Quality sleep is imperative for the maintenance of good health. There is a growing recognition of sex disparities in sleep and rhythm disorders. Complaints such as insufficient sleep and insomnia are ~40% more prevalent in women than men. Yet, historically, women and female animals are underrepresented in studies of sleep and its disorders. While gonadal steroids and gender are implicated as risk factors for sleep disruptions and insomnia, the relationship between ovarian steroids and sleep is poorly understood. The broad, long-term objective of the application is to understand the cellular mechanisms and functional consequences of estrogen- mediated changes in vigilance states. Findings from the previous grant have established that estradiol (E2) acting in the median preoptic nucleus (MnPO) is necessary and sufficient to reduce total sleep by ~50% of baseline in females. However, our data present an intriguing paradox: E2 suppresses sleep while markedly increasing extracellular adenosine, a potent inducer of sleep in the MnPO. Recent findings and preliminary data have begun to shed light on this apparent contradiction. Our findings in the MnPO show that (1) E2 blocks the sleep inducing actions of adenosine A2A receptor activation and activation of the adenosine A1 receptors or chemogenetic activation of MnPO astrocytes mimic E2 suppression of sleep. Together, these findings have led us to our central hypothesis that the E2-induced high levels of extracellular adenosine initiate a shift in adenosinergic balance from an A2A excitatory tone that activates MnPO sleep neurons to an A1R inhibitory tone that inhibits the sleep-neurons and E2 stimulation of MnPO astrocytes is responsible the increase in adenosine. We propose four independent, but related aims to address the following gaps in our knowledge that are critical to understanding the mechanisms underlying estrogenic regulation of sleep: (1) Is the increase in MnPO extracellular adenosine required for E2-induced suppression of sleep? (2) Is the activation of MnPO adenosine A1 receptors required for E2-induced suppression of sleep? (3) Is the suppression of MnPO adenosine A2A receptors required for E2-induced suppression of sleep? and (4) Is E2 stimulation of MnPO astrocytes required for increases in adenosine and sleep suppression? We approach these questions through a combination of modern neuroscience techniques that include a GAD1-Cre transgenic rat line, fast scan cyclic voltammetry (FSCV) of adenosine, fiber photometry and chemogenetics to investigate estrogenic regulation of sleep. There is heuristic value in comparing and contrasting cellular mechanisms between the sexes as this approach has frequently reveal novel and previously unknown mechanisms of neural plasticity. Thus, we have included a comparison of sex as a biological factor in addition to estrogenic influences on sleep mechanisms. The significance of advancing our understanding of the mechanisms underlying E2 modulation of s...