Multiplexed Neuromodulatory-Type-Annotated EM-Reconstruction of Larval Zebrafish

The brain is complementarily assembled by the sensorimotor and neuromodulatory (NM) pathways. Mapping the neural connectome is essential for elucidating the synaptic organization principles of this bi-pathway architecture. However, most electron microscopy (EM) reconstructions provide limited information about cell types, in particular NM neurons. Here we present a synapse-level, multiplexed molecular annotated reconstruction of an intact larval zebrafish brain, comprising over 170 thousand cells and 25 million synapses. Noradrenergic, dopaminergic, serotonergic, hypocretinergic/orexinergic, and glycinergic neurons were identified using subcellular localization of APEX2, while glutamatergic and GABAergic neurons were inferred from the Zebrafish Mesoscopic Atlas. NM neurons with varying indegree scale-distinctly innervate various sensory-motor brain regions, exhibiting heterogeneity in synapse number and strength. As a critical NM system, individual locus coeruleus noradrenergic (LC-NE) neurons integrate extensive brain-wide inputs displaying modality-specific spatial organization. Motor-related and sensory-related inputs preferentially target proximal and distal dendrites of these neurons, respectively. While the majority of inputs are one-to-one, approximately 16% synapse onto multiple targets, forming one-to-many connections. Notably, these shared input patterns extend across different monoaminergic systems, serving as a structural basis for coordinated neuromodulation. Our results demonstrate the organization principles of the NM system's input architecture, particularly within the LC-NE system. This brain-wide, multiplexed molecular annotated microscale reconstruction of a vertebrate brain, combined with multi-modal mesoscopic datasets, offers a reliable resource for the precise identification of diverse neuronal types within EM connectomes, and provides critical reference for elucidating the synaptic architecture principles underlying sensorimotor and NM pathways in the vertebrate brain.