70 million Americans suffer from some sort of sleep disorder. Behavior, mood and memory deteriorate with sleep loss and it gets worse with continuing sleep deprivation. There is considerable amount of data on arousal neurons whereas very little is known about the neurons that make us fall asleep. Indeed, current network models of sleep-wake regulation list many arousal neuronal populations compared to only one sleep group located in the preoptic area. Better hypnotics will emerge if there was direct evidence linking specific phenotypes of neurons to sleep. To provide this evidence, we will use optogenetics to selectively manipulate sleep- active neurons. One group of sleep-active neurons is located in the preoptic area and contains galanin. Using galanin-Cre mice, the gene for the light-sensitive excitatory opsin, channelrhodopsin-2 (ChR2), or the inhibitory opsin, enhanced Archaerhodopsin (eArch3.0), will be inserted into the galanin neurons and effects on sleep measured. Galanin neurons in the preoptic area will be optogenetically stimulated or inhibited during the night or day and under conditions that alter the animal's internal drive to stay awake (24h fasting) or sleep (6h sleep deprivation). In the same mouse, the second group of sleep-active neurons located in the lateral hypothalamus that contain melanin concentrating hormone (MCH) will be stimulated/inhibited with light. Recently, we published data indicating that stimulation of the MCH neurons robustly increases both non-REM and REM sleep. Now that there are two sleep- active neuronal groups it is necessary to simultaneously activate/inhibit both groups, or activate one while inhibiting th other to determine potency of each to sleep. To demonstrate that the stimulated neurons release the peptide an ELISA assay will be used to detect galanin and MCH in the CSF after 6h of optogenetic stimulation. Separate electrophysiology studies will directly monitor neuronal activity in the brain and determine the pattern of activity as the sleep-inducing signal propagates across the network. Does stimulation of the preoptic sleep-active neurons activate the distally located MCH sleep-active neurons? Do neurons adjacent to the stimulation site respond earlier or at the same time as distal arousal neurons? What is the temporal response of specific sleep versus wake neurons to the stimulation and to the emergence of sleep? Such brain activity maps are essential to identify functional connectivity and are consistent with the Brain Activity Map Project. From a translational perspective these studies are potentially useful in sleep disorders, such as insomnia, where sleep needs to be triggered against a strong arousal drive.
These aims will provide a framework for integrating the sleep-active neurons within an overall model of sleep- wake regulation.
New pharmacological approaches for treating insomnia are needed. The challenge is to discover new phenotypes of neurons that can induce sleep. This has been difficult to do because the sleep-active neurons are intermingled with neurons that serve other behaviors. In the last five years new tools have been developed that permit selective manipulation of specific neurons. We will utilize these tools to selectively manipulate specific phenotypes of neurons in the same mouse and demonstrate that it induces sleep against a strong drive to stay awake. These results will have a strong translational potential for sleep disorders, such as insomnia and jet lag, where sleep needs to be triggered against a strong waking drive.
