Discovery of Hypocretin/Orexin Ushers in a New Era of Sleep Research

Prior to the 21st century, genetic mechanisms that regulate sleep were largely unknown. In 1998, de Lecea et al. [1] (Proc. Natl. Acad. Sci. U. S. A. 1998; 95:322–327) and Sakurai et al. [2] (Cell 1998; 92: 573–585) reported the discovery of a gene they named hypocretin and orexin, respectively, which led to a revolution in our understanding of genetic and neuronal mechanisms that regulate sleep

Discovery of Hypocretin/Orexin Ushers in a New Era of Sleep Research

Prior to the 21st century, genetic mechanisms that regulate sleep were largely unknown. In 1998, de Lecea et al. [1] (Proc. Natl. Acad. Sci. U. S. A. 1998; 95:322–327) and Sakurai et al. [2] (Cell 1998; 92: 573–585) reported the discovery of a gene they named hypocretin and orexin, respectively, which led to a revolution in our understanding of genetic and neuronal mechanisms that regulate sleep

Attacking sleep from a new angle: contributions from zebrafish

Sleep consumes a third of our lifespan, but we are far from understanding how it is initiated, maintained and terminated, or what purposes it serves. To address these questions, alternative model systems have recently been recruited. The diurnal zebrafish holds the promise of bridging the gap between simple invertebrate systems, which show little neuroanatomical conservation with mammals, and well-established, but complex and nocturnal, murine systems. Zebrafish larvae can be monitored in a high-throughput fashion, pharmacologically tested by adding compounds into the water, genetically screened using transient transgenesis, and optogenetically manipulated in a non-invasive manner. Here we discuss work that has established the zebrafish as a powerful system for the study of sleep, as well as novel insights gained by exploiting its particular advantages

Ras1 Promotes Cellular Growth in the Drosophila Wing

The Ras GTPase links extracellular mitogens to intracellular mechanisms that control cell proliferation. To understand how Ras regulates proliferation in vivo, we activated or inactivated Ras in cell clones in the developing Drosophila wing. Cells lacking Ras were smaller, had reduced growth rates, accumulated in G1, and underwent apoptosis due to cell competition. Conversely, activation of Ras increased cell size and growth rates and promoted G1/S transitions. Ras upregulated the growth driver dMyc, and both Ras and dMyc increased levels of cyclin E posttranscriptionally. We propose that Ras primarily promotes growth and that growth is coupled to G1/S progression via cyclin E. Interestingly, upregulation of growth by Ras did not deregulate G2/M progression or a developmentally regulated cell cycle exit

Regulation of zebrafish sleep and arousal states: current and prospective approaches

Every day, we shift among various states of sleep and arousal to meet the many demands of our bodies and environment. A central puzzle in neurobiology is how the brain controls these behavioral states, which are essential to an animal's well-being and survival. Mammalian models have predominated sleep and arousal research, although in the past decade, invertebrate models have made significant contributions to our understanding of the genetic underpinnings of behavioral states. More recently, the zebrafish has emerged as a promising model system for sleep and arousal research. Here we review experimental evidence that the zebrafish, a diurnal vertebrate, exhibits fundamental behavioral and neurochemical characteristics of mammalian sleep and arousal. We also propose how specific advantages of the zebrafish can be harnessed to advance the field. These include tractable genetics to identify and manipulate molecular and cellular regulators of behavioral states, optical transparency to facilitate in vivo observation of neural structure and function, and amenability to high-throughput drug screens to discover novel therapies for neurological disorders

Norepinephrine is required to promote wakefulness and for hypocretin-induced arousal in zebrafish

Pharmacological studies in mammals suggest that norepinephrine (NE) plays an important role in promoting arousal. However, the role of endogenous NE is unclear, with contradicting reports concerning the sleep phenotypes of mice lacking NE due to mutation of dopamine β-hydroxylase (dbh). To investigate NE function in an alternative vertebrate model, we generated dbh mutant zebrafish. In contrast to mice, these animals exhibit dramatically increased sleep. Surprisingly, despite an increase in sleep,dbh mutant zebrafish have a reduced arousal threshold. These phenotypes are also observed in zebrafish treated with small molecules that inhibit NE signaling, suggesting that they are caused by the lack of NE. Using genetic overexpression of hypocretin (Hcrt) and optogenetic activation of hcrt-expressing neurons, we also find that NE is important for Hcrt-induced arousal. These results establish a role for endogenous NE in promoting arousal and indicate that NE is a critical downstream effector of Hcrt neurons

Neuropeptide VF neurons promote sleep via the serotonergic raphe

Although several sleep-regulating neuronal populations have been identified, little is known about how they interact with each other to control sleep/wake states. We previously identified neuropeptide VF (NPVF) and the hypothalamic neurons that produce it as a sleep-promoting system (Lee et al., 2017). Here we show using zebrafish that npvf-expressing neurons control sleep via the serotonergic raphe nuclei (RN), a hindbrain structure that is critical for sleep in both diurnal zebrafish and nocturnal mice. Using genetic labeling and calcium imaging, we show that npvf-expressing neurons innervate and can activate serotonergic RN neurons. We also demonstrate that chemogenetic or optogenetic stimulation of npvf-expressing neurons induces sleep in a manner that requires NPVF and serotonin in the RN. Finally, we provide genetic evidence that NPVF acts upstream of serotonin in the RN to maintain normal sleep levels. These findings reveal a novel hypothalamic-hindbrain neuronal circuit for sleep/wake control

Melatonin Is Required for the Circadian Regulation of Sleep

Sleep is an evolutionarily conserved behavioral state whose regulation is poorly understood. A classical model posits that sleep is regulated by homeostatic and circadian mechanisms. Several factors have been implicated in mediating the homeostatic regulation of sleep, but molecules underlying the circadian mechanism are unknown. Here we use animals lacking melatonin due to mutation of arylalkylamine N-acetyltransferase 2 (aanat2) to show that melatonin is required for circadian regulation of sleep in zebrafish. Sleep is dramatically reduced at night in aanat2 mutants maintained in light/dark conditions, and the circadian regulation of sleep is abolished in free-running conditions. We find that melatonin promotes sleep downstream of the circadian clock as it is not required to initiate or maintain circadian rhythms. Additionally, we provide evidence that melatonin may induce sleep in part by promoting adenosine signaling, thus potentially linking circadian and homeostatic control of sleep