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.

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.

A workshop was held at the National Institute for Diabetes and Digestive and Kidney Diseases with a focus on the impact of sleep and circadian disruption on energy balance and diabetes. The workshop identified a number of key principles for research in this area and a number of specific opportunities. Studies in this area would be facilitated by active collaboration between investigators in sleep/circadian research and investigators in metabolism/diabetes. There is a need to translate the elegant findings from basic research into improving the metabolic health of the American public. There is also a need for investigators studying the impact of sleep/circadian disruption in humans to move beyond measurements of insulin and glucose and conduct more in-depth phenotyping. There is also a need for the assessments of sleep and circadian rhythms as well as assessments for sleep-disordered breathing to be incorporated into all ongoing cohort studies related to diabetes risk. Studies in humans need to complement the elegant short-term laboratory-based human studies of simulated short sleep and shift work etc. with studies in subjects in the general population with these disorders. It is conceivable that chronic adaptations occur, and if so, the mechanisms by which they occur needs to be identified and understood. Particular areas of opportunity that are ready for translation are studies to address whether CPAP treatment of patients with pre-diabetes and obstructive sleep apnea (OSA) prevents or delays the onset of diabetes and whether temporal restricted feeding has the same impact on obesity rates in humans as it does in mice.

Although it is widely accepted that sleep must serve an essential biological function, little is known about molecules that underlie sleep regulation. Given that insomnia is a common sleep disorder that disrupts the ability to initiate and maintain restorative sleep, a better understanding of its molecular underpinning may provide crucial insights into sleep regulatory processes. Thus, we created a line of flies using laboratory selection that share traits with human insomnia. After 60 generations insomnia-like (ins-l) flies sleep 60 min a day, exhibit difficulty initiating sleep, difficulty maintaining sleep, and show evidence of daytime cognitive impairment. ins-l flies are also hyperactive and hyper responsive to environmental perturbations. In addition they have difficulty maintaining their balance, have elevated levels of dopamine, are short-lived and show increased levels of triglycerides, cholesterol, and free fatty acids. While their core molecular clock remains intact, ins-l flies lose their ability to sleep when placed into constant darkness. Whole genome profiling identified genes that are modified in ins-l flies. Among those differentially expressed transcripts genes involved in metabolism, neuronal activity, and sensory perception constituted over-represented categories. We demonstrate that two of these genes are upregulated in human subjects following acute sleep deprivation. Together these data indicate that the ins-l flies are a useful tool that can be used to identify molecules important for sleep regulation and may provide insights into both the causes and long-term consequences of insomnia.

Recent human studies suggest that genetic polymorphisms allow an individual to maintain optimal cognitive functioning during sleep deprivation. If such polymorphisms were not associated with additional costs, selective pressures would allow these alleles to spread through the population such that an evolutionary alternative to sleep would emerge. To determine whether there are indeed costs associated with resiliency to sleep loss, we challenged natural allelic variants of the foraging gene (for) with either sleep deprivation or starvation. Flies with high levels of Protein Kinase G (PKG) (for R ) do not display deficits in short-term memory following 12 h of sleep deprivation. However, short-term memory is significantly disrupted when for R flies are starved overnight. In contrast, flies with low levels of PKG (for s , for s2 ) show substantial deficits in short-term memory following sleep deprivation but retain their ability to learn after 12 h of starvation. We found that for R phenotypes could be largely recapitulated in for s flies by selectively increasing the level of PKG in the α/β lobes of the mushroom bodies, a structure known to regulate both sleep and memory. Together, these data indicate that whereas the expression of for may appear to provide resilience in one environmental context, it may confer an unexpected vulnerability in other situations. Understanding how these tradeoffs confer resilience or vulnerability to specific environmental challenges may provide additional clues as to why an evolutionary alternative to sleep has not emerged.A lthough sleep is a behavioral state that is conserved across a diverse range of species, the biological functions of sleep remain unknown. Sleep deprivation (SD) has been shown to negatively impact cognition, but individual responses to sleep loss can vary significantly within a population (1). Recent studies suggest that a portion of this variability may be influenced by genetic factors (2). For example, polymorphisms for PERIOD 3 (PER3), a circadian clock gene, can predict the magnitude of cognitive impairment and sleep homeostasis in response to a night of SD in humans (2). Although these genetic contributions may attenuate impairments following SD, the tradeoffs associated with resistance to sleep loss remain unknown. Presumably, the potential costs must be substantial. Thus, it is likely that the price of protection from sleep loss that can be conferred by allelic variation in one environment may induce a cost when manifested in a different environment. To date, putative costs of resiliency to sleep loss have not been identified in humans or any model organism.foraging (for), which codes for Protein Kinase G (PKG), is maintained in wild-type populations as a genetic polymorphism that results in either higher or lower levels of PKG activity (3). The allele associated with higher levels of PKG ("rover"; for R ) results in larvae with longer foraging trails between food patches, whereas the allele associated with lower levels of PKG ("sitter"; for s ) results i...

SUMMARY The SLC6 family comprises proteins that move extracellular neurotransmitters, amino acids and osmolytes across the plasma membrane into the cytosol. In mammals, deletion of SLC6 family members has dramatic physiologic consequences, but in the model organism Drosophila melanogaster, little is known about this family of proteins. Therefore,in this study we carried out an initial analysis of 21 known or putative SLC6 family members from the Drosophila genome. Protein sequences from these genes segregated into either well-defined subfamilies, including the novel insect amino acid transporter subfamily, or into a group of weakly related sequences not affiliated with a recognized subfamily. Reverse transcription-polymerase chain reaction analysis and in situhybridization showed that seven of these genes are expressed in the CNS. In situ hybridization revealed that two previously cloned SLC6 members, the serotonin and dopamine transporters, were localized to presumptive presynaptic neurons that previously immunolabelled for these transmitters. RNA for CG1732 (the putative GABA transporter) and CG15088 (a member of the novel insect amino acid transporter family)was localized in cells likely to be subtypes of glia, while RNA for CG5226, CG10804 (both members of the orphan neurotransmitter transporter subfamily) and CG5549 (a putative glycine transporter)were expressed broadly throughout the cellular cortex of the CNS. Eight of the 21 sequences were localized outside the CNS in the alimentary canal,Malpighian tubules and reproductive organs. Localization for six sequences was not found or not attempted in the adult fly. We used the Drosophilaortholog of the mammalian vesicular monoamine transporter 2, CG33528,to independently identify monoaminergic neurons in the adult fly. RNA for CG33528 was detected in a limited number of cells in the central brain and in a beaded stripe at the base of the photoreceptors in the position of glia, but not in the photoreceptors themselves. The SLC6 localization observations in conjunction with likely substrates based on phylogenetic inferences are a first step in defining the role of Na/Cl-dependent transporters in Drosophila physiology.

Although it has been hypothesized that contraction of hepatic stellate cells (HSC) regulates blood flow by modulating sinusoidal resistance, neither HSC contraction nor relaxation has been directly quantitated. To test this hypothesis, a force transducer was employed to directly measure the magnitude and rate of contraction and relaxation by primary rat HSC (4.7 × 105 ± 0.2 × 105 cells) cultured within a collagen gel. Serial exposures to 10% fetal bovine serum stimulated 81 ± 14 and 82 ± 10 dyn of contractile force, respectively. Subsequent stimulation with 2 nM endothelin-1 (ET-1) resulted in the development of 185 ± 25 dyn of force. Contractions began within 10 s of ET-1 stimulation, and the half time of maximal force development was <5 min. Removal of agonist resulted in complete or nearly complete relaxation within 45 min. These results suggest that the magnitude and rate of HSC contraction and relaxation are capable of modulating blood flow via sinusoidal constriction.

Individuals frequently find themselves confronted with a variety of challenges that threaten their wellbeing. While some individuals face these challenges efficiently and thrive (resilient) others are unable to cope and may suffer persistent consequences (vulnerable). Resilience/vulnerability to sleep disruption may contribute to the vulnerability to individuals exposed to challenging conditions. With that in mind we exploited individual differences in a fly’s ability to form short-term memory (STM) following 3 different types of sleep disruption to identify the underlying genes. Our analysis showed that in each category of flies examined, there are individuals that form STM in the face of sleep loss (resilient) while other individuals show dramatic declines in cognitive behavior (vulnerable). Molecular genetic studies revealed that Antimicrobial Peptides, factors important for innate immunity, were candidates for conferring resilience/vulnerability to sleep deprivation. Specifically, Metchnikowin (Mtk), drosocin (dro) and Attacin (Att) transcript levels seemed to be differentially increased by sleep deprivation in glia (Mtk), neurons (dro) or primarily in the head fat body (Att). Follow-up genetic studies confirmed that expressing Mtk in glia but not neurons, and expressing dro in neurons but not glia, disrupted memory while modulating sleep in opposite directions. These data indicate that various factors within glia or neurons can contribute to individual differences in resilience/vulnerability to sleep deprivation.