Detection of mitotic neuroblasts provides additional evidence of steady state neurogenesis in the adult small intestinal myenteric plexus.

Maintenance of normal structure of the enteric nervous system (ENS), which regulates key gastrointestinal functions, requires robust homeostatic mechanisms, since by virtue of its location within the gut wall, the ENS is subject to constant mechanical, chemical, and biological stressors. Using transgenic and thymidine analogue-based experiments, we previously discovered that neuronal turnover - where continual neurogenesis offsets ongoing neuronal loss at steady state - represents one such mechanism. Although other studies confirmed that neuronal death continues into adulthood in the myenteric plexus of the enteric nervous system (ENS), the complicated nature of thymidine analogue presents challenges in substantiating the occurrence of adult neurogenesis. Therefore, it's vital to employ alternative, well-recognized techniques to substantiate the existence of adult enteric neurogenesis in the healthy gut. Here, by using established methods of assessing nuclear DNA content and detecting known mitotic marker phosphor-histone H3 (pH3) in Hu⁺ adult ENS cells, we show that ∼10% of adult small intestinal myenteric Hu⁺ cells in mice, and ∼20% of adult human small intestinal myenteric Hu⁺ cells show evidence of mitosis and hence are cycling neuroblasts. We observe that proportions of Hu⁺ cycling neuroblasts in the adult murine ENS neither vary with ganglia size, nor do they differ significantly between two intestinal regions - duodenum and ileum, or between sexes. Confocal microscopy provides further evidence of cytokinesis in Hu⁺ cells. The presence of a significant population of cycling neuroblasts in adult ENS provide further evidence of steady state neurogenesis in the adult ENS.Significance statement Using 3-dimensional confocal microscopy, immunohistochemical detection of cell cycle marker phosphor-Histone H3, and DNA content assessments using flow cytometry in Hu+ cells from adult small intestinal murine myenteric plexus, we show that ∼10% of myenteric Hu+ cells in adult gut are mitotic neuroblasts, whose proportional representation does not significantly differ between sexes or small intestinal regions. We further test and observe mitotic marker pH3 also immunolabels ∼23% of adult human myenteric Hu+ cells suggesting that presence of mitotic neuroblasts also extends to the adult human gut. These data further evidence of steady state adult enteric neurogenesis in the healthy gut and provide important cellular details in understanding how precursor cells continually generate large numbers of adult neurons in healthy gut.