Transiently catecholaminergic (TC) cells in the bowel of the fetal rat: precursors of noncatecholaminergic enteric neurons

Experiments were done to study the fate of transient catecholaminergic (TC) cells that develop in the rodent gut during ontogeny. When they are first detected, at Day E11 in rats, TC cells are distributed along the vagal pathway, in advance of the descending fibers of the vagus nerves, and in the foregut. The early TC cells coexpress the immunoreactivities of several neural markers, including 150-kDa neurofilament protein, peripherin, microtubule associated protein (MAP) 5, and growth-associated protein (GAP)-43, with those of the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DBH). All cells in the fetal rat bowel at Day E11 that express neural markers also express TH immunoreactivity. The primitive TC cells also express the immunoreactivities of neural cell adhesion molecule (N-CAM), neuropeptide Y (NPY), and nerve growth factor (NGF) receptor (and NGF receptor mRNA). By Day E12 TC cells are found along the vagal pathway and throughout the entire preumbilical bowel. At this age TC cells acquire additional characteristics, including MAP 2 and synaptophysin immunoreactivities and acetylcholinesterase activity, which indicate that they continue to mature as neurons. In addition, TC cells of the rat are immunostained at Day E12 by the NC-1 monoclonal antibody, which in rats labels multiple cell types including migrating cells of neural crest origin. Despite their neural properties, at least some TC cells divide and therefore are neural precursors and not terminally differentiated neurons. At Day E10 TH mRNA-containing cells were not detected by in situ hybridization; however, by Day E11 TH mRNA was detected in sympathetic ganglia and in scattered cells in the mesenchyme of the foregut and vagal pathway. At this age, the number of enteric and vagal cells containing TH mRNA is about 30% less than the number of cells containing TH immunoreactivity in adjacent sections. The ratio of TH mRNA-containing cells to TH-immunoreactive vagal and enteric cells is even less at Day E12, especially in more caudal regions of the preumbilical bowel. A similar decline in the ratio of TH mRNA-containing to TH-immunoreactive cells was not observed in sympathetic ganglia. After Day E12 TH mRNA cannot be detected in enteric or vagal cells by in situ hybridization; nevertheless, TH immunoreactivity continues to be present through Day E14. DBH, NPY, and NGF receptor immunoreactivities are expressed by TH-immunoreactive transitional cells in the fetal rat gut after TH mRNA is no longer detectable.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immunocytochemical detection of neuronal nitric oxide synthase (nNOS)-IR in embryonic rat stomach between days 13 and 21 of gestation.

In this study, the localization and appearance of neuronal nitric oxide synthase-immunoreactive (nNOS-IR) nerve cells and their relationships with the developing gastric layers were studied by immunocytochemistry techniques and light microscopy in embryonic rat stomach. The stomachs of Wistar rat embryos aged 13-21 days were used. The first nerve cells containing nNOS-IR were seen on embryonic Day 14. The occurrence of mesenchymal cell condensation near nNOS-IR neuroblasts on embryonic Day 15 may reflect an active nerve element-specific mesenchymal cell induction causing the morphogenesis of muscle cells. Similarly, the appearance of glandular structures after nNOS-IR neuroblasts, on embryonic Day 18, suggests that the epithelial differentiation may depend on inputs coming from nNOS-IR neuroblasts, as well as other factors. Observation of nNOS-IR nerve fibers on embryonic Day 21 demonstrates that at this stage they contribute to nonadrenergic noncholinergic relaxation. In conclusion, depending on this study's results, it can be said that cells and tissues might be affected by NO secreted by nNOS-IR nerve cells during the development and differentiation of embryonic rat stomach.     

Transient catecholaminergic (TC) cells in the vagus nerves and bowel of fetal mice: relationship to the development of enteric neurons

Catecholaminergic cells are transiently present during development of the fetal murine bowel. These transient catecholaminergic (TC) cells appear at Day E10, but by Day E13 can no longer be detected. In order to evaluate the hypothesis that these cells are the precursors of enteric neurons, we investigated the possibilities that TC cells coexpress neuronal and catecholaminergic markers, that they can be found along the presumed path followed by crest-derived cells migrating to the gut, and that they are proliferating. TC cells were identified immunocytochemically using polyclonal or monoclonal antibodies to tyrosine hydroxylase (TH). At Day E9.5, TH-immunoreactive cells were observed to be present along the wall of the primordial esophagus in lines that extended from the developing nodose ganglia down to the boundary of the stomach. At Day E9.5, TC cells were absent from the remaining foregut. These lines of esophageal TH-immunoreactive cells became continuous with similar cells in the wall of the stomach and duodenum on Day E10. Coincident expression of neurofilament immunoreactivity was seen in all of the esophageal TH-immunoreactive cells present at Day E9.5, as well as in the entire set of esophageal and lower enteric TH-immunoreactive cells present at Day E10 (or later); moreover, at Days E9.5 and E10, all of the neurofilament-immunoreactive cells in the esophagus, stomach, or duodenum were also TH-immunoreactive. In contrast, neurofilament immunoreactivity was not expressed by the endodermally derived pancreatic duct and islet cells, which were also TH-immunoreactive; nor could expression of neurofilament immunoreactivity be detected in the TH-immunoreactive cells of the nodose ganglia. It was not until Day E11 that neurofilament-immunoreactive cells, which did not coexpress TH immunoreactivity (the definitive phenotype of enteric neurons) began to appear in the gut. Vagal axons reached as far distally as the nodose ganglion on Day E9.5, the esophagogastric junction on Day E10, and did not enter the stomach until Day E11. When the vagus nerves reached their level, the TH-immunoreactive cells in the wall of the esophagus came to lie among the nerve fibers. TH-immunoreactive cells are thus present on the pathway ultimately followed by the vagus nerves, but they develop before vagal fibers reach their level. The vagal TH-immunoreactive cells, therefore, are probably not initially migrating on vagal fibers, but appear instead to be overtaken by the descending vagus nerves.(ABSTRACT TRUNCATED AT 400 WORDS)     

Human gallbladder mucosa ultrastructure: evidence of intraepithelial nerve structures

The zinc iodide-osmium tetroxide (ZIO) fixative-staining method was used along with topographical ultrastructure to investigate cholecystectomized human gallbladders under light and electron microscopic techniques. This method delineated neuronal structures which may be involved in controlling the functions of the gallbladder epithelium. Three epithelial cell types were described in the surface epithelium: 1) Columnar clear cells; 2) dark, tuft osmiophilic cells; and 3) basal clear cells with electron-dense granules and showing intense ZIOphilic staining properties. While mucous granules were delineated in the first two cell types, the columnar epithelial clear cells are of uncertain function(s) and content but are probably absorptive cells. The small basal clear cells displaying intense ZIOphilia are associated with intraepithelial nerve endings. These nerve structures may have a sensory and/or motor function(s); they were detected throughout the gallbladder epithelial lining and mucosa.     

Immunohistochemical localization of superoxide dismutases in fetal and neonatal rat tissues.

We investigated the developmental profile of copper-zinc and manganese superoxide dismutase (CuZnSOD and MnSOD) in tissue sections obtained from fetal (Day 12 to 21 of gestation) and neonatal (Day 0 and 6) rats. Tissues were stained immunohistochemically with specific antisera against the respective rat SODs. There was a general trend towards richness of SODs in the epithelial linings and metabolically active sites, although differential distribution between the two SODs also existed. At Day 12 of gestation, immunoreactivity for both SODs was detected in the cardiomyocytes but not in other tissues. Hepatocytes expressed CuZnSOD at Day 14 and MnSOD at Day 17. By Day 18 CuZnSOD was detected in the epithelial cells of the gastrointestinal tract, respiratory tract, pancreatic islets, kidneys, and adrenals. These tissues exhibited MnSOD staining at Day 19. CuZnSOD occurred in the epithelia of the thyroid, thymus, and salivary glands at Day 19, while MnSOD was seen at Day 21. The increase in intensity of the staining for SODs occurred no later than postnatal Day 0, indicating that most tissues accumulated SODs during late gestation. Breathing atmospheric oxygen during early extrauterine life did not appreciably intensify the SOD staining. These results suggest that perinatal increase in SODs occurs as a general mechanism of preparation for birth.     

Histochemical characterization of myenteric plexus in domestic fowl small intestine

The myenteric plexus of the domestic fowl (Gallus domesticus) small intestine was studied by means of silver staining, glyoxylic acid-induced fluorescence, the modified Koelle-Friedenwald method for the detection of acetylcholinesterase, NADH-diaphorase techniques and the unlabelled antibody method involving the use of an antiserum raised against GABA conjugated by glutaraldehyde to bovine serum albumin. The majority of the perikarya were in the ganglia, with an average density of 3370 +/- 942 nerve cells/cm2. Cholinesterase-positive and a few GABA-immunoreactive nerve cell bodies were seen in the myenteric ganglia, while fluorescent ganglion cells were not observed. In addition to AChE and GABA-positive nerve fibres, a rich fluorescent network of varicose and nonvaricose nerve fibres was detected, pointing to the presence of an extrinsic aminergic system in the domestic fowl myenteric plexus. Electron microscopic observations on nerve cells, axon profiles and varicosites with various vesicle populations were in good agreement with the histochemical findings.     

Changes in synaptic potential properties during acetylcholine receptor accumulation and neurospecific interactions in Xenopus nerve-muscle cell culture

Acetylcholine receptors in the muscle cell membrane accumulate at the nerve contact area in Xenopus cell cultures. The correlation between spontaneous synaptic potential properties and extent of acetylcholine receptor accumulation was studied. Small and infrequent miniature endplate potentials were measured before acetylcholine receptor accumulation which was observed with fluorescence microscopy using tetramethylrhodamine-conjugated α-bungarotoxin. As acetylcholine receptors accumulate at the nerve contact area, these synaptic potentials become larger and their frequency increases dramatically. In nerve-contacted muscle cells where spontaneous synaptic activity could not be detected, extensive acetylcholine receptor accumulation was not found at sites of nerve contact. Furthermore, muscle cells which exhibited extensive acetylcholine receptor accumulation along the nerve always produced miniature endplate potentials. Thus acetylcholine receptor accumulation and the presence of miniature endplate potentials were strongly correlated. Noncholinergic neurons from dorsal root ganglia did not form functional synaptic contacts with muscle cells nor acetylcholine receptor accumulation along the path of contact. Furthermore, explants from tadpole spinal cord formed functional synaptic contacts with muscle cells but rarely caused AChR localization. These data are discussed in terms of developmental processes during neuromuscular junction formation.     

Role of the "little brain" in the gut in water and electrolyte homeostasis

The enteric nervous system plays a key role in maintenance of body fluid homeostasis by regulating the transport of ions by the intestinal epithelium. The epithelial cells normally absorb large volumes of fluid and ions daily, but tonically active submucosal neurons continuously suppress ion transport and limit the absorptive capacity of the intestine. Specialized nerve endings detect chemical, osmotic, or thermal alterations of the luminal contents or mechanical activity of the gut wall and encode this information as action potentials that propagate along nerve processes to the ganglia. Information transfer within the ganglia occurs at nicotinic cholinergic or other synapses. Ion transport is altered when neurotransmitters released from motor neurons interact with receptors on epithelial cells to initiate stimulus-response coupling. The signals that transduce changes in epithelial ion transport are largely unknown, except for acetylcholine, but may include vasoactive intestinal peptide or other peptides. These trigger changes in intracellular messengers that influence the state of ionic channels in the epithelial cells and thereby inhibit absorptive processes or stimulate secretory mechanisms. When conservation of salt and water is necessary, command signals from the central nervous system, and perhaps from the myenteric ganglia, will shut down the synaptic circuits in the submucosal ganglia and enhance the absorptive capacity of the bowel.     

Immunohistochemical analysis of proteoglycan biosynthesis during early development of the chicken cornea.

Antibodies to core proteins of chicken corneal keratan sulfate proteoglycan and chondroitin sulfate proteoglycan were prepared and purified by use of an affinity column. Using these antibodies and monoclonal antibody 5-D-4 to keratan sulfate (commercial), the localization of proteoglycans in developing corneas (Days 5 to 17 of embryonic age and 2 days after hatching) was determined immunohistochemically. Keratan sulfate proteoglycan antigen was not detected in cornea on Day 5, but it was detected uniformly over the whole stroma on Day 6, ca. 12 h after invasion of the primary stroma by mesenchymal cells. The absence of the antigen in cornea of Day 5 was confirmed by Western blotting of the corneal extract. Immunohistochemistry with 5-D-4 antibody revealed that the keratan sulfate chain was undersulfated in corneas of Days 6 to 7, because the staining was much weaker than that in cornea of Day 8. In addition, keratan sulfate proteoglycan antigen was detected uniformly over the whole stroma on Days 7 to 17 and 2 days after hatching, but not in the epithelial layer on Day 13 and after: because the epithelial layer was clearly not observed on photomicrographs until Day 13, it is not known whether keratan sulfate proteoglycan was synthesized by the epithelium during Days 6 to 12. In contrast, chondroitin sulfate proteoglycan antigen was detected in cornea on Day 5 and also, like keratan sulfate proteoglycan, uniformly over the whole stroma on Day 6 through 2 days after hatching. Furthermore, the chondroitin sulfate proteoglycan was not detected in the epithelial layer on Day 13 and after. These results show that keratan sulfate proteoglycan is synthesized by the stromal cells which invade the primary stroma between Day 5.5 and 6, while chondroitin sulfate proteoglycan is synthesized by epithelial and/or endothelial cells before the invasion, and also by the stromal cells after the invasion.     

Immunohistochemical localization of prostaglandin H synthase in the sheep placenta from early pregnancy to term.

Prostaglandin H synthase (PGHS) activity within intrauterine tissues is considered to catalyze a critical step in prostaglandin (PG) biosynthesis at parturition. In sheep, the placenta is a major site of PG production throughout pregnancy, but little information is available concerning the cells that are responsible. Therefore we determined the distribution of immunoreactive (IR-) PGHS in ovine placental tissue obtained at different times of pregnancy using immunohistochemical techniques. In placentomes from early pregnancy (Days 30-54), IR-PGHS was present in maternal epithelial syncytium, but was not detectable in trophoblast cells. Between Day 54 and Day 100, the number of cells that stained positive for PGHS declined in the maternal epithelial layer in the body of the placenta, but IR-PGHS was present in maternal epithelial cells overlying the vascular cones of the placental hemophagous zone. It was also present in the chorionic fibroblasts, but remained undetectable from all classes of trophoblast cells. IR-PGHS was first detectable in the trophoblastic epithelium by Day 114. Between Day 119 and term the trophoblast mononuclear epithelial cells were intensely immunopositive for PGHS, although immunonegative binucleate cells were present. The maternal epithelium was immunonegative except during the last 7-10 days of pregnancy when PGHS immunostaining appeared in both basal and apical regions of the placenta. Thus, the cellular localization of IR-PGHS changes during ovine pregnancy, from predominantly maternal during the first half of gestation to undetectable and then to predominantly trophoblastic between Day 114 and term, suggesting a gestation-dependent change in sites of PG production during ovine pregnancy. Appearance of IR-PGHS in the trophoblast precedes activation of the fetal hypothalamic-pituitary-adrenal axis, generally considered to provide the trigger to the onset of parturition in sheep, and would therefore appear to be regulated through alternative pathways or mechanisms.     

Isolation of fetal mouse motor neurons on discontinuous percoll density gradients

Summary The spinal cords of fetal NIH∶CR mice, gestational age Day 12 to 14, were dissected free of meninges and dorsal root ganglia, chemically dissociated, and layered onto discontinuous Percoll gradients at densities 1.040, 1.050, and 1.060 g/ml. After centrifugation (800 Xg for 15 min at 4° C), three morphologically, biochemically, and immunohistologically distinct cell populations were collected from the gradient interfaces. The first interface, located at a density of 1.040 g/ml, was choline acetyltransferase enriched (0.86±0.08) compared to the second and third fractions (0.42±0.01 and 0 pmol acetylcholine synthesized/μg protein, respectively). When simultaneously cultured with fetal mouse cardiac muscle on a gelatin-polylysine-laminin substrate in serum-free medium, these cells developed the characteristics of motor neurons. Dr. Strong is supported in part by a Research Fellowship from the Medical Research Council of Canada.     

Ontogeny of the distribution and colocalization of calbindin D28K within neural and endocrine cells of the gastrointestinal tract of fetal and neonatal sheep.

Using immunocytochemical techniques we have demonstrated that Calbindin D28K (CaBP) is present in the gastrointestinal tract of ovine fetuses early in development (by day 45). At day 45, CaBP was limited to neuronal elements in the developing intestine. By day 100, CaBP immunoreactivity was abundant in both epithelial endocrine cells and nerves of the submucous and myenteric ganglia. The location of CaBP containing cells and fibers was similar in duodenal sections taken from day 100 and term (145 days), as well as those taken from 24-48 h postnatal lambs. CaBP is colocalized in endocrine cells containing gastrin, glucagon, somatostatin and neurotensin, but not glucose dependent insulinotrophic peptide (GIP). Furthermore, it is extensively colocalized in nerve fibers and cells containing neurotensin but not somatostatin or vasoactive intestinal peptide. The colocalization of CaBP within various endocrine and nerve cells does not change in fetal sheep over the last one-third of gestation and there is no difference between fetal and neonatal sheep.     

Isolation and characterization of mouse neural precursor cells in primary culture

Summary Primary cultures of mouse neural precursor cells were established by enzymatic dissociation of embryonic Day 10 fetal heads followed by negative selection of non-neural contaminating cells. The latter were allowed to attach and spread on a plastic substrate under conditions that permitted neural precursor cells to remain suspended in the culture medium. The resulting neuroepithelial cell enriched suspension then was plated on dishes coated with poly-d-lysine. Growth of fibroblastic cells was inhibited in a selective medium. Cell proliferation was measured by immunoperoxidase staining of nuclei after bromodeoxyuridine labeling. The proportion of labeled cells declined from 50% on Day 1 until Day 5 when it approached zero, and after 7 days in culture a fourfold increase in cell number was achieved in medium containing 1% fetal bovine serum, transferrin, insulin, cholera toxin, and sodium selenite. Differentiation of neural precursor cells was studied by indirect immunofluorescence microscopy for the appearance of neuron- and astrocyte-specific cytoskeletal proteins at successive intervals in culture. Cells bearing neuritic processes and expressing neurofilaments as well as microtubule-associated protein 2 were present in low numbers on Day 1, increasing through Day 14. Stellate cells with morphologic features of astrocytes and immunoreactive for glial fibrillary acidic protein were not detected until Day 5 and did not become abundant until Day 11. No differences in morphology or immunocytochemical staining characteristics were found between neural precursor cells processed by enzymatic dissociation of whole fetal heads and those recovered by manual dissection of fetal neuroepithelia. The large number of neural precursor cells obtained by this rapid, simple method makes possible the production of mass cultures for molecular analysis of the regulatory factors that control proliferation and differentiation during early development of the mouse central nervous system. This study was entrusted to the Institute of Physical and Chemical Research (RIKEN) by the Science and Technology Agency (STA) and was financially supported by the Special Coordination Funds for Promoting Science and Technology. R. Shiurba is a recipient of a STA fellowship award from the Japanese Union of Scientists and Engineers.     

An immunohistochemical study on the cytogenesis of adenohypophysial cells in fetal rats.

An immunohistochemical study was undertaken in fetal rats to determine the time of differentiation of various types of adenohypophysial cells and the site where they first appear and proliferate during development. A cell count was carried out on the first and second days of cytodifferentiation in each type of cell. Both the time and the site of cytodifferentiation were peculiar to respective cell types. ACTH cells appeared on Day 15 of gestation in the ventral region of the pars distalis where it faces mesenchymal tissue. While TSH cells first appeared exclusively in the posterior half on Day 16, further proliferation of this cell type also occurred predominantly in the posterocentral portion of the gland. LH and FSH cells appeared on Days 17 and 19, respectively; both cell types showed a similar localization, i.e., they were almost concentrated in the ventral region in the anterior half and posteriorly they were distributed sparsely and homogeneously. GH cells appeared on Day 18 in the central region of the pars distalis. Prolactin cells failed to be seen in the fetal adenohypophysis, and even in newborns 0–1 days of age, this type of cell was not consistently seen, although sometimes a few cells were encountered in the central region of the gland. Of these cell types, TSH, LH, and FSH cells persisted to be concentrated even after birth in the area where they first appeared. These observations are discussed in relation to data previously reported by other investigators.     

Ultrasonographic and histological characterization of the placenta of somatic nuclear transfer-derived pregnancies in dairy cattle

The high incidence of pregnancy loss and prenatal morbidity and mortality in cloned animals may be due to placental insufficiency, thereby compromising fetal survival. Our objective was to characterize morphological changes in fetal membranes of cloned bovine pregnancies. Two groups of cows with cloned fetuses, produced by two cloning techniques, a commercial group (n=16) and a hand-made group (n=4), and control fetuses derived from traditional embryo transfer (n=6) or AI (n=6), were compared at various stages of gestation (Days 80, 120, 150, 180, 210, and 240; Day 0=estrus). Thickness and shape of the amniotic membrane, placentome shape and length, umbilical cord shape and diameter, and fetal fluid echodensities were assessed by ultrasonography, and the placenta was evaluated histologically. Only eight (40%) of cloned pregnancies reached term and seven calves (35%) were alive at birth. Both placentome length and umbilical cord diameter were larger (P<0.05) in clones than in normal fetuses at all stages of gestation. Amniotic membrane abnormalities (Day 120) including focal edema and the presence of a series of nodules were detected in 38% of the clones and were always accompanied by hyper-echodense spikes or irregularities (detected ultrasonographically) around the umbilical cord. Histopathology revealed degenerate inflammatory cells, edematous chorioallantoic membranes, and decreased epithelial thickness. We inferred that these morphological anomalies of placentomes compromised fetal development, and we concluded that ultrasonographic monitoring of pregnancies enabled characterization of changes in the placentae and may be useful to assess fetal well-being.     

Fluorescent Histochemistry and Cholinesterase Staining of Sympathetic Ganglia in a Teleost,Gadus morrhua

The anatomy and histochemistry of the sympathetic nervous system in the cod were studied by osmic acid staining, cholinesterase staining and fluorescent histochemistry of ganglia and nerve fibres. Large bundles of fluorescent fibres from the sympathetic ganglia in the head enter the cranial nerves and run with these. These bundles are exceptionally large to the vagi, and the cod vagi may therefore be regarded as vago-sympathetic trunks. All the sympathetic ganglion cells contain specific (acetyl-) cholinesterase, although the degree of staining was variable. The vast majority of cells in the ganglion coeliacum and other anterior ganglia show specific fluorescence of variable intensity. Ganglion cells completely devoid of specific fluorescence are scarce in the anterior ganglia, but abundant in the posterior ganglia associated with the vesicular nerve. A separate and distinct bundle of medullated fibres leaves the sympathetic chain on the left side and spreads in the wall of the left posterior cardinal vein, presumably innervating the chromaffin tissue. Similar fibres on the right side are also present, but do not form a distinct nerve.     

Nerve growth factor changes the relative levels of neuropeptides in developing sensory and sympathetic ganglia of the chick embryo

The effects of chronic nerve growth factor administration on the development of neuropeptides in the embryonic chick peripheral nervous system were quantitated by radioimmunoassays. Starting at embryonic Day 3.5, daily doses of 20 micrograms of nerve growth factor (NGF) increased the substance P content of lumbosacral spinal sensory ganglia at all ages studied (Days 10-14), while having no effect on substance P levels of thoracic sensory ganglia. In contrast, the contents of somatostatin were increased in both thoracic and lumbosacral ganglia, but only at comparatively late time points (Day 14). Nerve growth factor administration was also found to decrease the somatostatin contents of lumbosacral paravertebral sympathetic ganglia at early time points (Day 8) while increasing levels at later stages (Day 14), thus acting to accelerate the normally occurring developmental changes in level of this peptide. These changes were shown to be specific for somatostatin by demonstrating that NGF increased tyrosine hydroxylase levels in sympathetic neurons at Day 8, and had no effect on sympathetic vasoactive intestinal polypeptide levels at Day 14. It has been concluded that exogenous NGF does not simply act to increase or prolong the expression of neuron-specific phenotypes in the chick, but rather its action is time and location dependent to accelerate development.     

Structure of neuro-endocrine and neuro-epithelial interactions in human foetal pancreas

In the pancreas of many mammals including humans, endocrine islet cells can be integrated with the nervous system components into neuro-insular complexes. The mechanism of the formation of such complexes is not clearly understood. The present study evaluated the interactions between the nervous system components, epithelial cells and endocrine cells in the human pancreas. Foetal pancreas, gestational age 19–23 weeks (13 cases) and 30–34 weeks (7 cases), were studied using double immunohistochemical labeling with neural markers (S100 protein and beta III tubulin), epithelial marker (cytokeratin 19 (CK19)) and antibodies to insulin and glucagon. We first analyse the structure of neuro-insular complexes using confocal microscopy and provide immunohistochemical evidences of the presence of endocrine cells within the ganglia or inside the nerve bundles. We showed that the nervous system components contact with the epithelial cells located in ducts or in clusters outside the ductal epithelium and form complexes with separate epithelial cells. We observed CK19-positive cells inside the ganglia and nerve bundles which were located separately or were integrated with the islets. Therefore, we conclude that neuro-insular complexes may forms as a result of integration between epithelial cells and nervous system components at the initial stages of islets formation.     

Observations on the structure of the subepithelial nerve plexus in the tongue

The intrinsic innervation of the anterior two thirds of the tongue in adult dogs of both sexes was studied in paraffin sections stained with Bodian, Holmes, cholinesterase and other stains. In all the sections, a subepithelial plexus of nerve fibres and cells was always seen on the dorsum of the tongue. Nerve endings were seen extending in between the epithelial cells on the dorsum of the tongue. The nerve cells were usually spindle-shaped and collected to form numerous ganglia in the submucosa. There were other ganglia in the tongue whose structure was very much similar to terminal autonomic ganglia. The significance of the ganglia consisting of biopolar nerve cells is being discussed.