Min-Tsai Liu

5-HT4 Receptor-Mediated Neuroprotection and Neurogenesis in the Enteric Nervous System of Adult Mice

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-HT4 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-HT4 receptor promotes enteric neuron survival and/or neurogenesis. In vitro, 5-HT4 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-HT4 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-HT4 receptors are required postnatally for ENS growth and maintenance.

Orexin Synthesis and Response in the Gut

Orexin (hypocretin) appears to play a role in the regulation of energy balances. Previous reports have indicated that orexin-containing neurons are found only in the lateral hypothalamic (LH) area. We show that a subset of neurons in the gut which also express leptin receptors display orexin-like immunoreactivity and express functional orexin receptors. Orexin excites secretomotor neurons in the guinea pig submucosal plexus and increases motility. Moreover, fasting upregulates the phosphorylated form of cAMP response element-binding protein (pCREB) in orexin-immunoreactive neurons, indicating a functional response to food status in these cells. Together, these data suggest that orexin in the gut may play an even more intimate role in regulating energy homeostasis than it does in the CNS.

Netrins and DCC in the guidance of migrating neural crest-derived cells in the developing bowel and pancreas

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.

Immunohistochemical localization of nicotinic acetylcholine receptors in the guinea pig bowel and pancreas.

Although nicotinic acetylcholine receptors (nAChRs) are known to be present on neural elements in both the bowel and the pancreas, the precise location of these receptors has not previously been determined. Immunocytochemistry, by using a rat monoclonal antibody (mAb35), which recognizes α‐bungarotoxin (α‐Bgt)‐insensitive nAChRs, and a polyclonal antibody raised against the α‐Bgt‐sensitive receptor subunit, α7, was used to locate receptor protein in guinea pig gut and pancreas. mAb35‐receptor (mAb35‐R) immunoreactivity was abundant in both enteric plexuses, enterochromaffin cells, and pancreatic ganglia. Immunostaining was associated with the cell membrane, and clusters of mAb35‐R were observed on cell somas and dendrites. Receptor immunoreactivity was also observed on terminals and axons, suggesting that a subset of nAChRs is presynaptic. Internal sites of mAb35‐R were observed in permeabilized ganglia. Cells expressing the receptors were closely associated with ChAT‐immunoreactive nerve fibers. In addition, the majority of ChAT‐positive neurons expressed both cell surface and internal stores of mAb35‐R. In the bowel, clusters of mAb35‐R were present on the soma and dendrites of Dogiel type I motorneurons and secretomotor neurons. Receptors were detected at the plasma membrane of calbindin‐immunoreactive myenteric neurons. In contrast, calbindin‐immunoreactive submucosal neurons did not express cell surface mAb35‐R, supporting the idea that they are sensory neurons. A subset of enteric neurons expressed both mAb35‐R and glutamate receptor (GluR1) immunoreactivity. In the pancreas, mAb35‐R immunoreactivity was only observed in ganglia. α7‐immunoreactivity was found on both enteric cell bodies and nerve fibers. Based on these results, it appears that visceral nAChRs are composed of at least four subunits and that both pre‐ and postsynaptic nAChRs are present in the gut and pancreas. J. Comp. Neurol. 390:497–514, 1998.

Identification of cells that express 5- hydroxytryptamine1A receptors in the nervous systems of the bowel and pancreas

Although serotonin (5‐HT)1A receptors are known to be present on neural elements in both the bowel and the pancreas, the precise location of these receptors has not previously been determined. Earlier investigations have suggested that 5‐HT1A receptors are synthesized in enteric, but not pancreatic ganglia, and that they mediate pre‐ and postjunctional inhibition. Wholemount in situ hybridization was used to identify cells that contain mRNA encoding 5‐HT1A receptors, and immunocytochemistry was employed to locate receptor protein. mRNA encoding 5‐HT1A receptors was found in the majority of neurons in both submucosal and myenteric plexuses. 5‐HT1A immunoreactivity, however, was abundant only on the surfaces of a limited subset of nerve cell bodies and processes. 5‐HT‐immunoreactive axons were found in close proximity to sites of 5‐HT1A immunoreactivity. Myenteric, but not submucosal calbindin‐immunoreactive neurons (with Dogiel type II morphology) were surrounded by rings of 5‐HT1A immunoreactivity. The cytoplasm of the cell bodies and dendrites of a small subset of Dogiel type I neurons was also intensely 5‐HT1A immunoreactive. Most of the Dogiel type I 5‐HT1A‐immunoreactive myenteric neurons, and some of the type II neurons that were ringed by 5‐HT1A immunoreactivity became doubly labeled following injections of the retrograde tracer, FluoroGold (FG), into the submucosal plexus. 5‐HT1A immunoreactive neurons in distant submucosal ganglia also became labeled by retrograde transport of FG. None of the 5‐HT1A‐immunoreactive cells were labeled by the intraluminal administration of the β‐subunit of cholera toxin, a marker for vasoactive intestinal peptide‐containing secretomotor neurons. These observations suggest that some of the myenteric 5‐HT1A‐immunoreactive neurons project to submucosal ganglia and that the submucosal 5‐HT1A‐immunoreactive cells are interneurons. In addition to neurons, a subset of 5‐HT‐containing enterochromaffin cells expressed 5‐HT1A immunoreactivity, which was co‐localized with 5‐HT in secretory granules. In the pancreas, 5‐HT1A immunoreactivity was observed in ganglia, acinar nerves, and glucagon‐immunoreactive islet cells. Serotonergic enteropancreatic axons have been found to terminate in close proximity to each of these structures, which may thus be the targets of this innervation. The abundance of 5‐HT1A receptor immunoreactivity on nerves of the gut and pancreas suggests that drugs designed to interact with these receptors may have unanticipated visceral actions.

Differential localization of Ca2+ channel ?1 subunits in the enteric nervous system: Presence of ?1B channel-like immunoreactivity in intrinsic primary afferent neurons

Immunocytochemistry was employed to locate calcium (Ca2+) channel proteins in the enteric nervous system (ENS) of the rat and guinea pig. Anti‐peptide antibodies that specifically recognize the α1 subunits of class A (P/Q‐type), B (N‐type), C and D (L‐type) Ca2+ channels were utilized. α1B channel‐like immunoreactivity was abundant in both enteric plexuses, the mucosa, and circular and longitudinal muscle layers. Immunoreactivity was predominantly found in cholinergic varicosities, supporting a role for Ca2+ channels, which contain the α1B subunit, in acetylcholine release. Immunoreactivity was also associated with the cell soma of calbindin‐immunoreactive submucosal and myenteric neurons, cells that have been proposed to be intrinsic primary afferent neurons. α1C channel‐like immunoreactivity was distributed diffusely in the cell membrane of a large subset of neuronal cell bodies and processes, whereas α1D was found mainly in the cell soma and proximal dendrites of vasoactive intestinal polypeptide‐immunoreactive neurons in the guinea pig gut. α1A channel‐like immunoreactivity was found in a small subset of cell bodies and processes in the rat ENS. The differential localization of the α1 subunits of Ca2+ channels in the ENS implies that they serve distinct roles in neuronal excitation and signaling within the bowel. The presence of α1B channel‐like immunoreactivity in putative intrinsic primary afferent neurons suggested that class B Ca2+ channels play a role in enteric sensory neurotransmission; therefore, we determined the effects of the N‐type Ca2+ channel blocker, ω‐conotoxin GVIA (ω‐CTx GVIA), on the reflex‐evoked activity of enteric neurons. Demonstrating the phosphorylation of cyclic AMP (cAMP)‐responsive element‐binding protein (pCREB) identified neurons that became active in response to distension. Distension elicited hexamethonium‐resistant pCREB immunoreactivity in calbindin‐immunoreactive neurons in each plexus; however, in preparations stimulated in the presence of ω‐CTx GVIA, pCREB immunoreactivity was found only in calbindin‐immunoreactive neurons in the submucosal plexus and not in myenteric ganglia. These data confirm that intrinsic primary afferent neurons are located in the submucosal plexus and that N‐type Ca2+ channels play a role in sensory neurotransmission. J. Comp. Neurol. 409:85–104, 1999.

Guinea pig pancreatic neurons: morphology, neurochemistry, electrical properties, and response to 5-HT

The morphology, neurochemistry, and electrical properties of guinea pig pancreatic neurons were determined. The majority of neurons expressed choline acetyltransferase (ChAT) immunoreactivity; however, ChAT-negative neurons were also found. Both cholinergic and noncholinergic neurons expressed nitric oxide synthase (NOS) immunoreactivity. Three types of pancreatic neurons were distinguished. Phasic neurons fired action potentials (APs) at the onset of depolarizing current pulse, tonic neurons spiked throughout the duration of a suprathreshold depolarizing pulse, and APs could not be generated in nonspiking neurons, even though they did receive synaptic input. APs were tetrodotoxin sensitive, and all types of neurons received fast and slow excitatory postsynaptic potentials (EPSPs). Fast EPSPs had cholinergic and noncholinergic components. The majority of pancreatic neurons appeared to innervate the acini. NOS- and/or neuropeptide Y-immunoreactive phasic and tonic neurons were found. Microejection of 5-hydroxytryptamine (5-HT) caused a slow depolarization that was inhibited by the 5-HT1P antagonist N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide and mimicked by the 5-HT1Pagonist 6-hydroxyindalpine. A pancreatic 5-HT transporter was located, and inhibition of 5-HT uptake by fluoxetine blocked slow EPSPs in 5-HT-responsive neurons by receptor desensitization.