ABSTRACT
Although the mature enteric nervous system (ENS) has been shown to retain stem cells, enteric neurogenesis has not previously been demonstrated in adults. The relative number of enteric neurons in wild-type (WT) mice and those lacking 5-HT(4) receptors [knock-out (KO)] was found to be similar at birth; however, the abundance of ENS neurons increased during the first 4 months after birth in WT but not KO littermates. Enteric neurons subsequently decreased in both WT and KO but at 12 months were significantly more numerous in WT. We tested the hypothesis that stimulation of the 5-HT(4) receptor promotes enteric neuron survival and/or neurogenesis. In vitro, 5-HT(4) agonists increased enteric neuronal development/survival, decreased apoptosis, and activated CREB (cAMP response element-binding protein). In vivo, in WT but not KO mice, 5-HT(4) agonists induced bromodeoxyuridine incorporation into cells that expressed markers of neurons (HuC/D, doublecortin), neural precursors (Sox10, nestin, Phox2b), or stem cells (Musashi-1). This is the first demonstration of adult enteric neurogenesis; our results suggest that 5-HT(4) receptors are required postnatally for ENS growth and maintenance.
Subject(s)
Enteric Nervous System/physiology , Neurogenesis/physiology , Neurons/physiology , Receptors, Serotonin, 5-HT4/metabolism , Adult Stem Cells/physiology , Aging , Animals , Apoptosis/drug effects , Apoptosis/physiology , Cell Proliferation , Cell Survival/drug effects , Cell Survival/physiology , Cells, Cultured , Cyclic AMP Response Element-Binding Protein/metabolism , Enteric Nervous System/drug effects , Enteric Nervous System/growth & development , Gastrointestinal Tract/innervation , Gastrointestinal Tract/physiology , Indoles/pharmacology , Mice , Mice, Knockout , Neurogenesis/drug effects , Neurons/cytology , Neurons/drug effects , Phosphorylation/drug effects , Receptors, Serotonin, 5-HT4/genetics , Serotonin 5-HT4 Receptor Agonists , Serotonin Receptor Agonists/pharmacology , Sulfonamides/pharmacologyABSTRACT
The aim of the current study was to identify enteric 5-HT(4) splice variants, locate enteric 5-HT(4) receptors, determine the relationship, if any, of the 5-HT(4) receptor to 5-HT(1P) activity, and to ascertain the function of 5-HT(4) receptors in enteric neurophysiology. 5-HT(4a), 5-HT(4b), 5-HT(4e), and 5-HT(4f) isoforms were found in mouse brain and gut. The ratio of 5-HT(4) expression to that of the neural marker, synaptophysin, was higher in gut than in brain but was similar in small and large intestines. Submucosal 5-HT(4) expression was higher than myenteric. Although transcripts encoding 5-HT(4a) and 5-HT(4b) isoforms were more abundant, those encoding 5-HT(4e) and 5-HT(4f) were myenteric plexus specific. In situ hybridization revealed the presence of transcripts encoding 5-HT(4) receptors in subsets of enteric neurons, interstitial cells of Cajal, and smooth muscle cells. IgY antibodies to mouse 5-HT(4) receptors were raised, affinity purified, and characterized. Nerve fibers in the circular muscle and the neuropil in ganglia of both plexuses were highly 5-HT(4) immunoreactive, although only a small subset of neurons contained 5-HT(4) immunoreactivity. No 5-HT(4)-immunoreactive nerves were detected in the mucosa. 5-HT and 5-HT(1P) agonists evoked a G protein-mediated long-lasting inward current that was neither mimicked by 5-HT(4) agonists nor blocked by 5-HT(4) antagonists. In contrast, the 5-HT(4) agonists renzapride and tegaserod increased the amplitudes of nicotinic evoked excitatory postsynaptic currents. Enteric neuronal 5-HT(4) receptors thus are presynaptic and probably exert their prokinetic effects by strengthening excitatory neurotransmission.
Subject(s)
Enteric Nervous System/physiology , Protein Isoforms/biosynthesis , Receptors, Serotonin, 5-HT4/physiology , Alternative Splicing , Animals , Enteric Nervous System/ultrastructure , Excitatory Postsynaptic Potentials/drug effects , Female , Male , Mice , Neurons/metabolism , Patch-Clamp Techniques , Receptors, Serotonin, 5-HT4/biosynthesis , Reverse Transcriptase Polymerase Chain Reaction , Synaptophysin/biosynthesisABSTRACT
Vagal neural crest-derived precursors of the enteric nervous system colonize the bowel by descending within the enteric mesenchyme. Perpendicular secondary migration, toward the mucosa and into the pancreas, result, respectively, in the formation of submucosal and pancreatic ganglia. We tested the hypothesis that netrins guide these secondary migrations. Studies using RT-PCR, in situ hybridization, and immunocytochemistry indicated that netrins (netrins-1 and -3 mice and netrin-2 in chicks) and netrin receptors [deleted in colorectal cancer (DCC), neogenin, and the adenosine A2b receptor] are expressed by the fetal mucosal epithelium and pancreas. Crest-derived cells expressed DCC, which was developmentally regulated. Crest-derived cells migrated out of explants of gut toward cocultured cells expressing netrin-1 or toward cocultured explants of pancreas. Crest-derived cells also migrated inwardly toward the mucosa of cultured rings of bowel. These migrations were specifically blocked by antibodies to DCC and by inhibition of protein kinase A, which interferes with DCC signaling. Submucosal and pancreatic ganglia were absent at E12.5, E15, and P0 in transgenic mice lacking DCC. Netrins also promoted the survival/development of enteric crest-derived cells. The formation of submucosal and pancreatic ganglia thus involves the attraction of DCC-expressing crest-derived cells by netrins.
Subject(s)
Cell Adhesion Molecules/metabolism , Enteric Nervous System/embryology , Nerve Growth Factors/metabolism , Nerve Tissue Proteins/metabolism , Neural Crest/cytology , Neural Crest/metabolism , Tumor Suppressor Proteins/metabolism , Animals , Base Sequence , Cell Adhesion Molecules/antagonists & inhibitors , Cell Adhesion Molecules/deficiency , Cell Adhesion Molecules/genetics , Cell Line , Cell Movement , Chick Embryo , DCC Receptor , DNA/genetics , Enteric Nervous System/cytology , Enteric Nervous System/metabolism , Ganglia/abnormalities , Ganglia/embryology , Gene Expression Regulation, Developmental , Humans , Immunohistochemistry , In Situ Hybridization , Mice , Mice, Transgenic , Nerve Growth Factors/genetics , Nerve Tissue Proteins/genetics , Netrin-1 , Netrins , Neural Crest/embryology , Pancreas/cytology , Pancreas/embryology , Pancreas/metabolism , Receptors, Cell Surface , Transfection , Tumor Suppressor Proteins/antagonists & inhibitors , Tumor Suppressor Proteins/deficiency , Tumor Suppressor Proteins/geneticsABSTRACT
The actions of enteric 5-HT are terminated by 5-HT transporter (SERT)-mediated uptake, and gastrointestinal motility is abnormal in SERT -/- mice. We tested the hypothesis that adaptive changes in enteric 5-HT(3) receptors help SERT -/- mice survive despite inefficient 5-HT inactivation. Expression of mRNA encoding enteric 5-HT(3A) subunits was similar in SERT +/+ and -/- mice, but that of 5-HT(3B) subunits was fourfold less in SERT -/- mice. 5-HT(3B) mRNA was found, by in situ hybridization, in epithelial cells and enteric neurons. 5-HT evoked a fast inward current in myenteric neurons that was pharmacologically identified as 5-HT(3) mediated. The EC(50) of the 5-HT response was lower in SERT +/+ (18 microM) than in SERT -/- (36 microM) mice and desensitized rapidly in a greater proportion of SERT -/- neurons; however, peak amplitudes, steady-state current, and decay time constants were not different. Adaptive changes thus occur in the subunit composition of enteric 5-HT(3) receptors of SERT -/- mice that are reflected in 5-HT(3) receptor affinity and desensitization.