Although the exact mechanisms by which anesthetics induce unconsciousness remain unknown, there is evidence that some anesthetics activate neural circuits regulating sleep and inhibit neural systems promoting waking. Despite general anesthesia and sleep both activating a subset of seemingly similar, if not identical, neurons, there are clear differences between the two unconscious states, including the degree of arousal threshold changes and the timescale of state transition. The neural mechanisms underlying these related, yet distinct unconscious states are poorly understood. The parafacial zone (PZ) has recently been identified as a non-rapid-eye-movement (non-REM) sleep-promoting region; specifically, GABAergic neurons in the PZ (PZ-GABA) are active during non-REM sleep. My preliminary data demonstrate that PZ-GABA are also active during isoflurane exposure, and ablation of PZ-GABA increases resistance to isoflurane. The results also suggest that non-GABAergic neurons within the PZ are also involved in isoflurane-induced hypnosis. The overarching question asks how the neural circuitry driving distinct states of non-REM sleep and isoflurane anesthesia converge and diverge by first examining in PZ-GABA neurons, and then expanding beyond the PZ to consider all cell types in the medulla. It is hypothesized that these distinct endogenous and drug-induced unconscious states are generated by partially overlapping shared circuits but that key state differences arise from distinctive cellular activation patterns. The three key questions we will address during this proposal are: 1) Does acute reversible activation/inhibition of the PZ sleep-promoting neurons alter anesthetic sensitivity? 2) What is the cellular makeup of the PZ, and which cells are activated during each unconscious state? and 3) What are the overlapping and different elements between the brainstem neural circuits engaged during isoflurane exposure and those engaged during non-REM sleep? These questions will be addressed by anesthetic and sleep phenotyping assays, the single-cell level transcriptomic analysis by single nucleus RNA sequencing followed by multiplex in situ hybridization, and side- by-side comparison of ensembles of active neurons by Targeted Recombination in Active Population (TRAP). The proposed projects will uncover the underlying mechanism of how the brainstem neural circuits, including PZ, mediate these two different unconscious states. Understanding how the brain controls states of unconsciousness is vital for clinical practice. It can lead to more effective and safer somnogens and new potential sedative hypnotic anesthetics that may one day be used for sleep disorders such as insomnia and narcolepsy.