POST-MITOTIC ENTERIC NEURONS ARE HIGHLY PLASTIC AND RETAIN THEIR ABILITY TO REFORM NETWORKS AND REINNERVATE THE ADULT INTESTINE

The wiring of the enteric nervous system (ENS) is a dynamic process throughout development and adulthood. In the embryo, enteric neural crest derived cells expand and differentiate into the glia and neurons that form the ENS. In mice, these early neurons are electrically active after 2 days and regulate smooth muscle contraction within 9 days, indicating rapid differentiation into neurons with functional properties. Notably, the birth of new enteric neurons occurs into adulthood and in response to injury or inflammation, supporting the potential of leveraging postnatal neurogenesis for the treatment of enteric neuropathies. Although the ENS is formed rapidly in the embryo, the environmental conditions present in the aged intestine are different in size, structure, cell composition, signalling milieu and extracellular matrix components. Despite advancing knowledge in the mechanisms of endogenous postnatal neurogenesis and the utilization of cultured enteric neural stem cells for regenerative medicine applications, the capabilities of neurons in the postnatal environment to form neuronal circuits and reinnervate the gut have not been resolved. Here, we employ the first pan neuronal-specific Cre driver mouse model (BAF53b-Cre) to understand the potential of post-mitotic enteric neurons to reinnervate the postnatal intestine and elicit functional motor activity. BAF53b expression specifically colocalized with neuronal cell bodies (HuC/D) and fibres (TUBB3), but not enteric glia (Plp1). Cell cultures derived from single cell suspensions of the intestinal muscularis indicated that BAF53b-tdT neurons reform ganglia and interconnected networks within one-week in vitro. Support from mesenchymal cells or glia was required for enteric neurons to project nerve fibres in vitro. BAF53b-Diphtheria Toxin (DT) Receptor mice were generated to perform targeted ablation of colorectal enteric neurons by local delivery of DT. RNA-Seq analysis and wholemount imaging indicated successful and specific neuronal ablation and mice exhibited delayed fecal pellet production. Transplantation of myenteric neurons to the aganglionic environment reinnervated the colon within two weeks, restored contractile activity, and reversed delayed pellet expulsion. This was not dependent on the intestinal region of neuron isolation (colon v. small bowel). Optogenetic experiments using enteric neurons generated from BAF53b- channelrhodopsin mice transplanted to the muscularis indicate that enteric neurons can innervate the muscle with functional activity within 7 days in the postnatal environment. Together, these results demonstrate that enteric neurons are proficient in forming new functional connections in the postnatal environment in a timeframe similar to that of the embryo which highlights the remarkable capabilities of neural network formation and reinnervation in the adult intestine.