A functional network of highly pure enteric neurons in a dish

Abstract

The enteric nervous system (ENS) is the intrinsic nervous system that innervates the entire digestive tract and regulates major digestive functions. Recent evidence has shown that functions of the ENS critically rely on enteric neuronal connectivity; however, experimental models to decipher the underlying mechanisms are limited. Compared to the central nervous system, for which pure neuronal cultures have been developed for decades and are recognized as a reference in the field of neuroscience, an equivalent model for enteric neurons is lacking. In this study, we developed a novel model of highly pure rat embryonic enteric neurons with dense and functional synaptic networks. The methodology is simple and relatively fast. We characterized enteric neurons using immunohistochemical, morphological, and electrophysiological approaches. In particular, we demonstrated the applicability of this culture model to multi-electrode array technology as a new approach for monitoring enteric neuronal network activity. This in vitro model of highly pure enteric neurons represents a valuable new tool for better understanding the mechanisms involved in the establishment and maintenance of enteric neuron synaptic connectivity and functional networks.

The regulation of enteric neuron connectivity by semaphorin 5A is affected by the autism-associated S956G missense mutation

“…To investigate whether the effects induced by Sema5A on synaptic vesicle exocytosis and synapsin phosphorylation could translate into changes in the electrophysiological properties of enteric neurons, cell-attached and whole-cell patch-clamp recordings were performed as previously established. 28 In the cell-attached configuration, we observed several changes induced by Sema5A-Fc exposure compared to control Fc, including a decreased number of spontaneously firing neurons (56% vs. 33% of total neurons upon Fc and Sema5A-Fc exposure, respectively; Figure 9 B). Moreover, among these spontaneously active neurons (Fc n = 9 and Sema5A-Fc n = 5), we observed a marked reduction (5-fold) in firing frequency upon exposure to Sema5A compared to control conditions ( Figures 9 A–9C).…”

“…To further investigate the direct response of enteric neurons to Sema5A stimulation on synapsin 1 phosphorylation, we used a culture model highly purified in enteric neurons. 28 COS cells transfected with Fc, Sema5A-Fc or Sema5A-Fc-S956G constructs were plated on Transwell inserts deposited above enteric neurons to allow the Fc and Sema5A proteins to diffuse from COS cells to enteric neurons. Using site-specific phospho-synapsin 1 antibodies, we analyzed by western blot the levels of synapsin 1 phosphorylation at Ser603 and Ser9, involved in synapsin 1 dissociation from synaptic vesicles, 25 , 26 and at Ser62/67, involved in synapsin 1 dissociation from actin filaments.…”

“… 25 , 26 , 27 Thus, we investigated the impact of Sema5A on synaptic vesicle exocytosis by imaging the presynaptic release of the dye FM1-43 as previously described in central and enteric neurons. 28 , 30 , 31 After FM1-43 loading in neurons within synaptic vesicles, enteric neurons were stimulated by KCl-induced depolarization for 4 min and the fluorescence decrease was recorded. The decrease of FM1-43 fluorescence intensity corresponds to the fusion of synaptic vesicles with the plasma membrane and the release of the dye, therefore providing an indicator of the synaptic vesicle exocytosis activity ( Figure 7 A).…”

“…Primary culture of rat enteric neuron was performed as previously described. 28 Briefly, the intestine of E15 rat embryos were cut into pieces of equal length (1–2 mm) and placed in a well of a 24-well culture plate previously coated with collagen type I (cat: 354236, Corning). After 5 days of culture in enteric glia-conditioned neurobasal medium (cat: 21103-049, Gibco) supplemented with B27 (cat: 17504-044-Gibco), 10 mM Glutamine (cat: 25030-024-Gibco), 50,000 IU Penicillin/50,000 IU Streptomycin (cat: 15140-122-Gibco) and GDNF (50 ng/mL) (cat: 512-GF-010/CF R&D Systems - bio-techne), the explants were delicately removed and the cells having migrated out of the explants were detached with Accutase (cat: 07920, StemCell technologies, Saint-Egrève, France).…”