The neurochemical phenotype of myenteric neurons that arise from cells that transiently express TH (YFP+/TH-negative cells) was investigated by immunohistochemical analysis of YFP+ neurons in the jejunum of adult TH-Cre;R26R-YFP mice using antibodies to the pan-neuronal marker, Hu, and the neuron subtype markers serotonin, calbindin, neurofilament-M, calretinin, and nNOS. The data are summarized in Figs 4 and 5. Only around 5% of YFP+ myenteric neurons were TH-IR (Fig. 5A), and thus 95% of myenteric YFP+ neurons expressed TH only transiently. For technical reasons, we were unable to examine the overlap between TH-immunoreactivity and the other neuron subtype markers in this study. However, previous studies have shown that the TH neurons in the mouse small intestine are a different subpopulation from the serotonin neurons, and that the TH neurons appear to be a rare, but separate, subpopulation from other characterized neuron subtypes.
All YFP+ cells in the myenteric plexus of the jejunum were Hu-IR, and thus TH-expressing cells give rise only to neurons and not glial cells. YFP+ neurons comprised only 2.7 ± 0.8% of the total (Hu-IR) neurons in the myenteric plexus (n = 7038 Hu-IR neurons from three preparations) (Figs 4A–C; 5), and hence only a small proportion of myenteric neurons in the adult jejunum are the progeny of transiently TH-expressing cells. Nearly 30% of serotonin-IR neurons (n = 82 serotonin-IR neurons from three mice) in the myenteric plexus was YFP+, but only 6.3 ± 1.2% of YFP+ cells (n = 376 YFP+ neurons from three mice) were serotonin-IR (Figs 4J–O; 5), indicating that serotonin neurons are not the main derivative of the TC cells. Calbindin-IR neurons represented about 50% of the YFP+ neurons, while neurofilament-M-IR and calretinin-IR neurons comprised around 20% and 10%, respectively, of the YFP+ neurons (Figs 4D–I; 5). Only 2% of YFP+ neurons were nNOS-IR (Fig. 5). Although, calbindin-IR, neurofilament-M-IR and calretinin-IR show the largest overlap with YFP expression, YFP+ neurons comprise only 8.3 ± 1.0%, 7.1 ± 2.5%, and 0.9 ± 0.1% of these neuron subtypes, respectively (Fig. 5). To determine if there are any major differences in the proportions of neuron subtypes in the myenteric plexus of TH-Cre;R26R-YFP mice, we quantified the proportions of total (Hu-IR) neurons that exhibit nNOS- or calretinin-immunoreactivity; nNOS-IR and calretinin-IR neurons are two of the best characterized myenteric neuronal subtypes in the mouse ENS.[3, 4, 12, 19, 23, 31, 41, 45, 46] The proportions of Hu-IR myenteric neurons in the jejunum of two TH-Cre;R26R-YFP mice that were nNOS-IR (33% and 31%, n = 832 and 692 Hu-IR neurons, respectively) or calretinin-IR (44% and 47%, n = 615 and 630 Hu-IR neurons) were similar to previous reports of wild-type mice.[3, 4, 19, 46]
In the submucosal plexus of the jejunum, 35% of Hu+ neurons were YFP+, and TH-IR/YFP+ neurons comprised 62% of the YFP+ submucosal neurons (n = 172 YFP+ submucosal neurons examined from two mice). Therefore, around 13% of submucosal neurons (YFP+/TH-negative neurons) arise from cells that transiently express TH.