"Almost all other animals are clearly observed to partake in sleep, since fishes of all kinds...have been seen sleeping"

-- Aristotle, On Sleep and Sleeplessness 350 B.C.E.

zebrafish
Adult Zebrafish (Danio Rerio)

Sleep has fascinated poets, playwrights, philosophers, and scientists for thousands of years.  More recently, increased public and clinical attention has been paid to the negative health consequences of the lack of adequate sleep. Despite this scrutiny, the mysteries of sleep’s function and regulation endure.  Why is sleep essential for animals as diverse as flies and humans?  And what are the regulatory genes and neuronal circuits that control the timing, amount, and duration of sleep? 

Zebrafish.  In the Rihel lab, we use zebrafish as a model system to investigate sleep. The zebrafish is an attractive model system for sleep studies because they develop rapidly, exhibiting a complex behavioral repertoire, including circadian rhythms, feeding, and sleep by the fifth day of development. The zebrafish larvae are also optically transparent, facilitating the direct study of neural circuits.  Furthermore, as discussed below, zebrafish possess much of the conserved brain circuitry thought to regulate sleep in humans.  Thus, the zebrafish model is uniquely suited for sleep studies, as it combines the genetic tractability of invertebrate models with the sleep-relevant neural anatomy and physiology of mammals.

GFP larva
Transparent zebrafish larva
expressing GFP in brain


How can we know that zebrafish are asleep?

Sleep is a specific behavioral state that is defined as a period of
inactivity
, usually associated with a specific posture or resting location,
that is accompanied by a change in arousal threshold. In other words,
a sleeping animal (or human) is less sensitive to environmental stimuli
than when they are awake. In addition, sleep is under both circadian and
homeostatic control. That is, the timing of sleep is governed by a 24-hour rhythm, but if you deprive an animal (or human) of sleep, they will also exhibit increased sleep pressure followed by longer or deeper sleep. Using these criteria, the pioneering work of Irene Tobler, Joan Hendricks, Paul Shaw, and others demonstrated that even insects like the fruit fly have a sleep-like state, thus paving the way for the experimental investigation of sleep in simple model systems.

Similarly, we and others have demonstrated that both adult and larval zebrafish have a sleep-like state. Using automated video-tracking software, we watch the sleep/wake behavior of hundreds of zebrafish larvae over several days and nights, up to two weeks.


This video shows zebrafish larvae automatically tracked in a 96-well plate. In this simple tracking method, each moving larva is colored with a red flash. By recording the movements of each fish simultaneously, we can observe how experimental manipulations alter the larva's long-term behavioral dynamics.

To watch larvae continuously during the day or night, the plate is illumintated with infrared light, which they cannot see, and maintained at a constant temperature with a water bath.

activity graphThe recorded activity of a single larva plotted over two days. The gray area is enlarged on the right (arrow indicates the timing of lights out). We take each larval dataset and extract parameters including sleep latency, number of sleep bouts, and sleep length. Combining this data into a behavioral fingerprint allows us to make statistical comparisons between datasets.

Drug Screening.  With our automated assay, we generated a large dataset from the behavioral fingerprints of nearly 6,000 small molecules and identified hundreds of pharmacological agents that robustly altered zebrafish sleep, including not only known sedatives and psychotropic compounds but also novel molecules not previously implicated in sleep/wake regulation. Some of the behavioral effects were unexpected. For example, a large class of ether-a-go-go related gene (ERG) potassium channel inhibitors increased wakefulness, but only at night. We also observed a large panel of anti-inflammatory compounds increased wakefulness only during the day. A major focus of the lab is now to combine molecular biology, genetics, and neural imaging techniques to identify the underlying neuronal circuits that are modulated by these sleep-altering compounds.

In a second screen, we have also identified a number of genes that may regulate sleep in zebrafish. Through on-going research, we would like to know how these genes functionally interact with known sleep-regulating neural circuits to orchestrate the observed behavioral dynamics of zebrafish sleep/wake cycles.

Sleep Neural Circuits.  The most famous sleep-regulating neurons express the hypocretin (orexin) neuropeptide. The loss of these neurons (or mutations in either the receptors or the peptide) leads to narcolepsy, a sleep disease characterized by increased daytime sleepiness and often accompanied by attacks of cataplexy, the sudden loss of muscle tone. The hypocretin neuropeptide is expressed in a small number (~10,000) of neurons in the mammalian hypothalamus. These neurons in turn project to many wake-promoting areas of the brain.

We have previously shown that zebrafish larvae express hypocretin in about 10 neurons of the hypothalamus. Furthermore, many other aspects of the zebrafish hypocretin system are similar to what has been observed in mammals. The hypocretin neurons project to many putative wake-promoting areas and are most active during periods of consolidated wakefulness. In addition, flooding the zebrafish brain with hypocretin dramatically increases wakefulness and arousability. Thus, the zebrafish hypocretin system shares many features with the mammalian system.

Current efforts in the lab aim to understand how the genes and drugs identified in our screens modulate and interact with the hypocretin neuronal circuit.

 

Hypocretin-GFP Expressing GFP in zebrafish hypocretin neurons allows us to study their connections to other brain areas.

Rihel Lab


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