S. Pompolo

Projections and chemical coding of neurons with immunoreactivity for nitric oxide synthase in the guinea-pig small intestine

The distribution of nitric oxide synthase (NOS) immunoreactivity was investigated in the guinea-pig small intestine. There were many immunoreactive nerve cell bodies in the myenteric plexus but very few in submucous ganglia. NOS immunoreactivity was not found in non-neuronal cells except for rare mucosal endocrine cells. Abundant immunoreactive nerve fibres in both myenteric and submucous ganglia, and in the circular muscle, arose from myenteric nerve cells whose axons projected anally along the intestine. NOS immunoreactivity coexisted with VIP-immunoreactivity, but not with substance P immunoreactivity. We conclude that nitric oxide synthase is located in a sub-population of enteric neurons, amongst which are inhibitory motor neurons that supply the circular muscle layer.

Plurichemical transmission and chemical coding of neurons in the digestive tract.

The enteric nervous system contains neurons with well-defined functions. However, when neurons of the same function are examined in different regions or species, they are found to show subtle differences in their pharmacologies of transmission and different chemical coding. Individual enteric neurons use more than one transmitter, i.e., transmission is plurichemical. For example, enteric inhibitory neurons have three or more primary transmitters, including nitric oxide, vasoactive intestinal peptide, and possibly adenosine triphosphate and pituitary adenylyl cyclase activating peptide. Primary transmitters are highly conserved, although their relative roles vary considerably between gut regions. Multiple substances, including transmitters and their synthesizing enzymes and nontransmitters (such as neurofilament proteins), provide neurons with a chemical coding through which their functions and projections can be identified. Although equivalent neurons in different regions have the same primary transmitters, other chemical markers differ substantially. Caution must be taken in extrapolating pharmacological and neurochemical observations between species or even between regions in the one species. On the other hand, careful interregion and interspecies comparisons lead to an understanding of the features of enteric neurons that are highly conserved and can be used in valid extrapolation.

Roles of peptides in transmission in the enteric nervous system.

Studies of the enteric nervous system have proved to be important in the development of new concepts of the chemical nature of transmission from neurons. In particular, they have revealed the multiplicity of influences that peptides can have on transmission, such as their action as primary transmitters, and the fact that they often act as co-transmitters in enteric neurons. However, in other cases no roles can be attributed to neuropeptides in enteric neurons, and their involvement in short-term changes in excitability seems minor.

Calbindin neurons of the guinea-pig small intestine: quantitative analysis of their numbers and projections

SummaryThe distribution of nerve cells with immunoreactivity for the calcium-binding protein, calbindin, has been studied in the small intestine of the guinea-pig, and the projections of these neurons have been analysed by tracing their processes and by examining the consequences of nerve lesions. The immunoreactive neurons were numerous in the myenteric ganglia; there were 3500±100 reactive nerve cells per cm2 of undistended intestine, which is 30% of all nerve cells. In contrast, reactive nerve cells were extremely rare in submucous ganglia. The myenteric nerve cells were oval in outline and gave rise to several long processes; this morphology corresponds to Dogiels type-II classification. Processes from the cell bodies were traced through the circular muscle in perforating nerve fibre bundles. Other processes ran circumferentially in the myenteric plexus. Removal of the myenteric plexus, allowing time for subsequent fibre degeneration, showed that reactive nerve fibres in the submucous ganglia and mucosa came from the myenteric cell bodies. Operations to sever longitudinal or circumferential pathways in the myenteric plexus indicated that most reactive nerve terminals in myenteric ganglia arise from myenteric cell bodies whose processes run circumferentially for 1.5 mm, on average. It is deduced that the calbindin-reactive neurons are multipolar sensory neurons, with the sensitive processes in the mucosa and with other processes innervating neurons of the myenteric plexus.

Oncostatin M promotes bone formation independently of resorption when signaling through leukemia inhibitory factor receptor in mice

Effective osteoporosis therapy requires agents that increase the amount and/or quality of bone. Any modification of osteoclast-mediated bone resorption by disease or drug treatment, however, elicits a parallel change in osteoblast-mediated bone formation because the processes are tightly coupled. Anabolic approaches now focus on uncoupling osteoblast action from osteoclast formation, for example, by inhibiting sclerostin, an inhibitor of bone formation that does not influence osteoclast differentiation. Here, we report that oncostatin M (OSM) is produced by osteoblasts and osteocytes in mouse bone and that it has distinct effects when acting through 2 different receptors, OSM receptor (OSMR) and leukemia inhibitory factor receptor (LIFR). Specifically, mouse OSM (mOSM) inhibited sclerostin production in a stromal cell line and in primary murine osteoblast cultures by acting through LIFR. In contrast, when acting through OSMR, mOSM stimulated RANKL production and osteoclast formation. A key role for OSMR in bone turnover was confirmed by the osteopetrotic phenotype of mice lacking OSMR. Furthermore, in contrast to the accepted model, in which mOSM acts only through OSMR, mOSM inhibited sclerostin expression in Osmr-/- osteoblasts and enhanced bone formation in vivo. These data reveal what we believe to be a novel pathway by which bone formation can be stimulated independently of bone resorption and provide new insights into OSMR and LIFR signaling that are relevant to other medical conditions, including cardiovascular and neurodegenerative diseases and cancer.

EphrinB2 regulation by PTH and PTHrP revealed by molecular profiling in differentiating osteoblasts.

With the aim of identifying new pathways and genes regulated by PTH(1–34) and PTH‐related protein 1–141 [PTHrP(1–141)] in osteoblasts, this study was carried out using a mouse marrow stromal cell line, Kusa 4b10, that acquires features of the osteoblastic phenotype in long‐term culture conditions. After the appearance of functional PTH receptor 1 (PTHR1) in Kusa 4b10 cells, they were treated with either PTH(1–34) or PTHrP(1–141), and RNA was subjected to Affymetrix whole mouse genome array. The microarray data were validated using quantitative real‐time RT‐PCR on independently prepared RNA samples from differentiated Kusa 4b10, UMR106 osteosarcoma cells, and primary mouse calvarial osteoblasts, as well as in vivo using RNA from metaphyseal bone after a single PTH injection to 3‐wk‐old and 6‐mo‐old ovariectomized rats. Of the 45,101 probes used on the microarray, 4675 were differentially expressed by ≥1.5 fold, with a false discovery rate <0.1. Among the regulated genes, ephrinB2 mRNA was upregulated in response to both PTH and PTHrP. This was confirmed by quantitative real‐time PCR in vitro and in vivo. Increased ephrinB2 protein was also shown in vitro by Western blotting, and immunostaining of femur sections showed ephrinB2 in both osteoclasts and osteoblasts. Production of ephrinB2, as well as other ephrins or Eph family members, did not change during differentiation of Kusa 4b10 cells. Blockade of ephrinB2/EphB4 interaction resulted in inhibition of mineralization of Kusa 4b10 cells. Together with the shown effect of ephrinB2 promoting osteoblast differentiation and bone formation through action on EphB4, the data raise the possibility that PTH or PTHrP might regulate ephrinB2 to act in a paracrine or autocrine manner on EphB4 or EphB2 in the osteoblast, contributing as a local event to the anabolic action of PTH or PTHrP.

Cardiotrophin‐1 Is an Osteoclast‐Derived Stimulus of Bone Formation Required for Normal Bone Remodeling

Cardiotrophin (CT‐1) signals through gp130 and the LIF receptor (LIFR) and plays a major role in cardiac, neurological, and liver biology. We report here that CT‐1 is also expressed within bone in osteoclasts and that CT‐1 is capable of increasing osteoblast activity and mineralization both in vitro and in vivo. Furthermore, CT‐1 stimulated CAAT/enhancer‐binding protein‐δ (C/EBPδ) expression and runt‐related transcription factor 2 (runx2) activation. In neonate CT‐1−/− mice, we detected low bone mass associated with reduced osteoblasts and many large osteoclasts, but increased cartilage remnants within the bone, suggesting impaired resorption. Cultured bone marrow (BM) from CT‐1−/− mice generated many oversized osteoclasts and mineralized poorly compared with wildtype BM. As the CT‐1−/− mice aged, the reduced osteoblast surface (ObS/BS) was no longer detected, but impaired bone resorption continued resulting in an osteopetrotic phenotype in adult bone. CT‐1 may now be classed as an essential osteoclast‐derived stimulus of both bone formation and resorption.

Immunohistochemical evidence for the presence of calcium-binding proteins in enteric neurons

SummaryImmunoreactivity for vitamin D-dependent calcium-binding protein (CaBP) has been localized in nerve cell bodies and nerve fibres in the gastrointestinal tracts of guinea-pig, rat and man. CaBP immunoreactivity was found in a high proportion of nerve cell bodies of the myenteric plexus, particularly in the small intestine. It was also found in submucous neurons of the small and large intestines. Immunoreactive nerve fibres were numerous in the myenteric ganglia, and were also common in the submucous ganglia and in the intestinal mucosa. Immunoreactive fibres were rare in the circular and longitudinal muscle coats. In the myenteric ganglia of the guinea-pig small intestine the immunoreactivity is restricted to one class of nerve cell bodies, type-II neurons of Dogiel, which display calcium action potentials in their cell bodies. These neurons were also immunoreactive with antibodies to spot 35 protein, a calcium-binding protein from the cerebellum. From the distribution of their terminals and the electrophysiological properties of these neurons it is suggested they might be sensory neurons, or perhaps interneurons. The discovery of CaBP in restricted sub-groups of enteric neurons may provide an important key for the analysis of their functions.

Evidence that enteric motility reflexes can be initiated through entirely intrinsic mechanisms in the guinea‐pig small intestine

Abstract Although motility reflexes can be elicited in the intestine in vivo after all neural connections with the central nervous system are cut, or in vitro in isolated intestinal segments, it is not proven that the cell bodies of the primary sensory neurons for these reflexes are in the intestinal wall. It is feasible that the nerve cells are in dorsal root ganglia and that axon reflexes are involved in the initiation of the reflexes. We have examined reflexes in segments of guinea‐pig intestine in which extrinsic denervation, 9–11 days before the intestine was removed, and isolation of the intestine in vitro were combined. The experimental segments were isolated from extrinsic inputs by severing nerves in the mesentery and those running in the gut wall that entered the segment. The effectiveness of denervation was confirmed histochemically. Ascending and descending reflexes were evoked by mucosal distortion or distension and responses were recorded by intracellular microelectrodes in the circular muscle. Reflex responses recorded after denervation were no different to those recorded from control tissue. It is concluded that, in the small intestine of the guinea‐pig, cell bodies of primary sensory neurons for mucosal and probably for distension reflexes are intrinsic to the organ.

Ultrastructure and synaptic relationships of calbindin-reactive, Dogiel type II neurons, in myenteric ganglia of guinea-pig small intestine.

SummaryImmunoreactivity for calbindin D 28K was localized ultrastructurally in nerve cell bodies and nerve fibres in myenteric ganglia of the guinea-pig small intestine. Reactive cell bodies had a characteristic ultrastructure: the cytoplasm contained many elongate, electron-dense mitochondria, numerous secondary lysosomes that were peripherally located, peripheral stacks of rough endoplasmic reticulum and dispersed Golgi apparatus. The cells were generally larger than other myenteric neurons and had mainly smooth outlines. The cytoplasmic features of these neurons were shared by a small group of immunonegative cells, but the majority of negative cells had clearly different ultrastructural appearances. Of 310 cells from 16 ganglia that were systematically examined, 38% were immunoreactive for calbindin, 10% were unreactive but similar in ultrastructure to the calbindin-reactive neurons and 51% were unreactive and dissimilar in the appearance of their cytoplasmic organelles. Immunoreactive varicosities with synaptic specializations were found on most unreactive neurons, but were markedly less frequent on the calbindin-immunoreactive cell bodies. Non-reactive presynaptic fibres were also more common on non-reactive neurons than on the calbindin-positive cell bodies. Numerous reactive varicosities, some showing synaptic specializations, were found adjacent to other fibres in the neuropil. Light microscopic studies show calbindin immunoreactive neurons to have Dogiel type-II morphology. Thus the present work links distinguishing ultrastructural features to a specific nerve cell type recognized by light microscopy in the enteric ganglia for the first time.