Optogenetic activation of entorhinal projection neurons alters the target recognition and circuit development without enhancing axon regeneration after axotomy in organotypic slices.

The central nervous system (CNS) has a limited intrinsic capacity for axonal regeneration, making functional recovery after injury extremely challenging. Numerous strategies have been explored to overcome this blockade, among others, molecular interventions or modulation of the inhibitory extracellular environment. Despite some advances, effective regeneration remains elusive, particularly in adult CNS neurons. To investigate these mechanisms in a controlled and reproducible setting, we employ organotypic slice cultures (OSCs), which retain key structural and cellular features of the intact brain while allowing for long-term in vitro experimentation. In particular, the entorhino-hippocampal (EH) co-culture model preserves the anatomical and functional connectivity of the perforant pathway, providing an excellent platform for studying axonal degeneration and regeneration. This model reproduces laminar specificity, axonal myelination, and inhibitory signaling after axotomy, closely mimicking in vivo conditions. Furthermore, EH co-cultures facilitate the application of optogenetic tools to monitor and manipulate neuronal activity. Our study explores whether enhancing activity in entorhinal cortex neurons can promote axonal regeneration after a EH lesion. Our results show that increased activity in entorhinal neurons alters the development of the EH connection and fails to enhance the regrowth of injured mature entorhinal axons. These findings suggest that both extrinsic and intrinsic factors shape the regenerative response and highlight the utility of EH OSCs as a versatile model for testing future pro-regenerative interventions.