Combining brain-wide activity imaging with electron microscopy reveals a distributed brain network for processing threatening somatosensory stimuli

To understand how brains work, it is necessary to connect neural activity to synaptic-resolution circuit architecture. Recent advances in light-sheet microscopy (LSM) enable whole-brain, cellular-resolution imaging of activity of all neuronal cell bodies, however, most neurons from such datasets cannot be identified. In most organisms, neurons are identifiable based on their projections (and not based on their cell body position) which, when densely labelled, cannot be resolved using LSM. Here, we present a novel methodology to overcome this by combining whole-brain activity imaging with subsequent volume electron microscopy imaging of the same brain to visualise neuronal projections and identify neurons with interesting activity. We used this approach to identify brain neurons that process input from multisensory (nociceptive and mechanosensory) Basin interneurons that trigger vigorous escape in Drosophila larvae in response to threatening somatosensory stimuli. After whole-brain imaging of neuronal activity during Basin activation, we imaged the same brain with an enhanced focused ion-beam electron microscope (eFIB-SEM). We registered the functional and anatomical volumes and reconstructed (in the eFIB-SEM volume) the projections of neurons that responded to Basin activation to determine their developmental lineage and identity. This revealed a distributed network for processing threatening somatosensory stimuli that trigger vigorous escape spanning 25 distinct lineages and many distinct brain areas, and included direct brain targets of Basin neurons that integrate somatosensory information with other modalities, as well as brain output neurons (descending neurons [DN]) that likely contribute to action-selection. Our workflow provides a powerful framework for mapping neuronal activity onto structure across an entire brain, yielding novel insights into the distributed central processing of noxious stimuli.