Causal mechanisms of anesthetic induction and emergence in human cortical organoids

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PROJECT SUMMARY This project aims to use human cortical organoids, which are cortex-like structures generated in vitro from human induced pluripotent stem cells (hiPSCs), to resolve outstanding questions in our understanding of causal mechanisms underlying the mesoscale phenomenology of anesthetic induction (AI) and anesthetic emergence (AE). Millions of patients undergo general anesthesia every year, but the mechanisms by which anesthetic drugs give rise to the hallmarks of AI remain unresolved. Even less well understood are the mechanisms by which the brain emerges from anesthesia - a process over which clinicians have almost no control, and which is frequently associated with complications such as emergence delirium, respiratory events, and delayed emergence, which results in prolonged hospital stays and increased cost of care. In addition, 1-2 per every 1000 patients will experience intraoperative awareness with explicit recall, for reasons that are not understood. While a number of hypotheses regarding the mechanisms of AI and AE have been put forward, these hypotheses are still debated because of complex inter-circuit interactions during AI and AE in the intact brain. In particular, it is widely believed that the major cause of AI is the potentiation of cortical GABAa receptors, but it has been difficult to disentangle the effects of cortical GABAa potentiation from the subcortical effects of anesthesia, which may likewise contribute to AI. Similarly, though it is generally believed that at least one, if not several cortically projecting neuromodulatory structures - including the histaminergic tuberomammillary nucleus of the hypothalamus, the cholinergic basal forebrain, the serotonergic raphe nuclei, the orexinergic lateral hypothalamus, and the noradrenergic locus coeruleus - directly drive emergence from anesthesia, these systems are densely interconnected and mutually excitatory. For this reason, in vivo research has been unable to resolve which, if any, of these systems directly cause AE. A promising but completely unexplored tool for resolving these questions are human cortical organoids. Our team has recently developed a protocol for fusing together networks of excitatory and inhibitory cortical-like neurons derived from hiPSCs. These fusion cortical organoids can recapitulate the oscillatory electric activity of the awake human cortex, and our preliminary results suggest that these cortical organoids can mimic the mesoscale hallmarks of AI when they are exposed to the anesthetic propofol. Importantly, cortical organoids consist of purely cortical- like human tissue, and lack any influence from subcortical structures or neuromodulatory systems. This allows us to use human cortical organoids to isolate cortical versus non-cortical causal mechanisms of both AI and AE. Successful modeling of AI and AE in brain organoids would illustrate the utility of these structures in high- throughput screening of novel drugs for inducing anesthesia or emergence from anesthesia, potentially even on a single-patient basis. Additionally, this project would establish the potential for human brain organoids in screening therapies for other states of unconsciousness, such as coma and persistent vegetative states.