Paul J. Shaw

Stress response genes protect against lethal effects of sleep deprivation in Drosophila.

Sleep is controlled by two processes: a homeostatic drive that increases during waking and dissipates during sleep, and a circadian pacemaker that controls its timing. Although these two systems can operate independently, recent studies indicate a more intimate relationship. To study the interaction between homeostatic and circadian processes in Drosophila, we examined homeostasis in the canonical loss-of-function clock mutants period (per01), timeless (tim01), clock (Clkjrk) and cycle (cyc01). cyc01 mutants showed a disproportionately large sleep rebound and died after 10 hours of sleep deprivation, although they were more resistant than other clock mutants to various stressors. Unlike other clock mutants, cyc01 flies showed a reduced expression of heat-shock genes after sleep loss. However, activating heat-shock genes before sleep deprivation rescued cyc01 flies from its lethal effects. Consistent with the protective effect of heat-shock genes, was the observation that flies carrying a mutation for the heat-shock protein Hsp83 (Hsp8308445) showed exaggerated homeostatic response and died after sleep deprivation. These data represent the first step in identifying the molecular mechanisms that constitute the sleep homeostat.

Waking Experience Affects Sleep Need in Drosophila

Sleep is a vital, evolutionarily conserved phenomenon, whose function is unclear. Although mounting evidence supports a role for sleep in the consolidation of memories, until now, a molecular connection between sleep, plasticity, and memory formation has been difficult to demonstrate. We establish Drosophila as a model to investigate this relation and demonstrate that the intensity and/or complexity of prior social experience stably modifies sleep need and architecture. Furthermore, this experience-dependent plasticity in sleep need is subserved by the dopaminergic and adenosine 3′,5′-monophosphate signaling pathways and a particular subset of 17 long-term memory genes.

Inducing sleep by remote control facilitates memory consolidation in Drosophila.

Inducing sleep in flies reverses deficits in long-term memory caused by social enrichment. Sleep is believed to play an important role in memory consolidation. We induced sleep on demand by expressing the temperature-gated nonspecific cation channel Transient receptor potential cation channel (UAS-TrpA1) in neurons, including those with projections to the dorsal fan-shaped body (FB). When the temperature was raised to 31°C, flies entered a quiescent state that meets the criteria for identifying sleep. When sleep was induced for 4 hours after a massed-training protocol for courtship conditioning that is not capable of inducing long-term memory (LTM) by itself, flies develop an LTM. Activating the dorsal FB in the absence of sleep did not result in the formation of LTM after massed training.

Use-Dependent Plasticity in Clock Neurons Regulates Sleep Need in Drosophila

Sleep is important for memory consolidation and is responsive to waking experience. Clock circuitry is uniquely positioned to coordinate interactions between processes underlying memory and sleep need. Flies increase sleep both after exposure to an enriched social environment and after protocols that induce long-term memory. We found that flies mutant for rutabaga, period, and blistered were deficient for experience-dependent increases in sleep. Rescue of each of these genes within the ventral lateral neurons (LNVs) restores increased sleep after social enrichment. Social experiences that induce increased sleep were associated with an increase in the number of synaptic terminals in the LNV projections into the medulla. The number of synaptic terminals was reduced during sleep and this decline was prevented by sleep deprivation.

D1 Receptor Activation in the Mushroom Bodies Rescues Sleep Loss Induced Learning Impairments in Drosophila

BACKGROUND Extended wakefulness disrupts acquisition of short-term memories in mammals. However, the underlying molecular mechanisms triggered by extended waking and restored by sleep are unknown. Moreover, the neuronal circuits that depend on sleep for optimal learning remain unidentified. RESULTS Learning was evaluated with aversive phototaxic suppression. In this task, flies learn to avoid light that is paired with an aversive stimulus (quinine-humidity). We demonstrate extensive homology in sleep-deprivation-induced learning impairment between flies and humans. Both 6 hr and 12 hr of sleep deprivation are sufficient to impair learning in Canton-S (Cs) flies. Moreover, learning is impaired at the end of the normal waking day in direct correlation with time spent awake. Mechanistic studies indicate that this task requires intact mushroom bodies (MBs) and requires the dopamine D1-like receptor (dDA1). Importantly, sleep-deprivation-induced learning impairments could be rescued by targeted gene expression of the dDA1 receptor to the MBs. CONCLUSIONS These data provide direct evidence that extended wakefulness disrupts learning in Drosophila. These results demonstrate that it is possible to prevent the effects of sleep deprivation by targeting a single neuronal structure and identify cellular and molecular targets adversely affected by extended waking in a genetically tractable model organism.

Sleep and the fruit fly

The function of sleep remains a long-standing mystery in neurobiology. The presence of a sleep-like state has recently been demonstrated in the fruit fly, Drosophila melanogaster, meeting the essential behavioral criteria for sleep and also showing pharmacological and molecular correlates of mammalian sleep. This development opens up the possibility of applying genetic analysis to the identification of key molecular components of sleep. A mutant of monoamine metabolism has already been found to affect the homeostatic regulation of sleep-like behavior in the fly. The record of Drosophila in laying the foundations for subsequent studies in mammals argues in favor of the force of this new approach.

Essentials of sleep recordings in Drosophila: moving beyond sleep time.

The power of Drosophila genetics can be used to facilitate the molecular dissection of sleep regulatory mechanisms. While evaluating total sleep time and homeostatic processes provides valuable information, other variables, such as sleep latency, sleep bout duration, sleep cycle length, and the time of day when the longest sleep bout is initiated, should also be used to explore the nature of a genetic lesion on sleep regulatory processes. Each of these variables requires that the recording interval used to identify periods of sleep and waking be determined accurately and empirically. This article describes the procedures for recording sleep in Drosophila and associated methodological constraints. In addition, it provides results from a normative data set of 1037 Canton-S female flies and 639 male flies to illustrate the nature and variability of sleep variables that one can extract from 24 h of data collection in Drosophila.

Identification of a biomarker for sleep drive in flies and humans

It is a common experience to sacrifice sleep to meet the demands of our 24-h society. Current estimates reveal that as a society, we sleep on average 2 h less than we did 40 years ago. This level of sleep restriction results in negative health outcomes and is sufficient to produce cognitive deficits and reduced attention and is associated with increased risk for traffic and occupational accidents. Unfortunately, there is no simple quantifiable marker that can detect an individual who is excessively sleepy before adverse outcomes become evident. To address this issue, we have developed a simple and effective strategy for identifying biomarkers of sleepiness by using genetic and pharmacological tools that dissociate sleep drive from wake time in the model organism Drosophila melanogaster. These studies have identified a biomarker, Amylase, that is highly correlated with sleep drive. More importantly, both salivary Amylase activity and mRNA levels are also responsive to extended waking in humans. These data indicate that the fly is relevant for human sleep research and represents a first step in developing an effective method for detecting sleepiness in vulnerable populations.

No evidence of brain cell degeneration after long-term sleep deprivation in rats

Sleep deprivation leads to cognitive impairments in humans and, if sustained for 2-3 weeks in rats, it is invariably fatal. It has been suggested that neural activity associated with waking, if it is not interrupted by periods of sleep, may damage brain cells through excitotoxic or oxidative mechanisms and eventually lead to cell death. To determine whether sustained waking causes brain cell degeneration, three parallel strategies were used. The presence and extent of DNA fragmentation was analyzed with the TUNEL technique on brain sections from rats sleep deprived for various periods of time (from 8 h to 14 days) and from their respective controls. Adjacent sections from the same animals were stained with a newly developed fluorochrome (Fluoro-Jade) specific for degenerating neurons. Finally, total RNA from the cerebral cortex of the same animals was used to determine whether the expression of several stress response genes and apoptosis-related genes is modified after sustained waking. In most long-term sleep deprived rats only a few scattered TUNEL positive nuclei (1-3) were found in any given brain section. The overall number, distribution, and morphology of TUNEL positive cells in long-term sleep deprived rats did not differ significantly from yoked controls, short-term sleep deprived rats, and sleep controls. No evidence of degenerating neurons as detected by Fluoro-Jade was found in any experimental group. mRNA levels of all the stress response genes and apoptosis-related genes tested did not differ between long-term sleep deprived rats and their yoked controls. These results argue against the hypothesis that sustained waking can significantly damage brain cells through excitotoxic or oxidative mechanisms and that massive cell death may explain the fatal consequences of sleep deprivation.

The perilipin homologue, lipid storage droplet 2, regulates sleep homeostasis and prevents learning impairments following sleep loss.

Starvation, which is common in the wild, appears to initiate a genetic program that allows fruitflies to remain awake without the sleepiness and cognitive impairments that typically follow sleep deprivation.