A Rat Embryonic Enteric Nervous System Culture Platform for Modeling Neuronal Subtype Diversity and Network Activity Maturation

This system gives rise to functional neuronal networks composed of multiple enteric neuronal subtypes and closely integrated with enteric glial cells. Single-cell RNA sequencing identifies diverse neuronal populations, including ChAT-positive excitatory neurons, NOS-positive inhibitory neurons, and VIP-positive interneurons, as well as a glial cell population.

Transcriptomic analysis further defines cell type-specific marker profiles and candidate regulators of enteric neuronal and glial development. Electrophysiological recordings demonstrate maturation of spontaneous network activity, with contributions from cholinergic, nitrergic, and VIPergic neuronal populations. Together, these findings establish a tractable in vitro platform for investigating neuronal subtype specification, the emergence of cellular diversity, neuro-glial interactions and functional electrophysiological network organization.