Glutamate Is a Wake-Active Neurotransmitter in Drosophila melanogaster.

Introduction: In mammals, there is evidence that glutamate has a role as a wake-active neurotransmitter. So using video-based analysis of Drosophila behavior, we undertook a study to examine if glutamate, which has been previously shown to have an excitatory role in neuromuscular junctions in Drosophila, may have a conserved wake-active role in the adult brain. Aims and Methods: Using 6- to 9-day-old female flies, we examined the effect of perturbations of the glutamatergic signaling on total wakefulness and wake bout architecture. We increased and decreased neuronal activity of glutamatergic neurons in the brains of adult flies using Upstream Activating Sequence (UAS) NaChBac and UAS EKO, respectively. We blocked neurotransmission from glutamatergic neurons in adult flies using the UAS-driven temperature-sensitive dynamin mutation shibirets. We examined the behavior of flies with loss of function mutations of individual subunits of brain-specific ionotropic glutamate receptors. Results: Increasing the activity of glutamatergic neurons in the adult brain led to a significant increase in wakefulness compared to the control groups both in the daytime and nighttime and decreasing the activity of these same neurons reduced wakefulness in the nighttime. Blocking neurotransmitter release in glutamatergic neurons significantly reduced wake in the nighttime. The ionotropic receptor mutants had significantly less wake in the nighttime than their respective genetic background controls. Conclusion: The results show the following: glutamate is indeed a wake-active neurotransmitter in Drosophila; there is a major time of day effect associated with loss of glutamatergic neurotransmission; and it is a major wake-active neurotransmitter in the nighttime.

Aging induced endoplasmic reticulum stress alters sleep and sleep homeostasis.

Alterations in the quality, quantity, and architecture of baseline and recovery sleep have been shown to occur during aging. Sleep deprivation induces endoplasmic reticular (ER) stress and upregulates a protective signaling pathway termed the unfolded protein response. The effectiveness of the adaptive unfolded protein response is diminished by age. Previously, we showed that endogenous chaperone levels altered recovery sleep in Drosophila melanogaster. We now report that acute administration of the chemical chaperone sodium 4-phenylbutyrate (PBA) reduces ER stress and ameliorates age-associated sleep changes in Drosophila. PBA consolidates both baseline and recovery sleep in aging flies. The behavioral modifications of PBA are linked to its suppression of ER stress. PBA decreased splicing of X-box binding protein 1 and upregulation of phosphorylated elongation initiation factor 2 α, in flies that were subjected to sleep deprivation. We also demonstrate that directly activating ER stress in young flies fragments baseline sleep and alters recovery sleep. Alleviating prolonged or sustained ER stress during aging contributes to sleep consolidation and improves recovery sleep or sleep debt discharge.

Epigenetic regulation of axonal growth of Drosophila pacemaker cells by histone acetyltransferase tip60 controls sleep.

Tip60 is a histone acetyltransferase (HAT) enzyme that epigenetically regulates genes enriched for neuronal functions through interaction with the amyloid precursor protein (APP) intracellular domain. However, whether Tip60-mediated epigenetic dysregulation affects specific neuronal processes in vivo and contributes to neurodegeneration remains unclear. Here, we show that Tip60 HAT activity mediates axonal growth of the Drosophila pacemaker cells, termed "small ventrolateral neurons" (sLNvs), and their production of the neuropeptide pigment-dispersing factor (PDF) that functions to stabilize Drosophila sleep-wake cycles. Using genetic approaches, we show that loss of Tip60 HAT activity in the presence of the Alzheimer's disease-associated APP affects PDF expression and causes retraction of the sLNv synaptic arbor required for presynaptic release of PDF. Functional consequence of these effects is evidenced by disruption of the sleep-wake cycle in these flies. Notably, overexpression of Tip60 in conjunction with APP rescues these sleep-wake disturbances by inducing overelaboration of the sLNv synaptic terminals and increasing PDF levels, supporting a neuroprotective role for dTip60 in sLNv growth and function under APP-induced neurodegenerative conditions. Our findings reveal a novel mechanism for Tip60 mediated sleep-wake regulation via control of axonal growth and PDF levels within the sLNv-encompassing neural network and provide insight into epigenetic-based regulation of sleep disturbances observed in neurodegenerative diseases like Alzheimer's disease.

Genetic background has a major impact on differences in sleep resulting from environmental influences in Drosophila.

STUDY OBJECTIVES: To determine the effect of different genetic backgrounds on demographic and environmental interventions that affect sleep and evaluate variance of these measures; and to evaluate sleep and variance of sleep behaviors in 6 divergent laboratory strains of common origin. DESIGN: Assessment of the effects of age, sex, mating status, food sources, and social experience using video analysis of sleep behavior in 2 different strains of Drosophila, white(1118ex) (w(1118ex)) and white Canton-S (w(CS10)). Sleep was also determined for 6 laboratory strains of Canton-S and 3 inbred lines. The variance of total sleep was determined for all groups and conditions. MEASUREMENTS AND RESULTS: The circadian periods and the effects of age upon sleep were the same between w(1118ex) and w(CS10) strains. However, the w(1118ex) and w(CS10) strains demonstrated genotype-dependent differences in the effects upon sleep of sex, mating status, social experience, and being on different foods. Variance of total sleep was found to differ in a genotype dependent manner for interventions between the w(1118ex) and w(CS10) strains. Six different laboratory Canton-S strains were found to have significantly different circadian periods (P < 0.001) and sleep phenotypes (P < 0.001). Three inbred lines showed reduced variance for sleep measurements. CONCLUSIONS: One must control environmental conditions in a rigorously consistent manner to ensure that sleep data may be compared between experiments. Genetic background has a significant impact upon changes in sleep behavior and variance of behavior due to demographic factors and environmental interventions. This represents an opportunity to discover new genes that modify sleep/wake behavior.

What are microarrays teaching us about sleep?

Many fundamental questions about sleep remain unanswered. The presence of sleep across phyla suggests that it must serve a basic cellular and/or molecular function. Microarray studies, performed in several model systems, have identified classes of genes that are sleep-state regulated. This has led to the following concepts: first, a function of sleep is to maintain synaptic homeostasis; second, sleep is a stage of macromolecule biosynthesis; third, extending wakefulness leads to downregulation of several important metabolic pathways; and, fourth, extending wakefulness leads to endoplasmic reticulum stress. In human studies, microarrays are being applied to the identification of biomarkers for sleepiness and for the common debilitating condition of obstructive sleep apnea.

A video method to study Drosophila sleep.

STUDY OBJECTIVES: To use video to determine the accuracy of the infrared beam-splitting method for measuring sleep in Drosophila and to determine the effect of time of day, sex, genotype, and age on sleep measurements. DESIGN: A digital image analysis method based on frame subtraction principle was developed to distinguish a quiescent from a moving fly. Data obtained using this method were compared with data obtained using the Drosophila Activity Monitoring System (DAMS). The location of the fly was identified based on its centroid location in the subtracted images. MEASUREMENTS AND RESULTS: The error associated with the identification of total sleep using DAMS ranged from 7% to 95% and depended on genotype, sex, age, and time of day. The degree of the total sleep error was dependent on genotype during the daytime (P < 0.001) and was dependent on age during both the daytime and the nighttime (P < 0.001 for both). The DAMS method overestimated sleep bout duration during both the day and night, and the degree of these errors was genotype dependent (P < 0.001). Brief movements that occur during sleep bouts can be accurately identified using video. Both video and DAMS detected a homeostatic response to sleep deprivation. CONCLUSIONS: Video digital analysis is more accurate than DAMS in fly sleep measurements. In particular, conclusions drawn from DAMS measurements regarding daytime sleep and sleep architecture should be made with caution. Video analysis also permits the assessment of fly position and brief movements during sleep.

The energy hypothesis of sleep revisited.

One of the proposed functions of sleep is to replenish energy stores in the brain that have been depleted during wakefulness. Benington and Heller formulated a version of the energy hypothesis of sleep in terms of the metabolites adenosine and glycogen. They postulated that during wakefulness, adenosine increases and astrocytic glycogen decreases reflecting the increased energetic demand of wakefulness. We review recent studies on adenosine and glycogen stimulated by this hypothesis. We also discuss other evidence that wakefulness is an energetic challenge to the brain including the unfolded protein response, the electron transport chain, NPAS2, AMP-activated protein kinase, the astrocyte-neuron lactate shuttle, production of reactive oxygen species and uncoupling proteins. We believe the available evidence supports the notion that wakefulness is an energetic challenge to the brain, and that sleep restores energy balance in the brain, although the mechanisms by which this is accomplished are considerably more complex than envisaged by Benington and Heller.

Molecular mechanisms of sleep and wakefulness.

Major questions on the biology of sleep include the following: what are the molecular functions of sleep; why can wakefulness only be sustained for defined periods before there is behavioral impairment; what genes contribute to the individual differences in sleep and the response to sleep deprivation? Behavioral criteria to define sleep have facilitated identification of sleep states in a number of different model systems: Drosophila, zebrafish, and Caenorhabditis elegans. Each system has unique strengths. Studies in these model systems are identifying conserved signaling mechanisms regulating sleep that are present in mammals. For example, the PKA-CREB signaling mechanism promotes wakefulness in Drosophila, mice, and C. elegans. Microarray studies indicate that genes whose expression is upregulated during sleep are involved in macromolecule biosynthesis (proteins, lipids [including cholesterol], heme). Thus, a key function of sleep is likely to be macromolecule synthesis. Moreover, in all species studied to date, there is upregulation of the molecular chaperone BiP with extended wakefulness. Sleep deprivation leads to cellular ER stress in brain and the unfolded protein response. Identification of genes regulating sleep has the potential for translational studies to elucidate the genetics of sleep and response to sleep deprivation in humans.

Conservation of sleep: insights from non-mammalian model systems.

The past 10 years have seen new approaches to elucidating genetic pathways regulating sleep. The emerging theme is that sleep-like states are conserved in evolution, with similar signaling pathways playing a role in animals as distantly related as flies and humans. We review the evidence for the presence of sleep states in non-mammalian species including zebrafish (Danio rerio), fruitflies (Drosophila melanogaster) and roundworms (Caenorhabditis elegans). We describe conserved sleep-regulatory molecular pathways with a focus on cAMP and epidermal growth factor signaling; neurotransmitters with conserved effects on sleep and wake regulation, including dopamine and GABA; and a conserved molecular response to sleep deprivation involving the chaperone protein BiP/GRP78.

Lethargus is a Caenorhabditis elegans sleep-like state.

There are fundamental similarities between sleep in mammals and quiescence in the arthropod Drosophila melanogaster, suggesting that sleep-like states are evolutionarily ancient. The nematode Caenorhabditis elegans also has a quiescent behavioural state during a period called lethargus, which occurs before each of the four moults. Like sleep, lethargus maintains a constant temporal relationship with the expression of the C. elegans Period homologue LIN-42 (ref. 5). Here we show that quiescence associated with lethargus has the additional sleep-like properties of reversibility, reduced responsiveness and homeostasis. We identify the cGMP-dependent protein kinase (PKG) gene egl-4 as a regulator of sleep-like behaviour, and show that egl-4 functions in sensory neurons to promote the C. elegans sleep-like state. Conserved effects on sleep-like behaviour of homologous genes in C. elegans and Drosophila suggest a common genetic regulation of sleep-like states in arthropods and nematodes. Our results indicate that C. elegans is a suitable model system for the study of sleep regulation. The association of this C. elegans sleep-like state with developmental changes that occur with larval moults suggests that sleep may have evolved to allow for developmental changes.

Macromolecule biosynthesis: a key function of sleep.

The function(s) of sleep remains a major unanswered question in biology. We assessed changes in gene expression in the mouse cerebral cortex and hypothalamus following different durations of sleep and periods of sleep deprivation. There were significant differences in gene expression between behavioral states; we identified 3,988 genes in the cerebral cortex and 823 genes in the hypothalamus with altered expression patterns between sleep and sleep deprivation. Changes in the steady-state level of transcripts for various genes are remarkably common during sleep, as 2,090 genes in the cerebral cortex and 409 genes in the hypothalamus were defined as sleep specific and changed (increased or decreased) their expression during sleep. The largest categories of overrepresented genes increasing expression with sleep were those involved in biosynthesis and transport. In both the cerebral cortex and hypothalamus, during sleep there was upregulation of multiple genes encoding various enzymes involved in cholesterol synthesis, as well as proteins for lipid transport. There was also upregulation during sleep of genes involved in synthesis of proteins, heme, and maintenance of vesicle pools, as well as antioxidant enzymes and genes encoding proteins of energy-regulating pathways. We postulate that during sleep there is a rebuilding of multiple key cellular components in preparation for subsequent wakefulness.

Multiple mechanisms limit the duration of wakefulness in Drosophila brain.

The functions of sleep and what controls it remain unanswered biological questions. According to the two-process model, a circadian process and a homeostatic process interact to regulate sleep. While progress has been made in understanding the molecular and cellular functions of the circadian process, the mechanisms of the homeostatic process remain undiscovered. We use the recently established sleep model system organism Drosophila melanogaster to examine dynamic changes in gene expression during sleep and during prolonged wakefulness in the brain. Our experimental design controls for circadian processes by killing animals at three matched time points from the beginning of the consolidated rest period [Zeitgeber time (ZT) 14)] under two conditions, sleep deprived and spontaneously sleeping. Using ANOVA at a false discovery rate of 5%, we have identified 252 genes that were differentially expressed between sleep-deprived and control groups in the Drosophila brain. Using linear trends analysis, we have separated the significant differentially expressed genes into nine temporal expression patterns relative to a common anchor point (ZT 14). The most common expression pattern is a decrease during extended wakefulness but no change during spontaneous sleep (n = 114). Genes in this category were involved in protein production (n = 47), calcium homeostasis, and membrane excitability (n = 5). Multiple mechanisms, therefore, act to limit wakefulness. In addition, by studying the effects of the mechanical stimulus used in our deprivation studies during the period when the animals are predominantly active, we provide evidence for a previously unappreciated role for the Drosophila immune system in the brain response to stress.

Glycogen in the brain of Drosophila melanogaster: diurnal rhythm and the effect of rest deprivation.

One function of sleep is thought to be the restoration of energy stores in the brain depleted during wakefulness. One such energy store found in mammalian brains is glycogen. Many of the genes involved in glycogen regulation in mammals have also been found in Drosophila melanogaster and rest behavior in Drosophila has recently been shown to have the characteristics of sleep. We therefore examined, in the fly, variation in the glycogen contents of the brain, the whole head and the body throughout the rest/activity cycle and after rest deprivation. Glycogen in the brain varies significantly throughout the day (p=0.001) and is highest during rest and lowest while flies are active. Glycogen levels in the whole head and body do not show diurnal variation. Brain glycogen drops significantly when flies are rest deprived for 3 h (p=0.034) but no significant differences are observed after 6 h of rest deprivation. In contrast, glycogen is significantly depleted in the body after both 3 and 6 h of rest deprivation (p<0.0001 and p<0.0001, respectively). Glycogen in the fly brain changes in relationship to rest and activity and demonstrates a biphasic response to rest deprivation similar to that observed in mammalian astrocytes in culture.

Enzymes of adenosine metabolism in the brain: diurnal rhythm and the effect of sleep deprivation.

Adenosine plays a role in promoting sleep, an effect that is thought to be mediated in the basal forebrain. Adenosine levels vary in this region with prolonged wakefulness in a unique way. The basis for this is unknown. We examined, in rats, the activity of the major metabolic enzymes for adenosine - adenosine deaminase, adenosine kinase, ecto- and cytosolic 5'-nucleotidase - in sleep/wake regulatory regions as well as cerebral cortex, and how the activity varies across the day and with sleep deprivation. There were robust spatial differences for the activity of adenosine deaminase, adenosine kinase, and cytosolic and ecto-5'-nucleotidase. However, the basal forebrain was not different from other sleep/wake regulatory regions apart from the tuberomammillary nucleus. All adenosine metabolic enzymes exhibited diurnal variations in their activity, albeit not in all brain regions. Activity of adenosine deaminase increased during the active period in the ventrolateral pre-optic area but decreased significantly in the basal forebrain. Enzymatic activity of adenosine kinase and cytosolic-5'-nucleotidase was higher during the active period in all brain regions tested. However, the activity of ecto-5'-nucleotidase was augmented during the active period only in the cerebral cortex. This diurnal variation may play a role in the regulation of adenosine in relationship to sleep and wakefulness across the day. In contrast, we found no changes specifically with sleep deprivation in the activity of any enzyme in any brain region. Thus, changes in adenosine with sleep deprivation are not a consequence of alterations in adenosine enzyme activity.