Prober Lab | California Institute of Technology

Larval zebrafish in situ hybridization for hypocretin at 24 and 120 hours post-fertilization, and transgenic zebrafish larvae in which hypocretin neurons are labeled with red fluorescent protein and QRFP neurons are labeled with green fluorescent protein.


A person can reject food, abstain from sex and control his or her thirst, but cannot keep from falling asleep. What is the genetic and neural basis for this insistent bodily need, why is it observed in essentially all multicellular animals, and how is it regulated?

Despite years of study, answers to these questions remain elusive. We are using zebrafish as a new model to discover and understand genetic and neural circuits that regulate sleep.

Zebrafish as a model system to study sleep

Yes, fish sleep.

We and others have shown that larval and adult zebrafish exhibit the three behavioral criteria that define sleep-like states:

·  periods of inactivity that occur with a circadian rhythm

·  elevated arousal threshold to sensory stimulation during this inactivity

·  compensatory sleep rebound following sleep deprivation

Sleep-like states have been studied in a variety of model organisms, including roundworms, fruit flies and rodents. Zebrafish offer several advantages:

·  anatomical and molecular similarities between zebrafish and mammalian brains suggest that the basic

     neural circuits that regulate zebrafish sleep are likely conserved in mammals

·  zebrafish embryos and larvae are transparent, which facilitates observing and manipulating neural circuits

     and monitoring neural activity

·  high-throughput screening techniques can be used to identify drugs, genes and neurons that regulate

     sleep and wakefulness

We developed high-throughput assays to measure locomotor activity and arousal threshold in zebrafish larvae, which we study using genetic, pharmacological and neurological approaches.

Genetic mechanisms that regulate sleep

Hcrt signaling promotes locomotor activity and inhibits sleep

As proof-of-principle, we studied the zebrafish hypocretin/orexin (Hcrt) ortholog, whose loss causes the sleep disorder narcolepsy. We found that Hcrt overexpression impairs both the initiation and maintenance of sleep, consolidates wakefulness and induces hyperarousal, as in mammals. The movie below shows that most Hcrt-overexpressing larvae are active at night, while most wild type larvae are inactive.

A genetic screen to identify vertebrate sleep regulators

Genetic screens have been a powerful approach to identify invertebrate genes that regulate sleep, but some genes identified in these screens lack clear vertebrate orthologs, and several genes that regulate vertebrate sleep lack invertebrate orthologs. To identify genes that regulate vertebrate sleep, we developed a novel genetic overexpression screening strategy and used it to perform the first large-scale screen for genes that affect vertebrate sleep. Overexpression of one gene identified in the screen, neuromedin U (Nmu), promotes locomotor activity (left) and inhibits sleep (middle), whereas nmu mutants are hypoactive. We found that Nmu-induced arousal requires nmu receptor 2 and signaling via corticotropin releasing hormone (Crh) receptor 1, and likely acts via brainstem crh-expressing neurons (right, circled regions). These results revealed an unexpected interaction between Nmu and a brainstem arousal system that represents a novel wake-promoting pathway. We are further exploring this system and characterizing other genes identified in the screen.

Melatonin is required for the circadian regulation of sleep

A classical model postulates that sleep is regulated by homeostatic (S) and circadian (C) processes. Adenosine and other factors are implicated in process S and the circadian clock is understood at the molecular level, but little is known about how the circadian clock regulates sleep. Melatonin is a good candidate to mediate this process because its production is regulated by the circadian clock and it can induce sleep in diurnal animals. However, melatonin's role in sleep is controversial because most nocturnal lab mouse strains do not synthesize it, and pinealectomy studies produced different phenotypes in different species, suggesting that melatonin has species-specific functions. However, melatonin levels peak at night in both diurnal and nocturnal animals, and exogenous melatonin only promotes sleep in diurnal animals. The discrepant pinealectomy results may be due to differences in the imprecise pinealectomy procedure in different species and labs. To address the function of melatonin in a diurnal vertebrate in a clean and reproducible manner, we generated zebrafish that lack melatonin due to mutation of arylalkylamine N-acetyltransferase 2 (aanat2). In these mutants, nighttime sleep is reduced by ~50% in light:dark conditions (top), and the circadian regulation of sleep is abolished in free-running conditions (middle). In contrast to some experiments using pinealectomy or exogenous melatonin, we found that endogenous melatonin is not required for normal circadian rhythms. Finally, we found that melatonin promotes sleep in part by promoting adenosine signaling (bottom), suggesting a simple mechanism that integrates circadian and homeostatic control of sleep. This work helps to explain how the circadian clock regulates sleep in a diurnal vertebrate, and provides a basis to explore how the circadian clock and melatonin regulate sleep.

Additional candidate gene studies

We are using candidate gene gain- and loss-of-function approaches to identify and explore additional genes that regulate sleep. For example, we recently found that zebrafish larvae that lack noradrenaline due to mutation of dopamine beta hydroxylase (dbh) exhibit dramatically decreased locomotor activity (top) and increased sleep (bottom). We also found that overexpression of the hypothalamic neuropeptide QRFP (also known as 26RFa and P518) inhibits locomotor activity, while mutation of qrfp or its receptors gpr103a and gpr103b results in increased locomotor activity and decreased sleep. We are using similar methods to characterize other known and novel sleep regulatory pathways.

Neurological mechanisms that regulate sleep

The transparency and relatively simple but conserved vertebrate brain of zebrafish larvae make it a powerful system to study neural circuits that regulate sleep. As proof-of-principle, we and others showed that zebrafish larvae only have ~10 hcrt-expressing neurons, compared to thousands of these neurons in mammals, thus providing a simpler system to study Hcrt neuron development and function. We showed that zebrafish Hcrt neurons project to wake-promoting brain regions and are active during periods of consolidated wakefulness, as they are in mammals. We are now exploiting the transparency and relatively simple neural circuits of zebrafish larvae to study the development and function of Hcrt neurons, and to discover additional neural populations that regulate sleep. We are characterizing these neural circuits at the single neuron level using Brainbow, testing the effects of activating and inhibiting specific neurons on behavior using optogenetics, and monitoring effects on neuronal activity using GCaMP6.

Stimulation of Hcrt neurons promotes locomotor activity

Because zebrafish larvae are transparent, all neurons are accessible to ambient light. We exploited this feature to develop a high-throughput and non-invasive optogenetic assay that allows manipulation of genetically specified neurons in 96 freely-behaving larvae while monitoring the behavior of each animal. Using this assay, we found that stimulating Hcrt neurons promotes locomotor activity and that this effect requires noradrenaline (left). We also combined optogenetics and GCaMP6s imaging in intact larvae to show that activation of Hcrt neurons stimulates the locus coeruleus (right), the main source of noradrenaline in the brain.

As an alternative method to manipulate neurons that does not require light, we found that heterologously expressed TRPV1, TRPM8 and TRPA1 can be used to stimulate or ablate genetically specified neurons in response to their chemical or thermal agonists in freely behaving larvae. Using this approach, we found that stimulating Hcrt neurons using TRPV1 and a low concentration of its small molecule agonist capsaicin results in increased locomotor activity and decreased sleep. Conversely, ablation of Hcrt neurons using a higher concentration of capsaicin results in decreased locomotor activity and increased sleep. These tools may be particularly useful for activating specific neural populations while simultaneously monitoring whole-brain neural activity using GCaMP6.

Activation of larval zebrafish hypocretin neurons using TRPV1 and capsaicin.
Ablation of larval zebrafish hypocretin neurons using TRPV1 and capsaicin.

A live 7-day old zebrafish in which each Hcrt neuron is labeled with a different color using Brainbow.