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

Abstract

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.

DOI: http://dx.doi.org/10.7554/eLife.07000.001

eLife digest

Although the neural circuits that regulate sleep and wakefulness have yet to be fully identified, the importance of at least two brain regions is well established. These are the hypothalamus, a structure deep within the brain that controls a number of basic activities including hunger, thirst and sleep; and the brainstem, which connects the brain with the spinal cord.

Specific neurons within the hypothalamus and brainstem regulate the sleep–wake cycle by signaling to one another using chemicals called neurotransmitters and neuropeptides. Throughout the day, some hypothalamic neurons release a neuropeptide called hypocretin, which helps maintain wakefulness. Hypocretin acts on neurons within the brainstem and causes them to release other neurotransmitters that promote wakefulness. However, the identity of these molecules is unclear.

One candidate is norepinephrine. Drugs that enhance the effects of norepinephrine increase wakefulness, whereas those that block norepinephrine signaling promote sleep. Despite this, mice that have been genetically modified to lack the enzyme that produces norepinephrine exhibit relatively normal sleep. This may be because in mammals, norepinephrine also has important roles outside the brain, thus complicating the effects of this genetic modification on behavior. Alternatively, while zebrafish that lack norepinephrine are healthy, mice containing this modification die early in development. Treating these mice with a specific drug allows them to survive, but might affect their behavior.

To clarify the role of norepinephrine and its interaction with hypocretin, Singh, Oikonomou and Prober created a new animal model by genetically modifying zebrafish. In contrast to mice, zebrafish that were unable to make norepinephrine slept more than normal fish, although they were also lighter sleepers and were more prone to being startled. A genetic modification that increases hypocretin signaling induces insomnia; Singh, Oikonomou and Prober found that this occurs only in animals with normal levels of norepinephrine. Thus, these experiments indicate that hypocretin does indeed promote wakefulness though norepinephrine.

The work of Singh, Oikonomou and Prober has clarified the role of norepinephrine in regulating the sleep–wake cycle. These findings could help in the development of drugs that target the neurons that make hypocretin, which may improve treatments for sleep disorders.

DOI: http://dx.doi.org/10.7554/eLife.07000.002