Neural ensemble fragmentation in the anesthetized Drosophila brain.

General anesthetics cause a profound loss of behavioral responsiveness in all animals. In mammals, general anesthesia is induced in part by the potentiation of endogenous sleep-promoting circuits, although 'deep' anesthesia is understood to be more similar to coma (Brown et al., 2011). Surgically relevant concentrations of anesthetics such as isoflurane and propofol have been shown to impair neural connectivity across the mammalian brain (Mashour and Hudetz, 2017; Yang et al., 2021), which presents one explanation why animals become largely unresponsive when exposed to these drugs. It remains unclear whether general anesthetics affect brain dynamics similarly in all animal brains, or whether simpler animals such as insects even display levels of neural connectivity that could be disrupted by these drugs. Here, we utilized whole-brain calcium imaging in behaving female Drosophila flies to investigate whether isoflurane anesthesia induction activates sleep-promoting neurons, and then inquired how all other neurons across the fly brain behave under sustained anesthesia. We were able to track the activity of hundreds of neurons simultaneously during waking and anesthetized states, for spontaneous conditions as well as in response to visual and mechanical stimuli. We compared whole-brain dynamics and connectivity under isoflurane exposure to optogenetically-induced sleep. Neurons in the Drosophila brain remain active during general anesthesia as well as induced sleep, even though flies become behaviorally inert under both treatments. We identified surprisingly dynamic neural correlation patterns in the waking fly brain, suggesting ensemble-like behavior. These become more fragmented and less diverse under anesthesia but remain wake-like during induced sleep. SIGNIFICANCE STATEMENT: When humans are rendered immobile and unresponsive by sleep or general anesthetics, their brains do not shut off - they just change how they operate. We tracked the activity of hundreds of neurons simultaneously in the brains of fruit flies that were anesthetized by isoflurane or genetically put to sleep, to investigate if these behaviorally inert states shared similar brain dynamics. We uncovered dynamic patterns of neural activity in the waking fly brain, with stimulus-responsive neurons constantly changing through time. Wake-like neural dynamics persisted during induced sleep but became more fragmented under isoflurane anesthesia. This suggests that, like larger brains, the fly brain might also display ensemble-like behavior, which becomes degraded rather than silenced under general anesthesia.