Brain Wide Anesthetic-Active Neuronal Network

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Abstract Anesthetics are low affinity drugs that interact with hundreds of molecular targets present throughout the nervous system at clinically significant concentrations. Despite this molecular-level promiscuity, the hypnotic effects of anesthetics depend critically on specific neural circuits. This assertion is supported by numerous results showing that direct modulation of specific sites distributed broadly throughout the brain can potentiate or, conversely, antagonize the anesthetic state. However, because previous experimental work focused upon one brain site at a time, the identification of the long-hypothesized brain-wide canonical anesthesia circuit has so far remained elusive. To fill this critical gap in knowledge, we reasoned that ultimately, the state of anesthesia must be imposed onto the brain by neurons that remain active under anesthesia, while most other neurons are suppressed. To identify anesthetic active neurons throughout the brain, we used tissue clearing and 3D c-Fos immunohistochemistry. We validated that putative anesthesia-active neurons are indeed physiologically active in vivo using two photon microscopy and fiber photometry. Having identified anesthesia- active neurons, we further reasoned that brain regions which project broadly are more likely to play a pivotal role in modulating the level of consciousness. Thus, we combined our brain activity map with whole brain connectivity analyses. The potent combination of these experimental and bioinformatics approaches allowed us to identify regions that contain a high density of anesthetic-active neurons and project broadly throughout the brain. Our unbiased approach culminated in the definition of a putative canonical anesthesia network comprised of nuclei in the ventral hypothalamus, thalamus, and the prefrontal cortex. In Aim 1, we will discover the specific cell types within prefrontal cortex, ventral hypothalamus and thalamus that remain active under anesthesia. This is of critical importance as all brain regions contain many cell subtypes with distinct neurophysiological properties, connectivity patterns, and ultimately, behavioral effects. In Aim 2, we will discover the brain-wide projections made by anesthetic-active neurons by combining anterograde and retrograde viral tracing in transgenic mice that specifically label distinct neuronal subtypes with 3D immunohistochemistry. In Aim 3, we will establish the functional roles of the canonical anesthesia network as a whole and each of its elements individually by combining chemo- and optogenetics with behavioral and neurophysiologic assessments of arousal. The ultimate result of the proposed research will be the identification of a canonical network of neurons sufficient to elicit hypnosis. This has fundamental implications for how the state of arousal is controlled in health and dysregulated in disease. Identification of this circuit may also suggest druggable targets for the development of more specific anesthetic agents with fewer undesirable effects. This has the potential to significantly improve the care of millions of patients requiring general anesthesia for life saving procedures.