Monosynaptically-interconnected Network Module (MNM) Approach for High-Resolution Brain Sub-Network Analysis

We introduce the Monosynaptically-interconnected Network Module (MNM) approach, an innovative method designed for efficiently analyzing the anatomical structure and functional dynamics of specific brain network modules in vivo . Utilizing an Intein-mediated split-Cre system combined with bidirectional adeno-associated viruses, this technique precisely targets and manipulates monosynaptically interconnected modular subnetworks in freely moving animals. We demonstrate its utility through anatomical and functional mapping of a specific MNM encompassing the prefrontal cortex (PFC), basolateral amygdala (BLA), and intermediary hub regions. Specifically, the MNM approach with Cre-reporter mice visualizes detailed network architecture and enables the tracing of axonal connections among the nodes in the network. Furthermore, integration of the MNM approach with Cre-dependent Ca2+ indicator and multi-fiber photometry in freely moving mice reveals enhanced correlative network activities in social contexts. This versatile technique offers significant potential for advancing our understanding of network functions that underlie complex behaviors, providing a modular network perspective. ### Competing Interest Statement The authors have declared no competing interest.