Dissecting the role of neuronal-astroglial interactions in sleep homeostasis

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PROJECT SUMMARY Insufficient sleep, sleep disorders, and resulting problems with health and cognition are increasingly common in the United States. Many sleep disorders may be associated with abnormal sleep homeostasis: an innate regulatory process that balances sleep need, sleep intensity, and sleep amount as a function of prior time spent awake. Sleep homeostasis requires a feedback circuit to maintain the system within defined limits. However, the cellular components and protein signaling pathways of this feedback circuit remain incompletely defined. Our understanding of sleep homeostasis thus far is primarily based on the study of neurons, but I showed that non-neuronal cells (i.e. astrocytes) also play a role. I posit that the homeostatic feedback circuit includes a neuronal waking signal that reflects sleep need and an astroglial integrator of the neuronal waking signal. I propose that the wake-promoting neurotransmitter noradrenaline (NA) is a candidate for the neuronal waking signal that interacts with astrocytes. I further propose that calcium (Ca2+) is the astroglial integrator of sleep need because 1) NA increases astroglial Ca2+ activity and 2) I showed that astroglial Ca2+ plays a role in sleep homeostasis. My overall hypothesis is that wake-promoting neurons increase astroglial Ca2+ signaling during elevated sleep need. I will test this hypothesis in two AIMS: 1) Determine how NA impacts astroglial Ca2+ dynamics before, during, and after sleep deprivation (SD); 2) Determine how sleep loss impacts astroglial protein signaling. For AIM 1, I will use a multifaceted approach to optogenetically inhibit or stimulate NA neurons while imaging Ca2+ dynamics in adjacent astrocytes and recording electroencephalographic brain state activity in freely behaving mice. Optogenetics, Ca2+ imaging, and electroencephalographic recordings will occur simultaneously under baseline conditions and during SD & recovery. Using this multimodal approach, I can temporally register cell-type specific neuronal activity and astroglial Ca2+ dynamics within distinct arousal states in freely behaving mice. For AIM 2, I will determine which astroglial proteins respond to changes in sleep need. I will use ultra-performance liquid chromatography-tandem mass spectrometry to quantify astroglial proteins from rested and SD mice using targeted and untargeted proteomics. Targeted proteomics will include NA- & Ca2+-related signaling proteins as well as synaptic, metabolic, and gap junction proteins because these proteins are implicated in sleep homeostasis. I will also determine the phosphorylation status of these proteins because phosphorylation status changes with sleep need and is an important post-translational modification in astrocytes. Astrocytes will be isolated from brains of wild type mice and mutant mice with reduced astroglial Ca2+ signaling. In this way, I can determine which astroglial proteins are 1) responsive to changes in sleep need and 2) Ca2+-dependent. The proposed studies use innovative methods to define biological substrates of sleep homeostasis. These findings, in turn, will further characterize the contribution of non-neuronal cells in the regulation of sleep-wake behavior and will expand our understanding of physiological and disordered sleep.