A New Way for the Hypothalamus to Control Sleep
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Material below summarizes the article QRFP and Its Receptors Regulate Locomotor Activity and Sleep in Zebrafish, published on February 10, 2016, in JNeurosci and authored by Audrey Chen, Cindy N. Chiu, Eric A. Mosser, Sohini Khan, Rory Spence, and David A. Prober.
Sleep is an evolutionarily conserved behavioral state whose regulation is poorly understood. The hypothalamus is thought to play a key role in regulating sleep in vertebrate animals, but few sleep-promoting signaling pathways have been identified.
RFamide neuropeptides, named for the presence of arginine (R) and phenylalanine (F) amino acids at the end of each peptide, are candidate sleep regulators. This family of peptides has been implicated in regulating feeding, locomotor activity, and energy homeostasis in vertebrate animals. Members of this family have also been shown to promote sleep in invertebrate animals, such as fruit flies and roundworms.
Given these findings, and the fact that regulation of feeding and sleep are often linked, we hypothesized that QRFP, a member of the vertebrate RFamide family, would play a role in sleep regulation.
In mammals, QRFP is exclusively expressed in the hypothalamus in thousands of neurons. We found that in the zebrafish, a diurnal vertebrate animal, QRFP is also expressed in the hypothalamus, but in only approximately 15 neurons at larval stages, when sleep is first observed, and in approximately 120 neurons in adult animals. QRFP is expressed in a subpopulation of hypothalamic neurons that also produce the excitatory neurotransmitter glutamate and are adjacent to neurons that produce the neuropeptide hypocretin/orexin, which promotes wakefulness and feeding in both mammals and zebrafish.
Mammalian QRFP signals via the G-protein coupled receptor Gpr103, which exists as two paralogs, Gpr103a and Gpr103b, in zebrafish. Gpr103b is expressed in neurons that produce the inhibitory neurotransmitter GABA in some parts of the hypothalamus, and in neurons that release the excitatory neurotransmitter glutamate in other areas. Gpr103a is expressed in several small nuclei in the forebrain, hypothalamus, and hindbrain.
While mammalian sleep is usually monitored using electrophysiology, sleep can also be defined using behavioral criteria:
- Sleep mostly occurs during a specific period of the circadian cycle.
- Sleep is characterized by an increased arousal threshold, indicating that a stronger stimulus is needed to arouse the animal, which distinguishes sleep from quiet wakefulness.
- Sleep is rapidly reversible, which distinguishes sleep from paralysis or coma.
- Sleep is homeostatically controlled, which can be demonstrated as an increased need for sleep following sleep deprivation.
Using these criteria, several labs have shown that a period of one or more minutes of rest corresponds to a sleep state in zebrafish larvae.
To test whether QRFP signaling plays a role in regulating sleep, we created transgenic zebrafish that allow inducible overexpression of QRFP, and mutant zebrafish in which we mutated qrfp or its receptors.
When we tested the behavior of qrfp mutant zebrafish larvae, we found that they exhibited more locomotor activity and less sleep during the day compared to normal larvae. Similarly, gpr103a and gpr103b double-mutant larvae were more active and slept less during the day. Conversely, overexpression of QRFP resulted in decreased locomotor activity during the day, and this effect was blocked in gpr103a; gpr103b double-mutant animals.
These findings demonstrate that QRFP/Gpr103 signaling is required to maintain normal daytime sleep levels.
Because QRFP gain-of-function and loss-of-function phenotypes were seen primarily during the day, we asked whether circadian rhythms influence qrfp expression or function. We found that the level of qrfp mRNA did not oscillate in a circadian manner over a 24-hour period, suggesting that the circadian clock does not regulate qrfp expression.
We next tested whether the QRFP overexpression phenotype persists in animals that lack circadian rhythms. We raised and tested zebrafish larvae in either constant light or constant dark, which prevents the development of circadian rhythms.
We found that entrained circadian rhythms are not required for QRFP-induced sedation. Use of an alternate lighting paradigm in which larvae were exposed to alternating one hour periods of light and darkness confirmed that the QRFP overexpression phenotype is specific to the day due to the presence of light and not due to circadian regulation of QRFP function.
Our study suggests that QRFP neurons compose a novel sleep-promoting center in the brain. As excitatory glutamatergic neurons, QRFP neurons differ from most previously described sleep-promoting neurons, which are inhibitory. This suggests that QRFP neurons may regulate sleep in a manner that is distinct from previously described sleep-promoting neurons.
Future studies will focus on how QRFP/Gpr103 signaling, as well as QRFP neurons, interact with other sleep signaling pathways and neurons to regulate sleep, and also whether this system has a similar function in mammals.
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QRFP and Its Receptors Regulate Locomotor Activity and Sleep in Zebrafish. Audrey Chen, Cindy N. Chiu, Eric A. Mosser, Sohini Khan, Rory Spence, David A. Prober. The Journal of Neuroscience Feb 2016, 36(6): 1823-1840; DOI: 10.1523/JNEUROSCI.2579-15.2016