A. L. Kirchgessner

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.

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.

Identification of Serotonin Receptors Recognized by Anti‐Idiotypic Antibodies

Anti‐idiotypic antibodies were generated by immunizing rabbits with affinity‐purified antibodies to serotonin (5‐hydroxytryptamine; 5‐HT). Anti‐5‐HT activity was removed from the resulting antisera by chromatography through a 5‐HT affinity column. The anti‐idiotypic antibodies were demonstrated by enzyme‐linked immunosorbent assay to bind to affinity‐purified whole anti‐5‐HT antibodies and their Fab fragments. Anti‐idiotypic antibodies, purified by affinity chromatography on columns to which antibodies to 5‐HT were coupled, competed with 5‐HT (covalently bound to protein) for the binding sites on anti‐5‐HT antibodies and serotonin binding protein. The anti‐idiotypic antibodies antagonized the binding of [3H]5‐HT to membranes isolated from the cerebral cortex, striatum, and raphe area more than to membranes from hippocampus or cerebellum. The anti‐ idiotypic antibodies also blocked the binding of the 5‐HT1B‐ selective ligand (‐)‐[125I]iodocyanopindolol (in the presence of 30 μM isoproterenol) to cortical membranes. In contrast, anti‐idiotypic antibodies failed to inhibit binding of the 5‐ HT1A‐selective ligand 8‐hydroxy‐2‐(di‐n)‐[3H]propylamino)‐ tetralin ([3H]8‐OH‐DPAT) to raphe area membranes or hippocampal membranes. These observations suggested that the anti‐idiotypic antibodies may recognize some 5‐HT receptor subtypes but not others. This hypothesis was tested by ascertaining the ability of anti‐idiotypic antibodies to immunostain cells transfected in vitro with cDNA encoding the 5‐ HT1C or 5‐HT2 receptor or with a genomic clone encoding the 5‐HT1A receptor. Punctate sites of immunofluorescence were found on the surfaces of fibroblasts that expressed 5‐ HT1C and 5‐HT2 receptors, but not on the surfaces of HeLa cells that expressed 5‐HT1A receptors. Immunostaining of cells by the anti‐idiotypic antibodies was inhibited by appropriate pharmacological agents: immunostaining of cells expressing 5‐HT1C receptors was blocked by mesulergine (but not ketanserin, 8‐OH‐DPAT. or spiperone), whereas that of cells expressing 5‐HT2 receptors was blocked by ketanserin or spiperone (but not mesulergine or 8‐OH‐DPAT). The anti‐ idiotypic antibodies failed to inhibit the uptake of [3H]5‐HT by serotonergic neurons. It is concluded that the anti‐idiotypic antibodies generated with anti‐5‐HT serum recognize the 5‐ HT1B, 5‐HTlC, and 5‐HT2 receptor subtypes; however, neither 5‐HT1A receptors nor 5‐HT uptake sites appear to react with these antibodies.

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.

5-HT1P Receptors in the Bowel: G Protein Coupling, Localization, and Function

Although serotinin (5-HT) has been demonstrated to mediate slow exicitatory postsynaptic potentials in neurons of the myenteric plexus of the bowel, its role in the physiology of gastrointestinal motility and secretion is not well understood. Five subtypes of 5-HT receptor have been reported in the gut. The 5-HT2 receptor appears to be present on smooth muscle, while the others, 5-HT1A, 5-HT1P„ 5-HT3, and 5-HT4 are neuronal. The present experiments were undertaken in order to obtain more information about the location, action, and function of 5-HT1P receptors. GTP-γ-S, was found to inhibit the binding of 3H-5-HT by the 5-HT1P receptor (the IC50 for GTP-γ-S was 1.8 ± 0.4 μM). GTP-γ-S was more potent than GTP, while ATP and GMP (100 μM) were without effect. These observations are compatible with the hypothesis that the 5-HT1P receptor is coupled to a G protein. 5-HT (0.1–10μM) was also found to elevate levels of cAMP in preparations of isolated myenteric ganglia. Renzapride also increased cAMP, but the response neither to 5-HT nor to renzapride was inhibited by the 5-HT1P antagonist, N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide (5-HTP-DP) or the 5-HT3/5-HT4 antagonist, ICS 205–930, even at 10 μM. The action of 5-HT on ganglionic cAMP, therefore, cannot now be attributed to 5-HT1P, or 5-HT4 receptors. Polyclonal anti-idiotypic antibodies were raised that recognize some, but not all, subtypes of 5-HT receptor. These antibodies were demonstrated to bind to 5-HT2, 5-HT1C, 5-HT1P, and 5-HT3, but not 5-HT1A receptors. When applied to enteric neurons, the anti-idiotypic antibodies transiently mimicked and then blocked the actions of 5-HT at 5-HT3 and 5-HT1P receptors. The blockage of responses to 5-HT that followed application of the antibody was specific, since neurons continued to respond as before to carbachol or substance P. The 5-HT1P agonist-like actions of the antibodies were prevented by desensitization of 5-HT receptors and by renzapride (1 μM). The anti-idiotypic antibodies were found to be useful for the immunocytochemical localization of specific 5-HT receptor subtypes, if they were applied to tissues in the presence of appropriate antagonists. Use of these antibodies indicated that 5-HT1P receptors are located, not only on neurons in myenteric and submucosal ganglia, but also on a subepithelial plexus of nerve fibers. Finally, 5-HT1P receptor antagonists (5-HTP-DP, renzapride, and anti-idiotypic antibodies), were found to prevent excitation of submucosal or myenteric neurons by mucosal application of cholera toxin. This observation, and the localization of 5-HT1P receptors, is consistent with the idea that they play a role in the excitation of enteric neurons during the peristaltic reflex.

Preparation and characterization of monoclonal antibodies to serotonin binding protein

Abstract: Serotonin binding protein (SBP) is a constituent of the synaptic vesicles of serotonergic neurons. Two types of SBP, with molecular masses of 45 kDa and 56 kDa, have been purified. To determine whether there are shared epitopes between the two forms of SBP, we raised and tested for cross‐reactivity monoclonal antibodies (MAbs) against each form of SBP. We obtained 12 MAbs, all of which recognize both forms of SBP. Hybridoma clones were produced by fusing P3 ± 63Ag8.653 mouse myeloma cells with spleen cells from a mouse that had been immunized with 45‐kDa or 56‐kDa SBP. Culture supernatants were screened for the presence of anti‐SBP antibodies. MAb isotypes were determined by immunodiffusion, using immunoglobulin type‐specific antisera. Each antibody to SBP consisted of only a single subclass of immunoglobulin (IgM). We obtained 12 MAbs, each of which interacted with both forms of SBP, as judged by enzyme‐linked immunosorbent assay and immunoblot analysis. Ascites fluid to one clone (44–10) was obtained and affinity‐purified. In the presence of goat anti‐mouse IgM, the partially purified 44–10 antibodies quantitatively immunoprecipitated SBP from crude brain extracts. Immunoblotting revealed two major bands corresponding to 45 kDa and 56 kDa and a minor band corresponding to 68 kDa. MAb 44–10 blocked the binding of [3H]serotonin ([3H]5‐HT) to 45‐kDa and 56‐kDa SBP in a concentration‐dependent manner. The 68‐kDa protein was found to bind [3H]5‐HT. Sites reacting with Mab 44–10 were located immunocytochemically in sections of rat brain. 5‐HT immunoreactivity was localized simultaneously in the same sections by using affinity‐purified rabbit anti‐5‐HT antibodies and species‐specific secondary antibodies coupled to a contrasting fluorophore. MAb 44–10 immunostaining involved neuronal cell bodies, neurites, and terminals. This immunostaining was intense within the nuclei of the median raphe and the B9 cell group. Coincident expression with 5‐HT was observed; however, MAb 44–10 also immunostained many neurons in which 5‐HT immunoreactivity was not seen. These observations may indicate that SBP is distributed more widely in the brain than 5‐HT; however, because SBP immunoreactivity is not found in nonserotonergic neurons when monospecific polyclonal antibodies are used for immunocytochemistry, it seems more likely that some nonserotonergic neurons contain another protein (such as the 68‐kDa SBP) that also contains an epitope recognized by MAb 44–10. Nevertheless, these data demonstrate that MAb 44–10 reacts with the 5‐HT binding domain of 45‐kDa and 56‐kDa SBP and will be a valuable tool for analyzing these proteins.