Figure 4.
5-HT stimulates the development and/or survival of total and dopaminergic enteric neurons in cultures of ENCDCs isolated from E16 fetal mouse gut. ENCDCs were immunoselected with antibodies to p75NTR and cultured for 6 d in the absence or presence of exogenous 5-HT (1 μm). The TPH inhibitor PCPA (1 μm) was added to the medium to prevent biosynthesis of endogenous 5-HT. A, TH was identified immunocytochemically and was used as a dopaminergic marker. The abundance of TH-immunoreactive neurons in 5-HT-treated cultures was significantly greater than that in control cultures exposed only to vehicle. B, HuC/D was identified immunocytochemically and was used as a neuronal marker. The abundance of neurons in 5-HT-treated cultures was significantly greater than in control cultures exposed only to vehicle. C, Although numbers of both total neurons and dopaminergic neurons increased after the addition of 5-HT, the proportion of neurons that were dopaminergic was not significantly different in 5-HT-treated cultures from that of control cultures exposed only to vehicle. D, E, HuC/D-immunoreactive neurons (red fluorescence) were found in small aggregates in both vehicle-treated (D) and 5-HT-treated (E) cultures. The numbers and the sizes of the aggregates, however, were both larger in 5-HT-treated cultures than in cultures exposed only to vehicle. F–I, Double-labeled immunocytochemistry was used to locate simultaneously the immunoreactivities of HuC/D and TH (green fluorescence). The cell bodies of TH-immunoreactive (dopaminergic) neurons were found within aggregates of HuC/D-immunoreactive neurons. F, Vehicle-treated culture. A single dopaminergic neuron appears within an aggregate of HuC/D-immunoreactive neurons. No dopaminergic neurites are visible. G, 5-HT-treated culture. Dopaminergic neurons are more numerous and extend TH-immunoreactive neurites that project to adjacent neuronal aggregates. H, I, 5-HT-treated cultures. H, Dopaminergic neurites project from neurons within an aggregate to the adjacent substrate, in which they terminate in a series of varicosities. I, Some dopaminergic neurites in 5-HT-treated cultures are very long and branch extensively. Scale bars: D, E, 100 μm; F–H, 20 μm; I, 50 μm. ns, Not significant.