation success when paired with wild-type females, suggesting that the male’s ability to temporally control song significantly affects female mating receptivity. These results depict the consecutive inter-sex behavioral decisions, in which a male smells the close proximity of a female as an indication of her increased receptivity and accordingly coordinates his song choice, which then enhances the probability of his successful copulation.
doi:10.1371/journal.pone.0046025
PMCID: PMC3458092  PMID: 23049926
21.  Imaging analysis of clock neurons: light buffers the wake-promoting effect of dopamine 
Nature neuroscience  2011;14(7):889-895.
How animals maintain proper amounts of sleep yet still be flexible to changes in the environmental conditions remains unknown. Here we showed that environmental light suppresses the wake-promoting effects of dopamine in fly brains. A subset of clock neurons, the 10 large lateral-ventral neurons (l-LNvs), are wake-promoting and respond to dopamine, octopamine as well as light. Behavioral and imaging analyses suggested that dopamine is a stronger arousal signal than octopamine. Surprisingly, light exposure not only suppressed the l-LNv responses but also synchronized responses of neighboring l-LNvs. This regulation occured by distinct mechanisms: light-mediated suppression of octopamine responses is regulated by the circadian clock, whereas light regulation of dopamine responses occurs by upregulation of inhibitory dopamine receptors. Plasticity therefore alters the relative importance of diverse cues based on the environmental mix of stimuli. The regulatory mechanisms described here may contribute to the control of sleep stability while still allowing behavioral flexibility.
doi:10.1038/nn.2860
PMCID: PMC3424274  PMID: 21685918
22.  Correction: High-Resolution Positional Tracking for Long-Term Analysis of Drosophila Sleep and Locomotion Using the “Tracker” Program 
PLoS ONE  2012;7(8):10.1371/annotation/4c62d454-931e-4c48-841a-a701cb658a1c.
doi:10.1371/annotation/4c62d454-931e-4c48-841a-a701cb658a1c
PMCID: PMC3414589
23.  Circadian Biology: The Supporting Cast Takes On a Starring Role 
Current Biology  2011;21(9):R313-R314.
Brain circuits are generally thought to consist solely of neurons communicating with other neurons. In Drosophila, glia-to-neuron signaling has now been shown to be critical to the function of the circadian circuit.
doi:10.1016/j.cub.2011.03.056
PMCID: PMC3397197  PMID: 21549951
24.  High-Resolution Positional Tracking for Long-Term Analysis of Drosophila Sleep and Locomotion Using the “Tracker” Program 
PLoS ONE  2012;7(5):e37250.
Drosophila melanogaster has been used for decades in the study of circadian behavior, and more recently has become a popular model for the study of sleep. The classic method for monitoring fly activity involves counting the number of infrared beam crosses in individual small glass tubes. Incident recording methods such as this can measure gross locomotor activity, but they are unable to provide details about where the fly is located in space and do not detect small movements (i.e. anything less than half the enclosure size), which could lead to an overestimation of sleep and an inaccurate report of the behavior of the fly. This is especially problematic if the fly is awake, but is not moving distances that span the enclosure. Similarly, locomotor deficiencies could be incorrectly classified as sleep phenotypes. To address these issues, we have developed a locomotor tracking technique (the “Tracker” program) that records the exact location of a fly in real time. This allows for the detection of very small movements at any location within the tube. In addition to circadian locomotor activity, we are able to collect other information, such as distance, speed, food proximity, place preference, and multiple additional parameters that relate to sleep structure. Direct comparisons of incident recording and our motion tracking application using wild type and locomotor-deficient (CASK-β null) flies show that the increased temporal resolution in the data from the Tracker program can greatly affect the interpretation of the state of the fly. This is especially evident when a particular condition or genotype has strong effects on the behavior, and can provide a wealth of information previously unavailable to the investigator. The interaction of sleep with other behaviors can also be assessed directly in many cases with this method.
doi:10.1371/journal.pone.0037250
PMCID: PMC3352887  PMID: 22615954
25.  High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster 
Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field. Studying locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.
doi:10.3791/1096
PMCID: PMC2762895  PMID: 19390509

Results 1-25 (37)