Pan-neuronal functional imaging and anesthesia

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Volatile anesthetics, such as isoflurane, produce all stages of general anesthesia including unconsciousness, amnesia, analgesia and muscle relaxation. Despite their ubiquity, the fundamental mechanisms of action of these drugs remains unknown. Elucidating the mechanisms by which clinical anesthesia is produced is the foundational unanswered research question in the specialty of anesthesiology. The routine use of volatile anesthetics is not without clinical risk. Multiple exposures to anesthetics in infancy leads to possible behavioral problems in later life, and persistent post-operative cognitive dysfunction is seen in the elderly after anesthesia. Historically, research in this field has proceeded along two tracks: either molecular analysis looking for specific receptors for the volatile anesthetics (an approach that has largely foundered due to diffuse interactions with many receptors whose effects do not combine appropriately), or the gross measurement of neuronal activity in entire regions of the brain using EEG and fMRI (which are fundamentally limited by resolution). Clearly, there is an enormous gap in length resolution between synaptic-scale and EEG-scale. We hypothesized that the onset of anesthesia, and hence the loss of consciousness, is due to disruption in the communication between neurons at the level of small neuronal networks that lie well below the resolution limit of EEG and fMRI. We study the effect of anesthetic agents on intercommunication within intact, living neural networks, with single neuron resolution. We use C. elegans, the creature with the simplest, most tractable neuronal architecture in which anesthesia is known to be inducible. Using light-sheet microscopy, a combination of fixed and calcium- sensitive fluorophores expressed under neuronal promoters, and our customized supercomputing toolchain for image analysis and signal extraction, we track and capture the activity of essentially the entire nervous system and examine its behavior under varying levels of anesthetic exposure normalized to comparable levels used in human surgery. Using two-photon imaging, we are able to perform similar experiments in the mouse and extract activity from selected regions of the somatosensory cortex in both awake and anesthetized states. We will use a system of differential expression of fixed neuronal fluorophores in C. elegans to allow the precise identification of individual neurons under light-sheet imaging. In combination with the C. elegans connectome, we will determine the neuronal pathways that underlie anesthetized vs conscious states, how anesthetics alter chemical and electrical synaptic connections to induce these states, the changes in neuronal connectivity that permit the anesthetized state to resolve back into consciousness, and hence delineate the mechanisms of clinical post-operative cognitive dysfunction in the old and neurodevelopmental impairment in the young. We will extend our imaging and analysis techniques into vertebrate zebrafish, building a phylogenetic bridge in the action of anesthetics from simple creatures to humans. We will demonstrate that these changes in neuronal activity are consistent with the gross statistical hallmarks of anesthesia seen in the EEG in humans.