Combining brainwide activity imaging and electron microscopy reveals novel nociceptive circuits

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 cannot be resolved in using LSM. Here, we present a novel methodology to overcome this by combining in vivo whole-brain activity imaging, with subsequent 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 involved in nociception in Drosophila larva. After whole-brain imaging of activity during nociceptive stimulation, 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 nociceptive stimulation to determine their developmental lineage and identity. This revealed a distributed nociceptive network spanning 25 distinct lineages and many distinct brain areas, and included direct brain targets of nociceptive projection neurons that integrate nociceptive information with other sensory modalities, as well as brain output neurons (descending neurons [DN]) that likely contribute to action-selection. Surprisingly, we also found neurons previously previously associated with olfaction and learning, such as Kenyon cells. Our workflow provides a powerful framework for mapping neuronal activity onto structure across an entire brain, yielding novel insights into the central processing of noxious stimuli.