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.     

Distribution of the Parathyroid Hormone-Related Peptide and Its Receptor in the Saccus Vasculosus and Choroid Plexus in the Red Stingray (Dasyatis akajei: Elasmobranch)

1. The exact role of the parathyroid hormone-related peptide (PTHrP) is not fully understood. We used immunohistochemistry to localize the PTHrP and its receptor in the brain of the red stingray, particularly in the saccus vasculosus (SV) and choroid plexus.2. Immunoreactive PTHrP and its receptor were detected in the epithelial cells of the SV and the choroid plexus. In addition, the neuronal perikarya in the nucleus of the SV located in the hypothalamus is positive for the PTHrP.3. No PTHrP-containing neurons were detected in the choroid plexus. Extracts of SV and choroid plexus showed positive reactions against the PTHrP and its receptor antibody in Western blot analysis.4. High levels of immunoreactive PTHrP were detected in the plasma equivalent to those present in human humoral malignant hypercalcemia. In contrast, the immunoreactive PTHrP concentration in the cerebrospinal fluid was below detectable levels.5. Our results suggest that the regulation of the PTHrP in the SV differs from that in the choroid plexus in the red stingray.     

Immunohistochemical study of localization of gamma-glutamyl transpeptidase in the rat brain

Gamma-glutamyl transpeptidase (gamma-GTP) is a membrane-bound enzyme which is known to play a crucial role in active transport of amino acids across membrane barriers. We prepared a monoclonal antibody recognizing specifically rat gamma-GTP and investigated localization of the enzyme in the rat brain by immunohistochemistry with this antibody. The antigen was localized on the ependyma, epithelia of the choroid plexus and microvessels. More precise localization of gamma-GTP was examined with immuno-electron microscopy. The antigen was recognized on the microvilli and cilia of the ependymal cells, microvilli of the choroid epithelial cells and luminal membranes of the vascular endothelial cells.     

A serum-free culture system for stydying solute exchanges in the choroid plexus

Summary Organ cultures of choroid plexus tissues from the lateral ventricle of juvenile rats have been maintained for periods up to 7 wk in a chemically defined, serum-free media. Of several media and various supplements evaluated, the best growth and survival was obtained with the Pasadena Foundation for Medical Research-4 media supplemented with three hormones: epidermal growth factor, insulin, and hydrocortisone. Autoradiographic studies demonstrated that the epithelial cells incorporated [³H]leucine and [³H]thymidine indicating active protein and DNA synthesis, respectively. The organ cultures were characterized by bulbous, vesicular outgrowths from the choroidal villi explants. The fluid-filled lumina of the vesccles reached diameters of 900 μm and were easily accessed by micropipettes. The walls of the vesicles were composed of single layers of epithelial cells in which the ultrastructural features in the in vivo tissue were well maintained. The in vivo polarity (apical end toward the media and basilar end of the cells toward the luminal cavity) was also maintained. This morphologically stable in vitro system seems to be a promising model for investigation of secretory mechanisms of choroidal tissue. This work was supported in part by National Institutes of Health Grant NS 12906-06.     

Fluorescence histochemical study on regional differences in the sympathetic nerve supply of the choroid plexus from various laboratory animals

Summary The adrenergic nerve supply of the choroid plexus in all four ventricles was studied by the Falck-Hillarp histofluorescence technique in nine different species, and the noradrenaline concentration in whole plexus tissue was determined by a radioenzymatic method. The nerve density was usually in the order: third > lateral > fourth ventricular plexuses. Plexuses of the pig and cat possessed the largest number of nerves; the innervation was intermediary in the baboon, guinea-pig, rat, rabbit and hamster, whereas only few fluorescent nerves were found in the cow and mouse plexuses. Sympathetic denervation showed an ipsilateral supply from the superior cervical ganglia to the lateral plexuses and a mixed contribution to the midline plexuses. The total noradrenaline concentration varied between 0.10 and 0.73 ng per mg protein.     

Cholinergic Modulation of the Release of [3H]Acetylcholine from Synaptosomes of the Myenteric Plexus

Abstract: The purpose of these experiments was to determine if cholinergic agents affected the release of acetylcholine (ACh) from a synaptosomal preparation of the guinea pig ileum myenteric plexus. The synaptosomal preparation was first incubated with the precursor [³H]choline; subsequently, release of the stored [³H]ACh was measured. The release was decreased by oxotremorine or exogenous ACh plus hexamethonium and increased by exogenous ACh plus atropine. The nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium (DMPP) evoked release that was inhibited by nicotinic antagonists or muscarinic agonists. Release was stimulated half-maximally by approximately 2 μ m - and maximally by 10 μ m -DMPP. Either in the absence of calcium or at 0°C, DMPP was without effect. The effect of 10 μ m -DMPP was brief, a significant stimulation occurring only within the first 2 min at 37°C. Tetrodotoxin also inhibited excitation by DMPP but not completely. Thus, the release of [³H]ACh appears to be presynaptically modulated, negatively by muscarinic agonists and positively by nicotinic agonists.     

The influence of fluid shear stress on the remodeling of the embryonic primary capillary plexus

The primary capillary plexus in early yolk sacs is remodeled into matured vitelline vessels aligned in the direction of blood flow at the onset of cardiac contraction. We hypothesized that the influence of fluid shear stress on cellular behaviors may be an underlying mechanism by which some existing capillary channels remain open while others are closed during remodeling. Using a recently developed E-Tmod knock-out/lacZ knock-in mouse model, we showed that erythroblasts exhibited rheological properties similar to those of a viscous cell suspension. In contrast, the non-erythroblast (NE) cells, which attach among themselves within the yolk sac, are capable of lamellipodia extension and cell migration. Isolated NE cells in a parallel-plate flow chamber exposed to fluid shear stress, however, ceased lamellipodia extension. Such response may minimize NE cell migration into domains exposed to fluid shear stress. A two-dimensional mathematical model incorporating these cellular behaviors demonstrated that shear stress created by the blood flow initiated by the embryonic heart contraction might be needed for the remodeling of primary capillary plexus.     

Choroid plexus recovery after transient forebrain ischemia: role of growth factors and other repair mechanisms

1. Transient forebrain ischemia in adult rats, induced by 10 min of bilateral carotid occlusion and an arterial hypotension of 40 mmHg, caused substantial damage not only to CA-1 neurons in hippocampus but also to epithelial cells in lateral ventricle choroid plexus.2. When transient forebrain ischemia was followed by reperfusion (recovery) intervals of 0 to 12 hr, there was moderate to severe damage to many frond regions of the choroidal epithelium. In some areas, epithelial debris was sloughed into cerebrospinal fluid (CSF). Although some epithelial cells were disrupted and necrotic, their neighbors exhibited normal morphology. This patchy response to ischemia was probably due to regional differences in reperfusion or cellular metabolism.3. Between 12 and 24 hr postischemia, there was marked restoration of the Na⁺, K⁺, water content, and ultrastructure of the choroid plexus epithelium. Since there was no microscopical evidence for mitosis, we postulate that healthy epithelial cells either were compressed together on the villus or migrated from the choroid plexus stalk to more distal regions, in order to fill in gaps along the basal lamina caused by necrotic epithelial cell disintegration.4. Epithelial cells of mammalian choroid plexus synthesize and secrete many growth factors and other peptides that are of trophic benefit following injury to regions of the cerebroventricular system. For example, several growth factors are upregulated in choroid plexus after ischemic and traumatic insults to the central nervous system.5. The presence of numerous types of growth factor receptors in choroid plexus allows growth factor mediation of recovery processes by autocrine and paracrine mechanisms.6. The capability of choroid plexus after acute ischemia to recover its barrier and CSF formation functions is an important factor in stabilizing brain fluid balance.7. Moreover, growth factors secreted by choroid plexus into CSF are distributed by diffusion and convection into brain tissue near the ventricular system, e.g., hippocampus. By this endocrine-like mechanism, growth factors are conveyed throughout the choroid plexus–CSF–brain nexus and can consequently promote repair of ischemia-damaged tissue in the ventricular wall and underlying brain.